Moderating effect of education on glymphatic function and cognitive performance in mild cognitive impairment.

Objective: This research aims to investigate putative mechanisms between glymphatic activity and cognition in mild cognitive impairment (MCI) and analyzes whether the relationship between cognitive reserve (CR) and cognition was mediated by glymphatic activity. Methods: 54 MCI patients and 31 NCs were enrolled to evaluate the bilateral diffusivity along the perivascular spaces and to acquire an index for diffusivity along the perivascular space (ALPS-index) on diffusion tensor imaging (DTI). The year of education was used as a proxy for CR. The ALPS-index was compared between two groups and correlation analyses among the ALPS-index, cognitive function, and CR were conducted. Mediation analyses were applied to investigate the correlations among CR, glymphatic activity and cognition. Results: MCI group had a significantly lower right ALPS-index and whole brain ALPS-index, but higher bilateral diffusivity along the y-axis in projection fiber area (Dyproj) than NCs. In MCI group, the left Dyproj was negatively related to cognitive test scores and CR, the whole brain ALPS-index was positively correlated with cognitive test scores and CR. Mediation analysis demonstrated that glymphatic activity partially mediated the correlations between CR and cognitive function. Conclusion: MCI exhibited decreased glymphatic activity compared to NCs. CR has a protective effect against cognitive decline in MCI, and this effect may be partially mediated by changes in glymphatic activity.

Optimized decomposition of fresh tomato remnants in facility soil.

To return vegetable remnants to soil in situ and understand parameters that determine their decomposition efficiency, the tomato remnant length, soil moisture, soil temperature and dosage of a microbial decomposer (MD) have been evaluated through a laboratory experiment using a nylon mesh bag in this study. The results showed that the residual remnant weight, and total carbon content increased 28.49 % and 32.65 %, respectively with two different remnant lengths (∼0.5 cm and ∼2.5 cm), while the decay rate and organic carbon breakdown rate decreased by 6.14 % and 7.48 %, respectively. When the relative water content in soil increased, the residual remnant weight and total carbon content first decreased and then increased, while the trend of the decay rate (16.94 % with 80 % soil water content) and organic carbon breakdown rate (9.96 % with 60 % soil water content) were opposite. At a high MD dosage (7 % or 9 % of the total compost weight), both rates of remnants were greater than those at the low dosage (1 %), with an increase of 38.63 % or 36.19 % and 15.89 % or 15.78 %, respectively. With an increase in soil temperature, both residual remnant weight and total carbon content decreased first and then increased, while both decomposition rate and organic carbon breakdown rate increased first and then decreased by 27.35 % and 22.78 %, respectively at 45 °C, compared with those at 30 °C. It was concluded that the decomposition rate was significantly correlated with the remnant length and the MD dosage, while the organic carbon breakdown rate was significantly associated with all four parameters evaluated. The optimal decomposing efficiency was achieved through cutting tomato remnants to a length of ∼0.5 cm, maintaining soil relative moisture content at 89 %, keeping soil temperature at 50 °C, and adding 7 % microbial decomposer MD to chopped tomato cuttings.

Correlations between cognitive reserve, gray matter, and cerebrospinal fluid volume in healthy elders and mild cognitive impairment patients.

Objective: To explore the effect of cognitive reserve (CR) on brain volume and cerebrospinal fluid (CSF) in patients with mild cognitive impairment (MCI) and healthy elders (HE). Methods: 31 HE and 50 MCI patients were collected in this study to obtain structural MRI, cognitive function, and composite CR scores. Educational attainment, leisure time, and working activity ratings from two groups were used to generate cognitive reserve index questionnaire (CRIq) scores. The different volumes of brain regions and CSF were obtained using uAI research portal in both groups, which were taken as the regions of interest (ROI), the correlation analysis between ROIs and CRIq scores were conducted. Results: The scores of CRIq, CRIq-leisure time, and CRIq-education in HE group were significantly higher than patients in MCI group, and the montreal cognitive assessment (MoCA) and minimum mental state examination (MMSE) scores were positively correlated with the CRIq, CRIq-education in both groups, and were positively correlated with CRIq-leisure time in MCI group. The scores of auditory verbal learning test (AVLT) and verbal fluency test (VFT) were also positively correlated with CRIq, CRIq-leisure time, and CRIq-education in MCI group, but the score of AVLT was only positively correlated with CRIq in HE group. Moreover, in MCI group, the volume of the right middle cingulate cortex and the right parahippocampal gyrus were negatively correlated with the CRIq, and the volume of CSF, peripheral CSF, and third ventricle were positively correlated with the CRIq-leisure time score. The result of mediation analysis suggested that right parahippocampal gryus mediated the main effect of the relationship between CRIq and MoCA score in MCI group. Conclusion: People with higher CR show better levels of cognitive function, and MCI patients with higher CR showed more severe volume atrophy of the right middle cingulate cortex and the right parahippocampal gyrus, but more CSF at a given level of global cognition.

Heterogeneous nuclear ribonucleoprotein F deficiency in mouse podocyte promotes podocytopathy mediated by methyltransferase-like 14 nuclear translocation resulting in Sirtuin 1 gene inhibition.

Heterogeneous nuclear ribonucleoprotein F (HnRNP F) is a key regulator for nucleic acid metabolism; however, whether HnRNP F expression is important in maintaining podocyte integrity is unclear. Nephroseq analysis from a registry of human kidney biopsies was performed. Age- and sex-matched podocyte-specific HnRNP F knockout (HnRNP FPOD KO) mice and control (HnRNP Ffl/fl) were studied. Podocytopathy was induced in male mice (more susceptible) either by adriamycin (ADR)- or low-dose streptozotocin treatment for 2 or 8 weeks. The mouse podocyte cell line (mPODs) was used in vitro. Nephroseq data in three human cohorts were varied greatly. Both sexes of HnRNP FPOD KO mice were fertile and appeared grossly normal. However, male 20-week-old HnRNP FPOD KO than HnRNP Ffl/fl mice had increased urinary albumin/creatinine ratio, and lower expression of podocyte markers. ADR- or diabetic- HnRNP FPOD KO (vs. HnRNP Ffl/fl) mice had more severe podocytopathy. Moreover, methyltransferase-like 14 (Mettl14) gene expression was increased in podocytes from HnRNP FPOD KO mice, further enhanced in ADR- or diabetic-treated HnRNP FPOD KO mice. Consequently, this elevated Mettl14 expression led to sirtuin1 (Sirt1) inhibition, associated with podocyte loss. In mPODs, knock-down of HnRNP F promoted Mettl14 nuclear translocation, which was associated with podocyte dysmorphology and Sirt1 inhibition-mediated podocyte loss. This process was more severe in ADR- or high glucose- treated mPODs. Conclusion: HnRNP F deficiency in podocytes promotes podocytopathy through activation of Mettl14 expression and its nuclear translocation to inhibit Sirt1 expression, underscoring the protective role of HnRNP F against podocyte injury.

NRF2 Deficiency Attenuates Diabetic Kidney Disease in Db/Db Mice via Down-Regulation of Angiotensinogen, SGLT2, CD36, and FABP4 Expression and Lipid Accumulation in Renal Proximal Tubular Cells.

The role(s) of nuclear factor erythroid 2-related factor 2 (NRF2) in diabetic kidney disease (DKD) is/are controversial. We hypothesized that Nrf2 deficiency in type 2 diabetes (T2D) db/db mice (db/dbNrf2 knockout (KO)) attenuates DKD progression through the down-regulation of angiotensinogen (AGT), sodium-glucose cotransporter-2 (SGLT2), scavenger receptor CD36, and fatty -acid-binding protein 4 (FABP4), and lipid accumulation in renal proximal tubular cells (RPTCs). Db/dbNrf2 KO mice were studied at 16 weeks of age. Human RPTCs (HK2) with NRF2 KO via CRISPR-Cas9 genome editing and kidneys from patients with or without T2D were examined. Compared with db/db mice, db/dbNrf2 KO mice had lower systolic blood pressure, fasting blood glucose, kidney hypertrophy, glomerular filtration rate, urinary albumin/creatinine ratio, tubular lipid droplet accumulation, and decreased expression of AGT, SGLT2, CD36, and FABP4 in RPTCs. Male and female mice had similar results. NRF2 KO attenuated the stimulatory effect of the Nrf2 activator, oltipraz, on AGT, SGLT2, and CD36 expression and high-glucose/free fatty acid (FFA)-stimulated lipid accumulation in HK2. Kidneys from T2D patients exhibited markedly higher levels of CD36 and FABP4 in RPTCs than kidneys from non-diabetic patients. These data suggest that NRF2 exacerbates DKD through the stimulation of AGT, SGLT2, CD36, and FABP4 expression and lipid accumulation in RPTCs of T2D.

Identification of transcription factors related to diabetic tubulointerstitial injury.

BACKGROUND: Diabetic nephropathy (DN) is a main cause of chronic renal failure. Despite decades of extensive study, the molecular mechanisms underlying diabetic tubulointerstitial injury remain unclear. We aim to identify key transcription factor genes involved in diabetic tubulointerstitial injury. METHODS: A microarray dataset (GSE30122) from Gene Expression Omnibus (GEO) was downloaded. A total of 38 transcription factor genes based on 166 differentially expressed genes (DEGs) were identified by UCSC_TFBS. RESULTS: The regulatory network showed connections between the top 10 transcription factors and their target DEGs. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of targeted DEGs indicated that extracellular space, extracellular exosome, cell surface and complement and coagulation cascades were most significantly enriched. Utilizing Nephroseq v5 online platform, the mRNA expression pattern analysis of transcription factor genes demonstrated that mRNA expression of CDC5, CEBPA, FAC1, HFH1, IRF1, NFE2 and TGIF1 increased in renal tubulointerstitium of DN patients compared with normal controls while that of CEBPB and FOXO4 decreased in renal tubulointerstitium of DN patients compared with normal controls. Correlation analysis between mRNA expression of transcription factor genes in renal tubulointerstitium and clinical features showed that AP1, BACH1, CDC5, FAC1, FOXD1, FOXJ2, FOXO1, FOXO4, HFH1, IRF1, POU3F2, SOX5, SOX9, RSRFC4, S8 and TGIF1 may be related to diabetic tubulointerstitial injury. CONCLUSIONS: (1) CDC5, FAC1, FOXO4, HFH1, IRF1 and TGIF1 may be key transcription factor genes. (2)Transcription factors involved in diabetic tubulointerstitial injury may become prospective targets for diagnosis and treatment of DN.

The Clostridium Metabolite P-Cresol Sulfate Relieves Inflammation of Primary Biliary Cholangitis by Regulating Kupffer Cells.

OBJECTIVE: To study the effect and mechanism of the Clostridium metabolite p-Cresol sulfate (PCS) in primary biliary cholangitis (PBC). METHODS: Gas chromatography-mass spectrometry (GC-MS) was used to detect differences in tyrosine, phenylalanine, tryptophan, PCS, and p-Cresyl glucuronide (PCG) between the serum of PBC patients and healthy controls. In vivo experiments, mice were divided into the normal control, PBC group, and PBC tyrosine group. GC-MS was used to detect PCS and PCG. Serum and liver inflammatory factors were compared between groups along with the polarization of liver Kupffer cells. Additionally, PCS was cultured with normal bile duct epithelial cells and Kupffer cells, respectively. PCS-stimulated Kupffer cells were co-cultured with lipopolysaccharide-injured bile duct epithelial cells to detect changes in inflammatory factors. RESULTS: Levels of tyrosine and phenylalanine were increased, but PCS level was reduced in PBC patients, with PCG showing a lower concentration distribution in both groups. PCS in PBC mice was also lower than those in normal control mice. After oral administration of tyrosine feed to PBC mice, PCS increased, liver inflammatory factors were decreased, and anti-inflammatory factors were increased. Furthermore, Kupffer cells in the liver polarized form M1 transitioned to M2. PCS can damage normal bile duct epithelial cells and suppress the immune response of Kupffer cells. But PCS protects bile duct epithelial cells damaged by LPS through Kupffer cells. CONCLUSIONS: PCS produced by Clostridium-metabolized tyrosine reduced PBC inflammation, suggesting that intervention by food, or supplementation with PCS might represent an effective clinical strategy for treating PBC.

Epac activation ameliorates tubulointerstitial inflammation in diabetic nephropathy.

Tubulointerstitial inflammation plays an important role in the progression of diabetic nephropathy (DN), and tubular epithelial cells (TECs) are crucial promoters of the inflammatory cascade. Exchange protein activated by cAMP (Epac) has been shown to suppress the angiotensin II (Ang-II)-induced release of inflammatory cytokines in tubular cells. However, the role of Epac in TEC-mediated tubulointerstitial inflammation in DN remains unknown. We found that administering the Epac agonist 8-pCPT-2'-O-Me-cAMP (8-O-cAMP) to db/db mice inhibited tubulointerstitial inflammation characterized by macrophage infiltration and increased inflammatory cytokine release and consequently alleviated tubulointerstitial fibrosis in the kidney. Furthermore, 8-O-cAMP administration restored CCAAT/enhancer binding protein ß (C/EBP-ß) expression and further upregulated the expression of Suppressor of cytokine signaling 3 (SOCS3), while inhibiting p-STAT3, MCP-1, IL-6, and TNF-α expression in the kidney cortex in db/db mice. And in vitro study showed that macrophage migration and MCP-1 expression induced by high glucose (HG, 30 mM) were notably reduced by 8-O-cAMP in human renal proximal tubule epithelial (HK-2) cells. In addition, 8-O-cAMP treatment restored C/EBP-ß expression in HK-2 cells and promoted C/EBP-ß translocation to the nucleus, where it transcriptionally upregulated SOCS3 expression, subsequently inhibiting STAT3 phosphorylation. Under HG conditions, siRNA-mediated knockdown of C/EBP-ß or SOCS3 in HK-2 cells partially blocked the inhibitory effect of Epac activation on the release of MCP-1. In contrast, SOCS3 overexpression inhibited HG-induced activation of STAT3 and MCP-1 expression in HK-2 cells. These findings indicate that Epac activation via 8-O-cAMP ameliorates tubulointerstitial inflammation in DN through the C/EBP-ß/SOCS3/STAT3 pathway.

Submucosal tunnel endoscopic resection for a large leiomyoma originating from the muscularis propria in the upper esophagus.

A 57-year-old female underwent esophago-gastroduodenoscopy (EGD) for dysphagia and a mass was found with a central depression in the esophagus, 20 cm from incisors. Contrast-enhanced computed tomography (CT) showed a well-defined 3 x 3 cm esophageal mass and endoscopic ultrasonography (EUS) revealed a heterogeneously hypoechoic mass originating from the muscularis propria.

Mitochondria-Associated Endoplasmic Reticulum Membranes (MAMs) and Their Prospective Roles in Kidney Disease.

Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) serve as essential hubs for interorganelle communication in eukaryotic cells and play multifunctional roles in various biological pathways. A defect in ER-mitochondria signaling or MAMs dysfunction has pleiotropic effects on a variety of intracellular events, which results in disturbances of the mitochondrial quality control system, Ca2+ dyshomeostasis, apoptosis, ER stress, and inflammasome activation, which all contribute to the onset and progression of kidney disease. Here, we review the structure and molecular compositions of MAMs as well as the experimental methods used to study these interorganellar contact sites. We will specifically summarize the downstream signaling pathways regulated by MAMs, mainly focusing on mitochondrial quality control, oxidative stress, ER-mitochondria Ca2+ crosstalk, apoptosis, inflammasome activation, and ER stress. Finally, we will discuss how alterations in MAMs integrity contribute to the pathogenesis of kidney disease and offer directions for future research.

Tacrolimus ameliorates tubulointerstitial inflammation in diabetic nephropathy via inhibiting the NFATc1/TRPC6 pathway.

Tubulointerstitial inflammation is crucial for the progression of diabetic nephropathy (DN), and tubular cells act as a driving force in the inflammatory cascade. Emerging data suggested that tacrolimus (TAC) ameliorates podocyte injury and macrophage infiltration in streptozotocin (STZ) mice. However, the effect of TAC on tubulointerstitial inflammation remains unknown. We found that albuminuria and tubulointerstitial damage improved in db/db mice treated with TAC. Macrophage infiltration and expression of IL-6, TNF-α, fibronectin, collagen 1 and cleaved caspase 3 were inhibited as well. In addition, the expression of nuclear factor of activated T cell 1 (NFATc1) and transient receptor potential channel 6 (TRPC6) was up-regulated in the kidneys of DN patients and correlated with tubular injury and inflammation. The expression of NFATc1 and TRPC6 also increased in the kidneys of db/db mice and HK-2 cells with high glucose (HG), while TAC inhibited these effects. HG-induced inflammatory markers and apoptosis were reversed by TAC and NFATc1 siRNA in HK-2 cells, which was abolished by TRPC6 plasmid. Furthermore, HG-induced TRPC6 expression was inhibited by NFATc1 siRNA, while NFATc1 nuclear translocation was inhibited by TAC, but was restored by TRPC6 plasmid in HK-2 cells under HG conditions. These findings suggest that TAC ameliorates tubulointerstitial inflammation in DN through NFATc1/TRPC6 feedback loop.

Analysis of the Effect of Nine Consecutive Years' Intensive Management and Number of Times Achieving the Target Control on Endpoint Events in T2DM Patients in Sanlitun Community Health Service Center in Beijing.

OBJECTIVE: To investigate the effect of intensive management and achieving the target control more than 3 times on endpoint events during 9 consecutive years' annual assessment in type 2 diabetes (T2DM) patients in the Sanlitun Community Health Service Center in Beijing, including blood glucose, blood pressure, lipids profiles, and the joint target control. METHODS: In Beijing Community Diabetes Study (BCDS), 224 patients with T2DM from the Sanlitun Community Health Service Center were enrolled in 2008. All patients were randomly assigned to the intensive management group (n = 113) and the standard management group (n = 113) and the standard management group (. RESULTS: During the nine-year follow-up, the abscission number was 35 (14.29%), among which 14 (12.39%) was in the intensive management group and 21 (18.92%) was in the standard management group. The incidence of diabetic retinopathy (6 cases, 5.41%) and diabetic nephropathy (13 cases, 11.71%) in the standard management group was significantly higher than that in the intensive management group (1 case, 0.88%; 5 cases, 4.42%), respectively (P < 0.05). However, there were no significant differences on the other endpoint events between the two groups (P < 0.05). However, there were no significant differences on the other endpoint events between the two groups (P < 0.05). However, there were no significant differences on the other endpoint events between the two groups (P < 0.05). However, there were no significant differences on the other endpoint events between the two groups (P < 0.05). However, there were no significant differences on the other endpoint events between the two groups (. CONCLUSIONS: The intensive management can effectively reduce the occurrence of microvascular complications. The incidence of all-cause death and the other endpoint events decreased in T2DM patients who achieved the joint target control more than 3 times during the nine-year management, which improved survival time and life quality. This trial is registered with ChiCTR-TRC-13003978 and ChiCTR-OOC-15006090.

Multiple-microarray analysis for identification of hub genes involved in tubulointerstial injury in diabetic nephropathy.

Diabetic nephropathy (DN) is a primary cause of renal failure. However, studies providing renal gene expression profiles of diabetic tubulointerstitial injury are scarce and its molecular mechanisms still await clarification. To identify vital genes involved in the diabetic tubulointerstitial injury, three microarray data sets from gene expression omnibus (GEO) were downloaded. A total of 127 differentially expressed genes (DEGs) were identified by limma package. Gene set enrichment analysis (GSEA) plots showed that sister chromatid cohesion was the most significant enriched gene set positively correlated with the DN group while retinoid X receptor binding was the most significant enriched gene set positively correlated with the control group. Enriched Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of DEGs mostly included extracellular matrix organization, extracellular space, extracellular matrix structural constituent, and Staphylococcus aureus infection. Twenty hub genes from three significant modules were ascertained by Cytoscape. Correlation analysis and subgroup analysis between hub genes and clinical features of DN showed that ALB, ANXA1, APOH, C3, CCL19, COL1A2, COL3A1, COL4A1, COL6A3, CXCL6, DCN, EGF, HRG, KNG1, LUM, SERPINA3, SPARC, SRGN, and TIMP1 may involve in diabetic tubulointerstitial injury. ConnectivityMap analysis indicated the most significant three compounds are 5182598, thapsigargin and 5224221. In conclusion, this study may provide new insights into the molecular mechanisms underlying diabetic tubulointerstitial injury as well as potential targets for diagnosis and therapeutics of DN.

Identification of key biomarkers in diabetic nephropathy via bioinformatic analysis.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease. Although intense efforts have been made to elucidate the pathogenesis, the molecular mechanisms of DN remain to be clarified. To identify the candidate genes in the progression of DN, microarray datasets GSE30122, GSE30528, and GSE47183 were downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified, and function enrichment analyses were performed. The protein-protein interaction network was constructed and the module analysis was performed using the Search Tool for the Retrieval of Interacting Genes and Cytoscape. A total of 61 DEGs were identified. The enriched functions and pathways of the DEGs included glomerulus development, extracellular exosome, collagen binding, and the PI3K-Akt signaling pathway. Fifteen hub genes were identified and biological process analysis revealed that these genes were mainly enriched in acute inflammatory response, inflammatory response, and blood vessel development. Correlation analysis between unexplored hub genes and clinical features of DN suggested that COL6A3, MS4A6A,PLCE1, TNNC1, TNNI1, TNN2, and VSIG4 may involve in the progression of DN. In conclusion, DEGs and hub genes identified in this study may deepen our understanding of molecular mechanisms underlying the progression of DN, and provide candidate targets for diagnosis and treatment of DN.

Ectopic lipid accumulation: potential role in tubular injury and inflammation in diabetic kidney disease.

Emerging studies suggest that lipid accumulates in the kidneys during diabetic kidney disease (DKD). However, the correlation between ectopic lipid accumulation with tubular damage has not been thoroughly elucidated to date. Using Oil Red staining, lipid accumulation was observed in the kidneys of type 2 DKD patients (classes II-III) and db/db mice compared with the control and was predominantly located in the proximal tubular compartment. Immunohistochemistry (IHC) staining showed that the intensity of adipose differentiation related protein (ADRP) and sterol regulatory element binding protein-1 (SREBP-1) was clearly up-regulated, which was positively correlated with the tubulointerstitial damage score and inflammation. Furthermore, the urine ADRP content significantly increased in DKD patients compared with the control, which positively correlated with abnormal lipid metabolism, serum creatinine, urine N-acetyl-ß-glucosaminidase (NAG), albumin excretion (albumin-to-creatinine ratio (ACR)), and tumor necrosis factor-α (TNF-α) expression. However, there was no significant difference observed in plasma ADRP levels. In addition, the expression of SREBP-1 protein was dramatically increased in peripheral blood mononuclear cells (PBMCs) isolated from DKD patients, which was also tightly correlated with urine NAG, ACR, and TNF-α levels. In vitro studies demonstrated increased ADRP and SREBP-1 expression accompanied by lipid accumulation in HK-2 cells cultured in high glucose (HG). HG induced high levels of TNF-α expression, which was partially blocked by transfection of ADRP siRNA or SREBP-1 siRNA. These data indicated that ADRP and SREBP-1 are crucial factors that mediate lipid accumulation with tubular damage and inflammation in DKD, and ectopic lipid accumulation may serve as a novel therapeutic target for amelioration of tubular injury in DKD.

Chemical composition and antimicrobial activity of Gannan navel orange (Citrus sinensis Osbeck cv. Newhall) peel essential oils.

The present investigation reported the chemical composition of cold pressed Gannan navel orange peel essential oil (EO) and its molecular distillation fraction (light phase EO), and examined their antimicrobial activity against spoiling and pathogenic microorganisms. Gas chromatography-mass spectrometry analysis identified 27 and 20 different chemical constituents in cold pressed EO and light phase EO, respectively. Limonene was the major constituent, accounting for 85.32% of cold pressed EO and 60.44% of light phase EO. Both EOs and some of their constituents showed good antimicrobial activity. Compared to cold pressed EO, light phase EO exhibited the better antimicrobial activity under weak acidic and neutral conditions. The light phase EO presented a higher antimicrobial activity after thermo-treatment at 60-100°C for 20 min than cold pressed EO. These results demonstrated that light phase EO had a potential to be used as a novel antimicrobial agent for food preservation and food processing.

Epsin deficiency promotes lymphangiogenesis through regulation of VEGFR3 degradation in diabetes.

Impaired lymphangiogenesis is a complication of chronic complex diseases, including diabetes. VEGF-C/VEGFR3 signaling promotes lymphangiogenesis, but how this pathway is affected in diabetes remains poorly understood. We previously demonstrated that loss of epsins 1 and 2 in lymphatic endothelial cells (LECs) prevented VEGF-C-induced VEGFR3 from endocytosis and degradation. Here, we report that diabetes attenuated VEGF-C-induced lymphangiogenesis in corneal micropocket and Matrigel plug assays in WT mice but not in mice with inducible lymphatic-specific deficiency of epsins 1 and 2 (LEC-iDKO). Consistently, LECs isolated from diabetic LEC-iDKO mice elevated in vitro proliferation, migration, and tube formation in response to VEGF-C over diabetic WT mice. Mechanistically, ROS produced in diabetes induced c-Src-dependent but VEGF-C-independent VEGFR3 phosphorylation, and upregulated epsins through the activation of transcription factor AP-1. Augmented epsins bound to and promoted degradation of newly synthesized VEGFR3 in the Golgi, resulting in reduced availability of VEGFR3 at the cell surface. Preclinically, the loss of lymphatic-specific epsins alleviated insufficient lymphangiogenesis and accelerated the resolution of tail edema in diabetic mice. Collectively, our studies indicate that inhibiting expression of epsins in diabetes protects VEGFR3 against degradation and ameliorates diabetes-triggered inhibition of lymphangiogenesis, thereby providing a novel potential therapeutic strategy to treat diabetic complications.

In vitro oxidative decarboxylation of free fatty acids to terminal alkenes by two new P450 peroxygenases.

BACKGROUND: P450 fatty acid decarboxylases represented by the unusual CYP152 peroxygenase family member OleTJE have been receiving great attention recently since these P450 enzymes are able to catalyze the simple and direct production of 1-alkenes for potential applications in biofuels and biomaterials. To gain more mechanistic insights, broader substrate spectra, and improved decarboxylative activities, it is demanded to discover and investigate more P450 fatty acid decarboxylases. RESULTS: Here, we describe for the first time the expression, purification, and in vitro biochemical characterization of two new CYP152 peroxygenases, CYP-Aa162 and CYP-Sm46Δ29, that are capable of decarboxylating straight-chain saturated fatty acids. Both enzymes were found to catalyze the decarboxylation and hydroxylation of a broad range of free fatty acids (C10-C20) with overlapping substrate specificity, yet distinct chemoselectivity. CYP-Sm46Δ29 works primarily as a fatty (lauric) acid decarboxylase (66.1 ± 3.9% 1-undecene production) while CYP-Aa162 more as a fatty (lauric) acid hydroxylase (72.2 ± 0.9% hydroxy lauric acid production). Notably, the optical spectroscopic analysis of functional CYP-Sm46Δ29 revealed no characteristic P450 band, suggesting a unique heme coordination environment. Active-site mutagenesis analysis showed that substitution with the proposed key decarboxylation-modulating residues, His85 and Ile170, enhanced the decarboxylation activity of CYP-Aa162 and P450BSß, emphasizing the importance of these residues in directing the decarboxylation pathway. Furthermore, the steady-state kinetic analysis of CYP-Aa162 and CYP-Sm46Δ29 revealed both cooperative and substrate inhibition behaviors which are substrate carbon chain length dependent. CONCLUSIONS: Our data identify CYP-Sm46Δ29 as an efficient OleTJE-like fatty acid decarboxylase. Oxidative decarboxylation chemoselectivity of the CYP152 decarboxylases is largely dependent upon the carbon chain length of fatty acid substrates and their precise positioning in the enzyme active site. Finally, the kinetic mode analysis of the enzymes could provide important guidance for future process design.

Mutagenesis and redox partners analysis of the P450 fatty acid decarboxylase OleTJE.

The cytochrome P450 enzyme OleTJE from Jeotgalicoccus sp. ATCC 8456 is capable of converting free long-chain fatty acids into α-alkenes via one-step oxidative decarboxylation in presence of H2O2 as cofactor or using redox partner systems. This enzyme has attracted much attention due to its intriguing but unclear catalytic mechanism and potential application in biofuel production. Here, we investigated the functionality of a select group of residues (Arg245, Cys365, His85, and Ile170) in the active site of OleTJE through extensive mutagenesis analysis. The key roles of these residues for catalytic activity and reaction type selectivity were identified. In addition, a range of heterologous redox partners were found to be able to efficiently support the decarboxylation activity of OleTJE. The best combination turned out to be SeFdx-6 (ferredoxin) from Synechococcus elongatus PCC 7942 and CgFdR-2 (ferredoxin reductase) from Corynebacterium glutamicum ATCC 13032, which gave the highest myristic acid conversion rate of 94.4%. Moreover, Michaelis-Menton kinetic parameters of OleTJE towards myristic acid were determined.