A genetic screen to isolate “Lariat” peptide inhibitors of protein function

Functional genomic analyses provide information that allows hypotheses to be formulated on protein function. These hypotheses, however, need to be validated using reverse genetic approaches, which are difficult to perform on a large scale and in diploid organisms. To address this problem, we developed a genetic screen to rapidly isolate “lariat” peptides that function as trans dominant inhibitors of protein function. We engineered intein proteins to genetically produce lariats. A lariat consists of a lactone peptide covalently attached to a linear peptide. Cyclizing peptides with a lactone bond imposes a constraint even within the reducing environment found inside of cells. The covalently attached linear peptide provides a site for fusing protein moieties. We fused a transcriptional activation domain to a combinatorial lactone peptide, which allowed combinatorial lariat libraries to be screened for protein interactions using the yeast two-hybrid assay. We confirmed that the intein processed in yeast using Western blot analysis. A chemoselective ring opening of the lactone bond with heavy water, followed by mass spectrometry analysis showed that ~ 44% of purified lariat contained an intact lactone bond. To improve the stability of the lactone bond, we introduced mutations into the engineered intein and analyzed their processing and stability by mass spectrometery. Several mutations were identified that increased the amount of intact lariat. Combinatorial libraries of lactone peptides were generated and screened using the yeast-two-hybrid interaction trap. Lactone cyclic peptides that bound to a number of different targets including LexA, Jak2, and Riz1 were isolated. A lactone cyclic peptide isolated against the bacterial repressor protein LexA was characterized. LexA regulates bacterial SOS response and LexA mutants that cannot undergo autoproteolyis make bacteria more sensitive to, and inhibit resistance against cytotoxic reagents. The anti-LexA lariat interacted with LexA with a dissociation constant of 37 µM by surface plasmon resonance. The lactone constraint was determined to be required for the interaction of the anti-LexA L2 lariat with LexA in the yeast-two-hybrid assay. Alanine scanning showed that only two amino acids (G8 and E9) in the anti-LexA L2 sequence (1-SRSWDLPGEY-10) were not required for the interaction with LexA. The interaction of the anti-LexA lariat with LexA in vivo was confirmed by chromatin precipitation of the lactone peptide-LexA-DNA complex. The anti-microbial properties of the anti-LexA lariat were also characterized. The anti-LexA lariat potentiated the activity of a DNA damaging agent mitomycin C and inhibited the cleavage of LexA, preventing the SOS response pathway from being activated. In summary, lariats possess desired traits for characterizing the function and therapeutic potential of proteins. The ability to genetically and chemically synthesize lariats allows the lariat transcription activation domain to be replaced by other peptide and chemical moieties such as affinity tags, fluorescent molecules, localization sequences, et cetera, which give them advantages over “head to tail” cyclized peptides, which have no free end to attach moieties

DAGS:Key encapsulation using dyadic GS codes

Code-based cryptography is one of the main areas of interest for NIST's Post-Quantum Cryptography Standardization call. In this paper, we introduce DAGS, a Key Encapsulation Mechanism (KEM) based on quasi-dyadic generalized Srivastava codes. The scheme is proved to be IND-CCA secure in both random oracle model and quantum random oracle model. We believe that DAGS will offer competitive performance, especially when compared with other existing code-based schemes, and represent a valid candidate for post-quantum standardization.</p

Assessing gastro-intestinal related quality of life in cystic fibrosis: Validation of PedsQL GI in children and their parents

Background: Most patients with cystic fibrosis (CF) suffer from pancreatic insufficiency, leading to fat malabsorption, malnutrition and abdominal discomfort. Until recently, no specific tool was available for assessing gastro-intestinal related quality of life (GI QOL) in patients with CF. As the Horizon2020 project MyCyFAPP aims to improve GI QOL by using a newly designed mobile application, a sensitive and reliable outcome measure was needed. We aimed to study the applicability of the existing child-specific Pediatric Quality of Life Inventory, Gastrointestinal Symptoms Scales and Module (PedsQL GI) in children with CF. Methods: A multicenter, prospective observational study was performed in 6 European centers to validate the PedsQL GI in children with CF during 3 months. Results: In total, 248 children and their parents were included. Within-patient variability of PedsQL GI was low (24.11), and there was reasonable agreement between children and parents (ICC 0.681). Nine of 14 subscales were informative (no ceiling effect). The PedsQL GI and the median scores for 4 subscales were significantly lower in patients compared to healthy controls. Positive associations were found between PedsQL GI and age (OR = 1.044, p = 0.004) and between PedsQL GI and BMI z-score (OR = 1.127, p = 0.036). PedsQL GI correlated with most CFQ-R subscales (r 0.268 to 0.623) and with a Visual Analogue Scale (r = 0.20). Conclusions: PedsQL GI is a valid and applicable instrument to assess GI QOL in children with CF. Future research efforts should examine the responsiveness of the CF PedsQL GI to change in the context of clinical interventions and trials

Where are we now with European forest multi-taxon biodiversity and where can we head to?

The European biodiversity and forest strategies rely on forest sustainable management (SFM) to conserve forest biodiversity. However, current sustainability assessments hardly account for direct biodiversity indicators. We focused on forest multi-taxon biodiversity to: i) gather and map the existing information; ii) identify knowledge and research gaps; iii) discuss its research potential. We established a research network to fit data on species, standing trees, lying deadwood and sampling unit description from 34 local datasets across 3591 sampling units. A total of 8724 species were represented, with the share of common and rare species varying across taxonomic classes: some included many species with several rare ones (e.g., Insecta); others (e.g., Bryopsida) were represented by few common species. Tree-related structural attributes were sampled in a subset of sampling units (2889; 2356; 2309 and 1388 respectively for diameter, height, deadwood and microhabitats). Overall, multitaxon studies are biased towards mature forests and may underrepresent the species related to other developmental phases. European forest compositional categories were all represented, but beech forests were overrepresented as compared to thermophilous and boreal forests. Most sampling units (94%) were referred to a habitat type of conservation concern. Existing information may support European conservation and SFM strategies in: (i) methodological harmonization and coordinated monitoring; (ii) definition and testing of SFM indicators and thresholds; (iii) data-driven assessment of the effects of environmental and management drivers on multi-taxon forest biological and functional diversity, (iv) multi-scale forest monitoring integrating in-situ and remotely sensed information. Forest biodiversity Multi-taxon Sustainable management Biodiversity conservation Forest stand structurepublishedVersio

Clinical validation of an evidence-based method to adjust Pancreatic Enzyme Replacement Therapy through a prospective interventional study in paediatric patients with Cystic Fibrosis

Background A method to adjust Pancreatic Enzyme Replacement Therapy in Cystic Fibrosis is not currently available. Objectives To assess the in vivo efficacy of a method to adjust the dose of enzymatic supplement in CF extrapolated from previous in vitro digestion studies (theoretical optimal dose, TOD). Secondly, to assess how individual patient characteristics influence the expected coefficient of fat absorption (CFA) and thus to identify an individual correction factor to improve TOD. Methods A prospective interventional study in 43 paediatric patients with CF from 5 European centres. They followed a 24h fixed diet with the theoretical optimal dose for each meal. Faecal collection was carried out between colorimetric markers in order to include all the faeces corresponding to the fixed diet. Beta regression models were applied to assess the associations of individual patient characteristics with the CFA. Results Median CFA was 90% (84, 94% 1st, 3rd Q.) with no significant differences among centres. Intestinal transit time was positively associated with CFA (p = 0.007), but no statistical associations were found with and age, gender, phenotype or BMI. Regression model showed no improvement of the in vitro predicted theoretical optimal dose when taking individual patient characteristics into account. Conclusion Strict adherence to the theoretical optimal dose of enzymatic supplement for a prescribed meal, led to median CFA levels at the clinical target of 90% with a low variability between patients. The proposed method can be considered as a first approach for an evidencebased method in PERT dosing based on food characteristics. Results have to be confirmed in free dietary settings

Omega-3 fatty acids for the primary and secondary prevention of cardiovascular disease

Background: Researchers have suggested that omega-3 polyunsaturated fatty acids from oily fish (long-chain omega-3 (LCn3), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)), as well as from plants (alpha-linolenic acid (ALA)) benefit cardiovascular health. Guidelines recommend increasing omega-3-rich foods, and sometimes supplementation, but recent trials have not confirmed this. Objectives: To assess effects of increased intake of fish- and plant-based omega-3 for all-cause mortality, cardiovascular (CVD) events, adiposity and lipids. Search methods: We searched CENTRAL, MEDLINE and Embase to April 2017, plus ClinicalTrials.gov and World Health Organization International Clinical Trials Registry to September 2016, with no language restrictions. We handsearched systematic review references and bibliographies and contacted authors. Selection criteria: We included randomised controlled trials (RCTs) that lasted at least 12 months and compared supplementation and/or advice to increase LCn3 or ALA intake versus usual or lower intake. Data collection and analysis: Two review authors independently assessed studies for inclusion, extracted data and assessed validity. We performed separate random-effects meta-analysis for ALA and LCn3 interventions, and assessed dose-response relationships through meta-regression. Main results: We included 79 RCTs (112,059 participants) in this review update and found that 25 were at low summary risk of bias. Trials were of 12 to 72 months' duration and included adults at varying cardiovascular risk, mainly in high-income countries. Most studies assessed LCn3 supplementation with capsules, but some used LCn3- or ALA-rich or enriched foods or dietary advice compared to placebo or usual diet. Meta-analysis and sensitivity analyses suggested little or no effect of increasing LCn3 on all-cause mortality (RR 0.98, 95% CI 0.90 to 1.03, 92,653 participants; 8189 deaths in 39 trials, high-quality evidence), cardiovascular mortality (RR 0.95, 95% CI 0.87 to 1.03, 67,772 participants; 4544 CVD deaths in 25 RCTs), cardiovascular events (RR 0.99, 95% CI 0.94 to 1.04, 90,378 participants; 14,737 people experienced events in 38 trials, high-quality evidence), coronary heart disease (CHD) mortality (RR 0.93, 95% CI 0.79 to 1.09, 73,491 participants; 1596 CHD deaths in 21 RCTs), stroke (RR 1.06, 95% CI 0.96 to 1.16, 89,358 participants; 1822 strokes in 28 trials) or arrhythmia (RR 0.97, 95% CI 0.90 to 1.05, 53,796 participants; 3788 people experienced arrhythmia in 28 RCTs). There was a suggestion that LCn3 reduced CHD events (RR 0.93, 95% CI 0.88 to 0.97, 84,301 participants; 5469 people experienced CHD events in 28 RCTs); however, this was not maintained in sensitivity analyses - LCn3 probably makes little or no difference to CHD event risk. All evidence was of moderate GRADE quality, except as noted. Increasing ALA intake probably makes little or no difference to all-cause mortality (RR 1.01, 95% CI 0.84 to 1.20, 19,327 participants; 459 deaths, 5 RCTs),cardiovascular mortality (RR 0.96, 95% CI 0.74 to 1.25, 18,619 participants; 219 cardiovascular deaths, 4 RCTs), and it may make little or no difference to CHD events (RR 1.00, 95% CI 0.80 to 1.22, 19,061 participants, 397 CHD events, 4 RCTs, low-quality evidence). However, increased ALA may slightly reduce risk of cardiovascular events (from 4.8% to 4.7%, RR 0.95, 95% CI 0.83 to 1.07, 19,327 participants; 884 CVD events, 5 RCTs, low-quality evidence), and probably reduces risk of CHD mortality (1.1% to 1.0%, RR 0.95, 95% CI 0.72 to 1.26, 18,353 participants; 193 CHD deaths, 3 RCTs), and arrhythmia (3.3% to 2.6%, RR 0.79, 95% CI 0.57 to 1.10, 4,837 participants; 141 events, 1 RCT). Effects on stroke are unclear. Sensitivity analysis retaining only trials at low summary risk of bias moved effect sizes towards the null (RR 1.0) for all LCn3 primary outcomes except arrhythmias, but for most ALA outcomes, effect sizes moved to suggest protection. LCn3 funnel plots suggested that adding in missing studies/results would move effect sizes towards null for most primary outcomes. There were no dose or duration effects in subgrouping or meta-regression. There was no evidence that increasing LCn3 or ALA altered serious adverse events, adiposity or lipids, although LCn3 slightly reduced triglycerides and increased HDL. ALA probably reduces HDL (high- or moderate-quality evidence). Authors' conclusions: This is the most extensive systematic assessment of effects of omega-3 fats on cardiovascular health to date. Moderate- and high-quality evidence suggests that increasing EPA and DHA has little or no effect on mortality or cardiovascular health (evidence mainly from supplement trials). Previous suggestions of benefits from EPA and DHA supplements appear to spring from trials with higher risk of bias. Low-quality evidence suggests ALA may slightly reduce CVD event risk, CHD mortality and arrhythmia

Emerging indicators of fish welfare in aquaculture

As aquaculture continues to grow and intensify, there is increasing public concern over the welfare of farmed fish. Stress and production-related pathologies and repressed growth are examples of the challenges facing aquaculture, and their impacts could be minimised by effective identification of the early signs of impaired welfare. Many welfare monitoring methods have been recommended, however, continuous and reliable welfare monitoring in aquaculture is not yet widespread and commonplace. The aim of this scoping review was to present an overview of the most recent developments in fish welfare assessments with a specific focus on practical translation to the aquaculture industry. A keyword-based search was undertaken to identify peer-reviewed papers published between 2014-2020 in which a novel method with the potential to be used for the assessment of fish welfare in aquaculture was introduced. The results were sorted into two categories: non-invasive and invasive methods. All methods were assessed for their advantages and disadvantages, potential applicability to aquaculture. Invasive methods were also ranked on their degree of impact. It is concluded that increased interest into fish welfare, in combination with more intelligent modern technology, has resulted in the development of newer and more refined alternatives to traditional methods of welfare assessment such as behaviour monitoring by 2D cameras and plasma cortisol evaluation. Although, in many cases, more research is needed before these methods are suitable for widespread industry use, studies that focus on increasing the precision, automation and practical applicability of these methods are a promising avenue for future research.Output Status: Forthcoming/Available Onlin

Imaging Immune Cells Using Fc Domain Probes in Mouse Cancer Xenograft Models

Tracking immune responses is complex due to the mixture of cell types, variability in cell populations, and the dynamic environment. Tissue biopsies and blood analysis can identify infiltrating and circulating immune cells; however, due to the dynamic nature of the immune response, these are prone to sampling errors. Non-invasive targeted molecular imaging provides a method to monitor immune response, which has advantages of providing whole-body images, being non-invasive, and allowing longitudinal monitoring. Three non-specific Fc-containing proteins were labeled with near-infrared dye IRDye800CW and used as imaging probes to assess tumor-infiltrating immune cells in FaDu and A-431 xenograft models. We showed that Fc domains localize to tumors and are visible by fluorescent imaging. This tumor localization appears to be based on binding tumor-associated immune cells and some xenografts showed higher fluorescent signals than others. The Fc domain alone bound to different human immune cell types. The Fc domain can be a valuable research tool to study innate immune response