Crystal structure of the human N-Myc downstream-regulated gene 2 protein provides insight into its role as a tumor suppressor

Considerable attention has recently been paid to the N-Myc downstream-regulated gene (NDRG) family because of its potential as a tumor suppressor in many human cancers. Primary amino acid sequence information suggests that the NDRG family proteins may belong to the α/β-hydrolase (ABH) superfamily; however, their functional role has not yet been determined. Here, we present the crystal structures of the human and mouse NDRG2 proteins determined at 2.0 and 1.7 Å resolution, respectively. Both NDRG2 proteins show remarkable structural similarity to the ABH superfamily, despite limited sequence similarity. Structural analysis suggests that NDRG2 is a nonenzymatic member of the ABH superfamily, because it lacks the catalytic signature residues and has an occluded substrate-binding site. Several conserved structural features suggest NDRG may be involved in molecular interactions. Mutagenesis data based on the structural analysis support a crucial role for helix α6 in the suppression of TCF/β-catenin signaling in the tumorigenesis of human colorectal cancer, via a molecular interaction.     

A novel pheromone dispenser for mating disruption of the leafminer Phyllocnistis citrella (Lepidoptera: Gracillariidae)

The sex pheromone of the leafminer Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae) was deployed in a Florida citrus (Citrus spp.) grove by using a novel deployment device (IFM-413) containing SPLAT, a flowable formulation of an emulsified wax compound designed to provide slow release of semiochemicals. The device consisted of two disks connected by string. Each disk was loaded with 1 g of SPLAT containing either 0.15% (Z,Z,E)-7,11,13-hexadecatrienal (triene) or 2% (Z,Z)-7,11-hexadecadienal (diene). The devices were deployed using a two-dimensional multivariate design to determine the optimal rate of pheromone per unit area and degree of aggregation of the deployment devices (number of trees treated per unit area). The IFM-413 device proved effective at becoming securely entangled in tree branches. Furthermore, the devices effectively delivered pheromone-loaded SPLAT that resulted in disruption of trap catch of male P. citrella. Response surfaces showed a quadratic response of trap catch disruption to both total amount of pheromone per unit area and the degree of aggregation of the deployed devices (number of treated trees per unit area). The response surfaces for 0.15% triene or 2.0% diene were similar. The diene produced an effect similar to that of the triene at approximately 13 times the rate of the triene. The greatest disruption of trap catch occurred when the number of treated trees per unit area was greatest (no aggregation of deployment devices). Manufacturing, packaging, and mechanical deployment of the devices remain to be investigated.     

Evolution of Metamorphism in Thymidylate Synthases Within the Primate Lineages

Crystal structures of human thymidylate synthase (hTS) revealed that the protein exists in active and inactive conformations, defined by the position of a loop containing the active site nucleophile. TS is highly homologous among diverse species; however, the residue at position 163 (hTS) differs among species. Arginine at this position is predicted by structural modeling to enable conformational switching. Arginine or lysine is reported at this position in all mammals in the GenBank and Ensembl databases, with arginine reported in only primates. Sequence analysis of the TS gene of representative primates revealed that arginine occurs at this relative position in all primates except a representative of prosimians. Mutant human proteins were created with residues at position 163 that occur in TSs from prokaryotes and eukaryotes. Catalytic constants (k _cat) of mutant enzymes were 45–149% of hTS, with the lysine mutant (R163K) exhibiting the highest k _cat. The effect of lysine substitution on solution structure and on ligand binding was investigated. R163K exhibited higher intrinsic fluorescence, a more negative molar ellipticity, and higher dissociation constants (K _d) for ligands that modulate protein conformation than hTS. Temperature effects on intrinsic fluorescence and catalytic activity of hTS and R163K are consistent with proteins populating different conformational states. The data indicate that the enzyme with arginine at the position corresponding to 163 (hTS) evolved after the divergence of prosimians and simians and that substitution of lysine by arginine confers unique structural and functional properties to the enzyme expressed in simian primates.     

Inhibitory effects of the HDAC inhibitor valproic acid on prostate cancer growth are enhanced by simultaneous application of the mTOR inhibitor RAD001

AimsTo analyze the combined impact of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell growth.Main methodsPC-3, DU-145 and LNCaP cells were treated with RAD001, VPA or with an RAD001–VPA combination for 3 or 5 days. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT assay, flow cytometry and Western blotting, respectively. Effects of drug treatment on cell signaling pathways were determined.Key findingsSeparate application of RAD001 or VPA distinctly reduced tumor cell growth and impaired cell cycle progression. Significant additive effects were evoked when both drugs were used in concert. Particularly, the cell cycle regulating proteins cdk1, cdk2, cdk4 and cyclin B were reduced, whereas p21 and p27 were enhanced by the RAD001–VPA combination. Signaling analysis revealed deactivation of EGFr, ERK1/2 and p70S6k. Phosphorylation of Akt was diminished in DU-145 but elevated in PC-3 and LNCaP cells.SignificanceThe RAD001–VPA combination exerted profound antitumor properties on a panel of prostate cancer cell lines. Therefore, simultaneous blockage of HDAC and mTOR related pathways should be considered when designing novel therapeutic strategies for treating prostate carcinoma.     

Suppurative thyroiditis caused by Salmonella enteritidis

Bacterial thyroiditis is a rare disease, and one of which the clinical symptoms and signs are frequently misleading. On the other hand, prompt diagnosis is crucial for successful treatment. We report the case of an 82 year-old man with diabetes mellitus type 2 and a history of steroid treatment who presented with severe odynophagia and dysphagia associated with fever, chills, sore throat and right ear pain. Based on the clinical picture, radiological studies, thyroid cytology, blood and thyroid aspirate culture, suppurative thyroiditis caused by Salmonella enteritidis was diagnosed. The patient was successfully treated with antibiotics and surgical drainage.     

Phosphorylation of RSK2 at Tyr529 by FGFR2-p38 enhances human mammary epithelial cells migration

The members of p90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases are downstream effectors of MAPK/ERK pathway that regulate diverse cellular processes including cell growth, proliferation and survival. In carcinogenesis, RSKs are thought to modulate cell motility, invasion and metastasis. Herein, we have studied an involvement of RSKs in FGF2/FGFR2-driven behaviours of mammary epithelial and breast cancer cells. We found that both silencing and inhibiting of FGFR2 attenuated phosphorylation of RSKs, whereas FGFR2 overexpression and/or its stimulation with FGF2 enhanced RSKs activity. Moreover, treatment with ERK, Src and p38 inhibitors revealed that p38 kinase acts as an upstream RSK2 regulator. We demonstrate for the first time that in FGF2/FGFR2 signalling, p38 but not MEK/ERK, indirectly activated RSK2 at Tyr529, which facilitated phosphorylation of its other residues (Thr359/Ser363, Thr573 and Ser380). In contrast to FGF2-triggered signalling, inhibition of p38 in the EGF pathway affected only RSK2-Tyr529, without any impact on the remaining RSK phosphorylation sites. p38-mediated phosphorylation of RSK2-Tyr529 was crucial for the transactivation of residues located at kinase C-terminal domain and linker-region, specifically, in the FGF2/FGFR2 signalling pathway. Furthermore, we show that FGF2 promoted anchorage-independent cell proliferation, formation of focal adhesions and cell migration, which was effectively abolished by treatment with RSKs inhibitor (FMK). These indicate that RSK2 activity is indispensable for FGF2/FGFR2-mediated cellular effects. Our findings identified a new FGF2/FGFR2-p38-RSK2 pathway, which may play a significant role in the pathogenesis and progression of breast cancer and, hence, may present a novel therapeutic target in the treatment of FGFR2-expressing tumours.     

Improved prediction of residue flexibility by embedding optimized amino acid grouping into RSA-based linear models

Knowledge of protein flexibility is vital for deciphering the corresponding functional mechanisms. This knowledge would help, for instance, in improving computational drug design and refinement in homology-based modeling. We propose a new predictor of the residue flexibility, which is expressed by B-factors, from protein chains that use local (in the chain) predicted (or native) relative solvent accessibility (RSA) and custom-derived amino acid (AA) alphabets. Our predictor is implemented as a two-stage linear regression model that uses RSA-based space in a local sequence window in the first stage and a reduced AA pair-based space in the second stage as the inputs. This method is easy to comprehend explicit linear form in both stages. Particle swarm optimization was used to find an optimal reduced AA alphabet to simplify the input space and improve the prediction performance. The average correlation coefficients between the native and predicted B-factors measured on a large benchmark dataset are improved from 0.65 to 0.67 when using the native RSA values and from 0.55 to 0.57 when using the predicted RSA values. Blind tests that were performed on two independent datasets show consistent improvements in the average correlation coefficients by a modest value of 0.02 for both native and predicted RSA-based predictions.     

Association of microRNAs and pathologic response to preoperative chemotherapy in triple negative breast cancer: preliminary report

Triple negative breast cancer (TNBC) has caught the attention of oncologists worldwide because of poor prognosis and paucity of targeted therapies. Gene pathways have been widely studied, but less is known about epigenetic factors such as microRNAs (miRNAs) and their role in tailoring an individual systemic and surgical approach for breast cancer patients. The aim of the study was to examine selected miRNAs in TNBC core biopsies sampled before preoperative chemotherapy and the subsequent pathologic response in mastectomy or breast conservation specimens. Prior to treatment, core needle biopsies were collected from 11 female patients with inoperable locally advanced TNBC or large resectable tumors suitable for down-staging. In all 11 TNBC core biopsies we analyzed 19 miRNAs per sample: 512, 190, 200, 346, 148, 449, 203, 577, 93, 126, 423, 129, 193, 182, 136, 135, 191, 122 and 222 (miRCURY LNA? Universal RT microRNA polymerase chain reaction Custom Pick & Mixpanels). The Wilcoxon signed-rank test was used to compare related samples. Ingenuity pathway analysis was used to evaluate potential functional significance of differentially expressed miRNAs. Statistical analysis showed that 3 of 19 miRNAs differed in relation to pathologic response i.e. good versus poor. These differences failed to reach statistical significance, although a trend was observed (p = 0.06). Among these miRNAs, we identified—miR-200b-3p, miR-190a and miR-512-5p. In summary, our results indicate that higher miR-200b-3p, higher miR-190a and lower miR-512-5p expression levels in core biopsies sampled from TNBC patients may be associated with better pathologic response to chemotherapy and the increased feasibility of breast conserving surgery in these patients. Although these results were from a small cohort, they provide an important basis for larger, prospective, multicenter studies to investigate the potential role of miRNAs in neoadjuvant setting.     

A Possible Link between Food and Mood: Dietary Impact on Gut Microbiota and Behavior in BALB/c Mice

Major depressive disorder is a debilitating disease in the Western World. A western diet high in saturated fat and refined sugar seems to play an important part in disease development. Therefore, this study is aimed at investigating whether saturated fat or sucrose predisposes mice to develop behavioral symptoms which can be interpreted as depression-like, and the possible influence of the gut microbiota (GM) in this. Fourty-two mice were randomly assigned to one of three experimental diets, a high-fat, a high-sucrose or a control diet for thirteen weeks. Mice on high-fat diet gained more weight (p = 0.00009), displayed significantly less burrowing behavior than the control mice (p = 0.034), and showed decreased memory in the Morris water maze test compared to mice on high-sucrose diet (p = 0.031). Mice on high-sucrose diet burrowed less goal-oriented, showed greater latency to first bout of immobility in the forced swim test when compared to control mice (p = 0.039) and high-fat fed mice (p = 0.013), and displayed less anxiety than mice on high-fat diet in the triple test (p = 0.009). Behavioral changes were accompanied by a significant change in GM composition of mice fed a high-fat diet, while no difference between diet groups was observed for sucrose preferences, LPS, cholesterol, HbA1c, BDNF and the cytokines IL-1α, IL-1β, IL-6, IL-10, IL-12(p70), IL-17 and TNF-α. A series of correlations was found between GM, behavior, BDNF and inflammatory mediators. In conclusion, the study shows that dietary fat and sucrose affect behavior, sometimes in opposite directions, and suggests a possible association between GM and behavior.