Nanostructured polystyrene well plates allow unbiased high-throughput characterization of circulating tumor cells.

Rapid, reliable and unbiased circulating tumor cell (CTC) isolation and molecular characterization methods are urgently required for implementation in routine clinical diagnostic and prognostic procedures. We report on the development of a novel unbiased CTC detection approach that combines high-throughput automated microscopy with a simple yet efficient approach for achieving a high level of tumor cell binding in standard tissue culture polystyrene (PS) well plates. A single 5 min high-power oxygen plasma treatment was used to create homogeneous nanoscale roughness on standard PS tissue culture plates and, in turn, drastically enhance the binding of a range of tumor cells.

Notch signaling promotes intestinal crypt fission in the infant rat.

Background: Growth of the small intestine in the infant rat is promoted by crypt fission and later by increased crypt cell proliferation. Notch signaling could promote crypt fission. Hes-1 is a Notch target gene.

A simple, cost-effective and flexible method for processing of snap-frozen tissue to prepare large amounts of intact RNA using laser microdissection.

Understanding the molecular basis of disease requires gene expression profiling of normal and pathological tissue. Although the advent of laser microdissection (LMD) has greatly facilitated the procurement of specific cell populations, often only small amounts of low quality RNA is recovered. This precludes the use of global approaches of gene expression profiling which require sizable amounts of high quality RNA.

Wnt blockade with dickkopf reduces intestinal crypt fission and intestinal growth in infant rats.

Objectives: Intestinal crypt fission peaks during infancy. In human and experimental familial polyposis coli, increased crypt fission is due to activation of Wnt/β-catenin signalling, but the molecular basis of crypt fission during intestinal growth has not been examined. The aim of this project was to investigate whether crypt fission and intestinal growth are affected by experimental blockade of the Wnt/β-catenin signalling pathway.

Stem cell marker olfactomedin 4: critical appraisal of its characteristics and role in tumorigenesis.

Olfactomedin 4 (OLFM4), a member of the olfactomedin domain-containing proteins, is a glycoprotein with molecular weight of approximately 64 kDa. The protein is a "robust marker" of Lgr5+ stem cells and has been localised to mitochondria, nuclei and cell membranes. The bulk of OLFM4 exists in a polymeric form which is held together by disulfide bonds and carbohydrate interactions.

A comparison of the binding of urinary calcium oxalate monohydrate and dihydrate crystals to human kidney cells in urine.

Objective: To compare the binding kinetics of urinary calcium oxalate monohydrate (COM) and dihydrate (COD) crystals to human kidney (HK-2) cells in ultra-filtered (UF), and centrifuged and filtered (CF) human urine; and to quantify the binding of COM and COD crystals to cultured HK-2 cells in UF and CF urine samples collected from different individuals.

Materials And Methods: Urine was collected from healthy subjects, pooled, centrifuged and filtered. (14) C-oxalate-labelled COM and COD crystals were precipitated from the urine by adding oxalate after adjustment of two aliquots of the urine to 2 and 8 mm Ca(2+), respectively.

The effects of intracrystalline and surface-bound proteins on the attachment of calcium oxalate monohydrate crystals to renal cells in undiluted human urine.

Objective: To compare the binding to Madin-Darby canine kidney (MDCK)-II cells of: (i) inorganic calcium oxalate monohydrate (iCOM) crystals and COM crystals precipitated from urine containing different concentrations of protein; and (ii) urinary COM crystals containing intracrystalline and intracrystalline + surface-bound protein.

Materials And Methods: Urinary COM crystals were generated in sieved (sCOM), centrifuged and filtered (cfCOM), and ultrafiltered (ufCOM) portions of a pooled human urine and their adhesion to MDCK-II cells was compared using six different ultrafiltered urine samples as the binding medium. Crystal matrix extract (CME) was prepared by demineralizing calcium oxalate crystals precipitated from human urine and used to prepare COM crystals with intracrystalline, and intracrystalline + surface-bound CME at protein concentrations of 0, 0.

Face-specific binding of prothrombin fragment 1 and human serum albumin to inorganic and urinary calcium oxalate monohydrate crystals.

Objective: To compare the intracrystalline distributions of prothrombin fragment 1 (PTF1) and human serum albumin (HSA) within inorganic and urinary calcium oxalate (CaOx) monohydrate (COM) crystals and to determine whether binding of PTF1 can be explained by interactions between particular gamma-carboxyglutamic (Gla) residues and atomic arrays on individual faces of the COM crystal. MATERIALS AND METHODS COM: crystals were precipitated from inorganic solutions and ultrafiltered urine containing fluorescent HSA or PTF1 at different relative concentrations and examined by fluorescence microscopy. Accelrys Materials Studio and Discovery Studio were used to model the binding of PTF1 to the top, side and apical faces of the COM crystal.

High calcium concentration and calcium oxalate crystals cause significant inaccuracies in the measurement of urinary osteopontin by enzyme linked immunosorbent assay.

Strong evidence that osteopontin (OPN) is a determinant of urolithiasis has prompted studies comparing the protein's urinary excretion in healthy subjects and stone formers. However, reported mean urinary values have varied widely, from <1 microg/mL to more than 20 times that value. Since OPN binds to CaOx crystals, the presence of crystals in urine may cause underestimation of its urinary levels.

Intracrystalline urinary proteins facilitate degradation and dissolution of calcium oxalate crystals in cultured renal cells.

We have previously proposed that intracrystalline proteins would increase intracellular proteolytic disruption and dissolution of calcium oxalate (CaOx) crystals. Chauvet MC, Ryall RL. J Struct Biol 151: 12-17, 2005; Fleming DE, van Riessen A, Chauvet MC, Grover PK, Hunter B, van Bronswijk W, Ryall RL.

Novel action of n-3 polyunsaturated fatty acids: inhibition of arachidonic acid-induced increase in tumor necrosis factor receptor expression on neutrophils and a role for proteases.

Objective: Neutrophils and tumor necrosis factor (TNF) play important roles in the pathogenesis of rheumatoid arthritis (RA). Modulation of TNF receptors (TNFRs) may contribute to the regulation of tissue damage, and n-6 polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA) can increase the expression of TNFRI and TNFRII on neutrophils. Because the n-3 PUFAs are antiinflammatory in RA, we examined whether, as a novel mechanism of action, n-3 PUFAs can antagonize the AA-induced increase in TNFR expression.

The importance of a clean face: the effect of different washing procedures on the association of Tamm-Horsfall glycoprotein and other urinary proteins with calcium oxalate crystals.

This study was undertaken to determine whether the use of different washing procedures could explain dissident findings in published studies examining the role of urinary macromolecules in urolithiasis. Calcium oxalate monohydrate (COM) crystals were deposited from or added to the same sieved urine, washed with copious or limited amounts of distilled water, or with methanol, and examined by field emission scanning electron microscopy (FESEM). Demineralized extracts were analysed by SDS-PAGE and Western blotting for Tamm-Horsfall glycoprotein (THG), human serum albumin (HSA), osteopontin (OPN) and prothrombin fragment 1 (PTF1).

Effect of urine fractionation on attachment of calcium oxalate crystals to renal epithelial cells: implications for studying renal calculogenesis.

Our aim was to determine whether fractionation of human urine affects the attachment of calcium oxalate monohydrate (COM) crystals to renal cells. Urine collected from six healthy subjects was fractionated into sieved (S), centrifuged (C), centrifuged and filtered (CF), or ultrafiltered (UF). Attachment of [(14)C]COM crystals to Madin-Darby canine kidney (MDCK) cells was studied after precoating the crystals or the cells with the urine fractions and by using the same fractions as the binding medium.

Intracrystalline proteins and urolithiasis: a comparison of the protein content and ultrastructure of urinary calcium oxalate monohydrate and dihydrate crystals.

Objective: To compare the ultrastructure and protein content, particularly prothrombin fragment 1 and osteopontin, of calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals precipitated from human urine, and their susceptibility to proteolysis, to try to clarify the role of intracrystalline proteins in urolithiasis, as differences between these types of crystal may determine whether calcium oxalate crystals nucleated in urine progress to stone formation.

Materials And Methods: Sodium dodecyl sulphate gel electrophoresis and Western blotting were used to analyse demineralized extracts of COM and/or COD crystals deposited from the same centrifuged and filtered urine (which contains abundant urinary proteins) by adjusting the calcium concentration to 2 and 7 mmol/L, respectively. Similar analyses were performed on COM and COD crystals deposited from ultrafiltered urine (which contains only proteins of < 10 kDa) and then incubated in centrifuged and filtered urine, as well as crystals generated in the presence of increasing concentrations of proteins derived from the organic matrix of urinary calcium oxalate crystals.

Renal prothrombin: quantification of mRNA using reverse transcription-polymerase chain reaction in a rat model.

Attempts to quantify renal prothrombin (PT) have failed due to interference of its hepatic counterpart. In order to gauge PT synthesized by the kidney, the expression of PT mRNA was compared in renal and hepatic tissues of rats. PT mRNAs were quantified, using quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR), from RNA extracts of kidneys and livers of ten hooded Wistar rats.

Unique effect of arachidonic acid on human neutrophil TNF receptor expression: up-regulation involving protein kinase C, extracellular signal-regulated kinase, and phospholipase A2.

Arachidonic acid (AA) regulates the function of many cell types, including neutrophils. Although much emphasis has been placed on agonist-induced down-regulation of TNFR, our data show that AA caused a rapid (10-20 min) and dose-dependent (0.5-30 micro M) increase in the surface expression of both classes of TNFR (TNFR1 and TNFR2) on human neutrophils.

Intracrystalline proteins and urolithiasis: a synchrotron X-ray diffraction study of calcium oxalate monohydrate.

Unlabelled: The existence of intracrystalline proteins and amino acids in calcium oxalate monohydrate was demonstrated by X-ray synchrotron diffraction studies. Their presence has implications for the destruction of calcium oxalate crystals formed in the urinary tract and the prevention of kidney stones.

Introduction: Although proteins are present in human kidney stones, their role in stone pathogenesis remains unknown.

Dissolved urate salts out calcium oxalate in undiluted human urine in vitro: implications for calcium oxalate stone genesis.

Hyperuricosuria has long been documented as a predisposing factor to calcium oxalate (CaOx) stone pathogenesis. However, its mechanism is still without sound scientific foundation. Previously, we showed that hyperuricosuria, simulated by the addition of dissolved sodium urate, promotes the crystallization of CaOx.

The effect of preincubation of seed crystals of uric acid and monosodium urate with undiluted human urine to induce precipitation of calcium oxalate in vitro : implications for urinary stone formation.

Background: Previous studies demonstrated that crystals of uric acid (UA) and sodium urate (NaU) can induce the precipitation of calcium oxalate (CaOx) from its inorganic metastable solutions, but similar effects have not been unequivocally shown to occur in urine. The aim of this investigation was to determine whether preincubation of these seeds with urine alter their ability to induce deposition of CaOx from solution and thus provide a possible explanation for discrepancy of results obtained from aqueous inorganic solutions and undiluted urine.

Materials And Methods: The effects of commercial seed crystals of UA, NaU and CaOx (6 mg/100 ml) on CaOx crystallization were compared in a solution with the same crystals that had been preincubated for 3 hours with healthy male urine.

A simple single-step method for the synthesis of recombinant non-homologous competitor cDNA and its implications for quantitative PCR in biological sciences and molecular diagnostics.

In recent years there has been great interest in quantitative polymerase chain reaction. Consequently, a large number of assays have been developed, of which the one using non-homologous competitors is arguably the most precise. Despite widespread applications, currently there is no simple method to synthesize such competitors.

The effect of seed crystals of hydroxyapatite and brushite on the crystallization of calcium oxalate in undiluted human urine in vitro: implications for urinary stone pathogenesis.

Background: The aim of this study was to determine whether crystals of hydroxyapatite (HA) or brushite (BR) formed in urine promote the epitaxial deposition of calcium oxalate (CaOx) from undiluted human urine in vitro and thereby explain the occurrence of phosphate in the core of urinary stones consisting predominantly of CaOx.

Materials And Methods: Crystals of HA, BR, and CaOx were generated from human urine and their identity confirmed by X-ray analysis. Standard quantities of each crystal were then added to separate aliquots of pooled undiluted human urine and CaOx crystallization was induced by the addition of identical loads of sodium oxalate.

Effect of prothrombin and its activation fragments on calcium oxalate crystal growth and aggregation in undiluted human urine in vitro: relationship between protein structure and inhibitory activity.

In recent years there has been great interest in the putative role of prothrombin and its activation peptides, especially the urinary form of prothrombin fragment 1, in the pathogenesis of calcium oxalate (CaOx) urolithiasis. Previously, we showed that prothrombin and its activation peptides inhibit CaOx crystallization in inorganic conditions in vitro. The aim of the present study was to determine if this inhibitory activity is retained in undiluted human urine and, therefore, whether it is likely to have any influence under physiological conditions.