Heart rate-associated mechanical stress impairs carotid but not cerebral artery compliance in dyslipidemic atherosclerotic mice

The cardiac cycle imposes a mechanical stress that dilates elastic carotid arteries, while shear stress largely contributes to the endothelium-dependent dilation of downstream cerebral arteries. In the presence of dyslipidemia, carotid arteries stiffen while the endothelial function declines. We reasoned that stiffening of carotid arteries would be prevented by reducing resting heart rate (HR), while improving the endothelial function would regulate cerebral artery compliance and function. Thus we treated or not 3-mo-old male atherosclerotic mice (ATX; LDLr(-/-):hApoB(+/+)) for 3 mo with the sinoatrial pacemaker current inhibitor ivabradine (IVA), the β-blocker metoprolol (METO), or subjected mice to voluntary physical training (PT). Arterial (carotid and cerebral artery) compliance and endothelium-dependent flow-mediated cerebral dilation were measured in isolated pressurized arteries. IVA and METO similarly reduced (P < 0.05) 24-h HR by ≈15%, while PT had no impact. As expected, carotid artery stiffness increased (P < 0.05) in ATX mice compared with wild-type mice, while cerebral artery stiffness decreased (P < 0.05); this paradoxical increase in cerebrovascular compliance was associated with endothelial dysfunction and an augmented metalloproteinase-9 (MMP-9) activity (P < 0.05), without changing the lipid composition of the wall. Reducing HR (IVA and METO) limited carotid artery stiffening, but plaque progression was prevented by IVA only. In contrast, IVA maintained and PT improved cerebral endothelial nitric oxide synthase-dependent flow-mediated dilation and wall compliance, and both interventions reduced MMP-9 activity (P < 0.05); METO worsened endothelial dysfunction and compliance and did not reduce MMP-9 activity. In conclusion, HR-dependent mechanical stress contributes to carotid artery wall stiffening in severely dyslipidemic mice while cerebrovascular compliance is mostly regulated by the endothelium.     

Discovery and Verification of Osteopontin and Beta-2-microglobulin as Promising Markers for Staging Human African Trypanosomiasis

Human African trypanosomiasis, or sleeping sickness, is a parasitic disease endemic in sub-Saharan Africa, transmitted to humans through the bite of a tsetse fly. The first or hemolymphatic stage of the disease is associated with presence of parasites in the bloodstream, lymphatic system, and body tissues. If patients are left untreated, parasites cross the blood-brain barrier and invade the cerebrospinal fluid and the brain parenchyma, giving rise to the second or meningoencephalitic stage. Stage determination is a crucial step in guiding the choice of treatment, as drugs used for S2 are potentially dangerous. Current staging methods, based on counting white blood cells and demonstrating trypanosomes in cerebrospinal fluid, lack specificity and/or sensitivity. In the present study, we used several proteomic strategies to discover new markers with potential for staging human African trypanosomiasis. Cerebrospinal fluid (CSF) samples were collected from patients infected with Trypanosoma brucei gambiense in the Democratic Republic of Congo. The stage was determined following the guidelines of the national control program. The proteome of the samples was analyzed by two-dimensional gel electrophoresis (n = 9), and by sixplex tandem mass tag (TMT) isobaric labeling (n = 6) quantitative mass spectrometry. Overall, 73 proteins were overexpressed in patients presenting the second stage of the disease. Two of these, osteopontin and β-2-microglobulin, were confirmed to be potential markers for staging human African trypanosomiasis (HAT) by Western blot and ELISA. The two proteins significantly discriminated between S1 and S2 patients with high sensitivity (68% and 78%, respectively) for 100% specificity, and a combination of both improved the sensitivity to 91%. The levels of osteopontin and β-2-microglobulin in CSF of S2 patients (μg/ml range), as well as the fold increased concentration in S2 compared with S1 (3.8 and 5.5 respectively) make the two markers good candidates for the development of a test for staging HAT patients.Human African trypanosomiasis (HAT), or sleeping sickness, is caused by an extracellular protozoan parasite of the genus Trypanosoma, which is transmitted through the bite of a tsetse fly (genus Glossina). Two morphologically identical subspecies of the parasite, are responsible for the two geographically and clinically different forms of HAT: a chronic form, widespread in West and Central Africa, caused by T. b. gambiense, and an acute form, endemic in eastern Africa, caused by T. b. rhodesiense (1). In both forms of the disease, parasites are initially localized in the blood stream, lymph, and peripheral tissues; this is the first or hemolymphatic stage (S1). During this stage, patients present generic clinical features that are common to other infectious diseases such as human immunodeficiency virus (HIV), malaria, and tuberculosis (TB), which can coexist with HAT, thus making its early diagnosis difficult (2). If treatment is not carried out, the disease progresses to the second or meningoencephalitic stage (S2) after trypanosomes cross the blood-brain barrier (BBB) and invade the central nervous system (CNS). This phase is characterized by a broad range of neurological signs that are indicative of CNS involvement (1). Diagnosis of HAT is based on parasitological demonstration of parasites in blood or lymph-node aspirate (3). All positive or suspect patients have to undergo a lumbar puncture and cerebrospinal fluid (CSF)1 examination, to determine whether they have second stage disease (4). According to the World Health Organization (WHO) guidelines, the meningoencephalitic stage is defined by the presence of parasites in CSF and/or a white blood cell (WBC) count of more than 5 cells per μl (5). Other parameters, such as intrathecal IgM production could also provide additional information to determine whether the CNS is involved (6, 7).Treatment of HAT patients varies depending on the infecting parasite and the stage of disease (5, 8). S2 drugs in current use, including melarsoprol, eflornithine, and a combination of nifurtimox and eflornithine have several limitations, such as a high rate of toxicity (melarsoprol causes death to 5% of treated patients) (9), complex logistics, and mode of administration (6, 10). Consequently, staging is a vital step in the diagnosis and treatment of HAT. However, the poor specificity or sensitivity of WBC counting and of parasitological techniques for demonstration of parasites in CSF, highlight the need for discovery of better tools for staging the disease.Several attempts have been made during the last decade to identify potential biomarkers able to discriminate between the two stages of sleeping sickness. Most of the efforts focused on cytokines and chemokines, because the patient''s immune system plays a crucial role in the brain pathology (11–14).Proteomic approaches are increasingly being applied in biomedical research and clinical medicine to investigate body fluids as a source of biomarkers (15), including the diagnosis of neurological disorders such as Alzheimer''s disease (16), Parkinson''s disease (17), and multiple sclerosis (18, 19). The protein composition of CSF is strictly regulated and can reflect the physiological or pathological state of the CNS (15). Thus in the present study, we addressed the challenge of staging HAT by analyzing CSF from T. b. gambiense patients using two complementary proteomic strategies: a classical approach based on two-dimensional gel electrophoresis (2-DE), and quantitative mass spectrometry (MS) using isobaric tandem mass tag (TMT) technology (sixplex TMT® MS/MS) (20).     

Establishment of an in vitro propagation protocol for <Emphasis Type="Italic">Thymus lotocephalus</Emphasis>, a rare aromatic species of the Algarve (Portugal)

The aim of this work was to develop an in vitro propagation protocol for the endangered species Thymus lotocephalus using seedlings as explants. Several macronutrient concentrations of Murashige and Skoog medium (MS), cytokinin types and concentrations, and cytokinin/auxin combinations were tested to assess the shoots’ proliferation capacity. Although the best proliferation results were obtained with 6-benzyladenine, high percentages of hyperhidric shoots were observed. Because high proliferation of healthy shoots was observed in MS medium that was free of plant growth regulators, this medium was chosen for proliferation studies. The best rooting results were achieved in ¼MS medium without auxins (92.00 ± 6.11%, 6.54 ± 0.52 and 1.60 ± 0.10 cm regarding rooting frequency, number of roots per shoot and longest roots, respectively) or supplemented with 0.5 mg l^?1 indole-3-acetic acid (98.00 ± 2.11%, 11.14 ± 0.75 and 2.40 ± 0.24 cm, respectively). Plantlets were successfully acclimatised to ex vitro conditions with a survival rate of 93.33%. With the development of this micropropagation protocol, an important contribution has been made to the conservation of the endangered species T. lotocephalus.     

Nitrogen fluxes from treefrogs to tank epiphytic bromeliads: an isotopic and physiological approach

Diverse invertebrate and vertebrate species live in association with plants of the large Neotropical family Bromeliaceae. Although previous studies have assumed that debris of associated organisms improves plant nutrition, so far little evidence supports this assumption. In this study we used isotopic (¹⁵N) and physiological methods to investigate if the treefrog Scinax hayii, which uses the tank epiphytic bromeliad Vriesea bituminosa as a diurnal shelter, contributes to host plant nutrition. In the field, bromeliads with frogs had higher stable N isotopic composition (δ¹⁵N) values than those without frogs. Similar results were obtained from a controlled greenhouse experiment. Linear mixing models showed that frog feces and dead termites used to simulate insects that eventually fall inside the bromeliad tank contributed, respectively, 27.7% (±0.07 SE) and 49.6% (±0.50 SE) of the total N of V. bituminosa. Net photosynthetic rate was higher in plants that received feces and termites than in controls; however, this effect was only detected in the rainy, but not in the dry season. These results demonstrate for the first time that vertebrates contribute to bromeliad nutrition, and that this benefit is seasonally restricted. Since amphibian–bromeliad associations occur in diverse habitats in South and Central America, this mechanism for deriving nutrients may be important in bromeliad systems throughout the Neotropics.     

Abundance and Distribution of Sperm Whales in the Canary Islands: Can Sperm Whales in the Archipelago Sustain the Current Level of Ship-Strike Mortalities?

Sperm whales are present in the Canary Islands year-round, suggesting that the archipelago is an important area for this species in the North Atlantic. However, the area experiences one of the highest reported rates of sperm whale ship-strike in the world. Here we investigate if the number of sperm whales found in the archipelago can sustain the current rate of ship-strike mortality. The results of this study may also have implications for offshore areas where concentrations of sperm whales may coincide with high densities of ship traffic, but where ship-strikes may be undocumented. The absolute abundance of sperm whales in an area of 52933 km², covering the territorial waters of the Canary Islands, was estimated from 2668 km of acoustic line-transect survey using Distance sampling analysis. Data on sperm whale diving and acoustic behaviour, obtained from bio-logging, were used to calculate g(0) = 0.92, this is less than one because of occasional extended periods when whales do not echolocate. This resulted in an absolute abundance estimate of 224 sperm whales (95% log-normal CI 120–418) within the survey area. The recruitment capability of this number of whales, some 2.5 whales per year, is likely to be exceeded by the current ship-strike mortality rate. Furthermore, we found areas of higher whale density within the archipelago, many coincident with those previously described, suggesting that these are important habitats for females and immature animals inhabiting the archipelago. Some of these areas are crossed by active shipping lanes increasing the risk of ship-strikes. Given the philopatry in female sperm whales, replacement of impacted whales might be limited. Therefore, the application of mitigation measures to reduce the ship-strike mortality rate seems essential for the conservation of sperm whales in the Canary Islands.     

Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice

Exercise is an effective approach for primary and secondary prevention of cardiovascular diseases (CVD) and loss of muscular mass and function. Its benefits are widely documented but incompletely characterized. It has been reported that exercise can induce changes in the expression of antioxidant enzymes including Sod2, Trx1, Prdx3 and Gpx1 and limits the rise in oxidative stress commonly associated with CVD. These enzymes can be subjected to epigenetic regulation, such as DNA methylation, in response to environmental cues. The aim of our study was to determine whether in the early stages of atherogenesis, in young severely dyslipidemic mice lacking LDL receptors and overexpressing human ApoB100 (LDLR^-/-; hApoB^+/+), exercise regulates differentially the expression of antioxidant enzymes by DNA methylation in the skeletal muscles that consume high levels of oxygen and thus generate high levels of reactive oxygen species. Expression of Sod2, Txr1, Prdx3 and Gpx1 was altered by 3 months of exercise and/or severe dyslipidemia in 6-mo dyslipidemic mice. Of these genes, only Gpx1 exhibited changes in DNA methylation associated with dyslipidemia and exercise: we observed both increased DNA methylation with dyslipidemia and a transient decrease in DNA methylation with exercise. These epigenetic alterations are found in the second exon of the Gpx1 gene and occur alongside with inverse changes in mRNA expression. Inhibition of expression by methylation of this specific locus was confirmed in vitro. In conclusion, Gpx1 expression in the mouse skeletal muscle can be altered by both exercise and dyslipidemia through changes in DNA methylation, leading to a fine regulation of free radical metabolism.