Consequences of the exposome to gestational diabetes mellitus.

The exposome is the cumulative measure of environmental influences and associated biological responses throughout the lifespan, including those from the environment, diet, behaviour, and endogenous processes. The exposome concept and the 2030 Agenda for the Sustainable Development Goals (SDGs) from the United Nations are the basis for understanding the aetiology and consequences of non-communicable diseases, including gestational diabetes mellitus (GDM). Pregnancy may be developed in an environment with adverse factors part of the immediate internal medium for fetus development and the external medium to which the pregnant woman is exposed. The placenta is the interface between maternal and fetal compartments and acts as a protective barrier or easing agent to transfer exposome from mother to fetus. Under and over-nutrition in utero, exposure to adverse environmental pollutants such as heavy metals, endocrine-disrupting chemicals, pesticides, drugs, pharmaceuticals, lifestyle, air pollutants, and tobacco smoke plays a determinant role in the development of GDM. This phenomenon is worsened by metabolic stress postnatally, such as obesity which increases the risk of GDM and other diseases. Clinical risk factors for GDM development include its aetiology. It is proposed that knowledge-based interventions to change the potential interdependent ecto-exposome and endo-exposome could avoid the occurrence and consequences of GDM.

Viability of ex-vivo myography as a diagnostic tool for rectus abdominis muscle electrical activity collected at Cesarean section within a diamater cohort study.

BACKGROUND: Ex-vivo myography enables the assessment of muscle electrical activity response. This study explored the viability of determining the physiological responses in muscles without tendon, as rectus abdominis muscle (RAM), through ex-vivo myography to assess its potential as a diagnostic tool. RESULTS: All tested RAM samples (five different samples) show patterns of electrical activity. A positive response was observed in 100% of the programmed stimulation. RAM 3 showed greater weight (0.47 g), length (1.66 cm), and width (0.77 cm) compared to RAM 1, RAM 2, RAM 4 and RAM 5 with more sustained electrical activity over time, a higher percentage of fatigue was analyzed at half the time of the electrical activity. The order of electrical activity (Mn) was RAM 3 > RAM 5 > RAM 1 > RAM 4 > RAM 2. No electrical activity was recorded in the Sham group. CONCLUSIONS: This study shows that it is feasible to assess the physiological responses of striated muscle without tendon as RAM, obtained at C-section, under ex vivo myography. These results could be recorded, properly analyzed, and demonstrated its potential as a diagnostic tool for rectus abdominis muscle electrical activity.

Reversal of diabetic-induced myopathy by swimming exercise in pregnant rats: a translational intervention study.

Gestational diabetes mellitus (GDM) plus rectus abdominis muscle (RAM) myopathy predicts long-term urinary incontinence (UI). Atrophic and stiff RAM are characteristics of diabetes-induced myopathy (DiM) in pregnant rats. This study aimed to determine whether swimming exercise (SE) has a therapeutic effect in mild hyperglycemic pregnant rats model. We hypothesized that SE training might help to reverse RAM DiM. Mild hyperglycemic pregnant rats model was obtained by a unique subcutaneous injection of 100 mg/kg streptozotocin (diabetic group) or citrate buffer (non-diabetic group) on the first day of life in Wistar female newborns. At 90 days of life, the rats are mated and randomly allocated to remain sedentary or subjected to a SE protocol. The SE protocol started at gestational day 0 and consisted of 60 min/day for 6 days/week in a period of 20 days in a swim tunnel. On day 21, rats were sacrificed, and RAM was collected and studied by picrosirius red, immunohistochemistry, and transmission electron microscopy. The SE protocol increased the fiber area and diameter, and the slow-twitch and fast-twitch fiber area and diameter in the diabetic exercised group, a finding was also seen in control sedentary animals. There was a decreased type I collagen but not type III collagen area and showed a similar type I/type III ratio compared with the control sedentary group. In conclusion, SE during pregnancy reversed the RAM DiM in pregnant rats. These findings may be a potential protocol to consider in patients with RAM damage caused by GDM.

Purified myelin lipids display a critical mixing point at low surface pressure.

Lipids extracted from Purified Myelin Membranes (LPMM) were spread as monomolecular films at the air/aqueous interface. The films were visualized by Brewster Angle Microscopy (BAM) at different lateral pressures (π) and ionic environments. Coexistence of Liquid-Expanded (LE) and cholesterol-enriched (CE) rounded domains persisted up to π ≈ 5 mN/m but the monolayers became homogeneous at higher surface pressures. Before mixing, the domains distorted to non-rounded domains. We experimentally measured the line tension (λ) for the lipid monolayers at the domain borders by a shape relaxation technique using non-homogeneous electric fields. Regardless of the subphase conditions, the obtained line tensions are of the order of pN and tended to decrease as lateral pressure increased toward the mixing point. From the mean square displacement of nested trapped domains, we also calculated the dipole density difference between phases (µ). A non-linear drop was detected in this parameter as the mixing point is approached. Here we quantitively evaluated the π-dependance of both parameters with proper power laws in the vicinity of the critical mixing surface pressure, and the exponents showed to be consistent with a critical phenomenon in the two-dimensional Ising universality class. This idea of bidimensionality was found to be compatible only for simplified lipidic systems, while for whole myelin monolayers, that means including proteins, no critical mixing point was detected. Finally, the line tension values were related with the thickness differences between phases (Δt) near the critical point.

Periodic bilayer organization in the complexes of Beta-2 Glycoprotein I with anionic lipid membranes.

ß2 glycoprotein I (ß2GPI) is a soluble protein that participates in blood coagulation, clearance of apoptotic bodies and generation of antigens in antiphospholipid syndrome among many other functions. We studied the aggregates formed by ß2GPI with the anionic phospholipids palmitoyloleoylphosphatidyl glycerol, dimyristoylphosphatidyl glycerol, dipalmitoylphosphatidyl glycerol and cardiolipin using small angle X-ray scattering. The complexes obtained in a medium containing 0.01 M NaCl showed Bragg peaks up to the sixth order in a well-defined integer sequence indicating the presence of a lamellar stacking with a periodicity of 17.8 nm and with largely reduced membrane fluctuations. Modeling the complex signal allowed us to conclude that the coherence length was only two bilayers and that about 15% of the total surface was actually stacked. The space between bilayers allows accommodating an extended ß2GPI molecule making a bridge between the interacting bilayers. The interactions between membranes mediated by ß2GPI was favored when the membranes were in the liquid crystalline state.

Ionic environment, thickness and line tension as determinants of phase separation in whole Purified Myelin Membranes monolayers.

Purified myelin membranes (PMM) were spread as monomolecular films at the air/aqueous solution interface, and visualized by Brewster Angle Microscopy (BAM) at different lateral pressures (π) on three specific aqueous solutions: absence of salts, physiological conditions and presence of calcium. Coexistence of Liquid-Expanded (LE) and Liquid Ordered (LO) phases persisted up to collapse in the presence of salts, whereas monolayers became homogeneous at π ≥ 35-40 mN/m when salts are absent. This PMM phase-mixing behavior in monolayers is similar to the previously reported behavior of PMM multilamellar vesicles. Reflectivities (Rp) of p-polarized light from both phases were assessed throughout the whole π -range, and film thicknesses (t) were calculated from the Rp values and measured film refractive indices (n). The LO phase was found to be more reflective and thicker than the LE phase at π ≤ 15 mN/m, but less reflective and thinner at higher π. We also determined the line tension (λ) of PMM monolayers at the domain boundaries from the rate of domain shape relaxation, which turned out to be of the order of picoNewtons (pN) and decreased as π increased. A correlation between λ and thickness differences (Δt) was found, suggesting that Δt is a molecular determinant for λ in PMM monolayers. Both λ and Δt were found to increase markedly when calcium was present in the subphase. This result corroborates the concept of divalent cations as a stabilizing factor for phase separation, in line with earlier studies on this mixture forming multilamellar membrane arrangements.

Unraveling the etiology of myelin disorders: the P2 case in Charcot-Marie-Tooth disease.

There are several examples of single mutations that lead to a well-defined disease through a well-known mechanism. In other cases, a collection of mutations of the same protein produces a pathology with different degrees of severity. The accompanying work by Uusitalo et al. studies several mutants of the fatty acid binding protein P2 of the peripheral nervous system myelin. They conserve the native tertiary structure but a remarkable difference in the capacity to interact with lipids. This could be a clue to unravel the complex way in which these mutations affect myelin structure and function in a variant of Charcot-Marie-Tooth disease. Comment on: https://doi.org/10.1111/febs.16079.

Unmodified Clay Nanosheets at the Air-Water Interface.

Quasi-two-dimensional (2D) nanolayers, such as graphene oxide or clay layers, adhere to gas-liquid or liquid-liquid interfaces. Particularly, clays are of wide general interest in this context because of their extensive and crucial use as Pickering emulsion stabilizers, as well as for their ability to provide colloidosome capsules. So far, clays could only be localized at oil-water or air-saline-water interfaces in aggregated states, while our results now show that clay nanosheets without any modification can be located at air-deionized-water interfaces. The clay mineral used in the present work is synthetic fluorohectorite with a very high aspect ratio and superior quality in homogeneity and charge distribution compared to other clay minerals. This clay mineral is more suitable for achieving unmodified clay anchoring to fluid interfaces compared to other clay minerals used in previous works. In this context, we studied clay nanosheet organization at the air-water interface by combining different experimental methods: Langmuir-Blodgett trough studies, scanning electron microscopy (SEM) studies of film deposits, grazing-incidence X-ray off-specular scattering (GIXOS), and Brewster angle microscopy (BAM). Clay films formed at the air-water interface could be transferred to solid substrates by the Langmuir-Schaefer method. The BAM results indicate a dynamic equilibrium between clay sheets on the interface and in the subphase. Because of this dynamic equilibrium, the Langmuir monolayer surface pressure does not change significantly when pure clay sheets are spread on the liquid surface. However, also, GIXOS results confirm that there are clay nanosheets at the air-water interface. In addition, we find that clay sheets modified by a branched polymer are much more likely to be confined to the interface.

Phase Diagram of Purified CNS Myelin Reveals Continuous Transformation between Expanded and Compacted Lamellar States.

Purified myelin membranes (PMMs) are the starting material for biochemical studies, from individual components up to the isolation of detergent-resistant membrane (DRM) fractions or detergent-insoluble glycosphingolipid (DIG) fractions, which are commonly believed to resemble physiological lipid rafts. The normal DIG isolation protocol involves the extraction of lipids under moderate cooling. The isolation of PMMs also involves the cooling of myelin as well as exposure to low ionic strength (IS). Here, we addressed the combined influence of cooling and IS on the structure of PMMs. The phase behaviour was investigated by small angle X-ray diffraction. Analysis of the diffraction peaks revealed the lamellar periodicity ( d ), the number of periodically correlated bilayers ( N ), and the relatives fractions of each phase. Departure from physiological conditions induced a phase separation in myelin. The effect of monovalent and divalent ions was also compared at equivalent IS, showing a differential effect, and phase diagrams for both ion types were established-Ca2+ induced the well-known over-compacted phase, but additionally we also found an expanded phase at low IS. Na+ promoted phase separation, and also induced over-compaction at sufficiently high IS. Finally, exploring the whole phase diagram, we found evidence for the direct isothermal transformation from the expanded to the compacted phase, suggesting that both phases could in fact originate from the identical primary lateral phase separation, whereas the apparent difference lies in the inter-bilayer interaction that is modulated by the ionic milieu.

Bacteriocin enterocin CRL35 is a modular peptide that induces non-bilayer states in bacterial model membranes.

The mechanism of action of the anti-Listeria peptide enterocin CRL35 was studied with biophysical tools by using lipid mixtures that mimicked Gram-positive plasma membranes. Langmuir monolayers and infrared spectroscopy indicated that the peptide readily interacted with phospholipid assembled in monolayers and bilayers to produce a dual effect, depending on the acyl chains. Indeed, short chain mixtures were disordered by enterocin CRL35, but the gel-phases of membranes composed by longer acyl chains were clearly stabilized by the bacteriocin. Structural and functional studies indicated that non-bilayer states were formed when liposomes were co-incubated with enterocin CRL35, whereas significant permeabilization could be detected when bilayer and non-bilayer states co-existed. Results can be explained by a two-step model in which the N-terminal of the peptide firstly docks enterocin CRL35 on the lipid surface by means of electrostatic interactions; then, C-terminal triggers membrane perturbation by insertion of hydrophobic α-helix.

Hopanoids Like Sterols Form Compact but Fluid Films.

Hopanoids are pentacyclic molecules present in membranes from some bacteria, recently proposed as sterol surrogates in these organisms. Diplopterol is an abundant hopanoid that, similar to sterols, does not self-aggregate in lamellar structures when pure, but forms monolayers at the air-water interface. Here, we analyze the interfacial behavior of pure diplopterol and compare it with sterols from different organisms: cholesterol from mammals, ergosterol from fungi, and stigmasterol from plants. We prepared Langmuir monolayers of the compounds and studied their surface properties using different experimental approaches and molecular dynamics simulations. Our results indicate that the films formed by diplopterol, despite being compact with low mean molecular areas, high surface potentials, and high refractive index, depict shear viscosity values similar to that for fluid films. Altogether, our results reveal that hopanoids have similar interfacial behavior than that of sterols, and thus they may have the capacity of modulating bacterial membrane properties in a similar way sterols do in eukaryotes.

Correction: Cooling induces phase separation in membranes derived from isolated CNS myelin.

[This corrects the article DOI: 10.1371/journal.pone.0184881.].

Cooling induces phase separation in membranes derived from isolated CNS myelin.

Purified myelin membranes (PMMs) are the starting material for biochemical analyses such as the isolation of detergent-insoluble glycosphingolipid-rich domains (DIGs), which are believed to be representatives of functional lipid rafts. The normal DIGs isolation protocol involves the extraction of lipids under moderate cooling. Here, we thus address the influence of cooling on the structure of PMMs and its sub-fractions. Thermodynamic and structural aspects of periodic, multilamellar PMMs are examined between 4°C and 45°C and in various biologically relevant aqueous solutions. The phase behavior is investigated by small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC). Complementary neutron diffraction (ND) experiments with solid-supported myelin multilayers confirm that the phase behavior is unaffected by planar confinement. SAXS and ND consistently show that multilamellar PMMs in pure water become heterogeneous when cooled by more than 10-15°C below physiological temperature, as during the DIGs isolation procedure. The heterogeneous state of PMMs is stabilized in physiological solution, where phase coexistence persists up to near the physiological temperature. This result supports the general view that membranes under physiological conditions are close to critical points for phase separation. In presence of elevated Ca2+ concentrations (> 10 mM), phase coexistence is found even far above physiological temperatures. The relative fractions of the two phases, and thus presumably also their compositions, are found to vary with temperature. Depending on the conditions, an "expanded" phase with larger lamellar period or a "compacted" phase with smaller lamellar period coexists with the native phase. Both expanded and compacted periods are also observed in DIGs under the respective conditions. The observed subtle temperature-dependence of the phase behavior of PMMs suggests that the composition of DIGs is sensitive to the details of the isolation protocol.

Refractive index and thickness determination in Langmuir monolayers of myelin lipids.

Langmuir monolayers at the air/water interface are widely used as biomembrane models and for amphiphilic molecules studies in general. Under controlled intermolecular organization (lateral molecular area), surface pressure, surface potential, reflectivity (R) and other magnitudes can be precisely determined on these planar monomolecular films. However, some physical parameters such as the refractive index of the monolayer (n) still remain elusive. The refractive index is very relevant because (in combination with R) it allows for the determination of the thickness of the film. The uncertainties of n determine important errors that propagate non-linearly into the calculation of monolayers thickness. Here we present an analytical method for the determination of n in monolayers based on refractive index matching. By using a Brewster angle microscopy (BAM) setup and monolayers spread over subphases with variable refractive index (n2), a minimum in R is search as a function of n2. In these conditions, n equals n2. The results shown correspond to monolayers of myelin lipids. The n values remain constant at 1.46 upon compression and equals the obtained value for myelin lipid bilayers in suspension. The values for n and R allow for the determination of thickness. We establish comparisons between these thicknesses for the monolayer and those obtained from two X-ray scattering techniques: 1) GIXOS for monolayers at the air/water interface and 2) SAXS for bilayers in bulk suspension. This allows us to conclude that the thickness that we measure by BAM includes the apolar and polar headgroup regions of the monolayer.

The Single Transmembrane Segment of Minimal Sensor DesK Senses Temperature via a Membrane-Thickness Caliper.

Thermosensors detect temperature changes and trigger cellular responses crucial for survival at different temperatures. The thermosensor DesK is a transmembrane (TM) histidine kinase which detects a decrease in temperature through its TM segments (TMS). Here, we address a key issue: how a physical stimulus such as temperature can be converted into a cellular response. We show that the thickness of Bacillus lipid membranes varies with temperature and that such variations can be detected by DesK with great precision. On the basis of genetic studies and measurements of in vitro activity of a DesK construct with a single TMS (minimal sensor DesK [MS-DesK]), reconstituted in liposomes, we propose an interplay mechanism directed by a conserved dyad, phenylalanine 8-lysine 10. This dyad is critical to anchor the only transmembrane segment of the MS-DesK construct to the extracellular water-lipid interphase and is required for the transmembrane segment of MS-DesK to function as a caliper for precise measurement of membrane thickness. The data suggest that positively charged lysine 10, which is located in the hydrophobic core of the membrane but is close to the water-lipid interface, pulls the transmembrane region toward the water phase to localize its charge at the interface. Nevertheless, the hydrophobic residue phenylalanine 8, located at the N-terminal extreme of the TMS, has a strong tendency to remain in the lipid phase, impairing access of lysine 10 to the water phase. The outcome of this interplay is a fine-tuned sensitivity to membrane thickness that elicits conformational changes that favor different signaling states of the protein. IMPORTANCE: The ability to sense and respond to extracellular signals is essential for cell survival. One example is the cellular response to temperature variation. How do cells "sense" temperature changes? It has been proposed that the bacterial thermosensor DesK acts as a molecular caliper measuring membrane thickness variations that would occur as a consequence of temperature changes and activates a pathway to restore membrane fluidity at low temperature. Here, we demonstrated that membrane thickness variations do occur at physiological temperatures by directly measuring Bacillus lipid membrane thickness. We also dissected the N-terminal sensing motif of MS-DesK at the molecular-biophysical level and found that the dyad phenylalanine-lysine at the water-lipid phase is critical for achievement of a fine-tuned sensitivity to temperature.

CNS myelin structural modification induced in vitro by phospholipases A2.

Myelin is the self-stacked membrane surrounding axons; it is also the target of several pathological and/or neurodegenerative processes like multiple sclerosis. These processes involve, among others, the hydrolytic attack by phospholipases. In this work we describe the changes in isolated myelin structure after treatment with several secreted PLA2 (sPLA2), by using small angle x-ray scattering (SAXS) measurements. It was observed that myelin treated with all the tested sPLA2s (from cobra and bee venoms and from pig pancreas) preserved the lamellar structure but displayed an enlarged separation between membranes in certain zones. Additionally, the peak due to membrane asymmetry was clearly enhanced. The coherence length was also lower than the non-treated myelin, indicating increased disorder. These SAXS results were complemented by Langmuir film experiments to follow myelin monolayer hydrolysis at the air/water interface by a decrease in electric surface potential at different surface pressures. All enzymes produced hydrolysis with no major qualitative difference between the isoforms tested.

Characterization of a Pt mirror to be used to deflect synchrotron radiation beam onto Langmuir monolayers.

A homemade mirror for X-rays has been built to prepare a diffraction beamline for liquid surface diffraction and scattering measurements. This simple approach is in operation at the XRD2 bending-magnet beamline at the Brazilian Synchrotron Light Laboratory.

Perception of local inhabitants regarding the socioeconomic impact of tourism focused on provisioning wild dolphins in Novo Airão, Central Amazon, Brazil.

Botos (Inia geoffrensis) are currently provisioned for use in tourist attractions in five sites in the Brazilian Amazon. Despite the known negative effects associated with human-wild dolphin interactions, this activity has been regulated and licensed in the Anavilhanas National Park in Novo Airão, Amazonas State, Brazil. We present an updated evaluation of the perception of the local community concerning the possible socioeconomic impacts of this tourism in Novo Airão. In April 2011, 45 interviews were conducted with inhabitants. A small segment of Novo Airão perceives currently itself as being economically dependent on the botos feeding tourism. Despite that, the economic benefits of this controversial activity apparently are not shared among most inhabitants, and botos feeding tourism is perceived as generating diverse negative effects. We conclude that if the activity was banned or modified into a less impacting tourist activity, this action would probably not majorly affect the lives of the general population.

2-Bromopalmitate reduces protein deacylation by inhibition of acyl-protein thioesterase enzymatic activities.

S-acylation, the covalent attachment of palmitate and other fatty acids on cysteine residues, is a reversible post-translational modification that exerts diverse effects on protein functions. S-acylation is catalyzed by protein acyltransferases (PAT), while deacylation requires acyl-protein thioesterases (APT), with numerous inhibitors for these enzymes having already been developed and characterized. Among these inhibitors, the palmitate analog 2-brompalmitate (2-BP) is the most commonly used to inhibit palmitoylation in cells. Nevertheless, previous results from our laboratory have suggested that 2-BP could affect protein deacylation. Here, we further investigated in vivo and in vitro the effect of 2-BP on the acylation/deacylation protein machinery, with it being observed that 2-BP, in addition to inhibiting PAT activity in vivo, also perturbed the acylation cycle of GAP-43 at the level of depalmitoylation and consequently affected its kinetics of membrane association. Furthermore, 2-BP was able to inhibit in vitro the enzymatic activities of human APT1 and APT2, the only two thioesterases shown to mediate protein deacylation, through an uncompetitive mechanism of action. In fact, APT1 and APT2 hydrolyzed both the monomeric form as well as the micellar state of the substrate palmitoyl-CoA. On the basis of the obtained results, as APTs can mediate deacylation on membrane bound and unbound substrates, this suggests that the access of APTs to the membrane interface is not a necessary requisite for deacylation. Moreover, as the enzymatic activity of APTs was inhibited by 2-BP treatment, then the kinetics analysis of protein acylation using 2-BP should be carefully interpreted, as this drug also inhibits protein deacylation.

Equivalent aqueous phase modulation of domain segregation in myelin monolayers and bilayer vesicles.

Purified myelin can be spread as monomolecular films at the air/aqueous interface. These films were visualized by fluorescence and Brewster angle microscopy, showing phase coexistence at low and medium surface pressures (<20-30 mN/m). Beyond this threshold, the film becomes homogeneous or not, depending on the aqueous subphase composition. Pure water as well as sucrose, glycerol, dimethylsulfoxide, and dimethylformamide solutions (20% in water) produced monolayers that become homogeneous at high surface pressures; on the other hand, the presence of salts (NaCl, CaCl(2)) in Ringer's and physiological solution leads to phase domain microheterogeneity over the whole compression isotherm. These results show that surface heterogeneity is favored by the ionic milieu. The modulation of the phase-mixing behavior in monolayers is paralleled by the behavior of multilamellar vesicles as determined by small-angle and wide-angle x-ray scattering. The correspondence of the behavior of monolayers and multilayers is achieved only at high surface pressures near the equilibrium adsorption surface pressure; at lower surface pressures, the correspondence breaks down. The equilibrium surface tension on all subphases corresponds to that of the air/alkane interface (27 mN/m), independently on the surface tension of the clean subphase.