Phenotypic lentivirus screens to identify functional single domain antibodies

Manipulation of proteins is key in assessing their in vivo function. Although genetic ablation is straightforward, reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as camelid-derived VHHs, can serve as inhibitors or activators of intracellular protein function, but functional testing of identified VHHs is laborious. To address this challenge, we have developed a lentiviral screening approach to identify VHHs that elicit a phenotype when expressed intracellularly. We identified 19 antiviral VHHs that protect human A549 cells from lethal infection with influenza A virus (IAV) or vesicular stomatitis virus (VSV), respectively. Both negative-sense RNA viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins. Antiviral VHHs prevented nuclear import of viral ribonucleoproteins or mRNA transcription, respectively, and may provide clues for novel antiviral reagents. In principle, the screening approach described here should be applicable to identify inhibitors of any pathogen or biological pathway. To identify the proteins essential to a biological pathway, small-molecule inhibitors or activators may be used to manipulate protein function transiently. Alternatively, screens involving mutagenesis, a reduction in levels or complete elimination of gene products are common. As applied to mammalian cells, these methods usually seek to achieve the removal of a protein from its normal biological context. Many proteins are multifunctional, or are components of multi-subunit complexes. Depletion of any single component may cause unexpected phenotypes due to the collapse of entire protein complexes. Small-molecule inhibitors often lack specificity and at best can target a fraction of all the proteins of interest. The screening of chemically diverse libraries must be paired with sophisticated methods to identify the molecular targets of any hit identified. Antibodies have been used as intracellular perturbants of protein function after microinjection or cytosolic expression of single-chain variable antibody fragments, but technical challenges have limited their application to few selected cases. In addition to conventional antibodies, the immune system of camelids generates heavy-chain-only antibodies. Their antigen binding site only consists of the variable domain of the heavy chain. This domain can be expressed on its own and is referred to as a VHH or nanobody, an entity that can retain its function in the reducing environment of the cytosol, independent of glycosylation. Many VHHs bind to their targets with affinities comparable to conventional antibodies. VHHs expressed in the cytosol can therefore act as molecular perturbants by occluding the interfaces involved in protein–protein interactions, by binding in the active sites of enzymes, or through the recognition or stabilization of distinct conformations of their targets. Both phage and yeast display, as well as mass spectrometry in combination with high-throughput sequencing, allow the identification of VHHs based on their binding properties. However, the identification of inhibitory VHHs remains a time-consuming process. VHHs obtained through biochemical screening methods must be expressed individually in the relevant cell type to test for the functional consequences of VHH expression. To address this challenge, we developed a phenotypic VHH screening method in living cells.National Institutes of Health (U.S.) (Health Pioneer Award

A novel role for XIAP in copper homeostasis through regulation of MURR1

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102131/1/emboj7600031.pd

<i>Fusobacterium </i>spp. target human CEACAM1 via the trimeric autotransporter adhesin CbpF

Neisseria meningitidis, Haemophilus influenzae, and Moraxella catarrhalis are pathogenic bacteria adapted to reside on human respiratory mucosal epithelia. One common feature of these species is their ability to target members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, especially CEACAM1, which is achieved via structurally distinct ligands expressed by each species. Beside respiratory epithelial cells, cells at the dentogingival junction express high levels of CEACAM1. It is possible that bacterial species resident within the oral cavity also utilise CEACAM1 for colonisation and invasion of gingival tissues. From a screen of 59 isolates from the human oral cavity representing 49 bacterial species, we identified strains from Fusobacterium bound to CEACAM1. Of the Fusobacterium species tested, the CEACAM1-binding property was exhibited by F. nucleatum (Fn) and F. vincentii (Fv) but not F. polymorphum (Fp) or F. animalis (Fa) strains tested. These studies identified that CEACAM adhesion was mediated using a trimeric autotransporter adhesin (TAA) for which no function has thus far been defined. We therefore propose the name CEACAM binding protein of Fusobacterium (CbpF). CbpF was identified to be present in the majority of unspeciated Fusobacterium isolates confirming a subset of Fusobacterium spp. are able to target human CEACAM1

Murfreesboro Addresses

https://digitalcommons.acu.edu/crs_books/1086/thumbnail.jp

Evaluation of online videos to engage viewers and support decision-making for COVID-19 vaccination: how narratives and race/ethnicity enhance viewer experiences

BackgroundVaccine hesitancy has hampered the control of COVID-19 and other vaccine-preventable diseases.MethodsWe conducted a national internet-based, quasi-experimental study to evaluate COVID-19 vaccine informational videos. Participants received an informational animated video paired with the randomized assignment of (1) a credible source (differing race/ethnicity) and (2) sequencing of a personal narrative before or after the video addressing their primary vaccine concern. We examined viewing time and asked video evaluation questions to those who viewed the full video.ResultsAmong 14,235 participants, 2,422 (17.0%) viewed the full video. Those who viewed a personal story first (concern video second) were 10 times more likely to view the full video (p &lt; 0.01). Respondent–provider race/ethnicity congruence was associated with increased odds of viewing the full video (aOR: 1.89, p &lt; 0.01). Most viewers rated the informational video(s) to be helpful, easy to understand, trustworthy, and likely to impact others' vaccine decisions, with differences by demographics and also vaccine intentions and concerns.ConclusionUsing peer-delivered, personal narrative, and/or racially congruent credible sources to introduce and deliver vaccine safety information may improve the openness of vaccine message recipients to messages and engagement

The Lawyer in the Dutch Interrogation Room: Influence on Police and Suspect

In many European countries, providing a suspect in custody with legal aid before the first police interrogation is a heavily debated issue. In this paper, we report on an exploratory study on the use of coercion by the police and the use of the right to silence by suspects in 70 Dutch homicide cases and their relation to prior consultation and presence of a lawyer. Analysis of the data indicates that there is a relation between the presence of a lawyer in the interrogation room and the way in which police interrogators use coercion. To gain insight into whether the police use coercion and how this is achieved, we looked at the extent to which the interrogators make use of certain interrogation techniques and how the interrogation techniques are used to exert coercion. We found that legal advice from a lawyer before and during the interrogation corresponds with suspects more often using their right to silence. It also appears that the police are inclined to use ‘hard coercion’ when confronted with a silent suspect. The research thus raises the question as to whether the presence of a lawyer is an adequate way to prevent false confessions. Copyright © 2012 John Wiley & Sons, Ltd

Genetic architecture of sporadic frontotemporal dementia and overlap with Alzheimer's and Parkinson's diseases.

BACKGROUND: Clinical, pathological and genetic overlap between sporadic frontotemporal dementia (FTD), Alzheimer's disease (AD) and Parkinson's disease (PD) has been suggested; however, the relationship between these disorders is still not well understood. Here we evaluated genetic overlap between FTD, AD and PD to assess shared pathobiology and identify novel genetic variants associated with increased risk for FTD. METHODS: Summary statistics were obtained from the International FTD Genomics Consortium, International PD Genetics Consortium and International Genomics of AD Project (n>75 000 cases and controls). We used conjunction false discovery rate (FDR) to evaluate genetic pleiotropy and conditional FDR to identify novel FTD-associated SNPs. Relevant variants were further evaluated for expression quantitative loci. RESULTS: We observed SNPs within the HLA, MAPT and APOE regions jointly contributing to increased risk for FTD and AD or PD. By conditioning on polymorphisms associated with PD and AD, we found 11 loci associated with increased risk for FTD. Meta-analysis across two independent FTD cohorts revealed a genome-wide signal within the APOE region (rs6857, 3'-UTR=PVRL2, p=2.21×10-12), and a suggestive signal for rs1358071 within the MAPT region (intronic=CRHR1, p=4.91×10-7) with the effect allele tagging the H1 haplotype. Pleiotropic SNPs at the HLA and MAPT loci associated with expression changes in cis-genes supporting involvement of intracellular vesicular trafficking, immune response and endo/lysosomal processes. CONCLUSIONS: Our findings demonstrate genetic pleiotropy in these neurodegenerative diseases and indicate that sporadic FTD is a polygenic disorder where multiple pleiotropic loci with small effects contribute to increased disease risk

Mutational spectrum in a worldwide study of 29,700 families with BRCA1 or BRCA2 mutations.

The prevalence and spectrum of germline mutations in BRCA1 and BRCA2 have been reported in single populations, with the majority of reports focused on White in Europe and North America. The Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) has assembled data on 18,435 families with BRCA1 mutations and 11,351 families with BRCA2 mutations ascertained from 69 centers in 49 countries on six continents. This study comprehensively describes the characteristics of the 1,650 unique BRCA1 and 1,731 unique BRCA2 deleterious (disease-associated) mutations identified in the CIMBA database. We observed substantial variation in mutation type and frequency by geographical region and race/ethnicity. In addition to known founder mutations, mutations of relatively high frequency were identified in specific racial/ethnic or geographic groups that may reflect founder mutations and which could be used in targeted (panel) first pass genotyping for specific populations. Knowledge of the population-specific mutational spectrum in BRCA1 and BRCA2 could inform efficient strategies for genetic testing and may justify a more broad-based oncogenetic testing in some populations