Genome-Wide Identification and Characterization of Effector Candidates with Conserved Motif in Falciphora oryzae.

Microbes employ effectors to disrupt immune responses and promote host colonization. Conserved motifs including RXLR, LFLAK-HVLVxxP (CRN), Y/F/WxC, CFEM, LysM, Chitin-bind, DPBB_1 (PNPi), and Cutinase have been discovered to play crucial roles in the functioning of effectors in filamentous fungi. Nevertheless, little is known about effectors with conserved motifs in endophytes. This research aims to discover the effector genes with conserved motifs in the genome of rice endophyte Falciphora oryzae. SignalP identified a total of 622 secreted proteins, out of which 227 were predicted as effector candidates by EffectorP. By utilizing HMM features, we discovered a total of 169 effector candidates with conserved motifs and three novel motifs. Effector candidates containing LysM, CFEM, DPBB_1, Cutinase, and Chitin_bind domains were conserved across species. In the transient expression assay, it was observed that one CFEM and one LysM activated cell death in tobacco leaves. Moreover, two CFEM and one Chitin_bind inhibited cell death induced by Bax protein. At various points during the infection, the genes' expression levels were increased. These results will help to identify functional effector proteins involving omics methods using new bioinformatics tools, thus providing a basis for the study of symbiosis mechanisms.

Pathological evaluation of renal complications in children following allogeneic hematopoietic stem cell transplantation: a retrospective cohort study.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for hematologic malignancies and non-malignant disorders, such as aplastic anemia, fanconi anemia, and certain immune deficiencies. Post-transplantation kidney injury is a common complication and involves a wide spectrum of structural abnormalities, including glomerular (MSPGN, mesangial proliferative glomerulonephritis; FSGS, focal segmental glomerulosclerosis; MPGN, membranoproliferative glomerulonephritis; MCD, minimal change disease), vascular (TMA, thrombotic microangiopathy), and/or tubulointerstitial (TIN, tubulointerstitial nephritis; ATI, acute tubular injury). Renal biopsy is the gold-standard examination for defining multiple etiologies of kidney impairment. Although kidney injury following HSCT has been studied, little is known about the effects of allo-HSCT on renal pathology in pediatric patients. METHODS: We retrospectively analyzed renal biopsy specimens from children with kidney injury after allo-HSCT and correlated results with clinical data in the last 10 years. RESULTS: Among 25 children (18 males and 7 females), three patients had proteinuria indicating nephrotic syndrome (24-hour urinary total protein/weight > 50 mg/kg/d), nine patients had severely reduced estimated glomerular filtration rate (eGFR < 30 ml/min/1.73 m2) and four patients received kidney replacement therapy (KRT). The main pathologies identified from kidney biopsies were MSPGN (n = 12), FSGS (n = 12), MPGN (n = 5), TMA (n = 4), MCD (n = 3), diffuse glomerular fibrosis (DGF, n = 2), ATI and TIN, in isolation or combined with other pathologies. The median follow-up time was 16.5 (0.5 ~ 68.0) months. Three patients died of recurrent malignancy and/or severe infection, one child developed to end-stage renal disease (ESRD), six patients (24%) had elevated serum creatinine (SCr > 100µmol/l) and nine patients (36%) still had proteinuria. CONCLUSIONS: This study evaluates histomorphologic findings from kidney biopsies of pediatric recipients following allo-HSCT. Detailed evaluation of renal biopsy samples is helpful to elucidate the nature of renal insult, and may potentially identify treatable disease processes.

Inflammation in the peripheral blood system of Crohn's Disease.

Crohn's disease (CD) is an inflammatory bowel disease that is characterized by chronic inflammation of digestive system and has a nickname "green cancer" because of its sustained alternation of periods of flares and remissions. Here, we investigated the inflammation changes in peripheral blood system of CD patients, which are less reported in China. Peripheral blood samples of 167 CD patients and 30 healthy people, as well as their clinical information, were collected at the Second Affiliated Hospital of Soochow University. Flow cytometry was performed to analyze the ratio of CD4 T cells to CD8 T cells. Cytometric Bead Array kit was used to detect the cytokines in peripheral blood in CD patients. Moreover, the expression of inflammasomes was also detected by RT-PCR. The percentage and cell number of lymphocytes in CD patients' peripheral blood system decreased significantly, while monocytes increased remarkably. Interestingly, there was an inversion of the CD4 T cells/CD8 T cells ratio in peripheral blood of CD patients. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) increased significantly in CD patients' peripheral blood, and lipopolysaccharide (LPS) stimulation aggravate inflammatory response. In addition, the expression of nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 1 (NLRP1) and NLRP3 in peripheral blood mononuclear cells (PBMC) of CD patients increased significantly after LPS stimulation. The inflammation in peripheral blood of CD patients had significant changes, including PBMC, cytokines and inflammasomes. These results are helpful to get a deeper understanding of CD and improve the efficiency of diagnosis and treatment in China.

A novel RIPK1 inhibitor reduces GVHD in mice via a nonimmunosuppressive mechanism that restores intestinal homeostasis.

Intestinal epithelial cells (IECs) are implicated in the propagation of T-cell-mediated inflammatory diseases, including graft-versus-host disease (GVHD), but the underlying mechanism remains poorly defined. Here, we report that IECs require receptor-interacting protein kinase-3 (RIPK3) to drive both gastrointestinal (GI) tract and systemic GVHD after allogeneic hematopoietic stem cell transplantation. Selectively inhibiting RIPK3 in IECs markedly reduces GVHD in murine intestine and liver. IEC RIPK3 cooperates with RIPK1 to trigger mixed lineage kinase domain-like protein-independent production of T-cell-recruiting chemokines and major histocompatibility complex (MHC) class II molecules, which amplify and sustain alloreactive T-cell responses. Alloreactive T-cell-produced interferon gamma enhances this RIPK1/RIPK3 action in IECs through a JAK/STAT1-dependent mechanism, creating a feed-forward inflammatory cascade. RIPK1/RIPK3 forms a complex with JAK1 to promote STAT1 activation in IECs. The RIPK1/RIPK3-mediated inflammatory cascade of alloreactive T-cell responses results in intestinal tissue damage, converting the local inflammation into a systemic syndrome. Human patients with severe GVHD showed highly activated RIPK1 in the colon epithelium. Finally, we discover a selective and potent RIPK1 inhibitor (Zharp1-211) that significantly reduces JAK/STAT1-mediated expression of chemokines and MHC class II molecules in IECs, restores intestinal homeostasis, and arrests GVHD without compromising the graft-versus-leukemia (GVL) effect. Thus, targeting RIPK1/RIPK3 in IECs represents an effective nonimmunosuppressive strategy for GVHD treatment and potentially for other diseases involving GI tract inflammation.

The peroxins BcPex8, BcPex10, and BcPex12 are required for the development and pathogenicity of Botrytis cinerea.

Peroxisomes have been proved playing roles in infection of several plant pathogens. Although the contribution of a portion of peroxins in pathogenicity was demonstrated, most of them are undocumented in fungi, especially, Botrytis cinerea. The homologs of Pex8, Pex10, and Pex12 in B. cinerea were functionally characterized in this work using gene disruption strategies. Compared with the wild-type strain (WT), the Δbcpex8, Δbcpex10, and Δbcpex12 mutants exhibited significant reduction in melanin production, fatty acid utilization, and decreased tolerance to high osmotic pressure and reactive oxygen species (ROS). The mycelial growth and conidiation of were significantly inhibited in Δbcpex8, Δbcpex10, and Δbcpex12 strains. The mycelial growth rates of Δbcpex8, Δbcpex10, and Δbcpex12 were reduced by 32, 35, and 34%, respectively, compared with WT and ectopic transformant (ET), and the conidiation was reduced by approximately 89, 27, and 88%, respectively. The conidial germination, germ tube elongation, and the formation of initiate infection structures (IFSs) were also reduced by the deletion of the genes. The pathogenicity was tested on the leaves of tobacco and strawberry, and fruits of tomato. On the leaves of tobacco and strawberry, the Δbcpex8, Δbcpex10, and Δbcpex12 mutants could not induce necrotic lesions, and the lesions on tomato fruits infected with the mutants were significantly reduced than those of the wide type. The results indicated that BcPEX8, BcPEX10, and BcPEX12 are indispensable for the development and pathogenicity of B. cinerea.

De Novo Purine Nucleotide Biosynthesis Pathway Is Required for Development and Pathogenicity in Magnaporthe oryzae.

Purine nucleotides are indispensable compounds for many organisms and participate in basic vital activities such as heredity, development, and growth. Blocking of purine nucleotide biosynthesis may inhibit proliferation and development and is commonly used in cancer therapy. However, the function of the purine nucleotide biosynthesis pathway in the pathogenic fungus Magnaporthe oryzae is not clear. In this study, we focused on the de novo purine biosynthesis (DNPB) pathway and characterized MoAde8, a phosphoribosylglycinamide formyltransferase, catalyzing the third step of the DNPB pathway in M. oryzae. MoAde8 was knocked out, and the mutant (∆Moade8) exhibited purine auxotroph, defects in aerial hyphal growth, conidiation, and pathogenicity, and was more sensitive to hyperosmotic stress and oxidative stress. Moreover, ∆Moade8 caused decreased activity of MoTor kinase due to blocked purine nucleotide synthesis. The autophagy level was also impaired in ∆Moade8. Additionally, MoAde5, 7, 6, and 12, which are involved in de novo purine nucleotide biosynthesis, were also analyzed, and the mutants showed defects similar to the defects of ∆Moade8. In summary, de novo purine nucleotide biosynthesis is essential for conidiation, development, and pathogenicity in M. oryzae.

miR-370-3p as a Novel Biomarker Promotes Breast Cancer Progression by Targeting FBLN5.

miRNAs play a crucial part in multiple biological processes of cell proliferation, migration, apoptosis, and chemoresistance. In cancer, miRNAs can be divided into oncogenes or tumor suppressors on the basis of their functions in the carcinogenic process. The purpose of this study was to explore the roles and clinical diagnostic value of miR-370-3p in breast cancer. Our results demonstrated that miR-370-3p significantly promoted proliferation, metastasis, and stemness of breast cancer in vitro and in vivo. In particular, clinical data revealed that high expression of serum miR-370-3p and exosomal miR-370-3p from breast cancer patients was remarkably correlated with lymphatic metastasis and tumor node metastasis (TNM) stages. Mechanistically, miR-370-3p inhibited FBLN5 expression and activated the NF-κB signaling pathway to promote breast cancer cell proliferation, migration, and stemness. FBLN5 expression was significantly decreased in breast cancer cells and tumor tissues of breast cancer patients. Our research identified that miR-370-3p promoted breast cancer progression by inhibiting FBLN5 expression and activating the NF-κB signaling pathway. Serum exosomal miR-370-3p would provide a potential biomarker for the diagnosis of breast cancer.

Dark septate endophyte Falciphora oryzae-assisted alleviation of cadmium in rice.

Dark septate endophytes (DSEs) are the typical representatives of root endophytic fungi in heavy metal (HM)-contaminated environments. However, little is known about their roles in the HMs tolerance of hosts and the underlying mechanism. Here, we investigated the biological roles and molecular mechanisms of a DSE strain Falciphora oryzae in alleviating cadmium (Cd) toxicities in rice. It was found that F. oryzae possessed a capacity of accumulating Cd in its vacuoles and chlamydospores. During symbiosis, F. oryzae conferred improved Cd tolerance to rice, decreasing Cd accumulation in roots and translocation to shoots. F. oryzae alleviated Cd toxicity to rice by sequestering Cd in its vacuoles. Further application of F. oryzae as fertilizer in the field could reduce Cd content in rice grains. We identified a SNARE Syntaxin 1 gene through proteomics, which participated in Cd tolerance of F. oryzae by regulating chlamydospore formation and vacuole enlargement. This study provided novel insights into how the DSEs and their host plants combat Cd stress.

Epstein-Barr virus-related hemophagocytic lymphohistiocytosis complicated with coronary artery dilation and acute renal injury in a boy with a novel X-linked inhibitor of apoptosis protein (XIAP) variant: a case report.

BACKGROUND: X-linked lymphoproliferative disease (XLP) is a rare inherited X-linked primary immunodeficiency diseases (PID). One such disease, X-linked inhibitor of apoptosis protein (XIAP) deficiency, is characterized by Epstein-Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH). However, EBV-HLH with coronary artery dilation and acute renal injury (AKI) in children is unusual. CASE PRESENTATION: We report the case of a young boy aged 17 months with a novel XIAP variant. He was initially diagnosed with EBV-HLH based on the HLH-2004 diagnostic criteria and the condition was accompanied by coronary artery dilation and acute renal injury. The comprehensive genetic analysis of peripheral blood-derived DNA revealed a hemizygous variant of the XIAP gene [c.116G > C(p.G39A)], which was inherited from his mother (heterozygous condition). After combined treatment with rituximab, intravenous immunoglobulin, corticosteroids, antiviral drugs, and mycophenolate mofetil (MMF) in addition to supportive therapy, his clinical manifestations and laboratory indexes were improved. The patient achieved complete remission with MMF treatment in the 8-month follow-up. CONCLUSIONS: We report the [c.116G > C(p.G39A)] variant in the XIAP gene for the first time in a case of XLP-2 associated with EBV-HLH. For male patients with severe EBV-HLH, the possibility of XLP should be considered and molecular genetic testing should be used early in auxiliary diagnosis. Reports of EBV-HLH with coronary artery dilation and AKI in children are rare. In the patients with EBV-HLH, color Doppler echocardiography and urine tests should be monitored regularly. If necessary, renal biopsy can be performed to clarify the pathology. Treatment with rituximab, immunosuppressors and supportive therapy achieved a good effect, but long-term follow-up is required.

The P5-type ATPase Spf1 is required for development and virulence of the rice blast fungus Pyricularia oryzae.

Pyricularia oryzae (synonym Magnaporthe oryzae) is a plant pathogen causing major yield losses in cultivated rice and wheat. The P-type ATPases play important roles in cellular processes of fungi, plants, and animals via transporting specific substrates through ATP hydrolysis. Here, we characterized the roles of a P5-ATPase, Spf1, in the development and virulence of P. oryzae. Deletion of SPF1 led to decreased hyphal growth and conidiation, delayed spore germination and appressorium formation, reduced penetration and invasive hyphal extension, and attenuated virulence. Appressorium turgor, however, was not affected by deletion of SPF1. The co-localization of Spf1-GFP and an endoplasmic reticulum (ER) marker protein, Lhs1-DsRed2, indicated that Spf1 is an ER-localized P5-ATPase. An ER stress factor, 0.5 µg/ml tunicamycin (TUNI), inhibited the growth of ∆spf1, but another ER stress factor, 5 mM dithiothreitol (DTT), promoted the growth of ∆spf1. Treatment with chemicals for oxidative stress (5 mM H2O2 and 0.8 mM paraquat) also promoted the growth of ∆spf1. Gene expression assays showed that unfolded protein response (UPR) components KAR2, OST1, PMT1, ERV29, PDI1, SCJ1, SEC61, a Ca2+ channel-related P-type ATPase gene PMR1, and a calcineurin-dependent transcription factor CRZ1 were significantly up-regulated in ∆spf1, suggesting activation of UPR in the mutant. These lines of experimental evidence indicate that SPF1 is involved in some basal ER mechanisms of P. oryzae including UPR pathway and responses to ER related stresses, therefore, affecting fungal development and virulence. However, the detailed mechanism between Spf1 and virulence still awaits future researches.

Identification of Novel Glycolysis-Related Gene Signatures Associated With Prognosis of Patients With Clear Cell Renal Cell Carcinoma Based on TCGA.

OBJECTIVE: The purpose of the present study was to detect novel glycolysis-related gene signatures of prognostic values for patients with clear cell renal cell carcinoma (ccRCC). METHODS: Glycolysis-related gene sets were acquired from the Molecular Signatures Database (V7.0). Gene Set Enrichment Analysis (GSEA) software (4.0.3) was applied to analyze glycolysis-related gene sets. The Perl programming language (5.32.0) was used to extract glycolysis-related genes and clinical information of patients with ccRCC. The receiver operating characteristic curve (ROC) and Kaplan-Meier curve were drawn by the R programming language (3.6.3). RESULTS: The four glycolysis-related genes (B3GAT3, CENPA, AGL, and ALDH3A2) associated with prognosis were identified using Cox proportional regression analysis. A risk score staging system was established to predict the outcomes of patients with ccRCC. The patients with ccRCC were classified into the low-risk group and high-risk group. CONCLUSIONS: We have successfully constructed a risk staging model for ccRCC. The model has a better performance in predicting the prognosis of patients, which may have positive reference value for the treatment and curative effect evaluation of ccRCC.

A 3-Protein Expression Signature of Neuroblastoma for Outcome Prediction.

Neuroblastoma (NB) is the most common extracranial solid tumor in children with contrasting outcomes. Precise risk assessment contributes to prognosis prediction, which is critical for treatment strategy decisions. In this study, we developed a 3-protein predictor model, including the neural stem cell marker Msi1, neural differentiation marker ID1, and proliferation marker proliferating cell nuclear antigen (PCNA), to improve clinical risk assessment of patients with NB. Kaplan-Meier analysis in the microarray data (GSE16476) revealed that low expression of ID1 and high expression of Msi1 and PCNA were associated with poor prognosis in NB patients. Combined application of these 3 markers to constitute a signature further stratified NB patients into different risk subgroups can help obtain more accurate prediction performance. Survival prognostic power of age and Msi1_ID1_PCNA signature by receiver operating characteristics analysis showed that this signature predicted more effectively and sensitively compared with classic risk stratification system, compensating for the deficiency of the prediction function of the age. Furthermore, we validated the expressions of these 3 proteins in neuroblastic tumor spectrum tissues by immunohistochemistry revealed that Msi1 and PCNA exhibited increased expression in NB compared with intermedial ganglioneuroblastoma and benign ganglioneuroma, whereas ID1 levels were reduced in NB. In conclusion, we established a robust risk assessment predictor model based on simple immunohistochemistry for therapeutic decisions of NB patients.

Pre-incubation with hucMSC-exosomes prevents cisplatin-induced nephrotoxicity by activating autophagy.

BACKGROUND: The administration of cisplatin is limited due to its nephrotoxic side effects, and prevention of this nephrotoxicity of cisplatin is difficult. Mesenchymal stem cell (MSC)-derived exosomes have been implicated as a novel therapeutic approach for tissue injury. In this study, we demonstrated that the pretreatment of human umbilical cord MSC-derived exosomes (hucMSC-Ex) can prevent the development of cisplatin-induced renal toxicity by activation of autophagy in vitro and in vivo. METHODS: In vitro, rat renal tubular epithelial (NRK-52E) cells were pre-incubated with exosomes from hucMSC or HFL1 (human lung fibroblast cells; as control) for 30 min, and 3-methyladenine (an autophagic inhibitor) and rapamycin (an autophagic inducer) for 1 h before cisplatin treatment for 8 h, respectively. Cells were harvested for apoptosis assay, enzyme-linked immunosorbent assay (ELISA), Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). In vivo, we constructed cisplatin-induced acute kidney injury rat models. Prior to treatment with cisplatin for 0.5 h, hucMSC-Ex or HFL1-Ex were injected into the kidneys via the renal capsule. 3-methyladenine and rapamycin were injected under the kidney capsule before hucMSC-Ex. All animals were sacrificed at 3 days after cisplatin injection. Renal function, Luminex assay, tubular apoptosis and proliferation, and autophagy response were evaluated. RESULTS: hucMSC-Ex inhibited cisplatin-induced mitochondrial apoptosis and secretion of inflammatory cytokines in renal tubular epithelial cells in vitro. hucMSC-Ex increased the expression of the autophagic marker protein LC3B and the autophagy-related genes ATG5 and ATG7 in NRK-52E cells. Rapamycin mimicked the effects of hucMSC-Ex in protecting against cisplatin-induced renal injury, while the effects were abrogated by the autophagy inhibitor 3-methyladenine in the animals. CONCLUSIONS: Our findings indicate that the activation of autophagy induced by hucMSC-Ex can effectively relieve the nephrotoxicity of cisplatin. Therefore, pre-treatment of hucMSC-Ex may be a new method to improve the therapeutic effect of cisplatin.

Inhibition of Dopamine Receptor D4 Impedes Autophagic Flux, Proliferation, and Survival of Glioblastoma Stem Cells.

Glioblastomas (GBM) grow in a rich neurochemical milieu, but the impact of neurochemicals on GBM growth is largely unexplored. We interrogated 680 neurochemical compounds in patient-derived GBM neural stem cells (GNS) to determine the effects on proliferation and survival. Compounds that modulate dopaminergic, serotonergic, and cholinergic signaling pathways selectively affected GNS growth. In particular, dopamine receptor D4 (DRD4) antagonists selectively inhibited GNS growth and promoted differentiation of normal neural stem cells. DRD4 antagonists inhibited the downstream effectors PDGFRß, ERK1/2, and mTOR and disrupted the autophagy-lysosomal pathway, leading to accumulation of autophagic vacuoles followed by G0/G1 arrest and apoptosis. These results demonstrate a role for neurochemical pathways in governing GBM stem cell proliferation and suggest therapeutic approaches for GBM.

Transarterial chemoembolization (TACE) plus percutaneous ethanol injection (PEI) for the treatment of unresectable hepatocellular carcinoma: a meta-analysis of randomized controlled trials.

Transarterial chemoembolization (TACE) plus percutaneous ethanol injection (PEI) have been used for patients with unresectable hepatocellular carcinoma (HCC). However, whether the combination therapy of TACE plus PEI is better than TACE or PEI alone in the treatment of HCC remains controversial. Thus, we conducted this meta-analysis to assess the efficacy of combined therapy for unresectable HCC compared with that of TACE or PEI alone. Randomized controlled trials (RCTs) published from Pubmed, Embase, Web Of Science, Chinese Biomedical Literature database (SinoMed), China National Knowledge Infrastructure (CNKI), and Wanfang database, were systematically reviewed to assess the survival benefits and tumor recurrence for HCC patients treated with TACE plus PEI. Pooled risk ratio (RR) with 95% confidence intervals (95% CIs) for survival rate and tumor recurrence rate were calculated using a random-effects or fixed-effects model, depending on the heterogeneity between the included studies. 19 RCTs met the inclusion criteria were included in this meta-analysis with a total number of 1948 patients. The pooled results showed that the combination therapy of TACE plus PEI significantly improved 1, 2, 3-year survival rate [RR1-year = 1.24, 95% CI: 1.17-1.31, P = 0.000; RR2-year = 1.64, 95% CI: 1.44-1.87, P = 0.000; RR3-year = 2.27, 95% CI: 1.93-2.67, P = 0.000] compared with that of TACE or PEI alone. The local tumor recurrence rate in HCC patients treated with TACE plus PEI was lower than that of monotherapy (RR = 0.53, 95% CI: 0.29-0.96; P = 0.035). The combined therapy of TACE with PEI also significantly reduced the AFP level (RR = 1.40, 95% CI: 1.19-1.66, P = 0.000) and tumor size (>50%) (RR = 1.61, 95% CI: 1.40-1.85, P = 0.000). This meta-analysis confirms the benefits of TACE + PEI in the treatment of unresectable HCC, with an improvement in survival rate, and a reduction in local tumor recurrence, AFP level, and tumor size.

Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity.

Glioblastoma (GBM) is a cancer comprised of morphologically, genetically, and phenotypically diverse cells. However, an understanding of the functional significance of intratumoral heterogeneity is lacking. We devised a method to isolate and functionally profile tumorigenic clones from patient glioblastoma samples. Individual clones demonstrated unique proliferation and differentiation abilities. Importantly, naïve patient tumors included clones that were temozolomide resistant, indicating that resistance to conventional GBM therapy can preexist in untreated tumors at a clonal level. Further, candidate therapies for resistant clones were detected with clone-specific drug screening. Genomic analyses revealed genes and pathways that associate with specific functional behavior of single clones. Our results suggest that functional clonal profiling used to identify tumorigenic and drug-resistant tumor clones will lead to the discovery of new GBM clone-specific treatment strategies.

Early B-cell factor 3 (EBF3) is a novel tumor suppressor gene with promoter hypermethylation in pediatric acute myeloid leukemia.

BACKGROUND: Pediatric acute myeloid leukemia (AML) comprises up to 20% of all childhood leukemia. Recent research shows that aberrant DNA methylation patterning may play a role in leukemogenesis. The epigenetic silencing of the EBF3 locus is very frequent in glioblastoma. However, the expression profiles and molecular function of EBF3 in pediatric AML is still unclear. METHODS: Twelve human acute leukemia cell lines, 105 pediatric AML samples and 30 normal bone marrow/idiopathic thrombocytopenic purpura (NBM/ITP) control samples were analyzed. Transcriptional level of EBF3 was evaluated by semi-quantitative and real-time PCR. EBF3 methylation status was determined by methylation specific PCR (MSP) and bisulfite genomic sequencing (BGS). The molecular mechanism of EBF3 was investigated by apoptosis assays and PCR array analysis. RESULTS: EBF3 promoter was hypermethylated in 10/12 leukemia cell lines. Aberrant EBF3 methylation was observed in 42.9% (45/105) of the pediatric AML samples using MSP analysis, and the BGS results confirmed promoter methylation. EBF3 expression was decreased in the AML samples compared with control. Methylated samples revealed similar survival outcomes by Kaplan-Meier survival analysis. EBF3 overexpression significantly inhibited cell proliferation and increased apoptosis. Real-time PCR array analysis revealed 93 dysregulated genes possibly implicated in the apoptosis of EBF3-induced AML cells. CONCLUSION: In this study, we firstly identified epigenetic inactivation of EBF3 in both AML cell lines and pediatric AML samples for the first time. Our findings also showed for the first time that transcriptional overexpression of EBF3 could inhibit proliferation and induce apoptosis in AML cells. We identified 93 dysregulated apoptosis-related genes in EBF3-overexpressing, including DCC, AIFM2 and DAPK1. Most of these genes have never been related with EBF3 over expression. These results may provide new insights into the molecular mechanism of EBF3-induced apoptosis; however, further research will be required to determine the underlying details. Our findings suggest that EBF3 may act as a putative tumor suppressor gene in pediatric AML.

Molecular targeting of the oncoprotein PLK1 in pediatric acute myeloid leukemia: RO3280, a novel PLK1 inhibitor, induces apoptosis in leukemia cells.

Polo-like kinase 1 (PLK1) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs. RO3280 was recently identified as a novel PLK1 inhibitor; however its therapeutic effects in leukemia treatment are still unknown. We found that the PLK1 protein was highly expressed in leukemia cell lines as well as 73.3% (11/15) of pediatric acute myeloid leukemia (AML) samples. PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001). Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor PLK1 expression (p = 0.002). The 50% inhibitory concentration (IC50) of RO3280 for acute leukemia cells was between 74 and 797 nM. The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM, respectively. RO3280 induced apoptosis and cell cycle disorder in leukemia cells. RO3280 treatment regulated several apoptosis-associated genes. The regulation of DCC, CDKN1A, BTK, and SOCS2 was verified by western blot. These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined.

Toll-like receptor 4 prompts human breast cancer cells invasiveness via lipopolysaccharide stimulation and is overexpressed in patients with lymph node metastasis.

Toll-like receptor (TLR)4-mediated signaling has been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study investigated the expression and biological role of TLR4 in human breast cancer metastasis. MCF-7 and MDA-MB-231 are human breast cancer cell lines with low and high metastatic potential, respectively. Using lipopolysaccharide (LPS) to stimulate MCF-7 and MDA-MB-231 cells, expression of TLR4 mRNA and protein increased compared with that in control cells. TLR4 activation notably up-regulated expression of matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor(VEGF) mRNA and their secretion in the supernatants of both cell lines. LPS enhanced invasion of MDA-MB-231 cells by transwell assay and MCF-7 cells by wound healing assay. LPS triggered increased expression of TLR4 downstream signaling pathway protein myeloid differentiation factor 88(MyD88) and resulted in interleukin (IL)-6 and IL-10 higher production by human breast cancer cells. Stimulation of TLR4 with LPS promoted tumorigenesis and formed metastatic lesions in liver of nude mice. Moreover, expression of TLR4 and MyD88 as well as invasiveness and migration of the cells could be blocked by TLR4 antagonist. Combined with clinicopathological parameters, TLR4 was overexpressed in human breast cancer tissue and correlated with lymph node metastasis. These findings indicated that TLR4 may participate in the progression and metastasis of human breast cancer and provide a new therapeutic target.

Extrarenal nephroblastomatosis in children: a report of two cases.

BACKGROUND: Extrarenal nephroblastomatosis is a rare entity which occurs in retroperitoneum and inguinal region predominantly. Here we report two cases of primary extrarenal nephroblastomatosis of Han Chinese in Asian in unusual locations, one is located in testis and paratestis, and the other is paraspinal cord. CASE PRESENTATION: Patient 1 was a 19-month-old boy with a hard and nodular mass adherent to the left testis in inguinal region. Patient 2 was a 9-month-old boy with a 1 × 0.7 × 0.4 cm mass in spinal canal at the midline thoracolumbar region. Histological examinations of the two patients after operations revealed extrarenall nephroblastomatosis with multiple nephrogenic foci, composed of immature glomeruli, tubules and blastemal cells.Then the patients were closely monitored without adjuvant chemotherapy, and has been alive and well without any recurrence for >6 months. CONCLUSIONS: Most nephrogenic rests remain subclinical, and thus, complete excision of the lesion with conservative treatment is recommended. Otherwise, nephrogenic rests are close associated with Wilms tumor and regular follow-up is required to ensure early detection of malignant transformation.