Controlled production of a polyhydroxyalkanoate (PHA) tetramer containing different mole fraction of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3 HV), 4 HV and 5 HV units by engineered Cupriavidus necator.

Polyhydroxyalkanoates (PHAs) are promising alternatives to existing petrochemical-based plastics because of their bio-degradable properties. However, the limited structural diversity of PHAs has hindered their application. In this study, high mole-fractions of Poly (39 mol% 3HB-co-17 mol% 3 HV-co-44 mol% 4 HV) and Poly (25 mol% 3HB-co-75 mol% 5 HV) were produced from 4- hydroxyvaleric acid and 5-hydroxyvaleric acid, using Cupriavidus necator PHB-4 harboring the gene phaCBP-M-CPF4 with modified sequences. In addition, the complex toxicity of precursor mixtures was tested, and it was confirmed that the engineered C. necator was capable of synthesizing Poly (32 mol% 3HB-co-11 mol% 3 HV-co-25 mol% 4 HV-co-32 mol% 5 HV) at low mixture concentrations. Correlation analyses of the precursor ratio and the monomeric mole fractions indicated that each mole fractions could be precisely controlled using the precursor proportion. Physical property analysis confirmed that Poly (3HB-co-3 HV-co-4 HV) is a rubber-like amorphous polymer and Poly (3HB-co-5 HV) has a high tensile strength and elongation at break. Poly (3HB-co-3 HV-co-4 HV-co-5 HV) had a much lower glass transition temperature than the co-, terpolymers containing 3 HV, 4 HV and 5 HV. This study expands the range of possible physical properties of PHAs and contributes to the realization of custom PHA production by suggesting a method for producing PHAs with various physical properties through mole-fraction control of 3 HV, 4 HV and 5 HV.

Maximization of 3-hydroxyhexanoate fraction in poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) using lauric acid with engineered Cupriavidus necator H16.

As polyhydroxybutyrate (P(3HB)) was struggling with mechanical properties, efforts have been directed towards increasing mole fraction of 3-hydroxyhexanoate (3HHx) in P(3HB-co-3HHx) to improve the properties of polyhydroxyalkanoates (PHAs). Although genetic modification had significant results, there were several issues related to cell growth and PHA production by deletion of PHA synthetic genes. To find out easier strategy for high 3HHx mole fraction without gene deletion, Cupriavidus necator H16 containing phaC2Ra-phaACn-phaJ1Pa was examined with various oils resulting that coconut oil gave the highest 3HHx mole fraction. When fatty acid composition analysis with GC-MS was applied, coconut oil was found to have very different composition from other vegetable oil containing very high lauric acid (C12) content. To find out specific fatty acid affecting 3HHx fraction, different fatty acids from caproic acid (C6) to stearic acid (C18) was evaluated and the 3HHx mole fraction was increased to 26.5 ± 1.6 % using lauric acid. Moreover, the 3HHx mole fraction could be controlled from 9 % to 31.1 % by mixing bean oil and lauric acid with different ratios. Produced P(3HB-co-3HHx) exhibited higher molecular than P(3HB-co-3HHx) from phaB-deletion mutant. This study proposes another strategy to increase 3HHx mole fraction with easier way by modifying substrate composition without applying deletion tools.

Tailoring the Nucleation and Crystallization Rate of Polyhydroxybutyrate by Copolymerization.

In the polyester family, the biopolymer with the greatest industrial potential could be poly(3-hydroxybutyrate) (PHB), which can be produced nowadays biologically or chemically. The scarce commercial use of PHB derives from its poor mechanical properties, which can be improved by incorporating a flexible aliphatic polyester with good mechanical performance, such as poly(ε-caprolactone) (PCL), while retaining its biodegradability. This work studies the structural, thermal, and morphological properties of block and random copolymers of PHB and PCL. The presence of a comonomer influences the thermal parameters following nonisothermal crystallization and the kinetics of isothermal crystallization. Specifically, the copolymers exhibit lower melting and crystallization temperatures and present lower overall crystallization kinetics than neat homopolymers. The nucleation rates of the PHB components are greatly enhanced in the copolymers, reducing spherulitic sizes and promoting transparency with respect to neat PHB. However, their spherulitic growth rates are depressed so much that superstructural growth becomes the dominating factor that reduces the overall crystallization kinetics of the PHB component in the copolymers. The block and random copolymers analyzed here also display important differences in the structure, morphology, and crystallization that were examined in detail. Our results show that copolymerization can tailor the thermal properties, morphology (spherulitic size), and crystallization kinetics of PHB, potentially improving the processing, optical, and mechanical properties of PHB.

Using an aromatic amide as nucleating agent to enhance the crystallization and dimensional stability of poly(3-hydroxybutyrate-co-3-hydroxyhexanate).

Biosynthesized poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) has emerged as a promising biodegradable polymer with a great potential to compete with traditional petroleum-based plastics, however, the poor crystallization ability makes it challenge to transform into high-performance products via common melt-processing methods. Herein, we demonstrate that N,N'-dicyclohexyl-2,6-naphthalenedicarboxamide (TMB) can serve as an efficient nucleating agent to significantly enhance the crystallization and resulting storage stability of PHBHHx. The results indicate that PHBHHx with small amounts of TMB (0.3-0.5 wt%) can crystallize completely even under a rapid cooling rate of 100 °C/min and the isothermal crystallization time is greatly reduced. As a result, the crystallinity of the injection-molded PHBHHx products is increased from 24.5 % to 39.5 %, without secondary crystallization after being stored at room temperature for 6 h. The products exhibit superior dimensional stability and the post-shrinkage can be decreased to as low as 0.1 %. Our work offers a feasible method to develop high-performance PHBHHx materials with remarkably enhanced crystallization ability.

Quantification and Proteomic Characterization of ß-Hydroxybutyrylation Modification in the Hearts of AMPKα2 Knockout Mice.

AMP-activated protein kinase alpha 2 (AMPKα2) regulates energy metabolism, protein synthesis, and glucolipid metabolism myocardial cells. Ketone bodies produced by fatty acid ß-oxidation, especially ß-hydroxybutyrate, are fatty energy-supplying substances for the heart, brain, and other organs during fasting and long-term exercise. They also regulate metabolic signaling for multiple cellular functions. Lysine ß-hydroxybutyrylation (Kbhb) is a ß-hydroxybutyrate-mediated protein posttranslational modification. Histone Kbhb has been identified in yeast, mouse, and human cells. However, whether AMPK regulates protein Kbhb is yet unclear. Hence, the present study explored the changes in proteomics and Kbhb modification omics in the hearts of AMPKα2 knockout mice using a comprehensive quantitative proteomic analysis. Based on mass spectrometry (LC-MS/MS) analysis, the number of 1181 Kbhb modified sites in 455 proteins were quantified between AMPKα2 knockout mice and wildtype mice; 244 Kbhb sites in 142 proteins decreased or increased after AMPKα2 knockout (fold change >1.5 or <1/1.5, p < 0.05). The regulation of Kbhb sites in 26 key enzymes of fatty acid degradation and tricarboxylic acid cycle was noted in AMPKα2 knockout mouse cardiomyocytes. These findings, for the first time, identified proteomic features and Kbhb modification of cardiomyocytes after AMPKα2 knockout, suggesting that AMPKα2 regulates energy metabolism by modifying protein Kbhb.

ß-hydroxybutyrate as an Anti-Aging Metabolite.

The ketone bodies, especially ß-hydroxybutyrate (ß-HB), derive from fatty acid oxidation and alternatively serve as a fuel source for peripheral tissues including the brain, heart, and skeletal muscle. ß-HB is currently considered not solely an energy substrate for maintaining metabolic homeostasis but also acts as a signaling molecule of modulating lipolysis, oxidative stress, and neuroprotection. Besides, it serves as an epigenetic regulator in terms of histone methylation, acetylation, ß-hydroxybutyrylation to delay various age-related diseases. In addition, studies support endogenous ß-HB administration or exogenous supplementation as effective strategies to induce a metabolic state of nutritional ketosis. The purpose of this review article is to provide an overview of ß-HB metabolism and its relationship and application in age-related diseases. Future studies are needed to reveal whether ß-HB has the potential to serve as adjunctive nutritional therapy for aging.

Microbiome-Metabolomics Reveals Endogenous Alterations of Energy Metabolism by the Dushen Tang to Attenuate D-Galactose-Induced Memory Impairment in Rats.

Aging affects the brain function in elderly individuals, and Dushen Tang (DST) is widely used for the treatment of senile diseases. In this study, the protective effect of DST against memory impairment was evaluated through the Morris water maze (MWM) test and transmission electron microscopy (TEM). A joint analysis was also performed using LC-MS metabolomics and the microbiome. The MWM test showed that DST could significantly improve the spatial memory and learning abilities of rats with memory impairment, and the TEM analysis showed that DST could reduce neuronal damage in the hippocampus of rats with memory impairment. Ten potential biomarkers involving pyruvate metabolism, the synthesis and degradation of ketone bodies, and other metabolic pathways were identified by the metabolomic analysis, and it was found that 3-hydroxybutyric acid and lactic acid were involved in the activation of cAMP signaling pathways. The 16S rDNA sequencing results showed that DST could regulate the structure of the gut microbiota in rats with memory impairment, and these effects were manifested as changes in energy metabolism. These findings suggest that DST exerts a good therapeutic effect on rats with memory impairment and that this effect might be mainly achieved by improving energy metabolism. These findings might lead to the potential development of DST as a drug for the treatment of rats with memory impairment.

Enhanced crystallization and storage stability of mechanical properties of biosynthesized poly (3-hydroxybutyrate-co-3-hydroxyhexanate) induced by self-nucleation.

The poor mechanical properties induced by unsatisfactory crystallization ability limit the widespread use of biosynthesized poly (3-hydroxybutyrate-co-3-hydroxyhexanate) (PHBH). In this work, poly (3-hydroxybutyrate) (PHB) with a high melting point was first used as a homogeneous nucleating agent to increase the crystallization rate of PHBH by a self-nucleation method with a wider processing temperature window and crystallization kinetics and storage stability of mechanical properties of the PHBH/PHB mixtures were systematically investigated. By controlling the processing temperature and PHB content, the crystal nucleus density and crystallization rate of PHBH could be greatly increased while secondary crystallization was inhibited. When the processing temperature is 185 °C and PHB content is 20 wt%, the half crystallization time is shortened by 96% and the crystallinity was increased to 37.2%. Meanwhile, the mechanical performance of PHBH and its storage stability are greatly improved. Therefore, this work provides a simple and efficient way to improve the crystallization and mechanical performance of PHBH, which is expected to be applied to industrial production on a large scale.

Cellulosic nanofibers filled poly(ß-hydroxybutyrate): Relations between viscoelasticity of composites and aspect ratios of nanofibers.

Dispersion states are vital for fibrous nanocelluloses to be used as reinforcements for polymers, which is highly dependent on geometry of nanocelluloses. Three types of nanocelluloses with various fiber aspect ratios were used to prepare target composite samples with poly(ß-hydroxybutyrate) in this work. Viscoelasticity/elastoplasticity were used as probes to detect the flexibility-morphology relations of nanocelluloses in polymer. Cellulose nanocrystals (aspect ratio = 8) were rigid in polymer, retaining their rod-like shape, whereas bacterial celluloses (aspect ratio = 600) fully flexible, forming closely networked structure, and cellulose nanofibers (aspect ratio = 70) semi-flexible, dispersing as loosely flocculated clusters. Owing to these differences, the viscoelastic flow and elastoplastic deformation of three kinds of composites differed from one another. The strain-scaling and hysteresis work-scaling behaviors were then used to establish relaxation scale-structure correlations of target samples. This work provides interesting information around regulating the dispersion of nanocelluloses in polymer composites by tailoring aspect ratios of nanocelluloses.

3-Hydroxybutyrate as a Metabolite and a Signal Molecule Regulating Processes of Living Organisms.

3-hydroxybutyrate (3-HB) as a very important metabolite occurs in animals, bacteria and plants. It is well known that in animals, 3-HB is formed as a product of the normal metabolism of fatty acid oxidation and can therefore be used as an energy source in the absence of sufficient blood glucose. In microorganisms, 3-HB mainly serves as a substrate for the synthesis of polyhydroxybutyrate, which is a reserve material. Recent studies show that in plants, 3-HB acts as a regulatory molecule that most likely influences the expression of genes involved in DNA methylation, thereby altering DNA methylation levels. Additionally, in animals, 3-HB is not only an intermediate metabolite, but also an important regulatory molecule that can influence gene expression, lipid metabolism, neuronal function, and overall metabolic rate. Some of these effects are the direct effects of 3-HB itself, while others are indirect effects, regulated by the metabolites into which 3-HB is converted. One of the most important regulatory functions of 3-HB is the inhibition of the activity of histone deacetylases and thus the epigenetic regulation of many genes. Due to the number of functions of this compound, it also shows promising therapeutic properties.

Rapid analysis of polyhydroxyalkanoate contents and its monomer compositions by pyrolysis-gas chromatography combined with mass spectrometry (Py-GC/MS).

Here, we report an analysis method for determining PHA (polyhydroxyalkanoates) contents and their monomer composition in microbial cells based on pyrolysis gas chromatography combined with mass spectrometry (Py-GC/MS). Various kinds of microbial cells accumulating different PHA contents and monomer compositions were prepared through the cultivation of Ralstonia eutropha and recombinant Escherichia coli. Py-GC/MS could analyse these samples in a short time without complicated pretreatment steps. Characteristic peaks such as 2-butenoic acid, 2-pentenoic acid, and hexadecanoic acid regarding PHA compositions and cell components were identified. Considering constituents of cells and ratios of peak areas of dehydrated monomers to hexadecanoic acid, a simple equation for estimation of PHA contents in microbial cells was derived. Also, monomer compositions of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in R. eutropha could be successfully determined based on peak area of 2-butenoic acid and 2-pentenoic acid of Py-GC/MS, which are the corresponding species of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) in PHBV. Correlation of results between GC-FID and Py-GC/MS could be fitted very well. This method shows similar results for the samples obtained from same experimental conditions, allowing rapid and reliable analysis. Py-GC/MS can be a promising tool to rapidly screen PHA-positive strains based on polymer contents along with monomer compositions.

Production of polyhydroxyalkanoate copolymers containing 4-hydroxybutyrate in engineered Bacillus megaterium.

Despite being used as a common platform for the commercial production of many biochemicals, Bacilli are often overlooked as a source of industrial polyhydroxyalkanoates (PHAs), biodegradable plastic replacements. In addition to having a robust expression system, the lack of lipopolysaccharides and ease of lysis make Bacilli an attractive host for the production of PHAs. In this work, a Bacillus megaterium strain was engineered to generate poly(3-hydroxybutyrate-co-4-hydroxybutryate) (P[3HB-co-4HB]) copolymers, which are among the most useful and industrially-relevant copolymers. These copolymers had lower modulus and increased toughness, thus making the copolymer suitable for a broader range of applications. Due to high metabolic flux through succinate, the engineered B. megaterium strain produced P(3HB-co-4HB) with >10% mol fraction 4HB from glucose, without the use of highly regulated and expensive precursors or potentially damaging truncation of central biochemical pathways.

In vitro and in vivo evaluation of poly-3-hydroxybutyric acid-sodium alginate as a core-shell nanofibrous matrix with arginine and bacitracin-nanoclay complex for dermal reconstruction of excision wound.

The protective layer of the body, the skin is often prone to damage due to several factors like trauma, accidents, stress and hazardous exposure. This requires the skin to regenerate itself which is a finely regulated process. To hasten the process and prevent further damage, the dressing material is of prime importance. Herein, we fabricated poly-3-hydroxybutyric acid (P)-sodium alginate (S)-(core-shell) nanofibrous matrix as protective scaffold for the skin tissue regeneration in excision wound model. The arginine (A) and layered double hydroxides-bacitracin (LB) were incorporated into the core and shell of the nanofibrous matrix using co-axial electrospinning. The core-shell nanofibers assist in the synergistic, controlled delivery of L-arginine, and bacitracin with major role in the protein synthesis, cell signaling and infection control at wound site respectively. In vitro biocompatibility was confirmed by testing on dermal fibroblasts. Furthermore, in vivo studies revealed the synergistic effect of both the components in active healing of wounds. The biochemical, histochemical and immunohistochemical studies reveal that the arginine loaded scaffold aided cellular migration and proliferation. These results suggest that the simultaneous existence of the drug bacitracin-nano clay complex and L-arginine in the shell and core respectively has conferred interesting dynamic properties to the scaffold towards wound healing.

Microbial oversecretion of (R)-3-hydroxybutyrate oligomer with diethylene glycol terminal as a macromonomer for polyurethane synthesis.

Poly((R)-3-hydroxybutyrate) (P(3HB)) is a polyester that is synthesized and accumulated in many prokaryotic cells. Recently, a new culture method for the secretion of the intracellularly synthesized (R)-3-hydroxybutyrate oligomer (3HBO) from recombinant Escherichia coli cells was developed. In this study, we attempted to produce microbial 3HBO capped with a diethylene glycol terminal (3HBO-DEG) as a macromonomer for polymeric materials. First, we prepared recombinant E. coli strains harboring genes encoding various polyhydroxyalkanoate (PHA) synthases (PhaC, PhaEC or PhaRC) that can incorporate chain transfer (CT) agents such as DEG into the polymer's terminal and generate CT end-capped oligomers. To this end, each strain was cultivated under DEG supplemental conditions, and the synthesis of 3HBO-DEG was confirmed. As a result, the highest secretory production of 3HBO-DEG was observed for the PHA synthase derived from Bacillus cereus YB-4 (PhaRCYB4). To evaluate the usability of the secreted 3HBO-DEG as a macromonomer, 3HBO-DEG was purified from the culture medium and polymerized with 4,4'-diphenylmethane diisocyanate as a spacer compound. Characterization of the polymeric products revealed that 3HBO-based polyurethane was successfully obtained and was a flexible and transparent noncrystalline polymer, unlike P(3HB). These results suggested that microbial 3HBO-DEG is a promising platform building block for synthesizing polyurethane and various other polymers.

Supernucleation, crystalline structure and thermal stability of bacterially synthesized poly(3-hydroxybutyrate) polyester tailored by thymine as a biocompatible nucleating agent.

Naturally occurring thymine (TM) was incorporated into bacterial poly(3-hydroxybutyrate) (PHB) polyester to fabricate a novel and green biocomposite. Both 0.5% and 1% TM exhibit supernucleation effect on PHB, and crystallization kinetics suggests TM significantly increased Tc and Xc, and substantially shortened t1/2 of PHB. Epitaxial nucleation caused by a perfect crystal lattice matching between PHB and TM, was proposed to elucidate nucleation mechanism of PHB. Hydrogen bond interaction exists between CO, C-O-C groups of PHB and -CH3 (or -CH)/-NH- group of TM. TM interacted with CO group of PHB crystalline phase rather than that of amorphous one. In addition, two new IR crystalline bands assigned to C-O-C group of PHB appeared in the presence of TM, which arises from shift of two amorphous ones, respectively. TM enhanced onset thermal degradation temperature of PHB, mainly attributed to increased degree of crystallinity of PHB and flame retardance effect of TM.

Properties of scaffolds prepared by fused deposition modeling of poly(hydroxyalkanoates).

Poly(hydroxyalkanoates) are biodegradable and biocompatible polymers suitable for tissue engineering. Fused deposition modeling (FDM) belongs to modern rapid prototyping techniques for the fabrication of scaffolds. In this work, poly(3-hydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) were tested for FDM. Thermal and rheological properties of industrial PHAs were compared with poly(lactic acid) (PLA), which is a biodegradable polymer commonly used for FDM. The massive decrease in viscosity and loss of molecular weight of PHB and PHBV precluded their use for FDM. On the other hand, the thermal stability of PHBH was comparable to that of PLA. PHBH scaffolds prepared by FDM exhibited excellent mechanical properties, no cytotoxicity and large proliferation of mouse embryonic fibroblast cells within 96 h. The hydrolytic degradation of PHBH and PLA scaffolds tested in synthetic gastric juice for 52 days confirmed a faster degradation of PHBH than PLA. The decrease in molecular weight confirmed the first-order kinetics with a slightly higher (0.0169 day-1) degradation rate constant for PHBH as compared to the value (0.0107 day-1) obtained for PLA. These results indicate that PHBH could be used to produce scaffolds by FDM with application in tissue engineering.

Ketone Body, 3-Hydroxybutyrate: Minor Metabolite - Major Medical Manifestations.

Ketone bodies - 3-hydroxybutyrate (3-OHB), acetoacetate, and acetone - are ancient, evolutionarily preserved, small fuel substrates, which uniquely can substitute and alternate with glucose under conditions of fuel and food deficiency. Once canonized as a noxious, toxic pathogen leading to ketoacidosis in patients with diabetes, it is now becoming increasingly clear that 3-OHB possesses a large number of beneficial, life-preserving effects in the fields of clinical science and medicine. 3-OHB, the most prominent ketone body, binds to specific hydroxyl-carboxylic acid receptors and inhibits histone deacetylase enzymes, free fatty acid receptors, and the NOD-like receptor protein 3 inflammasome, tentatively inhibiting lipolysis, inflammation, oxidative stress, cancer growth, angiogenesis, and atherosclerosis, and perhaps contributing to the increased longevity associated with exercise and caloric restriction. Clinically ketone bodies/ketogenic diets have for a long time been used to reduce the incidence of seizures in epilepsy and may have a role in the treatment of other neurological diseases such as dementia. 3-OHB also acts to preserve muscle protein during systemic inflammation and is an important component of the metabolic defense against insulin-induced hypoglycemia. Most recently, a number of studies have reported that 3-OHB dramatically increases myocardial blood flow and cardiac output in control subjects and patients with heart failure. At the moment, scientific interest in ketone bodies, in particular 3-OHB, is in a hectic transit and, hopefully, future, much needed, controlled clinical studies will reveal and determine to which extent the diverse biological manifestations of 3-OHB should be introduced medically.

Facilitated vascularization and enhanced bone regeneration by manipulation hierarchical pore structure of scaffolds.

Sufficient vascularization is quite important for preventing cell death and promoting host integration during the repair of the critical sized bone defects. Porous structure providing enough space for the ingrowth of vessels is an essential consideration during the scaffold's development. In this study, we designed and fabricated three kinds of porous structured scaffolds based on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), such as mono-structured PHBHHx scaffolds with macro pores (PH-1), di-structured PHBHHx scaffolds with macro-meso pores (PHS-2), and tri-structured PHBHHx scaffolds with macro-micro-meso pores (PHS-3), respectively. In vitro effects of the hierarchical porous scaffolds on human umbilical vein endothelial cells (HUVECs), such as cell attachment, glucose and lactate detection, relative gene expressions of endothelial markers were investigated. The PHS-3 scaffolds exhibited preferential potency of inducing better angiogenesis in vitro. Consequently, the hierarchical porous scaffolds were applied to load rhBMP-2 and repair the critical sized bone defect (15 mm) in rabbits. Microangiography analysis by three dimensional micro-computed tomographic (micro-CT) demonstrated that the volume of blood vessels within the defect area was higher in the rhBMP-2 loaded PHS-3 (PHS-3/rhBMP-2) than that in other rhBMP-2 loaded porous scaffolds with simplex or double scaled pores (PH-1/rhBMP-2 or PHS-2/rhBMP-2) at 4 weeks and 8 weeks, which implied that multi-level porous structure was conducive to nutrition transmission and revascularization. Further investigations of orthotopic bone formation by micro-CT, histological and immunohistochemistry analysis confirmed the most accelerated new bone formation rate in the PHS-3/rhBMP-2 group. The maximum load value of the regenerated bone induced by PHS-3/rhBMP-2 at 12 weeks was 258.47 ± 14.77 N which did not show significant difference from the normal bone of 268.81 ± 12.05 N. These results highlighted that introducing multi-level pores into the biocompatible scaffolds may be an effective approach to promote angiogenesis and bone regeneration.

Preparation and characterization of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/pullulan-gelatin electrospun nanofibers with shell-core structure.

In this study, hydrophilic pullulan, which is favorable for cell adhesion, proliferation, and differentiation, was selected as a modifier for the preparation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB))/pullulan nanofibers via electrospinning to improve the biocompatibility of P(3HB-co-4HB) and increase the drug loading of composite fibers. Alkyl polyglycoside was used as the emulsifying agent to promote emulsification and stabilize the P(3HB-co-4HB)/pullulan composite solution. Drug-loading property of the nanofibers with a shell-core structure is increased because gelatin was not formed into fibers via electrospinning, thereby forming a stable drug-containing gelatin solution in the core layer. Finally, P(3HB-co-4HB)/pullulan-gelatin shell-core nanofibers were prepared. The intermolecular interaction, morphology, crystallization properties, mechanical properties, morphology, sustained release, and biocompatibility of composite nanofibers were characterized. Results show that the crystallization property of P(3HB-co-4HB)/pullulan composite nanofibers increases continuously with an increase in the pullulan content. As the pullulan content increases, the strain and stress of P(3HB-co-4HB)/pullulan nanofibers increase initially and decrease later. At the mass ratio of P(3HB-co-4HB) to pullulan of 10:2, P(3HB-co-4HB)/pullulan composite nanofibers exhibit a uniform morphology with an average diameter of 590 nm and porosity of 70.71%. At this mass ratio, the P(3HB-co-4HB)/pullulan-gelatin/drug shell-core structure, which sustained a release effect for more than 180 h, has potential applications as biomaterials without cytotoxicity.

Melanoma-Time to fast or time to feast? An interplay between PPARs, metabolism and immunity.

Development and progression of melanoma can be accelerated by intensification of particular metabolic pathways, such as aerobic glycolysis and avid amino acid catabolism, and is accompanied by aberrant immune responses within the tumor microenvironment. Contrary to other cancer types, melanoma reveals some unique tissue-specific features, such as melanogenesis, which is intertwined with metabolism. Nuclear peroxisome proliferator-activated receptors (PPARs) take part in regulation of systemic and cellular metabolism, inflammation and melanogenesis. They appear as a focal regulatory point for these three distinct processes by occupying the intersection among AMP-dependent protein kinase (AMPK), mammalian target of rapamycin (mTOR) and PPAR gamma coactivator 1-alpha (PGC-1α) signalling pathways. When deregulated, they may accelerate melanoma malignant growth. Presenting the contribution of PPARα and PPARγ in melanoma biology, we attempt to ask how two contrasting metabolic states: obesity and fasting, can change progression of the disease and possible outcome of the treatment. This short essay is aimed to provoke a discussion about some practical implications for melanoma prevention and treatment, especially: how metabolic manipulation may be exploited to overcome immunosuppression and support immune checkpoint blockade efficacy.