A Real-Time Phosphatidylinositol 4-Phosphate 5-Kinase Assay Using Fluorescence Spectroscopy.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is an enzyme that converts phosphatidylinositol 4-phosphate [PI4P] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. PIP5K plays a key role in the regulation of vesicular transport, cytoskeleton reorganization, and cell division. In general, to investigate an enzymatic activity of PIP5K, the amount of incorporated [P32] ATP into PI(4,5)P2 fraction is measured in in vitro reconstitution experiments. However, tools to monitor dynamic changes in its activity in real time have been lacking. Recently, we have developed a novel PIP5K assay using fluorescence spectroscopy. Compared to conventional methods in which lipids extraction steps are needed, our method is easy and quick to perform and enables a real-time analysis. This chapter provides a protocol to set up and perform the novel PIP5K assay we have recently established.

Distribution and localization of phosphatidylinositol 5-phosphate, 4-kinase alpha and beta in the brain.

Phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2 ) is critical for synaptic vesicle docking and fusion and generation of the second messengers, diacylglycerol and inositol-1,4,5-trisphosphate. PI-4,5-P2 can be generated by two families of kinases: type 1 phosphatidylinositol-4-phosphate 5-kinases, encoded by PIP5K1A, PIP5K1B and PIP5K1C, and type 2 phosphatidylinositol-5-phosphate 4-kinases, encoded by PIP4K2A, PIP4K2B, and PIP4K2C. While the roles of the type 1 enzymes in brain function have been extensively studied, the roles of the type 2 enzymes are poorly understood. Using selective antibodies validated by genetic deletion of pip4k2a or pip4k2b in mouse brain, we characterized the location of the enzymes, PI5P4Kα and PI5P4Kß, encoded by these genes. In mice, we demonstrate that PI5P4Kα is expressed in adulthood, whereas PI5P4Kß is expressed early in development. PI5P4Kα localizes to white matter tracts, especially the corpus callosum, and at a low level in neurons, while PI5P4Kß is expressed in neuronal populations, especially hippocampus and cortex. Dual labeling studies demonstrate that PI5P4Kα co-localizes with the oligodendrocyte marker, Olig2, whereas PI5P4Kß co-localizes with the neuronal marker, NeuN. Ultrastructural analysis demonstrates that both kinases are contained in axon terminals and dendritic spines adjacent to the synaptic membrane, which support a potential role in synaptic transmission. Immunoperoxidase analysis of macaque and human brain tissue demonstrate a conserved pattern for PI5P4Kα and PI5P4Kß. These results highlight the diverse cell-autonomous expression of PI5P4Kα and PI5P4Kß and support further exploration into their role in synaptic function in the brain.

Mechanistic regulation of SPHK1 expression and translocation by EMAP II in pulmonary smooth muscle cells.

Phosphorylation of sphingosine by sphingosine kinase 1 (SPHK1) produces the bioactive sphingolipid sphingosine-1-phosphate (S1P), a microvascular and immuno-modulator associated with vascular remodeling in pulmonary arterial hypertension (PAH). The low intracellular concentration of S1P is under tight spatial-temporal control. Molecular mechanisms that mediate S1P burden and S1P regulation of vascular remodeling are poorly understood. Similarities between two early response pro-inflammatory cytokine gene transcript activation profiles, S1P and Endothelial Monocyte Activating Polypeptide II (EMAP II), suggested a strategic link between their signaling pathways. We determined that EMAP II triggers a bimodal phosphorylation, transcriptional regulation and membrane translocation of SPHK1 through a common upstream process in both macrophages and pulmonary artery smooth muscle cells (PASMCs). EMAP II initiates a dual function of ERK1/2: phosphorylation of SPHK1 and regulation of the transcription factor EGR1 that induces expression of SPHK1. Activated ERK1/2 induces a bimodal phosphorylation of SPHK1 which reciprocally increases S1P levels. This identified common upstream signaling mechanism between a protein and a bioactive lipid initiates cell specific downstream signaling representing a multifactorial mechanism that contributes to inflammation and PASMC proliferation which are cardinal histopathological phenotypes of PAH.

Sphingosine Kinase 1 is Associated With Immune Cell-Related Gene Expressions in Human Breast Cancer.

BACKGROUND: Although previous experiments have implicated sphingosine-1-phosphate (S1P) as a links between immune reactions and cancer progression, the exact mechanism of this interaction has not comprehensively studied in clinical human samples. This study sought to evaluate the S1P regulation by sphingosine kinase 1 (SPHK1), an S1P-producing enzyme, in the immunity/immuno-reactivity of clinical human breast cancer surgical specimens. METHODS: S1P levels were examined in tumor, peritumoral, and normal human breast samples using mass spectrometry. Genomics Data Commons data portal of The Cancer Genome Atlas cohort was used to assess the expression of S1P-related and immune-related genes. RESULTS: S1P levels were significantly higher in tumor samples compared to peritumoral (P < 0.05) or normal human breast samples (P < 0.001). SPHK1 gene expression was elevated in tumoral samples compared to normal breast samples (P < 0.01). Furthermore, the elevated expression of SPHK1 in breast cancer tissue was associated with an increased expression of the different kinds of immune-related genes, such as CD68, CD163, CD4, and FOXP3 (forkhead box P3), in HER2-negative breast cancer. Network analysis showed the central role of SPHK1 in the interaction of S1P signaling and expression of immune cell-related proteins. CONCLUSIONS: We demonstrated that S1P is mainly produced by tumor tissue, rather than peritumoral tissue, in breast cancer patients. Our data revealed the involvement of S1P signaling in the regulation of immune-related genes, suggesting the links between S1P and complicated immune-cancer interactions in breast cancer patients.

Sphingosine-1-phosphate signal transducer and activator of transcription 3 signaling pathway contributes to baicalein-mediated inhibition of dextran sulfate sodium-induced experimental colitis in mice.

BACKGROUND: Baicalein has been shown to have anti-inflammatory and anti-tumor activities. However, the mechanisms underlying its anti-inflammatory effect on colitis remain unclear. METHODS: A dextran sodium sulfate (DSS)-induced model of acute colitis was established in BALB/c mice (6-8 weeks old, weighing 18-22 g). Six groups of mice received: (1) water for 10 days (control), n = 6; (2) DSS 4% solution in the drinking water for 7 days, followed by normal water for 3 days, n = 7; (3), (4), and (5) as for group 2 plus baicalein (10, 20, 40 mg/kg) administered once daily starting on day 1, n = 6; and (6) as for (2) plus 5-aminosalicylic acid (50 mg/kg) administered once daily starting on day 1, n = 6. Body weights, stool consistency, and hematochezia were recorded, and the severity of colitis was evaluated using a disease activity index. On day 11, the mice were euthanized, and organs and blood were collected for analysis. Serum inflammatory factors were detected by enzyme-linked immunosorbent assay; CD11b-positive cells were analyzed by immunofluorescence microscopy; expression of retinoic-acid-receptor-related orphan nuclear receptor gamma, sphingosine kinase 1 (SPHK1), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) was detected by immunohistochemistry; and expression of nucleotide-binding oligomerization domain 2 (NOD2), SPHK1, sphingosine 1-phosphate receptor 1 (S1PR1), total STAT3, and p-STAT3 were detected by western blotting analysis. Inter-group differences were compared using Student's t test. RESULTS: Baicalein treatment dose-dependently reduced DSS-induced weight loss (P < 0.01 or P < 0.05), splenomegaly (P < 0.01), and colonic damage, as reflected by amelioration of diarrhea, rectal bleeding, and colonic ulceration, congestion, edema (shown as colon length, P < 0.05 or P < 0.01), and inflammatory cell infiltration. Baicalein also significantly decreased the levels of inflammatory mediators in the serum (P < 0.01) and colon, and significantly inhibited expression of NOD2 SPHK1, S1PR1, and p-STAT3 in the colon (P < 0.05). CONCLUSIONS: Baicalein treatment ameliorated colitis in mice by inhibiting S1P-STAT3 signaling, suggesting that this flavonoid might be beneficial in the treatment of colitis.

Different Roles of Sphingosine Kinase 1 and 2 in Pancreatic Cancer Progression.

BACKGROUND: Pancreatic cancer is a disease with poor prognosis, and development of new treatments is necessary. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinases (SphK1 and SphK2), plays a critical role in progression of many types of cancer. However, little is known about the role of sphingosine kinases in pancreatic cancer. This study investigated the roles of sphingosine kinases in pancreatic cancer progression. MATERIALS AND METHODS: S1P levels in pancreatic cancer and noncancerous pancreatic tissue were measured in 10 patients. We generated PAN02 murine pancreatic cancer cell lines with a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system genes 9 (Cas9)-mediated deletion of SphK1 or SphK2 and assessed cell growth and migration. In an animal model, we assessed the survival of mice injected with PAN02 cells intraperitoneally. RESULTS: S1P levels in the pancreatic cancer tissue were significantly higher than those in noncancerous tissue. SphK1 knockout (KO) cells showed greater proliferation and migration than wild type (WT) cells, and SphK2 KO cells showed less proliferation and migration than WT cells. Animal experiments showed that the survival of mice injected with SphK1 KO cells was significantly shorter than those injected with WT cells, and the survival of mice injected with SphK2 KO cells was longer than those injected with WT cells. Surprisingly, cytotoxic assay using gemcitabine showed that SphK1 KO cells survived less than WT cells, and SphK2 KO cells survived more than WT cells. CONCLUSIONS: S1P produced by SphK1 and SphK2 may have different functions in pancreatic cancer cells. Targeting both SphK1 and SphK2 may be a potential strategy for pancreatic cancer treatment.

Inositol 1,4,5-trisphosphate 3-kinase A overexpressed in mouse forebrain modulates synaptic transmission and mGluR-LTD of CA1 pyramidal neurons.

Inositol 1,4,5-trisphosphate 3-kinase A (IP3K-A) regulates the level of the inositol polyphosphates, inositol trisphosphate (IP3) and inositol tetrakisphosphate to modulate cellular signaling and intracellular calcium homeostasis in the central nervous system. IP3K-A binds to F-actin in an activity-dependent manner and accumulates in dendritic spines, where it is involved in the regulation of synaptic plasticity. IP3K-A knockout mice exhibit deficits in some forms of hippocampus-dependent learning and synaptic plasticity, such as long-term potentiation in the dentate gyrus synapses of the hippocampus. In the present study, to further elucidate the role of IP3K-A in the brain, we developed a transgenic (Tg) mouse line in which IP3K-A is conditionally overexpressed approximately 3-fold in the excitatory neurons of forebrain regions, including the hippocampus. The Tg mice showed an increase in both presynaptic release probability of evoked responses, along with bigger synaptic vesicle pools, and miniature excitatory postsynaptic current amplitude, although the spine density or the expression levels of the postsynaptic density-related proteins NR2B, synaptotagmin 1, and PSD-95 were not affected. Hippocampal-dependent learning and memory tasks, including novel object recognition and radial arm maze tasks, were partially impaired in Tg mice. Furthermore, (R,S)-3,5-dihydroxyphenylglycine-induced metabotropic glutamate receptor long-term depression was inhibited in Tg mice and this inhibition was dependent on protein kinase C but not on the IP3 receptor. Long-term potentiation and depression dependent on N-methyl-d-aspartate receptor were marginally affected in Tg mice. In summary, this study shows that overexpressed IP3K-A plays a role in some forms of hippocampus-dependent learning and memory tasks as well as in synaptic transmission and plasticity by regulating both presynaptic and postsynaptic functions.

Immunohistochemical Detection of Sphingosine-1-Phosphate and Sphingosine Kinase-1 in Human Tissue Samples and Cell Lines.

Sphingosine-1-phosphate (S1P) and the enzyme primarily responsible for its production, sphingosine kinase-1 (SphK-1), are dysregulated in multiple human diseases including cancer, multiple sclerosis (MS), diabetes, neurological diseases, fibrosis, and certain pathologies associated with impaired angiogenesis such as age-related macular degeneration (AMD). Antibody-based techniques to identify and localize S1P and SphK-1 within cells and tissue specimens represent a powerful tool, not only to understand biological role of these molecules but also to validate these unique in-class targets in multiple state diseases. Consequently, the potential applications of these molecules for therapy and diagnostic purposes are currently under investigation. Here, we describe a new improved technique, Agitated Low Temperature Epitope Retrieval (ALTER) for staining procedures, to identify expression of S1P and SphK-1 in human frozen tissue samples. The challenges encountered in the process of localization in tissue samples of lipid molecules such as S1P are discussed.

Spectrophotometric assays for measuring redox biomarkers in blood and tissues: the NADPH network.

Nicotinamide adenine dinucleotide (NAD+/NADH) along with its phosphorylated form (NADP+/NADPH) are two molecules ubiquitously present in all organisms, and they play key roles as cofactors in fundamental catabolic and anabolic processes, respectively. The oxidation of NADPH to NADP+ initiates a cascade of reactions, where a network of molecules is implicated. The molecules of this cascade form a network with eminent translational potential in redox metabolism. A special point of interest is that spectrophotometric assays have been developed both for NADH/NADPH and the molecules directly regulated by them. Therefore, crucial molecules of the NADPH-dependent redox network can be measured, and the results can be used to assess the bioenergetic and/or oxidative stress status. The main aim of this review is to collectively present the NADPH-related molecules, namely NADPH, NADH, NAD+ kinase, NADPH oxidase, peroxiredoxin, thioredoxin, thioredoxin reductase, and nitric oxide synthase, that can be measured in blood and tissues with the use of a spectrophotometer, which is probably the most simple, inexpensive and widely used tool in biochemistry. We are providing the researchers with reliable and valid spectrophotometric assays for the measurement of the most important biomarkers of the NADPH network in blood and other tissues, thus allowing the opportunity to follow the redox changes in response to a stimulus.

Crosstalk between TLR2 and Sphk1 in microglia in the cerebral ischemia/reperfusion-induced inflammatory response.

Stroke is associated with high morbidity and mortality, and much remains unknown about the injury-related mechanisms that occur following reperfusion. This study aimed to explore the roles of Toll-like receptor 2 (TLR2) and sphingosine kinase 1 (Sphk1) in microglial cells in inflammatory responses induced by cerebral ischemia/reperfusion (I/R). For this purpose, C57BL/6 mice were randomly divided into 4 groups as follows: the sham-operated group, the I/R group, the I/R group treated with TLR2 antibody, and the I/R group treated with N,N-dimethylsphingosine. Focal cerebral I/R was induced by middle cerebral artery occlusion. Double-labeling immunofluorescence was used to observe the protein expression of TLR2 and Sphk1 in the ischemic brain tissue. Quantitative polymerase chain reaction was performed to determine the mRNA levels of TLR2 and Sphkl in ischemic brain tissue. Enzyme-linked immunosorbent assay was carried out to detect the protein contents of interleukin (IL)-1ß, tumor necrosis factor-α (TNF­α), IL-17 and IL-23 in ischemic brain tissue. The results revealed that I/R upregulated TLR2 and Sphk1 expression in microglial cells, and the inhibition of either TLR2 or Sphk1 inhibited the expression of the pro-inflammatory cytokines, IL-1ß, TNF-α, IL-17 and IL-23. Notably, the inhibition of TLR2 activity also decreased Sphk1 expression. These results thus indicate that the activation of microglial cells, via a TLR2→Sphk1→pro-inflammatory cytokine (IL-1ß, TNF-α, IL-17 and IL-23) pathway, may participate in I/R injury.

Predictive Value of Sphingosine Kinase 1 Expression in Papillary Thyroid Carcinoma.

BACKGROUND/AIM: Sphingolipid metabolites are emerging as key signaling molecules in cancer. Sphingosine kinase 1 is up-regulated in many different types of human malignancies and plays a crucial role in cancer development and progression. The utility of sphingosine kinase 1 to act as a predictive biomarker in thyroid cancer remains unclear. MATERIALS AND METHODS: Sphingosine kinase 1 expression was evaluated using immunohistochemical staining in 110 formalin-fixed, paraffin-embedded papillary thyroid carcinoma tissue samples. RESULTS: Sphingosine kinase 1 expression in papillary thyroid carcinoma tissue was significantly higher than in nodular goiter (p<0.001) or normal thyroid (p<0.001) tissue. Sphingosine kinase 1 was observed in the cytoplasm of tumor cells. Thirty-four (30.9%) of 110 papillary thyroid carcinomas exhibited high sphingosine kinase 1 expression, that was significantly associated with tumor multiplicity (p=0.004), extrathyroidal extension (p=0.013), presence of lymph node metastasis (p<0.001), and number of metastatic lymph nodes (p=0.042). In addition, high sphingosine kinase 1 expression was the only independent predictor of lymph node metastasis (p<0.001). CONCLUSION: Sphingosine kinase 1 is involved in papillary thyroid carcinoma development and progression and can serve as a potential biomarker predictive of lymph node metastasis.

Role of phosphatidylinositol-4,5-bisphosphate 3-kinase signaling in vesicular trafficking.

Phosphatidylinositol-4,5-bisphosphate 3-kinases (PI3Ks) are regulatory enzymes involved in the generation of lipid species that modulate cellular signaling pathways through downstream effectors to influence a variety of cellular functions. Years of intensive study of PI3Ks have produced a significant body of literature in many areas, including that PI3K can mediate intracellular vesicular trafficking and through these actions contribute to a number of important physiological functions. This review focuses on the crucial roles that PI3K and AKT, a major downstream partner of PI3K, play in the regulation of vesicle trafficking during various forms of vesicular endocytosis and exocytosis.

Impairment of Angiogenic Sphingosine Kinase-1/Sphingosine-1-Phosphate Receptors Pathway in Preeclampsia.

Preeclampsia (PE), is a serious pregnancy disorder characterized in the early gestation by shallow trophoblast invasion, impaired placental neo-angiogenesis, placental hypoxia and ischemia, which leads to maternal and fetal morbidity and mortality. Here we hypothesized that angiogenic sphingosine kinase-1 (SPHK1)/sphingosine-1-phosphate (S1P) receptors pathway is impaired in PE. We found that SPHK1 mRNA and protein expression are down-regulated in term placentae and term chorionic villous explants from patients with PE or severe PE (PES), compared with controls. Moreover, mRNA expression of angiogenic S1PR1 and S1PR3 receptors were decreased in placental samples of PE and PES patients, whereas anti-angiogenic S1PR2 was up-regulated in chorionic villous tissue of PES subjects, pointing to its potential atherogenic and inflammatory properties. Furthermore, in in vitro (JAR cells) and ex vivo (chorionic villous explants) models of placental hypoxia, SPHK1 mRNA and protein were strongly up-regulated under low oxygen tension (1% 02). In contrast, there was no change in SPHK1 expression under the conditions of placental physiological hypoxia (8% 02). In both models, nuclear protein levels of HIF1A were increased at 1% 02 during the time course, but there was no up-regulation at 8% 02, suggesting that SPHK1 and HIF1A might be the part of the same canonical pathway during hypoxia and that both contribute to placental neovascularization during early gestation. Taken together, this study suggest the SPHK1 pathway may play a role in the human early placentation process and may be involved in the pathogenesis of PE.

Bioluminescence Methods for Assaying Kinases in Quantitative High-Throughput Screening (qHTS) Format Applied to Yes1 Tyrosine Kinase, Glucokinase, and PI5P4Kα Lipid Kinase.

Assays in which the detection of a biological phenomenon is coupled to the production of bioluminescence by luciferase have gained widespread use. As firefly luciferases (FLuc) and kinases share a common substrate (ATP), coupling of a kinase to FLuc allows for the amount of ATP remaining following a kinase reaction to be assessed by quantitating the amount of luminescence produced. Alternatively, the amount of ADP produced by the kinase reaction can be coupled to FLuc through a two-step process. This chapter describes the bioluminescent assays that were developed for three classes of kinases (lipid, protein, and metabolic kinases) and miniaturized to 1536-well format, enabling their use for quantitative high-throughput (qHTS) of small-molecule libraries.

Sphingosine Kinase 1 urothelial expression is increased in patients with neurogenic detrusor overactivity

Objectives: To evaluate the expression of sphingosine kinase 1 (SPK1) in the bladder wall in patients with neurogenic lower urinary tract dysfunction and its association with clinical, urodynamic and pathological features. Materials and Methods: The expression of SPK1 was studied in bladder wall specimens obtained from cystectomy using immunohistochemistry in ten patients with spinal cord injury (n=8) or multiple sclerosis (n=2) with urodynamically proven neuropathic bladder dysfunction, and in controls (n=5). Inflammation and fibrosis were analysed with histological criteria and SPK1 expression was determined by individual immunohistochemical staining. Results: Significant increased SPK1 urothelial immunoreactivity was shown in patients compared to control group (p=0.03). By contrast, SPK1 immunoreactivity in patients was significantly decreased in the sub-urothelium, muscles and nerves, p=0.02; 0.01 and 0.003, respectively. Patients with neurogenic detrusor overactivity (NDO) had higher SPK1 urothelium expression than those without any DO (p=0.04). Conclusions: SPK1 is expressed in the human bladder wall, specifically the urothelium, in bladder specimens from patients with NDO. The role of SPK1 in the pathophysiology of NDO needs further elucidation.

Improvement of prognostic performance in severely injured patients by integrated clinico-transcriptomics: a translational approach.

INTRODUCTION: Severe trauma triggers a systemic inflammatory response that contributes to secondary complications, such as nosocomial infections, sepsis or multi-organ failure. The present study was aimed to identify markers predicting complications and an adverse outcome of severely injured patients by an integrated clinico-transcriptomic approach. METHODS: In a prospective study, RNA samples from circulating leukocytes from severely injured patients (injury severity score ≥ 17 points; n = 104) admitted to a Level I Trauma Center were analyzed for dynamic changes in gene expression over a period of 21 days by quantitative RT-PCR. Transcriptomic candidates were selected based on whole genome screening of a representative discovery set (n = 10 patients) or known mechanisms of the immune response, including mediators of inflammation (IL-8, IL-10, TNF-α, MIF, C5, CD59, SPHK1), danger signaling (HMGB1, TLR2, CD14, IL-33, IL-1RL1), and components of the heme degradation pathway (HP, CD163, HMOX1, BLVRA, BLVRB). Clinical markers comprised standard physiological and laboratory parameters and scoring systems routinely determined in trauma patients. RESULTS: Leukocytes, thrombocytes and the expression of sphingosine kinase-1 (SPHK1), complement C5, and haptoglobin (HP) have been identified as markers with the best performance. Leukocytes showed a biphasic course with peaks on day 0 and day 11 after trauma, and patients with sepsis exhibited significantly higher leukocyte levels. Thrombocyte numbers showed a typical profile with initial thrombopenia and robust thrombocytosis in week 3 after trauma, ranging 2- to 3-fold above the upper normal value. 'Relative thrombocytopenia' was associated with multi-organ dysfunction, the development of sepsis, and mortality, the latter of which could be predicted within 3 days prior to the time point of death. SPHK1 expression at the day of admission indicated mortality with excellent performance. C5-expression on day 1 after trauma correlated with an increased risk for the development of nosocomial infections during the later course, while HP was found to be a marker for the development of sepsis. CONCLUSIONS: The combination of clinical and transcriptomic markers improves the prognostic performance and may represent a useful tool for individual risk stratification in trauma patients.

ITPKA expression is a novel prognostic factor in hepatocellular carcinoma.

BACKGROUND: Inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) has recently been found to be implicated in the tumor progression of various cancers. However, the expression and the prognostic value of ITPKA in hepatocellular carcinoma (HCC) remains unexplored. The aim of this study is to investigate the clinical significance of ITPKA expression in HCC. METHODS: We determined the expression level of ITPKA in 135 cases of HCC tissues and the matched adjacent nontumorous tissues by quantitative real-time RT-PCR. The correlation between ITPKA expression and prognosis of HCC patients was further evaluated by univariate and multivariate analysis. Multivariate analysis of the prognostic factors was performed with Cox proportional hazards model. RESULTS: Up-regulation of ITPKA occurred in 48.9% of primary HCCs compared with their nontumor counterparts (P < 0.001). In addition, high expression of ITPKA was significantly associated with vascular invasion (P = 0.001) and TNM stage (P = 0.005). Kaplan-Meier analysis showed that the 5-year overall survival (OS) and relapse-free survival (RFS) rate in the group with high expression of ITPKA is poorer than that in low expression group (32.2 and 26.8% versus 59.2 and 57.7%). Univariate and multivariate analyses revealed that ITPKA was an independent prognostic factor for OS and RFS. Moreover, Stratified analysis revealed that its prognostic significance still existed within the subgroup of patients with early clinical stage (TNM stage I) or normal serum AFP level (≤25 µg/L). CONCLUSION: Our data indicated that ITPKA expression was significantly up-regulated in HCC and could serve as a potential novel prognostic biomarker for HCC patients after surgery.

Quantification of mevalonate-5-phosphate using UPLC-MS/MS for determination of mevalonate kinase activity.

OBJECTIVES: Mevalonate kinase deficiency, a rare autosomal recessive autoinflammatory disease, is caused by mutations in the MVK gene encoding mevalonate kinase (MK). MK catalyzes the phosphorylation of mevalonic acid to mevalonate-5-phosphate (MVAP) in the pathway of isoprenoid and sterol synthesis. The disease phenotype correlates with residual activity ranging from <0.5% for mevalonic aciduria to 1-7% for the milder hyperimmunoglobulinemia D and periodic fever syndrome (HIDS). Hence, assessment of loss-of-function requires high accuracy measurements. We describe a method using isotope dilution UPLC-MS/MS for precise and sensitive determination of MK activity. DESIGN AND METHODS: Wild-type MK and the variant V261A, which is associated with HIDS, were recombinantly expressed in Escherichia coli. Enzyme activity was determined by formation of MVAP over time quantified by isotope dilution UPLC-MS/MS. The method was validated according to the FDA Guidance for Bioanalytical Method Validation. RESULTS: Sensitivity for detection of MAVP by UPLC-MS/MS was improved by derivatization with butanol-HCl (LLOQ, 5.0 fmol) and the method was linear from 0.5 to 250 µmol/L (R(2) > 0.99) with a precision of ≥ 89% and an accuracy of ± 2.7%. The imprecision of the activity assay, including the enzymatic reaction and the UPLC-MS/MS quantification, was 8.3%. The variant V261A showed a significantly decreased activity of 53.1%. CONCLUSION: Accurate determination of MK activity was enabled by sensitive and reproducible detection of MVAP using UPLC-MS/MS. The novel method may improve molecular characterization of MVK mutations, provide robust genotype-phenotype correlations, and accelerate compound screening for drug candidates restoring variant MK activity.

Characterization of lipidic markers of chondrogenic differentiation using mass spectrometry imaging.

Mesenchymal stem cells (MSC) are an interesting alternative for cell-based therapy of cartilage defects attributable to their capacity to differentiate toward chondrocytes in the process termed chondrogenesis. The metabolism of lipids has recently been associated with the modulation of chondrogenesis and also with the development of pathologies related to cartilage degeneration. Information about the distribution and modulation of lipids during chondrogenesis could provide a panel of putative chondrogenic markers. Thus, the discovery of new lipid chondrogenic markers could be highly valuable for improving MSC-based cartilage therapies. In this work, MS imaging was used to characterize the spatial distribution of lipids in human bone marrow MSCs during the first steps of chondrogenic differentiation. The analysis of MSC micromasses at days 2 and 14 of chondrogenesis by MALDI-MSI led to the identification of 20 different lipid species, including fatty acids, sphingolipids, and phospholipids. Phosphocholine, several sphingomyelins, and phosphatidylcholines were found to increase during the undifferentiated chondrogenic stage. A particularly detected lipid profile was verified by TOF secondary ion MS. Using this technology, a higher intensity of phosphocholine-related ions was observed in the peripheral region of the micromasses collected at day 14.

Sphingosine Kinase 1 urothelial expression is increased in patients with neurogenic detrusor overactivity.

UNLABELLED: To evaluate the expression of sphingosine kinase 1 (SPK1) in the bladder wall in patients with neurogenic lower urinary tract dysfunction and its association with clinical, urodynamic and pathological features. MATERIALS AND METHODS: The expression of SPK1 was studied in bladder wall specimens obtained from cystectomy using immunohistochemistry in ten patients with spinal cord injury (n=8) or multiple sclerosis (n=2) with urodynamically proven neuropathic bladder dysfunction, and in controls (n=5). Inflammation and fibrosis were analysed with histological criteria and SPK1 expression was determined by individual immunohistochemical staining. RESULTS: Significant increased SPK1 urothelial immunoreactivity was shown in patients compared to control group (p=0.03). By contrast, SPK1 immunoreactivity in patients was significantly decreased in the sub-urothelium, muscles and nerves, p=0.02; 0.01 and 0.003, respectively. Patients with neurogenic detrusor overactivity (NDO) had higher SPK1 urothelium expression than those without any DO (p=0.04). CONCLUSIONS: SPK1 is expressed in the human bladder wall, specifically the urothelium, in bladder specimens from patients with NDO. The role of SPK1 in the pathophysiology of NDO needs further elucidation.