Mass Spectrometry Defines Lysophospholipids as Ligands for Chicken MHCY Class I Molecules.

Chicken (Gallus gallus) MHCY class I molecules are highly polymorphic yet substantially different from polymorphic MHC class I molecules that bind peptide Ags. The binding grooves in MHCY class I molecules are hydrophobic and too narrow to accommodate peptides. An earlier structural study suggested that ligands for MHCY class I might be lipids, but the contents of the groove were not clearly identified. In this study, lysophospholipids have been identified by mass spectrometry as bound in two MHCY class I isoforms that differ substantially in sequence. The two isoforms, YF1*7.1 and YF1*RJF34, differ by 35 aa in the α1 and α2 domains that form the MHC class I ligand binding groove. Lyso-phosphatidylethanolamine (lyso-PE) 18:1 was the dominant lipid identified in YF1*7.1 and YF1*RJF34 expressed as recombinant molecules and renatured with ß2-microglobulin in the presence of a total lipid extract from Escherichia coli. Less frequently detected were lyso-PE 17:1, lyso-PE 16:1, and lysophosphatidylglycerols 17:1 and 16:0. These data provide evidence that lysophospholipids are candidate ligands for MHCY class I molecules. Finding that MHCY class I isoforms differing substantially in sequence bind the same array of lysophospholipids indicates that the amino acid polymorphism that distinguishes MHCY class I molecules is not key in defining ligand specificity. The polymorphic positions lie mostly away from the binding groove and might define specificity in interactions of MHCY class I molecules with receptors that are presently unidentified. MHCY class I molecules are distinctive in bound ligand and in display of polymorphic residues.

A simple means for MHC-Y genotyping in chickens using short tandem repeat sequences.

Described here is a new, more efficient method for defining major histocompatibility complex-Y (MHC-Y) genotypes in chickens. The MHC-Y region is genetically independent from the classical MHC (MHC-B) region. MHC-Y is highly polymorphic and potentially important in the genetics of disease resistance. MHC-Y haplotypes contain variable numbers of specialized MHC class I-like genes, along with members of four additional gene families. Previously, MHC-Y haplotypes were defined by patterns of restriction fragments (RF) generated in labor-intensive procedures that were difficult to use to define MHC-Y genotypes for large numbers of samples. The method reported here is much simpler. MHC-Y genotypes are distinguished via patterns of PCR products generated from heritable short tandem repeat (STR) regions found immediately upstream of the MHC class I-like genes located throughout MHC-Y haplotypes. To validate the method, fully pedigreed families were analyzed for STR-defined haplotypes in light of haplotypes defined previously by RF patterns. STR-defined MHC-Y patterns segregate in fully pedigreed families as expected and correspond with haplotypes assigned by RF patterns. The patterns provided in STR chromatograms generated by capillary electrophoresis are distinct for different haplotypes and can be scored easily. Investigations into the influence of MHC-Y genetics on immune responses can now realistically be conducted with large sample sets.

A high-density SNP panel reveals extensive diversity, frequent recombination and multiple recombination hotspots within the chicken major histocompatibility complex B region between BG2 and CD1A1.

BACKGROUND: The major histocompatibility complex (MHC) is present within the genomes of all jawed vertebrates. MHC genes are especially important in regulating immune responses, but even after over 80 years of research on the MHC, much remains to be learned about how it influences adaptive and innate immune responses. In most species, the MHC is highly polymorphic and polygenic. Strong and highly reproducible associations are established for chicken MHC-B haplotypes in a number of infectious diseases. Here, we report (1) the development of a high-density SNP (single nucleotide polymorphism) panel for MHC-B typing that encompasses a 209,296 bp region in which 45 MHC-B genes are located, (2) how this panel was used to define chicken MHC-B haplotypes within a large number of lines/breeds and (3) the detection of recombinants which contributes to the observed diversity. METHODS: A SNP panel was developed for the MHC-B region between the BG2 and CD1A1 genes. To construct this panel, each SNP was tested in end-point read assays on more than 7500 DNA samples obtained from inbred and commercially used egg-layer lines that carry known and novel MHC-B haplotypes. One hundred and one SNPs were selected for the panel. Additional breeds and experimentally-derived lines, including lines that carry MHC-B recombinant haplotypes, were then genotyped. RESULTS: MHC-B haplotypes based on SNP genotyping were consistent with the MHC-B haplotypes that were assigned previously in experimental lines that carry B2, B5, B12, B13, B15, B19, B21, and B24 haplotypes. SNP genotyping resulted in the identification of 122 MHC-B haplotypes including a number of recombinant haplotypes, which indicate that crossing-over events at multiple locations within the region lead to the production of new MHC-B haplotypes. Furthermore, evidence of gene duplication and deletion was found. CONCLUSIONS: The chicken MHC-B region is highly polymorphic across the surveyed 209-kb region that contains 45 genes. Our results expand the number of identified haplotypes and provide insights into the contribution of recombination events to MHC-B diversity including the identification of recombination hotspots and an estimation of recombination frequency.

Brief review of the chicken Major Histocompatibility Complex: the genes, their distribution on chromosome 16, and their contributions to disease resistance.

Nearly all genes presently mapped to chicken chromosome 16 (GGA 16) have either a demonstrated role in immune responses or are considered to serve in immunity by reason of sequence homology with immune system genes defined in other species. The genes are best described in regional units. Among these, the best known is the polymorphic major histocompatibility complex-B (MHC-B) region containing genes for classical peptide antigen presentation. Nearby MHC-B is a small region containing two CD1 genes, which encode molecules known to bind lipid antigens and which will likely be found in chickens to present lipids to specialized T cells, as occurs with CD1 molecules in other species. Another region is the MHC-Y region, separated from MHC-B by an intervening region of tandem repeats. Like MHC-B, MHC-Y is polymorphic. It contains specialized class I and class II genes and c-type lectin-like genes. Yet another region, separated from MHC-Y by the single nucleolar organizing region (NOR) in the chicken genome, contains olfactory receptor genes and scavenger receptor genes, which are also thought to contribute to immunity. The structure, distribution, linkages and patterns of polymorphism in these regions, suggest GGA 16 evolves as a microchromosome devoted to immune defense. Many GGA 16 genes are polymorphic and polygenic. At the moment most disease associations are at the haplotype level. Roles of individual MHC genes in disease resistance are documented in only a very few instances. Provided suitable experimental stocks persist, the availability of increasingly detailed maps of GGA 16 genes combined with new means for detecting genetic variability will lead to investigations defining the contributions of individual loci and more applications for immunogenetics in breeding healthy poultry.

Mapping genes to chicken microchromosome 16 and discovery of olfactory and scavenger receptor genes near the major histocompatibility complex.

Trisomy mapping is a powerful method for assigning genes to chicken microchromosome 16 (GGA 16). The single chicken nucleolar organizer region (NOR), the 2 major histocompatibility complex regions (MHC-Y and MHC-B), and CD1 genes were all previously assigned to GGA 16 using trisomy mapping. Here, we combined array comparative genomic hybridization with trisomy mapping to screen unassigned genomic scaffolds (consigned temporarily to chrUn_random) for sequences originating from GGA 16. A number of scaffolds mapped to GGA 16. Among these were scaffolds that contain genes for olfactory (OR) and cysteine-rich domain scavenger (SRCR) receptors, along with a number of genes that encode putative immunoglobulin-like receptors and other molecules. We used high-resolution cytogenomic analyses to confirm assignment of OR and SRCR genes to GGA 16 and to pinpoint members of these gene families to the q-arm in partially overlapping regions between the centromere and the NOR. Southern blots revealed sequence polymorphism within the OR/SRCR region and linkage with the MHC-Y region, thereby providing evidence for conserved linkage between OR genes and the MHC within birds. This work localizes OR genes to the vicinity of the chicken MHC and assigns additional genes, including immune defense genes, to GGA 16.

Structure of a classical MHC class I molecule that binds "non-classical" ligands.

Chicken YF1 genes share a close sequence relationship with classical MHC class I loci but map outside of the core MHC region. To obtain insights into their function, we determined the structure of the YF1*7.1/ß(2)-microgloblin complex by X-ray crystallography at 1.3 Å resolution. It exhibits the architecture typical of classical MHC class I molecules but possesses a hydrophobic binding groove that contains a non-peptidic ligand. This finding prompted us to reconstitute YF1*7.1 also with various self-lipids. Seven additional YF1*7.1 structures were solved, but only polyethyleneglycol molecules could be modeled into the electron density within the binding groove. However, an assessment of YF1*7.1 by native isoelectric focusing indicated that the molecules were also able to bind nonself-lipids. The ability of YF1*7.1 to interact with hydrophobic ligands is unprecedented among classical MHC class I proteins and might aid the chicken immune system to recognize a diverse ligand repertoire with a minimal number of MHC class I molecules.

Genetic variation at the MHC in a population of introduced wild turkeys.

Genetic variation in the major histocompatibility complex (MHC) is known to affect disease resistance in many species. Investigations of MHC diversity in populations of wild species have focused on the antigen presenting class IIß molecules due to the known polymorphic nature of these genes and the role these molecules play in pathogen recognition. Studies of MHC haplotype variation in the turkey ( Meleagris gallopavo ) are limited. This study was designed to examine MHC diversity in a group of Eastern wild turkeys ( Meleagris gallopavo silvestris ) collected during population expansion following reintroduction of the species in southern Wisconsin, USA. Southern blotting with BG and class IIß probes and single nucleotide polymorphism (SNP) genotyping was used to measure MHC variation. SNP analysis focused on single copy MHC genes flanking the highly polymorphic class IIß genes. Southern blotting identified 27 class IIß phenotypes, whereas SNP analysis identified 13 SNP haplotypes occurring in 28 combined genotypes. Results show that genetic diversity estimates based on RFLP (Southern blot) analysis underestimate the level of variation detected by SNP analysis. Sequence analysis of the mitochondrial D-loop identified 7 mitochondrial haplotypes (mitotypes) in the sampled birds. Results show that wild turkeys located in southern Wisconsin have a genetically diverse MHC and originate from several maternal lineages.

BG1 has a major role in MHC-linked resistance to malignant lymphoma in the chicken.

Pathogen selection is postulated to drive MHC allelic diversity at loci for antigen presentation. However, readily apparent MHC infectious disease associations are rare in most species. The strong link between MHC-B haplotype and the occurrence of virally induced tumors in the chicken provides a means for defining the relationship between pathogen selection and MHC polymorphism. Here, we verified a significant difference in resistance to gallid herpesvirus-2 (GaHV-2)-induced lymphomas (Marek's disease) conferred by two closely-related recombinant MHC-B haplotypes. We mapped the crossover breakpoints that distinguish these haplotypes to the highly polymorphic BG1 locus. BG1 encodes an Ig-superfamily type I transmembrane receptor-like protein that contains an immunoreceptor tyrosine-based inhibition motif (ITIM), which undergoes phosphorylation and is recognized by Src homology 2 domain-containing protein tyrosine phosphatase (SHP-2). The recombinant haplotypes are identical, except for differences within the BG1 3'-untranslated region (3'-UTR). The 3'-UTR of the BG1 allele associated with increased lymphoma contains a 225-bp insert of retroviral origin and showed greater inhibition of luciferase reporter gene translation compared to the other allele. These findings suggest that BG1 could affect the outcome of GaHV-2 infection through modulation of the lymphoid cell responsiveness to infection, a condition that is critical for GaHV-2 replication and in which the MHC-B haplotype has been previously implicated. This work provides a mechanism by which MHC-B region genetics contributes to the incidence of GaHV-2-induced malignant lymphoma in the chicken and invites consideration of the possibility that similar mechanisms might affect the incidence of lymphomas associated with other oncogenic viral infections.

Research Note: MHCY haplotype impacts Campylobacter jejuni colonization in a backcross [(Line 61 x Line N) x Line N] population.

MHCY is a candidate region for influencing immune responses in chickens. MHCY contains multiple specialized, polymorphic MHC class I loci along with loci belonging to 4 additional gene families. In this study, MHCY haplotypes were tested for association with cecal colonization after Campylobacter jejuni infection of a backcross [(Line 61 × Line N) × Line N] population derived from 2 White Leghorn research lines, Line 61 and Line N, that were previously shown to exhibit heritable differences in colonization. Samples were obtained for 51 birds challenged with 108 CFU Campylobacter jejuni at 3 wk of age. Viable C. jejuni in the ceca were enumerated 5 d postinfection and counts were log-transformed for analysis. Birds were assigned to either low or high colonization groups based on the individual count being below or above the mean bacterial count for all birds. The mean bacterial count of the low infection group differed significantly from the high infection group. Sex and MHCB haplotype had similar distributions within the 2 groups. Overall, 7 MHCY haplotypes were found to be segregating. Two were significantly associated with C. jejuni colonization. MHCY Y18 was associated with low colonization (P = 3.00 × 10-5); whereas MHCY Y11a was associated with high colonization (P = 0.008). The MHCY haplotype impacted the mean bacterial count among all birds with MHCY Y18 having the lowest bacterial count compared with MHCY Y11a and all other MHCY (Y5, Y7, Y8, Y11b, and Y11c) haplotypes. These findings support further investigation of the contribution of chicken MHCY in resistance to Campylobacter colonization.

Association of MHCY genotypes in lines of chickens divergently selected for high or low antibody response to sheep red blood cells.

The chicken MHCY region contains members of several gene families including a family of highly polymorphic MHC class I genes that are structurally distinct from their classical class I gene counterparts. Genetic variability at MHCY could impart variability in immune responses, but robust tests for whether or not this occurs have been lacking. Here we defined the MHCY genotypes present in 2 sets of chicken lines selected for high or low antibody response, the Virginia Tech (VT) HAS and LAS, and the Wageningen University (WU) HA and LA lines. Both sets were developed under long-term bidirectional selection for differences in antibody responses following immunization with the experimental antigen sheep red blood cells. Lines in which selection was relaxed (VT HAR and LAR) or lacking (WU C) provided controls. We looked for evidence of association between MHCY genotypes and antibody titers. Chickens were typed for MHCY using a recently developed method based on a multilocus short tandem repeat sequence found across MHCY haplotypes. Five MHCY haplotypes were found segregating in the VT HAS and LAS lines. One haplotype was present only in HAS chickens, and another was present only in LAS chickens with distribution of the remaining 3 haplotypes differing significantly between the lines. In the WU HA and LA lines, there was a similar MHCY asymmetry. The control populations lacked similar asymmetries. These observations support the likelihood of MHCY genetics affecting heritable antibody responses and provide a basis for further investigations into the role of MHCY region genes in guiding immune responses in chickens.

The Gallus gallus RJF reference genome reveals an MHCY haplotype organized in gene blocks that contain 107 loci including 45 specialized, polymorphic MHC class I loci, 41 C-type lectin-like loci, and other loci amid hundreds of transposable elements.

MHCY is a second major histocompatibility complex-like gene region in chickens originally identified by the presence of major histocompatibility complex class I-like and class II-like gene sequences. Up to now, the MHCY gene region has been poorly represented in genomic sequence data. A high density of repetitive sequence and multiple members of several gene families prevented the accurate assembly of short-read sequence data for MHCY. Identified here by single-molecule real-time sequencing sequencing of BAC clones for the Gallus gallus Red Jungle Fowl reference genome are 107 MHCY region genes (45 major histocompatibility complex class I-like, 41 c-type-lectin-like, 8 major histocompatibility complex class IIß, 8 LENG9-like, 4 zinc finger protein loci, and a single only zinc finger-like locus) located amid hundreds of retroelements within 4 contigs representing the region. Sequences obtained for nearby ribosomal RNA genes have allowed MHCY to be precisely mapped with respect to the nucleolar organizer region. Gene sequences provide insights into the unusual structure of the MHCY class I molecules. The MHCY class I loci are polymorphic and group into 22 types based on predicted amino acid sequences. Some MHCY class I loci are full-length major histocompatibility complex class I genes. Others with altered gene structure are considered gene candidates. The amino acid side chains at many of the polymorphic positions in MHCY class I are directed away rather than into the antigen-binding groove as is typical of peptide-binding major histocompatibility complex class I molecules. Identical and nearly identical blocks of genomic sequence contribute to the observed multiplicity of identical MHCY genes and the large size (>639 kb) of the Red Jungle Fowl MHCY haplotype. Multiple points of hybridization observed in fluorescence in situ hybridization suggest that the Red Jungle Fowl MHCY haplotype is made up of linked, but physically separated genomic segments. The unusual gene content, the evidence of highly similar duplicated segments, and additional evidence of variation in haplotype size distinguish polymorphic MHCY from classical polymorphic major histocompatibility complex regions.

Proteomic analysis of surface and endosomal membrane proteins from the avian LMH epithelial cell line.

Proteins at the cell surface and within the endocytic pathway are increasingly being recognized for their roles in a wide variety of intercellular interactions. Here we used the inherent hydrophobicity and N-glycosylation of membrane proteins to enrich these proteins from the surface and endosome of avian LMH epithelial cells for mass spectrometric analysis. The cycling of many different types of proteins from the cell surface into the endosome and sometimes back to the surface again makes it appropriate to analyze these two membranous cellular components together. Stringent searches of the International Protein Index (IPI) entries for Gallus gallus identified 318 unique integral membrane proteins (IMPs) (201 bearing N-glycosylation sites), 265 unique membrane-associated proteins (MAPs), and an additional group of 784 non-membrane proteins (NMPs) among TX-114 detergent and aqueous phase-enriched proteins. Capture of N-glycosylated tryptic peptides revealed 36 additional glycoproteins most of which were CD antigens, receptors, and molecules for cell adhesion and immune response. IMPs and MAPs present at the surface and within the endosome included proteins involved in transport (255), metabolism (285), communication (108), adhesion (47), and immune responses (42). Among these were 355 putative uncharacterized and hypothetical IMPs, MAPs, and NMPs for which highly similar annotated sequences were found in standard protein-protein BLAST searches.

Contribution of mutation, recombination, and gene conversion to chicken MHC-B haplotype diversity.

The Mhc is a highly conserved gene region especially interesting to geneticists because of the rapid evolution of gene families found within it. High levels of Mhc genetic diversity often exist within populations. The chicken Mhc is the focus of considerable interest because of the strong, reproducible infectious disease associations found with particular Mhc-B haplotypes. Sequence data for Mhc-B haplotypes have been lacking thereby hampering efforts to systematically resolve which genes within the Mhc-B region contribute to well-defined Mhc-B-associated disease responses. To better understand the genetic factors that generate and maintain genomic diversity in the Mhc-B region, we determined the complete genomic sequence for 14 Mhc-B haplotypes across a region of 59 kb that encompasses 14 gene loci ranging from BG1 to BF2. We compared the sequences using alignment, phylogenetic, and genome profiling methods. We identified gene structural changes, synonymous and non-synonymous polymorphisms, insertions and deletions, and allelic gene rearrangements or exchanges that contribute to haplotype diversity. Mhc-B haplotype diversity appears to be generated by a number of mutational events. We found evidence that some Mhc-B haplotypes are derived by whole- and partial-allelic gene conversion and homologous reciprocal recombination, in addition to nucleotide mutations. These data provide a framework for further analyses of disease associations found among these 14 haplotypes and additional haplotypes segregating and evolving in wild and domesticated populations of chickens.

CXCR2 specific endocytosis of immunomodulatory peptide LL-37 in human monocytes and formation of LL-37 positive large vesicles in differentiated monoosteophils.

Immunomodulatory peptide cathelicidin/LL-37 induces human monocyte differentiation into a novel bone repair cell, the monoosteophil. We now demonstrate that LL-37 is endocytosed by monocytes over a period of 6 days producing large (10 × 2 µm), specialized LL-37 and integrin α3 positive vesicles. CXCR2, a membrane receptor previously associated with the binding of LL-37 to neutrophils, was co-endocytosed with LL-37 where both markers remained within the cytosol over a 16 h observation period. Endocytosis of LL-37 was mediated by a clathrin- and cavoelin/lipid raft-dependent pathway into early Rab5+ endosomes expressing APPL1 and EEA1. From 4 to 16 h, LL-37 vesicles co-localized with the Golgi, mitochondria, and to a lesser extent lysosomes and ER. By day 6, LL-37 was associated with large (>10 µm) vesicles, adjacent to Golgi, mitochondria, ER and lysosomes. LL-37 co-stained with integrin α3, tetraspanin CD9, GPI-linked CD59 and costimulatory molecule CD276 (B7-H3) in these vesicles. Continuous tracking of LL-37 with its associated vesicles over 6 days indicates that LL-37 is an extremely stable, membrane-associated peptide that plays a critical role in the differentiation of monocytes into monoosteophils.

Low- and high-thermogenic brown adipocyte subpopulations coexist in murine adipose tissue.

Brown adipose tissue (BAT), as the main site of adaptive thermogenesis, exerts beneficial metabolic effects on obesity and insulin resistance. BAT has been previously assumed to contain a homogeneous population of brown adipocytes. Utilizing multiple mouse models capable of genetically labeling different cellular populations, as well as single-cell RNA sequencing and 3D tissue profiling, we discovered a brown adipocyte subpopulation with low thermogenic activity coexisting with the classical high-thermogenic brown adipocytes within the BAT. Compared with the high-thermogenic brown adipocytes, these low-thermogenic brown adipocytes had substantially lower Ucp1 and Adipoq expression, larger lipid droplets, and lower mitochondrial content. Functional analyses showed that, unlike the high-thermogenic brown adipocytes, the low-thermogenic brown adipocytes have markedly lower basal mitochondrial respiration, and they are specialized in fatty acid uptake. Upon changes in environmental temperature, the 2 brown adipocyte subpopulations underwent dynamic interconversions. Cold exposure converted low-thermogenic brown adipocytes into high-thermogenic cells. A thermoneutral environment had the opposite effect. The recruitment of high-thermogenic brown adipocytes by cold stimulation is not affected by high-fat diet feeding, but it does substantially decline with age. Our results revealed a high degree of functional heterogeneity of brown adipocytes.

Expression, purification and preliminary X-ray crystallographic analysis of the chicken MHC class I molecule YF1*7.1.

YF1*7.1 is an allele of a polymorphic major histocompatibility complex (MHC) class I-like locus within the chicken Y gene complex. With the aim of understanding the possible role of the YF1*7.1 molecule in antigen presentation, the complex of YF1*7.1 heavy chain and beta(2)-microglobulin was reconstituted and purified without a peptide. Crystals diffracted synchrotron radiation to 1.32 A resolution and belonged to the monoclinic space group P2(1). The phase problem was solved by molecular replacement. A detailed examination of the structure may provide insight into the type of ligand that could be bound by the YF1*7.1 molecule.

Architecture and organization of chicken microchromosome 16: order of the NOR, MHC-Y, and MHC-B subregions.

Here we present a high-resolution cytogenomic analysis of chicken microchromosome 16. We established the location of the major histocompatibility complex (MHC)-B and -Y subregions relative to each other and to the nucleolus organizer region (NOR) encoding the 18S-5.8S-28S ribosomal DNA. To do so, we employed multicolor fluorescence in situ hybridization using large-insert bacterial artificial chromosome clones with fully sequenced inserts or repetitive sequence probes specific for the subregion of interest. We show that the MHC-Y and -B regions are located on the same side of the NOR, rather than opposite ends, as previously proposed. On the q arm, the MHC-Y is closely adjacent to the NOR, whereas the MHC-B is distal near the q-terminus. A relatively large GC-rich region separates the 2 MHC subregions and includes a specialized structure, a secondary constriction. We propose that the GC-rich large physical distance is the basis for the lack of genetic linkage between the NOR and MHC-B and between the MHC-Y and -B. An integrated model for GGA 16 is presented that incorporates gene complex order in the context of key architectural features including p and q arms, primary (centromere) and secondary constrictions, telomeres, as well as AT- and GC-rich regions.

Mhc-B haplotypes in "Campero-Inta" chicken synthetic line.

The major histocompatibility complex-B (MHC-B) in chickens is a cluster of genes located on chromosome 16. The chicken MHC-B is known to be highly associated with resistance to numerous diseases caused by viruses, bacteria, and parasitic pathogens. Since the level of resistance varies with MHC-B haplotypes, identification and classification of different haplotypes within lines is important for sustaining lines. The "Campero-INTA" chicken breed is a meat-type free-range poultry breed that was developed specifically for small producers in Argentina. Campero-INTA was started by selection in populations produced by crosses between a variety of established lines. MHC-B variation was examined in 65 samples obtained in 2002 using the VNTR marker LEI0258, a marker for MHC-B region. These samples plus and an additional 55 samples from 2018 were examined for variation using the MHC-B specific SNP panel that encompasses ∼230,000 bp of the MHC-B region. Eleven MHC-B SNP haplotypes with 6 LEI0258 alleles were identified in the 120 samples representing the Campero-INTA AH (male) line. Seven haplotypes originate from the breeds originally used in the development of Campero-INTA AH line. Two appear to be recombinant haplotypes. The origin of the remaining 2 is not known, but may be associated with genes introduced from crosses with the Fayoumi breed conducted more recently to sustain the line.

Single molecule characterization of individual extracellular vesicles from pancreatic cancer.

Biofluid-accessible extracellular vesicles (EVs) may represent a new means to improve the sensitivity and specificity of detecting disease. However, current methods to isolate EVs encounter challenges when they are used to select specific populations. Moreover, it has been difficult to comprehensively characterize heterogeneous EV populations at the single vesicle level. Here, we robustly assessed heterogeneous EV populations from cultured cell lines via nanoparticle tracking analysis, proteomics, transcriptomics, transmission electron microscopy, and quantitative single molecule localization microscopy (qSMLM). Using qSMLM, we quantified the size and biomarker content of individual EVs. We applied qSMLM to patient plasma samples and identified a pancreatic cancer-enriched EV population. Our goal is to advance single molecule characterization of EVs for early disease detection. Abbreviations: EV: Extracellular Vesicle; qSMLM: quantitative Single Molecule Localization Microscopy; PDAC: Pancreatic Ductal Adenocarcinoma; EGFR: epidermal growth factor receptor 1; CA19-9: carbohydrate antigen 19-9; SEC: size exclusion chromatography; WGA: wheat germ agglutinin; AF647: Alexa Fluor 647; Ab: antibody; HPDEC: Healthy Pancreatic Ductal Epithelial Cell; TEM: Transmission Electron Microscopy.