Exogenous enzymes and probiotics alter digestion kinetics, volatile fatty acid content and microbial interactions in the gut of Nile tilapia.

Sustainable aquafeed production requires fishmeal replacement, leading to an increasing use of plant-derived ingredients. As a consequence, higher levels of antinutritional substances, such as non-starch polysaccharides and phytate, are present in aquafeeds, with negative effects on fish performance, nutrient digestibility and overall gut health. To alleviate these negative effects, providing exogenous digestive enzymes and/or probiotics can be an effective solution. In this study, we tested the effect of dietary supplementation of enzymes (phytase and xylanase) and probiotics (three strains of Bacillus amyloliquefaciens) on nutrient digestion kinetics and volatile fatty acid content along the gut, and the distal gut microbiome diversity in Nile tilapia. Chyme volatile fatty content was increased with probiotic supplementation in the proximal gut, while lactate content, measured for the first time in vivo in fish, decreased with enzymes along the gut. Enzyme supplementation enhanced crude protein, Ca and P digestibility in proximal and middle gut. Enzymes and probiotics supplementation enhanced microbial interactions as shown by network analysis, while increased the abundance of lactic acid bacteria and Bacillus species. Such results suggest that supplementation with exogenous enzymes and probiotics increases nutrient availability, while at the same time benefits gut health and contributes to a more stable microbiome environment.

Virus-Derived Peptides for Hepatic Enzyme Delivery.

Recently, a lipopeptide derived from the hepatitis B virus (HBV) large surface protein has been developed as an HBV entry inhibitor. This lipopeptide, called MyrcludexB (MyrB), selectively binds to the sodium taurocholate cotransporting polypeptide (NTCP) on the basolateral membrane of hepatocytes. Here, the feasibility of coupling therapeutic enzymes to MyrB was investigated for the development of enzyme delivery strategies. Hepatotropic targeting shall enable enzyme prodrug therapies and detoxification procedures. Here, horseradish peroxidase (HRP) was conjugated to MyrB via maleimide chemistry, and coupling was validated by SDS-PAGE and reversed-phase HPLC. The specificity of the target recognition of HRP-MyrB could be shown in an NTCP-overexpressing liver parenchymal cell line, as demonstrated by competitive inhibition with an excess of free MyrB and displayed a strong linear dependency on the applied HRP-MyrB concentration. In vivo studies in zebrafish embryos revealed a dominating interaction of HRP-MyrB with scavenger endothelial cells vs xenografted NTCP expressing mammalian cells. In mice, radiolabeled 125I-HRP-MyrBy, as well as the non-NTCP targeted control HRP-peptide-construct (125I-HRP-alaMyrBy) demonstrated a strong liver accumulation confirming the nonspecific interaction with scavenger cells. Still, MyrB conjugation to HRP resulted in an increased and NTCP-mediated hepatotropism, as revealed by competitive inhibition. In conclusion, the model enzyme HRP was successfully conjugated to MyrB to achieve NTCP-specific targeting in vitro with the potential for ex vivo diagnostic applications. In vivo, target specificity was reduced by non-NTCP-mediated interactions. Nonetheless, tissue distribution experiments in zebrafish embryos provide mechanistic insight into underlying scavenging processes indicating partial involvement of stabilin receptors.

Improved pharmacokinetic characteristics and bioactive effects of anticancer enzyme delivery systems.

INTRODUCTION: Anticancer enzymes play important roles in cancer treatment. The anticancer enzyme has been in clinical use to treat acute lymphoblastic leukemia for many years. Other types of anticancer enzymes have been investigated in laboratory studies and clinical trials. Area covered: This paper will provide perspectives on the indications, anticancer mechanisms, enzymatic characteristics (such as molecular weight, organism source, and kinetic parameters) and pharmacokinetic behaviors of anticancer enzymes and their delivery systems by systematically analyzing available literature. The pharmacodynamics of anticancer enzyme delivery systems has also been summarized. Expert opinion: Anticancer enzymes kill cancer cells by depleting important nutrients required for growth or producing metabolite toxic to tumor cells. Suitable enzyme delivery systems have demonstrated promising effects on the pharmacokinetics, bioactivity and application of anticancer enzymes. Their current limitations and future potential are analyzed.[Figure: see text].

Therapeutic Enzymes: Applications and Approaches to Pharmacological Improvement.

BACKGROUND: Among therapeutic proteins, enzymes represent small and of course profitable market. They can be used to treat important, rare, and deadly diseases. Enzyme therapy is the only available treatment for certain disorders. METHOD: Here, pharmaceutical enzymes are reviewed. They are categorized in four main groups, enzymes in replacement therapy, enzymes in cancer treatment, enzymes for fibrinolysis, and finally enzymes that are used topically for various treatments. Furthermore, enzyme gene therapy and future perspective of therapeutic enzymes are mentioned in brief. RESULTS: There are many important approved enzymes in pharmaceutical market. Several approaches such as point mutation, fusion protein designing, glycoengineering, and PEGylation were used to achieve improved enzymes. Although sometimes enzymes were engineered to facilitate production and purification process, appropriate delivery to target sites, extending half-life, and reducing immunogenicity are among the main goals of engineering approaches. CONCLUSION: Overall, enzymes play a critical role in treatment of common and rare diseases. Evaluation of new enzymes as well as improvement of approved enzymes are of the most important challenges in biotechnology.

Lessons from Enzyme Kinetics Reveal Specificity Principles for RNA-Guided Nucleases in RNA Interference and CRISPR-Based Genome Editing.

RNA-guided nucleases (RGNs) provide sequence-specific gene regulation through base-pairing interactions between a small RNA guide and target RNA or DNA. RGN systems, which include CRISPR-Cas9 and RNA interference (RNAi), hold tremendous promise as programmable tools for engineering and therapeutic purposes. However, pervasive targeting of sequences that closely resemble the intended target has remained a major challenge, limiting the reliability and interpretation of RGN activity and the range of possible applications. Efforts to reduce off-target activity and enhance RGN specificity have led to a collection of empirically derived rules, which often paradoxically include decreased binding affinity of the RNA-guided nuclease to its target. We consider the kinetics of these reactions and show that basic kinetic properties can explain the specificities observed in the literature and the changes in these specificities in engineered systems. The kinetic models described provide a foundation for understanding RGN targeting and a necessary conceptual framework for their rational engineering.

Approaches for the estimation of timescales in nonlinear dynamical systems: Timescale separation in enzyme kinetics as a case study.

The derivation of timescales is frequently introduced as an art form in papers and textbooks. The best scaling techniques require the application of physical intuition to identify dimensionless variables that are one unit order of magnitude and small parameters, which can simplify nonlinear differential equations. However, physical intuition requires prior knowledge of the solution to the dynamical systems under investigation. There are problems where the application of physical intuition is not straightforward. Therefore, it is necessary to apply mathematical techniques to estimate scales for the separation of timescales and simplification. In this review, we present three mathematical techniques - determination of pairwise balances, principle of minimum simplification and scaling by inverse rates - to scale dynamical systems with limited prior knowledge of model behavior. We illustrate the application of these techniques with the Michaelis-Menten reaction, which is widely studied to introduce scaling and simplification techniques in textbooks. We show that the pairwise balance approach, though commonly introduced as a method for nondimensionalization, can fail to derive a separation between timescales. The other techniques we review here can be applied to a number of dynamical systems, where the separation of timescales can lead to the simplification of a complex nonlinear problem.

[The use of the enzymes for the enhancement of the effectiveness of antimicrobial photodynamic treatment of the patients presenting with chronic tonsillitis].

The objective of the present study was to enhance the effectiveness of the treatment of the patients presenting with various forms of chronic tonsillitis (CT) by means of antimicrobial photodynamic therapy (APDT). The study included 48 patients at the age from 18 to 55 years divided into three groups; all of them suffered from various forms of CT. Group 1 was comprised of 12 patients given a course of traditional conservative therapy. Group 2 consisted of 17 patients treated by APDT while group 3 included 19 patients in whom a course of antimicrobial photodynamic therapy was preceded by the treatment of the mucous membrane of the palatine amygdalae with a lidase solution. The microbiological testing was performed on days 5, 12, and 24 after APDT and also within the next 6-9 months. The results of the microbiological and clinical studies give evidence of the possibility to improve the effectiveness of the treatment of chronic tonsillitis by means of antimicrobial photodynamic therapy with the use of the preventive treatment of palatine tonsillar mucosa with a lidase solution. Such treatment was found to facilitate degradation of theintercellular matrix of the biofilm and reduced its resistance to the photodynamic impact.

Nanofibers as novel drug carrier--An overview.

Presently polymer nanofibers have received much attention due to their unique properties such as large surface area, high porosity, small pore size, superior mechanical properties and ease of addition of surface functionalities compared with any other material. Nanofibers particularly polymeric nanofiber prepared by electrospinning process can be used as carriers for the controlled drug delivery of bioactive molecules such as cytokines, growth factors, anticancer drugs, enzymes and certain vitamins. This article presents an overview of nanofibers, various techniques involved in fabrication of nanofibers, their characterization, parameters affecting electrospinning process and their applications.

Stationary distribution of self-organized states and biological information generation.

Self-organization, where spontaneous orderings occur under driven conditions, is one of the hallmarks of biological systems. We consider a statistical mechanical treatment of the biased distribution of such organized states, which become favored as a result of their catalytic activity under chemical driving forces. A generalization of the equilibrium canonical distribution describes the stationary state, which can be used to model shifts in conformational ensembles sampled by an enzyme in working conditions. The basic idea is applied to the process of biological information generation from random sequences of heteropolymers, where unfavorable Shannon entropy is overcome by the catalytic activities of selected genes. The ordering process is demonstrated with the genetic distance to a genotype with high catalytic activity as an order parameter. The resulting free energy can have multiple minima, corresponding to disordered and organized phases with first-order transitions between them.

Are hepatic impairment studies necessary for therapeutic proteins?

OBJECTIVE: This study assessed whether trials to investigate the effect of hepatic impairment on the pharmacokinetics of therapeutic proteins (TPs), which are conducted for small molecule drugs, are necessary. METHODS: The product labeling for 91 TPs that have been approved by the US Food and Drug Administration were reviewed. A PubMed search was also conducted to identify completed studies that assessed the effect of hepatic impairment on the pharmacokinetics of TPs. Biologic License Applications were subsequently reviewed to gather further descriptions of the trials conducted in patients with hepatic impairment and data analyses. RESULTS: No dedicated pharmacokinetics trials were conducted in patients with hepatic impairment for these approved TPs, but subgroup (n = 2 [2.2%]) or population (n = 5 [5.5%]) pharmacokinetic analyses were performed for 7 TPs. The pharmacokinetics of these proteins were not affected by the hepatic dysfunction, with the exception that the clearance of drotrecogin alfa seemed 25% higher in patients with hepatic impairment than in patients without hepatic impairment; however, no dose reduction was recommended. Thus, the effect of hepatic impairment on the pharmacokinetics of TPs is unclear based on the limited analyses completed to date. CONCLUSIONS: A dedicated pharmacokinetics trial for TPs in patients with hepatic impairment is not necessary. Recognizing that the data are very limited, it would be important to continue collecting pharmacokinetic data of TP in patients with hepatic impairment and using population pharmacokinetic analyses to evaluate the effect of hepatic impairment on the pharmacokinetics of TP.

[Erythrocytes - the new application in medicine]. / Erytrocyty - nowe zastosowanie w medycynie.

The new forms of drugs with better proprieties from traditional ones were sought for a long time. Erythrocytes applied as carriers of therapeutic substances are among promising. They are characterized by slower release of active substances, less toxicity, as well as better biocompatibility and biodegradation in the organism. It is especially important in administration of drugs with numerous side effects in therapy of chronic diseases e.g. malignancies. Investigations conducted from over twenty years showed, that erythrocytes are universal carriers in which different therapeutic substances were successfully closed, e.g. cytostatics, antibiotics, hormones and vitamins, as well as enzymes and vaccines. Some of the erythrocyte drug delivery systems are now studied at the clinical level, e.g. dexamthasone 21-phosphate in treatment of inflammatory bowel disease and chronic obstructive pulmonary disease. This substance encapsulated in human erythrocytes was also officially registered by European Medicines Agency, as the orphan drug in treatment of cystic fibrosis: Reports on application of carrier erythrocytes in patients with rare genetic diseases have also appeared.

Enzyme replacement therapy for lysosomal storage diseases.

PURPOSE OF REVIEW: Enzyme replacement therapy (ERT) for type 1 Gaucher has been highly successful. ERT is now available for other lysosomal storage disorders (LSDs) but none of these highly expensive treatments has had the same efficacy. This review explores why these newer treatments have failed to live up to expectations and how future products might be made more effective. RECENT FINDINGS: In Gaucher, the target cells for ERT are macrophages, which are efficiently accessed by intravenously injected recombinant enzyme. The target tissues in other LSDs receive much lower doses of enzyme and intravenous ERT does not enter the brain at all. Uptake of recombinant enzyme is via the mannose-6-phosphate receptor (M6PR). Recent work has looked at improving the efficiency of enzyme delivery to tissues by altering both the ligand on the infused enzyme and the expression of the M6PR on cells. For delivery to the central nervous system, intrathecal routes of administration have been explored. SUMMARY: Work in tissue culture and in animal models has shown increased efficiency of enzyme delivery and clinical trials of second-generation products and novel delivery systems are now underway.

Novel mathematical models for cell-mediated cytotoxicity assays without applying enzyme kinetics but with combinations and probability: bystanders in bulk effector cells influence results of cell-mediated cytotoxicity assays.

Cell-mediated cytotoxicity assays are widely implemented to evaluate cell-mediated cytotoxic activity, and some assays are analyzed using the analogy of enzyme kinetics. In the analogy, the effector cell is regarded as the enzyme, the target cell as the substrate, the effector cell-target cell conjugate as the enzyme-substrate complex and the dead target cell as the product. However, the assumptions analogous to those of enzyme kinetics are not always true in cell-mediated cytotoxicity assays, and the parameter analogous to the Michaelis-Menten constant is not constant but is dependent on the number of effector cells. Therefore I present novel mathematical models for cell-mediated cytotoxicity assays without applying enzyme kinetics. I instead use combinations and probability, because analysis of cell-mediated cytotoxicity assays by applying enzyme kinetics seems controversial. With my original models, I demonstrate simulations of the data in previously published papers. The results are exhibited in the same forms as the corresponding data. Comparing the simulation results with the published data, the results seem to agree well with the data. From simulations of cytotoxic assays with bulk effector cells, it appears that bystanders in bulk effector cells increase both the cytotoxic activity and the motility of effector cells.

Síntesis y evaluación de nuevos inhibidores de las HDAC / Synthesis and evaluation of new HDAC inhibitors

La acetilación de residuos de lisina en las histonas está mediada por las enzimas denominadas histona acetiltransferasas (HAT). Los grupos acetilo son eliminados de las e-N-acetil-lisinas por la actividadde las histonas desacetilasas (HDAC). El balance entre las actividades opuestas de las HAT y las HDAC regula el estado de acetilación de las histonas. Este tipo de modificaciones regulan en la célula procesos fundamentales clave en respuesta a señales extracelulares. En general, altos niveles de acetilación (hiperacetilación) se asocian a un incremento de la actividad transcripcional, mientras que bajos niveles de acetilación (hipoacetilación) se asocian a la represión de la expresión genética. Actualmente se conocen diversos tipos de inhibidores de las HDAC que pueden reactivar la expresión genética e inhibir el crecimiento de las células tumorales, por lo que se investiga su uso en el tratamiento frente al cáncer. Sería deseable identificar nuevos inhibidores de las enzimas HDAC para su utilización en el tratamiento o profilaxis de enfermedades en las que la inhibición de dichas enzimas HDAC está implicada. Se han obtenido 10 nuevos inhibidores de las HDAC y se ha evaluado su actividad frente a HDAC aislada. Se discute la importancia de las modificaciones realizadas en el espaciador(AU)

Lysine residues acetylation on histones is mediated by histone acetyltransferase (HAT). The acetyl groups are removed from e-N-acetyl-lysine by the histone deacetylase (HDAC) activity. The balance between the HATs and HDACs activities regulates the histone acetylation status. Such changes regulate key processes in the cell in response to extracellular signals. Mostly, high levels of acetylation(hyperacetylation) are associated with increased transcriptional activity. Low levels of acetylation (hypoacetylation) are associated with repression of gene expression. Currently, different types of HDAC inhibitors are known to reactivate gene expression and inhibit tumor cell growth. We aim at identifying novel HDAC inhibitors for the treatment or prophylaxis of cancer diseases. Ten new HDAC inhibitors have been obtained and their potency as HDAC inhibitors has been evaluated. A structure-activity relationship discussion has been focused on the structural changes made in the spacer(AU)

Imaging of enzyme replacement therapy using PET.

Direct enzyme replacement therapy (ERT) has been introduced as a means to treat a number of rare, complex genetic conditions associated with lysosomal dysfunction. Gaucher disease was the first for which this therapy was applied and remains the prototypical example. Although ERT using recombinant lysosomal enzymes has been shown to be effective in altering the clinical course of Gaucher disease, Fabry disease, Hurler syndrome, Hunter syndrome, Maroteaux-Lamy syndrome, and Pompe disease, the recalcitrance of certain disease manifestations underscores important unanswered questions related to dosing regimes, tissue half-life of the recombinant enzyme and the ability of intravenously administered enzyme to reach critical sites of known disease pathology. We have developed an innovative method for tagging acid beta-glucocerebrosidase (GCase), the recombinant enzyme formulated in Cerezyme(R) used to treat Gaucher disease, using an (18)F-labeled substrate analogue that becomes trapped within the active site of the enzyme. Using micro-PET we show that the tissue distribution of injected enzyme can be imaged in a murine model and that the PET data correlate with tissue (18)F counts. Further we show that PET imaging readily monitors pharmacokinetic changes effected by receptor blocking. The ability to (18)F-label GCase to monitor the enzyme distribution and tissue half-life in vivo by PET provides a powerful research tool with an immediate clinical application to Gaucher disease and a clear path for application to other ERTs.

Stabilization and release of enzymes from silk films.

A significant challenge remains to protect protein drugs from inactivation during production, storage, and use. In the present study, the stabilization and release of horseradish peroxidase (HRP) in silk films was investigated. Water-insoluble silk films were prepared under mild aqueous conditions, maintaining the activity of the entrapped enzyme. Depending on film processing and post-processing conditions, HRP retained more than 90% of the initial activity at 4 degrees C, room temperature and 37 degrees C over two months. The stability of protein drugs in silk films is attributed to intermolecular interactions between the silk and the enzymes, based on Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The unique structural feature of silk molecules, periodic hydrophobic-hydrophilic domains, enabled strong interactions with proteins. The entrapped protein was present in two states, untrapped active and trapped inactive forms. The ratio between the two forms varied according to processing conditions. Proteolytic degradation and dissolution of the silk films resulted in the release of the bound enzyme which was otherwise not released by diffusion; enzyme recovered full activity upon release. There was a linear relationship between silk degradation/dissolution and the release of entrapped enzyme. Modifying the secondary structure of the silk matrix and the interactions with the non-crystalline domains resulted in control of the film degradation or dissolution rate, and therefore the release rate of the entrapped enzyme. Based on the above results, silk materials are an intriguing carrier for proteins in terms of both retention of activity and controllable release kinetics from the films.

Biopharmaceutical drug targeting to the brain.

Biopharmaceuticals are large molecule drugs that do not cross the blood-brain barrier (BBB). The limiting factor in the drug development of biopharmaceuticals as new drugs for the human brain is the engineering of effective brain drug targeting technology platforms. Recombinant proteins, enzymes, and monoclonal antibodies can be re-engineered for transport across the human BBB with the molecular Trojan horse technology. The most active BBB molecular Trojan horse is a monoclonal antibody to the human insulin receptor. The genetic engineering of IgG fusion proteins has been demonstrated for neurotrophic factors, decoy receptors, therapeutic enzymes, single chain Fv antibodies, and avidin. The IgG fusion proteins are not toxic on repeated administration in high doses to primates and do not interfere with glycemic control in plasma or brain. IgG fusion proteins contain amino acid sequences that induce immune tolerance, and show low immunogenicity in primates. The IgG fusion proteins are new bifunctional biopharmaceuticals that are both targeted to brain via transport on endogenous BBB receptors, and exert pharmacological effects in brain at the cognate receptor, ligand, or enzyme substrate.

Evolution of complex probability distributions in enzyme cascades.

Unusual probability distribution profiles, including transient multi-peak distributions, have been observed in computer simulations of cell signaling dynamics. The emergence of these complex distributions cannot be explained using either deterministic chemical kinetics or simple Gaussian noise approximation. To develop physical insights into the origin of complex distributions in stochastic cell signaling, we compared our approximate analytical solutions of signaling dynamics with the exact numerical simulations. Our results are based on studying signaling in 2-step and 3-step enzyme amplification cascades that are among the most common building blocks of cellular protein signaling networks. We have found that while the multi-peak distributions are typically transient, and eventually evolve into single peak distributions, in certain cases these distributions may be stable in the limit of long times. We also have shown that introducing positive feedback loops results in diminution of the probability distribution complexity.

Quasi-steady-state kinetics at enzyme and substrate concentrations in excess of the Michaelis-Menten constant.

In vitro enzyme reactions are traditionally conducted under conditions of pronounced substrate excess since this guarantees that the bound enzyme is at quasi-steady-state (QSS) with respect to the free substrate, thereby justifying the Briggs-Haldane approximation (BHA). In contrast, intracellular reactions, amplification assays, allergen digestion assays and industrial applications span a range of enzyme-to-substrate ratios for which the BHA is invalid, including the extreme of enzyme excess. The quasi-equilibrium approximation (QEA) is valid for a subset of enzyme excess states. Previously, we showed that the total QSSA (tQSSA) overlaps and extends the validity of the BHA and the QEA, and that it is at least roughly valid for any total substrate and enzyme concentrations. The analysis of the tQSSA is hampered by square root nonlinearity. Previous simplifications of the tQSSA rate law are valid in a parameter domain that overlaps the validity domains of the BHA and the QEA and only slightly extends them. We now integrate the tQSSA rate equation in closed form, without resorting to further approximations. Moreover, we introduce a complimentary simplification of the tQSSA rate law that is valid in states of enzyme excess when the absolute difference between total enzyme and substrate concentrations greatly exceeds the Michaelis-Menten constant. This includes a wide range of enzyme and substrate concentrations where both the BHA and the QEA are invalid and allows us to define precisely the conditions for zero-order and first-order product formation. Remarkably, analytical approximations provided by the tQSSA closely match the expected stochastic kinetics for as few as 15 reactant molecules, suggesting that the conditions for the validity of the tQSSA and for its various simplifications are also of relevance at low molecule numbers.