Discovery and biocatalytic characterization of opine dehydrogenases by metagenome mining.

Enzymatic processes play an increasing role in synthetic organic chemistry which requires the access to a broad and diverse set of enzymes. Metagenome mining is a valuable and efficient way to discover novel enzymes with unique properties for biotechnological applications. Here, we report the discovery and biocatalytic characterization of six novel metagenomic opine dehydrogenases from a hot spring environment (mODHs) (EC 1.5.1.X). These enzymes catalyze the asymmetric reductive amination between an amino acid and a keto acid resulting in opines which have defined biochemical roles and represent promising building blocks for pharmaceutical applications. The newly identified enzymes exhibit unique substrate specificity and higher thermostability compared to known examples. The feature that they preferably utilize negatively charged polar amino acids is so far unprecedented for opine dehydrogenases. We have identified two spatially correlated positions in their active sites that govern this substrate specificity and demonstrated a switch of substrate preference by site-directed mutagenesis. While they still suffer from a relatively narrow substrate scope, their enhanced thermostability and the orthogonality of their substrate preference make them a valuable addition to the toolbox of enzymes for reductive aminations. Importantly, enzymatic reductive aminations with highly polar amines are very rare in the literature. Thus, the preparative-scale enzymatic production, purification, and characterization of three highly functionalized chiral secondary amines lend a special significance to our work in filling this gap. KEY POINTS: • Six new opine dehydrogenases have been discovered from a hot spring metagenome • The newly identified enzymes display a unique substrate scope • Substrate specificity is governed by two correlated active-site residues.

Novel Approach for the Isolation and Immobilization of a Recombinant Transaminase: Applying an Advanced Nanocomposite System.

The increasing application of recombinant enzymes demands not only effective and sustainable fermentation, but also highly efficient downstream processing and further stabilization of the enzymes by immobilization. In this study, a novel approach for the isolation and immobilization of His-tagged transaminase from Chromobacterium violaceum (CvTA) has been developed. A recombinant of CvTA was simultaneously isolated and immobilized by binding on silica nanoparticles (SNPs) with metal affinity linkers and additionally within poly(lactic acid) (PLA) nanofibers. The linker length and the nature of the metal ion significantly affected the enzyme binding efficiency and biocatalytic activity of CvTA-SNPs. The formation of PLA nanofibers by electrospinning enabled rapid embedding of CvTA-SNPs biocatalysts and ensured enhanced stability and activity. The developed advanced immobilization method reduces the time required for enzyme isolation, purification and immobilization by more than fourfold compared to a classical stepwise technique.

Opine dehydrogenases, an underexplored enzyme family for the enzymatic synthesis of chiral amines.

Opines and opine-type chemicals are valuable natural products with diverse biochemical roles, and potential synthetic building blocks of bioactive compounds. Their synthesis involves reductive amination of ketoacids with amino acids. This transformation has high synthetic potential in producing enantiopure secondary amines. Nature has evolved opine dehydrogenases for this chemistry. To date, only one enzyme has been used as biocatalyst, however, analysis of the available sequence space suggests more enzymes to be exploited in synthetic organic chemistry. This review summarizes the current knowledge of this underexplored enzyme class, highlights key molecular, structural, and catalytic features with the aim to provide a comprehensive general description of opine dehydrogenases, thereby supporting future enzyme discovery and protein engineering studies.

Immobilization of the Aspartate Ammonia-Lyase from Pseudomonas fluorescens R124 on Magnetic Nanoparticles: Characterization and Kinetics.

Aspartate ammonia-lyases (AALs) catalyze the non-oxidative elimination of ammonia from l-aspartate to give fumarate and ammonia. In this work the AAL coding gene from Pseudomonas fluorescens R124 was identified, isolated, and cloned into the pET-15b expression vector and expressed in E. coli. The purified enzyme (PfAAL) showed optimal activity at pH 8.8, Michaelis-Menten kinetics in the ammonia elimination from l-aspartate, and no strong dependence on divalent metal ions for its activity. The purified PfAAL was covalently immobilized on epoxy-functionalized magnetic nanoparticles (MNP), and effective kinetics of the immobilized PfAAL-MNP was compared to the native solution form. Glycerol addition significantly enhanced the storability of PfAAL-MNP. Inhibiting effect of the growing viscosity (modulated by addition of glycerol or glucose) on the enzymatic activity was observed for the native and immobilized form of PfAAL, as previously described for other free enzymes. The storage stability and recyclability of PfAAL-MNP is promising for further biocatalytic applications.

Ivermectin for causal malaria prophylaxis: a randomised controlled human infection trial.

OBJECTIVE: Ivermectin is safe and widely used for treating helminth infections. It also kills arthropods feeding on treated subjects, including malaria vectors. Thus, ivermectin mass drug administration as an additional tool for malaria control is being evaluated by WHO. As in vitro data, animal experiments and epidemiological observations suggest that ivermectin has a direct effect on the liver stages of the malaria parasite, this study was designed to assess the prophylactic effect of ivermectin on Plasmodium falciparum controlled human malaria infection. METHODS: A total of 4 volunteers were randomised to placebo, and 8 volunteers were randomised to receive ivermectin 0.4 mg/kg, orally, once 2 h before being experimentally infected intravenously with 3200 P. falciparum sporozoites. The primary endpoint was time to parasitaemia detected by positive thick blood smear; RT-qPCR was performed in parallel. RESULTS: All but one volunteer became thick blood smear positive between day 11 and day 12 after infection, and there was no significant effect of ivermectin on parasitaemia. CONCLUSION: Ivermectin - at the dose used - has no clinically relevant activity against the pre-erythrocytic stages of P. falciparum.

OBJECTIF: L'ivermectine est sûr et largement utilisé pour traiter les helminthiases. Il tue également les arthropodes se nourrissant sur les sujets traités, y compris les vecteurs du paludisme. Ainsi, l'administration en masse d'ivermectine en tant qu'outil supplémentaire de lutte contre le paludisme est actuellement évaluée par l'OMS. Comme les données in vitro, les expériences sur animaux et les observations épidémiologiques suggèrent que l'ivermectine a un effet direct sur les stades hépatiques du parasite du paludisme, cette étude a été conçue pour évaluer l'effet prophylactique de l'ivermectine sur l'infection paludéenne humaine par Plasmodium falciparum contrôlée. MÉTHODES: Quatre volontaires ont été randomisés pour un placebo et 8 volontaires ont été randomisés pour recevoir de l'ivermectine à 0,4 mg/kg en une fois par voie orale, 2 heures avant d'être expérimentalement infectés par voie intraveineuse avec 3.200 sporozoïtes de P. falciparum. Le critère d'évaluation principal était le temps à la parasitémie détectée par un frottis sanguin épais positif. Une RT-qPCR a été réalisée en parallèle. RÉSULTATS: Tous les volontaires sauf un sont devenus positifs pour les frottis sanguins épais entre le jour 11 et le jour 12 après l'infection et il n'y avait aucun effet significatif de l'ivermectine sur la parasitémie. CONCLUSION: L'ivermectine - à la dose utilisée - n'a aucune activité cliniquement pertinente contre les stades pré-érythrocytaires de P. falciparum.

"Fishing and Hunting"-Selective Immobilization of a Recombinant Phenylalanine Ammonia-Lyase from Fermentation Media.

This article overviews the numerous immobilization methods available for various biocatalysts such as whole-cells, cell fragments, lysates or enzymes which do not require preliminary enzyme purification and introduces an advanced approach avoiding the costly and time consuming downstream processes required by immobilization of purified enzyme-based biocatalysts (such as enzyme purification by chromatographic methods and dialysis). Our approach is based on silica shell coated magnetic nanoparticles as solid carriers decorated with mixed functions having either coordinative binding ability (a metal ion complexed by a chelator anchored to the surface) or covalent bond-forming ability (an epoxide attached to the surface via a proper linker) enabling a single operation enrichment and immobilization of a recombinant phenylalanine ammonia-lyase from parsley fused to a polyhistidine affinity tag.

The Possible Role of Mycotoxins in the Pathogenesis of Endometrial Cancer.

Endometrial cancer is one of the most common cancer types among women. Many factors can contribute to the development of this disease, including environmental factors and, thus, eating habits. Our study aims to determine the levels of various mycotoxins and their metabolites in the blood serum and endometrial tissue samples of participants with previously proven endometrial cancer and to find possible contributions to cancer development. In the cohort clinical trial, 52 participants aged between 44 and 86 were studied. The participants were divided into two groups: patients or matched controls. All patients had previously histologically diagnosed endometrial cancer. The cancer patients were divided into low-grade endometrioid and low- plus high-grade endometrioid groups. Controls had no history of endometrial malignancy or premalignancy. Blood serum and endometrial tissue samples were obtained from all study patients. We compared the concentrations of total Aflatoxins (Afs), Deoxynivalenol (DON), Ochratoxin-A (OTA), T2-toxin and HT2 toxin (T2/HT2 toxin), Zearalenone (ZEN), alpha-Zearalenol (α-ZOL), and Fumonisin B1 (FB1) in the serum and endometrium between the different study groups. As a result, we can see a significant correlation between the higher levels of Afs and zearalenone and the presence of endometrial cancer. In the case of Afs, DON, OTA, T2/HT2 toxins, ZEN, and alpha-ZOL, we measured higher endometrial concentrations than in serum. Considering the effect of mycotoxins and eating habits on cancer development, our results might lead to further research exploring the relationship between certain mycotoxins and endometrium cancer.

First Results on the Presence of Mycotoxins in the Liver of Pregnant Fallow Deer (Dama dama) Hinds and Fetuses.

Reproductive abnormalities have been observed in fallow deer populations in Hungary. We supposed mycotoxin contamination to be one of the possible causes because multi-mycotoxin contamination is known to be dangerous even at low toxin levels, especially for young animals. We investigated the spatial pattern of mycotoxin occurrences and the relationship between maternal and fetal mycotoxin levels. A total of 72 fallow deer embryos and their mothers were sampled in seven forested regions in Hungary in the 2020/2021 hunting season. We analyzed Aflatoxin (AF), Zearalenone (ZEA), Fumonizin B1 (FB1), DON, and T2-toxin concentrations in maternal and fetal livers by ELISA. AF was present in 70% and 82%, ZEA in 41% and 96%, DON in 90% and 98%, T2-toxin in 96% and 85%, and FB1 in 84% and 3% of hind and fetus livers, respectively. All mycotoxins passed into the fetus, but only Fumonizin B1 rarely passed. The individual variability of mycotoxin levels was extremely high, but the spatial differences were moderate. We could not prove a relation between the maternal and fetal mycotoxin concentrations, but we found an accumulation of ZEA and DON in the fetuses. These results reflect the possible threats of mycotoxins to the population dynamics and reproduction of wild fallow deer.

Immobilization of human tyrosine hydroxylase onto magnetic nanoparticles - A novel formulation of a therapeutic enzyme.

Human tyrosine hydroxylase (hTH) has key role in the production of catecholamine neurotransmitters. The structure, function and regulation of hTH has been extensively researched area and the possibility of enzyme replacement therapy (ERT) involving hTH through nanocarriers has been raised as well. However, our understanding on how hTH may interact with nanocarriers is still lacking. In this work, we attempted to investigate the immobilization of hTH on magnetic nanoparticles (MNPs) with various surface linkers in quantitative and mechanistic detail. Our results showed that the activity of hTH was retained after immobilization via secondary and covalent interactions as well. The colloidal stability of hTH could be also enhanced proved by Dynamic light scattering and Zeta potential analysis and a homogenous enzyme layer could be achieved, which was investigated by Raman mapping. The covalent attachment of hTH on MNPs via aldehyde or epoxy linkers provide irreversible immobilization and 38.1 % and 16.5 % recovery (ER). The hTH-MNPs catalyst had 25 % ER in average in simulated nasal electrolyte solution (SNES). This outcome highlights the relevance of immobilization applying MNPs as a potential formulation tool of sensitive therapeutic enzymes offering new opportunities for ERT related to neurodegenerative disorders.

Mindfulness and inhibitory control: Insights from the stop signal task with neutral and reward-associated stimuli.

Mindfulness has been linked to enhanced inhibitory control, yet the dynamics of this relationship, especially in reward situations, are not well understood. Our cross-sectional study aimed to explore the relationship between trait mindfulness and a performance measure of inhibitory control as a function of reward context operationalized by stimuli characteristics, and temporal inhibitory demands. Thirty-six individuals aged 19 to 41 filled out the Mindful Attention Awareness Scale (MAAS) and performed a stop signal task (SST), that included both neutral (stone images) and reward-related (money images) stimuli. The SST encompassed four conditions: neutral go/neutral stop, neutral go/reward stop, reward go/neutral stop, and reward go/reward stop, requiring participants to suppress reactions either during or after encountering reward-related stimuli. The relevant index of inhibitory control is the stop signal reaction time (SSRT), a performance measure of inhibitory control. Our findings showed no notable variation in the relationship between the MAAS score and SSRT across the different conditions. However, there was an overall significant effect of MAAS score on SSRT, irrespective of condition. Results may reflect the benefit of mindfulness on inhibitory control after generalized reward exposure.

[Possible applications of poultry immunoglobulins focusing on mycotoxin environmental loads and human influence]. / Baromfi-immunglobulinok lehetséges alkalmazásai a mikotoxin-környezetterhelések és a humánérintettség fókuszában.

In addition to their role, immunoglobulins can be used in animal and human diagnostic (immunoassay-based) measurements, prophylaxis and (immuno)therapy. For these purposes, today's "alternative" that is advantageous from an animal ethical point of view is the bird immunoglobulin Y isolated from egg yolk. Its development and production are cost-effective, the complexity is low, and due to its advantageous properties, it can be used in assays or even more so in medical therapies (primarily passive immunization). It is widely used (against pathogens or their toxins) in treatments of intestinal or metabolic diseases and inflammations. Its application in human diagnostics is limited, some markers are measured using immunoglobulin Y as assay component. In this study, a possible application, which is less common today, is presented. The problem of environmental impacts is becoming significant. Due to human activities, industrialization, environmental changes increase the appearance of natural environmental pollutants, including the effects of mycotoxins produced by molds locally and/or globally, which (mainly through nutrition) affect humans. Such agents often appear together, several mycotoxins affect the individual. As a result of their persistence, mycotoxins absorbed in the intestinal tract and accumulated in organs, can already reach levels that can cause physiological and/or behavioral effects. Although the examination of sources (contaminated foods) is regulated by law, the extent of accumulation has not been or cannot be examined and is often insufficiently taken into account. Due to the nature of the technique, the anti-mycotoxin avian immunoglobulin Y could be used both for detection of (deposited) mycotoxin(s) and/or even for immunotherapy (e.g., mycotoxin neutralization). Demonstrating the endocrine-disrupting mycotoxins using the example of zearalenone (with an explanation of its reproductive and immunological effects), we present generation of zearalenone (and mycotoxin-specific) avian immunoglobulin developments, advocate its use in human detection, urging the development of measurements that are suitable for detecting (multiple) accumulation. Orv Hetil. 2023; 164(39): 1527-1536.

Seeing attractive faces challenges inhibitory control, especially when mindful.

Previous studies have suggested positive effects of mindfulness on inhibitory control (stopping behaviour). However, scarce previous studies suggest the relationship may depend on context. We provide first evidence that inhibitory control is challenged when perceiving attractive faces, especially when being mindful. Specifically, we investigated the relationship between mindfulness and inhibitory control and the moderating role of a social reward context (being exposed to attractive opposite sex faces). Participants (n = 50) between 18-43 years old (M = 25, SD = 5.4) filled out questionnaires assessing standard demographic variables and dispositional mindfulness. Subsequently, they performed a Go/No-go task with a neutral condition and attractive faces condition. Results showed that inhibitory control was challenged in the attractive condition relative to the neutral condition, p = 0.019. Dispositional mindfulness was negatively correlated with inhibitory performance, but only in the attractive faces condition (r = -0.32, p = 0.024). Results did not support a moderating role of gender. Finally, though post-hoc, higher mindfulness was associated with reduced perceived attractiveness of presented faces (r = -0.33, p = 0.019). However, the relationship between mindfulness and reduced inhibitory control could not be explained by mindfulness associated reduced attractiveness. Taken together, results show that mindfulness challenges inhibitory control when perceiving attractive faces. This implies that mindfulness interventions aimed at enhancing inhibitory control, may not render the desired effect in a context of being exposed to attractive faces. Though certainly plausible, it remains an open question whether results generalize to other reward contexts as well.

Influence of Venoarterial Extracorporeal Membrane Oxygenation Integrated Hemoadsorption on the Early Reversal of Multiorgan and Microcirculatory Dysfunction and Outcome of Refractory Cardiogenic Shock.

Background: The purpose of this investigation was to evaluate the impact of venoarterial extracorporeal membrane oxygenation (VA−ECMO) integrated hemoadsorption on the reversal of multiorgan and microcirculatory dysfunction, and early mortality of refractory cardiogenic shock patients. Methods: Propensity score−matched cohort study of 29 pairs of patients. Subjects received either VA−ECMO supplemented with hemoadsorption or standard VA−ECMO management. Results: There was a lower mean sequential organ failure assessment score (p = 0.04), lactate concentration (p = 0.015), P(v−a)CO2 gap (p < 0.001), vasoactive inotropic score (p = 0.007), and reduced delta C−reactive protein level (p = 0.005) in the hemoadsorption compared to control groups after 72 h. In−hospital mortality was similar to the predictions in the control group (62.1%) and was much lower than the predicted value in the hemoadsorption group (44.8%). There were less ECMO-associated bleeding complications in the hemoadsorption group compared to controls (p = 0.049). Overall, 90-day survival was better in the hemoadsorption group than in controls without statistical significance. Conclusion: VA−ECMO integrated hemoadsorption treatment was associated with accelerated recovery of multiorgan and microcirculatory dysfunction, mitigated inflammatory response, less bleeding complications, and lower risk for early mortality in comparison with controls.

Dispositional Mindfulness and Inhibitory Control after Perceiving Neutral, Food and Money Related Stimuli.

Previous studies have shown that dispositional mindfulness is positively associated with cognitive performance, including the ability to stop behavioral actions (formally called inhibitory control). However, some studies suggest that the relationship may be context dependent. The current study addressed previous limitations and focused on the role of reward context regarding the relationship between mindfulness and inhibitory control. Seventy-five participants (31 men, 44 women) between 18-50 years old (M = 30, SD = 9) were included in the final sample. Participants filled out a self-report measure of mindfulness and performed a stop signal task with three conditions that varied in terms of reward context. In the neutral condition, go stimuli (stimuli to which a response was required) were letters; in the food and money condition, these were pictures of food and money, respectively. Results showed that inhibitory control was reduced in the money condition relative to the neutral condition (p = 0.012). Mindfulness was positively correlated with inhibitory control, but only in the money condition (p = 0.001). However, results might differ when inhibitory control is required while perceiving a learned reward-related stimulus. The latter represents an open question for future research.

A PfSPZ vaccine immunization regimen equally protective against homologous and heterologous controlled human malaria infection.

Immunization with radiation-attenuated Plasmodium falciparum (Pf) sporozoites (SPZ) in PfSPZ Vaccine, has provided better vaccine efficacy (VE) against controlled human malaria infection (CHMI) with the same parasites as in the vaccine (homologous) than with genetically distant parasites (heterologous). We sought to identify an immunization regimen that provided similar VE against CHMI with homologous and heterologous Pf for at least 9 weeks in malaria-naïve adults. Such a regimen was identified in part 1 (optimization), an open label study, and confirmed in part 2 (verification), a randomized, double-blind, placebo-controlled study in which VE was assessed by cross-over repeat CHMI with homologous (PfNF54) and heterologous (Pf7G8) PfSPZ at 3 and 9-10 weeks. VE was calculated using Bayesian generalized linear regression. In part 1, vaccination with 9 × 105 PfSPZ on days 1, 8, and 29 protected 5/5 (100%) subjects against homologous CHMI at 3 weeks after the last immunization. In part 2, the same 3-dose regimen protected 5/6 subjects (83%) against heterologous CHMI at both 3 and 9-10 weeks after the last immunization. Overall VE was 78% (95% predictive interval: 57-92%), and against heterologous and homologous was 79% (95% PI: 54-95%) and 77% (95% PI: 50-95%) respectively. PfSPZ Vaccine was safe and well tolerated. A 4-week, 3-dose regimen of PfSPZ Vaccine provided similar VE for 9-10 weeks against homologous and heterologous CHMI. The trial is registered with ClinicalTrials.gov, NCT02704533.

Transaminase-mediated synthesis of enantiopure drug-like 1-(3',4'-disubstituted phenyl)propan-2-amines.

Transaminases (TAs) offer an environmentally and economically attractive method for the direct synthesis of pharmaceutically relevant disubstituted 1-phenylpropan-2-amine derivatives starting from prochiral ketones. In this work, we report the application of immobilised whole-cell biocatalysts with (R)-transaminase activity for the synthesis of novel disubstituted 1-phenylpropan-2-amines. After optimisation of the asymmetric synthesis, the (R)-enantiomers could be produced with 88-89% conversion and >99% ee, while the (S)-enantiomers could be selectively obtained as the unreacted fraction of the corresponding racemic amines in kinetic resolution with >48% conversion and >95% ee.

Green synthesis of gold nanoparticles by thermophilic filamentous fungi.

Alternative methods, including green synthetic approaches for the preparation of various types of nanoparticles are important to maintain sustainable development. Extracellular or intracellular extracts of fungi are perfect candidates for the synthesis of metal nanoparticles due to the scalability and cost efficiency of fungal growth even on industrial scale. There are several methods and techniques that use fungi-originated fractions for synthesis of gold nanoparticles. However, there is less knowledge about the drawbacks and limitations of these techniques. Additionally, identification of components that play key roles in the synthesis is challenging. Here we show and compare the results of three different approaches for the synthesis of gold nanoparticles using either the extracellular fraction, the autolysate of the fungi or the intracellular fraction of 29 thermophilic fungi. We observed the formation of nanoparticles with different sizes (ranging between 6 nm and 40 nm) and size distributions (with standard deviations ranging between 30% and 70%) depending on the fungi strain and experimental conditions. We found by using ultracentrifugal filtration technique that the size of reducing agents is less than 3 kDa and the size of molecules that can efficiently stabilize nanoparticles is greater than 3 kDa.

Haloperidol 2 mg impairs inhibition but not visuospatial attention.

RATIONALE: The dopaminergic system has been implicated in visuospatial attention and inhibition, but the exact role has yet to be elucidated. Scarce literature suggests that attenuation of dopaminergic neurotransmission negatively affects attentional focusing and inhibition. To the best of our knowledge, this is the first study that evaluated the effect of dopaminergic antagonism on stopping performance. METHODS: Dopaminergic neurotransmission was attenuated in 28 healthy male participants by using 2 mg haloperidol. A repeated-measures placebo-controlled crossover design was implemented, and performance indices of attention and inhibition were assessed in the visual spatial cueing task (VSC) and stop signal task (SST). Additionally, the effect of haloperidol on motoric parameters was assessed. It was expected that haloperidol as contrasted to placebo would result in a reduction of the "validity effect," the benefit of valid cueing as opposed to invalid cueing of a target in terms of reaction time. Furthermore, an increase in stop signal reaction time (SSRT) in the SST was expected. RESULTS AND CONCLUSION: Results partially confirmed the hypothesis. Haloperidol negatively affected inhibitory motor control in the SST as indexed by SSRT, but there were no indications that haloperidol affected bias or disengagement in the VSC task as indicated by a lack of an effect on RTs. Pertaining to secondary parameters, motor activity increased significantly under haloperidol. Haloperidol negatively affected reaction time variability and errors in both tasks, as well as omissions in the SST, indicating a decreased sustained attention, an increase in premature responses, and an increase in lapses of attention, respectively.