Apple bZIP transcription factor MdbZIP44 regulates abscisic acid-promoted anthocyanin accumulation.

Phytohormone abscisic acid (ABA) induces anthocyanin biosynthesis; however, the underlying molecular mechanism is less known. In this study, we found that the apple MYB transcription factor MdMYB1 activated anthocyanin biosynthesis in response to ABA. Using a yeast screening technique, we isolated MdbZIP44, an ABA-induced bZIP transcription factor in apple, as a co-partner with MdMYB1. MdbZIP44 promoted anthocyanin accumulation in response to ABA by enhancing the binding of MdMYB1 to the promoters of downstream target genes. Furthermore, we identified MdBT2, a BTB protein, as an MdbZIP44-interacting protein. A series of molecular, biochemical, and genetic analysis suggested that MdBT2 degraded MdbZIP44 protein through the Ubiquitin-26S proteasome system, thus inhibiting MdbZIP44-modulated anthocyanin biosynthesis. Taken together, we reveal a novel working mechanism of MdbZIP44-mediated anthocyanin biosynthesis in response to ABA.

An apple NAC transcription factor enhances salt stress tolerance by modulating the ethylene response.

It is known that ethylene signaling is involved in the regulation of the salt stress response. However, the molecular mechanism of ethylene-regulated salt stress tolerance remains largely unclear. In this study, an apple NAM ATAF CUC transcription factor, MdNAC047, was isolated and functionally characterized to be involved in ethylene-modulated salt tolerance. MdNAC047 gene was significantly induced by salt treatment and its overexpression conferred increased tolerance to salt stress and facilitated the release of ethylene. Quantitative real-time-PCR analysis demonstrated that overexpression of MdNAC047 increased the expression of ethylene-responsive genes. Electrophoretic mobility shift assay, yeast one-hybrid and dual-luciferase assays suggested that MdNAC047 directly binds to the MdERF3 (ETHYLENE RESPONSE FACTOR) promoter and activates its transcription. In addition, genetic analysis assays indicated that MdNAC047 regulates ethylene production at least partially in an MdERF3-dependent pathway. Overall, we found a novel 'MdNAC047-MdERF3-ethylene-salt tolerance' regulatory pathway, which provide new insight into the link between ethylene and salt stress.

[Patterns of recurrence in patients with stage pT3N0M0 thoracic esophageal squamous cell carcinoma after two-field esophagectomy].

OBJECTIVE: To evaluate the patterns of recurrence and the related factors in patients with pT3N0M0 thoracic esophageal squamous cell carcinoma (ESCC) after two-field esophagectomy. METHODS: From Jan 2008 to Dec 2009, 208 patients with stage pT3N0M0(2002, UICC) thoracic ESCC were treated with two-field esophagectomy in our hospital. There were 138 males and 70 females, and the median age was 60 years old (range 33-78). There were 33 patients in the upper-, 134 in the middle-, and 41 in the lower-thoracic esophagus, with a median length of lesion of 5 cm. There were 32 patients with no-, 78 with mild- and 98 patients with severe adhesions at surgery. The median number of dissected lymph nodes was 9 (range 1-27). 98 patients were treated with surgery alone and 110 with postoperative adjuvant chemotherapy. The statistical analysis was conducted using SPSS 13.0 software. RESULTS: The follow-up was ended on July 2013. In the total group of 208 patients, the total recurrence rate was 41.8% (87/208). Among them, 52 patients had locoregional recurrence (LR), 15 had distant metastasis (DM) and 20 patients had both local recurrence and distant metastasis. 40.2% (35/87) of all recurrences were found within one year after operation, 67.8% (59/87) within 2 years, 86.2% (75/87) within 3 years, and 100% (87/87) within 4 years. The 1-, 3-, and 5-year progression-free survival (PFS) rate was 83.0%, 62.8% and 56.3%, respectively. The overall locoregional recurrence rate was 34.6% (72/208), among them, 9 cases had recurrence in the cervix (all were supraclavicular lymph node metastasis), 66 cases in the mediastinum and 4 cases had para-aortic lymph node metastasis. 83.3% (60/72) of the locoregional recurrence was located in the carinal region or upper area. The 1-, 3-, 5-year locoregional recurrence rate was 15.6%, 32.2%, and 36.8%, respectively, and the median time of recurrence was 15.5 months. The overall distant metastasis (DM) rate was 16.8% (35/208). The 1-, 3-, and 5-year DM rate was 4.4%, 15.3%, and 20.1%, respectively, and the median time of DM was 24 months. The most common site of DM was the lung and bone. The univariate analysis showed that age and tumor site were associated with PFS, tumor site and small lymph node in the mediastinum (diamter <1 cm) before surgery were related with LR (P<0.05 for all), and tumor site, histological differentiation and LR were related with distant metastasis after surgery (P<0.05). Multivariate analysis showed that the tumor site was an independent prognostic factor affecting the progression-free survival and locoregional recurrence (P<0.05), and histological differentiation and LR were independent factors associated with distant metastasis (P<0.05 for all). CONCLUSIONS: The recurrence rate is very high in patients with pT3N0M0 thoracic ESCC after surgery, and most of them occur within 3 years after operation. Locoregional recurrence occurs more frequently and shortly than distant metastasis, and most of LR is located in the carinal region or upper-mediastinum. LR rate in upper-thoracic ESCC is very high, therefore, postoperative radiotherapy (PORT) is strongly suggested. LR rate in middle thoracic ESCC is also rather high and PORT is suggested. LR occur much less in the lower-thoracic ESCC, thus, PORT is not suggested routinely. Patients with poorly differentiated ESCC and LR have a high rate of distant metastasis.

Cardiac tamponade, a rare complication of gastric cardia cancer resection after neoadjuvant chemotherapy combined with immunotherapy: a case report and literature review.

Transthoracic cardia resection is a technically well-established surgical procedure. However, acute cardiac tamponade in the early postoperative period is extremely rare. The occurrence is life-threatening to the patient. It also poses a great clinical challenge for perioperative management. To date, few cases of pericardial tamponade have been reported in gastric cancer resection performed after neoadjuvant chemotherapy combined with immunotherapy. We present the case of a 62-year-old woman who received neoadjuvant chemotherapy combined with immunotherapy before surgery, followed by transthoracic surgery. A life-threatening complication, pericardial tamponade, occurred in the early postoperative period. The successful outcome was achieved in through multidisciplinary collaboration.

Corrigendum: Cardiac tamponade, a rare complication of gastric cardia cancer resection after neoadjuvant chemotherapy combined with immunotherapy: a case report and literature review.

[This corrects the article DOI: 10.3389/fonc.2023.1189500.].

Comparison of neoadjuvant immunotherapy plus chemotherapy versus chemotherapy alone for patients with locally advanced esophageal squamous cell carcinoma: A propensity score matching.

Objectives: Clinical studies on immune checkpoint inhibitors (ICIs) combined with neoadjuvant chemotherapy (nCT) have been carried out for the resectable esophageal squamous cell carcinoma (ESCC). So far, few studies have compared the survival outcomes of nCT plus ICIs and nCT alone. This study aimed to compare the efficacy and safety of neoadjuvant ICIs combined with nCT versus nCT followed by esophagectomy for patients with resectable locally advanced ESCC. Methods: A retrospective analysis of ESCC patients underwent nCT or nCT combined with ICIs followed by esophagectomy (from March 2013 to April 2021) was performed. A 1:1 propensity score matching (PSM) with a caliper 0.01 was conducted to balance potential bias. Results: A total of 47 comparable pairs of ESCC patients receiving nCT and nCT combined with ICIs were selected for the final analysis. The tumor regression grade (TRG) 0 and pathologic complete response (pCR) rates in the nCT+ICIs group were significantly higher than those of the nCT group (21.7% vs. 4.5%, P=0.016; and 17.0% vs. 2.1%, P=0.035, respectively). The rate of nerve invasion was 4.3% in the nCT+ICIs group, significantly lower than 23.4% of the nCT group (P=0.007). The incidences of adverse events in the nCT+ICIs group were similar compared with the nCT group and there was no grade 5 toxicity in either group. The 1-, 2-year disease-free survival rates (DFS) were 95.7%, 80.7% and 76.1%, 63.8% in the two groups (P=0.001, and P=0.046, respectively). The 1-year OS was improved in the nCT+ICIs group, which was close to a statistical difference (95.7% vs. 84.8%, P=0.074). Local recurrence rate in the nCT+ICIs group was 6.4%, significantly lower than 21.3% of the nCT group (P=0.036), while there was no significant difference in the distant metastasis. Conclusions: Compared with nCT alone, neoadjuvant immunotherapy plus nCT for patients with locally advanced ESCC has an advantage in pathological response, and could improve DFS with a good safety and feasibility, while long term survival validation is still needed further.

BTB protein MdBT2 inhibits anthocyanin and proanthocyanidin biosynthesis by triggering MdMYB9 degradation in apple.

MdMYB9 is a positive regulator in the biosynthesis of anthocyanin and proanthocyanidin in apple. However, its posttranslational regulation is unclear. Here, we demonstrated that the BTB protein MdBT2 had a negative role in the biosynthesis of anthocyanin and proanthocyanidin. MdBT2 interacted with MdMYB9 and negatively regulated the abundance of MdMYB9 protein through the 26S proteasome system. The degradation of MdMYB9 by MdBT2 reduced the expression of MdMYB9-mediated anthocyanin and proanthocyanidin-related genes and reduced the accumulation of anthocyanin and proanthocyanidin, which functioned in an MdCUL3-independent pathway. Our results indicated that MdBT2 negatively regulated the stability of MdMYB9, which provides new insight into the homeostasis of anthocyanin and proanthocyanidin in apple.

MdHY5 positively regulates cold tolerance via CBF-dependent and CBF-independent pathways in apple.

Cold stress is a major external stimulator that affects crop quality and productivity. The CBF cold regulatory pathway has been regarded as a master regulator in the response to cold stress. In this study, we found that the apple bZIP transcription factor, MdHY5, was responsive to cold treatment both at the transcriptional and at the post-translational levels. Moreover, overexpression of MdHY5 enhanced cold tolerance in apple calli and Arabidopsis. Subsequently, EMSA assay and transient expression assay demonstrated that MdHY5 positively regulated the transcript of MdCBF1 by binding to G-Box motif of its promoter. Furthermore, MdHY5 also regulated the expression of CBF-independent cold-regulated genes. Taken together, our data suggest that MdHY5 positively modulates plant cold tolerance through CBF-dependent and CBF-independent pathways, providing a deeper understanding of MdHY5-regulated cold tolerance in apple.

The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple.

The basic leucine zipper (bZIP) transcription factor HY5 plays a multifaceted role in plant growth and development. Here the apple MdHY5 gene was cloned based on its homology with Arabidopsis HY5. Expression analysis demonstrated that MdHY5 transcription was induced by light and abscisic acid treatments. Electrophoretic mobility shift assays and transient expression assays subsequently showed that MdHY5 positively regulated both its own transcription and that of MdMYB10 by binding to E-box and G-box motifs, respectively. Furthermore, we obtained transgenic apple calli that overexpressed the MdHY5 gene, and apple calli coloration assays showed that MdHY5 promoted anthocyanin accumulation by regulating expression of the MdMYB10 gene and downstream anthocyanin biosynthesis genes. In addition, the transcript levels of a series of nitrate reductase genes and nitrate uptake genes in both wild-type and transgenic apple calli were detected. In association with increased nitrate reductase activities and nitrate contents, the results indicated that MdHY5 might be an important regulator in nutrient assimilation. Taken together, these results indicate that MdHY5 plays a vital role in anthocyanin accumulation and nitrate assimilation in apple.

Erratum: The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple.

[This corrects the article DOI: 10.1038/hortres.2017.23.].

Detection of cancer specific mutations in early-stage non-small cell lung cancer using cell-free DNA by targeted sequencing.

Non-small cell lung cancer (NSCLC) is a major public health problem worldwide and leads to a high mortality. NSCLC is always diagnosed in late stages because of its unapparent symptoms. However, cell-free DNA (cfDNA) may serve as a new potential biomarker to detect early stage of non­small cell lung cancer. Here we recruited 10 non-small cell lung cancer patients to obtain fresh tumor tissue, peripheral blood lymphocytes (PBLs), and plasma. CfDNAs from 13 elderly people and 7 middle-age smokers were also extracted as controls. Illumina HiSeq X10 was used to perform next-generation sequencing to evaluate differences in mutations among different samples. The result indicated that tumor DNA and its matched plasma cfDNA samples showed high concordance in their mutation patterns. Mutation rate of cfDNA was generally lower than that of tumor tissue and higher than that of PBLs. The plasma cfDNA concentration of NSCLC patients (69.2±46.9 ng/ml) is significantly higher than that of elderly people (32.5±5.2 ng/ml, t=2.96, p=0.007) and middle-aged smokers (17.9±9.1 ng/ml, t=2.83, p=0.013). Five mutations (PTEN_c.1375A>G, TP53_c.94G>A, STK11_c.816C>T, PIK3CA_c.1633 G>A, PIK3CA_c.2038G>C) were only identified in NSCLC patients but not in healthy people. Our conclusion was that cfDNA has a similar mutation pattern with its matched tumor tissue DNA. A high concentration of cfDNA and tumor specific mutations in cfDNA may serve as potential non-invasive biomarkers to detect early-stage non­small cell lung cancer.

Factors on prognosis in patients of stage pT3N0M0 thoracic esophageal squamous cell carcinoma after two-field esophagectomy.

AIMS: To evaluate prognosis and its related factors in patients of pT3N0M0 thoracic esophageal squamous cell carcinoma (ESCC). SUBJECTS AND METHODS: From January 2008 to December 2009, 249 patients of stage T3N0M0 thoracic ESCC were treated with two-field esophagectomy. Among them, 171 males and 78 females; the median age was 60 years old (range 33-78 years). Site of lesion: 39 in upper-, 166 in middle-, and 44 patients in lower-thoracic esophagus; the median length of lesion was 5 cm (range 2-12 cm); the degree of adhesion during surgery: No adhesions in 35, mild adhesions in 90, and severe adhesions in 124 patients; the median number of dissected lymph node (LN) was 9 (range 1-27). Ninety-eight patients are treated with surgery alone, 151 patients with postoperative adjuvant treatment (20 with radiotherapy, 110 with chemotherapy, and 21 patients with radiotherapy and chemotherapy). Statistical analysis was performed using SPSS 13.0 statistical software. RESULTS: Follow-up deadline was July 2013. The 1-, 3-, 5-year overall survival (OS) were 90.0%, 68.7% and 55.2%; Univariate analysis identified gender, location of lesion, differentiation of pathology and hemoglobin (Hb) levels were prognostic factors for OS (P < 0.05); multivariate analysis showed, location of lesion, Hb levels, mediastinal small LN (<1 cm in diameter) in computed tomography (CT) image before surgery, and number of removed LN were independent prognostic factors for OS. The 1-, 3-, 5-year progress-free survival (PFS) were 82.1%, 61.7%, and 53.9%; univariate analysis identified, age, site of lesion and degree of adhesion during surgery were associated with PFS (P < 0.05); multivariate analysis showed, site of lesion was the only independent prognostic factor. Postoperative adjuvant therapy did not effect on OS and PFS. CONCLUSION: For patients of pT3N0M0 thoracic ESCC, the independent factors were the site of a lesion for OS and PFS, Hb levels, small LN in CT, and number of removed LN for OS. The value of postoperative adjuvant therapy need be further proved.

[The role of transbronchial needle aspiration in the staging of bronchogenic carcinoma].

BACKGROUND: To explore the role of transbronchial needle aspiration (TBNA) in the staging of bronchogenic carcinoma. METHODS: To 42 cases of primary bronchogenic carcinoma with suspected lymph node metastasis by X-ray and CT scan of chest, the TBNA was performed before operation. The cytological results and c-TNM by TBNA were compared with the pathological ones and p-TNM after operation. RESULTS: The diagnosis of 10 cases with N2 metastasis was completely corresponding by TBNA and pathological examination after operation. Nineteen out of 22 cases with N1 metastasis were confirmed by TBNA, and the false negative results ocurred in 3 cases. The results of TBNA in lymph nodes' size from 2 to 3 cm was completely accordant with pathological ones after operation. For 1 to 2 cm lymphnodes, the accurate rate of TBNA was 88.5% (23/26). The overall accurate rate of c-TNM by TBNA was 85.7% (36/42) compared with p-TNM. A small amount of hemoptysis ocurred in 3 cases, no pneumothorax and other serious complications were observed. CONCLUSIONS: The TBNA for staging of bronchogenic carcinoma is a simple and economic method with high correct rate and high clinical applicable value.