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Graphical abstract, Highlights • Novel cell-free protein synthesis reaction buffer improves performance by 400%. • Enhanced performance is maintained across the synthesis of different proteins. • Protein synthesis performance is robust across different cell lysate batches and E. coli strains. • Buffer components affect aspects of reaction kinetics in differing ways., Cell-free protein synthesis (CFPS) reactions have grown in popularity with particular interest in applications such as gene construct prototyping, biosensor technologies and the production of proteins with novel chemistry. Work has frequently focussed on optimising CFPS protocols for improving protein yield, reducing cost, or developing streamlined production protocols. Here we describe a statistical Design of Experiments analysis of 20 components of a popular CFPS reaction buffer. We simultaneously identify factors and factor interactions that impact on protein yield, rate of reaction, lag time and reaction longevity. This systematic experimental approach enables the creation of a statistical model capturing multiple behaviours of CFPS reactions in response to components and their interactions. We show that a novel reaction buffer outperforms the reference reaction by 400% and importantly reduces failures in CFPS across batches of cell lysates, strains of E. coli, and in the synthesis of different proteins. Detailed and quantitative understanding of how reaction components affect kinetic responses and robustness is imperative for future deployment of cell-free technologies.

The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2 (SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors. The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phosphatase domain of SHP2 (SHP2-PTP) and allosteric inhibitors. Of note, this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76A. After initial screening of our in-house compound library (∼2300 compounds), we identified 4 new SHP2-PTP inhibitors (0.17% hit rate) and 28 novel allosteric SHP2 inhibitors (1.22% hit rate), of which SYK-85 and WS-635 effectively inhibited SHP2-PTP (SYK-85: IC50 = 0.32 μmol/L; WS-635: IC50 = 4.13 μmol/L) and thus represent novel scaffolds for designing new SHP2-PTP inhibitors. TK-147, an allosteric inhibitor, inhibited SHP2 potently (IC50 = 0.25 μmol/L). In structure, TK-147 could be regarded as a bioisostere of the well characterized SHP2 inhibitor SHP-099, highlighting the essential structural elements for allosteric inhibition of SHP2. The principle underlying the cross-validation protocol is potentially feasible to identify allosteric inhibitors or those inactivating mutants of other proteins., Graphical abstract This work established a cross-validation screening protocol based on fluorescence-based enzyme assay and conformation-dependent thermal shift assay. An in-house compound library consisting of about 2300 compounds was screened based on the protocol, leading to the discovery of 4 new SHP2-PTP inhibitors and 28 novel allosteric SHP2 inhibitors.Image 1

15 pags, 5 figs, 3 tabs. -- Supplementary data to this article can be found online at https://doi.org/10.1016/j.csbj.2021.11.027., Many microorganisms feed on the tissue and recalcitrant bone materials from dead animals, however little is known about the collaborative effort and characteristics of their enzymes. In this study, microbial metagenomes from symbionts of the marine bone-dwelling worm Osedax mucofloris, and from microbial biofilms growing on experimentally deployed bone surfaces were screened for specialized bone-degrading enzymes. A total of 2,043 taxonomically (closest match within 40 phyla) and functionally (1 proteolytic and 9 glycohydrolytic activities) diverse and non-redundant sequences (median pairwise identity of 23.6%) encoding such enzymes were retrieved. The taxonomic assignation and the median identity of 72.2% to homologous proteins reflect microbial and functional novelty associated to a specialized bone-degrading marine community. Binning suggests that only one generalist hosting all ten targeted activities, working in synergy with multiple specialists hosting a few or individual activities. Collagenases were the most abundant enzyme class, representing 48% of the total hits. A total of 47 diverse enzymes, representing 8 hydrolytic activities, were produced in Escherichia coli, whereof 13 were soluble and active. The biochemical analyses revealed a wide range of optimal pH (4.0-7.0), optimal temperature (5-65 °C), and of accepted substrates, specific to each microbial enzyme. This versatility may contribute to a high environmental plasticity of bone-degrading marine consortia that can be confronted to diverse habitats and bone materials. Through bone-meal degradation tests, we further demonstrated that some of these enzymes, particularly those from Flavobacteriaceae and Marinifilaceae, may be an asset for development of new value chains in the biorefinery industry., We acknowledge financial support of ERA-NET Marine Biotechnology (GA no.: 604814) funded under the FP7 ERA-NET scheme and nationally managed from the German Federal Ministry of Education and Research and Norwegian Research Council, the grants PCIN-2017-078 (within the Marine Biotechnology ERA-NET), BIO2017-85522-R, PID2020-112758RB-I00 and PDC2021-121534-I00 from the Ministerio de Economía, Industria y Competitividad, Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (AEI) (Digital Object Identifier 10.13039/501100011033), Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (“NextGenerationEU/PRTR”), and the grant 2020AEP061 from the Agencia Estatal CSIC. F.J.P and J.S-A. acknowledges grants PID2019-105838RB-C31 and PID2019-105838RB-C33 from the Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (AEI), Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (EU). Additional funding was received from the Norwegian Biodiversity Information Centre (PA 809116 knr. 47-14) and NORCE Norwegian Research Centre. J.W. acknowledge the use of de.NBI cloud and the support by the High Performance and Cloud Computing Group at the Zentrum für Datenverarbeitung of the Eberhard Karls University of Tübingen and the Federal Ministry of Education and Research (BMBF) through grant no 031 A535A. K.S.M. is supported by intramural funds of the US Department of Health and Human Services (to the National Library of Medicine).

Highlights • Fluorimetric quantification of entrapped FITC-BSA is possible. • Two separate methods allow its quantification in the nanogram range. • Dequenching of FITC-BSA by enzymatic degradation or alkaline unfolding. • Calcium phosphate increases FITC-BSA loading of PLGA nanoparticles by 2.7-fold. • Nanoparticle characteristics were optimized for inner ear drug delivery., Although therapeutically active proteins are highly efficacious, their content in protective nanoparticles is often too low to elicit adequate plasma levels. A strategy to increase protein loading is the in-situ generation of calcium phosphate as a protein adsorbent. To verify this approach, a highly sensitive and reliable fluorimetric method for quantification of incorporated fluorescein-labelled bovine serum albumin (FITC-BSA) as a model protein drug was developed. Dequenching the fluorescein label by pronase E, which digests the protein backbone, and dissolving the nanoparticle matrix in acetonitrile enabled FITC-BSA quantification in the nanogram per milliliter range. This test was confirmed by a second assay involving alkaline hydrolysis of FITC-BSA and the matrix. Nanoparticles prepared with calcium phosphate contained 40 µg FITC-BSA/mg and nanoparticles without calcium phosphate only 15 µg FITC-BSA/mg, representing a 2.7-fold increase in model protein loading. In this work the nanoparticle preparation procedure was optimized in terms of size for administration in the inner ear, but the range of applications is not limited.

Graphical abstract, Kinases are signalling proteins which have proven to be successful targets for the treatment of a variety of diseases, predominantly in cancers. However, only a small proportion of kinases (

Aldolases catalyze the reversible reactions of aldol condensation and cleavage and have strong potential for the synthesis of chiral compounds, widely used in pharmaceuticals. Here, we investigated a new Class II metal aldolase from the p-hydroxyphenylacetate degradation pathway in Acinetobacter baumannii, 4-hydroxy-2-keto-heptane-1,7-dioate aldolase (AbHpaI), which has various properties suitable for biocatalysis, including stereoselectivity/stereospecificity, broad aldehyde utilization, thermostability, and solvent tolerance. Notably, the use of Zn2+ by AbHpaI as a native cofactor is distinct from other enzymes in this class. AbHpaI can also use other metal ion (M2+) cofactors, except Ca2+, for catalysis. We found that Zn2+ yielded the highest enzyme complex thermostability (Tm of 87 °C) and solvent tolerance. All AbHpaI•M2+ complexes demonstrated preferential cleavage of (4R)-2-keto-3-deoxy-D-galactonate ((4R)-KDGal) over (4S)-2-keto-3-deoxy-D-gluconate ((4S)-KDGlu), with AbHpaI•Zn2+ displaying the highest R/S stereoselectivity ratio (sixfold higher than other M2+ cofactors). For the aldol condensation reaction, AbHpaI•M2+ only specifically forms (4R)-KDGal and not (4S)-KDGlu and preferentially catalyzes condensation rather than cleavage by ∼40-fold. Based on 11 X-ray structures of AbHpaI complexed with M2+ and ligands at 1.85 to 2.0 A resolution, the data clearly indicate that the M2+ cofactors form an octahedral geometry with Glu151 and Asp177, pyruvate, and water molecules. Moreover, Arg72 in the Zn2+-bound form governs the stereoselectivity/stereospecificity of AbHpaI. X-ray structures also show that Ca2+ binds at the trimer interface via interaction with Asp51. Hence, we conclude that AbHpaI•Zn2+ is distinctive from its homologues in substrate stereospecificity, preference for aldol formation over cleavage, and protein robustness, and is attractive for biocatalytic applications.

The extracellular matrix is a key component of tissues, yet it is underrepresented in proteomic datasets. Identification and evaluation of proteins in the extracellular matrix (ECM) has proved challenging due to the insolubility of many ECM proteins in traditional protein extraction buffers. Here we separate the decellularization and ECM extraction steps of several prominent methods for evaluation under real-world conditions. The results are used to optimize a two-fraction ECM extraction method. Approximately one dozen additional parameters are tested, and recommendations for analysis based on overall ECM coverage or specific ECM classes are given. Compared with a standard in-solution digest, the optimized method yielded a fourfold improvement in unique ECM peptide identifications., Graphical abstract, Highlights • Decellularization drastically improves ECM coverage. • ECM coverage is the highest with a dual chemical/enzymatic digestion method. • SPEED is the best tested single-shot method for ECM identification. • A fourfold improvement in ECM peptide IDs was achieved compared with standard methods., Due to their insolubility, many extracellular matrix (ECM) proteins are resistant to typical extraction and require optimized extraction methods for proteomic analysis. A wide variety of decellularization and ECM extraction methods have been published, but it remains unclear how these methods perform compared with one another on a complex, ECM-rich sample. A direct comparison of both cell and ECM extraction methods on a whole organism sample and four additional organs serves as a reference for future ECM proteomics.

Voltage-gated sodium channels (Nav1s) are responsible for the initiation and propagation of action potentials in neurons, muscle, and endocrine cells. Many clinically used drugs such as local anesthetics and antiarrhythmics inhibit Nav1s, and a variety of inherited human disorders are caused by mutations in Nav1 genes. Nav1s consist of the main α subunit and several auxiliary β subunits. Detailed information on the structure-function relationships of Nav1 subunits has been obtained through heterologous expression experiments and analyses of protein structures. The basic properties of Nav1s, including their gating and ion permeation, were classically described in the squid giant axon and other invertebrates. However, heterologous functional expression of Nav1s from marine invertebrates has been unsuccessful. Ascidians belong to the Urochordata, a sister group of vertebrates, and the larval central nervous system of ascidians shows a similar plan to that of vertebrates. Here, we report the biophysical properties of ascidian Ciona Nav1 (CiNav1a) heterologously expressed in Xenopus oocytes. CiNav1a exhibited tetrodotoxin-insensitive sodium currents with rapid gating kinetics of activation and inactivation. Furthermore, consistent with the fact that the Ciona genome lacks orthologous genes to vertebrate β subunits, the human β1 subunit did not influence the gating properties when coexpressed with CiNav1a. Interestingly, CiNav1a contains an ankyrin-binding motif in the II-III linker, which can be targeted to the axon initial segment of mammalian cortical neurons. Our findings provide a platform to gain insight into the evolutionary and biophysical properties of Nav1s, which are important for the development of targeted therapeutics.

Graphical abstract, Highlights • First structure of a glycoside hydrolase from a bacterial symbiont isolated from the digestive tract of the notorious termite pest Reticulitermes flavipes. • First example of a GH5 glycoside hydrolase that features a GH42-type homotrimeric structure. • High exo-type specificity for the terminal ®-1,4-mannosidic linkages in mannooligosaccharides and unsubstituted®-mannans. • Verrucomicrobial gut symbiont with high potential for hemicellulose degradation., The termite Reticulitermes flavipes causes extensive damage due to the high efficiency and broad specificity of the ligno- and hemicellulolytic enzyme systems produced by its symbionts. Thus, the R. flavipes gut microbiome is expected to constitute an excellent source of enzymes that can be used for the degradation and valorization of plant biomass. The symbiont Opitutaceae bacterium strain TAV5 belongs to the phylum Verrucomicrobia and thrives in the hindgut of R. flavipes. The sequence of the gene with the locus tag opit5_10225 in the Opitutaceae bacterium strain TAV5 genome has been classified as a member of glycoside hydrolase family 5 (GH5), and provisionally annotated as an endo-β-mannanase. We characterized biochemically and structurally the opit5_10225 gene product, and show that the enzyme, Op5Man5, is an exo-β-1,4-mannosidase [EC 3.2.1.25] that is highly specific for β-1,4-mannosidic bonds in mannooligosaccharides and ivory nut mannan. The structure of Op5Man5 was phased using electron cryo-microscopy and further determined and refined at 2.2 Å resolution using X-ray crystallography. Op5Man5 features a 200-kDa large homotrimer composed of three modular monomers. Despite insignificant sequence similarity, the structure of the monomer, and homotrimeric assembly are similar to that of the GH42-family β-galactosidases and the GH164-family exo-β-1,4-mannosidase Bs164 from Bacteroides salyersiae. To the best of our knowledge Op5Man5 is the first structure of a glycoside hydrolase from a bacterial symbiont isolated from the R. flavipes digestive tract, as well as the first example of a GH5 glycoside hydrolase with a GH42 β-galactosidase-type homotrimeric structure.

The combination of paclitaxel (PTX) and doxorubicin (DOX) has been widely used in the clinic. However, it remains unsatisfied due to the generation of severe toxicity. Previously, we have successfully synthesized a prodrug PTX-S-DOX (PSD). The prodrug displayed comparable in vitro cytotoxicity compared with the mixture of free PTX and DOX. Thus, we speculated that it could be promising to improve the anti-cancer effect and reduce adverse effects by improving the pharmacokinetics behavior of PSD and enhancing tumor accumulation. Due to the fact that copper ions (Cu2+) could coordinate with the anthracene nucleus of DOX, we speculate that the prodrug PSD could be actively loaded into liposomes by Cu2+ gradient. Hence, we designed a remote loading liposomal formulation of PSD (PSD LPs) for combination chemotherapy. The prepared PSD LPs displayed extended blood circulation, improved tumor accumulation, and more significant anti-tumor efficacy compared with PSD NPs. Furthermore, PSD LPs exhibited reduced cardiotoxicity and kidney damage compared with the physical mixture of Taxol and Doxil, indicating better safety. Therefore, this novel nano-platform provides a strategy to deliver doxorubicin with other poorly soluble antineoplastic drugs for combination therapy with high efficacy and low toxicity., Graphical abstract The authors designed a remote loading paclitaxel–doxorubicin prodrug liposomal formulation for combination of PTX and DOX. The prodrug could respond to high ROS/GSH of the tumor microenvironment. The stimuli-responsive liposomes were proved to be a high-efficacy and low-toxicity formulation. This technology provides a novel pathway for cancer combination therapy.Image 1

Graphical abstract, Highlights • Feed supplemented with GP or OMW increased GST activity in liver and spleen tissue. • Feed supplemented with GP increased γ-GCS expression in liver tissue. • Dietary administration with OMW decreased γ-GCS expression in liver tissue. • Experimental diet with GP or OMW did not affect SOD activity in both tissues. • The beneficial effects of the feeds were tissue- and developmental stage-specific., The aim of the present study was to investigate the effects of livestock feed supplemented with grape pomace (GP) or olive oil mill wastewater (OMW) byproducts on the enzymatic activity and protein expression of antioxidants enzymes, in liver and spleen tissue of sheep. Thus, 36 male sheep of Chios breed were divided into 3 homogeneous groups, control group (n = 12), GP group (n = 12) and OMW group (n = 12), receiving standard or experimental feed. Liver and spleen tissues were collected at 42 and 70 days post-birth. The enzymatic activity of superoxide dismutase (SOD) and glutathione-s-transferase (GST) and also the protein expression of γ-synthase glutamyl custeine (γ-GCS) were determined in these tissues. The results showed GP group exhibited increased enzymatic activity of GST and protein expression of γ-GCS in liver compared to control group. In GP group’s spleen, GST activity was increased compared to control but γ-GCS expression was not affected. In OMW group’s liver, GST activity was increased and γ-GCS expression was reduced compared to control. In OMW group’s spleen, GST activity was increased but GCS expression was not affected. SOD activity was not affected in both tissues either in GP or OMW group.

Polyethylene glycols (PEGs) in general use are polydisperse molecules with molecular weight (MW) distributed around an average value applied in their designation e.g., PEG 4000. Previous research has shown that PEGs can act as P-glycoprotein (P-gp) inhibitors with the potential to affect the absorption and efflux of concomitantly administered drugs. However, questions related to the mechanism of cellular uptake of PEGs and the exact role played by P-gp has not been addressed. In this study, we examined the mechanism of uptake of PEGs by MDCK-mock cells, in particular, the effect of MW and interaction with P-gp by MDCK-hMDR1 and A549 cells. The results show that: (a) the uptake of PEGs by MDCK-hMDR1 cells is enhanced by P-gp inhibitors; (b) PEGs stimulate P-gp ATPase activity but to a much lesser extent than verapamil; and (c) uptake of PEGs of low MW (5000 Da) occurs by a combination of passive diffusion and caveolae-mediated endocytosis. These findings suggest that PEGs can engage in P-gp-based drug interactions which we believe should be taken into account when using PEGs as excipients and in PEGylated drugs and drug delivery systems., Graphical abstract PEGs are taken up by cells and act as P-gp substrates. Low molecular weight (MW) PEGs cross cell membranes by passive diffusion, whereas those high MW PEGs enter cells by a combination of passive diffusion and caveolae-mediated endocytosis.Image 1

There is abundant evidence that H2O2 can act as an endothelium-derived hyperpolarizing factor in the resistance vasculature. However, whilst scavenging H2O2 can abolish endothelial dependent hyperpolarization (EDH) and the associated vascular relaxation in some arteries, EDH-dependent vasorelaxation can often be mimicked only by using relatively high concentrations of H2O2. We have examined the role of H2O2 in EDH-dependent vasodilatation by simultaneously measuring vascular diameter and changes in endothelial cell (EC) [Ca2+]i during the application of H2O2 or carbachol, which triggers EDH. Carbachol (10 µM) induced dilatation of phenylephrine-preconstricted rat cremaster arterioles was largely (73%) preserved in the presence of indomethacin (3 µM) and l-NAME (300 µM). This residual NO- and prostacyclin-independent dilatation was reduced by 89% upon addition of apamin (0.5 µM) and TRAM-34 (10 µM), and by 74% when an extracellular ROS scavenging mixture of SOD and catalase (S&C; 100 U ml−1 each) was present. S&C also reduced the carbachol-induced EC [Ca2+]i increase by 74%. When applied in Ca2+-free external medium, carbachol caused a transient increase in EC [Ca2+]i. This was reduced by catalase, and was enhanced when 1 µM H2O2 was present in the bath. H2O2 -induced dilatation, which occurred only at concentrations ≥100 µM, was reduced by a blocking antibody to TRPM2, which had no effect on carbachol-induced responses. Similarly, iberotoxin and Rp-8bromo cGMP reduced the vasodilatation induced by H2O2, but not by carbachol. Inhibiting PLC, PLA2 or CYP450 2C9 each greatly reduced the carbachol-induced increase in EC [Ca2+]i and vasodilatation, but adding 10 µM H2O2 during PLA2 or CYP450 2C9 inhibition completely restored both responses. The nature of the effective ROS species was investigated by using Fe2+ chelators to block the formation of ∙OH. A cell permeant chelator was able to inhibit EC Ca2+ store release, but cell impermeant chelators reduced both the vasodilatation and EC Ca2+ influx, implying that ∙OH is required for these responses. The results indicate that rather than mediating EDH by acting directly on smooth muscle, H2O2 promotes EDH by acting within EC to enhance Ca2+ release., Graphical abstract fx1, Highlights • We recorded carbachol- evoked endothelial cell (EC) [Ca2+]i rises in rat arterioles. • Ca2+ rises were reduced by catalase, cPLA2 and CYP29C blockers, and iron chelators. • EC Ca2+ release was potentiated by 30 pM IL-1β or 1 μM exogenous H2O2. • H2O2 and carbachol evoked vasodilation, but by dissimilar mechanisms. • H2O2 promotes hyperpolarization-dependent dilatation by increasing EC Ca2+ release.

Graphical abstract, Highlights • Toxoplasma infection of neurosecretory cells increases the production of dopamine. • Infection did not change host TH or DDC. • Host DDC was observed in the parasitic vacuole in infected brain cells. • Import of host DDC may facilitate DA synthesis in the parasitic vacuole. • This would prevent toxicity due to cytosolic unpackaged dopamine., Infection by the neurotropic agent Toxoplasma gondii alters rodent behavior and can result in neuropsychiatric symptoms in humans. Little is understood regarding the effects of infection on host neural processes but alterations to dopaminergic neurotransmission are implicated. We have previously reported elevated levels of dopamine (DA) in infected dopaminergic cells however the involvement of the host enzymes and fate of the produced DA were not defined. In order to clarify the effects of infection on host DA biosynthetic enzymes and DA packaging we examined enzyme levels and activity and DA accumulation and release in T. gondii-infected neurosecretory cells. Although the levels of the host tyrosine hydroxylase (TH) and DOPA decarboxylase and AADC (DDC) did not change significantly in infected cultures, DDC was found within the parasitophorous vacuole (PV), the vacuolar compartment where the parasites reside, as well as in the host cytosol in infected dopaminergic cells. Strikingly, DDC was found within the intracellular parasite cysts in infected brain tissue. This finding could provide some explanation for observations of DA within tissue cysts in infected brain as a parasite-encoded enzyme with TH activity was also localized within tissue cysts. In contrast, cellular DA packaging appeared unchanged in single-cell microamperometry experiments and only a fraction of the increased DA was accessible to high potassium-induced release. This study provides some understanding of how this parasite produces elevated DA within dopaminergic cells without the toxic ramifications of free cytosolic DA. The mechanism for synthesis and packaging of DA by T. gondii-infected dopaminergic cells may have important implications for the effects of chronic T. gondii infection on humans and animals.

Highlights • TROX-1 exhibits activation state-dependent inhibition of Cav2.2 in vitro. • TROX-1 selectively attenuates neuronal responses to mechanical stimulation. • Anti-nociceptive effect of TROX-1 dependent on pathophysiological state., Prialt, a synthetic version of Cav2.2 antagonist ω-conotoxin MVIIA derived from Conus magus, is the first clinically approved voltage-gated calcium channel blocker for refractory chronic pain. However, due to the narrow therapeutic window and considerable side effects associated with systemic dosing, Prialt is only administered intrathecally. N-triazole oxindole (TROX-1) is a novel use-dependent and activation state-selective small-molecule inhibitor of Cav2.1, 2.2 and 2.3 calcium channels designed to overcome the limitations of Prialt. We have examined the neurophysiological and behavioral effects of blocking calcium channels with TROX-1. In vitro, TROX-1, in contrast to state-independent antagonist Prialt, preferentially inhibits Cav2.2 currents in rat dorsal root ganglia (DRG) neurons under depolarized conditions. In vivo electrophysiology was performed to record from deep dorsal horn lamina V/VI wide dynamic range neurons in non-sentient spinal nerve-ligated (SNL) and sham-operated rats. In SNL rats, spinal neurons exhibited reduced responses to innocuous and noxious punctate mechanical stimulation of the receptive field following subcutaneous administration of TROX-1, an effect that was absent in sham-operated animals. No effect was observed on neuronal responses evoked by dynamic brushing, heat or cold stimulation in SNL or sham rats. The wind-up response of spinal neurons following repeated electrical stimulation of the receptive field was also unaffected. Spinally applied TROX-1 dose dependently inhibited mechanically evoked neuronal responses in SNL but not sham-operated rats, consistent with behavioral observations. This study confirms the pathological state-dependent actions of TROX-1 through a likely spinal mechanism and reveals a modality selective change in calcium channel function following nerve injury.

Recent studies indicate multiple roles for integrin αvβ3 in adult neurons, including response to pharmacological agents such as cocaine and selective serotonin reuptake inhibitors. In this study, we examined the role of the integrin β3 gene (Itgb3) in the response to environmental stimuli by subjecting Itgb3+/+ and Itgb3−/− mice to unpredictable chronic mild stressors. We found that genetic abrogation of integrin β3 expression elicits an exaggerated vulnerability to chronic unpredictable stress in the open field test. In this test, chronic stress elicited significant decreases in stereotypic behavior and horizontal locomotor activity, including increases in anxiety behaviors. Mild chronic stress led to reductions in dopamine turnover in midbrains of Itgb3+/+, but not Itgb3−/− mice, suggesting a disruption of stress-dependent regulation of DA homeostasis. Chronic stress elicited altered synaptic expression of syntaxin and synaptophysin in midbrains of Itgb3−/− mice, when compared to Itgb3+/+. Semi-quantitative Western blot studies revealed that the synaptic expression, but not total tissue expression, of multiple signaling proteins is correlated with integrin αv levels in the midbrain. Moreover, loss of integrin β3 expression modifies this correlation network. Together, these findings demonstrate that Itgb3−/− mice display a pattern of changes indicating disrupted regulation of midbrain synaptic systems involved in conferring resilience to mild stressors.

Graphical abstract, Measurement of ortho-phosphate in soil extracts usually involves sending dried samples of soil to a laboratory for analysis and waiting several weeks for the results. Phosphate determination methods often involve use of strong acids, heavy metals, and organic dyes. To overcome limitations of this approach, we have developed a phosphate determination method which can be carried out in the field to obtain results on the spot. This new method uses: • Small volumes. • An enzymatic reaction. • Green chemistry. First, the soil sample is extracted with deionized water and filtered. Next, an aliquot of the soil extract (0.5 mL) is transferred to a disposable cuvette, containing 0.5 mL of reaction mixture [200 mM HEPES, pH 7.6, 20 mM MgCl2, with 80 nmol 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) and 1 unit of recombinant purine nucleoside phosphorylase (PNP; EC 2.4.2.1)], mixed, and incubated for 10 min at field temperature. Absorbance of the completed reaction is measured at 360 nm in open-source, portable photometer linked by bluetooth to a smartphone. The phosphate and phosphorus content of the soil is determined by comparison of its absorbance at 360 nm to a previously prepared standard phosphate curve, which is stored in the smartphone app.

Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells., Highlights • Insulin acutely improves mitochondrial function of skeletal muscle cells. • Mitochondrial proton leak of skeletal muscle cells is attenuated by insulin. • Insulin sensitises anaerobic glycolysis of skeletal muscle cells to glucose. • Bioenergetic insulin effects offer novel assays for cellular insulin sensitivity.

The flavin-dependent homotetrameric enzyme pyranose 2-oxidase (P2O) is found mostly, but not exclusively, in lignocellulose-degrading fungi where it catalyzes the oxidation of β-d-glucose to the corresponding 2-keto sugar concomitantly with hydrogen peroxide formation during lignin solubilization. Here, we present crystal structures of P2O from the efficient lignocellulolytic basidiomycete Phanerochaete chrysosporium. Structures were determined of wild-type PcP2O from the natural fungal source, and two variants of recombinant full-length PcP2O, both in complex with the slow substrate 3-deoxy-3-fluoro-β-d-glucose. The active sites in PcP2O and P2O from Trametes multicolor (TmP2O) are highly conserved with identical substrate binding. Our structural analysis suggests that the 17 °C higher melting temperature of PcP2O compared to TmP2O is due to an increased number of intersubunit salt bridges. The structure of recombinant PcP2O expressed with its natural N-terminal sequence, including a proposed propeptide segment, reveals that the first five residues of the propeptide intercalate at the interface between A and B subunits to form stabilizing, mainly hydrophobic, interactions. In the structure of mature PcP2O purified from the natural source, the propeptide segment in subunit A has been replaced by a nearby loop in the B subunit. We propose that the propeptide in subunit A stabilizes the A/B interface of essential dimers in the homotetramer and that, upon maturation, it is replaced by the loop in the B subunit to form the mature subunit interface. This would imply that the propeptide segment of PcP2O acts as an intramolecular chaperone for oligomerization at the A/B interface of the essential dimer., Highlights • Structures of pyranose 2-oxidase from Phanerochaete chrysosporium were determined. • The N-terminus may act as a propeptide with a role in homotetramer assembly. • A large number of salt bridges between subunits provides thermostability. • The substrate is bound in the productive binding mode for oxidation at C2.

Excessive production of unsaturated aldehydes from oxidized lipoproteins and membrane lipids is a characteristic feature of cardiovascular disease. Our previous studies show that unsaturated lipid peroxidation-derived aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) promote autophagy in rat aortic smooth muscle cells (RASMC). In this study, we examined the mechanism by which HNE induces autophagy. Exposure of RASMC to HNE led to the modification of several proteins, most of which were identified by mass spectrometry and confocal microscopy to be localized to the endoplasmic reticulum (ER). HNE stimulated the phosphorylation of PKR-like ER kinase and eukaryotic initiation factor 2α and increased heme oxygenase-1 (HO-1) abundance. HNE treatment also increased LC3-II formation and the phosphorylation of JNK and p38. Pharmacological inhibition of JNK, but not p38, prevented HNE-induced HO-1 expression and LC3-II formation. Inhibition of JNK increased cell death in HNE-treated cells. Pretreatment with the chemical chaperone phenylbutryic acid prevented LC3-II formation as well as JNK phosphorylation and HO-1 induction. Taken together, these data suggest that autophagic responses triggered by unsaturated aldehydes could be attributed, in part, to ER stress, which stimulates autophagy by a JNK-dependent mechanism and promotes cell survival during oxidative stress., Graphical abstract Highlights ► The lipid peroxidation product 4-hydroxynonenal (HNE) modifies endoplasmic reticulum (ER) proteins in vascular smooth muscle cells. ► HNE activates autophagy. ► PKR-like ER kinase (PERK) and the stress kinase JNK are activated by HNE. ► Pharmacological inhibition of JNK or ER stress prevents autophagy in vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) develop a highly proliferative and synthetic phenotype in arterial diseases. Because such phenotypic changes are likely integrated with the energetic state of the cell, we hypothesized that changes in cellular metabolism regulate VSMC plasticity. VSMCs were exposed to platelet-derived growth factor-BB (PDGF) and changes in mitochondrial morphology, proliferation, contractile protein expression, and mitochondrial metabolism were examined. Exposure of VSMCs to PDGF resulted in mitochondrial fragmentation and a 50% decrease in the abundance of mitofusin 2. Synthetic VSMCs demonstrated a 20% decrease in glucose oxidation, which was accompanied by an increase in fatty acid oxidation. Results of mitochondrial function assays in permeabilized cells showed few changes due to PDGF treatment in mitochondrial respiratory chain capacity and coupling. Treatment of VSMCs with Mdivi-1—an inhibitor of mitochondrial fission—inhibited PDGF-induced mitochondrial fragmentation by 50% and abolished increases in cell proliferation; however, it failed to prevent PDGF-mediated activation of autophagy and removal of contractile proteins. In addition, treatment with Mdivi-1 reversed changes in fatty acid and glucose oxidation associated with the synthetic phenotype. These results suggest that changes in mitochondrial morphology and bioenergetics underlie the hyperproliferative features of the synthetic VSMC phenotype, but do not affect the degradation of contractile proteins. Mitochondrial fragmentation occurring during the transition to the synthetic phenotype could be a therapeutic target for hyperproliferative vascular disorders., Graphical abstract, Highlights • PDGF promotes mitochondrial fragmentation in vascular smooth muscle cells. • PDGF increases metabolic reliance on fatty acids. • Mitochondrial fragmentation regulates proliferation and bioenergetics. • PDGF-induced bioenergetic and autophagic responses regulate de-differentiation.

Long-term exposure to ascorbate is known to enhance endothelial nitric oxide synthase (eNOS) activity by stabilizing the eNOS cofactor tetrahydrobiopterin (BH4). We investigated acute effects of ascorbate on eNOS function in primary (HUVEC) and immortalized human endothelial cells (EA.hy926), aiming to provide a molecular explanation for the rapid vasodilatation seen in vivo upon administration of ascorbate. Enzymatic activity of eNOS and intracellular BH4 levels were assessed by means of an arginine–citrulline conversion assay and HPLC analysis, respectively. Over a period of 4 h, ascorbate steadily increased eNOS activity, although endothelial BH4 levels remained unchanged compared to untreated control cells. Immunoblot analyses revealed that as early as 5 min after treatment ascorbate dose-dependently increased phosphorylation at eNOS-Ser1177 and concomitantly decreased phosphorylation at eNOS-Thr495, a phosphorylation pattern indicative of increased eNOS activity. By employing pharmacological inhibitors, siRNA-mediated knockdown approaches, and overexpression of the catalytic subunit of protein phosphatase 2A (PP2A), we show that this effect was at least partly owing to reduction of PP2A activity and subsequent activation of AMP-activated kinase. In this report, we unravel a novel mechanism for how ascorbate rapidly activates eNOS independent of its effects on BH4 stabilization., Graphical abstract Highlights ► Ascorbate can enhance the activity of endothelial NO synthase (eNOS) within minutes. ► This effect occurs independent of stabilization of tetrahydrobiopterin. ► Ascorbate modulates eNOS phosphorylation in a PP2A- and AMPK-dependent manner.

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1E,6E-heptadiene-3,5-dione or diferuloyl methane) is a polyphenol derived from the Curcuma longa plant, commonly known as turmeric. This substance has been used extensively in Ayurvedic medicine for centuries for its anti-oxidant, analgesic, anti-inflammatory and antiseptic activity. More recently curcumin has been found to possess anti-cancer properties linked to its pro-apoptotic and anti-proliferative actions. The underlying mechanisms of these diverse effects are complex, not fully elucidated and subject of intense scientific debate. Despite increasing evidence indicating that different cation channels can be a molecular target for curcumin, very little is known about the effect of curcumin on chloride channels. Since, (i) the molecular structure of curcumin indicates that the substance could potentially interact with chloride channels, (ii) chloride channels play a role during the apoptotic process and regulation of the cell volume, and (iii) apoptosis is a well known effect of curcumin, we set out to investigate whether or not curcumin could (i) exert a modulatory effect (direct or indirect) on the swelling activated chloride current ICl(swell) in a human cell system, therefore (ii) affect cell volume regulation and (iii) ultimately modulate cell survival. The ICl(swell) channels, which are essential for regulating the cell volume after swelling, are also known to be activated under isotonic conditions as an early event in the apoptotic process. Here we show that long-term exposure of a human kidney cell line to extracellular 0.1-10 μM curcumin modulates ICl(swell) in a dose-dependent manner (0.1 μM curcumin is ineffective, 0.5-5.0 μM curcumin increase, while 10 μM curcumin decrease the current), and short-term exposure to micromolar concentrations of curcumin does not affect ICl(swell) neither if applied from the extracellular nor from the intracellular side - therefore, a direct effect of curcumin on ICl(swell) can be ruled out. Furthermore, we show that curcumin exposure induces apoptosis in human kidney cells, and at a concentration of 5.0-10 μM induces the appearance of a sub-population of cells with a dramatically increased volume. In these cells the regulation of the cell volume seems to be impaired, most likely as a consequence of the ICl(swell) blockade. Similarly, 50 μM curcumin induced apoptosis, caused cell cycle arrest in G1-phase and increased the volume of human colorectal adenocarcinoma HT-29 cells. The cell cycle arrest in G1 phase may be the mechanism underlying the volume increase observed in this cell line after exposure to curcumin.

Graphical abstract Highlights ► Three phases of the oxidative folding of RNase A were temporally separated using DHSox. ► Thermodynamic and kinetic parameters of the four des intermediates were obtained using DHSox. ► Each SS linkage in the native RNase A has different thermodynamic and kinetic roles. ► Unfolded des species (desU) gain native-like structures via X-Pro isomerization. ► All four des species were isolated and allowed to fold to N under aerobic conditions., Redox-coupled folding pathways of bovine pancreatic ribonuclease A (RNase A) with four intramolecular disulfide (SS) bonds comprise three phases: (I) SS formation to generate partially oxidized intermediate ensembles with no rigid folded structure; (II) SS rearrangement from the three SS intermediate ensemble (3S) to the des intermediates having three native SS linkages; (III) final oxidation of the last native SS linkage to generate native RNase A. We previously demonstrated that DHSox, a water-soluble selenoxide reagent for rapid and quantitative SS formation, allows clear separation of the three folding phases. In this study, the main conformational folding phase (phase II) has been extensively analyzed at pH 8.0 under a wide range of temperatures (5–45 °C), and thermodynamic and kinetic parameters for the four des intermediates were determined. The free-energy differences (ΔG) as a function of temperature suggested that the each SS linkage has different thermodynamic and kinetic roles in stability of the native structure. On the other hand, comparison of the rate constants and the activation energies for 3S → des with those reported for the conformational folding of SS-intact RNase A suggested that unfolded des species (desU) having three native SS linkages but not yet being folded are involved in very small amounts (

Despite the fact that non-viral nucleic acid delivery systems are generally considered to be less efficient than viral vectors, they have gained much interest in recent years due to their superior safety profile compared to their viral counterpart. Among these synthetic vectors are cationic polymers, branched dendrimers, cationic liposomes and cell-penetrating peptides (CPPs). The latter represent an assortment of fairly unrelated sequences essentially characterised by a high content of basic amino acids and a length of 10–30 residues. CPPs are capable of mediating the cellular uptake of hydrophilic macromolecules like peptides and nucleic acids (e.g. siRNAs, aptamers and antisense-oligonucleotides), which are internalised by cells at a very low rate when applied alone. Up to now, numerous sequences have been reported to show cell-penetrating properties and many of them have been used to successfully transport a variety of different cargos into mammalian cells. In recent years, it has become apparent that endocytosis is a major route of internalisation even though the mechanisms underlying the cellular translocation of CPPs are poorly understood and still subject to controversial discussions. In this review, we will summarise the latest developments in peptide-based cellular delivery of nucleic acid cargos. We will discuss different mechanisms of entry, the intracellular fate of the cargo, correlation studies of uptake versus biological activity of the cargo as well as technical problems and pitfalls.

High glucose and fatty acid levels impair pancreatic beta cell function. We have recently shown that palmitate-induced loss of INS-1E insulinoma cells is related to increased reactive oxygen species (ROS) production as both toxic effects are prevented by palmitoleate. Here we show that palmitate-induced ROS are mostly mitochondrial: oxidation of MitoSOX, a mitochondria-targeted superoxide probe, is increased by palmitate, whilst oxidation of the equivalent non-targeted probe is unaffected. Moreover, mitochondrial respiratory inhibition with antimycin A stimulates palmitate-induced MitoSOX oxidation. We also show that palmitate does not change the level of mitochondrial uncoupling protein-2 (UCP2) and that UCP2 knockdown does not affect palmitate-induced MitoSOX oxidation. Palmitoleate does not influence MitoSOX oxidation in INS-1E cells ±UCP2 and largely prevents the palmitate-induced effects. Importantly, UCP2 knockdown amplifies the preventive effect of palmitoleate on palmitate-induced ROS. Consistently, viability effects of palmitate and palmitoleate are similar between cells ±UCP2, but UCP2 knockdown significantly augments the palmitoleate protection against palmitate-induced cell loss at high glucose. We conclude that UCP2 neither mediates palmitate-induced mitochondrial ROS generation and the associated cell loss, nor protects against these deleterious effects. Instead, UCP2 dampens palmitoleate protection against palmitate toxicity., Highlights • Palmitate exposure induces mitochondrial superoxide in INS-1E cells. • Mitochondrial superoxide correlates inversely with INS-1E cell number. • Palmitate does not change uncoupling protein-2 (UCP2) level in INS-1E cells. • UCP2 does not affect palmitate-induced mitochondrial superoxide or INS-1E cell loss. • UCP2 dampens palmitoleate protection against palmitate toxicity.

Highlights • Sucrose is transported symplastically towards developing and growing nodosities. • Starch is accumulated and metabolized during nodosities growth and development. • Nodosity formation has systemic effects on non-infected root tips of phylloxerated plants. • Gall formation reprograms processes of the secondary metabolism as demonstrated transciptionally., Gall forming phylloxera may compete for nutrients with meristematic tissues and develop heterotrophic structures that act as carbon sinks. In this work, we studied the underlying starch metabolism, sink-source translocation of soluble sugars towards and within root galls. We demonstrated that nodosities store carbohydrates by starch accumulation and monitored the expression of genes involved in the starch metabolic. Thereby we proved that the nodosity is symplastically connected to the source tissues through its development and that the starch metabolism is significantly affected to synthesize and degrade starch within the gall. Genes required for starch biosynthesis and degradation are up-regulated. Among the carbohydrate transporters the expression of a glucose-6-phosphate translocater, one sucrose transporter and two SWEET proteins were increases, whereas hexose transporters, tonoplast monosaccharide transporter and Erd6-like sugar transporters were decreased. We found general evidence for plant response to osmotic stress in the nodosity as previously suggested for gall induction processes. We conclude that nodosities are heterogenous plant organs that accumulate starch to serve as temporary storage structure that is gradually withdrawn by phylloxera. Phylloxera transcriptionally reprograms gall tissues beyond primary metabolism and included downstream secondary processes, including response to osmotic stress.

Nucleotide pools in mammalian cells change due to the influence of antitumor drugs, which may help in evaluating the drug effect and understanding the mechanism of drug action. In this study, an ion-pair RP-HPLC method was used for a simple, sensitive and simultaneous determination of the levels of 12 nucleotides in mammalian cells treated with antibiotic antitumor drugs (daunorubicin, epirubicin and dactinomycin D). Through the use of this targeted metabolomics approach to find potential biomarkers, UTP and ATP were verified to be the most appropriate biomarkers. Moreover, a holistic statistical approach was put forward to develop a model which could distinguish 4 categories of drugs with different mechanisms of action. This model can be further validated by evaluating drugs with different mechanisms of action. This targeted metabolomics study may provide a novel approach to predict the mechanism of action of antitumor drugs., Graphical abstract A RP-HPLC method was used for determination 12 nucleotides in cells treated with antibiotic antitumor drugs. Four categories of drugs with different mechanisms of action were investigated by principal component analysis to develop a recognition model to predict a possible mechanism of an antitumor-compound action.