High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated,Low-Density Plasma Membrane Fragments

Principal Findings

HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C³⁵¹I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025–0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [³²P]GTPase and [³⁵S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound G_i1α within δ-opioid receptor-G_i1α fusion protein, but it was also valid for PTX-sensitive G proteins of G_i/G_o family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the “wobble in cone” model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye.

Summary

Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.     

Mother-to-Child HIV-1 Transmission Events Are Differentially Impacted by Breast Milk and Its Components from HIV-1-Infected Women

Breast milk is a vehicle of infection and source of protection in post-natal mother-to-child HIV-1 transmission (MTCT). Understanding the mechanism by which breast milk limits vertical transmission will provide critical insight into the design of preventive and therapeutic approaches to interrupt HIV-1 mucosal transmission. However, characterization of the inhibitory activity of breast milk in human intestinal mucosa, the portal of entry in postnatal MTCT, has been constrained by the limited availability of primary mucosal target cells and tissues to recapitulate mucosal transmission ex vivo. Here, we characterized the impact of skimmed breast milk, breast milk antibodies (Igs) and non-Ig components from HIV-1-infected Ugandan women on the major events of HIV-1 mucosal transmission using primary human intestinal cells and tissues. HIV-1-specific IgG antibodies and non-Ig components in breast milk inhibited the uptake of Ugandan HIV-1 isolates by primary human intestinal epithelial cells, viral replication in and transport of HIV-1- bearing dendritic cells through the human intestinal mucosa. Breast milk HIV-1-specific IgG and IgA, as well as innate factors, blocked the uptake and transport of HIV-1 through intestinal mucosa. Thus, breast milk components have distinct and complementary effects in reducing HIV-1 uptake, transport through and replication in the intestinal mucosa and, therefore, likely contribute to preventing postnatal HIV-1 transmission. Our data suggests that a successful preventive or therapeutic approach would require multiple immune factors acting at multiple steps in the HIV-1 mucosal transmission process.     

Health-Related Quality of Life in Relation to Obesity Grade,Type 2 Diabetes,Metabolic Syndrome and Inflammation

Background

Health-related quality of life (HR-QoL) may be compromised in obese individuals, depending on the presence of other complications. The aim of this study is to assess the effect of obesity-related conditions on HR-QoL. These conditions are i) grade of obesity with and without type 2 diabetes (T2D), ii) metabolic syndrome (MetS), and iii) level of inflammation.

Methods

From the Dutch LifeLines Cohort Study we included 13,686 obese individuals, aged 18–80 years. HR-QoL was measured with the RAND 36-Item Health Survey which encompasses eight health domains. We calculated the percentage of obese individuals with poor HR-QoL, i.e. those scoring below the domain and sex specific cut-off value derived from the normal weight population. Logistic regression analysis was used to calculate the probability of having poor domain scores according to the conditions under study.

Results

Higher grades of obesity and the additional presence of T2D were associated with lower HR-QoL, particularly in the domains physical functioning (men: odds ratios (ORs) 1.48–11.34, P<0.005, and women: ORs 1.66–5.05, P<0.001) and general health (men: ORs 1.44–3.07, P<0.005, and women: ORs 1.36–3.73, P<0.001). A higher percentage of obese individuals with MetS had a poor HR-QoL than those without MetS. Furthermore, we observed a linear trend between inflammation and the percentage of obese individuals with poor scores on the HR-QoL domains. Individuals with MetS were more likely to have poor scores in the domains general health, vitality, social functioning and role limitations due to emotional problems. Obese women with increased inflammation levels were more likely to have poor scores on all domains except role limitations due to emotional problems and mental health.

Conclusions

The impact of obesity on an individual’s quality of life is enhanced by grade of obesity, T2D, MetS and inflammation and are mainly related to reduced physical health. The mental well-being is less often impaired.     

Prediction of Intra-Species Protein-Protein Interactions in Enteropathogens Facilitating Systems Biology Study

Protein-protein interactions in Escherichia coli (E. coli) has been studied extensively using high throughput methods such as tandem affinity purification followed by mass spectrometry and yeast two-hybrid method. This can in turn be used to understand the mechanisms of bacterial cellular processes. However, experimental characterization of such huge amount of interactions data is not available for other important enteropathogens. Here, we propose a support vector machine (SVM)-based prediction model using the known PPIs data of E. coli that can be used to predict PPIs in other enteropathogens, such as Vibrio cholerae, Salmonella Typhi, Shigella flexneri and Yersinia entrocolitica. Different features such as domain-domain association (DDA), network topology, and sequence information were used in developing the SVM model. The proposed model using DDA, degree and amino acid composition features has achieved an accuracy of 82% and 62% on 5-fold cross validation and blind E. coli datasets, respectively. The predicted interactions were validated by Gene Ontology (GO) semantic similarity measure and String PPIs database (experimental PPIs only). Finally, we have developed a user-friendly webserver named EnPPIpred to predict intra-species PPIs in enteropathogens, which will be of great help for the experimental biologists. The webserver EnPPIpred is freely available at http://bicresources.jcbose.ac.in/ssaha4/EnPPIpred/.     

Long-Term Effects of Interprofessional Biopsychosocial Rehabilitation for Adults with Chronic Non-Specific Low Back Pain: A Multicentre,Quasi-Experimental Study

Background

Improvement of the long-term effectiveness of multidisciplinary ortho-paedic rehabilitation (MOR) in the management of chronic non-specific low back pain (CLBP) remains a central issue for health care in Germany. We developed an interprofessional and interdisciplinary, biopsychosocial rehabilitation concept named “PASTOR” to promote self-management in adults with CLBP and compared its effectiveness with the current model of MOR.

Methods

A multicentre quasi-experimental study with three measurement time points was implemented. 680 adults aged 18 to 65 with CLBP were assed for eligibil-ity in three inpatient rehabilitation centres in Germany. At first the effects of the MOR, with a total extent of 48 hours (control group), were assessed. Thereafter, PASTOR was implemented and evaluated in the same centres (intervention group). It consisted of six interprofessional modules, which were provided on 12 days in fixed groups, with a total extent of 48 hours. Participants were assessed with self-report measures at baseline, discharge, and 12 months for functional ability (primary outcome) using the Hannover Functional Ability Questionnaire (FFbH-R) and vari-ous secondary outcomes (e.g. pain, health status, physical activity, pain coping, pain-related cognitions).

Results

In total 536 participants were consecutively assigned to PASTOR (n=266) or MOR (n=270). At 12 months, complete data of 368 participants was available. The adjusted between-group difference in the FFbH-R at 12 months was 6.58 (95% CI 3.38 to 9.78) using complete data and 3.56 (95% CI 0.45 to 6.67) using available da-ta, corresponding to significant small-to-medium effect sizes of d=0.42 (p<0.001) and d=0.10 (p=0.025) in favour of PASTOR. Further improvements in secondary out-comes were also observed in favour of PASTOR.

Conclusion

The interprofessional and interdisciplinary, biopsychosocial rehabilita-tion program PASTOR shows some improvements of the long-term effectiveness of inpatient rehabilitation in the management of adults with CLBP. Further insights into mechanisms of action of complex intervention programs are required.

Trial Registration

ClinicalTrials.gov NCT02056951     

Ecological differentiation,lack of hybrids involving diploids,and asymmetric gene flow between polyploids in narrow contact zones of Senecio carniolicus (syn. Jacobaea carniolica,Asteraceae)

Areas of immediate contact of different cytotypes offer a unique opportunity to study evolutionary dynamics within heteroploid species and to assess isolation mechanisms governing coexistence of cytotypes of different ploidy. The degree of reproductive isolation of cytotypes, that is, the frequency of heteroploid crosses and subsequent formation of viable and (partly) fertile hybrids, plays a crucial role for the long‐term integrity of lineages in contact zones. Here, we assessed fine‐scale distribution, spatial clustering, and ecological niches as well as patterns of gene flow in parental and hybrid cytotypes in zones of immediate contact of di‐, tetra‐, and hexaploid Senecio carniolicus (Asteraceae) in the Eastern Alps. Cytotypes were spatially separated also at the investigated microscale; the strongest spatial separation was observed for the fully interfertile tetra‐ and hexaploids. The three main cytotypes showed highly significant niche differences, which were, however, weaker than across their entire distribution ranges in the Eastern Alps. Individuals with intermediate ploidy levels were found neither in the diploid/tetraploid nor in the diploid/hexaploid contact zones indicating strong reproductive barriers. In contrast, pentaploid individuals were frequent in the tetraploid/hexaploid contact zone, albeit limited to a narrow strip in the immediate contact zone of their parental cytotypes. AFLP fingerprinting data revealed introgressive gene flow mediated by pentaploid hybrids from tetra‐ to hexaploid individuals, but not vice versa. The ecological niche of pentaploids differed significantly from that of tetraploids but not from hexaploids.     

Two widespread green Neottia species (Orchidaceae) show mycorrhizal preference for Sebacinales in various habitats and ontogenetic stages

Plant dependence on fungal carbon (mycoheterotrophy) evolved repeatedly. In orchids, it is connected with a mycorrhizal shift from rhizoctonia to ectomycorrhizal fungi and a high natural ¹³C and ¹⁵N abundance. Some green relatives of mycoheterotrophic species show identical trends, but most of these remain unstudied, blurring our understanding of evolution to mycoheterotrophy. We analysed mycorrhizal associations and ¹³C and ¹⁵N biomass content in two green species, Neottia ovata and N. cordata (tribe Neottieae), from a genus comprising green and nongreen (mycoheterotrophic) species. Our study covered 41 European sites, including different meadow and forest habitats and orchid developmental stages. Fungal ITS barcoding and electron microscopy showed that both Neottia species associated mainly with nonectomycorrhizal Sebacinales Clade B, a group of rhizoctonia symbionts of green orchids, regardless of the habitat or growth stage. Few additional rhizoctonias from Ceratobasidiaceae and Tulasnellaceae, and ectomycorrhizal fungi were detected. Isotope abundances did not detect carbon gain from the ectomycorrhizal fungi, suggesting a usual nutrition of rhizoctonia‐associated green orchids. Considering associations of related partially or fully mycoheterotrophic species such as Neottia camtschatea or N. nidus‐avis with ectomycorrhizal Sebacinales Clade A, we propose that the genus Neottia displays a mycorrhizal preference for Sebacinales and that the association with nonectomycorrhizal Sebacinales Clade B is likely ancestral. Such a change in preference for mycorrhizal associates differing in ecology within the same fungal taxon is rare among orchids. Moreover, the existence of rhizoctonia‐associated Neottia spp. challenges the shift to ectomycorrhizal fungi as an ancestral pre‐adaptation to mycoheterotrophy in the whole Neottieae.