HR/MS-based lipidome analysis of rat brain modulated by tolcapone.

Lipids play key roles in the body, influencing cellular regulation, function, and signalling. Tolcapone, a potent catechol-O-methyltransferase (COMT) inhibitor described to enhance cognitive performance in healthy subjects, was previously shown to impact fatty acid ß-oxidation and oxidative phosphorylation. However, its impact on the brain lipidome remains unexplored. Hence, this study aimed to assess how tolcapone affects the lipidome of the rat pre-frontal cortex (PFC), a region of the brain highly relevant to tolcapone therapeutic effect, while evaluating its influence on operant behaviour. Tolcapone at 20 mg/kg was chronically administered to Wistar rats during a behavioural task and an untargeted liquid chromatography high-resolution mass spectrometry (LC-HR/MS) approach was employed to profile lipid species. The untargeted analysis identified 7227 features, of which only 33% underwent statistical analysis following data pre-processing. The results revealed an improved cognitive performance and a lipidome remodelling promoted by tolcapone. The lipidomic analysis showed 32 differentially expressed lipid species in tolcapone-treated animals (FC ≥ 1.2, p-value ≤ 0.1), and among these several triacylglycerols, cardiolipins and N-acylethanolamine (NAE 16:2) were found upregulated whereas fatty acids, hexosylceramides, and several phospholipids including phosphatidylcholines and phosphatidylethanolamines were downregulated. These preliminary findings shed light on tolcapone impact on lipid pathways within the brain. Although tolcapone improved cognitive performance and literature suggests the significance of lipids in cognition, this study did not conclusively establish that lipids directly drove or contributed to this outcome. Nevertheless, it underscores the importance of lipid modulation and encourages further exploration of tolcapone-associated mechanisms in the central nervous system (CNS).

Salt-inducible kinases: new players in pulmonary arterial hypertension?

Salt-inducible kinases (SIKs) are serine/threonine kinases belonging to the AMP-activated protein kinase (AMPK) family. Accumulating evidence indicates that SIKs phosphorylate multiple targets, including histone deacetylases (HDACs) and cAMP response element-binding protein (CREB)-regulated transcriptional coactivators (CRTCs), to coordinate signaling pathways implicated in metabolism, cell growth, proliferation, apoptosis, and inflammation. These pathways downstream of SIKs are altered not only in pathologies like cancer, systemic hypertension, and inflammatory diseases, but also in pulmonary arterial hypertension (PAH), a multifactorial disease characterized by pulmonary vasoconstriction, inflammation and remodeling of pulmonary arteries owing to endothelial dysfunction and aberrant proliferation of smooth muscle cells (SMCs). In this opinion article, we present evidence of SIKs as modulators of key signaling pathways involved in PAH pathophysiology and discuss the potential of SIKs as therapeutic targets for PAH, emphasizing the need for deeper molecular insights on PAH.

The role of salt-inducible kinases on the modulation of renal and intestinal Na+,K+-ATPase activity during short- and long-term high-salt intake.

Type 1 salt-inducible kinases (SIK1) has been shown to act as a mediator during the cellular adaptation to variations in intracellular sodium in a variety of cell types. Type 2 SIK (SIK2) modulates various biological functions and acts as a signal transmitter in various pathways. To evaluate the role of both SIK isoforms in renal and intestinal Na+,K+-ATPase (NKA) activity, we made use of constitutive sik1-/- (SIK1-KO), sik2-/- (SIK2-KO), double sik1-/-sik2-/- (double SIK1*2-KO) knockout and wild-type (WT) mice challenged to a standard (0.3% NaCl) or chronic high-salt (HS, 8% NaCl) diet intake for 48 h or 12 weeks. Long-term HS intake in WT was accompanied by 2-fold increase in jejunal NKA activity and slight (~30% reduction) decreases in NKA in the ileum and cecum; none of these changes was accompanied by changes in the expression of α1-NKA. The ablation of SIK1 and SIK2 prevented the marked increase in jejunal NKA activity following the long-term HS intake. The ablation of SIK1 and SIK2 in mice on a long-term HS intake impacted differently in the ileum and cecum. The most interesting finding is that in SIK2-KO mice marked reductions in NKA activity were observed in the ileum and cecum when compared to WT mice, both on normal and long-term HS intake. In summary, SIK1 or SIK2 ablation on chronic high-salt intake is accompanied by modulation of NKA along the intestinal tract, which differ from those after an acute high-salt intake, and this may represent an absorptive compensatory mechanism to keep electrolyte homeostasis.

Antagonistic modulation of SIK1 and SIK2 isoforms in high blood pressure and cardiac hypertrophy triggered by high-salt intake.

Salt-inducible kinases (SIKs) represent a subfamily of AMPK family kinases. SIK1 has been shown to act as a mediator during the cellular adaptation to variations in intracellular sodium in a variety of cell types. SIK2, as an isoform of the SIK family, modulates various biological functions and acts as a signal transmitter in various pathways. To evaluate the role of both SIK1 and SIK2 isoforms in blood pressure (BP), body fluid regulation and cardiac hypertrophy development, we made use of constitutive sik1-/- (SIK1-KO), sik2-/- (SIK2-KO), double sik1-/-sik2-/- (double SIK1*2-KO) knockout and wild-type (WT) mice challenged to a standard (0.3% NaCl) or chronic high-salt (HS, 8% NaCl) diet intake for 12 weeks.Mice, under a standard diet intake, had similar and normal BP. On a chronic HS intake, SIK1-KO and double SIK1*2-KO mice showed increased BP, but not WT and SIK2-KO mice. A chronic HS intake led to the development of cardiac left ventricle hypertrophy (LVH) in normotensive WT and hypertensive SIK1-KO mice, but not in SIK2-KO mice. Double SIK1*2-KO mice under standard diet intake show normal BP but an increased LV mass. Remarkably, in response to a dietary stress condition, there is an increase in BP but LVH remained unchanged in double SIK1*2-KO mice.In summary, SIK1 isoform is required for maintaining normal BP in response to HS intake. LVH triggered by HS intake requires SIK2 isoform and is independent of high BP.

Recent methods and biosensors for foodborne pathogen detection in fish: progress and future prospects to sustainable aquaculture systems.

The aquaculture industry has advanced toward sustainable recirculating systems, in where parameters of food quality are strictly monitored. Despite that, as in the case of conventional aquaculture practices, the recirculating systems also suffer threats from Aeromonas spp., Vibrio spp., Streptococcus spp., among other foodborne pathogens infecting farmed fish. The aquaculture pathogens are routinely detected by conventional PCR methods or antibody-based tests, with the detection protocols confined to laboratory use. Emerging assay technologies and biosensors recently reported in the literature open new opportunities to the development of sensitive, specific, and portable analytical devices to use in the field. Techniques of DNA/RNA analysis, immunoassays and other nanomolecular technologies have been facing important advances in response time, sensitivity, and enhanced power of discrimination among and within species. Moreover, the recent developments of electrochemical and optical signal transduction have facilitated the incorporation of the innovative assays to practical miniaturized devices. In this work, it is provided a critical review over foodborne pathogen detection by existing and promising methods and biosensors applied to fish samples and extended to other food matrices. While isothermal DNA/RNA amplification methods can be highlighted among the assay methods for their promising analytical performance and suitability for point-of-care testing, the electrochemical transduction provides a way to achieve cost-effective biosensors amenable to use in the aquaculture field. The adoption of new methods and biosensors would constitute a step forward in securing sustainable aquaculture systems.

Preclinical pharmacological evaluation of the fatty acid amide hydrolase inhibitor BIA 10-2474.

BACKGROUND AND PURPOSE: In 2016, one person died and four others had mild-to-severe neurological symptoms during a phase I trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474. EXPERIMENTAL APPROACH: Pharmacodynamic and pharmacokinetic studies were performed with BIA 10-2474, PF-04457845 and JNJ-42165279 using mice, rats and human FAAH expressed in COS cells. Selectivity was evaluated by activity-based protein profiling (APBB) in rats. BIA 10-2474 effect in stroke-prone spontaneously hypertensive rats (SHRSP) was investigated. KEY RESULTS: BIA 10-2474 was 10-fold less potent than PF-04457845 in inhibiting human FAAH in situ but inhibited mouse brain and liver FAAH with ED50 values of 13.5 and 6.2 µg·kg-1 , respectively. Plasma and brain BIA 10-2474 levels were consistent with in situ potency and neither BIA 10-2474 nor its metabolites accumulated following repeat administration. FAAH and α/ß-hydrolase domain containing 6 were the primary targets of BIA 10-2474 and, at higher exposure levels, ABHD11, PNPLA6, PLA2G15, PLA2G6 and androgen-induced protein 1. At 100 mg·kg-1 for 28 days, the level of several lipid species containing arachidonic acid increased. Daily treatment of SHRSP with BIA 10-2474 did not affect mortality rate or increased the incidence of haemorrhage or oedema in surviving animals. CONCLUSIONS AND IMPLICATIONS: BIA 10-2474 potently inhibits FAAH in vivo, similarly to PF-04457845 and interacts with a number of lipid processing enzymes, some previously identified in human cells as off-targets particularly at high levels of exposure. These interactions occurred at doses used in toxicology studies, but the implication of these off-targets in the clinical trial accident remains unclear.

A Fluorescence Sensing Method with Reduced DNA Typing and Low-Cost Instrumentation for Detection of Sample Tampering Cases in Urinalysis.

This work presents a method to unequivocally detect urine sample tampering in cases where integrity of the sample needs to be verified prior to urinalysis. The technique involves the detection of distinct patterns of a triplex short tandem repeats system in DNA extracted from human urine. The analysis is realized with single-dye fluorescence detection and using a regular smartphone camera. The experimental results had demonstrated the efficacy of the analytical approach to obtaining distinct profiles of amplicons in urine from different sample providers. Reproducibility tests with fresh and stored urine have revealed a maximum variation in the profiles within an interval of 5 to 9%. Cases of urine sample tampering via mixture were simulated in the study, and the experiments have identified patterns of mixed genotypes from dual mixtures of urine samples. Moreover, sample adulteration by mixing a non-human fluid with urine in a volume ratio over 25% can be detected. The low cost of the approach is accompanied by the compatibility of the technique to use with different DNA sample preparation protocols and PCR instrumentation. Furthermore, the possibility of realizing the method in an integrated microchip system open great perspectives to conducting sample integrity tests at the site of urine sample reception and/or at resource-limited settings.

Design and characterisation of a graphene-based FET for ultrasensitive detection of clinical biomarkers.

The high demand for point-of-care devices for the convenient detection and follow-up of chronic diseases is posing demands to the development of novel low-cost sensors. The chronic obstructive pulmonary disease (COPD) is one of the most worldwide spread diseases, due to cigarette smoking and air pollution. Owing to the unstable and spontaneous characteristics of this disease is essential to have a sensitive, rapid, and easy-to-use device for the detection of diseases biomarkers. The research of emerging materials such as graphene monolayer and perovskite may revolutionise the field of point-of-care devices. These materials can boost the sensitivity and specificity of the detection, and therefore the detection can be performed in samples taken non-invasively, such saliva, and with less sample quantity. A graphene field effect transistor (GFET) coated with PEDOT:PSS and perovskite, bring advantages to the photodetection field, due to the unique proprieties of 2D materials and the structure of perovskite. This work presents a study of material characteristics comprising a GFET, with perspective to detect biomarkers of COPD.

Effects of zamicastat treatment in a genetic model of salt-sensitive hypertension and heart failure.

Hyperactivity of sympathetic nervous system plays an important role in the development and progression of cardiovascular diseases. An approach to mitigate the enhanced sympathetic nervous system drive is restricting the biosynthesis of noradrenaline via inhibition of the enzyme dopamine ß-hydroxylase (DßH), that catalyzes the hydroxylation of dopamine to noradrenaline in sympathetic nerves. The aim of the present study was to evaluate the effects of zamicastat, a novel DßH inhibitor that decreases noradrenaline and increases dopamine levels in peripheral sympathetically innervated tissues, on the hemodynamic and cardiometabolic parameters in salt-induced hypertension and heart failure in the Dahl salt-sensitive (SS) rat. Zamicastat (10, 30 and 100 mg/kg body weight) was tested acutely against salt-induced hypertension in the Dahl SS rat. Chronic zamicastat treatment (30 mg/kg/day) was evaluated against salt-induced cardiac hypertrophy and biomarkers of cardiometabolic risk and inflammation in Dahl SS rats and upon the survival rate in aged Dahl SS rats fed a high-salt diet. The reduction in the sympathetic tone attained with zamicastat shaped a dose- and time-dependent effect on blood pressure. Prolonged treatment with zamicastat ameliorated end-organ damage, metabolic syndrome and inflammation hallmarks in hypertensive Dahl SS rats. Survival rate of Dahl SS rats fed a high-salt diet demonstrated that zamicastat increased median survival of Dahl SS rats fed a high-salt diet. The use of DßH inhibitors, like zamicastat, is a promising approach to treat hypertension, heart failure and cardiovascular diseases where a reduction in the sympathetic tone has beneficial effects.

The Role of Temperature, Ammonia and Nitrite to bioluminescence of Aliivibrio fischeri: towards a new sensor for aquaculture.

Recirculating Aquaculture Systems (RAS) present an innovative, clean and practical way of producing fish intensively. Stress caused by high concentrations of chemical species such as nitrite and un-ionized ammonia, affects fish health and growth and therefore the sustainability of RAS would require an online monitoring for those chemical stressors. This work reveals a study on the suitability of Aliivibrio fischeri as a toxicity sensor for un-ionized ammonia and nitrite. Temperature variation effects were also considered. An EC50 of 0.17 mg/L was found for nitrite and 0.57 mg/L for un-ionized ammonia. It was concluded that Allivibrio fischeri is suitable as an indicator for nitrite in aquaculture at optimal salinity and temperature conditions.

Discovery of a Potent, Long-Acting, and CNS-Active Inhibitor (BIA 10-2474) of Fatty Acid Amide Hydrolase.

Fatty acid amide hydrolase (FAAH) can be targeted for the treatment of pain associated with various medical conditions. Herein we report the design and synthesis of a novel series of heterocyclic-N-carboxamide FAAH inhibitors that have a good alignment of potency, metabolic stability and selectivity for FAAH over monoacylglycerol lipase (MAGL) and carboxylesterases (CEs). Lead optimization efforts carried out with benzotriazolyl- and imidazolyl-N-carboxamide series led to the discovery of clinical candidate 8 l (3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide; BIA 10-2474) as a potent and long-acting inhibitor of FAAH. However, during a Phase I clinical trial with compound 8 l, unexpected and unpredictable serious neurological adverse events occurred, affecting five healthy volunteers, including the death of one subject.

Monitoring Aquaculture Water Quality: Design of an Early Warning Sensor with Aliivibrio fischeri and Predictive Models.

A novel toxicity-warning sensor for water quality monitoring in recirculating aquaculture systems (RAS) is presented. The design of the sensor system mainly comprises a whole-cell biosensor. Aliivibrio fischeri, a luminescent bacterium widely used in toxicity analysis, was tested for a mixture of known fish-health stressors, namely nitrite, un-ionized ammonia, copper, aluminum and zinc. Two toxicity predictive models were constructed. Correlation, root mean squared error, relative error and toxic behavior were analyzed. The linear concentration addition (LCA) model was found suitable to ally with a machine learning algorithm for prediction of toxic events, thanks to additive behavior near the limit concentrations for these stressors, with a root-mean-squared error (RMSE) of 0.0623, and a mean absolute error of 4%. The model was proved to have a smaller relative deviation than other methods described in the literature. Moreover, the design of a novel microfluidic chip for toxicity testing is also proposed, which is to be integrated in a fluidic system that functions as a bypass of the RAS tank to enable near-real time monitoring. This chip was tested with simulated samples of RAS water spiked with zinc, with an EC50 of 6,46E-7 M. Future work will be extended to the analysis of other stressors with the novel chip.

Profiling a multiplex short tandem repeat loci from human urine with use of low cost on-site technology for verification of sample authenticity.

This work focuses on the development of a sophisticated technique via STR typing to unequivocally verify the authenticity of urine samples before sent to laboratories. STR profiling was conducted with the CSF1PO, TPOX, TH01 Multiplex System coupled with a smartphone-based detection method. The promising capability of the method to identify distinct STR profiles from urine of different persons opens the possibility to conduct sample authenticity tests. On-site STR profiling could be realized with a self-contained autonomous device with an integrated PCR microchip shown hereby.

Highly sensitive detection of human cancer antigens by an immunogold-silver assay chip coupled with a polythiophene-based optical sensor.

This work presents a novel method for protein or cancer antigen detection in clinical samples by an immunogold-silver assay microfluidic biochip coupled with a polythiophene-based organic photodetector. The method has showed a detection limit below 1ng/mL and the low cost and high sensitivity of both organic photodetector and immunogold-silver assay make this method amenable for realization in a portable handheld probe tip biosensor.

Polycarbazole-based organic photodiodes for highly sensitive chemiluminescent immunoassays.

It is reported the development of a polycarbazole-based organic photodetector for chemiluminescent immunoassays. The optical detector comprised a 1∶4 blend by weight of poly [N-9'-heptadecanyl-2,7-carbazole-alt-5,5-(4',7'-di-2-thienyl-2',1',3'-benzothiadiazole)] (PCDTBT) and [6,6]-phenyl C71-butyric acid methyl ester (PC70BM). Optimization of the photodetector design was conducted aiming to maximize photosensitivity and reduce the background level. Quantitation of recombinant human thyroid stimulating hormone indicated good linearity and yielded a detection sensitivity of ∼3.7 nA × nM(-1) and a detection limit of 80 pg/ml.

Detection of stress hormones by a microfluidic-integrated polycarbazole/fullerene photodetector.

A novel photodetector integrated microfluidic system for chemiluminescence (CL) detection is reported. The system incorporates a polycarbazole/fullerene photodiode whose optical characteristics (i.e. dark current, external quantum efficiency and photosensitivity) are described here. Using a CL immunoassay for detecting the stress hormone cortisol, the integrated photodetector achieved a detection sensitivity of 1.775 pA × nM(-1) and a detection limit of less than 0.28 nM. The device would be a powerful low-cost alternative to silicon photodiode and photomultiplier tube for bioanalytical assays, with potentially wide-ranging applications within point-of-care diagnostics.

An infrared radiation based thermal biosensor for enzymatic biochemical reactions.

In this paper, a thermal biosensor based on the infrared radiation energy is proposed for calorimetric measurement of biochemical reactions. Having a good structure design combined with MEMS technology as well as employing the Si /SiGe quantum well sensing material with a high TCR and low 1/f noise, the sensor shows potentials to be high sensitive and real-time. The urea enzymatic reaction was tested to verify the performance of sensor, which demonstrates a linear detection range from 0.5mM to 150mM and a relative standard deviation less than 1%. For the sensor fabrication, wafer-level transfer bonding is a key process, which makes the integration of quantum well material and a free standing structure possible. It reduces the heat loss from the sensor to the surrounding environment.

6-mercaptopurine inhibits atherosclerosis in apolipoprotein e*3-leiden transgenic mice through atheroprotective actions on monocytes and macrophages.

OBJECTIVE: 6-Mercaptopurine (6-MP), the active metabolite of the immunosuppressive prodrug azathioprine, is commonly used in autoimmune diseases and transplant recipients, who are at high risk for cardiovascular disease. Here, we aimed to gain knowledge on the action of 6-MP in atherosclerosis, with a focus on monocytes and macrophages. METHODS AND RESULTS: We demonstrate that 6-MP induces apoptosis of THP-1 monocytes, involving decreased expression of the intrinsic antiapoptotic factors B-cell CLL/Lymphoma-2 (Bcl-2) and Bcl2-like 1 (Bcl-x(L)). In addition, we show that 6-MP decreases expression of the monocyte adhesion molecules platelet endothelial adhesion molecule-1 (PECAM-1) and very late antigen-4 (VLA-4) and inhibits monocyte adhesion. Screening of a panel of cytokines relevant to atherosclerosis revealed that 6-MP robustly inhibits monocyte chemoattractant chemokine-1 (MCP-1) expression in macrophages stimulated with lipopolysaccharide (LPS). Finally, local delivery of 6-MP to the vessel wall, using a drug-eluting cuff, attenuates atherosclerosis in hypercholesterolemic apolipoprotein E*3-Leiden transgenic mice (P<0.05). In line with our in vitro data, this inhibition of atherosclerosis by 6-MP was accompanied with decreased lesion monocyte chemoattractant chemokine-1 levels, enhanced vascular apoptosis, and reduced macrophage content. CONCLUSIONS: We report novel, previously unrecognized atheroprotective actions of 6-MP in cultured monocytes/macrophages and in a mouse model of atherosclerosis, providing further insight into the effect of the immunosuppressive drug azathioprine in atherosclerosis.

Vitamin D receptor: a new risk marker for clinical restenosis after percutaneous coronary intervention.

OBJECTIVE: Restenosis is the main drawback of percutaneous coronary intervention (PCI). Inherited factors may explain part of the risk of restenosis. Recently, the vitamin D receptor (VDR) has been shown to be involved not only in bone metabolism but also in modulating immune responses and cell proliferation. Since the inflammatory response is implicated in restenosis, VDR-gene variants could therefore contribute to the risk of restenosis. METHODS/RESULTS: Systematic genotyping for 15 haplotype tagging single-nucleotide polymorphisms (SNPs) of the VDR gene was performed with the high throughput TaqMan allelic discrimination assays in the Genetic Determinants of Restenosis (GENDER) population. A haplotype-based survival analysis revealed an association of haplotypes in blocks 2, 3 and 4 of the VDR-gene with the risk of clinical restenosis (p-values 0.01, 0.04 and 0.02 respectively). After adjustment for clinical risk factors for restenosis, the individual effect of the block 2 AA haplotype (p = 0.011) persisted. CONCLUSIONS: The present study indicates that VDR plays a role in restenosis after PCI. Therefore, VDR genotype may be used as risk marker for restenosis and may contribute to individual patient screening prior to PCI in clinical practice.