Biological nitrogen fixation maintains carbon/nitrogen balance and photosynthesis at elevated CO2.

Understanding crop responses to elevated CO2 is necessary to meet increasing agricultural demands. Crops may not achieve maximum potential yields at high CO2 due to photosynthetic downregulation, often associated with nitrogen limitation. Legumes have been proposed to have an advantage at elevated CO2 due to their ability to exchange carbon for nitrogen. Here, the effects of biological nitrogen fixation (BNF) on the physiological and gene expression responses to elevated CO2 were examined at multiple nitrogen levels by comparing alfalfa mutants incapable of nitrogen fixation to wild-type. Elemental analysis revealed a role for BNF in maintaining shoot carbon/nitrogen (C/N) balance under all nitrogen treatments at elevated CO2, whereas the effect of BNF on biomass was only observed at elevated CO2 and the lowest nitrogen dose. Lower photosynthetic rates at were associated with the imbalance in shoot C/N. Genome-wide transcriptional responses were used to identify carbon and nitrogen metabolism genes underlying the traits. Transcription factors important to C/N signalling were identified from inferred regulatory networks. This work supports the hypothesis that maintenance of C/N homoeostasis at elevated CO2 can be achieved in plants capable of BNF and revealed important regulators in the underlying networks including an alfalfa (Golden2-like) GLK ortholog.

Are Rural Areas Holdouts in the Second Demographic Transition? Evidence From Canada and the United States.

A central premise of the first demographic transition theory is that demographic change would occur more slowly in rural than urban areas. Few studies, however, have investigated whether rural areas remain holdouts during the second demographic transition. To address this gap, this study (1) examines trends among rural and urban families in Canada and the United States over a 30-year period and (2) determines whether compositional differences in demographic, socioeconomic, and religious factors explain current differences between rural and urban families. We find that rural Canadian women continue to have, on average, 0.6 more children than urban women. However, rural families do not trail behind urban families on any other indicator of family change. In fact, rural women in both countries are now significantly more likely to cohabit and roughly 10 percentage points more likely to have children outside of marriage than urban women. These differences are largely explained by lower levels of education and income among rural American women and fewer immigrants in rural Canada. Examining family change through a rural-urban lens fills important empirical gaps and yields novel insights into current debates on the fundamental causes of ongoing family change in high-income countries.

Nitrogen sensing and regulatory networks: it's about time and space.

A plant's response to external and internal nitrogen signals/status relies on sensing and signaling mechanisms that operate across spatial and temporal dimensions. From a comprehensive systems biology perspective, this involves integrating nitrogen responses in different cell types and over long distances to ensure organ coordination in real time and yield practical applications. In this prospective review, we focus on novel aspects of nitrogen (N) sensing/signaling uncovered using temporal and spatial systems biology approaches, largely in the model Arabidopsis. The temporal aspects span: transcriptional responses to N-dose mediated by Michaelis-Menten kinetics, the role of the master NLP7 transcription factor as a nitrate sensor, its nitrate-dependent TF nuclear retention, its "hit-and-run" mode of target gene regulation, and temporal transcriptional cascade identified by "network walking." Spatial aspects of N-sensing/signaling have been uncovered in cell type-specific studies in roots and in root-to-shoot communication. We explore new approaches using single-cell sequencing data, trajectory inference, and pseudotime analysis as well as machine learning and artificial intelligence approaches. Finally, unveiling the mechanisms underlying the spatial dynamics of nitrogen sensing/signaling networks across species from model to crop could pave the way for translational studies to improve nitrogen-use efficiency in crops. Such outcomes could potentially reduce the detrimental effects of excessive fertilizer usage on groundwater pollution and greenhouse gas emissions.

Polyvictimization, polystrengths, and their contribution to subjective well-being and posttraumatic growth.

OBJECTIVE: The negative biopsychosocial outcomes associated with exposure to victimization are well-known, however, limited research has examined the protective factors that can enhance well-being and growth following polyvictimization from in-person and digital sources. This study examines the contribution of adversities and a range of psychological and social strengths on perceptions of subjective well-being and posttraumatic growth (PTG). METHOD: A sample of 478 individuals aged 12-75 (57.5% female; Mage = 36.44) from a largely rural Appalachian region of the United States completed a survey on victimization experiences, other adversities, psychosocial strengths, subjective well-being, and PTG. RESULTS: Approximately 93.3% of individuals reported at least one digital or in-person victimization, with 82.8% reporting two or more forms of victimization. Hierarchical logistic regression analyses indicated that strengths explained more than three times the variance in subjective well-being and PTG compared to adversities, with both models explaining about half of the variance in these outcomes (49% and 50%, respectively). Psychological endurance, sense of purpose, teacher support, and polystrengths were significantly associated with better well-being and/or PTG. CONCLUSION: Some strengths hold more promise than others for promoting well-being and PTG following polyvictimization. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

First report of severe tolpyralate sensitivity in corn (Zea mays) discovers a novel genetic factor conferring crop response to a herbicide.

BACKGROUND: Tolpyralate, a relatively new inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPPD), is registered for postemergence use in all types of corn (Zea mays L.) and has a record of excellent crop tolerance. A report of severe crop injury to sweet corn inbred (XSEN187) led to the following objectives: (i) determine whether sensitivity to tolpyralate in XSEN187 exists, and if confirmed, (ii) determine the genetic basis of tolpyralate sensitivity, and (iii) screen other corn germplasm for sensitivity to tolpyralate. RESULTS: Inbred XSEN187 was confirmed sensitive to tolpyralate. Inclusion of methylated seed oil or nonionic surfactant in the spray volume was necessary for severe crop injury. Tolpyralate sensitivity in XSEN187 is not conferred by alleles at Nsf1, a cytochrome P450-encoding gene (CYP81A9) conferring tolerance to many corn herbicides. Evidence suggests that tolpyralate sensitivity in XSEN187 is conferred by a single gene mapped to the Chr05: 283 240-1 222 909 bp interval. Moreover, tolpyralate sensitivity was observed in 48 other sweet corn and field corn inbreds. CONCLUSIONS: Severe sensitivity to tolpyralate exists in sweet corn and field corn germplasm when the herbicide is applied according to label directions. Whereas the corn response to several other herbicides, including HPPD-inhibitors, is conferred by the Nsf1 locus, corn sensitivity to tolpyralate is the result of a different locus. The use of tolpyralate should consider herbicide tolerance in inbred lines from which corn hybrids were derived, whereas alleles that render corn germplasm sensitive to tolpyralate should be eliminated from breeding populations, inbreds, and commercial cultivars. © 2023 Illinois Foundation Seeds, Inc and The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

DamID-seq: A Genome-Wide DNA Methylation Method that Captures Both Transient and Stable TF-DNA Interactions in Plant Cells.

Capturing the dynamic and transient interactions of a transcription factor (TF) with its genome-wide targets whose regulation leads to plants' adaptation to their changing environment is a major technical challenge. This is a widespread problem with biochemical methods such as chromatin immunoprecipitation-sequencing (ChIP-seq) which are biased towards capturing stable TF-target gene interactions. Herein, we describe how DNA adenine methyltransferase identification and sequencing (DamID-seq) can be used to capture both transient and stable TF-target interactions by DNA methylation. The DamID technique uses a TF protein fused to a DNA adenine methyltransferase (Dam) from E. coli. When expressed in a plant cell, the Dam-TF fusion protein will methylate adenine (A) bases near the sites of TF-DNA interactions. In this way, DamID results in a permanent, stable DNA methylation mark on TF-target gene promoters, even if the target gene is only transiently "touched" by the Dam-TF fusion protein. Here we provide a step-by-step protocol to perform DamID-seq experiments in isolated plant cells for any Dam-TF fusion protein of interest. We also provide information that will enable researchers to analyze DamID-seq data to identify TF-binding sites in the genome. Our protocol includes instructions for vector cloning of the Dam-TF fusion proteins, plant cell protoplast transfections, DamID preps, library preparation, and sequencing data analysis. The protocol outlined in this chapter is performed in Arabidopsis thaliana, however, the DamID-seq workflow developed in this guide is broadly applicable to other plants and organisms.

Building High-Confidence Gene Regulatory Networks by Integrating Validated TF-Target Gene Interactions Using ConnecTF.

Many methods are now available to identify or predict the target genes of transcription factors (TFs) in plants. These include experimental approaches such as in vivo or in vitro TF-target gene-binding assays and various methods for identifying regulated targets in mutants, transgenics, or isolated plant cells. In addition, computational approaches are used to infer TF-target gene interactions from the regulatory elements or gene expression changes across treatments. While each of these approaches has now been applied to a large number of TFs from many species, each method has its own limitations which necessitates that multiple data types are integrated to build the most accurate representation of the gene regulatory networks operating in plants. To make the analyses of TF-target interaction datasets available to the broader research community, we have developed the ConnecTF web platform ( https://connectf.org/ ). In this chapter, we describe how ConnecTF can be used to integrate validated and predicted TF-target gene interactions in order to dissect the regulatory role of TFs in developmental and stress response pathways. Using as our examples KN1 and RA1, two well-characterized maize TFs involved in developing floral tissue, we demonstrate how ConnecTF can be used to (1) compare the target genes between TFs, (2) identify direct vs. indirect targets by combining TF-binding and TF-regulation datasets, (3) chart and visualize network paths between TFs and their downstream targets, and (4) prune inferred user networks for high-confidence predicted interactions using validated TF-target gene data. Finally, we provide instructions for setting up a private version of ConnecTF that enables research groups to store and analyze their own TF-target gene interaction datasets.

Rapid RGR-dependent visual pigment recycling is mediated by the RPE and specialized Müller glia.

In daylight, demand for visual chromophore (11-cis-retinal) exceeds supply by the classical visual cycle. This shortfall is compensated, in part, by the retinal G-protein-coupled receptor (RGR) photoisomerase, which is expressed in both the retinal pigment epithelium (RPE) and in Müller cells. The relative contributions of these two cellular pools of RGR to the maintenance of photoreceptor light responses are not known. Here, we use a cell-specific gene reactivation approach to elucidate the kinetics of RGR-mediated recovery of photoreceptor responses following light exposure. Electroretinographic measurements in mice with RGR expression limited to either cell type reveal that the RPE and a specialized subset of Müller glia contribute both to scotopic and photopic function. We demonstrate that 11-cis-retinal formed through photoisomerization is rapidly hydrolyzed, consistent with its role in a rapid visual pigment regeneration process. Our study shows that RGR provides a pan-retinal sink for all-trans-retinal released under sustained light conditions and supports rapid chromophore regeneration through the photic visual cycle.

Persistent disparities in affordable rental housing among America's ethnoracial groups.

Housing and residential outcomes in the United States are significantly stratified by ethnoracial group, but the extent to which disparities exist in affordable renting over time is less clear. In this study, I explore affordable housing disparities among White, Black, Hispanic, and Asian renters and test hypotheses regarding variation by education, local ethnoracial composition, and chosen measurement of affordability. Chiefly, I find that White households have higher rates of affordable housing than Black and Hispanic households with disparities remaining nearly identical between 2005 and 2019 and become larger when considering household's ability to afford other basic needs. Nevertheless, returns to education are not uniformly larger for White renters, in that Black and Asian renters experience larger marginal increases in residual income based affordable housing at higher levels of education. The effects of county ethnoracial composition effects are consistent with all groups-including White households-experiencing declining affordability when living in counties with large coethnic populations.

The TARGET System: Rapid Identification of Direct Targets of Transcription Factors by Gene Regulation in Plant Cells.

The TARGET system allows for the rapid identification of direct regulated gene targets of transcription factors (TFs). It employs the transient transformation of plant protoplasts with inducible nuclear entry of the TF and subsequent transcriptomic and/or ChIP-seq analysis. The ability to separate direct TF-target gene regulatory interactions from indirect downstream responses and the significantly shorter amount of time required to perform the assay, compared to the generation of transgenics, make this plant cell-based approach a valuable tool for a higher throughput approach to identify the genome-wide targets of multiple TFs, to build validated transcriptional networks in plants. Here, we describe the use of the TARGET system in Arabidopsis seedling root protoplasts to map the gene regulatory network downstream of transcription factors-of-interest.

Expression and subcellular localization of USH1C/harmonin in human retina provides insights into pathomechanisms and therapy.

Usher syndrome (USH) is the most common form of hereditary deaf-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinae uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Müller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones and outer segments (OS) of rods as well as at adhesive junctions between MGCs and photoreceptor cells (PRCs) in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in PRCs and MGCs and rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. We also demonstrate that primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our studies thus provide novel insights into PRC cell biology, USH1C pathophysiology and development of gene therapy treatment(s).

Validation of a high-confidence regulatory network for gene-to-NUE phenotype in field-grown rice.

Nitrogen (N) and Water (W) - two resources critical for crop productivity - are becoming increasingly limited in soils globally. To address this issue, we aim to uncover the gene regulatory networks (GRNs) that regulate nitrogen use efficiency (NUE) - as a function of water availability - in Oryza sativa, a staple for 3.5 billion people. In this study, we infer and validate GRNs that correlate with rice NUE phenotypes affected by N-by-W availability in the field. We did this by exploiting RNA-seq and crop phenotype data from 19 rice varieties grown in a 2x2 N-by-W matrix in the field. First, to identify gene-to-NUE field phenotypes, we analyzed these datasets using weighted gene co-expression network analysis (WGCNA). This identified two network modules ("skyblue" & "grey60") highly correlated with NUE grain yield (NUEg). Next, we focused on 90 TFs contained in these two NUEg modules and predicted their genome-wide targets using the N-and/or-W response datasets using a random forest network inference approach (GENIE3). Next, to validate the GENIE3 TF→target gene predictions, we performed Precision/Recall Analysis (AUPR) using nine datasets for three TFs validated in planta. This analysis sets a precision threshold of 0.31, used to "prune" the GENIE3 network for high-confidence TF→target gene edges, comprising 88 TFs and 5,716 N-and/or-W response genes. Next, we ranked these 88 TFs based on their significant influence on NUEg target genes responsive to N and/or W signaling. This resulted in a list of 18 prioritized TFs that regulate 551 NUEg target genes responsive to N and/or W signals. We validated the direct regulated targets of two of these candidate NUEg TFs in a plant cell-based TF assay called TARGET, for which we also had in planta data for comparison. Gene ontology analysis revealed that 6/18 NUEg TFs - OsbZIP23 (LOC_Os02g52780), Oshox22 (LOC_Os04g45810), LOB39 (LOC_Os03g41330), Oshox13 (LOC_Os03g08960), LOC_Os11g38870, and LOC_Os06g14670 - regulate genes annotated for N and/or W signaling. Our results show that OsbZIP23 and Oshox22, known regulators of drought tolerance, also coordinate W-responses with NUEg. This validated network can aid in developing/breeding rice with improved yield on marginal, low N-input, drought-prone soils.

How a Medical Orchestra Cultivates Creativity, Joy, Empathy, and Connection.

Background: Inspired by research indicating that exposure to humanities correlates with reduced burnout, the Nebraska Medical Orchestra was founded in 2018 as a collaboration between the University of Nebraska Medical Center and the University of Nebraska at Omaha School of Music. Methods: Semistructured interviews about orchestra participants' experiences were conducted with 9 musicians and recorded and transcribed. Transcripts were analyzed using the constant comparative method. Results: The interviews suggested that participants are drawn to the orchestra to pursue a love of music, to be part of an ensemble, and to connect with others in an environment that provides a lighthearted, nonjudgmental, noncompetitive forum in which to create and enjoy music for its own sake. Conclusions: This study has implications for designing arts-based wellness activities for clinicians and scaling them nationwide.

Wellness Through the Lens of a Medical Orchestra.

Stress and burnout afflict medical students and professionals at alarming rates, which has led institutions to invest in counseling services and other traditional wellness programming. However, the stigma of utilizing these services permeates the medical community. This narrative explores the founding of the Nebraska Medical Orchestra-an orchestra created as a nontraditional antidote to reduce stress and burnout among health care students and professionals-and also examines the concept of wellness through interactions between the orchestra's director and health care-related musicians.

Developmental genome-wide occupancy analysis of bZIP transcription factor NRL uncovers the role of c-Jun in early differentiation of rod photoreceptors in the mammalian retina.

The basic motif-leucine zipper (bZIP) transcription factor neural retina leucine zipper (NRL) determines rod photoreceptor cell fate during retinal development, and its loss leads to cone-only retina in mice. NRL works synergistically with homeodomain protein Cone-Rod Homeobox and other regulatory factors to control the transcription of most genes associated with rod morphogenesis and functional maturation, which span over a period of several weeks in the mammalian retina. We predicted that NRL gradually establishes rod cell identity and function by temporal and dynamic regulation of stage-specific transcriptional targets. Therefore, we mapped the genomic occupancy of NRL at four stages of mouse photoreceptor differentiation by CUT&RUN analysis. Dynamics of NRL binding revealed concordance with the corresponding changes in transcriptome of the developing rods. Notably, we identified c-Jun proto-oncogene as one of the targets of NRL, which could bind to specific cis-elements in the c-Jun promoter and modulate its activity in HEK293 cells. Coimmunoprecipitation studies showed the association of NRL with c-Jun, also a bZIP protein, in transfected cells as well as in developing mouse retina. Additionally, shRNA-mediated knockdown of c-Jun in the mouse retina in vivo resulted in altered expression of almost 1000 genes, with reduced expression of phototransduction genes and many direct targets of NRL in rod photoreceptors. We propose that c-Jun-NRL heterodimers prime the NRL-directed transcriptional program in neonatal rod photoreceptors before high NRL expression suppresses c-Jun at later stages. Our study highlights a broader cooperation among cell-type restricted and widely expressed bZIP proteins, such as c-Jun, in specific spatiotemporal contexts during cellular differentiation.

Cost of Living Variation, Nonmetropolitan America, and Implications for the Supplemental Poverty Measure.

Poverty scholarship in the United States is increasingly reliant upon the Supplemental Poverty Measure (SPM) as opposed to the Official Poverty Measure of the United States for research and policy analysis. However, the SPM still faces several critiques from scholars focused on poverty in nonmetropolitan areas. Key among these critiques is the geographic adjustment for cost of living employed in the SPM, which is based solely upon median rental costs and pools together all nonmetropolitan counties within each state. Here, we evaluate the current geographic adjustment of the SPM using both microdata and aggregate data from the American Community Survey for 2014-2018. By comparing housing costs, tenure, and commuting, we determine that median rent is likely an appropriate basis for geographic adjustment. However, by demonstrating the wide variability between median rents of nonmetropolitan counties within the same state, we show that the current operationalization of this geographic adjustment could be improved through the use of more-specific categories such as metropolitan adjacency or Rural Urban Continuum Codes.

Multiomics analyses reveal early metabolic imbalance and mitochondrial stress in neonatal photoreceptors leading to cell death in Pde6brd1/rd1 mouse model of retinal degeneration.

Retinal diseases exhibit extensive genetic heterogeneity and complex etiology with varying onset and severity. Mutations in over 200 genes can lead to photoreceptor dysfunction and/or cell death in retinal neurodegeneration. To deduce molecular pathways that initiate and/or drive cell death, we adopted a temporal multiomics approach and examined molecular and cellular events in newborn and developing photoreceptors before the onset of degeneration in a widely-used Pde6brd1/rd1 (rd1) mouse, a model of autosomal recessive retinitis pigmentosa caused by PDE6B mutations. Transcriptome profiling of neonatal and developing rods from the rd1 retina revealed early downregulation of genes associated with anabolic pathways and energy metabolism. Quantitative proteomics of rd1 retina showed early changes in calcium signaling and oxidative phosphorylation, with specific partial bypass of complex I electron transfer, which precede the onset of cell death. Concurrently, we detected alterations in central carbon metabolism, including dysregulation of components associated with glycolysis, pentose phosphate and purine biosynthesis. Ex vivo assays of oxygen consumption and transmission electron microscopy validated early and progressive mitochondrial stress and abnormalities in mitochondrial structure and function of rd1 rods. These data uncover mitochondrial overactivation and related metabolic alterations as determinants of early pathology and implicate aberrant calcium signaling as an initiator of higher mitochondrial stress. Our studies thus provide a mechanistic framework with mitochondrial damage and metabolic disruptions as early drivers of photoreceptor cell death in retinal degeneration.

Testing Kissick's Iron Triangle-Structural Equation Modeling Analysis of a Practical Theory.

BACKGROUND/PURPOSE: The purpose of this research is to determine if the tradeoffs that Kissick proposed among cost containment, quality, and access remain as rigidly interconnected as originally conceived in the contemporary health care context. Although many have relied on the Kissick model to advocate for health policy decisions, to our knowledge the model has never been empirically tested. Some have called for policy makers to come to terms with the premise of the Kissick model tradeoffs, while others have questioned the model, given the proliferation of quality-enhancing initiatives, automation, and information technology in the health care industry. One wonders whether these evolutionary changes alter or disrupt the originality of the Kissick paradigms themselves. METHODS: Structural equation modeling (SEM) was used to evaluate the Kissick hypothetical relationships among the unobserved constructs of cost, quality, and access in hospitals for the year 2018. Hospital data were obtained from Definitive Healthcare, a subscription site that contains Medicare data as well as non-Medicare data for networks, hospitals, and clinics (final n = 2766). RESULTS: Reporting significant net effects as defined by our chosen study variables, we find that as quality increases, costs increase, as access increases, quality increases, and as access increases, costs increase. Policy and Practice Implications: Our findings lend continued relevance to a balanced approach to health care policy reform efforts. Simultaneously bending the health care cost curve, increasing access to care, and advancing quality of care is as challenging now as it was when the Kissick model was originally conceived.

Unequal From the Start? Poverty Across Immigrant Generations of Hispanic Children.

Recent cohorts of U.S. children increasingly consist of immigrants or the immediate descendants of immigrants, a demographic shift that has been implicated in high rates of child poverty. Analyzing data from the 2014-2018 Current Population Survey and using the U.S. Census Bureau's Supplemental Poverty Measure, we describe differences in child poverty rates across immigrant generations and assess how these disparities are rooted in generational differences in the prevalence and impact of key poverty risk factors. Our estimates show that poverty rates among Hispanic children are very high, particularly among first-generation children and second-generation children with two foreign-born parents. Low family employment is the most significant risk factor for poverty, but the prevalence of this risk varies little across immigrant generations. Differences in parental education account for the greatest share of observed intergenerational disparities in child poverty. Supplemental comparisons with third+-generation non-Hispanic White children underscore the disadvantages faced by all Hispanic children, highlighting the continued salience of race and ethnicity within the U.S. stratification system. Understanding the role of immigrant generation vis-à-vis other dimensions of inequality has significant policy implications given that America's population continues to grow more diverse along multiple social axes.

A Report Card on Prevention Efforts of COVID-19 Deaths in US.

COVID-19 (otherwise known as coronavirus disease 2019) is a life-threatening pandemic that has been combatted in various ways by the government, public health officials, and health care providers. These interventions have been met with varying levels of success. Ultimately, we question if the preventive efforts have reduced COVID-19 deaths in the United States. To address this question, we analyze data pertaining to COVID-19 deaths drawn from the Centers for Disease Control and Prevention (CDC). For this purpose, we employ incidence rate restricted Poisson (IRRP) as an underlying analysis methodology and evaluate all preventive efforts utilized to attempt to reduce COVID-19 deaths. Interpretations of analytic results and graphical visualizations are used to emphasize our various findings. Much needed modifications of the public health policies with respect to dealing with any future pandemics are compiled, critically assessed, and discussed.