Virulence-inhibitory activity of the degradation product 3-hydroxybutyrate explains the protective effect of poly-β-hydroxybutyrate against the major aquaculture pathogen Vibrio campbellii

The bacterial storage compound poly-beta-hydroxybutyrate, a polymer of the short-chain fatty acid 3-hydroxybutyrate, has been reported to protect various aquatic animals from bacterial disease. In order to obtain a better mechanistic insight, we aimed to (1) investigate whether 3-hydroxybutyrate is released from poly-beta-hydroxybutyrate within sterile brine shrimp larvae, (2) determine the impact of 3-hydroxybutyrate on the virulence of Vibrio campbellii to brine shrimp larvae and on its cell density in the shrimp, and (3) determine the impact of this compound on virulence factor production in the pathogen. We detected 3-hydroxybutyrate in poly-beta-hydroxybutyrate-fed brine shrimp, resulting in 24 mM 3-hydroxybutyrate in the intestinal tract of shrimp reared in the presence of 1000 mg l(-1) poly-beta-hydroxybutyrate. We further demonstrate that this concentration of 3-hydroxybutyrate does not affect the growth of V. campbellii, whereas it decreases the production of different virulence factors, including hemolysin, phospholipase and protease activities, and swimming motility. We hypothesize that by affecting all these virulence factors at once, 3-hydroxybutyrate (and thus also poly-beta-hydroxybutyrate) can exert a significant impact on the virulence of V. campbellii. This hypothesis was confirmed in a challenge test showing that 3-hydroxybutyrate protected gnotobiotic brine shrimp from pathogenic V. campbellii, without affecting the number of host-associated vibrios

Poly(3-hydroxybutyrate): Promising biomaterial for bone tissue engineering

Poly(3-hydroxybutyrate) is a natural polymer, produced by different bacteria, with good biocompatibility and biodegradability. Cardiovascular patches, scaffolds in tissue engineering and drug carriers are some of the possible biomedical applications of poly(3-hydroxybutyrate). In the past decade, many researchers examined the different physicochemical modifications of poly(3-hydroxybutyrate) in order to improve its properties for use in the field of bone tissue engineering. Poly(3-hydroxybutyrate) composites with hydroxyapatite and bioglass are intensively tested with animal and human osteoblasts in vitro to provide information about their biocompatibility, biodegradability and osteoinductivity. Good bone regeneration was proven when poly(3-hydroxybutyrate) patches were implanted in vivo in bone tissue of cats, minipigs and rats. This review summarizes the recent reports of in vitro and in vivo studies of pure poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate) composites with the emphasis on their bioactivity and biocompatibility with bone cells

Hair testing of GHB: an everlasting issue in forensic toxicology

In this paper, the authors present a critical review of different studies regarding hair testing of endogenous γ-hydroxybutyrate (GHB), concentrations in chronic users, and values measured after a single GHB exposure in drug facilitated sexual assault (DFSA) cases together with the role of a recently identified GHB metabolite, GHB-glucuronide. Content: The following databases (up to March 2017) PubMed, Scopus and Web of Science were used, searching the following key words: γ-hydroxybutyrate, GHB, GHB glucuronide, hair. The main key words “GHB” and “γ-hydroxybutyrate” were searched singularly and then associated individually to each of the other keywords. Summary: Of the 2304 sources found, only 20 were considered appropriate for the purpose of this paper. Summing up all the studies investigating endogenous GHB concentration in hair, a very broad concentration range from 0 to 12 ng/mg was found. In order to detect a single GHB dose in hair it is necessary to commonly wait 1 month for collecting hair and a segmental analysis of 3 or 5 mm fragments and the calculation of a ratio between the targeted segment and the others represent a reliable method to detect a single GHB intake considering that the ratios presently proposed vary from 3 and 10. The only two studies so far performed, investigating GHB-Glucuronide in hair, show that the latter does not seem to provide any diagnostic information regarding GHB exposure

Superparamagnetic Poly (3-hydroxybutyrate-co-3 hydroxyvalerate) (PHBV) nanoparticles for biomedical applications

Indexación: ScieloBackground: The progress in material science and the recent advances in biodegradable/biocompatible polymers and magnetic iron oxide nanoparticles have led to develop innovative diagnostic and therapeutic strategies for diseases based on multifunctional nanoparticles, which include contrast medium for magnetic resonance imaging, agent for hyperthermia and nanocarriers for targeted drug delivery. The aim of this work is to synthesize and characterize superparamagnetic iron oxide (magnetite), and to encapsulate them into poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanoparticles for biomedical applications. Results: The magnetite nanoparticles were confirmed by X-ray diffraction and exhibited a size of 22.3 ± 8.8 nm measured by transmission electron microscopy (TEM). Polymeric PHBV nanoparticles loaded with magnetite (MgNPs) were analyzed using dynamic light scattering and showed a size of 258.6 ± 35.7 nm and a negative zeta potential (-10.8 ± 3.5 mV). The TEM examination of MgNPs exhibited a spherical core-shell structure and the magnetic measurements showed in both, non-encapsulated magnetite and MgNPs, a superparamagnetic performance. Finally, the in vitro studies about the magnetic retention of MgNPs in a segment of small intestine of rats showed an active accumulation in the region of the magnetic field. Conclusions: The results obtained make the MgNPs suitable as potential magnetic resonance imaging contrast agents, also promoting hyperthermia and even as potential nanocarriers for site-specific transport and delivery of drugs. Keywords: hyperthermia, magnetic resonance image (MRI), magnetite, PHBV, polymeric nanoparticles.http://ref.scielo.org/cxt57

Single-Stage Biosynthesis Of Poly(3-Hydroxybutyrate-co-4- Hydroxybutyrate) Using Cupriavidus sp. Usmaa1020 Via Mixed-Substrate Cultivation Strategy

Kopolimer poli(3-hidroksibutirat-ko-4-hidroksibutirat) [P(3HB-ko-4HB)] merupakan polihidroksialkanoat (PHA) yang penting dalam bidang biofarmaseutikal dan perubatan Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer is polyhydroxyalkanoate (PHA) with biopharmaceutical and medical significanc

Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate): Structure, Property, and Fiber

Poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] are produced by various microorganisms as an intracellular carbon and energy reserve from agricultural feedstocks such as sugars and plant oils under unbalanced growth conditions. P(3HB) and P(3HB-co-3HV) have attracted the attention of academia and industry because of its biodegradability, biocompatibility, thermoplasticity, and plastic-like properties. This review first introduced the isodimorphism, spherulites, and molecular interaction of P(3HB) and P(3HB-co-3HV). In addition, the effects of 3HV content on the melting temperature and crystallization rate were discussed. Then the drawbacks of P(3HB) and P(3HB-co-3HV) including brittleness, narrow melt processing window, low crystallization rate, slow biodegradation rate in body, and so on were summarized. At last, the preparation, structure, and properties of P(3HB) and P(3HB-co-3HV) fiber were introduced

Controlled biotechnological production of polyhydroxyalkanoates

Předložená diplomová práce se zabývá produkcí polyhydroxyalkanoátů (PHA) bakterií Cupriavidus necator H16. Cílem práce byla příprava, selekce a charakterizace mutantních kmenů schopných vyšší produkce PHA. V teoretické části byla zpracována literární rešerše zabývající se nejdůležitějšími typy PHA, bakterií Cupriavidus necator a způsoby indukce mutageneze. V experimentální části byly připraveny mutantní kmeny pomocí fyzikální a chemické mutageneze. Mutantní kmeny schopné nadprodukce PHA byly selektovány pomocí kultivace na minerálním médium s olejem. Pro další studium byly vybrány 4 mutantní kmeny schopné nadprodukce PHA. Tyto mutantní kmeny byly dále podrobeny biochemické charakterizaci. Byly naměřeny specifické aktivity vybraných intracelulárních enzymů včetně enzymů podílejících se na biosyntéze PHA. Také byla naměřena resistence mutantů vůči oxidačnímu stresu. Bylo zjištěno, že mutantní kmeny schopné nadprodukce PHA mají vyšší aktivity enzymů produkujících NADPH. NADPH je jeden z klíčových substrátů ovlivňujících směr toku acetyl-CoA metabolizmem. Vyšší intracelulární koncentrace NADPH parciálně inhibuje Krebsův cyklus a aktivuje akumulaci PHA. Aktivity acetoacetyl-CoA reduktázy a PHA syntázy, enzymů zapojených do syntézy PHA, těchto mutantů proto byly také vyšší stejně jako molekulová hmotnost připravených polymerů. Aplikace fyzikálních a chemických mutagenů je způsob, kterým lze připravit biotechnologicky perspektivní mutantní kmeny schopné nadprodukce PHA.This diploma thesis deals with production of polyhydroxyalkanoates (PHA) by bacterial strain Cupriavidus necator H16. Goal of this work was preparation, selection and characterization of mutant strains overproducing PHA. Theoretical focuses on the most important PHA, bacteria Cupriavidus necator and mutagenesis techniques. In practical part mutant strains were prepared through physical and chemical mutagenesis. Mutant strains overproducing PHA were selected by cultivation in mineral medium with oil. For further study, 4 mutant strains overproducing PHA were selected. These mutants were biochemically characterized. Specific activities of several intracellular enzymes including enzymes involved in PHA biosynthesis were measured. Resistance of mutants against oxidative stress was measured as well. Mutant strains overproducing PHA revealed higher enzymatic activities of NADPH producing enzymes. Generally, NADPH is one of the substrates influencing flux of acetyl-CoA throughout the metabolism; higher intracellular concentration of NADPH partially inhibits TCA cycle and activates accumulation of PHA. Therefore, activities of acetoacetyl-CoA reductase and PHB synthase, enzymes directly involved in PHA synthesis were higher as compared to wild strain as well as molecular weight of produced materials. It can be concluded that biotechnologically perspective mutagens capable of PHA overproduction can be prepared by application of chemical and physical mutagens.

A Forward-Design Approach to Increase the Production of Poly-3-Hydroxybutyrate in Genetically Engineered Escherichia coli

Biopolymers, such as poly-3-hydroxybutyrate (P(3HB)) are produced as a carbon store in an array of organisms and exhibit characteristics which are similar to oil-derived plastics, yet have the added advantages of biodegradability and biocompatibility. Despite these advantages, P(3HB) production is currently more expensive than the production of oil-derived plastics, and therefore, more efficient P(3HB) production processes would be desirable. In this study, we describe the model-guided design and experimental validation of several engineered P(3HB) producing operons. In particular, we describe the characterization of a hybrid phaCAB operon that consists of a dual promoter (native and J23104) and RBS (native and B0034) design. P(3HB) production at 24 h was around six-fold higher in hybrid phaCAB engineered Escherichia coli in comparison to E. coli engineered with the native phaCAB operon from Ralstonia eutropha H16. Additionally, we describe the utilization of non-recyclable waste as a low-cost carbon source for the production of P(3HB)

Inhibition of the mitochondrial pyruvate carrier protects from excitotoxic neuronal death.

Glutamate is the dominant excitatory neurotransmitter in the brain, but under conditions of metabolic stress it can accumulate to excitotoxic levels. Although pharmacologic modulation of excitatory amino acid receptors is well studied, minimal consideration has been given to targeting mitochondrial glutamate metabolism to control neurotransmitter levels. Here we demonstrate that chemical inhibition of the mitochondrial pyruvate carrier (MPC) protects primary cortical neurons from excitotoxic death. Reductions in mitochondrial pyruvate uptake do not compromise cellular energy metabolism, suggesting neuronal metabolic flexibility. Rather, MPC inhibition rewires mitochondrial substrate metabolism to preferentially increase reliance on glutamate to fuel energetics and anaplerosis. Mobilizing the neuronal glutamate pool for oxidation decreases the quantity of glutamate released upon depolarization and, in turn, limits the positive-feedback cascade of excitotoxic neuronal injury. The finding links mitochondrial pyruvate metabolism to glutamatergic neurotransmission and establishes the MPC as a therapeutic target to treat neurodegenerative diseases characterized by excitotoxicity