A London experience 1995-2012: demographic, dietary and biochemical characteristics of a large adult cohort of patients with renal stone disease.

BACKGROUND: Kidney stone disease has an estimated prevalence of around 10%. Genetic as well as environmental factors are thought to play an important role in the pathogenesis of renal stones. AIM: The aim of our study was to analyse and report the main characteristics of patients with kidney stones attending a large UK metabolic stone clinic in London between 1995 and 2012. DESIGN: A cross-sectional study. METHODS: Analysis of data from stone formers attending the University College and Royal Free Hospitals' metabolic stone clinic from 1995 to 2012. Demographic, clinical, dietary and biochemical characteristics have been summarized and analysed for men and women separately; trends over time have also been analysed. RESULTS: Of the 2861 patients included in the analysis, 2016 (70%) were men with an average age of 47 years (range 18-87 years) and median duration of disease of 6 years (range 0-60 years). The prevalence of low urine volume, hypercalciuria, hyperoxaluria, hyperuricosuria and hypocitraturia was 5.6%, 38%, 7.9%, 18% and 23%, respectively. The prevalence of several risk factors for stones increased over time. The majority of stones were mixed, with around 90% composed of calcium salts in varying proportion. CONCLUSION: Our findings in a large cohort of patients attending a London-based stone clinic over the past 20 years show differences in distributions of risk factors for stones for men and women, as well as metabolic profiles and stone composition. The impact of most risk factors for stones appeared to change over time.

Label-free electronic detection of bio-toxins using aligned carbon nanotubes.

A facile route for sensitive label-free detection of bio-toxins using aligned single walled carbon nanotubes is described. This approach involves patterning of a catalyst on the surface of a quartz substrate using a sub-100 µm stripe-patterned polydimethylsiloxane stamp for aligned carbon nanotube generation followed by fabrication of field effect transistor (FET). Atomic force microscopy, field emission scanning electron microscopy and Raman spectroscopy are employed to characterize the synthesized nanotubes. Unlike previous reports, the adopted approach enables direct electronic detection of bio-toxins with sensitivities comparable to ELISA. As a proof of concept, the fabricated FET responds to nM concentration levels (with a LOD of ∼2 nM) of epsilon toxin produced by Clostridium perfringens and a prominent food toxin. This facile approach could be customized to detect other classes of toxins and biomarkers upon appropriate functionalization of the aligned carbon nanotubes. Finally, we demonstrate the use of the FET-platform for detection of toxin in more complex matrices such as orange juice.

Microarray-based enzyme profiling: Recent advances and applications (Review).

Enzymes are an integral part of biological systems. They constitute a significant majority of all proteins expressed (an estimated 18%-29%) within eukaryotic genomes. It thus comes as no major surprise that enzymes have been implicated in many diseases and form the second largest group of drug targets, after receptors. Despite their involvement in a multitude of physiological processes, only a limited number of enzymes have thus far been well-characterized. Consequently, little is understood about the physiological roles, substrate specificity, and downstream targets of the vast majority of these important proteins. In order to facilitate the biological characterization of enzymes, as well as their adoption as drug targets, there is a need for global "-omics" solutions that bridge the gap in understanding these proteins and their interactions. Herein the authors showcase how microarray methods can be adopted to facilitate investigations into enzymes and their properties, in a high-throughput manner. They will focus on several major classes of enzymes, including kinases, phosphatases, and proteases. As a result of research efforts over the last decade, these groups of enzymes have become readily amenable to microarray-based profiling methods. The authors will also describe the specific design considerations that are required to develop the appropriate chemical tools and libraries to characterize each enzyme class. These include peptide substrates, activity-based probes, and chemical compound libraries, which may be rapidly assembled using efficient combinatorial synthesis or "click chemistry" strategies. Taken together, microarrays offer a powerful means to study, profile, and also discover potent small molecules with which to modulate enzyme activity.

Aligned carbon nanotubes on quartz substrate for liquid gated biosensing.

A facile and high performance biosensing platform using aligned carbon nanotubes on quartz substrate is reported in this communication. Single walled carbon nanotubes are grown on quartz substrates by a chemical vapor deposition process and are characterized with field emission scanning electron microscopy and atomic force microscopy in order to verify the quality of the material. The quartz substrate is then directly used as a biosensor in a field effect transistor configuration. In order to demonstrate the sensing capabilities of the fabricated sensor devices, electronic detection of prostate specific antigen, a potential cancer biomarker, is carried out by adopting liquid gated configuration. A conductivity change due to the specific binding of target antigen with the immobilized receptor antibody demonstrates the sensing capabilities of the fabricated device. Sub-nM detection sensitivities have been obtained using the adopted direct immunoassay approach, which shows that the device responds to clinically relevant concentration regimes.

Renal calcium stones: insights from the control of bone mineralization.

Extracellular pyrophosphate (PPi) plays a central role in the control of normal bone mineralization since it antagonizes inorganic phosphate in the promotion of hydroxyapatite deposition. Studies using knock-out mice have established the functional importance of PPi generation via nucleotide pyrophosphatase phosphodiesterases (NPP) and of PPi transmembrane transport by the progressive ankylosis (ANK) protein. Tissue non-specific alkaline phosphatase activity counteracts this by hydrolysis of PPi to inorganic phosphate. The molecular nature and transport function of ANK are reviewed. A close parallel is drawn between the controlled mineralization of bone and the prevention of abnormal calcium crystal deposition within the kidney, especially when concentrated urine is produced. Pyrophosphate is present in urine, and ANK is expressed in the cortical collecting duct where PPi transport to both the tubular lumen and the renal interstitium may occur. Pyrophosphate may also be generated here by nucleoside triphosphate diphosphohydrolases (NTPD2 and 3) together with NPP1. Alkaline phosphatase activity is restricted to the proximal nephron, remote from these sites of PPi generation, transport and function. The physiological importance of PPi generation and transport in preventing idiopathic calcium renal stone disease and nephrocalcinosis now needs to be established.

How we estimate GFR--a pitfall of using a serum creatinine-based formula.

Chronic kidney disease (CKD) is defined using the estimated glomerular filtration rate (eGFR). This has led to a large increase in the diagnosis of CKD in the United Kingdom, the majority of which is in its earlier stages and is detected in non-hospital settings. It is important to be aware that eGFR calculations will reflect inaccuracies in the measured serum creatinine, as the latter is an important component of the calculation. We report a case in which a patient with high muscle-mass who had consumed large quantities of a creatine-containing nutritional supplement presented with apparently reduced renal function on the basis of the serum creatinine and therefore also the eGFR calculation (MDRD equation). Creatine is an amino acid which is a precursor of creatinine, and is known to transiently increase serum creatinine. 6 weeks after discontinuing creatine ingestion, serum creatinine had fallen but still gave rise to an apparently abnormal calculated eGFR. In fact, renal function was shown to be normal when estimated using 24-hour urinary creatinine clearance. This case demonstrates that the upper extreme of muscle mass and ingestion of creatine can affect not only serum creatinine but also the calculated eGFR. Knowledge of common confounding factors and their effects on serum creatinine and eGFR will allow appreciation of the limitations of these measures of renal function, and can prevent unnecessary over-investigation of such patients.

Synergism between hydrogen sulfide (H(2)S) and nitric oxide (NO) in vasorelaxation induced by stonustoxin (SNTX), a lethal and hypotensive protein factor isolated from stonefish Synanceja horrida venom.

Stonustoxin (SNTX) is a 148 kDa, dimeric, hypotensive and lethal protein factor isolated from the venom of the stonefish Synanceja horrida. SNTX (10-320 ng/ml) progressively causes relaxation of endothelium-intact, phenylephrine (PE)-precontracted rat thoracic aortic rings. The SNTX-induced vasorelaxation was inhibited by L-N(G)-nitro arginine methyl ester (L-NAME), suggesting that nitric oxide (NO) contributes to the SNTX-induced response. Interestingly, D, L-proparglyglycine (PAG) and beta-cyano-L-alanine (BCA), irreversible and competitive inhibitors of cystathionine-gamma-lyase (CSE) respectively, also inhibited SNTX-induced vasorelaxation, indicating that H(2)S may also play a part in the effect of SNTX. The combined use of L-NAME with PAG or BCA showed that H(2)S and NO act synergistically in effecting SNTX-induced vasorelaxation.

Immunomodulation of Japanese encephalitis vaccine through CpG oligodeoxynucleotides in mice.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine guanine (CpG) dinucleotides motifs act as immune adjuvant and provide means of modulation to immune responses when co-delivered with antigens. They stimulate both innate and adaptive immune responses and induce T helper 1 (Th1) immune responses. We investigated the immunomodulation of Japanese encephalitis (JE) vaccine using CpG ODN as an adjuvant. Mice were immunized with one dose of JE vaccine 0.1 ml with different concentrations (10, 25 and 100 microg) of CpG ODN. The serum antibody level and cytokines were evaluated and compared with mice immunized with two doses of JE vaccine alone. Our studies revealed that anti-JE antibody level in mice immunized with single dose of 0.1 ml JE vaccine and 100 microg CpG ODN were almost equal to mice immunized with two doses of JE vaccine alone. Furthermore, CpG ODN enhanced the production of TNF-alpha and Th1-mediated cytokines, including IFN-gamma and IL-2 compared with JE vaccine alone. In addition, absence of any significant changes in biochemical, haematological and histological studies suggest that CpG ODN are safe adjuvants for JE vaccine. Therefore, it is inferred that CpG ODN are effective and improve the efficacy of JE vaccine.

Melatonin attenuates stress-induced defecation: lesson from a rat model of stress-induced gut dysfunction.

Melatonin is known to alleviate stress and modulate gut motility. We investigated the modulating effects of melatonin on stress-induced gut dysfunction. One hundred Wistar rats were randomly assigned to five equal groups, receiving intraperitoneal injections of 0, 1, 10, 100 or 1000 microg kg(-1) melatonin, respectively. Fifteen minutes later, each group was divided again into four subgroups receiving no treatment, 0.25 mg luzindole (a non-selective melatonin receptor antagonist) intraperitoneally, wrap-restraint stress, and 10 mg kg(-1) serotonin intraperitoneally, respectively. Two hours later, serum serotonin, corticotropin-releasing factor (CRF) and melatonin levels, and faecal output were recorded. Results showed that intraperitoneal melatonin increased faecal output, but this effect was abolished by luzindole. In wrap-restraint group, prior intraperitoneal melatonin at doses of 100 or 1000 microg kg(-1) significantly inhibited stress-induced defecation. This effect was associated with corresponding reductions in serum serotonin and CRF concentrations. In serotonin-treated group, serotonin-induced defecation was also inhibited by melatonin. In conclusion, melatonin exhibited an excitatory effect on bowel output in rats placed under resting state, while attenuated defecation in those subjected to wrap-restraint stress or serotonin treatment. The inhibitory effects of melatonin on stress-induced defecation may stem from its antagonistic effect on stress-induced enhancement of serotonin and CRF secretion.

Influence of diabetes on peripheral bone mineral density in men: a controlled study.

The aims of the study were to (1) compare peripheral bone mineral density (BMD) in men with diabetes to peripheral BMD in non-diabetic men, and (2) explore factors which may predict BMD in diabetic men. Ninety men with type 2 diabetes and 35 men with type 1 diabetes were randomly selected for participation via a computerised database. Fifty healthy males were also recruited. All patients had peripheral BMD measured by dual energy Xray absorptiometry (DEXA) at the non-dominant distal radius. Information on a number of clinical parameters was obtained by direct questioning, and from patient case notes. The mean age (95% confidence interval (CI)) of the type 1 diabetic group, type 2 diabetic group and control group were, respectively: 49.3 years (44.6-53.9), 62.8 years (60.7-64.8) and 38.5 (34.9-42.1) years. Median (95% CI) Z-scores for the three groups were: -0.18 (-0.68 to +0.32), +0.19 (-0.14 to +0.49) and -0.02 (-0.4 to +0.31), respectively (p=not significant). Only body mass index (BMI) was correlated with BMD in the type 1 diabetic group, and only BMI and age were correlated with BMD in type 2 diabetics. There was no correlation between BMD and glycosylated haemoglobin concentration (HbA(1)c), disease duration or presence of microvascular or macrovascular complications in either of the diabetic groups. We did not find any significant difference in peripheral BMD between patients with type 1 diabetes, type 2 diabetes and controls.

Melatonin improves abdominal pain in irritable bowel syndrome patients who have sleep disturbances: a randomised, double blind, placebo controlled study.

BACKGROUND AND AIMS: Melatonin, a sleep promoting agent, is involved in the regulation of gastrointestinal motility and sensation. We aimed to determine if melatonin was effective in improving bowel symptoms and sleep disturbances in irritable bowel syndrome (IBS) patients with sleep disturbance. METHODS: Forty IBS patients (aged 20-64 years; 24 female) with sleep disturbances were randomly assigned to receive either melatonin 3 mg (n = 20) or matching placebo (n = 20) at bedtime for two weeks. Immediately before and after the treatment, subjects completed bowel, sleep, and psychological questionnaires, and underwent rectal manometry and overnight polysomnography. RESULTS: Compared with placebo, melatonin taken for two weeks significantly decreased mean abdominal pain score (2.35 v 0.70; p<0.001) and increased mean rectal pain threshold (8.9 v -1.2 mm Hg; p<0.01). Bloating, stool type, stool frequency, and anxiety and depression scores did not significantly differ after treatment in both groups. Data from sleep questionnaires and polysomnography showed that the two week course of melatonin did not influence sleep parameters, including total sleep time, sleep latency, sleep efficiency, sleep onset latency, arousals, duration of stages 1-4, rapid eye movement (REM) sleep, and REM onset latency. CONCLUSIONS: Administration of melatonin 3 mg at bedtime for two weeks significantly attenuated abdominal pain and reduced rectal pain sensitivity without improvements in sleep disturbance or psychological distress. The findings suggest that the beneficial effects of melatonin on abdominal pain in IBS patients with sleep disturbances are independent of its action on sleep disturbances or psychological profiles.

Cytokine changes in the horizontal diagonal band of Broca in the septum after running and stroke: a correlation to glial activation.

The relationship between running, glial cell activation and pro-inflammatory cytokines was studied in the context of neuroprotection against ischemic stroke induced by middle cerebral artery occlusion (MCAO). This was investigated in four groups of rats, namely, (1) nonrunner, (2) runner after 12 weeks of treadmill running, (3) nonrunner with MCAO and (4) runner with MCAO. The horizontal diagonal band of Broca (HDB) in the septum was scrutinized for qualitative cum quantitative changes in the microglia and astrocytes. Reverse transcription-polymerase chain reaction and immunoblot work were carried out in the forebrain homogenate to determine, respectively, the gene and protein expression of several pro-inflammatory cytokines. Our results indicated that the runner exhibited less immunoreactivity and reduced numbers of glial cells within the HDB compared with the nonrunner. Interestingly, the mRNA and protein levels of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and interferon-gamma, were significantly downregulated in the runner. Our data also suggest albeit with some inconsistency that the runner/MCAO rats had benefited from running. These observations suggest that running can result in changes to the microenvironment, in which the microglia and astrocytes exist in a state of quiescence concomitant with a reduced expression of pro-inflammatory cytokines, that may lead to beneficial effects seen in ischemic stroke induced by MCAO.

Analysis of strain difference in behavior to Cholecystokinin (CCK) receptor mediated drugs in PVG hooded and Sprague-Dawley rats using elevated plus-maze test apparatus.

This study investigated the behavioral mechanisms underlying the anxiogenic, or anxiolytic mediated effects of CCK(2) receptor mediated agonist (CCK-4) and antagonist drugs (LY225910, LY288513, CR2945) in PVG hooded and Sprague-Dawley (SD) rats using the elevated plus maze test apparatus. In addition, the effects of a CCK(1) antagonist (CR1409) were investigated for its possible mediation in anxiety behavior between PVG hooded and SD rats. PVG hooded rats treated with CCK-4, decreased the time spent in the open arm and increased the time spent in the closed arm and correspondingly showed increase in the number of entries in the open arms while the number of entries in closed arm was insignificant, whereas SD rats decreased the time spent in the closed arm, while other parameters remained insignificant. PVG hooded rats administered with various CCK(2) antagonists (LY225910, LY288513, and CR2945) significantly increased the time spent in the open arm and correspondingly decreased the time spent in the closed arm, while the number of entries in the open or closed arm was insignificant, in contrast, SD rats failed to show any reliable significance. PVG hooded rats administered with the CCK(1) antagonist (CR1409), failed to show any reliable significance, in contrast, SD rats significantly increased the time spent in the open arm. The strain differences observed in this study suggests that CCK plays mainly as a neuromodulator, in which the various CCK(2) antagonists may not affect baseline anxiety state, but instead they modulate heightened states of anxiety through differential effects of CCK(1)/CCK(2) receptors.

Spermine reduces infarction and neurological deficit following a rat model of middle cerebral artery occlusion: a magnetic resonance imaging study.

The role of nitric oxide (NO) in post-ischemic cerebral infarction has been extensively examined, but few studies have investigated its role on the neurological deficit. In the present study, we investigated the effect of spermine on the temporal evolution of infarct volume, NO production and neurological deficit using magnetic resonance imaging in a model of permanent focal cerebral ischemia in rats. Spermine given at 10 mg/kg 2 h after ischemia reduced the infarct volume by 40% and abolished brain NO production and improved the neurological score 24 h, 48 h and 72 h after ischemia. Spermine also reduced the neurological deficit as evaluated by rotamex, grip strength and neurological severity score tests.

The CCK2 agonist BC264 reverses freezing behavior habituation in PVG hooded rats on repeated exposures to a cat.

Our previous studies (NeuroReport 12 (2001) 2717) showed that PVG hooded and not Sprague-Dawley (SD) rats exhibit remarkable freezing behavior on exposure to a cat in the cat freezing test apparatus. In the present study, we further examined the differences between these two strains of rats in response to repeated daily exposure to a cat in the cat freezing test apparatus. Freezing behavior habituation was observed in both PVG hooded (days 5-7) and SD rats (days 3-7). A selective CCK(2) agonist (BC264, 0.3 microg/kg, i.p.) on day 8 reversed habituated freezing behavior and locomotor activity in PVG hooded rats, but not in SD rats. These results suggest that CCK2 receptors mediate habituation to an anxiety-inducing stimulus in PVG hooded rats and further suggest that differential expression of these CCK2 receptors underlies this strain difference.

Role of phenytoin in wound healing: microarray analysis of early transcriptional responses in human dermal fibroblasts.

Wound healing is a complex process involving a number of related genes and receptors. Using cDNA microarrays, we explored the global gene expression profile of phenytoin (20microg/ml) induced changes to human dermal fibroblasts. Microarray data analysis revealed approximately 1500 genes were differentially expressed by 2.5-fold. At 3, 6, 12, and 24h, the transcripts of the major growth factors involved in wound healing and their receptors were increased. This was further confirmed by RT-PCR. Genes encoding other proteins with roles in signal transduction (NFkappaB), extracellular matrix (MMP1) including type I collagen, fibronectin, and laminin were strongly induced at 6h and onwards. Genes involved in cell cycle regulation (CCND1 and CDKN1A) were down-regulated consistent with our finding that phenytoin per se did not have cell proliferation activity. Notably, phenytoin accelerates the autocrine and paracrine activity of growth factors by up-regulating the related receptors.

Distinct regions of periaqueductal gray (PAG) are involved in freezing behavior in hooded PVG rats on the cat-freezing test apparatus.

The periaqueductal gray (PAG) is considered to be an exit relay for defensive responses. Studies have shown that the ventrolateral periaqueductal gray (vlPAG) plays a role in the expression of freezing behavior whereas dorsolateral periaqueductal gray (dlPAG) is involved on both freezing and active forms of defensive behaviors. To further elucidate this theory, lesioned vlPAG and dlPAG rats were exposed to a cat in the cat-freezing test apparatus. Subsequently, a 7-day repeated exposure to a cat was done on the vlPAG and dlPAG lesioned rats. Results showed that the vlPAG lesioned rats demonstrated significant decrease in freezing behavior and corresponding increase in locomotor activity, while the dlPAG lesioned rats failed to show any significance. Subsequent repeated exposure of the vlPAG lesioned rats to a cat for 7 days showed a gradual decrease in freezing behavior with significance shown at days 5, 6 and 7 while the dlPAG lesioned rats failed to show any changes. These results suggest that vlPAG regulates freezing behavior in hooded PVG rats.

cDNA microarray analysis of gene expression in anxious PVG and SD rats after cat-freezing test.

To identify genes involved in the development of anxiety or fear, we analyzed the gene expression profiles of the cortex of anxious hooded PVG and Sprague-Dawley (SD) rats after exposure to the cat-freezing test apparatus. These two rat strains showed a marked difference in the extent of anxious behavior on the cat-freezing test; the hooded PVG rats showed highly anxious behavior while a low anxiety state was observed in SD rats. A cDNA microarray consisting of 5,931 genes was employed to investigate the global mRNA expression profiles of anxiety-related genes. According to the assumption that an abundance ratio of > or =1.5 is indicative of a change in gene expression, we detected 16 upregulated and 38 downregulated genes in PVG hooded and SD rats. Some of these genes have not yet been associated with anxiety (e.g. FGF), while other genes were recently found to be expressed in an anxious state (e.g., rat nerve growth factor-induced gene, NGFI-A). Our study also focused on the expression of some neurotransmitter receptors that have already been proven to be relevant to anxiety or fear, e.g., gamma-aminobutyric acid (GABA), cholecystokinin (CCK) and 5-HT(3) receptors. To further confirm the microarray data, the mRNA expressions of three genes: rat activity-regulated cytoskeleton-associated gene (Arc), rat NGFI-A gene and rat 5-HT(3) receptor (5-HT(3)R) mRNA, were studied by reverse transcription-polymerase chain reaction (RT-PCR). The results of RT-PCR were basically consistent with those from cDNA microarray. Our study therefore demonstrated that the microarray technique is an efficient tool for analyzing global expression profiles of anxiety-related genes, which may also provide further insight into the molecular mechanisms underlying the states of anxiety and fear.

Genetic variations in CCK2 receptor in PVG hooded and Sprague-Dawley rats and its mRNA expression on cat exposure.

This study examined differential freezing behavior, mediated by cholecystokinin-2 (CCK2) receptors (J. M. Farook et al., 2001), in PVG hooded and Sprague-Dawley (SD) rats exposed to a predator. The authors confirmed by reverse transcription polymerase chain reaction that CCK2 receptor expression in the PVG rats was increased in the hippocampus and cerebral cortex compared with that of SD rats. In addition, 4 variations in the coding region of the CCK2 receptor gene were detected between the PVG hooded and SD rats: 1 in Exon 4, 1 in Intron 2, and 2 in Intron 3. Acute treatment with a CCK2 agonist (CCK-4) or antagonist (LY225910) did not alter the level of CCK2 receptor expression, indicating no difference between the 2 strains in sensitivity of the CCK2 receptor to drugs.

Neuroprotection associated with running: is it a result of increased endogenous neurotrophic factors?

The possible neuroprotective effect of physical exercise was investigated in rats after middle cerebral artery occlusion (MCAO), a focal stroke model. It was found that physical exercise in the form of a 12-week treadmill running programme reduced the volume of infarction caused by MCAO. At the molecular level, reverse transcription polymerase chain reaction revealed that the runner had increased gene expression for nerve growth factor (NGF) over the nonrunner with or without MCAO. Expression of the NGF receptors, p75, was increased only in the absence of MCAO. In addition, runners showed a significantly higher number of cholinergic neurons, which constitutively expressed p75, in the horizontal diagonal band of Broca. The present findings suggest that neuroprotection after physical exercise may be a result of an increase in an endogenous neurotrophic factor nerve growth factor and the proliferation of its receptive cholinergic neurons.