[Effect of TSC2 gene expression downregulation by lentivirus induced RNA interference on U937 cell line and its mechanism].

Objective: To investigate the effect of biology and mTOR pathway activity of down-regulated TSC2 gene expression on U937 leukemia cells. Methods: Gene expression was down-regulated by lentivirus induced RNA interference on TSC2 high expressed U937 cell line; the proliferation, apoptosis and differentiation were detected by CCK-8 assay, colony formation assay and flow cytometry; the gene expression level and protein kinase activity were detected by qRT-PCR and Western blot. Results: Down-regulated expression of TSC2 gene promoted U937 cell proliferation and colony formation ability (P<0.05) . The proportion in G(0)/G(1) phase of TSC2 down-regulated U937 cell was much lower than that of the control cells [ (52.53±3.75) % vs (75.10±4.33) %, t=6.829, P=0.002], the S phase [ (22.43±1.00) % vs (15.47±1.20) %, t=-5.581, P=0.019] and G(2)/M phase [ (25.03±4.34) % vs (14.33±0.91) %, t=-5.413, P=0.013] was remarkably higher than that of the control cells (P<0.05) . There were no statistically significant differences in cell apoptosis and differentiation (P>0.05) . Down-regulation of TSC2 led to the increased activity of mTOR, 4EBP1 and S6K1, but did not influence the activity of AKT. The expressions of proliferation related cyclinD1, c-myc and PTEN were also up-regulated after TSC2 silenced, but the expressions of P27KIP and BCL-XL were not changed. Conclusion: Downregulation of TSC2 could promote the proliferation of U937 cells through up-regulation of mTOR activity.

[Frequency and clinical features of ASXL2 gene mutation in acute myeloid leukemia patients with AML1- ETO fusion gene positive].

OBJECTIVE: To investigate frequency and clinical features of additional sex combs-like 2 (ASXL2) gene mutation in acute myeloid leukemia (AML) patients with AML1-ETO fusion gene and to analyze the relationship between ASXL2 gene mutation and c- kit gene mutation. METHODS: Mutation analysis of exon 11 and 12 of ASXL2 gene in 59 de novo AML patients was performed by using polymerase chain reaction (PCR) followed by sequence analysis. The clinical features, survival curve and c-kit gene mutation in ASXL2 gene mutation positive and negative patients were compared. RESULTS: In a total of 59 AML patients with AML1-ETO fusion gene positive, 11.9% (7/59) patients harboured ASXL2 gene mutations. The hemoglobin levels of patients with mutated ASXL2 gene [56.2 (38.0- 72.0) g/L] were significantly lower than those with wild type ASXL2 [69.0(37.2-154.0) g/L] (P=0.038). Differences were not observed in white blood cell counts, platelet counts, the proportion of acidophilic cell, and the proportion of primitive cell in the marrow between patients with mutant ASXL2 and ones without mutant ASXL2 (P>0.05). None of all 59 patients suffered from liver, spleen, central nervous system metastases in both groups. Moreover, enlarged lymph nodes was similar between patients with mutant ASXL2 and ones without mutant ASXL2 (P=0.859). Immunophenotypic analysis: in positive group CD33 positive expression was significantly lower than that of negative group (P=0.033). cCD3 was not expressed in both groups. Expression levels of CD117, cMPO, HLA-DR, CD34, CD38, CD13, CD44, CD15, CD64, CD11b, CD56, CD19, cCD79a and CD7 were similar between patients with mutant ASXL2 and ones without mutant ASXL2 (P>0.05). All of 59 patients were in remission (P=0.577). Overall survival was similar between patients with mutant ASXL2 and ones without mutant ASXL2 (P=0.631). The mutation rates of c- kit in positive group and negative group were 14.3% and 29.4%, without statistical significance (P= 0.697). CONCLUSIONS: ASXL2 mutation may be a new event that can cooperate with AML1-ETO to induce leukemia. Patients in AML1- ETO positive AML with ASXL2 mutation show specific clinical characteristics of hemoglobin levels and expression level of CD33. ASXL2 gene mutations and c-kit gene mutations may not have a specific correlation between them.

[Discovery of a novel spliceosome of ABL gene (ABL(Δexon7+35INS)) and its association with TKIs resistance in chronic myeloid leukemia].

OBJECTIVE: To explore whether the ABL(Δexon7) and ABL(35INS) spliceosome contributed to TKIs resistance. METHODS: Screening ABL(Δexon7) and ABL(35INS) in 74 normal people and 76 CML patients (53 patients in remission and 23 patients with TKIs resistance) by using polyacrylamide gel electrophoresis combined with cloning sequencing. RESULTS: A novel spliceosome ABL(Δexon7+ 35INS) (ABL(Δexon7) and ABL(3)5INS existed at the same time) was identified and the mutation was detected in 8 (10.8%) of 74 normal people, 4 (7.5%) of 53 remission patients and 2 (8.7%) of 23 resistant patients. While 47 (63.5%) cases expressed ABL(Δexon7) and 8 (10.8% ) cases expressed ABL(35INS) in 74 healthy people, 30 (56.6%) cases expressed ABL(Δexon7) and 5 (9.4% ) cases expressed ABL(35INS) in 53 remission patients, 12 (52.2%) cases expressed ABL(Δexon7) and 3(13.0%) cases expressed ABL(35INS) in 23 resistant patients. Three kinds of spliceosome in all groups had no statistical difference. CONCLUSION: ABL(Δexon7+ 35INS), ABL(Δexon7) and ABL(35INS) may be not uncommon in ABL gene and were unrelated to resistance in CML with TKIs treatment. ABL(35INS) were often accompanying with exon 7 deletion.

Mechanism of curcumin analog MHMD-induced cell death in A549 lung cancer cells.

OBJECTIVE: To investigate the anticancer properties of a chemosynthetic curcumin analog, (1E,6E)-4-((furan-2-yl)methylene)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (C26H22O7, abbreviated MHMD) in A549 cells. MATERIALS AND METHODS: Inverted microscope was used to observe the alteration on cytomorphology. MTT assay was used to detect cell viability. Acridine-orange staining was used to measure autophagy, and AnnexinV/PI staining and Hoechst/PI staining to measure apoptosis and necrosis. RESULTS: MTT assays showed that at 12 h, 24 h, 48 h, MHMD reduced cell viability with an IC50 of 27.46 µM, 18.86 µM, and 11.23 µM, respectively. Typical characteristics were observed in concert with cell death, including treated-cells getting brighter, rounder, and becoming non-adherent gradually. Additionally, acridine-orange staining suggested that autophagy didn't involve in MHMD-induced cell death. However, apoptosis and necrosis played important roles in MHMD-induced cell death by Hoechst33342/PI staining. It showed apoptosis was the main cause at low concentrations (≤ 4 µM), while with the concentrations rising, necrosis was the leading role. AnnexinV/PI staining again indicated the occurrence of apoptosis at 4 µM. Furthermore, the caspases inhibitor z-VAD-fmk could prevent MHMD-induced cell death, which showed much higher cell viability than those only treated with MHMD (4 µM). Moreover, MTT assay also demonstrated that MHMD did possess a greater anti-proliferative ability than curcumin. CONCLUSIONS: The curcumin analog MHMD is able to induce A549 cell death in a time and dose-dependent manner via apoptosis and necrosis. And MHMD could be a more effective drug than curcumin.

Segmental dyschromatosis with blue naevi and cherry angiomas: a new syndrome?

The dyschromatoses are a group of disorders characterized by the presence of both hyperpigmented and hypopigmented macules, many of which are small in size and irregular in shape. Localized dyschromatosis is a rare manifestation of dyschromatosis. Localized dyschromatosis showing segmental distribution may be a result of degeneration of localized cutaneous nerves. We report a case of localized dyschromatosis, with segmental distribution, with co-occurrence of blue naevi and cherry angiomas.

Widespread white matter alterations in patients with end-stage renal disease: a voxelwise diffusion tensor imaging study.

BACKGROUND AND PURPOSE: ESRD results in excessive accumulation of urea and toxic metabolites. Hemodialysis is usually performed to maintain health in patients with ESRD; however, it may cause silent white matter alterations in the earlier stages. Hence, this study aimed to perform voxelwise diffusion tensor analysis for global detection of subtle white matter alterations in patients with ESRD. MATERIALS AND METHODS: Twenty-eight patients with ESRD and 25 age-matched control subjects were enrolled in this study. Each subject underwent CASI assessment and DTI. After spatial normalization of DTI images, voxelwise statistical analyses were performed to compare DTI parameters between the 2 groups. RESULTS: In patients with ESRD, AD, RD, and MD values were significantly increased, whereas the FA value was significantly decreased, mostly in the corpus callosum, bilateral sagittal stratum, and pons. Multiple regression analysis further revealed that both RD and MD were positively correlated with the duration of hemodialysis in the pons; however, no significant correlation was observed with FA. Negative correlations of RD and MD and a positive correlation of FA with the CASI score were observed in the corona radiata. CONCLUSIONS: We concluded that voxelwise DTI analysis is helpful in the detection of white matter alterations caused by hemodialysis.

First Report of Fusarium mangiferae Causing Mango Malformation in China.

Mango (Mangifera indica L.) malformation caused by Fusarium mangiferae has been reported in many mango-growing regions of the world (3). The disease was also observed in Yunnan and Sichuan provinces of China (1). Typical symptoms in seedlings included loss of apical dominance, hyperplasia and hypertrophy of vegetative buds, shortened internodes, and leaves that were more brittle than those of healthy plants. Inflorescences were abnormally branched and thickened, with panicles producing as much as two to five times the normal number of flowers. Flowers in the malformed inflorescence were much more enlarged and crowded than the generally hypertrophied axes of the panicle, thus producing no fruit or aborting early. To identify the pathogen, samples of malformed and healthy mango seedlings were collected from the affected plantings. For isolation, portions of stems were cut into 3- to 4-mm segments, surface disinfested, dried, and then plated on potato dextrose agar and incubated at 25°C. Within 5 days, white, fluffy, aerial mycelium developed. With the aid of an inverted microscope, single conidia were transferred to carnation leaf agar (CLA) medium. After 10 days of incubation, morphological characteristics were found to be identical to those of F. mangiferae (4). Aerial mycelium was white with no pigmentation observed on potato sucrose agar. Pigmentation on rice medium was pink. On CLA medium, conidia grew in branched conidiophores with false heads bearing monophialides or polyphialides. No conidiospores in chains were observed. Microconidia were ovate to long and oval, 0 to 1 septate, and 3.1 to 10.2 × 1.5 to 2.2 µm. Macroconidia are falculate, 3 to 5 septate, and 18 to 38 × 1.8 to 2.4 µm. Chlamydospores were not observed. Pathogenicity studies were conducted with 7-month-old asymptomatic mango seedlings. These seedlings, except for the controls, were inoculated by injection of the isolated fungus in the axillary or apical bud position. A 1-ml spore suspension (1 × 106 spores/ml) was injected slowly into the stems using a microsyringe with three buds per seedling, for a total of 10 seedlings. Typical malformation symptoms developed within 3 to 4 months, and none of the plants inoculated with sterile water resulted in malformation symptoms. Reisolations from the induced malformed shoots yielded the same fungus, and no fungal growth was observed to be growing from the control plants. To confirm identity of the causal fungus, the gene encoding translation elongation factor 1 alpha (EF-1α) was amplified and sequenced (2). The EF-1α sequence was 660 bp long. The sequence (GenBank Accession No. HM068871) was 99.68% similar to sequences of FD_01167 in the Fusarium ID database. On the basis of symptoms, fungal morphology, the EF-1α region sequence, and pathogenicity testing, this fungus was identified as F. mangiferae. To our knowledge, this is the first report of F. mangiferae causing mango malformation in China. This report will establish a foundation for further study of F. mangiferae and effectively addressing the disease. References: (1) X. H. Chen. Pract. Technol. (in Chinese) 6:5, 1992. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (3) J. Kumar et al. Annu. Rev. Phytopathol. 31:217, 1993. (4) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.

Intraperitoneal bladder rupture presenting as acute bloody ascites and oliguric acute renal failure in an alcoholic liver cirrhosis patient.

Intraperitoneal bladder rupture is a rare cause of acute abdomen with bloody ascites. We report herein the case of a patient who had alcoholic liver cirrhosis and multiple liver nodules, and experienced acute bloody ascites and oliguric acute renal failure in association with intraperitoneal bladder rupture. A 33-year-old male suffered from acute abdominal pain and oliguria following consumption of a large amount of alcohol and after blunt abdominal trauma. He was also found to have acute renal failure and newly onset bloody ascites that rapidly subsided following transurethral catheter drainage. Computed tomography cystography revealed intraperitoneal extravasation of contrast from the dome of the bladder, suggestive of intraperitoneal bladder rupture. The patient received surgical repair and was discharged with full recovery. This case shows that it is important for physicians to be aware of the possibility of intraperitoneal bladder rupture after alcohol consumption accompanied with abdominal blunt trauma. In particular, it has diagnostic complications for underlying liver tumors.

Involvement of GRP78 in inhibition of HBV secretion by Boehmeria nivea extract in human HepG2 2.2.15 cells.

Previous studies showed that the root extract of Boehmeria nivea (BNE) can significantly suppress the production of hepatitis B virus (HBV) in vitro and in vivo. In this study, viral core and large-surface proteins accompanied with their encapsidated viral DNA were observed to accumulate within the cells. Notably, 78-kDa glucose-regulated protein (GRP78) was found to be suppressed by BNE, and stimulation of the GRP78 expression by thapsigargin could rescue virus production initially inhibited by BNE. The antiviral effect of BNE was reversible, which also coincided with the level of GRP78. Furthermore, we synthesized the GRP78 siRNA to knockdown the expression of GRP78 protein, and the production of supernatant HBV DNA was reduced simultaneously. Moreover, combined treatment of BNE and 3TC exhibited an additive anti-hepatitis B virus effect. In conclusion, the inhibitory effect of BNE on blocking assembled virion secretion might be via the reduction of GRP78.

Aristolochic acid nephropathy due to herbal drug intake manifested differently as Fanconi's syndrome and end-stage renal failure--a 7-year follow-up.

Aristolochic acid (AA) may reduce glomerular or proximal tubular function, or both. We report a married couple taking AA-containing herbal drugs. The man developed Fanconi's syndrome (FS) whereas his wife reached end-stage renal failure (ESRF). He was a 36-year-old alcoholic cirrhotic patient who had taken the Chinese herbal drugs for 6 years, presenting with muscle weakness and laboratory findings of FS; the renal pathological findings were compatible with the diagnosis of aristolochic acid nephropathy (AAN). His 38-year-old wife, who took a lower cumulative amount of the same herbal drug for a shorter duration, developed advanced renal failure and severe anemia with pathological findings of extensive tubular atrophy, interstitial fibrosis but spared glomeruli. AA-I was detected in one of the herbal drugs. The wife has been on hemodialysis for 7 years, but the husband is still at the stage of slowly progressive chronic renal failure and persistent FS. None of their 5 children ever took the herbal drug, and none had renal problems during follow-up. It is important to trace the history of herbal drug intake in all the family members because of the possibility of sharing of drugs within a family. In addition to the effect of cumulative doses of AAs and the potentially higher susceptibility of females to AAN, the roles of liver cirrhosis and related vasodilators in the protection of the renal interstitium from fibrosis are questions that warrant further study.

Pictorial review: Unusual tumours involving the prostate: radiological-pathological findings.

The appearance of several unusual tumours in the prostate has resulted in questions being raised concerning their histogenesis; moreover, some of these tumours have prognoses that are quite unlike those of prostatic adenocarcinoma. Unusual neoplasms involving the prostate have been described in recent years, including mucinous cystadenocarcinoma, neuroendocrine cancer, lymphoma, spindle cell neoplasm, squamous cell carcinoma and transitional cell carcinoma. Radiological findings can overlap, and play limited roles in the diagnoses of these entities. However, knowledge of the radiological findings in these conditions can be helpful in making differential diagnoses. Images of prostate lesions using several imaging modalities, including transrectal ultrasound, MRI and CT, as well as available pathological images of such lesions, are presented in this article.

Decreased B cells and IgA-secreting B cells partially explain the high prevalence of IgA deficiency in dialysis patients.

AIMS: We previously reported 2 hemodialysis (HD) patients with recurrent infections and selective immunoglobulin A deficiency (IgAD). We further demonstrated that serum IgA levels were lower and the prevalence of IgAD was higher in uremic patients. The exact mechanisms of IgAD in uremic patients largely remained unclear. In some patients, it was caused by anti-IgA antibody neutralization and subsequent destruction. We performed the present study to survey if there is any defect in IgA production. MATERIALS AND METHODS: 288 patients were initially included for examination of serum immunoglobulins. 16 normal persons, 16 dialysis patients without IgAD, and 12 dialysis patients with IgAD were enrolled after the initial examination. Blood was drawn into heparinized tubes. WBC counts and lymphocyte percentage were examined by a CBC counter. Lymphocytes were separated by the Ficoll-Paque method. Flow cytometry was utilized to isolate the B cell and IgA-secreting B cell after staining with CD 19 phycoerythrin and FITC-conjugated rabbit anti-human IgA antibody. RESULTS: There is no significant difference between WBC counts or total lymphocyte counts of these 3 groups. However, we found a lower percentage of total lymphocyte counts in dialysis patients, either with or without IgAD. The total B cell numbers were lower in dialysis patients with IgAD. In addition, there were fewer IgA-secreting B cells in dialysis patients with IgAD. CONCLUSION: Decreased B cell and IgA-secreting B cell counts are seen in uremic patients with IgAD. This, in turn, indicates that there might be a defect of IgA production in some patients, rather than IgA destruction by anti-IgA antibodies as seen in some other patients. Further study is needed to investigate the mechanisms of decreased B cells and IgA-secreting B cells.