Robust optimization of SWATH-MS workflow for human blood serum proteome analysis using a quality by design approach

Background It is not enough to optimize proteomics assays. It is critical those assays are robust to operating conditions. Without robust assays, proteomic biomarkers are unlikely to translate readily into the clinic. This study outlines a structured approach to the identification of a robust operating window for proteomics assays and applies that method to Sequential Window Acquisition of all Theoretical Spectra Mass Spectroscopy (SWATH-MS). Methods We used a sequential quality by design approach exploiting a fractional screening design to first identify critical SWATH-MS parameters, then using response surface methods to identify a robust operating window with good reproducibility, before validating those settings in a separate validation study. Results The screening experiment identified two critical SWATH-MS parameters. We modelled the number of proteins and reproducibility as a function of those parameters identifying an operating window permitting robust maximization of the number of proteins quantified in human serum. In a separate validation study, these settings were shown to give good proteome-wide coverage and high quantification reproducibility. Conclusions Using design of experiments permits identification of a robust operating window for SWATH-MS. The method gives a good understanding of proteomics assays and greater data-driven confidence in SWATH-MS performance.

Antagonistic Properties and Screening of Bacillus Velezensis Nhw-B72 against Wood Fungal Decay

(1) Background: Wood decay is a serious issue that results from the presence of wood-destroying fungi and has a great influence on the international wood industry. The utilization of biological control methods offers good prospects for wood preservation. (2) Methods: The plate-screening experiment, the soil block test of the Chinese stand method (GB/T 13942.1), and the characterization of wood blocks were used to achieve biological control of brown rot and white rot. (3) Results: Through isolation, screening, and identification, the antagonistic bacterium Bacillus velezensis Nhw-B72 strain was obtained. In the plate-screening experiment, the inhibition zone diameter of Nhw-B72 for Gloeophyllum trabeum was 1.68 cm and that for Coriolus versicolor was 2.33 cm. After inhibition, the morphology of mycelia was distorted, malformed, and broken. In the soil block test, the average weight loss percentage of wood blocks in the control group was 61.66%. In the treatment group, the average weight loss percentage of the wood blocks with drying was 28.18% and that of the wood blocks without drying was 34.97%. (4) Conclusions: The strain has an obvious antagonistic effect on the wood-destroying fungi and the sterile fermentative liquid can effectively inhibit wood decay. In addition, compared to the drying of wood blocks, the air-drying of blocks after impregnation with the fermentative liquid had a better inhibition effect.

Antibacterial activity of Lippia alba, Myrcia lundianaand Ocimum basilicumessential oils against six food-spoiling pathogenic microorganisms

The aim of this study was to undertake a screening experiment on essential oils (EO) of Myrcia lundiana, Ocimum basilicum and Lippia alba against six food-spoiling pathogenic bacteria. Seventy-two (72) samples were initially analyzed for antimicrobial activity based on the agar diffusion test. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations were determined for the 12 samples which showed greatest antimicrobial potential in this stage. Two samples of L. alba, three samples of M. lundiana and seven samples of O. basilicum showed a MIC of 0.12-125 μL/mL for the six tested bacteria. Of these, the EO of O. basilicum cultivar Maria Bonita stood out with the lowest MIC and MBC. Thus, a mixture simulating this essential oil was prepared from commercial standards of the compounds (±)-linalool, geraniol and 1,8-cineole. Significantly higher MIC and MBC were detected in the simulation compared to the respective EO, suggesting a synergistic effectbetween compounds.

Bacterial Predation on Cyanobacteria

Predatory bacteria gained interest in the last 20 years. Nevertheless, only a few species are well characterized. The endobiotic predator <i>Bdellovibrio bacteriovorus</i> invades its prey to consume it from the inside, whereas <i>Myxococcus xanthus</i> hunts as a whole group to overcome its prey. Both species were described to prey on cyanobacteria as well. This minireview summarizes the findings of the last 20 years of predatory bacteria of cyanobacteria and is supplemented by new findings from a screening experiment for bacterial predators of the model organism <i>Anabaena variabilis</i> PCC 7937. Known predatory bacteria of cyanobacteria belong to the phyla Proteobacteria, Bacteroidetes, and Firmicutes and follow different hunting strategies. The underlying mechanisms are in most cases not known in much detail. Isolates from the screening experiment were clustered after predation behaviour and analyzed with respect to their size. The effect of predation in high nitrate levels and the occurrence of nitrogen-fixing cells, called heterocysts, are addressed.

Prioritisation of Compounds for 3CLpro Inhibitor Development on SARS-CoV-2 Variants

COVID-19 represents a new potentially life-threatening illness caused by severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 pathogen. In 2021, new variants of the virus with multiple key mutations have emerged, such as B.1.1.7, B.1.351, P.1 and B.1.617, and are threatening to render available vaccines or potential drugs ineffective. In this regard, we highlight 3CLpro, the main viral protease, as a valuable therapeutic target that possesses no mutations in the described pandemically relevant variants. 3CLpro could therefore provide trans-variant effectiveness that is supported by structural studies and possesses readily available biological evaluation experiments. With this in mind, we performed a high throughput virtual screening experiment using CmDock and the “In-Stock” chemical library to prepare prioritisation lists of compounds for further studies. We coupled the virtual screening experiment to a machine learning-supported classification and activity regression study to bring maximal enrichment and available structural data on known 3CLpro inhibitors to the prepared focused libraries. All virtual screening hits are classified according to 3CLpro inhibitor, viral cysteine protease or remaining chemical space based on the calculated set of 208 chemical descriptors. Last but not least, we analysed if the current set of 3CLpro inhibitors could be used in activity prediction and observed that the field of 3CLpro inhibitors is drastically under-represented compared to the chemical space of viral cysteine protease inhibitors. We postulate that this methodology of 3CLpro inhibitor library preparation and compound prioritisation far surpass the selection of compounds from available commercial “corona focused libraries”.

Unsupervised Inference of Protein Fitness Landscape from Deep Mutational Scan

The recent technological advances underlying the screening of large combinatorial libraries in high-throughput mutational scans deepen our understanding of adaptive protein evolution and boost its applications in protein design. Nevertheless, the large number of possible genotypes requires suitable computational methods for data analysis, the prediction of mutational effects, and the generation of optimized sequences. We describe a computational method that, trained on sequencing samples from multiple rounds of a screening experiment, provides a model of the genotype–fitness relationship. We tested the method on five large-scale mutational scans, yielding accurate predictions of the mutational effects on fitness. The inferred fitness landscape is robust to experimental and sampling noise and exhibits high generalization power in terms of broader sequence space exploration and higher fitness variant predictions. We investigate the role of epistasis and show that the inferred model provides structural information about the 3D contacts in the molecular fold.