Bomb radiocarbon evidence for strong global carbon uptake and turnover in terrestrial vegetation.

Vegetation and soils are taking up approximately 30% of anthropogenic carbon dioxide emissions because of small imbalances in large gross carbon exchanges from productivity and turnover that are poorly constrained. We combined a new budget of radiocarbon produced by nuclear bomb testing in the 1960s with model simulations to evaluate carbon cycling in terrestrial vegetation. We found that most state-of-the-art vegetation models used in the Coupled Model Intercomparison Project underestimated the radiocarbon accumulation in vegetation biomass. Our findings, combined with constraints on vegetation carbon stocks and productivity trends, imply that net primary productivity is likely at least 80 petagrams of carbon per year presently, compared with the 43 to 76 petagrams per year predicted by current models. Storage of anthropogenic carbon in terrestrial vegetation is likely more short-lived and vulnerable than previously predicted.

Comparison of polystyrene and hydrogel microcarriers for optical imaging of adherent cells.

Significance: The ability to observe and monitor cell density and morphology has been imperative for assessing the health of a cell culture and for producing high quality, high yield cell cultures for decades. Microcarrier-based cultures, used for large-scale cellular expansion processes, are not compatible with traditional visualization-based methods, such as widefield microscopy, due to their thickness and material composition. Aim: Here, we assess the optical imaging compatibilities of commercial polystyrene microcarriers versus custom-fabricated gelatin methacryloyl (gelMA) microcarriers for non-destructive and non-invasive visualization of the entire microcarrier surface, direct cell enumeration, and sub-cellular visualization of mesenchymal stem/stromal cells. Approach: Mie scattering and wavefront error simulations of the polystyrene and gelMA microcarriers were performed to assess the potential for elastic scattering-based imaging of adherent cells. A Zeiss Z.1 light-sheet microscope was adapted to perform light-sheet tomography using label-free elastic scattering contrast from planar side illumination to achieve optical sectioning and permit non-invasive and non-destructive, in toto, three-dimensional, high-resolution visualization of cells cultured on microcarriers. Results: The polystyrene microcarrier prevents visualization of cells on the distal half of the microcarrier using either fluorescence or elastic scattering contrast, whereas the gelMA microcarrier allows for high fidelity visualization of cell morphology and quantification of cell density using light-sheet fluorescence microscopy and tomography. Conclusions: The combination of optical-quality gelMA microcarriers and label-free light-sheet tomography will facilitate enhanced control of bioreactor-microcarrier cell culture processes.

PEARR tool training and implementation: building awareness of violence and human trafficking in a hospital system.

Introduction: Health professionals have an opportunity to assist patients who are experiencing many types of violence, including human trafficking; however, current approaches are often not person-centered. The Provide privacy, Educate, Ask, Respect and Respond (PEARR) Tool, a recognized screening tool in the U.S., is a structured conversation guide for health professionals on how to provide trauma-sensitive assistance to patients who may be experiencing such violence, including human trafficking. This is the first study to evaluate the PEARR Tool and its use in hospital settings. Methods: A U.S.-based health system adopted the PEARR Tool as part of its Abuse, Neglect, and Violence policy and procedure. To support successful adoption, the health system also developed educational modules on human trafficking and trauma-informed approaches to patient care, including a module on the PEARR steps. In October 2020 and June 2021, a voluntary "PEARR Tool Training and Implementation Survey" was distributed to emergency department staff in three hospitals. The survey consisted of 22 questions: eight demographic and occupation related questions; five questions related to the education provided to staff; and, nine questions related to the use of the PEARR Tool in identifying and assisting patients. Results: The overall findings demonstrate a general increase in awareness about the prevalence of human trafficking, as well as a significant increase in awareness about the implementation of the PEARR Tool. However, the findings demonstrate that most respondents were not utilizing the PEARR Tool between October 2020 and June 2021. Most reported that the reason for this was because they had not suspected any of their patients to be victims of abuse, neglect, or violence, including human trafficking. Of those that had utilized the PEARR Tool, there was a marked increase in staff that reported its usefulness and ease of access when caring for patients. Discussion: The COVID-19 pandemic posed many challenges during this study, including delays in staff education, changes in education format and delivery, and strains on staff. Initial data regarding the use of the PEARR Tool is promising; and additional research is recommended.

V2a neurons restore diaphragm function in mice following spinal cord injury.

The specific roles that different types of neurons play in recovery from injury is poorly understood. Here, we show that increasing the excitability of ipsilaterally projecting, excitatory V2a neurons using designer receptors exclusively activated by designer drugs (DREADDs) restores rhythmic bursting activity to a previously paralyzed diaphragm within hours, days, or weeks following a C2 hemisection injury. Further, decreasing the excitability of V2a neurons impairs tonic diaphragm activity after injury as well as activation of inspiratory activity by chemosensory stimulation, but does not impact breathing at rest in healthy animals. By examining the patterns of muscle activity produced by modulating the excitability of V2a neurons, we provide evidence that V2a neurons supply tonic drive to phrenic circuits rather than increase rhythmic inspiratory drive at the level of the brainstem. Our results demonstrate that the V2a class of neurons contribute to recovery of respiratory function following injury. We propose that altering V2a excitability is a potential strategy to prevent respiratory motor failure and promote recovery of breathing following spinal cord injury.

Agricultural intensification and childhood cancer in Brazil.

Over the last several decades, Brazil has become both the world's leading soy producer and the world's leading consumer of hazardous pesticides. Despite identified links between pesticide exposure and carcinogenesis, there has been little population-level research on the effects of pesticide intensification on broader human health in Brazil. We estimate the relationship between expanded soy production-and related community exposure to pesticides-on childhood cancer incidence using 15 y of data on disease mortality. We find a statistically significant increase in pediatric leukemia following expanded local soy production, but timely access to treatment mitigates this relationship. We show that pesticide exposure likely occurs via water supply penetration. Our findings represent only the tip of the iceberg for substantial health externalities of high-input crop production and land use change. Our results are of particular interest in developing contexts with demand for intensified food production systems and underscore the need for stronger regulation of pesticides and increased public health attention to exposure in the broader community.

Reporting reproducible imaging protocols.

A reproducible imaging protocol should include four main detailed sections. The first should describe the sample preparation and include details about the tissue and/or cell culture preparation, the staining procedure, the optical grade of the coverslip, and the type of mounting media used to mount the sample. The second section should describe the configuration and components of the microscope and include the type of stand, stage, illumination, and detector, as well as the emission (EM) and excitation (EX) filters, objective, and immersion medium specifications. Specialized microscopes may have other important components in the optical path to include. The third section should describe the settings used to acquire an image like the exposure and/or dwell time, final magnification and optical resolution, the pixel and field of view (FOV) sizes, time intervals for any time lapse, total power at the objective (i.e., directed at your sample) and number of planes and step size used to collect a 3-dimensional image, and order of operations used in multi-dimensional image acquisitions. The final section should include details about the image analysis workflow such as the image processing steps, segmentation and measurement methods used to extract information from the image, data size, and necessary computing hardware and networking requirements if data sets are >1 GB, as well as citations and versions for the software and code used to perform any of these steps. Every effort should be made to make an example dataset with accurate metadata available online. Finally, specifics about the type of replicates included in the experiment and details about the statistical analysis conducted are also necessary.

Volumetric imaging of human mesenchymal stem cells (hMSCs) for non-destructive quantification of 3D cell culture growth.

The adoption of cell-based therapies into the clinic will require tremendous large-scale expansion to satisfy future demand, and bioreactor-microcarrier cultures are best suited to meet this challenge. The use of spherical microcarriers, however, precludes in-process visualization and monitoring of cell number, morphology, and culture health. The development of novel expansion methods also motivates the advancement of analytical methods used to characterize these microcarrier cultures. A robust optical imaging and image-analysis assay to non-destructively quantify cell number and cell volume was developed. This method preserves 3D cell morphology and does not require membrane lysing, cellular detachment, or exogenous labeling. Complex cellular networks formed in microcarrier aggregates were imaged and analyzed in toto. Direct cell enumeration of large aggregates was performed in toto for the first time. This assay was successfully applied to monitor cellular growth of mesenchymal stem cells attached to spherical hydrogel microcarriers over time. Elastic scattering and fluorescence lightsheet microscopy were used to quantify cell volume and cell number at varying spatial scales. The presented study motivates the development of on-line optical imaging and image analysis systems for robust, automated, and non-destructive monitoring of bioreactor-microcarrier cell cultures.

Pipeline for fluorescent imaging and volumetric analysis of neurons in cleared mouse spinal cords.

Precisely measuring the number and somatic volume of neurons in the central nervous system at single-cell resolution is technically challenging. Here, we combine multiple techniques to address this challenge in optically cleared mouse spinal cords. We describe in vivo neuron labeling approaches, tissue-clearing technology, light sheet fluorescence microscopy, and machine learning-guided imaging analysis. This combination provides a precise determination of the cell number and somatic volume of any neuron population in the spinal cords.

Quantifiable Intravital Light Sheet Microscopy.

Live imaging of zebrafish embryos that maintains normal development can be difficult to achieve due to a combination of sample mounting, immobilization, and phototoxicity issues that, once overcome, often still results in image quality sufficiently poor that computer-aided analysis or even manual analysis is not possible. Here, we describe our mounting strategy for imaging the zebrafish midbrain-hindbrain boundary (MHB) with light sheet fluorescence microscopy (LSFM) and pilot experiments to create a study-specific set of parameters for semiautomatically tracking cellular movements in the embryonic midbrain primordium during zebrafish segmentation.

Environmental outcomes of the US Renewable Fuel Standard.

The Renewable Fuel Standard (RFS) specifies the use of biofuels in the United States and thereby guides nearly half of all global biofuel production, yet outcomes of this keystone climate and environmental regulation remain unclear. Here we combine econometric analyses, land use observations, and biophysical models to estimate the realized effects of the RFS in aggregate and down to the scale of individual agricultural fields across the United States. We find that the RFS increased corn prices by 30% and the prices of other crops by 20%, which, in turn, expanded US corn cultivation by 2.8 Mha (8.7%) and total cropland by 2.1 Mha (2.4%) in the years following policy enactment (2008 to 2016). These changes increased annual nationwide fertilizer use by 3 to 8%, increased water quality degradants by 3 to 5%, and caused enough domestic land use change emissions such that the carbon intensity of corn ethanol produced under the RFS is no less than gasoline and likely at least 24% higher. These tradeoffs must be weighed alongside the benefits of biofuels as decision-makers consider the future of renewable energy policies and the potential for fuels like corn ethanol to meet climate mitigation goals.

Dietary change in high-income nations alone can lead to substantial double climate dividend.

A dietary shift from animal-based foods to plant-based foods in high-income nations could reduce greenhouse gas emissions from direct agricultural production and increase carbon sequestration if resulting spared land was restored to its antecedent natural vegetation. We estimate this double effect by simulating the adoption of the EAT-Lancet planetary health diet by 54 high-income nations representing 68% of global gross domestic product and 17% of population. Our results show that such dietary change could reduce annual agricultural production emissions of high-income nations' diets by 61% while sequestering as much as 98.3 (55.6-143.7) GtCO2 equivalent, equal to approximately 14 years of current global agricultural emissions until natural vegetation matures. This amount could potentially fulfil high-income nations' future sum of carbon dioxide removal (CDR) obligations under the principle of equal per capita CDR responsibilities. Linking land, food, climate and public health policy will be vital to harnessing the opportunities of a double climate dividend.

Navigating the Light-Sheet Image Analysis Software Landscape: Concepts for Driving Cohesion From Data Acquisition to Analysis.

From the combined perspective of biologists, microscope instrumentation developers, imaging core facility scientists, and high performance computing experts, we discuss the challenges faced when selecting imaging and analysis tools in the field of light-sheet microscopy. Our goal is to provide a contextual framework of basic computing concepts that cell and developmental biologists can refer to when mapping the peculiarities of different light-sheet data to specific existing computing environments and image analysis pipelines. We provide our perspective on efficient processes for tool selection and review current hardware and software commonly used in light-sheet image analysis, as well as discuss what ideal tools for the future may look like.

Building a three-dimensional model of early-stage zebrafish embryo brain.

We introduce a computational approach to build three-dimensional (3D) surface mesh models of the early-stage zebrafish brain primordia from time-series microscopy images. The complexity of the early-stage brain primordia and lack of recognizable landmarks pose a distinct challenge for feature segmentation and 3D modeling. Additional difficulty arises because of noise and variations in pixel intensity. We overcome these by using a hierarchical approach in which simple geometric elements, such as "beads" and "bonds," are assigned to represent local features and their connectivity is used to smoothen the surface while retaining high-curvature regions. We apply our method to build models of two zebrafish embryo phenotypes at discrete time points between 19 and 28 h post-fertilization and collect measurements to quantify development. Our approach is fast and applicable to building models of other biological systems, as demonstrated by models from magnetic resonance images of the human fetal brain. The source code, input scripts, sample image files, and generated outputs are publicly available on GitHub.

The Sterol Trafficking Pathway in Arabidopsis thaliana.

In plants, the trafficking mechanisms by which sterols move through the plant and into target cells are unknown. Earlier studies identified endosomes as primary candidates for internalization of sterols in plants, but these results have come into question. Here, we show that in elongating root cells, the internalization of sterol occurs primarily by a non-endocytic mechanism. Added fluorescent sterols [dehydroergosterol (DHE) and BODIPY-cholesterol (BCh)] do not initially label endosomes identified by fluorescent protein markers or by internalized FM4-64. Instead, the nuclear envelope, an organelle not associated with the endocytic pathway but part of the endoplasmic reticulum (ER), becomes labeled. This result is supported by experiments with the inducible overexpression of auxilin-2-like protein (AUX2 line), which blocks most endocytosis upon induction. Internalization and nuclear envelope labeling still occur in induced AUX2 cells. Longer-term incubation labels the oil body, a site involved in sterol storage. Although the first site of localization, the nuclear envelope, is part of the ER, other domains of the ER do not accumulate the label. The trafficking pathway differs from vesicular endocytosis and points toward a different pathway of sterol transport possibly involving other mechanisms, such as ER-plasma membrane contact sites and cytoplasmic transport.

Extended focal depth Fourier domain optical coherence microscopy with a Bessel-beam - LP02 mode - from a higher order mode fiber.

We present a robust fiber-based setup for Bessel-like beam extended depth-of-focus Fourier-domain optical coherence microscopy, where the Bessel-like beam is generated in a higher order mode fiber module. In this module a stable guided LP02 core mode is selectively excited by a long period grating written in the higher order mode fiber. Imaging performance of this system in terms of lateral resolution and depth of focus was analyzed using samples of suspended microbeads and compared to the case where illumination is provided by the fundamental LP01 mode of a single mode fiber. Illumination with the LP02 mode allowed for a lateral resolution down to 2.5 µm as compared to 4.5 µm achieved with the LP01 mode of the single mode fiber. A three-fold enhancement of the depth of focus compared to a Gaussian beam with equally tight focus is achieved with the LP02 mode. Analysis of the theoretical lateral point spread functions for the case of LP01 and LP02 illumination agrees well with the experimental data. As the design space of waveguides and long-period gratings allows for further optimization of the beam parameters of the generated Bessel-like beams in an all-fiber module, this approach offers a robust and yet flexible alternative to free-space optics approaches or the use of conical fiber tips.

Cropland expansion in the United States produces marginal yields at high costs to wildlife.

Recent expansion of croplands in the United States has caused widespread conversion of grasslands and other ecosystems with largely unknown consequences for agricultural production and the environment. Here we assess annual land use change 2008-16 and its impacts on crop yields and wildlife habitat. We find that croplands have expanded at a rate of over one million acres per year, and that 69.5% of new cropland areas produced yields below the national average, with a mean yield deficit of 6.5%. Observed conversion infringed upon high-quality habitat that, relative to unconverted land, had provided over three times higher milkweed stem densities in the Monarch butterfly Midwest summer breeding range and 37% more nesting opportunities per acre for waterfowl in the Prairie Pothole Region of the Northern Great Plains. Our findings demonstrate a pervasive pattern of encroachment into areas that are increasingly marginal for production, but highly significant for wildlife, and suggest that such tradeoffs may be further amplified by future cropland expansion.