Exposure-efficacy and exposure-safety analyses of ropeginterferon alfa-2b treatment in patients with polycythaemia vera.

AIMS: To investigate the exposure-response (E-R) relationship, including exposure-efficacy and exposure-safety, of ropeginterferon alfa-2b treatment in patients with polycythaemia vera (PV). METHODS: Based on the results of the phase II trial A20-202 regarding ropeginterferon alfa-2b in patients with PV, E-R analyses were performed to evaluate the efficacy and safety of the given dosing regimen. The E-R analyses were based on logistic and linear regression and the relationship between exposure to ropeginterferon alfa-2b and key efficacy and safety variables. The key efficacy variables included complete haematologic response (CHR) and reduction of the driver mutation JAK2V617F. The safety variable was treatment-related adverse events (TRAEs). RESULTS: A clear relationship between the exposure to ropeginterferon alfa-2b and CHR was observed, with an increase in drug exposure resulting in an increased probability of achieving CHR. Similar CHR probabilities were observed in the third and fourth quantiles of the average concentration at Week 24. The results from the exposure-JAK2V617F model indicated that the JAK2V617F allele burden decreased with increasing exposure to ropeginterferon alfa-2b and baseline body surface area. Exposure-safety analysis revealed a risk of AEs associated with transaminase abnormalities, which were not associated with clinical significance. CONCLUSIONS: Our analyses have shown that patients with PV treated with ropeginterferon alfa-2b had an increased probability of achieving CHR and a molecular response with acceptable safety risks at the 250-350-500 µg titration dosing regimen. This study has provided the relevant data for the application of a biologics licence of ropeginterferon alfa-2b for PV treatment in China.

A retrospective observational study of vCare: a virtual emergency clinical advisory and transfer service in rural and remote Australia.

BACKGROUND: Provision of critical care in rural areas is challenging due to geographic distance, smaller facilities, generalist skill mix and population characteristics. Internationally, the amalgamation telemedicine and retrieval medicine services are developing to overcome these challenges. Virtual emergency clinical advisory and transfer service (vCare) is one of these novel services based in New South Wales, Australia. We aim to describe patient encounters with vCare from call initiation at the referring site to definitive care at the accepting site. METHODS: This retrospective observational study reviewed all patients using vCare in rural and remote Australia for clinical advice and/or inter-hospital transfer for higher level of care between February and March 2021. Data were extracted from electronic medical records and included remoteness of sites, presenting complaint, triage category, camera use, patient characteristics, transfer information, escalation of therapeutic intervention and outcomes. Data were summarised using cross tabulation. RESULTS: 1,678 critical care patients were supported by vCare, with children (12.5%), adults (50.6%) and older people (36.9%) evenly split between sexes. Clinicians mainly referred to vCare for trauma (15.1%), cardiac (16.1%) and gastroenterological (14.8%) presentations. A referral to vCare led to an escalation of invasive intervention, skill, and resources for patient care. vCare cameras were used in 19.8% of cases. Overall, 70.5% (n = 1,139) of patients required transfer. Of those, 95.1% were transferred to major regional hospitals and 11.7% required secondary transfer to higher acuity hospitals. Of high-urgency referrals, 42.6% did not receive high priority transport. Imaging most requested included CT and MRI scans (37.2%). Admissions were for physician (33.1%) and surgical care (23.3%). The survival rate was 98.6%. CONCLUSION: vCare was used by staff in rural and remote facilities to support decision making and care of patients in a critical condition. Issues were identified including low utilisation of equipment, heavy reliance on regional sites and high rates of secondary transfer. However, these models are addressing a key gap in the health workforce and supporting rural and remote communities to receive care.

Cell death in the lateral geniculate nucleus, and its possible relationship with nicotinic receptors and sudden infant death syndrome (SIDS).

The role of the lateral geniculate nucleus (LGN) in vision has been extensively studied, yet its extraretinal capacities are still being investigated, including its role in arousal from sleep. The ß2 nicotinic acetylcholine receptor (nAChR) subunit is involved in the laminal organisation of the LGN with magnocellular (MC) and parvocellular (PC) neurons. Sudden infant death syndrome (SIDS) occurs during a sleep period and, neuropathologically, is associated with increased neuronal cell death and altered nAChRs. A recent qualitative pilot study from our group implicates the possibility of increased neuronal death/apoptosis in the SIDS LGN. The present study used quantitative analysis to report the baseline expression of apoptotic and nAChR subunits α7 and ß2 in the PC and MC layers of the LGN, to determine correlations amongst these markers within layers and across layers, and to evaluate changes in the expression of these markers in the LGN of SIDS infants, along with associations with SIDS risk factors, such as age, sex, cigarette smoke exposure, bed-sharing, and presence of an upper respiratory tract infection (URTI). Tissue was immunohistochemically stained for cell death markers of active caspase-3 (Casp-3) and TUNEL, and for the α7 and ß2 nAChR subunits. Amongst 43 cases of sudden and unexpected deaths in infancy (SUDI), classifications included explained deaths (eSUDI, n = 9), SIDS I (n = 5) and SIDS II (n = 29). Results indicated a strong correlation of the apoptotic markers and ß2 nAChR subunit between the LGN layers, but not across the markers within the layers. Amongst the diagnostic groups, compared to eSUDI, the SIDS II cases had decreased Casp-3 expression while ß2 nAChR expression was increased in both PC and MC layers. Amongst the SIDS risk factors, URTI and bed-sharing were associated with changes in neuronal death but not in the α7 and ß2 markers. In conclusion, our findings do not support a role for the α7 and ß2 nAChRs in apoptotic regulation of the LGN layers during infancy. However, for SIDS victims, an inverse correlation between the changes for markers of apoptosis and the ß2 nAChR subunit expression suggests altered LGN function.

Exploring the impact of trait number and type on functional diversity metrics in real-world ecosystems.

The use of trait-based approaches to understand ecological communities has increased in the past two decades because of their promise to preserve more information about community structure than taxonomic methods and their potential to connect community responses to subsequent effects of ecosystem functioning. Though trait-based approaches are a powerful tool for describing ecological communities, many important properties of commonly-used trait metrics remain unexamined. Previous work in studies that simulate communities and trait distributions show consistent sensitivity of functional richness and evenness measures to the number of traits used to calculate them, but these relationships have yet to be studied in actual plant communities with a realistic distribution of trait values, ecologically meaningful covariation of traits, and a realistic number of traits available for analysis. Therefore, we propose to test how the number of traits used and the correlation between traits used in the calculation of functional diversity indices impacts the magnitude of eight functional diversity metrics in real plant communities. We will use trait data from three grassland plant communities in the US to assess the generality of our findings across ecosystems and experiments. We will determine how eight functional diversity metrics (functional richness, functional evenness, functional divergence, functional dispersion, kernel density estimation (KDE) richness, KDE evenness, KDE dispersion, Rao's Q) differ based on the number of traits used in the metric calculation and on the correlation of traits when holding the number of traits constant. Without a firm understanding of how a scientist's choices impact these metric, it will be difficult to compare results among studies with different metric parametrization and thus, limit robust conclusions about functional composition of communities across systems.

Risk Factors Associated With SARS-CoV-2 Infection Among Farmworkers in Monterey County, California.

Importance: Essential workers in agriculture and food production have been severely affected by the ongoing COVID-19 pandemic. Objective: To identify risk factors associated with SARS-CoV-2 infection among farmworkers in California. Design, Setting, and Participants: This cross-sectional study invited farmworkers in California's Salinas Valley (Monterey County) receiving transcription-mediated amplification (TMA) tests for SARS-CoV-2 infection at federally qualified community clinics and community sites to participate. Individuals were eligible if they were not pregnant, were 18 years or older, had conducted farmwork since the pandemic started, and were proficient in English or Spanish. Survey data were collected and SARS-CoV-2 tests were conducted among participants from July 16 to November 30, 2020. Exposures: Sociodemographic, household, community, and workplace characteristics. Main Outcomes and Measures: TMA- and immunoglobulin G (IgG)-positive SARS-CoV-2 infection. Results: A total of 1107 farmworkers (581 [52.5%] women; mean [SD] age, 39.7 [12.6] years) were included in these analyses. Most participants were born in Mexico (922 [83.3%]), were married or living with a partner (697 [63.0%]), and worked in the fields (825 [74.5%]). Overall, 118 of 911 (13.0%) had a positive result on their TMA test for SARS-CoV-2 infection, whereas 201 of 1058 (19.0%) had antibody evidence of infection. In multivariable analyses accounting for recruitment venue and enrollment period, the incidence of TMA-positive SARS-CoV-2 infection was higher among those with lower than primary school-level education (adjusted relative risk [aRR], 1.32; 95% CI, 0.99-1.76; non-statistically significant finding), who spoke an Indigenous language at home (aRR, 1.30; 95% CI, 0.97-1.73; non-statistically significant finding), who worked in the fields (aRR, 1.60; 95% CI, 1.03-2.50), and who were exposed to a known or suspected COVID-19 case at home (aRR, 2.98; 95% CI, 2.06-4.32) or in the workplace (aRR, 1.59; 95% CI, 1.18-2.14). Positive results on IgG tests for SARS-CoV-2 infection were more common among those who lived in crowded housing (aRR, 1.23; 95% CI, 0.98-1.53; non-statistically significant finding), with children aged 5 years or younger (aRR, 1.40; 95% CI, 1.11-1.76), with unrelated roommates (aRR, 1.40; 95% CI, 1.19-1.64), and with an individual with known or suspected COVID-19 (aRR, 1.59; 95% CI, 1.13-2.24). The risk of IgG positivity was also higher among those with body mass index of 30 or greater (aRR, 1.65; 95% CI, 1.01-2.70) or diabetes (aRR, 1.31; 95% CI, 0.98-1.75; non-statistically significant finding). Conclusions and Relevance: In this cross-sectional study of farmworkers in California, both residential and workplace exposures were associated with SARS-CoV-2 infection. Urgent distribution of COVID-19 vaccines and intervention on modifiable risk factors are warranted given this population's increased risk of infection and the essential nature of their work.

Initiating Students' Reflections on Life's Passing in the Anatomy Course - an International Observation at 14 Universities.

BACKGROUND: Medical and dental students' feelings and thoughts about the topic of death and life's passing are often associated with learning in the gross anatomy course, when students begin working with a deceased body donor in order to study human anatomy. Little is known of whether the format of anatomy teaching has an impact on these experiences. An observational study was performed to capture the initiation of students' sentiments on the topic of life's passing during the anatomy course at 14 international universities, identify common themes regarding these thoughts, and to study the connection to variations in anatomy course formats and included elements. METHOD: Preclinical anatomy students reflected on one question (i.e., "How did your experience in the anatomy laboratory bring about your reflections on the meaning of life and human existence as well as the sanctity of one's passing?"). Written assignments were collected and anonymously coded. Information on anatomy courses was obtained via faculty questionnaires. RESULT: A variety of themes were identified at the different schools, correlated with different anatomy formats and elements. Results indicate that the courses that offer hands-on cadaveric dissections may play an important role in triggering these sentiments. DISCUSSION: The initiation of students' sentiments about the topic of death varies and includes several themes. There can be a connection to the way anatomy is taught, particularly if hands-on comprehensive cadaveric dissection or prosections are included. CONCLUSION: In summary, anatomy courses can initiate students' thinking about life's passing - particularly in schools that offer hands-on cadaveric dissections or prosections.

Demystifying dominant species.

The pattern of a few abundant species and many rarer species is a defining characteristic of communities worldwide. These abundant species are often referred to as dominant species. Yet, despite their importance, the term dominant species is poorly defined and often used to convey different information by different authors. Based on a review of historical and contemporary definitions we develop a synthetic definition of dominant species. This definition incorporates the relative local abundance of a species, its ubiquity across the landscape, and its impact on community and ecosystem properties. A meta-analysis of removal studies shows that the loss of species identified as dominant by authors can significantly impact ecosystem functioning and community structure. We recommend two metrics that can be used jointly to identify dominant species in a given community and provide a roadmap for future avenues of research on dominant species. In our review, we make the case that the identity and effects of dominant species on their environments are key to linking patterns of diversity to ecosystem function, including predicting impacts of species loss and other aspects of global change on ecosystems.

Morphological Plant Modeling: Unleashing Geometric and Topological Potential within the Plant Sciences.

The geometries and topologies of leaves, flowers, roots, shoots, and their arrangements have fascinated plant biologists and mathematicians alike. As such, plant morphology is inherently mathematical in that it describes plant form and architecture with geometrical and topological techniques. Gaining an understanding of how to modify plant morphology, through molecular biology and breeding, aided by a mathematical perspective, is critical to improving agriculture, and the monitoring of ecosystems is vital to modeling a future with fewer natural resources. In this white paper, we begin with an overview in quantifying the form of plants and mathematical models of patterning in plants. We then explore the fundamental challenges that remain unanswered concerning plant morphology, from the barriers preventing the prediction of phenotype from genotype to modeling the movement of leaves in air streams. We end with a discussion concerning the education of plant morphology synthesizing biological and mathematical approaches and ways to facilitate research advances through outreach, cross-disciplinary training, and open science. Unleashing the potential of geometric and topological approaches in the plant sciences promises to transform our understanding of both plants and mathematics.

Reshaping Plant Biology: Qualitative and Quantitative Descriptors for Plant Morphology.

An emerging challenge in plant biology is to develop qualitative and quantitative measures to describe the appearance of plants through the integration of mathematics and biology. A major hurdle in developing these metrics is finding common terminology across fields. In this review, we define approaches for analyzing plant geometry, topology, and shape, and provide examples for how these terms have been and can be applied to plants. In leaf morphological quantifications both geometry and shape have been used to gain insight into leaf function and evolution. For the analysis of cell growth and expansion, we highlight the utility of geometric descriptors for understanding sepal and hypocotyl development. For branched structures, we describe how topology has been applied to quantify root system architecture to lend insight into root function. Lastly, we discuss the importance of using morphological descriptors in ecology to assess how communities interact, function, and respond within different environments. This review aims to provide a basic description of the mathematical principles underlying morphological quantifications.

Integrating succession and community assembly perspectives.

Succession and community assembly research overlap in many respects, such as through their focus on how ecological processes like dispersal, environmental filters, and biotic interactions influence community structure. Indeed, many recent advances have been made by successional studies that draw on modern analytical techniques introduced by contemporary community assembly studies. However, community assembly studies generally lack a temporal perspective, both on how the forces structuring communities might change over time and on how historical contingency (e.g. priority effects and legacy effects) and complex transitions (e.g. threshold effects) might alter community trajectories. We believe a full understanding of the complex interacting processes that shape community dynamics across large temporal scales can best be achieved by combining concepts, tools, and study systems into an integrated conceptual framework that draws upon both succession and community assembly theory.

HLA micropolymorphisms strongly affect peptide-MHC multimer-based monitoring of antigen-specific CD8+ T cell responses.

Peptide-MHC (pMHC) multimers have become one of the most widely used tools to measure Ag-specific T cell responses in humans. With the aim of understanding the requirements for pMHC-based personalized immunomonitoring, in which individuals expressing subtypes of the commonly studied HLA alleles are encountered, we assessed how the ability to detect Ag-specific T cells for a given peptide is affected by micropolymorphic differences between HLA subtypes. First, analysis of a set of 10 HLA-A*02:01-restricted T cell clones demonstrated that staining with pMHC multimers of seven distinct subtypes of the HLA-A*02 allele group was highly variable and not predicted by sequence homology. Second, to analyze the effect of minor sequence variation in a clinical setting, we screened tumor-infiltrating lymphocytes of an HLA-A*02:06 melanoma patient with either subtype-matched or HLA-A*02:01 multimers loaded with 145 different melanoma-associated Ags. This revealed that of the four HLA-A*02:06-restricted melanoma-associated T cell responses observed in this patient, two responses were underestimated and one was overlooked when using subtype-mismatched pMHC multimer collections. To our knowledge, these data provide the first demonstration of the strong effect of minor sequence variation on pMHC-based personalized immunomonitoring, and they provide tools to prevent this issue for common variants within the HLA-A*02 allele group.

Conditional ligands for Asian HLA variants facilitate the definition of CD8+ T-cell responses in acute and chronic viral diseases.

Conditional ligands have enabled the high-throughput production of human leukocyte antigen (HLA) libraries that present defined peptides. Immunomonitoring platforms typically concentrate on restriction elements associated with European ancestry, and such tools are scarce for Asian HLA variants. We report 30 novel irradiation-sensitive ligands, specifically targeting South East Asian populations, which provide 93, 63, and 79% coverage for HLA-A, -B, and -C, respectively. Unique ligands for all 16 HLA types were constructed to provide the desired soluble HLA product in sufficient yield. Peptide exchange was accomplished for all variants as demonstrated by an ELISA-based MHC stability assay. HLA tetramers with redirected specificity could detect antigen-specific CD8(+) T-cell responses against human cytomegalovirus, hepatitis B (HBV), dengue virus (DENV), and Epstein-Barr virus (EBV) infections. The potential of this population-centric HLA library was demonstrated with the characterization of seven novel T-cell epitopes from severe acute respiratory syndrome coronavirus, HBV, and DENV. Posthoc analysis revealed that the majority of responses would be more readily identified by our unbiased discovery approach than through the application of state-of-the-art epitope prediction. This flow cytometry-based technology therefore holds considerable promise for monitoring clinically relevant antigen-specific T-cell responses in populations of distinct ethnicity.

Invasion of an intact plant community: the role of population versus community level diversity.

To improve the understanding of how native plant diversity influences invasion, we examined how population and community diversity may directly and indirectly be related to invasion in a natural field setting. Due to the large impact of the dominant C(4) grass species (Andropogon gerardii) on invasion resistance of tallgrass prairie, we hypothesized that genetic diversity and associated traits within a population of this species would be more strongly related to invasion than diversity or traits of the rest of the community. We added seeds of the exotic invasive C(4) grass, A. bladhii, to 1-m(2) plots in intact tallgrass prairie that varied in genetic diversity of A. gerardii and plant community diversity, but not species richness. We assessed relationships among genetic diversity and traits of A. gerardii, community diversity, community aggregated traits, resource availability, and early season establishment and late-season persistence of the invader using structural equation modeling (SEM). SEM models suggested that community diversity likely enhanced invasion indirectly through increasing community aggregated specific leaf area as a consequence of more favorable microclimatic conditions for seedling establishment. In contrast, neither population nor community diversity was directly or indirectly related to late season survival of invasive seedlings. Our research suggests that while much of diversity-invasion research has separately focused on the direct effects of genetic and species diversity, when taken together, we find that the role of both levels of diversity on invasion resistance may be more complex, whereby effects of diversity may be primarily indirect via traits and vary depending on the stage of invasion.

Engineering T cells specific for a dominant severe acute respiratory syndrome coronavirus CD8 T cell epitope.

Severe acute respiratory syndrome (SARS) is a highly contagious and life threatening disease, with a fatality rate of almost 10%. The etiologic agent is a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), with animal reservoirs found in bats and other wild animals and thus the possibility of reemergence. In this study, we first investigated at 6 years postinfection whether SARS-specific memory T cells persist in SARS-recovered individuals, demonstrating that these subjects still possess polyfunctional SARS-specific memory CD4+ and CD8+ T cells. A dominant memory CD8+ T cell response against SARS-CoV nucleocaspid protein (NP; amino acids 216 to 225) was then defined in SARS-recovered individuals carrying HLA-B*40:01, a HLA-B molecule present in approximately one-quarter of subjects of Asian ethnicities. To reconstitute such a CD8+ T cell response, we isolated the alpha and beta T cell receptors of the HLA-B*40:01-restricted SARS-specific CD8+ T cells. Using T cell receptor gene transfer, we generated SARS-specific redirected T cells from the lymphocytes of normal individuals. These engineered CD8+ T cells displayed avidity and functionality similar to that of natural SARS-specific memory CD8+ T cells. They were able to degranulate and produce gamma interferon, tumor necrosis factor alpha, and macrophage inflammatory proteins 1α and 1ß after antigenic stimulation. Since there is no effective treatment against SARS, these transduced T cells specific for an immunodominant SARS epitope may provide a new avenue for treatment during a SARS outbreak.

Stability screening of arrays of major histocompatibility complexes on combinatorially encoded flow cytometry beads.

The binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellular immune responses. We have developed a versatile high throughput cell-free method to measure MHC stability by capturing a variety of MHC products on the surface of streptavidin-coated particles followed by flow cytometry analysis. Arrays of peptide-MHC combinations, generated by exchanging conditional ligand-loaded MHC, could be probed in a single experiment, thus combining the molecular precision of biochemically purified MHCs with high content multiparametric flow cytometry-based assays. Semiquantitative determination of the peptide affinity for the restriction element could also be accomplished through competition experiments using this bead-based assay. Furthermore, the generated peptide-MHC reagents could directly be applied to antigen-specific CD8(+) T lymphocyte analysis. The combinatorial labeling of beads allowed straightforward identification by their unique fluorescent signatures and provided a convenient means for extended assay multiplexing.

Sources of diversity in T cell epitope discovery.

CD8-positive T cells respond to small antigenic peptide fragments presented on class I major histocompatibility complexes (MHCs). Those specific T cell epitopes capable of precipitating a cellular immune response are either derived from (altered) self (i.e. they are autoimmune- or cancer-associated) or come from foreign sources (i.e. they are pathogen-associated). Identification of T cell epitopes provides elementary information that can be employed in technologies that monitor and predict the likely outcome of an immune response, as well as in therapeutic and vaccine development efforts. The coexistence between host and pathogen has largely driven the diversification of both their systems of immune surveillance and their antigenic determinants, respectively. In this review, we discuss the multitude of factors that introduce diversity to the T cell response from both sides of the host-pathogen interaction. Furthermore, we provide an overview of a variety of commonly employed methods and tools to characterize class I MHC restricted antigen presentation and recent endeavors towards the harmonization of reporting data concerning T cell responses.

Patterns of selection of two North American native and nonnative populations of monkeyflower (Phrymaceae).

To better understand invasion dynamics, it is essential to determine the influence of genetics and ecology in species persistence in both native and nonnative habitats. One approach is to assess patterns of selection on floral and growth traits of individuals in both habitats. Mimulus guttatus (Phrymaceae) has a mixed mating system and grows under variable water conditions across its native and nonnative range in North America. Field investigations of patterns of selection of floral and plant size traits were conducted in two native and two nonnative populations. Field-collected seed was grown and crossed in the glasshouse using a paternal half-sib design. The resulting offspring were grown in saturated and dry-down low-water conditions and the same traits were measured in both environments. Patterns of selection varied across years in the native range. Nonnative populations exhibited selection for increased floral size, consistent with the hypothesis that selection favors larger size in nonnative habitats. In the glasshouse, we detected genetic variation for traits across population/treatment combinations. However, size hierarchy in the glasshouse was dependent on water conditions. Our results suggest that both variable selection pressures and local adaptation probably influence the persistence of both native and nonnative populations.

Large-scale, high-throughput validation of short hairpin RNA sequences for RNA interference.

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for position specific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNA interference (RNAi).