Persistence of PFOA pollution at a PTFE production site and occurrence of replacement PFASs in English freshwaters revealed by sentinel species, the Eurasian otter (Lutra lutra)

Concentrations of 33 PFASs were determined in 20 Eurasian otters, sampled 2015–2019, along a transect away from a factory, which used PFOA in PTFE manufacture. Despite cessation of usage in 2012, PFOA concentrations remained high near the factory (>298 μg/kg ww 150 km away). Long-chain legacy PFASs dominated the Σ33PFAS profile, particularly PFOS, PFOA, PFDA, and PFNA. Replacement compounds, PFECHS, F-53B, PFBSA, PFBS, PFHpA, and 8:2 FTS, were detected in ≥19 otters, this being the first report of PFBSA and PFECHS in the species. Concentrations of replacement PFASs were generally lower than legacy compounds (max: 70.3 μg/kg ww and 4,640 μg/kg ww, respectively). Our study underscores the utility of otters as sentinels for evaluating mitigation success and highlights the value of continued monitoring to provide insights into the longevity of spatial associations with historic sources. Lower concentrations of replacement, than legacy, PFASs likely reflect their lower bioaccumulation potential, and more recent introduction. Continued PFAS use will inevitably lead to increased environmental and human exposure if not controlled. Further research is needed on fate, toxicity, and bioaccumulation of replacement compounds

Anthropogenic drivers of variation in concentrations of perfluoroalkyl substances in otters (Lutra lutra) from England and Wales

Per- and polyfluoroalkyl substances (PFASs) are ubiquitous environmental contaminants that have been linked to adverse health effects in wildlife and humans. Here, we report the presence of PFASs in Eurasian otters (Lutra lutra) in England and Wales and their association with anthropogenic sources. The following 15 compounds were analyzed: 10 perfluoroalkyl carboxylic acids (PFCAs), 4 perfluoroalkyl sulfonic acids (PFSAs), and perfluorooctane sulfonamide, in livers of 50 otters which died between 2007 and 2009. PFASs were detected in all otters analyzed, with 12/15 compounds detected in ≥80% of otters. Perfluorooctane sulfonate (PFOS) accounted for 75% of the ΣPFAS profile, with a maximum concentration of 6800 μg/kg wet weight (ww). Long-chain (≥C8) PFCAs accounted for 99.9% of the ΣPFCA profile, with perfluorodecanoic acid and perfluorononanoic acid having the highest maxima (369 μg/kg ww and 170 μg/kg ww, respectively). Perfluorooctanoic acid (PFOA) concentrations were negatively associated with the distance from a factory that used PFOA in polytetrafluoroethylene manufacture. Most PFAS concentrations in otters were positively associated with load entering wastewater treatment works (WWTW) and with arable land, suggesting that WWTW effluent and sewage sludge-amended soils are significant pathways of PFASs into freshwaters. Our results reveal the widespread pollution of British freshwaters with PFASs and demonstrate the utility of otters as effective sentinels for spatial variation in PFAS concentrations

DNA methylation patterns identify subgroups of pancreatic neuroendocrine tumors with clinical association

Here we report the DNA methylation profile of 84 sporadic pancreatic neuroendocrine tumors (PanNETs) with associated clinical and genomic information. We identified three subgroups of PanNETs, termed T1, T2 and T3, with distinct patterns of methylation. The T1 subgroup was enriched for functional tumors and ATRX, DAXX and MEN1 wild-type genotypes. The T2 subgroup contained tumors with mutations in ATRX, DAXX and MEN1 and recurrent patterns of chromosomal losses in half of the genome with no association between regions with recurrent loss and methylation levels. T2 tumors were larger and had lower methylation in the MGMT gene body, which showed positive correlation with gene expression. The T3 subgroup harboured mutations in MEN1 with recurrent loss of chromosome 11, was enriched for grade G1 tumors and showed histological parameters associated with better prognosis. Our results suggest a role for methylation in both driving tumorigenesis and potentially stratifying prognosis in PanNETs

Socializing One Health: an innovative strategy to investigate social and behavioral risks of emerging viral threats

In an effort to strengthen global capacity to prevent, detect, and control infectious diseases in animals and people, the United States Agency for International Development’s (USAID) Emerging Pandemic Threats (EPT) PREDICT project funded development of regional, national, and local One Health capacities for early disease detection, rapid response, disease control, and risk reduction. From the outset, the EPT approach was inclusive of social science research methods designed to understand the contexts and behaviors of communities living and working at human-animal-environment interfaces considered high-risk for virus emergence. Using qualitative and quantitative approaches, PREDICT behavioral research aimed to identify and assess a range of socio-cultural behaviors that could be influential in zoonotic disease emergence, amplification, and transmission. This broad approach to behavioral risk characterization enabled us to identify and characterize human activities that could be linked to the transmission dynamics of new and emerging viruses. This paper provides a discussion of implementation of a social science approach within a zoonotic surveillance framework. We conducted in-depth ethnographic interviews and focus groups to better understand the individual- and community-level knowledge, attitudes, and practices that potentially put participants at risk for zoonotic disease transmission from the animals they live and work with, across 6 interface domains. When we asked highly-exposed individuals (ie. bushmeat hunters, wildlife or guano farmers) about the risk they perceived in their occupational activities, most did not perceive it to be risky, whether because it was normalized by years (or generations) of doing such an activity, or due to lack of information about potential risks. Integrating the social sciences allows investigations of the specific human activities that are hypothesized to drive disease emergence, amplification, and transmission, in order to better substantiate behavioral disease drivers, along with the social dimensions of infection and transmission dynamics. Understanding these dynamics is critical to achieving health security--the protection from threats to health-- which requires investments in both collective and individual health security. Involving behavioral sciences into zoonotic disease surveillance allowed us to push toward fuller community integration and engagement and toward dialogue and implementation of recommendations for disease prevention and improved health security

Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR

Current approaches for generating major histocompatibility complex (MHC) Class-I proteins with desired bound peptides (pMHC-I) for research, diagnostic and therapeutic applications are limited by the inherent instability of empty MHC-I molecules. Using the properties of the chaperone TAP-binding protein related (TAPBPR), we have developed a robust method to produce soluble, peptide-receptive MHC-I molecules in Chinese Hamster Ovary cells at high yield, completely bypassing the requirement for laborious refolding from inclusion bodies expressed in E.coli. Purified MHC-I/TAPBPR complexes can be prepared for multiple human allotypes, and exhibit complex glycan modifications at the conserved Asn 86 residue. As a proof of concept, we demonstrate both HLA allele-specific peptide binding and MHC-restricted antigen recognition by T cells for two relevant tumor-associated antigens. Our system provides a facile, high-throughput approach for generating pMHC-I antigens to probe and expand TCR specificities present in polyclonal T cell repertoires

Structural insights into the broad protection against H1 influenza viruses by a computationally optimized hemagglutinin vaccine

Abstract Influenza virus poses an ongoing human health threat with pandemic potential. Due to mutations in circulating strains, formulating effective vaccines remains a challenge. The use of computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) proteins is a promising vaccine strategy to protect against a wide range of current and future influenza viruses. Though effective in preclinical studies, the mechanistic basis driving the broad reactivity of COBRA proteins remains to be elucidated. Here, we report the crystal structure of the COBRA HA termed P1 and identify antigenic and glycosylation properties that contribute to its immunogenicity. We further report the cryo-EM structure of the P1-elicited broadly neutralizing antibody 1F8 bound to COBRA P1, revealing 1F8 to recognize an atypical receptor binding site epitope via an unexpected mode of binding

Screen of MN glycan mutant supernatants for improvements to bN-mAb binding.

<p>A FIA was used to identify 293 GnTI^- expressed MN-rgp120 glycan variants exhibiting improved bN-mAb binding profiles as compared to the wildtype MN sequence expressed in GnTI^- (MN358). Recombinant gp120s were expressed in GnTI^- 293 cells via transient transfection, and transfection supernatants were normalized to contain ~2μg/mL. MN-rgp120 variants were captured onto 96 well black plates using a 1μg/mL concentration of mouse monoclonal antibody to an N-terminal gD tag. Binding curves to the VRCO1 bN-mAb, which binds a conformation dependent epitope in the CD4 binding site, were used to assay for maintenance of overall secondary and tertiary structure. All screening assays were performed in duplicate. MN-rgp120 glycan variants were assayed for improved antigenicity to a panel of glycan dependent bN-mAbs to be considered for further analysis.</p

Endo H digest and immunoblot of A244 gp120 glycan variants.

<p>A244-rgp120s containing either N332 or N334 based PNGS were expressed in either CHO-S (lanes 1–4) or HEK 293 GnTI^- cells (lanes 5–8) via transient transfection. Purified protein was subjected to Endo H or mock digest (digest buffer alone), and analyzed for mobility on 4–12% reducing SDS-PAGE gels. Immunoblots were probed with the mouse monoclonal 34.1 that binds a conformation independent epitope in the N-terminal gD tag of all expressed proteins, and visualized with goat-anti-mouse HRP conjugated polyclonal sera.</p

Binding of MN glycan variants to extended panel of bN-mAbs.

<p>The MN-rgp120 variants MN358 and MN1320 expressed in GnTI^- cells were compared to MN_GNE for improved binding to an array of bN-mAbs. Recombinant gp120s were expressed in GnTI^- 293 cells via transient transfection. Purified MN_GNE or transfection supernatants were normalized to contain 4μg/mL rgp120 and captured using 2μg/mL of mouse monoclonal antibody 34.1, then assayed by FIA for bN-mAb binding. Results are reported in μg/mL as (EC50), the concentration of antibody required for a half-maximal binding, measured in Relative Fluorescence Units (RFU) on a titration-binding curve. Values are reported as ≥2.5μg/mL if titration curves did not plateau or if mean EC50 was ≥2.5μg/mL. Binding curves to bN-mAbs were performed in quadruplicate. The rp120 constructs exhibiting statistically significant differences in EC50 values (p<0.05) from the MN_GNE are noted in bold. Human IgGK polyclonal antibody was used as a negative control and purified goat polyclonal antibody raised against rgp120 (PB94) was used as a coating control.</p

Modification of N-linked glycosylation sites in MN- and A244-rgp120.

<p>(<b>A</b>) The A244-rgp120 or MN-rgp120 sequences were analyzed for the presence of highly conserved glycans known to be important for bN-mAb binding within the C2-C3 domains. Glycosylation sites are represented as either black (present in RV144 immunogen) or grey (absent in original RV144 immunogen) structures. (<b>B</b>) A ribbon diagram depicts the 3-dimensional arrangement of the N289, N301, N332, and N334 PNGS. The structure is based on crystal structure of the BG505 SOSIP.664 gp140 trimer (in gold) bound to the PGT122 bN-mAb (in grey) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196370#pone.0196370.ref041" target="_blank">41</a>]. The N301 and N332 glycan structures immobilized by the PGT122 antibody are indicated in green, while the asparagine residues at the base of relevant PNGS are indicated in red. (<b>C</b>) Site directed mutagenesis was used to create MN- or A244-rgp120 variants introducing one or more of the indicated PNGS. A summary of the PNGS variant constructs assayed is shown. The constructs with identical number and location of PNGS to the RV144 rgp120 immunogens are marked with an asterisk.</p