Three-dimensional simulation of clouds of multi-disperse evaporating saliva droplets in a train cabin

In line with recent ongoing efforts to collect crucial information about the mechanisms of virus diffusion and put them in relation to the effective complexity of the several natural or artificial environments where human beings leave and operate, the present study deals with the dispersion of evaporating saliva droplets in the cabin of an interregional train. A relevant physical model is constructed taking into account the state of the art in terms of existing paradigms and their ability to represent some fundamental aspects related to the evolution in time of a cloud of multi-disperse droplets. Conveniently, such a theoretical framework is turned into a computational one that relies on low Mach-number asymptotics and can therefore take advantage of the typical benefits (relatively low computational cost) associated with pressure-based methods. Numerical simulations are used to predict the flow established in the cabin as a result of the ventilation systems and related settings dictated by considerations on passenger comfort. The solution of two-way coupled Lagrangian evolution equations is used to capture the associated dynamics of the dispersed phase and predict its transport in conjunction with the peculiar topology of the considered flow and morphology of solid surfaces, which bound it (including the human beings). Typical physiological processes such as talking or coughing are considered. An analysis on the impact of the multiplicity of droplet sources is also conducted, thereby providing some indications in terms of potential risks for the cabin occupants

Functional divergence in the role of N-linked glycosylation in smoothened signaling

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice

ACE2-Mediated Reduction of Oxidative Stress in the Central Nervous System Is Associated with Improvement of Autonomic Function

Oxidative stress in the central nervous system mediates the increase in sympathetic tone that precedes the development of hypertension. We hypothesized that by transforming Angiotensin-II (AngII) into Ang-(1–7), ACE2 might reduce AngII-mediated oxidative stress in the brain and prevent autonomic dysfunction. To test this hypothesis, a relationship between ACE2 and oxidative stress was first confirmed in a mouse neuroblastoma cell line (Neuro2A cells) treated with AngII and infected with Ad-hACE2. ACE2 overexpression resulted in a reduction of reactive oxygen species (ROS) formation. In vivo, ACE2 knockout (ACE2−/y) mice and non-transgenic (NT) littermates were infused with AngII (10 days) and infected with Ad-hACE2 in the paraventricular nucleus (PVN). Baseline blood pressure (BP), AngII and brain ROS levels were not different between young mice (12 weeks). However, cardiac sympathetic tone, brain NADPH oxidase and SOD activities were significantly increased in ACE2−/y. Post infusion, plasma and brain AngII levels were also significantly higher in ACE2−/y, although BP was similarly increased in both genotypes. ROS formation in the PVN and RVLM was significantly higher in ACE2−/y mice following AngII infusion. Similar phenotypes, i.e. increased oxidative stress, exacerbated dysautonomia and hypertension, were also observed on baseline in mature ACE2−/y mice (48 weeks). ACE2 gene therapy to the PVN reduced AngII-mediated increase in NADPH oxidase activity and normalized cardiac dysautonomia in ACE2−/y mice. Altogether, these data indicate that ACE2 gene deletion promotes age-dependent oxidative stress, autonomic dysfunction and hypertension, while PVN-targeted ACE2 gene therapy decreases ROS formation via NADPH oxidase inhibition and improves autonomic function. Accordingly, ACE2 could represent a new target for the treatment of hypertension-associated dysautonomia and oxidative stress

Hedgehog partial agonism drives warburg-lie metabolism in muscle and brown fat

Diabetes, obesity, and cancer affect upward of 15% of the world&rsquo;s population. Interestingly, all three diseases juxtapose dysregulated intracellular signaling with altered metabolic state. Exactly which genetic factors define stable metabolic set points in vivo remains poorly understood. Here, we show that hedgehog signaling rewires cellular metabolism. We identify a cilium-dependent Smo-Ca2+-Ampk axis that triggers rapid Warburg-like metabolic reprogramming within minutes of activation and is required for proper metabolic selectivity and flexibility. We show that Smo modulators can uncouple the Smo-Ampk axis from canonical signaling and identify cyclopamine as one of a new class of &ldquo;selective partial agonists,&rdquo; capable of concomitant inhibition of canonical and activation of noncanonical hedgehog signaling. Intriguingly, activation of the Smo-Ampk axis in vivo drives robust insulin-independent glucose uptake in muscle and brown adipose tissue. These data identify multiple noncanonical endpoints that are pivotal for rational design of hedgehog modulators and provide a new therapeutic avenue for obesity and diabetes.<br /

Gi/o-protein coupled receptors in the aging brain

Cells translate extracellular signals to regulate processes such as differentiation, metabolism and proliferation, via transmembranar receptors. G protein-coupled receptors (GPCRs) belong to the largest family of transmembrane receptors, with over 800 members in the human species. Given the variety of key physiological functions regulated by GPCRs, these are main targets of existing drugs. During normal aging, alterations in the expression and activity of GPCRs have been observed. The central nervous system (CNS) is particularly affected by these alterations, which results in decreased brain functions, impaired neuroregeneration, and increased vulnerability to neuropathologies, such as Alzheimer's and Parkinson diseases. GPCRs signal via heterotrimeric G proteins, such as Go, the most abundant heterotrimeric G protein in CNS. We here review age-induced effects of GPCR signaling via the Gi/o subfamily at the CNS. During the aging process, a reduction in protein density is observed for almost half of the Gi/o-coupled GPCRs, particularly in age-vulnerable regions such as the frontal cortex, hippocampus, substantia nigra and striatum. Gi/o levels also tend to decrease with aging, particularly in regions such as the frontal cortex. Alterations in the expression and activity of GPCRs and coupled G proteins result from altered proteostasis, peroxidation of membranar lipids and age-associated neuronal degeneration and death, and have impact on aging hallmarks and age-related neuropathologies. Further, due to oligomerization of GPCRs at the membrane and their cooperative signaling, down-regulation of a specific Gi/o-coupled GPCR may affect signaling and drug targeting of other types/subtypes of GPCRs with which it dimerizes. Gi/o-coupled GPCRs receptorsomes are thus the focus of more effective therapeutic drugs aiming to prevent or revert the decline in brain functions and increased risk of neuropathologies at advanced ages.This work was supported by Fundação para a Ciência e Tecnologia, Centro 2020 and Portugal 2020, the COMPETE program, QREN, and the European Union (FEDER program) via the GoBack project (PTDC/CVT-CVT/32261/2017), the pAGE program (Centro-01-0145-FEDER-000003), and Institute for Biomedicine iBiMED (UID/BIM/04501/2013; UID/BIM/04501/2019).publishe

The heme-containing N-fragment (residues 1-56) of cytochrome c is a bis-histidine functional system

The structural and redox properties of a heme-containing fragment (1-56 residues) of cytochrome c have been investigated by spectroscopic (circular dichroism, electronic absorption, and EPR) and voltammetric techniques. The results indicate that the N-fragment lacks ordered secondary structure and has two histidines axially bound to the heme-iron (the native His18 and a misligated His26 or His33). Despite the absence of ordered secondary structure, the peptide chain shields the heme group from solvent, as shown by (i) the pK(a) of protonation of the nonnative histidine ligand (5.18 +/- 0.05), lower than that of the bis-histidine guanidine-unfolded cytochrome c (5.58 +/- 0.05), and (ii) the redox potential, E-o = 0 +/- 5 mV versus NHE, close to that of bis-histidine cytochrome c mutants but less negative than that of bis-histidine complexes of microperoxidase with short peptides. The electroactive N-fragment may be taken as a "minichrome c" model, with interesting potential for application to biosensor technology; further, the system provides useful information far a deeper understanding of cytochrome c folding and structural/functional organization. (C) 2000 Academic Press

Role of the dimeric structure in Cu,Zn superoxide dismutase - pH-dependent, reversible denaturation of the monomeric enzyme from Escherichia coli

To investigate the structural/functional role of the dimeric structure in Cu,Zn superoxide dismutases, we have studied the stability to a variety of agents of the Escherichia coli enzyme, the only monomeric variant of this class so far isolated, Differential scanning calorimetry of the native enzyme showed the presence of two well defined peaks identified as the metal free and holoprotein, Unlike dimeric Cu,Zn superoxide dismutases, the unfolding of the monomeric enzyme was found to be highly reversible, a behavior that may be explained by the absence of free cysteines and the highly polar nature of its molecular surface. The melting temperature of the E. coli enzyme was found to be pH-dependent with the holoenzyme transition centered at 66 degrees C at pH 7.8 and at 79.3 degrees C at pH 6.0, The active-site metals, which were easily displaced from the active site by EDTA, were found to enhance the thermal stability of the monomeric apoprotein but to a lower extent than in the dimeric enzymes from eukaryotic sources, Apo-superoxide dismutase from E. coli was shown to be nearly as stable as the bovine apoenzyme, whose hole form is much more stable and less sensitive to pH variations. The remarkable pH susceptibility of the E. coli enzyme structure was paralleled by the slow decrease in activity of the enzyme incubated at alkaline pH and by modification of the EPR spectrum at lower pH values than in the case of dimeric enzymes, Unlike eukaryotic Cu,Zn superoxide dismutases, the active-site structure of the E. coli enzyme was shown to be reversibly perturbed by urea, These observations suggest that the conformational stability of Cu,Zn superoxide dismutases is largely due to the intrinsic stability of the beta-barrel fold rather than to the dimeric structure and that pH sensitivity and weak metal binding of the E. coli enzyme are due to higher flexibility and accessibility to the solvent of its active-site region

The mitochondrial oxoglutarate carrier: Structural and dynamic properties of transmembrane segment IV studied by site-directed spin labeling

The structural and dynamic features of the fourth transmembrane segment of the mitochondrial oxoglutarate carrier were investigated using site-directed spin labeling and electron paramagnetic resonance (EPR). Using a functional carrier protein with native cysteines replaced with serines, the 18 consecutive residues from S184 to S201 which are believed to form the transmembrane segment IV were substituted individually with cysteine and labeled with a thiol-selective nitroxide reagent. Most of the labeled mutants exhibited significant oxoglutarate transport in reconstituted liposomes, where they were examined by EPR as a function of the incident microwave power in the presence and absence of two paramagnetic perturbants, i.e., the hydrophobic molecular oxygen or the hydrophilic chromium oxalate complex. The periodicity of the sequence-specific variation in the spin-label mobility and the O2 accessibility parameters unambiguously identifies the fourth transmembrane segment of the mitochondrial oxoglutarate carrier as an α-helix. The accessibility to chromium oxalate is out of phase with oxygen accessibility, indicating that the helix is amphipatic, with the hydrophilic face containing the residues found to be important for transport activity by site-directed mutagenesis and chemical modification. The helix is strongly packed, as indicated by the values of normalized mobility, which also suggest that the conformational changes occurring during transport probably involve the N-terminal region of the helix