Diagnostics of SARS-CoV-2 infection using electrical impedance spectroscopy with an immunosensor to detect the spike protein.

Mass testing for the diagnostics of COVID-19 has been hampered in many countries owing to the high cost of the methodologies to detect genetic material of SARS-CoV-2. In this paper, we report on a low-cost immunosensor capable of detecting the spike protein of SARS-CoV-2, including in samples of inactivated virus. Detection is performed with electrical impedance spectroscopy using an immunosensor that contains a monolayer film of carboxymethyl chitosan as matrix, coated with an active layer of antibodies specific to the spike protein. In addition to a low limit of detection of 0.179 fg/mL within an almost linear behavior from 10-20 g/mL to 10-14 g/mL, the immunosensor was highly selective. For the samples with the spike protein could be distinguished in multidimensional projection plots from samples with other biomarkers and analytes that could be interfering species for healthy and infected patients. The excellent analytical performance of the immunosensors was validated with the distinction between control samples and those containing inactivated SARS-CoV-2 at different concentrations. The mechanism behind the immunosensor performance is the specific antibody-protein interaction, as confirmed with the changes induced in C-H stretching and protein bands in polarization-modulated infrared reflection absorption spectra (PM-IRRAS). Because impedance spectroscopy measurements can be made with low-cost portable instruments, the immunosensor proposed here can be applied in point-of-care diagnostics for mass testing even in places with limited resources.

The longevity gene mIndy (I'm Not Dead, Yet) affects blood pressure through sympathoadrenal mechanisms.

Reduced expression of the plasma membrane citrate transporter INDY (acronym I'm Not Dead, Yet) extends life span in lower organisms. Deletion of the mammalian Indy (mIndy) gene in rodents improves metabolism via mechanisms akin to caloric restriction, known to lower blood pressure (BP) by sympathoadrenal inhibition. We hypothesized that mIndy deletion attenuates sympathoadrenal support of BP. Continuous arterial BP and heart rate (HR) were reduced in mINDY-KO mice. Concomitantly, urinary catecholamine content was lower, and the decreases in BP and HR by mIndy deletion were attenuated after autonomic ganglionic blockade. Catecholamine biosynthesis pathways were reduced in mINDY-KO adrenals using unbiased microarray analysis. Citrate, the main mINDY substrate, increased catecholamine content in pheochromocytoma cells, while pharmacological inhibition of citrate uptake blunted the effect. Our data suggest that deletion of mIndy reduces sympathoadrenal support of BP and HR by attenuating catecholamine biosynthesis. Deletion of mIndy recapitulates beneficial cardiovascular and metabolic responses to caloric restriction, making it an attractive therapeutic target.

Nanostructured functional peptide films and their application in C-reactive protein immunosensors.

Peptides with an active redox molecule are incorporated into nanostructured films for electrochemical biosensors with stable and controllable physicochemical properties. In this study, we synthesized three ferrocene (Fc)-containing peptides with the sequence Fc-Glu-(Ala)n-Cys-NH2, which could form self-assembled monolayers on gold and be attached to antibodies. The peptide with two alanines (n = 2) yielded the immunosensor with the highest performance in detecting C-reactive protein (CRP), a biomarker of inflammation. Using electrochemical impedance-derived capacitive spectroscopy, the limit of detection was 240 pM with a dynamic range that included clinically relevant CRP concentrations. With a combination of electrochemical methods and polarization-modulated infrared reflection-absorption spectroscopy, we identified the chemical groups involved in the antibody-CRP interaction, and were able to relate the highest performance for the peptide with n = 2 to chain length and efficient packing in the organized films. These strategies to design peptides and methods to fabricate the immunosensors are generic, and can be applied to other types of biosensors, including in low cost platforms for point-of-care diagnostics.

Electrochemical and optical detection and machine learning applied to images of genosensors for diagnosis of prostate cancer with the biomarker PCA3.

The development of simple detection methods aimed at widespread screening and testing is crucial for many infections and diseases, including prostate cancer where early diagnosis increases the chances of cure considerably. In this paper, we report on genosensors with different detection principles for a prostate cancer specific DNA sequence (PCA3). The genosensors were made with carbon printed electrodes or quartz coated with layer-by-layer (LbL) films containing gold nanoparticles and chondroitin sulfate and a layer of a complementary DNA sequence (PCA3 probe). The highest sensitivity was reached with electrochemical impedance spectroscopy with the detection limit of 83 pM in solutions of PCA3, while the limits of detection were 2000 pM and 900 pM for cyclic voltammetry and UV-vis spectroscopy, respectively. That detection could be performed with an optical method is encouraging, as one may envisage extending it to colorimetric tests. Since the morphology of sensing units is known to be affected in detection experiments, we applied machine learning algorithms to classify scanning electron microscopy images of the genosensors and managed to distinguish those exposed to PCA3-containing solutions from control measurements with an accuracy of 99.9%. The performance in distinguishing each individual PCA3 concentration in a multiclass task was lower, with an accuracy of 88.3%, which means that further developments in image analysis are required for this innovative approach.

Eco-friendly gelatin films with rosin-grafted cellulose nanocrystals for antimicrobial packaging.

We report on gelatin films incorporating rosin-grafted cellulose nanocrystals (r-CNCs), which fulfill the most relevant requirements for antimicrobial packaging applications. Transparent gelatin/r-CNCs bionanocomposite films (0.5-6 wt% r-CNCs) were obtained by solution casting and displayed high UV-barrier properties, which were superior to the most used plastic packaging films. The gelatin/r-CNCs films exhibited a moderate water vapor permeability (0.09 g mm/m2 h kPa), and high tensile strength (40 MPa) and Young's modulus (1.9 GPa). The r-CNCs were more efficient in improving the optical, water vapor barrier and tensile properties of gelatin films than conventional CNCs. Grafting of rosin on CNCs resulted in an antimicrobial nanocellulose that inhibited the growth of Staphylococcus aureus and Escherichia coli. The antibacterial properties of r-CNCs were sustained in the gelatin films, as demonstrated by agar diffusion tests and proof-of-principle experiments involving cheese storage. Overall, the incorporation of r-CNCs as active fillers in gelatin films is a suitable approach for producing novel eco-friendly, antimicrobial packaging materials.

Electronic Nose Based on Carbon Nanocomposite Sensors for Clove Essential Oil Detection.

This work describes the development of an electronic nose (e-nose) based on carbon nanocomposites to detect clove essential oil (CEO), eugenol (EUG), and eugenyl acetate (EUG.ACET). Our e-nose system comprises an array of six sensing units modified with nanocomposites of poly(aniline), graphene oxide, and multiwalled carbon nanotubes doped with different acids, dodecyl benzene sulfonic acid, camphorsulfonic acid, and hydrochloric acid. The e-nose presented an excellent analytical performance to the detected analytes (CEO, EUG, and EUG.ACET) with high sensitivity and reversibility. The limit of detection was lower than 1.045 ppb, with response time (<13.26 s) and recovery time (<106.29 s) and low hysteresis. Information visualization methods (PCA and IDMAP) demonstrated that the e-nose was efficient to discriminate the different concentrations of analyte volatile oil compounds. PM-IRRAS measurements suggest that the doping mechanism of molecular architectures is composed of a change in the oscillation energy of the characteristic dipoles and changes in the molecular orientation dipoles C═C and C═O at 1615 and 1740 cm-1, respectively. The experimental results indicate that our e-nose system is promising for a rapid analysis method to monitor the quality of essential oils.

Experimental models and methods for cutaneous wound healing assessment.

Wound healing studies are intricate, mainly because of the multifaceted nature of the wound environment and the complexity of the healing process, which integrates a variety of cells and repair phases, including inflammation, proliferation, reepithelialization and remodelling. There are a variety of possible preclinical models, such as in mice, rabbits and pigs, which can be used to mimic acute or impaired for example, diabetic and nutrition-related wounds. These can be induced by many different techniques, with excision or incision being the most common. After determining a suitable model for a study, investigators need to select appropriate and reproducible methods that will allow the monitoring of the wound progression over time. The assessment can be performed by non-invasive protocols such as wound tracing, photographic documentation (including image analysis), biophysical techniques and/or by invasive protocols that will require wound biopsies. In this article, we provide an overview of some of the most often needed and used: (a) preclinical/animal models including incisional, excisional, burn and impaired wounds; (b) methods to evaluate the healing progression such as wound healing rate, wound analysis by image, biophysical assessment, histopathological, immunological and biochemical assays. The aim is to help researchers during the design and execution of their wound healing studies.

Unexpectedly high leprosy seroprevalence detected using a random surveillance strategy in midwestern Brazil: A comparison of ELISA and a rapid diagnostic test.

BACKGROUND: Leprosy diagnosis is mainly based on clinical evaluation, although this approach is difficult, especially for untrained physicians. We conducted a temporary campaign to detect previously unknown leprosy cases in midwestern Brazil and to compare the performance of different serological tests. METHODS: A mobile clinic was stationed at the main bus terminal in Brasília, Brazil. Volunteers were quizzed and given a clinical exam to allow categorization as either patients, known contacts of patients or non-contacts, and blood was collected to determine anti-PGL-I and anti-LID-1 antibody titers by ELISA and by the NDO-LID rapid test. New cases of leprosy and the impact of performing this broad random surveillance strategy were evaluated. Accuracy values and concordance between the test results were evaluated among all groups. RESULTS: Four hundred thirty-four individuals were evaluated, and 44 (10.1%) were diagnosed with leprosy. Borderline forms were the most frequent presentation. Both tests presented higher positivity in those individuals with multibacillary disease. Serological tests demonstrated specificities arround 70% for anti-PGL-1 and anti-LID ELISA; and arround 40% for NDO-LID. Sensitivities ranged from 48 to 62%. A substantial agreement between NDO-LID and ELISA with concomitant positive results was found within leprosy patients (Kappa index = 0.79 CI95% 0.36-1.22). CONCLUSIONS: The unexpectedly high leprosy prevalence in this population indicates ongoing community-based exposure to Mycobacterium leprae antigens and high rates of subclinical infection. All tests showed low specificity and sensitivity values and therefore cannot be considered for use as stand-alone diagnostics. Rather, considering their positivity among MB patients and non-patients, these tests can be considered effective tools for screening and identifying individuals at high risk who might benefit from regular monitoring.

Immunosensor for Pancreatic Cancer Based on Electrospun Nanofibers Coated with Carbon Nanotubes or Gold Nanoparticles.

We report the fabrication of immunosensors based on nanostructured mats of electrospun nanofibers of polyamide 6 and poly(allylamine hydrochloride) coated either with multiwalled carbon nanotubes (MWCNTs) or gold nanoparticles (AuNPs), whose three-dimensional structure was suitable for the immobilization of anti-CA19-9 antibodies to detect the pancreatic cancer biomarker CA19-9. Using impedance spectroscopy, the sensing platform was able to detect CA19-9 with a detection limit of 1.84 and 1.57 U mL-1 for the nanostructured architectures containing MWCNTs and AuNPs, respectively. The high sensitivity achieved can be attributed to the irreversible adsorption between antibodies and antigens, as confirmed with polarization-modulated infrared reflection absorption spectroscopy. The adsorption mechanism was typical Langmuir-Freundlich processes. The high sensitivity and selectivity of the immunosensors were also explored in tests with blood serum from patients with distinct concentrations of CA19-9, for which the impedance spectra data were processed with a multidimensional projection technique. The robustness of the immunosensors in dealing with patient samples without suffering interference from analytes present in biological fluids is promising for a simple, effective diagnosis of pancreatic cancer at early stages.

Clinical Manifestation, Histopathology, and Imaging of Traumatic Injuries Caused by Brazilian Porcupine (Sphiggurus villosus) Quills.

Injuries to humans caused by porcupines are rare. However, they may occur due to the proximity of urban areas and the animal's habitat in areas such as the Floresta da Tijuca in Rio de Janeiro. Outdoor sports and leisure activities in areas close to forests or in the rain forest are also relevant for incidents of this kind and a better knowledge of the local forest fauna would prevent such undesirable accidents. Porcupine quills have microscopic barbs at their tips which facilitate skin penetration, but hampering their removal. Once the spines are lodged in tissue, the microscopic backward-facing deployable barbs at the tips cause trauma if anyone tries to remove them. Local haemorrhage and an inflammatory response to the contaminated foreign body occur. Depending on the time lapse in removing the spines either septic or sterile foreign body reactions may occur. There is also the risk of migration of the spines, where fatal cases have been reported in human and veterinary medicine. Herein we report two unusual cases of accidents involving humans and the South American porcupine. The Sphiggurus villosus spines removed from scalp skin were also documented through Scanning Electron Microscopy.

C-type natriuretic peptide and natriuretic peptide receptor B signalling inhibits cardiac sympathetic neurotransmission and autonomic function.

AIMS: B-type natriuretic peptide (BNP)-natriuretic peptide receptor A (NPR-A) receptor signalling inhibits cardiac sympathetic neurotransmission, although C-type natriuretic peptide (CNP) is the predominant neuropeptide of the nervous system with expression in the heart and vasculature. We hypothesized that CNP acts similarly to BNP, and that transgenic rats (TGRs) with neuron-specific overexpression of a dominant negative NPR-B receptor would develop heightened sympathetic drive. METHODS AND RESULTS: Mean arterial pressure and heart rate (HR) were significantly (P < 0.05) elevated in freely moving TGRs (n = 9) compared with Sprague Dawley (SD) controls (n = 10). TGR had impaired left ventricular systolic function and spectral analysis of HR variability suggested a shift towards sympathoexcitation. Immunohistochemistry demonstrated co-staining of NPR-B with tyrosine hydroxylase in stellate ganglia neurons. In SD rats, CNP (250 nM, n = 8) significantly reduced the tachycardia during right stellate ganglion stimulation (1-7 Hz) in vitro whereas the response to bath-applied norepinephrine (NE, 1 µM, n = 6) remained intact. CNP (250 nM, n = 8) significantly reduced the release of 3H-NE in isolated atria and this was prevented by the NPR-B antagonist P19 (250 nM, n = 6). The neuronal Ca2+ current (n = 6) and intracellular Ca2+ transient (n = 9, using fura-2AM) were also reduced by CNP in isolated stellate neurons. Treatment of the TGR (n = 9) with the sympatholytic clonidine (125 µg/kg per day) significantly reduced mean arterial pressure and HR to levels observed in the SD (n = 9). CONCLUSION: C-type natriuretic peptide reduces cardiac sympathetic neurotransmission via a reduction in neuronal calcium signalling and NE release through the NPR-B receptor. Situations impairing CNP-NPR-B signalling lead to hypertension, tachycardia, and impaired left ventricular systolic function secondary to sympatho-excitation.

Femtosecond Laser Patterning of the Biopolymer Chitosan for Biofilm Formation.

Controlling microbial growth is crucial for many biomedical, pharmaceutical and food industry applications. In this paper, we used a femtosecond laser to microstructure the surface of chitosan, a biocompatible polymer that has been explored for applications ranging from antimicrobial action to drug delivery. The influence of energy density on the features produced on chitosan was investigated by optical and atomic force microscopies. An increase in the hydrophilic character of the chitosan surface was attained upon laser micromachining. Patterned chitosan films were used to observe Staphylococcus aureus (ATCC 25923) biofilm formation, revealing an increase in the biofilm formation in the structured regions. Our results indicate that fs-laser micromachining is an attractive option to pattern biocompatible surfaces, and to investigate basic aspects of the relationship between surface topography and bacterial adhesion.

Wound healing modulation by a latex protein-containing polyvinyl alcohol biomembrane.

In a previous study, we performed the chemical characterization of a polyvinyl alcohol (PVA) membrane supplemented with latex proteins (LP) displaying wound healing activity, and its efficacy as a delivery system was demonstrated. Here, we report on aspects of the mechanism underlying the performance of the PVA-latex protein biomembrane on wound healing. LP-PVA, but not PVA, induced more intense leukocyte (neutrophil) migration and mast cell degranulation during the inflammatory phase of the cicatricial process. Likewise, LP-PVA induced an increase in key markers and mediators of the inflammatory response (myeloperoxidase activity, nitric oxide, TNF, and IL-1ß). These results demonstrated that LP-PVA significantly accelerates the early phase of the inflammatory process by upregulating cytokine release. This remarkable effect improves the subsequent phases of the healing process. The polyvinyl alcohol membrane was fully absorbed as an inert support while LP was shown to be active. It is therefore concluded that the LP-PVA is a suitable bioresource for biomedical engineering.

Controlled Film Architectures to Detect a Biomarker for Pancreatic Cancer Using Impedance Spectroscopy.

The need for analytical devices for detecting cancer at early stages has motivated research into nanomaterials where synergy is sought to achieve high sensitivity and selectivity in low-cost biosensors. In this study, we developed a film architecture combining self-assembled monolayer (SAM) and layer-by-layer (LbL) films of polysaccharide chitosan and the protein concanavalin A, on which a layer of anti-CA19-9 antibody was adsorbed. Using impedance spectroscopy with this biosensor, we were capable of detecting low concentrations of the antigen CA19-9, an important biomarker for pancreatic cancer. The limit of detection of 0.69U/mL reached is sufficient for detecting pancreatic cancer at very early stages. The selectivity of the biosensor was inferred from a series of control experiments with samples of cell lines that were tested positive (HT29) and negative (SW620) for the biomarker CA19-9, in addition to the lack of changes in the capacitance value for other analytes and antigen that are not related to this type of cancer. The high sensitivity and selectivity are ascribed to the very specific antigen-antibody interaction, which was confirmed with PM-IRRAS and atomic force microscopy. Also significant is that used information visualization methods to show that different cell lines and commercial samples containing distinct concentrations of CA19-9 and other analytes can be easily distinguished from each other. These computational methods are generic and may be used in optimization procedures to tailor biosensors for specific purposes, as we demonstrated here by comparing the performance of two film architectures in which the concentration of chitosan was varied.

Cholinergic Pathway Suppresses Pulmonary Innate Immunity Facilitating Pneumonia After Stroke.

BACKGROUND AND PURPOSE: Temporary immunosuppression has been identified as a major risk factor for the development of pneumonia after acute central nervous system injury. Although overactivation of the sympathetic nervous system was previously shown to mediate suppression of systemic cellular immune responses after stroke, the role of the parasympathetic cholinergic anti-inflammatory pathway in the antibacterial defense in lung remains largely elusive. METHODS: The middle cerebral artery occlusion model in mice was used to examine the influence of the parasympathetic nervous system on poststroke immunosuppression. We used heart rate variability measurement by telemetry, vagotomy, α7 nicotinic acetylcholine receptor-deficient mice, and parasympathomimetics (nicotine, PNU282987) to measure and modulate parasympathetic activity. RESULTS: Here, we demonstrate a rapidly increased parasympathetic activity in mice after experimental stroke. Inhibition of cholinergic signaling by either vagotomy or by using α7 nicotinic acetylcholine receptor-deficient mice reversed pulmonary immune hyporesponsiveness and prevented pneumonia after stroke. In vivo and ex vivo studies on the role of α7 nicotinic acetylcholine receptor on different lung cells using bone marrow chimeric mice and isolated primary cells indicated that not only macrophages but also alveolar epithelial cells are a major cellular target of cholinergic anti-inflammatory signaling in the lung. CONCLUSIONS: Thus, cholinergic pathways play a pivotal role in the development of pulmonary infections after acute central nervous system injury.

Phototherapy improves wound healing in rats subjected to high-fat diet.

This study aimed to compare the phototherapy effects on wound healing in rats submitted to normal and high-fat diets. Thirty-six rats received normal lipidic diet (NL) and 36 high lipidic (HL) diet for 45 days. The nutritional status was measured by body mass, blood glucose, total cholesterol, and triglycerides levels. Four experimental groups were performed according light (L) therapy applied "on" or "off" (660 nm, 100 mW, 70 J/cm(2), 2 J) on 1.5-mm-punched dorsum skin wounds as NLL+, NLL-, HLL+, and HLL-. The wound healing rate (WHR) and oxidative stress markers were analyzed on 2nd, 7th, and 14th days. Despite no difference among body mass, the HL rats presented higher blood glucose, total cholesterol, and triglycerides levels than NL rats. Respectively, on the 2nd and 14th days, the HLL+ group presented the highest WHRs (0.38 ± 0.16/0.97 ± 0.02) among all groups, while the HLL- (-0.002 ± 0.12/0.81 ± 12.1) the lowest WHRs. Hydroxyproline level was lower in HLL- (6.41 ± 1.09 µg/mg) than HLL+ (7.71 ± 0.61 µg/mg) and also NLL+ (9.33 ± 0.84 µg/mg). HLL+ presented oxidative stress markers similar to normal control group (NLL-) during follow up and highest antioxidant defense on 7th day. The results showed phototherapy accelerated the cutaneous wound healing by modulating oxidative stress in rats with metabolic disorders under a high-fat diet.

Preserved functional autonomic phenotype in adult mice overexpressing moderate levels of human alpha-synuclein in oligodendrocytes.

Mice overexpressing human alpha-synuclein in oligodendrocytes (MBP1-α-syn) recapitulate some key functional and neuropathological features of multiple system atrophy (MSA). Whether or not these mice develop severe autonomic failure, which is a key feature of human MSA, remains unknown. We explored cardiovascular autonomic regulation using long-term blood pressure (BP) radiotelemetry and pharmacological testing. We instrumented 12 MBP1-α-syn mice and 11 wild-type mice aged 9 months for radiotelemetry. Animals were tested with atropine, metoprolol, clonidine, and trimethaphan at 9 and 12 months age. We applied spectral and cross-spectral analysis to assess heart rate (HR) and BP variability. At 9 months of age daytime BP (transgenic: 101 ± 2 vs. wild type: 99 ± 2 mmHg) and HR (497 ± 11 vs. 505 ± 16 beats/min) were similar. Circadian BP and HR rhythms were maintained. Nighttime BP (109 ± 2 vs. 108 ± 2 mmHg) and HR (575 ± 15 vs. 569 ± 14 beats/min), mean arterial BP responses to trimethaphan (-21 ± 8 vs. -10 ± 5 mmHg, P = 0.240) and to clonidine (-8 ± 3 vs. -5 ± 2 mmHg, P = 0.314) were similar. HR responses to atropine (+159 ± 24 vs. +146 ± 22 beats/min), and to clonidine (-188 ± 21 vs. -163 ± 33 beats/min) did not differ between strains. Baroreflex sensitivity (4 ± 1 vs. 4 ± 1 msec/mmHg) and HR variability (total power, 84 ± 17 vs. 65 ± 21 msec²) were similar under resting conditions and during pharmacological testing. Repeated measurements at 12 months of age provided similar results. In mice, moderate overexpression of human alpha-synuclein in oligodendrocytes is not sufficient to induce overt autonomic failure. Additional mechanisms may be required to express the autonomic failure phenotype including higher levels of expression or more advanced age.

NFκB activation in cutaneous lesions of leprosy is associated with development of multibacillary infection.

BACKGROUND: Nuclear factor kappa B (NFκB) transcription factors play a central role in controlling the expression of genes involved in inflammatory reactions, proliferation, and survival of human cells. However, the in situ evaluation of NFκB activity in leprosy has not been completed previously. The aim of this study was to determine whether NFκB activity correlates with susceptibility or resistance to Mycobacterium leprae infection in biopsies from skin lesions of 38 patients with the clinical and laboratory diagnosis of leprosy. METHODS: The NFκB activation profile was evaluated in biopsies from skin lesions of 38 patients with the clinical and laboratory diagnosis of leprosy. NFκB activation was evaluated and quantified by Southwestern histochemistry, and its activation index (range, 0-4) was calculated according to the percentage of nuclear positivity by the histochemistry. Activation index >1 was considered representative of activation of NFκB. RESULTS: Fifteen patients (39.5%) demonstrated activated NFκB. Multibacillary leprosy was associated with activated NFκB (54.5%, P=0.028). Borderline leprosy was most strongly associated with NFκB activation (80%), with an odds ratio of 32.7 (P=0.016). These clinical forms are characterized by increased susceptibility to M. leprae and by immunological instability. Activation of NFκB was absent in the granulomas in tuberculoid leprosy, which represents an effective inflammatory reaction pattern against M. leprae. CONCLUSION: These results indicate that NFκB activation could favor susceptibility and immunological instability to M. leprae infection, potentially by the stimulation of phagocytosis and the regulation of apoptotic mechanisms of infected cells, leading to the proliferation of this intracellular bacillus. Further studies are needed to evaluate if inhibition of NFκB activation in multibacillary leprosy could favor resistance and an effective granulomatous immune response.

AVE 0991, a non-peptide Mas-receptor agonist, facilitates penile erection.

The renin-angiotensin system plays a crucial role in erectile function. It has been shown that elevated levels of angiotensin II contribute to the development of erectile dysfunction both in humans and in aminals. On the contrary, the heptapeptide angiotensin-(1-7) appears to mediate penile erection by activation of the Mas receptor. Recently, we have shown that the erectile function of Mas gene-deleted mice was substantially reduced, which was associated with a marked increase in fibrous tissue in the corpus cavernosum. We have hypothesized that the synthetic non-peptide Mas agonist, AVE 0991, would potentiate penile erectile function. We showed that intracavernosal injection of AVE 0991 potentiated the erectile response of anaesthetized Wistar rats, measured as the ratio between corpus cavernosum pressure and mean arterial pressure, upon electrical stimulation of the major pelvic ganglion. The facilitatory effect of AVE 0991 on erectile function was dose dependent and completely blunted by the nitric oxide synthesis inhibitor, l-NAME. Importantly, concomitant intracavernosal infusion of the specific Mas receptor blocker, A-779, abolished the effect of AVE 0991. We demonstrated that AVE 0991 potentiates the penile erectile response through Mas in an NO-dependent manner. Importantly, these results suggest that Mas agonists, such as AVE 0991, might have significant therapeutic benefits for the treatment of erectile dysfunction.

Spinophilin regulates central angiotensin II-mediated effect on blood pressure.

Central angiotensin II (AngII) plays an important role in the regulation of the sympathetic nervous system. The underlining molecular mechanisms are largely unknown. Spinophilin (SPL) is a regulator of G protein-coupled receptor signaling. Deletion of SPL induces sympathetically mediated arterial hypertension in mice. We tested the hypothesis that SPL restrains blood pressure (BP) by regulating AngII activity. We equipped SPL(-/-) and SPL(+/+) mice with telemetric devices and applied AngII (1.0 mg kg(-1) day(-1), minipumps) or the AngII subtype 1 receptor (AT1-R) blocker valsartan (50 mg kg(-1) day(-1), gavage). We assessed autonomic nervous system activity through intraperitoneal application of trimethaphan, metoprolol, and atropine. We also tested the effect of intracerebroventricular (icv) AngII on blood pressure in SPL(-/-) and in SPL(+/+) mice. Chronic infusion of AngII upregulates SPL expression in the hypothalamus of SPL(+/+) mice. Compared with SPL(+/+) mice, SPL(-/-) mice showed a greater increase in daytime BP with AngII (19.2 ± 0.8 vs. 13.5 ± 1.6 mmHg, p < 0.05). SPL(-/-) showed a greater depressor response to valsartan. BP and heart rate decreased more with trimethaphan and metoprolol in AngII-treated SPL(-/-) than in AngII-treated SPL(+/+) mice. SPL(-/-) mice responded more to icv AngII. Furthermore, brainstem AT1-R and AngII type 2 receptor (AT2-R) expression was reduced in SPL(-/-) mice. AngII treatment normalized AT1-R and AT2-R expression levels. In summary, our findings suggest that SPL restrains AngII-mediated sympathetic nervous system activation. SPL is a hitherto unrecognized molecule with regard to central blood pressure control and may pave the way to novel strategies for the treatment of hypertension.