Increased Grey Matter Associated with Long-Term Sahaja Yoga Meditation: A Voxel-Based Morphometry Study

Objectives

To investigate regional differences in grey matter volume associated with the practice of Sahaja Yoga Meditation.

Design

Twenty three experienced practitioners of Sahaja Yoga Meditation and twenty three non-meditators matched on age, gender and education level, were scanned using structural Magnetic Resonance Imaging and their grey matter volume were compared using Voxel-Based Morphometry.

Results

Grey matter volume was larger in meditators relative to non-meditators across the whole brain. In addition, grey matter volume was larger in several predominantly right hemispheric regions: in insula, ventromedial orbitofrontal cortex, inferior temporal and parietal cortices as well as in left ventrolateral prefrontal cortex and left insula. No areas with larger grey matter volume were found in non-meditators relative to meditators.

Conclusions

The study shows that long-term practice of Sahaja Yoga Meditation is associated with larger grey matter volume overall, and with regional enlargement in several right hemispheric cortical and subcortical brain regions that are associated with sustained attention, self-control, compassion and interoceptive perception. The increased grey matter volume in these attention and self-control mediating regions suggests use-dependent enlargement with regular practice of this meditation.     

Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter

We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state.The amino acid/polyamine/organocation (APC)² superfamily comprises about 250 members that occur in all phyla from prokaryotes to higher eukaryotes. These membrane proteins function as solute/cation symporters or solute/solute antiporters (1). One APC subfamily is established by l-amino acid transporters (LATs), which correspond to the light subunits of eukaryotic heteromeric amino acid transporters (2, 3). Heteromeric amino acid transporters are composed of a light subunit that provides transport activity and a disulfide-linked heavy subunit that shows responsibility for plasma membrane targeting. Genetic defects in light and heavy subunits cause a number of inherited human diseases. Mutations in the light as well as the heavy subunit of system b^0,+ lead to cystinuria (4, 5), whereas mutations in the light subunit y⁺LAT1 cause lysinuric protein intolerance (6, 7). Another light subunit, xCT that mediates cysteine uptake and glutamate efflux (8, 9), is involved in vivo in cocaine relapse (10) and maintenance of the plasma redox balance (11). LAT1, the light subunit of system L, is overexpressed in certain primary human tumors. It transports essential neutral amino acids with long, branched, or aromatic side chains required by tumor cells to support their unabated growth (12). Therefore, amino acid transporters like LAT1 are attractive anticancer drug targets.So far a high resolution structure of a eukaryotic LAT family member is not available. However, studies on xCT revealed a membrane topology of 12 transmembrane helices (TMHs) with cytosolic N and C termini and a re-entrant loop structure between TMHs II and III (13). The identified first prokaryotic member of the LAT family, SteT from Bacillus subtilis, is a serine/threonine antiporter, which shows high sequence identity (∼30%) to the light subunits of eukaryotic heteromeric amino acid transporters. Moreover SteT exhibits a similar putative membrane topology and sequential mode of obligate exchange (14). Thus, SteT is an excellent model for studying the structure-function relationship of LAT family members.According to current models, transport proteins undergo functionally related conformational changes. Transporters alternate between two conformations to expose their binding sites to the cytoplasmic and extracellular side (15–22). However, prior to conformational changes substrates have to be recognized and bound. If substrates are amino acids, three main features can be used for specific selection and binding: (i) the negatively charged α-carboxyl group, (ii) the positively charged α-amino group, and (iii) the electrostatic, hydrophobic, or spatial properties of the side chain (22–24). α-Carboxyl and α- amino groups of l-amino acids possess similar structural and chemical characteristics (except for proline); however, their side chains differ in shape, size, and electrostatic properties. Combinations of these features are assumed to establish different interactions within the side chain binding pocket, which determines the substrate specificity of the transporter. The two main substrates of SteT, l-serine and l-threonine, differ by only one methylene group in their side chain; thus they have similar properties. Additionally SteT transports aromatic l-amino acids (Trp, Tyr, and Phe) albeit less efficiently (14).Since its invention, the atomic force microscope (AFM) (25) has evolved from a surface imaging device to a versatile tool for studying interactions of manifold biological systems (26–31). Introduced to characterize interactions between receptor-ligand complexes (32, 33) and complementary DNA strands (34), AFM-based single molecule force spectroscopy (SMFS) has been exploited to explore antibody-antigen recognition (35) and unfolding and refolding of soluble proteins (29, 36) and to probe the adhesion of living cells at molecular resolution (37). Applied to membrane proteins, SMFS uses the AFM stylus to exert a mechanical pulling force to the terminal end of a protein that is embedded and anchored by the lipid membrane (see Fig. 1A) (38). Sufficiently high stretching forces initiate sequential unfolding of the membrane protein with each step indicating the unfolding of a structural segment (39). Recording the applied force over pulling distance results in a force-distance (F-D) curve in which individual force peaks represent the rupture of intra- and intermolecular interactions. The height of a force peak measures the strength of an interaction with piconewton accuracy, and the pulling distance, at which the force peak occurs, allows the interaction within the membrane protein structure to be located (38).Open in a separate windowFIGURE 1.SMFS of SteT. A, pushing the AFM stylus onto the proteoliposomes promotes contacting single transporters to the stylus. This molecular link allows exertion of a mechanical pulling force that initiates stepwise unfolding of SteT. During the experiments, sample and cantilever are immersed in buffer solution. B, F-D curves recorded while unfolding single substrate-free SteT molecules. C, superimpositions of F-D curves recorded while unfolding SteT in buffer lacking any substrate (top) and supplemented with 5 mm l-serine (middle) or 5 mm l-threonine (bottom). Superimpositions are represented as density plots, each calculated from 60 F-D curves. Gray lines represent WLC curves with a persistence length of 0.4 nm and contour length (in amino acids) as indicated by the numbers next to the lines. The contour lengths were obtained from the Gaussian fits shown in D. F-D curves were obtained at room temperature at a pulling velocity of 654 nm/s in buffer solution (150 mm NaCl, 20 mm Tris-HCl, pH 8.0, substrate as indicated). D, frequency of force peaks detected at different positions of the stretched polypeptide. Every force peak detected in individual F-D curves (B) was fitted using the WLC model with the contour length of the stretched polypeptide as the only fitting parameter. The frequency at which the force peaks appeared is plotted in the histogram: substrate-free, n = 132; 5 mm l-serine, n = 128; and 5 mm l-threonine, n = 127. The bin size of the histograms is 3 aa and reflects the accuracy of fitting the WLC model (55) to individual force peaks. Error bars representing the S.E. were calculated using S.E. = (p(1 − p)/n)^0.5 where p is the probability and n is the total number of F-D curves. The width of each force peak distribution is given by the experimental noise, conformational variability of the structural segments, and fitting accuracy of the force peaks (53, 99–102). The gray solid curve represents the sum of seven Gaussian fits to the seven main peaks from the histograms and superimpositions (C). Numbers next to peaks denote peak positions (measured in amino acids) obtained from Gaussian fits.Besides quantification and localization of molecular interactions in membrane proteins, SMFS provides information about their energy landscape. For that purpose, the interactions of membrane proteins are probed over a range of different time scales by dynamic force spectroscopy (DFS). Bell (40) and Evans and co-worker (41, 42) provided the most commonly used theoretical framework to analyze DFS data. Their model describes the deformation of the energy landscape by an externally applied force, F. Such force-induced deformations reduce the energy barriers that separate bound and unbound states (see Fig. 2). Consequently transition rates over such energy barriers are force-dependent. Probing the interactions at different pulling velocities and thus at different force loading rates, r_f, leads to a so-called dynamic force spectrum in which the most probable force, F*, of rupture is plotted versus the logarithm of r_f. In these dynamic force spectra, each linear regime represents an energy barrier. Energy barriers located closer to the bound state are probed at higher pulling velocities because the energy barriers located further from the bound state are suppressed by increasingly applied forces (see Fig. 2) (41). The slope of each linear regime measures the distance from the ground state to the transition state, whereas extrapolation of a linear regime to zero force provides the rate constant of crossing the corresponding barrier in the absence of any load. These two parameters allow an estimate of the rigidity of the probed structure (43, 44).Open in a separate windowFIGURE 2.Energy landscape tilted by force. Schematic representation of the free energy profile along the reaction coordinate and applied force according to the Bell-Evans theory (40–42). The potential along the reaction coordinate (vector of force) in the absence of force (black curve) exhibits two energy barriers separating the folded from the unfolded state. Application of an external force, F, changes the thermal likelihood of reaching the top of the energy barrier(s). Although for a sharp barrier the position, x_u, of the energy barrier relative to the folded state is not changed, the thermally averaged projection of the energy profile along the pulling direction is tilted by the mechanical energy (−F·cos θ)x (long-dashed line). This tilt decreases the energy barriers (short-dashed curve). Consequently the relevant energy barrier that has to be overcome is the outermost barrier. At slow pulling velocities, the thermal contribution is higher, and therefore, the mechanical energy required to overcome the barrier is smaller. With increasing pulling velocities, the barriers are further lowered. At some velocity, the height of the outer barrier will be lower than that of the inner barrier (short-dashed curve), which then becomes the relevant energy barrier to be overcome. Each energy barrier manifests as a linear regime in dynamic force spectra (Fig. 3).In this study, we applied SMFS to characterize molecular interactions that stabilize SteT in the absence and in the presence of its substrates, l-serine and l-threonine. We used DFS to characterize how substrate binding changes the energy landscape and the mechanical properties of the antiporter. It was observed that the structural regions stabilized within SteT did not depend on substrate binding. However, substrate binding dynamically changed the energy landscape of these structures. In the absence of substrate all structural regions within SteT were stabilized by a narrow inner energy barrier and co-stabilized by a second outer energy barrier. The unique properties of these energy barriers restricted the conformation of SteT thereby trapping the antiporter in a kinetically instable and mechanically rigid conformation. In contrast, substrate binding sets SteT into a different energy minimum that significantly increased the kinetic stability and conformational flexibility of the antiporter.     

Projection Structure of DtpD (YbgH), a Prokaryotic Member of the Peptide Transporter Family

Cellular uptake of di- and tripeptides has been characterized in numerous organisms, and various transporters have been identified. In contrast, structural information on peptide transporters is very sparse. Here, we have cloned, overexpressed, purified, and biochemically characterized DtpD (YbgH) from Escherichia coli, a prokaryotic member of the peptide transporter family. Its homologues in mammals, PEPT1 (SLC15A1) and PEPT2 (SLC15A2), not only transport peptides but also are of relevance for uptake of drugs as they accept a large spectrum of peptidomimetics such as β-lactam antibiotics, antivirals, peptidase inhibitors, and others as substrates. Uptake experiments indicated that DtpD functions as a canonical peptide transporter and is, therefore, a valid model for structural studies of this family of proteins. Blue native polyacrylamide gel electrophoresis, gel filtration, and transmission electron microscopy of single-DtpD particles suggest that the transporter exists in a monomeric form when solubilized in detergent. Two-dimensional crystallization of DtpD yielded first tubular crystals that allowed the determination of a projection structure at better than 19 Å resolution. This structure of DtpD represents the first structural view of a member of the peptide transporter family.     

Optimization of purification and refolding of the human chemokine receptor CXCR1 improves the stability of proteoliposomes for structure determination

The human chemokine receptor CXCR1 is a G-protein coupled receptor that has been successfully expressed in E. coli as inclusion bodies, and purified and refolded in multi-milligram quantities required for structural studies. Expression in E. coli enables selective and uniform isotopic labeling with (13)C and (15)N for NMR studies. Long-term chemical and conformational stability and oligomeric homogeneity of CXCR1 in phospholipid bilayers are crucial for structural studies under physiological conditions. Here we describe substantial refinements in our previously described purification and reconstitution procedures for CXCR1 in phospholipid bilayers. These refinements have led to the preparation of highly purified, completely monomeric, proteoliposome samples that are stable for months at 35°C while subject to the high power radiofrequency irradiations of solid-state NMR experiments. The principal changes from the previously described methods include: 1) ensure that CXCR1 is pure and homogeneously monomeric within the limits of detection (>98%); 2) monitor and control the pH at all times especially following the addition of TCEP, which serves as a reducing agent but also changes the pH; 3) slowly refold CXCR1 with the complete removal of all traces of SDS using a KCl precipitation/dialysis method; and 4) ensure that the molar ratio between the CXCR1 and the phospholipids does not change during refolding and detergent removal. NMR samples prepared with these protocols yield reproducible results over a period of many months at 35°C. This purification and refolding protocol is likely to be applicable with minimal changes to other GPCRs as well as other membrane proteins.     

Recovery of soil phosphorus on former bauxite mines through tropical forest restoration

Soil phosphorus (P) is a major driver of forest development and a critically limited nutrient in tropical soils, especially when topsoil is removed by mining. This nutrient can be present in soils in the form of different fractions, which have direct consequences for P availability to plants and, consequently, for restoration success. Therefore, understanding how the stocks of different soil P fractions change over the restoration process can be essential for guiding restoration interventions, monitoring, and adaptive management. Here, we investigated the recovery of soil P fractions by forest restoration interventions on bauxite mine sites in the Brazilian Atlantic Forest. We assessed the concentration of different fractions of soil organic and inorganic P at (1) a bauxite mine prepared for restoration; (2) two former bauxite mines undergoing forest restoration for 6 and 24 years; and (3) an old‐growth forest remnant. Overall, restored areas recovered levels of labile organic P (P_o‐NaHCO₃) at 5–40 cm and of moderately labile organic P (P_o‐NaOH) at different depths, exhibiting concentrations similar to those found in a conserved forest. The use of P‐rich fertilizers and forest topsoil may have greatly contributed to this outcome. Some other fractions, however, recovered only after 24 years of restoration. Other inorganic P fractions did not differ among mined, restored, and conserved sites: nonlabile P_i (residual P and P‐HCl), labile P_i (P_i‐NaHCO₃), and moderately labile P_i (P_i‐NaOH). Forest restoration was able to promote efficient recovery of important soil P fractions, highlighting the value of restoration efforts to mitigate soil degradation by mining.     

Identification of potential hosts plants of Cowpea aphid‐borne mosaic virus

Among the major pathogens affecting passion fruit orchards, the cowpea aphid‐borne mosaic virus (CABMV), also known as the fruit‐hardening virus, has gained prominence owing to its role in the drastic reduction in fruit production in yellow passion fruit orchards (Passiflora edulis f. flavicarpa) from the second year of cultivation. To mitigate the damage, several regions adopt the annual planting system where a sanitary void is maintained from August to September. However, the virus is believed to remain dormant in weeds. This study aimed to identify potential weed hosts of CABMV. The study was conducted with a randomized design with four replications in Londrina, PR. Twenty‐eight weed species were tested, and a sample of yellow passion fruit leaves symptomatic for CABMV infection was used as the virus inoculum source. Mechanical inoculation was performed using the extract from the symptomatic plant. Symptoms were visually evaluated every 3 days. For molecular confirmation, total RNA was extracted, followed by RT‐PCR with CABMV‐specific oligonucleotides, reinoculation in passion fruit plants and sequencing. CABMV infection was observed in southern sandbur (Cenchrus echinatus), Siberian motherwort (Leonurus sibiricus), showy rattlepod (Crotalaria spectabilis) and yellow passion fruit (Passiflora edulis). The CABMV‐positive weed species extract was able to infect yellow passion fruit plant when a fresh mechanical inoculation was performed. Showy rattlepod (Crotalaria spectabilis) was the only weed species to exhibit observable symptoms of CABMV. C. echinatus, L. sibiricus and C. spectabilis act as a source of CABMV inoculum.     

Continuous fill intermittent decant type sequencing batch reactor application to upgrade the UASB treated sewage

The performance of continuous flow intermittent decant type sequencing batch (CFID) reactor treating the effluent of an UASB reactor treating domestic wastewater and operated at 8 h hydraulic retention time (HRT) was investigated. The CFID was operated at three different HRTs (22, 8 and 6 h) and three different dissolved oxygen (DO) patterns (<0.5, 2.5–3.5 and 3.5–4.5 mg/L). The highest effluent quality was observed at the 8 h HRT and 2.5–3.5 mg/L DO concentration. At this operational condition, the average BOD, TSS, ammonia nitrogen and fecal coliform removal efficiencies were 83, 90, 74 and 99 %, respectively. The CFID is a promising post-treatment option for existing UASB systems, with a final effluent quality that comply with receiving water and effluent reuse criteria.     

MiR-216a: a link between endothelial dysfunction and autophagy

Endothelial dysfunction and impaired autophagic activity have a crucial role in aging-related diseases such as cardiovascular dysfunction and atherosclerosis. We have identified miR-216a as a microRNA that is induced during endothelial aging and, according to the computational analysis, among its targets includes two autophagy-related genes, Beclin1 (BECN1) and ATG5. Therefore, we have evaluated the role of miR-216a as a molecular component involved in the loss of autophagic function during endothelial aging. The inverse correlation between miR-216a and autophagic genes was conserved during human umbilical vein endothelial cells (HUVECs) aging and in vivo models of human atherosclerosis and heart failure. Luciferase experiments indicated BECN1, but not ATG5 as a direct target of miR-216a. HUVECs were transfected in order to modulate miR-216a expression and stimulated with 100 μg/ml oxidized low-density lipoprotein (ox-LDL) to induce a stress repairing autophagic process. We found that in young HUVECs, miR-216a overexpression repressed BECN1 and ATG5 expression and the ox-LDL induced autophagy, as evaluated by microtubule-associated protein 1 light chain 3 (LC3B) analysis and cytofluorimetric assay. Moreover, miR-216a stimulated ox-LDL accumulation and monocyte adhesion in HUVECs. Conversely, inhibition of miR-216a in old HUVECs rescued the ability to induce a protective autophagy in response to ox-LDL stimulus. In conclusion, mir-216a controls ox-LDL induced autophagy in HUVECs by regulating intracellular levels of BECN1 and may have a relevant role in the pathogenesis of cardiovascular disorders and atherosclerosis.     

Variation of a carotenoid-based trait in relation to oxidative stress and endocrine status during the breeding season in the Eurasian kestrel: a multi-factorial study

Carotenoid-based skin colorations vary seasonally in many bird species and are thought to be honest sexually selected signals. In order to provide more insight in the potential signal function and underlying mechanisms of such colorations we here quantified patterns of variation of leg coloration in adult male and female Eurasian kestrels (Falco tinnunculus tinnunculus) over the breeding season, and evaluated the relationship between coloration and levels of carotenoids, androgens and estrogens, oxidative damage and plasma non-enzymatic antioxidant capacity. We studied both reproducing wild and non-reproducing captive birds to test for the effect of diet and breeding effort. Males were more colored than females only during mating, and independently of diet, suggesting that leg-color is a sexually selected trait. Seasonal variation in leg color was associated with circulating carotenoids, but concentrations of these molecules were not related to antioxidant capacity, body condition or oxidative damage. These results indicate that carotenoid-based colorations may not be an honest signal of health status in this species. Production of carotenoid rich eggs coincided with low levels of circulating carotenoids in females, indicating that carotenoids might be a limited resource for laying female kestrels. Finally, young rearing males had higher levels of oxidative damage than females, and wild birds of both sexes had higher levels of these parameters than captive birds. These results may indicate that parental effort and physical activity are costly, independently from hormonal status. Since androgens did not explain carotenoid variation we suggest that multiple interacting factors can regulate carotenoid levels along the season.