Oculocerebrorenal syndrome of Lowe (OCRL) controls leukemic T-cell survival by preventing excessive PI(4,5)P2 hydrolysis in the plasma membrane.

T-cell acute lymphoblastic leukemia (T-ALL) is one of the deadliest and most aggressive hematological malignancies, but its pathological mechanism in controlling cell survival is not fully understood. Oculocerebrorenal syndrome of Lowe is a rare X-linked recessive disorder characterized by cataracts, intellectual disability, and proteinuria. This disease has been shown to be caused by mutation of oculocerebrorenal syndrome of Lowe 1 (OCRL1; OCRL), encoding a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase involved in regulating membrane trafficking; however, its function in cancer cells is unclear. Here, we uncovered that OCRL1 is overexpressed in T-ALL cells, and knockdown of OCRL1 results in cell death, indicating the essential role of OCRL in controlling T-ALL cell survival. We show OCRL is primarily localized in the Golgi and can translocate to plasma membrane (PM) upon ligand stimulation. We found OCRL interacts with oxysterol-binding protein-related protein 4L, which facilitates OCRL translocation from the Golgi to the PM upon cluster of differentiation 3 stimulation. Thus, OCRL represses the activity of oxysterol-binding protein-related protein 4L to prevent excessive PI(4,5)P2 hydrolysis by phosphoinositide phospholipase C ß3 and uncontrolled Ca2+ release from the endoplasmic reticulum. We propose OCRL1 deletion leads to accumulation of PI(4,5)P2 in the PM, disrupting the normal Ca2+ oscillation pattern in the cytosol and leading to mitochondrial Ca2+ overloading, ultimately causing T-ALL cell mitochondrial dysfunction and cell death. These results highlight a critical role for OCRL in maintaining moderate PI(4,5)P2 availability in T-ALL cells. Our findings also raise the possibility of targeting OCRL1 to treat T-ALL disease.

Characterization and expression pattern of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase gene families in Populus.

Trehalose plays an important role in plant metabolism, growth development, and stress tolerance. Trehalose-6-phosphate synthase gene (TPS) and trehalose-6-phosphate phosphatase gene (TPP) are vital for the synthesis of trehalose. Populus is a prominent perennial woody plant, in which systematic genome-wide analysis of the TPS and TPP family is limited. In this study, 13 PtTPS and 10 PtTPP genes were identified in the Populus genome. Phylogenetic analysis indicated PtTPS and PtTPP genes were both divided into two subfamilies, and gene members of each subfamily have highly conserved intron structures. Analysis of cis-acting elements showed that PtTPS and PtTPP genes were involved in plant hormones and environmental stress responses. Expression profiles also found PtTPSs and PtTPPs expressed differently in response to salt stress, cold, mechanical damage, salicylic acid, and methyl jasmonate treatment. Furthermore, reverse transcription quantitative real-time PCR results found PtTPSs and PtTPPs displayed a specific expression pattern in the seven developmental stages of Populus male and female floral buds. This work will not only lead a foundation on reveal the functions of PtTPS and PtTPP gene families in trehalose regulation of poplar but also provide references to related trehalose research in other perennial plants.

High-level extracellular production and immobilisation of methyl parathion hydrolase from Plesiomonas sp. M6 expressed in Pichia pastoris.

Methyl parathion hydrolase (MPH) hydrolyses methyl parathion efficiently and specifically. Herein, we produced MPH from Plesiomonas sp. M6 using a Pichia pastoris multi-copy expression system. The original signal peptide sequence of the target gene was removed, and a modified coding sequence was synthesised. Multi-copy expression plasmids containing MPH were constructed using pHBM905BDM, and used to generate recombinant strains containing 1, 2, 3 or 4 copies of the MPH gene. The results showed that a higher target gene copy number increased the production of recombinant MPH (MPH-R), as anticipated. The expression level of the recombinant strain containing four copies of the MPH gene was increased to 1.9 U/ml using 500 ml shake flasks, and the specific activity was 15.8 U/mg. High-density fermentation further increased the target protein yield to 18.4 U/ml. Several metal ions were tested as additives, and Ni2+, Co2+ and Mg2+ at a concentration of 1 mM enhanced MPH-R activity by 196%, 201% and 154%, respectively. Enzyme immobilisation was then applied to overcome the difficulties in recovery, recycling and long-term stability associated with the free enzyme. Immobilised MPH-R exhibited significantly enhanced thermal and long-term stability, as well as broad pH adaptability. In the presence of inhibitors and chelating agents such as sodium dodecyl sulphate (SDS), immobilised MPH-R displayed 2-fold higher activity than free MPH-R, demonstrating its potential for industrial application.

Sirtuin 3 Alleviates Diabetic Cardiomyopathy by Regulating TIGAR and Cardiomyocyte Metabolism.

Background Impairment of glycolytic metabolism is suggested to contribute to diabetic cardiomyopathy. In this study, we explored the roles of SIRT3 (Sirtuin 3) on cardiomyocyte glucose metabolism and cardiac function. Methods and Results Exposure of H9c2 cardiomyocyte cell lines to high glucose (HG) (30 mmol/L) resulted in a gradual decrease in SIRT3 and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) expression together with increases in p53 acetylation and TP53-induced glycolysis and apoptosis regulator (TIGAR) expression. Glycolysis was significantly reduced in the cardiomyocyte exposed to HG. Transfection with adenovirus-SIRT3 significantly increased PFKFB3 expression and reduced HG-induced p53 acetylation and TIGAR expression. Overexpression of SIRT3 rescued impaired glycolysis and attenuated HG-induced reactive oxygen species formation and apoptosis. Knockdown of TIGAR in cardiomyocytes by using siRNA significantly increased PFKFB3 expression and glycolysis under hyperglycemic conditions. This was accompanied by a significant suppression of HG-induced reactive oxygen species formation and apoptosis. In vivo, overexpression of SIRT3 by an intravenous jugular vein injection of adenovirus-SIRT3 resulted in a significant reduction of p53 acetylation and TIGAR expression together with upregulation of PFKFB3 expression in the heart of diabetic db/db mice at day 14. Overexpression of SIRT3 further reduced reactive oxygen species formation and blunted microvascular rarefaction in the diabetic db/db mouse hearts. Overexpression of SIRT3 significantly blunted cardiac fibrosis and hypertrophy and improved cardiac function at day 14. Conclusions Our study demonstrated that SIRT3 attenuated diabetic cardiomyopathy via regulating p53 acetylation and TIGAR expression. Therefore, SIRT3 may be a novel target for abnormal energy metabolism in diabetes mellitus.

MutT Homolog1 has multifaceted role in glioma and is under the apparent orchestration by Hypoxia Inducible factor1 alpha.

AIMS: The study focused on the expression and role of a recent potential cancer therapeutic target protein, MutT Homolog1 (MTH1). MTH1 gets activated in an increased reactive oxygen species (ROS) environment and removes the oxidized nucleotides from the cell. The study aimed to check the role of MTH1 in DNA damage and apoptosis, migration and angiogenesis and also to examine its regulation in glioma. MAIN METHODS: The experiments were carried out in human glioma tissue samples and brain tissues of epilepsy patients (non-tumor control). We used two human glioblastomas cell lines, U87MG and U251MG cells. In order to study the role of MTH1 in glioma and to analyze the relation of MTH1 with Hif1α, we have used MTH1 siRNA and Hif1α siRNA respectively. KEY FINDINGS: We found an increased expression of MTH1 in glioma tissues compared to the non-tumor brain tissues. Correlation analysis revealed that those samples showing reduced expression of MTH1 also had high levels of DNA damage and apoptotic markers, while diminished expression of angiogenesis regulators and levels of migration. MTH1 knockdown in vitro by siRNA in tumor cell lines corroborates the above observation. This justifies the emergence of MTH1 inhibitors as potential first-in-class drugs. Mechanistically, our observations suggest that Hif1α may modulate MTH1 expression. SIGNIFICANCE: We found elevated MTH1 expression in glioma irrespective of their grades, while its inhibition affects multiple tumor progression pathways, and that targeting Hif1α could simulate the same.

Phosphatase orphan 1 inhibits myoblast proliferation and promotes myogenic differentiation.

Myogenesis includes sequential stages of progenitor cell proliferation, myogenic commitment and differentiation, myocyte fusion, and myotube maturation. Different stages of myogenesis are orchestrated and regulated by myogenic regulatory factors and various downstream cellular signaling. Here we identify phosphatase orphan 1 (Phospho1) as a new player in myogenesis. During activation, proliferation, and differentiation of quiescent satellite cells, the expression of Phospho1 gradually increases. Overexpression of Phospho1 inhibits myoblast proliferation but promotes their differentiation and fusion. Conversely, knockdown of Phospho1 accelerates myoblast proliferation but impairs myotube formation. Moreover, knockdown of Phospho1 decreases the OXPHO protein levels and mitochondria density, whereas overexpression of Phospho1 upregulates OXPHO protein levels and promotes mitochondrial oxygen consumption. Finally, we show that Phospho1 expression is controlled by myogenin, which binds to the promoter of Phospho1 to regulate its transcription. These results indicate a key role of Phospho1 in regulating myogenic differentiation and mitochondrial function.

Bioprospecting plant growth promoting endophytic bacteria isolated from Himalayan yew (Taxus wallichiana Zucc.).

The present study aims to investigate the endophytic bacteria, isolated from the roots of Taxus wallichiana Zucc. and designated as GBPI_TWL and GBPI_TWr, for their plant growth promoting traits. On the basis of phenotypic and molecular characters, the bacteria are identified as species of Burkholderia and Enterobacter, respectively. Both the bacteria could grow at wide range of temperature (5-40 °C, opt=25 °C) and pH (1.5-11.0, opt = 6-7), and tolerate salt concentration up to 12 %. While both the bacterial endophytes possessed siderophore, HCN, ammonia, and salicyclic acid producing abilities, GBPI_TWL showed IAA and ACC deaminase producing abilities, in addition. The bacteria were found to be potential phosphate solubilizers at wide temperature range (5-35 °C) by utilizing tricalcium, iron, and aluminium phosphate as substrate. Further, the bacterial isolates produced phytase and phosphatase enzymes in both acidic and alkaline conditions. Positive influence of the inoculation with the bioformulations of GBPI_TWL and GBPI_TWr was demonstrated on the test crops namely rice (Oryza sativa) and soybean (Glycine max) with respect to physico-chemical and plant growth parameters in net house experiments. The study will have implications in developing bioformulations, specifically for low temperature environments, in view of environmental sustainability.

Human alkaline ceramidase 2 promotes the growth, invasion, and migration of hepatocellular carcinoma cells via sphingomyelin phosphodiesterase acid-like 3B.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. It has a poor prognosis because it is often diagnosed at the advanced stage when treatments are limited. In addition, HCC pathogenesis is not fully understood, and this has affected early diagnosis and treatment of this disease. Human alkaline ceramidase 2 (ACER2), a key enzyme that regulates hydrolysis of cellular ceramides, affects cancer cell survival, however its role in HCC has not been well characterized. Our results showed that ACER2 is overexpressed in HCC tissues and cell lines. In addition, high ACER2 protein expression was associated with tumor growth; ACER2 knockdown resulted in decreased cell growth and migration. Sphingomyelin phosphodiesterase acid-like 3B (SMPDL3B) promoted HCC cell growth, invasion, and migration; SMPDL3B knockdown had a significant inhibitory effect on HCC tumor growth in vivo. Moreover, ACER2 positively regulated the protein level of SMPDL3B. Of note, ACER2/SMPDL3B promoted ceramide hydrolysis and S1P production. This axis induced HCC survival and could be blocked by inhibition of S1P formation. In conclusion, ACER2 promoted HCC cell survival and migration, possibly via SMPDL3B. Thus, inhibition of ACER2/SMPDL3B may be a novel therapeutic target for HCC treatment.

Expression and purification of soluble recombinant SapM from Mycobacterium tuberculosis.

SapM from Mycobacterium tuberculosis is a secreted phosphatase critical for pathogen survival inside the host, representing an attractive target for the development of anti-tuberculosis drugs. The main limitation to biochemical and structural studies of SapM has been the lack of a suitable protocol to produce soluble recombinant protein. The aim of the present work was to produce SapM in Escherichia coli in a soluble and catalytically active form. We describe here the construct design, expression and purification of soluble SapM using Sarkosyl as a solubility-enhancing agent and auto-induction media. We demonstrate that solubilisation of the recombinant protein with Sarkosyl, and further purification, yields a catalytically active enzyme with high purity and monodisperse. The identity and molecular weight of the recombinant SapM was confirmed by mass spectrometry analyses, and we provide evidence that SapM behaves as a monomer in solution. Overall, this work lays the foundation for further studies to exploit SapM as a drug target, and provides a protocol for producing active and soluble recombinant enzymes that are hard to solubilise in E. coli.

Nudix hydrolase 1 is a prognostic biomarker in hepatocellular carcinoma.

We investigated the prognostic significance of Nudix hydrolase 1 (NUDT1) in hepatocellular carcinoma (HCC). NUDT1 mRNA and protein levels were significantly higher in HCC tissues than normal liver tissues. The level of NUDT1 expression correlated with tumor grade, stage, size, differentiation, degree of vascular invasion, overall survival (OS), and disease-free survival (DFS) in HCC patients. Multivariate analysis showed that NUDT1 expression was an independent prognostic factor for OS and DFS in HCC patients. We constructed a prognostic nomogram with NUDT1 expression, AFP levels, vascular invasion, Child-Pugh classification, age, sex, AJCC staging, and tumor differentiation as variables. This nomogram was highly accurate in predicting the 5-year OS of HCC patients (c-index= 0.709; AUC= 0.740). NUDT1 silencing in HCC cells significantly reduced their survival, colony formation, migration, and invasiveness. Gene set enrichment analysis showed that biological pathways related to cell cycle, fatty acid metabolism, bile acid and bile salt metabolism, and PLK1 signaling were associated with NUDT1, as were the gene ontology terms "DNA binding transcription activator activity," "RNA polymerase II," "nuclear division," and "transmembrane transporter activity." Our study thus demonstrates that NUDT1 is a prognostic biomarker with therapeutic potential in HCC patients.

EDTP/MTMR14: A novel target for improved survivorship to prolonged anoxia and cellular protein aggregates.

Drosophila egg-derived tyrosine phosphatase (EDTP), a lipid phosphatase that removes 3-position phosphate at the inositol ring, has dual functions in oogenesis and muscle performance in adults. A mammalian homologous gene MTMR14, which encodes the myotubularin-related protein 14, negatively regulates autophagy. Mutation of EDTP/MTMR14, however, causes at least three deleterious consequences: (1) the lethality in early embryogenesis in Drosophila; (2) a "jumpy" phenotype with apparently impaired motor functions; and (3) an association with a rare genetic disorder called centronuclear myopathy. The potential benefit of EDTP/MTMR14 downregulation is likely masked by the lethality or severe muscle defects due to ubiquitous loss of this gene. Here we show that flies carrying a heterozygous EDTP mutation had increased survivorship to prolonged anoxia; tissue-specific downregulation of EDTP in non-muscle tissues, particularly motoneurons, extended lifespan and improved survivorship to beta-amyloid peptides (Aß42) and polyglutamine protein aggregates. These data highlight the significance of selective downregulation of EDTP in non-muscles for beneficial consequences. MTMR14 expression was evident in the hippocampus and cortex in C57BL/6 J and APP/PS1 mice. Compared with C57BL/6 J mice, APP/PS1 mice had reduced MTMR14 in the cortex. Hippocampal expression of MTMR14 was increased and plateaued at 9-17 months compared with 2-6 months in C57BL/6 J mice. Additionally, MTMR14 was inducible by Aß42 in the rat primarily hippocampal neurons and mouse Neuro2a neuroblasts. We demonstrate a novel approach of tissue-specific downregulation of the disease-associated gene EDTP/MTMR14 for extended lifespan and improved survivorship to cellular protein aggregates. This approach could be extended from insects to mammals.

Expression and purification of human phosphatase and actin regulator 1 (PHACTR1) in plant-based systems.

Cardiovascular diseases are a prevalent cause of morbidity and mortality especially in industrialized countries. The human phosphatase and actin regulator 1 (PHACTR1) may be involved in such diseases, but its precise regulatory function remains unclear due to the large number of potential interaction partners. The same phenomenon makes this protein difficult to express in mammalian cells, but it is also an intrinsically disordered protein that likely aggregates when expressed in bacteria due to the absence of chaperones. We therefore used a design of experiments approach to test the suitability of three plant-based systems for the expression of satisfactory quantities of recombinant PHACTR1, namely transient expression in tobacco (Nicotiana tabacum) BY-2 plant cell packs (PCPs), whole N. benthamiana leaves and BY-2 cell lysate (BYL). The highest yield was achieved using the BYL: up to 120 mg product kg-1 biomass equivalent within 48 h of translation. This was 1.3-fold higher than transient expression in N. benthamiana together with the silencing inhibitor p19, and 6-fold higher than the PCP system. The presence of Triton X-100 in the extraction buffer increased the recovery of PHACTR1 by 2-200-fold depending on the conditions. PHACTR1 was incompatible with biomass blanching and was stable for less than 16 h in raw plant extracts. Purification using a DDK-tag proved inefficient whereas 15% purity was achieved by immobilized metal affinity chromatography.

Cloning and Expression of the Organophosphate Pesticide-Degrading α-ß Hydrolase Gene in Plasmid pMK-07 to Confer Cross-Resistance to Antibiotics.

Pesticide residual persistence in agriculture soil selectively increases the pesticide-degrading population and transfers the pesticide-degrading gene to other populations, leading to cross-resistance to a wide range of antibiotics. The enzymes that degrade pesticides can also catabolize the antibiotics by inducing changes in the gene or protein structure through induced mutations. The present work focuses on the pesticide-degrading bacteria isolated from an agricultural field that develop cross-resistance to antibiotics. This cross-resistance is developed through catabolic gene clusters present in an extrachromosomal plasmid. A larger plasmid (236.7 Kbp) isolated from Bacillus sp. was sequenced by next-generation sequencing, and important features such as α-ß hydrolase, DNA topoisomerase, DNA polymerase III subunit beta, reverse transcriptase, plasmid replication rep X, recombination U, transposase, and S-formylglutathione hydrolase were found in this plasmid. Among these, the α-ß hydrolase enzyme is known for the degradation of organophosphate pesticides. The cloning and expression of the α-ß hydrolase gene imply nonspecific cleavage of antibiotics through a cross-resistance phenomenon in the host. The docking of α-ß hydrolase with a spectrum of antibiotics showed a high G-score against chloramphenicol (-3.793), streptomycin (-2.865), cefotaxime (-5.885), ampicillin (-4.316), and tetracycline (-3.972). This study concludes that continuous exposure to pesticide residues may lead to the emergence of multidrug-resistant strains among the wild microbial flora.

INPP4B overexpression and c-KIT downregulation in human achalasia.

BACKGROUND: Achalasia is a rare motility disorder characterized by myenteric neuron and interstitial cells of Cajal (ICC) abnormalities leading to deranged/absent peristalsis and lack of relaxation of the lower esophageal sphincter. The mechanisms contributing to neuronal and ICC changes in achalasia are only partially understood. Our goal was to identify novel molecular features occurring in patients with primary achalasia. METHODS: Esophageal full-thickness biopsies from 42 (22 females; age range: 16-82 years) clinically, radiologically, and manometrically characterized patients with primary achalasia were examined and compared to those obtained from 10 subjects (controls) undergoing surgery for uncomplicated esophageal cancer (or upper stomach disorders). Tissue RNA extracted from biopsies of cases and controls was used for library preparation and sequencing. Data analysis was performed with the "edgeR" option of R-Bioconductor. Data were validated by real-time RT-PCR, western blotting and immunohistochemistry. KEY RESULTS: Quantitative transcriptome evaluation and cluster analysis revealed 111 differentially expressed genes, with a P ≤ 10-3 . Nine genes with a P ≤ 10-4 were further validated. CYR61, CTGF, c-KIT, DUSP5, EGR1 were downregulated, whereas AKAP6 and INPP4B were upregulated in patients vs controls. Compared to controls, immunohistochemical analysis revealed a clear increase in INPP4B, whereas c-KIT immunolabeling resulted downregulated. As INPP4B regulates Akt pathway, we used western blot to show that phospho-Akt was significantly reduced in achalasia patients vs controls. CONCLUSIONS & INFERENCES: The identification of altered gene expression, including INPP4B, a regulator of the Akt pathway, highlights novel signaling pathways involved in the neuronal and ICC changes underlying primary achalasia.

Anticancer effect of (S)-crizotinib on osteosarcoma cells by targeting MTH1 and activating reactive oxygen species.

MTH1 has become a new rising star in the field of 'cancer phenotypic lethality' and can be targeted in many kinds of tumors. This study aimed to explore the anticancer effect of MTH1-targeted drug (S)-crizotinib on osteosarcoma (OS) cells. We detected MTH1 expression in OS tissues and cells using immunohistochemistry and western blot. The effects of MTH1 on OS cell viability were explored using the siRNA technique and CCK8. The anticancer effects of the MTH1-targeted drug (S)-crizotinib on OS cells were explored by in-vitro assays. The intracellular 8-oxo-dGTP level and oxygen reactive species (ROS) of OS cells were detected by Cy3-conjugated avidin staining and dichlorofluorescein diacetate staining, respectively. The expression of MTH1 was significantly higher in OS tissues and cell lines than that in the corresponding adjacent tissues and osteoblastic cell line. The proliferation of OS cells was significantly inhibited through knockdown of MTH1 by siRNA technology. (S)-Crizotinib could inhibit the proliferation of OS cells with an increase in the apoptosis levels and causing G0/G1 arrest by targeting MTH1 and activating ROS. In addition, (S)-crizotinib could inhibit the migration of OS cells. (S)-Crizotinib could suppress the proliferation and migration, cause G0/G1 arrest, and increase the apoptosis level of OS cells by targeting MTH1 and activating ROS. This study will provide a promising therapeutic target and the theoretical basis for the clinical application of (S)-crizotinib in OS.

miR-1290 Contributes to Colorectal Cancer Cell Proliferation by Targeting INPP4B.

Colorectal cancer (CRC) is one of the most common oncological conditions worldwide, to date. MicroRNA-1290 (miR-1290) has been demonstrated to regulate its progression. We studied the role of miR-1290 in CRC progression. The gene was upregulated in CRC tissues and cells. Its overexpression promoted CRC cell proliferation analyzed by MTT assay, colony formation assay, and soft agar growth assay. In addition, miR-1290 knockdown inhibited CRC cell proliferation. We also found that miR-1290 overexpression reduced the p27 level and increased cyclin D1 at both the mRNA and protein levels, whereas miR-1290 knockdown increased p27 and reduced cyclin D1, confirming miR-1290 promoted CRC cell proliferation. Inositol polyphosphate 4-phosphatase B (INPP4B) was the target of miR-1290. Luciferase reporter assay revealed that miR-1290 directly bound to the 3'-UTR of INPP4B; the mutated seed sites in miR-1290 abrogated this effect. Double knockdown of INPP4B and miR-1290 promoted CRC cell proliferation, suggesting miR-1290 promoted CRC cell proliferation by targeting INPP4B.

MicroRNA-940 Targets INPP4A or GSK3ß and Activates the Wnt/ß-Catenin Pathway to Regulate the Malignant Behavior of Bladder Cancer Cells.

In this report, we aimed to explore the role and regulatory mechanism of microRNA-940 (miR-940) in bladder cancer development. The expressions of miR-940 in bladder cancer tissues and cells were measured. miR-940 mimics, miR-940 inhibitor small interference RNA against INPP4A (si-INPP4A), and GSK3ß (si-GSK3ß) and their corresponding controls were then transfected into cells. We investigated the effects of miR-940, INPP4A, or GSK3ß on cell proliferation, migration, invasion, and apoptosis. Additionally, target prediction and luciferase reporter assays were performed to investigate the targets of miR-940. The regulatory relationship between miR-940 and the Wnt/ß-catenin pathway was also investigated. miR-940 was upregulated in bladder cancer tissues and cells. Overexpression of miR-940 significantly increased bladder cancer cell proliferation, promoted migration and invasion, and inhibited cell apoptosis. INPP4A and GSK3ß were the direct targets of miR-940, and knockdown of INPP4A or GSK3ß significantly increased cancer cell proliferation, migration, and invasion and inhibited cell apoptosis. After miR-940 overexpression, the protein expression levels of c-Myc, cyclin D1, and ß-catenin were significantly increased, and the expression levels of p27 and p-ß-catenin were markedly decreased. The opposite effects were obtained after suppression of miR-940. XAV939, a tankyrase 1 inhibitor that could inhibit Wnt/ß-catenin signaling, significantly reversed the effects of miR-940 overexpression on cell migration and invasion. Our results indicate that overexpression of miR-940 may promote bladder cancer cell proliferation, migration, and invasion and inhibit cell apoptosis via targeting INPP4A or GSK3ß and activating the Wnt/ß-catenin pathway. Our findings imply the key roles of suppressing miRNA-940 in the therapy of bladder cancer.

Manipulation of GES and ERG20 for geraniol overproduction in Saccharomyces cerevisiae.

Manipulation of monoterpene synthases to maximize flux towards targeted products from GPP (geranyl diphosphate) is the main challenge for heterologous monoterpene overproduction, in addition to cell toxicity from compounds themselves. In our study, by manipulation of the key enzymes geraniol synthase (GES) and farnesyl diphosphate synthase (Erg20), geraniol (a valuable acyclic monoterpene alcohol) overproduction was achieved in Saccharomyces cerevisiae with truncated 3-hydroxy-3-methylglutaryl-coenzyme reductase (tHMGR) and isopentenyl diphosphate isomerase (IDI1) overexpressed. The expressions of all above engineered genes were under the control of Gal promoter for alleviating product toxicity. Geraniol production varied from trace amount to 43.19mg/L (CrGES, GES from Catharanthus roseus) by screening of nine GESs from diverse species. Further through protein structure analysis and site-directed mutation in CrGES, it was firstly demonstrated that among the high-conserved amino acid residues located in active pocket, Y436 and D501 with strong affinity to diphosphate function group, were critical for the dephosphorylation (the core step for geraniol formation). Moreover, the truncation position of the transit peptide from the N-terminus of CrGES was found to influence protein expression and activity significantly, obtaining a titer of 191.61mg/L geraniol in strain with CrGES truncated at S43 (t3CrGES). Furthermore, directed by surface electrostatics distribution of t3CrGES and Erg20WW (Erg20F96W-N127W), co-expression of the reverse fusion of Erg20ww/t3CrGES and another copy of Erg20WW promoted the geraniol titer to 523.96mg/L at shakes flask level, due to enhancing GPP accessibility led by protein interaction of t3CrGES-Erg20WW and the free Erg20WW. Eventually, a highest reported titer of 1.68g/L geraniol in eukaryote cells was achieved in 2.0L fed-batch fermentation under carbon restriction strategy. Our research opens large opportunities for other microbial production of monoterpenes. It also sets a good reference for desired compounds overproduction in microorganisms in terms of manipulation of key enzymes by protein engineering and metabolic engineering.

Altered Expression of Sphingosine-1-Phosphate Metabolizing Enzymes in Oral Cancer Correlate With Clinicopathological Attributes.

We have determined the gene expression of sphingosine-1-phosphate (S1P) metabolizing enzymes (SphK1, SphK2, SGPL1, SGPP1, SGPP2, PPAP2A, PPAP2B, and PPAP2C) by quantitative real-time polymerase chain reaction in tumor tissues and adjacent normal tissues of 50 oral squamous cell carcinoma (OSCC) patients. Expression of SphK1 and SGPP1 genes was up-regulated significantly in 70% and 75% OSCC tumors respectively. Importantly, expression of SphK2 and PPAP2B was down-regulated in the tumor tissues of 70% OSCC patients. Expression of SphK2 and PPAP2B negatively correlated with tumor-node-metastasis (TNM) staging and tumor volume respectively. Furthermore, LPP1 is an independent predictor of TNM staging and lymph node ratio.

Basal biomarkers nestin and INPP4b identify intrinsic subtypes accurately in breast cancers that are weakly positive for oestrogen receptor.

AIMS: Recent evidence indicates that weakly positive immunohistochemical staining of oestrogen receptor (ER) is not associated reliably with a luminal subtype, with the majority reclassified as basal-like by gene expression profile. In this study we assessed the capacity of recently identified immunohistochemical markers of basal-like subtype not dependent upon ER status - positive expression of nestin or loss of inositol polyphosphate-4-phosphatase (INPP4b) - to discriminate intrinsic subtypes, focusing on clinically problematic cases with weak ER positivity. METHODS AND RESULTS: Formalin-fixed paraffin-embedded blocks, enriched for large proportions of ER-negative and ER weakly positive breast cancers, were selected from two previous studies conducted in the period 2008-13 and used for (i) RNA extraction for 50-gene subtype predictor (PAM50) intrinsic subtyping and (ii) tissue microarray construction for immunohistochemical assessment of nestin and INPP4b. Fifty-eight cases were weakly positive for ER (Allred 3-5), among which 28 (48%) were assigned as basal-like by PAM50 gene expression. In these 58 cases, the nestin/INPP4b panel identified 23 basal-like cases with a positive predictive value of 87% [95% confidence interval (CI) 78-95%] and excluded luminal subtype with a negative predictive value of 95% (95% CI 88-100%). Weakly positive ER patients assigned as basal-like by nestin/INPP4b definition demonstrated a median survival time of 45.8 months, significantly lower than 65 months among other ER weakly positive cases (P = 0.012). CONCLUSIONS: Immunohistochemical assessment of nestin and INPP4b provides an accurate, accessible and inexpensive tool to identify basal-like breast cancer subtype in the clinically problematic setting of weak ER positivity. This panel identifies poor prognosis patients who might need strong considerations for non-endocrine-based therapies.