Umeå University's logo

The main protease M^pro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with M^pro. These investigations resulted in the four-armed compound 35b that binds directly to M^pro. 35b could be cocrystallized with M^pro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.

Nitrogen can be taken up by trees in the form of nitrate, ammonium and amino acids, but the influence of the different forms on tree growth and development is poorly understood in angiosperm species like Populus. We studied the effects of both organic and inorganic forms of nitrogen on growth and wood formation of hybrid aspen trees in experimental conditions that allowed growth under four distinct steady-state nitrogen levels. Increased nitrogen availability had a positive influence on biomass accumulation and the radial dimensions of both xylem vessels and fibers, and a negative influence on wood density. An optimal level of nitrogen availability was identified where increases in biomass accumulation outweighed decreases in wood density. None of these responses depended on the source of nitrogen except for shoot biomass accumulation, which was stimulated more by treatments complemented with nitrate than by ammonium alone or the organic source arginine. The most striking difference between the nitrogen sources was the effect on lignin composition, whereby the abundance of H-type lignin increased only in the presence of nitrate. The differential effect of nitrate is possibly related to the well-known role of nitrate as a signaling compound. RNA-sequencing revealed that while the lignin-biosynthetic genes did not significantly (FDR <0.01) respond to added NO_3- , the expression of several laccases, catalysing lignin polymerization, was dependent on N-availability. These results reveal a unique role of nitrate in wood formation and contribute to the knowledge basis for decision-making in utilizing hybrid aspen as a bioresource.

The recalcitrant structures either from substrate or microbial biomass contained in digestates after anaerobic digestion (AD) highly influence digestate valorization. To properly assess the microbial biomass contribution to the digested organic matter (OM), a combination of characterization methods and the use of various substrate types in anaerobic continuous reactors was required. The use of totally biodegradable substrates allowed detecting soluble microbial products via fluorescence spectroscopy at emission wavelengths of 420 and 460 nm while the protein-like signature was enhanced by the whey protein. During reactors' operation, a transfer of complex compounds to the dissolved OM from the particulate OM was observed through fluorescence applied on biochemical fractionation. Consequently, the fluorescence complexity index of the dissolved OM increased from 0.59–0.60 to 1.06–1.07, whereas it decreased inversely for the extractable soluble from the particulate OM from 1.16–1.19 to 0.42–0.54. Accordingly, fluorescence regional integration showed differences among reactors based on visual inspection and orthogonal partial latent structures (OPLS) analysis. Similarly, the impact of the substrate type and operation time on the particulate OM was revealed by 13C nuclear magnetic resonance using OPLS, providing a good model (R2X = 0.93 and Q2 = 0.8) with a clear time-trend. A high signal resonated at ∼30 ppm attributed to CH2-groups in the aliphatic chain of lipid-like structure besides carbohydrates intensities at 60–110 ppm distinguished the reactor fed with whey protein from the other, which was mostly biomass related. Indeed, this latter displayed a higher presence of peptidoglycan (δH/C: 1.6–2.0/20–25 ppm) derived from microbial biomass by 1H-13C heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance. Interestingly, the sample distribution obtained by non-metric multidimensional scaling of bacterial communities resembled the attained using 13C NMR properties, opening new research perspectives. Overall, this study discloses the microbial biomass contribution to digestates composition to improve the OM transformation mechanism knowledge.

The global production of fossil-based plastics has reached critical levels, and their substitution with bio-based polymers is an urgent requirement. Poly(3-hydroxybutyrate) (PHB) is a biopolymer that can be produced via microbial cultivation, but efficient microorganisms and low-cost substrates are required. Halomonas boliviensis LC1, a moderately halophilic bacterium, is an effective PHB producer, and hydrolysates of the residual stalks of quinoa (Chenopodium quinoa Willd.) can be considered a cheap source of sugars for microbial fermentation processes in quinoa-producing countries. In this study, H. boliviensis LC1 was adapted to a cellulosic hydrolysate of quinoa stalks obtained via acid-catalyzed hydrothermal pretreatment and enzymatic saccharification. The adapted strain was cultivated in hydrolysates and synthetic media, each of them with two different initial concentrations of glucose. Cell growth, glucose consumption, and PHB formation during cultivation were assessed. The cultivation results showed an initial lag in microbial growth and glucose consumption in the quinoa hydrolysates compared to cultivation in synthetic medium, but after 33 h, the values were comparable for all media. Cultivation in hydrolysates with an initial glucose concentration of 15 g/L resulted in a higher glucose consumption rate (0.15 g/(L h) vs. 0.14 g/(L h)) and volumetric productivity of PHB (14.02 mg/(L h) vs. 10.89 mg/(L h)) than cultivation in hydrolysates with 20 g/L as the initial glucose concentration. During most of the cultivation time, the PHB yield on initial glucose was higher for cultivation in synthetic medium than in hydrolysates. The produced PHBs were characterized using advanced analytical techniques, such as high-performance size-exclusion chromatography (HPSEC), Fourier transform infrared (FTIR) spectroscopy, 1H nuclear magnetic resonance (NMR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). HPSEC revealed that the molecular weight of PHB produced in the cellulosic hydrolysate was lower than that of PHB produced in synthetic medium. TGA showed higher thermal stability for PHB produced in synthetic medium than for that produced in the hydrolysate. The results of the other characterization techniques displayed comparable features for both PHB samples. The presented results show the feasibility of producing PHB from quinoa stalks with H. boliviensis.

Wood is the most important repository of assimilated carbon in the biosphere, in the form of large polymers (cellulose, hemicelluloses including glucuronoxylan, and lignin) that interactively form a composite, together with soluble extractives including phenolic and aliphatic compounds. Molecular interactions among these compounds are not fully understood.

We have targeted the expression of a fungal α-glucuronidase to the wood cell wall of aspen (Populus tremula L. × tremuloides Michx.) and Arabidopsis (Arabidopsis thaliana (L.) Heynh), to decrease contents of the 4-O-methyl glucuronopyranose acid (mGlcA) substituent of xylan, to elucidate mGlcA's functions.

The enzyme affected the content of aliphatic insoluble cell wall components having composition similar to suberin, which required mGlcA for binding to cell walls. Such suberin-like compounds have been previously identified in decayed wood, but here, we show their presence in healthy wood of both hardwood and softwood species. By contrast, γ-ester bonds between mGlcA and lignin were insensitive to cell wall-localized α-glucuronidase, supporting the intracellular formation of these bonds.

These findings challenge the current view of the wood cell wall composition and reveal a novel function of mGlcA substituent of xylan in fastening of suberin-like compounds to cell wall. They also suggest an intracellular initiation of lignin–carbohydrate complex assembly.

Demand for sustainable packaging and building materials has increased the need for biobased additives. Biocomposites can often be exposed to different weather conditions and UV irradiation. Thus, additives to prevent the negative impact of weathering are generally added to composites. This study aims to evaluate using esterified lignin as an additive against weathering effects in polylactic-acid (PLA) composites. Lignin is extracted from construction and demolition waste (CDW) wood using a deep eutectic solvent then esterified and tested as an additive in the fabrication of biobased composites. For comparison, lignin from birch is used as a raw material for an additive. Esterification is confirmed by solid-state N MR analysis. Samples are exposed to artificial weathering for 700 hours and their impact strength and color change properties are measured. The results indicate that esterified lignin from CDW (CDW e-lignin) as an additive protects the biocomposite from the weathering impact. The sample containing the CDW e-lignin as an additive suffers only a 4.3% of reduction of impact strength, while the samples that contain commercial additives lose clearly more of their impact strength (from 23.1% to 61.1%). Based on the results CDW e-lignin is a good additive to prevent weathering. As a conclusion, the esterified lignin from CDW, is a versatile additive for composite production.

This study aims to elucidate the role of sulfide and its precursors in anaerobic digestion (i.e., cysteine, representing sulfur-containing amino acids, and sulfate) on microbial oleate conversion to methane. Serine, with a similar structure to cysteine but with a hydroxyl group instead of a thiol, was included as a control to assess potential effects on methane formation that were not related to sulfur functionalities. The results showed that copresence of sulfide and oleate in anaerobic batch assays accelerated the methane formation compared to assays with only oleate and mitigated negative effect on methane formation caused by increased sulfide level. Nuclear magnetic resonance spectroscopy of sulfide-exposed oleate suggested that sulfide reaction with oleate double bonds likely contributed to negation of the negative effect on the methanogenic activity. Methane formation from oleate was also accelerated in the presence of cysteine or serine, while sulfate decreased the cumulative methane formation from oleate. Neither cysteine nor serine was converted to methane, and their accelerating effects was associated to different mechanisms due to establishment of microbial communities with different structures, as evidenced by high-throughput sequencing of 16S rRNA gene. These outcomes contribute with new knowledge to develop strategies for optimum use of sulfur- and lipid-rich wastes in anaerobic digestion processes.

A halotolerant, exopolysaccharide-producing bacterium isolated from the Salar de Uyuni salt flat in Bolivia was identified as Bacillus atrophaeus using next-generation sequencing. Comparisons indicate that the genome most likely (p-value: 0.0024) belongs to a subspecies previously not represented in the database. The growth of the bacterial strain and its ability to produce exopolysaccharides (EPS) in synthetic media with glucose or xylose as carbon sources, and in hydrolysates of quinoa stalks, was investigated. The strain grew well in all synthetic media, but the growth in glucose was better than that in xylose. Sugar consumption was better when initial concentrations were low. The growth was good in enzymatically produced cellulosic hydrolysates but was inhibited in hemicellulosic hydrolysates produced using hydrothermal pretreatment. The EPS yields were up to 0.064 g/g on initial glucose and 0.047 g/g on initial xylose, and was higher in media with relatively low sugar concentrations. The EPS was isolated and purified by a sequential procedure including centrifugation, cold ethanol precipitation, trichloroacetic acid treatment, dialysis, and freeze-drying. Glucose and mannose were the main sugars identified in hydrolyzed EPS. The EPS was characterized by size-exclusion chromatography, Fouriertransform infrared (FTIR) spectroscopy, heteronuclear single-quantum coherence nuclear magnetic resonance (HSQC NMR) spectroscopy, scanning electron microscopy, X-ray diffraction, and thermogravimetric analysis. No major differences were elucidated between EPS resulting from cultivations in glucoseor-xylose-based synthetic media, while some divergences with regard to molecular-weight averages and FTIR and HSQC NMR spectra were detected for EPS from hydrolysate-based media.

Hydrolysis lignin, i.e., the hydrolysis residue of cellulosic ethanol plants, was extracted with the green solvent γ-valerolactone (GVL). Treatments at 170–210 °C were performed with either non-acidified GVL/water mixtures (NA-GVL) or with mixtures containing sulfuric acid (SA-GVL). SA-GVL treatment at 210 °C resulted in the highest lignin solubilization (64% (w/w) of initial content), and 76% of the solubilized mass was regenerated by water-induced precipitation. Regenerated lignins were characterized through compositional analysis with sulfuric acid, as well as using pyrolysis–gas chromatography/mass spectrometry (Py-GC/MS), high-performance size-exclusion chromatography (HPSEC), solid-state cross-polarization/magic angle spinning ¹³C nuclear magnetic resonance (CP/MAS ¹³C NMR) spectroscopy, ¹H^–13C heteronuclear single-quantum coherence NMR (HSQC NMR), and Fourier-transform infrared (FTIR) spectroscopy. The characterization revealed that the main difference between regenerated lignins was their molecular weight. Molecular weight averages increased with treatment temperature, and they were higher and had broader distribution for SA-GVL lignins than for NA-GVL lignins.

The potential drug target choline acetyltransferase (ChAT) catalyses the production of the neurotransmitter acetylcholine in cholinergic neurons, T-cells, and B-cells. Herein, we show that arylvinylpyridiniums (AVPs), the most widely studied class of ChAT inhibitors, act as substrate in an unusual coenzyme A-dependent hydrothiolation reaction. This in situ synthesis yields an adduct that is the actual enzyme inhibitor. The adduct is deeply buried in the active site tunnel of ChAT and interactions with a hydrophobic pocket near the choline binding site have major implications for the molecular recognition of inhibitors. Our findings clarify the inhibition mechanism of AVPs, establish a drug modality that exploits a target-catalysed reaction between exogenous and endogenous precursors, and provide new directions for the development of ChAT inhibitors with improved potency and bioactivity.