A bioenergetically-active ploy (glycerol sebacate)-based multiblock hydrogel improved diabetic wound healing through revitalizing mitochondrial metabolism.

Diabetic wounds impose significant burdens on patients' quality of life and healthcare resources due to impaired healing potential. Factors like hyperglycemia, oxidative stress, impaired angiogenesis and excessive inflammation contribute to the delayed healing trajectory. Mounting evidence indicates a close association between impaired mitochondrial function and diabetic complications, including chronic wounds. Mitochondria are critical for providing energy essential to wound healing processes. However, mitochondrial dysfunction exacerbates other pathological factors, creating detrimental cycles that hinder healing. This study conducted correlation analysis using clinical specimens, revealing a positive correlation between mitochondrial dysfunction and oxidative stress, inflammatory response and impaired angiogenesis in diabetic wounds. Restoring mitochondrial function becomes imperative for developing targeted therapies. Herein, we synthesized a biodegradable poly (glycerol sebacate)-based multiblock hydrogel, named poly (glycerol sebacate)-co-poly (ethylene glycol)-co-poly (propylene glycol) (PEPGS), which can be degraded in vivo to release glycerol, a crucial component in cellular metabolism, including mitochondrial respiration. We demonstrate the potential of PEPGS-based hydrogels to improve outcomes in diabetic wound healing by revitalizing mitochondrial metabolism. Furthermore, we investigate the underlying mechanism through proteomics analysis, unravelling the regulation of ATP and nicotinamide adenine dinucleotide metabolic processes, biosynthetic process and generation during mitochondrial metabolism. These findings highlight the therapeutic potential of PEPGS-based hydrogels as advanced wound dressings for diabetic wound healing.

The effects of feeding sows at onset of farrowing supplemental energy (blend of carbohydrates and glycerol) on farrowing kinetics and piglet vitality.

Delivering piglets is one of the most energy-demanding activities sows undergo in their lifetime. Sows can have myometrial contractions from 2 to 12 h before the first piglet is expelled as well as a nest-building behavior. Thus, when the first piglet is delivered, the female has already used part of her energy supply. When the sow gets exhausted due to lack of energy, the farrowing process can be interrupted, causing damage to the viability and vitality of the piglets. In the present study, we evaluated the effects of feeding sows an energy supplement at the onset of farrowing on farrowing kinetics and piglet vitality. The energy supplement consisted of a blend of carbohydrates and glycerol which provides 439 kJ of metabolizable energy per kg of metabolic weight. A total of 180 sows were used. At the onset of farrowing, sows were assigned to one of the following treatments: sows that were not supplied energy at the onset of farrowing, serving as controls (CON, n = 85); sows fed the energy supplement at the onset of farrowing (ESP, n = 95). Farrowing kinetics, blood glucose concentration, and piglet vitality were recorded for each sow. Blood glucose concentration was assessed by puncturing the auricular vein and using a portable glucometer at four different time points: after the birth of the 1st piglet (T0), and at 20 (T20), 40 (T40), 80 (T80), and 180 (T180) min after the birth of the 1st piglet. The vitality of the 1st, 6th, 12th, 17th, and 20th piglet born was evaluated using the Apgar score. Piglet birth weight and average colostrum intake were measured. The farrowing duration was 20 min shorter (P < 0.05) for ESP sows in comparison with CON sows. Sows from ESP treatment had higher (P ≤ 0.05) blood glucose concentration at T20 and T40 compared to the CON sows. The inter-piglet birth interval was shortened (P < 0.05) by 14 min between the 1st and 2nd piglet for the ESP treatment. The 17th and 20th piglets born from ESP sows had higher (P < 0.05) Apgar score compared to piglets of the same birth order from CON sows. Colostrum intake was higher (P < 0.01) for piglets born from ESP sows. Litter growth performance did not differ (P > 0.05). In conclusion, feeding a blend of carbohydrates and glycerol as an energy supplement for farrowing sows improved farrowing kinetics and piglet vitality score.

Itaconate Production from Crude Substrates with U. maydis: Scale-up of an Industrially Relevant Bioprocess.

BACKGROUND: Industrial by-products accrue in most agricultural or food-related production processes, but additional value chains have already been established for many of them. Crude glycerol has a 60% lower market value than commercial glucose, as large quantities are produced in the biodiesel industry, but its valorisation is still underutilized. Due to its high carbon content and the natural ability of many microorganisms to metabolise it, microbial upcycling is a suitable option for this waste product. RESULTS: In this work, the use of crude glycerol for the production of the value-added compound itaconate is demonstrated using the smut fungus Ustilago maydis. Starting with a highly engineered strain, itaconate production from an industrial glycerol waste stream was quickly established on a small scale, and the resulting yields were already competitive with processes using commercial sugars. Adaptive laboratory evolution resulted in an evolved strain with a 72% increased growth rate on glycerol. In the subsequent development and optimisation of a fed-batch process on a 1.5-2 L scale, the use of molasses, a side stream of sugar beet processing, eliminated the need for other expensive media components such as nitrogen or vitamins for biomass growth. The optimised process was scaled up to 150 L, achieving an overall titre of 72 g L- 1, a yield of 0.34 g g- 1, and a productivity of 0.54 g L- 1 h- 1. CONCLUSIONS: Pilot-scale itaconate production from the complementary waste streams molasses and glycerol has been successfully established. In addition to achieving competitive performance indicators, the proposed dual feedstock strategy offers lower process costs and carbon footprint for the production of bio-based itaconate.

The inhibitory effects of Xiao-Gao-Jiang-Zhuo-containing serum on adipogenesis in 3T3-L1 preadipocytes.

BACKGROUND: Obesity and related metabolic diseases are becoming a worldwide epidemic, leading to increased mortality and heavy medical costs. Our Chinese herbal formula Xiao-Gao-Jiang-Zhuo (XGJZ) has remarkable effects on curing obese patients in the clinic, but the cellular and molecular basis remains unknown. This study aimed to reveal the molecular mechanism involved in adipogenesis in vitro. METHODS: Chinese herbal formula XGJZ-containing serum was prepared from XGJZ-treated obesity model rats. The function of XGJZ-containing serum was validated in 3T3-L1 preadipocytes. Oil O staining was performed to determine intracellular lipid accumulation in differentiated 3T3-L1 cells. The expression of pro-adipogenic transcription factors was measured to further validate the adipogenesis of 3T3-L1 adipocytes. The contents of triglyceride (TG), free fatty acid (FFA), and glycerin, along with the activities of lipid metabolism-related enzymes (including FAT, FATP1, DGAT, GPAT, ATGL, and HSL) were measured to study the lipogenesis in 3T3-L1 adipocytes. RESULTS: XGJZ-containing serum inhibited 3T3-L1 differentiation, decreased intracellular lipid accumulation, and suppressed the expression of pro-adipogenic transcription factors in differentiated 3T3-L1 cells. The contents of TG, FFA, and glycerin were decreased when treated with XGJZ-containing serum, which also modulated lipid metabolism-related enzyme activities. The activities of fatty acid transporters (FAT, FATP1) and lipid mobilization enzymes (ATGL, HSL) were promoted, while activities of triglyceride biosynthesis enzymes (DGAT, GPAT) were attenuated in differentiated 3T3-L1 cells. CONCLUSION: XGJZ-containing serum has inhibitory effects on adipogenesis in 3T3-L1 preadipocytes, affirming the effect of XGJZ in treating obesity. It provides evidence for the mechanism of obesity.

Glycerol tributylate (Triacylglycerol tributanoate) promoted the liver lipid metabolism by cultivating the intestinal flora of grass carp (Ctenopharyngodon idellus).

To investigate the effects of glycerol tributyrin (TB) (Triacylglycerol tributanoate) on the regulation of liver lipid metabolism by intestinal flora of grass carp (Ctenopharyngodon idellus). The compound feed with soybean oil 2.8% + fish oil 1.8%, soybean oil 6.3% + fish oil 1.8%, and soybean oil 6.2% + fish oil 1.8% + TB 0.1% was added to the basal diet as a fat source and fed to the basal (control) group, high lipid (HL) group, and tributyrin (TB) group for 12 weeks. We tested the growth performance, fat content, diversity, and abundance of gut flora and other related indexes of grass carp by Soxhlet extraction, liver tissue enzyme activity, oil red O staining, and 16S rRNA high-throughput sequencing. The results showed that the liver fat number and liver fat content of grass carp in the TB group were lower than those in the HL group, while the fattening degree was significantly higher than those in the other two groups; according to the indices such as Shannon, Ace, and Coverage, it was found that the grass carp in the TB group had the highest abundance and diversity of intestinal microflora; at the portal level, Proteobacteria and Fusobacteria were the main dominant flora in the TB group, with the number of unique OUTs accounting for about 59. 9% of the total number measured; at the genus level, the relative abundance of lipase-producing, short-chain fatty acid-associated bacteria, such as Bacillus-Lactobacillus and Bifidobacterium, was significantly lower (p < 0.05). Thus, we conclude that the addition of TB to high-fat diets can alter the structure of the intestinal microbial community and promote hepatic lipid metabolism in grass carp. TB can alleviate fatty liver in grass carp by increasing the relative abundance of short-chain fatty acids in the intestine. Meanwhile, TB inhibits the conversion of primary bile acids to secondary bile acids in the host, which can block intestinal FXR signaling and the hepatic FXR-SHP pathway, thus slowing down fat synthesis and alleviating the accumulation of liver lipids in grass carp.

Glycerol-3-Phosphate Acyltransferase GPAT9 Enhanced Seed Oil Accumulation and Eukaryotic Galactolipid Synthesis in Brassica napus.

Glycerol-3-phosphate acyltransferase GPAT9 catalyzes the first acylation of glycerol-3-phosphate (G3P), a committed step of glycerolipid synthesis in Arabidopsis. The role of GPAT9 in Brassica napus remains to be elucidated. Here, we identified four orthologs of GPAT9 and found that BnaGPAT9 encoded by BnaC01T0014600WE is a predominant isoform and promotes seed oil accumulation and eukaryotic galactolipid synthesis in Brassica napus. BnaGPAT9 is highly expressed in developing seeds and is localized in the endoplasmic reticulum (ER). Ectopic expression of BnaGPAT9 in E. coli and siliques of Brassica napus enhanced phosphatidic acid (PA) production. Overexpression of BnaGPAT9 enhanced seed oil accumulation resulting from increased 18:2-fatty acid. Lipid profiling in developing seeds showed that overexpression of BnaGPAT9 led to decreased phosphatidylcholine (PC) and a corresponding increase in phosphatidylethanolamine (PE), implying that BnaGPAT9 promotes PC flux to storage triacylglycerol (TAG). Furthermore, overexpression of BnaGPAT9 also enhanced eukaryotic galactolipids including monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), with increased 36:6-MGDG and 36:6-DGDG, and decreased 34:6-MGDG in developing seeds. Collectively, these results suggest that ER-localized BnaGPAT9 promotes PA production, thereby enhancing seed oil accumulation and eukaryotic galactolipid biosynthesis in Brassica napus.

Genome-Wide Analysis of Glycerol-3-Phosphate Acyltransferase (GPAT) Family in Perilla frutescens and Functional Characterization of PfGPAT9 Crucial for Biosynthesis of Storage Oils Rich in High-Value Lipids.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step in triacylglycerol (TAG) biosynthesis. However, GPAT members and their functions remain poorly understood in Perilla frutescens, a special edible-medicinal plant with its seed oil rich in polyunsaturated fatty acids (mostly α-linolenic acid, ALA). Here, 14 PfGPATs were identified from the P. frutescens genome and classified into three distinct groups according to their phylogenetic relationships. These 14 PfGPAT genes were distributed unevenly across 11 chromosomes. PfGPAT members within the same subfamily had highly conserved gene structures and four signature functional domains, despite considerable variations detected in these conserved motifs between groups. RNA-seq and RT-qPCR combined with dynamic analysis of oil and FA profiles during seed development indicated that PfGPAT9 may play a crucial role in the biosynthesis and accumulation of seed oil and PUFAs. Ex vivo enzymatic assay using the yeast expression system evidenced that PfGPAT9 had a strong GPAT enzyme activity crucial for TAG assembly and also a high substrate preference for oleic acid (OA, C18:1) and ALA (C18:3). Heterogeneous expression of PfGPAT9 significantly increased total oil and UFA (mostly C18:1 and C18:3) levels in both the seeds and leaves of the transgenic tobacco plants. Moreover, these transgenic tobacco lines exhibited no significant negative effect on other agronomic traits, including plant growth and seed germination rate, as well as other morphological and developmental properties. Collectively, our findings provide important insights into understanding PfGPAT functions, demonstrating that PfGPAT9 is the desirable target in metabolic engineering for increasing storage oil enriched with valuable FA profiles in oilseed crops.

Promoting FADH2 Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli.

Hydroxytyrosol is a natural polyphenolic compound widely used in the food and drug industries. The current commercial production of hydroxytyrosol relies mainly on plant extracts, which involve long extraction cycles and various raw materials. Microbial fermentation has potential value as an environmentally friendly and low-cost method. Here, a de novo biosynthetic pathway of hydroxytyrosol has been designed and constructed in an Escherichia coli strain with released tyrosine feedback inhibition. By introduction of hpaBC from E. coli and ARO10 and ADH6 from Saccharomyces cerevisiae, the de novo biosynthesis of hydroxytyrosol was achieved. An important finding in cofactor engineering is that the introduction of L-amino acid deaminase (LAAD) promotes not only cofactor regeneration but also metabolic flow redistribution. To further enhance the hydroxylation process, different 4-hydroxyphenylacetate 3-monooxygenase (hpaB) mutants and HpaBC proteins from different sources were screened. Finally, after optimization of the carbon source, pH, and seed medium, the optimum engineered strain produced 9.87 g/L hydroxytyrosol in a 5 L bioreactor. This represents the highest titer reported to date for de novo biosynthesis of hydroxytyrosol in microorganisms.

Aromatic secondary metabolite production from glycerol was enhanced by amino acid addition in Pichia pastoris.

Aromatic secondary metabolites are widely used in various industries, including the nutraceutical, dietary supplement, and pharmaceutical industries. Their production currently relies on plant extraction. Microbe-based processes have recently attracted attention as sustainable alternatives to plant-based processes. We previously showed that the yeast Pichia pastoris (Komagataella phaffii) is an optimal host for producing aromatic secondary metabolites. Additionally, titers of resveratrol, an aromatic secondary metabolite, increased by 156 % when glycerol was used as a carbon source instead of glucose. However, the mechanisms by which glycerol resulted in higher production has remained unclear. In this study, we aimed to elucidate how P. pastoris produces higher levels of aromatic secondary metabolites from glycerol than from glucose. Titers of p-coumarate, naringenin, and resveratrol increased by 103 %, 118 %, and 157 %, respectively, in natural complex media containing glycerol compared with that in media containing glucose. However, the titers decreased in minimal synthetic medium without amino acids, indicating that P. pastoris cells used the amino acids only when glycerol was the carbon source. Fermentation with the addition of single amino acids showed that resveratrol titers from glycerol varied depending on the amino acid supplemented. In particular, addition of aspartate or tryptophan into the medium improved resveratrol titers by 146 % and 156 %, respectively. These results suggest that P. pastoris could produce high levels of aromatic secondary metabolites from glycerol with enhanced utilization of specific amino acids. This study provides a basis for achieving high-level production of aromatic secondary metabolites by P. pastoris. KEY POINTS: • P. pastoris can produce high levels of aromatic metabolites from glycerol • P. pastoris cells use amino acids only when glycerol is the carbon source • Aromatic metabolite titers from glycerol increase with amino acids utilization.

Effect of pre- and postpartum supplementation of a pure glycerol product to dairy cows on feed intake, metabolic markers, and milk yield and components.

The objective of this study was to quantify the effects of supplementing transition dairy cows with a low inclusion dry glycerol product in the pre- and postpartum periods on feed intake, metabolic markers, and milk yield and components. Multiparous Holstein dairy cows (n = 60) were enrolled in a 2-by-2 factorial design study. Starting 21 d before expected parturition, cows individually received a dry cow diet with (1) 250 g/d glycerol product supplementation [66% pure glycerol (United States Pharmacopeia grade); GLY], or (2) no supplementation (CON) mixed to their total mixed ration. After parturition, cows, again, were individually assigned to either GLY, or (2) no supplementation (CON) to their partial mixed ration for the first 21 d in milk (DIM). Cows were milked by an automated milking system and offered a target of 5.4 kg DM/d pellet (23% of target total dry matter intake, DMI) in the automated milking system and followed for 42 d into lactation. Blood samples were collected 6.3 ± 3.47 d before calving for all blood measures and 3, 7, 10, and 14 DIM for analysis of glucose and ß-hydroxybutyrate, as well as 3 and 7 DIM for nonesterified fatty acids (NEFA) and haptoglobin. Initial dry cow body weight (BW), calf birth weight, previous 305-d milk, and month of parturition were used as covariates in the statistical model. Cows supplemented with GLY prepartum lost less BW and consumed more DMI pre- and postpartum, as well as had lower postpartum blood ß-hydroxybutyrate and NEFA concentrations compared with those fed the CON treatment prepartum. Cows supplemented with GLY postpartum had lesser DMI in the first 42 DIM than cows fed CON postpartum, but also had reduced blood NEFA concentrations, odds of a high haptoglobin test, odds of a low blood glucose test, and lesser preformed fatty acid concentrations and yields in their milk. Cows supplemented glycerol both pre- and postpartum lost the least total BW from -21 to 21 DIM. No treatment effects were detected for milk yield; however, cows receiving GLY postpartum had lower milk fat. Overall, glycerol supplementation during the transition period, particularly during the 21 d before calving, was associated with markers of improved metabolic status.

Effect of crude glycerin supplementation via drinking water on feed intake, water intake and productive performance of dairy ewes.

This study was performed to determine the effects of crude glycerin (CG) supplementation in drinking water on DM and nutrient intake, milk production, milk composition, and serum glucose. Twenty multiparous Lacaune × East Friesian ewes were randomly distributed into four dietary treatments throughout the lactation cycle. Treatments consisted of doses of CG supplementation via drinking water as follows: (1) no CG supplementation, (2) 15.0 g CG/kg DM, (3) 30.0 g CG/kg DM, and (4) 45.0 g CG/kg DM. DM and nutrient intake were reduced linearly with CG supplementation. CG linearly reduced water intake when expressed as kg d-1. However, no effect of CG was observed when it was expressed as a percentage of body weight or metabolic body weight. The water to DM intake ratio was increased linearly with CG supplementation. No effect of CG doses on serum glucose was observed. The production of standardized milk decreased linearly with the experimental doses of CG. Protein, fat, and lactose yield were linearly reduced with the experimental doses of CG. Milk urea concentration was quadratically increased with CG doses. Feed conversion was quadratically increased by treatments during the pre-weaning period (P < 0.05), in which the worst values were observed when the ewes were supplemented with 15 and 30 g CG/kg DM. The N-efficiency was linearly increased with CG supplementation in drinking water. Our results suggest that dairy sheep can be supplemented with CG up to 15 g/kg DM in drinking water. Greater doses are not beneficial for feed intake, milk production, and the yield of milk components.

Phytochemical Characterization of Purple Coneflower Roots (Echinacea purpurea (L.) Moench.) and Their Extracts.

Echinacea purpurea is a perennial plant that belongs to the Asteraceae family. It has a wide range of applications mainly in the treatment and prevention of inflammations in the respiratory system. The current study aimed to perform a phytochemical characterization of purple coneflower (Echinacea purpurea) roots and their extracts (water, 40%, 50%, 60% ethanol, and 60% glycerol). Phytochemical characterization was carried out by gravimetric, spectrophotometric, and chromatographic methods. Echinacea roots were characterized by a low lipid (0.8%) content. In contrast, carbohydrates (45%) and proteins (20%) occupied a large part of the dry matter. Amongst the extracts, the highest yield was obtained using water as a solvent (53%). Water extract was rich in protein and carbohydrates as fructans (inulin) were the most abundant carbohydrate constituent. The most exhaustive recovery of the phenolic components was conducted by extraction with 40% ethanol and 60% glycerol. It was found that water is the most suitable extractant for obtaining a polysaccharide-containing complex (PSC) (8.87%). PSC was composed mainly of fructans (inulin) and proteins with different molecular weight distributions. The yield of PSC decreased with an increasing ethanol concentration (40% > 50% > 60%) but the lowest yield was obtained from 60% glycerol extract. The obtained results showed that Echinacea roots contained a large amount of biologically active substances-phenolic components and polysaccharides and that glycerol was equally efficient to ethanol in extracting caffeic acid derivatives from purple coneflower roots. The data can be used for the preparation of extracts having different compositions and thus easily be incorporated into commercial products.

Mycobacterial phosphodiesterase Rv0805 is a virulence determinant and its cyclic nucleotide hydrolytic activity is required for propionate detoxification.

Cyclic AMP (cAMP) signaling is essential to Mycobacterium tuberculosis (Mtb) pathogenesis. However, the roles of phosphodiesterases (PDEs) Rv0805, and the recently identified Rv1339, in cAMP homeostasis and Mtb biology are unclear. We found that Rv0805 modulates Mtb growth within mice, macrophages and on host-associated carbon sources. Mycobacterium bovis BCG grown on a combination of propionate and glycerol as carbon sources showed high levels of cAMP and had a strict requirement for Rv0805 cNMP hydrolytic activity. Supplementation with vitamin B12 or spontaneous genetic mutations in the pta-ackA operon restored the growth of BCGΔRv0805 and eliminated propionate-associated cAMP increases. Surprisingly, reduction of total cAMP levels by ectopic expression of Rv1339 restored only 20% of growth, while Rv0805 complementation fully restored growth despite a smaller effect on total cAMP levels. Deletion of an Rv0805 localization domain also reduced BCG growth in the presence of propionate and glycerol. We propose that localized Rv0805 cAMP hydrolysis modulates activity of a specialized pathway associated with propionate metabolism, while Rv1339 has a broader role in cAMP homeostasis. Future studies will address the biological roles of Rv0805 and Rv1339, including their impacts on metabolism, cAMP signaling and Mtb pathogenesis.

Expression of glycerol-3-phosphate acyltransferase increases non-polar lipid accumulation in Nannochloropsis oceanica.

Microalgae are considered a suitable production platform for high-value lipids and oleochemicals. Several species including Nannochloropsis oceanica produce large amounts of essential [Formula: see text]-3 polyunsaturated fatty acids (PUFAs) which are integral components of food and feed and have been associated with health-promoting effects. N. oceanica can further accumulate high contents of non-polar lipids with chemical properties that render them a potential replacement for plant oils such as palm oil. However, biomass and lipid productivities obtained with microalgae need to be improved to reach commercial feasibility. Genetic engineering can improve biomass and lipid productivities, for instance by increasing carbon flux to lipids. Here, we report the overexpression of glycerol-3-phosphate acyltransferase (GPAT) in N. oceanica during favorable growth conditions as a strategy to increase non-polar lipid content. Transformants overproducing either an endogenous (NoGPAT) or a heterologous (Acutodesmus obliquus GPAT) GPAT enzyme targeted to the endoplasmic reticulum had up to 42% and 51% increased non-polar lipid contents, respectively, compared to the wild type. Biomass productivities of transformant strains were not substantially impaired, resulting in lipid productivities that were increased by up to 37% and 42% for NoGPAT and AoGPAT transformants, respectively. When exposed to nutrient stress, transformants and wild type had similar lipid contents, suggesting that GPAT enzyme exerts strong flux control on lipid synthesis in N. oceanica under favorable growth conditions. NoGPAT transformants further accumulated PUFAs in non-polar lipids, reaching a total of 6.8% PUFAs per biomass, an increase of 24% relative to the wild type. Overall, our results indicate that GPAT is an interesting target for engineering of lipid metabolism in microalgae, in order to improve non-polar lipid and PUFAs accumulation in microalgae.

Identification of GmGPATs and their effect on glycerolipid biosynthesis through seed-specific expression in soybean.

BACKGROUND: Genetic improvement of soybean oil content depends on in-depth study of the glycerolipid biosynthesis pathway. The first acylation reaction catalysed by glycerol-3-phosphate acyltransferase (GPAT) is the rate-limiting step of triacylglycerol biosynthesis. However, the genes encoding GPATs in soybean remain unknown. METHODS: We used a novel yeast genetic complementation system and seed-specific heterologous expression to identify GmGPAT activity and molecular function in glycerolipid biosynthesis. RESULTS: Sixteen GmGPAT genes were cloned by reverse transcription-PCR for screening in yeast genetic complementation. The results showed that GmGPAT9-2 could restore the conditional lethal double knockout mutant strain ZAFU1, and GmGPAT1-1 exhibited low acyltransferase activity in serial dilution assays. In addition, the spatiotemporal expression pattern of GmGPAT9-2 exhibited tissue specificity in leaves, flowers and seeds at different developmental stages. Furthermore, both the proportion of arachidic acid and erucic acid were significantly elevated in Arabidopsis thaliana transgenic lines containing the seed-specific GmGPAT9-2 compared wild type, but the oil content was not affected. CONCLUSION: Together, our results provide reference data for future engineering of triacylglycerol biosynthesis and fatty acid composition improvement through GPATs in soybean.

Antifungal activity and potential mechanism of berberine hydrochloride against fluconazole-resistant Candida albicans.

Introduction. The emergence of resistance to fluconazole in Candida albicans has made the clinical treatment of this microbe difficult. A potential strategy to address this problem involves diminishing fungal resistance to antimicrobial drugs.Hypothesis. Berberine hydrochloride (BH), the primary active ingredient of the traditional Chinese medicine (TCM) Coptis, inhibits the growth of fluconazole-resistant C. albicans through its action on the high-osmolarity glycerol mitogen-activated protein kinase (HOG-MAPK) pathway.Aim. To examine the effect of BH on the HOG-MAPK pathway to assess the potential molecular mechanism by which BH inhibits fluconazole-resistant C. albicans.Methodology. The minimum inhibitory concentration (MIC) of BH to fluconazole-resistant C. albicans was measured using the broth microdilution approach to determine the concentration of effective drug intervention. Changes in physiological functions regulated by the HOG-MAPK pathway in response to BH treatment were measured, as well as the expression of central signalling pathway genes and key downstream factors by qRT-PCR and Western blotting, respectively.Results. BH inhibited fluconazole-resistant C. albicans and the sensitivity to fluconazole increased after BH treatment. At a concentration of 256 and 64 µg ml-1 BH may affect key downstream factors that regulate several physiological functions of C. albicans by upregulating the core genes expression of SLN1, SSK2, HOG1, and PBS2 in the HOG-MAPK pathway. Upregulation of GPD1, the key gene for glycerol synthesis, increased cell osmotic pressure. BH treatment increased the accumulation of reactive oxygen species by upregulating the expression of the key respiratory metabolism gene ATP11 and downregulating the expression of the superoxide dismutase gene SOD2. Furthermore, downregulation of mycelial-specific HWP1 hindered the morphological transformation of C. albicans and inhibition of the chitin synthase gene CHS3 and the ß-(1,3) glucan synthase gene GSC1 impaired cytoderm integrity.Conclusion. BH affects multiple target genes in diminishing the resistance of C. albicans strains to fluconazole. This effect may be related to the action of BH on the HOG-MAPK pathway.

Media engineering in marine diatom Phaeodactylum tricornutum employing cost-effective substrates for sustainable production of high-value renewables.

Phaeodactylum tricornutum is a marine diatom, rich in omega-3 polyunsaturated fatty acids especially eicosapentaenoic acid (EPA) and brown pigment, that is, fucoxanthin. These high-value renewables (HVRs) have a high commercial and nutritional relevance. In this study, our focus was to enhance the productivities of such renewables by employing media engineering strategy via., photoautotrophic (P1, P2, P3) and mixotrophic (M1, M2, M3, M4) modes of cultivation with varying substrate combinations of carbon (glycerol: 0.1 m) and nitrogen (urea: 441 mm and/or sodium nitrate: 882 mm). Our results demonstrate that mixotrophic [M4] condition supplemented with glycerol (0.1 m) and urea (441 mm) feed enhanced productivities (mg L-1  day-1 ) as follows: biomass (770.0), total proteins (36.0), total lipids (22.0), total carbohydrates (23.0) with fatty acid methyl esters (9.6), EPA (2.7), and fucoxanthin (1.1), respectively. The overall yield of EPA represents 28% of total fatty acids in the mixotrophic [M4] condition. In conclusion, our improved strategy of feeding urea to a glycerol-supplemented medium defines a new efficient biomass valorization paradigm with cost-effective substrates for the production of HVRs in oleaginous diatoms P. tricornutum.

Metabolic engineering of Pichia pastoris for myo-inositol production by dynamic regulation of central metabolism.

BACKGROUND: The methylotrophic budding yeast Pichia pastoris GS115 is a powerful expression system and hundreds of heterologous proteins have been successfully expressed in this strain. Recently, P. pastoris has also been exploited as an attractive cell factory for the production of high-value biochemicals due to Generally Recognized as Safe (GRAS) status and high growth rate of this yeast strain. However, appropriate regulation of metabolic flux distribution between cell growth and product biosynthesis is still a cumbersome task for achieving efficient biochemical production. RESULTS: In this study, P. pastoris was exploited for high inositol production using an effective dynamic regulation strategy. Through enhancing native inositol biosynthesis pathway, knocking out inositol transporters, and slowing down carbon flux of glycolysis, an inositol-producing mutant was successfully developed and low inositol production of 0.71 g/L was obtained. The inositol production was further improved by 12.7% through introduction of heterologous inositol-3-phosphate synthase (IPS) and inositol monophosphatase (IMP) which catalyzed the rate-limiting steps for inositol biosynthesis. To control metabolic flux distribution between cell growth and inositol production, the promoters of glucose-6-phosphate dehydrogenase (ZWF), glucose-6-phosphate isomerase (PGI) and 6-phosphofructokinase (PFK1) genes were replaced with a glycerol inducible promoter. Consequently, the mutant strain could be switched from growth mode to production mode by supplementing glycerol and glucose sequentially, leading to an increase of about 4.9-fold in inositol formation. Ultimately, the dissolved oxygen condition in high-cell-density fermentation was optimized, resulting in a high production of 30.71 g/L inositol (~ 40-fold higher than the baseline strain). CONCLUSIONS: The GRAS P. pastoris was engineered as an efficient inositol producer for the first time. Dynamic regulation of cell growth and inositol production was achieved via substrate-dependent modulation of glycolysis and pentose phosphate pathways and the highest inositol titer reported to date by a yeast cell factory was obtained. Results from this study provide valuable guidance for engineering of P. pastoris for the production of other high-value bioproducts.

Creatine supplementation reduces the cerebral oxidative and metabolic stress responses to acute in utero hypoxia in the late-gestation fetal sheep.

Prophylactic creatine treatment may reduce hypoxic brain injury due to its ability to sustain intracellular ATP levels thereby reducing oxidative and metabolic stress responses during oxygen deprivation. Using microdialysis, we investigated the real-time in vivo effects of fetal creatine supplementation on cerebral metabolism following acute in utero hypoxia caused by umbilical cord occlusion (UCO). Fetal sheep (118 days' gestational age (dGA)) were implanted with an inflatable Silastic cuff around the umbilical cord and a microdialysis probe inserted into the right cerebral hemisphere for interstitial fluid sampling. Creatine (6 mg kg-1  h-1 ) or saline was continuously infused intravenously from 122 dGA. At 131 dGA, a 10 min UCO was induced. Hourly microdialysis samples were obtained from -24 to 72 h post-UCO and analysed for percentage change of hydroxyl radicals (• OH) and interstitial metabolites (lactate, pyruvate, glutamate, glycerol, glycine). Histochemical markers of protein and lipid oxidation were assessed at post-mortem 72 h post-UCO. Prior to UCO, creatine treatment reduced pyruvate and glycerol concentrations in the microdialysate outflow. Creatine treatment reduced interstitial cerebral • OH outflow 0 to 24 h post-UCO. Fetuses with higher arterial creatine concentrations before UCO presented with reduced levels of hypoxaemia ( PO2${P_{{{\rm{O}}_{\rm{2}}}}}$ and SO2${S_{{{\rm{O}}_{\rm{2}}}}}$ ) during UCO which associated with reduced interstitial cerebral pyruvate, lactate and • OH accumulation. No effects of creatine treatment on immunohistochemical markers of oxidative stress were found. In conclusion, fetal creatine treatment decreased cerebral outflow of • OH and was associated with an improvement in cerebral bioenergetics following acute hypoxia. KEY POINTS: Fetal hypoxia can cause persistent metabolic and oxidative stress responses that disturb energy homeostasis in the brain. Creatine in its phosphorylated form is an endogenous phosphagen; therefore, supplementation is a proposed prophylactic treatment for fetal hypoxia. Fetal sheep instrumented with a cerebral microdialysis probe were continuously infused with or without creatine-monohydrate for 10 days before induction of 10 min umbilical cord occlusion (UCO; 131 days' gestation). Cerebral interstitial fluid was collected up to 72 h following UCO. Prior to UCO, fetal creatine supplementation reduced interstitial cerebral pyruvate and glycerol concentrations. Fetal creatine supplementation reduced cerebral hydroxyl radical efflux up to 24 h post-UCO. Fetuses with higher arterial creatine concentrations before UCO and reduced levels of systemic hypoxaemia during UCO were associated with reduced cerebral interstitial pyruvate, lactate and • OH following UCO. Creatine supplementation leads to some improvements in cerebral bioenergetics following in utero acute hypoxia.

An integrated multi-omics approach revealed the regulation of melatonin on age-dependent mitochondrial function impair and lipid dyshomeostasis in mice hippocampus.

Melatonin can improve mitochondrial dysfunction associated with the aging process by removing active oxygen, as well as inhibiting lipid peroxidation to maintain biofilm fluidity and resist free radical attack. However, there is poor understanding of the effect of melatonin on age-dependent mitochondrial function and lipid profile changes in brain. In this study, we investigated the energy metabolism of the whole body and brain of mice at 9 months, 13 months, and 25 months of continuous gastric administration of 3 mg/kg/d melatonin once per day morning for two months. In addition, we performed transcriptomic, proteomic and lipidomic analysis in the hippocampus of mice at different ages. Proteomics showed that melatonin regulated mitochondrial electron transport and leucine degradation in mouse hippocampus. Lipomics suggested that the long-chain unsaturated glycerol phospholipids in mouse hippocampus increased in an age-dependent manner, while ceramide and glycerol phospholipids decreased significantly in hippocampus of mouse chronically exposed to melatonin. The combined analysis of proteome and liposome demonstrated that Mpst, Ccsap, Hdhd5, Rpl5 and Flna were the key proteins of the network which involved in the regulation of numerous lipids. Furthermore, ultrastructure observation results illustrated that melatonin could improve the damaged mitochondrial and morphologies of 25-month-old mice hippocampus. In conclusion, we describe a mechanism that age-dependent up-regulation of long-chain unsaturated lipids is a driving risk factor for mitochondrial damage and this effect could be reversed by chronic supplement of low-dose melatonin.