Changes in Microbiota Composition during the Anaerobic Digestion of Macroalgae in a Three-Stage Bioreactor.

The use of microalgae as a raw material for biogas production is promising. Macroalgae were mixed with cattle manure, wheat straw, and an inoculant from sewage sludge. Mixing macroalgae with co-substrates increased biogas and methane yield. The research was carried out using a three-stage bioreactor. During biogas production, the dynamics of the composition of the microbiota in the anaerobic chamber of the bioreactor was evaluated. The microbiota composition at different organic load rates (OLRs) of the bioreactor was evaluated. This study also demonstrated that in a three-stage bioreactor, a higher yield of methane in biogas was obtained compared to a single-stage bioreactor. It was found that the most active functional pathway of methane biosynthesis is PWY-6969, which proceeds via the TCA cycle V (2-oxoglutarate synthase). Microbiota composition and methane yield depended on added volatile solids (VSadded). During the research, it was found that after reducing the ORL from 2.44 to 1.09 kg VS/d, the methane yield increased from 175.2 L CH4/kg VSadded to 323.5 L CH4/kg VSadded.

Changes in the Bacterial Communities of Biocomposites with Different Flame Retardants.

In today's world, the use of environmentally friendly materials is strongly encouraged. These materials derive from primary raw materials of plant origin, like fibrous hemp, flax, and bamboo, or recycled materials, such as textiles or residual paper, making them suitable for the growth of microorganisms. Here, we investigate changes in bacterial communities in biocomposites made of hemp shives, corn starch, and either expandable graphite or a Flovan compound as flame retardants. Using Next Generation Sequencing (NGS), we found that after 12 months of incubation at 22 °C with a relative humidity of 65%, Proteobacteria accounted for >99.7% of the microbiome in composites with either flame retardant. By contrast, in the absence of flame retardants, the abundance of Proteobacteria decreased to 32.1%, while Bacteroidetes (36.6%), Actinobacteria (8.4%), and Saccharobacteria (TM7, 14.51%) appeared. Using the increasing concentrations of either expandable graphite or a Flovan compound in an LB medium, we were able to achieve up to a 5-log reduction in the viability of Bacillus subtilis, Pseudomonas aeruginosa, representatives of the Bacillus and Pseudomonas genera, the abundance of which varied in the biocomposites tested. Our results demonstrate that flame retardants act on both Gram-positive and Gram-negative bacteria and suggest that their antimicrobial activities also have to be tested when producing new compounds.

Bacterial Viability in Self-Healing Concrete: A Case Study of Non-Ureolytic Bacillus Species.

Cracking is an inevitable feature of concrete, typically leading to corrosion of the embedded steel reinforcement and massive deterioration because of the freezing-thawing cycles. Different means have been proposed to increase the serviceability performance of cracked concrete structures. This case study deals with bacteria encapsulated in cementitious materials to "heal" cracks. Such a biological self-healing system requires preserving the bacteria's viability in the cement matrix. Many embedded bacterial spores are damaged during concrete curing, drastically reducing efficiency. This study investigates the viability of commonly used non-ureolytic bacterial spores when immobilized in calcium alginate microcapsules within self-healing cementitious composites. Three Bacillus species were used in this study, i.e., B. pseudofirmus, B. cohnii, and B. halodurans. B. pseudofirmus demonstrated the best mineralization activity; a sufficient number of bacterial spores remained viable after the encapsulation. B. pseudofirmus and B. halodurans spores retained the highest viability after incorporating the microcapsules into the cement paste, while B. halodurans spores retained the highest viability in the mortar. Cracks with a width of about 0.13 mm were filled with bacterial calcium carbonate within 14 to 28 days, depending on the type of bacteria. Larger cracks were not healed entirely. B. pseudofirmus had the highest efficiency, with a healing coefficient of 0.497 after 56 days. This study also revealed the essential role of the cement hydration temperature on bacterial viability. Thus, further studies should optimize the content of bacteria and nutrients in the microcapsule structure.

The Impact of Microorganisms on the Performance of Linseed Oil and Tung Tree Oil Impregnated Composites Made of Hemp Shives and Corn Starch.

In this study, the performance characteristics of hemp shives impregnated with linseed oil and tung tree oil (HS)- and corn starch (CS)-based biocomposites containing flame retardants were evaluated before and after treatment with the mixture of bacterium Pseudomonas putida and fungus Rhizopus oryzae. Enzymatic activities and physical-mechanical properties such as water absorption, thickness swelling, compressive strength, and thermal conductivity were tested to evaluate the suitability of selected composites for thermal insulation purposes. In addition, electron microscopy was used to investigate the impact of microorganisms on the microstructure of the material. It was determined that the type of oil used for impregnation significantly affects the properties of biocomposites after 6 months of incubation with mixture of bacterium P. putida and fungus Rh. oryzae. Biocomposites impregnated with linseed oil and after treatment with a mixture of microorganisms had cellulase activity of 25 U/mL, endo ß-1-4-glucanase activity of 26 U/mL, lipase activity of 101 U/mL, only a 10% decrease in compressive strength, 50% higher short-term water absorption, unchanged swelling in thickness, and slightly decreased thermal conductivity compared to control biocomposites. At the same time, biocomposites with tung tree oil had a much more pronounced deterioration of the properties tested, cellulase activity of 28 U/mL, endo ß-1-4-glucanase activity of 37 U/mL, lipase activity of 91 U/mL, two times lower compressive strength and two times higher short-term water absorption, 2.5 times greater thickness swelling, and a slightly increased thermal conductivity. We conclude that linseed oil provides better protection against the action of microorganisms compared to impregnation with tung tree oil.

Impact of Cellulolytic Fungi on Biodegradation of Hemp Shives and Corn Starch-Based Composites with Different Flame-Retardants.

Biocomposite boards (BcBs) composed of hemp shives and corn starch are known as thermal insulating or structural building materials. Therefore, they must be stable during exploitation. However, BcBs are exposed to microorganisms present in the environment, and it is of great interest to investigate the biodegradation behaviour of these materials. This work identified microorganisms growing on BcBs that contain either Flovan CGN or expandable graphite as flame retardants and selected fungi such as Rhizopus oryzae and Aspergillus fumigatus to test the way they affect the materials of interest. For this purpose, the enzymatic activity of cellulases and amylases produced by these organisms were determined. In addition, the apparent density as well as compressive strength of the affected boards were evaluated. The results showed that apparent density and compressive strength deteriorated in BcB composition with the Flovan CGN flame retardant. At the same time, the level of deterioration was lower when the expandable graphite was used, suggesting that it also acts as an antimicrobial agent. A scanning electronic microscopy analysis was employed to monitor the growth of microorganisms in the BcBs. Such analysis demonstrated that, regardless of BcB composition, fungi easily penetrate into the middle layers of the material.

Microbial community dynamics in the mesophilic and thermophilic phases of textile waste composting identified through next-generation sequencing.

Composting is a promising source of mesophilic and thermophilic microorganisms directly involved in the decay of organic matter. However, there is a paucity of information related to bacterial and fungal diversity in compost and their enzymatic activities during the composting process. In this work, bacterial and fungal diversity during the mesophilic and thermophilic phases of textile waste composting was investigated as a way to explain the physical-chemical results obtained during the composting process. This was accomplished using a next-generation sequencing approach that targets either the 16S rRNA or ITS genomic regions of bacteria and fungi, respectively. It was observed that Proteobacteria, Bacteroidetes, and Actinobacteria were the dominant bacterial phyla present at the mesophilic phase but not at the thermophilic one. Composting textile waste exhibits a sustained thermophilic profile (above 55 °C) that usually precludes fungal activity. Nonetheless, the presence of fungi at the thermophilic phase was observed. Rozellomycota, Basidiomycota, and Ascomycota were the most dominant phyla during both composting phases. Such thermophilic fungi with great ability to decay organic matter could be isolated as pure cultures and used for the bioaugmentation of textile waste composting to achieve an advanced maturity level of textile waste compost.

A Study on Machine Learning Methods' Application for Dye Adsorption Prediction onto Agricultural Waste Activated Carbon.

The adsorption of dyes using 39 adsorbents (16 kinds of agro-wastes) were modeled using random forest (RF), decision tree (DT), and gradient boosting (GB) models based on 350 sets of adsorption experimental data. In addition, the correlation between variables and their importance was applied. After comprehensive feature selection analysis, five important variables were selected from nine variables. The RF with the highest accuracy (R2 = 0.9) was selected as the best model for prediction of adsorption capacity of agro-waste using the five selected variables. The results suggested that agro-waste characteristics (pore volume, surface area, agro-waste pH, and particle size) accounted for 50.7% contribution for adsorption efficiency. The pore volume and surface area are the most important influencing variables among the agro-waste characteristics, while the role of particle size was inconspicuous. The accurate ability of the developed models' prediction could significantly reduce experimental screening efforts, such as predicting the dye removal efficiency of agro-waste activated carbon according to agro-waste characteristics. The relative importance of variables could provide a right direction for better treatments of dyes in the real wastewater.

A direct electron transfer formaldehyde dehydrogenase biosensor for the determination of formaldehyde in river water.

In this work, we report the construction of a direct electron transfer (DET) biosensor based on NAD-dependent formaldehyde dehydrogenase from Pseudomonas sp. (FDH) immobilized on the gold nanoparticle-modified gold electrode. To the best of our knowledge, a DET for FDH was achieved for the first time - the oxidation of formaldehyde started at a low electrode potential of -190 mV vs. Ag/AgCl and reached a maximum current density of 1100 nA cm-2 at 200 mV vs. Ag/AgCl. Also, the designed electrode was insensitive to substrate inhibition (in comparison to the free enzyme) and operated in solutions with formaldehyde concentrations up to 10 mM. The electrode was used and characterized as a mediatorless biosensor for the detection of formaldehyde. The biosensor demonstrated a limit of detection (0.05 mM), linear range from 0.25 to 2.0 mM, the sensitivity of 178.9 nA mM cm-2, high stability and selectivity. The biosensor has been successfully tested for the determination of added formaldehyde concentration in river water samples, thus the developed electrode could be applied for a fast, inexpensive and simple measurement of formaldehyde in various media.

Prolonging Bacterial Viability in Biological Concrete: Coated Expanded Clay Particles.

One of the biggest challenges in the development of a biological self-healing concrete is to ensure the long-term viability of bacteria that are embedded in the concrete. In the present study, a coated expanded clay (EC) is investigated for its potential use as a bacterial carrier in biological concrete. Eight different materials for coatings were selected considering cost, workability and accessibility in the construction industry. Long-term (56 days) viability analysis was conducted with a final evaluation of each coating performance. Our results indicate that healing efficiency in biological concrete specimens is strongly related to viable bacteria present in the healing agent. More viable bacteria-containing specimens exhibited a higher crack closure ratio. Our data suggest that the additional coating of EC particles improves long-term bacterial viability and, consequently, provides efficient crack healing in biological concrete.

Structural Evidence for a [4Fe-5S] Intermediate in the Non-Redox Desulfuration of Thiouracil.

We recently discovered a [Fe-S]-containing protein with in vivo thiouracil desulfidase activity, dubbed TudS. The crystal structure of TudS refined at 1.5 Šresolution is reported; it harbors a [4Fe-4S] cluster bound by three cysteines only. Incubation of TudS crystals with 4-thiouracil trapped the cluster with a hydrosulfide ligand bound to the fourth non-protein-bonded iron, as established by the sulfur anomalous signal. This indicates that a [4Fe-5S] state of the cluster is a catalytic intermediate in the desulfuration reaction. Structural data and site-directed mutagenesis indicate that a water molecule is located next to the hydrosulfide ligand and to two catalytically important residues, Ser101 and Glu45. This information, together with modeling studies allow us to propose a mechanism for the unprecedented non-redox enzymatic desulfuration of thiouracil, in which a [4Fe-4S] cluster binds and activates the sulfur atom of the substrate.

Biochemical Pathways Leading to the Formation of Wyosine Derivatives in tRNA of Archaea.

Tricyclic wyosine derivatives are present at position 37 in tRNAPhe of both eukaryotes and archaea. In eukaryotes, five different enzymes are needed to form a final product, wybutosine (yW). In archaea, 4-demethylwyosine (imG-14) is an intermediate for the formation of three different wyosine derivatives, yW-72, imG, and mimG. In this review, current knowledge regarding the archaeal enzymes involved in this process and their reaction mechanisms are summarized. The experiments aimed to elucidate missing steps in biosynthesis pathways leading to the formation of wyosine derivatives are suggested. In addition, the chemical synthesis pathways of archaeal wyosine nucleosides are discussed, and the scheme for the formation of yW-86 and yW-72 is proposed. Recent data demonstrating that wyosine derivatives are present in the other tRNA species than those specific for phenylalanine are discussed.

The Effect of Different Plant Oil Impregnation and Hardening Temperatures on Physical-Mechanical Properties of Modified Biocomposite Boards Made of Hemp Shives and Corn Starch.

In this study, tung tree and linseed drying oils, as well as semi-drying hempseed oil, were analyzed as the protective coatings for biocomposite boards (BcB) made of hemp shives, corn starch binder, and the performance-enhancing additives. The hydrophobization coatings were formed at 40, 90, and 120 °C temperatures, respectively. The physical-mechanical properties such as the compressive strength, thermal conductivity, dimensional stability, water absorption, and swelling were tested. In addition, scanning electron microscopy (SEM) was employed for the analysis of the board microstructure to visualize the oil fills and impregnation in pores and voids. It was demonstrated that the compressive strength of oil-modified BcBs compared to uncoated BcBs (at 10% of relative deformation) increased by up to 4.5-fold and could reach up to 14 MPa, water absorption decreased up to 4-fold (from 1.34 to 0.37 kg/m2), swelling decreased up to 48% (from 8.20% to 4.26%), whereas the thermal conductivity remained unchanged with the thermal conductivity coefficient of around 0.085 W/m·K. Significant performance-enhancing properties were obtained due to the formation of a protective oil film when the tung tree oil was used.

Isocytosine deaminase Vcz as a novel tool for the prodrug cancer therapy.

BACKGROUND: The cytosine deaminase (CD)/5-fluorocytosine (5-FC) system is among the best explored enzyme/prodrug systems in the field of the suicide gene therapy. Recently, by the screening of the environmental metagenomic libraries we identified a novel isocytosine deaminase (ICD), termed Vcz, which is able of specifically converting a prodrug 5-fluoroisocytosine (5-FIC) into toxic drug 5-fluorouracil (5-FU). The aim of this study is to test the applicability of the ICD Vcz / 5-FIC pair as a potential suicide gene therapy tool. METHODS: Vcz-expressing human glioblastoma U87 and epithelial colorectal adenocarcinoma Caco-2 cells were treated with 5-FIC, and the Vcz-mediated cytotoxicity was evaluated by performing an MTT assay. In order to examine anti-tumor effects of the Vcz/5-FIC system in vivo, murine bone marrow-derived mesenchymal stem cells (MSC) were transduced with the Vcz-coding lentivirus and co-injected with 5-FIC or control reagents into subcutaneous GL261 tumors evoked in C57/BL6 mice. RESULTS: 5-FIC alone showed no significant toxic effects on U87 and Caco-2 cells at 100 µM concentration, whereas the number of cells of both cell lines that express Vcz cytosine deaminase gene decreased by approximately 60% in the presence of 5-FIC. The cytotoxic effects on cells were also induced by media collected from Vcz-expressing cells pre-treated with 5-FIC. The co-injection of the Vcz-transduced mesenchymal stem cells and 5-FIC have been shown to augment tumor necrosis and increase longevity of tumorized mice by 50% in comparison with control group animals. CONCLUSIONS: We have confirmed that the novel ICD Vcz together with the non-toxic prodrug 5-FIC has a potential of being a new enzyme/prodrug system for suicide gene therapy.

Identification of a 2'-O-Methyluridine Nucleoside Hydrolase Using the Metagenomic Libraries.

Ribose methylation is among the most ubiquitous modifications found in RNA. 2'-O-methyluridine is found in rRNA, snRNA, snoRNA and tRNA of Archaea, Bacteria, and Eukaryota. Moreover, 2'-O-methylribonucleosides are promising starting materials for the production of nucleic acid-based drugs. Despite the countless possibilities of practical use for the metabolic enzymes associated with methylated nucleosides, there are very few reports regarding the metabolic fate and enzymes involved in the metabolism of 2'-O-alkyl nucleosides. The presented work focuses on the cellular degradation of 2'-O-methyluridine. A novel enzyme was found using a screening strategy that employs Escherichia coli uracil auxotroph and the metagenomic libraries. A 2'-O-methyluridine hydrolase (RK9NH) has been identified together with an aldolase (RK9DPA)-forming a part of a probable gene cluster that is involved in the degradation of 2'-O-methylated nucleosides. The RK9NH is functional in E. coli uracil auxotroph and in vitro. The RK9NH nucleoside hydrolase could be engineered to enzymatically produce 2'-O-methylated nucleosides that are of great demand as raw materials for production of nucleic acid-based drugs. Moreover, RK9NH nucleoside hydrolase converts 5-fluorouridine, 5-fluoro-2'-deoxyuridine and 5-fluoro-2'-O-methyluridine into 5-fluorouracil, which suggests it could be employed in cancer therapy.

Discovery of Bacterial Deaminases That Convert 5-Fluoroisocytosine Into 5-Fluorouracil.

Cytosine is one of the four letters of a standard genetic code, found both in DNA and in RNA. This heterocyclic base can be converted into uracil upon the action of the well-known cytosine deaminase. Isocytosine (2-aminouracil) is an isomer of cytosine, yet the enzymes that could convert it into uracil were previously mainly overlooked. In order to search for the isocytosine deaminases we used a selection strategy that is based on uracil auxotrophy and the metagenomic libraries, which provide a random pool of genes from uncultivated soil bacteria. Several genes that encode isocytosine deaminases were found and two respective recombinant proteins were purified. It was established that both novel deaminases do not use cytosine as a substrate. Instead, these enzymes are able to convert not only isocytosine into uracil, but also 5-fluoroisocytosine into 5-fluorouracil. Our findings suggest that novel isocytosine deaminases have a potential to be efficiently used in targeted cancer therapy instead of the classical cytosine deaminases. Use of isocytosine instead of cytosine would produce fewer side effects since deaminases produced by the commensal E. coli gut flora are ten times less efficient in degrading isocytosine than cytosine. In addition, there are no known homologs of isocytosine deaminases in human cells that would induce the toxicity when 5-fluoroisocytosine would be used as a prodrug.

A gene encoding a DUF523 domain protein is involved in the conversion of 2-thiouracil into uracil.

Modified nucleotides are present in many RNA species in all Domains of Life. While the biosynthetic pathways of such nucleotides are well studied, much less is known about the degradation of RNAs and the return to the metabolism of modified nucleotides, their respective nucleosides or heterocyclic bases. Using an E. coli uracil auxotroph, we screened the metagenomic libraries for genes, which would allow the conversion of 2-thiouracil to uracil and thereby lead to the growth on a defined synthetic medium. We show that a gene encoding a protein consisting of previously uncharacterized Domain of Unknown Function 523 (DUF523) is responsible for such phenotype. We have purified this recombinant protein and demonstrated that it contains a FeS cluster. The substitution of cysteines, which have been predicted to form such clusters, with alanines abolished the growth phenotype. We conclude that DUF523 is involved in the conversion of 2-thiouracil into uracil in vivo.

Evolution of tRNAPhe:imG2 methyltransferases involved in the biosynthesis of wyosine derivatives in Archaea.

Tricyclic wyosine derivatives are found at position 37 of eukaryotic and archaeal tRNAPhe In Archaea, the intermediate imG-14 is targeted by three different enzymes that catalyze the formation of yW-86, imG, and imG2. We have suggested previously that a peculiar methyltransferase (aTrm5a/Taw22) likely catalyzes two distinct reactions: N1-methylation of guanosine to yield m1G; and C7-methylation of imG-14 to yield imG2. Here we show that the recombinant aTrm5a/Taw22-like enzymes from both Pyrococcus abyssi and Nanoarchaeum equitans indeed possess such dual specificity. We also show that substitutions of individual conservative amino acids of P. abyssi Taw22 (P260N, E173A, and R174A) have a differential effect on the formation of m1G/imG2, while replacement of R134, F165, E213, and P262 with alanine abolishes the formation of both derivatives of G37. We further demonstrate that aTrm5a-type enzyme SSO2439 from Sulfolobus solfataricus, which has no N1-methyltransferase activity, exhibits C7-methyltransferase activity, thereby producing imG2 from imG-14. We thus suggest renaming such aTrm5a methyltransferases as Taw21 to distinguish between monofunctional and bifunctional aTrm5a enzymes.

Yeast ß-1,6-glucan is a primary target for the Saccharomyces cerevisiae K2 toxin.

Certain Saccharomyces cerevisiae strains secrete different killer proteins of double-stranded-RNA origin. These proteins confer a growth advantage to their host by increasing its survival. K2 toxin affects the target cell by binding to the cell surface, disrupting the plasma membrane integrity, and inducing ion leakage. In this study, we determined that K2 toxin saturates the yeast cell surface receptors in 10 min. The apparent amount of K2 toxin, bound to a single cell of wild type yeast under saturating conditions, was estimated to be 435 to 460 molecules. It was found that an increased level of ß-1,6-glucan directly correlates with the number of toxin molecules bound, thereby impacting the morphology and determining the fate of the yeast cell. We observed that the binding of K2 toxin to the yeast surface receptors proceeds in a similar manner as in case of the related K1 killer protein. It was demonstrated that the externally supplied pustulan, a poly-ß-1,6-glucan, but not the glucans bearing other linkage types (such as laminarin, chitin, and pullulan) efficiently inhibits the K2 toxin killing activity. In addition, the analysis of toxin binding to the intact cells and spheroplasts confirmed that majority of K2 protein molecules attach to the ß-1,6-glucan, rather than the plasma membrane-localized receptors. Taken together, our results reveal that ß-1,6-glucan is a primary target of K2 toxin and is important for the execution of its killing property.

Biosynthesis of wyosine derivatives in tRNA(Phe) of Archaea: role of a remarkable bifunctional tRNA(Phe):m1G/imG2 methyltransferase.

The presence of tricyclic wyosine derivatives 3'-adjacent to anticodon is a hallmark of tRNA(Phe) in eukaryotes and archaea. In yeast, formation of wybutosine (yW) results from five enzymes acting in a strict sequential order. In archaea, the intermediate compound imG-14 (4-demethylwyosine) is a target of three different enzymes, leading to the formation of distinct wyosine derivatives (yW-86, imG, and imG2). We focus here on a peculiar methyltransferase (aTrm5a) that catalyzes two distinct reactions: N(1)-methylation of guanosine and C(7)-methylation of imG-14, whose function is to allow the production of isowyosine (imG2), an intermediate of the 7-methylwyosine (mimG) biosynthetic pathway. Based on the formation of mesomeric forms of imG-14, a rationale for such dual enzymatic activities is proposed. This bifunctional tRNA:m(1)G/imG2 methyltransferase, acting on two chemically distinct guanosine derivatives located at the same position of tRNA(Phe), is unique to certain archaea and has no homologs in eukaryotes. This enzyme here referred to as Taw22, probably played an important role in the emergence of the multistep biosynthetic pathway of wyosine derivatives in archaea and eukaryotes.

Screening the budding yeast genome reveals unique factors affecting K2 toxin susceptibility.

BACKGROUND: Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. PRINCIPAL FINDINGS: We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. SIGNIFICANCE: Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action.