Characterizing and predicting the functional and conformational diversity of seven-transmembrane proteins

The activation of seven-transmembrane receptors (7TMRs) allows cells to sense their environment and convert extracellular signals (like hormone binding) into intracellular signals (through G protein-coupled and/or β arrestin-coupled pathways). A single 7TMR is capable of transducing a wide spectrum of physiological responses inside a cell by coupling to these pathways. This intracellular pleiotropic action is enabled by multiple conformations exhibited by these receptors. Developments in membrane protein structure determination technologies have led to a rapid increase in crystal structures for many 7TMRs. Majority of these receptors have been crystallized in their inactive conformation and, for some, one of the many active conformations has also been crystallized. Given the topological constraints of a lipid bilayer that results in a single fold of seven almost parallel TM helices connected by mostly unstructured loops, these structures exhibit a diversity of conformations not only across the receptors but also across the different functional forms for receptors with structures for one of the functionally active conformations. Here we present a method to characterize this conformational diversity in terms of transmembrane helix topology (TMHTOP) parameters and how to use these helix orientation parameters to predict functionally-distinct multiple conformations for these receptors. The TMHTOP parameters enable a quantification of the structural changes that underlie 7TMR activation and also sheds a unique mechanistic light on the pleiotropic nature of these receptors. It provides a common language to describe the 7TMR activation mechanisms as well as differences across many receptors in terms of visually intuitive structural parameters. Protein structure prediction methods can use these parameters to describe 7TMR conformational ensembles, which coupled to experimental data can be used to develop testable hypotheses for the structural basis of 7TMR functions

Does implementation matter if comprehension is lacking? A qualitative investigation into perceptions of advance care planning in people with cancer

Purpose: While advance care planning holds promise, uptake is variable and it is unclear how well people engage with or comprehend advance care planning. The objective of this study was to explore how people with cancer comprehended Advance Care Plans and examine how accurately advance care planning documentation represented patient wishes. Methods: This study used a qualitative descriptive design. Data collection comprised interviews and an examination of participants’ existing advance care planning documentation. Participants included those who had any diagnosis of cancer with an advance care plan recorded: Refusal of Treatment Certificate; Statement of Choices; and/or Enduring Power of Attorney (Medical Treatment) at one cancer treatment centre. Results: Fourteen participants were involved in the study. Twelve participants were female (86%). The mean age was 77 (range: 61-91) and participants had completed their advance care planning documentation between 8 and 72 weeks prior to the interview (mean 33 weeks). Three themes were evident from the data: Incomplete advance care planning understanding and confidence; Limited congruence for attitude and documentation; Advance care planning can enable peace of mind. Complete advance care planning understanding was unusual; most participants demonstrated partial comprehension of their own advance care plan, and some indicated very limited understanding. Participants’ attitudes and their written document congruence was limited, but advance care planning was seen as helpful. Conclusions: This study highlighted advance care planning was not a completely accurate representation of patient wishes. There is opportunity to improve how patients comprehend their own advance care planning documentation

Comparison of polymerase chain reaction and bacterial culture for Salmonella detection in the Muscovy duck in Trinidad and Tobago

Objectives: The purpose of this study was to investigate the presence and serovar identity of Salmonella, at the national level, in farmed Muscovy ducks (Cairina moschata) in Trinidad and Tobago, and to compare the relative benefits of bacterial culture to those of polymerase chain reaction (PCR) for use in the routine detection and surveillance of Salmonella in these ducks. Methods: From March-September 2003, 110 fecal samples were collected from 82 farms across the islands of Trinidad and Tobago. Salmonella was isolated from fresh and frozen samples and the serotype of each was determined through bacterial culture. An in-house, nested PCR that detects all pathogenic Salmonella species was utilized in analyzing the samples. Results: Five samples were positive for Salmonella by bacterial culture, whereas 44 were positive by the nested PCR. Serovars isolated were Kiambu, Orion, Uganda, and two isolates from Group E1 whose H antigens could not be fully characterized. Of the samples, 87 (79%) gave equivalent PCR results for both enrichment broths-28 were positive for both and 59 were negative for both). However, 16 samples were positive for one broth, but not for the other, with the majority (14 of the 16) resulting positive for Selenite broth. PCR results for seven samples were inconclusive due to ambiguous band size or multiple bands near the expected band size. Conclusions: In Trinidad and Tobago, the Muscovy duck does not appear to be a significant source of S. typhimurium or S. enteritidis, but it does harbor other Salmonella species. In-house, nested PCR represents a simple, relatively inexpensive and potentially more sensitive method than bacterial culture for the routine surveillance of pathogenic Salmonella in the Muscovy duck

Genetic Parameters and Effect of WUR Genotype on Piglet Response to Co-Infection with PRRS and PCV2b, with or without Vaccination for PRRS

Commercial crossbred nursery piglets were either vaccinated or not using a modified live porcine reproductive and respiratory syndrome (PRRS) virus vaccine and all pigs were co-infected with PRRS virus (PRRSV) and porcine circovirus type 2b (PCV2b) 28 days later. Genetic correlations indicate that traits associated with primary exposure to PRRSV infection (PRRS viral load (VL) of vaccinated pigs prior to co-infection and PRRS VL of non-vaccinated pigs post co-infection) are genetically the same trait. The WUR single nucleotide polymorphism on chromosome 4, previously associated with reduced PRRS VL under PRRSV-only infection, was associated with significantly reduced PRRS VL following vaccination and co-infection (for non-vaccinated pigs), but also with reduced PCV2b VL of vaccinated pigs. These results indicate a significant effect of WUR genotype on PRRS VL upon primary PRRS exposure, whether in a PRRSV-only or PRRS and PCV2b co-infected population, but also with PCV2b VL of vaccinated pigs under PRRS and PCV2b co- infection

Pigs Selected for Increased Feed Efficiency Are Less Affected by Experimental Infection with the PRRS Virus

Analyses of average daily gain (ADG) and viral load (VL) suggest that selection for increased feed efficiency based on residual feed intake (RFI) does not increase the impact of Porcine Reproductive and Respiratory Syndrome (PRRS) infection on these two traits. In fact, the results show that growth of the more efficient pigs was less affected by PRRS infection than that of the inefficient line. These findings provide commercial farmers with additional incentives to invest in feed-efficient pigs

Effect of WUR Genotype and PRRS Vaccination on Pigs Co-Infected with PRRS and PCV2b

Average daily gain (ADG) and viral load (VL) were evaluated for pigs co-infected with porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) and porcine circovirus type 2b (PCV2b). Pigs were pre-selected for WUR genotype (a marker on chromosome 4 associated with weight gain and VL under PRRSV-challenge), half were vaccinated for PRRS, and half were not. Results indicate that vaccination for PRRS resulted in slower growth prior to co-infection and that the AB WUR genotype was associated with faster growth prior to co-infection, lower PRRS VL, and lower PCV2b VL in vaccinated pigs

An Open, Large-Scale, Collaborative Effort to Estimate the Reproducibility of Psychological Science

Reproducibility is a defining feature of science. However, because of strong incentives for innovation and weak incentives for confirmation, direct replication is rarely practiced or published. The Reproducibility Project is an open, large-scale, collaborative effort to systematically examine the rate and predictors of reproducibility in psychological science. So far, 72 volunteer researchers from 41 institutions have organized to openly and transparently replicate studies published in three prominent psychological journals in 2008. Multiple methods will be used to evaluate the findings, calculate an empirical rate of replication, and investigate factors that predict reproducibility. Whatever the result, a better understanding of reproducibility will ultimately improve confidence in scientific methodology and findings

Senolytic Intervention Improves Cognition, Metabolism, and Adiposity in Female APP

Senescent cells accumulate throughout the body and brain contributing to unhealthy aging and Alzheimer\u27s disease (AD). The APPNL-F/NL-F amyloidogenic AD mouse model exhibits increased markers of senescent cells and the senescence-associated secretory phenotype (SASP) in visceral white adipose tissue and the hippocampus before plaque accumulation and cognitive decline. We hypothesized that senolytic intervention would alleviate cellular senescence thereby improving spatial memory in APPNL-F/NL-F mice. Thus, 4-month-old male and female APPNL-F/NL-F mice were treated monthly with vehicle, 5 mg/kg dasatinib + 50 mg/kg quercetin, or 100 mg/kg fisetin. Blood glucose levels, energy metabolism, spatial memory, amyloid burden, and senescent cell markers were assayed. Dasatinib + quercetin treatment in female APPNL-F/NL-F mice increased oxygen consumption and energy expenditure resulting in decreased body mass. White adipose tissue mass was decreased along with senescence markers, SASP, blood glucose, and plasma insulin and triglycerides. Hippocampal senescence markers and SASP were reduced along with soluble and insoluble amyloid-β (Aβ)42 and senescence-associated-β-gal activity leading to improved spatial memory. Fisetin had negligible effects on these measures in female APPNL-F/NL-F mice while neither senolytic intervention altered these parameters in the male mice. Considering women have a greater risk of dementia, identifying senotherapeutics appropriate for sex and disease stage is necessary for personalized medicine

Type I interferons augment regulatory T cell polarization in concert with ancillary cytokine signals

In the transplant community, research efforts exploring endogenous alternatives to inducing tolerogenic allo-specific immune responses are much needed. In this regard, CD4 +FoxP3+ regulatory T cells (Tregs) are appealing candidates due to their intrinsic natural immunosuppressive qualities. To date, various homeostatic factors that dictate Treg survival and fitness have been elucidated, particularly the non-redundant roles of antigenic CD3ζ/T-cell-receptor, co-stimulatory CD28, and cytokine interleukin (IL-)2 dependent signaling. Many of the additional biological signals that affect Tregs remain to be elucidated, however, especially in the transplant context. Previously, we demonstrated an unexpected link between type I interferons (IFNs) and Tregs in models of multiple myeloma (MM)—where MM plasmacytes escaped immunological surveillance by enhancing type I IFN signaling and precipitating upregulated Treg responses that could be overturned with specific knockdown of type I IFN signaling. Here, we elaborated on these findings by assessing the role of type I IFN signaling (IFN-α and -β) on Treg homeostasis within an alloimmune context. Specifically, we studied the induction of Tregs from naïve CD4 T cells. Using in vitro and in vivo models of murine skin allotransplantation, we found that type I IFN indeed spatiotemporally enhanced the polarization of naïve CD4 T cells into FoxP3+ Tregs. Notably, however, this effect was not independent of, and rather co-dependent on, ancillary cytokine signals including IL-2. These findings provide evidence for the relevance of type I IFN pathway in modulating FoxP3+ Treg responses and, by extension, stipulate an additional means of facilitating Treg fitness via type I IFNs.</p