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

The allocative effectiveness of market protocols under intelligent trading

Abstract. We study the performance of four market protocols that lead to allocative ef-ficiency: batch auction, continuous double auction, specialist dealership, and a hybrid of these last two. In a former study, we compared them with respect to several additional performance criteria under the assumption of zero intelligence. This paper analyzes three performance criteria under different ways to remove the assumption of zero intelligence. The following conclusions are robust. The number of wasteful transaction is minimized by the batch auction and the dealership. Moreover, the former minimizes price dispersion and the latter minimizes time to convergence

Uncoupling vaccination from politics: a call to action

Political polarisation in the USA is impeding vaccination of the population against SARS-CoV-2. Today, the lowest COVID-19 vaccination rates in the USA are overwhelmingly in Republican-leaning states and counties. At a time when the delta variant is spreading, these are also the areas experiencing surges in admissions to hospital and intensive care.1 If political divides on COVID-19 vaccination become ingrained, the consequences could include greater resistance to all vaccination and outbreaks of other vaccine-preventable diseases. Understanding and countering this trend are urgent public health priorities

Night optimised care technology for users needing assisted lifestyles

There is growing interest in the development of ambient assisted living services to increase the quality of life of the increasing proportion of the older population. We report on the Night Optimised Care Technology for UseRs Needing Assisted Lifestyles project, which provides specialised night time support to people at early stages of dementia. This article explains the technical infrastructure, the intelligent software behind the decision-making driving the system, the software development process followed, the interfaces used to interact with the user, and the findings and lessons of our user-centred approach

Cosmological distance indicators

We review three distance measurement techniques beyond the local universe: (1) gravitational lens time delays, (2) baryon acoustic oscillation (BAO), and (3) HI intensity mapping. We describe the principles and theory behind each method, the ingredients needed for measuring such distances, the current observational results, and future prospects. Time delays from strongly lensed quasars currently provide constraints on $H_0$ with < 4% uncertainty, and with 1% within reach from ongoing surveys and efforts. Recent exciting discoveries of strongly lensed supernovae hold great promise for time-delay cosmography. BAO features have been detected in redshift surveys up to z <~ 0.8 with galaxies and z ~ 2 with Ly-$\alpha$ forest, providing precise distance measurements and $H_0$ with < 2% uncertainty in flat $\Lambda$CDM. Future BAO surveys will probe the distance scale with percent-level precision. HI intensity mapping has great potential to map BAO distances at z ~ 0.8 and beyond with precisions of a few percent. The next years ahead will be exciting as various cosmological probes reach 1% uncertainty in determining $H_0$, to assess the current tension in $H_0$ measurements that could indicate new physics.Comment: Review article accepted for publication in Space Science Reviews (Springer), 45 pages, 10 figures. Chapter of a special collection resulting from the May 2016 ISSI-BJ workshop on Astronomical Distance Determination in the Space Ag

South Atlantic Interbasin Exchanges of Mass, Heat, Salt and Anthropogenic Carbon

The exchange of mass, heat, salt and anthropogenic carbon (Cant) between the South Atlantic, south of 24°S, and adjacent ocean basins is estimated from hydrographic data obtained during 2008-2009 using an inverse method. Transports of anthropogenic carbon are calculated across the western (Drake Passage), eastern (30°E) and northern (24°S) boundaries. The freshwater overturning transport of 0.09 Sv is southward, consistent with an overturning circulation that exports freshwater from the North Atlantic, and consistent with a bistable Meridional Overturning Circulation (MOC), under conditions of excess freshwater perturbation. At 30°E, net eastward Antarctic Circumpolar Current (ACC) transport, south of the Subtropical Front, is compensated by a 15.9±2.3 Sv westward flow along the Antarctic boundary. The region as a whole is a substantial sink for atmospheric anthropogenic carbon of 0.51±0.37 PgC yr-1, of which 0.18±0.12 PgC yr-1 accumulates and is stored within the water column. At 24°S, a 20.2 Sv meridional overturning is associated with a 0.11 PgC yr-1 Cant overturning. The remainder is transported into the Atlantic Ocean north of 24°S (0.28±0.16 PgC yr-1) and Indian sector of Southern Ocean (1.12±0.43 PgC yr-1), having been enhanced by inflow through Drake Passage (1.07±0.44 PgC yr-1). This underlines the importance of the South Atlantic as a crucial element of the anthropogenic carbon sink in the global oceans

Associations of common breast cancer susceptibility alleles with risk of breast cancer subtypes in BRCA1 and BRCA2 mutation carriers

Introduction: More than 70 common alleles are known to be involved in breast cancer (BC) susceptibility, and several exhibit significant heterogeneity in their associations with different BC subtypes. Although there are differences in the association patterns between BRCA1 and BRCA2 mutation carriers and the general population for several loci, no study has comprehensively evaluated the associations of all known BC susceptibility alleles with risk of BC subtypes in BRCA1 and BRCA2 carriers. Methods: We used data from 15,252 BRCA1 and 8,211 BRCA2 carriers to analyze the associations between approximately 200,000 genetic variants on the iCOGS array and risk of BC subtypes defined by estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and triple-negative- (TN) status; morphologic subtypes; histological grade; and nodal involvement. Results: The estimated BC hazard ratios (HRs) for the 74 known BC alleles in BRCA1 carriers exhibited moderate correlations with the corresponding odds ratios from the general population. However, their associations with ER-positive BC in BRCA1 carriers were more consistent with the ER-positive as

Comprehensive analysis of epigenetic clocks reveals associations between disproportionate biological ageing and hippocampal volume

The concept of age acceleration, the difference between biological age and chronological age, is of growing interest, particularly with respect to age-related disorders, such as Alzheimer’s Disease (AD). Whilst studies have reported associations with AD risk and related phenotypes, there remains a lack of consensus on these associations. Here we aimed to comprehensively investigate the relationship between five recognised measures of age acceleration, based on DNA methylation patterns (DNAm age), and cross-sectional and longitudinal cognition and AD-related neuroimaging phenotypes (volumetric MRI and Amyloid-β PET) in the Australian Imaging, Biomarkers and Lifestyle (AIBL) and the Alzheimer’s Disease Neuroimaging Initiative (ADNI). Significant associations were observed between age acceleration using the Hannum epigenetic clock and cross-sectional hippocampal volume in AIBL and replicated in ADNI. In AIBL, several other findings were observed cross-sectionally, including a significant association between hippocampal volume and the Hannum and Phenoage epigenetic clocks. Further, significant associations were also observed between hippocampal volume and the Zhang and Phenoage epigenetic clocks within Amyloid-β positive individuals. However, these were not validated within the ADNI cohort. No associations between age acceleration and other Alzheimer’s disease-related phenotypes, including measures of cognition or brain Amyloid-β burden, were observed, and there was no association with longitudinal change in any phenotype. This study presents a link between age acceleration, as determined using DNA methylation, and hippocampal volume that was statistically significant across two highly characterised cohorts. The results presented in this study contribute to a growing literature that supports the role of epigenetic modifications in ageing and AD-related phenotypes

Genome-Wide Association Study in BRCA1 Mutation Carriers Identifies Novel Loci Associated with Breast and Ovarian Cancer Risk

BRCA1-associated breast and ovarian cancer risks can be modified by common genetic variants. To identify further cancer risk-modifying loci, we performed a multi-stage GWAS of 11,705 BRCA1 carriers (of whom 5,920 were diagnosed with breast and 1,839 were diagnosed with ovarian cancer), with a further replication in an additional sample of 2,646 BRCA1 carriers. We identified a novel breast cancer risk modifier locus at 1q32 for BRCA1 carriers (rs2290854, P = 2.7×10-8, HR = 1.14, 95% CI: 1.09-1.20). In addition, we identified two novel ovarian cancer risk modifier loci: 17q21.31 (rs17631303, P = 1.4×10-8, HR = 1.27, 95% CI: 1.17-1.38) and 4q32.3 (rs4691139, P = 3.4×10-8, HR = 1.20, 95% CI: 1.17-1.38). The 4q32.3 locus was not associated with ovarian cancer risk in the general population or BRCA2 carriers, suggesting a BRCA1-specific associat