Identification and characterization of apple MdNLP7 transcription factor in the nitrate response.

Nitrogen is an essential nutrient for plant growth and development. Low utilization of nitrogen fertilizer during agricultural production causes a series of environmental problems, such as water eutrophication, soil acidity, and air pollution. Investigating the patterns and mechanisms of crop NO3- absorption and utilization therefore key to fully improving crop nitrogen utilization rates and promoting sustainable agricultural development. Apple is one of the most important horticultural crops in the world. Its nitrogen demand by apple during the growth period is very high, but few studies have been performed on apple genes, that regulate the NO3- response. Here, we found that the apple transcription factor MdNLP7 promoted nitrogen absorption and assimilation by activating the expression of MdNIA2 and MdNRT1.1. MdNLP7 also regulated H2O2 content by increasing catalase activity, which may also influence nitrate utilization. Our findings provide insight into the mechanisms by which MdNLP7 controls nitrate utilization in apple.

The BTB protein MdBT2 recruits auxin signaling components to regulate adventitious root formation in apple.

Ubiquitination is an important post-translational protein modification. Although BROAD-COMPLEX, TRAMTRACK AND BRIC A BRAC and TRANSCRIPTION ADAPTOR PUTATIVE ZINC FINGER domain protein 2 (BT2) is involved in many biological processes, its role in apple (Malus domestic) root formation remains unclear. Here, we revealed that MdBT2 inhibits adventitious root (AR) formation through interacting with AUXIN RESPONSE FACTOR8 (MdARF8) and INDOLE-3-ACETIC ACID INDUCIBLE3 (MdIAA3). MdBT2 facilitated MdARF8 ubiquitination and degradation through the 26S proteasome pathway and negatively regulated GRETCHEN HAGEN 3.1 (MdGH3.1) and MdGH3.6 expression. MdARF8 regulates AR formation through inducing transcription of MdGH3s (MdGH3.1, MdGH3.2, MdGH3.5, and MdGH3.6). In addition, MdBT2 facilitated MdIAA3 stability and slightly promoted its interaction with MdARF8. MdIAA3 inhibited AR formation by forming heterodimers with MdARF8 as well as other MdARFs (MdARF5, MdARF6, MdARF7, and MdARF19). Our findings reveal that MdBT2 acts as a negative regulator of AR formation in apple.

Low nitrate alleviates iron deficiency by regulating iron homeostasis in apple.

Iron (Fe) is an essential element for plant growth, development and metabolism. Due to its lack of solubility and low bioavailability in soil, Fe levels are usually far below the optimum amount for most plants' growth and development. In apple production, excessive use of nitrogen fertilizer may cause iron chlorosis symptoms in the newly growing leaves, but the regulatory mechanisms underlying this phenomenon are unclear. In this study, low nitrate (NO3- , LN) application alleviated the symptoms of Fe deficiency and promoted lower rhizosphere pH, which was beneficial for root Fe acquisition. At the same time, LN treatment increased citrate and abscisic acid accumulation in roots, which promoted Fe transport from root to shoot and maintained Fe homeostasis. Moreover, qRT-PCR analysis showed that nitrate application caused differential expression of genes related to Fe uptake and transport, as well as transcriptional regulators. In summary, our data reveal that low nitrate alleviated Fe deficiency through multiple pathways, demonstrating a new option for minimizing Fe deficiency by regulating the balance between nutrients.

NIN-like protein 7 promotes nitrate-mediated lateral root development by activating transcription of TRYPTOPHAN AMINOTRANSFERASE RELATED 2.

Nitrate is essential for plant growth and development. When nitrate availability is low, plants produce more lateral roots (LRs) to seek nitrate from the soil. In this study, by DNA electrophoretic mobility shift and luciferase assays, it was showed that NIN-like protein 7 (NLP7) transcription factor activated expression of TAR2 by directly binding to its promoter. Finally, through genetic analysis, it was speculated that NLP7 regulated LR development through TAR2. In conclusion, NLP7 binds to the TAR2 promoter and activates TAR2 expression, thereby promoting nitrate-dependent LR development.

The BTB-TAZ protein MdBT2 negatively regulates the drought stress response by interacting with the transcription factor MdNAC143 in apple.

Drought stress is a severe source of abiotic stress that can affect apple yield and quality, yet the underlying molecular mechanism of the drought stress response and the role of MdBT2 in the process remain unclear. Here, we find that MdBT2 negatively regulates the drought stress response. Both in vivo and in vitro assays indicated that MdBT2 interacted physically with and ubiquitinated MdNAC143, a member of the NAC TF family that is a positive regulator under drought stress. In addition, MdBT2 promotes the degradation of MdNAC143 via the 26S proteasome system. A series of transgenic assays in apple calli and Arabidopsis verify that MdBT2 confers susceptibility to drought stress at least in part by the regulation of MdNAC143. Overall, our findings provide new insight into the mechanism of MdBT2, which functions antagonistically to MdNAC143 in regulating drought stress by regulating the potential downstream target protein MdNAC143 for proteasomal degradation in apple.

microRNA-33a prevents epithelial-mesenchymal transition, invasion, and metastasis of gastric cancer cells through the Snail/Slug pathway.

Invasion and metastasis are responsible for the majority of deaths in gastric cancer (GC). microRNA-33a (miR-33a) might function as a tumor suppressor in multiple cancers. Here, we describe the regulation and function of miR-33a in GC and mechanisms involved in epithelial-mesenchymal transition (EMT) and metastasis. First, GC tissues and adjacent normal tissues were collected. miR-33a upregulation or SNAI2 depletion on GC cells were introduced to assess the detailed regulatory mechanism of them. We assessed the expression of miR-33a, SNAI2, Snail/Slug signaling pathway-related genes, and EMT-related markers in GC tissues and cells. miR-33a distribution in GC tissues and adjacent normal tissues was measured. Cell proliferation, migration and invasion, and cell cycle distribution were assessed. In nude mice, GC tumor growth and lymph node metastasis were observed. Furthermore, the predicative value of miR-33a in the prognosis of GC patients was evaluated. The obtained results indicated that lowly expressed miR-33a, highly expressed SNAI2, activated Snail/Slug, and increased EMT were identified in GC tissues. miR-33a was located mainly in the cytoplasm. miR-33a targeted and negatively regulated SNAI2. MKN-45 and MKN-28 cell lines were selected for in vitro experiments. Upregulated miR-33a expression or siRNA-mediated silencing of SNAI2 suppressed the activation of Snail/Slug, whereby GC cell proliferation, invasion and migration, EMT, tumor growth, and lymph node metastasis were inhibited. High expression of miR-33a was a protective factor influencing the prognosis of GC. This study suggests that miR-33a inhibited EMT, invasion, and metastasis of GC through the Snail/Slug signaling pathway by modulating SNAI2 expression.NEW & NOTEWORTHY miR-33a targets and inhibits the expression of SNAI2, overexpression of SNAI2 activates the Snail/Slug signaling pathway, the Snail/Slug signaling pathway promotes GC cell proliferation, invasion, and metastasis, and overexpression of miR-33a inhibits cell proliferation, invasion, and metastasis. This study provides a new therapeutic target for the treatment of GC.

Long non-coding RNA-ENST00000434223 suppresses tumor progression in gastric cancer cells through the Wnt/ß-catenin signaling pathway.

BACKGROUND: Gastric cancer (GC) develops from the lining of the stomach. The present study aimed to explore the effects of long non-coding RNA-ENST00000434223 (lncRNA ENST00000434223) on gastric cancer (GC) cells. METHODS: One hundred and four GC tissues and paracancerous tissues were collected from GC patients, and expression of ENST00000434223, Wnt2b, ß-catenin, cyclinD1, E-cadherin, N-cadherin, vimentin, and snail was subsequently assessed. Morphological changes in cells were assessed using an inverted microscope, and expression of Bcl-2, Bax and caspase-3 was examined. RESULTS: We found that expression of Wnt2b, ß-catenin, cyclinD1, N-cadherin, vimentin, and snail was increased in GC tissues, while expression of ENST00000434223 and E-cadherin was decreased. SGC-7901 cells were closely arranged, and expression of Wnt2b, ß-catenin, CyclinD1, N-cadherin, Vimentin, snail and Bcl-2 was increased, whereas expression of ENST00000434223, E-cadherin, Bax and caspase-3 was decreased. Furthermore, the rate of apoptosis was decreased and cell proliferation, invasion and migration were increased in response to downregulation of ENST00000434223. By contrast, upregulation of ENST00000434223 exhibited the opposite effects in MKN-45 cells. CONCLUSION: The results of this study provide a promising experimental basis for the treatment of gastric cancer through interventional targeting of lncRNA ENST00000434223.

microRNA­372 inhibits proliferation and induces apoptosis in human breast cancer cells by directly targeting E2F1.

Breast cancer is the most prevalent cancer and the leading cause of cancer­associated mortalities among women worldwide today. Accumulating evidence suggested that miR­372 may serve important roles in the initiation and development of various human cancers. However, the role of miR­372 in breast cancer remains unknown. The present study demonstrated that the expression level of miR­372 in human breast cancer tissues and cell lines is significantly reduced compared with normal breast tissues cell lines. Furthermore, results of functional assays indicated that miR­372 inhibits cell proliferation and induces apoptosis in the MCF­7 human breast cancer cell line. E2F1 was identified as a direct functional target of miR­372 in breast cancer. In conclusion, the findings revealed that miR­372 may have the potential to act as a novel molecule for the diagnosis and therapy of patients with breast cancer.

Gossypol induces cell death by activating apoptosis and autophagy in HT-29 cells.

Gossypol is a polyphenolic, yellowish compound derived from cottonseed extract. The present study examined the effects of gossypol on the apoptosis and autophagy of HT­29 cells. A Cell Counting Kit­8 assay, Annexin V­FITC, JC­1 staining and western blotting were used to identify the viability of cells, stages of apoptosis and the expression levels of the signaling proteins. Gossypol promoted apoptosis and induced the loss of mitochondrial membrane potential. Further investigation of the apoptotic mechanism revealed that gossypol increased the ratio of B­cell lymphoma 2 (Bcl-2)-associated X protein/Bcl­2 protein levels and upregulated the expression of caspase­3. Gossypol also enhanced the activity of microtubule­associated protein light chain 3 LC3­II and Beclin­1 and downregulated LC3­I, in a dose­dependent manner. Together, these finding suggested that gossypol may be a novel and potential antitumor agent.

Romidepsin induces G2/M phase arrest via Erk/cdc25C/cdc2/cyclinB pathway and apoptosis induction through JNK/c-Jun/caspase3 pathway in hepatocellular carcinoma cells.

The aim of the study is to demonstrate the effect of Romidepsin in hepatocellular carcinoma (HCC) by inducing G2/M phase arrest via Erk/cdc25C/cdc2/cyclinB pathway and apoptosis through JNK/c-Jun/caspase3 pathway in vitro and in vivo. Human HCC cell lines were cultured with Romidepsin and DMSO (negative control) and 5-fluorouracil (positive control). Then the cells' viability and apoptosis were determined by cell proliferation assay and flow cytometry. Protein concentrations and expression changes were measured by Western blot. Subsequently, Huh7 cells were subcutaneously inoculated into the nude mice, which were employed to further probe the tumor-suppressive effect of Romidepsin in vivo. Romidepsin treatment led to a time- and dose-dependent induction of cell cycle arrest in the G2/M phase and apoptosis. G2/M phase arrest inhibited the proliferation of HCC cells by alterations in p21/cdc25C/cdc2/cyclinB proteins. Increased concentrations of Erk and JNK phosphorylations were observed in a dose-dependent manner in the Romidepsin group, but p38 phosphorylation was not affected. G2/M phase arrest and the apoptosis of HCC cells induced by Romidepsin were mediated by the activation of Erk/MAPK pathways and JNK/MAPK pathways. The tumor size was significantly larger in the negative control group compared to Romidepsin group and no significant loss in body weight was observed in the Romidepsin group. Our findings offer proof-of-concept for use of Romidepsin as a novel class of chemotherapy in the treatment of HCC.

Expression of AQP1 Was Associated with Apoptosis and Survival of Patients in Gastric Adenocarcinoma.

RATIONALE AND OBJECTIVE: Recently, interest in the role of aquaporin 1 (AQP1) in human gastrointestinal carcinogenesis has developed. However, to date no studies have examined relationships between AQP1 expression and specific characteristics of gastric adenocarcinoma. METHODS: We investigated 109 specimens of primary gastric adenocarcinoma and their corresponding normal gastric mucosa using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) to determine AQP1 expression. We then evaluated disease free survival (DFS) and overall survival (OS) in these patients in association with AQP1 expression. RESULTS: Both immunohistochemical and RT-PCR analyses identified increased AQP1 expression in tumors from patients with gastric adenocarcinoma (p < 0.001). The 3-year DFS and OS rates were higher in the AQP1-negative group than in the positive group (DFS: 77.2 vs. 52.8%, p < 0.001; OS: 85.1 vs. 70.7%, p < 0.001). The 5-year DFS and OS rates exhibited a similar trend (p < 0.001). Subgroup analysis of patients with early gastric adenocarcinoma (stages I and II) revealed a total 5-year OS of 90.0%, with 5-year OS being higher in the AQP1-negative group than in the positive group (95.2 vs. 84.2%). Furthermore, incidence of tumor recurrence following surgical treatment was significantly higher in the AQP1-positive group (4/19, 21.1%) compared with the negative group (0/21, 0%). CONCLUSIONS: Our study demonstrates that AQP1 plays an important role in gastric adenocarcinoma and may therefore represent a novel therapeutic target and prognostic marker in this disease.

Clinical features, surgical treatment, and long-term outcome in pediatric patients with moyamoya disease in China.

BACKGROUND: There was few detailed demographic and clinical data about Chinese patients with moyamoya disease. Here we describe the clinical features, surgical treatment, and long-term outcome of pediatric patients with moyamoya disease at a single institution in China. METHODS: Our cohort included 288 pediatric patients with moyamoya disease. The demographic and clinical characteristics were obtained by retrospective chart review and long-term outcome was evaluated using the stroke status. Univariate and multivariate logistic regression analyses were performed to determine the risk factors for clinical outcome. The risk of subsequent stroke was determined using the Kaplan-Meier method. RESULTS: The median age for the onset of symptoms was 8.0 years. The ratio of female to male patients was 1:1. Familial occurrence of moyamoya disease was 9.4%. The incidence of postoperative complications was 4.2%. Postoperative ischemic events were identified as predictors of unfavorable clinical outcome, while older age of symptom onset was associated with a favorable clinical outcome. The Kaplan-Meier estimate stroke risk was 5% in the first 2 years, and the 5-year-Kaplan-Meier risk of stroke was 9% after surgery for all patients treated with surgical revascularization. Overall, 86% of patients had an independent life with no significant disability. CONCLUSION: This long-term survey demonstrated that most surgically treated pediatric patients with MMD maintain good outcomes. Our results indicate that an early diagnosis and active intervention before the establishment of irreversible hemodynamic change are essential to achieve a favorable clinical outcome.

Efficacy of docetaxel combined with oxaliplatin and fluorouracil against stage III/IV gastric cancer.

AIM: To investigate the clinical efficacy and toxic effects of neoadjuvant chemotherapy using docetaxel combined with oxaliplatin and fluorouracil for treating stage III/IV gastric cancer. METHODS: A total of 53 stage III/IV gastric cancer patients were enrolled into the study and treated with neoadjuvant chemotherapy. Two of the cases were excluded. The program was as follows: 75 mg/m(2) docetaxel and 85 mg/m(2) oxaliplatin on day 1 and 1500 mg/m(2) fluorouracil on days 1 to 3 for three weeks. RESULTS: The tumour changes, postoperative remission rate, changes in the symptoms and adverse reactions were observed. The overall clinical efficacy (complete remission + partial remission) of the neoadjuvant chemotherapy was 62.7%. R0 radical resection was performed on 60.8% of the patients, with a remission rate (pathological complete response + pathological subtotal response + pathological partial response) of 74.2%. The Karnofksy score improved in 42 cases. The toxicity reactions mostly included myelosuppression, followed by gastrointestinal mucosal lesions, nausea, vomiting and diarrhoea. CONCLUSION: Neoadjuvant chemotherapy consisting of docetaxel combined with oxaliplatin and fluorouracil is effective for stage III/IV gastric cancer. However, the treatment is associated with a high incidence of bone marrow suppression, which should be managed clinically.

Latexin inhibits the proliferation of CD133+ miapaca-2 pancreatic cancer stem-like cells.

BACKGROUND: An increasing number of evidence suggests that pancreatic cancer contains cancer stem cells (CSCs), which may be relevant to the resistance of chemotherapy. Latexin (Lxn) is a negative regulator of stem cell proliferation and we investigate the effects of Lxn on CD133+ pancreatic cancer stem-like cells. METHODS: CD133+ miapaca-2 cells, a human pancreatic carcinoma cell line, were isolated and sorted by magnetic activated cell sorting and flow cytometry. The capacity for self-renewal, proliferation, and tumorigenicity of CD133+ miapaca-2 cells was determined by the floating spheres test and tumor xenograft assays. Protein and mRNA expression of Lxn in CD133+ and CD133- miapaca-2 cells were detected by Western blotting and qRT-PCR, respectively. After CD133+ miapaca-2 cells were treated with Lxn in serum-free medium (SFM), cell proliferation was assayed with a Cell Counting Kit 8 (CCK-8) and apoptosis was analyzed by flow cytometry. The protein and mRNA expression levels of Bcl-2, bax, and c-myc were also analyzed. RESULTS: We successfully isolated CD133+ miapaca-2 cells that exhibited the capacity for self-renewal in SFM, a proliferation potential in DMEM supplemented with FBS, and high tumorigenicity in nude mice. Lxn protein and mRNA expression levels in CD133+ miapaca-2 cells were significantly lower than those in CD133- cells. Lxn-treated CD133+ miapaca-2 cells exhibited increased apoptosis and low proliferation activity, down-regulation of Bcl-2 and c-myc expression, and up-regulation of Bax expression in a dose-dependent manner. CONCLUSIONS: Lxn induces apoptosis and inhibits the proliferation of CD133+ miapaca-2 cells. These changes are associated with down-regulation of Bcl-2 and c-myc and up-regulation of Bax.

Artesunate inhibits the growth of gastric cancer cells through the mechanism of promoting oncosis both in vitro and in vivo.

This study aims to investigate the significance and mechanism of artesunate involved in suppressing the proliferation of gastric cancer in vitro and in vivo. In the in-vitro experiments, artesunate inhibited the growth of gastric cancer cell lines (SGC-7901, BGC-823, and AGS) with concentration-dependent activity, with no significant effect on GES-1 cells. BGC-823 cells treated with artesunate showed the typical morphologic features of oncosis rather than apoptosis. Meanwhile, we observed calcium overload, downregulation of vascular endothelial growth factor expression, and upregulation of calpain-2 expression in the artesunate-treated BGC-823 cells. In addition, the in-vivo study showed that artesunate produced a dose-dependent tumor regression in nude mice. The antitumor activity of 240 mg/kg artesunate was similar to that of 10 mg/kg docetaxel. Furthermore, compared with the control group, no significant difference was observed in the body weight of artesunate-treated nude mice other than docetaxel-treated nude mice. These observations show that artesunate has concentration-dependent inhibitory activities against gastric cancer in vitro and in vivo by promoting cell oncosis through an impact of calcium, vascular endothelial growth factor, and calpain-2 expression.

Clinical features, surgical treatment and long-term outcome in adult patients with Moyamoya disease in China.

BACKGROUND: Moyamoya disease (MMD) develops mostly in Asian countries including Japan, Korea, mainland China and Taiwan. However, there are few detailed demographic and clinical data about Chinese patients with MMD. Currently, the most effective treatment in adult patients with MMD is unknown. There have only been a few small case series reporting on encephaloduroarteriosynangiosis (EDAS) in an adult population. Here we describe the clinical features, surgical treatment and long-term outcome of adults with MMD treated at a single institution in China. METHODS: Our cohort included 470 adult patients with MMD. The demographic and clinical characteristics were obtained by retrospective chart review and long-term outcome was evaluated using the stroke status. The modified Rankin Scale (mRS) was used to determine the neurological functional outcome. Univariate and multivariate logistic regression analyses were performed to determine risk factors for postoperative morbidity and functional outcome. The risk of subsequent stroke was determined using the Kaplan-Meier method and Cox regression was used to determine risk factors for postoperative or subsequent strokes. RESULTS: The median age for the onset of symptoms was 36.8 (range, 18-59) years. The ratio of female to male patients was 1:1 (231/239). Familial occurrence of MMD was 2.3%. The most common initial symptom was a cerebral ischemic event. The incidence of postoperative ischemic events or hemorrhage was 5.9% (9.8% of patients). Older age at symptom onset, posterior cerebral artery (PCA) involvement and the presence of transient ischemic attack (TIA) were identified as predictors of adverse postoperative events. The Kaplan-Meier estimate stroke risk was 10.1% in the first 2 years, and the 5-year Kaplan-Meier risk of stroke was 13% after surgery for all patients treated with surgical revascularization. Older age at symptom onset, PCA involvement and the presence of TIA were identified as predictors of postoperative or subsequent strokes. Overall, 73.2% of patients had an independent life with no significant disability, with the strongest predictor being the preoperative mRS score. CONCLUSION: Clinical characteristics of adult MMD in China are different from those in other Asian countries. EDAS in adult patients with MMD carries a low risk, is effective at preventing future ischemic events and improves quality of life.

Lysine acetylation: enzymes, bromodomains and links to different diseases.

Lysine acetylation refers to transfer of the acetyl moiety from acetyl-CoA to the ε-amino group of a lysine residue on a protein. This has recently emerged as a major covalent modification and interplays with other modifications, such as phosphorylation, methylation, ubiquitination (addition of a small protein called ubiquitin) and SUMOylation [addition of a ubiquitin-like protein known as SUMO (small ubiquitin-related modifier)], to form multisite modification programmes for cellular regulation in diverse organisms. This modification is post-translational (i.e. after synthesis of a protein) and reversible, with its level being dynamically balanced by two groups of enzymes known as lysine acetyltransferases and deacetylases. The acetyltransferases belong to three major families, whereas deacetylases have been divided into the classical and sirtuin [Sir-tu-in, for Sir2 (silent information regulator 2)-like protein; named after the yeast protein Sir2] families. In addition to these enzymes, proteins containing the bromodomain, a protein module named after the fly protein Brahma (God of creation in Hindu), are relevant to lysine acetylation biology due to their ability to recognize acetyl-lysine-containing peptides. Importantly, recent studies have made intimate links between these three different groups of proteins to different pathological conditions. In this chapter, we provide a brief overview of these proteins and emphasize their direct links to related human diseases.

Histone acetyltransferases and deacetylases: molecular and clinical implications to gastrointestinal carcinogenesis.

Histone acetyltransferases and deacetylases are two groups of enzymes whose opposing activities govern the dynamic levels of reversible acetylation on specific lysine residues of histones and many other proteins. Gastrointestinal (GI) carcinogenesis is a major cause of morbidity and mortality worldwide. In addition to genetic and environmental factors, the role of epigenetic abnormalities such as aberrant histone acetylation has been recognized to be pivotal in regulating benign tumorigenesis and eventual malignant transformation. Here we provide an overview of histone acetylation, list the major groups of histone acetyltransferases and deacetylases, and cover in relatively more details the recent studies that suggest the links of these enzymes to GI carcinogenesis. As potential novel therapeutics for GI and other cancers, histone deacetylase inhibitors are also discussed.