Relationships of crystallinity and reaction rates for enzymatic degradation of poly (ethylene terephthalate), PET.

Biocatalytic degradation of plastic waste is anticipated to play an important role in future recycling systems. However, enzymatic degradation of crystalline poly (ethylene terephthalate) (PET) remains consistently poor. Herein, we employed functional assays to elucidate the molecular underpinnings of this limitation. This included utilizing complementary activity assays to monitor the degradation of PET disks with varying crystallinity (XC ), as well as determining enzymatic kinetic parameters for soluble PET fragments. The results indicate that an efficient PET-hydrolase, LCCICCG , operates through an endolytic mode of action, and that its activity is limited by conformational constraints in the PET polymer. Such constraints become more pronounced at high XC values, and this limits the density of productive sites on the PET surface. Endolytic chain-scissions are the dominant reaction type in the initial stage, and this means that little or no soluble organic product are released. However, endolytic cuts gradually and locally promote chain mobility and hence the density of attack sites on the surface. This leads to an upward concave progress curve; a behavior sometimes termed lag-phase kinetics.

Metabolites, ions, and the mechanisms behind seasonal cold hardening of Pyrochroa coccinea (Pyrochroidae) larvae.

The larvae of the black headed cardinal beetle Pyrochroa coccinea, overwinters under the bark of dead logs in northern European dioecious forests, and are thus exposed to temperatures below the melting point of their bodily fluids. Here we explore the mechanisms behind their seasonal cold hardening by characterising field samples collected monthly throughout the year. Both the lower lethal temperature and supercooling point dropped as much as 10℃ in the second half of November, reaching values around -15℃ by the beginning of December. This change was accompanied by a 320 mosmol/kg increase in hemolymph osmolality, which is a doubling compared to the summer levels. We used NMR metabolomics to identify and measure the absolute concentrations of the responsible cryoprotective C-H containing metabolites in the hemolymph. The largest increase was found to be in either glucose or trehalose, with an average total increase of 120 mM. Proline, alanine, and choline concentrations were found to increase by around 10 mM each. Contrarily, phosphocholine and phosphoethanolamine were halved, resulting in a total decrease of around 50 mM. These measurements were complemented with ion exchange chromatography measurements. This allowed us to account for all the osmotic pressure in the summer hemolymph, and the measured concentration changes explained as much as 40 % of the observed osmolality increase upon cold hardening. Preliminary results indicate that the remainder may be explained by non-colligative protein contributions.

Identification of early stage and metastatic prostate cancer using electrochemical detection of beta-2-microglobulin in urine samples from patients.

To improve prostate cancer (PCa) diagnosis, it is imperative to identify novel biomarkers and establish effective screening techniques. Here, we introduce electrochemical biosensing of ß-2-Microglobulin (ß2M) in urine as a potential diagnostic tool for PCa. The immunosensor is composed of a screen-printed graphene electrode coated with anti ß2M antibodies. The sensor is capable of detecting the protein directly in urine without any sample pretreatment within 45 min including sample incubation and a lower limit of detection of 204 µg/L. The sensor demonstrated a significant difference in the ß2M-creatinine ratio in urine between control and both local- and metastatic PCa (mPCa) (P = 0.0302 and P = 0.0078 respectively), and between local- and mPCa (P = 0.0302). This first example of electrochemical sensing of ß2M for the diagnosis of PCa may set the stage for an affordable, on-site screening technique for PCa.

Morphology-Dependent Interactions between α-Synuclein Monomers and Fibrils.

Amyloid fibrils may adopt different morphologies depending on the solution conditions and the protein sequence. Here, we show that two chemically identical but morphologically distinct α-synuclein fibrils can form under identical conditions. This was observed by nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence spectroscopy, as well as by cryo-transmission electron microscopy (cryo-TEM). The results show different surface properties of the two morphologies, A and B. NMR measurements show that monomers interact differently with the different fibril surfaces. Only a small part of the N-terminus of the monomer interacts with the fibril surface of morphology A, compared to a larger part of the monomer for morphology B. Differences in ThT binding seen by fluorescence titrations, and mesoscopic structures seen by cryo-TEM, support the conclusion of the two morphologies having different surface properties. Fibrils of morphology B were found to have lower solubility than A. This indicates that fibrils of morphology B are thermodynamically more stable, implying a chemical potential of fibrils of morphology B that is lower than that of morphology A. Consequently, at prolonged incubation time, fibrils of morphology B remained B, while an initially monomorphic sample of morphology A gradually transformed to B.

Metabolomics of early myocardial ischemia.

INTRODUCTION: Diagnosing myocardial infarction is difficult during the initial phase. As, acute myocardial ischemia is associated with changes in metabolic pathways, metabolomics may provide ways of identifying early stages of ischemia. We investigated the changes in metabolites after induced ischemia in humans using nuclear magnetic resonance spectroscopy (NMR). METHODS: We included patients undergoing elective coronary angiography showing normal coronary arteries. These were randomized into 4 groups and underwent coronary artery occlusion for 0, 30, 60 or 90 s. Blood was collected over the next 3 h and analyzed using NMR. We used 2-way ANOVA of time from baseline- and treatment group to find metabolites that changed significantly following the intervention and principal component analysis (PCA) to investigate changes between the 90 s ischemia- and control groups at 15 and 60 min after intervention. RESULTS: We included 34 patients. The most pronounced changes were observed in the lipid metabolism where 38 of 112 lipoprotein parameters (34%) showed a significant difference between the patients exposed to ischemia and the control group. There was a decrease in total plasma triglycerides over the first hour followed by a normalization. The principal component analysis showed a effects of the treatment after just 15 min. These effects were dominated by changes in high-density lipoprotein. An increase in lactic acid levels was detected surprisingly late, 1-2 h after the ischemia. CONCLUSION: We investigated the earliest changes in metabolites of patients undergoing brief myocardial ischemia and found that ischemia led to changes throughout the lipid metabolism as early as 15 min post-intervention.

Identification of Distinct Soluble States During Fibril Formation Using Multilinear Analysis of NMR Diffusion Data.

Protein misfolding and self-assembling into amyloid structures are associated with a number of diseases. Characterization of protein amyloid formation reactions is a challenging task as transient populations of multiple species are involved. Here we outline a method for identification and characterization of the individual soluble states during protein amyloid formation. The method combines NMR translational diffusion measurements with multilinear data analysis.

A Drosophila melanogaster model for TMEM43-related arrhythmogenic right ventricular cardiomyopathy type 5.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a severe cardiac disease that leads to heart failure or sudden cardiac death (SCD). For the pathogenesis of ARVC, various mutations in at least eight different genes have been identified. A rare form of ARVC is associated with the mutation TMEM43 p.S358L, which is a fully penetrant variant in male carriers. TMEM43 p.S358 is homologous to CG8111 p.S333 in Drosophila melanogaster. We established CRISPR/Cas9-mediated CG8111 knock-out mutants in Drosophila, as well as transgenic fly lines carrying an overexpression construct of the CG8111 p.S333L substitution. Knock-out flies developed normally, whereas the overexpression of CG8111 p.S333L caused growth defects, loss of body weight, cardiac arrhythmias, and premature death. An evaluation of a series of model mutants that replaced S333 by selected amino acids proved that the conserved serine is critical for the physiological function of CG8111. Metabolomic and proteomic analyses revealed that the S333 in CG8111 is essential to proper energy homeostasis and lipid metabolism in the fly. Of note, metabolic impairments were also found in the murine Tmem43 disease model, and fibrofatty replacement is a hallmark of human ARVC5. These findings contribute to a more comprehensive understanding of the molecular functions of CG8111 in Drosophila, and can represent a valuable basis to assess the aetiology of the human TMEM43 p.S358L variant in more detail.

Correlation between stage of prostate cancer and tyrosine and tryptophan in urine samples measured electrochemically.

Prostate cancer (PCa) is the second most common cancer in men and one of the leading causes of cancer-related deaths. Early detection is the key to successful treatment and provides the greatest chance to cure the patient. Currently, early detection involves screening for prostate-specific antigen levels in blood, which is not a tumor-specific biomarker. There is a critical need to develop clinically useful methods for screening for more reliable biomarkers. Here, we introduce an electrochemical biosensor that measures the concentrations of the amino acids tyrosine and tryptophan, and propose it as a possible diagnostic and prognostic tool for PCa. The limits of detection of tyrosine and tryptophan using the electrochemical sensors were 1.15 and 1.13 µmol/L in 1:10 urine: PBS, respectively. This study is the first to present electrochemical measurements of tyrosine and tryptophan directly in patient urine samples. We demonstrated an inverse correlation between the measured electrochemical signals and the severity of PCa. The most notable observation was a significant difference between controls and metastatic PCa patients (P ≤ 0.001). This observation was further validated using Liquid-Chromatography-Mass Spectrometry. Our data provides the basis for further research with electrochemical measurements of tyrosine and tryptophan as potential biomarkers for PCa.

Adsorption of bio-organic eco-corona molecules reduces the toxic response to metallic nanoparticles in Daphnia magna.

As the use of engineered nanomaterials increases, so does the risk of them spreading to natural ecosystems. Hitherto, knowledge regarding the toxic properties of nanoparticles (NP's) and their potential interactions with natural bio-organic molecules adsorbed to them, and thereby forming surface coronas, is limited. However, we show here that the toxic effect of NPs of tungsten carbide cobalt (WC-Co) and cobalt (Co) on the crustacean Daphnia magna is postponed in the presence of natural biological degradation products (eco-corona biomolecules). For Daphnia exposed to WC-Co NPs the survival time increased with 20-25% and for Co NPs with 30-47% after mixing the particles with a solution of eco-corona biomolecules before exposure. This suggests that an eco-corona, composed of biomolecules always present in natural ecosystems, reduces the toxic potency of both studied NPs. Further, the eco-coronas did not affect the particle uptake, suggesting that the reduction in toxicity was related to the particle-organism interaction after eco-corona formation. In a broader context, this implies that although the increasing use and production of NPs may constitute a novel, global environmental threat, the acute toxicity and long-term effects of some NPs will, at least under certain conditions, be reduced as they enter natural ecosystems.

Charge Regulation during Amyloid Formation of α-Synuclein.

Electrostatic interactions play crucial roles in protein function. Measuring pKa value perturbations upon complex formation or self-assembly of e.g. amyloid fibrils gives valuable information about the effect of electrostatic interactions in those processes. Site-specific pKa value determination by solution NMR spectroscopy is challenged by the high molecular weight of amyloid fibrils. Here we report a pH increase during fibril formation of α-synuclein, observed using three complementary experimental methods: pH electrode measurements in water; colorimetric changes of a fluorescent indicator; and chemical shift changes for histidine residues using solution state NMR spectroscopy. A significant pH increase was detected during fibril formation in water, on average by 0.9 pH units from 5.6 to 6.5, showing that protons are taken up during fibril formation. The pH upshift was used to calculate the average change in the apparent pKaave value of the acidic residues, which was found to increase by at least 1.1 unit due to fibril formation. Metropolis Monte Carlo simulations were performed on a comparable system that also showed a proton uptake due to fibril formation. Fibril formation moreover leads to a significant change in proton binding capacitance. Parallel studies of a mutant with five charge deletions in the C-terminal tail revealed a smaller pH increase due to fibril formation, and a smaller change (0.5 units on average) in the apparent pKaave values of the acidic residues. We conclude that the proton uptake during the fibril formation is connected to the high density of acidic residues in the C-terminal tail of α-synuclein.

Prediction of complex phenotypes using the Drosophila melanogaster metabolome.

Understanding the genotype-phenotype map and how variation at different levels of biological organization is associated are central topics in modern biology. Fast developments in sequencing technologies and other molecular omic tools enable researchers to obtain detailed information on variation at DNA level and on intermediate endophenotypes, such as RNA, proteins and metabolites. This can facilitate our understanding of the link between genotypes and molecular and functional organismal phenotypes. Here, we use the Drosophila melanogaster Genetic Reference Panel and nuclear magnetic resonance (NMR) metabolomics to investigate the ability of the metabolome to predict organismal phenotypes. We performed NMR metabolomics on four replicate pools of male flies from each of 170 different isogenic lines. Our results show that metabolite profiles are variable among the investigated lines and that this variation is highly heritable. Second, we identify genes associated with metabolome variation. Third, using the metabolome gave better prediction accuracies than genomic information for four of five quantitative traits analyzed. Our comprehensive characterization of population-scale diversity of metabolomes and its genetic basis illustrates that metabolites have large potential as predictors of organismal phenotypes. This finding is of great importance, e.g., in human medicine, evolutionary biology and animal and plant breeding.

Impacts of thermal fluctuations on heat tolerance and its metabolomic basis in Arabidopsis thaliana, Drosophila melanogaster, and Orchesella cincta.

Temperature varies on a daily and seasonal scale and thermal fluctuations are predicted to become even more pronounced under future climate changes. Studies suggest that plastic responses are crucial for species' ability to cope with thermal stress including variability in temperature, but most often laboratory studies on thermal adaptation in plant and ectotherm organisms are performed at constant temperatures and few species included. Recent studies using fluctuating thermal regimes find that thermal performance is affected by both temperature mean and fluctuations, and that plastic responses likely will differ between species according to life strategy and selective past. Here we investigate how acclimation to fluctuating or constant temperature regimes, but with the same mean temperature, impact on heat stress tolerance across a plant (Arabidopsis thaliana) and two arthropod species (Orchesella cincta and Drosophila melanogaster) inhabiting widely different thermal microhabitats and with varying capability for behavioral stress avoidance. Moreover, we investigate the underlying metabolic responses of acclimation using NMR metabolomics. We find increased heat tolerance for D. melanogaster and A. thaliana exposed to fluctuating acclimation temperatures, but not for O. cincta. The response was most pronounced for A. thaliana, which also showed a stronger metabolome response to thermal fluctuations than both arthropods. Generally, sugars were more abundant across A. thaliana and D. melanogaster when exposed to fluctuating compared to constant temperature, whereas amino acids were less abundant. This pattern was not evident for O. cincta, and generally we do not find much evidence for similar metabolomics responses to fluctuating temperature acclimation across species. Differences between the investigated species' ecology and different ability to behaviorally thermoregulate may have shaped their physiological responses to thermal fluctuations.

Ultrastructural evidence for self-replication of Alzheimer-associated Aß42 amyloid along the sides of fibrils.

The nucleation of Alzheimer-associated Aß peptide monomers can be catalyzed by preexisting Aß fibrils. This leads to autocatalytic amplification of aggregate mass and underlies self-replication and generation of toxic oligomers associated with several neurodegenerative diseases. However, the nature of the interactions between the monomeric species and the fibrils during this key process, and indeed the ultrastructural localization of the interaction sites have remained elusive. Here we used NMR and optical spectroscopy to identify conditions that enable the capture of transient species during the aggregation and secondary nucleation of the Aß42 peptide. Cryo-electron microscopy (cryo-EM) images show that new aggregates protrude from the entire length of the progenitor fibril. These protrusions are morphologically distinct from the well-ordered fibrils dominating at the end of the aggregation process. The data provide direct evidence that self-replication through secondary nucleation occurs along the sides of fibrils, which become heavily decorated under the current solution conditions (14 µM Aß42, 20 mM sodium phosphate, 200 µM EDTA, pH 6.8).

Sensory Assessment of Fish and Chicken Protein Hydrolysates. Evaluation of NMR Metabolomics Profiling as a New Prediction Tool.

Nuclear magnetic resonance (NMR) metabolomics profiling was evaluated as a new tool in sensory assessment of protein hydrolysates. Hydrolysates were produced on the basis of different raw materials (cod, salmon, and chicken), enzymes (Food Pro PNL and Bromelain), and hydrolysis time (10 and 50 min). The influence of raw material and hydrolysis parameters on sensory attributes was determined by traditional descriptive sensory analysis and 1H NMR spectroscopy. The raw material had a major influence on the attribute intensity and metabolite variation, followed by enzyme and hydrolysis time. However, the formation of bitter taste was not affected by the raw material. Partial least-squares regression (PLSR) on 1H NMR and sensory data provided good models (Q2 = 0.55-0.89) for 11 of the 17 evaluated attributes, including bitterness. Significant metabolite-attribute associations were identified. The study confirms the potential prediction of the sensory properties of protein hydrolysates from cod, salmon, and chicken based on 1H NMR metabolomics profiling.

Revealing Well-Defined Soluble States during Amyloid Fibril Formation by Multilinear Analysis of NMR Diffusion Data.

Amyloid fibril formation is a hallmark of neurodegenerative disease caused by protein aggregation. Oligomeric protein states that arise during the process of fibril formation often coexist with mature fibrils and are known to cause cell death in disease model systems. Progress in this field depends critically on development of analytical methods that can provide information about the mechanisms and species involved in oligomerization and fibril formation. Here, we demonstrate how the powerful combination of diffusion NMR and multilinear data analysis can efficiently disentangle the number of involved species, their kinetic rates of formation or disappearance, spectral contributions, and diffusion coefficients, even without prior knowledge of the time evolution of the process or chemical shift assignments of the various species. Using this method we identify oligomeric species that form transiently during aggregation of human superoxide dismutase 1 (SOD1), which is known to form misfolded aggregates in patients with amyotrophic lateral sclerosis. Specifically, over a time course of 42 days, during which SOD1 fibrils form, we detect the disappearance of the native monomeric species, formation of a partially unfolded intermediate in the dimer to tetramer size range, subsequent formation of a distinct similarly sized species that dominates the final spectrum detected by solution NMR, and concomitant appearance of small peptide fragments.

In-hospital metabolite changes in infective endocarditis-a longitudinal 1H NMR-based study.

Treatment of infective endocarditis (IE) is a 4-6-week provided course of intravenously administered antibiotics. The aim of this study was to investigate how serum metabolites as measured by proton nuclear magnetic resonance (1H NMR) spectroscopy are changing over time during the active phase of IE, and to see whether these metabolite changes might be used to monitor recovery in these patients. Patients hospitalized with first-time IE at Herlev Hospital, Denmark, from September 2015 to June 2017 were included. Longitudinal blood sampling was performed and serum was analyzed using 1H NMR. Orthogonal projection to latent structures discriminant analysis (OPLS-DA) was used to separate sample groups and analyze differences in metabolite profiles. Thirteen patients were included in the study (77% men, median age 62 years (IQR 53-77)). All patients were cured during the hospitalization without any relapse during 6 months of follow-up. We analyzed 61 serum samples (median 5 samples, range 2-8 per person) drawn in the treatment period after IE diagnosis. The main changes during the in-hospital period were decreased levels of glucose, mannose, leucine, isoleucine, phenylalanine, tyrosine, and signals from polyols and N-acetylated protein. The metabolomic changes could in contrast to the routinely used parameters CRP and leucocyte levels distinguish between the early and late stages of disease treatment. We present the first longitudinal study of 1H NMR metabolomics in patients with infective endocarditis. The metabolomic changes show a promising strength compared to routinely used clinical parameters.

A method of predicting the in vitro fibril formation propensity of Aß40 mutants based on their inclusion body levels in E. coli.

Overexpression of recombinant proteins in bacteria may lead to their aggregation and deposition in inclusion bodies. Since the conformational properties of proteins in inclusion bodies exhibit many of the characteristics typical of amyloid fibrils. Based on these findings, we hypothesize that the rate at which proteins form amyloid fibrils may be predicted from their propensity to form inclusion bodies. To establish a method based on this concept, we first measured by SDS-PAGE and confocal microscopy the level of inclusion bodies in E. coli cells overexpressing the 40-residue amyloid-beta peptide, Aß40, wild-type and 24 charge mutants. We then compared these results with a number of existing computational aggregation propensity predictors as well as the rates of aggregation measured in vitro for selected mutants. Our results show a strong correlation between the level of inclusion body formation and aggregation propensity, thus demonstrating the power of this approach and its value in identifying factors modulating aggregation kinetics.

pKa Determination of a Histidine Residue in a Short Peptide Using Raman Spectroscopy.

Determining the pKa of key functional groups is critical to understanding the pH-dependent behavior of biological proteins and peptide-based biomaterials. Traditionally, ¹H NMR spectroscopy has been used to determine the pKa of amino acids; however, for larger molecules and aggregating systems, this method can be practically impossible. Previous studies concluded that the C-D stretches in Raman are a useful alternative for determining the pKa of histidine residues. In this study, we report on the Raman application of the C2-D probe on histidine's imidazole side chain to determining the pKa of histidine in a short peptide sequence. The pKa of the tripeptide was found via difference Raman spectroscopy to be 6.82, and this value was independently confirmed via ¹H NMR spectroscopy on the same peptide. The C2-D probe was also compared to other Raman reporters of the protonation state of histidine and was determined to be more sensitive and reliable than other protonation-dependent signals. The C2-D Raman probe expands the tool box available to chemists interested in directly interrogating the pKa's of histidine-containing peptide and protein systems.

Metabolic control of PPAR activity by aldehyde dehydrogenase regulates invasive cell behavior and predicts survival in hepatocellular and renal clear cell carcinoma.

BACKGROUND: Changes in cellular metabolism are now recognized as potential drivers of cancer development, rather than as secondary consequences of disease. Here, we explore the mechanism by which metabolic changes dependent on aldehyde dehydrogenase impact cancer development. METHODS: ALDH7A1 was identified as a potential cancer gene using a Drosophila in vivo metastasis model. The role of the human ortholog was examined using RNA interference in cell-based assays of cell migration and invasion. 1H-NMR metabolite profiling was used to identify metabolic changes in ALDH7A1-depleted cells. Publically available cancer gene expression data was interrogated to identify a gene-expression signature associated with depletion of ALDH7A1. Computational pathway and gene set enrichment analysis was used to identify signaling pathways and cellular processes that were correlated with reduced ALDH7A1 expression in cancer. A variety of statistical tests used to evaluate these analyses are described in detail in the methods section. Immunohistochemistry was used to assess ALDH7A1 expression in tissue samples from cancer patients. RESULTS: Depletion of ALDH7A1 increased cellular migration and invasiveness in vitro. Depletion of ALDH7A1 led to reduced levels of metabolites identified as ligands for Peroxisome proliferator-activated receptor (PPARα). Analysis of publically available cancer gene expression data revealed that ALDH7A1 mRNA levels were reduced in many human cancers, and that this correlated with poor survival in kidney and liver cancer patients. Using pathway and gene set enrichment analysis, we establish a correlation between low ALDH7A1 levels, reduced PPAR signaling and reduced patient survival. Metabolic profiling showed that endogenous PPARα ligands were reduced in ALDH7A1-depleted cells. ALDH7A1-depletion led to reduced PPAR transcriptional activity. Treatment with a PPARα agonist restored normal cellular behavior. Low ALDH7A1 protein levels correlated with poor clinical outcome in hepatocellular and renal clear cell carcinoma patients. CONCLUSIONS: We provide evidence that low ALDH7A1 expression is a useful prognostic marker of poor clinical outcome for hepatocellular and renal clear cell carcinomas and hypothesize that patients with low ALDH7A1 might benefit from therapeutic approaches addressing PPARα activity.

Amyloid ß42 peptide is toxic to non-neural cells in Drosophila yielding a characteristic metabolite profile and the effect can be suppressed by PI3K.

The human Aß42 peptide is associated with Alzheimer's disease through its deleterious effects in neurons. Expressing the human peptide in adult Drosophila in a tissue- and time-controlled manner, we show that Aß42 is also toxic in non-neural cells, neurosecretory and epithelial cell types in particular. This form of toxicity includes the aberrant signaling by Wingless morphogen leading to the eventual activation of Caspase 3. Preventing Caspase 3 activation by means of p53 keeps epithelial cells from elimination but maintains the Aß42 toxicity yielding more severe deleterious effects to the organism. Metabolic profiling by nuclear magnetic resonance (NMR) of adult flies at selected ages post Aß42 expression onset reveals characteristic changes in metabolites as early markers of the pathological process. All morphological and most metabolic features of Aß42 toxicity can be suppressed by the joint overexpression of PI3K.