Sphingosylphosphorylcholine alleviates hypoxia-caused apoptosis in cardiac myofibroblasts via CaM/p38/STAT3 pathway.

Blockade of hypoxia-caused nonmyocytes apoptosis helps improve survival and mitigate ventricular remodeling and dysfunction during the chronic stage of myocardial infarction. But tools affecting nonmyocyte apoptosis are very rare. Sphingosylphosphorylcholine (SPC), a naturally occurring bioactive sphingolipid in plasma, was proved to protect cardiomyocyte against apoptosis in an ischemic model in our previous study. Here, we showed that SPC also inhibited hypoxia-induced apoptosis in myofibroblasts, an important type of nonmyocytes in the heart. Calmodulin (CaM) is an identified receptor of SPC. We clarified that SPC inhibited myofibroblast apoptosis through CaM as evidenced by decreased cleaved caspase 3, PARP1 and condensed nucleus. Furthermore, the employment of inhibitor and agonist of p38 and STAT3 suggests that SPC inhibits myofibroblast apoptosis by regulating the phosphorylation of p38 and STAT3, and they act as downstream of CaM. The present work may provide new evidence on the regulation of myofibroblasts apoptosis by SPC and a novel target for heart remodeling after hypoxia.

Dysbiosis of intestinal microbiota mediates tubulointerstitial injury in diabetic nephropathy via the disruption of cholesterol homeostasis.

Background: Our previous study demonstrated that the disruption of cholesterol homeostasis promotes tubulointerstitial injury in diabetic nephropathy (DN). This study aimed to further investigate the effects of gut microbiota dysbiosis on this process and explored its potential mechanism. Methods: Diabetic rats treated with broad-spectrum oral antibiotics or faecal microbiota transplantation (FMT) from the healthy donor group and human kidney 2 (HK-2) cells stimulated with sodium acetate were used to observe the effects of gut microbiota on cholesterol homeostasis. The gut microbiota distribution was measured by 16S rDNA sequencing with faeces. Serum acetate level was examined by gas chromatographic analysis. Protein expression of G protein coupled receptor 43 (GPR43) and molecules involved in cholesterol homeostasis were assessed by immunohistochemical staining, immunofluorescence staining, and Western Blotting. Results: Depletion of gut microbiota significantly attenuated albuminuria and tubulointerstitial injury. Interestingly, serum acetate levels were also markedly decreased in antibiotics-treated diabetic rats and positively correlated with the cholesterol contents in kidneys. An in vitro study demonstrated that acetate significantly increased cholesterol accumulation in HK-2 cells, which was caused by increased expression of proteins mainly modulating cholesterol synthesis and uptake. As expected, FMT effectively decreased serum acetate levels and alleviated tubulointerstitial injury in diabetic rats through overriding the disruption of cholesterol homeostasis. Furthermore, GPR43 siRNA treatment blocked acetate-mediated cholesterol homeostasis dysregulation in HK-2 cells through decreasing the expression of proteins governed cholesterol synthesis and uptake. Conclusion: Our studies for the first time demonstrated that the acetate produced from gut microbiota mediated the dysregulation of cholesterol homeostasis through the activation of GPR43, thereby contributing to the tubulointerstitial injury of DN, suggesting that gut microbiota reprogramming might be a new strategy for DN prevention and therapy.

Gut microbiota dysbiosis-induced activation of the intrarenal renin-angiotensin system is involved in kidney injuries in rat diabetic nephropathy.

Some studies have shown that gut microbiota along with its metabolites is closely associated with diabetic mellitus (DM). In this study we explored the relationship between gut microbiota and kidney injuries of early diabetic nephropathy (DN) and its underlying mechanisms. Male SD rats were intraperitoneally injected with streptozotocin to induce DM. DM rats were orally administered compound broad-spectrum antibiotics for 8 weeks. After the rats were sacrificed, their blood, urine, feces, and renal tissues were harvested for analyses. We found that compared with the control rats, DM rats had abnormal intestinal microflora, increased plasma acetate levels, increased proteinuria, thickened glomerular basement membrane, and podocyte foot process effacement in the kidneys. Furthermore, the protein levels of angiotensin II, angiotensin-converting enzyme, and angiotensin II type 1 receptor in the kidneys of DM rats were significantly increased. Administration of broad-spectrum antibiotics in DM rats not only completely killed most intestinal microflora, but also significantly lowered the plasma acetate levels, inhibited intrarenal RAS activation, and attenuated kidney damage. Finally, we showed that plasma acetate levels were positively correlated with intrarenal angiotensin II protein expression (r = 0.969, P < 0.001). In conclusion, excessive acetate produced by disturbed gut microbiota might be involved in the kidney injuries of early DN through activating intrarenal RAS.

Urinary levels of podocyte-derived microparticles are associated with the progression of chronic kidney disease.

BACKGROUND: Podocyte-derived microparticles (MPs) could be secreted from activated or apoptotic podocytes. An increased number of podocyte-derived MPs in the urine might reflect podocyte injury in renal diseases. This study aimed to observe the change of urinary podocyte-derived MP levels in patients with chronic kidney disease (CKD) and to further explore its correlation with the progression of CKD. METHODS: A prospective, longitudinal study was conducted in eighty patients with biopsy-proven CKD. Podocyte-derived MPs (annexin V and podocalyxin positive) were detected by flow cytometry. The number of urinary podocyte-derived MPs was analyzed to evaluate the association with biochemical measurements and pathological glomerulosclerosis assessment. Patients with idiopathic membranous nephropathy (IMN) were followed up after the six-month treatment of prednisone combined with tacrolimus to evaluate the association of urinary podocyte-derived MP levels and the remission of IMN. RESULTS: The CKD patients had higher urinary podocyte-derived MP levels compared with healthy controls (HCs). Baseline urinary levels of podocyte-derived MPs were positively correlated with 24-hour proteinuria, while were inversely correlated with the percentage of global glomerulosclerosis. The urinary podocyte-derived MPs levels had good discrimination for glomerulosclerosis [area under curve (AUC), 0.66]. The urinary podocyte-derived MPs levels in IMN patients were significantly decreased accompanied with the recovery of abnormal clinical parameters after six-month treatment. CONCLUSIONS: The urinary levels of podocyte-derived MPs were closely associated with podocyte injury and glomerulosclerosis, which could be useful for monitoring disease activity in CKD patients. Urinary podocyte-derived MPs might be a non-invasive biomarker for the evaluation of early CKD progression.

Role of Podocyte Injury in Glomerulosclerosis.

Finding new therapeutic targets of glomerulosclerosis treatment is an ongoing quest. Due to a living environment of various stresses and pathological stimuli, podocytes are prone to injuries; moreover, as a cell without proliferative potential, loss of podocytes is vital in the pathogenesis of glomerulosclerosis. Thus, sufficient understanding of factors and underlying mechanisms of podocyte injury facilitates the advancement of treating and prevention of glomerulosclerosis. The clinical symptom of podocyte injury is proteinuria, sometimes with loss of kidney functions progressing to glomerulosclerosis. Injury-induced changes in podocyte physiology and function are actually not a simple passive process, but a complex interaction of proteins that comprise the anatomical structure of podocytes at molecular levels. This chapter lists several aspects of podocyte injuries along with potential mechanisms, including glucose and lipid metabolism disorder, hypertension, RAS activation, micro-inflammation, immune disorder, and other factors. These aspects are not technically separated items, but intertwined with each other in the pathogenesis of podocyte injuries.

Urinary podocyte microparticles are associated with disease activity and renal injury in systemic lupus erythematosus.

BACKGROUND: New non-invasive biomarkers are demanded to identify renal damage in various autoimmune-associated kidney diseases. Glomerular podocyte damage mediated by systemic lupus erythematosus (SLE) plays an important role in the pathogenesis and progression of lupus nephritis (LN). This study evaluated whether the podocyte-derived microparticles (MPs) were novel biomarkers of clinical and histological features in SLE patients with LN. METHODS: A cross-sectional study, including 34 SLE patients and 16 healthy controls, was designed. Urinary annexin V+ podocalyxin+ MPs of all participants were quantified by flow cytometry. The correlation of podocyte-derived MPs with clinical and histological parameters of SLE patients was analysed. RESULTS: The number of annexin V+ podocalyxin+ MPs from urine samples were markly increased in patients with SLE. Furthermore, the level of urinary podocyte-derived MPs was positively correlated with the SLE Disease Activity Index (SLEDAI) score, anti-dsDNA antibody titre, erythrocyte sedimentation rate, and proteinuria. Conversely, it was negatively correlated with the level of complement C3 and serum albumin. The number of urinary podocyte-derived MPs was significantly increased in SLE patients with high activity indices. Receiver operating characteristic (ROC) curves were calculated to assess the power for podocyte-derived MP levels in differentiating between SLE patients with and without LN. Podocyte-derived MP levels were able to differentiate between SLE patients with mild disease activity, as well as those with moderate and above disease activity. SLE patients showed increased podocyte-derived MP excretion into the urine. CONCLUSIONS: These findings suggest that the change in urinary podocyte-derived MP levels could be useful for evaluating and monitoring SLE disease activity.

Publisher Correction: Inflammation-activated CXCL16 pathway contributes to tubulointerstitial injury in mouse diabetic nephropathy.

The REFERENCES 1-35 are wrong because of the error in the process of typesetting.

Caspase 3/ROCK1 pathway mediates high glucose-induced platelet microparticles shedding.

BACKGROUND: Platelet microparticles (PMPs) are closely associated with diabetic macrovascular complications. This study aimed to explore the underlying mechanisms of high glucose-induced PMPs generation. METHODS: Washed platelets, obtained from the plasma of healthy male Sprague-Dawley rats, were incubated with high glucose. PMPs were isolated using gradient centrifugation and counted by flow cytometry. Expression and activity of ROCK1 and caspase3 were evaluated by real-time PCR, Western blotting, and activity assay kit. RESULTS: High glucose enhanced PMPs shedding in the presence of collagen. The mRNA and protein levels of ROCK1, but not ROCK2, were increased in platelets incubated with high glucose. Y-27632, an inhibitor of ROCK, blocked the increased PMPs shedding induced by high glucose. Expression and activity of caspase3 were elevated in platelets under the high glucose conditions. Z-DVED-FMK, a caspase3 inhibitor, inhibited ROCK1 activity and decreased the PMPs generation under high glucose. CONCLUSION: High glucose increased PMPs shedding via caspase3-ROCK1 signal pathway.

Aspirin attenuates podocyte injury in diabetic rats through overriding cyclooxygenase-2-mediated dysregulation of LDL receptor pathway.

AIM: This study aimed to investigate the effects of aspirin on podocyte injury and its underlying mechanisms in diabetic nephropathy (DN). METHODS: Eight-week-old male Sprague-Dawley rats were divided into three groups: non-diabetic rats (Control), streptozotocin-induced diabetic rats (DM), and diabetic rats treated with aspirin (DM + Aspirin) for 12 weeks. Intracellular lipid accumulation was evaluated by Oil Red O staining and quantitative free cholesterol assays. Podocyte injury and the levels of COX-2, inflammatory cytokines, and low-density lipoprotein receptor (LDLr) pathway-related proteins were evaluated by electron microscopy, immunohistochemical staining, and Western blotting, respectively. RESULTS: Lipid levels and urinary albumin-creatinine ratios were higher in the DM rats than in the Control rats. Periodic acid-Schiff staining showed glomerular hypertrophy and mild mesangial area widening in the DM rats. Electron microscopy showed that the podocyte foot processes were significantly flattened or absent in the DM rats. The protein expression levels of WT-1 and nephrin in the podocytes of DM rats were reduced. Interestingly, lipid accumulation in the kidneys of DM rats was significantly increased due to increased protein expression levels of LDLr, sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), SREBP-2, cyclooxygenase-2 (COX-2), and inflammatory cytokines. Confocal immunofluorescent staining showed that COX-2 and WT-1 were co-expressed. Furthermore, COX-2 protein expression levels were positively correlated with LDLr protein expression levels. However, when COX-2 expression was inhibited by aspirin, these changes in the DM rats were significantly attenuated. CONCLUSION: Aspirin attenuates podocyte injury in DN, which may be through COX-2-mediated dysregulation of LDLr pathway.

The Release of Monocyte-Derived Tissue Factor-Positive Microparticles Contributes to a Hypercoagulable State in Idiopathic Membranous Nephropathy.

AIM: Idiopathic membranous nephropathy (IMN) is an immune-mediated inflammatory disease characterized by a high risk of thromboembolic complications. Microparticles (MPs), a type of extracellular vesicles, have procoagulant properties, especially when they display tissue factor (TF). This study aimed to investigate whether circulating TF-positive MPs contributed to the hypercoagulable state in patients with IMN. METHODS: Twenty adult IMN patients and fourteen healthy subjects were included in the study. The basic indexes of a routine biochemical examination and coagulative function were determined. The plasma levels of MPs were detected by flow cytometry, and TF activity of MPs was examined using an assay kit. The plasma levels of lipopolysaccharide (LPS) were measured by an enzyme-linked immunosorbent assay. RESULTS: Total circulating MPs were not increased in patients with IMN compared with healthy controls. Circulating CD14＋/TF＋MPs were significantly increased in IMN patients, but this achieved significance was not observed in CD41＋/TF＋MPs between the two groups. Interestingly, the circulating TF-positive MPs were increased significantly. Plasma MPs TF assays revealed high procoagulant activity, which was positively associated with the D-dimer level in IMN. In addition, circulating LPS in IMN patients were significantly higher than those in the controls. Furthermore, after two hours' incubation with healthy whole blood, LPS enhanced the release of circulating TF-positive MPs and the TF activity of MPs. CONCLUSION: Increased circulating LPS may mediate the release of monocyte-derived TF-positive MPs, which further contributes to the hypercoagulable state in IMN patients. These findings provide an additional mechanism by which patients with IMN have a higher risk of thromboembolic complication.

Platelet microparticles contribute to aortic vascular endothelial injury in diabetes via the mTORC1 pathway.

Platelet microparticles (PMPs) are closely associated with diabetic macrovascular complications. The present study aimed to investigate the effects of PMPs in diabetes on aortic vascular endothelial injury and to explore the underlying mechanisms. Peritoneal injection of streptozotocin was used to generate a diabetic rat model in vivo, and human umbilical vein endothelial cells (HUVECs) treated with PMPs were used in vitro. PMP levels in the circulation and aorta tissues were time-dependently increased in streptozotocin-induced diabetic rats at weeks 4, 8, and 12 (P < 0.05). Aspirin significantly inhibited the PMP levels at each time point (P < 0.05). In diabetic rats, the endothelial nitric oxide levels were decreased significantly combined with increased endothelial permeability. PMPs were internalized by HUVECs and primarily accumulated around the nuclei. PMPs inhibited endothelial nitric oxide levels to about 50% and caused approximately twofold increase in reactive oxygen species production. Furthermore, PMPs significantly decreased the endothelial glycocalyx area and expression levels of glypican-1 and occludin (P < 0.05). Interestingly, the PMP-induced endothelial injuries were prevented by raptor siRNA and rapamycin. In conclusion, increased PMPs levels contribute to aortic vascular endothelial injuries in diabetes through activating the mTORC1 pathway.

Platelet Microparticles Mediate Glomerular Endothelial Injury in Early Diabetic Nephropathy.

BACKGROUND: Glomerular endothelium dysfunction, which plays a crucial role in the pathogenesis of early diabetic nephropathy, might be caused by circulating metabolic abnormalities. Platelet microparticles, extracellular vesicles released from activated platelets, have recently emerged as a novel regulator of vascular dysfunction. METHODS: We studied the effects of platelet microparticles on glomerular endothelial injury in early diabetic nephropathy in rats with streptozotocin-induced diabetes and primary rat glomerular endothelial cells. Isolated platelet microparticles were measured by flow cytometry. RESULTS: Plasma platelet microparticles were significantly increased in diabetic rats, an effect inhibited in aspirin-treated animals. In cultured glomerular endothelial cells, platelet microparticles induced production of reactive oxygen species, decreased nitric oxide levels, inhibited activities of endothelial nitric oxide synthase and SOD, increased permeability of the glomerular endothelium barrier, and reduced thickness of the endothelial surface layer. Conversely, inhibition of platelet microparticles in vivo by aspirin improved glomerular endothelial injury. Further analysis showed that platelet microparticles activated the mammalian target of rapamycin complex 1 (mTORC1) pathway in glomerular endothelial cells; inhibition of the mTORC1 pathway by rapamycin or raptor siRNA significantly protected against microparticle-induced glomerular endothelial injury in vivo and in vitro. Moreover, platelet microparticle-derived chemokine ligand 7 (CXCL7) contributed to glomerular endothelial injury, and antagonizing CXCL7 using CXCL7-neutralizing antibody or blocking CXCL7 receptors with a competitive inhibitor of CXCR1 and CXCR2 dramatically attenuated such injury. CONCLUSIONS: These findings demonstrate a pathogenic role of platelet microparticles in glomerular endothelium dysfunction, and suggest a potential therapeutic target, CXCL7, for treatment of early diabetic nephropathy.

Lipoprotein(a) accelerated the progression of atherosclerosis in patients with end-stage renal disease.

BACKGROUND: Increased plasma level of lipoprotein(a) (Lpa) is a risk factor of cardiovascular diseases. This study aimed to explore the role of Lpa in the progression of atherosclerosis in patients with end-stage renal disease (ESRD) and to investigate whether its potential mechanism is mediated by CXC chemokine ligand 16 (CXCL16) and low-density lipoprotein receptor (LDLr). METHODS: This is a retrospective clinical study. From January 2015 to April 2016, forty-six ESRD patients from Danyang First People's Hospital were investigated. The patients were grouped according to their plasma Lpa levels: control group (Lpa < 300 mg/l, n = 23) and high Lpa group (Lpa ≥ 300 mg/l, n = 23). ESRD Patients with acute infective diseases, cancer, and/or chronic active hepatitis were excluded. Biochemical indexes and lipid profiles of the patients were measured. Surgically removed tissues from the radial arteries of ESRD patients receiving arteriovenostomy were used for the preliminary evaluation of atherosclerosis. Haematoxylin-eosin (HE) and filipin staining were used to observe foam cell formation. Protein expression levels of Lpa, CXCL16, and LDLr were detected by immunohistochemistry staining and immunofluorescent staining. RESULTS: There was more foam cell formation and cholesterol accumulation in the radial arteries of the high Lpa group than in those of the control group. Furthermore, the expression levels of Lpa, CXCL16, and LDLr were significantly increased in the radial arteries of the high Lpa group. Correlation analyses showed that the protein expression levels of Lpa (r = 0.72, P < 0.01), LDLr (r = 0.54, P < 0.01), and CXCL16 (r = 0.6, P < 0.01) in the radial arteries of ESRD patients were positively correlated with the plasma Lpa levels. Further analyses showed that the co-expression of Lpa with LDLr or CXCL16 was increased in the high Lpa group. CONCLUSIONS: High plasma Lpa levels accelerated the progression of atherosclerosis in ESRD through inducing Lpa accumulation in the arteries, which was associated with LDLr and CXCL16. These two lipoproteins could both be major lipoprotein components that regulate the entry of Lpa into arterial cells.

ANXA2 promotes esophageal cancer progression by activating MYC-HIF1A-VEGF axis.

BACKGROUND: ANXA2 (Annexin A2) is a pleiotropic calcium-dependent phospholipid binding protein that is abnormally expressed in various cancers. We previously found that ANXA2 is upregulated in esophageal squamous cell carcinoma (ESCC). This study was designed to investigate the functional significance of ANXA2 dysregulation and underlying mechanism in ESCC. METHODS: Proliferation, migration, invasion and metastasis assay were performed to examine the functional roles of ANXA2 in ESCC cells in vitro and in vivo. Real-time RT-PCR, immunoblotting, ChIP, reporter assay, confocal-immunofluorescence staining, co-immunoprecipitation and ubiquitination assay were used to explore the molecular mechanism underlying the actions of deregulated ANXA2 in ESCC cells. RESULTS: Overexpression of ANXA2 promoted ESCC cells migration and invasion in vitro and metastasis in vivo through activation of the MYC-HIF1A-VEGF cascade. Notably, ANXA2 phosphorylation at Tyr23 by SRC led to its translocation into the nucleus and enhanced the metastatic potential of ESCC cells. Phosphorylated ANXA2 (Tyr23) interacted with MYC and inhibited ubiquitin-dependent proteasomal degradation of MYC protein. Accumulated MYC directly potentiated HIF1A transcription and then activated VEGF expression. Correlation between these molecules were also found in ESCC tissues. Moreover, dasatinib in combination with bevacizumab or ANXA2-siRNA produced potent inhibitory effects on the growth of ESCC xenograft tumors in vivo. CONCLUSIONS: This study provides evidence that highly expressed p-ANXA2 (Tyr23) contributes to ESCC progression by promoting migration, invasion and metastasis, and suggests that targeting the SRC-ANXA2-MYC-HIF1A-MYC axis may be an efficient strategy for ESCC treatment.

Intestinal dysbiosis activates renal renin-angiotensin system contributing to incipient diabetic nephropathy.

Considerable interest nowadays has focused on gut microbiota owing to their pleiotropic roles in human health and diseases. This intestinal community can arouse a variety of activities in the host and function as "a microbial organ" by generating bioactive metabolites and participating in a series of metabolism-dependent pathways. Alternations in the composition of gut microbiota, referred to as intestinal dysbiosis, are reportedly associated with several diseases, especially diabetes mellitus and its complications. Here we focus on the relationship between gut microbiota and diabetic nephropathy (DN), as the latter is one of the major causes of chronic kidney diseases. The activation of renin angiotensin system (RAS) is a critical factor to the onset of DN, and emerging data has demonstrated a provoking and mediating role of gut microbiota for this system in the context of metabolic diseases. The purpose of the current review is to highlight some research updates about the underlying interplay between gut microbiota, their metabolites, and the development and progression of DN, along with exploring innovative approaches to targeting this intestinal community as a therapeutic perspective in clinical management of DN patients.

Inflammation-activated CXCL16 pathway contributes to tubulointerstitial injury in mouse diabetic nephropathy.

Inflammation and lipid disorders play crucial roles in synergistically accelerating the progression of diabetic nephropathy (DN). In this study we investigated how inflammation and lipid disorders caused tubulointerstitial injury in DN in vivo and in vitro. Diabetic db/db mice were injected with 10% casein (0.5 mL, sc) every other day for 8 weeks to cause chronic inflammation. Compared with db/db mice, casein-injected db/db mice showed exacerbated tubulointerstitial injury, evidenced by increased secretion of extracellular matrix (ECM) and cholesterol accumulation in tubulointerstitium, which was accompanied by activation of the CXC chemokine ligand 16 (CXCL16) pathway. In the in vitro study, we treated HK-2 cells with IL-1ß (5 ng/mL) and high glucose (30 mmol/L). IL-1ß treatment increased cholesterol accumulation in HK-2 cells, leading to greatly increased ROS production, ECM protein expression levels, which was accompanied by the upregulated expression levels of proteins in the CXCL16 pathway. In contrast, after CXCL16 in HK-2 cells was knocked down by siRNA, the IL-1ß-deteriorated changes were attenuated. In conclusion, inflammation accelerates renal tubulointerstitial lesions in mouse DN via increasing the activity of CXCL16 pathway.

The Emerging Roles of Microparticles in Diabetic Nephropathy.

Microparticles (MPs) are a type of extracellular vesicles (EVs) shed from the outward budding of plasma membranes during cell apoptosis and/or activation. These microsized particles then release specific contents (e.g., lipids, proteins, microRNAs) which are active participants in a wide range of both physiological and pathological processes at the molecular level, e.g., coagulation and angiogenesis, inflammation, immune responses. Research limitations, such as confusing nomenclature and overlapping classification, have impeded our comprehension of these tiny molecules. Diabetic nephropathy (DN) is currently the greatest contributor to end-stage renal diseases (ESRD) worldwide, and its public health impact will continue to grow due to the persistent increase in the prevalence of diabetes mellitus (DM). MPs have recently been considered as potentially involved in DN onset and progression, and this review juxtaposes some of the research updates about the possible mechanisms from several relevant aspects and insights into the therapeutic perspectives of MPs in clinical management and pharmacological treatment of DN patients.

Spatial intratumoral heterogeneity and temporal clonal evolution in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is among the most common malignancies, but little is known about its spatial intratumoral heterogeneity (ITH) and temporal clonal evolutionary processes. To address this, we performed multiregion whole-exome sequencing on 51 tumor regions from 13 ESCC cases and multiregion global methylation profiling for 3 of these 13 cases. We found an average of 35.8% heterogeneous somatic mutations with strong evidence of ITH. Half of the driver mutations located on the branches of tumor phylogenetic trees targeted oncogenes, including PIK3CA, NFE2L2 and MTOR, among others. By contrast, the majority of truncal and clonal driver mutations occurred in tumor-suppressor genes, including TP53, KMT2D and ZNF750, among others. Interestingly, phyloepigenetic trees robustly recapitulated the topological structures of the phylogenetic trees, indicating a possible relationship between genetic and epigenetic alterations. Our integrated investigations of spatial ITH and clonal evolution provide an important molecular foundation for enhanced understanding of tumorigenesis and progression in ESCC.

Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae.

Phytophthora sojae is a devastating pathogen that causes soybean Phytophthora root rot. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay targeting the A3aPro element for visual detection of P. sojae. The A3aPro-LAMP assay efficiently amplified the target element in < 80 min at 64 °C and was evaluated for specificity and sensitivity. The specificity was evaluated against P. sojae, Phytophthora spp., Pythium spp., and true fungi isolates. Magnesium pyrophosphate resulting from the LAMP of P. sojae could be detected by real-time measurement of turbidity. Phytophthora sojae DNA products were visualized as a ladder-like banding pattern on 2% gel electrophoresis. A positive colour (sky blue) was only observed in the presence of P. sojae with the addition of hydroxynaphthol blue prior to amplification, whereas none of other isolates showed a colour change. The detection limit of the A3aPro-specific LAMP assay for P. sojae was 10 pg µL(-1) of genomic DNA per reaction. The assay also detected P. sojae from diseased soybean tissues and residues. These results suggest that the A3aPro-LAMP assay reported here can be used for the visual detection of P. sojae in plants and production fields.