Isoenzyme characterization of Leishmania infantum toward checking the antioxidant activity of superoxide dismutase and glutathione peroxidase.

BACKGROUND: Leishmania infantum is the major causative agent of visceral leishmaniasis in Mediterranean regions. Isoenzyme electrophoresis (IE), as a biochemical technique, is applied in the characterization of Leishmania species. The current study attempted to investigate the isoenzyme patterns of logarithmic and stationary promastigotes and axenic amastigotes (amastigote-like) of L. infantum using IE. The antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX) was also checked in the aforementioned forms. METHOD: After L. infantum cultivation and obtaining logarithmic and stationary promastigotes, axenic amastigotes were achieved by incubation of stationary promastigotes at 37 °C for 48 h. The lysate samples were prepared and examined for six enzymatic systems including glucose-6-phosphate dehydrogenase (G6PD), nucleoside hydrolase 1 (NH1), malate dehydrogenase (MDH), glucose-phosphate isomerase (GPI), malic enzyme (ME), and phosphoglucomutase (PGM). Additionally, the antioxidant activity of SOD and GPX was measured. RESULTS: GPI, MDH, NH1, and G6PD enzymatic systems represented different patterns in logarithmic and stationary promastigotes and axenic amastigotes of L. infantum. PGM and ME showed similar patterns in the aforementioned forms of parasite. The highest level of SOD activity was determined in the axenic amastigote form and GPX activity was not detected in different forms of L. infantum. CONCLUSION: The characterization of leishmanial-isoenzyme patterns and the measurement of antioxidant activity of crucial antioxidant enzymes, including SOD and GPX, might reveal more information in the biology, pathogenicity, and metabolic pathways of Leishmania parasites and consequently drive to designing novel therapeutic strategies in leishmaniasis treatment.

Hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC in conjunction with Lgl.

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here, we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell-cell contacts. In 3D Matrigel culture of human hepatocytic HepG2 cells, which mimics a process of liver development and regeneration, depletion of Par3, Cdc42, or aPKC results in an impaired establishment of apicobasolateral polarity and a loss of subsequent apical lumen formation. The aPKC activity is also required for bile canalicular (apical) elongation in mouse primary hepatocytes. The lateral membrane-associated proteins Lgl1 and Lgl2, major substrates of aPKC, seem to be dispensable for hepatocyte polarity establishment because Lgl-depleted HepG2 cells are able to form a single apical lumen in 3D culture. On the other hand, Lgl depletion leads to lateral invasion of aPKC, and overexpression of Lgl1 or Lgl2 prevents apical lumen formation, indicating that they maintain proper lateral integrity. Thus, hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC; Par3 cooperates with Cdc42 to recruit aPKC, which plays a crucial role in apical membrane development and regulation of the lateral maintainer Lgl.

Analytical evaluation of the Nittobo Medical tartrate resistant acid phosphatase isoform 5b (TRACP-5b) EIA and comparison with IDS iSYS in different clinically defined populations.

OBJECTIVES: Tartrate-resistant acid phosphatase, isoform 5b (TRACP-5b) is a bone resorption marker not influenced by renal function or food intake. TRACP-5b can be measured with Nittobo Medical enzymatic-immunoassay and IDS-iSYS automated immunoassay. We evaluated the Nittobo assay and established reference ranges for a Western-European population. We compared Nittobo and IDS results in different well-defined clinical populations. METHODS: We established the limits of detection and quantification (LOD-LOQ), linearity, imprecision and the reference ranges in 119 males, 50 women (<45 years) and 120 women (>60 years) for TRACP-5b with the Nittobo assay. We compared both assays in 30 hemodialyzed (HD), and 40 stage 3-5 patients suffering from chronic kidney disease (CKD), 40 patients suffering from rheumatoid arthritis and osteoporosis and 80 post-menopausal women. We measured TRACP-5b, ß-crosslaps (ß-CTX), bone alkaline phosphatase (B-ALP) and PTH in 20 hemodialyzed (HD) and 40 CKD patients. RESULTS: LOD and LOQ were 0.02 and 0.35 U/L. CV ranged from 8.3 to 4.3% (2/5 samples presenting CV > desirable CV). Method was linear up to of 11.3 U/L. Upper and lower limits of normality were 0.8-7.6 U/L in men, 0.9-4.7 U/L in women <45 and 0.9-7.1 U/L in women >60. The regression equation between the 2 methods was Nittobo = 1.13 (95% CI: 1.09-1.16) × iSYS - 0.4 (95% CI: -0.5; -0.3). TRACP-5b and b-ALP were in their respective reference ranges for most of CKD and HD patients. That was not the case for ß-CTX, which increased with decreasing eGFR. CONCLUSIONS: Nittobo TRACP-5b presents interesting analytical features and a good concordance with IDS iSYS. These methods could thus potentially be harmonized.

Disposable stochastic sensors obtained by nanolayer deposition of copper, graphene, and copper-graphene composite on silk for the determination of isocitrate dehydrogenases 1 and 2.

Three disposable stochastic sensors designed using nanolayer deposition of copper (Cu), graphene (GR), and copper-graphene (Cu-GR) composite on the silk textile, as substrate, were modified with chitosan (n=371-744), for biomedical analysis. Isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) served as model analytes for molecular recognition and quantification in biological samples such as whole blood and brain tumor tissue samples. The best sensitivities (3.77×107s µg mL-1 for IDH1, and 1.88×107s µg mL-1 for IDH2) and the lowest limits of quantification (10-2fg mL-1 for IDH1, and 5×10-2fg mL-1 for IDH2) for both IDH1 and IDH2 were recorded for the disposable stochastic sensors based on chitosan/graphene nanolayer. Very good correlations between the screening method based on disposable stochastic sensors and enzyme-linked immunosorbent assay (ELISA) were obtained; this was also proved by the results obtained using the paired t-test.

NAT1 is a critical prognostic biomarker and inhibits proliferation of colorectal cancer through modulation of PI3K/Akt/mTOR.

The aim of this study was to analyze the correlations between NAT1 and clinicopathological features of and prognosis in colorectal cancer (CRC). RNA sequencing data and clinical information were retrieved from The Cancer Genome Atlas database. Wilcoxon test, logistic regression and Kaplan-Meier method were used to estimate the association between NAT1 and prognosis in CRC. In vitro experiments were conducted to confirm the role of NAT1. NAT1 is significantly less expressed in CRC and independently associated with poor prognosis in CRC patients. The authors further confirmed that expression of NAT1 was significantly lower in SW116 colon cancer cells than in NCM460 cells. Overexpressed NAT1 obviously inhibited the growth of CRC cells by downregulating phosphorylation of the PI3K/Akt/mTOR signaling pathway. NAT1 may be a potential therapeutic target for CRC.

Lay abstract Colorectal cancer (CRC) is a common malignancy worldwide. Because of the limited understanding of the pathogenesis and prognostic factors associated with CRC, the treatment effect in CRC remains poor. In the present study, the authors demonstrate that NAT1 is significantly less expressed in CRC and independently associated with poor prognosis in CRC patients. NAT1 may exert antitumor activity by inhibiting phosphorylation of the PI3K/Akt/mTOR signaling pathway. These results suggest that NAT1 may be a prognostic factor in and therapeutic target for CRC.

Changes of adenosine deaminase activity in serum and saliva around parturition in sows with and without postpartum dysgalactia syndrome.

BACKGROUND: Postpartum dysgalactia syndrome (PDS) is associated with a significantly higher activation of the inflammatory and stress response at parturition than in the healthy sow. Therefore, reliable and possibly non-invasive biomarkers for substantial increases of inflammation are searched to support the PDS diagnosis. This report studies the possible changes of the inflammatory marker enzyme adenosine deaminase (ADA) in serum and saliva of 38 PDS positive sows (PDS+) and 38 healthy sows (PDS-). Sampling was performed every 24 h from 60 h before to 36 h after parturition. Isoenzyme 1 (ADA1) and isoenzyme 2 (ADA2), as well as total ADA (tADA), were measured and their statistical association with several serum and saliva biomarkers of inflammation and stress was investigated. RESULTS: Compared to a baseline (60 to 36h prepartum), salivary activities of ADA1, ADA2 and tADA increased significantly over time in both PDS+ and PDS- sows, reaching their peaks after parturition. In serum from PDS- sows, no changes were observed over time in either ADA1, ADA2 or tADA. In PDS+ sows, serum ADA2 activity decreased temporarily after parturition followed by a significant increase compared to baseline. ADA1, ADA2 and tADA were all significantly associated with several inflammatory biomarkers and ADA1 in serum was associated with serum cortisol. Although serum activity was higher in PDS+ than in PDS- sows, the differences were not statistically significant. Further, no difference was noted between the groups in the analyses of saliva. CONCLUSIONS: Salivary ADA1 and ADA2 increased in all sows after parturition, potentially as a response to the postpartum inflammation. However, no difference in the activity of ADA1, ADA2 and tADA were found between PDS+ and PDS- sows indicating inability to diagnose PDS under the conditions described in this report.

Cytotoxicity, metabolism, and isozyme mapping of the synthetic cannabinoids JWH-200, A-796260, and 5F-EMB-PINACA studied by means of in vitro systems.

Intake of synthetic cannabinoids (SC), one of the largest classes of new psychoactive substances, was reported to be associated with acute liver damage but information about their hepatotoxic potential is limited. The current study aimed to analyze the hepatotoxicity including the metabolism-related impact of JWH-200, A-796260, and 5F-EMB-PINACA in HepG2 cells allowing a tentative assessment of different SC subclasses. A formerly adopted high-content screening assay (HCSA) was optimized using a fully automated epifluorescence microscope. Metabolism-mediated effects in the HCSA were additionally investigated using the broad CYP inhibitor 1-aminobenzotriazole. Furthermore, phase I metabolites and isozymes involved were identified by in vitro assays and liquid chromatography-high-resolution tandem mass spectrometry. A strong cytotoxic potential was observed for the naphthoylindole SC JWH-200 and the tetramethylcyclopropanoylindole compound A-796260, whereas the indazole carboxamide SC 5F-EMB-PINACA showed moderate effects. Numerous metabolites, which can serve as analytical targets in urine screening procedures, were identified in pooled human liver microsomes. Most abundant metabolites of JWH-200 were formed by N-dealkylation, oxidative morpholine cleavage, and oxidative morpholine opening. In case of A-796260, most abundant metabolites included an oxidative morpholine cleavage, oxidative morpholine opening, hydroxylation, and dihydroxylation followed by dehydrogenation. Most abundant 5F-EMB-PINACA metabolites were generated by ester hydrolysis plus additional steps such as oxidative defluorination and hydroxylation. To conclude, the data showed that a hepatotoxicity of the investigated SC cannot be excluded, that metabolism seems to play a minor role in the observed effects, and that the extensive phase I metabolism is mediated by several isozymes making interaction unlikely.

Advances in Activity-Based Sensing Probes for Isoform-Selective Imaging of Enzymatic Activity.

Until recently, there were no generalizable methods for assessing the effects of post-translational regulation on enzymatic activity. Activity-based sensing (ABS) has emerged as a powerful approach for monitoring small-molecule and enzyme activities within living systems. Initial examples of ABS were applied for measuring general enzymatic activity; however, a recent focus has been placed on increasing the selectivity to monitor a single enzyme or isoform. The highest degree of selectivity is required for differentiating between isoforms, where the targets display significant structural similarities as a result of a gene duplication or alternative splicing. This Minireview highlights key examples of small-molecule isoform-selective probes with a focus on the relevance of isoform differentiation, design strategies to achieve selectivity, and applications in basic biology or in the clinic.

Soluble guanylate cyclase isoenzymes: The expression of α1, α2, ß1, and ß2 subunits in the benign and malignant breast tumors.

Soluble guanylate cyclase (sGC) encompasses α and ß subunits. This study examined the expression of α1, α2, ß1, and ß2 subunits in the malignant and benign breast tumors using the Western blot analysis. Both benign and malignant tumors showed a significantly higher expression of the α1 subunit in comparison with normal tissues (p < 0.0001). In contrast, the expression of α2 and ß2 sGC were significantly lower in these tumors than normal tissues (p < .0015 and p < .001, p < .007 and p < .0001, respectively). The expression level of α1 sGC was significantly correlated with ER + PR+ (p < .0001). A significant correlation was also detected for sGC-α1 and -α2 expression with c-erbB2-negative status (p < .01). However, the expression level of sGC was not associated with tumor stage, tumor grade, or other clinicopathological features. In conclusion, as the expression of α1 sGC is upregulated and α2 and ß2 sGC are downregulated in malignant breast tumors. Variations in the expression of sGC isoenzymes may be suggested as an indicator to confirm the enzyme antitumor activity.

A Novel Sandwich ELISA for Tartrate-Resistant Acid Phosphatase 5a and 5b Protein Reveals that Both Isoforms are Secreted by Differentiating Osteoclasts and Correlate to the Type I Collagen Degradation Marker CTX-I In Vivo and In Vitro.

Tartrate-resistant acid phosphatase type 5 (TRAP) exists as two isoforms, 5a and 5b. 5b is a marker of osteoclast number and 5a of chronic inflammation; however, its association with bone resorption is unknown. In this study, a double-TRAP 5a/5b sandwich ELISA measuring 5a and 5b protein in the same sample was developed. TRAP 5a and 5b protein levels were evaluated as osteoclast differentiation/activity markers in serum and in culture, and their correlation to the resorption marker CTX-I was examined. Serum TRAP 5a and 5b concentrations in healthy men were 4.4 ± 0.6 ng/ml and 1.3 ± 0.2 ng/ml, respectively, and they correlated moderately to each other suggesting that their secretion is coupled under healthy conditions. A correlation was also observed between serum TRAP 5a and 5b with CTX-I, suggesting that both TRAP isoforms associate with osteoclast number. During osteoclast differentiation on plastic/bone, predominantly 5b increased in media/lysate from M-CSF/RANKL-stimulated CD14+ PBMCs. However, substantial levels of 5a were detected at later stages suggesting that both isoforms are secreted from differentiating OCs. More TRAP 5b was released on bone indicating a connection to osteoclast resorptive activity, and a peak in TRAP 5b/5a-ratio coincided with rapid CTX-I release. At the end of the culture period of M-CSF + RANKL-stimulated CD14+ PBMCs, there was a correlation between the secretion of TRAP 5a and 5b proteins with CTX-I. The correlation of not only 5b but also 5a with collagen degradation, both in serum and osteoclast cultures indicates that a considerable proportion of the TRAP 5a originates from osteoclasts and may reflect a hitherto undisclosed regulatory mechanism during bone resorption and bone remodeling.

Expression of cholinesterases and their anchoring proteins in rat heart.

Acetylcholine (ACh)-mediated vagal transmission as well as nonneuronal ACh release are considered cardioprotective in pathological situations with increased sympathetic drive such as ischemia-reperfusion and cardiac remodeling. ACh action is terminated by hydrolysis by the cholinesterases (ChEs), acetylcholinesterase, and butyrylcholinesterase. Both ChEs exist in multiple molecular variants either soluble or anchored by specific anchoring proteins like collagen Q (ColQ) anchoring protein and proline-rich membrane anchoring protein (PRiMA). Here we assessed the expression of specific ChE molecular forms in different heart compartments using RT-qPCR. We show that both ChEs are expressed in all heart compartments but display different expression patterns. The acetylcholinesterase-T variant together with PRiMA and ColQ is predominantly expressed in rat atria. Butylcholinesterase is found in all heart compartments and is accompanied by both PRiMA and ColQ anchors. Its expression in the ventricular system suggests involvement in the nonneuronal cholinergic system. Additionally, two PRiMA variants are detected throughout the rat heart.

Improved immunohistochemical detection of phosphorylated mitogen-activated protein kinases in the injured rat optic nerve head.

The activity of mitogen-activated protein kinases (MAPKs) is largely controlled by addition or removal of phosphate groups, which are carried out by kinase or phosphatase enzymes, respectively. Determining the phosphorylation status of MAPK isoenzymes, therefore, aids elucidation of the physiological and pathological roles of this enzyme. In practical terms, however, end-point procurement of appropriate experimental tissues produces conditions where MAPK phosphorylation status can rapidly alter, thus giving rise to aberrant data. We therefore attempted to instigate a means of stabilising end-point MAPK phosphorylation levels when procuring tissues for analysis. We employed a well-described rat model of ocular hypertension in which MAPK isoenzyme activation occurs in the optic nerve head (ONH), but can vary according to the level of resultant tissue pathology. Animals were appropriately treated and after 3 days were perfused in the presence or absence of a cocktail of phosphatase inhibitors (PIs), immediately prior to tissue fixation, in order to prevent dephosphorylation of phosphorylated MAPKs. Immunohistochemical labelling for phosphorylated MAPKs in untreated ONH sections was unaffected by the presence of PIs in the perfusate. MAPK activation was detected by immunohistochemistry in the treated ONH, but findings varied considerably, particularly in animals with less extensive tissue damage. The presence of PIs in the perfusate, however, significantly reduced this variation and enabled consistent changes to be detected, particularly in the animals with less extensive tissue damage. Thus, the addition of PIs to the perfusate is suggested when studying MAPK activation by immunohistochemistry, especially in the ONH.

The development and characterization of a chemical probe targeting PRMT1 over PRMT5.

Protein arginine methyltransferases (PRMTs) are a family of mammalian enzymes catalyzing the symmetric dimethylation (Type I), asymmetric dimethylation (Type II), or monomethylation (Type III) of arginine residues within proteins. This family is composed of 11 isozymes, however the vast majority of asymmetric and symmetric dimethylation in mammals is completed by either PRMT1 or PRMT5, respectively. In recent years, a number of chemical probes targeting this family of enzymes have been developed, but the majority of these probes lack isozyme specificity. Herein, we report the development of a chemical probe, based on a non-natural peptide sequence, which specifically labels PRMT1 over PRMT5 with high selectivity and sensitivity.

Isoenzyme and molecular approach for authenticating and monitoring of animal cell lines.

Authentication of cell lines is of paramount importance to validate the results from their use in biomedical research. Although isoenzyme polymorphism is the standard method, molecular methods based on mitochondrial DNA (mtDNA) have been developed to replace it. The aim of this study was the improvement of our isoenzyme electrophoretic analysis and the validation of one molecular technique targeted at mtDNA for the authentication of our animal cell lines. The combined method of cellular lysing through osmotic shock, followed by freezing-thawing in N2 to obtain isoenzyme extracts, and with 42 × 106 cells maintained the best efficiency. The superior electrophoretic conditions were PAGE run at 200 V. All cell lines had isoenzymatic mobility corresponding to their species to lactate dehydrogenase, malate-dehydrogenase, and glucose-6-phosphate dehydrogenase isoenzymes, and could be distinguished from each other. Two molecular techniques based on mtDNA were tested, one on the cytochrome b gene and other on cytochrome c oxidase I subunit gene. Due to difficulties in distinguishing all cell lines using only one these techniques, we merged the primers of two methods in such a way that there was a sufficient differentiation of all DNA fragments. The sequencing of these PCR products was also performed to validate these data.

Stellate cells and mesenchymal stem cells in benign mammary stroma are associated with risk factors for breast cancer - an observational study.

BACKGROUND: It is not known whether stromal cells in benign breast tissue can mediate risk of breast cancer. We recently described aldehyde dehydrogenase 1 A1 (ALDH1) positive (+) cells in morphologically normal breast stroma of premenopausal women, and the data indicated that their distribution is associated with clinical risk factors for breast cancer. The aim of the present study was to define the identities of these cells using histologic and immunohistologic methods, and to investigate associations between those cells and hormonal and genetic risk factors in pre- and postmenopausal women. METHODS: Stroma of morphologically normal tissue was analyzed in samples from 101 well-characterized women whose breasts had been operated. Morphology and immunolabeling were applied to determine cell identities based on the putative stem cell markers ALDH1 and stage-specific embryonic antigen-3 (SSEA3), and immunophenotypes indicating mast cells or stellate cells. The results were compared with the patients' risk factors using regression analysis (two-tailed). RESULTS: ALDH1+ round/oval cells were associated with low parity in BRCA1/2 carriers (p = 0.022), while in non-BRCA1/2-carriers they were negatively associated with nulliparity (p = 0.057). In premenopausal women ALDH1+ round/oval cells were associated with family history (p = 0.058). SSEA3+ round/oval cells were morphologically and immunohistologically consistent with multilineage stress-enduring (Muse) cells, and these cells were independently associated with the breast cancer risk factors low parity (p = 0.015), family history (p = 0.021), and hormone use after menopause (p = 0.032). ALDH1+ spindle-shaped/polygonal cells were immunohistologically consistent with stellate cells, and were negatively associated with family history of breast cancer (p = 0.001). CONCLUSION: This study identified novel stromal cell types in benign breast tissue that have a potential for stratifying women for breast cancer risk.

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2) displays an unexpected subcellular localization in the plasma membrane.

BACKGROUND: Heparan sulfate (HS) 3-O-sulfation can be catalysed by seven 3-O-sulfotransferases (HS3STs) in humans, still it is the rarest modification in HS and its biological function is yet misunderstood. HS3ST2 and HS3ST3B exhibit the same activity in vitro. They are however differently expressed in macrophages depending on cell environment, which suggests that they may be involved in distinct cellular processes. Here, we hypothesized that both isozymes might also display distinct subcellular localizations. METHODS: The subcellular distribution of HS3ST2 and HS3ST3B was analysed by using overexpression systems in HeLa cells. The localization of endogenous HS3ST2 was confirmed by immunostaining in primary macrophages. RESULTS: We found that HS3ST3B was only localized in the Golgi apparatus and no difference between full-length enzyme and truncated construct depleted of its catalytic domain was observed. In contrast, HS3ST2 was clearly visualized at the plasma membrane. Its truncated form remained in the Golgi apparatus, meaning that the catalytic domain might support correct addressing of HS3ST2 to cell surface. Moreover, we found a partial co-localization of HS3ST2 with syndecan-2 in HeLa cells and primary macrophages. Silencing the expression of this proteoglycan altered the localization of HS3ST2, which suggests that syndecan-2 is required to address the isozyme outside of the Golgi apparatus. CONCLUSIONS: We demonstrated that HS3ST3B is a Golgi-resident isozyme, while HS3ST2 is addressed to the plasma membrane with syndecan-2. GENERAL SIGNIFICANCE: The membrane localization of HS3ST2 suggests that this enzyme may participate in discrete processes that occur at the cell surface.

Alterations in cancer stem-cell marker CD44 expression predict oncologic outcome in soft-tissue sarcomas.

BACKGROUND: Cancer stem cells (CSCs) have been shown to resist chemotherapy and promote metastasis after cytotoxic therapies. We sought to determine if the expression of CSC markers (aldehyde dehydrogenase [ALDH], CD44, and epidermal growth factor receptor [EGFR]) predicted outcomes in soft-tissue sarcoma (STS) patients. METHODS: We queried an institutional database of 23 STS patients and evaluated immunohistochemical expression of CSC markers ALDH, CD44, and EGFR. The Cancer Genome Atlas (TCGA) was also queried for STS clinical and genomic data. Disease-specific (DSS) and overall survival (OS) were assessed by univariate and Kaplan-Meier analysis. RESULTS: Of the 23 institutional patients, the majority was female, had high-grade tumors and had extremity tumors. With a median follow-up of 27 months, nine patients (39%) experienced distant recurrence, and four (17%) died of disease. Mean H-scores at diagnosis (±standard error of the mean) for CD44, ALDH1, and EGFR were 169 ± 27, 77 ± 15, and 144 ± 23, respectively. On univariate analysis, there was a trend for increased CD44 score to predict both worse DSS and OS (hazard ratio = 1.01, 95% confidence interval 1-1.02, P = 0.056), whereas ALDH and EGFR scores did not. Analysis of 74 TCGA STS cases with complete clinical and genomic data revealed that CD44 copy number alterations predicted worse DSS (9.89 months versus 72.5 months, P = 0.007) and a trend for worse OS (14.03 months versus 38.6 months, P = 0.12), whereas ALDH1 and EGFR copy number alteration did not. Multivariate analysis of the combined data sets was consistent with worse DSS among patients with higher CD44 expression. CONCLUSIONS: Institutional and national TCGA data show the association of elevated baseline CD44 expression with worse STS outcomes. Further study of CD44 as a possible novel STS biomarker appears indicated.

The role of circulating tumor cells in metastatic breast cancer: prognostic and predictive value.

The aim of the current study was to assess the prognostic value of circulating tumor cells (CTCs) and their related markers at different points of chemotherapy regimens in metastatic breast cancer (MBC) patients. The impact of CTCs on progression free survival (PFS) and overall survival (OS) rates were also assessed. Peripheral blood samples were obtained from 66 female patients with MBC at different time intervals for evaluation of CTCs by flow cytometry (FC). cytokeratin 19 (CK19), mammaglobin, prolactin inducible peptide (PIP), aldehyde dehydrogenase 1 (ALDH1) and human chorionic gonadotropin (hCG) were also assessed by qRT-PCR. Analysis of different CTC levels (at 4, 5, and 6 cells/7 ml), showed statistically significant values at 4 cells/7 ml blood. The presence of baseline CTCs < 4 cells/7 ml, associated significantly with higher PFS (P value = 0.03). Patients showing a decrease in the CTCs level after treatment had significantly prolonged median PFS and OS rates compared to those whose CTCs level increased (P = 0.007 and P = 0.014; respectively). Mammaglobin, CK19, PIP, ALDH1 and hCG expression did not affect PFS or OS. However, patients with CTCs ≥ 4 at diagnosis had higher rates of progression compared to those with CTCs < 4 (1.9 times, P = 0.07), and who metastasized before 4 years showed a worse decrease outcomes (they were 2.4 time more progressed than those who metastasized after 4 years; P = 0.029). CTCs could be an independent prognostic and predictive biomarker for MBC patients' outcomes. Although none of the assessed genes (mammaglobin, CK19, PIP, ALDH1 and hCG) showed correlation with PFS or OS rates, further studies on a larger number of patients are required to validate the current results.

Urinary biomarkers predict advanced acute kidney injury after cardiovascular surgery.

BACKGROUND: Acute kidney injury (AKI) after cardiovascular surgery is a serious complication. Little is known about the ability of novel biomarkers in combination with clinical risk scores for prediction of advanced AKI. METHODS: In this prospectively conducted multicenter study, urine samples were collected from 149 adults at 0, 3, 6, 12 and 24 h after cardiovascular surgery. We measured urinary hemojuvelin (uHJV), kidney injury molecule-1 (uKIM-1), neutrophil gelatinase-associated lipocalin (uNGAL), α-glutathione S-transferase (uα-GST) and π-glutathione S-transferase (uπ-GST). The primary outcome was advanced AKI, under the definition of Kidney Disease: Improving Global Outcomes (KDIGO) stage 2, 3 and composite outcomes were KDIGO stage 2, 3 or 90-day mortality after hospital discharge. RESULTS: Patients with advanced AKI had significantly higher levels of uHJV and uKIM-1 at 3, 6 and 12 h after surgery. When normalized by urinary creatinine level, uKIM-1 in combination with uHJV at 3 h post-surgery had a high predictive ability for advanced AKI and composite outcome (AUC = 0.898 and 0.905, respectively). The combination of this biomarker panel (normalized uKIM-1, uHJV at 3 h post-operation) and Liano's score was superior in predicting advanced AKI (AUC = 0.931, category-free net reclassification improvement of 1.149, and p <  0.001). CONCLUSIONS: When added to Liano's score, normalized uHJV and uKIM-1 levels at 3 h after cardiovascular surgery enhanced the identification of patients at higher risk of progression to advanced AKI and composite outcomes.

Differential subcellular distribution of four phospholipase C isoforms and secretion of GPI-PLC activity.

Phospholipase C (PLC) is an important enzyme of signal transduction pathways by generation of second messengers from membrane lipids. PLCs are also indicated to cleave glycosylphosphatidylinositol (GPI)-anchors of surface proteins thus releasing these into the environment. However, it remains unknown whether this enzymatic activity on the surface is due to distinct PLC isoforms in higher eukaryotes. Ciliates have, in contrast to other unicellular eukaryotes, multiple PLC isoforms as mammals do. Thus, Paramecium represents a perfect model to study subcellular distribution and potential surface activity of PLC isoforms. We have identified distinct subcellular localizations of four PLC isoforms indicating functional specialization. The association with different calcium release channels (CRCs) argues for distinct subcellular functions. They may serve as PI-PLCs in microdomains for local second messenger responses rather than free floating IP3. In addition, all isoforms can be found on the cell surface and they are found together with GPI-cleaved surface proteins in salt/ethanol washes of cells. We can moreover show them in medium supernatants of living cells where they have access to GPI-anchored surface proteins. Among the isoforms we cannot assign GPI-PLC activity to specific PLC isoforms; rather each PLC is potentially responsible for the release of GPI-anchored proteins from the surface.