Plant Copper Amine Oxidases: Key Players in Hormone Signaling Leading to Stress-Induced Phenotypic Plasticity

Polyamines are ubiquitous, low-molecular-weight aliphatic compounds, present in living organisms and essential for cell growth and differentiation. Copper amine oxidases (CuAOs) oxidize polyamines to aminoaldehydes releasing ammonium and hydrogen peroxide, which participates in the complex network of reactive oxygen species acting as signaling molecules involved in responses to biotic and abiotic stresses. CuAOs have been identified and characterized in different plant species, but the most extensive study on a CuAO gene family has been carried out in Arabidopsis thaliana. Growing attention has been devoted in the last years to the investigation of the CuAO expression pattern during development and in response to an array of stress and stress-related hormones, events in which recent studies have highlighted CuAOs to play a key role by modulation of a multilevel phenotypic plasticity expression. In this review, the attention will be focused on the involvement of different AtCuAOs in the IAA/JA/ABA signal transduction pathways which mediate stress-induced phenotypic plasticity events.     

Implication of the NLRP3 Inflammasome in Bovine Age-Related Sarcopenia

Sarcopenia is defined as the age-related loss of skeletal muscle mass, quality, and strength. The pathophysiological mechanisms underlying sarcopenia are still not completely understood. The aim of this work was to evaluate, for the first time, the expression of NLRP3 inflammasome in bovine skeletal muscle in order to investigate the hypothesis that inflammasome activation may trigger and sustain a pro-inflammatory environment leading to sarcopenia. Samples of skeletal muscle were collected from 60 cattle belonging to three age-based groups. Morphologic, immunohistochemical and molecular analysis were performed to assess the presence of age-related pathologic changes and chronic inflammation, the expression of NLRP3 inflammasome and to determine the levels of interleukin-1β, interleukin-18 and tumor necrosis factor alpha in muscle tissue. Our results revealed the presence of morphologic sarcopenia hallmark, chronic lymphocytic inflammation and a type II fibers-selective NLRP3 expression associated to a significant decreased number of immunolabeled-fibers in aged animals. Moreover, we found a statistically significant age-related increase of pro-inflammatory cytokines such as interleukin-1β and interleukin-18 suggesting the activation of NLRP3 inflammasome. Taken together, our data suggest that NLRP3 inflammasome components may be normally expressed in skeletal muscle, but its priming and activation during aging may contribute to enhance a pro-inflammatory environment altering normal muscular anabolism and metabolism.     

Sofosbuvir Selects for Drug-Resistant Amino Acid Variants in the Zika Virus RNA-Dependent RNA-Polymerase Complex In Vitro

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure significantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40–80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half- maximal inhibitory concentration (IC₅₀) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC₅₀ shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofosbuvir as a possible lead for anti-ZIKV drug development.     

Deep venous thrombosis: epidemiologic, diagnostic and therapeutic aspects

Deep vein thrombosis (DVT) has a high social and economic cost disease being its prevalence in the general population elevated and producing possibly fatal (pulmonary embolism) or disabling (post-thrombotic syndrome) complications. Thus, it appears of great importance to know the epidemiological and clinical characteristics of DVT in order to perform the best diagnosis, therapy and prophylaxis. The study population is composed by 146 patients (84 males and 62 females, mean age 60.9 +/- 15.3 years, range 19.92 years), arrived in our Vascular Echography Laboratory with the clinical suspect of DVT confirmed by means of echo color Doppler. The most frequent clinical signs were skin hyperthermia in 118 patients (80.8%) and edema in 116 patients (79.5%), while the most common symptom was pain, 89 patients (61.0%). Eleven patients (7.5%) were asymptomatic. The echo criteria utilized were direct thrombus visualization, vessel diameter higher than the contralateral, reduced or absent vessel wall ability to be compressed, reduced or absent color Doppler venous flow, lack or reduction of respiratory flow modulation, visualization of collateral circulation. DVT was located in 131 patients (89.7%) in inferior limbs (proximal in 122 patients, isolated distal in 9 patients), in 14 patients (9.6%) in superior limbs and in 3 patients (2.1%) in the internal jugular vein. In 130 patients a risk factor or a predisposing condition was identified: secondary DVT; in 16 patients the DVT was considered idiopathic. The most frequent risk factors were: previous surgery 28.1%, immobilization 19.9% trauma 17.1%, tumors 9.6%. A hypercoagulation was detected in 4 patients: antithrombin III deficit in 2, post-splenectomy thrombocytosis in 1 and antiphospholipid antibodies syndrome in the last one. The Pisa territory epidemiologic data showed a male 0.51 and female 0.38/1000 subject/year DVT incidence, with significantly higher values in older than 45-54 males and 55-64 females. One hundred and thirty one patients were treated with 5-11 day heparin infusion and thereafter with warfarin at least for 6 months, 1 year or indefinitely depending on thromboembolic risk. Six patients with distal DVT and 9 patients with hemorrhagic risk were treated with subcutaneous calcic or low weight heparin. In 1 patient with a mobile thrombus judged as at very high risk of embolization, a caval filter was positioned. Anticoagulant therapy complications were: 2 minor bleedings, 1 alopecia, 1 thrombocytopenia. Two patients died for neoplastic complications. Fifty-seven patients completed a 6-month follow-up and were submitted to a control each study that evidenced: total recanalization in 15 (26.3%), partial recanalization in 25 (43.9%) and no recanalization in 17 patients (29.8%). In 6 patients there was a DVT relapse and in 9 pulmonary embolization: almost all these patients were in the partial recanalization group.     

Experimental proof of Doppler bandwidth invariance

A line flow of scatterers crossing the sound field produced by a focused circular transducer at uniform velocity originates a quasi-triangular Doppler spectrum. It is known that the spectrum shape and width depend on the line flow to beam axis angle, as well as on the transducer geometry. It has recently been theoretically predicted that this spectrum width is independent of the flow line location in the sound field. Experimental verification of the new theorem, based on the use of a thread phantom operated at various orientations, ranges, and offsets, with respect to the ultrasound transducer, is presented. The tests were made with a computerized pulsed Doppler system designed to perform optimal real-time spectral analysis of data obtained in this application. The prototype system and the experimental procedure adopted for demonstrating in vitro the invariance of the Doppler spectral bandwidth are described.     

Neuroprotective Effects of Testosterone in the Hypothalamus of an Animal Model of Metabolic Syndrome

Metabolic syndrome (MetS) is known to be associated to inflammation and alteration in the hypothalamus, a brain region implicated in the control of several physiological functions, including energy homeostasis and reproduction. Previous studies demonstrated the beneficial effects of testosterone treatment (TTh) in counteracting some MetS symptoms in both animal models and clinical studies. This study investigated the effect of TTh (30 mg/kg/week for 12 weeks) on the hypothalamus in a high-fat diet (HFD)-induced animal model of MetS, utilizing quantitative RT-PCR and immunohistochemical analyses. The animal model recapitulates the human MetS features, including low testosterone/gonadotropin plasma levels. TTh significantly improved MetS-induced hypertension, visceral adipose tissue accumulation, and glucose homeostasis derangements. Within hypothalamus, TTh significantly counteracted HFD-induced inflammation, as detected in terms of expression of inflammatory markers and microglial activation. Moreover, TTh remarkably reverted the HFD-associated alterations in the expression of important regulators of energy status and reproduction, such as the melanocortin and the GnRH-controlling network. Our results suggest that TTh may exert neuroprotective effects on the HFD-related hypothalamic alterations, with positive outcomes on the circuits implicated in the control of energy metabolism and reproductive tasks, thus supporting a possible role of TTh in the clinical management of MetS.     

Synthesis and Evaluation of Voltage-Gated Sodium Channel Blocking Pyrroline Derivatives Endowed with Both Antiarrhythmic and Antioxidant Activities

Under the hypothesis that cardioprotective agents might benefit from synergism between antiarrhythmic activity and antioxidant properties, a small series of mexiletine analogues were coupled with the 2,2,5,5-tetramethylpyrroline moiety, known for its antioxidant effect, in order to obtain dual-acting drugs potentially useful in the protection of the heart against post-ischemic reperfusion injury. The pyrroline derivatives reported herein were found to be more potent as antiarrhythmic agents than mexiletine and displayed antioxidant activity. The most interesting tetramethylpyrroline congener, a tert-butyl-substituted analogue, was at least 100 times more active as an antiarrhythmic than mexiletine.     

High-Fat Diet Leads to Reduced Protein O-GlcNAcylation and Mitochondrial Defects Promoting the Development of Alzheimer’s Disease Signatures

The disturbance of protein O-GlcNAcylation is emerging as a possible link between altered brain metabolism and the progression of neurodegeneration. As observed in brains with Alzheimer’s disease (AD), flaws of the cerebral glucose uptake translate into reduced protein O-GlcNAcylation, which promote the formation of pathological hallmarks. A high-fat diet (HFD) is known to foster metabolic dysregulation and insulin resistance in the brain and such effects have been associated with the reduction of cognitive performances. Remarkably, a significant role in HFD-related cognitive decline might be played by aberrant protein O-GlcNAcylation by triggering the development of AD signature and mitochondrial impairment. Our data support the impairment of total protein O-GlcNAcylation profile both in the brain of mice subjected to a 6-week high-fat-diet (HFD) and in our in vitro transposition on SH-SY5Y cells. The reduction of protein O-GlcNAcylation was associated with the development of insulin resistance, induced by overfeeding (i.e., defective insulin signaling and reduced mitochondrial activity), which promoted the dysregulation of the hexosamine biosynthetic pathway (HBP) flux, through the AMPK-driven reduction of GFAT1 activation. Further, we observed that a HFD induced the selective impairment of O-GlcNAcylated-tau and of O-GlcNAcylated-Complex I subunit NDUFB8, thus resulting in tau toxicity and reduced respiratory chain functionality respectively, highlighting the involvement of this posttranslational modification in the neurodegenerative process.