NAT10-mediated ac4C modification promotes stemness and chemoresistance of colon cancer by stabilizing NANOGP8.

Background: Colon cancer (CC) stem cells can self-renew as well as expand, thereby promoting tumor progression and conferring resistance to chemotherapeutic agents. The acetyltransferase NAT10 mediates N4-acetylcytidine (ac4C) modification, which in turn drives tumorigenesis, metastasis, stemness properties maintenance, and cell fate decisions. Nonetheless, the specific involvement of ac4C modification mediated by NAT10 in regulating stemness and chemosensitivity in CC remains undetermined. Methods: The levels of NAT10 in normal colon and chemoresistant CC tissues were determined utilizing quantitative real-time polymerase chain reaction alongside immunohistochemistry. Assessing cancer cell stemness and chemosensitivity was conducted by various methods including spheroid and colony formation, western blotting, and flow cytometry. RNA-Seq was used to identify target genes, and RNA immunoprecipitation analysis was used to explore the potential mechanisms. Results: We observed NAT10 overexpression and increased ac4C modification levels in chemoresistant CC tissues. The in vivo and in vitro analysis findings suggested that NAT10 promoted CC cell stemness while suppressing their chemosensitivity. Conversely, Remodelin, a NAT10-specific inhibitor, enhanced CC cell chemosensitivity. Mechanistically, NAT10 increased the level of NANOGP8 ac4C modification and promoted NANOGP8 mRNA stability. Conclusions: NAT10 promotes the maintenance of stemness and chemoresistance in CC cells by augmenting the mRNA stability of NANOGP8. The inhibition of NAT10 via Remodelin improves chemotherapeutic efficacy and impedes CC progression.

Efficacy of ScopeGuide-Assisted Training in Enhancing Colonoscopy Competence and Reducing Patient Discomfort.

OBJECTIVE: In this study, we aimed to evaluate the efficacy of the Magnetic Scope Guide Assist (ScopeGuide) in enhancing the procedural competence of endoscopists and reducing patient discomfort during colonoscopy. METHODS: This was a retrospective study with 88 trainee participants. The study participants were trained on patients who underwent colonoscopy without anesthesia. Both ScopeGuide-assisted training and conventional training (without ScopeGuide) were utilized for colonoscopy instruction. The outcomes of training were compared, with a particular emphasis on the competency of looping resolution. RESULTS: ScopeGuide-assisted training was superior to conventional training in multiple aspects, including looping resolution ( Z =-3.681, P <0.001), pain scores ( Z =-4.211, P <0.001), time to reach the cecum ( Z =-4.06, P <0.001), willingness to undergo repeat colonoscopy ( Z =-4.748, P <0.001), competence of positional changes ( Z =-4.079, P <0.001), and the effectiveness of assisted compression ( Z =-3.001, P =0.003). Further stratified analysis revealed that the ScopeGuide-assisted training mode was more beneficial for junior endoscopists ( P <0.05 in all parameters) but not for intermediate endoscopists ( P >0.05) and partially beneficial for senior endoscopists ( P <0.05 for all parameters except looping resolution). CONCLUSION: ScopeGuide-assisted training can significantly facilitate endoscopists in resolving loops and reducing patient pain, thereby enhancing their colonoscopy abilities.

Global Profiles of Acetylated Proteins in Brains of Scrapie Agents 139A- and ME7-Infected Mice Collected at Mid-Early, Mid-Late, and Terminal Stages.

Objective: To describe the global profiles of acetylated proteins in the brains of scrapie agents 139A- and ME7-infected mice collected at mid-early, mid-late, and terminal stages. Methods: The acetylated proteins from the cortex regions of scrapie agent (139A- and ME7)-infected mice collected at mid-early (80 days postinfection, dpi), mid-late (120 dpi), and terminal (180 dpi) stages were extracted, and the global profiles of brain acetylated proteins were assayed with proteomic mass spectrometry. The proteins in the infected mice showing 1.5-fold higher or lower levels than that of age-matched normal controls were considered as differentially expressed acetylated peptides (DEAPs). Results: A total of 118, 42, and 51 DEAPs were found in the brains of 139A-80, 139A-120, and 139A-180 dpi mice, respectively. Meanwhile, 390, 227, and 75 DEAPs were detected in the brains of ME7-80, ME7-120, and ME7-180 dpi mice, respectively. The overwhelming majority of DEAPs in the mid-early stage were down-regulated, and more portions of DEAPs in the mid-late and late stages were up-regulated. Approximately 22.1% (328/1,485) of acetylated peptides mapped to 74 different proteins were mitochondrial associated. Kyoto Encyclopedia of Genes and Genomes pathway analysis identified 39 (80 dpi), 13 (120 dpi), and 10 (180 dpi) significantly changed pathways in 139A-infected mice. Meanwhile, 55, 25, and 18 significantly changed pathways were observed in the 80, 120, and 180 dpi samples of 139A- and ME7-infected mice ( P < 0.05), respectively. Six pathways were commonly involved in all tested samples. Moreover, many steps in the citrate cycle (tricarboxylic acid cycle) were affected, represented by down-regulated acetylation for relevant enzymes in the mid-early stage and up-regulated acetylation in the mid-late and late stages. Conclusion: Our data here illustrated the changes in the global profiles for brain acetylated proteins during prion infection, showing remarkably inhibited acetylation in the early stage and relatively enhanced acetylation in the late stage.

Synergistic effects of hIAPP and Aß1-42 impaired the olfactory function associated with the decline of adult neurogenesis in SVZ.

According to many in the field，the prevalence of Alzheimer's disease (AD) in type II diabetes (T2DM) populations is considerably higher than that in the normal population. Human islet amyloid polypeptide (hIAPP) is considered to be a common risk factor for T2DM and AD. Preliminary observations around T2DM animal model show that the decrease of adult neural stem cells (NSCs) in the subventricular zone (SVZ) is accompanied by olfactory dysfunction. Furthermore, impaired olfactory function could serve as to an early predictor of neurodegeneration，which is associated with cognitive impairment. However, the synergistic effects between hIAPP and amyloid-beta (Aß) 1-42 in the brain and the neurodegeneration remains to be further clarified. In this study, olfactory capacity, synaptic density, status of NSC in SVZ, and status of newborn neurons in olfactory bulb (OB) were assessed 6 months after stereotactic injection of oligomer Aß1-42 into the dens gyrus (DG) of hIAPP-/+ mice or wild-type homogenous mice. Our results set out that Aß42 and amylin co-localized into OB and raised Aß42 deposition in hIAPP-/+ mice compared with wild-type brood mice. In addition, 6 months after injection of Aß1-42 in hIAPP-/+ mice, these mice showed increased olfactory dysfunction, significant loss of synapses, depletion of NSC in SVZ, and impaired cell renewal in OB. Our present study suggested that the synergistic effects between hIAPP and Aß1-42 impairs olfactory function and was associated with decreased neurogenesis in adults with SVZ.

Pancreatic neuroendocrine carcinoma in a pregnant woman: A case report and review of the literature.

BACKGROUND: Portal venous thromboembolism caused by malignant pancreatic neuroendocrine tumor metastasis, as the initial presentation of portal hypertension and upper gastrointestinal bleeding, is a rare entity. To our knowledge, there are no reports of this entity in pregnant women. We describe a case of pancreatic neuroendocrine carcinoma during pregnancy with hematemesis and hematochezia as the initial presentation and review the literature to analyze the demographic, clinical, and pathological features to provide a reference for clinical diagnosis and treatment. CASE SUMMARY: A 40-year-old woman presented with hematemesis and hematochezia at 26-wk gestation; she had no other remarkable medical history. The physical examination revealed normal vital signs, an anemic appearance, and lower abdominal distension. Abdominal color Doppler ultrasonography showed portal vein thrombosis, splenomegaly, intrauterine pregnancy, and intrauterine fetal death. Esophagogastroduodenoscopy revealed esophageal and gastric varicose veins and portal hypertensive gastropathy. Contrast-enhanced computed tomography demonstrated multiple emboli formation in the portal and splenic veins, multiple round shadows in the liver with a slightly lower density, portal vein broadening, varicose veins in the lower esophagus and gastric fundus, splenomegaly, bilateral pleural effusion, ascites and pelvic effusion, broadening of the common bile duct, and increased uterine volume. According to the results of Positron emission tomography-computed tomography and immunohistochemical staining, the final diagnoses were that the primary lesion was a pancreatic neuroendocrine tumor and that there were secondary intrahepatic metastases and venous cancer thrombogenesis. CONCLUSION: Upper gastrointestinal bleeding in a pregnant woman may be caused by portal hypertension due to a malignant pancreatic neuroendocrine tumor.

Triterpene saponins from tea seed pomace (Camellia oleifera Abel) and their cytotoxic activity on MCF-7 cells in vitro.

Triterpenoid saponins are the main active ingredients extracted from Camellia oleifera Abel. In this study, crude saponins (Tc) was extracted from tea seed pomace and purified to obtain total saponins (T0). We used a COSMOSIL C18-OPN to separate T0 into three fractions-highly polar saponins (T1), moderately polar saponins (T2), and weakly polar saponins (T3). HPLC-ESI-MS analysis revealed that T2 were mainly composed of components with m/z ([M-H]-) of 1201.5617, 1187.5822, 1245.5862, and 1215.5779. Cell cycle analysis showed that both T0 and T2 inhibited proliferation and induced S phase arrest of MCF-7 cells. Further cell invasion assays demonstrated T0 and T2 also significantly reduced the invasive potential of MCF-7 cells. So T2 extracted from tea seed pomace (Camellia oleifera) may have effective antitumor activity.

Interaction of human IAPP and Aß1-42 aggravated the AD-related pathology and impaired the cognition in mice.

Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) have a common pathology. Both diseases are characterized by local deposition of amyloid proteins in the brain or islet organ, but their phenotypes and clinical manifestation vary widely. Although the sources of islet amyloid polypeptide (IAPP) and amyloid beta (Aß) are independent, their fibrillar sequences are highly homologous. The prevalence of AD in T2DM populations is considerably higher than that in the normal population, but a mechanistic linkage remains elusive. Therefore, the present study aimed to explore the effects of Aß42 deposition in the brain on the persistently expression of human IAPP (hIAPP). Additionally, cognitive ability, synaptic plasticity, the state of neural stem cells and mitochondrial function were evaluated at 2 or 6 months after stereotaxically injected the oligomer Aß1-42 into the dentate gyrus of hIAPP (-/+) mice or the wild-type littermates. We found that Aß42 and amylin were co-located in hippocampus and Aß42 levels increased when Aß1-42 was injected in hIAPP transgenic mice compared with that of the wild-type littermates. Furthermore, at 6 months after Aß1-42 injection in hIAPP (-/+) mice, it exhibits exacerbated AD-related pathologies including Aß42 deposition, cognitive impairment, synapse reduction, neural stem cells exhaustion and mitochondrial dysfunction. Our present study suggested that hIAPP directly implicated the Aß42 production and deposition as an important linkage between T2DM and AD.

Noncanonical Roles of hα-syn (A53T) in the Pathogenesis of Parkinson's Disease: Synaptic Pathology and Neuronal Aging.

The motor and nonmotor symptoms of PD involve several brain regions. However, whether α-syn pathology originating from the SNc can directly lead to the pathological changes in distant cerebral regions and induce PD-related symptoms remains unclear. Here, AAV9-synapsin-mCherry-human SNCA (A53T) was injected into the unilateral SNc of mice. Motor function and olfactory sensitivity were evaluated. Our results showed that AAV9-synapsin-mCherry-human SNCA was continuously expressed in SNc. The animals showed mild motor and olfactory dysfunction at 7 months after viral injection. The pathology in SNc was characterized by the loss of dopaminergic neurons accompanied by ER stress. In the striatum, hα-syn expression was high, CaMKß-2 and NR2B expression decreased, and active synapses reduced. In the olfactory bulb, hα-syn expression was high, and aging cells in the mitral layer increased. The results suggested that hα-syn was transported in the striatum and OB along the nerve fibers that originated from the SNc and induced pathological changes in the distant cerebral regions, which contributed to the motor and nonmotor symptoms of PD.

Risk score based on expression of five novel genes predicts survival in soft tissue sarcoma.

In this study, The Cancer Genome Atlas and Genotype-Tissue Expression databases were used to identify potential biomarkers of soft tissue sarcoma (STS) and construct a prognostic model. The model was used to calculate risk scores based on the expression of five key genes, among which MYBL2 and FBN2 were upregulated and TSPAN7, GCSH, and DDX39B were downregulated in STS patients. We also examined gene signatures associated with the key genes and evaluated the model's clinical utility. The key genes were found to be involved in the cell cycle, DNA replication, and various cancer pathways, and gene alterations were associated with a poor prognosis. According to the prognostic model, risk scores negatively correlated with infiltration of six types of immune cells. Furthermore, age, margin status, presence of metastasis, and risk score were independent prognostic factors for STS patients. A nomogram that incorporated the risk score and other independent prognostic factors accurately predicted survival in STS patients. These findings may help to improve prognostic prediction and aid in the identification of effective treatments for STS patients.

Fibrillation of human islet amyloid polypeptide and its toxicity to pancreatic ß-cells under lipid environment.

BACKGROUND: Previous studies suggested that fibrillar human IAPP (hIAPP) is more likely to deposit in ß-cells, resulting in ß-cell injury. However, the changes in the conformation of hIAPP in lipid environment and the mechanism involved in ß-cell damage are unclear. METHODS: Synthetic hIAPP was incubated with five types of free fatty acids and phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), which constitute the cell membrane. Thioflavin-T fluorescence assay was conducted to analyze the degree of hIAPP fibrosis, and circular dichroism spectroscopy was performed to detect the ß-fold formation of hIAPP. Furthermore, INS-1 cells were infected with human IAPP delivered by a GV230-EGFP plasmid. The effects of endogenous hIAPP overexpression induced by sodium palmitate on the survival, endoplasmic reticulum (ER) stress, and apoptosis of INS-1 cells were evaluated. RESULTS: The five types of free fatty acids can accelerate the fibrosis of hIAPP. Sodium palmitate also maintained the stability of fibrillar hIAPP. POPS, not POPC, accelerated hIAPP fibrosis. Treatment of INS-1 cells with sodium palmitate increased the expression of hIAPP, activated ER stress and ER stress-dependent apoptosis signaling pathways, and increased the apoptotic rate. CONCLUSION: Free fatty acids and anionic phospholipid can promote ß-fold formation and fibrosis in hIAPP. High lipid induced the overexpression of hIAPP and aggravated ER stress and apoptosis in INS-1 cells, which caused ß-cell death in high lipid environment. GENERAL SIGNIFICANCE: Our study reveals free fatty acids and hIAPP synergistically implicated in endoplasmic reticulum stress and apoptosis of islet ß-cells.

High-Fat Diet Increases Amylin Accumulation in the Hippocampus and Accelerates Brain Aging in hIAPP Transgenic Mice.

The accumulation of human islet amyloid polypeptide (hIAPP) in pancreatic islets under induction by a high-fat diet plays a critical role in the development of type-2 diabetes mellitus (T2DM). T2DM is a risk factor of late-onset Alzheimer's disease (AD). Nevertheless, whether hIAPP in combination with hyperlipidemia may lead to AD-like pathological changes in the brain remains unclear. hIAPP transgenic mice were fed with a high-fat diet for 6 or 12 months to establish the T2DM model. The accumulation of amylin, the numbers of Fluoro-Jade C (FJC)-positive and ß-gal positive cells, and the deposition level of Aß42 in the hippocampi of the transgenic mice were detected by using brain sections. Cytoplasmic and membrane proteins were extracted from the hippocampi of the transgenic mice, and the ratio of membrane GLUT4 expression to cytoplasmic GLUT4 expression was measured through Western blot analysis. Changes in the cognitive functions of hIAPP transgenic mice after 12 months of feeding with a high-fat diet were evaluated. hIAPP transgenic mice fed with a high-fat diet for 6 or 12 months showed elevated blood glucose levels and insulin resistance; increased amylin accumulation, number of FJC-positive and ß-gal positive cells, and Aß42 deposition in the hippocampi; and reduced membrane GLUT4 expression levels. hIAPP transgenic mice fed with a high-fat diet for 12 months showed reductions in social cognitive ability and passive learning ability. A high-fat diet increased amylin accumulation in the hippocampi of hIAPP transgenic mice, which presented AD-like pathology and behavior characterized by neural degeneration, brain aging, Aß42 deposition, and impaired glucose utilization and cognition.

Stimulations of the Culture Medium of Activated Microglia and TNF-Alpha on a Scrapie-Infected Cell Line Decrease the Cell Viability and Induce Marked Necroptosis That Also Occurs in the Brains from the Patients of Human Prion Diseases.

Activation of microglia and increased expression of TNF-α are frequently observed in the brains of human and animal prion diseases. As an important cytokine, TNF-α participates in not only pro-inflammatory responses but also in cellular communication, cell differentiation, and cell death. However, the role of TNF-α in the pathogenesis of prion disease remains ambiguous. In this study, the activities of a scrapie-infected cell line SMB-S15 and its normal partner SMB-PS exposed to the supernatant of a LPS-activated microglia cell line BV2 were evaluated. After it was exposed to the LPS-stimulated supernatant of BV2 cells, the cell viability of SMB-S15 cells was markedly decreased, whereas that of the SMB-PS cells remained unchanged. The level of TNF-α was significantly increased in the LPS-stimulated supernatant of BV2 cells. Further, we found that the recombinant TNF-α alone induced the decreased cell viability of SMB-S15 and the neutralizing antibody for TNF-α completely antagonized the decreased cell viability caused by the LPS-stimulated supernatant of BV2 cells. Stimulation with TNF-α induced the remarkable increases of apoptosis-associated proteins in SMB-PS cells, such as cleaved caspase-3 and RIP1, whereas an obvious increase of necroptosis-associated protein in SMB-S15 cells, such as p-MLKL. Meanwhile, the upregulation of caspase-8 activity in SMB-PS cells was more significant than that of SMB-S15 cells. The decreased cell viability of SMB-S15 and the increased expression of p-MLKL induced by TNF-α were completely rescued by Necrostatin-1. Moreover, we verified that removal of PrPSc propagation in SMB-S15 cells by resveratrol partially rescues the cell tolerance to the stimulation of TNF-α. These data indicate that the prion-infected cell line SMB-S15 is more vulnerable to the stimulations of activated microglia and TNF-α, which is likely due to the outcome of necroptosis rather than apoptosis. Furthermore, significant upregulation of p-MLKL, MLKL, and RIP3 was detected in the post-mortem cortical brains of the patients of various types of human prion diseases, including sporadic Creutzfeldt-Jakob disease (sCJD), G114 V-genetic CJD (gCJD), and fatal familial insomnia (FFI).

Apatinib for treating advanced intrahepatic cholangiocarcinoma after failed chemotherapy: A case report and literature review.

RATIONALE: Intrahepatic cholangiocarcinoma (ICC) originates from the secondary branch of the bile duct and the intrahepatic bile duct epithelial cells, and is a rare pathological type of primary liver cancer. Recently, apatinib has been successfully used for a variety of malignancies. PATIENT CONCERNS: A 23-year-old female was noted with intermittent right upper abdominal distension, abdominal pain, and vomiting after eating for more than 1 month. The enhanced CT scan revealed multiple intrahepatic lesions, portal vein and right branch tumor emboli were present. DIAGNOSIS: Combined with the patient's medical history and pathology and immunohistochemistry, the diagnosis was confirmed as locally advanced unresectable ICC (cT4N1M1, Stage IVB). INTERVENTIONS: The disease progressed after six cycles of gemcitabine plus capecitabine chemotherapy. She received oral apatinib treatment since September 30, 2017. Due to related adverse reactions, the patient could not tolerate the treatment, and the subsequent reduction therapy was given. OUTCOMES: On April 11, 2018, the review of CT evaluation suggested that the disease was progressed. Hence, in this patient, apatinib as second-line treatment for advanced ICC showed a progression-free survival with 6 months. LESSONS: Apatinib as second-line treatment for advanced ICC is effective, and the adverse effects are tolerable. However, the efficacy and safety of apatinib in the treatment of ICC need to be further confirmed by large sample of prospective randomized controlled trials.

Spermidine ameliorates the neuronal aging by improving the mitochondrial function in vitro.

Changes in mitochondrial structure and function are the initial factors of cell aging. Spermidine has an antiaging effect, but its effect on neuronal aging and mitochondrial mechanisms is unclear. In this study, mouse neuroblastoma (N2a) cells were treated with d­galactose (d­Gal) to establish cell aging to investigate the antiaging effect and mechanisms of spermidine. Changes in the cell cycle and ß-galactosidase activity were analyzed to evaluate the extent of cell aging. Stabilities of mitochondrial mRNA and mitochondrial membrane potential (MMP) were evaluated in the process of cell aging under different treatments. The mitochondrial function was also evaluated using the Seahorse Metabolic Analysis System combined with ATP production. The unfolded protein response (UPR) of the N2a cells was analyzed under different treatments. Results showed that spermidine pretreatment could delay the cell aging and could maintain the mitochondrial stability during d­Gal treatment. Spermidine increased the proportion of cells in the S phase and maintained the MMP. The oxygen utilization and ATP production in the N2a cells were reduced by d­Gal treatment but were partially rescued by the spermidine pretreatment. Spermidine ameliorated the N2a cell aging by promoting the autophagy and inhibiting the apoptosis except the UPR. These results showed that spermidine could ameliorate the N2a cell aging by maintaining the mitochondrial mRNA transcription, MMP and oxygen utilization during the d­Gal treatment.

Lipid accelerating the fibril of islet amyloid polypeptide aggravated the pancreatic islet injury in vitro and in vivo.

BACKGROUND: The fibrillation of islet amyloid polypeptide (IAPP) triggered the amyloid deposition, then enhanced the loss of the pancreatic islet mass. However, it is not clear what factor is the determinant in development of the fibril formation. The aim of this study is to investigate the effects of lipid on IAPP fibril and its injury on pancreatic islet. METHODS: The fibril form of human IAPP (hIAPP) was tested using thioflavin-T fluorescence assay and transmission electron microscope technology after incubated with palmitate for 5 h at 25 °C. The cytotoxicity of fibril hIAPP was evaluated in INS-1 cells through analyzing the leakage of cell membrane and cell apoptosis. Type 2 diabetes mellitus (T2DM) animal model was induced with low dose streptozotocin combined the high-fat diet feeding for two months in rats. Plasma biochemistry parameters were measured before sacrificed. Pancreatic islet was isolated to evaluate their function. RESULTS: The results showed that co-incubation of hIAPP and palmitate induced more fibril form. Fibril hIAPP induced cell lesions including cell membrane leakage and cell apoptosis accompanied insulin mRNA decrease in INS-1 cell lines. In vivo, Plasma glucose, triglyceride, rIAPP and insulin increased in T2DM rats compared with the control group. In addition, IAPP and insulin mRNA increased in pancreatic islet of T2DM rats. Furthermore, T2DM induced the reduction of insulin receptor expression and cleaved caspase-3 overexpression in pancreatic islet. CONCLUSIONS: Results in vivo and in vitro suggested that lipid and IAPP plays a synergistic effect on pancreatic islet cell damage, which implicated in enhancing the IAPP expression and accelerating the fibril formation of IAPP.

Metabolic Characterization of the Anthocyanidin Reductase Pathway Involved in the Biosynthesis of Flavan-3-ols in Elite Shuchazao Tea (Camellia sinensis) Cultivar in the Field.

Anthocyanidin reductase (ANR) is a key enzyme in the ANR biosynthetic pathway of flavan-3-ols and proanthocyanidins (PAs) in plants. Herein, we report characterization of the ANR pathway of flavan-3-ols in Shuchazao tea (Camellia sinesis), which is an elite and widely grown cultivar in China and is rich in flavan-3-ols providing with high nutritional value to human health. In our study, metabolic profiling was preformed to identify two conjugates and four aglycones of flavan-3-ols: (-)-epigallocatechin-gallate [(-)-EGCG], (-)-epicatechin-gallate [(-)-ECG], (-)-epigallocatechin [(-)-EGC], (-)-epicatechin [(-)-EC], (+)-catechin [(+)-Ca], and (+)-gallocatechin [(+)-GC], of which (-)-EGCG, (-)-ECG, (-)-EGC, and (-)-EC accounted for 70-85% of total flavan-3-ols in different tissues. Crude ANR enzyme was extracted from young leaves. Enzymatic assays showed that crude ANR extracts catalyzed cyanidin and delphinidin to (-)-EC and (-)-Ca and (-)-EGC and (-)-GC, respectively, in which (-)-EC and (-)-EGC were major products. Moreover, two ANR cDNAs were cloned from leaves, namely CssANRa and CssANRb. His-Tag fused recombinant CssANRa and CssANRb converted cyanidin and delphinidin to (-)-EC and (-)-Ca and (-)-EGC and (-)-GC, respectively. In addition, (+)-EC was observed from the catalysis of recombinant CssANRa and CssANRb. Further overexpression of the two genes in tobacco led to the formation of PAs in flowers and the reduction of anthocyanins. Taken together, these data indicate that the majority of leaf flavan-3-ols in Shuchazao's leaves were produced from the ANR pathway.

Use of human aortic extracellular matrix as a scaffold for construction of a patient-specific tissue engineered vascular patch.

Synthetic or biologic materials are usually used to repair vascular malformation in congenital heart defects; however, non-autologous materials show both mismatch compliance and antigenicity, as well as a lack of recellularization on its surface. Here, we constructed a tissue-engineered vascular patch (TEVP) using decellularized extracellular matrix (ECM) scaffold obtained from excised human aorta during surgery, which was seeded with patient-derived bone marrow CD34-positive (CD34+) progenitor cells. While cellular components were removed, the decellularized ECM scaffold retained native ECM composition, similar mechanical performance to undecellularized aortic tissue, and supported the adhesion, survival and proliferation of CD34+ progenitor cells. Interestingly, after in vitro seeding of decellularized aortic ECM scaffold for 21 d, CD34+ progenitor cells differentiated into mature vascular endothelial cells without addition of any growth factors, as confirmed by the increased levels of endothelial surface markers (CD31, Von Willebrand factor (VWF), VE-cadherin and ICAM-2) and upregulated gene levels (CD31, VWF and eNOS) concurrently with decreased expression of stem cell markers (CD133 and CD34), thus, resulting in surface endothelialization of decellularized ECM scaffold. Consequently, the patient-specific TEVP constructed in this study holds great potential for clinical use in pediatric patients with vascular malformation.

Role of Arcuate Nucleus in the Regulation of Feeding Behavior in the Process of Altitude Acclimatization in Rats.

Liu, Xiang-Wen, Jie Yin, Qi-Sheng Ma, Chu-Chu Qi, Ji-Ying Mu, Lang Zhang, Li-Ping Gao, and Yu-Hong Jing. Role of arcuate nucleus in the regulation of feeding behavior in the process of altitude acclimatization in rats. High Alt Med Biol. 18:234-241, 2017.-Highly efficient energy utilization and metabolic homeostasis maintenance rely on neuromodulation. Altitude exposure is known to stimulate neuroendocrine systems to respond to acute hypoxia and adaptive acclimatization. However, limited data on how the adaptive regulation of the arcuate nucleus performs in the process of altitude acclimatization are available. In the present study, male Sprague Dawley rats were transported to Huashixia, Qinghai (with an altitude of 4400 m) from Xian (with an altitude of 300 m) by air; rats were consistently raised in Xian as control. Food uptake and body weight were measured consecutively after being subjected to high-altitude condition. Contents of plasma leptin and ghrelin were analyzed by the Enzyme Linked Immunosorbent Assay (ELISA) Kits. Brain coronal sections were obtained, and neuropeptide Y (NPY), proopiomelanocotin (POMC), and c-fos immunoreactivity in arcuate nucleus were observed. Arcuate nucleus was isolated from the hypothalamus, and the mRNA of NPY and POMC were measured by quantitative real-time polymerase chain reaction. Our results showed both food consumption and body weight decreased in the high plateau compared with rats raised in the low-altitude condition. Plasma leptin increased at the early stage, and ghrelin decreased at a later stage after reaching the high plateau. The peak of c-fos immunoreactivity in the arcuate nucleus was at day 3 after reaching the high plateau. The expression level of NPY increased, and POMC decreased in the arcuate nucleus at day 7 after reaching the high plateau compared with the plain control group. These results indicate that the arcuate nucleus of hypothalamus performs an important function in regulating feeding behavior during altitude acclimatization. Our study suggested that altitude acclimation is regulated by the hypothalamus that received leptin and ghrelin signals to response by its microcircuit, including NPY- and POMC-neurons in the arcuate nucleus.

Autophagy plays beneficial effect on diabetic encephalopathy in type 2 diabetes: studies in vivo and in vitro.

OBJECTIVES: The hypothalamus regulates metabolism and feeding behavior by perceiving the levels of peripheral insulin. However, little is known about the hypothalamic changes after aberrant metabolism. In this study, we investigated the changes of insulin and autophagy relevant signals of hypothalamus under diabetes mellitus. METHODS: C57B/L mice were injected with low-dose streptozotocin (STZ) and fed with high-fat diet to induce type 2 diabetes mellitus. In vitro, PC12 cells were treated with oleic acid to mimic lipotoxicity. RESULTS: Results showed that the cholesterol level in the hypothalamus of the diabetic mice was higher than that of the normal mice. The expression of insulin receptors and insulin receptor substrate-1 were downregulated and the number of Fluoro-Jade C positive cells significantly increased in the hypothalamic arcuate nucleus of the diabetic mice. Furthermore, Upregulation of mammalian target of rapamycin (mTOR) and downregulation of LC 3II were obvious in the hypothalamus of the diabetic mice. In vitro, results showed that high-lipid caused PC12 cell damage and upregulated LC3 II expression. Pretreatment of cells with 3-methyladenine evidently downregulated LC3 II expression and aggravated PC12 cell death under high lipid conditions. By contrast, pretreatment of cells with rapamycin upregulated LC3 II expression and ameliorated PC12 cell death caused by lipotoxicity. CONCLUSION: These results demonstrate that autophagy activation confers protection to neurons under aberrant metabolism and that autophagy dysfunction in the hypothalamus occurs in the chronic metabolic disorder such as T2DM.

Functional analysis of flavonoid 3',5'-hydroxylase from tea plant (Camellia sinensis): critical role in the accumulation of catechins.

BACKGROUND: Flavonoid 3',5'-hydroxylase (F3'5'H), an important branch point enzyme in tea plant flavan-3-ol synthesis, belongs to the CYP75A subfamily and catalyzes the conversion of flavones, flavanones, dihydroflavonols and flavonols into 3',4',5'-hydroxylated derivatives. However, whether B-ring hydroxylation occurs at the level of flavanones and/or dihydroflavonols, in vivo remains unknown. RESULTS: The Camellia sinensis F3'5'H (CsF3'5'H) gene was isolated from tea cDNA library. Expression pattern analysis revealed that CsF3'5'H expression was tissue specific, very high in the buds and extremely low in the roots. CsF3'5'H expression was enhanced by light and sucrose. Over-expression of CsF3'5'H produced new-delphinidin derivatives, and increased the cyanidin derivative content of corollas of transgenic tobacco plants, resulting in the deeper transgenic plant flower color. Heterologous expressions of CsF3'5'H in yeast were carried out to demonstrate the function of CsF3'5'H enzyme in vitro. Heterologous expression of the modified CsF3'5'H (CsF3'5'H gene fused with Vitis vinifera signal peptide, FSI) revealed that 4'-hydroxylated flavanone (naringenin, N) is the optimum substrate for CsF3'5'H, and was efficiently converted into both 3'4'- and 3'4'5'-forms. The ratio of 3'4'5'- to 3'4'-hydroxylated products in FSI transgenic cells was significantly higher than VvF3'5'H cells. CONCLUSIONS: CsF3'5'H is a key controller of tri-hydroxyl flavan-3-ol synthesis in tea plants, which can effectively convert 4'-hydroxylated flavanone into 3'4'5'- and/or 3'4'-hydroxylated products. These findings provide animportant basis for further studies of flavonoid biosynthesis in tea plants. Such studies would help accelerate flavonoid metabolic engineering in order to increase B-ring tri-hydroxyl product yields.