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Abstract Background There are persistent racial and ethnic health disparities in end-of-life health outcomes in the United States. African American patients are less likely than White patients to access palliative care, enroll in hospice care, have documented goals of care discussions with their healthcare providers, receive adequate symptom control, or die at home. We developed Community Health Worker Intervention for Disparities in Palliative Care (DeCIDE PC) to address these disparities. DeCIDE PC is an integrated community health worker (CHW) palliative care intervention that uses community health workers (CHWs) as care team members to enhance the receipt of palliative care for African Americans with advanced cancer. The overall objectives of this study are to (1) assess the effectiveness of the DeCIDE PC intervention in improving palliative care outcomes amongst African American patients with advanced solid organ malignancy and their informal caregivers, and (2) develop generalizable knowledge on how contextual factors influence implementation to facilitate dissemination, uptake, and sustainability of the intervention. Methods We will conduct a multicenter, randomized, assessor-blind, parallel-group, pragmatic, hybrid type 1 effectiveness-implementation trial at three cancer centers across the United States. The DeCIDE PC intervention will be delivered over 6 months with CHW support tailored to the individual needs of the patient and caregiver. The primary outcome will be advance care planning. The treatment effect will be modeled using logistic regression. The secondary outcomes are quality of life, quality of communication, hospice care utilization, and patient symptoms. Discussion We expect the DeCIDE PC intervention to improve integration of palliative care, reduce multilevel barriers to care, enhance clinic and patient linkage to resources, and ultimately improve palliative care outcomes for African American patients with advanced cancer. If found to be effective, the DeCIDE PC intervention may be a transformative model with the potential to guide large-scale adoption of promising strategies to improve palliative care use and decrease disparities in end-of-life care for African American patients with advanced cancer in the United States. Trial registration Registered on ClinicalTrials.gov (NCT05407844). First posted on June 7, 2022.

Abstract Methane is a potent greenhouse gas, which has contributed to approximately a fifth of global warming since pre-industrial times. The agricultural sector produces significant methane emissions, especially from livestock, waste management and rice cultivation. Rice fields alone generate around 9% of total anthropogenic emissions. Methane is produced in waterlogged paddy fields by methanogenic archaea, and transported to the atmosphere through the aerenchyma tissue of rice plants. Thus, bioengineering rice with catalysts to detoxify methane en route could contribute to an efficient emission mitigation strategy. Particulate methane monooxygenase (pMMO) is the predominant methane catalyst found in nature, and is an enzyme complex expressed by methanotrophic bacteria. Recombinant expression of pMMO has been challenging, potentially due to its membrane localization, multimeric structure, and polycistronic operon. Here we show the first steps towards the engineering of plants for methane detoxification with the three pMMO subunits expressed in the model systems tobacco and Arabidopsis. Membrane topology and protein–protein interactions were consistent with correct folding and assembly of the pMMO subunits on the plant ER. Moreover, a synthetic self-cleaving polypeptide resulted in simultaneous expression of all three subunits, although low expression levels precluded more detailed structural investigation. The work presents plant cells as a novel heterologous system for pMMO allowing for protein expression and modification.

Abstract Objective Wendler's glottoplasty (WG) is a pitch‐elevating surgery performed by laryngologists providing gender‐affirming care. The surgery creates an anterior glottic web that could theoretically cause airway concerns, either perioperatively or at the time of future procedures; such concerns are not well‐described in the literature. We seek to assess surgeon opinions on airway concerns regarding WG. Method A survey of laryngologists assessing opinions on airway considerations in glottoplasty. Results A total of 19 physicians responded, representing approximately 193 surgeries. 52.6% performed glottoplasty and the remainder responded based on experience with anterior glottic webs. Two perioperative airway complications were reported, both mild stridor that did not prevent same‐day discharge. No long‐term sequela was reported. All surveyed laryngologists endorsed an altered general anesthetic approach for future procedures, with 73.7% advocating for use of a smaller endotracheal tube. 72.2% did not have “major concerns” about future intubations, and only 5.3% thought the immediate risk of airway compromise was a “real concern.” 91.9% counsel their patients routinely but briefly on airway concerns. Open‐ended comments conveyed themes of concern for post‐operative disruption of the web more than of airway compromise. Conclusion Because glottoplasty is performed in the anterior glottis and does not significantly impact airway patency, the risk of serious airway complications appears to be minimal. Laryngologists believe future intubations require a modified approach with a smaller tube, partly due to concern for glottic web trauma. Based on this pilot study, the topic deserves greater work to standardize care and anesthetic alterations for patients with WG. Level of Evidence 5

Purpose: The study aimed to (1) examine the feasibility of providing a training course on auricular point acupressure (APA) for clinical oncology nurses to integrate APA into real-world nursing care settings, and (2) examine the effectiveness of APA on cancer-related pain (CRP) under usual inpatient oncology ward conditions. Methods: This was a 2-phase feasibility study. Phase 1, an in-person, 8 hour training program was provided to oncology nurses. Phase 2, a prospective and feasibility study was conducted to evaluate the integration of APA into nursing care activities to manage CRP. Oncology patients were included if their pain was rated at ≥4 on a 0 to 10 numeric rating scale in the past 24 hours. Patients received 1 APA treatment administered by the nurses and were instructed to stimulate the points for 3 days. Study outcomes (pain intensity, fatigue, and sleep disturbance), pain medication use, and APA practice were measured by a phone survey daily. Results: Ten oncology nurses received APA training in phase 1. APA had been added to the hospital’s electronic health records (EHRs) as a pain treatment. In phase 2, 33 oncology patients received APA treatment with a 100% adherence rate (pressing the seeds 3 times per day, 3 minutes per time based on the suggestion). The side effects of APA were minimal (~8%-12% felt tenderness on the ear). After 3 days of APA, patients reported 38% pain relief, 39% less fatigue, and 45% improvement in sleep disturbance; 24% reduced any type of pain medication use and 19% reduced opioid use (10 mg opioids using milligram morphine equivalent). The major barrier to integrating APA into routine nursing practice was time management (how to include APA in a daily workflow). Conclusion: It is feasible to provide 8-hour training to oncology nurses for mastering APA skill and then integrating APA into their daily nursing care for patients with CRP. Based on the promising findings (decreased pain, improved fatigue and sleep disturbance, and less opioid use), the next step is to conduct a randomized clinical trial with a larger sample to confirm the efficacy of APA for oncology nurses to treat CRP in real-world practice. ClinicalTrial.gov identifier number: NCT04040140.

Background: The specialty of hospice and palliative medicine struggles to merge the fast-paced technological consultative work of acute hospital palliative care with slower paced and home-based hospice. Each has equal if different merits. Here, we describe creation of a position that allowed half-time hospice employment, alongside academic hospital-based palliative care. Methods: Johns Hopkins Medicine and Gilchrist, Inc., a large nonprofit hospice, partnered to form a joint position with time spent equally between the two locations. Results: Created as a university position with ?leasing? to the hospice, specific attention has been paid to mentoring at both sites to allow professional advancement. Both organizations have benefited in terms of recruitment, and more physicians have chosen this dual pathway suggesting that it is working well. Discussion: Hybrid positions are possible and may be desired by those who wish to practice both palliative medicine and hospice. Creation of one successful position helped recruit a second and a third candidate a year later. The original recipient has been promoted within Gilchrist to direct the inpatient unit. Such positions require careful mentoring and coordination to allow success at both sites and this can be done with foresight.

Intracortical microstimulation (ICMS) of the somatosensory cortex via penetrating microelectrode arrays (MEAs) can evoke cutaneous and proprioceptive sensations for restoration of perception in individuals with spinal cord injuries. However, ICMS current amplitudes needed to evoke these sensory percepts tend to change over time following implantation. Animal models have been used to investigate the mechanisms by which these changes occur and aid in the development of new engineering strategies to mitigate such changes. Non-human primates are commonly the animal of choice for investigating ICMS, but ethical concerns exist regarding their use. Rodents are a preferred animal model due to their availability, affordability, and ease of handling, but there are limited choices of behavioral tasks for investigating ICMS. In this study, we investigated the application of an innovative behavioral go/no-go paradigm capable of estimating ICMS-evoked sensory perception thresholds in freely moving rats. We divided animals into two groups, one receiving ICMS and a control group receiving auditory tones. Then, we trained the animals to nose-poke – a well-established behavioral task for rats – following either a suprathreshold ICMS current-controlled pulse train or frequency-controlled auditory tone. Animals received a sugar pellet reward when nose-poking correctly. When nose-poking incorrectly, animals received a mild air puff. After animals became proficient in this task, as defined by accuracy, precision, and other performance metrics, they continued to the next phase for perception threshold detection, where we varied the ICMS amplitude using a modified staircase method. Finally, we used non-linear regression to estimate perception thresholds. Results indicated that our behavioral protocol could estimate ICMS perception thresholds based on ~95% accuracy of rat nose-poke responses to the conditioned stimulus. This behavioral paradigm provides a robust methodology for evaluating stimulation-evoked somatosensory percepts in rats comparable to the evaluation of auditory percepts. In future studies, this validated methodology can be used to study the performance of novel MEA device technologies on ICMS-evoked perception threshold stability using freely moving rats or to investigate information processing principles in neural circuits related to sensory perception discrimination.

NMR and native mass spectrometry reveal that the major capsid VP1 protein from murine and human norovirus exhibit distinct behaviors and are differentially regulated by the binding of glycochenodeoxycholic acid.

Abstract This study highlighted how particular intersections of personal characteristics were related to Motivation to Learn (MtL) among adults. MtL is a prerequisite for adult education and training participation. However, little is known about MtL across subpopulations due to several methodological limitations. This study developed a national profile of MtL by key subpopulations that are defined by combinations of age, gender, education level, and literacy proficiency in the United States. Data were obtained from 2012/2014/2017 Program for International Assessment of Adult Competencies (PIAAC) restricted use file (N = 8400). The alignment optimization (AO) method was employed to estimate subpopulation means of a PIAAC-based latent MtL construct. Subpopulations with younger age, greater educational attainment, and higher literacy proficiency showed significantly greater MtL.

Abstract Background The recommendation from national documents and reports to promote inquiry-related science activities has not been supported by recent studies, which have found the overall frequency of inquiry activities to be negatively associated with student learning outcomes. This study was inspired by such conflicting reports and aimed to clarify the associations of science-specific, inquiry-related activities and epistemological beliefs with students’ mathematical and scientific literacies. Results A secondary analysis of the database from the Programme for International Student Assessment 2015 of Australia (N 1 = 14,530) and Taiwan (N 2 = 7708) utilizing structural equation modelling revealed that these two countries exhibited similar data patterns. Results suggested that open-inquiry activities (such as debating and planning experiments) had a negative relationship with secondary students’ mathematical and scientific literacies. Structured inquiry learning (such as students explaining their ideas and teacher explaining how an idea can be applied to different phenomena) and epistemological beliefs about science were significant and positive predictors of student mathematical and scientific literacy performance. Conclusions The current study further highlights and provides empirical evidence that the teacher’s role in structured inquiry (especially pertaining to the relevance and applicability of these ideas) appears to be essential to the development of student literacy. Educational implications and recommendations are discussed.

Noroviruses are the leading cause of severe childhood diarrhea and foodborne disease worldwide. While they are a major cause of disease in all age groups, infections in the very young can be quite severe with annual estimates of 50,000-200,000 fatalities in children under 5 years old. In spite of the remarkable disease burden associated with norovirus infections in people, very little is known about the pathogenic mechanisms underlying norovirus diarrhea, principally because of the lack of tractable small animal models. We recently demonstrated that wild-type neonatal mice are susceptible to murine norovirus (MNV)-induced acute self-resolving diarrhea in a time course mirroring human norovirus disease. Using this robust pathogenesis model system, we demonstrate that virulence is regulated by the responsiveness of the viral capsid to environmental cues that trigger contraction of the VP1 protruding (P) domain onto the particle shell, thus enhancing receptor binding and infectivity. The capacity of a given MNV strain to undergo this contraction positively correlates with infection of cells expressing low abundance of the virus receptor CD300lf, supporting a model whereby virion contraction triggers infection of CD300lflo cell types that are responsible for diarrhea induction. These findings directly link environmentally-influenced biophysical features with norovirus disease severity.

Background: Systematic identification of data essential for outcome prediction in metastatic prostate cancer (mPC) would accelerate development of precision oncology. Objective: To identify novel phenotypes and features associated with mPC outcome, and to identify biomarker and data requirements to be tested in future precision oncology trials. Design, setting, and participants: We analyzed deep longitudinal clinical, neuroendocrine expression, and autopsy data of 33 men who died from mPC between 1995 and 2004 (PELICAN33), and related findings to mPC biomarkers reported in the literature. Intervention: Thirty-three men prospectively consented to participate in an integrated clinical-molecular rapid autopsy study of mPC. Outcome measurements and statistical analysis: Data exploration with correction for multiple testing and survival analysis from the time of diagnosis to time to death and time to first occurrence of severe pain as outcomes were carried out. The effect of seven complications on the modeled probability of dying within 2 yr after presenting with the complication was evaluated using logistic regression. Results and limitations: Feature exploration revealed novel phenotypes related to mPC outcome. Four complications (pleural effusion, severe anemia, severe or controlled pain, and bone fracture) predict the likelihood of death within 2 yr. Men with Gleason grade group 5 cancers developed severe pain sooner than those with lower-grade tumors. Surprisingly, neuroendocrine (NE) differentiation was frequently observed in the setting of high serum prostate-specific antigen (PSA) levels (≥30 ng/ml). In 4/33 patients, no controlled (requiring analgesics) or severe pain was detected, and strikingly, 14/15 metastatic sites studied in these men did not express NE markers, suggesting an inverse relationship between NE differentiation and pain in mPC. Intracranial subdural metastasis is common (36%) and is usually clinically undetected. Categorization of “skeletal-related events” complications used in recent studies likely obscures the understanding of spinal cord compression and fracture. Early death from prostate cancer was identified in a subgroup of men with a low longitudinal PSA bandwidth. Cachexia is common (body mass index

Abstract Manufacturing and resource industries are the key drivers for economic growth with a huge environmental cost (e.g. discharge of industrial effluents and post-mining substrates). Pollutants from waste streams, either organic or inorganic (e.g. heavy metals), are prone to interact with their physical environment that not only affects the ecosystem health but also the livelihood of local communities. Unlike organic pollutants, heavy metals or trace metals (e.g. chromium, mercury) are non-biodegradable, bioaccumulate through food-web interactions and are likely to have a long-term impact on ecosystem health. Microorganisms provide varied ecosystem services including climate regulation, purification of groundwater, rehabilitation of contaminated sites by detoxifying pollutants. Recent studies have highlighted the potential of methanotrophs, a group of bacteria that can use methane as a sole carbon and energy source, to transform toxic metal (loids) such as chromium, mercury and selenium. In this review, we synthesise recent advances in the role of essential metals (e.g. copper) for methanotroph activity, uptake mechanisms alongside their potential to transform toxic heavy metal (loids). Case studies are presented on chromium, selenium and mercury pollution from the tanneries, coal burning and artisanal gold mining, respectively, which are particular problems in the developing economy that we propose may be suitable for remediation by methanotrophs. Video Abstract

Abstract Background Inpatient palliative care is associated with lower inpatient costs; however, this has yet to be studied using a more nuanced, multi-tiered measure of inpatient palliative care and a national population-representative dataset. Using a population-based cohort of Canadians who died in hospital, our objectives were to: describe patients’ receipt of palliative care and active interventions in their terminal hospitalization; and examine the relationship between inpatient palliative care and hospitalization costs. Methods Retrospective cohort study using data from the Discharge Abstract Database in Canada between fiscal years 2012 and 2015. The cohort were Canadian adults (age ≥ 18 years) who died in hospital between April 1st, 2012 and March 31st, 2015 (N = 250,640). The exposure was level of palliative care involvement defined as: medium-high, low, or no palliative care. The main measure was acute care costs calculated using resource intensity weights multiplied by the cost of standard hospital stay, represented in 2014 Canadian dollars (CAD). Descriptive statistics were represented as median (IQR), and n(%). We modelled cost as a function of palliative care using a gamma generalized estimating equation (GEE) model, accounting for clustering by hospital. Results There were 250,640 adults who died in hospital. Mean age was 76 (SD 14), 47% were female. The most common comorbidities were: metastatic cancer (21%), heart failure (21%), and chronic obstructive pulmonary disease (16%). Of the decedents, 95,450 (38%) had no palliative care involvement, 98,849 (38%) received low involvement, and 60,341 (24%) received medium to high involvement. Controlling for age, sex, province and predicted hospital mortality risk at admission, the cost per day of a terminal hospitalization was: $1359 (95% CI 1323: 1397) (no involvement), $1175 (95% CI 1146: 1206) (low involvement), and $744 (95% CI 728: 760) (medium-high involvement). Conclusions Increased involvement of palliative care was associated with lower costs. Future research should explore whether this relationship holds for non-terminal hospitalizations, and whether palliative care in other settings impacts inpatient costs.

Existing literature attests to the importance of assessing the learning enjoyment and learning interest of students toward socio-scientific issues (SSI). However, there are few existing studies that examine how ecological stimuli, which are crucial to young learners' perceptual development and the shaping of ethical judgment, predict their learning enjoyment and learning interest in SSI. This investigation addresses this gap in the available literature by investigating and assessing the effects of self-perceived influences of three ecological stimuli constructs--textbooks, family/classmates, and news media--on a learning interest in SSI construct and a learning enjoyment from SSI construct among Taiwanese high school students. A structural equation model consisting of these five constructs was fitted to data collected from 966 students. Results show that influence on students' ethical judgments from textbooks and news media directly predict learning interest in SSI, with effects partially mediated by learning enjoyment from SSI. The influence of family/classmates on students' learning interest was fully mediated by learning enjoyment. The role of enjoyment and learning interest as predictors of these outcomes is discussed within the context of genuine interest in learning SSI content. The value and implications of these results for science education specialists and interest researchers are forwarded and suggested directions of future investigation submitted.

The present study examined the 2015 Trends in International Mathematics and Science Study “Advanced” data to examine how the educational credentials of maths teachers and other teacher characteristics were related to attitude towards advanced mathematics and perceptions of engaged teaching among 12th-grade students enrolled in advanced mathematics courses in the U.S. As attitudinal outcomes in this study, two measures of attitude towards mathematics were employed – the Students Like Learning Advanced Mathematics scale and the Students Value Advanced Mathematics scale, and one measure of student perception of engaged teaching – the Students’ Views on Engaging Teaching in Advanced Mathematics Lessons. A set of multilevel regression analyses were conducted predicting each of these aforementioned outcomes. No statistically significant effects on the attitudinal outcomes were observed for teacher variables. Positive effects were noted for parental education on students’ valuing of advanced mathematics. A prominent finding was that higher levels of parental education were associated with higher student levels of valuing mathematics, which likely reflects a family/home culture that implicitly or explicitly places high value on science and mathematics. Identifying factors that might facilitate positive attitude is important to increase the likelihood that students will choose, and be retained in, mathematics and STEM education and careers.

Abstract Introduction Management of cancer is often characterized by difficult decisions. The National Coalition for Cancer Survivorship (NCCS) has developed the “Know Yourself” tool, a question prompt list (QPL) to enable patients to participate in these decisions. Methods We investigated the feasibility of using the NCCS tool by oncologists and their patients with cancer in a before-and-after pilot study at a tertiary medical center. We also measured patient reported decision preparedness, anxiety, satisfaction with care, trust in physician, discussion of care with their primary care physician (PCP), and general state of health, and solicited feedback from clinicians and patients on use of the form. Results Ninety patients and fifteen clinicians participated. Most patients reported the Tool was easy to use (91%) and would recommend it to others (73%) however fewer reported discussing the Tool at the visit (31%) or felt that it improved the quality of care (45%) or communication with the oncologist (56%). Clinicians reported Tool use in only 16 of 60 visits (27%); in these visits the Tool was helpful in identifying areas of concern (74%), guiding the clinical interaction (67%), promoting communication (62%), identifying areas of need (70%), and improving quality of care (71%). Decision preparedness, trust in physicians, uncertainty about care, anxiety, patient satisfaction and discussion of care with the PCP was unchanged with Tool use compared to non-use. Conclusions The Know Yourself tool had poor uptake but was favorably received among both patients and clinicians who used it. These findings suggest some patients could benefit from QPLs. Future work should test how implementation strategies might achieve greater use.

NSCLC treatment includes targeting of EGFR with tyrosine kinase inhibitors (TKIs) such as Erlotinib; however, resistance to TKIs is commonly acquired through T790M EGFR mutations or overexpression of vascular endothelial growth factor receptor-2 (VEGFR-2). We investigated the mechanisms of EGFR-TKI resistance in NSCLC cell lines with EGFR mutations or acquired resistance to Erlotinib. These studies showed upregulated gene and protein expression of VEGF, VEGFR-2, and a VEGF co-receptor neuropilin-1 (NP-1) in Erlotinib-resistant (1.4–5.3-fold) and EGFR double-mutant (L858R and T790M; 4.1–8.3-fold) NSCLC cells compared to parental and EGFR single-mutant (L858R) NSCLC cell lines, respectively. Immunofluorescence and FACS analysis revealed increased expression of VEGFR-2 and NP-1 in EGFR-TKI-resistant cell lines compared to TKI-sensitive cell lines. Cell proliferation assays showed that treatment with a VEGFR-2 inhibitor combined with Erlotinib lowered cell survival in EGFR double-mutant NSCLC cells to 9% compared to 72% after treatment with Erlotinib alone. Furthermore, Kaplan–Meier analysis revealed shorter median survival in late-stage NSCLC patients with high vs. low VEGFR-2 expression (14 mos vs. 21 mos). The results indicate that VEGFR-2 may play a key role in EGFR-TKI resistance and that combined treatment of Erlotinib with a VEGFR-2 inhibitor may serve as an effective therapy in NSCLC patients with EGFR mutations.

Noroviruses are responsible for almost a fifth of all cases of gastroenteritis worldwide. The calicivirus capsid is composed of 180 copies of VP1 with a molecular weight of ~58 kDa. This coat protein is divided into the N-terminus (N), the shell (S) and C-terminal protruding (P) domains. The S domain forms a shell around the viral RNA genome, while the P domains dimerize to form protrusions on the capsid surface. The P domain is subdivided into P1 and P2 subdomains, with the latter containing the binding sites for cellular receptors and neutralizing antibodies. Reviewed here are studies on murine norovirus (MNV) showing that the capsid responds to several physiologically relevant cues; bile, pH, Mg2+, and Ca2+. In the initial site of infection, the intestinal tract, high bile and metal concentrations and low pH cause two significant conformational changes: (1) the P domain contracts onto the shell domain and (2) several conformational changes within the P domain lead to enhanced receptor binding while blocking antibody neutralization. In contrast, the pH is neutral, and the concentrations of bile and metals are low in the serum. Under these conditions, the loops at the tip of the P domain are in the open conformation with the P domain floating on a linker or tether above the shell. This conformational state favors antibody binding but reduces interactions with the receptor. In this way, MNV uses metabolites and environmental cues in the intestine to optimize cellular attachment and escape antibody binding but presents a wholly different structure to the immune system in the serum. To our knowledge, this is the first example of a virus shapeshifting in this manner to escape the immune response.

The purpose of this qualitative case study was to explore how youth's engineering identity has changed in the process of productive struggle in Making activities in the Midwestern U.S in a summer program. The researchers conducted focus-group interviews, and also administered an Engineering Identity survey as well as the Draw an Engineer Test to the students at two time points (pre/post). Additionally, the researchers observed the level of youths' interaction by video-recording each session, conducted debriefing sessions, and examined youth artifacts. The findings revealed two themes. The first theme is that the boys and girls who were engaged in the process of productive struggle showed changes in their identity with engineering. The second theme is that the boys and girls who weren't fully engaged in the process of productive struggle (i.e., demonstrated unproductive struggle) showed little or no change in their identity with engineering. This study showed that regardless of whether the students went through the process of productive struggle or not, they developed neutral or positive attitudes toward engineering. This finding also added to the existing literature on that productive struggle in Making activities has potential to enhance these youth' learning of engineering knowledge and engineering tools.

Protein-shelled viruses have been thought as “tin cans” that merely carry the genomic cargo from cell to cell. However, through the years, it has become clear that viruses such as rhinoviruses and caliciviruses are active and dynamic structures waiting for the right environmental cues to deliver their genomic payload to the host cell. In the case of human rhinoviruses, the capsid has empty cavities that decrease the energy required to cause conformational changes, resulting in the capsids “breathing”, waiting for the moment when the receptor binds for it to release its genome. Most strikingly, the buried N-termini of VP1 and VP4 are transiently exposed during this process. A more recent example of a “living” protein capsid is mouse norovirus (MNV). This family of viruses have a large protruding (P) domain that is loosely attached to the shell via a single-polypeptide tether. Small molecules found in the gut, such as bile salts, cause the P domains to rotate and collapse onto the shell surface. Concomitantly, bile alters the conformation of the P domain itself from one that binds antibodies to one that recognizes receptors. In this way, MNV appears to use capsid flexibility to present one face to the immune system and a completely different one to attack the host tissue. Therefore, it appears that even protein-shelled viruses have developed an impressive array of tricks to dodge our immune system and efficiently attack the host.

DNA copies of many non-retroviral RNA virus genes or portions thereof (NIRVs) are present in the nuclear genomes of many eukaryotes. These have often been preserved for millions of years of evolution, suggesting that they play an important cellular function. One possible function is resistance to infection by related viruses. In some cases, this appears to occur through the piRNA system, but in others by way of counterfeit viral proteins encoded by NIRVs. In the fungi, NIRVs may be as long as 1,400 uninterrupted codons. In one such case in the yeast Debaryomyces hansenii, one of these genes provides immunity to a related virus by virtue of expression of a counterfeit viral capsid protein, which interferes with assembly of viral capsids by negative complementation. The widespread occurrence of non-retroviral RNA virus genes in eukaryotes may reflect an underappreciated method of host resistance to infection. This work demonstrates for the first time that an endogenous host protein encoded by a gene that has been naturally acquired from a virus and fixed in a eukaryote can interfere with the replication of a related virus and do so by negative complementation.

ABSTRACT Ideal antiviral vaccines elicit antibodies (Abs) with broad strain recognition that bind to regions that are difficult to mutate for escape. Using 10 murine norovirus (MNV) strains and 5 human norovirus (HuNoV) virus-like particles (VLPs), we identified monoclonal antibody (MAb) 2D3, which broadly neutralized all MNV strains tested. Importantly, escape mutants corresponding to this antibody were very slow to develop and were distal to those raised against our previously studied antibody, A6.2. To understand the atomic details of 2D3 neutralization, we determined the cryo-electron microscopy (cryo-EM) structure of the 2D3/MNV1 complex. Interestingly, 2D3 binds to the top of the P domain, very close to where A6.2 binds, but the only escape mutations identified to date fall well outside the contact regions of both 2D3 and A6.2. To determine how mutations in distal residues could block antibody binding, we used molecular dynamics flexible fitting simulations of the atomic structures placed into the density map to examine the 2D3/MNV1 complex and these mutations. Our findings suggest that the escape mutant, V339I, may stabilize a salt bridge network at the P-domain dimer interface that, in an allostery-like manner, affects the conformational relaxation of the P domain and the efficiency of binding. They further highlight the unusual antigenic surface bound by MAb 2D3, one which elicits cross-reactive antibodies but which the virus is unable to alter to escape neutralization. These results may be leveraged to generate norovirus (NoV) vaccines containing broadly neutralizing antibodies. IMPORTANCE The simplest and most common way for viruses to escape antibody neutralization is by mutating residues that are essential for antibody binding. Escape mutations are strongly selected for by their effect on viral fitness, which is most often related to issues of protein folding, particle assembly, and capsid function. The studies presented here demonstrated that a broadly neutralizing antibody to mouse norovirus binds to an exposed surface but that the only escape mutants that arose were distal to the antibody binding surface. To understand this finding, we performed an in silico analysis that suggested that those escape mutations blocked antibody binding by affecting structural plasticity. This kind of antigenic region—one that gives rise to broadly neutralizing antibodies but that the virus finds difficult to escape from—is therefore ideal for vaccine development.

Global climate change poses challenges to areas such as low-lying coastal zones, where sea level rise (SLR) and storm-surge overwash events can have long-term effects on vegetation and on soil and groundwater salinities, posing risks of habitat loss critical to native species. An early warning system is urgently needed to predict and prepare for the consequences of these climate-related impacts on both the short-term dynamics of salinity in the soil and groundwater and the long-term effects on vegetation. For this purpose, the U.S. Geological Survey’s spatially explicit model of vegetation community dynamics along coastal salinity gradients (MANHAM) is integrated into the USGS groundwater model (SUTRA) to create a coupled hydrology–salinity–vegetation model, MANTRA. In MANTRA, the uptake of water by plants is modeled as a fluid mass sink term. Groundwater salinity, water saturation and vegetation biomass determine the water available for plant transpiration. Formulations and assumptions used in the coupled model are presented. MANTRA is calibrated with salinity data and vegetation pattern for a coastal area of Florida Everglades vulnerable to storm surges. A possible regime shift at that site is investigated by simulating the vegetation responses to climate variability and disturbances, including SLR and storm surges based on empirical information.

Exposure to vehicle exhaust has been associated with cardiac and respiratory disease, lung cancer and greater overall mortality. We investigated whether amino-polycyclic aromatic hydrocarbon (amino-PAH) metabolites of nitro-PAHs could be used as biomarkers of these exposures. Pre- and post-shift urine samples were collected at the beginning and end of a work week from 82 male U.S. trucking industry workers. We used repeated-measures analysis to examine associations of total 1- and 2-aminonaphthalene (1 & 2-AN) and 1-aminopyrene (1-AP) urinary concentrations with microenvironment exposures to particulate matter (PM2.5), elemental and organic carbon and between 1 & 2-AN and 1-AP with urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG). There was an association between work week mean PM2.5 levels and post-shift 1 & 2-AN (141.8 pg/mL increase (95% CI: 53.3, 230.2) for each IQR increase (5.54 µg/m3) in PM2.5), but no associations with other exposure measures. There was a statistically significant increase in 8-OHdG concentrations with 1 & 2-AN (2.38 µg/mg creatinine (95% CI: 0.19, 4.58) per 242.85 pg/mg creatinine increase in 1 & 2-AN) and suggestive associations with all other exposure measures. Our findings suggest associations between urinary amino-PAHs with vehicle exhaust-related PM2.5, as well as with a biomarker of oxidative DNA damage.

In this work, we report on the development of an electrochemical biosensor for high selectivity and rapid detection of Hg2+ and Pb2+ ions using DNA-based specific aptamer probes labeled with ferrocene (or methylene blue) and thiol groups at their 5′ and 3′ termini, respectively. Aptamers were immobilized onto the surface of screen-printed gold electrodes via the SH (thiol) groups, and then cyclic voltammetry and impedance spectra measurements were performed in buffer solutions with the addition of HgCl2 and PbCl2 salts at different concentrations. Changes in 3D conformation of aptamers, caused by binding their respective targets, e.g., Hg2+ and Pb2+ ions, were accompanied by an increase in the electron transfer between the redox label and the electrode. Accordingly, the presence of the above ions can be detected electrochemically. The detection of Hg2+ and Pb2+ ions in a wide range of concentrations as low as 0.1 ng/mL (or 0.1 ppb) was achieved. The study of the kinetics of aptamer/heavy metal ions binding gave the values of the affinity constants of approximately 9.10−7 mol, which proved the high specificity of the aptamers used.

Noroviruses are responsible for almost a fifth of all cases of gastroenteritis worldwide. New strains evolve every 2–4 years by escaping herd immunity and cause worldwide epidemics. In the US alone, noroviruses are responsible for ~20 million cases and more than 70,000 hospitalizations of infected children, annually. Efforts towards a vaccine have been hindered by a lack of detailed structural information about antibody binding and the mechanisms of antibody escape. Caliciviruses have 180 copies of the major capsid protein (VP1; ~58 kDa), that is divided into the N-terminus (N), the shell (S) and C-terminal protruding (P) domains. The S domain forms a shell around the viral RNA genome, while the P domains dimerize to form protrusions on the capsid surface. The P domain is subdivided into P1 and P2 subdomains, with the latter containing the binding sites for cellular receptors and neutralizing antibodies. There is increasing evidence that these viruses are extremely dynamic and this flexibility is critical for viral replication. There are at least two modes of flexibility; the entire P domain relative to the shell and within the P domain itself. Here, the details and possible roles for this remarkable flexibility will be reviewed.

OBJECTIVES/SPECIFIC AIMS: Central neuropathic pain is a severely disabling consequence of conditions that cause tissue damage in the central nervous system (CNS) such as multiple sclerosis (MS) and neuromyelitis optica (NMO). It impacts mood, mobility and quality of life, but is often refractory to common treatments. Scrambler Therapy is an emerging non-invasive pain modifying technique that utilizes transcutaneous electrical stimulation of nociceptive fibers with the intent of re-organizing maladaptive signaling pathways. It has been examined for treatment of peripheral neuropathy with favorable safety and efficacy outcomes, but its use in central neuropathic pain has not been reported. We aim to explore acceptability and safety of Scrambler Therapy through a Phase II sham-controlled trial in NMO, and describe its use to date in central neuropathic pain. METHODS/STUDY POPULATION: Two patients with longstanding central neuropathic pain who failed multiple drug trials were treated as proof-of-concept, supporting the recent launch of a Phase II randomized controlled trial in NMO where patients receive 10 daily Scrambler treatments versus sham. Safety and acceptability from those recruited to date will be reported. Acceptability is measured by adherence and responses to patient surveys. RESULTS/ANTICIPATED RESULTS: We plan to recruit 22 patients, randomized 1:1 into experimental and sham arms. We will present acceptability and safety data for Scrambler use in patients with NMO who have been recruited by the time of this conference, as well as effectiveness data from two cases that have been completed outside of the trial. One case involved a 65-year-old woman with a 4-year history of central neuropathic pain following a C3-C5 TM. Her numerical rating scale (NRS) pain score was reduced to 0/10 from a baseline score of 5/10. The second case involved a 52-year-old woman with a 13-year history of pain following a medullary cavernoma bleed. Her baseline NRS pain score was 9/10, which was reduced to 0.5/10 post-treatment. No adverse events were reported. Pain relief was sustained at 30 days’ post-treatment. DISCUSSION/SIGNIFICANCE OF IMPACT: We are investigating the acceptability and efficacy of Scrambler Therapy for central neuropathic pain treatment in NMO. Proof-of-concept was supported by two patients whose pain scores improved considerably more in response to this treatment than with previous pharmacologic and non-pharmacologic interventions. Results from this trial may support future investigation in other disorders that cause damage in the CNS, including MS and TM.

Objective: Social adjustment, including alcohol use, directly affects the success of college students. Due to an increased reliance on computer-delivered alcohol interventions (CDIs) a need has emerged to further investigate alcohol use and web-based interventions. Methods: In-depth focus group interviews were conducted with 51 undergraduate students to elicit information from students on the shared experience of participating in a CDI. Results: Participants identified the influence of gender, culture, parents, and family on alcohol use behavior. A difference in personal factors, previous exposure, and experiences can affect the attitudes, behaviors, and outcomes of a CDI. Conclusion: Multiple approaches geared towards a wide variety of students from different backgrounds and environments are needed to be truly successful in preventing alcohol misuse.