Cross Pharmacological, Biochemical and Computational Studies of a Human Kv3.1b Inhibitor from Androctonus australis Venom

The voltage-gated K+ channels Kv3.1 display fast activation and deactivation kinetics and are known to have a crucial contribution to the fast-spiking phenotype of certain neurons. AahG50, as a natural product extracted from Androctonus australis hector venom, inhibits selectively Kv3.1 channels. In the present study, we focused on the biochemical and pharmacological characterization of the component in AahG50 scorpion venom that potently and selectively blocks the Kv3.1 channels. We used a combined optimization through advanced biochemical purification and patch-clamp screening steps to characterize the peptide in AahG50 active on Kv3.1 channels. We described the inhibitory effect of a toxin on Kv3.1 unitary current in black lipid bilayers. In silico, docking experiments are used to study the molecular details of the binding. We identified the first scorpion venom peptide inhibiting Kv3.1 current at 170 nM. This toxin is the alpha-KTx 15.1, which occludes the Kv3.1 channel pore by means of the lysine 27 lateral chain. This study highlights, for the first time, the modulation of the Kv3.1 by alpha-KTx 15.1, which could be an interesting starting compound for developing therapeutic biomolecules against Kv3.1-associated diseases.

The neural G protein Gαo tagged with GFP at an internal loop is functional in C. elegans

Gαo is the alpha subunit of the major heterotrimeric G protein in neurons and mediates signaling by every known neurotransmitter, yet the signaling mechanisms activated by Gαo remain to be fully elucidated. Genetic analysis in Caenorhabditis elegans has shown that Gαo signaling inhibits neuronal activity and neurotransmitter release, but studies of the molecular mechanisms underlying these effects have been limited by lack of tools to complement genetic studies with other experimental approaches. Here we demonstrate that inserting the green fluorescent protein (GFP) into an internal loop of the Gαo protein results in a tagged protein that is functional in vivo and that facilitates cell biological and biochemical studies of Gαo. Transgenic expression of Gαo-GFP rescues the defects caused by loss of endogenous Gαo in assays of egg laying and locomotion behaviors. Defects in body morphology caused by loss of Gαo are also rescued by Gαo-GFP. The Gαo-GFP protein is localized to the plasma membrane of neurons, mimicking localization of endogenous Gαo. Using GFP as an epitope tag, Gαo-GFP can be immunoprecipitated from C. elegans lysates to purify Gαo protein complexes. The Gαo-GFP transgene reported in this study enables studies involving in vivo localization and biochemical purification of Gαo to complement the already well-developed genetic analysis of Gαo signaling.

The neural G protein Gαo tagged with GFP at an internal loop is functional in C. elegans

Gαo is the alpha subunit of the major heterotrimeric G protein in neurons and mediates signaling by every known neurotransmitter, yet the signaling mechanisms activated by Gαo remain to be fully elucidated. Genetic analysis in Caenorhabditis elegans has shown that Gαo signaling inhibits neuronal activity and neurotransmitter release, but studies of the molecular mechanisms underlying these effects have been limited by lack of tools to complement genetic studies with other experimental approaches. Here we demonstrate that inserting the green fluorescent protein (GFP) into an internal loop of the Gαo protein results in a tagged protein that is functional in vivo and that facilitates cell biological and biochemical studies of Gαo. Transgenic expression of Gαo-GFP rescues the defects caused by loss of endogenous Gαo in assays of egg laying and locomotion behaviors. Defects in body morphology caused by loss of Gαo are also rescued by Gαo-GFP. The Gαo-GFP protein is localized to the plasma membrane of neurons, mimicking localization of endogenous Gαo. Using GFP as an epitope tag, Gαo-GFP can be immunoprecipitated from C. elegans lysates to purify Gαo protein complexes. The Gαo-GFP transgene reported in this study enables studies involving in vivo localization and biochemical purification of Gαo to complement the already well-developed genetic analysis of Gαo signaling.

EXPERIMENTAL STUDIES OF BIOCHEMICAL GAS TREATMENT IN THE WORKING AREA OF CLOSED WASTEWATER TREATMENT PLANTS

The results of the study of gas emissions from blocked wastewater treatment plants are presented. During the experiment, high concentrations of sulfur dioxide were detected. The analysis of methods for reducing the concentration of pollutants in the air of the working area of the aeration station was carried out. It is proposed to use a biochemical purification using a biofilter with a load of bark and biohumus. A model of the installation was constructed. To determine the technological parameters of the biofilter considered different types of combinations (height, moisture, particle size) of the carrier biomass and supporting layer of gravel, the effective processing time of the gas mixture. A decrease in sulfur dioxide, hydrogen sulfide, and formaldehyde was found. Additional advantages of the applied biologically active filter material are given.

Modeling of hydrogen sulfide removal under biomethane production in the concept of renewable energy potential growth of Ukraine

Today, the global trend in the development of renewable energy sources is the implementation of integrated processing of organic waste with the production of biogenic gases such as biomethane. In this case, an essential focus is the study of biogas purification processes to methane. This paper focuses on the process of modeling biochemical purification of biogas from hydrogen sulfide to develop the direction of biomethane production. Simulation of hydrogen sulfide bio-oxidation process with the use of granulated carrier based on phosphogypsum was conducted using experimental data from previous studies to verify the adequacy of the proposed mathematical model. Thus, to implement the process of phosphogypsum utilization in technological systems of biogas purification, it is important to consider the level of bioactivity in the immobilization of bacteria on the loading surface of phosphogypsum and the degree of biotransformation of phosphogypsum components in the oxidation of carbon dioxide and hydrogen sulfide impurities to achieve the highest ecological effect. Also, the use of overlay visualization allowed to form the main clusters of development of research potential in the field of biogas technologies for Ukraine.

INVESTIGATION OF SUSPENDED SOLIDS IN WATER FOR COKE EXTINCTION

The article is devoted to the results of work for the study of the composition and quantitative content of suspended solids (SS) in the wastewater of coke plants before and after biochemical purification plants (BPP), as well as in other waters involved in the process of coke extinction. The characteristic indicators have been investigated of waters before and after BPP of five coke-chemical enterprises of Ukraine, as well as the chemical composition of suspended solids in water after BPP, etc. It is shown that SS in water after BPP is represented mainly by activated sludge, which is partially oxidized to CO2 when water is supplied to hot coke. The rest activated sludge is returned with water condensate to the settling tank of the extinction tower and, thus, is not emitted into the atmosphere in the form of SS. In addition to sludge, the particles of coke, coal and products of the equipment corrosion are present in the purified water after the BPP and in the water of the extinction tower settling tank. The possibility of the presence of concrete particles is also not excluded. To establish the nature of solid particles, their elemental composition and ash composition has been determined. In particular, it was found that the ash composition of suspended solids from water after BPP quantitatively differs from the both of ash composition of coal concentrates and the composition of concrete. For example, the mass fraction of silicon dioxide in the suspended solids of water after BTH is much lower than in concrete and in ash of coal concentrates. The detected predominance of iron (III) oxide in the composition of SS ash after BPP is explained by the presence in the selected samples of products of the equipment corrosion. Based on our research, it is proposed to make changes in the normative document "Technological standards of permissible emissions of pollutants from coke ovens", namely in chapter IV "Operating conditions of coke ovens" par. 8: to exclude such a normative value as the content of suspended solids in water after BPP, which is submitted for coke extinction. Keywords: water for coke extinction, water after biochemical purification, suspended solids (SS), content, chemical composition, elemental composition, ash composition. Corresponding author A.L. Borisenko, e-mail: [email protected]

A SARS-CoV-2 – host proximity interactome

Viral replication is dependent on interactions between viral polypeptides and host proteins. Identifying virus-host protein interactions can thus uncover unique opportunities for interfering with the virus life cycle via novel drug compounds or drug repurposing. Importantly, many viral-host protein interactions take place at intracellular membranes and poorly soluble organelles, which are difficult to profile using classical biochemical purification approaches. Applying proximity-dependent biotinylation (BioID) with the fast-acting miniTurbo enzyme to 27 SARS-CoV-2 proteins in a lung adenocarcinoma cell line (A549), we detected 7810 proximity interactions (7382 of which are new for SARS-CoV-2) with 2242 host proteins (results available at covid19interactome.org). These results complement and dramatically expand upon recent affinity purification-based studies identifying stable host-virus protein complexes, and offer an unparalleled view of membrane-associated processes critical for viral production. Host cell organellar markers were also subjected to BioID in parallel, allowing us to propose modes of action for several viral proteins in the context of host proteome remodelling. In summary, our dataset identifies numerous high confidence proximity partners for SARS-CoV-2 viral proteins, and describes potential mechanisms for their effects on specific host cell functions.

Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq

Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods sensitive to both changes in replication fork structure and the formation of recombinogenic DNA ends. Here we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single stranded DNA 3’ ends genome-wide and with base pair resolution. TrAEL-seq labels DNA breaks, and profiles both stalled and processive replication forks in yeast and mammalian cells. Replication forks stalling at defined barriers and expressed genes are detected by TrAEL-seq with exceptional signal-to-noise, most likely through labelling of DNA 3’ ends exposed during fork reversal. TrAEL-seq also labels unperturbed processive replication forks to yield maps of replication fork direction similar to those obtained by Okazaki fragment sequencing, however TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3’ ends also allows accurate detection of double strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double strand break hotspots in a dmc1Δ mutant. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.