Enhanced Detection of Early Pulmonary Fibrosis Disease Using 68Ga-FAPI-LM3 PET.

Early detection of pulmonary fibrosis is a critical yet insufficiently met clinical necessity. This study evaluated the effectiveness of FAPI-LM3, a 68Ga-radiolabeled heterobivalent molecular probe that targets fibroblast activating protein (FAP) and somatostatin receptor 2 (SSTR2), in the early detection of pulmonary fibrosis, leveraging its potential for early disease identification. A bleomycin-induced early pulmonary fibrosis model was established in C57BL/6 mice for 7 days. FAP and SSTR2 expression levels were quantitatively assessed in human idiopathic pulmonary fibrosis lung tissue samples and bleomycin-treated mouse lung tissues by using western blotting, real-time quantitative PCR (RT-qPCR), and immunofluorescence techniques. The diagnostic performance of FAPI-LM3 was investigated by synthesizing monomeric radiotracers 68Ga-FAPI-46 and 68Ga-DOTA-LM3 alongside the heterobivalent probe 68Ga-FAPI-LM3. These imaging radiopharmaceuticals were used in small-animal PET to compare their uptake in fibrotic and normal lung tissues. Results indicated significant upregulation of FAP and SSTR2 at both RNA and protein levels in fibrotic lung tissues compared with that in normal controls. PET imaging demonstrated significantly enhanced uptake of the 68Ga-FAPI-LM3 probe in fibrotic lung tissues, with superior visual effects compared to monomeric tracers. At 60 min postinjection, early stage fibrotic tissues (day 7) demonstrated low-to-medium uptake of monomeric probes, including 68Ga-DOTA-LM3 (0.45 ± 0.04% ID/g) and 68Ga-FAPI-46 (0.78 ± 0.09% ID/g), whereas the uptake of the heterobivalent probe 68Ga-FAPI-LM3 (1.90 ± 0.10% ID/g) was significantly higher in fibrotic lesions than in normal lung tissue. Blockade experiments confirmed the specificity of 68Ga-FAPI-LM3 uptake, which was attributed to synergistic targeting of FAP and SSTR2. This study demonstrates the potential of 68Ga-FAPI-LM3 for early pulmonary fibrosis detection via molecular imaging, offering significant benefits over monomeric tracers 68Ga-FAPI-46 and 68Ga-DOTA-LM3. This strategy offers new possibilities for noninvasive and precise early detection of pulmonary fibrosis.

Evaluation of the role of postoperative radiotherapy in locally invasive thymoma: A propensity-matched study based on the SEER database.

OBJECTIVES: No consensus was reached on the efficacy of postoperative radiotherapy (PORT) in locally invasive thymomas because of the rarity of the thymic epithelial and the variations of study results. Therefore, we aimed to explore the efficacy of PORT in locally invasive thymomas using the Surveillance, Epidemiology, and End Results (SEER) database. METHODS: Patients diagnosed with thymomas from 2004 to 2016 were identified using the SEER database. Prognostic factors of cancer-specific survival (CSS) and overall survival (OS) were identified using univariate and multivariate Cox regression analyses.Propensity score matching (PSM) was performed to balance the baseline characteristics. RESULTS: A total of 700 eligible patients were identified. After PSM, 262 paired patients were selected from the two groups, those who received or did not receive PORT. Receiving PORT improved CSS and OS before and after PSM. In the matched population, the multivariate analyses showed that tumour invasion into adjacent organs/structures and non-utilisation of PORT were independent poor prognostic factors for CSS, whereas age ≥62 years,tumour invasion into adjacent organs/structures, and non-utilisation of PORT were independently associated with poorer OS. The subgroup analysis revealed that PORT improved CSS and OS in Masaoka-Koga stage III thymoma, but showed no OS benefit in Masaoka-Koga stage IIB thymoma. CONCLUSION: Based on the SEER database, we found that PORT provides a significant survival benefit in Masaoka-Koga stage III thymoma with complete or incomplete resection. The role of PORT in thymoma requires further evaluation.

B-box containing protein 1 from Malus domestica (MdBBX1) is involved in the abiotic stress response.

B-box proteins (BBXs), which act as transcription factors, mainly regulate photomorphogenesis. However, the molecular functions underlying the activity of plant BBXs in response to abiotic stress remain largely unclear. In this investigation, we found that a BBX from Malus domestica (MdBBX1) was involved in the response to various abiotic stresses. The expression of MdBBX1 was significantly upregulated in response to abiotic stresses and abscisic acid (ABA). Recombinant MdBBX1 increased stress tolerance in Escherichia coli cells. In addition, overexpression of MdBBX1 in Arabidopsis decreased sensitivity to exogenous ABA, resulting in a germination rate and root length that were greater and longer, respectively, than those of wild-type (WT) plants. Moreover, the expression of ABI5 was decreased in MdBBX1-overexpressing lines under ABA treatment. After salt and drought treatments, compared with the WT plants, the MdBBX1 transgenic plants displayed enhanced tolerance and had a higher survival rate. Furthermore, under salt stress, increased proline (PRO) contents, decreased levels of malondialdehyde (MDA), increased activity of antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX)) and decreased accumulation of reactive oxygen species (ROS) were observed in the MdBBX1-overexpressing plants. Overall, our results provide evidence that MdBBX1 might play a critical role in the regulation of abiotic stress tolerance by reducing the generation of ROS.

Pineal Yolk Sac Tumor Producing α-Fetoprotein Detected by 68Ga-FAPI PET/MRI.

ABSTRACT: Pineal yolk sac tumors (YSTs) are a rare type of extragonadal YST. They make up a small fraction of all intracranial germ cell tumors and an even small fraction of pineal masses overall. This study reported a case of pineal YST with α-fetoprotein production revealed by 18F-FDG and 68Ga-FAPI PET/MRI. In the PET images, 68Ga-FAPI showed a far better tumor-to-background ratio than 18F-FDG in the pineal YST because there is little 68Ga-FAPI uptake in the brain. This case indicates that 68Ga-FAPI PET/MRI may be a useful tool for evaluating intracranial YST and other types of tumors in central nervous system.

68Ga-fibroblast activation protein inhibitor PET/CT on gross tumour volume delineation for radiotherapy planning of oesophageal cancer.

BACKGROUND AND PURPOSE: To compare 68Ga-fibroblast activation protein inhibitor (FAPI) and 18F-FDG PET/CT in imaging locally advanced oesophageal cancer, and evaluate the potential usefulness of 68Ga-FAPI PET/CT on gross target volume (GTV) delineation aimed at radiotherapy planning for oesophageal cancer as compared with contrast-enhanced CT (CE-CT) and 18F-FDG PET/CT. MATERIALS AND METHODS: Twenty-one patients with newly diagnosed oesophageal cancer who underwent both 18F-FDG and 68Ga-FAPI PET/CT scans were selected. GTVs of the primary tumours based on CE-CT (GTVCT), PET/CT, and CE-CT plus PET/CT were delineated. Gross tumour lengths were measured by GTVs and endoscopy and recorded. RESULTS: The 68Ga-FAPI PET showed significantly higher radiotracer uptake than 18F-FDG PET (median SUVmax 16.71 vs. 11.23; P = 0.002) in the primary tumours. SUV thresholds of FAPI ×20%, 30%, 40%, and FDG ×40% showed similar lesion lengths compared with that in endoscopic examination (P > 0.05). GTVCT demonstrated the largest volume (median: 48.80 mm3, range: 14.83-162.23 mm3) than PET-based GTVs. For PET/CT-guided complementary contouring of GTVCT, four patients (19%) were increased by FAPI ×20% and 30%, two patients (9.5%) were increased by FAPI ×40%, and only one patient was increased by FDG ×40%. Furthermore, the volume of GTV based on CE-CT plus FAPI ×20%, 30%, and 40% showed no significant difference with GTVCT and planning target volume based CE-CT plus FAPI-PET and meets the organ at risk standard. CONCLUSION: The 68Ga-FAPI PET/CT methodology showed favourable tumour-to-background contrast in oesophageal cancer and might provide additional information for target volume delineation and help avoid tumour geographic misses.

Delayed postoperative radiotherapy might improve the long-term prognosis of locally advanced esophageal squamous cell carcinoma.

OBJECTIVE: There is no consensus on the optimal timing of postoperative radiotherapy (PORT) for locally advanced esophageal squamous cell carcinoma (ESCC). We aimed to determine whether the timing of PORT affects the long-term prognosis of ESCC, and plotted nomograms to predict survival. METHODS: We retrospectively analyzed 351 ESCC patients who underwent radical surgery and PORT. Receiver operating characteristic curves were used to estimate the optimal cutoff point of the time interval between surgery and PORT. Cox proportional hazards regression was used to identify prognostic predictors. Overall survival (OS) and progression-free survival (PFS) were predicted using nomograms. RESULTS: The median follow-up was 53 months (range: 3-179 months). Compared to early PORT, PORT at >48 days after surgery was associated with better OS (adjusted hazard ratio [HR]: 1.406, p = 0.037) and PFS (adjusted HR: 1.475, p = 0.018). In the chemotherapy subgroup, incorporation of chemotherapy timing into the analysis suggested that 2-4 chemotherapy cycles followed by PORT was the optimal treatment schedule as compared to 0-1 chemotherapy cycle followed by PORT and concurrent chemoradiotherapy (5-year PFS: 65.9% vs. 51.0% vs. 50.1%; p = 0.049). The nomograms for OS and PFS were superior to the TNM classification (concordance indices: 0.721 vs. 0.626 and 0.716 vs. 0.610, respectively). CONCLUSIONS: Delayed PORT (>48 days) provides better survival benefit than early PORT among ESCC patients. PORT following 2-4 chemotherapy cycles might lead to the best survival rate. The nomogram plotted in this study effectively predicted survival and may help guide treatment.

Usefulness of [68Ga]Ga-DOTA-FAPI-04 PET/CT in patients presenting with inconclusive [18F]FDG PET/CT findings.

PURPOSE: This prospective study aimed to evaluate the potential usefulness of [68Ga]Ga-DOTA-FAPI-04 positron emission tomography/computed tomography (PET/CT) in the oncological evaluation of patients presenting with inconclusive [18F]FDG PET/CT findings. METHODS: [68Ga]Ga-DOTA-FAPI-04 was performed in patients presenting with inconclusive [18F]FDG PET/CT findings. Tumour uptake was quantified by the maximum standard uptake value (SUV). Histopathology or follow-up imaging served as the standard for the final diagnosis. RESULTS: A total of 68 patients with inconclusive [18F]FDG PET/CT findings underwent additional [68Ga]Ga-DOTA-FAPI-04 PET/CT. Of them, 18 (26.5%) were for discrimination of mass lesions detected on conventional imaging, 6 (8.8%) for detection of the unknown primary site in biopsy-proven metastatic malignancy, 21 (30.9%) for the staging of cancer, and the other 23 (33.8%) for evaluation of suspected disease recurrence. Most of the primary and metastatic lesions demonstrated higher uptake of [68Ga]Ga-DOTA-FAPI-04 than did [18F]FDG, which resulted in favourable tumour-to-background contrast in various types of cancer. As a result, [68Ga]Ga-DOTA-FAPI-04 PET/CT identified suspicious mass lesions with an accuracy of 12/18 (66.7%), detected the primary site in 4/6 patients (66.7%) with unknown malignancy, upgraded tumour staging in 7/21 patients (33.3%), and detected disease recurrence in 20/23 patients (87.0%). CONCLUSIONS: In patients undergoing oncological evaluation with inconclusive [18F]FDG PET/CT findings, [68Ga]Ga-DOTA-FAPI-04 may have a complementary role in discriminating mass lesions on conventional imaging, locating the primary site of unknown malignancy, modifying tumour staging, and detecting suspected disease recurrence. Nevertheless, careful attention should be paid when reading the [68Ga]Ga-DOTA-FAPI-04 PET/CT images in tumours complicated with inflammation.

Impact of Extracapsular Lymph Node Involving the Esophagus in Esophageal Perforation During and After Radiotherapy: A Propensity Score-Matched Analysis.

BACKGROUND: This study aimed to analyze the risk factors for esophageal squamous cell carcinoma (ESCC), especially extracapsular lymph node involving the esophagus (ECLNIE), occurring during or after radiotherapy (RT) in patients with esophageal perforation (EP). METHODS: In total, 306 patients with ESCC who received RT and/or chemotherapy between January 2016 and December 2017 in our hospital and who met the inclusion criteria of the study were recruited. The continuous variables were converted into classification variables using the receiver operating characteristic curve or common clinical parameters. Risk factors for EP were examined by univariable analysis using the chi-square test or Fisher's exact and by multivariable analysis using logistic regression model. Propensity score matching (PSM) was used to compensate for the differences in baseline characteristics, and the incidence of EP was compared after matching. RESULTS: EP was observed in 26 patients (incidence rate, 8.5%). Univariable analysis revealed that age, BMI, T4 stage, tumor length, esophageal wall thickness, ECLNIE, necrotic areas, niche sign by esophagogram before RT, neutrophil-to-lymphocyte ratio, and prognostic nutritional index were significantly associated with EP among patients with ESCC who received radiotherapy. Multivariable analysis demonstrated that age, ECLNIE, esophageal wall thickness, and niche sign by esophagogram before RT were independent risk factors for EP. After PSM, compared with patients without ECLNIE, patients with ESCC and ECLNIE had a significantly higher risk of EP. CONCLUSION: The presence of ECLNIE could be a strong risk factor of EP during and after RT.

Prognostic factors and treatment comparison in small cell neuroendocrine carcinoma of the uterine cervix based on population analyses.

OBJECTIVE: We aimed to assess the impact of the treatment modality on the outcome of small cell neuroendocrine cervical carcinoma (SCNEC) using the Surveillance Epidemiology and End Results (SEER) database. METHODS: Patients from the SEER program between 1981 and 2014 were identified. Significant factors for cancer-specific survival (CSS) and overall survival (OS) were analyzed using the Kaplan-Meier survival and Cox regression methods. RESULTS: A total of 503 SCNEC patients were identified. The 5-year CSS and OS were 36.6% and 30.6%, respectively. The International Federation of Gynecology and Obstetrics (FIGO) stage I to IV distributions was 189 (37.6%), 108 (21.5%), 95 (18.9%), and 111 patients (22.0%), respectively. Within the patients with known treatment strategies, 177 (45.9%) were treated with radical surgery and 209 (54.1%) underwent primary radiotherapy. Local treatment strategies were independent prognostic factor for CSS and OS. The 5-year CSS for radical surgery and primary radiotherapy was 50.0% and 27.9%, respectively (P < .001). The 5-year OS for those who received radical surgery and primary radiotherapy was 57.8%, and 29.6%, respectively (P < .001). In FIGO stage I SCNEC, patients treated with radical surgery had superior CSS (P = .001) and OS (P = .003) than those with primary radiotherapy. However, in FIGO stage II and III SCNEC, there were no differences in CSS and OS with respect to different local treatment strategies. Our results also found that the addition of brachytherapy impacted OS in the FIGO stage III SENCE (P = .002). The 5-year CSS and OS of patients with FIGO IV were only 11.7% and 7.1%, respectively. CONCLUSIONS: SCNEC is a rare disease with aggressive clinical behavior. The findings indicate that radical surgery should be suggested for early-stage SCNEC and combining radiation therapy with brachytherapy should be suitable for patients with advanced stage.

Concordance of PD-L1 Status Between Image-Guided Percutaneous Biopsies and Matched Surgical Specimen in Non-Small Cell Lung Cancer.

OBJECTIVE: Programmed death-ligand 1 (PD-L1) expression status is a crucial index for identifying patients who will benefit from anti-programmed cell death protein 1 (PD-1)/PD-L1 therapy for non-small cell lung cancer (NSCLC). However, the concordance of Tumor Proportion Score (TPS) between biopsies and matched surgical specimens remains controversial. This study aims to evaluate the concordance of PD-L1 expression between image-guided percutaneous biopsies and matched surgical specimens. METHOD: We evaluated 157 patients diagnosed with operable NSCLC on both surgical tissue sections and matched lung biopsies retrospectively. The patients underwent either regular computed tomography (CT)-guided biopsy (n = 82) or positron emission tomography (PET)/CT-guided biopsy (n = 75). The concordance between surgical specimens and lung biopsies for PD-L1 TPS was evaluated using Cohen's kappa (κ) coefficient. RESULTS: Immunohistochemical expression of PD-L1 was evaluated in both surgical resected specimens and matched biopsies in the eligible 138 patients. The concordance rate of PD-L1 expression between surgical tissue sections and matched biopsies was fairly high at 84.1% (116/138), and the κ value was 0.73 (95% CI: 0.63-0.83, P < 0.001). The concordance rate was higher for tissue sections from PET/CT-guided biopsy than for tissue sections from CT-guided biopsy [88.6% (62/70, κ value: 0.81) vs 79.4% (54/68, κ value: 0.66)]. CONCLUSION: PD-L1 TPS was strongly concordant between surgical specimens and matched lung biopsies. Thus, the routine evaluation of PD-L1 expression in diagnostic percutaneous biopsies could be reliable for identifying patients who will benefit from anti-PD-1/PD-L1 immunotherapy.

Postoperative chemoradiotherapy is superior to postoperative chemotherapy alone in squamous cell lung cancer patients with limited N2 lymph node metastasis.

BACKGROUND: The aim of the present study was to assess the efficacy of postoperative chemoradiotherapy (POCRT) following surgery in non-small-cell lung cancer patients with N2 lymph node metastasis (N2-NSCLC). METHODS: The clinical data of patients with N2-NSCLC treated with POCRT or postoperative chemotherapy (pCT) alone were retrospectively collected and reviewed. The overall survival (OS) rates were analyzed utilizing the Kaplan-Meier method and compared by the log-rank test. Cox regression analysis was used to determine factors significantly associated with survival. Propensity score matching (PSM) analysis was used to compensate for differences in baseline characteristics and OS was compared after matching. RESULTS: Between 2004 and 2014, a total of 175 patients fulfilled the inclusion criteria, 60 of whom were treated with POCRT, while 115 were administered pCT. The 1, 3 and 5-year OS rates in the POCRT and pCT groups were 98.3 vs. 86.1%, 71.7 vs. 53.0% and 45.7 vs. 39.0%, respectively (P = 0.019). Compared with pCT, POCRT improved OS in patients with squamous cell subtype (P = 0.010), no lymphovascular invasion (P = 0.006), pN2a (P = 0.006) or total number of metastatic lymph nodes ≤7 (P = 0.016). After PSM, these survival differences between POCRT and pCT remained significant in patients with squamous cell lung cancer (P = 0.010). CONCLUSIONS: POCRT following complete resection may be beneficial for patients with squamous cell lung cancer, particularly those with limited nodal involvement.

The long-term impact of tumor burden in pT3N0M0 esophageal squamous cell carcinoma: A propensity score-matched analysis.

The purpose of this study was to assess the impact of tumor burden on the survival of patients with pathologic T3N0M0 (pT3N0M0) esophageal squamous cell carcinoma (ESCC).A total of 84 patients with pathologic T3N0M0 ESCC treated with radical esophagectomy and 3-field lymphadenectomy (3-FL) from January 2008 to December 2009 in our center were analyzed. Receiver-operating characteristic (ROC) curve analysis was performed to calculate the optimal cutoff value. The Kaplan-Meier method and log-rank test were used to assess the overall survival (OS) differences between groups. A regression model was applied to identify prognostic factors for OS. Propensity score matching (PSM) was performed to adjust for the imbalance and indication biases in the 2 groups.The median follow-up time was 62 months (range, 1-84 months), and the 5-year OS rate was 62% (95% confidence interval, 52.2-71.8%). According to the ROC curve analysis, the optimal cutoff values for the maximal esophageal wall thickness, tumor length, and tumor volume were 1.3 cm, 5.9 cm, and 18.6 cc, respectively. Univariate analysis revealed that maximal esophageal wall thickness >1.3 cm (P = .014), tumor volume >18.6 cc (P < .001), and vascular invasion (P < .001) were significantly associated with OS. The multivariate Cox regression model identified tumor volume and vascular invasion as factors affecting OS. After propensity matching, patients with a tumor volume ≤18.6 cc had a better OS than those with a tumor volume >18.6 cc (5-year OS, 85% vs 50%, P = .008).Tumor volume may serve as a good prognostic factor for patients with pT3N0M0 ESCC treated with radical esophagectomy and 3-FL. Larger-scale studies are warranted to validate these findings.

Preoperative systemic inflammation score (SIS) is superior to neutrophil to lymphocyte ratio (NLR) as a predicting indicator in patients with esophageal squamous cell carcinoma.

BACKGROUND: The aim of this study was to assess the prognostic significance of preoperative systemic inflammation score (SIS) on patients with esophageal squamous cell carcinoma (ESCC). METHODS: A total of 357 ESCC patients who accepted radical esophagectomy between January 2008 and December 2009 at our institution were recruited in the analysis. The cut-off finder application was used to calculate the optimal cutoff values. The Chi-squared test or Fisher's exact test were used to analyze categorical variables. Overall survival (OS) was calculated using the Kaplan-Meier method and the log-rank test. Multivariate analysis was calculated using Cox regression analysis model. A model combining SIS was created and its performance was evaluated using the Akaike information criterion (AIC) and concordance index (C-index). RESULTS: The median follow-up time was 58 months (range, 1-84 months). The 5-year OS rate was 50% (95% CI, 49.94-50.06%). The optimal cut-off values for preoperative neutrophil to lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR) and serum albumin (Alb) were 2.27, 3.79 and 36.55, respectively. Univariate analyses revealed that gender (P = 0.047), T stage (P < 0.001), N stage (P < 0.001), vascular invasion (P < 0.001), tumor location (P = 0.018), tumor length(P < 0.001), NLR (P = 0.006), LMR (P = 0.007), serum Alb (P = 0.001), and SIS (P < 0.001) were significantly associated with OS. Independent prognostic factors for OS were T stage, N stage, tumor location, tumor length, and SIS. However, NLR was not an independent prognostic factor in multivariate analysis. The model combining SIS had smaller AIC and higher C-index compared to the model without SIS, which suggesting that the adding the SIS to the multivariate model increasing the predictive accuracy of the OS in the ESCC patients treated with radical esophagectomy and 3-field lymphadenectomy (3-FL). CONCLUSIONS: SIS may treat as a novel prognostic factor than NLR for ESCC patients who underwent radical esophagectomy and 3-FL. However, Larger-scale studies are needed to validate these findings.

Long-term impact of prognostic nutritional index in cervical esophageal squamous cell carcinoma patients undergoing definitive radiotherapy.

BACKGROUND: The prognostic nutritional index (PNI) has been found to have prognostic value in several cancers, and we aimed to determine its predictive value for the long-term prognosis of cervical esophageal squamous cell carcinoma (CESCC) patients treated with chemoradiotherapy (CRT). METHODS: The data for 106 CESCC patients who received radiotherapy with or without chemotherapy at the Cancer Hospital of Fujian Medical University from June 1, 2000 to December 31, 2015 were retrospectively analyzed. According to serum measurements taken before therapy, the PNI was calculated as albumin (g/L) + 5 × total lymphocyte count. The association between PNI and overall survival (OS) was determined by the Kaplan-Meier method and Cox proportional regression model analysis. RESULTS: The median follow-up time was 19 months. The optimal cutoff value for PNI was calculated to be 48.15, and patients were divided into a low PNI group (<48.15) and high PNI group (≥48.15). Univariate analysis showed that a low survival rate was significantly associated with male gender (P=0.004), tumor length ≥5.0 cm (P=0.043), radiotherapy technique (P=0.016), synchronous chemotherapy (P=0.012), lymphocyte-monocyte ratio (LMR) (P=0.007), neutrophil-lymphocyte ratio (NLR) (P=0.007), lung cancer inflammation index (ALI) (P=0.008), cervical esophageal carcinoma inflammation index (CEI) (P=0.043), and PNI (P<0.001). The OS was higher in the high PNI group than in the low PNI group. On multivariate analysis, gender (P=0.004), radiotherapy technique (P=0.029), and PNI (P=0.007) were independent prognostic factors in CESCC treated with CRT. CONCLUSIONS: The PNI value is a simple, reliable, and reproducible indicator for improving the accuracy of patient prognosis. And larger-scale studies are warranted to validate these findings.

Members of B-box Protein Family from Malus domestica Enhanced Abiotic Stresses Tolerance in Escherichia coli.

The B-box proteins (BBXs) are zinc finger proteins containing one or two B-box domain(s) and involved in regulation of development processes as transcription factors in plants. Here, seven BBX genes in Malus domestica genome (MdBBXs) were identified and found to be up-regulated under abiotic stresses, with 2-12 folds in roots. All recombinant MdBBXs expressed in Escherichia coli (E. coli) enhanced the cell's tolerance to salt and osmotic stresses, respectively. Deficiency of B-box domain of MdBBX10 led to the loss of anti-stress functions. Five conservative cysteines in B-box domain played crucial roles in stress resistance, which are involved in two of metal iron binding sites of zinc finger motifs in BBXs. All the above results suggested MdBBXs confer stress tolerance to E. coli cell against abiotic stresses.

Genomewide analysis of homeobox gene family in apple (Malus domestica Borkh.) and their response to abiotic stress.

Homeobox proteins (HOXs) comprise a large family in eukaryotes and share a highly conserved DNA-binding motif, the homeodomain (HD). HOXs play an important role in the regulation of plant growth, development and stress response. However, systematic analysis and expression profiling of these genes have not been reported in Malus domestica. In this study, a total of 207 HOXs of M. domestica (MdHOXs) were identified and classified into 11 distinct subfamilies, and an unclassified group according to their functional domains. The MdHOXs were localized in all 17 chromosomes with various densities and a majority of them tended to formgene clusters. Analysis of the Ka/Ks ratios suggested that the duplicated genes of MdHOXs mainly underwent purifying selection with restrictive functional divergence after the duplication events. The expression of MdHOXs has organ specific characteristics and were divided into seven different groups. Stress-related cis-acting elements were prevalent in the upstream sequence of MdHOXs by systematic analysis. To explore the response to abiotic stress, eight MdHOXs were randomly selected to investigate their expression using quantitative real-time polymerase chain reaction. Transcription levels of MdHOXs were upregulated in the leaves and roots under cold, osmotic, high salinity or exogenous ABA treatments, which suggested that they may take part in the plant response to abiotic stress. These results provided basic information of HOXs in apple and will further contribute to the functional research of MdHOXs, especially the response to abiotic stress.

A B-box zinc finger protein, MdBBX10, enhanced salt and drought stresses tolerance in Arabidopsis.

KEY MESSAGE: The expression of MdBBX10 was significantly induced by different stresses and ABA treatments. Overexpression of MdBBX10 in Arabidopsis significantly enhanced abiotic stresses tolerance by ABA signalling. The roles of B-box domain(s) containing proteins (BBXs) in regulation of flowering and light morphogenesis of plants were intensively studied. However, the roles of plant BBXs in abiotic stresses are poorly understood. A B-box protein encoding gene from apple (MdBBX10) was found to be up-regulated from gene expression profile under salt stress. qRT-PCR analysis indicated that the expression of MdBBX10 was significantly induced by different stresses and exogenous abscisic acid (ABA) in apple roots and leaves. The ß-glucuronidase activity driven by the promoter of MdBBX10 was also strongly induced by NaCl, H2O2, polyethylene glycol and exogenous ABA, which was consistent to the existence of rich cis-acting elements related to the abiotic stresses in the promoter sequence. Over-expression of MdBBX10 in Arabidopsis significantly enhanced tolerance to abiotic stresses, with higher germination ratio and longer length of roots than the wild type plants. Transgenic plants of over-expressing MdBBX10 lines were more sensitive to exogenous ABA than the wild type plants. Under abiotic stress treatments, the transcript levels of ABA- and stress-related genes were higher in MdBBX10-overexpressing plants than wild type plants. Over-expression of MdBBX10 could enhance plant's ability to scavenge reactive oxygen species (ROS) under stresses, which is correlated with the expression of ROS-scavenging genes. These results provided the evidences that MdBBX10 plays an important role in enhanced plant tolerance to abiotic stresses, which were involved in ABA-mediated response and ROS response.

Identification and Functional Analysis of a Protein Disulfide Isomerase (AtPDI1) in Arabidopsis thaliana.

Protein disulfide isomerase (PDI) catalyzes the conversion of thiol-disulfide and plays an important role in various physiological events in animals. A PDI (OaPDI) from a tropical plant was detailed studied and it was found to be involved in response of biotic stress (Gruber et al., 2007). However, the activities of PDI related to physiological functions in plants are poorly understood. In the present study, a homolog of human PDI in Arabidopsis (AtPDI1), encoded by the gene (At3g54960), was characterized. The recombinant AtPDI1 protein had disulfide isomerase activity in vitro and two pairs of conservative cysteines in catalytic domains play a crucial role in the PDI activities. Expression of AtPDI1 in Escherichia coli significantly enhanced stress tolerance of cells and the mutations of critical cysteines almost lose this function. In plants, AtPDI1 was strongly induced by abiotic stresses and exogenous abscisic acid. An ArabidopsisAtPDI1 knockdown mutant (pdi1) and overexpression lines of transgenic plants obtained by this investigation were used to further examine the function of AtPDI1. The mutant line was more sensitive to stresses than the wild-type, while overexpressing AtPDI1 increased tolerance of seedlings to abiotic stresses, with a higher germination ratio and longer length of roots than the wild-type. Our results suggested AtPDI1 played roles in anti-stresses in Arabidopsis, which relate to the activities of PDI.

Genome-wide identification and expression analysis of the B-box gene family in the Apple (Malus domestica Borkh.) genome.

The B-box proteins (BBXs) are a family of zinc finger proteins containing one/two B-box domain(s). Compared with intensive studies of animal BBXs, investigations of the plant BBX family are limited, though some specific plant BBXs have been demonstrated to act as transcription factors in the regulation of flowering and photomorphogenesis. In this study, using a global search of the apple (Malus domestica Borkh.) genome, a total of 64 members of BBX (MdBBX) were identified. All the MdBBXs were divided into five groups based on the phylogenetic relationship, numbers of B-boxes contained and whether there was with an additional CCT domain. According to the characteristics of organ-specific expression, MdBBXs were divided into three groups based on the microarray information. An analysis of cis-acting elements showed that elements related to the stress response were prevalent in the promoter sequences of most MdBBXs. Twelve MdBBX members from different groups were randomly selected and exposed to abiotic stresses. Their expressions were up-regulated to some extent in the roots and leaves. Six among 12 MdBBXs were sensitive to osmotic pressure, salt, cold stress and exogenous abscisic acid treatment, with their expressions enhanced more than 20-fold. Our results suggested that MdBBXs may take part in response to abiotic stress.

Encapsulation artocarpanone and ascorbic acid in O/W microemulsions: Preparation, characterization, and antibrowning effects in apple juice.

The aim of this study is to improve artocarpanone solubility by developing an O/W microemulsion with the evaluation of its antibrowning effects. The chemical and physical stabilities as well as antibrowning effects in apple juice were also evaluated. The formulation of artocarpanone microemulsion consisted of 4% w/w of ethyl butyrate, 10.67% w/w of Tween 80, 5.33% w/w of polyethylene glycol 400, and 80% w/w of water, with a maximum solubility of artocarpanone up to 10.54 ± 0.01 mg/mL, at least 3000-folds increase in solubility compared that in water. Encapsulating artocarpanone and ascorbic acid (VC) into microemulsion simultaneously decreased modest artocarpanone solubility whereas improving its stability in long-term storage. Blank, artocarpanone and artocarpanone-Vc-loaded microemulsions demonstrated steadily during accelerated and long-term storage. Artocarpanone-Vc-loaded microemulsion showed strong antibrowning effects in apple juice at room temperature in 24h, suggesting that artocarpanone-Vc-loaded microemulsion is a good antibrowning agent for apple juices.