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All-cellulose composites (ACCs) have been fabricated by using a variety of cellulosic sources, versatile technologies, and are sustainable alternatives for traditional composites. In this study, nonwoven-woven ACC laminates were created from wood-based Spinnova short fibers and Lyocell fabrics via partial dissolution and an NaOH-urea solvent system. The less-known wood-based Spinnova fiber is created for the textile industry, but it also has great potential for the composite industry. To identify the mechanical properties of ACCs—which greatly influence the range of material application—tensile, impact, and flexural tests were conducted. The mechanical properties indicated only moderate properties, which are influenced by high porosity and weak fiber bonding. Despite this, valuable information on the nonwoven-woven structured ACCs was obtained. To improve the ACC laminate’s ability to resist moisture, bio-based coatings (e.g., commercially available birch bark betulin and suberin acid mixture) were applied on the surface of ACCs and it successfully improved the wetting resistance. The results of contact angle analyses demonstrated that the highest contact angle of 128° was measured for betulin-coated laminates and the best stable hydrophobicity calculated a minute after the beginning of the experiment were observed at 109° for the uncommercial pressurized hot ethanol (PHE) extract of birch bark.

Cold seeps release methane (CH4) from the seafloor to the water column, which fuels microbially mediated aerobic methane oxidation (MOx). Methane-oxidising bacteria (MOB) utilise excess methane, and the MOB biomass serves as a carbon source in the food web. Yet, it remains unclear if and how MOx modifies the composition of dissolved organic matter (DOM) in cold seeps. We investigated MOx rates, DOM compositions and the microbial community during ex-situ incubations of seawater collected from a cold seep site at Norskebanken (north of the Svalbard archipelago) in the Arctic Ocean. Samples were incubated with and without methane amendments. Samples amended with methane (∼1 µM final concentration) showed elevated rates of MOx in both seep and non-seep incubations. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) analyses showed that the number of DOM formulas (i.e., molecular diversity) increased by up to 39% in these incubations. In contrast, the number of formulas decreased by 20% in samples not amended with methane, both from non-seep and seep locations. DOM composition was thus altered towards a more diverse and heterogeneous composition along with elevated methanotrophic activity in methane-amended conditions. In addition to microbial DOM production, abating microbial diversity indicates that elevated DOM diversity was potentially related to grazing pressure on bacteria. The diversity of DOM constituents, therefore, likely increased with the variety of decaying cells contributing to DOM production. Furthermore, based on a principal coordinate analysis, we show that the final DOM composition of non-seep samples amended with methane became more resemblant to that of seep samples. This suggests that methane intrusions will affect water column DOM dynamics similarly, irrespective of the water column’s methane history.

Mycobacterium tuberculosis (Mtb), which is responsible for more than a million deaths annually, uses lipids as the source of carbon and energy for its survival in the latent phase of infection. Mtb cannot synthesize all of the lipid molecules required for its growth and pathogenicity. Therefore, it relies on transporters such as the mammalian cell entry (Mce) complexes to import lipids from the host across the cell wall. Despite their importance for the survival and pathogenicity of Mtb, information on the structural properties of these proteins is not yet available. Each of the four Mce complexes in Mtb (Mce1–4) comprises six substrate-binding proteins (SBPs; MceA–F), each of which contains four conserved domains (N-terminal transmembrane, MCE, helical and C-terminal unstructured tail domains). Here, the properties of the various domains of Mtb Mce1A and Mce4A, which are involved in the import of mycolic/fatty acids and cholesterol, respectively, are reported. In the crystal structure of the MCE domain of Mce4A (MtMce4A39–140) a domain-swapped conformation is observed, whereas solution studies, including small-angle X-ray scattering (SAXS), indicate that all Mce1A and Mce4A domains are predominantly monomeric. Further, structural comparisons show interesting differences from the bacterial homologs MlaD, PqiB and LetB, which form homohexamers when assembled as functional transporter complexes. These data, and the fact that there are six SBPs in each Mtb mce operon, suggest that the MceA–F SBPs from Mce1–4 may form heterohexamers. Also, interestingly, the purification and SAXS analysis showed that the helical domains interact with the detergent micelle, suggesting that when assembled the helical domains of MceA–F may form a hydrophobic pore for lipid transport, as observed in EcPqiB. Overall, these data highlight the unique structural properties of the Mtb Mce SBPs.

The pyroligneous acids (PAs) of woody biomass produced by torrefaction have pesticidal properties. Thus, PAs are potential alternatives to synthetic plant protection chemicals. Although woody biomass is a renewable feedstock, its use must be efficient. The efficiency of biomass utilization can be improved by applying a cascading use principle. This study is novel because we evaluate for the first time the pesticidal potential of PAs derived from the bark of hybrid aspen (Populus tremula L. × Populus tremuloides Michx.) and examine simultaneously how the production of the PAs can be interlinked with the cascade processing of hybrid aspen biomass. Hybrid aspen bark contains valuable extractives that can be separated before the hemicellulose is thermochemically converted into plant protection chemicals. We developed a cascade processing scheme, where these extractives were first extracted from the bark with hot water (HWE) or with hot water and alkaline alcohol (HWE+AAE) prior to their conversion into PAs by torrefaction. The herbicidal performance of PAs was tested using Brassica rapa as the test species, and the fungicidal performance was proven using Fusarium culmorum. The pesticidal activities were compared to those of the PAs of debarked wood and of commercial pesticides. According to the results, extractives can be separated from the bark without overtly diminishing the weed and fungal growth inhibitor performance of the produced PAs. The HWE of the bark before its conversion into PAs appeared to have an enhancing effect on the herbicidal activity. In contrast, HWE+AAE lowered the growth inhibition performance of PAs against both the weeds and fungi. This study shows that hybrid aspen is a viable feedstock for the production of herbicidal and fungicidal active chemicals, and it is possible to utilize biomass according to the cascading use principle.

Abstract The encapsulation and/or surface modification can stabilize and protect the phosphorescence bio‐probes but impede their intravenous delivery across biological barriers. Here, a new class of biocompatible rhenium (ReI) diimine carbonyl complexes is developed, which can efficaciously permeate normal vessel walls and then functionalize the extravascular collagen matrixes as in situ oxygen sensor. Without protective agents, ReI‐diimine complex already exhibits excellent emission yield (34%, λem = 583 nm) and large two‐photon absorption cross‐sections (σ2 = 300 GM @ 800 nm) in water (pH 7.4). After extravasation, remarkably, the collagen‐bound probes further enhanced their excitation efficiency by increasing the deoxygenated lifetime from 4.0 to 7.5 µs, paving a way to visualize tumor hypoxia and tissue ischemia in vivo. The post‐extravasation functionalization of extracellular matrixes demonstrates a new methodology for biomaterial‐empowered phosphorescence sensing and imaging.

Summary: Porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis, catalyzes the sequential coupling of four porphobilinogen (PBG) molecules into a heme precursor. Mutations in PBGD are associated with acute intermittent porphyria (AIP), a rare metabolic disorder. We used Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to demonstrate that wild-type PBGD and AIP-associated mutant R167W both existed as holoenzymes (Eholo) covalently attached to the dipyrromethane cofactor, and three intermediate complexes, ES, ES2, and ES3, where S represents PBG. In contrast, only ES2 was detected in AIP-associated mutant R173W, indicating that the formation of ES3 is inhibited. The R173W crystal structure in the ES2-state revealed major rearrangements of the loops around the active site, compared to wild-type PBGD in the Eholo-state. These results contribute to elucidating the structural pathogenesis of two common AIP-associated mutations and reveal the important structural role of Arg173 in the polypyrrole elongation mechanism.

This study assessed the pyrolysis liquids obtained by slow pyrolysis of industrial hemp leaves, hurds, and roots. The liquids recovered between a pyrolysis temperature of 275–350 °C, at two condensation temperatures 130 °C and 70 °C, were analyzed. Aqueous and bio-oil pyrolysis liquids were produced and analyzed by proton nuclear magnetic resonance (NMR), gas chromatography–mass spectrometry (GC-MS), and atmospheric pressure photoionization Fourier transform ion cyclotron resonance mass spectrometry (APPI FT-ICR MS). NMR revealed quantitative concentrations of the most abundant compounds in the aqueous fractions and compound groups in the oily fractions. In the aqueous fractions, the concentration range of acetic acid was 50–241 gL−1, methanol 2–30 gL−1, propanoic acid 5–20 gL−1, and 1-hydroxybutan-2-one 2 gL−1. GC-MS was used to compare the compositions of the volatile compounds and APPI FT-ICR MS was utilized to determine the most abundant higher molecular weight compounds. The different obtained pyrolysis liquids (aqueous and oily) had various volatile and nonvolatile compounds such as acetic acid, 2,6-dimethoxyphenol, 2-methoxyphenol, and cannabidiol. This study provides a detailed understanding of the chemical composition of pyrolysis liquids from different parts of the industrial hemp plant and assesses their possible economic potential.

Flucloxacillin (FLU), an isoxazolyl penicillin, is widely used for the treatment of different bacterial infections in intensive care units (ICU). Being highly bound to plasma proteins, FLU is prone to drug-drug interactions (DDI) when administered concurrently with other drugs. As FLU is binding to both Sudlow’s site I and site II of human serum albumin (HSA), competitive and allosteric interactions with other drugs, highly bound to the same sites, seem conceivable. Knowledge about interaction(s) of FLU with the widely used anticancer agents paclitaxel (PAC), imatinib (IMA), and 5-fluorouracil (5-FU is scarce. The effects of the selected anticancer agents on the unbound fraction of FLU were evaluated in pooled plasma as well as in HSA and α-1-acid glycoprotein (AGP) samples, the second major drug carrier in plasma. FLU levels in spiked samples were analyzed by LC-MS/MS after ultrafiltration. Significant increase in FLU unbound fraction was observed when in combination with PAC and IMA and to a lesser extent with 5-FU. Furthermore, significant binding of FLU to AGP was observed. Collectively, this is the first study showing the binding of FLU to AGP as well as demonstrating a significant DDI between PAC/IMA/5-FU and FLU.

Background Carbonic anhydrases (CAs) are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H+ ions. Vertebrate genomes generally contain gene loci for 15–21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only secretory protein of the enzymatically active isoforms. We discovered that non-mammalian CA VI contains a C-terminal pentraxin (PTX) domain, a novel combination for both CAs and PTXs. Methods We isolated and sequenced zebrafish (Danio rerio) CA VI cDNA, complete with the sequence coding for the PTX domain, and produced the recombinant CA VI–PTX protein. Enzymatic activity and kinetic parameters were measured with a stopped-flow instrument. Mass spectrometry, analytical gel filtration and dynamic light scattering were used for biophysical characterization. Sequence analyses and Bayesian phylogenetics were used in generating hypotheses of protein structure and CA VI gene evolution. A CA VI–PTX antiserum was produced, and the expression of CA VI protein was studied by immunohistochemistry. A knock-down zebrafish model was constructed, and larvae were observed up to five days post-fertilization (dpf). The expression of ca6 mRNA was quantitated by qRT-PCR in different developmental times in morphant and wild-type larvae and in different adult fish tissues. Finally, the swimming behavior of the morphant fish was compared to that of wild-type fish. Results The recombinant enzyme has a very high carbonate dehydratase activity. Sequencing confirms a 530-residue protein identical to one of the predicted proteins in the Ensembl database (ensembl.org). The protein is pentameric in solution, as studied by gel filtration and light scattering, presumably joined by the PTX domains. Mass spectrometry confirms the predicted signal peptide cleavage and disulfides, and N-glycosylation in two of the four observed glycosylation motifs. Molecular modeling of the pentamer is consistent with the modifications observed in mass spectrometry. Phylogenetics and sequence analyses provide a consistent hypothesis of the evolutionary history of domains associated with CA VI in mammals and non-mammals. Briefly, the evidence suggests that ancestral CA VI was a transmembrane protein, the exon coding for the cytoplasmic domain was replaced by one coding for PTX domain, and finally, in the therian lineage, the PTX-coding exon was lost. We knocked down CA VI expression in zebrafish embryos with antisense morpholino oligonucleotides, resulting in phenotype features of decreased buoyancy and swim bladder deflation in 4 dpf larvae. Discussion These findings provide novel insights into the evolution, structure, and function of this unique CA form.

In vitro toxicological studies together with atomistic molecular dynamics simulations show that occupational co-exposure with C60 fullerene may strengthen the health effects of organic industrial chemicals. The chemicals studied are acetophenone, benzaldehyde, benzyl alcohol, m-cresol, and toluene which can be used with fullerene as reagents or solvents in industrial processes. Potential co-exposure scenarios include a fullerene dust and organic chemical vapor, or a fullerene solution aerosolized in workplace air. Unfiltered and filtered mixtures of C60 and organic chemicals represent different co-exposure scenarios in in vitro studies where acute cytotoxicity and immunotoxicity of C60 and organic chemicals are tested together and alone by using human THP-1-derived macrophages. Statistically significant co-effects are observed for an unfiltered mixture of benzaldehyde and C60 that is more cytotoxic than benzaldehyde alone, and for a filtered mixture of m-cresol and C60 that is slightly less cytotoxic than m-cresol. Hydrophobicity of chemicals correlates with co-effects when secretion of pro-inflammatory cytokines IL-1β and TNF-α is considered. Complementary atomistic molecular dynamics simulations reveal that C60 co-aggregates with all chemicals in aqueous environment. Stable aggregates have a fullerene-rich core and a chemical-rich surface layer, and while essentially all C60 molecules aggregate together, a portion of organic molecules remains in water.

Avidins are a family of proteins widely employed in biotechnology. We have previously shown that functional chimeric mutant proteins can be created from avidin and avidin-related protein 2 using a methodology combining random mutagenesis by recombination and selection by a tailored biopanning protocol (phage display). Here, we report the crystal structure of one of the previously selected and characterized chimeric avidin forms, A/A2-1. The structure was solved at 1.8 Å resolution and revealed that the protein fold was not affected by the shuffled sequences. The structure also supports the previously observed physicochemical properties of the mutant. Furthermore, we improved the selection and screening methodology to select for chimeric avidins with slower dissociation rate from biotin than were selected earlier. This resulted in the chimeric mutant A/A2-B, which showed increased thermal stability as compared to A/A2-1 and the parental proteins. The increased stability was especially evident at conditions of extreme pH as characterized using differential scanning calorimetry. In addition, amino acid sequence and structural comparison of the chimeric mutants and the parental proteins led to the rational design of A/A2-B I109K. This mutation further decreased the dissociation rate from biotin and yielded an increase in the thermal stability.

The avidin protein family members are well known for their high affinity towards D-biotin and high structural stability. These properties make avidins valuable tools for a wide range of biotechnology applications. We have identified a new member of the avidin family in the zebrafish (Danio rerio) genome, hereafter called zebavidin. The protein is highly expressed in the gonads of both male and female zebrafish and in the gills of male fish, but our data suggest that zebavidin is not crucial for the developing embryo. Biophysical and structural characterisation of zebavidin revealed distinct properties not found in any previously characterised avidins. Gel filtration chromatography and native mass spectrometry suggest that the protein forms dimers in the absence of biotin at low ionic strength, but assembles into tetramers upon binding biotin. Ligand binding was analysed using radioactive and fluorescently labelled biotin and isothermal titration calorimetry. Moreover, the crystal structure of zebavidin in complex with biotin was solved at 2.4 Å resolution and unveiled unique ligand binding and subunit interface architectures; the atomic-level details support our physicochemical observations.

Our previous microarray study showed that the non-specific cytotoxic cell receptor protein 1 (Nccrp1) transcript is significantly upregulated in the gastric mucosa of carbonic anhydrase IX (CA IX)-deficient (Car9(-/-)) mice. In this paper, we aimed to characterize human NCCRP1 and to elucidate its relationship to CA IX. Recombinant NCCRP1 protein was expressed in Escherichia coli, and a novel polyclonal antiserum was raised against the purified full-length protein. Immunocytochemistry showed that NCCRP1 is expressed intracellularly, even though it has previously been described as a transmembrane protein. Using bioinformatic analyses, we identified orthologs of NCCRP1 in 35 vertebrate genomes, and up to five paralogs per genome. These paralogs are FBXO genes whose protein products are components of the E3 ubiquitin ligase complexes. NCCRP1 proteins have no signal peptides or transmembrane domains. NCCRP1 has mainly been studied in fish and was thought to be responsible for the cytolytic function of nonspecific cytotoxic cells (NCCs). Our analyses showed that in humans, NCCRP1 mRNA is expressed in tissues containing squamous epithelium, whereas it shows a more ubiquitous tissue expression pattern in mice. Neither human nor mouse NCCRP1 expression is specific to immune tissues. Silencing CA9 using siRNAs did not affect NCCRP1 levels, indicating that its expression is not directly regulated by CA9. Interestingly, silencing NCCRP1 caused a statistically significant decrease in the growth of HeLa cells. These studies provide ample evidence that the current name, "non-specific cytotoxic cell receptor protein 1," is not appropriate. We therefore propose that the gene name be changed to FBXO50.

The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (strept)avidin to improve the existing applications. Even so, (strept)avidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces.

BACKGROUND: Allergen-mediated cross-linking of IgE antibodies bound to the FcepsilonRI receptors on the mast cell surface is the key feature of the type I allergy. If an allergen is a homodimer, its allergenicity is enhanced because it would only need one type of antibody, instead of two, for cross-linking. METHODOLOGY/PRINCIPAL FINDINGS: An analysis of 55 crystal structures of allergens showed that 80% of them exist in symmetric dimers or oligomers in crystals. The majority are transient dimers that are formed at high protein concentrations that are reached in cells by colocalization. Native mass spectrometric analysis showed that native allergens do indeed form transient dimers in solution, while hypoallergenic variants of them exist almost solely in the monomeric form. We created a monomeric Bos d 5 allergen and show that it has a reduced capability to induce histamine release. CONCLUSIONS/SIGNIFICANCE: The results suggest that dimerization would be a very common and essential feature for allergens. Thus, the preparation of purely monomeric variants of allergens could open up novel possibilities for specific immunotherapy.

Pyrogallarene shows concentration-dependent instability in dilute solutions resulting in elimination of two ketene molecules and formation of pyrogallarene lactones. This unexpected phenomenon, which is not observed with resorcinarenes, highlights the significance of the four hydroxyl groups at 2-position for the molecular characteristics of pyrogallarenes. [ABSTRACT FROM AUTHOR]