Nuclear Run-On Assay Using Biotin Labeling, Magnetic Bead Capture and Analysis by Fluorescence-Based RT-PCR

In this report, we present a fluorescencebased approach to the assessment of cellular gene expression and transcription rates. Nuclear run-on was performed by supplying biotin-16-UTP to nuclei, and labeled transcripts were bound to streptavidin-coated magnetic beads. Total cDNA was then synthesized by means of random hexamer primed reverse transcription of captured molecules. To monitor transcript abundance in cDNA, both from nuclear run-on and total RNA, we propose a semiquantitative PCR approach based on the use of fluorescent primers

The web of the Giant: spectroscopic confirmation of a Large Scale Structure around the z=6.31 quasar SDSS J1030+0524

We report on the spectroscopic confirmation of a large scale structure around the luminous, z=6.31 QSO SDSS~J1030+0524, that is powered by a billion solar mass black hole. The structure is populated by at least six members, four Lyman Break Galaxies (LBGs) and two Lyman Alpha Emitters (LAEs). The four LBGs have been identified among a sample of 21 i-band dropouts with z{AB}<25.5 selected up to projected separations of 5 physical Mpc (15 arcmin) from the QSO. Their redshifts have been determined through up to 8hr-long multi-object spectroscopic observations at 8-10m class telescopes. The two LAEs have been identified in a 6hr VLT/MUSE observation centered on the QSO. The redshifts of the six galaxies cover the range 6.129-6.355. Assuming that peculiar velocities are negligible, this range corresponds to radial separations of +/-5 physical Mpc from the QSO, that is comparable to the projected scale of the observed LBG distribution on the sky. We conservatively estimate that this structure is significant at >3.5 sigma level, and that the level of the galaxy overdensity is at least 1.5-2 within the large volume sampled (~780 physical Mpc^3). The spectral properties of the six member galaxies (Lyalpha strength and UV luminosity) are similar to those of field galaxies at similar redshifts. This is the first spectroscopic identification of a galaxy overdensity around a super-massive black hole in the first billion years of the Universe. Our finding lends support to the idea that the most distant and massive black holes form and grow within massive (>10^{12} Msun) dark matter halos in large scale structures, and that the absence of earlier detections of such systems was likely due to observational limitations.Comment: 8 pages including Appendix, 5 figures, accepted as a letter on Astronomy & Astrophysics. v2: minor changes in Table 1 caption and Figs. 2 & 3 label

May-Hegglin Anomaly, Sebastian Syndrome, Fechtner Syndrome, and Epstein Syndrome Are not Distinct Entities but Represent a Variable Expression of a Single Illness

May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are autosomal dominant macrothrombocytopenias distinguished by different combinations of clinical and laboratory signs, such as sensorineural hearing loss, cataract, nephritis, and polymorphonuclear Döhle-like bodies. Mutations in the MYH9 gene encoding for the nonmuscle myosin heavy chain IIA (NMMHC-IIA) have been identified in all these syndromes. To understand the role of the MYH9 mutations, we report the molecular defects in 12 new cases, which together with our previous works represent a cohort of 19 families. Since no genotype-phenotype correlation was established, we performed an accurate clinical and biochemical re-evaluation of patients. In addition to macrothrombocytopenia, an abnormal distribution of NMMHC-IIA within leukocytes was observed in all individuals, including those without Döhle-like bodies. Selective, high-tone hearing deficiency and cataract was diagnosed in 83% and 23%, respectively, of patients initially referred as having May-Hegglin anomaly or Sebastian syndrome. Kidney abnormalities, such as hematuria and proteinuria, affected not only patients referred as Fechtner syndrome and Epstein syndrome but also those referred as May-Hegglin anomaly and Sebastian syndrome. These findings allowed us to conclude that May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but rather a single disorder with a continuous clinical spectrum varying from mild macrothrombocytopenia with leukocyte inclusions to a severe form complicated by hearing loss, cataracts, and renal failure. For this new nosologic entity, we propose the term "MHY9-related disease," which better interprets the recent knowledge in this field and identifies all patients at risk of developing renal, hearing, or visual defects

The double-sided of human leukocyte antigen-G molecules in type 1 autoimmune hepatitis

The immunomodulatory effects of HLA-G expression and its role in cancers, human liver infections and liver transplantation are well documented, but so far, there are only a few reports addressing autoimmune liver diseases, particularly autoimmune hepatitis (AIH). Method and materialsWe analyzed the genetic and phenotypic characteristics of HLA-G in 205 type 1 AIH patients (AIH-1) and a population of 210 healthy controls from Sardinia (Italy). ResultsAnalysis of the HLA-G locus showed no substantial differences in allele frequencies between patients and the healthy control population. The HLA-G UTR-1 haplotype was the most prevalent in both AIH-1 patients and controls (40.24% and 34.29%). Strong linkage was found between the HLA-G UTR-1 haplotype and HLA-DRB1*03:01 in AIH-1 patients but not controls (D' = 0.92 vs D' = 0.50 respectively; P = 1.3x10(-8)). Soluble HLA-G (sHLA-G) levels were significantly lower in AIH-1 patients compared to controls [13.9 (11.6 - 17.4) U/mL vs 21.3 (16.5 - 27.8) U/mL; P = 0.011]. Twenty-four patients with mild or moderate inflammatory involvement, as assessed from liver biopsy, showed much higher sHLA-G levels compared to the 28 patients with severe liver inflammation [33.5 (23.6 - 44.8) U/mL vs 8.8 (6.1 - 14.5) U/mL; P = 0.003]. Finally, immunohistochemistry analysis of 52 liver biopsies from AIH-1 patients did not show expression of HLA-G molecules in the liver parenchyma. However, a percentage of 69.2% (36/52) revealed widespread expression of HLA-G both in the cytoplasm and the membrane of plasma cells labeled with anti-HLA-G monoclonal antibodies. ConclusionThis study highlights the positive immunomodulatory effect of HLA-G molecules on the clinical course of AIH-1 and how this improvement closely correlates with plasma levels of sHLA-G. However, our results open the debate on the ambiguous role of HLA-G molecules expressed by plasma cells, which are pathognomonic features of AIH-1

Cepheid Metallicity in the Leavitt Law (C-MetaLL) survey: I. HARPS-N@TNG spectroscopy of 47 Classical Cepheid and 1 BL Her variables

Classical Cepheids (DCEPs) are the most important primary indicators of the extragalactic distance scale. Establishing the dependence on metallicity of their period--luminosity and period--Wesenheit ($PLZ$/$PWZ$) relations has deep consequences on the calibration of secondary distance indicators that lead to the final estimate of the Hubble constant (H$_0$). We collected high-resolution spectroscopy for 47 DCEPs plus 1 BL Her variables with HARPS-N@TNG and derived accurate atmospheric parameters, radial velocities and metal abundances. We measured spectral lines for 29 species and characterized their chemical abundances, finding very good agreement with previous results. We re-determined the ephemerides for the program stars and measured their intensity-averaged magnitudes in the $V,I,J,H,K_s$ bands. We complemented our sample with literature data and used the Gaia Early Data Release 3 (EDR3) to investigate the $PLZ$/$PWZ$ relations for Galactic DCEPs in a variety of filter combinations. We find that the solution without any metallicity term is ruled out at more than the 5 $\sigma$ level. Our best estimate for the metallicity dependence of the intercept of the $PLK_s$, $PWJK_s$, $PWVK_s$ and $PWHVI$ relations with three parameters, is $-0.456\pm$0.099, $-0.465\pm$0.071, $-0.459\pm$0.107 and $-0.366\pm$0.089 mag/dex, respectively. These values are significantly larger than the recent literature. The present data are still inconclusive to establish whether or not also the slope of the relevant relationships depends on metallicity. Applying a correction to the standard zero point offset of the Gaia parallaxes has the same effect of reducing by $\sim$22\% the size of the metallicity dependence on the intercept of the PLZ/PWZ relations

Redshift identification of X-ray selected active galactic nuclei in the J1030 field: searching for large-scale structures and high-redshift sources

We publicly release the spectroscopic and photometric redshift catalog of the sources detected with Chandra in the field of the $z$=6.3 quasar SDSS J1030+0525. This is currently the fifth deepest X-ray field, and reaches a 0.5-2 keV flux limit $f_{\rm 0.5-2}$=6$\times$10$^{-17}$ erg s$^{-1}$ cm$^{-2}$. By using two independent methods, we measure a photometric redshift for 243 objects, while 123 (51%) sources also have a spectroscopic redshift, 110 of which coming from an INAF-Large Binocular Telescope (LBT) Strategic Program. We use the spectroscopic redshifts to determine the quality of the photometric ones, and find it in agreement with that of other X-ray surveys which used a similar number of photometric data-points. In particular, we measure a sample normalized median absolute deviation $\sigma_{NMAD}$=1.48||$z_{phot}$-$z_{spec}$||/(1+$z_{spec}$)=0.065. We use these new spectroscopic and photometric redshifts to study the properties of the Chandra J1030 field. We observe several peaks in our spectroscopic redshift distribution between $z$=0.15 and $z$=1.5, and find that the sources in each peak are often distributed across the whole Chandra field of view. This evidence confirms that X-ray selected AGN can efficiently track large-scale structures over physical scales of several Mpc. Finally, we computed the Chandra J1030 $z>$3 number counts: while the spectroscopic completeness at high-redshift of our sample is limited, our results point towards a potential source excess at $z\geq$4, which we plan to either confirm or reject in the near future with dedicated spectroscopic campaigns

Overexpression of the Cytokine BAFF and Autoimmunity Risk

$\textbf{BACKGROUND}$: Genomewide association studies of autoimmune diseases have mapped hundreds of susceptibility regions in the genome. However, only for a few association signals has the causal gene been identified, and for even fewer have the causal variant and underlying mechanism been defined. Coincident associations of DNA variants affecting both the risk of autoimmune disease and quantitative immune variables provide an informative route to explore disease mechanisms and drug-targetable pathways. $\textbf{METHODS}$: Using case-control samples from Sardinia, Italy, we performed a genomewide association study in multiple sclerosis followed by TNFSF13B locus-specific association testing in systemic lupus erythematosus (SLE). Extensive phenotyping of quantitative immune variables, sequence-based fine mapping, cross-population and cross-phenotype analyses, and gene-expression studies were used to identify the causal variant and elucidate its mechanism of action. Signatures of positive selection were also investigated. $\textbf{RESULTS}$: A variant in TNFSF13B, encoding the cytokine and drug target B-cell activating factor (BAFF), was associated with multiple sclerosis as well as SLE. The disease-risk allele was also associated with up-regulated humoral immunity through increased levels of soluble BAFF, B lymphocytes, and immunoglobulins. The causal variant was identified: an insertion-deletion variant, GCTGT→A (in which A is the risk allele), yielded a shorter transcript that escaped microRNA inhibition and increased production of soluble BAFF, which in turn up-regulated humoral immunity. Population genetic signatures indicated that this autoimmunity variant has been evolutionarily advantageous, most likely by augmenting resistance to malaria. $\textbf{CONCLUSIONS}$: A TNFSF13B variant was associated with multiple sclerosis and SLE, and its effects were clarified at the population, cellular, and molecular levels. (Funded by the Italian Foundation for Multiple Sclerosis and others.).Supported by grants (2011/R/13 and 2015/R/09, to Dr. Cucca) from the Italian Foundation for Multiple Sclerosis; contracts (N01-AG-1-2109 and HHSN271201100005C, to Dr. Cucca) from the Intramural Research Program of the National Institute on Aging, National Institutes of Health (NIH); a grant (FaReBio2011 “Farmaci e Reti Biotecnologiche di Qualità,” to Dr. Cucca) from the Italian Ministry of Economy and Finance; a grant (633964, to Dr. Cucca) from the Horizon 2020 Research and Innovation Program of the European Union; a grant (U1301.2015/AI.1157.BE Prat. 2015-1651, to Dr. Cucca) from Fondazione di Sardegna; grants (“Centro per la ricerca di nuovi farmaci per malattie rare, trascurate e della povertà” and “Progetto collezione di composti chimici ed attività di screening,” to Dr. Cucca) from Ministero dell’Istruzione, dell’Università e della Ricerca; grants (HG005581, HG005552, HG006513, and HG007022, to Dr. Abecasis) from the National Human Genome Research Institute; a grant (9-2011-253, to Dr. Todd) from JDRF; a grant (091157, to Dr. Todd) from the Wellcome Trust; a grant (to Dr. Todd) from the National Institute for Health Research (NIHR); and the NIHR Cambridge Biomedical Research Centre. Dr. Idda was a recipient of a Master and Back fellowship from the Autonomous Region of Sardinia

Population- and individual-specific regulatory variation in Sardinia

Genetic studies of complex traits have mainly identified associations with noncoding variants. To further determine the contribution of regulatory variation, we combined whole-genome and transcriptome data for 624 individuals from Sardinia to identify common and rare variants that influence gene expression and splicing. We identified 21,183 expression quantitative trait loci (eQTLs) and 6,768 splicing quantitative trait loci (sQTLs), including 619 new QTLs. We identified high-frequency QTLs and found evidence of selection near genes involved in malarial resistance and increased multiple sclerosis risk, reflecting the epidemiological history of Sardinia. Using family relationships, we identified 809 segregating expression outliers (median z score of 2.97), averaging 13.3 genes per individual. Outlier genes were enriched for proximal rare variants, providing a new approach to study large-effect regulatory variants and their relevance to traits. Our results provide insight into the effects of regulatory variants and their relationship to population history and individual genetic risk.M.P. is supported by the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement 633964 (ImmunoAgeing). Z.Z. is supported by the National Science Foundation (NSF) GRFP (DGE- 114747) and by the Stanford Center for Computational, Evolutionary, and Human Genomics (CEHG). Z.Z., J.R.D., and G.T.H. also acknowledge support from the Stanford Genome Training Program (SGTP; NIH/NHGRI T32HG000044). J.R.D. is supported by the Stanford Graduate Fellowship. K.R.K. is supported by Department of Defense, Air Force Office of Scientific Research, National Defense Science and Engineering Graduate (NDSEQ) Fellowship 32 CFR 168a. S.J.S. is supported by the NIHR Cambridge Biomedical Research Centre. The SardiNIA project is supported in part by the intramural program of the National Institute on Aging through contract HHSN271201100005C to the Consiglio Nazionale delle Ricerche of Italy. The RNA sequencing was supported by the PB05 InterOmics MIUR Flagship grant; by the FaReBio2011 “Farmaci e Reti Biotecnologiche di Qualità” grant; and by Sardinian Autonomous Region (L.R. no. 7/2009) grant cRP3-154 to F. Cucca, who is also supported by the Italian Foundation for Multiple Sclerosis (FISM 2015/R/09) and by the Fondazione di Sardegna (ex Fondazione Banco di Sardegna, Prot. U1301.2015/AI.1157.BE Prat. 2015-1651). S.B.M. is supported by the US National Institutes of Health through R01HG008150, R01MH101814, U01HG007436, and U01HG009080. All of the authors would like to thank the CRS4 and the SCGPM for the computational infrastructure supporting this project

Fermentation process of apple juice investigated by NMR spectroscopy

Cider is a slightly alcoholic beverage and is an important and promising segment of the fruit industry. In this study, a still cider produced by fermentation of apples juice was investigated. A simultaneous characterization of bioactive compounds in apple juice and the intermediate products of the cidermaking process was performed for the first time using Nuclear Magnetic Resonance spectroscopy. Alongside sugar consumption, a progressive increase in trigonelline, chalcone, fumarate, caffeic acid, uracil, tyrosol and xanthine content was detected during the alcoholic fermentation process. Of note was the relatively high tyrosol content which imparts health benefits in the cider. The concomitant changes in phenol, amino acid and organic acid (malic, lactic, quinic, pyruvic, citric and succinic) content, was important when monitoring the fermentation process and assessing quality control of the final product

NMR-based metabolic profiling of different yeast fermented apple juices

NMR based metabolic profiling was used to investigate metabolic changes in ciders fermented by yeast species as valuable tool for the identification of metabolites responsible for yeast activities. 1H NMR spectra of apple ciders produced by Saccharomyces cerevisiae EC1118, Torulaspora delbrueckii TD291, Starmerella bacillaris YR21, Hanseniaspora osmophila HO16, Hanseniaspora uvarum Y4M, and Saccharomyces uvarum SU3, revealed to be dominated by glycerol and organic acids signals. The aromatic region showed the presence of low intense signals referred to epicatechin, catechin, chlorogenate, xanthine, uracil, tyrosol, fumarate, histamine, and histidine. The anomeric region showed signals related to xylose, arabinose, glucose, fructose and sucrose. Finally, in the aliphatic region, signals due to amino acids like alanine, aspartate, asparagine, isoleucine and threonine, organic acids like acetate, lactate, malate, pyruvate, quinate, succinate, and alcohols like 2,3 butanediol, ethanol, and glycerol and sterol were detected. Principal component analysis performed considering NMR data from either all spectral regions and only aromatic region revealed the potentiality to discriminate the yeast action. Signals due to glucose, fructose, glycerol, malate, fumarate, tyrosol, histidine, and histamine resulted discriminant for sample differentiation. This study, the first one by NMR, provides preliminary insights on the metabolic differences among yeast species involved in cider production