A novel hepatocyte ketone production assay to help the selection of nutrients for the ketogenic diet treatment of epilepsy.

The classic ketogenic diet is an effective treatment option for drug-resistant epilepsy, but its high fat content challenges patient compliance. Optimizing liver ketone production guided by a method comparing substrates for their ketogenic potential may help to reduce the fat content of the diet without loss in ketosis induction. Here, we present a liver cell assay measuring the ß-hydroxybutyrate (ßHB) yield from fatty acid substrates. Even chain albumin-conjugated fatty acids comprising between 4 and 18 carbon atoms showed a sigmoidal concentration-ßHB response curve (CRC) whereas acetate and omega-3 PUFAs produced no CRC. While CRCs were not distinguished by their half-maximal effective concentration (EC50), they differed by maximum response, which related inversely to the carbon chain length and was highest for butyrate. The assay also suitably assessed the ßHB yield from fatty acid blends detecting shifts in maximum response from exchanging medium chain fatty acids for long chain fatty acids. The assay further detected a dual role for butyrate and hexanoic acid as ketogenic substrate at high concentration and ketogenic enhancer at low concentration, augmenting the ßHB yield from oleic acid and a fatty acid blend. The assay also found propionate to inhibit ketogenesis from oleic acid and a fatty acid blend at low physiological concentration. Although the in vitro assay shows promise as a tool to optimize the ketogenic yield of a fat blend, its predictive value requires human validation.

Epithelial-mesenchymal transition-related signaling pathways in gastric Cancer cells distinctively respond to long-term experimental ketosis.

BACKGROUND: The interrelationship between cellular metabolism and the epithelial-to-mesenchymal transition (EMT) process has made it an interesting topic to investigate the adjuvant effect of therapeutic diets in the treatment of cancers. However, the findings are controversial. In this study, the effects of glucose limitation along and with the addition of beta-hydroxybutyrate (bHB) were examined on the expression of specific genes and proteins of EMT, Wnt, Hedgehog, and Hippo signaling pathways, and also on cellular behavior of gastric cancer stem-like (MKN-45) and non-stem-like (KATO III) cells. METHODS AND RESULTS: The expression levels of chosen genes and proteins studied in cancer cells gradually adopted a low-glucose condition of one-fourth, along and with the addition of bHB, and compared to the unconditioned control cells. The long-term switching of the metabolic fuels successfully altered the expression profiles and behaviors of both gastric cancer cells. However, the results for some changes were the opposite. Glucose limitation along and with the addition of bHB reduced the CD44+ population in MKN-45 cells. In KATO III cells, glucose restriction increased the CD44+ population. Glucose deprivation alleviated EMT-related signaling pathways in MKN-45 cells but stimulated EMT in KATO III cells. Interestingly, bHB enrichment reduced the beneficial effect of glucose starvation in MKN-45 cells, but also alleviated the adverse effects of glucose restriction in KATO III cells. CONCLUSIONS: The findings of this research clearly showed that some controversial results in clinical trials for ketogenic diet in cancer patients stemmed from the different signaling responses of various cells to the metabolic changes in a heterogeneous cancer mass.

Hepatic ketone body regulation of renal gluconeogenesis.

OBJECTIVES: During fasting, liver pivotally regulates blood glucose levels through glycogenolysis and gluconeogenesis. Kidney also produces glucose through gluconeogenesis. Gluconeogenic genes are transactivated by fasting, but their expression patterns are chronologically different between the two organs. We find that renal gluconeogenic gene expressions are positively correlated with the blood ß-hydroxybutyrate concentration. Thus, we herein aim to investigate the regulatory mechanism and its physiological implications. METHODS: Gluconeogenic gene expressions in liver and kidney were examined in hyperketogenic mice such as high-fat diet (HFD)-fed and ketogenic diet-fed mice, and in hypoketogenic PPARα knockout (PPARα-/-) mice. Renal gluconeogenesis was evaluated by rise in glycemia after glutamine loading in vivo. Functional roles of ß-hydroxybutyrate in the regulation of renal gluconeogenesis were investigated by metabolome analysis and RNA-seq analysis of proximal tubule cells. RESULTS: Renal gluconeogenic genes were transactivated concurrently with blood ß-hydroxybutyrate uprise under ketogenic states, but the increase was blunted in hypoketogenic PPARα-/- mice. Administration of 1,3-butandiol, a ketone diester, transactivated renal gluconeogenic gene expression in fasted PPARα-/- mice. In addition, HFD-fed mice showed fasting hyperglycemia along with upregulated renal gluconeogenic gene expression, which was blunted in HFD-fed PPARα-/- mice. In vitro experiments and metabolome analysis in renal tubular cells showed that ß-hydroxybutyrate directly promotes glucose and NH3 production through transactivating gluconeogenic genes. In addition, RNA-seq analysis revealed that ß-hydroxybutyrate-induced transactivation of Pck1 was mediated by C/EBPß. CONCLUSIONS: Our findings demonstrate that ß-hydroxybutyrate mediates hepato-renal interaction to maintain homeostatic regulation of blood glucose and systemic acid-base balance through renal gluconeogenesis regulation.

Hormonal effects on glucose and ketone metabolism in a perfused liver of an elasmobranch, the North Pacific spiny dogfish, Squalus suckleyi.

Hormonal influence on hepatic function is a critical aspect of whole-body energy balance in vertebrates. Catecholamines and corticosteroids both influence hepatic energy balance via metabolite mobilization through glycogenolysis and gluconeogenesis. Elasmobranchs have a metabolic organization that appears to prioritize the mobilization of hepatic lipid as ketone bodies (e.g. 3-hydroxybutyrate [3-HB]), which adds complexity in determining the hormonal impact on hepatic energy balance in this taxon. Here, a liver perfusion was used to investigate catecholamine (epinephrine [E]) and corticosteroid (corticosterone [B] and 11-deoxycorticosterone [DOC]) effects on the regulation of hepatic glucose and 3-HB balance in the North Pacific Spiny dogfish, Squalus suckleyi. Further, hepatic enzyme activity involved in ketogenesis (3-hydroxybutyrate dehydrogenase), glycogenolysis (glycogen phosphorylase), and gluconeogenesis (phosphoenolpyruvate carboxykinase) were assessed in perfused liver tissue following hormonal application to discern effects on hepatic energy flux. mRNA transcript abundance key transporters of glucose (glut1 and glut4) and ketones (mct1 and mct2) and glucocorticoid function (gr, pepck, fkbp5, and 11ßhsd2) were also measured to investigate putative cellular components involved in hepatic responses. There were no changes in the arterial-venous difference of either metabolite in all hormone perfusions. However, perfusion with DOC increased gr transcript abundance and decreased flow rate of perfusions, suggesting a regulatory role for this corticosteroid. Phosphoenolpyruvate carboxykinase activity increased following all hormone treatments, which may suggest gluconeogenic function; E also increased 3-hydroxybutyrate dehydrogenase activity, suggesting a function in ketogenesis, and decreased pepck and fkbp5 transcript abundance, potentially showing some metabolic regulation. Overall, we demonstrate hormonal control of hepatic energy balance using liver perfusions at various levels of biological organization in an elasmobranch.

Effects of poly(3-hydroxybutyrate) [P(3HB)] coating on the bacterial communities of artificial structures.

The expanding urbanization of coastal areas has led to increased ocean sprawl, which has had both physical and chemical adverse effects on marine and coastal ecosystems. To maintain the health and functionality of these ecosystems, it is imperative to develop effective solutions. One such solution involves the use of biodegradable polymers as bioactive coatings to enhance the bioreceptivity of marine and coastal infrastructures. Our study aimed to explore two main objectives: (1) investigate PHA-degrading bacteria on polymer-coated surfaces and in surrounding seawater, and (2) comparing biofilm colonization between surfaces with and without the polymer coating. We applied poly(3-hydroxybutyrate) [P(3HB)) coatings on concrete surfaces at concentrations of 1% and 6% w/v, with varying numbers of coating cycles (1, 3, and 6). Our findings revealed that the addition of P(3HB) indeed promoted accelerated biofilm growth on the coated surfaces, resulting in an occupied area approximately 50% to 100% larger than that observed in the negative control. This indicates a remarkable enhancement, with the biofilm expanding at a rate roughly 1.5 to 2 times faster than the untreated surfaces. We observed noteworthy distinctions in biofilm growth patterns based on varying concentration and number of coating cycles. Interestingly, treatments with low concentration and high coating cycles exhibited comparable biofilm enhancements to those with high concentrations and low coating cycles. Further investigation into the bacterial communities responsible for the degradation of P(3HB) coatings identified mostly common and widespread strains but found no relation between the concentration and coating cycles. Nevertheless, this microbial degradation process was found to be highly efficient, manifesting noticeable effects within a single month. While these initial findings are promising, it's essential to conduct tests under natural conditions to validate the applicability of this approach. Nonetheless, our study represents a novel and bio-based ecological engineering strategy for enhancing the bioreceptivity of marine and coastal structures.

A maternal ketogenic diet alters oviduct fluid nutrients and embryo histone acetylation in mice.

In brief: A ketogenic diet (KD) elevates blood ß-hydroxybutyrate to concentrations that are known to perturb the development, metabolism, histone acetylation and viability of preimplantation mouse embryos in culture. This study shows that a maternal KD changes available nutrient levels in the oviduct, leading to altered embryo development and epigenetic state in vivo. Abstract: A ketogenic diet elevates blood ß-hydroxybutyrate to concentrations that perturb the development, metabolism, histone acetylation (H3K27ac) and viability of preimplantation mouse embryos in vitro. However, whether a ketogenic diet alters ß-hydroxybutyrate concentrations within female reproductive fluid is unknown. This study aimed to quantify glucose and ß-hydroxybutyrate within mouse blood and oviduct fluid following standard diet and ketogenic diet consumption and to assess whether a maternal periconceptional ketogenic diet impacts in vivo embryo development and blastocyst H3K27ac. Female C57BL/6 × CBA mice were fed a standard or ketogenic diet (n = 24 each) for 24-27 days. Glucose and ß-hydroxybutyrate were quantified in blood via an electronic monitoring system and in oviduct fluid via ultramicrofluorescence. The developmental grade of flushed blastocysts was recorded, and blastocyst cell number and H3K27ac were assessed via immunofluorescence. A maternal ketogenic diet elevated ß-hydroxybutyrate in day 24 blood (P < 0.001) and oviduct fluid (P < 0.05) compared with a standard diet, whereas glucose was unchanged. A periconceptional ketogenic diet did not impact blastocyst cell number; however, it significantly delayed blastocyst development (P < 0.05) and reduced trophectoderm-specific H3K27ac (P < 0.05) compared with standard diet-derived embryos. Maternal ketogenic diet consumption is, therefore, associated with reproductive tract nutrient changes and altered embryonic development and epigenetics in vivo. Future studies to assess whether periconceptional/gestational ketogenic diet consumption impacts human preimplantation, fetal, and long-term offspring development and health are warranted.

Protein acylations induced by a ketogenic diet demonstrate diverse patterns depending on organs and differ between histones and global proteins.

An essential ketone body, ß-hydroxybutyrate (BOHB), plays various roles in physiological regulations via protein acylations such as lysine acetylation and ß-hydroxybutyrylation. Here, to understand how BOHB systemically regulates acylations from an overarching perspective, we administered a ketogenic diet to mice to increase BOHB concentration and examined acylations. We found that global acetylation and ß-hydroxybutyrylation dramatically increase in various organs except for the brains, where the increase was much smaller than in the other organs. Interestingly, we observe no increase in histone acetylation in the organs where significant global protein acetylation occurs despite a substantial rise in histone ß-hydroxybutyrylation. Finally, we compared the transcriptome data of the mice's liver after the ketogenic diet to the public databases, showing that upregulated genes are enriched in those related to histone ß-hydroxybutyrylation in starvation. Our data indicate that a ketogenic diet induces diverse patterns of acylations depending on organs and protein localizations, suggesting that different mechanisms regulate acylations and that the ketogenic diet is associated with starvation in terms of protein modifications.

Cardiac mitochondrial metabolism during pregnancy and the postpartum period.

The goal of the present study was to characterize changes in mitochondrial respiration in the maternal heart during pregnancy and after birth. Timed pregnancy studies were performed in 12-wk-old female FVB/NJ mice, and cardiac mitochondria were isolated from the following groups of mice: nonpregnant (NP), midpregnancy (MP), late pregnancy (LP), and 1-wk postbirth (PB). Similar to our previous studies, we observed increased heart size during all stages of pregnancy (e.g., MP and LP) and postbirth (e.g., PB) compared with NP mice. Differential cardiac gene and protein expression analyses revealed changes in several mitochondrial transcripts at LP and PB, including several mitochondrial complex subunits and members of the Slc family, important for mitochondrial substrate transport. Respirometry revealed that pyruvate- and glutamate-supported state 3 respiration was significantly higher in PB vs. LP mitochondria, with respiratory control ratio (RCR) values higher in PB mitochondria. In addition, we found that PB mitochondria respired more avidly when given 3-hydroxybutyrate (3-OHB) than mitochondria from NP, MP, and LP hearts, with no differences in RCR. These increases in respiration in PB hearts occurred independent of changes in mitochondrial yield but were associated with higher abundance of 3-hydroxybutyrate dehydrogenase 1. Collectively, these findings suggest that, after birth, maternal cardiac mitochondria have an increased capacity to use 3-OHB, pyruvate, and glutamate as energy sources; however, increases in mitochondrial efficiency in the postpartum heart appear limited to carbohydrate and amino acid metabolism.NEW & NOTEWORTHY Few studies have detailed the physiological adaptations that occur in the maternal heart. We and others have shown that pregnancy-induced cardiac growth is associated with significant changes in cardiac metabolism. Here, we examined mitochondrial respiration and substrate preference in isolated mitochondria from the maternal heart. We show that following birth, cardiac mitochondria are "primed" to respire on carbohydrate, amino acid, and ketone bodies. However, heightened respiratory efficiency is observed only with carbohydrate and amino acid sources. These results suggest that significant changes in mitochondrial respiration occur in the maternal heart in the postpartum period.

Elevation of hypothalamic ketone bodies induces a decrease in energy expenditures and an increase risk of metabolic disorder.

OBJECTIVE: Ketone bodies (such as ß-hydroxybutyrate or BHB) have been recently proposed as signals involved in brain regulation of energy homeostasis and obesity development. However, the precise role of ketone bodies sensing by the brain, and its impact on metabolic disorder development remains unclear. Nevertheless, partial deletion of the ubiquitous ketone bodies transporter MCT1 in mice (HE mice) results in diet-induced obesity resistance, while there is no alteration under normal chow diet. These results suggest that ketone bodies produced during the high fat diet would be important signals involved in obesity onset. METHODS: In the present study we used a specific BHB infusion of the hypothalamus and analyzed the energy homeostasis of WT or HE mice fed a normal chow diet. RESULTS: Our results indicate that high BHB levels sensed by the hypothalamus disrupt the brain regulation of energy homeostasis. This brain control dysregulation leads to peripheral alterations of energy expenditure mechanisms. CONCLUSIONS: Altogether, the changes induced by high ketone bodies levels sensed by the brain increase the risk of obesity onset in mice.

Microbially synthesized poly(hydroxybutyrate-co-hydroxyhexanoate) with low to moderate hydroxyhexanoate content: Properties and applications.

Plastic pollution is the biggest environmental concern of our time. Breakdown products like micro- and nano-plastics inevitably enter the food chain and pose unprecedented health risks. In this scenario, bio-based and biodegradable plastic alternatives have been given a momentum aiming to bridge a transition towards a more sustainable future. Polyhydroxyalkanoates (PHAs) are one of the few thermoplastic polymers synthesized 100 % via biotechnological routes which fully biodegrade in common natural environments. Poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] is a PHA copolymer with great potential for the commodity polymers industry, as its mechanical properties can be tailored through fine-tuning of its molar HHx content. We have recently developed a strategy that enables for reliable tailoring of the monomer content of P(HB-co-HHx). Nevertheless, there is often a lack of comprehensive investigation of the material properties of PHAs to evaluate whether they actually mimic the functionalities of conventional plastics. We present a detailed study of P(HB-co-HHx) copolymers with low to moderate hydroxyhexanoate content to understand how the HHx monomer content influences the thermal and mechanical properties and to link those to their abiotic degradation. By increasing the HHx fractions in the range of 2 - 14 mol%, we impart an extension of the processing window and application range as the melting temperature (Tm) and glass temperature (Tg) of the copolymers decrease from Tm 165 °C to 126 °C, Tg 4 °C to -5.9 °C, accompanied by reduced crystallinity from 54 % to 20 %. Elongation at break was increased from 5.7 % up to 703 % at 14 mol% HHx content, confirming that the range examined was sufficiently large to obtain ductile and brittle copolymers, while tensile strength was maintained throughout the studied range. Finally, accelerated abiotic degradation was shown to be slowed down with an increasing HHx fraction decreasing from 70 % to 55 % in 12 h.

Relationship between serum ß-hydroxybutyrate and hepatic fatty acid oxidation in individuals with obesity and NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excess lipid accumulation that can progress to inflammation (nonalcoholic steatohepatitis, NASH), and fibrosis. Serum ß-hydroxybutyrate (ß-HB), a product of the ketogenic pathway, is commonly used as a surrogate marker for hepatic fatty acid oxidation (FAO). However, it remains uncertain whether this relationship holds true in the context of NAFLD in humans. We compared fasting serum ß-HB levels with direct measurement of liver mitochondrial palmitate oxidation in humans stratified based on NAFLD severity (n = 142). Patients were stratified based on NAFLD activity score (NAS): NAS = 0 (no disease), NAS = 1-2 (mild), NAS = 3-4 (moderate), and NAS ≥ 5 (advanced). Moderate and advanced NAFLD is associated with reductions in liver 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), serum ß-HB, but not 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL) mRNA, relative to no disease. Worsening liver mitochondrial complete palmitate oxidation corresponded with lower HMGCS2 mRNA but not total (complete + incomplete) palmitate oxidation. Interestingly, we found that liver HMGCS2 mRNA and serum ß-HB correlated with liver mitochondrial ß-hydroxyacyl-CoA dehydrogenase (ß-HAD) activity and CPT1A mRNA. Also, lower mitochondrial mass and markers of mitochondrial turnover positively correlated with lower HMGCS2 in the liver. These data suggest that liver ketogenesis and FAO occur at comparable rates in individuals with NAFLD. Our findings support the utility of serum ß-HB to serve as a marker of liver injury and hepatic FAO in the context of NAFLD.NEW & NOTEWORTHY Serum ß-hydroxybutyrate (ß-HB) is frequently utilized as a surrogate marker for hepatic fatty acid oxidation; however, few studies have investigated this relationship during states of liver disease. We found that the progression of nonalcoholic fatty liver disease (NAFLD) is associated with reductions in circulating ß-HB and liver 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2). As well, decreased rates of hepatic fatty acid oxidation correlated with liver HMGCS2 mRNA and serum ß-HB. Our work supports serum ß-HB as a potential marker for hepatic fatty acid oxidation and liver injury during NAFLD.

Exercise-induced ß-hydroxybutyrate promotes Treg cell differentiation to ameliorate colitis in mice.

Increasing attention is being paid to the mechanistic investigation of exercise-associated chronic inflammatory disease improvement. Ulcerative colitis (UC) is one type of chronic inflammatory bowel disease with increasing incidence and prevalence worldwide. It is known that regular moderate aerobic exercise (RMAE) reduces the incidence or risk of UC, and attenuates disease progression in UC patients. However, the mechanisms of this RMAE's benefit are still under investigation. Here, we revealed that ß-hydroxybutyrate (ß-HB), a metabolite upon prolonged aerobic exercise, could contribute to RMAE preconditioning in retarding dextran sulfate sodium (DSS)-induced mouse colitis. When blocking ß-HB production, RMAE preconditioning-induced colitis amelioration was compromised, whereas supplementation of ß-HB significantly rescued impaired ß-HB production-associated defects. Meanwhile, we found that RMAE preconditioning significantly caused decreased colonic Th17/Treg ratio, which is considered to be important for colitis mitigation; and the downregulated Th17/Treg ratio was associated with ß-HB. We further demonstrated that ß-HB can directly promote the differentiation of Treg cell rather than inhibit Th17 cell generation. Furthermore, ß-HB increased forkhead box protein P3 (Foxp3) expression, the core transcriptional factor for Treg cell, by enhancing histone H3 acetylation in the promoter and conserved noncoding sequences of the Foxp3 locus. In addition, fatty acid oxidation, the key metabolic pathway required for Treg cell differentiation, was enhanced by ß-HB treatment. Lastly, administration of ß-HB without exercise significantly boosted colonic Treg cell and alleviated colitis in mice. Together, we unveiled a previously unappreciated role for exercise metabolite ß-HB in the promotion of Treg cell generation and RMAE preconditioning-associated colitis attenuation.

Pre-treatment with ß-hydroxybutyrate mitigates cisplatin-induced acute kidney injury.

ß-Hydroxybutyrate (ß-HB), the primary circulating ketone body, plays a dual role as both a metabolic fuel and an endogenous signaling molecule, offering diverse systemic benefits. Recent studies have highlighted the renoprotective effects of exogenous ß-HB therapy in various animal models of kidney disease. In this investigation, our goal was to assess whether pre-treatment with exogenous ß-HB could alleviate kidney damage in a mouse model of cisplatin-induced acute kidney injury (AKI). Prior to cisplatin administration, intraperitoneal administration of ß-HB was carried out, and the groups were classified into four: Sham, ß-HB, cisplatin, and ß-HB + cisplatin. The tubular damage score and serum creatinine levels were significantly lower in the ß-HB + cisplatin group compared to the cisplatin group. Furthermore, the expression of phosphorylated NF-κB, inflammatory cytokines, and the quantity of F4/80-positive macrophages in the ß-HB + cisplatin group were reduced compared to those in the cisplatin group. Additionally, oxidative stress markers for DNA, protein, and lipid in the ß-HB + cisplatin group were markedly diminished compared to those in the cisplatin group. The number of TUNEL-positive and cleaved caspase 3-positive tubular cells in the ß-HB + cisplatin group was lower than in the cisplatin group. Pre-treating with exogenous ß-HB effectively mitigated kidney damage by suppressing inflammation, oxidative stress, and tubular apoptosis in cisplatin-induced AKI. Therefore, exogenous ß-HB as a pre-treatment emerges as a promising and novel strategy for preventing cisplatin-induced AKI.

The therapeutic potential of ketones in cardiometabolic disease: impact on heart and skeletal muscle.

ß-Hydroxybutyrate (ßOHB) is the major ketone in the body, and it is recognized as a metabolic energy source and an important signaling molecule. While ketone oxidation is essential in the brain during prolonged fasting/starvation, other organs such as skeletal muscle and the heart also use ketones as metabolic substrates. Additionally, ßOHB-mediated molecular signaling events occur in heart and skeletal muscle cells, and via metabolism and/or signaling, ketones may contribute to optimal skeletal muscle health and cardiac function. Of importance, when the use of ketones for ATP production and/or as signaling molecules becomes disturbed in the presence of underlying obesity, type 2 diabetes, and/or cardiovascular diseases, these changes may contribute to cardiometabolic disease. As a result of these disturbances in cardiometabolic disease, multiple approaches have been used to elevate circulating ketones with the goal of optimizing either ketone metabolism or ketone-mediated signaling. These approaches have produced significant improvements in heart and skeletal muscle during cardiometabolic disease with a wide range of benefits that include improved metabolism, weight loss, better glycemic control, improved cardiac and vascular function, as well as reduced inflammation and oxidative stress. Herein, we present the evidence that indicates that ketone therapy could be used as an approach to help treat cardiometabolic diseases by targeting cardiac and skeletal muscles.

Enhancing poly(3-hydroxybutyrate) production in halophilic bacteria through improved salt tolerance.

Polyhydroxyalkanoates (PHA) have emerged as a promising bio-compound in the industrial application due to their potential to replace conventional petroleum-based plastics with sustainable bioplastics. This study focuses on Halomonas sp. YJPS3-3, a halophilic bacterium, and presents a novel approach to enhance PHA production by exploiting its salt tolerance toward PHA biosynthesis. Through gamma irradiation-induced mutants with enhanced salt tolerance from 15% NaCl to 20% NaCl, mutant halo6 showing a significant 11% increase in PHA yield, was achieved. Moreover, the mutants displayed not only higher PHA content but also remarkable cell morphology with elongation. In addition, this research unravels the genetic determinants behind the elevated PHA content and identifies a corresponding shift in fatty acid composition favoring PHA accumulation. This novel mutant obtained from gamma irradiation with enhanced salt tolerance in halophilic bacteria opens up new avenues not only for the bioplastic industry but also for applications in the production of high-value metabolites.

Metabolic engineering of Halomonas bluephagenesis for production of five carbon molecular chemicals derived from L-lysine.

5-Aminovaleric acid (5-AVA), 5-hydroxyvalerate (5HV), copolymer P(3HB-co-5HV) of 3-hydroxybutyrate (3HB) and 5HV were produced from L-lysine as a substrate by recombinant Halomonas bluephagenesis constructed based on codon optimization, deletions of competitive pathway and L-lysine export protein, and three copies of davBA genes encoding L-lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) inserted into its genome to form H. bluephagenesis YF117ΔgabT1+2, which produced 16.4 g L-1 and 67.4 g L-1 5-AVA in flask cultures and in 7 L bioreactor, respectively. It was able to de novo synthesize 5-AVA from glucose by L-lysine-overproducing H. bluephagenesis TD226. Corn steep liquor was used instead of yeast extract for cost reduction during the 5-AVA production. Using promoter engineering based on Pporin mutant library for downstream genes, H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC produced 6 g L-1 5HV in shake flask growth, while H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC-Pporin278-phaCRE-abfT synthesized 42 wt% P(3HB-co-4.8 mol% 5HV) under the same condition. Thus, H. bluephagenesis was successfully engineered to produce 5-AVA and 5HV in supernatant and intracellular P(3HB-co-5HV) utilizing L-lysine as the substrate.

Impact of fasting & ketogenic interventions on the NLRP3 inflammasome: A narrative review.

Overactivation of the NLRP3 inflammasome is implicated in chronic low-grade inflammation associated with various disease states, including obesity, type 2 diabetes, atherosclerosis, Alzheimer's disease, and Parkinson's disease. Emerging evidence, mostly from cell and animal models of disease, supports a role for ketosis in general, and the main circulating ketone body beta-hydroxybutyrate (BHB) in particular, in reducing NLRP3 inflammasome activation to improve chronic inflammation. As a result, interventions that can induce ketosis (e.g., fasting, intermittent fasting, time-restricted feeding/eating, very low-carbohydrate high-fat ketogenic diets) and/or increase circulating BHB (e.g., exogenous ketone supplementation) have garnered increasing interest for their therapeutic potential. The purpose of the present review is to summarize our current understanding of the literature on how ketogenic interventions impact the NLRP3 inflammasome across human, rodent and cell models. Overall, there is convincing evidence that ketogenic interventions, likely acting through multiple interacting mechanisms in a cell-, disease- and context-specific manner, can reduce NLRP3 inflammasome activation. The evidence supports a direct effect of BHB, although it is important to consider the myriad of other metabolic responses to fasting or ketogenic diet interventions (e.g., elevated lipolysis, low insulin, stable glucose, negative energy balance) that may also impact innate immune responses. Future research is needed to translate promising findings from discovery science to clinical application.

Bone repair potential of collagen-poly(3-hydroxybutyrate)-carbon nanotubes scaffold loaded with mesenchymal stem cells for the reconstruction of critical-sized mandibular defects.

The poor structural stability of collagen (COL) upon hydration poses a significant challenge in tissue engineering (TE). To overcome this limitation, the incorporation of hydrophobic polymers such as poly(3-hydroxybutyrate) (PHB), and nanomaterials such as carbon nanotubes (CNTs) has been explored. In this study, we investigated the physical, chemical, and biological characteristics of COL-based scaffolds modified with PHB and CNTs for bone tissue engineering (BTE) applications. The tensile strength analysis revealed a substantial improvement in the ultimate tensile strength with the addition of 10 % PHB and 4 % CNTs. Scanning electron microscopy (SEM) images depicted a denser and more compact structure resulting from the presence of PHB and CNTs, enhancing the scaffold's mechanical properties. Fourier-transform infrared spectroscopy (FTIR) confirmed the successful incorporation of PHB and CNTs into the composite scaffold, maintaining the chemical integrity of COL. Stereological studies also conducted in a rat model with induced critical-sized bone defects in the mandibular bone further emphasize the substantial increase in bone formation and reduction in defect volume achieved by the scaffold loaded with stem cells. These findings underscore the promising approach to enhance bone healing, using COL-based scaffolds loaded with stem cells, and the favorable results obtained in this study can contribute to the advancement of BTE strategies.

Production of poly(3-hydroxybutyrate)/poly(lactic acid) from industrial wastewater by wild-type Cupriavidus necator H16.

The massive production of urban and industrial wastes has created a clear need for alternative waste management processes. One of the more promising strategies is to use waste as raw material for the production of biopolymers such as polyhydroxyalkanoates (PHAs). In this work, a lactate-enriched stream obtained by anaerobic digestion (AD) of wastewater (WW) from a candy production plant was used as a feedstock for PHA production in wild-type Cupriavidus necator H16. Unexpectedly, we observed the accumulation of poly(3-hydroxybutyrate)/poly(lactic acid) (P(3HB)/PLA), suggesting that the non-engineered strain already possesses the metabolic potential to produce these polymers of interest. The systematic study of factors, such as incubation time, nitrogen and lactate concentration, influencing the synthesis of P(3HB)/PLA allowed the production of a panel of polymers in a resting cell system with tailored lactic acid (LA) content according to the GC-MS of the biomass. Further biomass extraction suggested the presence of methanol soluble low molecular weight molecules containing LA, while 1 % LA could be detected in the purified polymer fraction. These results suggested that the cells are producing a blend of polymers. A proteomic analysis of C. necator resting cells under P(3HB)/PLA production conditions provides new insights into the latent pathways involved in this process. This study is a proof of concept demonstrating that LA can polymerize in a non-modified organism and paves the way for new metabolic engineering approaches for lactic acid polymer production in the model bacterium C. necator H16.

ß-hydroxybutyrate administered at reperfusion reduces infarct size and preserves cardiac function by improving mitochondrial function through autophagy in male mice.

Ischemia/reperfusion (I/R) injury after revascularization contributes ∼50% of infarct size and causes heart failure, for which no established clinical treatment exists. ß-hydroxybutyrate (ß-OHB), which serves as both an energy source and a signaling molecule, has recently been reported to be cardioprotective when administered immediately before I/R and continuously after reperfusion. This study aims to determine whether administering ß-OHB at the time of reperfusion with a single dose can alleviate I/R injury and, if so, to define the mechanisms involved. We found plasma ß-OHB levels were elevated during ischemia in STEMI patients, albeit not to myocardial protection level, and decreased after revascularization. In mice, compared with normal saline, ß-OHB administrated at reperfusion reduced infarct size (by 50%) and preserved cardiac function, as well as activated autophagy and preserved mtDNA levels in the border zone. Our treatment with one dose ß-OHB reached a level achievable with fasting and strenuous physical activity. In neonatal rat ventricular myocytes (NRVMs) subjected to I/R, ß-OHB at physiologic level reduced cell death, increased autophagy, preserved mitochondrial mass, function, and membrane potential, in addition to attenuating reactive oxygen species (ROS) levels. ATG7 knockdown/knockout abolished the protective effects of ß-OHB observed both in vitro and in vivo. Mechanistically, ß-OHB's cardioprotective effects were associated with inhibition of mTOR signaling. In conclusion, ß-OHB, when administered at reperfusion, reduces infarct size and maintains mitochondrial homeostasis by increasing autophagic flux (potentially through mTOR inhibition). Since ß-OHB has been safely tested in heart failure patients, it may be a viable therapeutic to reduce infarct size in STEMI patients.