Severe hypoglycaemia-induced microglial inflammation damages microvascular endothelial cells, leading to retinal destruction.

Human microglia (HMC) are stress-induced inflammatory cells of the retina. It is unknown whether severe hypoglycaemia causes inflammation in microglia, affects the permeability of human retinal microvascular endothelial cells (HRMECs), and causes retinal damage. This study aimed to explore the effects of severe hypoglycaemia on retinal microglial inflammation and endothelial cell permeability and evaluate the damage caused by hypoglycaemia to the retina. The CCK-8 assay was used to measure cell viability. Western blotting was used to detect IL-1ß, IL-6, TNF- α, claudin-1, and occludin expression. ELISA was used to detect IL-1ß, IL-6, and TNF- α. Transmission electron microscopy (TEM) and haematoxylin and eosin staining were used to observe the retinal structure. Immunohistochemistry and immunofluorescence staining assays were also used to detect IL-1ß, IL-6, TNF- α, claudin-1, and occludin expression. Severe hypoglycaemia promoted inflammation in HMC3 cells. Inflammation caused by hypoglycaemia leads to the decreased expression of tight junction proteins. In vivo, severe hypoglycaemia induced structural damage to the retina, increased the expression of inflammatory factors, and decreased the expression of tight junction proteins. Our results suggest that severe hypoglycaemia leads to acute retinal inflammation, affecting the permeability of HRMECs and causing retinal damage.

Dapagliflozin ameliorated retinal vascular permeability in diabetic retinopathy rats by suppressing inflammatory factors.

BACKGROUND: Diabetic retinopathy is a common microvascular complication of diabetes and one of the major causes of blindness in the working-age population. Emerging evidence has elucidated that inflammation drives the key mechanism of diabetes-mediated retinal disturbance. As a new therapeutic drug targeting diabetes, whether dapagliflozin could improve vascular permeability from the perspective of anti-inflammatory effect need to be further explored. METHODS: Type 2 diabetic retinopathy rat model was established and confirmed by fundus fluorescein angiography (FFA). ELISA detected level of plasma inflammatory factors and C-peptide. HE staining, immunohistochemistry and western blot detected histopathology changes of retina, expression of retinal inflammatory factors and tight junction proteins. RESULTS: Dapagliflozin exhibited hypoglycemic effect comparable to insulin, but did not affect body weight. By inhibiting expression of inflammatory factors (NLRP3, Caspase-1, IL-18, NF-κB) in diabetic retina and plasma, dapagliflozin reduced damage of retinal tight junction proteins and improved retinal vascular permeability. The anti-inflammatory effect of dapagliflozin was superior to insulin. CONCLUSIONS: Dapagliflozin improved retinal vascular permeability by reducing diabetic retinal and plasma inflammatory factors. The anti-inflammatory mechanism of dapagliflozin is independent of hypoglycemic effect and superior to insulin.

Association between area under the C-peptide curve during an oral glucose tolerance test and diabetic retinopathy in type 2 diabetes patients.

AIMS/INTRODUCTION: To evaluate the relative contributions of the area under the C-peptide curve (AUCC ) in diabetic retinopathy (DR) during an oral glucose tolerance test and C-peptide release test in patients with type 2 diabetes. MATERIALS AND METHODS: We retrospectively analyzed the data of 969 patients. Their general characteristics were retrieved. A series of parameters for assessing pancreatic ß-cells function, such as the AUCC for six time periods: 0-60 min (AUCC0-60 ), 0-120 min (AUCC0-120 ), 0-180 min (AUCC0-180 ), 60-120 min (AUCC60-120 ), 60-180 min (AUCC60-180 ) and 120-180 min (AUCC120-180 ); the area under the glucose-time curve for six time periods: 0-60 min (AUCG0-60 ), 0-120 min (AUCG0-120 ), 0-180 min (AUCG0-180 ), 60-120 min (AUCG60-120 ), 60-180 min (AUCG60-180 ) and 120-180 min (AUCG120-180 ) and their related indexes, were calculated through 0-180 min oral glucose tolerance test and C-peptide release test. We used univariate analysis to examine the potential factors affecting DR. Spearman's correlation was used to analyze the correlation between AUCC -related indexes and DR. The logistic regression model was used to investigate AUCC and its related indexes' contribution to incidence DR. A smooth curve fitting model was used to determine the correlation, non-linear relationship, and threshold effect between AUCC and DR. RESULTS: Of the 969 patients with type 2 diabetes, 469 (48.40%) and 500 (51.60%) were classified as the DR group and non-DR group. Compared with the non-DR group, the DR patients had lower AUCC and AUCC /AUCG . Spearman's correlation analysis showed that AUCC -related indexes were all negatively correlated with DR. The logistic regression analysis determined that there were associations between AUCC and DR in the adjusted models. The odds ratio values of AUCC0-60 , AUCC0-120 , AUCC0-180 , AUCC0-60 /AUCG0-60 , AUCC0-120 /AUCG0-120 , AUCC0-180 /AUCG0-180 , AUCC60-120 , AUCC60-180 , AUCC120-180 , AUCC60-120 /AUCG60-120 , AUCC60-180 /AUCG60-180 and AUCC120-180 /AUCG120-180 were 0.817 (0.750, 0.890), 0.925 (0.895, 0.955), 0.951 (0.932, 0.970), 0.143 (0.060, 0.340), 0.194 (0.093, 0.406), 0.223 (0.116, 0.427), 0.886 (0.842, 0.933), 0.939 (0.915, 0.963), 0.887 (0.846, 0.930), 0.253 (0.133, 0.479), 0.282 (0.160, 0.497) and 0.355 (0.220, 0.573), respectively. AUCC showed a non-linear relationship with DR, with an inflection point. The inflection points of AUCC180 /AUCG180 , AUCC60-120 , AUCC60-180 , AUCC120-180 , AUCC60-120 /AUCG60-120 , AUCC60-180 /AUCG60-180 , AUCC120-180 /AUCG120-180 and DR were 17.51, 0.542, 6.6, 15.7, 8.23, 0.534, 0.593 and 0.808 (P < 0.0001). When the indexes related to the AUCC were less than the inflection point value, they were significantly negatively associated with DR. CONCLUSIONS: The indexes related to the AUCC for six time periods during an oral glucose tolerance test and C-peptide release test was closely associated with the incidence to DR in patients with type 2 diabetes. AUCC has the added advantage of being a cheap and convenient risk assessment over traditional ophthalmic screening.

The miR156/SPL12 module orchestrates fruit colour change through directly regulating ethylene production pathway in blueberry.

Colour change is an important event during fruit ripening in blueberry. It is well known that miR156/SPLs act as regulatory modules mediating anthocyanin biosynthesis and ethylene plays critical roles during colour change, but the intrinsic connections between the two pathways remain poorly understood. Previously, we demonstrated that blueberry VcMIR156a/VcSPL12 affects the accumulation of anthocyanins and chlorophylls in tomato and Arabidopsis. In this study, we first showed that VcMIR156a overexpression in blueberry led to enhanced anthocyanin biosynthesis, decreased chlorophyll accumulation, and, intriguingly, concomitant elevation in the expression of ethylene biosynthesis genes and the level of the ethylene precursor ACC. Conversely, VcSPL12 enhanced chlorophyll accumulation and suppressed anthocyanin biosynthesis and ACC synthesis in fruits. Moreover, the treatment with ethylene substitutes and inhibitors attenuated the effects of VcMIR156a and VcSPL12 on pigment accumulation. Protein-DNA interaction assays indicated that VcSPL12 could specifically bind to the promoters and inhibit the activities of the ethylene biosynthetic genes VcACS1 and VcACO6. Collectively, our results show that VcMIR156a/VcSPL12 alters ethylene production through targeting VcACS1 and VcACO6, therefore governing fruit colour change. Additionally, VcSPL12 may directly interact with the promoter region of the chlorophyll biosynthetic gene VcDVR, thereby activating its expression. These findings established an intrinsic connection between the miR156/SPL regulatory module and ethylene pathway.

Liraglutide intervention improves high-glucose-induced reactive gliosis of Müller cells and ECM dysregulation.

Reactive gliosis of Müller cells plays an important role in the pathogenesis of diabetic retinopathy (DR). Liraglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist, has been shown to improve DR by inhibiting reactive gliosis. However, the mechanism of inhibition has yet to be elucidated. This study investigated the effects of liraglutide on Müller glia reactivity in the early stages of DR and the underlying mechanisms. Proteomics combined with bioinformatics analysis, HE staining, and immunofluorescence staining revealed ganglion cell loss, reactive gliosis of Müller cells, and extracellular matrix (ECM) imbalance in rats with early stages of DR. High glucose (HG) exposure up-regulated GFAP and TNF-α expression and down-regulated ITGB1 expression and FN1 content in extracellular fluid in rMC1 cells, thereby promoting reactive gliosis. GLP-1R knockdown and HG+DAPT inhibition experiments show that liraglutide balances ECM levels by inhibiting activation of the Notch1/Hes1 pathway and ameliorates high-glucose-induced Müller glia reactivity. Thus, the study provides new targets and ideas for improvement of DR in early stages.

Association between high-density lipoprotein cholesterol to apolipoprotein A ratio and diabetic retinopathy: A cross-sectional study.

AIMS: Our study is aimed to investigate the relationship between high-density lipoprotein cholesterol to apolipoprotein A ratio (HDL-C/ApoA) and diabetic retinopathy (DR) in subjects with type 2 diabetes mellitus (T2DM). METHODS: We retrospect the consecutive medical files of 1058 subjects with T2DM and recorded their clinical information and laboratory findings. Subjects with T2DM were divided into DR group (n = 522) and non-DR group (n = 536). We compared the lipids values of the two groups. Meanwhile we also observed the prevalence of DR at different HDL-C/ApoA levels. Binary logistic regression was used to correct confounding factors. Smooth curve fitting model and subgroup analysis were used to determine the correlation, non-linear relationship and threshold effect between HDL/ApoA and DR. RESULTS: HDL-C/ApoA value of DR group was significantly higher than non-DR group (0.88 ± 0.17 vs 0.84 ± 0.13, P < 0.05). The prevalence of DR significantly increased as HDL-C/ApoA level increased. There was association between HDL/ApoA levels and DR in the adjusted models (OR 1.55, 95%CI 0.60 to 4.02). After full adjustments for other relevant clinical covariates, patients with HDL/ApoA values in quartile 3 (Q3) had 1.50 times (95 % CI 1.00 to 2.17) and in Q4 had 2.39 times (95%CI 1.65 to 3.47) as high as the risk of DR compared with patients in Q1. HDL/ApoA showed a non-linear relationship with DR, with an inflection point value of 0.759. When HDL/ApoA>0.759, HDL/ApoA was significantly positively associated with DR (HR = 26.508, 95 % CI 7.623-92.174; P < 0.0001). Compared to patients with age < 60, HDL/ApoA was obviously associated with DR when age ≥ 60 (OR = 38.05, 95 % CI 8.06-179.69; P < 0.001). CONCLUSIONS: HDL-C/ApoA was found to be associated with the incidence of DR in patients with T2DM. After adjusting potential related factors HDL-C/ApoA OR value was 1.55 (95%CI 0.60 to 4.02). A non-linear association between HDL/ApoA and DR was observed in T2DM. Subgroup analysis showed that age could alter the relationship between HDL/ApoA and DR.

Autophagy Modulates the Migration of Retinal Pericytes Induced by Advanced Glycation End Products.

Retinal pericyte migration occurs in the early stage of diabetic retinopathy (DR), which is one of the important causes of pericyte loss. Autophagy has been found to play essential roles in the regulation of many types of cell migration. In this study, we explored the relationship between autophagy and retinal pericyte migration. In diabetic rats, the retinas became thinner, and the level of autophagy in each cell layer increased. In the primary culture of bovine retinal pericytes, we found that advanced glycation end products (AGEs) increased the migratory cell ability without influencing cell viability, which also increased the phosphorylation of focal adhesion kinase (FAK) and the expression of matrix metalloproteinase (MMP)-2 and decreased the expression of vinculin. AGEs-induced retinal pericyte autophagy and the inhibition of autophagy with chloroquine significantly inhibited cell migration, reversed AGEs-induced FAK phosphorylation, and changed vinculin and MMP-2 protein expression. These results provide a new insight into the migration mechanism of retinal pericytes. The early control of autophagy has a potential effect on regulating pericyte migration, which may contribute to keeping the integrity of retinal vessels in DR.

Proteomics identifies new potential therapeutic targets of diabetic retinopathy.

Retinal pigment epithelium (RPE) is an important component of the outer blood-retinal barrier and plays a critical role in maintaining retinal homeostasis. Alterations in RPE can be detected during the early stages of diabetic retinopathy (DR). However, the molecular mechanisms underlying these early changes remain unclear. We investigated the molecular changes induced in the RPE by high glucose concentrations by constructing a high glucose-induced ARPE-19 cell injury model simulating the DR environment in vitro. Proteomic analysis was conducted to measure differences in protein expression between cells treated with normal (5 mM) and high (25 mM) glucose concentrations, and bioinformatics techniques were used to analyze the mechanism of action. The results of the proteomic analyses were validated using western blotting. High glucose levels inhibited the proliferation of ARPE-19 cells. We identified 88 upregulated proteins and 114 downregulated proteins. Six of these proteins were selected for further validation. Changes in the proteome mainly affected the lysosome and cell cycle pathways. Proteomic differences between ARPE-19 cells treated with normal and high glucose concentrations indicate that damage to the RPE in DR may be caused by specific mechanisms. Our study verified protein changes in ARPE-19 cells in a high-glucose environment and may provide new strategies for understanding the molecular mechanisms underlying DR.

Dapagliflozin Reduces Apoptosis of Diabetic Retina and Human Retinal Microvascular Endothelial Cells Through ERK1/2/cPLA2/AA/ROS Pathway Independent of Hypoglycemic.

Introduction: It is known that the metabolic disorder caused by high glucose is one of pathogenesis in diabetic retinopathy (DR), the leading cause of blindness, due to the main pathological change of apoptosis of endothelial cells (ECs). In previous studies, the potential impact of sodium glucose cotransporter-2 (SGLT-2), whose inhibitors slow the progression of DR, has not been elucidated. The purpose of the presented study was to explore the effect of SGLT-2 inhibitors dapagliflozin (DAPA) on apoptosis of diabetic mice retina and human retinal microvascular endothelial cells (HRMECs), examine the effects of dapagliflozin on HRMECs metabolism, and explore the molecular processes that affect DR. Methods and Results: The eyeballs of male streptozotocin (STZ)-induced diabetic C57BL/6N mice were evaluated. C57BL/6N mice were divided into control group (CON), diabetic untreated group (DM), diabetic dapagliflozin treatment group (DM + DAPA) and diabetic insulin treatment group (DM + INS). Hematoxylin-Eosin (HE) staining was performed to observe the pathological structure of the mice retina, and TUNEL staining to detect apoptosis of mice retinal cells. In vitro, DCFH-DA and western blot (WB) were used to evaluate ROS, Bcl-2, BAX, cleaved-caspase 3 in HRMECs and metabolomics detected the effect of dapagliflozin on the metabolism of HRMECs. And then, we performed correlation analysis and verification functions for significantly different metabolites. In vivo, dapagliflozin reduced the apoptosis of diabetic mice retina independently of hypoglycemic. In vitro, SGLT-2 protein was expressed on HRMECs. Dapagliflozin reduced the level of ROS caused by high glucose, decreased the expression of cleaved-caspase3 and the ratio of BAX/Bcl-2. Metabolomics results showed that dapagliflozin did not affect the intracellular glucose level. Compared with the high glucose group, dapagliflozin reduced the production of arachidonic acid (AA) and inhibited the phosphorylation of ERK1/2, therefore, reducing the phosphorylation of cPLA2, which is a key enzyme for arachidonic acid release. Conclusion: Collectively, results unearthed for the first time that dapagliflozin reduced apoptosis of retina induced by DM whether in vivo or in vitro. Dapagliflozin did not affect the glucose uptake while mitigated intracellular arachidonic acid in HRMECs. Dapagliflozin alleviated HRMECs apoptosis induced by high glucose through ERK/1/2/cPLA2/AA/ROS pathway.

Switching Reactive Oxygen Species into Reactive Nitrogen Species by Photocleaved O2 -Released Nanoplatforms Favors Hypoxic Tumor Repression.

In various reactive oxygen species (ROS)-based antitumor approaches (e.g., photodynamic therapy), increasing attentions are made to improve ROS level, but the short lifetime that is another decisive hurdle of ROS-based antitumor outcomes is not even explored yet. To address it, a photocleaved O2 -released nanoplatform is constructed to release and switch ROS into reactive nitrogen species (RNS) for repressing hypoxic breast tumor. Systematic explorations validate that the nanoplatforms can attain continuous photocontrolled O2 release, alleviate hypoxia, and elevate ROS level. More significantly, the entrapped PDE5 inhibitor (PDE5-i) in this nanoplatform can be enzymatically decomposed into nitric oxide that further combines with ROS to generate RNS, enabling the persistent antitumor effect since RNS features longer lifetime than ROS. Intriguingly, ROS conversion into RNS can help ROS to evade the hypoxia-induced resistance to ROS-based antitumor. Eventually, RNS production unlocks robust antitumor performances along with ROS elevation and hypoxia mitigation. Moreover, this extraordinary conversion from ROS into RNS also can act as a general method to solve the short lifetime of ROS.

Comprehensive Analysis of the SBP Family in Blueberry and Their Regulatory Mechanism Controlling Chlorophyll Accumulation.

SQUAMOSA Promoter Binding Protein (SBP) family genes act as central players to regulate plant growth and development with functional redundancy and specificity. Addressing the diversity of the SBP family in crops is of great significance to precisely utilize them to improve agronomic traits. Blueberry is an important economic berry crop. However, the SBP family has not been described in blueberry. In the present study, twenty VcSBP genes were identified through data mining against blueberry transcriptome databases. These VcSBPs could be clustered into eight groups, and the gene structures and motif compositions are divergent among the groups and similar within each group. The VcSBPs were differentially expressed in various tissues. Intriguingly, 10 VcSBPs were highly expressed at green fruit stages and dramatically decreased at the onset of fruit ripening, implying that they are important regulators during early fruit development. Computational analysis showed that 10 VcSBPs were targeted by miR156, and four of them were further verified by degradome sequencing. Moreover, their functional diversity was studied in Arabidopsis. Noticeably, three VcSBPs significantly increased chlorophyll accumulation, and qRT-PCR analysis indicated that VcSBP13a in Arabidopsis enhanced the expression of chlorophyll biosynthetic genes such as AtDVR, AtPORA, AtPORB, AtPORC, and AtCAO. Finally, the targets of VcSBPs were computationally identified in blueberry, and the Y1H assay showed that VcSBP13a could physically bind to the promoter region of the chlorophyll-associated gene VcLHCB1. Our findings provided an overall framework for individually understanding the characteristics and functions of the SBP family in blueberry.

Bicyclol Regulates Hepatic Gluconeogenesis in Rats with Type 2 Diabetes and Non-alcoholic Fatty Liver Disease by Inhibiting Inflammation.

Hepatic gluconeogenesis plays an important role in maintaining the body's glucose metabolism homeostasis. Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver diseases, when combined with type 2 diabetes mellitus (T2DM), it can cause severe glucose metabolism disorders. Studies have confirmed that chronic liver inflammatory lesions are the basis of T2DM combined with NAFLD (T2DM-NAFLD), inhibiting liver inflammation can improve glucose metabolism disorders. It is essential to explore safe and effective drugs to inhibit liver inflammation to improve the body's glucose metabolism disorders. Bicyclol is a biphenyl derivative that has anti-oxidative and anti-inflammatory properties. In the present study, the hepatoprotective effects and underlying mechanisms of bicyclol in T2DM-NAFLD were investigated, and T2DM-NAFLD with/without bicyclol treatment models were established. The results revealed that bicyclol alleviated fasting blood glucose, serum transaminase levels, insulin resistance, hepatic adipogenesis, lipid accumulation and markedly reduced T2DM-NAFLD rat histological alterations of livers. Not only that, bicyclol markedly attenuated T2DM-NAFLD induced production of inflammation factors (IL-1ß and TNF-α). Moreover, bicyclol suppressed the expression of insulin/gluconeogenesis signaling pathway (Akt, PGC-1α and PEPCK). These findings suggested that bicyclol might be a potentially effective drug for the treatment of T2DM-NAFLD and other metabolic disorders.

Vertically oriented mesoporous silica film modified fluorine-doped tin oxide electrode for enhanced electrochemiluminescence detection of lidocaine in serum.

Owing to a nanochannel-based enrichment effect and anti-fouling ability, highly ordered and vertically oriented mesoporous silica thin film (VMSF) modified electrodes have demonstrated their great potential in direct and highly sensitive analysis of complex samples. In this work, a VMSF modified fluorine-doped tin oxide (FTO) electrode (VMSF/FTO) is fabricated for enhanced electrochemiluminescence (ECL) analysis of lidocaine in serum. VMSF with good integrity and mechanical stability can be rapidly and conveniently grown on FTO in a few seconds at room temperature using an electrochemically assisted self-assembly (EASA) method. Due to the strong electrostatic attraction between the cationic ECL probe and negatively charged nanochannel, the VMSF/FTO electrode shows significant enrichment of tris(2,2-bipyridine) ruthenium(ii) (Ru(bpy)3 2+), leading to ∼10 times enhancement of its ECL signal in comparison to the bare FTO electrode. Lidocaine, an anesthetic and antiarrhythmic drug, can act as the co-reactant of Ru(bpy)3 2+ and promote its ECL signal. Sensitive ECL detection of lidocaine is achieved by the sensor in a wide linear range from 10 nM to 50 µM with a low limit-of-detection (LOD) of 8 nM. Combined with the antifouling ability of VMSF, the VMSF/FTO electrode also realizes the accurate and rapid analysis of lidocaine in real serum samples.

Contrast-enhanced ultrasound for needle biopsy of thoracic lesions.

Two-dimensional ultrasound (US) and color doppler flow imaging are associated with certain limitations in the preprocedural evaluation and design of the puncture path for biopsies of thoracic lesions, such as a poorly defined boundary between the tumor and the atelectatic lesions in central lung cancer with atelectasis. Contrast-enhanced ultrasound (CEUS) can be valuable in the preoperative evaluation of the biopsy site and in increasing the accuracy of the biopsy. The present study investigated the value of clinical application of CEUS in US-guided core needle biopsy (US-CNB) in improving the diagnostic accuracy in thoracic lesions. A total of 120 patients with first-stage thoracic lesions from the Affiliated Tumor Hospital of Guangxi Medical University who underwent US-CNB were recruited and randomnly assigned to a conventional US group (n=66) and a CEUS group (n=54). All patients underwent preoperative evaluation and US-guided puncture of thoracic lesions. The intergroup differences in sonographic features, biopsy duration, biopsy success rate and complications were assessed. The CEUS group had a higher rate of detection of necrotic tissue (40.7% vs. 16.7%; χ2=8.633; P=0.003) and change of initial puncture path (48.1%) compared with the US group. In central lung cancer with atelectasis, the ability to distinguish between tumor and atelectasis was higher in the CEUS group compared with the conventional US group (31.5 vs. 7.6%; χ2=11.336; P=0.001). In addition, the CEUS group had a higher puncture success (96.3 vs. 80.3%; χ2=6.946; P=0.008) and a lower complication rate (3.7% vs. 18.2%; χ2=6.041; P=0.014) compared with the US group. CEUS can identify necrotic areas and occult tumors within atelectatic lung tissue and can be used for guiding puncture biopsy of thoracic lesions to improve the diagnostic accuracy with greater comparative clinical utility than conventional US. Pre-biopsy CEUS is especially useful for patients undergoing repeated US-CNB and those with hypovascular lesions, atelectasis or necrosis.

A blueberry MIR156a-SPL12 module coordinates the accumulation of chlorophylls and anthocyanins during fruit ripening.

Color change is an important event during fruit maturation in blueberry, usually depending on chlorophyll degradation and anthocyanin accumulation. MicroRNA156 (miR156)-SPL modules are an important group of regulatory hubs involved in the regulation of anthocyanin biosynthesis. However, little is known regarding their roles in blueberry or in chlorophyll metabolism during color change. In this study, a MIR156 gene (VcMIR156a) was experimentally identified in blueberry (Vaccinium corymbosum). Overexpression of VcMIR156a in tomato (Solanum lycopersicum) enhanced anthocyanin biosynthesis and chlorophyll degradation in the stem by altering pigment-associated gene expression. Further investigation indicated that the VcSPL12 transcript could be targeted by miR156, and showed the reverse accumulation patterns during blueberry fruit development and maturation. Noticeably, VcSPL12 was highly expressed at green fruit stages, while VcMIR156a transcripts mainly accumulated at the white fruit stage when expression of VcSPL12 was dramatically decreased, implying that VcMIR156a-VcSPL12 is a key regulatory hub during fruit coloration. Moreover, VcSPL12 decreased the expression of several anthocyanin biosynthetic and regulatory genes, and a yeast two-hybrid assay indicated that VcSPL12 interacted with VcMYBPA1. Intriguingly, expression of VcSPL12 significantly enhanced chlorophyll accumulation and altered the expression of several chlorophyll-associated genes. Additionally, the chloroplast ultrastructure was altered by the expression of VcMIR156a and VcSPL12. These findings provide a novel insight into the functional roles of miR156-SPLs in plants, especially in blueberry fruit coloration.

Neuroprotective Role of GLP-1 Analog for Retinal Ganglion Cells via PINK1/Parkin-Mediated Mitophagy in Diabetic Retinopathy.

GLP-1 analogs have been widely used to treat patients with type 2 diabetes in recent years and studies have found that GLP-1 analogs have multiple organ benefits. However, the role of GLP-1 analogs in diabetic retinopathy (DR), a common complication of diabetes mellitus (DM), remains controversial. Retinal ganglion cells (RGCs) are the only afferent neurons responsible for transmitting visual information to the visual center and are vulnerable in the early stage of DR. Protection of RGC is vital for visual function. The incretin glucagon-like peptide-1 (GLP-1), which is secreted by L-cells after food ingestion, could lower blood glucose level through stimulating the release of insulin. In the present study, we evaluated the effects of GLP-1 analog on RGCs both in vitro and in vivo. We established diabetic rat models in vivo and applied an RGC-5 cell line in vitro. The results showed that in high glucose conditions, GLP-1 analog alleviated the damage of RGCs. In addition, GLP-1 analog prevented mitophagy through the PINK1/Parkin pathway. Here we demonstrated the neuroprotective effect of GLP-1 analog, which may be beneficial for retinal function, and we further elucidated a novel mechanism in GLP-1 analog-regulated protection of the retina. These findings may expand the multi-organ benefits of GLP-1 analogs and provide new insights for the prevention of DR.

Identification and functional characterization of the Aux/IAA gene VcIAA27 in blueberry.

Aux/IAA genes are an important class of players in diverse developmental processes in plants, which generally exert their functions through the auxin signaling pathway. Blueberry is an economically and nutritionally important berry-bearing crop. However, Aux/IAA genes remain unknown in blueberry. In the present study, an Aux/IAA gene (VcIAA27) was identified and characterized in blueberry, and it is most closely related to IAA27 in other plant species. Expression analysis indicated that VcIAA27 transcripts accumulate highly in shoot, flower and fruit. Interestingly, VcIAA27 was highly expressed at early fruit developmental stages, and dramatically decreased from the onset of fruit ripening, implying that VcIAA27 possibly plays important roles during fruit enlargement. Meanwhile, the analysis of promoter activity in Arabidopsis showed that strong GUS signal was detected in the trichome and hydathodes of leaves, receptacle of silique, and lateral roots of seedling. Overexpression of VcIAA27 in Arabidopsis leads to auxin-related defects such as downward-curled leaves, short or sterile siliques, shorter stature, and more shoot branches. Moreover, qPCR analysis indicated that VcIAA27 is able to alter the expression patterns of the auxin-related genes BRU6, SAG13, SAUR26 in Arabidopsis, suggesting that VcIAA27 might be negatively involved in the auxin signaling pathway. The findings will greatly contribute to future investigation of Aux/IAA-mediated mechanisms that control blueberry development, especially fruit development and ripening.

Preoperative color Doppler ultrasonography predicts early recurrence in AFP-positive hepatocellular carcinoma.

The present study aimed to evaluate the hepatic hemodynamics of patients with α-fetoprotein (AFP)-positive hepatocellular carcinoma (HCC) and early recurrence (ER), as determined by color Doppler ultrasound. Screening with color Doppler ultrasound was performed in 150 patients with AFP-positive HCC, yielding 43 cases with postoperative ER. In addition, 35 healthy controls were enrolled in the study for comparison of the blood flow parameters between the healthy control and the HCC groups. Receiver operating characteristic curve analysis was performed to evaluate the predicted effect of Doppler for the ER of HCC. The average mean flow velocity of portal vein in patients with HCC (14.686±5.873 cm/s) was significantly lower in the HCC group compared with the healthy control group (17.631±3.569 cm/s; P=0.005). Additionally, the preoperative portal vein diameter in the HCC group was significantly higher compared with that in the healthy control group (P=0.001). Regarding the effect of surgery, the portal vein diameter was significantly increased in the patients postoperatively compared with preoperatively (P=0.003), while the pulsation and resistance indexes exhibited a decreasing trend in the postoperative group (P=0.001 and P=0.003, respectively). Notably, patients with HCC and ER presented with a higher resistance index (1.163±0.342) compared with the ER-free group (1.023±0.176; P=0.004). Furthermore, a significant difference in hepatic artery pulsation index was also observed between the ER group (0.673±0.075) and the ER-free group (0.624±0.056; P=0.018), indicating that an increased hepatic artery pulsation index may significantly predict the ER of HCC. In addition, areas under the curve of 0.683 and 0.700, respectively, suggested that the hepatic artery resistance and pulsation indexes may be used to diagnose ER in HCC (P=0.009 and P=0.004, respectively). Taken together, Doppler ultrasound provides a reliable and accurate quantification of hepatic hemodynamics for detecting ER in HCC with a good diagnostic accuracy.

Contrast-Enhanced Ultrasound to Monitor Early Recurrence of Primary Hepatocellular Carcinoma after Curative Treatment.

OBJECTIVE: To evaluate contrast-enhanced ultrasound (CEUS) for monitoring early intrahepatic recurrence of primary hepatocellular carcinoma (HCC) after curative treatment. METHODS: We prospectively analyzed 97 patients (124 nodules) with primary HCC who underwent hepatic resection or radiofrequency ablation and subsequently experienced intrahepatic recurrence. Patients were assessed with conventional ultrasound and CEUS. They were also assessed with contrast-enhanced computed tomography (CECT) and/or magnetic resonance imaging (MRI). The image characteristics of CEUS of recurrent hepatocellular carcinoma and high-grade dysplastic nodules (HGDNs) were analyzed. In addition, the ability of CEUS and CECT/MRI to assess internal artery vascularization in recurrent disease was compared. RESULTS: CEUS of recurrent hepatocellular carcinoma showed hyperenhancement in the arterial phase in 96 of 99 nodules, and it showed hypo- or isoenhancement for portal venous and delayed phases. The most common enhancement patterns were "fast-in and slow-out" and "fast-in and fast-out". Based on the arterial hyperenhancement of lesions and with clinical data such as patient history of HCC and increased level of serum alpha-fetoprotein, the diagnostic accuracy of CEUS for recurrent HCC was significantly higher than that based on the enhancement pattern of "fast-in and fast-out". CEUS of HGDNs showed local or global hyperenhancement during the arterial phase, isoenhancement during the portal venous phase, and isoenhancement or slight hypoenhancement during the delayed phase. The enhancement pattern was "fast-in and slow-out". In some cases, it was difficult to differentiate HGDNs from recurrent disease using CEUS. Vascularization in recurrent disease was significantly higher when assessed by CEUS than when assessed with CECT/MRI (P < 0.05). For detecting recurrent disease, CEUS showed sensitivity of 97.0%, specificity of 68.0%, positive predictive value of 92.3%, and negative predictive value of 85.0%. The corresponding parameters for CECT/MRI were 71.7%, 72.0%, 88.8%, and 39.1%. CONCLUSION: Intrahepatic recurrent HCC and HGDNs with diameter ≤ 3.0 cm have a characteristic appearance on CEUS. This imaging modality may be effective for monitoring early intrahepatic recurrence after curative treatment of primary HCC.

Therapeutic Evaluation of Radiotherapy with Contrast-Enhanced Ultrasound in Non-Resectable Hepatocellular Carcinoma Patients with Portal Vein Tumor Thrombosis.

BACKGROUND Therapeutic evaluation of 3-dimensional conformal radiotherapy (3DCRT) is rarely reported for non-resectable hepatocellular carcinoma (HCC) with portal vein tumor thrombus (PVTT). The aim of this study was to determine the value of contrast-enhanced ultrasound (CEUS) in evaluating the therapeutic response of HCC with PVTT treated with 3DCRT. MATERIAL AND METHODS PVTT reduction rate in the study was determined after 3DCRT using time intensity curve (TIC) analysis software before and after radiotherapy. Seventy-nine HCC patients with PVTT treated with 3DCRT were studied. HCC and PVTT were performed by CEUS, before and after 3DCRT, over time. The parameters of blood flow, including arrival time (AT), time to peak (TTP), peak intensity (PI), washout time (WT), and area under the curve (AUC), were quantified and evaluated on still images by CEUS. RESULTS After 3DCRT, typing and staging of PVTT in 38 patients was decreased, the reduction rate was 48.1%. HCC was effective in 45 patients, the effective rate was 57%; No differences were found between the PVTT reduction rate and the HCC effective rate (χ2=2.96, P>0.05). In the effective group, the PI and AUC of HCCs and PVTTs after 3DCRT were significantly lower than before 3DCRT, while the other parameters of TIC were not significantly different before and after 3DCRT. CONCLUSIONS CEUS might be a useful monitoring option for the evaluation of HCC with PVTT treated with 3DCRT. CEUS might be useful as an important choice for monitoring and evaluation HCC with PVTT after 3DCRT. TIC parameters might provide quantitative data for efficacy evaluation, which helps to modify treatment strategies timely and accurately.