Complete biosynthesis of QS-21 in engineered yeast.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Development of a 2-pyrrolidone biosynthetic pathway in Corynebacterium glutamicum by engineering an acetyl-CoA balance route.

2-Pyrrolidone is widely used in the textile and pharmaceutical industries. Here, we established a 2-pyrrolidone biosynthesis pathway in Corynebacterium glutamicum, by expressing glutamate decarboxylase (Gad) mutant and ß-alanine CoA transferase (Act) which activates spontaneous dehydration cyclization of GABA to form 2-pyrrolidone. Also, the 5' untranslated regions (UTR) strategy was used to increase the expression of protein. Furthermore, considering the importance of acetyl-CoA in the 2-pyrrolidone synthesis pathway, the acetyl-CoA synthetase (acsA) gene was introduced to convert acetate into acetyl-CoA thus achieving the recyclability of the economy. Finally, the fed-batch fermentation of the final strain in a 5 L bioreactor produced 10.5 g/L 2-pyrrolidone within 78 h, which increased by 42.5% by altering the level of gene expression. This is the first time to build the basic chemical 2-pyrrolidone from glucose in one step in C. glutamicum.

Efficient D-allulose synthesis under acidic conditions by auto-inducing expression of the tandem D-allulose 3-epimerase genes in Bacillus subtilis.

BACKGROUND: D-allulose, a hexulose monosaccharide with low calorie content and high sweetness, is commonly used as a functional sugar in food and nutrition. However, enzyme preparation of D-allulose from D-frutose was severely hindered by the non-enzymatic browning under alkaline and high-temperature, and the unnecessary by-products further increased the difficulties in separation and extraction for industrial applications. Here, to address the above issue during the production process, a tandem D-allulose 3-epimerase (DPEases) isomerase synergistic expression strategy and an auto-inducible promoter engineering were levered in Bacillus subtilis 168 (Bs168) for efficient synthesis of D-allulose under the acidic conditions without browning. RESULTS: First, based on the dicistron expression system, two DPEases with complementary functional characteristics from Dorea sp. CAG:317 (DSdpe) and Clostridium cellulolyticum H10 (RCdpe) were expressed in tandem under the promoter HpaII in one cell. A better potential strain Bs168/pMA5-DSdpe-RCdpe increases enzyme activity to 18.9 U/mL at acidic conditions (pH 6.5), much higher than 17.2 and 16.7 U/mL of Bs168/pMA5-DSdpe and Bs168/pMA5-RCdpe, respectively. Subsequently, six recombinant strains based on four constitutive promoters were constructed in variable expression cassettes for improving the expression level of protein. Among those engineered strains, Bs168/pMA5-PspoVG-DSdpe-PsrfA-RCdpe exhibited the highest enzyme activity with 480.1 U/mL on fed-batch fermentation process in a 5 L fermenter at pH 6.5, about 2.1-times higher than the 228.5 U/mL of flask fermentation. Finally, the maximum yield of D-allulose reached as high as 163.5 g/L at the fructose concentration (50% w/v) by whole-cell biocatalyst. CONCLUSION: In this work, the engineered recombinant strain Bs168/pMA5-PspoVG-DSdpe-PsrfA-RCdpe was demonstrated as an effective microbial cell factory for the high-efficient synthesis of D-allulose without browning under acidic conditions. Based on the perspectives from this research, this strategy presented here also made it possible to meet the requirements of the industrial hyper-production of other rare sugars under more acidic conditions in theory.

Chronic developmental exposure to low-dose ([C8mim][PF6]) induces neurotoxicity and behavioural abnormalities in rats.

Ionic liquids (ILs) are widely used for their physical and chemical properties. Toxicological assessments of ILs could help to avoid their threat to human health, but these are rarely reported, and no assessments of IL neurotoxicity in mammals have been performed. Here, we aimed to evaluate the neurotoxicity of chronic 1-octyl-3-methylimidazolium hexafluorophosphate ([C8mim][PF6]) (0, 1 mg/kg) exposure during development on rats. Our results indicated that chronic exposure to low-dose ([C8mim][PF6]) induces behavioural abnormalities, including cognitive deficits, social communication disorders, and sensory gating function impairment. Moreover, rats subjected to chronic ([C8mim][PF6]) exposure showed hypofunction of glutamatergic excitatory synapses, including increased expression of NMDA receptor subunits, increased density and immaturity of dendritic spines, and increased expression of PSD95. Additionally, ([C8mim][PF6]) exposure resulted in hippocampal-specific inflammatory activation, indicated by increased levels of proinflammatory factors, elevated nuclear localisation of NF-κB, and activation of microglia and astrocytes. In conclusion, chronic exposure to low-dose ([C8mim][PF6]) induced neurotoxicity, including damage to glutamatergic excitatory synapses and inflammatory activation, which may illuminate the associated behavioural abnormalities. The results presented here may be helpful for the safe use of ILs in the future.

PII Signal Transduction Protein GlnK Alleviates Feedback Inhibition of N-Acetyl-l-Glutamate Kinase by l-Arginine in Corynebacterium glutamicum.

PII signal transduction proteins are ubiquitous and highly conserved in bacteria, archaea, and plants and play key roles in controlling nitrogen metabolism. However, research on biological functions and regulatory targets of PII proteins remains limited. Here, we illustrated experimentally that the PII protein Corynebacterium glutamicum GlnK (CgGlnK) increased l-arginine yield when glnK was overexpressed in Corynebacterium glutamicum Data showed that CgGlnK regulated l-arginine biosynthesis by upregulating the expression of genes of the l-arginine metabolic pathway and interacting with N-acetyl-l-glutamate kinase (CgNAGK), the rate-limiting enzyme in l-arginine biosynthesis. Further assays indicated that CgGlnK contributed to alleviation of the feedback inhibition of CgNAGK caused by l-arginine. In silico analysis of the binding interface of CgGlnK-CgNAGK suggested that the B and T loops of CgGlnK mainly interacted with C and N domains of CgNAGK. Moreover, F11, R47, and K85 of CgGlnK were identified as crucial binding sites that interact with CgNAGK via hydrophobic interaction and H bonds, and these interactions probably had a positive effect on maintaining the stability of the complex. Collectively, this study reveals PII-NAGK interaction in nonphotosynthetic microorganisms and further provides insights into the regulatory mechanism of PII on amino acid biosynthesis in corynebacteria.IMPORTANCE Corynebacteria are safe industrial producers of diverse amino acids, including l-glutamic acid and l-arginine. In this study, we showed that PII protein GlnK played an important role in l-glutamic acid and l-arginine biosynthesis in C. glutamicum Through clarifying the molecular mechanism of CgGlnK in l-arginine biosynthesis, the novel interaction between CgGlnK and CgNAGK was revealed. The alleviation of l-arginine inhibition of CgNAGK reached approximately 48.21% by CgGlnK addition, and the semi-inhibition constant of CgNAGK increased 1.4-fold. Furthermore, overexpression of glnK in a high-yield l-arginine-producing strain and fermentation of the recombinant strain in a 5-liter bioreactor led to a remarkably increased production of l-arginine, 49.978 g/liter, which was about 22.61% higher than that of the initial strain. In conclusion, this study provides a new strategy for modifying amino acid biosynthesis in C. glutamicum.

LysR-Type Transcriptional Regulator MetR Controls Prodigiosin Production, Methionine Biosynthesis, Cell Motility, H2O2 Tolerance, Heat Tolerance, and Exopolysaccharide Synthesis in Serratia marcescens.

Prodigiosin, a secondary metabolite produced by Serratia marcescens, has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. However, information on the regulatory mechanism behind prodigiosin biosynthesis in S. marcescens remains limited. In this work, a prodigiosin-hyperproducing strain with the BVG90_22495 gene disrupted (ZK66) was selected from a collection of Tn5G transposon insertion mutants. Using real-time quantitative PCR (RT-qPCR) analysis, ß-galactosidase assays, transcriptomics analysis, and electrophoretic mobility shift assays (EMSAs), the LysR-type regulator MetR encoded by the BVG90_22495 gene was found to affect prodigiosin synthesis, and this correlated with MetR directly binding to the promoter region of the prodigiosin-synthesis positive regulator PigP and hence negatively regulated the expression of the prodigiosin-associated pig operon. More analyses revealed that MetR regulated some other important cellular processes, including methionine biosynthesis, cell motility, H2O2 tolerance, heat tolerance, exopolysaccharide synthesis, and biofilm formation in S. marcescens Although MetR protein is highly conserved in many bacteria, we report here on the LysR-type regulator MetR exhibiting novel roles in negatively regulating prodigiosin synthesis and positively regulating heat tolerance, exopolysaccharide synthesis, and biofilm formation.IMPORTANCESerratia marcescens, a Gram-negative bacterium, is found in a wide range of ecological niches and can produce several secondary metabolites, including prodigiosin, althiomycin, and serratamolide. Among them, prodigiosin shows diverse functions as an immunosuppressant, antimicrobial, and anticancer agent. However, the regulatory mechanisms behind prodigiosin synthesis in S. marcescens are not completely understood. Here, we adapted a transposon mutant library to identify the genes related to prodigiosin synthesis, and the BVG90_22495 gene encoding the LysR-type regulator MetR was found to negatively regulate prodigiosin synthesis. The molecular mechanism of the metR mutant hyperproducing prodigiosin was investigated. Additionally, we provided evidence supporting new roles for MetR in regulating methionine biosynthesis, cell motility, heat tolerance, H2O2 tolerance, and exopolysaccharide synthesis in S. marcescens Collectively, this work provides novel insight into regulatory mechanisms of prodigiosin synthesis and uncovers novel roles for the highly conserved MetR protein in regulating prodigiosin synthesis, heat tolerance, exopolysaccharide (EPS) synthesis, and biofilm formation.

Optimization of l-arginine purification from Corynebacterium crenatum fermentation broth.

l-Arginine has many special physiological and biochemical functions, with wide applications in the food and pharmaceutical industry. Few studies on the purification of l-arginine from fermentation broth have been conducted; however, none of them were systematic enough for industrial scale-up. Therefore, it is necessary to develop a highly efficient and systematic process for the purification of l-arginine from fermentation broth. In this study, we screened out a cation exchange resin, D155, having high exchange capacity, high selectivity, and easy elution capacity, and analyzed its adsorption isotherm, thermodynamics, and kinetics using different models. Further, the process parameters of fixed-bed ion exchange adsorption and elution were optimized, and the penetration curve during the operation was modeled. Based on the fixed-bed ion-exchange parameters, a 30-column continuous ion-exchange system was designed, and the flow velocity in each zone was optimized. Finally, to obtain a high purity of l-arginine, the purification tests were conducted using anion exchange resin 711, and an l-arginine yield of 99.1% and purity of 98.5% was obtained. This effective and economical method also provides a promising strategy for separation of other amino acids from the fermentation broth, which is of great significance to the l-arginine fermentation industry.

Synthetic engineering of Corynebacterium crenatum to selectively produce acetoin or 2,3-butanediol by one step bioconversion method.

BACKGROUND: Acetoin (AC) and 2,3-butanediol (2,3-BD) as highly promising bio-based platform chemicals have received more attentions due to their wide range of applications. However, the non-efficient substrate conversion and mutually transition between AC and 2,3-BD in their natural producing strains not only led to a low selectivity but also increase the difficulty of downstream purification. Therefore, synthetic engineering of more suitable strains should be a reliable strategy to selectively produce AC and 2,3-BD, respectively. RESULTS: In this study, the respective AC (alsS and alsD) and 2,3-BD biosynthesis pathway genes (alsS, alsD, and bdhA) derived from Bacillus subtilis 168 were successfully expressed in non-natural AC and 2,3-BD producing Corynebacterium crenatum, and generated recombinant strains, C. crenatum SD and C. crenatum SDA, were proved to produce 9.86 g L-1 of AC and 17.08 g L-1 of 2,3-BD, respectively. To further increase AC and 2,3-BD selectivity, the AC reducing gene (butA) and lactic acid dehydrogenase gene (ldh) in C. crenatum were then deleted. Finally, C. crenatumΔbutAΔldh SD produced 76.93 g L-1 AC in one-step biocatalysis with the yield of 0.67 mol mol-1. Meanwhile, after eliminating the lactic acid production and enhancing 2,3-butanediol dehydrogenase activity, C. crenatumΔldh SDA synthesized 88.83 g L-1 of 2,3-BD with the yield of 0.80 mol mol-1. CONCLUSIONS: The synthetically engineered C. crenatumΔbutAΔldh SD and C. crenatumΔldh SDA in this study were proved as an efficient microbial cell factory for selective AC and 2,3-BD production. Based on the insights from this study, further synthetic engineering of C. crenatum for AC and 2,3-BD production is suggested.

Identification of steroid C27 monooxygenase isoenzymes involved in sterol catabolism and stepwise pathway engineering of Mycobacterium neoaurum for improved androst-1,4-diene-3,17-dione production.

Cholesterol oxidase, steroid C27 monooxygenase and 3-ketosteroid-Δ1-dehydrogenase are key enzymes involved in microbial catabolism of sterols. Here, three isoenzymes of steroid C27 monooxygenase were firstly characterized from Mycobacterium neoaurum as the key enzyme in sterol C27-hydroxylation. Among these three isoenzymes, steroid C27 monooxygenase 2 exhibits the strongest function in sterol catabolism. To improve androst-1,4-diene-3,17-dione production, cholesterol oxidase, steroid C27 monooxygenase 2 and 3-ketosteroid-Δ1-dehydrogenase were coexpressed to strengthen the metabolic flux to androst-1,4-diene-3,17-dione, and 3-ketosteroid 9α-hydroxylase, which catalyzes the androst-1,4-diene-3,17-dione catabolism, was disrupted to block the androst-1,4-diene-3,17-dione degradation pathway in M. neoaurum JC-12. Finally, the recombinant strain JC-12S2-choM-ksdd/ΔkshA produced 20.1 g/L androst-1,4-diene-3,17-dione, which is the highest reported production with sterols as substrate. Therefore, this work is hopes to pave the way for efficient androst-1,4-diene-3,17-dione production through metabolic engineering.

Intracellular Environment Improvement of Mycobacterium neoaurum for Enhancing Androst-1,4-Diene-3,17-Dione Production by Manipulating NADH and Reactive Oxygen Species Levels.

As one of the most significant steroid hormone precursors, androst-1,4-diene-3,17-dione (ADD) could be used to synthesize many valuable hormone drugs. The microbial transformation of sterols to ADD has received extensive attention in recent years. In a previous study, Mycobacterium neoaurum JC-12 was isolated and converted sterols to the major product, ADD. In this work, we enhanced ADD yield by improving the cell intracellular environment. First, we introduced a nicotinamide adenine dinucleotide (NADH) oxidase from Bacillus subtilis to balance the intracellular NAD+ availability in order to strengthen the ADD yield. Then, the catalase gene from M. neoaurum was also over-expressed to simultaneously scavenge the generated H2O2 and eliminate its toxic effects on cell growth and sterol transformation. Finally, using a 5 L fermentor, the recombinant strain JC-12yodC-katA produced 9.66 g/L ADD, which increased by 80% when compared with the parent strain. This work shows a promising way to increase the sterol transformation efficiency by regulating the intracellular environment.

A Novel 3-Phytosterone-9α-Hydroxylase Oxygenation Component and Its Application in Bioconversion of 4-Androstene-3,17-Dione to 9α-Hydroxy-4-Androstene-3,17-Dione Coupling with A NADH Regeneration Formate Dehydrogenase.

9α-Hydroxy-4-androstene-3,17-dione (9-OH-AD) is one of the significant intermediates for the preparation of ß-methasone, dexamethasone, and other steroids. In general, the key enzyme that enables the biotransformation of 4-androstene-3,17-dione (AD) to 9-OH-AD is 3-phytosterone-9α-hydroxylase (KSH), which consists of two components: a terminal oxygenase (KshA) and ferredoxin reductase (KshB). The reaction is carried out with the concomitant oxidation of NADH to NAD+. In this study, the more efficient 3-phytosterone-9α-hydroxylase oxygenase (KshC) from the Mycobacterium sp. strain VKM Ac-1817D was confirmed and compared with reported KshA. To evaluate the function of KshC on the bioconversion of AD to 9-OH-AD, the characterization of KshC and the compounded system of KshB, KshC, and NADH was constructed. The optimum ratio of KSH oxygenase to reductase content was 1.5:1. An NADH regeneration system was designed by introducing a formate dehydrogenase, further confirming that a more economical process for biological transformation from AD to 9-OH-AD was established. A total of 7.78 g of 9-OH-AD per liter was achieved through a fed-batch process with a 92.11% conversion rate (mol/mol). This enzyme-mediated hydroxylation method provides an environmentally friendly and economical strategy for the production of 9-OH-AD.

Acute ethanol exposure-induced autophagy-mediated cardiac injury via activation of the ROS-JNK-Bcl-2 pathway.

Binge drinking is associated with increased cardiac autophagy, and often triggers heart injury. Given the essential role of autophagy in various cardiac diseases, this study was designed to investigate the role of autophagy in ethanol-induced cardiac injury and the underlying mechanism. Our study showed that ethanol exposure enhanced the levels of LC3-II and LC3-II positive puncta and promoted cardiomyocyte apoptosis in vivo and in vitro. In addition, we found that ethanol induced autophagy and cardiac injury largely via the sequential triggering of reactive oxygen species (ROS) accumulation, activation of c-Jun NH2-terminal kinase (JNK), phosphorylation of Bcl-2, and dissociation of the Beclin 1/Bcl-2 complex. By contrast, inhibition of ethanol-induced autophagic flux with pharmacologic agents in the hearts of mice and cultured cells significantly alleviated ethanol-induced cardiomyocyte apoptosis and heart injury. Elimination of ROS with the antioxidant N-acetyl cysteine (NAC) or inhibition of JNK with the JNK inhibitor SP600125 reduced ethanol-induced autophagy and subsequent autophagy-mediated apoptosis. Moreover, metallothionein (MT), which can scavenge reactive oxygen and nitrogen species, also attenuated ethanol-induced autophagy and cell apoptosis in MT-TG mice. In conclusion, our findings suggest that acute ethanol exposure induced autophagy-mediated heart toxicity and injury mainly through the ROS-JNK-Bcl-2 signaling pathway.

miR-23b promotes cutaneous wound healing through inhibition of the inflammatory responses by targeting ASK1.

Wound healing is a complicated event that develops in three overlapping phases: inflammatory, proliferative, and remodeling. MicroRNAs (miRNAs) have been proved to play an important role in the healing process of skin trauma, and alteration of specific miRNA expression during different phases may be associated with abnormal wound healing. In this study, we determined the variation of miR-23b expression after trauma in normal mice and in cultured cells exposed to lipopolysaccharide. We further demonstrated that excessive miR-23b could significantly accelerate wound healing in vivo. Up-regulation of miR-23b decreases infiltration of inflammatory cells, as evidenced by pathologic staining. Meanwhile, miR-23b could significantly inhibit the expression of pro-inflammatory cytokines, including TNF-α, IL-1ß, IL-6, and Ccl2, and significantly increase anti-inflammatory factor IL-10. Furthermore, miR-23b could also promote α-SMA expression in a fiber pattern and increase the expression of Col1a1 and Col3a1. Importantly, we also showed that miR-23b could inhibit inflammation to promote wound healing by targeting apoptotic signal-regulating kinase 1 (ASK1). Notably, knockdown of ASK1 could reduce inflammation factor expression in vitro. Together, our data reveal that miR-23b is a potent therapeutic agent for cutaneous wound healing that shortens the period of inflammatory responses and promotes keratinocyte migration for the re-epithelialization of wound sites.

A Novel CXCR4 antagonist enhances angiogenesis via modifying the ischaemic tissue environment.

Endothelial progenitor cells (EPCs) play a capital role in angiogenesis via directly participating in neo-vessel formation and secreting pro-angiogenic factors. Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 play a critical role in the retention and quiescence of EPCs within its niche in the bone marrow. Disturbing the interaction between SDF-1 and CXCR4 is an effective strategy for EPC mobilization. We developed a novel CXCR4 antagonist P2G, a mutant protein of SDF-1ß with high antagonistic activity against CXCR4 and high potency in enhancing ischaemic angiogenesis and blood perfusion. However, its direct effects on ischaemic tissue remain largely unknown. In this study, P2G was found to possess a robust capability to promote EPC infiltration and incorporation in neo-vessels, enhance the expression and function of pro-angiogenic factors, such as SDF-1, vascular endothelial growth factor and matrix metalloprotein-9, and activate cell signals involved in angiogenesis, such as proliferating cell nuclear antigen, protein kinase B (Akt), extracellular regulated protein kinases and mammalian target of rapamycin, in ischaemic tissue. Moreover, P2G can attenuate fibrotic remodelling to facilitate the recovery of ischaemic tissue. The capability of P2G in direct augmenting ischaemic environment for angiogenesis suggests that it is a potential candidate for the therapy of ischaemia diseases.

A mutant form of 3-ketosteroid-Δ(1)-dehydrogenase gives altered androst-1,4-diene-3, 17-dione/androst-4-ene-3,17-dione molar ratios in steroid biotransformations by Mycobacterium neoaurum ST-095.

Mycobacterium neoaurum ST-095 and its mutant M. neoaurum JC-12, capable of transforming phytosterol to androst-1,4-diene-3,17-dione (ADD) and androst-4-ene-3,17-dione (AD), produce very different molar ratios of ADD/AD. The distinct differences were related to the enzyme activity of 3-ketosteroid-Δ(1)-dehydrogenase (KSDD), which catalyzes the C1,2 dehydrogenation of AD to ADD specifically. In this study, by analyzing the primary structure of KSDDI (from M. neoaurum ST-095) and KSDDII (from M. neoaurum JC-12), we found the only difference between KSDDI and KSDDII was the mutation of Val(366) to Ser(366). This mutation directly affected KSDD enzyme activity, and this result was confirmed by heterologous expression of these two enzymes in Bacillus subtilis. Assay of the purified recombinant enzymes showed that KSDDII has a higher C1,2 dehydrogenation activity than KSDDI. The functional difference between KSDDI and KSDDII in phytosterol biotransformation was revealed by gene disruption and complementation. Phytosterol transformation results demonstrated that ksdd I and ksdd II gene disrupted strains showed similar ADD/AD molar ratios, while the ADD/AD molar ratios of the ksdd I and ksdd II complemented strains were restored to their original levels. These results proved that the different ADD/AD molar ratios of these two M. neoaurum strains were due to the differences in KSDD. Finally, KSDD structure analysis revealed that the Val(366)Ser mutation could possibly play an important role in stabilizing the active center and enhancing the interaction of AD and KSDD. This study provides a reliable theoretical basis for understanding the structure and catalytic mechanism of the Mycobacteria KSDD enzyme.

The Indeterminate Domain Protein ROC1 Regulates Chilling Tolerance via Activation of DREB1B/CBF1 in Rice.

Abiotic stress, including salinity, drought and cold, severely affect diverse aspects of plant development and production. Rice is an important crop that does not acclimate to cold; therefore, it is relatively sensitive to low temperature stress. Dehydration-responsive element-binding protein 1s (DREB1s)/C-repeat binding factors (CBFs) are well known for their function in cold tolerance, but the transcriptional regulation of CBFs remains elusive, especially in rice. Here, we performed a yeast one-hybrid assay using the promoter of CBF1, a cold-induced gene, to isolate transcriptional regulators of CBF1. Among the seven candidates identified, an indeterminate domain (IDD) protein named ROC1 (a regulator of CBF1) was further analyzed. The ROC1 transcript was induced by exogenously-treated auxin, while it was not altered by cold or ABA stimuli. ROC1-GFP was localized at the nucleus, and ROC1 showed trans-activation activity in yeast. The electrophoretic mobility shift assay (EMSA) and ChIP analyses revealed that ROC1 directly bound to the promoter of CBF1. Furthermore, ROC1 mutants exhibited chilling-sensitive symptoms and inhibited cold-mediated induction of CBF1 and CBF3, indicating that ROC1 is a positive regulator of cold stress responses. Taken together, this study identified the CBF1 regulator, and the results are important for rice plant adaptation to chilling stress.

Fibroblast growth factor 21 protects the heart from apoptosis in a diabetic mouse model via extracellular signal-regulated kinase 1/2-dependent signalling pathway.

AIMS/HYPOTHESIS: This study investigated fibroblast growth factor 21 (FGF21)-mediated cardiac protection against apoptosis caused by diabetic lipotoxicity and explored the protective mechanisms involved. METHODS: Cardiac Fgf21 mRNA expression was examined in a diabetic mouse model using real-time PCR. After pre-incubation of palmitate-treated cardiac H9c2 cells and primary cardiomyocytes with FGF21 for 15 h, apoptosis and Fgf21-induced cell-survival signalling were investigated using small interfering (si)RNA and/or pharmacological inhibitors. We also examined the cardiac apoptotic signalling and structural and functional indices in wild-type and Fgf21-knockout (Fgf21-KO) diabetic mice. RESULTS: In a mouse model of type 1 diabetes, cardiac Fgf21 expression was upregulated about 40-fold at 2 months and 3-1.5-fold at 4 and 6 months after diabetes. FGF21 significantly reduced palmitate-induced cardiac apoptosis. Mechanistically, palmitate downregulated, but FGF21 upregulated, phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2, mitogen-activated protein kinase 14 (p38 MAPK) and AMP-activated protein kinase (AMPK). Inhibition of each kinase with its inhibitor and/or siRNA revealed that FGF21 prevents palmitate-induced cardiac apoptosis via upregulating the ERK1/2-dependent p38 MAPK-AMPK signalling pathway. In vivo administration of FGF21, but not FGF21 plus ERK1/2 inhibitor, to diabetic or fatty-acid-infused mice significantly prevented cardiac apoptosis and reduced inactivation of ERK1/2, p38 MAPK and AMPK and prevented cardiac remodelling and dysfunction. The Fgf21-KO mice were more susceptible to diabetes-induced cardiac apoptosis, and this could be prevented by administration of FGF21. Deletion of Fgf21 did not further exacerbate cardiac dysfunction. CONCLUSIONS/INTERPRETATION: These results demonstrate that FGF21 prevents lipid- or diabetes-induced cardiac apoptosis by activating the ERK1/2-p38 MAPK-AMPK pathway. FGF21 may be a therapeutic target for the treatment of diabetes-related cardiac damage.

Additive protection by LDR and FGF21 treatment against diabetic nephropathy in type 2 diabetes model.

The onset of diabetic nephropathy (DN) is associated with both systemic and renal changes. Fibroblast growth factor (FGF)-21 prevents diabetic complications mainly by improving systemic metabolism. In addition, low-dose radiation (LDR) protects mice from DN directly by preventing renal oxidative stress and inflammation. In the present study, we tried to define whether the combination of FGF21 and LDR could further prevent DN by blocking its systemic and renal pathogeneses. To this end, type 2 diabetes was induced by feeding a high-fat diet for 12 wk followed by a single dose injection of streptozotocin. Diabetic mice were exposed to 50 mGy LDR every other day for 4 wk with and without 1.5 mg/kg FGF21 daily for 8 wk. The changes in systemic parameters, including blood glucose levels, lipid profiles, and insulin resistance, as well as renal pathology, were examined. Diabetic mice exhibited renal dysfunction and pathological abnormalities, all of which were prevented significantly by LDR and/or FGF21; the best effects were observed in the group that received the combination treatment. Our studies revealed that the additive renal protection conferred by the combined treatment against diabetes-induced renal fibrosis, inflammation, and oxidative damage was associated with the systemic improvement of hyperglycemia, hyperlipidemia, and insulin resistance. These results suggest that the combination treatment with LDR and FGF21 prevented DN more efficiently than did either treatment alone. The mechanism behind these protective effects could be attributed to the suppression of both systemic and renal pathways.

Lactate levels in the brain and blood of schizophrenia patients: A systematic review and meta-analysis.

BACKGROUND: The pathophysiological mechanisms of schizophrenia are still unclear. Converging evidence suggests that energy metabolism abnormalities are involved in schizophrenia, and support its role in the pathophysiology of this disease. Lactate plays an important role in energy metabolism. Many studies have reported changes in the levels of lactate in the brain and serum of schizophrenia patients; however, the results from these studies are not consistent. To overcome this limitation, the goal of the present meta-analysis is to analyze the changes in lactate levels in the brain and blood of schizophrenia patients. METHODS: For this systematic review and meta-analysis, we performed a thorough search of relevant literature in the English language, using the MEDLINE, Cochrane, and Embase databases. RESULTS: In the present meta-analysis, 20 studies were scrutinized, including 13 studies on brain lactate levels, which involved 322 schizophrenia patients and 324 healthy individuals as controls. 7 studies on blood lactate levels, involving 234 schizophrenia patients and 238 healthy individuals, were also included. Brain lactate levels were elevated in schizophrenia patients, both in vivo and in post-mortem studies. Nevertheless, blood lactate levels in schizophrenia patients have revealed no statistically significant difference, as compared with control individuals. CONCLUSIONS: In comparison with healthy individuals, schizophrenia patients had higher lactate levels in the brain, rather than in the blood. These findings suggest independent regulatory mechanisms of lactate levels in the brain and peripheral tissues. Abnormal lactate metabolism in the brain may be an important pathological mechanism in schizophrenia.

Decreased CNNM2 expression in prefrontal cortex affects sensorimotor gating function, cognition, dendritic spine morphogenesis and risk of schizophrenia.

Genome-wide association studies (GWASs) have reported multiple single nucleotide polymorphisms (SNPs) associated with schizophrenia, yet the underlying molecular mechanisms are largely unknown. In this study, we aimed to identify schizophrenia relevant genes showing alterations in mRNA and protein expression associated with risk SNPs at the 10q24.32-33 GWAS locus. We carried out the quantitative trait loci (QTL) and summary data-based Mendelian randomization (SMR) analyses, using the PsychENCODE dorsolateral prefrontal cortex (DLPFC) expression QTL (eQTL) database, as well as the ROSMAP and Banner DLPFC protein QTL (pQTL) datasets. The gene CNNM2 (encoding a magnesium transporter) at 10q24.32-33 was identified to be a robust schizophrenia risk gene, and was highly expressed in human neurons according to single cell RNA-seq (scRNA-seq) data. We further revealed that reduced Cnnm2 in the mPFC of mice led to impaired cognition and compromised sensorimotor gating function, and decreased Cnnm2 in primary cortical neurons altered dendritic spine morphogenesis, confirming the link between CNNM2 and endophenotypes of schizophrenia. Proteomics analyses showed that reduced Cnnm2 level changed expression of proteins associated with neuronal structure and function. Together, these results identify a robust gene in the pathogenesis of schizophrenia.