Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ? 98 %). All samples were analyzed following injection (100 ?l) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 ?m, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2-30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min(-1) flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients

Clinical course, therapeutic responses and outcomes in relapsing MOG antibody-associated demyelination.

Abstract OBJECTIVE: We characterised the clinical course, treatment and outcomes in 59 patients with relapsing myelin oligodendrocyte glycoprotein (MOG) antibody-associated demyelination. METHODS: We evaluated clinical phenotypes, annualised relapse rates (ARR) prior and on immunotherapy and Expanded Disability Status Scale (EDSS), in 218 demyelinating episodes from 33 paediatric and 26 adult patients. RESULTS: The most common initial presentation in the cohort was optic neuritis (ON) in 54% (bilateral (BON) 32%, unilateral (UON) 22%), followed by acute disseminated encephalomyelitis (ADEM) (20%), which occurred exclusively in children. ON was the dominant phenotype (UON 35%, BON 19%) of all clinical episodes. 109/226 (48%) MRIs had no brain lesions. Patients were steroid responsive, but 70% of episodes treated with oral prednisone relapsed, particularly at doses <10\u2009mg daily or within 2 months of cessation. Immunotherapy, including maintenance prednisone (P=0.0004), intravenous immunoglobulin, rituximab and mycophenolate, all reduced median ARRs on-treatment. Treatment failure rates were lower in patients on maintenance steroids (5%) compared with non-steroidal maintenance immunotherapy (38%) (P=0.016). 58% of patients experienced residual disability (average follow-up 61 months, visual loss in 24%). Patients with ON were less likely to have sustained disability defined by a final EDSS of 652 (OR 0.15, P=0.032), while those who had any myelitis were more likely to have sustained residual deficits (OR 3.56, P=0.077). CONCLUSION: Relapsing MOG antibody-associated demyelination is strongly associated with ON across all age groups and ADEM in children. Patients are highly responsive to steroids, but vulnerable to relapse on steroid reduction and cessation

Patient-reported outcomes in metastatic castration-resistant prostate cancer

Many novel therapies are available for use in patients with metastatic castration-resistant prostate cancer (mCRPC), some of which convey substantial progression-free survival and overall survival benefits. Delaying disease progression and providing palliation of symptoms are primary therapeutic aims of treating patients with mCRPC; therefore, ensuring that the benefit-to-harm ratios are acceptable to patients, through systematic measurement of patient-reported outcomes (PROs) using validated tools, is vital. In this Perspectives, we appraised the published reports from clinical trials testing treatments of mCRPC over the past 5 years and found that PROs were either not being measured routinely, or if used, were often not reported adequately, thus hampering evaluation of the true effects of many of these treatments on patients' quality of life. Improvements are needed because data collected directly from patients, not just physician-collected safety data and adverse events, are crucial to inform clinical decision-making on treatment options

Intracranial injection of dengue virus induces interferon stimulated genes and CD8(+) T cell infiltration by sphingosine kinase 1 independent pathways

We have previously reported that the absence of sphingosine kinase 1 (SK1) affects both dengue virus (DENV) infection and innate immune responses in vitro. Here we aimed to define SK1-dependancy of DENV-induced disease and the associated innate responses in vivo. The lack of a reliable mouse model with a fully competent interferon response for DENV infection is a challenge, and here we use an experimental model of DENV infection in the brain of immunocompetent mice. Intracranial injection of DENV-2 into C57BL/6 mice induced body weight loss and neurological symptoms which was associated with a high level of DENV RNA in the brain. Body weight loss and DENV RNA level tended to be greater in SK1-/- compared with wildtype (WT) mice. Brain infection with DENV-2 is associated with the induction of interferon-β (IFN-β) and IFN-stimulated gene (ISG) expression including viperin, Ifi27l2a, IRF7, and CXCL10 without any significant differences between WT and SK1-/- mice. The SK2 and sphingosine-1-phosphate (S1P) levels in the brain were unchanged by DENV infection or the lack of SK1. Histological analysis demonstrated the presence of a cellular infiltrate in DENV-infected brain with a significant increase in mRNA for CD8 but not CD4 suggesting this infiltrate is likely CD8+ but not CD4+ T-lymphocytes. This increase in T-cell infiltration was not affected by the lack of SK1. Overall, DENV-infection in the brain induces IFN and T-cell responses but does not influence the SK/S1P axis. In contrast to our observations in vitro, SK1 has no major influence on these responses following DENV-infection in the mouse brain.Wisam H. Al-Shujairi, Jennifer N. Clarke, Lorena T. Davies, Mohammed Alsharifi, Stuart M. Pitson, Jillian M. Car

Seasonal variations in the nitrogen isotopic composition of settling particles at station K2 in the western subarctic North Pacific

Intensive observations using hydrographical cruises and moored sediment trap deployments during 2010 and 2012 at station K2 in the North Pacific western subarctic gyre (WSG) revealed seasonal changes in δ15N of both suspended and settling particles. Suspended particles (SUS) were collected from depths between the surface and 200 m; settling particles by drifting traps (DST; 100-200 m) and moored traps (MST; 200 and 500 m). All particles showed higher δ15N values in winter and lower in summer, contrary to the expected by isotopic fractionation during phytoplankton nitrate consumption. We suggest that these observed isotopic patterns are due to ammonium consumption via light-controlled nitrification, which could induce variations in δ15N(SUS) of 0.4-3.1 ‰ in the euphotic zone (EZ). The δ15N(SUS) signature was reflected by δ15 N(DST) despite modifications during biogenic transformation from suspended particles in the EZ. δ15 N enrichment (average: 3.6 ‰) and the increase in C:N ratio (by 1.6) in settling particles suggests year-round contributions of metabolites from herbivorous zooplankton as well as TEPs produced by diatoms. Accordingly, seasonal δ15 N(DST) variations of 2.4-7.0 ‰ showed a significant correlation with primary productivity (PP) at K2. By applying the observed δ15 N(DST) vs. PP regression to δ15 N(MST) of 1.9-8.0 ‰, we constructed the first annual time-series of PP changes in the WSG. Moreover, the monthly export ratio at 500 m was calculated using both estimated PP and measured organic carbon fluxes. Results suggest a 1.6 to 1.8 times more efficient transport of photosynthetically-fixed carbon to the intermediate layers occurs in summer/autumn rather than winter/spring

H2S biosynthesis and catabolism: new insights from molecular studies

Hydrogen sulfide (H2S) has profound biological effects within living organisms and is now increasingly being considered alongside other gaseous signalling molecules, such as nitric oxide (NO) and carbon monoxide (CO). Conventional use of pharmacological and molecular approaches has spawned a rapidly growing research field that has identified H2S as playing a functional role in cell-signalling and post-translational modifications. Recently, a number of laboratories have reported the use of siRNA methodologies and genetic mouse models to mimic the loss of function of genes involved in the biosynthesis and degradation of H2S within tissues. Studies utilising these systems are revealing new insights into the biology of H2S within the cardiovascular system, inflammatory disease, and in cell signalling. In light of this work, the current review will describe recent advances in H2S research made possible by the use of molecular approaches and genetic mouse models with perturbed capacities to generate or detoxify physiological levels of H2S gas within tissue

CSF neopterin and quinolinic acid are biomarkers of neuroinflammation and neurotoxicity in FIRES and other infection-triggered encephalopathy syndromes.

OBJECTIVE: Infection-triggered encephalopathy syndromes (ITES) are potentially devastating neuroinflammatory conditions. Although some ITES syndromes have recognisable MRI neuroimaging phenotypes, there are otherwise few biomarkers of disease. Early detection to enable immune modulatory treatments could improve outcomes. METHODS: We measured CSF neopterin, quinolinic acid, kynurenine and kynurenine/tryptophan ratio using a liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) system. The CSF of 18 children with ITES were compared with acute encephalitis (n = 20), and three control groups, namely epilepsy (n = 20), status epilepticus (n = 18) and neurogenetic controls (n = 20). RESULTS: The main ITES phenotypes in 18 patients were acute encephalopathy with biphasic seizures and late restricted diffusion (AESD, n = 4), febrile infection-related epilepsy syndrome (FIRES n = 4) and other ITES phenotypes. Influenza A was the most common infectious trigger (n = 5), and 50% of patients had a preceding notable neurodevelopmental or family history. CSF neopterin, quinolinic acid and kynurenine were elevated in ITES group compared to the three control groups (all p < 0.0002). The ROC (area under curve) for CSF neopterin (99.3%, CI 98.1-100) was significantly better than CSF pleocytosis (87.3% CI 76.4-98.2) (p = 0.028). Elevated CSF neopterin could discriminate ITES from other causes of seizures, status epilepticus and febrile status epilepticus (all p < 0.0002). The elevated CSF metabolites normalised during longitudinal testing in two patients with FIRES. INTERPRETATION: CSF neopterin and quinolinic acid are neuroinflammatory and excitotoxic metabolites. This CSF metabolomic inflammatory panel can discriminate ITES from other causes of new onset seizures or status epilepticus, and rapid results (4 h) may facilitate early immune modulatory therapy

Silica burial enhanced by iron limitation in oceanic upwelling margins

In large swaths of the ocean, primary production by diatoms may be limited by the availability of silica, which in turn limits the biological uptake of carbon dioxide. The burial of biogenic silica in the form of opal is the main sink of marine silicon. Opal burial occurs in equal parts in iron-limited open-ocean provinces and upwelling margins, especially the eastern Pacific upwelling zone. However, it is unclear why opal burial is so efficient in this margin. Here we measure fluxes of biogenic material, concentrations of diatom-bound iron and silicon isotope ratios using sediment traps and a sediment core from the Gulf of California upwelling margin. In the sediment trap material, we find that periods of intense upwelling are associated with transient iron limitation that results in a high export of silica relative to organic carbon. A similar correlation between enhanced silica burial and iron limitation is evident in the sediment core, which spans the past 26,000 years. A global compilation also indicates that hotspots of silicon burial in the ocean are all characterized by high silica to organic carbon export ratios, a diagnostic trait for diatoms growing under iron stress. We therefore propose that prevailing conditions of silica limitation in the ocean are largely caused by iron deficiency imposing an indirect constraint on oceanic carbon uptake

Born Knowing: Tentacled Snakes Innately Predict Future Prey Behavior

Background: Aquatic tentacled snakes (Erpeton tentaculatus) can take advantage of their prey’s escape response by startling fish with their body before striking. The feint usually startles fish toward the snake’s approaching jaws. But when fish are oriented at a right angle to the jaws, the C-start escape response translates fish parallel to the snake’s head. To exploit this latter response, snakes must predict the future location of the fish. Adult snakes can make this prediction. Is it learned, or are tentacled snakes born able to predict future fish behavior? Methods and Findings: Laboratory-born, naïve snakes were investigated as they struck at fish. Trials were recorded at 250 or 500 frames per second. To prevent learning, snakes were placed in a water container with a clear transparency sheet or glass bottom. The chamber was placed over a channel in a separate aquarium with fish below. Thus snakes could see and strike at fish, without contact. The snake’s body feint elicited C-starts in the fish below the transparency sheet, allowing strike accuracy to be quantified in relationship to the C-starts. When fish were oriented at a right angle to the jaws, naïve snakes biased their strikes to the future location of the escaping fish’s head, such that the snake’s jaws and the fish’s translating head usually converged. Several different types of predictive strikes were observed. Conclusions: The results show that some predators have adapted their nervous systems to directly compensate for the future behavior of prey in a sensory realm that usually requires learning. Instead of behavior selected during their lifetime