DeepSinse: deep learning-based detection of single molecules.

MOTIVATION: Imaging single molecules has emerged as a powerful characterization tool in the biological sciences. The detection of these under various noise conditions requires the use of algorithms that are dependent on the end-user inputting several parameters, the choice of which can be challenging and subjective. RESULTS: In this work, we propose DeepSinse, an easily trainable and useable deep neural network that can detect single molecules with little human input and across a wide range of signal-to-noise ratios. We validate the neural network on the detection of single bursts in simulated and experimental data and compare its performance with the best-in-class, domain-specific algorithms. AVAILABILITYAND IMPLEMENTATION: Ground truth ROI simulating code, neural network training, validation code, classification code, ROI picker, GUI for simulating, training and validating DeepSinse as well as pre-trained networks are all released under the MIT License on www.github.com/jdanial/DeepSinse. The dSTORM dataset processing code is released under the MIT License on www.github.com/jdanial/StormProcessor. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SAR and molecular mechanism studies of monoamine oxidase inhibition by selected chalcone analogs.

The present study describes the synthesis of a series of 22 chalcone analogs. These compounds were evaluated as potential human MAO-A and MAO-B inhibitors. The compounds showed varied selectivity against the two isoforms. The IC50 values were found to be in the micromolar to submicromolar range. The Ki values of compound 16 were determined to be 0.047 and 0.020 µM for the inhibition of MAO-A and MAO-B, respectively. Dialysis of enzyme-inhibitor mixtures indicated a reversible competitive mode of inhibition. Most of the synthesized chalcone analogs showed a better selectivity toward MAO-B. However, introducing of 2,4,6-trimethoxy substituents on ring B shifted the selectivity toward MAO-A. In addition, we investigated the molecular mechanism of MAO-B inhibition by selected chalcone analogs. Our results revealed that these selected chalcone analogs increased dopamine levels in the rat hepatoma (H4IIE) cells and decreased the relative mRNA expression of the MAO-B enzyme.

Pyroptosis Induction and Visualization at the Single-Cell Level Using Optogenetics.

Pyroptosis has been identified as a pro-inflammatory form of programmed cell death. It can be triggered by different stimuli including pathogen invasion or cell stress/danger signals releasing hundreds of proteins upon lysis that cause complex responses in neighboring cells. Pyroptosis is executed by the gasdermin (GSDM) family of proteins which, upon cleavage by caspases, form transmembrane pores that release cytokines to induce inflammation. However, despite the importance of gasdermins in the development of inflammatory diseases and cancer, a lot is still to be understood in the downstream consequences of this cell death pathway. Currently, conventional methods, such as drug treatments or chemically forced oligomerization, are limited in the spatiotemporal analysis of pyroptosis signaling in the cellular population, since all cells are primed for undergoing pyroptosis. Here, we provide a protocol for the application of a novel optogenetics tool called NLS_PhoCl_N-GSDMD_mCherry that enables precise temporal and spatial pyroptosis induction in a confocal microscopy setup, followed by imaging of the cell death process and subsequent quantitative analysis of the experiment. This tool opens new opportunities for the study of pyroptosis activation and of its effects on the bystander cell responses.

Visualization of BOK pores independent of BAX and BAK reveals a similar mechanism with differing regulation.

BOK is a poorly understood member of the BCL-2 family of proteins that has been proposed to function as a pro-apoptotic, BAX-like effector. However, the molecular mechanism and structural properties of BOK pores remain enigmatic. Here, we show that the thermal stability and pore activity of BOK depends on the presence of its C-terminus as well as on the mitochondrial lipid cardiolipin. We directly visualized BOK pores in liposomes by electron microscopy, which appeared similar to those induced by BAX, in line with comparable oligomerization properties quantified by single molecule imaging. In addition, super-resolution STED imaging revealed that BOK organized into dots and ring-shaped assemblies in apoptotic mitochondria, also reminiscent of those found for BAX and BAK. Yet, unlike BAX and BAK, the apoptotic activity of BOK was limited by partial mitochondrial localization and was independent of and unaffected by other BCL-2 proteins. These results suggest that, while BOK activity is kept in check by subcellular localization instead of interaction with BCL-2 family members, the resulting pores are structurally similar to those of BAX and BAK.

Systematic Assessment of the Accuracy of Subunit Counting in Biomolecular Complexes Using Automated Single-Molecule Brightness Analysis.

Analysis of single-molecule brightness allows subunit counting of high-order oligomeric biomolecular complexes. Although the theory behind the method has been extensively assessed, systematic analysis of the experimental conditions required to accurately quantify the stoichiometry of biological complexes remains challenging. In this work, we develop a high-throughput, automated computational pipeline for single-molecule brightness analysis that requires minimal human input. We use this strategy to systematically quantify the accuracy of counting under a wide range of experimental conditions in simulated ground-truth data and then validate its use on experimentally obtained data. Our approach defines a set of conditions under which subunit counting by brightness analysis is designed to work optimally and helps in establishing the experimental limits in quantifying the number of subunits in a complex of interest. Finally, we combine these features into a powerful, yet simple, software that can be easily used for the analysis of the stoichiometry of such complexes.

The Mysteries around the BCL-2 Family Member BOK.

BOK is an evolutionarily conserved BCL-2 family member that resembles the apoptotic effectors BAK and BAX in sequence and structure. Based on these similarities, BOK has traditionally been classified as a BAX-like pro-apoptotic protein. However, the mechanism of action and cellular functions of BOK remains controversial. While some studies propose that BOK could replace BAK and BAX to elicit apoptosis, others attribute to this protein an indirect way of apoptosis regulation. Adding to the debate, BOK has been associated with a plethora of non-apoptotic functions that makes this protein unpredictable when dictating cell fate. Here, we compile the current knowledge and open questions about this paradoxical protein with a special focus on its structural features as the key aspect to understand BOK biological functions.

Determinants of BH3 Sequence Specificity for the Disruption of Bcl-xL/cBid Complexes in Membranes.

The prosurvival Bcl-2 proteins exhibit a specific pattern of interactions with BH3-only proteins that determines the cellular dependence on apoptotic stress. This specificity is crucial for the development of BH3 mimetics, a class of anticancer molecules based on the BH3 domain with promising activity in clinical trials. Although complex formation mainly takes place in the mitochondrial outer membrane, most studies so far addressed the interaction between BH3 peptides and truncated Bcl-2 proteins in solution. As a consequence, quantitative understanding of the sequence specificity determinants of BH3 peptides in the membrane environment is missing. Here, we tackle this issue by systematically quantifying the ability of BH3 peptides to compete for the complexes between cBid and Bcl-xL in giant unilamellar vesicles and compare it with solution and mitochondria. We show that the BH3 peptides derived from Hrk, Bim, Bid, and Bad are the most efficient in disrupting cBid/Bcl-xL complexes in the membrane, which correlates with their activity in mitochondria. Our findings support the targeting to the membrane of small molecules that bind Bcl-2 proteins as a strategy to improve their efficiency.

3D Printing of Biomolecular Models for Research and Pedagogy.

The construction of physical three-dimensional (3D) models of biomolecules can uniquely contribute to the study of the structure-function relationship. 3D structures are most often perceived using the two-dimensional and exclusively visual medium of the computer screen. Converting digital 3D molecular data into real objects enables information to be perceived through an expanded range of human senses, including direct stereoscopic vision, touch, and interaction. Such tangible models facilitate new insights, enable hypothesis testing, and serve as psychological or sensory anchors for conceptual information about the functions of biomolecules. Recent advances in consumer 3D printing technology enable, for the first time, the cost-effective fabrication of high-quality and scientifically accurate models of biomolecules in a variety of molecular representations. However, the optimization of the virtual model and its printing parameters is difficult and time consuming without detailed guidance. Here, we provide a guide on the digital design and physical fabrication of biomolecule models for research and pedagogy using open source or low-cost software and low-cost 3D printers that use fused filament fabrication technology.

Design and synthesis of novel chalcones as potent selective monoamine oxidase-B inhibitors.

A novel series of substituted chalcones were designed and synthesized to be evaluated as selective human MAO-B inhibitors. A combination of either methylsulfonyl or trifluoromethyl substituents on the aromatic ketone moiety with a benzodioxol ring on the other end of the chalcone scaffold was investigated. The compounds were tested for their inhibitory activities on both human MAO-A and B. All compounds appeared to be selective MAO-B inhibitors with Ki values in the micromolar to submicromolar range. Molecular modeling studies have been performed to get insight into the binding mode of the synthesized compounds to human MAO-B active site.

New inhibitors of VEGFR-2 targeting the extracellular domain dimerization process.

We are reporting the discovery of small molecule inhibitors for vascular endothelial growth factor receptor type 2 (VEGFR-2) extracellular domain. The VEGFR-2 extracellular domain is responsible for the homo-dimerization process, which has been recently reported as a main step in VEGFR signal transduction cascade. This cascade is essential for the vascularization and survival of most types of cancers. Two main design strategies were used; Molecular docking-based Virtual Screening and Fragment Based Design (FBD). A virtual library of drug like compounds was screened using a cascade of docking techniques in order to discover an inhibitor that binds to this new binding site. Rapid docking methodology was used first to filter the large number of compounds followed by more accurate and slow ones. Fragment based molecular design was adopted afterwards due to unsatisfactory results of screening process. Screening and design process resulted in a group of inhibitors with superior binding energies exceeding that of the natural substrate. Molecular dynamics simulation was used to test the stability of binding of these inhibitors and finally the drug ability of these compounds was assisted using Lipinski rule of five. By this way the designed compounds have shown to possess high pharmacologic potential as novel anticancer agents.