Genome-wide analysis of basic helix-loop-helix (bHLH) transcription factors in Aquilaria sinensis.

The basic helix-loop-helix (bHLH) transcription factors are involved in several biological processes both in plant development and stress responses. Agarwood, a major active and economical product, is only induced and accumulated when the roots, stems, or branches are wounded in Aquilaria sinensis. Although genome-wide comprehensive analyses of the bHLH family have been identified in many plants, no systematic study of the genes in this family has been conducted in A. sinensis. In this study, 105 bHLH genes were identified in A. sinensis through genome-wide analysis and named according to their chromosomal locations. Based on a phylogenetic tree, AsbHLH family proteins were classified into 18 subfamilies. Most of them were distributed on eight chromosomes, with the exception of two genes. Based on the tissue-specific expression characteristics and expression patterns in response to methyl jasmonate (MeJA) treatment, seven AsbHLH genes were likely involved in wound-induced agarwood formation. The results provide comprehensive information on AsbHLHs that can be used to elucidate the molecular functions and physiological roles of these proteins in A. sinensis.

Effects of Baihui electroacupuncture in a rat model of depression.

BACKGROUND: This study aimed to investigate the effect of electroacupuncture (EA) on behavior in a rat model of chronic unpredictable mild stress (CUMS) and to explore the underlying molecular mechanisms. METHODS: A total of 45 adult male Sprague-Dawley rats were randomly divided into three groups: the control, CUMS, and CUMS plus EA groups. Rats in the CUMS and EA groups were subjected to a 3-week CUMS condition, while rats in the EA group received EA at the Baihui (GV 20) acupoint (2 Hz, 0.6 mA) for 10 min once daily before being subjected to the CUMS condition. The sucrose preference test (SPT) was used as a measure to infer activation of the pleasure response to depression-like behaviour. After the behavioral test, 5-bromodeoxyuridine (BrdU) was intraperitoneally injected (100 mg/kg) and brain samples were collected 24 h later for the detection of hippocampal BrdU. Cell proliferation was determined according to the proportion of BrdU-positive cells. Brain-derived neurotrophic factor (BDNF) expression was detected. RESULTS: The severity of anhedonia, BDNF+ cells, and BrdU+ neurons in DG significantly decreased in CUMS rats, and was accompanied by a reduced BDNF and BrdU+ expression (P<0.05). After EA, the low levels of BDNF+ cells and BrdU+ expression and the depression-like behavior increased markedly (P<0.05). CONCLUSIONS: EA contributes to neuroprotection against CUMS by enhancing BDNF expression and improving hippocampal neurogenesis.

WRKY44 represses expression of the wound-induced sesquiterpene biosynthetic gene ASS1 in Aquilaria sinensis.

Agarwood is derived from wounds in Aquilaria trees and is widely used in traditional medicine, incense, and perfume. Sesquiterpenes are one of the main active components in agarwood and are known to be induced by wounding or injury; However, the molecular mechanisms by which wounding leads to sesquiterpene formation remain largely unknown. Agarwood sesquiterpene synthase 1 (ASS1) is one of key enzymes responsible for the biosynthesis of sesquiterpenes and is a crucial jasmonate (JA)-responsive wound-inducible synthase. However, it is not known why ASS1 is not expressed in healthy trees and how its expression is induced as a result of wounding. Here, we report that ASS1 is a wound-induced gene with a promoter in which a 242-bp region (-973 to -731bp) is identified as the core sequence for responding to wound signals. AsWRKY44 binds directly to this region and represses ASS1 promoter activity. Down-regulation or disruption of AsWRKY44 can relieve the inhibition and activate ASS1 expression. In addition, AsWRKY44 is degraded and the expression of ASS1 is significantly up-regulated in response to exogenous application of methyl jasmonate. Thus, AsWRKY44 is a crucial negative regulator of wound-induced ASS1 transcription, and is central to the mechanism of sesquiterpene biosynthesis in agarwood.

Anti-atherosclerotic effects between a combined treatment with simvastatin plus hirudin and single simvastatin therapy in patients with early type 2 diabetes mellitus.

BACKGROUND: This study aimed to investigate the efficacy and safety of simvastatin plus hirudin in preventing atherosclerosis in the patients with early type 2 diabetes mellitus (T2DM). METHODS: This was a 24-week, randomized, open-label and controlled study in which 150 outpatients initially diagnosed with T2DM were randomly assigned into either simvastatin (40 mg daily at night) plus hirudin (3 g thrice daily) group [combined group (CG) n=75] or simvastatin (40 mg once daily) group [monotherapy group (MG) n=75]. The therapeutic efficacy was evaluated by the score of carotid artery atherosclerosis, plaque size, peak systolic velocity (PSV) and end-diastolic velocity (EDV) on carotid ultrasonography at three and six months after treatment. Logistic regression analysis was used to investigate the correlation between treatment and carotid atherosclerosis. RESULTS: One hundred and thirty-one patients completed this study, and there were no significant differences in the dropout rate in the CG (14.67%) and the MG (10.67%). Significant difference was found in the incidence of adverse events in the CG compared with the MG (37.50% vs. 17.91%, P<0.05) due to the higher risk of hemorrhage (12.50% vs. 1.49%, P<0.05), which did not affect the treatment compliance. The efficacy of combined treatment was better than monotherapy in the enhancement of carotid artery atherosclerosis scores (P<0.01), the plaque thickness (P<0.05) and the change of PSV (P<0.05) and EDV (P<0.05) since three months after treatment, which maintained to the end of observation. In addition, hirudin treatment was able to independently predict the carotid artery atherosclerosis scores (ß=2.37, P<0.05), the plaque thickening (ß=3.51, P<0.01) and the change of PSV (ß=1.69, P<0.05) and EDV (ß=1.79, P<0.05). CONCLUSIONS: Combined use of simvastatin and hirudin is well tolerated and possesses better anti-atherosclerotic effects than simvastatin alone in patients with early T2DM.

Transcription Factor AsMYC2 Controls the Jasmonate-Responsive Expression of ASS1 Regulating Sesquiterpene Biosynthesis in Aquilaria sinensis (Lour.) Gilg.

Sesquiterpenes are one of the most important defensive secondary metabolite components of agarwood. Agarwood, which is a product of the Aquilaria sinensis response to external damage, is a fragrant and resinous wood that is widely used in traditional medicines, incense and perfume. We previously reported that jasmonic acid (JA) plays an important role in promoting agarwood sesquiterpene biosynthesis and induces expression of the sesquiterpene synthase ASS1, which is a key enzyme that is responsible for the biosynthesis of agarwood sesquiterpenes in A. sinensis. However, little is known about this molecular regulation mechanism. Here, we characterized a basic helix-loop-helix transcription factor, AsMYC2, from A. sinensis as an activator of ASS1 expression. AsMYC2 is an immediate-early jasmonate-responsive gene and is co-induced with ASS1. Using a combination of yeast one-hybrid assays and chromatin immunoprecipitation analyses, we showed that AsMYC2 bound the promoter of ASS1 containing a G-box motif. AsMYC2 activated expression of ASS1 in tobacco epidermis cells and up-regulated expression of sesquiterpene synthase genes (TPS21 and TPS11) in Arabidopsis, which was also promoted by methyl jasmonate. Our results suggest that AsMYC2 participates in the regulation of agarwood sesquiterpene biosynthesis in A. sinensis by controlling the expression of ASS1 through the JA signaling pathway.

The effect of nilotinib plus arsenic trioxide on the proliferation and differentiation of primary leukemic cells from patients with chronic myoloid leukemia in blast crisis.

AIM: To determine the effects of arsenic trioxide (ATO) and nilotinib (AMN107, Tasigna) alone or in combination on the proliferation and differentiation of primary leukemic cells from patients with chronic myeloid leukemia in the blast crisis phase (CML-BC). METHODS: Cells were isolated from the bone marrow of CML-BC patients and were treated with 1 µM ATO and 5 nM nilotinib, either alone or in combination. Cell proliferation was evaluated using a MTT assay. Cell morphology and the content of hemoglobin were examined with Wright-Giemsa staining and benzidine staining, respectively. The expression of cell surface markers was determined using flow cytometric analysis. The levels of mRNA and protein were analyzed using RT-PCR and Western blotting, respectively. RESULTS: ATO and nilotinib alone or in combination suppressed cell proliferation in a dose- and time-dependent pattern (P < 0.01 vs. control). Drug treatments promoted erythroid differentiation of CML-BC cells, with a decreased nuclei/cytoplasm ratio but increased hemoglobin content and glycophorin A (GPA) expression (P < 0.01 compared with control). In addition, macrophage and granulocyte lineage differentiation was also induced after drug treatment. The mRNA and protein levels of basic helix-loop-helix (bHLH) transcription factor T-cell acute lymphocytic leukemia protein 1 (TAL1) and B cell translocation gene 1 (BTG1) were both upregulated after 3 days of ATO and Nilotinib treatment. CONCLUSIONS: Our findings indicated that ATO and nilotinib treatment alone or in combination greatly suppressed cell proliferation but promoted the differentiation of CML-BC cells towards multiple-lineages. Nilotinib alone preferentially induced erythroid differentiation while combined treatment with ATO preferentially induced macrophage and granulocyte lineage differentiation.

Down-regulation of HDAC5 inhibits growth of human hepatocellular carcinoma by induction of apoptosis and cell cycle arrest.

Histone deacetylases (HDACs) play a critical role in the proliferation, differentiation, and apoptosis of cancer cells. An obstacle for the application of HDAC inhibitors as effective anti-cancer therapeutics is that our current knowledge on the contributions of different HDACs in various cancer types remains scarce. The present study reported that the mRNA and protein levels of HDAC5 were up-regulated in human hepatocellular carcinoma (HCC) tissues and cells as shown by quantitative real-time PCR and Western blot. MTT assay and BrdU incorporation assay showed that the down-regulation of HDAC5 inhibited cell proliferation in HepG2, Hep3B, and Huh7 cell lines. Data from in vivo xenograft tumorigenesis model also demonstrated the anti-proliferative effect of HDAC5 depletion on tumor cell growth. Furthermore, the suppression of HDAC5 promoted cell apoptosis and induced G1-phase cell cycle arrest in HCC cells. On the molecular level, we observed altered expression of apoptosis-related proteins such as p53, bax, bcl-2, cyto C, and caspase 3 in HDAC5-shRNA-transfected cells. Knockdown of HDAC5 led to a significant up-regulation of p21 and down-regulation of cyclin D1 and CDK2/4/6. We also found that the down-regulation of HDAC5 substantially increased p53 stability and promoted its nuclear localization and transcriptional activity. Our study suggested that knockdown of HDAC5 could inhibit cancer cell proliferation by the induction of cell cycle arrest and apoptosis; thus, suppression of HDAC5 may be a viable option for treating HCC patients.

Preliminary study on the role of virtual touch tissue quantification combined with a urinary ß2-microglobulin test on the early diagnosis of gouty kidney damage.

The goal of the work described here was to evaluate the role of virtual touch tissue quantification (VTQ) combined with urinary ß2-microglobulin (ß2-MG) measurement in the early diagnosis of gouty kidney damage. Two hundred fifty-nine patients with gouty kidney damage and 200 healthy control subjects were tested. The shear wave velocity (SWV) of the renal parenchyma and sinus as determined with VTQ and the urinary ß2-MG level of the two groups were analyzed. Although there were no significant differences in age, body mass index, creatinine level and blood urea nitrogen between the two groups (all p's > 0.05), the aforementioned parameters were higher in the group with gouty kidney damage than in the control group. Urinary ß2-MG levels of the patients with kidney damage were significantly higher than those of the control subjects (t = 6.38, p < 0.01). The SWV of the renal parenchyma was higher than that of the sinus in both groups. Compared with controls, patients with kidney damage had significantly increased renal parenchyma and sinus SWVs (all p-values < 0.05). Urinary ß2-MG level was positively linearly correlated with the SWV of renal parenchyma in patients with kidney damage (r = 0.442, p < 0.0001). However, there was no correlation between urinary ß2-MG level and the SWV of the sinus in patients with kidney damage (r = 0). In the control group, there was no correlation between urinary ß2-MG level and the SWV of the renal parenchyma or sinus. The elasticity of the kidney as determined with VTQ, combined with the urinary ß2-MG level, may be helpful in the early diagnosis of gouty kidney damage.

FoxO3a and nilotinib-induced erythroid differentiation of CML-BC cells.

We explored the potential involvement of FoxO3a activation in erythroid and granulocytic differentiation for Ph(+) cells of chronic myeloid leukemia blast crisis (CML BC). We demonstrate that FoxO3a activation in CML blast crisis (BC) cells by overexpressing FoxO3a leads to the maturation of CML BC cells. Hemoglobin production significantly increased upon FoxO3a activation in CML BC cells. FoxO3a activation upregulated erythroid surface protein (glycophorin A, GPA), but did not significantly modulate granulocytic markers (CD11b). Additionally, FoxO3a activation reduced the mRNA and protein expression of Tal1. Similar results were observed in cells that were given nilotinib. Our results indicate that FoxO3a activation may promote erythroid differentiation of BC cells via down-regulating Tal1 expression.