Description of a marine peritrichous ciliate, Pseudovorticella sinensis n. sp. (Ciliophora, Peritrichia) from China

A new marine peritrich ciliate, Pseudovorticella sinensis n. sp. was isolated from a shrimp-farming pond in the littoral area of Qingdao, China. The morphology, infraciliature, and silverline system were studied based on living and silver-impregnated specimens. This species is characterized by (1) an elongated bell-shaped body that measures 50-60 x 35-45 microm in vivo, (2) one large, ventrally located contractile vacuole, and (3) a pellicle covered by a layer of transparent, cortical vesicles. The number of transverse silverlines from the peristomial area to the aboral ciliary wreath is 26-32, and from the aboral ciliary wreath to the scopula is 12-15. The stalk measures about 160-250 microm long x 5-6 microm wide. The spasmoneme has one row of conspicuous thecoplasmic granules, which are about 0.8 microm in diameter.     

Nonredundant role of Bax and Bak in Bid-mediated apoptosis

Animal models suggest that Bax and Bak play an essential role in the implementation of apoptosis and as a result can hinder tumorigenesis. We analyzed the expression of these proteins in 50 human glioblastoma multiforme (GBM) tumors. We found that all the tumors expressed Bak, while three did not express Bax. In vitro, Bax-deficient GBM (BdGBM) exhibited an important resistance to various apoptogenic stimuli (e.g., UV, staurosporine, and doxorubicin) compared to the Bax-expressing GBM (BeGBM). Using an antisense strategy, we generated Bak(-) BeGBM and Bak(-) BdGBM, which enabled us to show that the remaining sensitivity of the BdGBM to apoptosis was due to the overexpression of Bak. Bax/Bak single or double deficiency had no influence on either the clonogenicity or the growth of tumors in Swiss nude mice. Of note, Bak(-) BeGBM cells were resistant to apoptosis induced by caspase 8 (C8) but not to that induced by granzyme B (GrB). Cells lacking both Bax and Bak (i.e., Bak(-) BdGBM) were completely resistant to all stimuli including the microinjection of C8 and GrB. We show that GrB-cleaved Bid and C8-cleaved Bid differ in size and utilize preferentially Bax and Bak, respectively, to promote cytochrome c release from mitochondria. Our results suggest that Bax deficiency is compensated by an increase of the expression of Bak in GBM and show, for the first time in human cancer, that the double Bax and Bak deficiency severely impairs the apoptotic program.     

Trophic factor withdrawal: p38 mitogen-activated protein kinase activates NHE1, which induces intracellular alkalinization

Trophic factor withdrawal induces cell death by mechanisms that are incompletely understood. Previously we reported that withdrawal of interleukin-7 (IL-7) or IL-3 produced a rapid intracellular alkalinization, disrupting mitochondrial metabolism and activating the death protein Bax. We now observe that this novel alkalinization pathway is mediated by the pH regulator NHE1, as shown by the requirement for sodium, blocking by pharmacological inhibitors or use of an NHE1-deficient cell line, and the altered phosphorylation of NHE1. Alkalinization also required the stress-activated p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK activity with pharmacological inhibitors or expression of a dominant negative kinase prevented alkalinization. Activated p38 MAPK directly phosphorylated the C terminus of NHE1 within a 40-amino-acid region. Analysis by mass spectroscopy identified four phosphorylation sites on NHE1, Thr 717, Ser 722, Ser 725, and Ser 728. Thus, loss of trophic cytokine signaling induced the p38 MAPK pathway, which phosphorylated NHE1 at specific sites, inducing intracellular alkalinization.     

Chemoradiotherapy or chemotherapy as adjuvant treatment for resected gastric cancer: should we use selection criteria?

BackgroundThe management of gastric adenocarcinoma is essentially based on surgery followed by adjuvant treatment. Adjuvant chemotherapy (CT) as well as chemoradiotherapy (CTRT) have proven their effectiveness in survival outcomes compared to surgery alone. However, there is little data comparing the two adjuvant approaches. This study aimed to compare the prognosis and survival outcomes of patients with gastric adenocarcinoma operated and treated by adjuvant radio-chemotherapy or chemotherapyMaterials and methodsWe retrospectively evaluated 80 patients with locally advanced gastric cancer (LGC) who received adjuvant treatment. We compared survival outcomes and patterns of recurrence of 53 patients treated by CTRT and those of 27 patients treated by CT.ResultsAfter a median follow-up of 38.48 months, CTRT resulted in a significant improvement of the 5-year PFS (60.9% vs. 36%, p = 0.03) and the 5-year OS (55.9% vs. 33%, p = 0.015) compared to adjuvant CT. The 5-year OS was significantly increased by adjuvant CTRT (p = 0.046) in patients with lymph node metastasis, and particularly those with advanced pN stage (p = 0.0078) and high lymph node ratio (LNR) exceeding 25% (p = 0.012). Also, there was a significant improvement of the PFS of patients classified pN2–N3 (p = 0.022) with a high LNR (p = 0.018). CTRT was also associated with improved OS and PFS in patients with lymphovascular and perineural invasion (LVI and PNI) compared to chemotherapy.ConclusionThere is a particular survival benefit of adding radiotherapy to chemotherapy in patients with selected criteria such as lymph node involvement, high LNR LVI, and PNI.     

MicroRNA Profiling in Intraocular Medulloepitheliomas

Purpose

To study the differential expression of microRNA (miRNA) profiles between intraocular medulloepithelioma (ME) and normal control tissue (CT).

Material and Methods

Total RNA was extracted from formalin fixed paraffin embedded (FFPE) intraocular ME (n=7) and from age matched ciliary body controls (n=8). The clinical history and phenotype was recorded. MiRNA profiles were determined using the Affymetrix GeneChip miRNA Arrays analyzed using expression console 1.3 software. Validation of significantly dysregulated miRNA was confimed by quantitaive real-time PCR. The web-based DNA Intelligent Analysis (DIANA)-miRPath v2.0 was used to perform enrichment analysis of differentially expressed (DE) miRNA gene targets in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway.

Results

The pathologic evaluation revealed one benign (benign non-teratoid, n=1) and six malignant tumors (malignant teratoid, n=2; malignant non-teratoid, n = 4). A total of 88 miRNAs were upregulated and 43 miRNAs were downregulated significantly (P<0.05) in the tumor specimens. Many of these significantly dysregulated miRNAs were known to play various roles in carcinogenesis and tumor behavior. RT-PCR validated three significantly upregulated miRNAs and three significantly downregulated miRNAs namely miR-217, miR-216a, miR-216b, miR-146a, miR-509-3p and miR-211. Many DE miRNAs that were significant in ME tumors showed dysregulation in retinoblastoma, glioblastoma, and precursor, normal and reactive human cartilage. Enriched pathway analysis suggested a significant association of upregulated miRNAs with 15 pathways involved in prion disease and several types of cancer. The pathways involving significantly downregulated miRNAs included the toll-like receptor (TLR) (p<4.36E-16) and Nuclear Factor kappa B (NF-κB) signaling pathways (p<9.00E-06).

Conclusions

We report significantly dysregulated miRNAs in intraocular ME tumors, which exhibited abnormal profiles in other cancers as well such as retinoblastoma and glioblastoma. Pathway analysis of all dysregulated miRNAs shared commonalities with other cancer pathways.     

Knockdown of CDK2AP1 in Primary Human Fibroblasts Induces p53 Dependent Senescence

Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) is known to be a tumor suppressor that plays a role in cell cycle regulation by sequestering monomeric CDK2, and targeting it for proteolysis. A reduction of CDK2AP1 expression is considered to be a negative prognostic indicator in patients with oral squamous cell carcinoma and also associated with increased invasion in human gastric cancer tissue. CDK2AP1 overexpression was shown to inhibit growth, reduce invasion and increase apoptosis in prostate cancer cell lines. In this study, we investigated the effect of CDK2AP1 downregulation in primary human dermal fibroblasts. Using a short-hairpin RNA to reduce its expression, we found that knockdown of CDK2AP1in primary human fibroblasts resulted in reduced proliferation and in the induction of senescence associated beta-galactosidase activity. CDK2AP1 knockdown also resulted in a significant reduction in the percentage of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Immunocytochemical analysis also revealed that the CDK2AP1 knockdown significantly increased the percentage of cells that exhibited γ-H2AX foci, which could indicate presence of DNA damage. CDK2AP1 knockdown also resulted in increased mRNA levels of p53, p21, BAX and PUMA and p53 protein levels. In primary human fibroblasts in which p53 and CDK2AP1 were simultaneously downregulated, there was: (a) no increase in senescence associated beta-galactosidase activity, (b) decrease in the number of cells in the G1-phase and increase in number of cells in the S-phase of the cell cycle, and (c) decrease in the mRNA levels of p21, BAX and PUMA when compared with CDK2AP1 knockdown only fibroblasts. Taken together, this suggests that the observed phenotype is p53 dependent. We also observed a prominent increase in the levels of ARF protein in the CDK2AP1 knockdown cells, which suggests a possible role of ARF in p53 stabilization following CDK2AP1 knockdown. Altogether, our results show that knockdown of CDK2AP1 in primary human fibroblasts reduced proliferation and induced premature senescence, with the observed phenotype being p53 dependent.     

Akhirin regulates the proliferation and differentiation of neural stem cells in intact and injured mouse spinal cord

Although the central nervous system is considered a comparatively static tissue with limited cell turnover, cells with stem cell properties have been isolated from most neural tissues. The spinal cord ependymal cells show neural stem cell potential in vitro and in vivo in injured spinal cord. However, very little is known regarding the ependymal niche in the mouse spinal cord. We previously reported that a secreted factor, chick Akhirin, is expressed in the ciliary marginal zone of the eye, where it works as a heterophilic cell‐adhesion molecule. Here, we describe a new crucial function for mouse Akhirin (M‐AKH) in regulating the proliferation and differentiation of progenitors in the mouse spinal cord. During embryonic spinal cord development, M‐AKH is transiently expressed in the central canal ependymal cells, which possess latent neural stem cell properties. Targeted inactivation of the AKH gene in mice causes a reduction in the size of the spinal cord and decreases BrdU incorporation in the spinal cord. Remarkably, the expression patterns of ependymal niche molecules in AKH knockout (AKH?/?) mice are different from those of AKH+/+, both in vitro and in vivo. Furthermore, we provide evidence that AKH expression in the central canal is rapidly upregulated in the injured spinal cord. Taken together, these results indicate that M‐AKH plays a crucial role in mouse spinal cord formation by regulating the ependymal niche in the central canal. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 494–504, 2015