Characterization of Heat-labile Uracil-DNA Glycosylase from Oncorhynchus mykiss and its Application for Carry-over Contamination Control in RT-qPCR.

BACKGROUND: Heat-labile uracil-DNA glycosylase (HL-UDG) is commonly employed to eliminate carry-over contamination in DNA amplifications. However, the prevailing HL-UDG is markedly inactivated at 50°C, rendering it unsuitable for specific one-step RT-qPCR protocols utilizing reverse transcriptase at an optimal temperature of 42°C. OBJECTIVE: This study aimed to explore novel HL-UDG with lower inactivation temperature and for recombinant expression. METHODS: The gene encoding an HL-UDG was cloned from the cold-water fish rainbow trout (Oncorhynchus mykiss) and expressed in Escherichia coli with high yield. The thermostability of this enzyme and other enzymatic characteristics were thoroughly examined. The novel HL-UDG was then applied for controlling carry-over contamination in one-step RT-qPCR. RESULTS: This recombinantly expressed truncated HL-UDG of rainbow trout (OmUDG) exhibited high amino acids similarity (84.1% identity) to recombinant Atlantic cod UDG (rcUDG) and was easily denatured at 40°C. The optimal pH of OmUDG was 8.0, and the optimal concentrations of both Na+ and K+ were 10 mM. Since its inactivation temperature was lower than that of rcUDG, the OmUDG could be used to eliminate carry-over contamination in one-step RT-qPCR with moderate reverse transcription temperature. CONCLUSION: We successfully identified and recombinantly expressed a novel HL-UDG with an inactivation temperature of 40°C. It is suitable for eliminating carry-over contamination in one-step RT-qPCR.

Metabolic regulation of forkhead box P3 alternative splicing isoforms and their impact on health and disease.

Forkhead Box P3 (FOXP3) is crucial for the development and suppressive function of human regulatory T cells (Tregs). There are two predominant FOXP3 splicing isoforms in healthy humans, the full-length isoform and the isoform lacking exon 2, with different functions and regulation mechanisms. FOXP3 splicing isoforms show distinct abilities in the cofactor interaction and the nuclear translocation, resulting in different effects on the differentiation, cytokine secretion, suppressive function, linage stability, and environmental adaptation of Tregs. The balance of FOXP3 splicing isoforms is related to autoimmune diseases, inflammatory diseases, and cancers. In response to environmental challenges, FOXP3 transcription and splicing can be finely regulated by T cell antigen receptor stimulation, glycolysis, fatty acid oxidation, and reactive oxygen species, with various signaling pathways involved. Strategies targeting energy metabolism and FOXP3 splicing isoforms in Tregs may provide potential new approaches for the treatment of autoimmune diseases, inflammatory diseases, and cancers. In this review, we summarize recent discoveries about the FOXP3 splicing isoforms and address the metabolic regulation and specific functions of FOXP3 splicing isoforms in Tregs.

Rapid visual detection of Vibrio parahaemolyticus by combining LAMP-CRISPR/Cas12b with heat-labile uracil-DNA glycosylase to eliminate carry-over contamination.

Vibrio parahaemolyticus is a major pathogen frequently found in seafood. Rapid and accurate detection of this pathogen is important for the control of bacterial foodborne diseases and to ensure food safety. In this study, we established a one-pot system that combines uracil-DNA glycosylase (UDG), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12b (Cas12b) for detecting V. parahaemolyticus in seafood. This detection system can effectively perform identification using a single tube and avoid the risk of carry-over contamination.

Rapid and accurate genotyping of human SNP rs671 in aldehyde dehydrogenase 2 gene using one-step CRISPR/Cas12b assay without DNA amplification.

BACKGROUND: The SNP rs671 of Human aldehyde dehydrogenase (ALDH) is G-A transition at 1510th nucleotides, which is an important clinical indicator of alcoholic liver disease, digestive tract cancer and some drug efficiency. The commonly used genotyping assay of this polymorphism is relatively time-consuming and costly. FINDING: This study develops a rapid and accurate one-step CRISPR/Cas12b assay to distinguish the G1510A polymorphism of human ALDH2 free of DNA amplification. The method we established requires only one step of adding 1 µl genomic DNA sample to premixed system, and waiting for the acquisition of fluorescent signal, taking approximate 30 min. CONCLUSIONS: This method provides a potential tool for more accurate and reliable nucleic acid detection with a single base difference and supports the relevant disease diagnosis and personalized medicine.

Improvement of duplex-specific nuclease salt tolerance by fusing DNA-binding domain of DNase from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix.

Salt tolerance is an important property of duplex-specific nuclease (DSN). DSN with high salt tolerance can be more widely used in genetic engineering, especially in the production of nucleic acid drugs. To improve the salt tolerance of DSN, we selected five DNA-binding domains from extremophilic organisms, which have been shown the ability to improve salt tolerance of DNA polymerases and nucleases. The experimental results demonstrated that the fusion protein TK-DSN produced by fusing a N-terminal DNA-binding domain, which comprised two HhH (helix-hairpin-helix) motifs domain from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix, has a significantly improved salt tolerance. TK-DSN can tolerate the concentration of NaCl up to 800 mM; in addition, the ability of digesting DNA was also enhanced during in vitro transcription and RNA purification. This strategy provides the method for the personalized customization of biological tool enzymes for different applications.

Manganese-independent Reverse Transcriptase Activity of Tth DNA Polymerase with Two Amino Acid Substitutions.

BACKGROUND: The DNA polymerase of Thermus thermophilus (Tth pol) presents reverse transcriptase activity with Mn2+, and can be used for one-step RT-qPCR. However, Mn2+ would reduce amplification fidelity and cause nonspecific products. OBJECTIVE: Eliminating the Mn2+ dependence of the reverse transcriptase activity of Tth pol by point mutations. METHODS: We constructed three variants I640F, I709K, and I640F/I709K, and measured their DNA polymerase and reverse transcriptase activities without Mn2+. Their enzymatic characteristics and PCR inhibitor resistance were also tested. Finally, these variants were applied in one-step RT-qPCR. RESULTS: All three variants presented reverse transcriptase activity with Mg2+ only and increased DNA polymerase activity. The variants, except I709K, showed no significant difference in thermostability, optimal pH, optimal NaCl concentration, storage stability and PCR inhibitor resistance compared to the wild type. Variant I640F/I709K had good performance in one-step RT-qPCR with Mg2+ only, whereas both variants with single substitution exhibited nonspecific amplification. CONCLUSION: We successfully constructed three Tth pol variants possessing Mn2+ independent reverse transcriptase activity. The variant I640F/I709K was suitable for one-step RT-qPCR because of its good performance.

Sodium-Glucose Cotransporter 2 Inhibitor Canagliflozin Antagonizes Salt-Sensitive Hypertension Through Modifying Transient Receptor Potential Channels 3 Mediated Vascular Calcium Handling.

Background Salt-sensitive hypertension is highly prevalent and associated with cardiorenal damage. Large clinical trials have demonstrated that SGLT2 (sodium-glucose cotransporter 2) inhibitors exert hypotensive effect and cardiorenal protective benefits in patients with hypertension with and without diabetes. However, the underlying mechanism remains elusive. Methods and Results Dahl salt-sensitive rats and salt-insensitive controls were fed with 8% high-salt diet and some of them were treated with canagliflozin. The blood pressure, urinary sodium excretion, and vascular function were detected. Transient receptor potential channel 3 (TRPC3) knockout mice were used to explain the mechanism. Canagliflozin treatment significantly reduced high-salt-induced hypertension and this effect was not totally dependent on urinary sodium excretion in salt-sensitive hypertensive rats. Assay of vascular function and proteomics showed that canagliflozin significantly inhibited vascular cytoplasmic calcium increase and vasoconstriction in response to high-salt diet. High salt intake increased vascular expression of TRPC3 in salt-sensitive rats, which could be alleviated by canagliflozin treatment. Overexpression of TRPC3 mimicked salt-induced vascular cytosolic calcium increase in vitro and knockout of TRPC3 erased the antihypertensive effect of canagliflozin. Mechanistically, high-salt-induced activation of NCX1 (sodium-calcium exchanger 1) reverse mode increased cytoplasmic calcium level and vasoconstriction, which required TRPC3, and this process could be blocked by canagliflozin. Conclusions We define a previously unrecognized role of TRPC3/NCX1 mediated vascular calcium dysfunction in the development of high-salt-induced hypertension, which can be improved by canagliflozin treatment. This pathway is potentially a novel therapeutic target to antagonize salt-sensitive hypertension.

Identification of the key genes controlling stomach adenocarcinoma stem cell characteristics via an analysis of stemness indices.

Background: In-depth research on tumors has shown that cancer stem cells (CSCs) play a crucial role in tumorigenesis. However, the mechanisms underlying the growth and maintenance of CSCs in stomach adenocarcinoma (STAD) are unclear. This study sought to investigate the expression of stem cell-related genes in STAD. Methods: We identified key genes related to STAD stem cell characteristics by combining gene expression data obtained from The Cancer Genome Atlas to define a messenger ribonucleic acid expression-based stemness index (mRNAsi) based on mRNA expression. The correlations between the mRNAsi and STAD clinical characteristics, including age, tumor grade, pathological stage, and survival status, were explored. Additionally, a weighted gene co-expression network analysis was conducted to identify relevant modules and key genes. The expression verification and functional analysis of the key genes was carried out using multiple databases, including the TIMER (https://cistrome.shinyapps.io/timer/), and Gene Expression Profiling Integrative Analysis, and Gene Expression Omnibus databases. Results: The mRNAsi score was closely related to the clinical characteristics of STAD, including age, tumor grade, pathological stage, and survival status. Similarly, the mRNAsi score was significantly higher in STAD tissues than normal tissues, and the score decreased with tumor stage. The higher the mRNAsi score, the higher the overall survival rate. We screened a module of interest and found a strong correlation between 19 key genes. Among these 19 key genes, 16 had previously been shown to be closely related to STAD survival. The functional analysis showed that these key genes were linked to cell-cycle events, such as chromosome separation, mitosis, and microtubule movement. Conclusions: We identified 19 key genes that play an important role in the maintenance of STAD stem cells. Among these genes, 16 play a role in predicting the prognosis of STAD patients. The cell-cycle pathway was the most important signaling pathway for the key genes associated with STAD stem cells. These findings may provide a new rationale for screening therapeutic targets and the characterization of STAD stem cells.

LncRNA HOXA11-AS promotes vascular endothelial cell injury in atherosclerosis by regulating the miR-515-5p/ROCK1 axis.

AIMS: Long non-coding RNA HOXA11-AS participated in heart disease. In this study, we aim to evaluate the potential roles of HOXA11-AS in atherosclerosis and its underlying mechanisms. METHODS AND RESULTS: The expression levels of HOXA11-AS in ox-LDL-treated HUVECs and arch tissues of high-fat diet-fed ApoE-/- mice (n = 10) were assessed by qRT-PCR. The effects of HOXA11-AS knockdown on the development of atherosclerosis were evaluated using in vitro and in vivo models. Luciferase reporter and RNA immunoprecipitation (RIP) assays verified the potential relationships between HOXA11-AS or ROCK1 and miR-515-5p. The interactive roles between HOXA11-AS and miR-515-5p and between miR-515-5p and ROCK1 were further characterized in ox-LDL-treated HUVECs. Our data showed that HOXA11-AS was significantly up-regulated (P < 0.001), whereas miR-515-5p was dramatically down-regulated in AS mice tissues (P < 0.001) and ox-LDL-treated HUVECs (P < 0.01). Ox-LDL could induce endothelial injuries by inhibiting cell proliferation (P < 0.001) and SOD synthesis (P < 0.001), promoting apoptosis (P < 0.01), ROS (P < 0.001), and MDA production (P < 0.001), increasing Bax (P < 0.001) and cleaved Caspase-3 (P < 0.001), and decreasing Bcl-2 (P < 0.001) and phosphorylated eNOS (P < 0.01). HOXA11-AS knockdown attenuated endothelial injuries via increasing eNOS phosphorylation. Luciferase assay and RIP results confirmed that miR-515-5p is directly bound to HOXA11-AS and ROCK1. HOXA11-AS promoted ox-LDL-induced HUVECs injury by directly inhibiting miR-515-5p from increasing ROCK1 expression and subsequently decreasing the expression and phosphorylation of eNOS. MiR-515-5p mimics could partially reverse the effects of HOXA11-AS knockdown. CONCLUSIONS: HOXA11-AS contributed to atherosclerotic injuries by directly regulating the miR-515-5p/ROCK1 axis. This study provided new evidence that HOXA11-AS might be a candidate for atherosclerosis therapy.

Use of ultra high performance liquid chromatography with high resolution mass spectrometry to analyze urinary metabolome alterations following acute kidney injury in post-cardiac surgery patients.

BACKGROUND: Cardiac surgery-associated acute kidney injury (AKI) can increase the mortality and morbidity, and the incidence of chronic kidney disease, in critically ill survivors. The purpose of this research was to investigate possible links between urinary metabolic changes and cardiac surgery-associated AKI. METHODS: Using ultra-high-performance liquid chromatography coupled with Q-Exactive Orbitrap mass spectrometry, non-targeted metabolomics was performed on urinary samples collected from groups of patients with cardiac surgery-associated AKI at different time points, including Before_AKI (uninjured kidney), AKI_Day1 (injured kidney) and AKI_Day14 (recovered kidney) groups. The data among the three groups were analyzed by combining multivariate and univariate statistical methods, and urine metabolites related to AKI in patients after cardiac surgery were screened. Altered metabolic pathways associated with cardiac surgery-induced AKI were identified by examining the Kyoto Encyclopedia of Genes and Genomes database. RESULTS: The secreted urinary metabolome of the injured kidney can be well separated from the urine metabolomes of uninjured or recovered patients using multivariate and univariate statistical analyses. However, urine samples from the AKI_Day14 and Before_AKI groups cannot be distinguished using either of the two statistical analyses. Nearly 4000 urinary metabolites were identified through bioinformatics methods at Annotation Levels 1-4. Several of these differential metabolites may also perform essential biological functions. Differential analysis of the urinary metabolome among groups was also performed to provide potential prognostic indicators and changes in signalling pathways. Compared with the uninjured kidney group, the patients with cardiac surgery-associated AKI displayed dramatic changes in renal metabolism, including sulphur metabolism and amino acid metabolism. CONCLUSIONS: Urinary metabolite disorder was observed in patients with cardiac surgery-associated AKI due to ischaemia and medical treatment, and the recovered patients' kidneys were able to return to normal. This work provides data on urine metabolite markers and essential resources for further research on AKI.

LncRNA LINC00461 exacerbates myocardial ischemia-reperfusion injury via microRNA-185-3p/Myd88.

OBJECTIVE: Long non-coding RNAs (lncRNAs) play critically in the pathogenesis of myocardial ischemia-reperfusion (I/R) injury. Thus, it was proposed to investigate the mechanism of LINC00461 in the disease through mediating microRNA-185-3p (miR-185-3p)/myeloid differentiation primary response gene 88 (Myd88) axis. METHODS: miR-185-3p, LINC00461 and Myd88 expression in mice with I/R injury was measured. Mice with I/R injury were injected with the gene expression-modified vectors, after which cardiac function, hemodynamics, myocardial enzyme, oxidative stress, and cardiomyocyte apoptosis were analyzed. RESULTS: I/R mice showed LINC00461 and Myd88 up-regulation and miR-185-3p down-regulation. Down-regulating LINC00461 or up-regulating miR-185-3p recovered cardiac function, reduced myocardial enzyme levels, and attenuated oxidative stress and cardiomyocyte apoptosis in mice with I/R. miR-185-3p overexpression rescued the promoting effect of LINC00461 upregulation on myocardial injury in I/R mice. CONCLUSION: LINC00461 knockdown attenuates myocardial I/R injury via elevating miR-185-3p expression to suppress Myd88 expression.

Lack of TRPV1 aggravates obesity-associated hypertension through the disturbance of mitochondrial Ca2+ homeostasis in brown adipose tissue.

The combination of obesity and hypertension is associated with high morbidity and mortality; however, the mechanism underlying obesity-induced hypertension remains unclear. In this study, we detected the possible effects of TRPV1, a previously identified antihypertensive calcium (Ca2+) channel in adipose tissue, on the occurrence of obesity and hypertension in mice lacking UCP1, a spontaneously genetically manipulated obesity model, by generating TRPV1 and UCP1 double knockout mice. In these mice, obesity and hypertension appeared earlier and were more severe than in mice with the knockout of UCP1 or TRPV1 alone. The knockout of TRPV1 in UCP1 knockout mice further reduced functional brown adipose tissue (BAT) generation; decreased resting oxygen consumption, heat production, and locomotor activities; and was accompanied by severe mitochondrial respiratory dysfunction in BAT. Mechanistically, TRPV1, UCP1, and LETM1 acted as a complex to maintain an appropriate mitochondrial Ca2+ level, and TRPV1 knockout caused a compensatory increase in mitochondrial Ca2+ uptake via LETM1 activation. However, the compensatory response was blocked in UCP1-/- mice, resulting in dramatically reduced mitochondrial Ca2+ uptake and higher production of ATP and oxidative stress. This study provides in vivo evidence for the critical role of BAT mitochondrial Ca2+ homeostasis in obesity-associated hypertension and indicates that the TRPV1/UCP1/LETM1 complex may be an alternative intervention target.

Inhibition of MicroRNA-92 alleviates atherogenesis by regulation of macrophage polarization through targeting KLF4.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease in which macrophage polarization plays an important role in contribution to atherosclerotic plaque formation and stability. Here we tested the effect of miR-92 regulation on the development of atherosclerosis beyond tumorigenesis and explored the potential mechanism. METHODS AND RESULTS: In the present study, bone marrow derived macrophages (BMDMs), mouse peritoneal macrophages (MPMs), and human macrophages were used to test the expression of miR-92. Here we noticed miR-92 levels were enhanced in classic M1 macrophage but decreased in alternative M2 macrophage, respectively. In vitro, we demonstrated that macrophages transfected with miR-92 inhibitor attenuated proinflammatory cytokine secretion represented by polarized M1 markers but promoted anti-inflammatory state that was indicative of an M2 phenotype. Mechanistically, miR-92 was found to directly interact with KLF4 and we further identified a requirement role of KLF4 in mediating the effect of miR-92 silencing macrophage polarization. Concomitantly, miR-92 inhibition treated ApoE-/- mice promoted macrophage polarization toward alternative M2 macrophage, thus protecting against atherosclerotic plaque formation and preventing a vulnerable phenotype. CONCLUSION: miR-92 inhibition promoted alternative macrophage activation and attenuated atherosclerosis regression partially regulated in a KLF4-dependent manner, which indicated that miR-92/KLF4 axis may serve as a promising strategy for prevention of atherosclerotic diseases.

Generation and Characterization of Monoclonal Antibodies Against Tth DNA Polymerase and its Application to Hot-Start PCR.

BACKGROUND: As a heat-resistant polymerase, Thermus thermophilus (Tth) DNA polymerase can be widely used in Polymerase Chain Reaction (PCR). However, its non-specific amplification phenomenon is serious, which greatly limits development. OBJECTIVE: In this study, we prepared Tth monoclonal antibodies against Tth DNA polymerase and researched their application in hot-start PCR. METHODS: Tth was recombinantly expressed and purified, and used as an antigen to immunize BALB/ c mice to obtain monoclonal antibodies. The qualified monoclonal antibody and Tth were incubated for a period of time at a certain temperature to obtain the hot-start Tth. We tested the polymerase activity and exonuclease activity blocking the performance of hot-start Tth. Finally, the hot-start Tth was applied to one-step RT-PCR. RESULTS: Tth with a purity of >95% was obtained, and ten monoclonal antibodies were obtained by immunization. After incubation, three monoclonal antibodies were identified that could inhibit the polymerase activity of Tth at low temperature. Furthermore, these three antibodies successfully eliminated non-specific amplification in practical applications. CONCLUSION: Three monoclonal antibodies were successfully validated. Among them, monoclonal antibody 9 had the best overall effect. They possess the function of inhibiting at low temperature and releasing at high temperature, which can be used as Tth polymerase inhibitors in the field of molecular diagnostics.

The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium for thermostability because of high-binding affinity to calcium.

The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium ions for thermostability, and is a promising alternative to commercially available α-amylases to increase the efficiency of industrial processes like the liquefaction of starch. We analyzed the amino acid sequence of this α-amylase by sequence alignments and structural modeling, and found that this α-amylase closely resembles the α-amylase from Pyrococcus woesei. The gene of this α-amylase was cloned in Escherichia coli and the recombinant α-amylase was overexpressed and purified with a combined renaturation-purification procedure. We confirmed thermostability and exogenous calcium ion independency of the recombinant α-amylase and further investigated the mechanism of the independency using biochemical approaches. The results suggested that the α-amylase has a high calcium ion binding affinity that traps a calcium ion that would not dissociate at high temperatures, providing a direct explanation as to why the addition of calcium ions is not required for thermostability. Understanding of the mechanism offers a strong base on which to further engineer properties of this α-amylase for better potential applications in industrial processes.

HyPRP1 Gene Suppressed by Multiple Stresses Plays a Negative Role in Abiotic Stress Tolerance in Tomato.

Many hybrid proline-rich protein (HyPRP) genes respond to biotic and abiotic stresses in plants, but little is known about their roles other than as putative cell-wall structural proteins. A HyPRP1 gene encodes a protein with proline-rich domain, and an eight-cysteine motif was identified from our previous microarray experiments on drought-tolerant tomato. In this study, the expression of the HyPRP1 gene in tomato was suppressed under various abiotic stresses, such as drought, high salinity, cold, heat, and oxidative stress. Transgenic functional analysis showed no obvious changes in phenotypes, but enhanced tolerance to various abiotic stresses (e.g., oxidative stress, dehydration, and salinity) was observed in RNAi transgenic plants. Interestingly, several SO2 detoxification-related enzymes, including sulfite oxidase, ferredoxins (Fds), and methionine sulfoxide reductase A (Msr A), were revealed in HyPRP1-interacting proteins identified by Yeast Two-Hybrid screening. More sulfates and transcripts of Msr A and Fds were accumulated in HyPRP1 knockdown lines when wild-type plants were exposed to SO2 gas. Our findings illustrate that the tomato HyPRP1 is a negative regulator of salt and oxidative stresses and is probably involved in sulfite metabolism.

Knockdown of a JmjC domain-containing gene JMJ524 confers altered gibberellin responses by transcriptional regulation of GRAS protein lacking the DELLA domain genes in tomato.

Plants integrate responses to independent hormonal and environmental signals to survive adversity. In particular, the phytohormone gibberellin (GA) regulates a variety of developmental processes and stress responses. In this study, the Jumonji-C (JmjC) domain-containing gene JMJ524 was characterized in tomato. JMJ524 responded to circadian rhythms and was upregulated by GA treatment. Knockdown of JMJ524 by RNAi caused a GA-insensitive dwarf phenotype with shrunken leaves and shortened internodes. However, in these transgenic plants, higher levels of endogenous GAs were detected. A genome-wide gene expression analysis by RNA-seq indicated that the expression levels of two DELLA-like genes, SlGLD1 ('GRAS protein Lacking the DELLA domain') and SlGLD2, were increased in JMJ524-RNAi transgenic plants. Nevertheless, only the overexpression of SlGLD1 in tomato resulted in a GA-insensitive dwarf phenotype, suggesting that SlGLD1 acts as a repressor of GA signalling. This study proposes that JMJ524 is required for stem elongation by altering GA responses, at least partially by regulating SlGLD1.

Members of the tomato FRUITFULL MADS-box family regulate style abscission and fruit ripening.

The tomato (Solanum lycopersicum) protein MADS-RIN plays important roles in fruit ripening. In this study, the functions of two homologous tomato proteins, FUL1 and FUL2, which contain conserved MIKC domains that typify plant MADS-box proteins, and which interact with MADS-RIN, were analysed. Transgenic functional analysis showed that FUL1 and FUL2 function redundantly in fruit ripening regulation, but exhibit distinct roles in the regulation of cellular differentiation and expansion. Over-expression of FUL2 in tomato resulted in a pointed tip at the blossom end of the fruit, together with a thinner pericarp, reduced stem diameter, and smaller leaves, but no obvious phenotypes resulted from FUL1 over-expression. Dual suppression of FUL1 and FUL2 substantially inhibited fruit ripening by blocking ethylene biosynthesis and decreasing carotenoid accumulation. In addition, the levels of transcript corresponding to ACC SYNTHASE2 (ACS2), which plays a key role in ethylene biosynthesis, were significantly decreased in the FUL1/FUL2 knock-down tomato fruits. Overall, our results suggest that FUL proteins can regulate tomato fruit ripening through fine-tuning ethylene biosynthesis and the expression of ripening-related genes.

Peroxisome proliferator-activated receptor delta protects against obesity-related glomerulopathy through the P38 MAPK pathway.

OBJECTIVE: Obesity is a prominent component of metabolic syndrome and a major risk factor for renal disease. The aim of this study was to explore the effect of cross-talk between peroxisome proliferator-activated receptor (PPAR)δ and p38 mitogen-activated protein kinase (p38 MAPK) on obesity-related glomerulopathy. DESIGN AND METHODS: Male Wistar rats were randomly assigned to standard laboratory chow or a high-fat diet for 32 weeks. Glomerular mesangial cells HBZY-1 and mature differentiation 3T3-L1 cells were cocultured and were transfected with PPARδ-expressing vectors or treated with agonist or inhibitor of PPARδ or p38 MAPK. RESULTS: Rats on a high-fat diet showed typical characteristics of metabolic syndrome including obesity, dyslipidemia, insulin resistance, and hypertension. Rats on a high-fat diet also had significant glomerular hypertrophy and extracellular matrix accumulation, which were accompanied by increased p38 MAPK phosphorylation and decreased PPARδ expression in the kidney tissue. The roles of p38 MAPK and PPARδ in a coculture system of mesangial cells and mature differentiation 3T3-L1 cells were further explored. PPARδ suppression promoted laminin and type IV collagen secretion through p38 MAPK phosphorylation in mesangial cells, whereas PPARδ overexpression or PPARδ agonist attenuated phosphorylation of p38 MAPK and laminin and type IV collagen secretion. CONCLUSIONS: The characteristics of obesity-related glomerulopathy, which might be partly caused by PPARδ suppression-induced p38 MAPK activation and laminin and type IV collagen secretion was demonstrated.

A STAY-GREEN protein SlSGR1 regulates lycopene and ß-carotene accumulation by interacting directly with SlPSY1 during ripening processes in tomato.

As a primary source of lycopene in the human diet, fleshy fruits synthesize this compound both de novo and via chlorophyll metabolism during ripening. SlSGR1 encodes a STAY-GREEN protein that plays a critical role in the regulation of chlorophyll degradation in tomato leaves and fruits. We report that SlSGR1 can regulate tomato (Solanum lycopersicum) lycopene accumulation through direct interaction with a key carotenoid synthetic enzyme SlPSY1, and can inhibit its activity. This interaction with SlSGR1 mediates lycopene accumulation during tomato fruit maturation. We confirmed this inhibitory activity in bacteria engineered to produce lycopene, where the introduction of SlSGR1 reduced dramatically lycopene biosynthesis. The repression of SlSGR1 in transgenic tomato fruits resulted in altered accumulation patterns of phytoene and lycopene, whilst simultaneously elevating SlPSY1 mRNA accumulation and plastid conversion at the early stages of fruit ripening, resulting in increased lycopene and ß-carotene (four- and nine-fold, respectively) in red ripe fruits. SlSGR1 influences ethylene signal transduction via the altered expression of ethylene receptor genes and ethylene-induced genes. Fruit shelf-life is extended significantly in SlSGR1-repressed tomatoes. Our results indicate that SlSGR1 plays a pivotal regulatory role in color formation and fruit ripening regulation in tomato, and further suggest that SlSGR1 activity is mediated through direct interaction with PSY1.