A decision analysis model for KEGG pathway analysis.

BACKGROUND: The knowledge base-driven pathway analysis is becoming the first choice for many investigators, in that it not only can reduce the complexity of functional analysis by grouping thousands of genes into just several hundred pathways, but also can increase the explanatory power for the experiment by identifying active pathways in different conditions. However, current approaches are designed to analyze a biological system assuming that each pathway is independent of the other pathways. RESULTS: A decision analysis model is developed in this article that accounts for dependence among pathways in time-course experiments and multiple treatments experiments. This model introduces a decision coefficient-a designed index, to identify the most relevant pathways in a given experiment by taking into account not only the direct determination factor of each Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway itself, but also the indirect determination factors from its related pathways. Meanwhile, the direct and indirect determination factors of each pathway are employed to demonstrate the regulation mechanisms among KEGG pathways, and the sign of decision coefficient can be used to preliminarily estimate the impact direction of each KEGG pathway. The simulation study of decision analysis demonstrated the application of decision analysis model for KEGG pathway analysis. CONCLUSIONS: A microarray dataset from bovine mammary tissue over entire lactation cycle was used to further illustrate our strategy. The results showed that the decision analysis model can provide the promising and more biologically meaningful results. Therefore, the decision analysis model is an initial attempt of optimizing pathway analysis methodology.

Value of low-dose and optimized-length computed tomography (CT) scan in CT-guided percutaneous transthoracic needle biopsy of pulmonary nodules.

OBJECTIVE: To investigate the value of application of low-dose and optimized length CT scan on puncture results, complications and patients' radiation dosage during CT-guided percutaneous biopsy of pulmonary nodules (PTNB). METHODS: A total of 231 patients with PTNB under CT guidance were collected. Low dose scanning utilized tube current of 20 mA as compared with 40 mA in conventional dosage. Optimized length in CT is defined as intentionally narrowing the range of CT scanning just to cover 25 mm (5 layers) around the target layer during needle adjustment. According to whether low-dose scans and optimized length scans techniques were utilized, patients were divided into three groups: conventional group (conventional sequence + no optimization), optimized length group (conventional sequence + optimized length), and low-dose optimized length group (low dose sequence + optimized length). The ED (effective dose), the DLP (dose length product), the average CTDIvol (Volume CT dose index), total milliampere second between subgroups were compared. RESULTS: Compared with the conventional group, ED, intraoperative guidance DLP, total milliseconds and operation time in the optimized length group were reduced by 18.2% (P=0.01), 37% (P=0.003), 17.5% (P=0.013) and 13.3% (P=0.021) respectively. Compared with the optimized length group, the ED was reduced by 87%, preoperative positioning, intraoperative guidance and postoperative review DLP were also reduced by 88%, total milliampere second was reduced by 79%, with an average CTDIvol was reduced by 86%, in the low-dose optimized length group (P<0.001 for all). CONCLUSION: Optimizing the length during CT scanning can effectively reduce the intraoperative radiation dose and reduce the operation time compared with conventional plan; low-dose and optimized length CT scan can further reduce the total radiation dose compared with optimized length group with no differences on intraoperative complications, biopsy results and operation time.

Inhibition of protein kinase C zeta blocks the attachment of stable microtubules to kinetochores leading to abnormal chromosome alignment.

The attachment of spindle microtubules to kinetochores is crucial for accurate segregation of chromosomes to daughter cells during mitosis. While a growing number of proteins involving this step are being identified, its molecular mechanisms are still not clear. Here we show that protein kinase C zeta (PKCzeta) is localized at the mitotic spindle during mitosis and plays a role in stable kinetochore-microtubule attachment. Striking staining for PKCzeta was observed at the mitotic spindle and spindle poles in cells at prometaphase and metaphase. PKCzeta molecules at these stages were phosphorylated at Thr-410, as detected by a phosphospecific antibody. PKCzeta was also detected at the spindle midzone and the midbody during anaphase and telophase, respectively, and PKCzeta at these stages was no longer phosphorylated at Thr-410. The polarity determinants Par3 and Par6, which are known to associate with PKCzeta, were also localized to the spindles and spindle poles at prometaphase and metaphase. Knockdown of PKCzeta by RNA interference affected normal chromosome alignment leading to generation of cells with aberrant nuclei. A specific PKCzeta inhibitor strongly blocked the formation of cold-sensitive stable kinetochore microtubules, and thus prevented microtubule-kinetochore attachment. Treatment of cells with the PKCzeta inhibitor also dislocated the minus-end directed motor protein dynein from kinetochores, but not the mitotic checkpoint proteins Mad2 and CENP-E. Prolonged exposure to the PKCzeta inhibitor eventually resulted in cell death. These results suggest a critical role of PKCzeta in spindle microtubule-kinetochore attachment and subsequent chromosomal separation.

[Effect of serum restriction on insulin like growth factor-1 expressions and invasiveness in human trophoblast HTR-8/SVneo cells in vitro].

OBJECTIVE: To explore the effect of serum restriction on the invasiveness and expressions of insulin-like growth factor-1 (IGF-1) and matrix metalloproteinase-2 (MMP-2) in human trophoblast HTR-8/SVneo cells in vitro. METHODS: HTR-8/SVneo cells were cultured in the presence of 1%, 5%, or 10% fetal bovine serum (FBS) for 48 h. Fluorescence quantitative PCR and immunofluorescence staining were employed to examine the changes in IGF-1 and MMP-2 expressions at both the mRNA and protein levels in HTR-8/SVneo cells; MTT assay and Transwell invasion assay were used to assess the changes of the cell proliferation and the cell invasion ability, respectively. MMP-2 expression, cell proliferation and invasiveness were also assessed in the cells treated with recombinant human IGF-1. RESULTS: HTR-8/SVneo cells exhibited significantly lowered cell proliferation in cultures containing low concentrations of FBS (P<0.05). The expressions of IGF-1 and MMP-2 at both mRNA and protein levels were significantly down-regulated and the invasiveness was significantly lowered in cells cultured in the medium containing 1% FBS as compared with those of cells cultured in the presence of 5% and 10% FBS (P<0.05). Treatment of the cells with recombinant human IGF-1 significantly up-regulated MMP-2 expression (P<0.05) and increased the cell invasiveness (P<0.05). CONCLUSIONS: FBS restriction down-regulates IGF-1 expression in human trophoblast HTR-8/SVneo cells and suppress the cell invasiveness possibly by suppressing MMP-2 expression. Treatment with recombinant human IGF-1 can up-regulate MMP-2 expression and promote the invasiveness of HTR-8/SVneo cells.

TIM, a Dbl-related protein, regulates cell shape and cytoskeletal organization in a Rho-dependent manner.

The Dbl-like guanine nucleotide exchange factors (GEFs) have been implicated in direct activation of the Rho family of small GTPases. We previously isolated transforming immortalized mammary (TIM) as a Dbl-like protein. Here, we show that, when expressed in cells, TIM was a potent activator of RhoA. Like activated Rho proteins, expression of TIM potentiated the serum response factor (SRF)- and AP-1-regualted transcriptional activities and activated the SAPK/JNK signaling pathway. In NIH 3T3 cells, TIM induced transforming foci, which was inhibited by the ROCK inhibitor Y-27632 or the dominant negative mutants of Rho proteins. Expression of TIM led to pronounced changes in cell shape and organization of the actin cytoskeleton, including the formation of thick stress fibers at the cell periphery and cell rounding. TIM also promoted redistribution of vinculin-enriched focal adhesions at the cell periphery and increased the phosphorylation of myosin light chain (MLC). These results, taken together, suggest that TIM acts as an upstream regulator for the RhoA/ROCK-mediated cellular functions.

Inhibition of cyclin-dependent kinase 1 induces cytokinesis without chromosome segregation in an ECT2 and MgcRacGAP-dependent manner.

Cleavage furrow formation marks the onset of cell division during early anaphase. The small GTPase RhoA and its regulators ECT2 and MgcRacGAP have been implicated in furrow ingression in mammalian cells, but the signaling upstream of these molecules remains unclear. We now show that the inhibition of cyclin-dependent kinase (Cdk)1 is sufficient to initiate cytokinesis. When mitotically synchronized cells were treated with the Cdk-specific inhibitor BMI-1026, the initiation of cytokinesis was induced precociously before chromosomal separation. Cytokinesis was also induced by the Cdk1-specific inhibitor purvalanol A but not by Cdk2/Cdk5- or Cdk4-specific inhibitors. Consistent with initiation of precocious cytokinesis by Cdk1 inhibition, introduction of anti-Cdk1 monoclonal antibody resulted in cells with aberrant nuclei. Depolymerization of mitotic spindles by nocodazole inhibited BMI-1026-induced precocious cytokinesis. However, in the presence of a low concentration of nocodazole, BMI-1026 induced excessive membrane blebbing, which appeared to be caused by formation of ectopic cleavage furrows. Depletion of ECT2 or MgcRacGAP by RNA interference abolished both of the phenotypes (precocious furrowing after nocodazole release and excessive blebbing in the presence of nocodazole). RNA interference of RhoA or expression of dominant-negative RhoA efficiently reduced both phenotypes. RhoA was localized at the cleavage furrow or at the necks of blebs. We propose that Cdk1 inactivation is sufficient to activate a signaling pathway leading to cytokinesis, which emanates from mitotic spindles and is regulated by ECT2, MgcRacGAP, and RhoA. Chemical induction of cytokinesis will be a valuable tool to study the initiation mechanism of cytokinesis.