We combined efficient sample preparation and ultra‐low‐flow liquid chromatography with a newly developed data acquisition and analysis scheme termed wide window acquisition (WWA) to quantify >3,000 ...proteins from single cells in rapid label‐free analyses. WWA employs large isolation windows to intentionally co‐isolate and co‐fragment adjacent precursors along with the selected precursor. Optimized WWA increased the number of MS2‐identified proteins by ≈40 % relative to standard data‐dependent acquisition. For a 40‐min LC gradient operated at ≈15 nL/min, we identified an average of 3,524 proteins per single‐cell‐sized aliquot of protein digest. Reducing the active gradient to 20 min resulted in a modest 10 % decrease in proteome coverage. Using this platform, we compared protein expression between single HeLa cells having an essential autophagy gene, atg9a, knocked out, with their isogenic WT parental line. Similar proteome coverage was observed, and 268 proteins were significantly up‐ or downregulated. Protein upregulation primarily related to innate immunity, vesicle trafficking and protein degradation.
A new data acquisition strategy for biological mass spectrometry, termed wide‐window acquisition, combined with efficient sample preparation and ultra‐low‐flow liquid chromatography, enables single‐cell proteome profiling to an unprecedented depth of >3000 proteins per cell. This advance allows additional classes of proteins to be studied within single cells.
Satellite-derived remote-sensing products are providing a modern circumpolar perspective of Arctic vegetation and its changes, but this new view is dependent on a long heritage of ground-based ...observations in the Arctic. Several products of the Conservation of Arctic Flora and Fauna are key to our current understanding. We review aspects of the PanArctic Flora, the Circumpolar Arctic Vegetation Map, the Arctic Biodiversity Assessment, and the Arctic Vegetation Archive (AVA) as they relate to efforts to describe and map the vegetation, plant biomass, and biodiversity of the Arctic at circumpolar, regional, landscape and plot scales. Cornerstones for all these tools are ground-based plant-species and plant-community surveys. The AVA is in progress and will store plot-based vegetation observations in a public-accessible database for vegetation classification, modeling, diversity studies, and other applications. We present the current status of the Alaska Arctic Vegetation Archive (AVA-AK), as a regional example for the panarctic archive, and with a roadmap for a coordinated international approach to survey, archive and classify Arctic vegetation. We note the need for more consistent standards of plot-based observations, and make several recommendations to improve the linkage between plot-based observations biodiversity studies and satellite-based observations of Arctic vegetation.
As genomics advances reveal the cancer gene landscape, a daunting task is to understand how these genes contribute to dysregulated oncogenic pathways. Integration of cancer genes into networks offers ...opportunities to reveal protein-protein interactions (PPIs) with functional and therapeutic significance. Here, we report the generation of a cancer-focused PPI network, termed OncoPPi, and identification of >260 cancer-associated PPIs not in other large-scale interactomes. PPI hubs reveal new regulatory mechanisms for cancer genes like MYC, STK11, RASSF1 and CDK4. As example, the NSD3 (WHSC1L1)-MYC interaction suggests a new mechanism for NSD3/BRD4 chromatin complex regulation of MYC-driven tumours. Association of undruggable tumour suppressors with drug targets informs therapeutic options. Based on OncoPPi-derived STK11-CDK4 connectivity, we observe enhanced sensitivity of STK11-silenced lung cancer cells to the FDA-approved CDK4 inhibitor palbociclib. OncoPPi is a focused PPI resource that links cancer genes into a signalling network for discovery of PPI targets and network-implicated tumour vulnerabilities for therapeutic interrogation.
Chronic wounds (e.g. diabetic foot ulcers) reduce the quality of life, yet treatments remain limited. Glucocorticoids (activated by the enzyme 11β-hydroxysteroid dehydrogenase type 1, 11β-HSD1) ...impair wound healing.
Efficacy, safety, and feasibility of 11β-HSD1 inhibition for skin function and wound healing.
Investigator-initiated, double-blind, randomized, placebo-controlled, parallel-group phase 2b pilot trial.
Single-center secondary care setting. Adults with type 2 diabetes mellitus without foot ulcers were administered 400 mg oral 11β-HSD1 inhibitor AZD4017 (n = 14) or placebo (n = 14) bi-daily for 35 days. Participants underwent 3-mm full-thickness punch skin biopsies at baseline and on day 28; wound healing was monitored after 2 and 7 days. Computer-generated 1:1 randomization was pharmacy-administered. Analysis was descriptive and focused on CI estimation. Of the 36 participants screened, 28 were randomized.
Exploratory proof-of-concept efficacy analysis suggested AZD4017 did not inhibit 24-h ex vivoskin 11β-HSD1 activity (primary outcome; difference in percentage conversion per 24 h 1.1% (90% CI: -3.4 to 5.5) but reduced systemic 11β-HSD1 activity by 87% (69-104%). Wound diameter was 34% (7-63%) smaller with AZD4017 at day 2, and 48% (12-85%) smaller after repeat wounding at day 30. AZD4017 improved epidermal integrity but modestly impaired barrier function. Minimal adverse events were comparable to placebo. Recruitment rate, retention, and data completeness were 2.9/month, 27/28, and 95.3%, respectively.
A phase 2 trial is feasible, and preliminary proof-of-concept data suggests AZD4017 warrants further investigation in conditions of delayed healing, for example in diabetic foot ulcers.
Stress hormone activation by the enzyme 11β-HSD type 1 impairs skin function (e.g. integrity) and delays wound healing in animal models of diabetes, but effects in human skin were previously unknown. Skin function was evaluated in response to treatment with a 11β-HSD type 1 inhibitor (AZD4017), or placebo, in people with type 2 diabetes. Importantly, AZD4017 was safe and well tolerated. This first-in-human randomized, controlled, clinical trial found novel evidence that 11β-HSD type 1 regulates skin function in humans, including improved wound healing, epidermal integrity, and increased water loss. Results warrant further studies in conditions of impaired wound healing, for example, diabetic foot ulcers to evaluate 11β-HSD type 1 as a novel therapeutic target forchronic wounds.
DNA methylation (DNAm) age acceleration (AgeAccel) and cardiac age by 12-lead advanced electrocardiography (A-ECG) are promising biomarkers of biological and cardiac aging, respectively. We aimed to ...explore the relationships between DNAm age and A-ECG heart age and to understand the extent to which DNAm AgeAccel relates to cardiovascular (CV) risk factors in a British birth cohort from 1946.
We studied four DNAm ages (AgeHannum, AgeHorvath, PhenoAge, and GrimAge) and their corresponding AgeAccel. Outcomes were the results from two publicly available ECG-based cardiac age scores: the Bayesian A-ECG-based heart age score of Lindow et al. 2022 and the deep neural network (DNN) ECG-based heart age score of Ribeiro et al. 2020. DNAm AgeAccel was also studied relative to results from two logistic regression-based A-ECG disease scores, one for left ventricular (LV) systolic dysfunction (LVSD), and one for LV electrical remodeling (LVER). Generalized linear models were used to explore the extent to which any associations between biological cardiometabolic risk factors (body mass index, hypertension, diabetes, high cholesterol, previous cardiovascular disease CVD, and any CV risk factor) and the ECG-based outcomes are mediated by DNAm AgeAccel. We derived the total effects, average causal mediation effects (ACMEs), average direct effects (ADEs), and the proportion mediated PM with their 95% confidence intervals CIs. 498 participants (all 60-64 years) were included, with the youngest ECG heart age being 27 and the oldest 90. When exploring the associations between cardiometabolic risk factors and Bayesian A-ECG cardiac age, AgeAccelPheno appears to be a partial mediator, as ACME was 0.23 years 0.01, 0.52 p = 0.028 (i.e., PM≈18%) for diabetes, 0.34 0.03, 0.74 p = 0.024 (i.e., PM≈15%) for high cholesterol, and 0.34 0.03, 0.74 p = 0.024 (PM≈15%) for any CV risk factor. Similarly, AgeAccelGrim mediates ≈30% of the relationship between diabetes or high cholesterol and the DNN ECG-based heart age. When exploring the link between cardiometabolic risk factors and the A-ECG-based LVSD and LVER scores, it appears that AgeAccelPheno or AgeAccelGrim mediate 10-40% of these associations.
By the age of 60, participants with accelerated DNA methylation appear to have older, weaker, and more electrically impaired hearts. We show that the harmful effects of CV risk factors on cardiac age and health, appear to be partially mediated by DNAm AgeAccelPheno and AgeAccelGrim. This highlights the need to further investigate the potential cardioprotective effects of selective DNA methyltransferases modulators.
Primate crop raiding is a major cause of human-wildlife conflict around the forests of western Uganda. In an attempt to ameliorate the situation a conflict mitigation strategy was established in ...villages around the Budongo Forest Reserve in 2001. Live-traps were constructed that allowed the identification of crop raiding animals; pest species could be disposed of and threatened species released unharmed. However, by 2004 none of the traps in the study area were functioning and interviews were conducted to assess the reasons for their decline and local people's acceptance of the intervention. Forty-one percent of respondents did not believe the strategy was effective and the majority of local farmers did not accept responsibility for the traps. This was because of operational failures in four areas: (1) the identification of key stakeholders, (2) objective evaluation to assess the efficacy and benefit of the intervention, (3) participatory monitoring and evaluation, and (4) long-term funding commitment by conservation agencies. We examine the impact of these four elements upon the sustainability of the live-trap programme and stress the importance of recognizing and reporting failures to develop effective and acceptable mitigation strategies.
A global clock distribution strategy used on several microprocessor chips is described. The clock network consists of buffered tunable trees or treelike networks, with the final level of trees all ...driving a single common grid covering most of the chip. This topology combines advantages of both trees and grids. A new tuning method was required to efficiently tune such a large strongly connected interconnect network consisting of up to 6 m of wire and modeled with 50000 resistors, capacitors, and inductors. Variations are described to handle different floor-planning styles. Global clock skew as low as 22 ps on large microprocessor chips has been measured.
Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic ...processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. High-resolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having α,β -unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme's catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS/PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.
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