Aspergillus fumigatus (Af) are a significant cause of invasive fungal disease and lead to high morbidity and mortality in diverse populations throughout the world. Currently available antifungal drugs are often ineffective, thus contributing to unacceptably high mortality rates in patients suffering from invasive fungal infections. The use of cytokines as adjunctive immune therapies holds the promise of significantly improving patient outcomes in the future. In recent studies, we identified an essential role for type I and III interferons as regulators of optimal antifungal responses by pulmonary neutrophils during infection with Af. Although various membrane and cytosolic nucleic acid sensors are known to regulate interferon production in response to viruses, the pathways that regulate the production of these cytokines during fungal infection remain uncovered. In the current study, we demonstrate that dectin-1-mediated recognition of β-glucan on the cell wall of the clinically relevant fungal pathogen Aspergillus fumigatus promotes the activation of a protective cascade of type I and III interferon expression. We further demonstrate that exogenous administration of type I and III interferons can rescue inadequate antifungal responses in dectin-1−/− mice, suggesting the potential therapeutic benefit of these cytokines as activators of antifungal defense in the context of innate defects.Pulmonary infections with  Aspergillus fumigatus (Af) is the most common etiological agent of invasive aspergillosis and remains a challenging infection to treat  are one class of pattern recognition receptors (PRRs) important for the recognition of carbohydrate pathogen-associated molecular patterns (PAMPs) expressed on the cell wall of Aspergillus and other fungi  to harvest lungs, or at 48 h post-infection (peak of IFN-λ and ROS expression) to harvest lungs and BALs. Mice were exsanginuated and lungs were perfused with 10 ml of phosphate-buffered saline (PBS) to remove excess blood.Mice were challenged via non-invasive intratracheal delivery  to obtain single cell suspensions, then lysed to remove red blood cells. Lung suspensions were divided for analysis by flow cytometry and extraction of total lung RNA by Trizol. 48 h suspensions were also plated on saboraud agar plates by serial dilution for CFUs.Mice were treated intratracheally with 100 μg curdlan (Sigma) suspended in PBS. Lungs were harvested at 48 h for analysis.6) from BALs were incubated with 1 μM of CM-H2DCFDA (Life Technologies) in pre-warmed Hank's Balanaced Salt Solution at 37°C for 45–50 min. Afterward, cells were surface stained for 10 min and analyzed by flow cytometry.Cells  were euthanized. Lungs were perfused in PBS and fixed in 10% formalin. Fixed lungs were paraffin embedded and stained with Gomori-methenamine silver stain by the Histology Core Facility at Rutgers NJMS. Stained tissue sections were imaged at 40x magnification using a Leica confocal microscope and Surveyor software.−/− mice were infected with live CEA10 conidia as outlined above. On the day of and 1 day following infection, a cohort of dectin-1−/− were injected intraperitoneally with 1 μg each of IFN-α2 (Novoprotein) and pegylated-IFN-λ3 (PBL). Mice were then euthanized at 48 h and lungs and BALs assessed as described. For survival experiments, mice were given doses of both IFN-α2 and pegylated-IFN-λ3 on the day of infection, and then every other day up to the end point.Wild-type and dectin-1GraphPad Prism software was used to conduct all statistical analysis using non-parametric tests.Af, we challenged mice with three distinct inoculums: heat-inactivated resting conidia, swollen heat-killed (HK) conidia, or live spores. These preparations have been previously shown to vary in their levels of β-glucan exposure  and/or by cytosolic sensors STING, RIG-I, and downstream MAVS are potent activators of type I and III interferon expression  and type III (IFN-λ3) IFNs was able to enhance the function of neutrophils in monocytopenic mice (Espinosa et al., All datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by Rutgers University Institutional Animal Care and Use Committee.AR and OD conceived the project, designed the experiments, analyzed the data, and made figures and co-wrote the manuscript. OD, VE, KW, and SA performed experiments and analyzed data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The hexaploid nature of the coast redwood genome makes it very challenging to successfully assemble and annotate the coast redwood transcriptome. The de novo transcriptome assembly generated by Trinity and CLC considering only reads with a minimum length of 180 bp and contigs no less than 200 bp long resulted in 634,772 and 788,464 unigenes (unique contigs), respectively.Coast redwood is a very important endemic conifer timber species in Southern Oregon and Northern California in the USA. Due to its good wood properties and fast growth rate it can be considered as a prospective timber species also in other countries with similar or changing toward similar climatic conditions due to global climate warming, such as Germany. In general, it is frost sensitive and suffers from freezing temperatures. To study genetic mechanisms of frost resistance in this species and to select the most frost tolerant trees we tested 17 clones in climate control chamber experiments and generated two  Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12554549 Sequoia sempervirens (D. Don) Endl.) is an endemic forest tree conifer species occupying a narrow range along the Pacific Northwest coast in southern Oregon and northern California, USA. It is a valuable timber species characterized by fast growth rate and good quality wood1. The species has been planted successfully in some other countries for commercial wood production2, but in Germany it is used currently rather as a decorative, exotic species, mainly due to its sensitivity to freezing temperatures. However, some coast redwood trees survived freezing temperatures in Germany demonstrating cold-tolerance. Considering global warming and climate change, this species can be potentially considered as a prospective commercial timber species for future German sustainable forestry. To study genetic mechanisms of cold-resistance and to select frost-resistant coast redwood trees we tested replicates of 17 different coast redwood clones of diverse origin , but it is still unpublished and not annotated. Published transcriptome data are also limited3. Two transcriptome assemblies obtained in our study provide additional invaluable genomic resources and can support further coast redwood genetic studies including those concerning response of this and other conifer species to frost stress or other environmental stresses in general. We also hope that our experience with de novo sequencing, assembling and annotating the transcriptome of this difficult non-model polyploid species can help other similar studies.Coast redwood . RNA quality and integrity were assessed using the Fragment Analyzer System and standard sensitivity RNA Analysis Kit DNF-471 . All samples selected for sequencing had an RNA integrity number over 8.Needle tissue was ground in liquid nitrogen, and its RNA was extracted following the CTAB protocol of Chang 6.Only clones with sufficient quality RNA at all three temperature treatments were used for sequencing. In total, ramets of 11 clones representing all three temperature treatments resulted in 93 samples that were equimolarily pooled into one sample and sequenced at the NGS Integrative Genomics Core Unit, University Medical Center, Göttingen Fig. . A pairede novo assemblies were generated using two programs - Trinity and CLC Genomics Workbench, respectively. The Trinity assembly was normalized to a maximum read coverage of 30X. This value is less than the Trinity default of 50X, but it is in agreement with a value of at least 30, which is recommended by the authors of this algorithm in the supplement (S4) to Haas et al.7 The CLC assembly was carried out with default settings considering a minimum unigene (contig) length of 200 bp. Assemblies from both softwares showed signs of inflation and overrepresentation (Table 8 was used with default parameters to identify microsatellite loci (Summary of the microsatellite loci identified by the MISA tool in the Trinity assembly based on the filtered reads9) with di-, tri-, tetra-, penta- and hexanucleotide motifs in this Trinity assembly, and PCR primers (PCR primers designed for the microsatellite loci identified by the MISA tool in the Trinity assembly based on the filtered reads using Primer3 online tool9) were designed for these loci using the Primer3 tool.The reads were trimmed using Phred quality score 30 and base call accuracy of 99.9%. As a result, 1.35% of the reads were trimmed, which reduced the average read length by about 0.5 bp. Then, only reads with a minimum length of 180 bp were used for transcriptome assembly. Based on these reads two transcriptome blastx search with gilist taxid option for “Green plants” homologs were identified for the contigs of the Trinity assembly in the GenBank nr database. Then, the blastx output data were sorted out by the Blast2GO PRO program using the “Gene Ontology Mapping” function.Using https://identifiers.org/ncbi/insdc.sra:SRP227297). The contigs for the Trinity10 and CLC11 transcriptome assemblies have been deposited as Transcriptome Shotgun Assembly (TSA) projects at DDBJ/EMBL/NCBI GenBank under the accession numbers GIBU00000000 (https://identifiers.org/ncbi/insdc:GIBU00000000) and GIDF00000000 (https://identifiers.org/ncbi/insdc:GIDF00000000), respectively. Functional annotation of the Trinity transcriptome assembly is available as a supplementary gff file at figshare . Summary of the microsatellite loci identified by the MISA tool in the Trinity assembly based on the filtered reads is available as a supplementary excel file at figshare (Summary of the microsatellite loci identified by the MISA tool in the Trinity assembly based on the filtered reads9). PCR primers designed for these microsatellite loci using Primer3 online tool are available as a supplementary excel file at figshare (PCR primers designed for the microsatellite loci identified by the MISA tool in the Trinity assembly based on the filtered reads using Primer3 online tool9).The filtered and cleaned original RNA sequencing data have been deposited at the NCBI Sequence Read Archive under the SRA study accession SRP227297  out of total 622,955 unigenes (contigs) were mapped and 176,683 (28%) annotated; 130,013 (21%) had no blast hits, and 316,259 (51%) had hits but were not annotated  terms demonstrated that within the biological processes the most frequent were metabolic and cellular processes that were represented by more than 90 000 unigene (contig) sequences. Response to stimulus was the third most common process represented by approximately 20 000 unigene (contig) sequences  annotation revealed that annotated sequences represented mostly carbohydrate, amino acid, cofactor, and vitamin related metabolism  and embryophyta_odb10  were used as lineage-specific datasets. The results are presented in Table To test transcriptome assemblies for completeness, a search for conserved orthologous genes was done in both transcriptome assemblies using the Benchmarking Universal Single-Copy Orthologs (BUSCO) programhttps://ncbi.github.io/magicblast/). In total, 96.9% and 98.5% of transcripts in the CLC and Trinity assemblies, respectively, were mapped to the genome. It is worth noting that we tried also a few other programs such as STAR, HISAT2, exonerate, and nucmer, but they could not handle mapping the large transcriptome assemblies to the large genome, and some of these programs (for example STAR) were designed to map short reads rather than relatively long transcripts. It is hard to predict how many genes can be expected in a coast redwood genome considering its hexaploid nature. The coast redwood draft genome assembly is neither annotated nor published yet. There are also only a few conifer species  with annotated genomes . Based on these data it ranges from 47,602 in Pinus taeda to 102,915 in Picea glauca. Therefore, we can easily expect as many as 600,000 genes. However, we have to emphasize that the presented assemblies are raw de novo ones and are likely highly redundant.Transcripts from both transcriptome assemblies were mapped to the reference coast redwood genome assembly (NCBI Assembly accession number GCA_007258455.1) using magic-blast (9). Using the online software Primer3 PCR primer pairs were successfully designed for 28,285 microsatellites: 14,806 with mononucleotide motifs, 6,226 - dinucleotide, 5,601 - trinucleotide, 432 - tetranucleotide, 77 - pentanucleotide, 95 – hexanucleotide, and 1,048 compound or complex motifs (PCR primers designed for the microsatellite loci identified by the MISA tool in the Trinity assembly based on the filtered reads using Primer3 online tool9). Twenty PCR primer pairs with unique single-copy annealing sites in both Trinity transcriptome and reference genome assemblies and with similar melting temperatures for multiplexing were selected, and respective oligos were synthesized with forward primers containing the M13 tail (5′-CACGACGTTGTAAACGAC-3′) and reverse primers containing the pig-tail (5′-GTTTCTT-3′). The M13 primer were labelled either by 6-FAM or HEX . The same touch-down PCR program was used for all 20 PCR primer pairs following the protocol described in Breidenbach et al.15 The PCR products were separated and visualized using the ABI Genetic Analyser 3130xl with GENSCAN ROX 500 as an internal size standard. The primers were tested in a population sample of eight trees. Their DNA was isolated from needles or cambium using the DNeasy Plant Kit  following the manufacturer’s instructions. The isolated DNA was diluted in ddH2O 1:10 for PCR amplification and stored at −20 °C. All primer pairs amplified alleles of expected size, and 14 markers were polymorphic (20 PCR primer pairs tested9) and can be used in different applications.The MISA search of the 622,955 unigene (contig) sequences found 37,164 microsatellite loci in 31,968 sequences. Among them, 19,048 SSRs represented microsatellite loci with mononucleotide motifs, 9,795 - dinucleotide, 7,346 - trinucleotide, 669 - tetranucleotide, 132 - pentanucleotide, and 174 - hexanucleotide motifs (Summary of the microsatellite loci identified by the MISA tool in the Trinity assembly based on the filtered reads


In this study we developed a dual therapeutic metal ion-releasing nanoparticle for advanced osteogenic differentiation of stem cells. In order to enhance the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and induce angiogenesis, zinc (Zn) and iron (Fe) were synthesized together into a nanoparticle with a pH-sensitive degradation property. Zn and Fe were loaded within the nanoparticles to promote early osteogenic gene expression and to induce angiogenic paracrine factor secretion for hMSCs. In vitro studies revealed that treating an optimized concentration of our zinc-based iron oxide nanoparticles to hMSCs delivered Zn and Fe ion in a controlled release manner and supported osteogenic gene expression  with improved vascular endothelial growth factor secretion. Simultaneous intracellular release of Zn and Fe ions through the endocytosis of the nanoparticles further modulated the mild reactive oxygen species generation level in hMSCs without cytotoxicity and thus improved the osteogenic capacity of the stem cells. Current results suggest that our dual ion releasing nanoparticles might provide a promising platform for future biomedical applications. Our body contains various metal ions such as sodium ,2, magneRUNX2/OSTERIX has been reported to show upregulation with Zn ion treatment . I. I2O4 anDissolution of ZIOs was treated in a weak acidic condition (pH 4.5) and neutral condition (pH 7.0), respectively. The dissolution amount of Zn and Fe was characterized by inductively coupled plasma (ICP). In the neutral condition, the ZIOs were hardly dissolved f. When w+) cells in 5 and 20 μg/mL groups as compared to the control group  in hMSCs after ZIOs treatment within 72 h (The Zn and Fe ions safely delivered to hMSCs in intracellular manner through the endocytosis of the ZIOs played a critical role to modulate cellular behaviors. The Zn ions delivered into hMSCs with our ZIOs promoted early osteogenic differentiation of hMSCs a. The F-ter 24 h c and 72 ter 24 h d. In add2 BMP-2, d and colr groups d. There hin 72 h c,d. Howehin 72 h e. It hashin 72 h , which hVEGF from hMSCs treated with ZIOs compared to control group (0 μg/mL group, The Fe ions are known to increase the secretion of angiogenic paracrine factors in stem cells by raising the ROS level in the cells . On the In this study, we have synthesized bimetallic ZIOs under relatively mild experimental conditions and used them for biomedical applications. ZIOs were introduced to hMSCs to modulate the osteogenic differentiation. The synthesized ZIOs dissolved under weak acid conditions which mimics the pH of the endosome condition. Through our dual ion releasing ZIOs, the Zn and Fe ions were successfully delivered to hMSCs without cytotoxicity and enhanced the osteogenic differentiation and angiogenic paracrine factor secretion. The results demonstrated in our study might support the future tissue engineering for bone regeneration based on dual ion delivery system.


The timing of puberty is highly variable and is associated with long-term health outcomes. To date, understanding of the genetic control of puberty timing is based largely on studies in women. Here, we report a multi-trait genome-wide association study for male puberty timing with an effective sample size of 205,354 men. We find moderately strong genomic correlation in puberty timing between sexes (rg = 0.68) and identify 76 independent signals for male puberty timing. Implicated mechanisms include an unexpected link between puberty timing and natural hair colour, possibly reflecting common effects of pituitary hormones on puberty and pigmentation. Earlier male puberty timing is genetically correlated with several adverse health outcomes and Mendelian randomization analyses show a genetic association between male puberty timing and shorter lifespan. These findings highlight the relationships between puberty timing and health outcomes, and demonstrate the value of genetic studies of puberty timing in both sexes. Age at voice-breaking is used to determine puberty timing in men, recall of which is considered less accurate than age at first menarche in women. Here, the authors perform multi-trait GWAS for male puberty timing by including both age at voice breaking and age of first facial hair for improved phenotype definition and power. Understanding the biological mechanisms underlying such variation is an important step towards understanding why earlier puberty timing is consistently associated with higher risks for a range of later life diseases, including several cancers, cardiovascular disease and Type 2 diabetes4.The timing of puberty varies widely in populations and is determined by a broad range of environmental and genetic factors5. In contrast, genetic studies of puberty timing in men are much fewer and smaller in scale, due to lack of data in many studies on male pubertal milestones. We previously reported a GWAS for recalled age at voice breaking in men  from a single study, 23andMe, which identified 14 genetic independent genetic association signals, and many signals with similar effect sizes on voice breaking as for AAM in women6. This overlapping genetic architecture between puberty timing in males (in 23andMe) and females was also reported by others7, and supports the use of recalled age at voice breaking in men as an informative measure of puberty timing for further genetic studies.Most of our understanding of the genetic determinants of puberty timing is based on studies in women, as the age of first menstrual bleeding  is a well-recalled and widely measured marker of female sexual development. A recent large-scale genome-wide association study (GWAS) for AAM, in ∼370,000 women, identified 389 independent signals, accounting for approximately one quarter of the estimated heritability for the traitrg = 0.74) and many genetic variants show similar effects sizes in both sexes, there are a number of genetic signals that differ between sexes. Most notably, at the SIM1/MCHR2 locus, the allele that promotes earlier puberty in one sex delays it in the other6. Sex-specific expression patterns for several of such highlighted genes was reported in the hypothalamus and pituitary of pre-pubertal mice8. Further work to identify the pubertal mechanisms that are divergent between sexes may shed light on differences in later life disease risk associations between sexes.Although the overall shared genetic architecture for pubertal timing between sexes is high  study. For each of these measures, participants were asked if the event occurred relative to their peers: younger than average, about average or older than average. We previously reported that signals for AAM in women show concordant associations with dichotomised voice breaking traits in these UKBB menN = 13,226) vs. average onset ; and (2) relatively late onset  vs. average onset. These facial hair traits were in concordance with similarly dichotomised voice breaking traits defined in the same individuals, although more men reported early or late facial hair onset  than early or late voice breaking  (Supplementary Table −31) and 276  showed directionally concordant individual associations with relatively early and relatively late facial hair, respectively. Furthermore, substantially more AAM signals showed at least nominally significant associations (GWAS P < 0.05) with relatively early  or relatively late facial hair  compared to ~16 expected by chance for each outcome  relatively early onset ( relativerg) aligned to later timing of voice breaking ranged 0.57–0.91; Supplementary Table rg 0.61–0.81). These high correlations supported the rationale to combine the GWAS data across all five strata using MTAG10. This approach enables genetic data from correlated traits and overlapping samples to be combined in a single analysis, provided three key assumptions are met: (1) homogeneity of variance-covariance matrix for effect sizes for all SNPs across all traits; (2) sampling variation can be ignored and (3) sample overlap is adequately captured. Simulations involving large numbers of traits with extreme sample overlap have demonstrated that assumptions (2) and (3) may safely be made for most applications of MTAG. Violation of the assumption of homogeneity of effect sizes may be plausible in this setting if some SNPs have an effect on voice breaking and not facial hair (and vice-versa). We therefore calculated the upper bound for the false discovery rate (FDR) in our meta-analysis using the ‘maxFDR’ calculation developed by the MTAG authors. As the value for maxFDR was 7.9 × 10−4, this indicates that test statistics are unlikely to be biased due to any violation of the homogeneity assumption.We analysed four GWAS models in UKBB men : two models (early and late) for each of relative timing of voice breaking and facial hair. The shared genetic architecture between these four traits was high (genetic correlations (−8), comprising 76 independent signals  was located in/near LIN28B, consistent with previously reported studies in men and women. Of these 76 signals, 29 were not in linkage disequilibrium  with a previously reported signal for puberty timing (either AAM in women or voice breaking in men)  by using MTAG to combine data from the four dichotomous UKBB puberty timing traits all aligned to later timing of voice breaking; this approach yielded an effective GWAS meta-analysis sample size of 205,354 men for continuous pubertal timing. In this combined MTAG dataset, we identified 7,897 variants associated with male puberty timing at the genome-wide statistical significance threshold , and was associated with lower likelihood of attaining various milestones of pubertal maturation at specific time-points (Pmin (pubic hair development at age 14.7 years old): = 4.6 × 10−10, univariate model r2 ~ 1.5%)6, we observed a moderately strong genome-wide genetic correlation in pubertal timing between males and females , with similar effect estimates in both sexes for many individual variants  heterogeneity between sexes in their effects on puberty timing  . Only one signal showed significant directionally opposite effects ; rs6931884 at SIM1/PRDM13/MCHR2, as previously reported . Two variants located near to genes that are disrupted in rare disorders of puberty13 showed no effect or weaker effect in males than in females: rs184950120, 5′UTR to MKRN3 , and rs62342064, one of 3 AAM variants in/near TACR3 .Consistent with our previous studynts Fig.  and 15/3nts Fig.  showed sKDM4C (rs913588), encoding a lysine-specific demethylase, and a male-specific signal in ALX4 (rs3824915), encoding a homeobox gene involved in fibroblast growth factor (FGF) signalling that is mutated in rare disorders of cranium/central neural system (CNS) development with male-specific hypogonadism14. A further 10 lead variants were in strong LD (r2 > 0.8) with one or more non-synonymous variants, of which three have not previously been associated with puberty timing: FGF11, which encodes a FGF expressed in the developing CNS and promotes peripheral androgen receptor expression15, TFAP4, which encodes a transcription factor of the basic helix-loop-helix-zipper family16, and GCKR, which encodes a regulatory protein that inhibits glucokinase in liver and pancreatic islets and is associated with a range of cardiometabolic traits17 (Supplementary Table ). A further seven are reported signals for AAM, but were not previously reported for voice-breaking. These missense variants are in the following genes: SRD5A2, encoding for Steroid 5-alpha-reductase, which converts testosterone into the more potent androgen dihydrotestosterone; LEPR, encoding the receptor for appetite and reproduction hormone leptin; SMARCAD1, encoding a mediator of histone H3/H4 deacetylation; BDNF, FNDC9, FAM118A and ZNF446.Two of the 76 lead variants associated with male pubertal timing were non-synonymous: a previously reported AAM signal in DM4C rs91588, encoConsistent with genetic analyses of AAM in females, tissues in the CNS were the most strongly enriched for genes co-located near to male puberty timing associated variants , we assessed the broader relationship between these traits. It is known that men have darker natural hair colour than women in European ancestry populations18 and this sex difference appears following the progressive darkening of hair and skin colour during adolescence20. However, a link between inter-individual variation in natural hair colour and puberty timing has not previously been described. We assessed this phenotypic relationship in up to 179,594 white males of European ancestry in UKBB in a model including 40 genetic principal components (to adjust for even minor subpopulation ethnic variations). Men with red, dark brown and black natural hair colours showed progressively higher odds of early puberty timing, relative to men with blond hair. Similarly, women with darker natural hair colours had earlier puberty timing relative to women with blond hair . Such an approach can provide more robust and explicit evidence that there is shared biology between these two traits. The findings infer that susceptibility to darker natural hair colour also confers earlier puberty timing in men  with no evidence of heterogeneity across signals (Cochrane Q: P = 0.99) or directional pleiotropy (MR-Egger Intercept: P = 0.99) , and with modest heterogeneity across signals (Cochrane Q: P = 0.038)  approach, we modelled 119 recently reported GWAS hair colour signals as a single instrumental variableking in 2andMe menPIVW = 1.72 × 10−19) and lower risk for late voice breaking (PIVW = 5.90 × 10−6); but we found no association with AAM in women (PIVW = 0.23) . In general, early genetically predicted puberty timing in men was correlated with adverse health outcomes. We then performed MR analyses to test the genetic association with two exemplar traits which have previously been associated with puberty timing: lifespan22 and prostate cancer5.To assess the extent of shared heritability between male puberty timing and other complex traits, we calculated genome-wide genetic correlations across 751 complex traits/datasets using LD score regression23. Here, using a similar two-sample multi-variate MR approach, we did not find a significant association with prostate cancer  or advanced prostate cancer  . We used a similar MR approach to test the relevance of male puberty timing genetics to lifespan (see Methods). The findings support a genetic association between later puberty timing in males and longer lifespan, corresponding to 9 months longer life per year later puberty (IVW P = 6.7 × 10−4) , and with methods robust to the effects of heterogeneous SNPs (weighted median P = 0.02) (Supplementary Table 7).We previously reported genetic associations between earlier AAM and higher risks for hormone sensitive cancers, when adjusting for the protective effects of higher BMIn = 55,871), although still smaller than similar studies in women . While there was overall moderately strong overlap between sexes in the genetic architecture of puberty timing (rg = 0.68), several findings point to mechanisms with particular relevance to men. The genes implicated in puberty timing include, ALX4 and SRD5A2, the disruption of which leads to male pseudohermaphroditism whereas higher 5-alpha-reductase activity has been reported in women with polycystic ovary syndrome24, and INHBB encoding the beta-B subunit of the hormone Inhibin B, which is secreted by testicular Sertoli cells in males. By contrast, the known female puberty timing locus at INHBA, which encodes the beta-A subunit of the hormone Inhibin A, showed little association with puberty timing in males.Here, we report the a genetic study of male puberty timing by performing a meta-analysis across closely related phenotypic traits in two large studies. The effective sample size of 205,354 men is substantially higher than the previous GWAS in men , is cleaved into several peptides with significant melanogenic activity  by the pro-hormone convertase enzymes, PC-1 and PC-2, both of which are implicated by previously reported loci for AAM in females5. The relationship in women is more complicated suggesting that while there is a relationship between pigmentation and puberty timing in both sexes, it may act in a sex-specific manner.Non-genetic observational studies have consistently reported darkening of hair and skin pigmentation in children of European ancestry during the peri-pubertal years26. In conclusion, our findings demonstrate the utility of multi-trait GWAS to combine data across studies with related measures to provide insights into the regulation and consequences of puberty timing.Finally, our findings substantiate links between puberty timing in men and later life health outcomes, including Type 2 diabetes and hypertension. Our observed genetic association between earlier puberty timing in males and reduced lifespan was consistent across three different MR analytical approaches, which reduces the likelihood of horizontal pleiotropy (where variants influence lifespan though mechanisms separate to puberty timing). Plausible interpretations are a causal effect of earlier male puberty timing on earlier mortality or widespread horizontal pleiotropy (affecting >50% of variants for puberty timing) whereby puberty variants affect biologic pathways, which on the one hand determine puberty timing and on the other hand influence the risk of mortality. We note that horizontal pleiotropy may have been underestimated in this analysis as the same UK Biobank population were included in both exposure and outcome samples27. SNPs were excluded prior to imputation based on the following criteria: Hardy-Weinberg equilibrium P < 10−20; call rate < 95%; or a large frequency discrepancy when compared with European 1000 Genomes reference data6. Imputation of genotypes was performed using the March 2012 ‘v3’ release of 1000 Genomes reference haplotype panel. Genetic associations with puberty timing were obtained by linear regression models using age and five genetically determined principal components to account for population structure as covariates, with additive allelic effects assumed. P values for SNP associations were computed using likelihood ratio tests. Participants provided informed consent to take part in this research under a protocol approved by Ethical and Independent Review Services, an institutional review board accredited by the Association for the Accreditation of Human Research Protection Programs.Genome-wide SNP data were available in up to 55,871 men aged 18 or older of European ancestry from the 23andMe study. Age at voice breaking was determined by response to the question ‘How old were you when your voice began to crack/deepen?’ in an online questionnaire. Participants chose from one of seven pre-defined age bins . These were then re-scaled to one year age bins post-analysis by a previously validated method9. For this analysis, we limited our sample to individuals of white European ancestry. Age at voice breaking for male participants from the UK Biobank cohort study was obtained by responses to the touchscreen question ‘When did your voice break?’ Participants were required to choose from one of five possible options . For age at first facial hair, respondents were asked to choose from one of the same five options in response to the touchscreen question ‘When did you start to grow facial hair?’ In total, data were available in up to 191,270 individuals for voice breaking and 198,731 individuals for facial hair. UK Biobank received ethical approval from the NHS National Research Ethics Service North West (11/NW/0382).Genotyping for UK Biobank participants has been described in detail previouslyRespondents who answered either ‘older than average’ or ‘younger than average’ were compared separately using the ‘about average’ group as a reference in a case-control design for both phenotypes. For both voice breaking and facial hair, relatively early and relatively late effect estimates were obtained using linear mixed models which were applied using BOLT-LMM software, which accounts for cryptic population structure and relatedness. Covariates included age, genotyping chip and 10 principle components.10. MTAG uses GWAS summary statistics from multiple correlated traits to effectively increase sample size and statistical power to detect genetic associations. Full details on the methodology have been described previously10. In brief, MTAG estimates a variance-covariance matrix to correlate the effect sizes of each trait using a moments-based method, with each trait and genotype standardised to have a mean of zero and variance of one. In addition, MTAG calculates a variance-covariance matrix for the GWAS estimation error using LD score regressions. The effect estimate for the association of each SNP on the trait of interest is then derived using a moments-based function, in a generalisation of standard inverse-variance weighted meta-analysis.GWAS summary results for each of the five strata  all aligned to later timing of voice breaking were meta-analysed using MTAGPrior to meta-analysis, we removed extremely rare variants (MAF < 0.01). In addition, for the four UK Biobank strata we calculated the effective sample size usingP < 5 × 10−8 to determine significant SNP associations. Independent signals were identified using distance-based clumping, with the SNP with the lowest P-value within a 1 MB window being considered the association signal at that locus.Effective sample sizes for early and late voice breaking were 15,711 and 21,217, respectively, and 17,391 and 23,011 for early and late facial hair, respectively. We used the genome-significant P-value threshold of r2 < 0.05). Heterogeneity between AAM and VB for each SNP was determined by the I2 statistic and P-value generated by the METAL software.For each independent signal identified in the voice breaking meta-analysis, we identified all previously reported age at menarche (AAM) and voice breaking (VB) loci within 1MB of that SNP. We assessed if each locus had previously been associated with puberty timing (AAM or VB) within 1MB, and if any such loci within 1MB were in LD . Full details of recruitment, follow-up and data collection have been reported previously11. Ethical approval for the study was obtained from the ALSPAC Ethics and Law committee and the Local Research Ethics Committees. A series of nine postal questionnaires regarding pubertal development was administered approximately annually from the time the participant was aged 8 until he was aged 17. The questionnaires, which were responded by either the parents or the participant, had schematic drawings and verbal descriptions of secondary sexual characteristics  based on the Tanner staging system, as well as information on armpit hair growth and voice change. Age at voice change was considered the age at which the adolescent reported his voice to be occasionally a lot lower or to have changed completely. Weight and height were measured annually up to age 13 years, then at ages 15 and 17 years by a trained research team. Age at peak height velocity (PHV) was estimated using Superimposition by Translation And Rotation (SITAR) mixed effects growth curve analysis28. The sample size available varied according to the phenotype, from 1126  to 2403 (for age at which armpit hair started to grow).The Avon Longitudinal Study of Parents and Children (ALSPAC) recruited pregnant women resident in the Avon area of the UK with an expected delivery date between 1 April 1991 and 31 December 1992. Since then, mothers, partners, and offspring have been followed up regularly through questionnaires and clinical assessmentsThe genetic risk score (GRS) was calculated based on 73 SNPs (genotypes at 3 SNPs were unavailable) weighted by the effect size reported for that SNP in the ReproGen Consortium. The GRS was standardised, and results are presented as increase in the phenotype per standard-deviation increase in the GRS. Linear (continuous phenotype) and logistic (binary phenotype) regression analyses were performed unadjusted and adjusted for age  and controlled for population stratification using the first 10 principal components.29. In brief, genes are mapped to an index SNP based on a 150 kb window, with a regression model applied to correct the P-value (gene score) for gene size, SNP density and LD-related properties. Gene scores are ranked, and the numbers of gene scores observed in a given pathway in the 75th and 95th percentiles are calculated. A P-value for gene-set enrichment analysis (GSEA) is calculated by comparing these values to one million randomly generated gene sets. Testing was completed on 3216 pathways from four databases . Significance was determined based on an FDR < 0.05 for genes in the 75th or 95th percentile.We used MAGENTA to investigate whether genetic associations in the meta-analysed dataset showed enrichment in any known biological pathways. MAGENTA has previously been described in detailP-value of 9.4 × 10−4 (=0.05/53) to determine significance.To determine tissue-specific expression of genes, we used information from the GTEx project. GTEx characterises transcription levels of RNA in a variety of tissue and cell types, using sample from over 1000 deceased individuals of European, African-American and Asian descent. We investigated transcription levels of significant genes identified in our meta-analysis of voice breaking in 53 different tissue types. We used a conservative Bonferroni-corrected Information on natural hair colour for UK Biobank participants was collected via touchscreen questionnaire, in response to the question ‘What best describes your natural hair colour? ’. Participants chose from one of 6 possible colours: blond, red, light brown, dark brown, black or other. For our analyses, we restricted this to include only non-related individuals of white European ancestry, totalling 190,845 men and 238,179 women. Hair colours were assigned numerical values from lightest (blond) to darkest (black) in order to perform ordered logistic regression of hair colour for both relative age at voice breaking in men and AAM in women. In both cases, blond hair was used as the reference group and models were adjusted for the top 10 principle components to account for population structure. In men this produces an effect estimate as an odds ratio for early puberty , while in women the effect estimate is on a continuous scale for AAM (in years) relative to the mean AAM for those with blond hair.rg) were calculated between age of puberty in males and 751 health-related traits which were publically available from the LD Hub database using LD Score Regression30.Genetic correlations , UK Biobank Irish, and UK Biobank other white European descent. The resultant hazard ratios and their standard errors were then taken forward for two-sample MR using the 74 male puberty loci which were available in both datasets. We then performed a sensitivity analysis after removing outlier SNPs on the basis of heterogeneity using MR-PRESSO33.GWAS summary statistics for longevity were obtained from Timmers et al.34. The analyses were repeated using summary statistics from a comparison of the subset of 9,640 cases with advanced disease versus 45,704 controls, where advanced cases were defined as those with at least one of: Gleason score 8+, prostate cancer death, metastatic disease or PSA > 100. Two-sample MR analyses were conducted using weighted linear regressions of the SNP-prostate cancer log odds ratios (logOR) on the SNP-puberty beta coefficients, using the variance of the logORs as weights. This is equivalent to an inverse-variance weighted meta-analysis of the variant-specific causal estimates. Because of evidence of over-dispersion , the residual standard error was estimated, making this equivalent to a random-effects meta-analysis. Unbalanced horizontal pleiotropy was tested based on the significance of the intercept term in MR-Egger regression. The total effect of puberty timing on prostate cancer risk was separated into a direct effect  and an indirect effect (operating via BMI), as described in Burgess et al.19.Summary statistics for the association between the genetic variants and risk of prostate cancer were obtained from the PRACTICAL/ELLIPSE consortium, based on GWAS analyses of 65,044 prostate cancer cases and 48,344 controls  genotyped using the iCOGS or OncoArray chips19) using the most recently reported GWAS for hair colour18 and 23andMe data for SNP effect estimates on age at voice breaking in men, and published ReproGen consortium data on AAM in women. However, there is partial overlap  and the samples used for puberty timing . Therefore, we also performed a sensitivity analysis in non-overlapping samples; we used a more limited 5-SNP instrument for darker hair colour (identified in an earlier hair colour GWAS that did not include UK Biobank35), and assessed its effects on puberty timing in UK Biobank men and women in an individual-level MR analysis, controlling for geographical (assessment centre) and genetic ancestry factors .Genetic associations between hair colour and puberty timing were assessed in two ways. First, we performed a two-sample MR analysis based on summary statistics (as described in Burgess et al.Further information on research design is available in the Peer Review FileReporting SummarySupplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Description of Additional Supplementary Files


Some PnuC proteins are known to transport nicotinamide riboside (NR), which is a component of the ubiquitous and abundant enzyme cofactor nicotinamide adenine dinucleotide (NAD+). Thus, we speculated that the newly found “pnuC motif” RNAs might function as aptamers for a novel class of NAD+-sensing riboswitches. RNA constructs that encompass the conserved nucleotides and secondary structure features that define the motif indeed selectively bind NAD+, nicotinamide mononucleotide (NMN), and NR. Mutations that disrupt strictly conserved nucleotides of the aptamer also disrupt ligand binding. These bioinformatic and biochemical findings indicate that pnuC motif RNAs are likely members of a second riboswitch class that regulates gene expression in response to NAD+ binding.A bacterial noncoding RNA motif almost exclusively associated with  Most known riboswitch classes sense compounds that are derived from RNA monomers or their precursors . This finadA motif RNAs  and limited to the phylum Acidobacteria . This name was chosen because representatives commonly reside in the 5′ untranslated regions of open reading frames annotated as pnuC. These genes code for membrane proteins that presumably transport the nucleoside derivative nicotinamide riboside (NR)  . Thus, tpnuC motif RNAs function as riboswitches selective for NAD+ and its NR-containing fragments. RNA constructs carrying the consensus pnuC motif undergo substantial folding changes that are induced only by their cognate ligands. Biochemical analysis of analogs indicates that the aptamer selectively recognizes functional groups on the nicotinamide-derived nucleobase. This latter finding demonstrates that natural RNAs directly recognize the redox-active moiety of this coenzyme and its immediate precursors. These findings add to the number and functional diversity of bacterial RNAs that monitor the levels of the ubiquitous coenzyme NAD+.In this report, we present bioinformatic and biochemical data indicating that pnuC motif . Given the small number of highly similar examples, most nucleotide positions appear to be highly conserved. Although we predict that three base-paired substructures are formed, called P1, P1a, and a possible pseudoknot (pk), only stem P1a is supported by evidence of nucleotide covariation consistent with base-pairing. This stem also appears to carry a variable loop region whose sequence is not important for the function of the RNA. Although the proposed P1 and pseudoknot substructures lack evidence of covariation, these structures are consistent with structural probing data as described in detail in a later section.The consensus model for the uC motif A was derpnuC genes associated with the newly discovered RNA motif are annotated as transporters for NR    B. Also, n (PnuT)  and ribon (PnuT) , or haven (PnuT) . The exiStreptococcus sp. HMSC070B10) was most similar to a protein proven to selectively transport NR .To assess this possibility, we conducted an analysis of the amino acid sequences of representative transporter proteins in this collection. Multiple sequence analysis using Clustal Omega  revealedsport NR  comparedsport NR C. Simila+. Therefore, we focused our biochemical analyses on assessing the ligand binding function of NAD+ and its derivatives with constructs carrying the pnuC motif consensus.Indeed, some of the more distantly related PnuT and PnuX proteins are the products of genes occasionally found associated with riboswitches for TPP and FMN, respectively (data not shown). Thus, the specificities of the PnuC-like transporters can be predicted by the riboswitch class regulating their respective genes, and therefore it is logical that genes coding for NR transporters would be associated with a riboswitch class for NADpnuC motif present in Streptococcus parasanguinis. RNA constructs called 85 pnuC or 65 pnuC . These results are consistent with the hypothesis that PnuC proteins associated with pnuC motif representatives are most likely NR transporters and that gene expression should be regulated by a compound indicative of the status of cellular NAD+ concentration.In-line probing reactions conducted with 85 scission B, which + and NMN only begin with nucleotides forming the junction between P1 and P1a of the 85 pnuC construct  . Both constructs exhibit a pattern of bands from in-line probing reactions that are consistent with the predicted base-pairing interactions, including the pk substructure. These results suggest that the aptamer domain is confined to the most highly conserved part of the RNA. Both the nicotinamide and ribose moieties appear to carry the most important contacts for recognition by the aptamer. Moreover, the structure formed in the presence of NAD+ is consistent with a riboswitch mechanism wherein the ribosome binding site (RBS) is sequestered to suppress gene expression when ligand is bound  value for the RNA-cofactor interaction , suggesting again that the shorter construct carries the complete aptamer domain. There is no indication from this data that the riboswitch aptamer senses the adenosyl moiety of NAD+.We subjected the 65 eraction C. The fry 100 µM D. Simila+, which is the predominant biochemical form of the nicotinamide moiety, has been measured at ∼2.6 mM in Escherichia coli cells  for this coenzyme also has been published recently  and M2 (A60U) fail to respond to 1 mM NMN . The 65 pnuC RNA construct strongly discriminates against all compounds tested that carry a modification to the nicotinamide ring   B. The RNnd NaMN) . As expeSupplemental Figs. S3. S4) indicate that the aptamer of NAD+-II riboswitches selectively recognizes the nicotinamide moiety joined to a ribose via a β-N-glycosidic linkage. In-line probing assays conducted at various concentrations of ligand were used to establish KD values for the three compounds that exhibit binding activity  and NMN moieties appear to be bound by separate but similar aptamer domains arranged in tandem  were reduced to 43 unique representatives that were bounded by the nucleotides beginning near the left shoulder of P1 and the start codon. Alignments were manually prepared and analyzed to establish the consensus sequence and structural model. Given the small number of unique representatives, base-pair annotations were made if any evidence of covariation was observed, without statistical analysis.The collection of 130 pnuC riboswitch candidate (Streptococcus sp. HMSC070B10) in comparison to transporters for NR (PnuC (Nicotinamide Riboside); Haemophilus influenzae), deoxynucleosides , thiamin , and riboflavin . An unrooted phylogenetic tree was established based on the resulting sequence alignments.Clustal Omega was used to conduct comparative sequence analyses of a PnuC protein associated with the pnuC motif RNA served as the query, which was compared pairwise with all other protein sequences .Pairwise analyses of the sequences of the same proteins were also conducted by using BLASTP . A FASTASupplemental Fig. S6). Template DNAs were hybridized to single-stranded T7 RNA polymerase promoter strands in transcription reactions, which were incubated at 37°C for 2–3 h. The resulting RNA transcripts were separated by using denaturing (8 M urea) 10% polyacrylamide gel electrophoresis (PAGE). RNAs were recovered from the gel, dephosphorylated using rAPid alkaline phosphatase (Roche Applied Science), and subsequently 5′ 32P-labeled using [γ-32P]-ATP and T4 polynucleotide kinase (New England Biolabs) according to the manufacturer's protocols.RNA oligonucleotides were prepared as previously described  using th32P-labeled RNAs (trace) were incubated in the absence or presence of ligand candidates as indicated in the presence of 20 mM MgCl2, 100 mM KCl, and 50 mM Tris-HCl (pH 8.3 at ∼23°C). The reaction products were separated by denaturing (8 M urea) 10% PAGE and were visualized by using a Typhoon phosphorimager . As described previously (KD.In-line probing reactions were performed as previously described . 32P-labeviously , band inSupplemental material is available for this article.


The breadth and cross-reactivity of these responses are important against rapidly mutating RNA viruses, such as dengue (DENV), yet how viral diversity affect T cell responses and their cross-reactivity against multiple variants of the virus remains poorly defined. In this study, an integrated analysis was performed to map experimentally validated CD8+ T cell epitopes onto the distribution of DENV genome sequences across the 4 serotypes worldwide. Despite the higher viral diversity observed within HLA-I restricted epitopes, mapping of 609 experimentally validated epitopes sequences on 3985 full-length viral genomes revealed 19 highly conserved epitopes across the four serotypes within the immunogenic regions of NS3, NS4B and NS5. These conserved epitopes were associated with a higher magnitude of IFN-γ response when compared to non-conserved epitopes and were restricted to 13 HLA class I genotypes, hence providing high coverage among human populations. Phylogeographic analyses showed that these epitopes are largely conserved in most of the endemic regions of the world, and with only some of these epitopes presenting distinct mutated variants circulating in South America and Asia.This study provides evidence for the existence of highly immunogenic and conserved epitopes across serotypes, which may impact design of new universal T-cell-inducing vaccine candidates that minimise detrimental effects of viral diversification and at the same time induce responses to a broad human population.Cytotoxic CD8 The incidence of DENV infection has increased 30 fold during recent decades2 posing an alarming risk on human health. A disease which was only restricted to nine countries prior to 1970, has now become endemic in more than 125 countries in both tropics and subtropics with 70% of the cases occurring in Asia3. The majority of DENV infections are asymptomatic, and only a small proportion of cases develops more severe forms including dengue hemorrhagic fever (DHF) and dengue shock syndrome5. To date, there is no effective vaccine or antiviral therapy for dengue and treatment is limited to supportive management.Dengue virus (DENV) has become a major public health problem placing half the global population at risk with an estimated 390 million annual infections worldwide6. Like other RNA viruses, high genome replication errors result in extensive genetic diversity within each serotype7. Four serotypes (DENV1-4) are known, that share 65–75% sequence homology with each other8. It is known that humoral adaptive response is strongly associated to clearance of infection from one serotype, which also provides long lasting immunity against that particular serotype9, but limited cross-protection against the remaining serotypes10. The substantial genomic diversity across the four DENV serotypes poses a challenge in the identification of immune targets across conserved epitopes which can provide protection against multiple infections.DENV is a single-stranded positive RNA virus with a genome size of 10.7 kb consisting of three structural  and seven non-structural proteins 12. Cytotoxic CD8+ T cells are known to be associated to clearance, via the production of pro-inflammatory cytokines such as IFN-γ and TNF-α, while CD4+ T cells have been found to contribute to enhancement of B and CD8+ T-cell responses. In DENV infections, both CD4+ and CD8+ T cells have shown to generate memory phenotype following clearance14. Several studies from human infections17 and animal models20 have demonstrated a protective role for CD8+ T cell responses. CD8+ T cell responses targeting several serotype specific as well as cross-reactive antigenic epitopes have been reported with cytotoxic activity in acute phase of DENV infections22. Similarly, T cell responses targeting non-structural regions has been detected with functional activity measured by IFN-γ production after DENV vaccination24. Furthermore, high magnitude and poly-functional CD8+ T cell responses, i.e., those that upon stimulation have degranulation capacity as well as interleukin secretions  have been found to be associated with protection from DENV infections19. In addition, several class I HLA alleles, e.g. the HLA-I B*35:01 and A*33:01, have been identified conferring protection against DENV infections26.The role of cellular immune responses in determining clearance of DENV infections and long-term immunity has been investigated, however the exact contribution of T cells to immune protection remains unclear+ T cell responses to immunological protection from re-infection remains unclear. For instance, there is limited knowledge on the memory CD8+ T cell responses that can be recalled during a secondary infection, and whether these reactivated cells are functional and contribute to protection. It has been postulated that antigenic sin may somewhat limit the functional response of memory reactivated cells against a secondary infection, however these theories have been mostly based on humoral responses27. Only a limited number of studies investigated the frequency of epitope-specific CD8+ T cells that occur during primary infection and how these contribute to the control or elimination of secondary infections. Notably, the limited studies so far were based on a small sample size and a limited number of epitopes which often were serotype specific29. Therefore, better characterization of the specificity of CD8+ T cell responses and the degree of diversification within epitope targets need to be further elucidated.Despite these findings, the exact contribution of dengue specific CD8+ T cells against these targets, utilizing 609 experimentally validated epitopes and also IFN-γ ELISPOT responses from two independent large cohort studies on healthy previously exposed subjects. With these data we discovered a subset of epitopes within conserved regions of the DENV genome that were associated to a high magnitude of IFN-γ production and that were restricted to a broad range of HLA-I genotypes, thus providing high population coverage.In this study, we have employed a bioinformatics approach to estimate viral diversity from a large set of full-length viral genome sequences, and to quantify the relationship between viral diversity within epitope targets and the magnitude of CD8www.viprbrc.org, accessed in August 2018). These sequences were 1736 DENV-1, 1241 DENV-2, 816 DENV-3, and 192 DENV-4 specific and covering a 60 years time-period. The number of sequences varied across serotypes and countries. All the sequences were aligned using the Geneious aligner in the Geneious-Prime v2019.0.3) (http://www.geneious.com)30.A total of 3985 full-length DENV sequences were retrieved from Virus Pathogen Database and Analysis Resource (31. Phylogenetic trees were estimated for each serotype separately using RAxML (version 8.2.11), employing the GTR + Γ nucleotide substitution model and 100 bootstrapping replicates32.DENV nucleotide sequences for full length coding regions were aligned using MAFFT, implementing the L-INS-I algorithm33. SE values were calculated from the distribution of SNVs with a frequency of occurrence 0.1% within the sequences analysed. SE was measured per genomic region and per amino acid position across the DENV to determine both intra-serotype and inter-serotype variability, respectively. SE values were normalised by the length of the genomic region, and these values were utilised to calculate the mean values within sub-genomic regions and also within and outside epitope segments. These values were calculated for the near full‐length ORF as well as for each viral protein coding region  separately.The diversity of the DENV genomes was measured using Shannon entropy (SE) calculated from the distribution of non-synonymous and synonymous single nucleotide variants (SNV) using a bioinformatics pipeline as previously described+ T cell responses were retrieved from the Immune Epitope Data Base . A total of 609 experimentally validated MHC class I epitopes  were found with a positive IFN-γ ELISPOT assay from one or more independent experiment. The selection criteria for conserved epitopes shown in Table 38.Experimentally validated epitopes eliciting antigen specific CD839, reporting population-based studies from Sri Lanka and Nicaragua  per positive donor . If the same epitope was tested in both studies with positive SFC responses, then the mean value was considered.The SFC values were retrieved from two previous publicationswww.allelefrequencies.net. The allele frequency was calculated as the total number of copies of the allele in the population  in decimal format. When multiple HLA population coverage were available within a region e.g. across more than one country within that region of interest, then an average value was considered for each HLA type and used it as the estimate of the coverage for that specific region.Allele frequencies across human populations were retrieved from the online data base Comparative analyses between Shannon entropy distributions as well as IFN-γ responses between conserved and serotype specific epitopes were performed using a Mann–Whitney U test for unpaired data. Graphs were generated using GraphPad Prism version 7.0 .To identify the level of genomic diversification across the four DENV serotypes circulating worldwide, a total of 3985 full-length DENV genomes  were analysed, representing a wide geographical area across the globe. The majority of these sequences (76%) were sampled from Asia. 59.6% of DENV1 sequences were sampled from Southeast Asia while 58.8% of DENV2 from South America and Central/North America Fig. .Figure 1As sub-genomic regions of the genome are different in size, the proportion of genomic sites carrying synonymous and non-synonymous single nucleotide variants (SNVs) were calculated for each sub-genomic region. Synonymous SNVs accounted for the majority of the genomic diversity, with more than half of the sites presenting synonymous SNV for each serotype  per position was calculated, which is a measure of viral diversity and can be used to identify patterns of mutations across each serotype. A higher SE value indicates high genomic variability, while a SE value of zero corresponds to an invariant genomic site. Overall, the distribution of SE values corresponding to non-synonymous and synonymous SNVs were heterogeneously distributed along the genome Fig. C,D. Furt+ T cells and the overall viral diversity, SE values from the distribution of non-synonymous SNVs between epitope and non-epitope regions were analysed across the full-length genomes. For this analysis, 609 experimentally validated HLA class I epitopes were retrieved from the Immune Epitope database (IEDB)  associated with conserved epitopes . These responses were significantly higher when compared to those targeting serotype specific epitopes  (Mann Whitney U test, p < 0.0001, Fig. To assess the magnitude of the CD8Amino acid differences within the epitope sequences of the 19 conserved regions were mapped to phylogenetic trees generated from the full length DENV genomes. This analysis revealed that only a few amino acid variants were specifically circulating in distinct geographic regions, while the majority of the epitopes were relatively conserved across the world Fig. . Notably15. Twelve out of the 19 conserved epitopes were presented by HLA-B alleles, which is consistent with the larger diversity of HLA-B types among human populations compared to HLA–A types. The analysis also revealed three alleles, i.e., HLA-A*02:01, A*11:01 and A*24:02, which were among the most prevalent in Asia and Oceania.The investigation of the distribution of HLA-restrictions of the 19 epitopes revealed a broad HLA coverage among human populations, with an average worldwide coverage of 60% and 41% for the HLA-A and HLA-B, respectively Table . Within + T cell responses that elicit strong IFN-γ response and can be further investigated in future designs of vaccine targets to increase response against a broad range of viral strains and yet maintaining broad population coverage.This study revealed the presence of highly conserved HLA-I restricted epitopes across all four serotypes that elicited a stronger IFN-γ response compared to serotype specific T cell responses. Despite the surprisingly higher viral diversity within epitope regions across the four serotypes, this analysis revealed highly conserved epitopes that were presented by a broad range of HLA alleles, including some with known associations to protection. These results provide further evidence for the existence of cross-serotype viral targets for CD8+ T cell responses that are retained from primary infections and utilised as memory recall in secondary responses40. Previous studies that investigated the extent of conservation of DENV genome and its effect on the T cell responses were performed only with a limited number of full-length sequences, which could significantly impact and overestimate the presence of cross-serotype responses43. In this study, the higher IFN-γ responses identified with CD8+ T cells targeting conserved epitopes across all four DENV serotypes may be the result of repeated exposure to DENV infections across different serotypes, which result in the selection and expansion of T cell responses that are recalled from precedent infection if the viral targets remain conserved. This is indeed supported by the fact that the majority of subjects from the Sri Lanka and Nicaragua cohorts investigated in this study were healthy adult donors, thus likely to have been exposed to more than one infection during their lives. High magnitude of CD8+ T cell responses is also consistent with the hypothesis that high-fitness costs are associated with mutations within conserved regions of the genome, which impairs viral replication or transmission of the mutated strains, as it has been already demonstrated for HIV45 and hepatitis C virus47. Impaired fitness of the virus may explain the evolutionary adaptation of the virus as a result of strong IFN-γ response against those regions of the genome. For instance, studies on influenza virus revealed the existence of several epitopes targeted by CD8+ T cells that remain conserved across strains, thus suggesting that this conserved set of epitopes could be exploited for vaccine targets48.Limited data exist on the magnitude and breadth of CD849, and is supported by recent findings using genome-wide mutagenesis, which revealed that these two genomic regions along with prM and NS2A are intolerant to insertional mutagenesis50. However, the striking observation of high viral diversity within epitope regions suggest ongoing adaptation of the virus to the host T cell response. Therefore, it is conceivable that during future epidemics, immunogenic epitope regions may eventually acquire novel variants, within these conserved regions, thus impacting viral recognition.The presence of conserved regions within NS3 and NS5 is consistent with previous findings which were limited to a smaller data set51. In contrast, recent work on Takeda vaccine TAK3, which contains non-structural regions of the DENV2 has shown efficacy across all serotypes52, and notably, this vaccine has been also tested for level of T cell responses, showing high level of IFN-γ production across serotypes53. A previous study also showed that vaccination with tetravalent live attenuated vaccine induce CD8+ T cell responses against conserved epitopes of the DENV genome24. Notably, in this study only 7% of the total epitopes identified was serotype specific, and the majority were conserved across all four serotypes within one amino acid. It should be noted that this previous study however considered only a limited number of DENV sequences, and a maximum of 10 sequences per geographic region, hence it is likely that many of the conserved epitopes identified carry major variants in other population.The conserved epitopes identified in this study could be specifically tested for their role in inducing a strong cross-reactive response, thus suggesting that antigenic sin may be an important contribution, rather than limitation to immune protection. These epitopes may be also useful for designing T-cell based vaccine targets that can minimise the effect of viral diversification and at the same time cover large human populations. Recent results with Dengvaxia vaccine trials, which lack non-structural dengue specific genome regions in its construct, have not reported significant success, rather an association with severe disease15, which are also known to be associated with protection54. Even though there are no data supporting how the cellular immune responses against DENV vary according to different ethnic groups, the higher heterogeneity of HLA-B compared to HLA-A alleles55, could limit the population coverage of T-cell-inducing vaccines, as in the case of African ethnicities, which have the highest genetic diversity among human populations56. Notably, the presence of strong CD8+ T cell responses against conserved regions of the viral genome and targeted by specific HLA has been previously reported for other viruses, e.g. in HIV43 and in influenza virus57. Overall, further studies should investigate in more details the strength of T cell responses against conserved regions of the DENV genomes, and specifically investigate the set of HLA restrictions in endangered communities with rare HLA types, such as indigenous populations.The observation of strong IFN-γ responses against multiple conserved epitopes restricted by HLA-B 35:01 and A-33*01 is consistent with previous findings of high frequency and magnitude of T cell responses restricted by these allelesThere are limitations of this study that should be acknowledged. Firstly, the degree of conservation of DENV genome could be evolving due to rapid adaptation and consequent escape from host natural responses. Due to viral genomes evolving during epidemics, conserved genomic regions may eventually mutate following host responses. Secondly, this study was based on a set of more than 600 experimentally validated epitopes. Other epitopes may exist, for instance in exposed individuals during acute phase of infection, which are difficult to identify after viral clearance due to low precursor frequencies in the blood. These responses could become low frequency in the blood post clearance and remain within lymph-node and skin, which is the major site of DENV specific immune response. Future studies should investigate the extent of poly-functional responses targeting conserved epitopes soon after acute infection or in vaccine trials.+ T cell responses play a role in controlling infections and be robust to viral diversity. A better understanding of T cell responses in current vaccine trials and natural responses may be helpful to better understand protection, while cellular responses can be integrated with neutralizing and non-neutralizing B cell responses to achieve high level of protection across human populations.In conclusion, this study provided strong evidence that cross-reactive CD8Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Figure 1.Supplementary Figure 2.Supplementary Figure Legends.


Traditionally, massive, life-threatening pulmonary embolism (PE) has been treated with systemic thrombolytic therapy while submassive and smaller acute PEs have been treated with systemic anticoagulation therapy. Given that thrombolytic therapy is associated with the risk of life-threatening complications including intracranial hemorrhage, it has not been routinely used or recommended for submassive PEs. In 2017, the Food and Drug administration (FDA) approved ultrasound-facilitated catheter-directed thrombolysis (USCDT) for acute massive and sub-massive pulmonary embolism. USCDT has primarily been performed using jugular or femoral venous access. There have been isolated reports of USCDT performed through upper extremity venous access. We present a case of USCDT in a submassive PE patient with dual right upper extremity venous access where both sheaths were advanced into the basilic vein (due to anatomic variation). Based on recent clinical trial data suggesting that shorted duration USCDT is as effective as longer duration, tissue plasminogen activator (tPA) was infused in this case for 6 hours. This strategy for intervention can enhance patient comfort with USCDT therapy and can be particularly helpful in patients at high risk for access site complications and those unable to lie supine for the long duration of infusion therapy. Pulmonary embolism (PE) was mentioned in the literature as early as the 1800s and was often associated with fatal outcomes. In 1960, a trial on the efficacy of systemic anticoagulation using intravenous heparin in pulmonary embolism showed a reduced mortality rate of 17% , advancePatients presenting with sub-massive acute PE and impending right-sided heart failure usually benefit from systemic fibrinolytic therapy. Unfortunately, systemic fibrinolytic therapy has been associated with increased risk for major bleeding including intracranial hemorrhage and hemorrhagic stroke . The advWe present a 33-year-old male patient with past medical history significant for morbid obesity, hypertriglyceridemia, protein S deficiency and lower extremity deep venous thrombosis (DVT) six years prior who presented with the acute onset of severe dyspnea and substernal pressure like exertional chest pain. The pain was non-radiating, not associated with nausea, vomiting or diaphoresis and alleviated partially with rest. On physical examination, he had a blood pressure of 124/60 mmHg, pulse of 118 beats/min, respiratory rate of 12 breaths/min and oxygen saturation of 97% on 2-liter nasal cannula. He was obese and demonstrated vesicular breathing on chest auscultation, normal heart sounds without any significant murmurs on cardiac examination and chronic left lower extremity edema secondary to his old DVT. The remainder of his physical examination was unremarkable. Patient’s surgical, social, and family histories were unremarkable.Laboratory findings were significant for elevated D-dimer of 4908 ng/dL (<250 ng/dL), Troponin T less than 0.02 ng/mL (<0.04 ng/mL), partial thromboplastin time (PTT) 27.9 seconds, white blood count of 11.4 (4.5-11), hemoglobin of 13.6 g/dL (13.5-17.5 g/dL) and platelets of 237 (150-450). Blood urea nitrogen of 12 mg/dL (7-20 mg/dL) and creatinine of 0.8 mg/dL (0.8-1.2 mg/dL). The remainder of his blood work was unremarkable. An initial electrocardiogram (EKG) demonstrated normal sinus rhythm at 87 beats/min without any significant ST-T wave changes. There were no electrocardiographic signs for possible right ventricular (RV) strain. Due to a high suspicion of pulmonary embolism, the patient underwent CT pulmonary arteriography that showed large bilateral central pulmonary embolism that extended distally, principally into both the left and right lower lobe branches  catheters were placed in the right antecubital fossa, one medial and one lateral. The intention was to advance one infusion catheter via the cephalic vein and the other via the basilic vein. Limited contrast venography performed via each of the PIVs demonstrated that the medial PIV was placed in the basilic vein and the lateral PIV was in a small antecubital vein that appeared to drain more directly to the larger basilica vein. The vein connection to the relatively diminutive cephalic vein involved sharp angulation . The lateral PIV was exchanged in the same technique. There was no difficulty or resistance noted in advancing the two introducer sheaths into the right basilic vein. Through the more medial sheath we performed a right heart catheterization with a 5F Swan Ganz catheter, demonstrating right atrial pressure of 14 mmHg, pulmonary artery pressure of 55/26 mmHg and pulmonary capillary wedge pressure of 21 mmHg. The Swan Ganz catheter was exchanged over a V18 wire for a 12cm USCDT infusion catheter , placed in the left lower lobe pulmonary artery branch. This sequence of steps was repeated through the more lateral sheath with an infusion catheter placed in the right lower lobe pulmonary artery branch Figure . tPA wasUltrasound-facilitated catheter-directed thrombolysis (USCDT) is a minimally invasive technique for intravascular thrombolysis, in this case for use in the pulmonary arteries for sub-massive bilateral pulmonary emboli. The mechanism of action is based on an acoustic field created by the ultrasonic core of the catheter, leading to thrombolysis through driving the anticoagulant drug deeper into the clot and unwinding the fibrin to expose plasminogen receptor sites. USCDT treatment utilizing the Ekosonic Endovascular System has been shown to be clinically superior to anticoagulation with heparin alone in patients suffering from intermediate risk and massive PE. In both ULTIMA  and SEATCommon femoral vein or internal jugular vein access has been used for catheter-directed thrombolysis most frequently -8. In SEThere was one case reported where USCDT catheters were introduced into the superficial veins of the right upper extremity with bilateral pulmonary artery catheters advanced through the cephalic and basilic veins separately to deliver tPA for 12 hours . In our In our case due to the patient’s body habitus and obesity, we decided to proceed with upper extremity access to avoid multiple attempts in the femoral region. Due to anatomical variation of the patient right upper extremity venous system, both access sheaths were advanced into the basilic vein, without difficulty or complication. The arm was immobilized to avoid any catheter dislodgement and tPA was delivered for 6 hours with successful improvement in the patient’s symptoms.To enhance patient comfort and to limit the risk of access site complications, superficial venous access of the upper extremity through basilic and cephalic veins can be utilized for treating pulmonary embolism using thrombolytic infusion catheters. Performing venography through the peripheral intravenous catheters prior to placing the introducer sheath allows us to visualize the venous anatomy of the upper extremity prior to sheath insertion thus limiting the risk of local complications including hematoma. In addition, coupling upper extremity venous access with the strategy of using a lower dose and shorter duration of catheter directed thrombolytic therapy will enhance patient comfort and may reduce the risk of bleeding complications.


Effective feedback is an important step in the acquisition of residents’ clinical skills and a key component of most adult learning strategies. Faculty-resident feedback discussions can facilitate resident self-assessment and reflection on their performance and motivate them to study and ask questions in areas where their knowledge may be evaluated as deficient. The flipped training model approach, a type of blended learning that reverses the traditional learning environment by delivering instructional content outside of the classroom, has garnered increased support within both graduate medical education (GME) and other healthcare disciplines.The overall purpose of this exploratory pilot project was to examine the pre-post impact of a faculty feedback flipped training model course provided to a convenience sample of community-based faculty learners. After receiving campus IRB approval, the authors developed a set of five primary course goals and objectives. A convenience sample of n = 17 community-based faculty who had completed the entire course were administered a pair of pre and post-course surveys regarding their overall feedback satisfaction and comfort levels for supervising residents.In summary, five of the 13 total survey items increased at statistically significant levels from pre-course levels. The majority of qualitative faculty comments also positively evaluated the flipped training model approach.These promising pilot findings suggest that a flipped GME faculty feedback skills training model can help improve faculty learners’ satisfaction and confidence as they supervise residents and/or medical students. The impact of these types of flipped training models for GME faculty needs to be more rigorously examined in project settings with larger samples to identify what specific types of curricular activities might prove to be most effective for diverse faculty learners in GME programs across the nation. Resident physicians frequently learn through direct clinical encounters with patients under faculty supervision.Effective feedback is an important step for residents to acquire clinical skills and a key component of most adult learning strategies.Studies have indicated that the impact of faculty feedback messages is often related to the manner in which the feedback was provided.The truth is that medical trainees in graduate medical education (GME) environments more often desire honest faculty feedback. For example, a study of over 1,500 residents found that 96% of respondents believed that instructor feedback was an important part of their learning, although most residents described their having received feedback as uncommon.The flipped training model approach, a type of blended learning that reverses traditional didactic learning techniques with delivering online content before classroom activities, has garnered increased support in GME and other healthcare disciplines.The overall purpose of this exploratory pilot project was to examine the pre-post course impact of a faculty feedback flipped training model course provided to a convenience sample of community-based faculty learners.After receiving campus IRB approval, the authors, all employed by the Michigan State University Statewide Campus System (SCS)To meet the course objectives, the authors formulated faculty learner self-assessments and summative course evaluations aligned to each of the different objectives. Using Bloom’s taxonomy,Desire2Learn learning management software.The online course objectives and assessments were used to create an online course using the After the online course components were finalized, the authors met with the University of Michigan’s Center for Research on Learning and Teaching (CRLT) PlayersAfter the formulation of the online course and face-to-face workshop were complete, approximately 2,000 SCS-affiliated core faculty, program directors, and directors of medical education were emailed course promotion materials. Nineteen (0.95%) faculty completed the course registration process.To evaluate the potential of the flipped training model approach to influence pre and post-course faculty learners’ feedback satisfaction and confidence levels, the authors administered a hard copy of the earlier-validated Faculty Feedback Survey (FFS)The authors sent instructions regarding how to access the online course content. Two registrants failed to complete the online course and were not invited to the subsequent in-class workshop. Each of the remaining 17 registered participants attended the event and completed the post-course survey to measure any changes in their feedback knowledge attainment.Transgender at time of the post-course survey. Six faculty indicated that they had been supervising residents and/or medical students for between Zero and Five Years, with six reporting Six to Ten Years and the remaining respondents reporting 11 years or More. Only two (11%) faculty indicated that they had received any prior formal feedback training.A total sample of 17 faculty respondents completed over 95% of FFSNot Every Day to this survey item, with the remaining 11 (58%) of faculty indicating Not Every Day but Every 2 to 5 Days. A total of 15 (88%) of sample faculty indicated that they had generally “enjoyed providing feedback” to residents/medical students.Two (11%) faculty indicated before the course that they “rarely” ever provided such feedback to residents/students, four (21%) indicated positive feedback and/or constructive criticism to residents/students . The Mean number of barriers reported by participants was 3.94 (SD 1.89) and ranged from two to seven. Cited barriers included: a) Don’t Feel Skilled at Giving Feedback. (n = 6), b) Residents/medical students don’t accept feedback well. (n = 5), c) I don’t want to harm my rapport with residents/students. (n = 16), d) Time constraints. (n = 11), e) I don’t think of it during shift. (n = 5) and f) Lack of a Private Place toTalk. (n = 4).Each of the 17 survey respondents indicated at least two “barriers” to their providing either The analyst author (WDC) generated descriptive analyses of the working data set to evaluate appropriate analytic options and compare pre-to-post-course feedback response data. As might be readily expected from a smaller self-selected sample, he confirmed that the distribution of summary and individual FFSquality of feedback (QF), amount of feedback (AF), timeliness offeedback (TF) “summary” feedback satisfaction items, the sample pre-course means were obtained: a) QF Mean 3.47 (SD 0.624), b) AF Mean 3.18 (SD 0.728), and c) TF Mean 3.47 (SD 0.717).  to”5” (“Very Satisfied”). These initial findings demonstrated that the average course participant felt a moderate level of pre-workshop satisfaction with the feedback patterns they currently provided residents and students. .A total convenience sample of 17 faculty completed the FFSPoor”) to”5” (“Excellent”) scale). Participants’ self-rating scores each averaged in the”3” (Good) or “4” (“Very Good”) rating categories. Two remaining comfort survey items  to”8” (“Extremely Comfortable”) averaged slightly higher than the satisfaction items in the 5 or 6 categories. (Table 1).The next eight individual pre-course feedback satisfaction item ratings data were fairly similar to the summary pre-course measures,  . (Table 2)Each of the three  pre-to-post-course feedback satisfaction ratings scores were found to have increased at statistically significant levels . (Table 1). Overall, however, the majority of individual feedback satisfaction (n = 8) or comfort (n = 2) score increases failed to reach statistical significance except for one item  also increased significantly  after the workshop. (Table 2) In summary, five of the 13 total post-course survey items were shown to have increased at statistically significant levels from pre-course levels.Readers should note that only two of these eight individual survey items had complete participant data, indicating that respondents may have experienced some difficulty answering some of these items. In addition, one of the two final feedback comfort survey items with complete survey data  participating faculty reported some degree of difficulty either accessing the online course and/or navigating online course content.Eleven additional items in the post-course survey were included to ask respondents to evaluate different aspects of the on-line feedback and in-class workshop components of the curriculum. In summary, at least 64% of respondents reported either In summary, five of the 13 total post-course FFSHawthorne/observer effect since respondents knew their study responses would be reviewed by the authors.Readers should consider these initial pilot results within the context of several clear project limitations. The results came from a smaller convenience sample of 17 GME faculty who already appeared from pre-course survey responses to be fairly satisfied and/or comfortable with their resident feedback behaviors. The project was certainly likely underpowered to detect some meaningful pre-post-course sample subgroup differences  that might be detectable in a larger diverse faculty sample. The authors acknowledge that measured increases in participants’ feedback satisfaction and confidence ratings may have been somewhat skewed by some degree of These promising project findings suggest that a flipped GME faculty-training model may be useful to improve faculty feedback satisfaction and comfort when supervising residents and/or medical students. The full impact of these types of curricular models for GME faculty requires studies more rigorously examined in settings with larger samples to tease out what specific types of curricular activities might be most effective for the diverse faculty learners.In hindsight, the authors could have more aggressively tried to address and discuss perceived barriers to providing resident feedback for faculty during the online pre-course training content. The authors could have also increased in-class feedback training activities to provide faculty with more opportunities to apply this content in a variety of hypothetical scenarios.These measured improvements in faculty learners’ perceived feedback satisfaction and comfort provide evidence suggesting that the more purposeful targeting and delivery of such educational curricula may contribute to community-based GME faculty development programs. Ideally, the curricular refinements developed from future studies will lead to improvements in the feedback patterns of GME faculty supervising residents and medical students across the nation.The authors declare no conflict of interest.


The phosphorylation pattern did not change during an oral glucose tolerance test (0–120 min). We further found that human Fet A binds preferentially saturated fatty acids (>90%) at the expense of mono- and poly-unsaturated fatty acids. Our results indicate that different molecular species of Fet A are present in human plasma and that these different modifications may determine the different biological effects of Fet A.The hepatokine fetuin A (Fet A) has been associated with diverse pathological states such as insulin resistance, type 2 diabetes, macrovascular disease, and systemic ectopic and vascular calcification. Fet A may also play a role in tumor growth and metastasis. The biological activity of Fet A may be affected by various modifications, including phosphorylation, O- and N-glycosylation and fatty acid binding. We developed an antibody-based assay for the detection of Fet A phosphorylated at serine 312. Fatty acid pattern was determined by gas chromatography. Using the antibody, we found that the phosphorylation was stable in human plasma or serum at room temperature for 8 h. We observed that Fet A is present in several glycosylation forms in human plasma, but the extent of Ser Furthermore, Fet A contains two N-linked (Asp 138 and Asp 158) and two O-linked glycosylation sites (Thr 238 and Thr 252) further emphasizing the large heterogeneity of human fetuin A  1019 × l2 × mol−2; data are shown as median [interquartile range]). Untreated samples show very different glycosylation and phosphorylation patterns  to be rather a chance finding.Since a previous report  indicateTo evaluate if short-term changes in metabolism affects pFet A/Fet A ratio; we studied serum samples of 12 non-diabetic subjects during an OGTT. As shown in Free fatty acid pattern of Fet A from Sigma-Aldrich and rhFet A were analyzed as previously described . A repre312, which is the most abundantly phosphorylated site. The antibody recognized a time-dependent enzymatic dephosphorylation of pFet A as well as rephosphorylation of Fet A with FAM20C supporting the specificity of the generated antibody. Furthermore, the finding that commercial Fet A isolated from human serum could be further phosphorylated confirms a previous report that Ser312 is not fully phosphorylated [Based on previous results showing that only the phosphorylated but not unphosphorylated Fet A inhibits insulin signal transduction, we hypothesized that the serum or plasma pFet A may be the biological form of Fet A exerting its metabolic function. We therefore set out to find a method to detect and to characterize pFet A. To achieve this goal, we generated antibodies directed against Fet A phosphorylated at Serorylated . Since Forylated , we used312 consensus site in Fet A. We found, besides other candidates, glycogen synthase kinase, which however did not phosphorylate Fet A at Ser312 in an in vitro assay. Then we tested FAM20C kinase and found that this kinase phosphorylates Fet A on Ser312 time-dependently. Previous reports have indicated that FAM20C is a single kinase generating most of the secreted phospho-proteome [In our search for the physiologic kinase responsible for Fet A phosphorylation we screened for kinases with the Serproteome . FAMC20-proteome . Howeverproteome . Thus, tBefore applying our assay to human blood species, we evaluated the performance of our novel assay format. Since preanalytical issues may influence the results, we studied the stability of pFet A in different routinely used sample materials. We found that pFet A levels were unchanged in serum or EDTA plasma over time (8 h) at 0 °C and at room temperature . This fi312 phosphorylation of Fet A is associated with BMI, serum glucose, serum insulin and HOMA-IR we did not observe any significant correlation between these anthropometric and metabolic parameters and pFet A forms  the established property of Fet A, particularly phosphorylated Fet A, to inhibit insulin signaling, thus promoting insulin resistance in humans ,25. Prev312 in one assay. The assay is specific and can be used for the detailed investigation of pFet A in human serum or plasma. We found that Fet A as well as pFet A are heterogeneous in human samples due to different glycosylation, and that glycosylation does not affect phosphorylation of Fet A. Other biochemical modifications, including fatty acids bound to Fet A may be more important for determining the biological effects of Fet A, and thus, its contribution to the pathophysiology of the chronic complications of diabetes.In conclusion, we have developed a Western blot-based immunoassay for the detection and quantification of both Fet A and pFet A phosphorylated at Ser