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Biochemistry
MaxQuant.Live enables global targeting of more than 25,000 peptides.
Feb 13, 2019   Molecular & Cellular Proteomics : MCP
Wichmann C, Meier F, Virreira Winter S, Brunner AD, Cox J, Mann M
MaxQuant.Live enables global targeting of more than 25,000 peptides.
Feb 13, 2019
Molecular & Cellular Proteomics : MCP
Mass spectrometry (MS)-based proteomics is often performed in a shotgun format, in which as many peptide precursors as possible are selected from full or MS1 scans so that their fragment spectra can be recorded in MS2 scans. While achieving great proteome depths, shotgun proteomics cannot guarantee that each precursor will be fragmented in each run. In contrast, targeted proteomics aims to reproducibly and sensitively record a restricted number of precursor/fragment combinations in each run, based on pre-scheduled mass-to-charge and retention time windows. Here we set out to unify these two concepts by a global targeting approach in which an arbitrary number of precursors of interest are detected in real-time, followed by standard fragmentation or advanced peptide-specific analyses. We made use of a fast application programming interface to a quadrupole Orbitrap instrument and real-time recalibration in mass, retention time and intensity dimensions to predict precursor identity. MaxQuant.Live is freely available (www.maxquant.live) and has a graphical user interface to specify many pre-defined data acquisition strategies. Acquisition speed is as fast as with the vendor software and the power of our approach is demonstrated with the acquisition of breakdown curves for hundreds of precursors of interest. We also uncover precursors that are not even visible in MS1 scans, using elution time prediction based on the auto-adjusted retention time alone. Finally, we successfully recognized and targeted more than 25,000 peptides in single LC-MS runs. Global targeting combines the advantages of two classical approaches in MS-based proteomics, while greatly expanding the analytical toolbox.
Pre-surgical neoadjuvant oncolytic virotherapy confers protection against rechallenge in a murine model of breast cancer.
Feb 13, 2019   Scientific Reports
Martin NT, Roy DG, Workenhe ST, van den Wollenberg DJM, Hoeben RC, Mossman KL, Bell JC, Bourgeois-Daigneault MC
Pre-surgical neoadjuvant oncolytic virotherapy confers protection against rechallenge in a murine model of breast cancer.
Feb 13, 2019
Scientific Reports
The use of oncolytic viruses (OVs) for cancer treatment is emerging as a successful strategy that combines the direct, targeted killing of the cancer with the induction of a long-lasting anti-tumor immune response. Using multiple aggressive murine models of triple-negative breast cancer, we have recently demonstrated that the early administration of oncolytic Maraba virus (MRB) prior to surgical resection of the primary tumor is sufficient to minimize the metastatic burden, protect against tumor rechallenge, cure a fraction of the mice and sensitize refractory tumors to immune checkpoint blockade without the need for further treatment. Here, we apply our surgical model to other OVs: Vesicular stomatitis virus (VSV), Adenovirus (Ad), Reovirus (Reo) and Herpes simplex virus (HSV) and show that all of the tested OVs could positively change the outcome of the treated animals. The growth of the primary and secondary tumors was differently affected by the various OVs and most of the viruses conferred survival benefits in this neoadjuvant setting despite the absence of direct treatment following rechallenge. This study establishes that OV-therapy confers long-term protection when administered in the pre-operative window of opportunity.
Tumor infiltrating mast cells determine oncogenic HIF-2α-conferred immune evasion in clear cell renal cell carcinoma.
Feb 13, 2019   Cancer Immunology, Immunotherapy : CII
Xiong Y, Liu L, Xia Y, Qi Y, Chen Y,   . . . . . .   , Bai Q, Zhang W, Yang Y, Guo J, Xu J
Tumor infiltrating mast cells determine oncogenic HIF-2α-conferred immune evasion in clear cell renal cell carcinoma.
Feb 13, 2019
Cancer Immunology, Immunotherapy : CII
PURPOSE: Hypoxia-inducible factor 2α (HIF-2α) overexpression leads to activation of angiogenic pathways. However, little is known about the association between HIF-2α expression and anti-tumor immunity in clear cell renal cell carcinoma (ccRCC). We aimed to explore how HIF-2α influenced the microenvironment and the underlying mechanisms. EXPERIMENTAL DESIGN: We immunohistochemically evaluated immune cells infiltrations and prognostic value of HIF-2α expression in a retrospective Zhongshan Hospital cohort of 280 ccRCC patients. Fresh tumor samples, non-tumor tissues and autologous peripheral blood for RT-PCR, ELISA and flow cytometry analyses were collected from patients who underwent nephrectomy in Zhongshan Hospital from September 2017 to April 2018. The TCGA KIRC cohort and SATO cohort were assessed to support our findings. RESULTS: We demonstrated that ccRCC patients with HIF-2αhigh tumors exhibited reduced overall survival (p = 0.025) and recurrence-free survival (p < 0.001). Functions of CD8+ T cells were impaired in HIF-2αhigh patients. In ccRCC patients, HIF-2α induced the expression of stem cell factor (SCF), which served as chemoattractant for mast cells. Tumor infiltrating mast cells impaired anti-tumor immunity partly by secreting IL-10 and TGF-β. HIF-2α mRNA level adversely associated with immunostimulatory genes expression in KIRC and SATO cohorts. CONCLUSIONS: HIF-2α contributed to evasion of anti-tumor immunity via SCF secretion and subsequent recruitment of mast cells in ccRCC patients.
Architectural principles for Hfq/Crc-mediated regulation of gene expression.
Feb 13, 2019   ELife
Pei XY, Dendooven T, Sonnleitner E, Chen S, Blasi U, Luisi BF
Architectural principles for Hfq/Crc-mediated regulation of gene expression.
Feb 13, 2019
ELife
In diverse bacterial species, the global regulator Hfq contributes to post-transcriptional networks that control expression of numerous genes. Hfq of the opportunistic pathogen Pseudomonas aeruginosa inhibits translation of target transcripts by forming a regulatory complex with the catabolite repression protein Crc. This repressive complex acts part of an intricate mechanism of preferred nutrient utilisation. We describe high-resolution cryo-EM structures of the assembly of Hfq and Crc bound to the translation initiation site of a target mRNA. The core of the assembly is formed through interactions of two cognate RNAs, two Hfq hexamers and a Crc pair. Additional Crc protomers are recruited to the core to generate higher-order assemblies with demonstrated regulatory activity in vivo. This study reveals how Hfq cooperates with a partner protein to regulate translation, and provides a structural basis for an RNA code that guides global regulators to interact cooperatively and regulate different RNA targets.
Reactivity Profiles of Diazo Amides, Esters, and Ketones in Transition-Metal-Free C-H Insertion Reactions.
Feb 13, 2019   Journal Of The American Chemical Society
Cleary SE, Li X, Yang LC, Houk KN, Hong X, Brewer M
Reactivity Profiles of Diazo Amides, Esters, and Ketones in Transition-Metal-Free C-H Insertion Reactions.
Feb 13, 2019
Journal Of The American Chemical Society
Vinyl cations derived from diazo ketones participate in transition-metal-free C-H insertion reactions, but the corresponding amide and ester analog exhibit divergent reactivity profiles. Whereas cations formed from diazo ketones undergo a rearrangement and C-H insertion sequence, those from diazo amides do so less efficiently and tend to be competitively trapped before the insertion step occurs. Diazo esters undergo several rearrangement steps and fail to insert. DFT calculations reveal that this disparity stems from two factors: differing levels of electrostatic stabilization of the initially formed vinyl cation by the adjacent carbonyl oxygen and predistortion of the ketone and amide systems toward C-H insertion. The computational data is in strong agreement with experimental results, and this study explains how structural and electronic factors determine the outcome of reactions of diazo carbonyl-derived vinyl cations.
3d-Metal Induced Magnetic Ordering on U(IV) Atoms as a Route toward U(IV) Magnetic Materials.
Feb 15, 2019   Journal Of The American Chemical Society
Klepov VV, Pace KA, Calder S, Felder JB, Loye HZ
3d-Metal Induced Magnetic Ordering on U(IV) Atoms as a Route toward U(IV) Magnetic Materials.
Feb 15, 2019
Journal Of The American Chemical Society
Uranium(IV) 5f2 magnetism is dominated by a transition from a triplet to a singlet ground state at low temperatures. For the first time, we achieved magnetic ordering of U(IV) atoms in a complex fluoride through the incorporation of 3 d transition metal cations. This new route allowed us to obtain an unprecedented series of U(IV) ferrimagnetic materials of the new composition Cs2MU3F16 (M = Mn2+, Co2+, and Ni2+), which were comprehensively characterized with respect to their structural and magnetic properties. Magnetic susceptibility measurements revealed ferromagnetic-like phase transitions at temperatures of ∼14.0, 3.5, and 4.8 K for M = Mn2+, Co2+, and Ni2+, respectively. The transition is not observed when the magnetic M cations are replaced by a diamagnetic cation, Zn2+. Neutron diffraction measurements revealed the magnetic moments of 0.91(6)-1.97(3) μB on the U atoms, which are only partially compensated by antiparallel moments of 1.53(14)-3.26(5) μB on the 3 d cations. This arrangement promotes suppression of the transition to a diamagnetic ground state characteristic of U(IV), and in doing so, induces magnetic ordering on uranium via 3 d-5 f exchange coupling.
Gene expression variability in human skeletal muscle transcriptome responses to acute resistance exercise.
Feb 13, 2019   Experimental Physiology
Bonafiglia JT, Menzies KJ, Gurd BJ
Gene expression variability in human skeletal muscle transcriptome responses to acute resistance exercise.
Feb 13, 2019
Experimental Physiology
NEW FINDINGS: What is the central question of this study? Does exercise, independent of random error and within-subject variability, contribute to the variability in gene expression responses to an acute bout of resistance exercise? What is the main finding and its importance? A reanalysis of publicly-available microarray data revealed that variability in observed gene expression responses for a subset of genes could be partially attributable to an effect of acute resistance exercise. These finding support the notion that individual responsiveness explains a portion of the variability in observed gene expression responses to acute resistance exercise. ABSTRACT: The purpose of this study was to use publicly available transcriptomic data to determine whether variability in gene expression responses to an acute bout of resistance exercise (AR) can be attributable to an effect AR per se. We examined microarray data from a previous study that collected skeletal muscle biopsies before and 24 hours after AR or a no-exercise time-matched control period (CTL). By subtracting the standard deviation (SD) in the observed responses to CTL from AR, we determined that AR contributed to the variability in the observed gene expression responses for many (∼31,000), but not all, transcripts included on the Affymetrix Human Genome chips. AR had a large effect on variability in the observed gene expression responses in 1,290 genes that was not attributed to any technical/biological variability associated with repeated measurements. Pathway analysis using WebGestalt revealed that several of these 1,290 genes are involved in pathways known to regulate skeletal muscle adaptations to chronic resistance training (CR). These results suggest that variability in the observed gene expression responses for a subset of genes could be partially attributable to an effect of AR. This article is protected by copyright. All rights reserved.
Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress.
Feb 13, 2019   The Plant Journal : For Cell And Molecular Biology
Luria G, Rutley N, Lazar I, Harper JF, Miller G
Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress.
Feb 13, 2019
The Plant Journal : For Cell And Molecular Biology
Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorofluorescin diacetate (i.e. H2 DCFDA-staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into 'low-ROS' and 'high-ROS' subpopulations. Pollen germination assays following fluorescence-activated cell sorting (FACS) revealed that the high-ROS pollen germinated with a frequency that was 35-fold higher than the low-ROS pollen, supporting a model in which a significant fraction of a flower's pollen remain in a low-metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose-dependent alterations in DCF-fluorescence in response to oxidative stress or antioxidant treatments. Heat stress treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry-based approaches to investigate metabolic changes during stress responses in pollen. This article is protected by copyright. All rights reserved.
The histone demethylase PHF8 facilitates alternative splicing of the histocompatibility antigen HLA-G.
Feb 13, 2019   FEBS Letters
Leisegang MS, Gu L, Preussner J, Günther S, Hitzel J, Ratiu C, Weigert A, Chen W, Schwarz EC, Looso M, Fork C, Brandes RP
The histone demethylase PHF8 facilitates alternative splicing of the histocompatibility antigen HLA-G.
Feb 13, 2019
FEBS Letters
Histone3-Lysine9 (H3K9) residues not only control gene expression, but also contribute to RNA splicing. Here, the H3K9 histone demethylase PHF8 was investigated in endothelial cells for its involvement in alternative splicing. An angiogenic sprouting assay shows the importance of PHF8 for endothelial cells. Immunoprecipitation reveals that PHF8 interacts with U1 spliceosomal proteins, such as SRPK1 and snRNP70. We identify the histocompatibility antigen HLA-G as a target of PHF8. The inclusion of HLA-G intron-4, with concomitant RNA Polymerase II accumulation at this intron, is controlled by PHF8 and H3K9. Soluble-HLA-G is generated after PHF8 knockdown, which leads to reduced T cell proliferation. Collectively, PHF8 knockdown generates the immunosuppressive alternative splice product soluble-HLA-G, which is secreted by endothelial cells to elicit a potential inhibitory effect on inflammation. This article is protected by copyright. All rights reserved.
In vitro activities of antimicrobial peptides and ceragenins against Legionella pneumophila.
Feb 13, 2019   The Journal Of Antibiotics
Birteksoz-Tan AS, Zeybek Z, Hacioglu M, Savage PB, Bozkurt-Guzel C
In vitro activities of antimicrobial peptides and ceragenins against Legionella pneumophila.
Feb 13, 2019
The Journal Of Antibiotics
Legionella pneumophila is a waterborne intracellular pathogenic bacterium, the most frequent cause of human legionellosis and a relatively common cause of community-acquired and nosocomial pneumonia. Some legionellosis outbreaks are related to the presence of biofilms, which provide a reservoir for L. pneumophila strains. We investigated the in vitro activities of antibiotics; erythromycin and doxycycline, antimicrobial peptides AMPs; melittin, LL-37 and CAMA (cecropin A (1-7)-Melittin A (2-9) and ceragenins; CSA-8, CSA-13, CSA-44, CSA-131 and CSA-138 against L. pneumophila. Isolation of Legionella strains was conducted according to ISO 1998. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and minimum biofilm eradication concentrations (MBECs) were determined using microbroth dilution techniques. MIC ranges for melittin, LL-37, and CAMA were 0.25-1, 1-4, and 2-8 µg ml-1, respectively. MIC ranges for CSA-8, 13, 44, 131, and 138 were 0.5-2, 0.5-1, 1-4, 0.5-2, and 1-2 µg ml-1, respectively, and MBEC values for the ceragenins were 10-160 µg ml-1. These results demonstrate that AMPs and ceragenins display broad-spectrum, in vitro activity against L. pneumophila. In particular, CSA-8, CSA-13 and melittin gave the lowest MICs and MBCs. We also observed that ceragenins are active against established L. pneumophila biofilms.
CFP suppresses breast cancer cell growth by TES-mediated upregulation of the transcription factor DDIT3.
Feb 13, 2019   Oncogene
Block I, Müller C, Sdogati D, Pedersen H, List M,   . . . . . .   , Thomassen M, Kruse TA, Karlskov Hansen SW, Kioschis P, Mollenhauer J
CFP suppresses breast cancer cell growth by TES-mediated upregulation of the transcription factor DDIT3.
Feb 13, 2019
Oncogene
Breast cancer is a heterogeneous genetic disease driven by the accumulation of individual mutations per tumor. Whole-genome sequencing approaches have identified numerous genes with recurrent mutations in primary tumors. Although mutations in well characterized tumor suppressors and oncogenes are overrepresented in these sets, the majority of the genetically altered genes have so far unknown roles in breast cancer progression. To improve the basic understanding of the complex disease breast cancer and to potentially identify novel drug targets or regulators of known cancer-driving pathways, we analyzed 86 wild-type genes and 94 mutated variants for their effect on cell growth using a serially constructed panel of MCF7 cell lines. We demonstrate in subsequent experiments that the metal cation transporter CNNM4 regulates growth by induction of apoptosis and identified a tumor suppressive role of complement factor properdin (CFP) in vitro and in vivo. CFP appears to induce the intracellular upregulation of the pro-apoptotic transcription factor DDIT3 which is associated with endoplasmic reticulum-stress response.
Destabilization of AETFC through C/EBPα-mediated repression of LYL1 contributes to t(8;21) leukemic cell differentiation.
Feb 13, 2019   Leukemia
Zhang MM, Liu N, Zhang YL, Rong B, Wang XL,   . . . . . .   , Roeder RG, Lan F, Wang L, Huang QH, Sun XJ
Egg White Hydrolysate as a functional food ingredient to prevent cognitive dysfunction in rats following long-term exposure to aluminum.
Feb 13, 2019   Scientific Reports
Martinez CS, Alterman CDC, Vera G, Márquez A, Uranga JA, Peçanha FM, Vassallo DV, Exley C, Mello-Carpes PB, Miguel M, Wiggers GA
Egg White Hydrolysate as a functional food ingredient to prevent cognitive dysfunction in rats following long-term exposure to aluminum.
Feb 13, 2019
Scientific Reports
Aluminum (Al), which is omnipresent in human life, is a potent neurotoxin. Here, we have tested the potential for Egg White Hydrolysate (EWH) to protect against changes in cognitive function in rats exposed to both high and low levels of Al. Indeed, EWH has been previously shown to improve the negative effects induced by chronic exposure to heavy metals. Male Wistar rats received orally: Group 1) Low aluminum level (AlCl3 at a dose of 8.3 mg/kg b.w. during 60 days) with or without EWH treatment (1 g/kg/day); Group 2) High aluminum level (AlCl3 at a dose of 100 mg/kg b.w. during 42 days) with or without EWH treatment (1 g/kg/day). After 60 or 42 days of exposure, rats exposed to Al and EWH did not show memory or cognitive dysfunction as was observed in Al-treated animals. Indeed, co-treatment with EWH prevented catalepsy, hippocampal oxidative stress, cholinergic dysfunction and increased number of activated microglia and COX-2-positive cells induced by Al exposure. Altogether, since hippocampal inflammation and oxidative damage were partially prevented by EWH, our results suggest that it could be used as a protective agent against the detrimental effects of long term exposure to Al.
Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1.
Feb 13, 2019   The Journal Of Cell Biology
Bass TE, Cortez D
Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1.
Feb 13, 2019
The Journal Of Cell Biology
The ATR kinase controls cell cycle transitions and the DNA damage response. ATR activity is regulated through two ATR-activating proteins, ETAA1 and TOPBP1. To examine how each activator contributes to ATR signaling, we used quantitative mass spectrometry to identify changes in protein phosphorylation in ETAA1- or TOPBP1-deficient cells. We identified 724, 285, and 118 phosphosites to be regulated by TOPBP1, ETAA1, or both ATR activators, respectively. Gene ontology analysis of TOPBP1- and ETAA1-dependent phosphoproteins revealed TOPBP1 to be a primary ATR activator for replication stress, while ETAA1 regulates mitotic ATR signaling. Inactivation of ATR or ETAA1, but not TOPBP1, results in decreased Aurora B kinase activity during mitosis. Additionally, ATR activation by ETAA1 is required for proper chromosome alignment during metaphase and for a fully functional spindle assembly checkpoint response. Thus, we conclude that ETAA1 and TOPBP1 regulate distinct aspects of ATR signaling with ETAA1 having a dominant function in mitotic cells.
Multilayer fabrication of unobtrusive poly(dimethylsiloxane) nanobrush for tunable cell adhesion.
Feb 13, 2019   Scientific Reports
Chae SS, Jung JH, Choi WJ, Park JK, Baik HK, Jung J, Ko HW
Multilayer fabrication of unobtrusive poly(dimethylsiloxane) nanobrush for tunable cell adhesion.
Feb 13, 2019
Scientific Reports
Precise modulation of polymer brush in its thickness and grafting density can cause unexpected cell behaviors and regulated bioactivities. Herein, a nanoscale poly(dimethylsiloxane) (PDMS) brush was employed to use as a controllable material for cell adhesion. Facile fabrication of ultrathin monolayer PDMS nanobrush on an underlying substrate facilitated regaining cell adhesion through long-range cell attractive forces such as the van der Waals forces. We showed that cell adhesion is diminished by increasing the number of nanobrush layers, causing a gradual decrease of the effectiveness of the long-range force. The result demonstrates that ultrathin PDMS nanobrush can either promote or inhibit cell adhesion, which is required for various biomedical fields such as tissue-engineering, anti-fouling coating, and implantable biomaterials and sensors.
Ventricular-Subventricular Zone Contact by Glioblastoma is Not Associated with Molecular Signatures in Bulk Tumor Data.
Feb 13, 2019   Scientific Reports
Mistry AM, Wooten DJ, Davis LT, Mobley BC, Quaranta V, Ihrie RA
Ventricular-Subventricular Zone Contact by Glioblastoma is Not Associated with Molecular Signatures in Bulk Tumor Data.
Feb 13, 2019
Scientific Reports
Whether patients with glioblastoma that contacts the ventricular-subventricular zone stem cell niche (VSVZ + GBM) have a distinct survival profile from VSVZ - GBM patients independent of other known predictors or molecular profiles is unclear. Using multivariate Cox analysis to adjust survival for widely-accepted predictors, hazard ratios (HRs) for overall (OS) and progression free (PFS) survival between VSVZ + GBM and VSVZ - GBM patients were calculated in 170 single-institution patients and 254 patients included in both The Cancer Genome (TCGA) and Imaging (TCIA) atlases. An adjusted, multivariable analysis revealed that VSVZ contact was independently associated with decreased survival in both datasets. TCGA molecular data analyses revealed that VSVZ contact by GBM was independent of mutational, DNA methylation, gene expression, and protein expression signatures in the bulk tumor. Therefore, while survival of GBM patients is independently stratified by VSVZ contact, with VSVZ + GBM patients displaying a poor prognosis, the VSVZ + GBMs do not possess a distinct molecular signature at the bulk sample level. Focused examination of the interplay between the VSVZ microenvironment and subsets of GBM cells proximal to this region is warranted.
Exposure to Nanoscale Particulate Matter from Gestation to Adulthood Impairs Metabolic Homeostasis in Mice.
Feb 13, 2019   Scientific Reports
Woodward NC, Crow AL, Zhang Y, Epstein S, Hartiala J,   . . . . . .   , Finch CE, Bouret SG, Sioutas C, Morgan TE, Allayee H
Exposure to Nanoscale Particulate Matter from Gestation to Adulthood Impairs Metabolic Homeostasis in Mice.
Feb 13, 2019
Scientific Reports
Emerging evidence from epidemiological and animal studies suggests that exposure to traffic-related air pollutants and particulate matter less than 2.5 µm in diameter (PM2.5) contributes to development of obesity and related metabolic abnormalities. However, it is not known whether nanoscale particulate matter (nPM) with aerodynamic diameter ≤200 nm have similar adverse metabolic effects. The goal of the present study was to determine the effects of prenatal and early life exposure to nPM on metabolic homeostasis in mice. C57BL/6 J mice were exposed to nPM or filtered air from gestation until 17 weeks of age and characterized for metabolic and behavioral parameters. In male mice, nPM exposure increased food intake, body weight, fat mass, adiposity, and whole-body glucose intolerance (p < 0.05). Consistent with these effects, male mice exposed to nPM displayed alterations in the expression of metabolically-relevant neuropeptides in the hypothalamus and decreased expression of insulin receptor signaling genes in adipose (p < 0.05). There were no differences in exploratory behavior or motor function, fasting lipid levels, or the inflammatory profile of adipose tissue. Our results provide evidence that chronic nPM exposure from gestation to early adulthood in male mice promotes metabolic dysregulation in part through modulation of feeding behavior and in the absence of an obesogenic diet.
Biallelic VARS variants cause developmental encephalopathy with microcephaly that is recapitulated in vars knockout zebrafish.
Feb 16, 2019   Nature Communications
Siekierska A, Stamberger H, Deconinck T, Oprescu SN, Partoens M,   . . . . . .   , Weckhuysen S, Francklyn C, Antonellis A, de Witte P, De Jonghe P
Biallelic VARS variants cause developmental encephalopathy with microcephaly that is recapitulated in vars knockout zebrafish.
Feb 16, 2019
Nature Communications
Aminoacyl tRNA synthetases (ARSs) link specific amino acids with their cognate transfer RNAs in a critical early step of protein translation. Mutations in ARSs have emerged as a cause of recessive, often complex neurological disease traits. Here we report an allelic series consisting of seven novel and two previously reported biallelic variants in valyl-tRNA synthetase (VARS) in ten patients with a developmental encephalopathy with microcephaly, often associated with early-onset epilepsy. In silico, in vitro, and yeast complementation assays demonstrate that the underlying pathomechanism of these mutations is most likely a loss of protein function. Zebrafish modeling accurately recapitulated some of the key neurological disease traits. These results provide both genetic and biological insights into neurodevelopmental disease and pave the way for further in-depth research on ARS related recessive disorders and precision therapies.
Costimulation of type-2 innate lymphoid cells by GITR promotes effector function and ameliorates type 2 diabetes.
Feb 16, 2019   Nature Communications
Galle-Treger L, Sankaranarayanan I, Hurrell BP, Howard E, Lo R,   . . . . . .   , Gilliland FD, Allayee H, Soroosh P, Sharpe AH, Akbari O
Costimulation of type-2 innate lymphoid cells by GITR promotes effector function and ameliorates type 2 diabetes.
Feb 16, 2019
Nature Communications
Metabolic syndrome is characterized by disturbances in glucose homeostasis and the development of low-grade systemic inflammation, which increase the risk to develop type 2 diabetes mellitus (T2DM). Type-2 innate lymphoid cells (ILC2s) are a recently discovered immune population secreting Th2 cytokines. While previous studies show how ILC2s can play a critical role in the regulation of metabolic homeostasis in the adipose tissue, a therapeutic target capable of modulating ILC2 activation has yet to be identified. Here, we show that GITR, a member of the TNF superfamily, is expressed on both murine and human ILC2s. Strikingly, we demonstrate that GITR engagement of activated, but not naïve, ILC2s improves glucose homeostasis, resulting in both protection against insulin resistance onset and amelioration of established insulin- resistance. Together, these results highlight the critical role of GITR as a novel therapeutic molecule against T2DM and its fundamental role as an immune checkpoint for activated ILC2s.
Three-dimensional alteration of neurites in schizophrenia.
Feb 16, 2019   Translational Psychiatry
Mizutani R, Saiga R, Takeuchi A, Uesugi K, Terada Y,   . . . . . .   , Ide S, Ikeda K, Oshima K, Itokawa M, Arai M
Three-dimensional alteration of neurites in schizophrenia.
Feb 16, 2019
Translational Psychiatry
Psychiatric symptoms of schizophrenia suggest alteration of cerebral neurons. However, the physical basis of the schizophrenia symptoms has not been delineated at the cellular level. Here, we report nanometer-scale three-dimensional analysis of brain tissues of schizophrenia and control cases. Structures of cerebral tissues of the anterior cingulate cortex were visualized with synchrotron radiation nanotomography. Tissue constituents visualized in the three-dimensional images were traced to build Cartesian coordinate models of tissue constituents, such as neurons and blood vessels. The obtained Cartesian coordinates were used for calculating curvature and torsion of neurites in order to analyze their geometry. Results of the geometric analyses indicated that the curvature of neurites is significantly different between schizophrenia and control cases. The mean curvature of distal neurites of the schizophrenia cases was ~1.5 times higher than that of the controls. The schizophrenia case with the highest neurite curvature carried a frame shift mutation in the GLO1 gene, suggesting that oxidative stress due to the GLO1 mutation caused the structural alteration of the neurites. The differences in the neurite curvature result in differences in the spatial trajectory and hence alter neuronal circuits. It has been shown that the anterior cingulate cortex analyzed in this study has emotional and cognitive functions. We suggest that the structural alteration of neurons in the schizophrenia cases should reflect psychiatric symptoms of schizophrenia.
Epigenomic analysis reveals DNA motifs regulating histone modifications in human and mouse.
Feb 16, 2019   Proceedings Of The National Academy Of Sciences Of The United States Of America
Ngo V, Chen Z, Zhang K, Whitaker JW, Wang M, Wang W
Epigenomic analysis reveals DNA motifs regulating histone modifications in human and mouse.
Feb 16, 2019
Proceedings Of The National Academy Of Sciences Of The United States Of America
Histones are modified by enzymes that act in a locus, cell-type, and developmental stage-specific manner. The recruitment of enzymes to chromatin is regulated at multiple levels, including interaction with sequence-specific DNA-binding factors. However, the DNA-binding specificity of the regulatory factors that orchestrate specific histone modifications has not been broadly mapped. We have analyzed 6 histone marks (H3K4me1, H3K4me3, H3K27ac, H3K27me3, K3H9me3, H3K36me3) across 121 human cell types and tissues from the NIH Roadmap Epigenomics Project as well as 8 histone marks (with addition of H3K4me2 and H3K9ac) from the mouse ENCODE Consortium. We have identified 361 and 369 DNA motifs in human and mouse, respectively, that are the most predictive of each histone mark. Interestingly, 107 human motifs are conserved between the two species. In human embryonic cell line H1, we mutated only the found DNA motifs at particular loci and the significant reduction of H3K27ac levels validated the regulatory roles of the perturbed motifs. The functionality of these motifs was also supported by the evidence that histone-associated motifs, especially H3K4me3 motifs, significantly overlap with the expression of quantitative trait loci SNPs in cancer patients more than the known and random motifs. Furthermore, we observed possible feedbacks to control chromatin dynamics as the found motifs appear in the promoters or enhancers associated with various histone modification enzymes. These results pave the way toward revealing the molecular mechanisms of epigenetic events, such as histone modification dynamics and epigenetic priming.
A Genome-Wide Analysis of Adhesion in Caulobacter crescentus Identifies New Regulatory and Biosynthetic Components for Holdfast Assembly.
Feb 13, 2019   MBio
Hershey DM, Fiebig A, Crosson S
A Genome-Wide Analysis of Adhesion in Caulobacter crescentus Identifies New Regulatory and Biosynthetic Components for Holdfast Assembly.
Feb 13, 2019
MBio
Due to their intimate physical interactions with the environment, surface polysaccharides are critical determinants of fitness for bacteria. Caulobacter crescentus produces a specialized structure at one of its cell poles called the holdfast that enables attachment to surfaces. Previous studies have shown that the holdfast is composed of carbohydrate-based material and identified a number of genes required for holdfast development. However, incomplete information about its chemical structure, biosynthetic genes, and regulatory principles has limited progress in understanding the mechanism of holdfast synthesis. We leveraged the adhesive properties of the holdfast to perform a saturating screen for genes affecting attachment to cheesecloth over a multiday time course. Using similarities in the temporal profiles of mutants in a transposon library, we defined discrete clusters of genes with related effects on cheesecloth colonization. Holdfast synthesis, flagellar motility, type IV pilus assembly, and smooth lipopolysaccharide (SLPS) production represented key classes of adhesion determinants. Examining these clusters in detail allowed us to predict and experimentally define the functions of multiple uncharacterized genes in both the holdfast and SLPS pathways. In addition, we showed that the pilus and the flagellum control holdfast synthesis separately by modulating the holdfast inhibitor hfiA. This report defines a set of genes contributing to adhesion that includes newly discovered genes required for holdfast biosynthesis and attachment. Our data provide evidence that the holdfast contains a complex polysaccharide with at least four monosaccharides in the repeating unit and underscore the central role of cell polarity in mediating attachment of C. crescentus to surfaces.IMPORTANCE Bacteria routinely encounter biotic and abiotic materials in their surrounding environments, and they often enlist specific behavioral programs to colonize these materials. Adhesion is an early step in colonizing a surface. Caulobacter crescentus produces a structure called the holdfast which allows this organism to attach to and colonize surfaces. To understand how the holdfast is produced, we performed a genome-wide search for genes that contribute to adhesion by selecting for mutants that could not attach to cheesecloth. We discovered complex interactions between genes that mediate surface contact and genes that contribute to holdfast development. Our genetic selection identified what likely represents a comprehensive set of genes required to generate a holdfast, laying the groundwork for a detailed characterization of the enzymes that build this specialized adhesin.
Nucleotide Resolution Comparison of Transcription of Human Cytomegalovirus and Host Genomes Reveals Universal Use of RNA Polymerase II Elongation Control Driven by Dissimilar Core Promoter Elements.
Feb 13, 2019   MBio
Parida M, Nilson KA, Li M, Ball CB, Fuchs HA, Lawson CK, Luse DS, Meier JL, Price DH
Nucleotide Resolution Comparison of Transcription of Human Cytomegalovirus and Host Genomes Reveals Universal Use of RNA Polymerase II Elongation Control Driven by Dissimilar Core Promoter Elements.
Feb 13, 2019
MBio
The large genome of human cytomegalovirus (HCMV) is transcribed by RNA polymerase II (Pol II). However, it is not known how closely this betaherpesvirus follows host transcriptional paradigms. We applied PRO-Seq and PRO-Cap methods to profile and quantify transcription initiation and productive elongation across the host and virus genomes in late infection. A major similarity between host transcription and viral transcription is that treatment of cells with the P-TEFb inhibitor flavopiridol preempts virtually all productive elongation, which otherwise covers most of the HCMV genome. The deep, nucleotide resolution identification of transcription start sites (TSSs) enabled an extensive analysis of core promoter elements. An important difference between host and viral transcription is that initiation is much more pervasive on the HCMV genome. The sequence preferences in the initiator region around the TSS and the utilization of upstream T/A-rich elements are different. Upstream TATA positions the TSS and boosts initiation in both the host and the virus, but upstream TATT has a significant stimulatory impact only on the viral template. The major immediate early (MIE) promoter remained active during late infection and was accompanied by transcription of both strands of the MIE enhancer from promoters within the enhancer. Surprisingly, we found that the long noncoding RNA4.9 is intimately associated with the viral origin of replication (oriLyt) and was transcribed to a higher level than any other viral or host promoter. Finally, our results significantly contribute to the idea that late in infection, transcription takes place on viral genomes that are not highly chromatinized.IMPORTANCE Human cytomegalovirus infects more than half of humans, persists silently in virtually all tissues, and produces life-threatening disease in immunocompromised individuals. HCMV is also the most common infectious cause of birth defects and the leading nongenetic cause of sensorineural hearing loss in the United States. Because there is no vaccine and current drugs have problems with potency, toxicity, and antiviral drug resistance, alternative treatment strategies that target different points of viral control are needed. Our current study contributes to this goal by applying newly developed methods to examine transcription of the HCMV and host genomes at nucleotide resolution in an attempt to find targetable differences between the two. After a thorough analysis of productive elongation and of core promoter element usage, we found that some mechanisms of regulating transcription are shared between the host and HCMV but that others are distinctly different. This suggests that HCMV transcription may be a legitimate target for future antiviral therapies and this might translate to other herpesviruses.
Influence of the familial Alzheimer's disease-associated T43I mutation on the transmembrane structure and γ-secretase processing of the C99 peptide.
Feb 13, 2019   The Journal Of Biological Chemistry
Tang TC, Kienlen-Campard P, Hu Y, Perrin F, Opsomer R, Octave JN, Constantinescu SN, Smith SO
Influence of the familial Alzheimer's disease-associated T43I mutation on the transmembrane structure and γ-secretase processing of the C99 peptide.
Feb 13, 2019
The Journal Of Biological Chemistry
Extracellular deposition of amyloid-β (Aβ) peptides in the brain is a hallmark of Alzheimer's disease (AD). Upon β-secretase-mediated cleavage of the β C-terminal fragment (β-CTF) from the Aβ precursor protein, the γ-secretase complex produces the amyloid β (Aβ) peptides associated with AD. The familial T43I mutation within the transmembrane domain of the β-CTF (also referred to as C99) increases the ratio between the Aβ42 and Aβ40 peptides largely due to a decrease in Aβ40 formation. Aβ42 is the principal component of amyloid deposits within the brain parenchyma, and an increase in the Aβ42:Aβ40 ratio is correlated with early-onset AD. Using NMR and FTIR spectroscopy, here we addressed how the T43I substitution influences the structure of C55, the minimal sequence containing the entire extracellular and transmembrane (TM) domains of C99 needed for γ-secretase processing. 13C NMR chemical shifts indicated that the T43I substitution increases helical structure within the TM domain of C55. These structural changes were associated with a shift of the C55 dimer to the monomer and an increase in the tilt of the TM helix relative to the membrane normal in the T43I mutant compared with that of wild-type C55. The A21G (Flemish) mutation was previously found to increase secreted Aβ40 levels; here, we combined this mutation in the extracellular domain of C99 with T43I and observed that the T43I/A21G double mutant decreases Aβ40 formation. We discuss how the observed structural changes in the T43I mutant may decrease Aβ40 formation and increase the Aβ42:Aβ40 ratio.
Lack of harmonization in high fluorescent cell automated counts with body fluids mode in ascitic, pleural, synovial, and cerebrospinal fluids.
Feb 13, 2019   International Journal Of Laboratory Hematology
Buoro S, Seghezzi M, Dominoni P, Moioli V, Manenti B, Previtali G, Ottomano C, Lippi G
Lack of harmonization in high fluorescent cell automated counts with body fluids mode in ascitic, pleural, synovial, and cerebrospinal fluids.
Feb 13, 2019
International Journal Of Laboratory Hematology
INTRODUCTION: Cellular analysis in body fluids (BFs) provides important diagnostic information in various pathological settings. This study was hence aimed at comparing automated cell count obtained with Mindray BC-6800 (BC-BF) vs Sysmex XN-series (XN-BF) and evaluating other quantitative and qualitative information provided by these analyzers in ascitic (AF), pleural (PF), synovial (SF), and cerebrospinal (CSF) fluids. METHODS: Three hundred and fifty-one samples (99 AFs, 45 PFs, 75 SFs, and 132 CSFs) were analyzed in parallel with BC-BF, XN-BF, and optical microscopy (OM). The study also included the assessment of diagnostic agreement among BC-BF, XN-BF, and OM. RESULTS: The comparison of BC-BF vs XN-BF yielded slopes of Passing and Bablok regression always comprised between 0.9 and 1.0 except for EO-BF and HF-BF, whilst the intercepts ranged from -0.4 for MN-BF and 12.0 for PMN-BF. The bias was comprised between -103.3% and 67.1% for HF-BF and EO-BF, respectively. A significant bias was found for TC-BF, WBC-BF, HF-BF (negative bias) and for PMN-BF and EO-BF (positive bias). The agreement (Cohen's kappa) between XN-BF and BC-BF was always ≥0.7, ranging between 0.87 in CSFs and 0.94 in AFs, and that with OM was similar (ie, 0.85 and 0.96). CONCLUSION: The cytometric analysis of BF samples using BC-BF and XN-BF is clinically favorable when appropriately combined with OM. Quantitative and qualitative parameters displayed optimal agreement, whilst instrument-specific cut-offs should be defined and implemented for HF-BF and EO-BF. Further efforts should be made for achieving better harmonization in cytometric analysis of BF samples.

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