Mapping immunological and host receptor binding determinants of SARS-CoV spike protein utilizing the Qubevirus platform.

The motifs involved in tropism and immunological interactions of SARS-CoV spike (S) protein were investigated utilizing the Qubevirus platform. We showed that separately, 14 overlapping peptide fragments representing the S protein (F1-14 of 100 residues each) could be inserted into the C terminus of A1 on recombinant Qubevirus without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F6 from S425-525 was found to contain the binding determinant of the recombinant human angiotensin-converting enzyme 2, with the shortest active binding motif situated between residues S437-492. Upstream, another fragment, F7, containing an overlapping portion of F6 would not bind to recombinant human angiotensin-converting enzyme 2, confirming that a contiguous stretch of residues could adopt the appropriate structural orientation of F6 as an insertion within the Qubevirus. The F6 (S441-460) and other inserts, including F7/F8 (S601-620) and F10 (S781-800), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes improved substantially the recognition and binding to their cognate antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of subunit vaccines or other biologics against SARS-CoV.

Specific antibody responses to Qß-displayed Plasmodium falciparum-derived UB05 and MSP3 proteins in mother-neonate couples.

Malaria blood-stage parasite is a critical pathogenic stage responsible for serious adverse outcomes in pregnant women and their neonates. Immunoglobulin G (IgG) antibody responses specific to various asexual blood-stage antigens were well reported in non-pregnant individuals. However, little is still known during placental malaria. To assess the antibody responses specific to Plasmodium falciparum-derived MSP3 and UB05 malaria vaccine candidates in mother-neonate couples, mother's peripheral blood and neonate's cord blood samples were collected at delivery. After malaria diagnostic, plasma levels of IgG and IgG subclass responses specific to UB05, MSP3 and UB05-MSP3 were determined using ELISA. As outcomes, both mothers and neonates had significantly higher IgG responses to UB05 and UB05-MSP3 compared to anti-MSP3 IgG (p < 0.05), irrespective of malaria status. Significant negative correlations were observed between IgG levels specific to the three antigens and parasitaemia (p < 0.01). Anti-UB05 and anti-UB05-MSP3 IgG levels in neonates showed a significant positive correlation with the corresponding mothers' antibodies (rs = 0.25 with p = 0.04; rs = 0.31 with p = 0.01, respectively). UB05MSP3-specific IgG3 and IgG1 subclass responses were significantly higher than the IgG4 subclass (p < 0.01). The neonates IgG1 and IgG3 levels positively correlated with the corresponding antibody subclasses of mothers. These findings suggest an association between UB05 and UB05-MSP3-specific antibody responses and malaria control during pregnancy. Maternal-foetal transfer of MSP3 and UB05-specific IgG occurs during pregnancy, suggesting the interest in the future malaria vaccination strategies in pregnant women to generate early protective immunity in baby against malaria.

Five financial incentives to revive the Gulf of Mexico dead zone and Mississippi basin soils.

A central challenge in the Mississippi River Basin is how to continue to support profitable agricultural production, provide water supply, flood control, transportation, and other benefits, while reducing the current burden of environmental degradation. Several practices have been shown to reduce nutrient runoff and water pollution, and improve soil fertility, while often yielding profits for farmers. Yet many of these beneficial practices remain underutilized. Participants at an expert workshop identified five candidate financial mechanisms that could increase adoption of these beneficial farming practices in four focal Midwest states in the next five years: crop insurance premium subsidies, transformation of the private service provider business model, expansion and targeting of 2019 U.S. Farm Bill funding, development of new state funds, and direction of post-disaster federal funds towards habitat restoration, particularly in floodplains. This study provides rough approximations of the change in nutrient runoff and greenhouse gas (GHG) emissions, the annualized costs, and the nutrient and GHG reductions per dollar likely to result from deployment of each financial mechanism. Based upon these approximations, the adoption of these programs could reduce annual nitrate flows at the outlet of the Ohio and Upper Mississippi River Basins by 25%, surpassing the intermediate 2025 target (20% reduction) and achieving more than half of the long-term target (45% reduction) set by the Mississippi River/Gulf of Mexico Hypoxia Task Force. These approximations also illustrate that these five mechanisms could provide the same GHG reductions (∼43 Tg CO2e yr-1) as taking 12 coal-fired energy plants offline. The total cost of these five financial mechanisms is estimated at ∼$2.6 billion, or 64 g of nitrates and ∼17 kg of CO2e per dollar spent. These proposed solutions all face political, financial, cultural or institutional challenges, but with industry support, creative political action, and continued communication of both private and public benefits, they can create meaningful nutrient reductions and rebuild soils by 2022.

Mapping immunological and host receptor binding determinants of SARS-CoV spike protein utilizing the Qubevirus platform.

The motifs involved in tropism and immunological interactions of SARS-CoV spike (S) protein were investigated utilizing the Qubevirus platform. We showed that separately, 14 overlapping peptide fragments representing the S protein (F1-14 of 100 residues each) could be inserted into the C-terminus of A1 on recombinant Qubevirus without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F6 from S 425-525 , was found to contain the binding determinant of the recombinant human angiotensin converting enzyme 2 (rhACE2), with the shortest active binding motif situated between residues S 437-492 . Upstream, another fragment, F7, containing an overlapping portion of F6 would not bind to rhACE2, confirming not just only that residues were linear but equally also the appropriate structural orientation of F6 upon the Qubevirus. The F6 (S 441-460 ) and other inserts, including F7/F8 (S 601-620 ) and F10 (S 781-800 ), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes, improved substantially the recognition and binding to their cognate antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of subunit vaccines or other biologics against SARS-CoV. Significance: Mapping epitopes within the receptor binding domains of viruses which are essential for viral tropism is critical for developing antiviral agents and subunit vaccines. In this study we have engineered the surface of Qubevirus to display a peptide library derived from the SARS-CoV S protein. In biopanning with S protein antibodies, we have identified three peptide fragments (EP1, EP2 and EP3) which reacted selectively with antibodies specific to the S protein. We demonstrated that all recombinant phage displayed peptide fragments both individually and as chimera exposed important immunological epitopes to their cognate antibodies. A peptide fragment F6 situated at S 425-525 , was found containing the binding determinant of the recombinant human angiotensin converting enzyme 2 (rhACE2), with the shortest active binding motif situated between residues S 437-492 . The platform is rapidly to identify epitopes and receptor binding sites within viral receptors found in target host cell. Thus, this platform holds great significance.

Encouragement from the right source: Evaluating the impact of the 4R Nutrient Stewardship Certification Program in the Ohio Western Lake Erie Basin.

Harmful algal blooms are a considerable environmental issue predominantly caused by runoff of nutrients from agricultural lands. One high-profile set of practices promoted to combat this threat is the 4Rs of nutrient stewardship that concern using the right source of nutrients at the right rate and right time in the right place. While outreach for agricultural conservation is often undertaken by governmental or nonprofit entities, there is increasing interest in engaging agricultural retailers to leverage the trust that already exists between farmers and their agribusiness professionals. The 4R Nutrient Stewardship Certification Program, implemented in the Western Lake Erie Basin (WLEB) in 2014, certifies crop advising companies and agronomy retailers or Nutrient Service Providers (NSPs) to promote best practices in nutrient management. This program has since grown and now exists in six US states and the province of Ontario and Prince Edward Island in Canada. Using a survey of farmers in the WLEB, we investigate the impact of working with certified NSPs over time on farmers' reported 4R-related behaviors. We find evidence that working with a certified NSP has a positive impact on 4R behaviors that is independent of other potential explanations for this change (e.g., farmer concern about nutrient loss, local regulatory efforts, and exposure to general 4R-related outreach). Overall, these results suggest that the 4R Nutrient Stewardship Certification Program is having an independent, positive impact on farmer behavior, and engaging with agricultural retailers and agronomists can be effective at advancing adoption of environmentally impactful nutrient management practices.

Gender, Police Culture, and Structured Ambivalence: Navigating 'Fit' with the Brotherhood, Boys' Club, and Sisterhood.

Women are increasingly represented in policing; however, inclusion alone will not eradicate existing structural and cultural barriers to meaningful change. Insights from interviews with ninety-one Canadian women police of varied rank and tenure, demonstrate women's experiences of structured ambivalence as they strategically deploy and resist gendered policing narratives of the Brotherhood, Boys' Club, and Sisterhood to negotiate their own 'fit.' In this way, they both challenge and reinforce gendered boundaries that create barriers to meaningful transformation. These findings demonstrate the need for change initiatives to address the complex and ever-shifting role of gender in policing organizations.

Preparation of the pancreas allograft for transplantation.

Meticulous preparation of the allograft on the back bench is one of the cornerstones of technically successful whole-organ pancreas transplantation. With evolving surgical approaches for organ retrieval and implantation, it has become routine to procure the pancreas in conjunction with other abdominal organs without sacrificing vasculature or graft quality and without compromising excellent outcomes. This review article summarizes the major steps required for proper back table preparation of the pancreas allograft with suggestions and tips whenever alternative approaches are presented.

Imagining value, imagining users: academic technology transfer for health innovation.

Governments have invested heavily in the clinical and economic promise of health innovation and express increasing concern with the efficacy and efficiency of the health innovation system. In considering strategies for 'better' health innovation, policy makers and researchers have taken a particular interest in the work of universities and related public research organizations: How do these organizations identify and transfer promising innovations to market, and do these efforts make best use of public sector investments? We conducted an ethnographic study of technology transfer offices (TTOs) in Ontario and British Columbia, Canada, to consider the place of health and health system imperatives in judgments of value in early-stage health innovation. Our analysis suggests that the valuation process is poorly specified as a set of task-specific judgments. Instead, we argue that technology transfer professionals are active participants in the construction of the innovation and assign value by 'imagining' the end product in its 'context of use'. Oriented as they are to the commercialization of health technology, TTOs understand users primarily as market players. The immediate users of TTOs' efforts are commercial partners (i.e., licensees, investors) who are capable of translating current discoveries into future commodities. The ultimate end users - patients, clinicians, health systems - are the future consumers of the products to be sold. Attention to these proximate and more distal users in the valuation process is a complex and constitutive feature of the work of health technology transfer. At the same time, judgements about individual technologies are made in relation to a broader imperative through which TTOs seek to imagine and construct sustainable innovation systems. Judgments of value are rendered sensible in relation to the logic of valuation for systems of innovation that, in turn, configure users of health innovation in systemic ways.

Function of the RNA Coliphage Qß Proteins in Medical In Vitro Evolution.

Qß is a positive (+) single-stranded RNA bacteriophage covered by a 25 nm icosahedral shell. Qß belongs to the family of Leviviridae and is found throughout the world (bacterial isolates and sewage). The genome of Qß is about 4.2 kb, coding for four proteins. This genome is surrounded by 180 copies of coat proteins (capsomers) each comprised of 132 residues of amino acids. The other proteins, the subunit II (ß) of a replicase, the maturation protein (A2) and the read-through or minor coat protein (A1), play a key role in phage infection. With the replicase protein, which lacks proofreading activity, as well as its short replication time, and high population size, Qß phage has attractive features for in vitro evolution. The A1 protein gene shares the same initiation codon with the coat protein gene and is produced during translation when the coat protein's UGA stop codon triplet (about 400 nucleotides from the initiation) is suppressed by a low level of ribosome misincorporation of tryptophan. Thus, A1 is termed the read-through protein. This RNA phage platform technology not only serves to display foreign peptides but is also exceptionally suited to address questions about in vitro evolution. The C-terminus of A1 protein confers to this RNA phage platform an exceptional feature of not only a linker for foreign peptide to be displayed also a model for evolution. This platform was used to present a peptide library of the G-H loop of the capsid region P1 of the foot-and-mouth disease virus (FMDV) called VP1 protein. The library was exposed on the exterior surface of Qß phages, evolved and selected with the monoclonal antibodies (mAbs) SD6 of the FMDV. These hybrid phages could principally be good candidates for FMDV vaccine development. Separately, the membrane proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) epitopes was fused with the A1 proteins and exposed on the Qß phage exterior surface. The engineered phages with MPER epitopes were recognized by anti-MPER specific antibodies. This system could be used to overcome the challenge of effective presentation of MPER to the immune system. A key portion of this linear epitope could be randomized and evolved with the Qß system. Overall, antigens and epitopes of RNA viruses relevant to public health can be randomized, evolved and selected in pools using the proposed Qß model to overcome their plasticity and the challenge of vaccine development. Major epitopes of a particular virus can be engineered or displayed on the Qß phage surface and used for vaccine efficacy evaluation, thus avoiding the use of live viruses.

Finding common ground in team-based qualitative research using the convergent interviewing method.

Research councils, agencies, and researchers recognize the benefits of team-based health research. However, researchers involved in large-scale team-based research projects face multiple challenges as they seek to identify epistemological and ontological common ground. Typically, these challenges occur between quantitative and qualitative researchers but can occur between qualitative researchers, particularly when the project involves multiple disciplinary perspectives. The authors use the convergent interviewing technique in their multidisciplinary research project to overcome these challenges. This technique assists them in developing common epistemological and ontological ground while enabling swift and detailed data collection and analysis. Although convergent interviewing is a relatively new method described primarily in marketing research, it compares and contrasts well with grounded theory and other techniques. The authors argue that this process provides a rigorous method to structure and refine research projects and requires researchers to identify and be accountable for developing a common epistemological and ontological position.

Gender differences in upper airway compliance during NREM sleep: role of neck circumference.

It has been proposed that the gender difference in sleep apnea prevalence is related to gender differences in upper airway structure and function. We hypothesized that men would have smaller retropalatal cross-sectional area and higher compliance during sleep compared with women. Using upper airway imaging, we measured upper airway cross-sectional area and retropalatal compliance in wakefulness and non-rapid eye movement (NREM) sleep in 15 men and 15 women without sleep-disordered breathing. Cross-sectional area at the beginning of inspiration tended to be larger in men compared with women in both wakefulness [194.5 +/- 21.3 vs. 138.8 +/- 12.0 (SE) mm(2)] and NREM sleep (111.1 +/- 17.6 vs. 83.3 +/- 11.9 mm(2); P = 0.058). There was no significant difference, however, after correction for body surface area. Retropalatal compliance also tended to be higher in men during both wakefulness (5.9 +/- 1.4 vs. 3.1 +/- 1.4 mm(2)/cmH(2)O; P = 0.006) and NREM sleep (12.6 +/- 2.7 vs. 4.7 +/- 2.6 mm(2)/cmH(2)O; P = 0.055). However, compliance was similar in men relative to women after correction for neck circumference. We conclude that the gender difference in retropalatal compliance is more accurately attributed to differences in neck circumference between the genders.

Early reexploration for suspected thrombosis after pancreas transplantation.

BACKGROUND: Graft thrombosis is the most common cause of early graft loss after pancreas transplantation. Early reexploration may permit salvage or timely removal of the thrombosed graft. METHODS: This was a retrospective review of 345 pancreas transplants performed at a single center between January 2003 and December 2009. Early reexploration was defined as within 1 week of pancreas transplantation. RESULTS: Of the 345 transplants, there were 35 early reexplorations. The graft was compromised in 20 cases (57%): 10 venous thromboses, 3 arterial thromboses, 2 combined arterial and venous thrombosis, 2 thromboses secondary to allograft pancreatitis, and 3 cases of positional ischemia without thrombosis. Of these allografts, three reperfused once repositioned and six were successfully thrombectomized for a graft salvage rate of 45%. One of the thrombectomized grafts remained perfused but never functioned and was removed at retransplantation. The 10 remaining compromised grafts that were deemed unsalvageable and required allograft pancreatectomy. Nine of these recipients were retransplanted (eight within 2 weeks) and one was not a retransplantation candidate. CONCLUSIONS: Reexploration for suspected graft thrombosis after pancreas transplantation resulted in a negative laparotomy rate of 43%, but permitted graft salvage in 45% of compromised grafts.

Use of extended criteria livers decreases wait time for liver transplantation without adversely impacting posttransplant survival.

INTRODUCTION: The use of extended criteria donors (ECDs) could minimize shortage of suitable donor livers for transplantation. In 3 years, the aggressive use of ECD livers has reduced the wait list at our center from 257 to 30 patients with a median wait time of 18 days without using living donors. This study compares the graft/patient survival from standard (SD) and ECD for our transplant population between 2001 and 2005. METHODS: Records of all adult liver transplant recipients over 4 years were reviewed (n = 571). ECD criteria included: age >59 years, BMI >34.9, maximum AST/ALT >500, maximum bilirubin >2.0, peak serum sodium >170, HBV/HCV/HTLV reactive, donation after cardiac death, cold ischemia time >12 hours, ICU stay >5 days, 3 or more pressors simultaneously, extensive alcohol abuse, cancer history (nonskin), active meningitis/bacteremia, or significant donor liver trauma. Outcomes included graft and patient survival at 90 days, 1 year, and 2 years. RESULTS: Sixty-eight percent of recipients (n = 388) received ECD livers. Primary factors accounting for ECD-liver status included: elevated liver function tests (20%), hypernatremia (12.6%), and extensive alcohol abuse (11.4%). Graft survival was (SD, ECD): 90-day 91%, 88%; 1-year 84%, 80%; 2-year 78%, 77%; patient survival was: 90-day 93%, 90%; 1-year 87%, 82%; 2-year 83%, 79%. Kaplan-Meier survival analysis failed to demonstrate an overall difference in graft or patient survival at any time point. Only donor age >60 years was associated with decreased graft and patient survival. CONCLUSIONS: Liver grafts from ECD can be used to dramatically reduce wait list time with outcomes comparable to those for SD without resorting to living donor liver transplantation.