In Silico Derivation of HLA-Specific Alloreactivity Potential from Whole Exome Sequencing of Stem Cell Transplant Donors and Recipients: Understanding the Quantitative Immuno-biology of Allogeneic Transplantation

Donor T cell mediated graft vs. host effects may result from the aggregate alloreactivity to minor histocompatibility antigens (mHA) presented by the HLA in each donor-recipient pair (DRP) undergoing stem cell transplantation (SCT). Whole exome sequencing has demonstrated extensive nucleotide sequence variation in HLA-matched DRP. Non-synonymous single nucleotide polymorphisms (nsSNPs) in the GVH direction (polymorphisms present in recipient and absent in donor) were identified in 4 HLA-matched related and 5 unrelated DRP. The nucleotide sequence flanking each SNP was obtained utilizing the ANNOVAR software package. All possible nonameric-peptides encoded by the non-synonymous SNP were then interrogated in-silico for their likelihood to be presented by the HLA class I molecules in individual DRP, using the Immune-Epitope Database (IEDB) SMM algorithm. The IEDB-SMM algorithm predicted a median 18,396 peptides/DRP which bound HLA with an IC50 of <500nM, and 2254 peptides/DRP with an IC50 of <50nM. Unrelated donors generally had higher numbers of peptides presented by the HLA. A similarly large library of presented peptides was identified when the data was interrogated using the Net MHCPan algorithm. These peptides were uniformly distributed in the various organ systems. The bioinformatic algorithm presented here demonstrates that there may be a high level of minor histocompatibility antigen variation in HLA-matched individuals, constituting an HLA-specific alloreactivity potential. These data provide a possible explanation for how relatively minor adjustments in GVHD prophylaxis yield relatively similar outcomes in HLA matched and mismatched SCT recipients.Comment: Abstract: 235, Words: 6422, Figures: 7, Tables: 3, Supplementary figures: 2, Supplementary tables:

Subclinical infection of macaques and baboons with a baboon simarterivirus

Simarteriviruses (Arteriviridae: Simarterivirinae) are commonly found at high titers in the blood of African monkeys but do not cause overt disease in these hosts. In contrast, simarteriviruses cause severe disease in Asian macaques upon accidental or experimental transmission. Here, we sought to better understand the host-dependent drivers of simarterivirus pathogenesis by infecting olive baboons (n = 4) and rhesus monkeys (n = 4) with the simarterivirus Southwest baboon virus 1 (SWBV-1). Surprisingly, none of the animals in our study showed signs of disease following SWBV-1 inoculation. Three animals (two rhesus monkeys and one olive baboon) became infected and sustained high levels of SWBV-1 viremia for the duration of the study. The course of SWBV-1 infection was highly predictable: plasma viremia peaked between 1 × 107 and 1 × 108 vRNA copies/mL at 3–10 days post-inoculation, which was followed by a relative nadir and then establishment of a stable set-point between 1 × 106 and 1 × 107 vRNA copies/mL for the remainder of the study (56 days). We characterized cellular and antibody responses to SWBV-1 infection in these animals, demonstrating that macaques and baboons mount similar responses to SWBV-1 infection, yet these responses are ineffective at clearing SWBV-1 infection. SWBV-1 sequencing revealed the accumulation of non-synonymous mutations in a region of the genome that corresponds to an immunodominant epitope in the simarterivirus major envelope glycoprotein GP5, which likely contribute to viral persistence by enabling escape from host antibodies

A Dynamic Programming Approach to De Novo Peptide Sequencing via Tandem Mass Spectrometry

The tandem mass spectrometry fragments a large number of molecules of the same peptide sequence into charged prefix and suffix subsequences, and then measures mass/charge ratios of these ions. The de novo peptide sequencing problem is to reconstruct the peptide sequence from a given tandem mass spectral data of k ions. By implicitly transforming the spectral data into an NC-spectrum graph G=(V,E) where |V|=2k+2, we can solve this problem in O(|V|+|E|) time and O(|V|) space using dynamic programming. Our approach can be further used to discover a modified amino acid in O(|V||E|) time and to analyze data with other types of noise in O(|V||E|) time. Our algorithms have been implemented and tested on actual experimental data.Comment: A preliminary version appeared in Proceedings of the 11th Annual ACM-SIAM Symposium on Discrete Algorithms, pages 389--398, 200

Regulation of the Neuron-specific Ras GTPase-activating Protein, synGAP, by Ca2+/Calmodulin-dependent Protein Kinase II

synGAP is a neuron-specific Ras GTPase-activating protein found in high concentration in the postsynaptic density fraction from mammalian forebrain. Proteins in the postsynaptic density, including synGAP, are part of a signaling complex attached to the cytoplasmic tail of the N-methyl-D-aspartate-type glutamate receptor. synGAP can be phosphorylated by a second prominent component of the complex, Ca2+/calmodulin-dependent protein kinase II. Here we show that phosphorylation of synGAP by Ca2+/calmodulin-dependent protein kinase II increases its Ras GTPase-activating activity by 70-95%. We identify four major sites of phosphorylation, serines 1123, 1058, 750/751/756, and 764/765. These sites together with other minor phosphorylation sites in the carboxyl tail of synGAP control stimulation of GTPase-activating activity. When three of these sites and four other serines in the carboxyl tail are mutated, stimulation of GAP activity after phosphorylation is reduced to 21 ± 5% compared with 70-95% for the wild type protein. We used phosphosite-specific antibodies to show that, as predicted, phosphorylation of serines 765 and 1123 is increased in cultured cortical neurons after exposure of the neurons to the agonist N-methyl-D-aspartate

A gas-liquid solid phase peptide and protein sequenator

A new miniaturized protein and peptide sequenator has been constructed which uses gas phase reagents at the coupling and cleavage steps of the Edman degradation. The sample is embedded in a matrix of Polybrene dried onto a porous glass fiber disc located in a small cartridge-style reaction cell. The protein or peptide, though not covalently attached to the support, is essentially immobile throughout the degradative cycle, since only relatively apolar, liquid phase solvents pass through the cell. This instrument can give useful sequence data on as little as 5 pmol or protein, can perform extended sequence runs (greater than 30 residues) on subnanomole quantities of proteins purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and can sequence hydrophobic peptides to completion. The sequenator is characterized by a high repetitive yield during the degradation, low reagent consumption, low maintenance requirements, and a degradative cycle time of only 50 min using a complete double cleavage program

The impact of sequence database choice on metaproteomic results in gut microbiota studies

Background: Elucidating the role of gut microbiota in physiological and pathological processes has recently emerged as a key research aim in life sciences. In this respect, metaproteomics, the study of the whole protein complement of a microbial community, can provide a unique contribution by revealing which functions are actually being expressed by specific microbial taxa. However, its wide application to gut microbiota research has been hindered by challenges in data analysis, especially related to the choice of the proper sequence databases for protein identification. Results: Here, we present a systematic investigation of variables concerning database construction and annotation and evaluate their impact on human and mouse gut metaproteomic results. We found that both publicly available and experimental metagenomic databases lead to the identification of unique peptide assortments, suggesting parallel database searches as a mean to gain more complete information. In particular, the contribution of experimental metagenomic databases was revealed to be mandatory when dealing with mouse samples. Moreover, the use of a "merged" database, containing all metagenomic sequences from the population under study, was found to be generally preferable over the use of sample-matched databases. We also observed that taxonomic and functional results are strongly database-dependent, in particular when analyzing the mouse gut microbiota. As a striking example, the Firmicutes/Bacteroidetes ratio varied up to tenfold depending on the database used. Finally, assembling reads into longer contigs provided significant advantages in terms of functional annotation yields. Conclusions: This study contributes to identify host- and database-specific biases which need to be taken into account in a metaproteomic experiment, providing meaningful insights on how to design gut microbiota studies and to perform metaproteomic data analysis. In particular, the use of multiple databases and annotation tools has to be encouraged, even though this requires appropriate bioinformatic resources

Molecular cloning of the cDNA encoding pp36, a tyrosine-phosphorylated adaptor protein selectively expressed by T cells and natural killer cells.

Activation of T and natural killer (NK) cells leads to the tyrosine phosphorylation of pp36 and to its association with several signaling molecules, including phospholipase Cgamma-1 and Grb2. Microsequencing of peptides derived from purified rat pp36 protein led to the cloning, in rat and man, of cDNA encoding a T- and NK cell-specific protein with several putative Src homology 2 domain-binding motifs. A rabbit antiserum directed against a peptide sequence from the cloned rat molecule recognized tyrosine phosphorylated pp36 from pervanadate-treated rat thymocytes. When expressed in 293T human fibroblast cells and tyrosine-phosphorylated, pp36 associated with phospholipase Cgamma-1 and Grb2. Studies with GST-Grb2 fusion proteins demonstrated that the association was specific for the Src homology 2 domain of Grb-2. Molecular cloning of the gene encoding pp36 should facilitate studies examining the role of this adaptor protein in proximal signaling events during T and NK cell activation

Experimental data from flesh quality assessment and shelf life monitoring of high pressure processed European sea bass (Dicentrarchus labrax) fillets

Fresh fish are highly perishable food products and their short shelf-life limits their commercial exploitation and leads to waste, which has a negative impact on aquaculture sustainability. New non-thermal food processing methods, such as high pressure (HP) processing, prolong shelf-life while assuring high food quality. The effect of HP processing (600MPa, 25 °C, 5min) on European sea bass (Dicentrarchus labrax) fillet quality and shelf life was investigated. The data presented comprises microbiome and proteome profiles of control and HP-processed sea bass fillets from 1 to 67 days of isothermal storage at 2 °C. Bacterial diversity was analysed by Illumina high-throughput sequencing of the 16S rRNA gene in pooled DNAs from control or HP-processed fillets after 1, 11 or 67 days and the raw reads were deposited in the NCBI-SRA database with accession number PRJNA517618. Yeast and fungi diversity were analysed by high-throughput sequencing of the internal transcribed spacer (ITS) region for control and HP-processed fillets at the end of storage (11 or 67 days, respectively) and have the SRA accession number PRJNA517779. Quantitative label-free proteomics profiles were analysed by SWATH-MS (Sequential Windowed data independent Acquisition of the Total High-resolution-Mass Spectra) in myofibrillar or sarcoplasmic enriched protein extracts pooled for control or HP-processed fillets after 1, 11 and 67 days of storage. Proteome data was deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD012737. These data support the findings reported in the associated manuscript "High pressure processing of European sea bass (Dicentrarchus labrax) fillets and tools for flesh quality and shelf life monitoring", Tsironi et al., 2019, JFE 262:83-91, doi.org/10.1016/j.jfoodeng.2019.05.010.FCT (Foundation of Science and Technology) COFASP/0002/2015; Portuguese Foundation for Science and Technology UID/Multi/04326/2019 POCI-01-0145-FEDER007440 UID/NEU/04539/2019info:eu-repo/semantics/publishedVersio