Thyroxine (T4) is a drug extensively utilized for the treatment of hypothyroidism. However, the oral absorption of T4 presents certain limitations. This research investigates the efficacy of CO2 ...nanobubbles in water as a potential oral carrier for T4 administration to C57BL/6 hypothyroid mice. Following 18 h of fasting, the formulation was administered to the mice, demonstrating that the combination of CO2 nanobubbles and T4 enhanced the drug’s absorption in blood serum by approximately 40%. To comprehend this observation at a molecular level, we explored the interaction mechanism through which T4 engages with the CO2 nanobubbles, employing molecular simulations, semi-empirical quantum mechanics, and PMF calculations. Our simulations revealed a high affinity of T4 for the water–gas interface, driven by additive interactions between the hydrophobic region of T4 and the gas phase and electrostatic interactions of the polar groups of T4 with water at the water–gas interface. Concurrently, we observed that at the water–gas interface, the cluster of T4 formed in the water region disassembles, contributing to the drug’s bioavailability. Furthermore, we examined how the gas within the nanobubbles aids in facilitating the drug’s translocation through cell membranes. This research contributes to a deeper understanding of the role of CO2 nanobubbles in drug absorption and subsequent release into the bloodstream. The findings suggest that utilizing CO2 nanobubbles could enhance T4 bioavailability and cell permeability, leading to more efficient transport into cells. Additional research opens the possibility of employing lower concentrations of this class of drugs, thereby potentially reducing the associated side effects due to poor absorption.
The occurrence of intercellular channels formed by pannexin1 has been challenged for more than a decade. Here, we provide an electrophysiological characterization of exogenous human pannexin1 ...(hPanx1) cell–cell channels expressed in HeLa cells knocked out for connexin45. The observed hPanx1 cell–cell channels show two phenotypes: O-state and S-state. The former displayed low transjunctional voltage (Vj) sensitivity and single-channel conductance of ∼175 pS, with a substate of ∼35 pS; the latter showed a peculiar dynamic asymmetry in Vj dependence and single-channel conductance identical to the substate conductance of the O-state. S-state hPanx1 cell–cell channels were also identified between TC620 cells, a human oligodendroglioma cell line that endogenously expresses hPanx1. In these cells, dye and electrical coupling increased with temperature and were strongly reduced after hPanx1 expression was knocked down by small interfering RNA or inhibited with Panx1 mimetic inhibitory peptide. Moreover, cell–cell coupling was augmented when hPanx1 levels were increased with a doxycycline-inducible expression system. Application of octanol, a connexin gap junction (GJ) channel inhibitor, was not sufficient to block electrical coupling between HeLa KO Cx45-hPanx1 or TC620 cell pairs. In silico studies suggest that several arginine residues inside the channel pore may be neutralized by hydrophobic interactions, allowing the passage of DAPI, consistent with dye coupling observed between TC620 cells. These findings demonstrate that endogenously expressed hPanx1 forms intercellular cell–cell channels and their unique properties resemble those described in innexin-based GJ channels. Since Panx1 is ubiquitously expressed, finding conditions to recognize Panx1 cell–cell channels in different cell types might require special attention.
High order oligomers are crucial for normal cell physiology, and protein function perturbed by missense mutations underlies several autosomal dominant diseases. Dynamin-2 is one of such protein ...forming helical oligomers that catalyze membrane fission. Mutations in this protein, where R465W is the most frequent, cause dominant centronuclear myopathy, but the molecular mechanisms underpinning the functional modifications remain to be investigated. To unveil the structural impact of this mutation in dynamin-2, we used full-atom molecular dynamics simulations and coarse-grained models and built dimers and helices of wild-type (WT) monomers, mutant monomers, or both WT and mutant monomers combined. Our results show that the mutation R465W causes changes in the interactions with neighbor amino acids that propagate through the oligomer. These new interactions perturb the contact between monomers and favor an extended conformation of the bundle signaling element (BSE), a dynamin region that transmits the conformational changes from the GTPase domain to the rest of the protein. This extended configuration of the BSE that is only relevant in the helices illustrates how a small change in the microenvironment surrounding a single residue can propagate through the oligomer structures of dynamin explaining how dominance emerges in large protein complexes.
Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated ...with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca
/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation-measured by changes in DAPI uptake rate-was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (∼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca
signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.
Endoxylanases belonging to family 10 of the glycoside hydrolases (GH10) are versatile in the use of different substrates. Thus, an understanding of the molecular mechanisms underlying substrate ...specificities could be very useful in the engineering of GH10 endoxylanases for biotechnological purposes. Herein, we analyzed XynA, an endoxylanase that contains a (β/α)
-barrel domain and an intrinsically disordered region (IDR) of 29 amino acids at its amino end. Enzyme activity assays revealed that the elimination of the IDR resulted in a mutant enzyme (XynAΔ29) in which two new activities emerged: the ability to release xylose from xylan, and the ability to hydrolyze
-nitrophenyl-β-d-xylopyranoside (pNPXyl), a substrate that wild-type enzyme cannot hydrolyze. Circular dichroism and tryptophan fluorescence quenching by acrylamide showed changes in secondary structure and increased flexibility of XynAΔ29. Molecular dynamics simulations revealed that the emergence of the pNPXyl-hydrolyzing activity correlated with a dynamic behavior not previously observed in GH10 endoxylanases: a hinge-bending motion of two symmetric regions within the (β/α)
-barrel domain, whose hinge point is the active cleft. The hinge-bending motion is more intense in XynAΔ29 than in XynA and promotes the formation of a wider active site that allows the accommodation and hydrolysis of pNPXyl. Our results open new avenues for the study of the relationship between IDRs, dynamics and activity of endoxylanases, and other enzymes containing (β/α)
-barrel domain.
Obesity is a common chronic comorbidity of patients with COVID-19, that has been associated with disease severity and mortality. COVID-19 at high altitude seems to be associated with increased rate ...of ICU discharge and hospital survival than at sea-level, despite higher immune levels and inflammation. The primary aim of this study was to investigate the survival rate of critically ill obese patients with COVID-19 at altitude in comparison with overweight and normal patients. Secondary aims were to assess the predictive factors for mortality, characteristics of mechanical ventilation setting, extubation rates, and analytical parameters.
This is a retrospective cohort study in critically ill patients with COVID-19 admitted to a hospital in Quito-Ecuador (2,850 m) from Apr 1, 2020, to Nov 1, 2021. Patients were cathegorized as normal weight, overweight, and obese, according to body mass index BMI).
In the final analysis 340 patients were included, of whom 154 (45%) were obese, of these 35 (22.7%) were hypertensive and 25 (16.2%) were diabetic. Mortality in obese patients (31%) was lower than in the normal weight (48%) and overweight (40%) groups, but not statistically significant (p = 0.076). At multivariable analysis, in the overall population, older age (> 50 years) was independent risk factor for mortality (B = 0.93, Wald = 14.94, OR = 2.54 95%CI = 1.58-4.07, p < 0.001). Ferritin and the neutrophil/lymphocyte ratio were independent predictors of mortality in obese patients. Overweight and obese patients required more positive and-expiratory pressure compared to normal-weight patients. In obese patients, plateau pressure and mechanical power were significantly higher, whereas extubation failure was lower as compared to overweight and normal weight.
This preliminary study suggests that BMI was not associated with mortality in critically ill patients at high altitude. Age was associated with an increase in mortality independent of the BMI. Biomarkers such as ferritin and neutrophils/lymphocytes ratio were independent predictors of mortality in obese patients with COVID-19 at high altitude.
Palaeoseismological studies play a crucial role in the seismic characterization of regions with slow-moving faults. This is the case in the central Betic Cordillera, a highly populated area for which ...the record of prehistoric earthquakes is very limited, despite being one of the regions with the greatest seismic hazard in Spain.
The present work reports the changes for the mesoporous materials SBA-15 and KIT-6 associated with the structural, textural, and chemical properties when they are subjected to thermo-alkaline ...treatment. Despite the fact that the silica supports have not a strong affinity for CO
2
adsorption, the adsorption enthalpy profiles (ΔH
ads
) reported that the substrates subjected to the thermo-alkaline treatment (S15H and K6H) have a greater energetic affinity towards CO
2
capture if compared to the precursory solids (S15 and K6). The ΔH
ads
is − 26.7 kJ mol
−1
at 0.15 mmol g
−1
by supported S15H and K6H while the ΔH
ads
is − 20. 7 kJ mol
−1
and − 18.7 kJ mol
−1
by K6 and S15, respectively, at the same CO
2
coverage. Furthermore, the CO
2
adsorption performances by the hydrolytic condensation between silica supports and the N´- (3-trimethoxysilylpropyl)diethylenetriamine (NAEPTES) or 3-aminopropiltriethoxysilane (APTES) are presented and it can be seen that the best performer for CO
2
adsorption is reported for the S15HN since it is able to absorb 0.93 mmol at 0.15 atm at 318 K. Thereby, the outcomes show that the effects of porous curvature and the magnitude of the amine species are parameters to be considered, as well as the thermo-alkaline treatment, in order to improve the subsequent surface reactions on silica supports. The materials were characterized by XRD, TEM, and N
2
adsorption at 77 K, NIR, and pyridine thermodesorption using Fourier Transform Infrared Spectroscopy (FTIR-Py), NMR for
29
Si and
13
C, DSC, and CO
2
adsorption.
Graphical abstract
This paper analyses the growth of the adsorbed layer during the course of
N
2
adsorption verified in two types of elliptical pores, oblates and prolates, by means of the Broekhoff de Boer theory. The ...information provided by the value of the thickness of the adsorbed layer permits to characterize the geometrical features of the liquid–vapour interface at every stage of the isotherm, prior and at the onset of capillary condensation. The features of the liquid–vapour interface that are characteristic of the instability at the onset of capillary condensation were identified. Additionally, the critical ellipsoidal pore sizes were calculated, thus identifying the limit of stability for the formation of the liquid–vapour interface during
N
2
adsorption.
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