MdBT2 regulates nitrogen-mediated cuticular wax biosynthesis via a MdMYB106-MdCER2L1 signalling pathway in apple.

Cuticular waxes play important roles in plant development and the interaction between plants and their environment. Researches on wax biosynthetic pathways have been reported in several plant species. Also, wax formation is closely related to environmental condition. However, the regulatory mechanism between wax and environmental factors, especially essential mineral elements, is less studied. Here we found that nitrogen (N) played a negative role in the regulation of wax synthesis in apple. We therefore analysed wax content, composition and crystals in BTB-TAZ domain protein 2 (MdBT2) overexpressing and antisense transgenic apple seedlings and found that MdBT2 could downregulate wax biosynthesis. Furthermore, R2R3-MYB transcription factor 16-like protein (MdMYB106) interacted with MdBT2, and MdBT2 mediated its ubiquitination and degradation through the 26S proteasome pathway. Finally, HXXXD-type acyl-transferase ECERIFERUM 2-like1 (MdCER2L1) was confirmed as a downstream target gene of MdMYB106. Our findings reveal an N-mediated apple wax biosynthesis pathway and lay a foundation for further study of the environmental factors associated with wax regulatory networks in apple.

Diagnostic performance of Neutrophil CD64 index, procalcitonin, and C-reactive protein for early sepsis in hematological patients.

BACKGROUND: Patients with hematological diseases are immunosuppressed due to various factors, including the disease itself and treatments, such as chemotherapy and immunotherapy, and are susceptible to infection. Infections in these patients often progress rapidly to sepsis, which is life-threatening. AIM: To evaluate the diagnostic efficacy of the neutrophil CD64 (nCD64) index, compared to procalcitonin (PCT) and high-sensitivity C-reactive protein (hs-CRP), for the identification of early sepsis in patients with hematological diseases. METHODS: This was a prospective analysis of patients with hematological diseases treated at the Fuxing Hospital affiliated with Capital Medical University, between March 2014 and December 2018. The nCD64 index was quantified by flow cytometry and the Leuko64 assay software. The factors which may affect the nCD64 index levels were compared between patients with different infection statuses (local infection, sepsis, and no infection), and the control group and the nCD64 index levels were compared among the groups. The diagnostic efficacy of the nCD64 index, PCT, and hs-CRP for early sepsis was evaluated among patients with hematological diseases. RESULTS: A total of 207 patients with hematological diseases (non-infected group, n = 50; locally infected group, n = 67; sepsis group, n = 90) and 26 healthy volunteers were analyzed. According to the absolute neutrophil count (ANC), patients with hematological diseases without infection were divided into the normal ANC, ANC reduced, and ANC deficiency groups. There was no statistically significant difference in the nCD64 index between these three groups (P = 0.586). However, there was a difference in the nCD64 index among the non-infected (0.74 ± 0.26), locally infected (1.47 ± 1.10), and sepsis (2.62 ± 1.60) groups (P < 0.001). The area under the diagnosis curve of the nCD64 index, evaluated as the difference between the sepsis and locally infected group, 0.777, which was higher than for PCT (0.735) and hs-CRP (0.670). The positive and negative likelihood ratios were also better for the nCD64 index than either PCT and hs-CRP. CONCLUSION: Our results indicate the usefulness of the nCD64 index as an inflammatory marker of early sepsis in hematological patients.

Review: The effects of hormones and environmental factors on anthocyanin biosynthesis in apple.

Fruit coloration is an appearance trait that directly affects the commercial value and market competitiveness of apples. The red color of apple fruit is mainly affected by anthocyanin accumulation, and the synthesis of anthocyanin is affected by various factors. The critical roles of hormones and environmental factors during apple anthocyanin biosynthesis are described. This review also elaborates the specific mechanisms of the responses of internal genes to stress and changes in anthocyanin when apples are exposed to different environmental stressors. This study provides direction for future research on apple anthocyanin and is a reference for anthocyanin studies in other species.

Identification and functional analysis of the MdLTPG gene family in apple.

Cuticular wax is synthesized from intracellular lipids that are exported by epidermal cells, and plant lipid transfer proteins (LTPs) play an important role in this process. The glycosylphosphatidylinositol (GPI)-anchored LTPs (LTPGs) are a large subgroup within the LTP family and function in lipid transport and wax formation. Although LTPG family members have been identified in several plant species, the LTPG gene family of apple (Malus domestica) remains uncharacterized. In this paper, we identified 26 potential LTPG genes by searching apple whole-genome annotation files using "GPI-anchored" and "lipid transferase" as keywords. Twenty of the 26 putative LTPG genes were confirmed as MdLTPG family members based on their subcellular localization predictions. The MdLTPGs were divided into four classes based on phylogenetic analysis and functional domain prediction. One member of each class was analyzed for subcellular localization, and all identified members were located on the plasma membrane. Most MdLTPG genes were induced by abiotic stress treatments such as low temperature, NaCl, and ABA. Finally, the MdLTPG17 protein was shown to interact with the lysine-rich arabinogalactan protein MdAGP18 to perform its function in wax transport during plant growth and development.

Cancer-associated fibroblasts induce epithelial-mesenchymal transition and cisplatin resistance in ovarian cancer via CXCL12/CXCR4 axis.

Aim: Cancer-associated fibroblasts (CAFs) are closely related to epithelial-mesenchymal transition (EMT) and chemoresistance in various cancers. Patients & methods: Experiments in vivo and retrospective studies were applied to explore the role of CAFs in epithelial ovarian cancer (EOC). Results: We found that CXCL12 expression was significantly increased in interstitial CAFs by immunofluorescence. CAF-derived CXCL12 induced EMT though CXCR4/Wnt/ß-catenin pathway in EOC cells. Inhibited EMT led to increased apoptosis and cisplatin sensitivity. Multivariate regression analysis shows that CXCL12 expression in the stromal cells and cytoreduction satisfaction are independent prognostic markers of platinum-containing chemotherapy sensitivity in 296 EOC patients. Conclusion: CAFs may activate the Wnt/ß-catenin pathway in EOC cells via CXCL12/CXCR4 axis, and then induce EMT and cisplatin resistance.

[Improvement of Method for Detecting Peripheral Blood Neutrophil CD64 Index in Patients with Hematologic Malignancies].

OBJECTIVE: To improve the method for detecting the neutrophil CD64 (nCD64) index and to enhance the detection rate and accuracy of nCD64 index in patients with hematologic malignancies. METHODS: The nCD64 index in peripheral blood of patients with hematologic malignancies combined with suspicious bacterial infection (255 cases-time) was detected by using array method. When the detection of nCD64 index in samples was interfered with abnormal cells in detection process of enrolled patients, the antibodies CD45, CD15 and CD10 were added into samples on the basis of routine detection by using the primary detection kit, in order to more accurately distinguish the neutrphils and obtain the nCD64 index. The nCD64 index as well as the efficiency of nCD64 index, PCT and CRP for diagnosis of sepsis before and after the improvement of deteation method were compared. RESULTS: The samples of 60 cases were interfered with abnormal cells in detection process, out of which the samples of 18 cases failed in detection, but these samples of 18 cases all got the effective results of detection after the detection method was improved. The detection showed that the nCD64 index before and after the improvement as well as the PCT and CRP levels in sepsis group were higher than those in non-sepsis group(P＜0.05). After improvement of method, the efficiency of nCD64 index for diagnosis of sepsis was suprior to the PCT and CRP, the nCD64 index obtained after the improvement of method possessed the diagnosis efficiency same as the efficiency obtained before improvement of method, moreover the detection results were more reliable. CONCLUSION: For the samples of patients with hematologic malignancies interfered with abnormal cells in the process of detecting the nCD64 index, the corresponding antibodies are added into detected samples according to the kinds of hemotologic diseases of patients, in order to more accurately gate the neutrophils in paripheral blood of patients, there by the detection rate and accuracy for detecting the nCD64 index are enhanced by accurately distinguishing the neutrophils.

CAFs enhance paclitaxel resistance by inducing EMT through the IL­6/JAK2/STAT3 pathway.

Carcinoma­associated fibroblasts (CAFs) are the major components of mesenchymal cells in the inflammatory tumor microenvironment. They are involved in epithelial­mesenchymal transition (EMT) and chemotherapy resistance by directly contacting with cancer cells or secretory cytokines. In the present study, we examined the role of CAFs in the induction of EMT in ovarian cancer. Primary ovarian cancer cells, CAFs and normal fibroblasts (NFs) were isolated from fresh cancer tissue and cultured for immunohistochemistry studies. Enzyme­linked immunosorbent assay (ELISA) was used to detect the expression of IL­6 in the culture supernatants of these cells. The expression of IL­6 at the mRNA level was examined by RT­PCR. The expression of IL­6 at the protein level in ovarian cancer tissues was determined using an immunofluorescence assay in both tissue sections and cell lobes. OVCAR3 cells were treated with the culture supernatants collected from CAFs and NFs. IL­6 monoclonal antibody (mAb) was employed to neutralize IL­6. The expression of phosphorylated STAT3 was assessed. Changes in EMT, proliferation, invasion and proapoptotic protein expression were also examined. Flow cytometry was performed to detect the changes in apoptosis resistance of OVCAR3 cells. The JAK2/STAT3 pathway­specific inhibitor AG490 was used to block this pathway and the ß­TGF inhibitor was used to inhibit EMT. The clinical data of patients treated in our hospital were collected between January 1st, 2009 and June 30th, 2013. The expression of interstitial IL­6 in paraffin­embedded tissues was detected by immunohistochemistry. The relationship between the expression of interstitial IL­6 and the treatment response was examined by linear regression and multiple linear regression analyses. We found that CAFs were the main source of IL­6 in ovarian cancer tissue. CAFs promoted the phosphorylation of STAT3 in ovarian cancer and enhanced the proliferation, invasion and EMT. Enhanced EMT may lead to apoptosis resistance, inhibitory expression of pro­apoptotic proteins and paclitaxel resistance. A total of 255 patients were enrolled in this retrospective study. Univariate and multivariate analyses revealed that age, CA125, interstitial IL­6 expression and cytoreduction satisfaction were closely related to the sensitivity of the TP (docetaxel plus cisplatin or carbopatin) regimen in ovarian cancer (P<0.05). These results demonstrated that CAFs highly secreted IL­6 and promoted ß­TGF­mediated EMT in ovarian cancer via the JAK2/STAT3 pathway, leading to inhibited apoptosis and subsequent paclitaxel resistance. Therefore, CAFs may be a new therapeutic target for the treatment of ovarian cancer.

[Expression and clinical significance of PDCD5 in patients with acute myeloid leukemia].

OBJECTIVE: This study was aimed to investigate the roles of PDCD5 (programmed cell death 5) in pathogenesis of acute myeloid leukemia (AML) and the relevance of PDCD5 with the clinical characteristics and prognosis of patients by testing the PDCD5 expression in adult AML patients. METHODS: The mRNA and intracellular protein levels of PDCD5 from 36 newly diagnosed AML patients were analyzed by real-time fluorescence quantitative polymerase chain reaction (RQ-PCR) and flow cytometry (FCM), respectively. The correlation of mRNA levels and intracellular protein levels of PDCD5 with the clinical characteristics and survival time of patients were analyzed. RESULTS: The intracellular protein expression levels of PDCD5 in AML patients were significantly higher than those in normal controls (P < 0.05). The PDCD5 mRNA levels were not significantly different between patients and controls (P > 0.05). The mRNA and protein levels of PDCD5 did not significantly correlate with sex, age, WBC count, FAB subtype, extramedullary infiltration, WT1 gene, NPM1 gene mutation and the patients response to induction therapy. The patients with positive FLT3/ITD mutation displayed higher protein levels of PDCD5 as compared with negative FLT3/ITD mutation patients (P < 0.05). CONCLUSION: The intracellular protein of PDCD5 significantly increased in AML patients. However, the increased PDCD5 does not exert the pro-apoptotic effects on AML cells. The patients with positive FLT3/ITD mutation show higher protein levels of PDCD5.

[Expression and clinical significance of B-cell lymphoma 2 (BCL-2) in hyperleukocytic acute myeloid leukemia].

This study was aimed to investigate the role of B-cell lymphoma 2 (BCL-2) in pathogenesis of hyperleukocytic acute myeloid leukemia (AML). The levels of intracellular BCL-2 in 48 AML patients were detected by flow cytometry (FCM). Serum levels of BCL-2 in 40 AML patients were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that the serum levels of BCL-2 in hyperleukocytic AML and non-hyperleukocytic AML patients were significantly higher than that in normal controls (P < 0.05), but intracellular BCL-2 levels were not significantly different, as compared with normal controls (P > 0.05). There were no difference of intracellular and serum BCL-2 levels between hyperleukocytic and non-hyperleukocytic AML patients (P > 0.05). The serum and intracellular levels of BCL-2 between hyperleukocytic AML, non-hyperleukocytic AML patients and normal controls were not statistically correlated. It is concluded that leukemic cells in AML patients produce and secrete too much BCL-2, which may be involved in the pathogenesis of leukemia disease. However, the anti-apoptosis effect of BCL-2 has no significant impact on the pathogenesis of hyperleukocytic AML.

[Effect of heat stress on expression of gp96 in K562 cell line of the chronic myeloid leukemia and its significance].

This study was purposed to investigate the effect of different heat stress conditions on expression level of heat shock protein gp96 in K562 cell line of chronic myeloid leukemia in order to provide experiment basis for seeking optimal heat stress condition increasing extraction amount of gp96 from K562 cells. The expression changes of gp96 in K562 cell line was detected by immunocytochemistry under 38, 40, 42, 44, 46 and 48 degrees C for 30 minutes in water, by flow cytometry under 40, 44, 48 and 52 degrees C for 30 minutes in water, by Western blot under 40, 44, 48 and 52 degrees C for 30 minutes in water. Immunocytochemistry assay showed that gp96 existed mainly in cytoplasm. The peak of gp96 expression was at 30 minutes after 48 degrees C in water. The result of flow cytometry was consistent to immunocytochemistry detection results under temperatures 40, 44, 48 and 52 degrees C (P < 0.01). Western blot showed that detection result was the same as the immunocytochemistry and flow cytometry detections. In conclusion, the expression of gp96 in K562 cell line reached peak at 30 minutes after 48 degrees C in water. This condition may be an effective preparative condition for extraction of gp96 from K562 cells.

[Differentiation and function of human dendritic cells influenced by heat shock protein gp96 purified from K562 cells].

This study was to establish the method of purifying heat shock protein GP96 from K562 cells and explore the differentiation and function of human DC influenced by heat shock prolein (HSP). Using ammonium sulfate precipitation, conA-sepharose affinity chromatography and DEAE-sephacel anion exchange chromatography GP96 from K562 cells lysate was isolated and purified. The identification of the purified protein was controlled by Western blot with anti-human GP96 IgG. Human dendritic cell derived from peripheral blood mononuclear cell were cultured with purified GP96. The phenotype changes of DC were analyzed by flow cytometry and MLR was detected by MTT. The results showed that 60-80 microg GP96 was purified successfully from 1 x 10(10) K562 cells. DC stimulated with HSP-GP96 had higher expression rates of CD83, CD86, HLA-DR and lower expression rates of CD1a and had stronger ability to induce T cells proliferation. It is concluded that heat shock protein GP96 can be isolated and purified from K562 cells and could induce maturation of dendritic cell. HSP-DC vaccine show stronger ability to induce T cell proliferation than DC.

[Dendritic cells cultured with human umbilical cord serum instead of fetal calf serum].

To investigate whether the dendritic cells (DC) could grow up in cultural system with umbilical cord serum (UCS), the UCS was used in the culture instead of fetal calf serum. The phenotype of dendritic cells was detected by flow cytometry and the antigen presenting ability of DC in allo-MLR was measured by MTT assay. The results showed that DC grown in UCS (UCS-DC) had higher expression rate of CD86, CD83 and HLA-DR than that in grown in FCS (FCS-DC). (P < 0.05), and their expression of CD1a was lower than that of FCS-DC. The ability to induce T cell proliferation had no difference between UCS-DC and FCS-DC. It is suggested that dendritic cells with more mature phenotype had been produced in the medium containing UCS than those in the medium containing FCS, and UCS-DC possessed potent ability to stimulate proliferation of allogeneic T cells.