Determination of the genetic mechanisms responsible for generating diversity in the cattle NK cell receptor repertoire

Cattle have expanded the KIR gene repertoire, a polymorphic and polygenic im- munoglobulin family that encode Natural Killer cell receptors specific to MHC class I ligands. In humans, KIR are important mediators of innate immunity to viral pathogens such as HCV and HIV, and there is potential for exploiting cattle KIR diversity as a means of improving animal health. Cattle KIR expansion has occurred independently to humans, the result is a cattle KIR haplotype (CKH) with a completely different gene content. Successful sequencing and assembly of the CKH using whole genome techniques has failed. To interrogate cattle KIR, their function and comparative evolution, the content of a CKH must be estab- lished, then the extent of polymorphism and gene presence/absence variation can be studied. In this project the first CKH has been sequenced and assembled using second generation sequencing of BAC clones. This has provided a reference sequence for whole genome sequence data to be aligned revealing the KIR content of different Bovidae species, including the aurochs, the ancestor to all domesticated cattle. Furthermore genome capture and enrichment was performed to determine polymorphic and polygenic KIR variation within 24 different cattle genomes. The sheep KIR haplotype (SKH) was sequenced using PacBio of BAC clones to enable comparative analysis with cattle. The CKH has expanded through block duplications resulting in 16 discrete KIR loci. The haplotype is dominated by functional inhibitory receptor genes and the attenuated remains of activating KIR. Predicted similarity between au- rochs and modern CKH suggests KIR blocks expanded through natural selection and not artificial selection generated through centuries of domestication. Com- parative analysis of the SKH and CKH reveals that sheep have independently expanded at least five of the shared KIR that cattle have expanded. Cattle KIR are extremely polymorphic, with diversity focused within the Ig domains, regions predicted to interact with ligand.Open Acces

Identification of Rare Membrane Antigen Specific Human B Cells

The experimentally well supported model that MG pathology is caused by antibodies of the IgG class that bind to AChR at the neuromuscular junction, activate complement, and possibly cause internalization of receptors or their functional blockade has enabled the development of a range of reasonably effective treatments. A better understanding of which B cells are responsible for producing these pathogenic antibodies, and why such B cells develop would enable the development of more targeted therapies. Studies of antibodies isolated from single B cells from patients have provided some of this information that was not available from studies of polyclonal antibodies in sera, but perhaps future studies of the B cells themselves will provide deeper insight into the causes of the disease and thereby enable its prevention

Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing

Whole genome reconstruction of bacterial pathogens has become an important tool for tracking transmission and antimicrobial resistance gene spread, but highly accurate and complete assemblies have largely only historically been achievable using hybrid long and short-read sequencing. We previously found the Oxford Nanopore Technologies (ONT) R10.4/kit12 flowcell/chemistry produced improved assemblies over the R9.4.1/kit10 combination, however long-read only assemblies contained more errors compared to Illumina-ONT hybrid assemblies. ONT have since released an R10.4.1/kit14 flowcell/chemistry upgrade and recommended the use of Bovine Serum Albumin (BSA) during library preparation, both of which reportedly increase accuracy and yield. They have also released updated basecallers trained using native bacterial DNA containing methylation sites intended to fix systematic basecalling errors, including common adenosine (A) to guanine (G) and cytosine (C) to thymine (T) substitutions. To evaluate these improvements, we successfully sequenced four bacterial reference strains, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, and nine genetically diverse E. coli bloodstream infection-associated isolates from different phylogroups and sequence types, both with and without BSA. These sequences were de novo assembled and compared against Illumina-corrected reference genomes. In this small evaluation of 13 isolates we found that nanopore long read-only R10.4.1/kit 14 assemblies with updated basecallers trained using bacterial methylated DNA produce accurate assemblies with ≥40x depth, sufficient to be cost-effective compared with hybrid ONT/Illumina sequencing in our setting

Using citizen science to identify Australia’s least known birds and inform conservation action

Citizen science is a popular approach to biodiversity surveying, whereby data that are collected by volunteer naturalists may help analysts to understand the distribution and abundance of wild organisms. In Australia, birdwatchers have contributed to two major citizen science programs, eBird (run by the Cornell Lab of Ornithology) and Birdata (run by Birdlife Australia), which collectively hold more than 42 million records of wild birds from across the country. However, these records are not evenly distributed across space, time, or taxonomy, with particularly significant variation in the number of records of each species in these datasets. In this paper, we explore this variation and seek to determine which Australian bird species are least known as determined by rates of citizen science survey detections. We achieve this by comparing the rates of survey effort and species detection across each Australian bird species? range, assigning all 581 species to one of the four groups depending on their rates of survey effort and species observation. We classify 56 species into a group considered the most poorly recorded despite extensive survey effort, with Coxen?s Fig Parrot Cyclopsitta coxeni, Letter-winged Kite Elanus scriptus, Night Parrot Pezoporus occidentalis, Buff-breasted Buttonquail Turnix olivii and Red-chested Buttonquail Turnix pyrrhothorax having the very lowest numbers of records. Our analyses provide a framework to identify species that are poorly represented in citizen science datasets. We explore the reasons behind why they may be poorly represented and suggest ways in which targeted approaches may be able to help fill in the gaps.Publisher PDFPeer reviewe

A functionally defined high-density NRF2 interactome reveals new conditional regulators of ARE transactivation

NRF2 (NFE2L2) is a cytoprotective transcription factor associated with >60 human diseases, adverse drug reactions and therapeutic resistance. To provide insight into the complex regulation of NRF2 responses, 1962 predicted NRF2-partner interactions were systematically tested to generate an experimentally defined high-density human NRF2 interactome. Verification and conditional stratification of 46 new NRF2 partners was achieved by co-immunoprecipitation and the novel integration of quantitative data from dual luminescence-based co-immunoprecipitation (DULIP) assays and live-cell fluorescence cross-correlation spectroscopy (FCCS). The functional impact of new partners was then assessed in genetically edited loss-of-function (NRF2−/−) and disease-related gain-of-function (NRF2T80K and KEAP1−/−) cell-lines. Of the new partners investigated >77% (17/22) modified NRF2 responses, including partners that only exhibited effects under disease-related conditions. This experimentally defined binary NRF2 interactome provides a new vision of the complex molecular networks that govern the modulation and consequence of NRF2 activity in health and disease

Cocapture of cognate and bystander antigens can activate autoreactive B cells

Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) are associated with autoimmune central nervous system diseases like acute disseminated encephalomyelitis (ADEM). For ADEM, it is speculated that a preceding infection is the trigger of the autoimmune response, but the mechanism connecting the infection to the production of MOG antibodies remains a mystery. We reasoned that the ability of B cells to capture cognate antigen from cell membranes, along with small quantities of coexpressed “bystander” antigens, might enable B-cell escape from tolerance. We tested this hypothesis using influenza hemagglutinin as a model viral antigen and transgenic, MOG-specific B cells. Using flow cytometry and live and fixed cell microscopy, we show that MOG-specific B cells take up large amounts of MOG from cell membranes. Uptake of the antigen from the membrane leads to a strong activation of the capturing B cell. When influenza hemagglutinin is also present in the membrane of the target cell, it can be cocaptured with MOG by MOG-specific B cells via the B-cell receptor. Hemagglutinin and MOG are both presented to T cells, which in turn are activated and proliferate. As a consequence, MOG-specific B cells get help from hemagglutinin-specific T cells to produce anti-MOG antibodies. In vivo, the transfer of MOG-specific B cells into recipient mice after the cocapture of MOG and hemagglutinin leads to the production of class-switched anti-MOG antibodies, dependent on the presence of hemagglutinin-specific T cells. This mechanism offers a link between infection and autoimmunity. Keywords: tolerance; autoantibodies; antigen capture; antigen presentation; influenz

Anti-MOG antibodies are present in a subgroup of patients with a neuromyelitis optica phenotype

Background: Antibodies against myelin oligodendrocyte glycoprotein (MOG) have been identified in a subgroup of pediatric patients with inflammatory demyelinating disease of the central nervous system (CNS) and in some patients with neuromyelitis optica spectrum disorder (NMOSD). The aim of this study was to examine the frequency, clinical features, and long-term disease course of patients with anti-MOG antibodies in a European cohort of NMO/NMOSD. Findings: Sera from 48 patients with NMO/NMOSD and 48 patients with relapsing-remitting multiple sclerosis (RR-MS) were tested for anti-aquaporin-4 (AQP4) and anti-MOG antibodies with a cell-based assay. Anti-MOG antibodies were found in 4/17 patients with AQP4-seronegative NMO/NMOSD, but in none of the AQP4-seropositive NMO/NMOSD (n = 31) or RR-MS patients (n = 48). MOG-seropositive patients tended towards younger disease onset with a higher percentage of patients with pediatric (<18 years) disease onset (MOG+, AQP4+, MOG-/AQP4-: 2/4, 3/31, 0/13). MOG-seropositive patients presented more often with positive oligoclonal bands (OCBs) (3/3, 5/29, 1/13) and brain magnetic resonance imaging (MRI) lesions during disease course (2/4, 5/31, 1/13). Notably, the mean time to the second attack affecting a different CNS region was longer in the anti-MOG antibody-positive group (11.3, 3.2, 3.4 years). Conclusions: MOG-seropositive patients show a diverse clinical phenotype with clinical features resembling both NMO (attacks mainly confined to the spinal cord and optic nerves) and MS with an opticospinal presentation (positive OCBs, brain lesions). Anti-MOG antibodies can serve as a diagnostic and maybe prognostic tool in patients with an AQP4-seronegative NMO phenotype and should be tested in those patients

Common loss of far-red light photoacclimation in cyanobacteria from hot and cold deserts: a case study in the Chroococcidiopsidales

Deserts represent an extreme challenge for photosynthetic life. Despite their aridity, they are often inhabited by diverse microscopic communities of cyanobacteria. These organisms are commonly found in lithic habitats, where they are partially sheltered from extremes of temperature and UV radiation. However, living under the rock surface imposes additional constraints, such as limited light availability, and enrichment of longer wavelengths than are typically usable for oxygenic photosynthesis. Some cyanobacteria from the genus Chroococcidiopsis can use this light to photosynthesize, in a process known as far-red light photoacclimation, or FaRLiP. This genus has commonly been reported from both hot and cold deserts. However, not all Chroococcidiopsis strains carry FaRLiP genes, thus motivating our study into the interplay between FaRLiP and extreme lithic environments. The abundance of sequence data and strains provided the necessary material for an in-depth phylogenetic study, involving spectroscopy, microscopy, and determination of pigment composition, as well as gene and genome analyses. Pigment analyses revealed the presence of red-shifted chlorophylls d and f in all FaRLiP strains tested. In addition, eight genus-level taxa were defined within the encompassing Chroococcidiopsidales, clarifying the phylogeny of this long-standing polyphyletic order. FaRLiP is near universally present in a generalist genus identified in a wide variety of environments, Chroococcidiopsis sensu stricto, while it is rare or absent in closely related, extremophile taxa, including those preferentially inhabiting deserts. This likely reflects the evolutionary process of gene loss in specialist lineages