Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance

Simultaneous labeling of multiple targets in a single sample, or multiplexing, is a powerful approach to directly compare the amount, localization and/or molecular properties of different targets in the same sample. Here we highlight the robust reliability of the simultaneous use of multiple mouse monoclonal antibodies (mAbs) of different immunoglobulin G (IgG) subclasses in a wide variety of multiplexing applications employing anti-mouse IgG subclass-specific secondary antibodies (2°Abs). We also describe the unexpected finding that IgG subclass-specific 2°Abs are superior to general anti-mouse IgG 2°Abs in every tested application in which mouse mAbs were used. This was due to a detection bias of general anti-mouse IgG-specific 2°Abs against mAbs of the most common mouse IgG subclass, IgG1, and to a lesser extent IgG2b mAbs. Thus, when using any of numerous mouse mAbs available through commercial and non-profit sources, for cleaner and more robust results each mAb should be detected with its respective IgG subclass-specific 2°Ab and not a general anti-mouse IgG-specific 2°Ab

A CRISPR screen identifies a pathway required for paraquat-induced cell death

Paraquat, a herbicide linked to Parkinson's disease, generates reactive oxygen species (ROS), which causes cell death. Because the source of paraquat-induced ROS production remains unknown, we conducted a CRISPR-based positive-selection screen to identify metabolic genes essential for paraquat-induced cell death. Our screen uncovered three genes, POR (cytochrome P450 oxidoreductase), ATP7A (copper transporter), and SLC45A4 (sucrose transporter), required for paraquat-induced cell death. Furthermore, our results revealed POR as the source of paraquat-induced ROS production. Thus, our study highlights the use of functional genomic screens for uncovering redox biology

Renal clearable catalytic gold nanoclusters for in vivo disease monitoring

Ultra-small gold nanoclusters (AuNCs) have emerged as agile probes for in vivo imaging, as they exhibit exceptional tumour accumulation and efficient renal clearance properties. However, their intrinsic catalytic activity, which can enable increased detection sensitivity, has yet to be explored for in vivo sensing. By exploiting the peroxidase-mimicking activity of AuNCs and the precise nanometer size filtration of the kidney, we designed multifunctional protease nanosensors that respond to disease microenvironments to produce a direct colorimetric urinary readout of disease state in less than 1 h. We monitored the catalytic activity of AuNCs in collected urine of a mouse model of colorectal cancer where tumour-bearing mice showed a 13-fold increase in colorimetric signal compared to healthy mice. Nanosensors were eliminated completely through hepatic and renal excretion within 4 weeks after injection with no evidence of toxicity. We envision that this modular approach will enable rapid detection of a diverse range of diseases by exploiting their specific enzymatic signatures

Multi-ancestry sleep-by-SNP interaction analysis in 126,926 individuals reveals lipid loci stratified by sleep duration.

Both short and long sleep are associated with an adverse lipid profile, likely through different biological pathways. To elucidate the biology of sleep-associated adverse lipid profile, we conduct multi-ancestry genome-wide sleep-SNP interaction analyses on three lipid traits (HDL-c, LDL-c and triglycerides). In the total study sample (discovery + replication) of 126,926 individuals from 5 different ancestry groups, when considering either long or short total sleep time interactions in joint analyses, we identify 49 previously unreported lipid loci, and 10 additional previously unreported lipid loci in a restricted sample of European-ancestry cohorts. In addition, we identify new gene-sleep interactions for known lipid loci such as LPL and PCSK9. The previously unreported lipid loci have a modest explained variance in lipid levels: most notable, gene-short-sleep interactions explain 4.25% of the variance in triglyceride level. Collectively, these findings contribute to our understanding of the biological mechanisms involved in sleep-associated adverse lipid profiles

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

A global horizon scan of the future impacts of robotics and autonomous systems on urban ecosystems

Technology is transforming societies worldwide. A major innovation is the emergence of robotics and autonomous systems (RAS), which have the potential to revolutionize cities for both people and nature. Nonetheless, the opportunities and challenges associated with RAS for urban ecosystems have yet to be considered systematically. Here, we report the findings of an online horizon scan involving 170 expert participants from 35 countries. We conclude that RAS are likely to transform land use, transport systems and human–nature interactions. The prioritized opportunities were primarily centred on the deployment of RAS for the monitoring and management of biodiversity and ecosystems. Fewer challenges were prioritized. Those that were emphasized concerns surrounding waste from unrecovered RAS, and the quality and interpretation of RAS-collected data. Although the future impacts of RAS for urban ecosystems are difficult to predict, examining potentially important developments early is essential if we are to avoid detrimental consequences but fully realize the benefits

A multi-ancestry genome-wide study incorporating gene-smoking interactions identifies multiple new loci for pulse pressure and mean arterial pressure

Elevated blood pressure (BP), a leading cause of global morbidity and mortality, is influenced by both genetic and lifestyle factors. Cigarette smoking is one such lifestyle factor. Across five ancestries, we performed a genome-wide gene-smoking interaction study of mean arterial pressure (MAP) and pulse pressure (PP) in 129 913 individuals in stage 1 and follow-up analysis in 480 178 additional individuals in stage 2. We report here 136 loci significantly associated with MAP and/or PP. Of these, 61 were previously published through main-effect analysis of BP traits, 37 were recently reported by us for systolic BP and/or diastolic BP through gene-smoking interaction analysis and 38 were newly identified (P <5 x 10(-8), false discovery rate <0.05). We also identified nine new signals near known loci. Of the 136 loci, 8 showed significant interaction with smoking status. They include CSMD1 previously reported for insulin resistance and BP in the spontaneously hypertensive rats. Many of the 38 new loci show biologic plausibility for a role in BP regulation. SLC26A7 encodes a chloride/bicarbonate exchanger expressed in the renal outer medullary collecting duct. AVPR1A is widely expressed, including in vascular smooth muscle cells, kidney, myocardium and brain. FHAD1 is a long non-coding RNA overexpressed in heart failure. TMEM51 was associated with contractile function in cardiomyocytes. CASP9 plays a central role in cardiomyocyte apoptosis. Identified only in African ancestry were 30 novel loci. Our findings highlight the value of multi-ancestry investigations, particularly in studies of interaction with lifestyle factors, where genomic and lifestyle differences may contribute to novel findings.Peer reviewe

HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates.

<p>(A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038313#pone-0038313-g004" target="_blank">Figure 4C</a>, and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′)₂ fragment of HL (<i>e.g.</i>, L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard & Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.</p