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The purpose was to describe the prevalence and effect of elevated milk β-hydroxybutyrate (BHB) as detected by routine Fourier-transform infrared analysis in Dairy Herd Improvement milk samples. Data collected over 4 yr included cow information as well as milk yield and composition from 498,310 samples from postparturient Holstein cows (5-35 d in milk) from 4,242 herds. The following thresholds were used to classify cows based on their early lactation milk BHB concentration: <0.15 mmol/L = negative; 0.15 to 0.19 mmol/L = suspect; and ≥0.20 mmol/L = positive. Overall prevalence (suspect + positive) was 22.6% and was higher for older cows (18.7, 19.5, and 27.6%, for cows in their first, second, and third or greater lactation, respectively). Distribution with regards to days in milk was different among parity groups, with first-lactation cows having highest prevalence (30%) in the first week after calving; cows in their second and third and greater parity had the highest prevalence in the second week after calving, at 25.8 and 34.6%, respectively. Season of calving affected the prevalence of elevated milk BHB, with cows calving in the fall and spring seasons showing higher prevalence. Distribution among herds was highly variable, as 45% of herds had a prevalence of 20% or less, 47% of herds had a prevalence between 21 and 40%, 6% of herds had a prevalence between 40 and 50%, and 2% of herds had a prevalence of 50% or above. Positive cows had lower milk yield, protein concentration and yield, and lower Transition Cow Index than negative cows, but also higher fat concentration and yield, as well as higher somatic cell count than negative cows. Suspect cows were generally intermediate. The present analysis highlights the opportunity for elevated milk BHB monitoring at the herd level through routine BHB testing in Dairy Herd Improvement milk samples. [ABSTRACT FROM AUTHOR]

β-Hydroxybutyrate (BHB) is an important indicator for metabolic disorders in dairy cows, such as ketosis and fatty liver. Dairy cows with ketosis display oxidative stress that may be associated with high levels of BHB. The purpose of this study was to demonstrate a correlation between the high levels of BHB and oxidative stress in dairy cows with ketosis, and to investigate the molecular mechanisms underlying oxidative damage in bovine hepatocytes. The results showed that dairy cows with ketosis exhibited oxidative stress and liver damage, which was significantly correlated with plasma BHB. Similarly, high concentrations of BHB increased the oxidative stress of cow hepatocytes in vitro, resulting in the phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK), which led to increased expression, nuclear localization, and transcriptional activity of p53 and decreased Nrf2 in bovine hepatocytes. High concentrations of BHB significantly increased the expression of proapoptotic genes and significantly inhibited the expression of antiapoptotic genes. Finally, high concentrations of BHB promoted apoptosis in bovine hepatocytes. N-Acetyl-l-cysteine, glucose, and SB203580 (p38 inhibitor) significantly attenuated BHB-induced apoptotic damage in hepatocytes. These results indicate that BHB induces bovine hepatocyte apoptosis through the ROS-p38-p53/Nrf2 signaling pathway. [ABSTRACT FROM AUTHOR]

There has been growing interest in biocomposites in recent years due to the adverse environmental impact of petroleum-based thermoplastics. This work focuses on manufacturing novel green composites using a bacterial polyester, poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), with fibers of invasive plants, namely, common reed (Phragmites australis), reed canary grass (Phalaris arundinacea), and wild or water celery (Vallisneria americana). Fabrication of the biocomposites, with a range of fiber loadings, was carried out by extrusion compounding followed by injection molding of ASTM type I parts, and the effects of the fibers on the morphological and thermal properties of PHBV were characterized. Thermal analysis indicated miscibility between the fibers and PHBV for various blends, while the midpoint glass transition shifted to 5°C from 0°C for pure PHBV. Water celery fibers, specifically, were finely dispersed in the PHBV matrices as visualized by SEM, indicating good compatibility. The finely ground water celery fibers increased the fiber-matrix interactions without the use of additives or compatibilizers, reducing the production cost of the composites. This new use for invasive species can provide a dual benefit of contributing to a novel composite and reducing environmental impact. [ABSTRACT FROM AUTHOR]

D-3-Hydroxybutyrate dehydrogenase catalyzes the reversible conversion of acetoacetate and D-3-hydroxybutyrate. These ketone bodies are both energystorage forms of acetyl-CoA. In order to clarify the structural mechanisms of the catalytic reaction with the cognate substrate D-3-hydroxybutyrate and of the inhibition of the reaction by inhibitors, the enzyme from Alcaligenes faecalis has been analyzed by X-ray crystallography in liganded states with the substrate and with two types of inhibitor: malonate and methylmalonate. In each subunit of the tetrameric enzyme, the substrate is trapped on the nicotinamide plane of the bound NAD+. An OMIT map definitively shows that the bound ligand is D-3-hydroxybutyrate and not acetoacetate. The two carboxylate O atoms form four hydrogen bonds to four conserved amino-acid residues. The methyl group is accommodated in the nearby hydrophobic pocket so that the formation of a hydrogen bond from the OH group of the substrate to the hydroxy group of Tyr155 at the active centre is facilitated. In this geometry, the H atom attached to the C³ atom of the substrate in the sp³ configuration is positioned at a distance of 3.1 Å from the nicotinamide C4 atom in the direction normal to the plane. In addition, the donor-acceptor relationship of the hydrogen bonds suggests that the Tyr155 OH group is allowed to ionize by the two donations from the Ser142 OH group and the ribose OH group. A comparison of the protein structures with and without ligands indicates that the Gln196 residue of the small movable domain participates in the formation of additional hydrogen bonds. It is likely that this situation can facilitate H-atom movements as the trigger of the catalytic reaction. In the complexes with inhibitors, however, their principal carboxylate groups interact with the enzyme in a similar way, while the interactions of other groups are changed. The crucial determinant for inhibition is that the inhibitors have no active H atom at C ³. A second determinant is the Tyr155 OH group, which is perturbed by the inhibitors to donate its H atom for hydrogen-bond formation, losing its nucleophilicity. [ABSTRACT FROM AUTHOR]

The substrate specificity of 3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis with a non-native substrate, levulinic acid, was studied by analysis of the enzyme-substrate molecular interactions. The relation between structural and kinetic parameters was investigated considering the catalytic mechanism of the enzyme. The effects of key positive mutations (H144L, H144L/W187F) on the catalytic activity of the enzyme were studied by employing a surface analysis of its interatomic contacts between the enzyme and substrate atoms. The results revealed that the alteration of hydrogen bond network and rearrangement of the hydrophobic interactions between the active site and substrate molecule are the key structural basis for the change of the substrate specificity of 3-hydroxybutyrate dehydrogenase toward levulinic acid. With this approach, the structural basis for the substrate specificity of the enzyme could be elucidated in a quantitative manner. [ABSTRACT FROM AUTHOR]

Mitochondria are the site of iron utilization, wherein imported iron is incorporated into heme or iron-sulfur clusters. Previously, we showed that a cytosolic siderophore, which resembles a bacterial siderophore, facilitates mitochondrial iron import in eukaryotes, including zebrafish. An evolutionarily conserved 3-hydroxy butyrate dehydrogenase, 3-hydroxy butyrate dehydrogenase 2 (Bdh2), catalyzes a rate-limiting step in the biogenesis of the eukaryotic siderophore. We found that inactivation of bdh2 in developing zebrafish embryo results in heme deficiency and delays erythroid maturation. The basis for this erythroid maturation defect is not known. Here we show that bdh2 inactivation results in mitochondrial dysfunction and triggers their degradation by mitophagy. Thus, mitochondria are prematurely lost in bdh2-inactivated erythrocytes. Interestingly, bdh2-inactivated erythroid cells also exhibit genomic alterations as indicated by transcriptome analysis. Reestablishment of bdh2 restores mitochondrial function, prevents premature mitochondrial degradation, promotes erythroid development, and reverses altered gene expression. Thus, mitochondrial communication with the nucleus is critical for erythroid development. [ABSTRACT FROM AUTHOR]

The growth promoting effect of the bacterial storage compound poly-β-hydroxybutyrate (PHB) has been studied for young fish of high trophic level (European sea bass) and intermediate trophic level (Siberian sturgeon). Here, the effect of PHB on growth, digestive enzyme activities, body composition and diseases resistance of juvenile Nile tilapia ( Oreochromis niloticus ) of low trophic level was investigated. Although dietary PHB supplementation (5, 25 and 50 g PHB kg −1 formulated semi-purified diet) during 28 days resulted in a trend of increased weight gain, there was no significant difference in the mean final body weight (258–284 mg) when compared to the fish from the control group (on average 218 mg). Lipase activity increased significantly with about 20–40% by the supplementation of PHB in the diet, which may have led to the significant increase in total lipid content with about 10% in the PHB treatment groups. However, the profile of total (n-6) fatty acids (FAs), total monounsaturated FAs and total saturated FAs relative to the total lipid was similar among various PHB treatments. An additional challenge test on gnotobiotic Nile tilapia larvae using the pathogen Edwardsiella ictaluri gly09R showed that feeding challenged larvae with PHB-enriched Artemia nauplii resulted in a 20% higher survival as compared to the challenged control larvae. Overall, it is suggested that the trend of increased body weight gain resulted from intestinal lipid digestion, absorption and deposition and that PHB is effective as an antimicrobial agent for application in Nile tilapia larviculture. [ABSTRACT FROM AUTHOR]

Methane (CH 4 ) is a readily available feedstock for production of polyhydroxyalkanoates (PHAs). The structure and PHA production capacity of a Methylocystis -dominated methanotrophic enrichment was stable in long-term operation (>175 days) when grown exponentially under non-aseptic conditions in fill-and-draw batch cultures with ammonium as nitrogen source. Cells harvested in the draw step were incubated in the absence of nitrogen with various combinations of CH 4 and valerate to assess capacity for synthesis of poly(3-hydroxybutyrate) (P3HB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). When fed CH 4 alone, only P3HB was produced. When fed CH 4 plus valerate, PHBV was synthesized. The mol% of 3-hydroxyvalerate (3HV) increased with added valerate. Oxidation of CH 4 was required for valerate assimilation, and the fraction of CH 4 oxidized increased with increased mol% 3HV. By separating PHA accumulation from cell replication, tailored PHA-rich biomass can be generated by addition of co-substrate, while retaining a large inoculum for the next cycle of cell division. [ABSTRACT FROM AUTHOR]

Short-chain dehydrogenases/reductases (SDR) form a large enzyme superfamily playing important roles in health and disease. Furthermore, they are useful tools in biocatalysis. Unveiling the nature of the transition state for hydride transfer is a crucial undertaking toward defining the physicochemical underpinnings of catalysis by SDR enzymes, including possible contributions from quantum mechanical tunneling. Primary deuterium kinetic isotope effects can uncover the contribution from chemistry to the rate-limiting step and potentially provide detailed information on the hydride-transfer transition state in SDR-catalyzed reactions. For the latter, however, one needs to determine the intrinsic isotope effect: that which would be measured if hydride transfer were rate determining. Alas, as is the case for many other enzymatic reactions, those catalyzed by SDRs are often limited by the rate of isotope-insensitive steps, such as product release and conformational changes, which masks the expression of the intrinsic isotope effect. This can be overcome by the powerful yet underexplored method of Palfey and Fagan via which intrinsic kinetic isotope effects can be extracted from pre-steady-state kinetics data. SDRs are ideal systems to which this method can be applied. We have employed this approach to elucidate the transition states for hydride transfer catalyzed by NADH-dependent cold- and warm-adapted (R)-3-hydroxybutyrate dehydrogenase. Experimental conditions which simplify the analysis are discussed., (Copyright © 2023 Elsevier Inc. All rights reserved.)

BACKGROUND Polyhydroxyalkanoates ( PHA) are very interesting biopolymers, their most promising applications lie in biomedical science. The wettability of the surface of PHA increases their biocompatibility properties, making them suitable for such applications. RESULTS The treatment of the surface of poly (3-hydroxybutyrate) ( PHB) and poly (3-hydroxybutyrate- co-3-hydroxyvalerate) ( PHBV) films with the cutinase TfCut2 from Thermobifida fusca KW3 resulted in decrease of the water contact angle of PHB films from 90° to 36° and PHBV films from 93-98° to 50-60° with 3-hydroxyvalerate content ranging from 6 to 9 mol%, respectively. CONCLUSION A linear dependence up to 9 mol% was found between the 3-hydroxyvalerate content of the PHBV and the resulting water contact angle decrease, caused by partial hydrolysis of the surface of the films by the cutinase. PHBV films with a 3-hydroxyvalerate content of 6 mol% showed the highest gain in surface hydophilicity following treatment with the enzyme. © 2014 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

A ( R )-3-hydroxyhexanoate (3HH) composition-regulating technology for poly (3-hydroxybutyrate- co -3-hydroxyhexanoate) (PHBH) production was developed using recombinant Cupriavidus necator H16 with butyrate as a co-substrate. A new ( R )-3-hydroxyhexanoyl-CoA (( R )-3HH-CoA) synthesis pathway was designed and enhanced by replacing the PHA synthase gene ( phaC1 ) of C. necator by the phaC Ac NSDG (encoding the N149S and D171G mutant of PHA synthase from Aeromonas caviae ) and deactivation of the phaA gene (encoding (β-ketothiolase) from C. necator H16 chromosome). The effect of butyrate as co-substrate was assessed in high-cell-density fed-batch cultures of several C. necator mutants, and the 3HH fraction was successfully increased by adding butyrate to the culture. Moreover, overexpression of BktB (encoding the second β-ketothiolase with broad substrate specificity) enhanced the ( R )-3HH-CoA synthesis pathway in the phaA deactivated mutant of C. necator by promoting the condensation of acetyl-CoA and butyryl-CoA into 3-ketohexanoyl-CoA. Consequently, PHBH containing 4.2–13.0 mol % 3HH was produced from butyrate and palm kernel oil by the genetically modified C. necator H16 strains. [ABSTRACT FROM AUTHOR]

The recent discovery of a coenzyme B12-dependent acyl-coenzyme A (acyl-CoA) mutase isomerizing 3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in the mesophilic bacterium Aquincola tertiaricarbonis L108 (N. Yaneva, J. Schuster, F. Schäfer, V. Lede, D. Przybylski, T. Paproth, H. Harms, R. H. Müller, and T. Rohwerder, J Biol Chem 287:15502-15511, 2012, http://dx.doi.org/10.1074/jbc.M111.314690) could pave the way for a complete biosynthesis route to the building block chemical 2-hydroxyisobutyric acid from renewable carbon. However, the enzyme catalyzes only the conversion of the stereoisomer (S)-3-hydroxybutyryl-CoA at reasonable rates, which seriously hampers an efficient combination of mutase and well-established bacterial poly-(R)-3-hydroxybutyrate (PHB) overflow metabolism. Here, we characterize a new 2-hydroxyisobutyryl-CoA mutase found in the thermophilic knallgas bacterium Kyrpidia tusciae DSM 2912. Reconstituted mutase subunits revealed highest activity at 55°C. Surprisingly, already at 30°C, isomerization of (R)-3-hydroxybutyryl-CoA was about 7,000 times more efficient than with the mutase from strain L108. The most striking structural difference between the two mutases, likely determining stereospecificity, is a replacement of active-site residue Asp found in strain L108 at position 117 with Val in the enzyme from strain DSM 2912, resulting in a reversed polarity at this binding site. Overall sequence comparison indicates that both enzymes descended from different prokaryotic thermophilic methylmalonyl-CoA mutases. Concomitant expression of PHB enzymes delivering (R)-3-hydroxybutyryl-CoA (beta-ketothiolase PhaA and acetoacetyl-CoA reductase PhaB from Cupriavidus necator) with the new mutase in Escherichia coli JM109 and BL21 strains incubated on gluconic acid at 37°C led to the production of 2-hydroxyisobutyric acid at maximal titers of 0.7 mM. Measures to improve production in E. coli, such as coexpression of the chaperone MeaH and repression of thioesterase II, are discussed. [ABSTRACT FROM AUTHOR]

The goal of this work was the investigation of the impact of two different excimer lasers on two biocompatible and biodegradable polymers (poly- l -lactide and poly hydroxybutyrate). Both polymers find usage in medical and pharmaceutical fields. The polymers were modified by KrF and ArF excimer lasers. Subsequently the impact on surface morphology, surface chemistry changes, and thermal properties was studied by means of confocal and AFM microscopy, FTIR and XPS spectroscopy and DSC calorimetry. Under the same conditions of laser treatment it was observed that ArF laser causes more significant changes on surface chemistry, surface morphology and pattern formation on the polymers under investigation. The data obtained in this work can be used for a wide range of possible applications, in tissue engineering or in combination with metallization in electronics, e.g. for biosensors. [ABSTRACT FROM AUTHOR]

A high-protein, low-carbohydrate diet has been regarded as a dietary intervention for weight loss in the obese population. We integrated metabolomics profiles and correlation-based network analysis to reveal the difference in metabolism under diets with different protein:carbohydrate ratios. Rats were fed a control diet (moderate-protein moderate-carbohydrate: MPMC; 20 % protein, 56 % carbohydrate) or HPLC diet (high-protein low-carbohydrate: 45 % protein, 30 % carbohydrate) for 6 weeks. The fat content was equal for both diets. HPLC feeding induced weight loss and reduced adipose weight and plasma triglyceride. Compared to the MPMC diet, HPLC significantly increased plasma α-tocopherol, pyruvate, 2-oxoisocaproate, and β-hydroxybutyrate, and reduced linoleate, palmitate, α-glycerophosphate and pyroglutamic acid. The HPLC-associated urinary metabolite profile was signified with an increase in palmitate and stearate and a reduction of citrate, 2-ketoglutarate, malate, and pantothenate. Pathway analysis implicated a significant alteration of the TCA cycle in urine. Biomarker screening demonstrated that individual metabolites, including plasma urea, pyruvate, and urinary citrate, robustly distinguished the HPLC group from the MPMC group. Correlation-based network analysis enabled to demonstrate that the correlation of plasma metabolite was strengthened after the HPLC diet, while the energy-metabolism relatives 2-ketoglutarate and fumarate correlated positively with phenylalanine, methionine, and serine. The correlation network between plasma-urinary metabolites revealed a negative correlation of plasma valine with urinary β-hydroxybutyrate in MPMC rats. In HPLC rats, plasma 2-oxoisocaproate negatively correlated with urinary pyruvate and glycine. This study using metabolomics analysis revealed the systemic metabolism in response to diet treatment and identified the significantly distinct profiles associated with a HPLC diet. [ABSTRACT FROM AUTHOR]

Gluconobacter oxydans is an industrially important bacterium that possesses many uncharacterized oxidoreductases, which might be exploited for novel biotechnological applications. In this study, gene gox1801 was homologously overexpressed in G. oxydans and it was found that the relative expression of gox1801 was 13-fold higher than that in the control strain. Gox1801 was predicted to belong to the 3-hydroxyisobutyrate dehydrogenase-type proteins. The purified enzyme had a native molecular mass of 134 kDa and forms a homotetramer. Analysis of the enzymatic activity revealed that Gox1801 is a succinic semialdehyde reductase that used NADH and NADPH as electron donors. Lower activities were observed with glyoxal, methylglyoxal, and phenylglyoxal. The enzyme was compared to the succinic semialdehyde reductase GsSSAR from Geobacter sulfurreducens and the γ-hydroxybutyrate dehydrogenase YihU from Escherichia coli K-12. The comparison revealed that Gox1801 is the first enzyme from an aerobic bacterium reducing succinic semialdehyde with high catalytic efficiency. As a novel succinic semialdehyde reductase, Gox1801 has the potential to be used in the biotechnological production of γ-hydroxybutyrate. [ABSTRACT FROM AUTHOR]

Background: Long-term and intensive physical effort causes metabolic and biochemical adaptations for both athletic and non-athletic objectives. Knowing the importance of aerobic training in football players, the aim of this study was to evaluate changes in the activity of: creatinine kinase (CK), creatine kinase MB (CKMB), lactate dehydrogenase (LDH), αhydroxybutyrate dehydrogenase (HBDH), cholinesterase (ChE) and alkaline phosphatase (ALP) in response to a semi-long distance outdoor run under aerobic conditions among both female and male football players. Methods: Sixteen participants aged 21.9±2 years (women) and 18.4±0.5 years (men), all of them voluntarily recruited football players, took part in an outdoor run, the women covering a distance of 7.4±0.3 km while men covered a distance of 10.7±1.0 km. Plasma activities of the studied enzymes were determined using an appropriate diagnostic assay kit. Results: Our results indicate that total LDH activity could be a useful tool in evaluating physical fitness among athletes. We simultaneously established that ChE could not be a marker useful in assessing metabolic response to physical effort in athletes. Moreover, our results suggest that post-effort changes in ALP activity might be used to estimate early symptoms of certain vitamin deficiencies in an athlete’s diet. Conclusions: We confirmed that the assessment of activity of selected traditional diagnostic enzymatic markers provides information about muscle state after physical effort. [ABSTRACT FROM AUTHOR]

Legionella pneumophila is a pathogen widespread in aquatic environment, able to multiply both within amoebae and human macrophages. The aim of this study was to identify genes differently expressed in a spontaneous avirulent Legionella pneumophila serogroup 6 mutant, named Vir−, respect the parental strain (Vir+), and to determine their role in the loss of virulence. Protein profiles revealed some differences in Vir− proteomic maps, and among the identified proteins the undetectable 3-hydroxybutyrate dehydrogenase (BdhA) and a down-produced lipase. Both Legionella enzymes were studied before and were here further characterized at genetic level. A significant down-regulation of both genes was observed in Vir− at the transcriptional level, but the use of defined mutants demonstrated that they did not affect the intracellular multiplication. A mutant ( MS1) showed an accumulation of poly-3-hydroxybutyrate ( PHB) granules suggesting a role of bdhA gene in its degradation process. The lipase deduced amino acid sequence revealed a catalytic triad, typical of the 'lipase box' characteristic of PHB de-polymerase enzymes, that let us suppose a possible involvement of lipase in the PHB granule degradation process. Our results revealed unexpected alterations in secondary metabolic pathways possibly linking the loss of virulence to Legionella lack of energy sources. [ABSTRACT FROM AUTHOR]

4-Hydroxybutyryl-coenzyme A (CoA) dehydratase (4HBD) from Clostridium aminobutyricum catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA and the irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. 4HBD is an oxygen-sensitive homotetrameric enzyme with one [4Fe-4S]2+ cluster and one flavin adenine dinucleotide (FAD) in each subunit. Upon the addition of crotonyl-CoA or the analogues butyryl-CoA, acetyl-CoA, and CoA, UV-visible light and electron paramagnetic resonance (EPR) spectroscopy revealed an internal one-electron transfer to FAD and the [4Fe-4S]2+ cluster prior to hydration. We describe an active recombinant 4HBD and variants produced in Escherichia coli. The variants of the cluster ligands (H292C [histidine at position 292 is replaced by cysteine], H292E, C99A, C103A, and C299A) had no measurable dehydratase activity and were composed of monomers, dimers, and tetramers. Variants of other potential catalytic residues were composed only of tetramers and exhibited either no measurable (E257Q, E455Q, and Y296W) hydratase activity or<1% (Y296F and T190V) dehydratase activity. The E455Q variant but not the Y296F or E257Q variant displayed the same spectral changes as the wild-type enzyme after the addition of crotonyl-CoA but at a much lower rate. The results suggest that upon the addition of a substrate, Y296 is deprotonated by E455 and reduces FAD to FADH., aided by protonation from E257 via T190. In contrast to FADH., the tyrosyl radical could not be detected by EPR spectroscopy. FADH. appears to initiate the radical dehydration via an allylic ketyl radical that was proposed 19 years ago. The mode of radical generation in 4HBD is without precedent in anaerobic radical chemistry. It differs largely from that in enzymes, which use coenzyme B12, S-adenosylmethionine, ATP-driven electron transfer, or flavin-based electron bifurcation for this purpose. [ABSTRACT FROM AUTHOR]

Uncontrollable displacements that greatly affect the concentration of active agents at the target tissues are among a major limitation of the use of microparticulate drug delivery systems (DDS). Under this context a biphasic injectable DDS combining poly(hydroxybutyrate- co -hydroxyvalerate) (PHBV) microparticles (MPs) and a gellan gum (GG) injectable hydrogel is herein proposed for the localized delivery and long-term retention of MPs carrying hydrophilic and hydrophobic model active agents. A double emulsion-solvent evaporation method was adopted to develop the PHBV MPs, carrying bovine serum albumin (BSA) or dexamethasone (Dex) as hydrophilic and hydrophobic active agents’ models, respectively. Moreover, this method was modified, together with the properties of the hydrogel to tailor the delivery profile of the active agents. Variations of the composition of the organic phase during the process allowed tuning surface topography, particle size distribution and core porosity of the PHBV MPs and, thus, the in vitro release profile of Dex but not of BSA. Besides, after embedding hydrogels of higher GG concentration led to a slower and more sustained release of both active agents, independently of the processing conditions of the microparticulate system. [ABSTRACT FROM AUTHOR]

The effect of hexahistidine-tag (His-tag) location at either the C or N-terminus on the catalytic activity of 3-hydroxybutyrate dehydrogenase (3HBDH) from Alcaligenes faecalis was studied. The kinetic parameters of 3HBDHs with C and N-terminal His-tags were investigated, and the enzyme with an N-terminal His-tag was found to have approximately 1,200-fold higher catalytic efficiency than its C-terminal counterpart. Furthermore, the effect of His-tag location on the catalytic activity of 3 engineered variants of 3HBDH that were previously developed for the conversion of levulinic acid to 4-hydroxyvaleric acid was also investigated. All of the N-terminal variants exhibited higher catalytic efficiency for levulinic acid than did the C-terminal counterparts. The structural basis of the His-tag effect was studied by investigating the structure of 3HBDH obtained from in silico His-tag modification, and the results revealed that the modification of the C-terminal structure could deform the hinge region of the active site entry loop, disrupting the catalytic motion of the enzyme. In contrast, due to the location of the N-terminus far from the active site of the enzyme, the catalytic activity of the enzyme was not severely affected by the N-terminal His-tag. [ABSTRACT FROM AUTHOR]

Siderophores are best known as small iron-binding molecules that facilitate iron uptake in bacteria and fungi. In our previous study, we demonstrated that eukaryotes also produce siderophore-like molecules via a remarkably conserved biosynthetic pathway. A member of the short-chain dehydrogenase family of reductases, 3-hydroxybutyrate dehydrogenase-2, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore. Physiologically, depletion of the mammalian siderophore by inhibiting expression of the 3-hydroxybutyrate dehydrogenase-2 gene ( Bdh2) results in abnormal accumulation of intracellular iron, increased oxidative stress, and mitochondrial iron deficiency. Thus, the mammalian siderophore is an important regulator of cellular iron homeostasis. The cellular iron storage protein ferritin also regulates iron metabolism and protects cells from oxidative stress. Depletion of ferritin results in intracellular iron accumulation, predisposes to oxidative stress, and confers a growth advantage to cells. We therefore hypothesize that the siderophore and ferritin coregulate cellular iron metabolism/homeostasis in eukaryotes. We tested this prediction by depleting both the siderophore and ferritin. This resulted in a marked accumulation of cellular iron, and caused increased sensitivity to oxidants. Interestingly, cells lacking both the siderophore and ferritin proliferated at a higher rate than cells lacking either of these components alone. Taken together, our findings suggest that the siderophore and ferritin synergistically regulate cellular iron levels. [ABSTRACT FROM AUTHOR]

Genes encoding 3-hydroxybutyrate oligomer hydrolase (PhaZc) and 3-hydroxybutyrate dehydrogenase (Hbd) were isolated from Paracoccus denitrificans. PhaZc and Hbd were overproduced as His-tagged proteins in Escherichia coli and purified by affinity and gel filtration chromatography. Purified His-tagged proteins had molecular masses of 31 kDa and 120 kDa (a tetramer of 29-kDa subunits). The His-tagged PhaZc hydrolyzed not only 3-hydroxybutyrate oligomers but also 3-hydroxyvalerate oligomers. The His-tagged Hbd catalyzed the dehydrogenation of 3-hydroxyvalerate as well as 3-hydroxybutyrate. When both enzymes were included in the same enzymatic reaction system with 3-hydroxyvalerate dimer, sequential reactions occurred, suggesting that PhaZc and Hbd play an important role in the intracellular degradation of poly(3-hydroxyvalerate). When the phaZc gene was disrupted in P. denitrificans by insertional inactivation, the mutant strain lost PhaZc activity. When the phaZc-disrupted P. denitrificans was complemented with phaZc, PhaZc activity was restored. These results suggest that P. denitrificans carries a single phaZc gene. Disruption of the phaZc gene in P. denitrificans affected the degradation rate of PHA. [ABSTRACT FROM AUTHOR]

Objectives: This study aimed to investigate the clinical and biochemical profiles of patients with imported malaria infection between 1 January 2011 and 30 April 2022 and admitted to the Fourth People's Hospital of Nanning., Methods: This cohort study enrolled 170 patients with conformed imported malaria infection. The clinical and biochemical profiles of these participants were analyzed with malaria parasite clearance, and signs and symptoms related to malaria disappearance were defined as the primary outcome. A multivariable logistic regression model was used to evaluate the odds ratios (ORs) with 95% confidence intervals (CIs) for cerebral malaria. The Cox model was used to estimate the hazard ratios (HRs) with 95% CIs for parasite clearance., Results: Adenosine deaminase and parasitemia were found to be independent risk factors for severe malaria in patients with imported malaria (OR = 1.0088, 95% CI: 1.0010-1.0167, p = 0.0272 and OR = 2.0700, 95% CI: 1.2584-3.4050, p = 0.0042, respectively). A 0.5-standard deviation (SD) increase of variation for urea (HR = 0.6714, 95% CI: 0.4911-0.9180), a 0.5-SD increase of variation for creatinine (HR = 0.4566, 95% CI: 0.2762-0.7548), a 0.25-SD increase of variation for albumin (HR = 0.4947, 95% CI: 0.3197-0.7653), a 0.25-SD increase of variation for hydroxybutyrate dehydrogenase (HR = 0.6129, 95% CI: 0.3995-0.9402), and a 1.0-SD increase of variation for ferritin (HR = 0.5887, 95% CI: 0.3799-0.9125) were associated with a higher risk for increased parasite clearance duration than a low-level change., Conclusions: Aspartate aminotransferase, urea, creatinine, albumin, hydroxybutyrate dehydrogenase, and ferritin are useful biochemical indicators in routine clinical practice to evaluate prognosis for imported malaria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bi, Lin, Luo, Lin, Tang, Luo, Lu and Huang.)

Background: Recent studies have pointed to the anti-tumour effects of a ketogenic diet (KD) in cancer. It is believed that patients with low ketolytic Enzymes gene expression levels are more sensitive and may respond better to the KD therapy. However, the ketolytic Enzymes gene expression levels and their association with mitochondrial activity and content in oral squamous cell carcinoma (OSCC) is not yet obvious. Therefore, the aim of this study was to explore the potential use of ketolytic enzymes as biomarkers for mitochondrial activity and content. Materials and Methods: Here we aimed to compare the mRNA expression levels of ketolytic enzymes (ACAT1, BDH1, BDH2 and OXCT1) between tumour and adjacent pre-tumor tissues of 16 OSCC patients. Additionally, we examined the association of the mitochondrial ketolytic enzymes, including ACAT1, OXCT1, and BDH1 gene expression with mitochondrial activity and content. Results: Our findings did not show any significant difference in ketolytic gene expression levels between tumour and pre-tumor tissues of OSCC patients. ACAT1 and BDH1 mRNA expression levels were significantly correlated with the mRNA level of ND2 in tumour of OSCC patients. The mRNA levels of ACAT1, BDH1 and BDH2 were not correlated with the mRNA expression of 16srRNA. Conclusion: Our data suggest that mRNA gene expression levels of BDH1 and ACAT1 correlate with the mitochondrial activity in tumour of OSCC patients. BDH2 mRNA level significantly anti-correlate with tumour grade. We offer clues on the potential of ACAT1 as a biomarker of mitochondrial activity, but future studies are needed to establish this concept.

Red coloration is a salient feature of the natural world. Many vertebrates produce red color by converting dietary yellow carotenoids into red ketocarotenoids via an unknown mechanism. Here, we show that two enzymes, cytochrome P450 2J19 (CYP2J19) and 3-hydroxybutyrate dehydrogenase 1-like (BDH1L), are sufficient to catalyze this conversion. In birds, both enzymes are expressed at the sites of ketocarotenoid biosynthesis (feather follicles and red cone photoreceptors), and genetic evidence implicates these enzymes in yellow/red color variation in feathers. In fish, the homologs of CYP2J19 and BDH1L are required for ketocarotenoid production, and we show that these enzymes are sufficient to produce ketocarotenoids in cell culture and when ectopically expressed in fish skin. Finally, we demonstrate that the red-cone-enriched tetratricopeptide repeat protein 39B (TTC39B) enhances ketocarotenoid production when co-expressed with CYP2J19 and BDH1L. The discovery of this mechanism of ketocarotenoid biosynthesis has major implications for understanding the evolution of color diversity in vertebrates., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Poly-3-hydroxybutyrate (PHB)-derived plastics are polymer materials with excellent biodegradability, being insoluble in water and relatively resistant to hydrolysis. There is a need for a method capable of synthesizing 3-hydroxybutyrate, a monomer of PHB, from a renewable material. In this work, visible-light driven 3-hydroxybutyrate from CO 2 and acetone with the system consisting of triethanolamine, water-soluble zinc porphyrin, pentamethylcyclopentadienyl coordinated rhodium complex, NAD + and a cell extract containing acetone carboxylase and 3-hydroxybutyrate dehydrogenase from Rhodobacter capsulatus SB1003 cultured in acetone-bicarbonate medium is established. In particular, the conversion yield for acetone to 3-hydroxybutyrate was improved up to 81% in this system after 7 h irradiation.

Abstract: Enzymatic reduction of levulinic acid (LA) was performed for the synthesis of 4-hydroxyvaleric acid (4HV) – a monomer of bio-polyester and a precursor of bio-fuels – using 3-hydroxybutyrate dehydrogenase (3HBDH) from Alcaligenes faecalis. Due to the catalytic inactivity of the wild-type enzyme toward LA, engineering of the substrate specificity of the enzyme was performed. A rational design approach with molecular docking simulation was applied, and a double mutant, His144Leu/Trp187Phe, which has catalytic activity (k cat/K m =578.0min−1 M−1) toward LA was generated. Approximately 57% conversion of LA to 4HV was achieved with this double mutant in 24h, while no conversion was achieved with the wild-type enzyme. [Copyright &y& Elsevier]

A disposable amperometric biosensor for ketone 3-β-hydroxybutyrate (3HB) has been developed successfully. The sensor is based on a screen-printed carbon electrode containing Meldola's Blue (MB) and sensing components containing nicotinamide adenine dinucleotide (NAD) and 3-β-hydroxybutyrate dehydrogenase (3HBDH) immobilized in mesoporous silica (FSM8.0) using an aqueous photo-cross-linkable polymer matrix of polyvinyl alcohol (O-391), and it requires only a small sample volume of 10 μL for the measurement. The behavior of a resulting biosensor, i.e., 3HBDH-FSM8.0/NAD/MB-SPCE, was examined in terms of NAD concentration for construction, pH, applied potential, operational range, selectivity, and storage stability. The sensor showed an optimum response at a pH of 7.6 and at an applied potential of −50 mV. The determination range and the response time for 3HB were from 30 μM to 8 mM and approximately 30 s, respectively. In addition, the sensor was quite stable and maintained >90 % of its initial response after being stored for over 6 months. This result implies that our method provides a novel biosensor for ketone 3-β-hydroxybutyrate which is easy-to-use, cost-effective, and has good reproducibility, which are vital for commercial purposes. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: A putative β-hydroxybutyrate dehydrogenase (βHBDH) ortholog was identified in Trypanosoma brucei, the unicellular eukaryotic parasite responsible for causing African Sleeping Sickness. The trypanosome enzyme has greater sequence similarity to bacterial sources of soluble βHBDH than to membrane-bound Type I βHBDH found in higher eukaryotes. The βHBDH gene was cloned from T. brucei genomic DNA and active, recombinant His-tagged enzyme (His10-TbβHBDH) was purified to approximate homogeneity from E. coli. βHBDH catalyzes the reversible NADH-dependent conversion of acetoacetate to d-3-hydroxybutyrate. In the direction of d-3-hydroxybutyrate formation, His10-TbβHBDH has a k cat value of 0.19s−1 and a K M value of 0.69mM for acetoacetate. In the direction of acetoacetate formation, His10-TbβHBDH has a k cat value of 11.2s−1 and a K M value of 0.65mM for d-3-hydroxybutyrate. Cofactor preference was examined and His10-TbβHBDH utilizes both NAD(H) and NADP(H) almost equivalently, distinguishing the parasite enzyme from other characterized βHBDHs. Furthermore, His10-TbβHBDH binds NAD(P)+ in a cooperative fashion, another unique characteristic of trypanosome βHBDH. The apparent native molecular weight of recombinant His10-TbβHBDH is 112kDa, corresponding to tetramer, as determined through size exclusion chromatography. RNA interference studies in procyclic trypanosomes were carried out to evaluate the importance of TbβHBDH in vivo. Upon knockdown of TbβHBDH, a small reduction in parasite growth was observed suggesting βHBDH has an important physiological role in T. brucei. [Copyright &y& Elsevier]