High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment

Identification of genetic risk loci and prioritization of genes and pathways for myasthenia gravis : a genome-wide association study

Myasthenia gravis is a chronic autoimmune disease characterized by autoantibody-mediated interference of signal transmission across the neuromuscular junction. We performed a genome-wide association study (GWAS) involving 1,873 patients diagnosed with acetylcholine receptor antibody-positive myasthenia gravis and 36,370 healthy individuals to identify disease-associated genetic risk loci. Replication of the discovered loci was attempted in an independent cohort from the UK Biobank. We also performed a transcriptome-wide association study (TWAS) using expression data from skeletal muscle, whole blood, and tibial nerve to test the effects of disease-associated polymorphisms on gene expression. We discovered two signals in the genes encoding acetylcholine receptor subunits that are the most common antigenic target of the autoantibodies: a GWAS signal within the cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) gene and a TWAS association with the cholinergic receptor nicotinic beta 1 subunit (CHRNB1) gene in normal skeletal muscle. Two other loci were discovered on 10p14 and 11q21, and the previous association signals at PTPN22, HLA-DQA1/HLA-B, and TNFRSF11A were confirmed. Subgroup analyses demonstrate that early-and late-onset cases have different genetic risk factors. Genetic correlation analysis confirmed a genetic link between myasthenia gravis and other autoimmune diseases, such as hypothyroidism, rheumatoid arthritis, multiple sclerosis, and type 1 diabetes. Finally, we applied Priority Index analysis to identify potentially druggable genes/proteins and pathways. This study provides insight into the genetic architecture underlying myasthenia gravis and demonstrates that genetic factors within the loci encoding acetylcholine receptor subunits contribute to its pathogenesis.Peer reviewe

Spider-Venom Peptides as Therapeutics

Spiders are the most successful venomous animals and the most abundant terrestrial predators. Their remarkable success is due in large part to their ingenious exploitation of silk and the evolution of pharmacologically complex venoms that ensure rapid subjugation of prey. Most spider venoms are dominated by disulfide-rich peptides that typically have high affinity and specificity for particular subtypes of ion channels and receptors. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides, making them a valuable resource for drug discovery. Here we review the structure and pharmacology of spider-venom peptides that are being used as leads for the development of therapeutics against a wide range of pathophysiological conditions including cardiovascular disorders, chronic pain, inflammation, and erectile dysfunction

CropPol: a dynamic, open and global database on crop pollination

Seventy five percent of the world's food crops benefit from insect pollination. Hence, there has been increased interest in how global change drivers impact this critical ecosystem service. Because standardized data on crop pollination are rarely available, we are limited in our capacity to understand the variation in pollination benefits to crop yield, as well as to anticipate changes in this service, develop predictions, and inform management actions. Here, we present CropPol, a dynamic, open and global database on crop pollination. It contains measurements recorded from 202 crop studies, covering 3,394 field observations, 2,552 yield measurements (i.e. berry weight, number of fruits and kg per hectare, among others), and 47,752 insect records from 48 commercial crops distributed around the globe. CropPol comprises 32 of the 87 leading global crops and commodities that are pollinator dependent. Malus domestica is the most represented crop (32 studies), followed by Brassica napus (22 studies), Vaccinium corymbosum (13 studies), and Citrullus lanatus (12 studies). The most abundant pollinator guilds recorded are honey bees (34.22% counts), bumblebees (19.19%), flies other than Syrphidae and Bombyliidae (13.18%), other wild bees (13.13%), beetles (10.97%), Syrphidae (4.87%), and Bombyliidae (0.05%). Locations comprise 34 countries distributed among Europe (76 studies), Northern America (60), Latin America and the Caribbean (29), Asia (20), Oceania (10), and Africa (7). Sampling spans three decades and is concentrated on 2001-05 (21 studies), 2006-10 (40), 2011-15 (88), and 2016-20 (50). This is the most comprehensive open global data set on measurements of crop flower visitors, crop pollinators and pollination to date, and we encourage researchers to add more datasets to this database in the future. This data set is released for non-commercial use only. Credits should be given to this paper (i.e., proper citation), and the products generated with this database should be shared under the same license terms (CC BY-NC-SA). This article is protected by copyright. All rights reserved

A Theoretical Analysis of the Geography of Schistosomiasis in Burkina Faso Highlights the Roles of Human Mobility and Water Resources Development in Disease Transmission

We study the geography of schistosomiasis across Burkina Faso by means of a spatially explicit model of water-based disease dynamics. The model quantitatively addresses the geographic stratification of disease burden in a novel framework by explicitly accounting for drivers and controls of the disease, including spatial information on the distributions of population and infrastructure, jointly with a general description of human mobility and climatic/ecological drivers. Spatial patterns of disease are analysed by the extraction and the mapping of suitable eigenvectors of the Jacobian matrix subsuming the stability of the disease-free equilibrium. The relevance of the work lies in the novel mapping of disease burden, a byproduct of the parametrization induced by regional upscaling, by model-guided field validations and in the predictive scenarios allowed by exploiting the range of possible parameters and processes. Human mobility is found to be a primary control at regional scales both for pathogen invasion success and the overall distribution of disease burden. The effects of water resources development highlighted by systematic reviews are accounted for by the average distances of human settlements from water bodies that are habitats for the parasite's intermediate host. Our results confirm the empirical findings about the role of water resources development on disease spread into regions previously nearly disease-free also by inspection of empirical prevalence patterns. We conclude that while the model still needs refinements based on field and epidemiological evidence, the proposed framework provides a powerful tool for large-scale public health planning and schistosomiasis management

Spider-Venom Peptides as Therapeutics

Spiders are the most successful venomous animals and the most abundant terrestrial predators. Their remarkable success is due in large part to their ingenious exploitation of silk and the evolution of pharmacologically complex venoms that ensure rapid subjugation of prey. Most spider venoms are dominated by disulfide-rich peptides that typically have high affinity and specificity for particular subtypes of ion channels and receptors. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides, making them a valuable resource for drug discovery. Here we review the structure and pharmacology of spider-venom peptides that are being used as leads for the development of therapeutics against a wide range of pathophysiological conditions including cardiovascular disorders, chronic pain, inflammation, and erectile dysfunction

High-throughput expression screening and purification of recombinant proteins in E-coli

The protocols outlined in this chapter allow for the small-scale test expression of a single or multiple proteins concurrently using several expression conditions to identify optimal strategies for producing soluble, stable proteins. The protocols can be performed manually without the need for specialized equipment, or can be translated to robotic platforms. The high-throughput protocols begin with transformation in a 96-well format, followed by small-scale test expression using auto-induction medium in a 24-well format, finishing with purification in a 96-well format. Even from such a small scale, there is the potential to use the purified proteins for characterization in pilot studies, for sensitive micro-assays, or for the quick detection of and differentiation of the expected size and oxidation state of the protein by mass spectrometry