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Background: Mammary adenocarcinomas are one of the most common tumour diseases in bitches. The relationship between oxidative stress and the degree of malignancy of the tumour has not been sufficiently researched in veterinary medicine., Objectives: The main objective was to investigate the potential role of MDA as a practice-relevant biomarker for the assessment of systemic oxidative stress and to determine whether this parameter can indicate the malignancy grade of a mammary adenocarcinoma., Methods: In the present pilot study, MDA plasma concentrations were analysed in 55 bitches with (n = 28) and without (n027) malignant adenocarcinomas of the mammary gland using two different measurement methods and the relationship to tumour size was investigated., Results: The mean MDA concentration measured by enzyme-linked immunosorbent assay (ELISA) was 289 ng/mL (range 365-634 ng/mL) in dogs with grade 1 adenocarcinoma (n = 13), 288.5 ng/mL (range 85-752 ng/mL) in dogs with grade 2 adenocarcinoma (n = 10), 332 ng/mL (range 239-947 ng/mL) in dogs with grade 3 (n = 5) adenocarcinoma and 293 ng/mL (range 175-549 ng/mL) in dogs without a mammary tumour (n = 27). When MDA was measured by HPLC, the average MDA concentration in the study group (n = 11) was 0.24 µmol/L (range 0.16-0.37) and that of the control group (n = 15) was 0.27 µmol/L (range 0.16-1.62). Thus, there were no significant differences between the study group with malignant adenocarcinomas and the control group in both examination methods (p > 0.05). Furthermore, there was no correlation between the MDA concentrations and the approximate volume of the mammary tumour., Conclusion: The results highlight the challenges of providing a prognosis for the malignancy of a mammary adenocarcinoma based on MDA concentrations in plasma using ELISA or HPLC. As a result, histopathological examination remains the gold standard for diagnosing and differentiating adenocarcinomas of the mammary gland., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Within the framework of plant biostimulation, a pivotal role is played by the achievement of low-cost, easily prepared nanoparticles for priming purposes. Therefore, in this report, two different synthetic strategies are described to engineer zinc oxide nanoparticles with an inulin coating. In both protocols, i.e., two-step and gel-like one-pot protocols, nanoparticles with a highly pure ZnO kernel are obtained when the reaction is carried out at T ≥ 40 °C, as ascertained by XRD and ATR/FTIR studies. However, a uniformly dispersed, highly homogeneous coating is achieved primarily when different temperatures, i.e., 60 °C and 40 °C, are employed in the two phases of the step-wise synthesis. In addition, a different binding mechanism, i.e., complexation, occurs in this case. When the gel-like process is employed, a high degree of coverage by the fructan is attained, leading to micrometric coated aggregates of nanometric particles, as revealed by SEM investigations. All NPs from the two-step synthesis feature electronic bandgaps in the 3.25-3.30 eV range in line with previous studies, whereas the extensive coating causes a remarkable 0.4 eV decrease in the bandgap. Overall, the global analysis of the investigations indicates that the samples synthesized at 60 °C and 40 °C are the best suited for biostimulation. Proof-of-principle assays upon Vicia faba seed priming with Zn5 and Zn5@inu indicated an effective growth stimulation of seedlings at doses of 100 mgKg -1 , with concomitant Zn accumulation in the leaves.

In the present work, a simple two-step method is proposed for mixed oxide synthesis aimed at the achievement of antibacterial nanomaterials. In particular, Cu, Zn and Co have been selected to achieve single-, double- and triple-cation oxides. The synthesized samples are characterized by XRD, IR, SEM and EDX, indicating the formation of either crystalline or amorphous hydrocarbonate precursors. The oxides present one or two crystalline phases, depending on their composition; the triple-cation oxides form a solid solution of tenorite. Also, the morphology of the samples varies with the composition, yielding nanoparticles, filaments and hydrangea-like microaggregates. The antibacterial assays are conducted against E. coli and indicate an enhanced efficacy, especially displayed by the oxide containing 3% Co and 9% Zn incorporated into the CuO lattice. The oxides with the highest antibacterial properties are tested for their cytotoxicity, indicating a low toxicity impact, in line with literature data.

The link between morphology and properties is well-established in the nanoparticle literature. In this report, we show that different approaches in the synthesis of copper oxide can lead to nanoparticles (NPs) of different size and morphology. The structure and properties of the synthesized NPs are investigated with powder X-ray diffraction, scanning electron microscopy (SEM), and diffuse reflectance spectroscopy (DRS). Through detailed SEM analyses, we were able to correlate the synthetic pathways with the particles' shape and aggregation, pointing out that bare hydrothermal pathways yield mainly spheroidal dandelion-like aggregates, whereas, if surfactants are added, the growth of the nanostructures along a preferential direction is promoted. The effect of the morphology on the electronic properties was evaluated through DRS, which allowed us to obtain the electron bandgap in every system synthesized, and to find that the rearrangement of threaded particles into more compact structures leads to a reduction in the energy difference. The latter result was compared with Density Functional Theory (DFT) computational models of small centrosymmetric CuO clusters, cut from the tenorite crystal structure. The computed UV-Vis absorption spectra obtained from the clusters are in good agreement with experimental findings.

In the quest to find powerful modifiers of screen-printed electrodes for sensing applications, a set of rare earth-doped Ca 10-x RE x (PO 4 ) 6 (OH) 2 (RE = La, Nd, Sm, Eu, Dy, and Tm and x = 0.01, 0.02, 0.10, and 0.20) hydroxyapatite (HAp) samples were subjected to an in-depth electrochemical characterization using electrochemical impedance spectroscopy and cyclic and square wave voltammetry. Among all of these, the inorganic phosphates doped with lanthanum proved to be the most reliable, revealing robust analytical performances in terms of sensitivity, repeatability, reproducibility, and reusability, hence paving the way for their exploitation in sensing applications. Structural data on La-doped HAp samples were also provided by using different techniques, including optical microscopy, X-ray diffraction, Rietveld refinement from X-ray data, Fourier transform infrared, and Raman vibrational spectroscopies, to complement the electrochemical characterization.

Extrinsic chemiluminescence can be an efficient tool for determining pesticides and fungicides, which do not possess any intrinsic fluorescent signal. On this basis, (3-aminopropyl) trimethoxysilane (APTMS)-coated ZnO (APTMS@ZnO) was synthesized and tested as an extrinsic probe for the fungicide penconazole. Several synthetic routes were probed using either a one-pot or two-steps method, in order to ensure both a green synthetic pathway and a good signal variation for the penconazole concentration. The synthesized samples were characterized using X-ray diffraction (XRD), infrared (IR), Raman and ultraviolet-visible (UV-Vis) spectroscopy, scanning electron microscopy (SEM) imaging and associated energy-dispersive X-ray (EDX) analysis. The average size of the synthesized ZnO nanoparticles (NPs) is 54 ± 10 nm, in line with previous preparations. Of all the samples, those synthesized in two steps, at temperatures ranging from room temperature (RT) to a maximum of 40 °C, using water solvent (G-APTMG@ZnO), appeared to be composed of nanoparticles, homogeneously coated with APTMS. Chemiluminescence tests of G-APTMG@ZnO, in the penconazole concentration range 0.7-1.7 ppm resulted in a quenching of the native signal between 6% and 19% with a good linear response, thus indicating a green pathway for detecting the contaminant. The estimated detection limit (LOD) is 0.1 ± 0.01 ppm.

Due to the increasing tattoo practicing in Eastern countries and general concern on tattoo ink composition and safety, the green tattoo inks Green Concentrate by Eternal, for European and "for Asia Market Only" were analyzed, under the premise that only the former falls under a composition regulation. A separation of the additives from the pigment was carried out by successive extraction in solvents of different polarities, i.e., water, acetone and dichloromethane. The solid residues were analyzed by IR and Raman spectroscopies, the liquid fractions by GC/mass spectrometry. The relative pigment load and element traces were also estimated. We found that the European and the Asian inks are based on the same pigment, PG7, restricted in Europe, though at different loads. They have a similar content of harmful impurities, such as Ni, As, Cd and Sb and both contain siloxanes, including harmful D4. Furthermore, they have different physical-chemical properties, the European ink being more hydrophilic, the Asian more hydrophobic. Additionally, the Asian ink contains harmful additives for the solubilization of hydrophobic matrices and by-products of the phthalocyanine synthesis. Teratogenic phthalates are present as well as chlorinated teratogenic and carcinogenic compounds usually associated to the laser treatment for removal purposes, to a larger extent in the European ink. The composition of the inks does not seem to reflect regulatory restrictions, where issued.

Dipeptides, the prototype peptides, exist in both linear ( l -) and cyclo ( c -) structures. Since the first mass spectrometry experiments, it has been observed that some l -structures may turn into the cyclo ones, likely via a temperature-induced process. In this work, combining several different experimental techniques (mass spectrometry, infrared and Raman spectroscopy, and thermogravimetric analysis) with tight-binding and ab initio simulations, we provide evidence that, in the case of l-phenylalanyl-l-alanine, an irreversible cyclization mechanism, catalyzed by water and driven by temperature, occurs in the condensed phase. This process can be considered as a very efficient strategy to improve dipeptide stability by turning the comparatively fragile linear structure into the robust and more stable cyclic one. This mechanism may have played a role in prebiotic chemistry and can be further exploited in the preparation of nanomaterials and drugs.

Deep Eutectic Solvents have gained a lot of attention in the last few years because of their vast applicability in a large number of technological processes, the simplicity of their preparation and their high biocompatibility and harmlessness. One of the fields where DES prove to be particularly valuable is the synthesis and modification of inorganic materials-in particular, nanoparticles. In this field, the inherent structural inhomogeneity of DES results in a marked templating effect, which has led to an increasing number of studies focusing on exploiting these new reaction media to prepare nanomaterials. This review aims to provide a summary of the numerous and most recent achievements made in this area, reporting several examples of the newest mixtures obtained by mixing molecules originating from natural feedstocks, as well as linking them to the more consolidated methods that use "classical" DES, such as reline.

The tattoos removal has become an issue upon spread of the tattooing practice worldwide and hindsight regrets. Lasers are typically used for the purpose, though some colours such as green are considered "recalcitrant" to the treatment. In the current investigation, we aim at determining the efficacy of removal of a green ink water dispersion, using 5 laser treatments: Nd:YAG nano- and picosecond lasers in normal and array mode and Ruby nanosecond laser, keeping the total irradiated energy constant. The UV-Vis spectroscopy of the treated samples indicate that Nd:YAG picosecond laser is most effective, and the Ruby nanosecond laser is the least efficient. Fragment compounds generated from the pigment and siloxanes are common to all treatments, whereas hydrocarbon emerge by a larger amount upon Nd:YAG nanosecond treatment. Fibres are formed upon picosecond treatments and when operating in array mode, and lamellae are achieved by Ruby nanosecond laser treatment. Residual particles suspensions are very heterogeneous upon nanosecond treatments., (© 2022. The Author(s).)

Gold nanoparticles (AuNPs) were functionalized by ruthenium porphyrins through a sulfur/gold covalent bond using a three-steps reaction. The catalyst was characterized by scanning electron microscopy (SEM) and thermogravimetric analysis (TGA) in order to control the binding of ruthenium porphyrin on AuNPs' surface. The catalyst was tested and compared with an analog system not bound to AuNPs in the oligomerization reaction using 1-phenylacetylene as the substrate.

Water pollution caused by hexavalent chromium (Cr(VI)) ions represents a serious hazard for human health due to the high systemic toxicity and carcinogenic nature of this metal species. The optical sensing of Cr(VI) through specifically engineered nanomaterials has recently emerged as a versatile strategy for the application to easy-to-use and cheap monitoring devices. In this study, a one-pot oxidative method was developed for the cage opening of C 60 fullerene and the synthesis of stable suspensions of N-doped carbon dots in water-THF solutions (N-CDs-W-THF). The N-CDs-W-THF selectively showed variations of optical absorbance in the presence of Cr(VI) ions in water through the arising of a distinct absorption band peaking at 550 nm, i.e., in the transparency region of pristine material. Absorbance increased linearly, with the ion concentration in the range 1-100 µM, thus enabling visual and ratiometric determination with a limit of detection (LOD) of 300 nM. Selectivity and possible interference effects were tested over the 11 other most common heavy metal ions. The sensing process occurred without the need for any other reactant or treatment at neutral pH and within 1 min after the addition of chromium ions, both in deionized and in real water samples.

In the present study, we successfully synthesized N-doped carbon quantum dots (N-CQDs) using a top-down approach, i.e., hydroxyl radical opening of fullerene with hydrogen peroxide, in basic ambient using ammonia for two different reaction times. The ensuing characterization via dynamic light scattering, SEM, and IR spectroscopy revealed a size control that was dependent on the reaction time, as well as a more pronounced -NH 2 functionalization. The N-CQDs were probed for metal ion detection in aqueous solutions and during bioimaging and displayed a Cr 3+ and Cu 2+ selectivity shift at a higher degree of -NH 2 functionalization, as well as HEK-293 cell nuclei marking.

Complete androgen insensitivity syndrome (CAIS)-resulting in 46,XY karyotype, but female phenotype-is a disorder of sex development and primary amenorrhea, but its effect on bone mineral density (BMD) is singular and difficult to manage. Androgens are an important modulator of bone remodeling and health, and the androgen receptor (AR) is pivotal for signaling within the bone cells. CAIS results in a severely disrupted AR throughout the body, causing an elevated risk of early osteoporosis. Timing of gonadectomy and hormone replacement therapy protocols are not established, creating a wide variety of treatment plans and BMD profiles. Our objective is to report a patient with CAIS status post prepubertal orchiectomy that developed early osteoporosis and to describe the lack of optimal strategies and consensus available to improve bone health in this population. Additionally, our case illustrates the fact there are no guidelines advocating the use of newer drugs for osteoporosis in this population., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Psychiatric inpatients constitute a population at considerably increased risk for suicide. Identifying those at imminent risk is still a challenging task for hospital staff. This retrospective case-control study focused on clinical risk factors related to the course of the hospital stay. Method: Inpatient suicide cases were identified by linking the Tyrol Suicide Register with the registers of three psychiatric hospitals in the state. Control subjects were patients who had also been hospitalized in the respective psychiatric unit but had not died by suicide. Matching variables included sex, age, hospital, diagnosis, and admission date. The study period comprised 7 years. Data were analyzed by the appropriate two-sample tests and by logistic regression. Results: A total of 30 inpatient suicide cases and 54 control patients were included. A number of factors differentiated cases from controls; after correction for multiple testing, the following retained significance: history of aborted suicide, history of attempted suicide, history of any suicidal behavior/threats, suicidal ideation continuing during hospitalization, no development of prospective plans, no improvement of mood during the hospital stay, and leaving ward without giving notice. Logistic regression identified the latter three variables and history of attempted suicide as highly significant predictors of inpatient suicide. Conclusions: Preventive measures during hospitalization include thorough assessment of suicidal features, an emphasis on the development of future perspectives, and a review of hospital regulations for patients who want to leave the ward., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Deisenhammer, Behrndt-Bauer, Kemmler, Haring and Miller.)

Objective: Pheochromocytoma is diagnosed biochemically by demonstrating an excessive production of catecholamines and their metabolites in the blood and urine. However, these tests are at times fraught with false-positive results due to drug effects. We report here a patient with markedly elevated urinary metanephrines associated with the use of eletriptan for migraine treatment., Methods: A literature search was conducted using the PubMed and Google Scholar databases for eletriptan and false positive metanephrine elevation. Urine and plasma metanephrine tests were performed via liquid chromatography/tandem mass-spectrometry., Results: A 29-year-old man with migraine recently started on eletriptan was evaluated for a worsening headache. Initially his blood pressure was 220/160 mm Hg with a creatinine of 1.9 mg/dL. He was treated with intravenous nicardipine. His lab tests showed normal aldosterone/plasma renin activity ratio, midnight salivary cortisol, thyroid function, and urinary drug screen. A 24-hour urine metanephrine level at 2,494 μg (normal, 45 to 290 μg) and normetanephrine level at 1,341 μg (normal, 82 to 500 μg) for secondary hypertension work-up were markedly elevated. In contrast, plasma metanephrines were at 27 pg/mL (normal, 0 to 62 pg/mL) and normetanephrines were at 255 pg/mL (normal, 0 to 145 pg/mL) were only mildly elevated. Adrenal CT and gallium-68 positron emission tomography/computed tomography showed no abnormalities. Within 1 week of eletriptan discontinuation, his urine and plasma metanephrine and normetanephrine levels completely normalized as well as a reduction of blood pressure (130's/80's mm Hg)., Conclusion: The discrepancy between plasma and urine studies in our patient suggests the possibility of false positive tests. It is possible that eletriptan may affect the urine assays, but the exact mechanism causing elevated urine metanephrines/normetanephrines is not clear., (Copyright © 2020 AACE.)

In the present paper, we investigate the effects of alkali and operational temperature on NiO capacitive and optical properties. The NiO samples were prepared by a straightforward, surfactant-free hydrothermal synthesis, employing Ni(NO 3 ) 2 and either urea or moderately sterically hindered triethylamine (TEA). The syntheses were followed by calcinations at either 400 or 600 °C. NiO samples were characterized by XRD, scanning electron microscopy, and nitrogen adsorption isotherms. The optical properties were investigated by reflectance spectroscopy, and the pseudocapacitance was studied by cyclic voltammetry and galvanostatic charge charge-discharge measurements. We found that the synthesis with TEA yielded nanoflowers whereas the morphology of the synthesis with urea varied with the calcination temperature and resulted in nanoparticles or nanoslices at calcination temperatures of 400 and 600 °C, respectively. The NiO samples prepared at a lower temperature displayed a favorable combination of surface area and porosity that allowed for high performance with capacitances of 502 and 520 F g -1 at a current density of 1 A g -1 for nanoflowers and nanoparticles, respectively. The band gaps of all the samples were compatible with the estimated nanoparticle sizes. Finally, we used the synthesized NiO samples for the preparation of screen-printed electrodes (SPEs) modified by drop-casting and probed them against a [Fe(CN) 6 ] 3-/4- probe.

The synthesis of organic-inorganic hybrid compounds based on phenylphosphonate and their use as precursors to form LiMn x Fe (1-x) PO₄ composites containing carbonaceous substances with sub-micrometric morphology are presented. The experimental procedure includes the preliminary synthesis of Fe 2+ and/or Mn 2+ phenylphosphonates with the general formula Fe (1-x) Mn x [(C₆H₅PO₃)(H₂O)] (with 0 < x < 1), which are then mixed at different molar ratios with lithium carbonate. In this way the carbon, obtained from in situ partial oxidation of the precursor organic part, coats the LiMn x Fe (1-x) PO₄ particles. After a structural and morphological characterization, the electrochemical behavior of lithium iron manganese phosphates has been compared to the one of pristine LiFePO₄ and LiMnPO₄, in order to evaluate the doping influence on the material., Competing Interests: The authors declare no conflict of interest.

Dermatomyositis is a rare autoimmune disease with a heterogeneous presentation that often has multiple extramuscular manifestations, although it does not typically involve the renal function. A 62-year-old female presented with proximal muscle weakness and rashes, which are classic symptoms of dermatomyositis without creatine kinase (CK) elevation. Initial serologic evaluation revealed a positive p-ANCA, although she did not develop renal failure for several months, at which point renal biopsy findings were consistent with microscopic polyangiitis. The patient was initially treated with cyclophosphamide, maintained with rituximab, and has been in remission for more than 2 years. Dermatomyositis and microscopic polyangiitis are both uncommon diseases, but are concomitantly present in this patient. A positive p-ANCA and development of renal insufficiency should be promptly evaluated in dermatomyositis patients., Competing Interests: Conflict of Interest: No conflict of interest was declared by the authors.

The epidermal permeability barrier (EPB) prevents organisms from dehydration and infection. The transcriptional regulation of EPB development is poorly understood. We demonstrate here that transcription factor COUP-TF-interacting protein 1 (CTIP1/BCL11A; hereafter CTIP1) is highly expressed in the developing murine epidermis. Germline deletion of Ctip1 (Ctip1 -/- ) results in EPB defects accompanied by compromised epidermal differentiation, drastic reduction in profilaggrin processing, reduced lamellar bodies in granular layers and significantly altered lipid composition. Transcriptional profiling of Ctip1 -/- embryonic skin identified altered expression of genes encoding lipid-metabolism enzymes, skin barrier-associated transcription factors and junctional proteins. CTIP1 was observed to interact with genomic elements within the regulatory region of the gene encoding the differentiation-associated gene, Fos-related antigen2 (Fosl2) and lipid-metabolism-related gene, Fatty acid elongase 4 (Elvol4), and the expression of both was altered in Ctip1 -/- mice. CTIP1 appears to play a role in EPB establishment of via direct or indirect regulation of a subset of genes encoding proteins involved in epidermal differentiation and lipid metabolism. These results identify potential, CTIP1-regulated avenues for treatment of skin disorders involving EBP defects.

Background: Clinical swollen joint examination of the obese rheumatoid arthritis (RA) patient can be difficult. Musculoskeletal Ultrasound (MSUS) has higher sensitivity than physical examination for swollen joints (SJ). The purpose of this study was to determine the joint-specific association between power Doppler (PDUS) and clinical SJ in RA across body mass index (BMI) categories., Methods: Cross-sectional clinical and laboratory data were collected on 43 RA patients. PDUS was performed on 9 joints (wrist, metacarpalphalangeal 2-5, proximal interphalgeal 2/3 and metatarsalphalangeal 2/5). DAS28 and clinical disease activity index (CDAI) were calculated. Patients were categorized by BMI: <25, 25-30, and >30. Demographic and clinical characteristics were compared across BMI groups with Kruskal-Wallis test and chi-square tests. Joint-level associations between PDUS and clinically SJ were evaluated with mixed effects logistic regression models., Results: While demographics and clinically-determined disease activity were similar among BMI groups, PDUS scores significantly differed (p = 0.02). Using PDUS activity as the reference standard for synovitis and clinically SJ as the test, the positive predictive value of SJ was significantly lower in higher BMI groups (0.71 in BMI < 25, 0.58 in BMI 25-30 and 0.44 in BMI < 30) (p = 0.02). The logistic model demonstrated that increased BMI category resulted in decreased likelihood of PDUS positivity (OR 0.52, p = 0.03)., Conclusions: This study suggests that in an obese RA patient, a clinically assessed SJ is less likely to represent true synovitis (as measured by PDUS). Disease activity in obese RA patients may be overestimated by CDAI/DAS28 calculations and clinicians when considering change in therapy.

Genome-wide association studies of schizophrenia (GWAS) have revealed the role of rare and common genetic variants, but the functional effects of the risk variants remain to be understood. Protein interactome-based studies can facilitate the study of molecular mechanisms by which the risk genes relate to schizophrenia (SZ) genesis, but protein-protein interactions (PPIs) are unknown for many of the liability genes. We developed a computational model to discover PPIs, which is found to be highly accurate according to computational evaluations and experimental validations of selected PPIs. We present here, 365 novel PPIs of liability genes identified by the SZ Working Group of the Psychiatric Genomics Consortium (PGC). Seventeen genes that had no previously known interactions have 57 novel interactions by our method. Among the new interactors are 19 drug targets that are targeted by 130 drugs. In addition, we computed 147 novel PPIs of 25 candidate genes investigated in the pre-GWAS era. While there is little overlap between the GWAS genes and the pre-GWAS genes, the interactomes reveal that they largely belong to the same pathways, thus reconciling the apparent disparities between the GWAS and prior gene association studies. The interactome including 504 novel PPIs overall, could motivate other systems biology studies and trials with repurposed drugs. The PPIs are made available on a webserver, called Schizo-Pi at http://severus.dbmi.pitt.edu/schizo-pi with advanced search capabilities.

Extracellular high-mobility group box 1 (HMGB1) (disulfide form), via activation of toll-like receptor 4 (TLR4)-dependent signaling, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4 signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A cell-based screening strategy led us to identify first generation HIV-protease inhibitors (PI) as potential inhibitors of HMGB1-TLR4 driven cytokine production. Here we report that the first-generation HIV-PI saquinavir (SQV), as well as a newly identified mammalian protease inhibitor STO33438 (334), potently block disulfide HMGB1-induced TLR4 activation, as assayed by the production of TNF-α by human monocyte-derived macrophages (THP-1). We further report on the identification of mammalian cathepsin V, a protease, as a novel target of these inhibitors. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between cathepsin V with TLR4 and its adaptor protein MyD88. Treatment with SQV, 334 or the known cathepsin inhibitor SID26681509 (SID) significantly improved survival in murine models of sepsis and reduced liver damage following warm liver ischemia/reperfusion (I/R) models, both characterized by strong HMGB1-TLR4 driven pathology. The current study demonstrates a novel role for cathepsin V in TLR4 signaling and implicates cathepsin V as a novel target for first-generation HIV-PI compounds. The identification of cathepsin V as a target to block HMGB1-TLR4-driven inflammation could allow for a rapid transition of the discovery from the bench to the bedside. Disulfide HMGB1 drives pathologic inflammation in many models by activating signaling through TLR4. Cell-based screening identified the mammalian protease cathepsin V as a novel therapeutic target to inhibit TLR4-mediated inflammation induced by extracellular HMGB1 (disulfide form). We identified two protease inhibitors (PIs) that block cathepsin V and thereby inhibit disulfide HMGB1-induced TLR4 activation: saquinavir (SQV), a first-generation PI targeting viral HIV protease and STO33438 (334), targeting mammalian proteases. We discovered that cathepsin V binds TLR4 under basal and HMGB1-stimulated conditions, but dissociates in the presence of SQV over time. Thus cathepsin V is a novel target for first-generation HIV PIs and represents a potential therapeutic target of pathologic inflammation.

Pulmonary hypertension (PH) is a progressive and fatal disease with no cure. Vascular remodeling in PH involves intraluminal growth of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Cell growth in these lesions is quasi-neoplastic, with evidence of monoclonality, apoptosis resistance and cancer-like metabolic derangements. Herein we tested the effect of human interferon alpha 2b (IFNα), a pleiotropic cytokine and anti-cancer therapeutic, on the development and progression of PH in the rat SU5416/hypoxia (SUH) model and mouse hypoxia model of the disease. In both models IFNα attenuated the development of PH and reversed established PH as assessed by measuring right ventricular systolic pressure and right ventricular hypertrophy. The effect of IFNα was dependent on the type I interferon receptor (IFNAR) since mice lacking a subunit of the IFNAR were not protected by IFNα. Morphometric analysis of pulmonary aterioles from hypoxic mice or SUH rats showed that IFNα inhibited pulmonary vascular remodeling in both models and that IFNα reversed remodeling in SUH rats with established disease. Immunohistochemical staining revealed that IFNα decreased the number of PCNA and Tunel positive cells in the wall of pulmonary arterioles. In vitro, IFNα inhibited proliferation of human pulmonary artery smooth muscle cells and as well as human pulmonary artery endothelial cell proliferation and apoptosis. Together these findings demonstrate that IFNα reverses established experimental PH and provide a rationale for further exploration of the use of IFNα and other immunotherpies in PH.

Rationale: Recent studies demonstrate a role for toll-like receptor 4 (TLR4) in the pathogenesis of pulmonary hypertension (PH); however, the cell types involved in mediating the effects of TLR4 remain unknown., Objectives: The objective of this study was to determine the contribution of TLR4 expressed on nonparenchymal cells to the pathogenesis of PH., Methods and Results: TLR4 bone marrow chimeric mice revealed an equal contribution of TLR4 on nonparenchymal and parenchymal cells in the pathogenesis of PH as determined by measuring right ventricular (RV) systolic pressure and RV hypertrophy. However, the deletion of TLR4 from myeloid lineage cells had no effect on the development of PH because we found no difference in RV systolic pressure or RV hypertrophy in wild-type versus LysM-TLR4(-/-) mice. To explore the potential role of platelet TLR4 in the pathogenesis of PH, platelet-specific TLR4(-/-) mice were generated (PF4-TLR4(-/-) mice). TLR4(-/-) platelets from either global TLR4(-/-) or PF4-TLR4(-/-) mice were functional but failed to respond to lipopolysaccharide, demonstrating a lack of TLR4. PF4-TLR4(-/-) mice demonstrated significant protection from hypoxia-induced PH, including attenuated increases in RV systolic pressure and RV hypertrophy, decreased platelet activation, and less pulmonary vascular remodeling. The deletion of TLR4 from platelets attenuated serotonin release after chronic hypoxia, and lipopolysaccharide-stimulated platelets released serotonin and promoted pulmonary artery smooth muscle cell proliferation in a serotonin-dependent manner., Conclusions: Our data demonstrate that TLR4 on platelets contributes to the pathogenesis of PH and further highlights the role of platelets in PH.

Survival rates for patients with pulmonary hypertension (PH) remain low, and our understanding of the mechanisms involved are incomplete. Here we show in a mouse model of chronic hypoxia (CH)-induced PH that the nuclear protein and damage-associate molecular pattern molecule (DAMP) high mobility group box 1 (HMGB1) contributes to PH via a Toll-like receptor 4 (TLR4)-dependent mechanism. We demonstrate extranuclear HMGB1 in pulmonary vascular lesions and increased serum HMGB1 in patients with idiopathic pulmonary arterial hypertension. The increase in circulating HMGB1 correlated with mean pulmonary artery pressure. In mice, we similarly detected the translocation and release of HMGB1 after exposure to CH. HMGB1-neutralizing antibody attenuated the development of CH-induced PH, as assessed by measurement of right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling and endothelial activation and inflammation. Genetic deletion of the pattern recognition receptor TLR4, but not the receptor for advanced glycation end products, likewise attenuated CH-induced PH. Finally, daily treatment of mice with recombinant human HMGB1 exacerbated CH-induced PH in wild-type (WT) but not Tlr4(-/-) mice. These data demonstrate that HMGB1-mediated activation of TLR4 promotes experimental PH and identify HMGB1 and/or TLR4 as potential therapeutic targets for the treatment of PH.

In pulmonary hypertension the loss of precapillary arterioles results from vascular injury causing endothelial dysfunction. Endothelial cell migration and proliferation are critical for vascular regeneration. This study focused on the effect of high mobility group box 1 protein (HMGB1) on these critical processes. HMGB1 had no effect on human pulmonary artery endothelial cell (HPAEC) proliferation. In contrast, treatment of HPAECs with HMGB1 dose-dependently inhibited VEGF-stimulated HPAEC migration. The effect of HMGB1 on HPAEC migration was TLR4-dependent because it was reversed by TLR4 siRNA or TLR4-neutralizing antibody. Exposure of HPAECs to hypoxia caused translocation and release of HMGB1 and inhibition of HPAEC migration. The effect of hypoxia on HPAEC migration was mediated by HMGB1 because HMGB1-neutralizing antibody but not control IgG restored HPAEC migration. Likewise, TLR4 siRNA but not control siRNA reversed the inhibitory effect of hypoxia in HPAECs. The canonical TLR4 signaling pathway requires the adaptor protein MyD88 and leads to downstream NFκB activation. Interestingly, HMGB1 failed to stimulate NFκB translocation to the nucleus, but instead activated an alternative pathway characterized by activation of interferon response factor 3 (IRF3). This was in contrast to human umbilical vein endothelial cells in which HMGB1 stimulated nuclear translocation of NFκB but not IRF3. IRF3 siRNA, but not MyD88 siRNA, reversed the inhibitory effect of HMGB1 on HPAEC migration. These data demonstrate that HMGB1 inhibits HPAEC migration, a critical process for vascular regeneration, via TLR4- and IRF3-dependent mechanisms.

Caveolin-1 (Cav-1)-/- mice develop mild pulmonary hypertension as they age. In this study, we sought to determine the effect of chronic hypoxia, an established model of pulmonary hypertension, on young Cav-1-/- mice with no measurable signs of pulmonary hypertension. Exposure of Cav-1-/- mice to chronic hypoxia resulted in an initial rise in right ventricular (RV) systolic pressure (RVSP) similar to wild-type (WT) mice. By three weeks RVSP decreased in the Cav-1-/- mice, whereas it was maintained in WT mice. The drop in RVSP in Cav-1-/- mice was accompanied by decreased cardiac output, increased RV hypertrophy, RV interstitial fibrosis, decreased RV sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a mRNA and decreased RV function compared with WT mice. Importantly, minimal differences were noted in pulmonary vascular remodeling between WT and Cav-1-/- mice, and left ventricular function was normal in hypoxic Cav-1-/- mice. Mechanistically, increased endothelial nitric oxide synthase uncoupling and increased tyrosine nitration of protein kinase G were detected in the RV of Cav-1-/- mice. These hemodynamic, histological, and molecular changes were prevented in Cav-1-/- mice expressing an endothelial-specific Cav-1 transgene or by nitric oxide synthase inhibition. These data suggest that, in Cav-1-/- mice, increased oxidative/nitrosative stress due to endothelial nitric oxide synthase uncoupling modifies the response of the RV to pressure overload, accelerating the deterioration of RV function.

Background: Gamma-glutamyltransferase (GTT), a known marker for apoptotic balance, seems to promote tumour progression, invasion and drug resistance. Recently, high GGT serum levels were shown to be associated with impaired prognosis in patients with cervical cancer. The aim of this study was to investigate the value of pre-therapeutic serum GGT levels as prognostic parameter in patients with endometrial cancer., Methods: Within the present multi-centre trial, clinical-pathological parameters and pre-therapeutic serum GGT levels were evaluated in 874 consecutive patients with endometrial cancer. Patients were stratified in GGT risk groups, and univariate and multivariable survival analyses were performed., Results: Mean pre-therapeutic serum GGT level was 30.8 (41.5) U l(-1). Elevated and highly elevated serum GGT levels (P=0.03 and P=0.005), tumour stage (P<0.001 and P<0.001), grade (P<0.001 and P=0.02) and age (P<0.001 and P<0.001) were independently associated with progression-free survival in univariate and multivariable survival analyses. Pre-therapeutic GGT was not associated with advanced tumour stage (P=0.6), higher histological grade (P=0.6) or unfavourable histological subtype (P=0.3)., Conclusion: Pre-therapeutic serum GGT is a novel and independent prognostic parameter for progression-free survival of patients with endometrial cancer. Stratifying patients into prognostic subgroups could be used for patient counselling and individualised treatment planning.

Aims: Pulmonary arterial hypertension (PAH) is a progressive lung disease characterized by pulmonary vasoconstriction and vascular remodelling, leading to increased pulmonary vascular resistance and right heart failure. Loss of nitric oxide (NO) signalling and increased endothelial nitric oxide synthase (eNOS)-derived oxidative stress are central to the pathogenesis of PAH, yet the mechanisms involved remain incompletely determined. In this study, we investigated the role activated CD47 plays in promoting PAH., Methods and Results: We report high-level expression of thrombospondin-1 (TSP1) and CD47 in the lungs of human subjects with PAH and increased expression of TSP1 and activated CD47 in experimental models of PAH, a finding matched in hypoxic human and murine pulmonary endothelial cells. In pulmonary endothelial cells CD47 constitutively associates with caveolin-1 (Cav-1). Conversely, in hypoxic animals and cell cultures activation of CD47 by TSP1 disrupts this constitutive interaction, promoting eNOS-dependent superoxide production, oxidative stress, and PAH. Hypoxic TSP1 null mice developed less right ventricular pressure and hypertrophy and markedly less arteriole muscularization compared with wild-type animals. Further, therapeutic blockade of CD47 activation in hypoxic pulmonary artery endothelial cells upregulated Cav-1, increased Cav-1CD47 co-association, decreased eNOS-derived superoxide, and protected animals from developing PAH., Conclusion: Activated CD47 is upregulated in experimental and human PAH and promotes disease by limiting Cav-1 inhibition of dysregulated eNOS.

Activation of bone morphogenetic protein (BMP) receptor II (BMPRII) promotes pulmonary artery endothelial cell (PAEC) survival, proliferation, and migration. Mutations to BMPRII are associated with the development of pulmonary arterial hypertension (PAH). Endothelial dysfunction, including decreased endothelial nitric-oxide synthase (eNOS) activity and loss of bioactive nitric oxide (NO), plays a prominent role in the development of PAH. We hypothesized that stimulation of BMPRII promotes normal PAEC function by activating eNOS. We report that BMPRII ligands, BMP2 and BMP4, (i) stimulate eNOS phosphorylation at a critical regulatory site, (ii) increase eNOS activity, and (iii) result in canonical changes in eNOS protein-protein interactions. The stimulation of eNOS activity by BMPRII ligands was largely dependent on protein kinase A (PKA) activation, as demonstrated using the PKA inhibitors H89 and myristoylated PKI(6-22) amide. PAEC migration stimulated by BMP2 and BMP4 was inhibited by the NOS inhibitor l-nitroarginine methyl ester, providing functional evidence of eNOS activation. Furthermore, BMP2 and BMP4 failed to stimulate eNOS phosphorylation when BMPRII was knocked down by siRNA. Most important to the pathophysiology of the disease, BMP2 and BMP4 failed to stimulate eNOS phosphorylation in PAECs isolated from patients with mutations in the BMPR2 gene. These data demonstrate a new action of BMPs/BMPRII in the pulmonary endothelium and provide novel mechanistic insight into the pathogenesis of PAH.

We previously reported that zinc thiolate signaling contributes to hypoxic contraction of small, nonmuscularized arteries of the lung. The present studies were designed to investigate mechanisms by which hypoxia-released zinc induces contraction in isolated pulmonary endothelial cells and to delineate the signaling pathways involved in zinc-mediated changes in the actin cytoskeleton. We used fluorescence-based imaging to show that hypoxia induced time-dependent increases in actin stress fibers that were reversed by the zinc chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). We further showed that hypoxia-induced phosphorylation of the contractile protein myosin light chain (MLC) and assembly of actin stress fibers were each TPEN sensitive. Hypoxia and zinc-induced inhibition of MLC phosphatase (MLCP) were independent of the regulatory subunit (MYPT1) of MLCP, and therefore hypoxia-released zinc likely inhibits MLCP at its catalytic (PP1) subunit. Inhibition of PKC by Ro-31-8220 and a dominant-negative construct of PKC-ε attenuated hypoxia-induced contraction of isolated pulmonary endothelial cells. Furthermore, zinc-induced phosphorylation of MLC (secondary to inhibition of MLCP) was PKC dependent, and hypoxia-released zinc promoted the phosphorylation of the PKC substrate, CPI-17. Collectively, these data suggest a link between hypoxia, elevations in labile zinc, and activation of PKC, which in turn acts through CPI-17 to inhibit MLCP activity and promote MLC phosphorylation, ultimately inducing stress fiber formation and endothelial cell contraction.

Background: Evidence suggests a role of both innate and adaptive immunity in the development of pulmonary arterial hypertension. The complement system is a key sentry of the innate immune system and bridges innate and adaptive immunity. To date there are no studies addressing a role for the complement system in pulmonary arterial hypertension., Methodology/principal Findings: Immunofluorescent staining revealed significant C3d deposition in lung sections from IPAH patients and C57Bl6/J wild-type mice exposed to three weeks of chronic hypoxia to induce pulmonary hypertension. Right ventricular systolic pressure and right ventricular hypertrophy were increased in hypoxic vs. normoxic wild-type mice, which were attenuated in C3-/- hypoxic mice. Likewise, pulmonary vascular remodeling was attenuated in the C3-/- mice compared to wild-type mice as determined by the number of muscularized peripheral arterioles and morphometric analysis of vessel wall thickness. The loss of C3 attenuated the increase in interleukin-6 and intracellular adhesion molecule-1 expression in response to chronic hypoxia, but not endothelin-1 levels. In wild-type mice, but not C3-/- mice, chronic hypoxia led to platelet activation as assessed by bleeding time, and flow cytometry of platelets to determine cell surface P-selectin expression. In addition, tissue factor expression and fibrin deposition were increased in the lungs of WT mice in response to chronic hypoxia. These pro-thrombotic effects of hypoxia were abrogated in C3-/- mice., Conclusions: Herein, we provide compelling genetic evidence that the complement system plays a pathophysiologic role in the development of PAH in mice, promoting pulmonary vascular remodeling and a pro-thrombotic phenotype. In addition we demonstrate C3d deposition in IPAH patients suggesting that complement activation plays a role in the development of PAH in humans.

Aims: Thrombospondin-1 (TSP1), via its necessary receptor CD47, inhibits nitric oxide (NO)-stimulated soluble guanylate cyclase activation in vascular smooth muscle cells, and TSP1-null mice have increased shear-dependent blood flow compared with wild-type mice. Yet, the endothelial basement membrane should in theory function as a barrier to diffusion of soluble TSP1 into the arterial smooth muscle cell layer. These findings suggested that endothelial-dependent differences in blood flow in TSP1-null mice may be the result of direct modulation of endothelial NO synthase (eNOS) activation by circulating TSP1. Here we tested the hypothesis that TSP1 inhibits eNOS activation and endothelial-dependent arterial relaxation., Methods and Results: Acetylcholine (ACh)-stimulated activation of eNOS and agonist-driven calcium transients in endothelial cells were inhibited by TSP1. TSP1 also inhibited eNOS phosphorylation at serine(1177). TSP1 treatment of the endothelium of wild-type and TSP1-null but not CD47-null arteries inhibited ACh-stimulated relaxation. TSP1-null vessels demonstrated greater endothelial-dependent vasorelaxation compared with the wild type. Conversely, TSP1-null arteries demonstrated less vasoconstriction to phenylephrine compared with the wild type, which was corrected upon inhibition of eNOS. In TSP1-null mice, intravenous TSP1 blocked ACh-stimulated decreases in blood pressure, and both intravenous TSP1 and a CD47 agonist antibody acutely elevated blood pressure in mice., Conclusion: TSP1, via CD47, inhibits eNOS activation and endothelial-dependent arterial relaxation and limits ACh-driven decreases in blood pressure. Conversely, intravenous TSP1 and a CD47 antibody increase blood pressure. These findings suggest that circulating TSP1, by limiting endogenous NO production, functions as a pressor agent supporting blood pressure.

Objective: To characterize postorthodontic settling of the posterior occlusion of patients wearing Hawley retainers vs patients who initially wore Perfector retainers and then switched to Hawley retainers., Materials and Methods: This follow-up study was based on 40 patients (25 Perfector and 15 Hawley), who were part of a larger sample of 50 patients randomly assigned to wear either Hawley or Perfector retainers. The Perfector patients were given Hawley retainers 2 months after retainer delivery. Occlusal bite registrations were scanned and traced to quantify posterior areas of contact and near contact (ACNC). A seven-item questionnaire was used to assess the patient's perception of occlusion. Measurements were obtained at the on the day of retainer delivery, 2 months post delivery, 6 months post delivery, and 8 months post delivery., Results: ACNC increased significantly (P < .05) during the first 6 months of retainer wear. The ACNC of the Hawley and Perfector/Hawley groups increased by 129% and 105%, respectively, over 8 months of retention. The greatest increases in ACNC occurred during the first 2 months. The ACNC further increased between 2 and 6 months in both groups. The Perfector/Hawley group also showed slight increases in ACNC between 6 and 8 months. Overall group differences were not statistically significant. The Perfector/Hawley group perceived greater improvements in occlusion than the Hawley group, but group differences after 8 months were small., Conclusions: Substantial amounts of settling occurred at decelerating rates during the first 6 months after retainer delivery. No significant differences in ACNC were found between the Hawley and Perfector/Hawley groups after 8 months of retainer wear.

Recent research and clinical experience with cochlear implants suggest that subjects' speech recognition with monopolar or broad bipolar stimulation might be equal to or better than that obtained with narrow bipolar stimulation or other spatially restricted electrode configurations. Furthermore, subjects often prefer the monopolar configurations. The mechanisms underlying these effects are not clear. Two hypotheses are (a) that broader configurations excite more neurons resulting in a more detailed and robust neural representation of the signal and (b) that broader configurations achieve a better spatial distribution of the excited neurons. In this study we compared the effects of electrode configuration and the effects of longitudinal placement and spacing of the active electrodes on speech recognition in human subjects. We used experimental processor maps consisting of 11 active electrodes in a 22-electrode scala tympani array. Narrow bipolar (BP), wide bipolar (BP + 6), and monopolar (MP2) configurations were tested with various locations of active electrodes. We tested basal, centered, and apical locations (with adjacent active electrodes) and spatially distributed locations (with every other electrode active) with electrode configuration held constant. Ten postlingually deafened adult human subjects with Nucleus prostheses were tested using the SPEAK processing strategy. The effects of electrode configuration and longitudinal place of stimulation on recognition of CNC phonemes and words in quiet and CUNY sentences in noise (+10 dB S/N) were similar. Both independent variables had large effects on speech recognition and there were interactions between these variables. These results suggest that the effects of electrode configuration on speech recognition might be due, in part, to differences among the various configurations in the spatial location of stimulation. Correlations of subjective judgments of sound quality with speech-recognition ability were moderate, suggesting that the mechanisms contributing to subjective quality and speech-recognition ability do not completely overlap.