bluuebunny/crossref_metadata_2025 huggingface dataset:
The Southern Ocean mixed layer and its boundary fluxes: fine-scale observational progress and future research priorities
Authors: Swart Sebastiaan , du Plessis Marcel D. , Nicholson Sarah-Anne , Monteiro Pedro M. S. , Dove Lilian A. , Thomalla Sandy , Thompson Andrew F. , Biddle Louise C. , Edholm Johan M. , Giddy Isabelle , Heywood Karen J. , Lee Craig , Mahadevan Amala , Shilling Geoff , de Souza Ronald Buss
Published: May 2023

Interactions between the upper ocean and air-ice-ocean fluxes in the Southern Ocean play a critical role in global climate by impacting the overturning circulation and oceanic heat and carbon uptake. Remote and challenging conditions have led to sparse observational coverage, while ongoing field programmes often fail to collect sufficient information in the right place or at the time-space scales required to constrain the variability occurring in the coupled ocean-atmosphere system. Only within the last 10 years have we been able to directly observe and assess the role of the fine-scale ocean and rapidly evolving atmospheric marine boundary layer on the upper limb of the Southern Ocean's overturning circulation. This review summarizes advances in mechanistic understanding, arising in part from observational programmes using autonomous platforms, of the fine-scale processes (1–100 km, hours-seasons) influencing the Southern Ocean mixed layer and its variability. We also review progress in observing the ocean interior connections and the coupled interactions between the ocean, atmosphere and cryosphere that moderate air-sea fluxes of heat and carbon. Most examples provided are for the ice-free Southern Ocean, while major challenges remain for observing the ice-covered ocean. We attempt to elucidate contemporary research gaps and ongoing/future efforts needed to address them.

This article is part of a discussion meeting issue 'Heat and carbon uptake in the Southern Ocean: the state of the art and future priorities'.
Sparse observations induce large biases in estimates of the global ocean CO
2
sink: an ocean model subsampling experiment
Authors: Hauck Judith , Nissen Cara , Landschützer Peter , Rödenbeck Christian , Bushinsky Seth , Olsen Are
Published: May 2023

Estimates of ocean

CO

2

uptake from global ocean biogeochemistry models and

p

CO

2

-based data products differ substantially, especially in high latitudes and in the trend of the

CO

2

uptake since 2000. Here, we assess the effect of data sparsity on two

p

CO

2

-based estimates by subsampling output from a global ocean biogeochemistry model. The estimates of the ocean

CO

2

uptake are improved from a sampling scheme that mimics present-day sampling to an ideal sampling scheme with 1000 evenly distributed sites. In particular, insufficient sampling has given rise to strong biases in the trend of the ocean carbon sink in the

p

CO

2

products. The overestimation of the

CO

2

flux trend by 20–35% globally and 50–130% in the Southern Ocean with the present-day sampling is reduced to less than

15
%

with the ideal sampling scheme. A substantial overestimation of the decadal variability of the Southern Ocean carbon sink occurs in one product and appears related to a skewed data distribution in

p

CO

2

space. With the ideal sampling, the bias in the mean

CO

2

flux is reduced from 9–12% to 2–9% globally and from 14–26% to 5–17% in the Southern Ocean. On top of that, discrepancies of about

0.4
 

PgC
 
yr

−
1

(15%) persist due to uncertainties in the gas-exchange calculation.

This article is part of a discussion meeting issue ‘Heat and carbon uptake in the Southern Ocean: the state of the art and future priorities’.
Process controlling iron–manganese regulation of the Southern Ocean biological carbon pump
Authors: Anugerahanti Prima , Tagliabue Alessandro
Published: May 2023

Iron (Fe) is a key limiting nutrient driving the biological carbon pump and is routinely represented in global ocean biogeochemical models. However, in the Southern Ocean, the potential role for other micronutrients has not received the same attention. For example, although manganese (Mn) is essential to photosynthetic oxygen production and combating oxidative stress, it is not included in ocean models and a clear understanding of its interaction with Fe in the region is lacking. This is especially important for the Southern Ocean because both Mn and Fe are strongly depleted. We use a hierarchical modelling approach to explore how the physiological traits associated with Fe and Mn contribute to driving the footprint of micronutrient stress across different phytoplankton functional types (PFTs). We find that PFT responses are driven by physiological traits associated with their physiological requirements and acclimation to environmental conditions. Southern Ocean-specific adaptations to prevailing low Fe, such as large photosynthetic antenna sizes, are of major significance for the regional biological carbon pump. Other traits more strongly linked to Mn, such as dealing with oxidative stress, may become more important under a changing Fe supply regime.

This article is part of a discussion meeting issue ‘Heat and carbon uptake in the Southern Ocean: the state of the art and future priorities’.
A clearer view of Southern Ocean air–sea interaction using surface heat flux asymmetry
Authors: Josey Simon A. , Grist Jeremy P. , Mecking Jennifer V. , Moat Ben I. , Schulz Eric
Published: May 2023

Progress in understanding Southern Ocean heat exchange and wind forcing is discussed and new results presented. These include a metric of the zonal asymmetry between surface ocean heat gain in the Atlantic/Indian sector and heat loss in the Pacific sector. The asymmetry arises from an intersector variation in the humidity gradient between the sea surface and near-surface atmosphere. This gradient increases by 60% in the Pacific sector enabling a 20 Wm−2stronger latent heat loss compared with the Atlantic/Indian sector. The new metric is used for intercomparison of atmospheric reanalyses and CMIP6 climate simulations. CMIP6 has weaker Atlantic/Indian sector heat gain compared with the reanalyses primarily due to Indian Ocean sector differences. The potential for surface flux buoys to provide an observation-based counterpart to the asymmetry metric is explored. Over the past decade, flux buoys have been deployed at two sites (south of Tasmania and upstream of Drake Passage). The data record provided by these moorings is assessed and an argument developed for a third buoy to sample the Atlantic/Indian sector of the asymmetry metric. To close, we assess evidence that the main westerly wind belt has strengthened and moved southward in recent decades using the ERA5 reanalysis.

This article is part of a discussion meeting issue 'Heat and carbon uptake in the Southern Ocean: the state of the art and future priorities'.
Southern Ocean phytoplankton dynamics and carbon export: insights from a seasonal cycle approach
Authors: Thomalla Sandy J. , Du Plessis Marcel , Fauchereau Nicolas , Giddy Isabelle , Gregor Luke , Henson Stephanie , Joubert Warren R. , Little Hazel , Monteiro Pedro M. S. , Mtshali Thato , Nicholson Sarah , Ryan-Keogh Thomas J. , Swart Sebastiaan
Published: May 2023

Quantifying the strength and efficiency of the Southern Ocean biological carbon pump (BCP) and its response to predicted changes in the Earth's climate is fundamental to our ability to predict long-term changes in the global carbon cycle and, by extension, the impact of continued anthropogenic perturbation of atmospheric CO
2
. There is little agreement, however, in climate model projections of the sensitivity of the Southern Ocean BCP to climate change, with a lack of consensus in even the direction of predicted change, highlighting a gap in our understanding of a major planetary carbon flux. In this review, we summarize relevant research that highlights the important role of fine-scale dynamics (both temporal and spatial) that link physical forcing mechanisms to biogeochemical responses that impact the characteristics of the seasonal cycle of phytoplankton and by extension the BCP. This approach highlights the potential for integrating autonomous and remote sensing observations of fine scale dynamics to derive regionally optimized biogeochemical parameterizations for Southern Ocean models. Ongoing development in both the observational and modelling fields will generate new insights into Southern Ocean ecosystem function for improved predictions of the sensitivity of the Southern Ocean BCP to climate change.

This article is part of a discussion meeting issue 'Heat and carbon uptake in the Southern Ocean: the state of the art and future priorities'.
Finale: impact of the ORCHESTRA/ENCORE programmes on Southern Ocean heat and carbon understanding
Authors: Meijers Andrew J. S. , Meredith Michael P. , Shuckburgh Emily F. , Kent Elizabeth C. , Munday David R. , Firing Yvonne L. , King Brian , Smyth Tim J. , Leng Melanie J. , George Nurser A. J. , Hewitt Helene T. , Povl Abrahamsen E. , Weiss Alexandra , Yang Mingxi , Bell Thomas G. , Alexander Brearley J. , Boland Emma J. D. , Jones Daniel C. , Josey Simon A. , Owen Robyn P. , Grist Jeremy P. , Blaker Adam T. , Biri Stavroula , Yelland Margaret J. , Pimm Ciara , Zhou Shenjie , Harle James , Cornes Richard C.
Published: May 2023

The 5-year Ocean Regulation of Climate by Heat and Carbon Sequestration and Transports (ORCHESTRA) programme and its 1-year extension ENCORE (ENCORE is the National Capability ORCHESTRA Extension) was an approximately 11-million-pound programme involving seven UK research centres that finished in March 2022. The project sought to radically improve our ability to measure, understand and predict the exchange, storage and export of heat and carbon by the Southern Ocean. It achieved this through a series of milestone observational campaigns in combination with model development and analysis. Twelve cruises in the Weddell Sea and South Atlantic were undertaken, along with mooring, glider and profiler deployments and aircraft missions, all contributing to measurements of internal ocean and air–sea heat and carbon fluxes. Numerous forward and adjoint numerical experiments were developed and supported by the analysis of coupled climate models. The programme has resulted in over 100 peer-reviewed publications to date as well as significant impacts on climate assessments and policy and science coordination groups. Here, we summarize the research highlights of the programme and assess the progress achieved by ORCHESTRA/ENCORE and the questions it raises for the future.

This article is part of a discussion meeting issue ‘Heat and carbon uptake in the Southern Ocean: the state of the art and future priorities’.
Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis inCaenorhabditis elegans
Authors: Warnhoff Kurt , Bhattacharya Sushila , Snoozy Jennifer , Breen Peter C. , Ruvkun Gary
Published: May 2023

Dedicated genetic pathways regulate cysteine homeostasis. For example, high levels of cysteine activate cysteine dioxygenase, a key enzyme in cysteine catabolism in most animal and many fungal species. The mechanism by which cysteine dioxygenase is regulated is largely unknown. In an unbiased genetic screen for mutations that activate cysteine dioxygenase (cdo-1) in the nematodeC. elegans,we isolated loss-of-function mutations inrhy-1andegl-9,which encode proteins that negatively regulate the stability or activity of the oxygen-sensing hypoxia inducible transcription factor (hif-1). EGL-9 and HIF-1 are core members of the conserved eukaryotic hypoxia response. However, we demonstrate that the mechanism of HIF-1-mediated induction ofcdo-1is largely independent of EGL-9 prolyl hydroxylase activity and the von Hippel-Lindau E3 ubiquitin ligase, the classical hypoxia signaling pathway components. We demonstrate thatC. elegans cdo-1is transcriptionally activated by high levels of cysteine andhif-1.hif-1-dependent activation ofcdo-1occurs downstream of an H2S-sensing pathway that includesrhy-1, cysl-1,andegl-9. cdo-1transcription is primarily activated in the hypodermis where it is also sufficient to drive sulfur amino acid metabolism. Thus, the regulation ofcdo-1byhif-1reveals a negative feedback loop that maintains cysteine homeostasis. High levels of cysteine stimulate the production of an H2S signal. H2S then acts through therhy-1/cysl-1/egl-9signaling pathway to increase HIF-1-mediated transcription ofcdo-1,promoting degradation of cysteine via CDO-1.
Exchange between compartments regulates steady states and stochastic switching of a multisite phosphorylation network
Authors: Schmidt Hannah N. , Leopin Emily E. , Abel Steven M.
Published: May 2023

The phosphoregulation of proteins with multiple phosphorylation sites is governed by biochemical reaction networks that can exhibit multistable behavior. However, the behavior of such networks is typically studied in a single reaction volume, while cells are spatially organized into compartments that can exchange proteins. In this work, we use stochastic simulations to study the impact of compartmentalization on a two-site phosphorylation network. We characterize steady states and fluctuation-driven transitions between them as a function of the rate of protein exchange between two compartments. Surprisingly, the rate of stochastic switching between states depends nonmonotonically on the protein exchange rate, with the highest rate of switching occurring at intermediate exchange rates. At sufficiently small exchange rates, the state of the system and rate of switching are controlled largely by fluctuations in the balance of enzymes in each compartment. This leads to negatively correlated states in the compartments. For large exchange rates, the two compartments behave as a single effective compartment. However, when the compartmental volumes are unequal, the behavior differs from a single compartment with the same total volume. These results demonstrate that exchange of proteins between distinct compartments can regulate the emergent behavior of a common signaling motif.
Benchmarking splice variant prediction algorithms using massively parallel splicing assays
Authors: Smith Cathy , Kitzman Jacob O.
Published: May 2023

Background

Variants that disrupt mRNA splicing account for a sizable fraction of the pathogenic burden in many genetic disorders, but identifying splice-disruptive variants (SDVs) beyond the essential splice site dinucleotides remains difficult. Computational predictors are often discordant, compounding the challenge of variant interpretation. Because they are primarily validated using clinical variant sets heavily biased to known canonical splice site mutations, it remains unclear how well their performance generalizes.

Results

We benchmarked eight widely used splicing effect prediction algorithms, leveraging massively parallel splicing assays (MPSAs) as a source of experimentally determined ground-truth. MPSAs simultaneously assay many variants to nominate candidate SDVs. We compared experimentally measured splicing outcomes with bioinformatic predictions for 3,616 variants in five genes. Algorithms’ concordance with MPSA measurements, and with each other, was lower for exonic than intronic variants, underscoring the difficulty of identifying missense or synonymous SDVs. Deep learning-based predictors trained on gene model annotations achieved the best overall performance at distinguishing disruptive and neutral variants. Controlling for overall call rate genome-wide, SpliceAI and Pangolin also showed superior overall sensitivity for identifying SDVs. Finally, our results highlight two practical considerations when scoring variants genome-wide: finding an optimal score cutoff, and the substantial variability introduced by differences in gene model annotation, and we suggest strategies for optimal splice effect prediction in the face of these issues.

Conclusion

SpliceAI and Pangolin showed the best overall performance among predictors tested, however, improvements in splice effect prediction are still needed especially within exons.
Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma
Authors: Hofman Damon A. , Ruiz-Orera Jorge , Yannuzzi Ian , Murugesan Rakesh , Brown Adam , Clauser Karl R. , Condurat Alexandra L. , van Dinter Jip T. , Engels Sem A.G. , Goodale Amy , van der Lugt Jasper , Abid Tanaz , Wang Li , Zhou Kevin N. , Vogelzang Jayne , Ligon Keith L. , Phoenix Timothy N. , Roth Jennifer A. , Root David E. , Hubner Norbert , Golub Todd R. , Bandopadhayay Pratiti , van Heesch Sebastiaan , Prensner John R.
Published: May 2023

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames. To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a step-wise approach to employ multiple CRISPR-Cas9 screens to elucidate functional non-canonical ORFs implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream open reading frames (uORFs) exhibited selective functionality independent of the main coding sequence. One of these, ASNSD1-uORF or ASDURF, was upregulated, associated with the MYC family oncogenes, and was required for medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future cancer genomics studies seeking to define new cancer targets.

Highlights

Ribo-seq reveals widespread translation of non-canonical ORFs in medulloblastoma

High-resolution CRISPR tiling reveals uORF functions in medulloblastoma

ASNSD1-uORF controls downstream pathways with the prefoldin-like complex

ASNSD1-uORF is necessary for medulloblastoma cell survival
Legume-nodulating rhizobia are widespread in soils and plants across the island of O‘ahu, Hawai‘i
Authors: Abe Jonathan N. A. , Dhungana Ishwora , Nguyen Nhu H.
Published: May 2023

Legumes and their interaction with rhizobia represent one of the most well-characterized symbioses that are widespread across both natural and agricultural environments. However, larger distribution patterns and host associations on isolated Pacific islands with many native and introduced hosts have not been well-documented. Here, we used molecular and culturing techniques to characterize rhizobia from soils and 24 native and introduced legume species on the island of O’ahu, Hawai’i. We chose two of these isolates to inoculated an endemic legume tree,Erythina sandwicensisto measure nodulation potentials and host benefits. We found that all rhizobia genera can be found in the soil, where onlyCupriaviduswas found at all sites, although at lower abundance relative to other more common genera such as Rhizobium (and close relatives),Bradyzhizobium, andDevosia. Bradyrhizobiumwas the most common nodulator of plants, where the strainBradyrhizobiumsp. strain JA1 is a generalist capable of forming nodules on nine different host species, including two native species. In greenhouse nursery inoculations, the two differentBradyrhizobiumstrains successfully nodulate the endemicE. sandwicensis;both strains equally and significantly increased seedling biomass in nursery inoculations. Overall, this work provides a molecular-based framework in which to study potential native and introduced rhizobia on one of the most isolated archipelagos on the planet.
Mapping the Morphology of DNA on Carbon Nanotube-Based Sensors in Solution using X-ray Scattering Interferometry
Authors: Rosenberg Daniel J. , Cunningham Francis J. , Hubbard Joshua D. , Goh Natalie S. , Wang Jeffrey Wei-Ting , Hayman Emily B. , Hura Greg L. , Landry Markita P. , Pinals Rebecca L.
Published: May 2023

Single-walled carbon nanotubes (SWCNTs) with adsorbed single-stranded DNA (ssDNA) are applied as sensors to investigate biological systems, with applications ranging from clinical diagnostics to agricultural biotechnology. Unique ssDNA sequences render SWCNTs selectively responsive to target analytes. However, it remains unclear how the ssDNA conformation on the SWCNT surface contributes to their ultimate functionality, as observations have been constrained to computational models or experiments under dehydrated states that differ substantially from the aqueous biological environments in which the nanosensors are applied. Herein, we demonstrate a direct mode of measuring in-solution ssDNA geometries on SWCNTs via X-ray scattering interferometry (XSI), which leverages the interference pattern produced by AuNP tags conjugated to ssDNA on the SWCNT surface. We employ XSI to quantify distinct surface-adsorbed morphologies for two ssDNA oligomer lengths, conformational changes as a function of ionic strength, and the mechanism of dopamine sensing for a previously established ssDNA-SWCNT nanosensor, with correspondingab initiomodeling for visualization. We show that the shorter oligomer, (GT)6, adopts a highly ordered structure of stacked rings along the SWCNT axis, compared to the longer, less periodic (GT)15wrapping. The presence of dopamine elicits a simultaneous axial elongation and radial constriction of the ssDNA closer to the SWCNT surface. Application of XSI to probe solution-phase morphologies of nanoparticle-based tools will yield insights into sensing mechanisms and inform future design strategies for polymer-functionalized SWCNT technologies.
Colonic mucosal associated invariant T cells in Crohn’s disease have a diverse and non-public T cell receptor beta chain repertoire
Authors: Konecny Andrew J , Shows Donna M , Lord James D
Published: May 2023

Objectives

Mucosal-Associated Invariant T (MAIT) cells are T cells with a semi-invariant T cell receptor (TCR), recognizing riboflavin precursors presented by a non-polymorphic MR1 molecule. As these precursors are produced by the gut microbiome, we characterized the frequency, phenotype and clonality of MAIT cells in human colons with and without Crohn’s disease (CD).

Methods

The transcriptome of MAIT cells sorted from blood and intestinal lamina propria cells from colectomy recipients were compared with other CD8+T cells. Colon biopsies from an additional ten CD patients and ten healthy controls (HC) were analyzed by flow cytometry. TCR genes were sequenced from individual MAIT cells from these biopsies and compared with those of MAIT cells from autologous blood.

Results

MAIT cells in the blood and colon showed a transcriptome distinct from other CD8 T cells, with more expression of the IL-23 receptor. MAIT cells were enriched in the colons of CD patients, with less NKG2D in inflamed versus uninflamed segments. Regardless of disease, most MAIT cells expressed integrin αEβ7 in the colon but not in the blood, where they were enriched for α4β7 expression. TCR sequencing revealed heterogeneity in the colon and blood, with few public sequences associated with cohorts.

Conclusion

MAIT cells are enriched in the colons of CD patients and disproportionately express molecules (IL-23R, integrin α4β7) targeted by CD therapeutics, to suggest a pathogenic role for them in CD. Public TCR sequences were neither common nor sufficiently restricted to a cohort to suggest protective or pathogenic antigen-specificities.
The Coviral Portal: Multi-Cohort Viral Loads and Antigen-Test Virtual Trials for COVID-19
Authors: Morgan Alexandra , Contreras Elisa , Yasuda Michie , Dutta Sanjucta , Hamel Donald , Shankar Tarini , Balallo Diane , Riedel Stefan , Kirby James E. , Kanki Phyllis J. , Arnaout Ramy
Published: May 2023

Background

Regulatory approval of new over-the-counter tests for infectious agents such as SARS-CoV-2 has historically required that clinical trials include diverse groups of specific patient populations, making the approval process slow and expensive. Showing that populations do not differ in their viral loads—the key factor determining test performance—could expedite the evaluation of new tests.

Methods

46,726 RT-qPCR-positive SARS-CoV-2 viral loads were annotated with patient demographics and health status. Real-world performance of two commercially available antigen tests was evaluated over a wide range of viral loads. An open-access web portal was created allowing comparisons of viral-load distributions across patient groups and application of antigen-test performance characteristics to patient distributions to predict antigen-test performance on these groups.

Findings

In several cases distributions were surprisingly similar where a difference was expected (e.g. smokers vs. non-smokers); in other cases there was a difference that was the opposite direction from expectations (e.g. higher in patients who identified as White vs. Black). Sensitivity and specificity of antigen tests for detecting contagiousness were similar across most groups. The portal is athttps://arnaoutlab.org/coviral/.

Conclusions

In silico analyses of large-scale, real-world clinical data repositories can serve as a timely evidence-based proxy for dedicated trials of antigen tests for specific populations. Free availability of richly annotated data facilitates large-scale hypothesis generation and testing.

Funding

Funded by the Reagan-Udall Foundation for the FDA (RA and JEK) and via a Novel Therapeutics Delivery Grant from the Massachusetts Life Sciences Center (JEK).
Imputed Gene Expression versus Single Nucleotide Polymorphism in Predicting Gray Matter Phenotypes
Authors: Chen Jiayu , Fu Zening , Iraji Armin , Calhoun Vince D. , Liu Jingyu
Published: May 2023

Genetics plays an important role in psychiatric disorders. A clinically relevant question is whether we can predict psychiatric traits from genetics, which holds promise for early detection and tailored intervention. Imputed gene expression, also known as genetically-regulated expression (GRE), reflects the tissue-specific regulatory effects of multiple single nucleotide polymorphisms (SNPs) on genes. In this work, we explored the utility of GRE for trait association studies and how the GRE-based polygenic risk score (gPRS) compared with SNP-based PRS (sPRS) in predicting psychiatric traits. A total of 13 schizophrenia-related gray matter networks identified in another study served as the target brain phenotypes for assessing genetic associations and prediction accuracies in 34,149 individuals from the UK Biobank cohort. GRE was computed leveraging MetaXcan and GTEx tools for 56,348 genes across 13 available brain tissues. We then estimated the effects of individual SNPs and genes separately on each tested brain phenotype in the training set. The effect sizes were then used to compute gPRS and sPRS in the testing set, whose correlations with the brain phenotypes were used to assess the prediction accuracy. The results showed that, with the testing sample size set to 1,138, for training sample sizes from 1,138 up to 33,011, overall both gPRS and sPRS successfully predicted the brain phenotypes with significant correlations observed in the testing set, and higher accuracies noted for larger training sets. In addition, gPRS outperformed sPRS by showing significantly higher prediction accuracies across 13 brain phenotypes, with greater improvement noted for training sample sizes below ∼15,000. These findings support that GRE may serve as the primary genetic variable in brain phenotype association and prediction studies. Future imaging genetic studies may consider GRE as an option depending on the available sample size.
Distinct lung cell signatures define the temporal evolution of diffuse alveolar damage in fatal COVID-19
Authors: Milross Luke , Hunter Bethany , McDonald David , Merces George , Thompson Amanda , Hilkens Catharien M.U. , Wills John , Rees Paul , Jiwa Kasim , Cooper Nigel , Majo Joaquim , Ashwin Helen , Duncan Christopher J.A. , Kaye Paul M. , Bayraktar Omer Ali , Filby Andrew , Fisher Andrew J.
Published: May 2023

Background

Lung damage in severe COVID-19 is highly heterogeneous however studies with dedicated spatial distinction of discrete temporal phases of diffuse alveolar damage (DAD) and alternate lung injury patterns are lacking. Existing studies have also not accounted for progressive airspace obliteration in cellularity estimates. We used an imaging mass cytometry (IMC) analysis with a novel airspace correction step to more accurately identify the cellular immune response that underpins the heterogeneity of severe COVID-19 lung disease.

Methods

Lung tissue was obtained at post-mortem from severe COVID-19 deaths. Pathologist-selected regions of interest (ROIs) were chosen by light microscopy representing the patho-evolutionary spectrum of DAD and alternate disease phenotypes were selected for comparison. Architecturally normal SARS-CoV-2-positive lung tissue and tissue from SARS-CoV-2-negative donors served as controls. ROIs were stained for 40 cellular protein markers and ablated using IMC before segmented cells were classified. Cell populations corrected by ROI airspace and their spatial relationships were compared across lung injury patterns.

Results

Forty patients (32M:8F, age:22-98), 345 ROIs and >900k single cells were analysed. DAD progression was marked by airspace obliteration and significant increases in mononuclear phagocytes (MnPs), T and B lymphocytes and significant decreases in alveolar epithelial and endothelial cells. Neutrophil populations proved stable overall although several interferon-responding subsets demonstrated expansion. Spatial analysis revealed immune cell interactions occur prior to microscopically appreciable tissue injury.

Conclusions

The immunopathogenesis of severe DAD in COVID-19 lung disease is characterised by sustained increases in MnPs and lymphocytes with key interactions occurring even prior to lung injury is established.
PiE: An open-source pipeline for home cage behavioral analysis
Authors: Benedict Jessie , Cudmore Robert H
Published: May 2023

Over the last two decades a growing number of neuroscience labs are conducting behavioral assays in rodents. The equipment used to collect this behavioral data must effectively limit environmental and experimenter disruptions, to avoid confounding behavior data. Proprietary behavior boxes are expensive, offer limited compatible sensors, and constrain analysis with closed-source hardware and software. Here, we introduce PiE, an open-source, end-to-end, user-configurable, scalable, and inexpensive behavior assay system. The PiE system includes the custom-built behavior box to hold a home cage, as well as software enabling continuous video recording and individual behavior box environmental control. To limit experimental disruptions, the PiE system allows the control and monitoring of all aspects of a behavioral experiment using a remote web browser, including real-time video feeds. To allow experiments to scale up, the PiE system provides a web interface where any number of boxes can be controlled, and video data easily synchronized to a remote location. For the scoring of behavior video data, the PiE system includes a standalone desktop application that streamlines the blinded manual scoring of large datasets with a focus on quality control and assay flexibility. The PiE system is ideal for all types of behavior assays in which video is recorded. Users are free to use individual components of this setup independently, or to use the entire pipeline from data collection to analysis. Alpha testers have included scientists without prior coding experience. An example pipeline is demonstrated with the PiE system enabling the user to record home cage maternal behavior assays, synchronize the resulting data, conduct blinded scoring, and import the data into R for data visualization and analysis.
A disease-associated gene desert orchestrates macrophage inflammatory responses via ETS2
Authors: Stankey CT , Bourges C , Turner-Stokes T , Piedade AP , Palmer-Jones C , Papa I , Dos Silva dos Santos M , Randzavola LO , Speidel L , Parkes EC , Edwards W , Rochford AP , Murray CD , MacRae JI , Skoglund P , Wallace C , Cader MZ , Thomas DC , Lee JC
Published: May 2023

Increasing global rates of autoimmune and inflammatory disease present a burgeoning threat to human health1. This is compounded by the limited efficacy of available treatments1and high failure rates during drug development2– underscoring an urgent need to better understand disease mechanisms. Here we show how genetics could address this challenge. By investigating an intergenic haplotype on chr21q22, independently linked to inflammatory bowel disease (IBD), ankylosing spondylitis, primary sclerosing cholangitis and Takayasu’s arteritis3–6, we discover that the causal gene,ETS2, is a master regulator of inflammatory responses in human macrophages and delineate how the risk haplotype increasesETS2expression. Genes regulated by ETS2 were prominently expressed in affected tissues from chr21q22-associated diseases and more enriched for IBD GWAS hits than almost all previously described pathways. OverexpressingETS2in resting macrophages produced an activated effector state that phenocopied intestinal macrophages from IBD7, with upregulation of multiple drug targets including TNFα and IL-23. Using a database of cellular signatures8, we identify drugs that could modulate this pathway and validate the potent anti-inflammatory activity of one class of small moleculesin vitroandex vivo. Together, this highlights the potential for common genetic associations to improve both the understanding and treatment of human disease.
Conserved cardiolipin-mitochondrial ADP/ATP carrier interactions assume distinct structural and functional roles that are clinically relevant
Authors: Senoo Nanami , Chinthapalli Dinesh K. , Baile Matthew G. , Golla Vinaya K. , Saha Bodhisattwa , Ogunbona Oluwaseun B. , Saba James A. , Munteanu Teona , Valdez Yllka , Whited Kevin , Chorev Dror , Alder Nathan N. , May Eric R. , Robinson Carol V. , Claypool Steven M.
Published: May 2023

The mitochondrial phospholipid cardiolipin (CL) promotes bioenergetics via oxidative phosphorylation (OXPHOS). Three tightly bound CLs are evolutionarily conserved in the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) which resides in the inner mitochondrial membrane and exchanges ADP and ATP to enable OXPHOS. Here, we investigated the role of these buried CLs in the carrier using yeast Aac2 as a model. We introduced negatively charged mutations into each CL-binding site of Aac2 to disrupt the CL interactions via electrostatic repulsion. While all mutations disturbing the CL-protein interaction destabilized Aac2 monomeric structure, transport activity was impaired in a pocket-specific manner. Finally, we determined that a disease-associated missense mutation in one CL-binding site in ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.
Elucidating the structural basis for ligand binding and translocation in conserved insect odorant receptor co-receptors
Authors: Pacalon Jody , Audic Guillaume , Magnat Justine , Philip Manon , Golebiowski Jérôme , Moreau Christophe J. , Topin Jérémie
Published: May 2023

In numerous insects, the olfactory receptor family forms a unique class of heteromeric cation channels. Recent progress in resolving the odorant receptor structures offers unprecedented opportunities for deciphering their molecular mechanisms of ligand recognition. Unexpectedly, these structures in apo or ligand-bound states did not reveal the pathway taken by the ligands between the extracellular space and the deep internal cavities. By combining molecular modeling with electrophysiological recordings, we identified amino acids involved in the dynamic entry pathway and the binding of VUAA1 toDrosophila melanogaster’s odorant receptor co-receptor (Orco). Our results provide new evidence for the exact location of the agonist binding site and a detailed and original mechanism of ligand translocation controlled by a network of conserved residues. These findings would explain the particularly high selectivity of Orcos for their ligands.
CRISPR/Cas9-mediated mutagenesis of the Asian Citrus Psyllid,Diaphorina citri
Authors: Chaverra-Rodriguez Duverney , Bui Michelle , Gilleland Cody L. , Rasgon Jason L. , Akbari Omar S.
Published: May 2023

The most devastating disease affecting the global citrus industry is Huanglongbing (HLB), caused by the pathogenCandidatus Liberibacter asiaticus. HLB is primarily spread by the insect vectorDiaphorina citri(Asian Citrus Psyllid). To counteract the rapid spread of HLB byD. citri, traditional vector control strategies such as insecticide sprays, the release of natural predators, and mass introductions of natural parasitoids are used. However, these methods alone have not managed to contain the spread of disease. To further expand the available tools forD. citricontrol via generating specific modifications of theD. citrigenome, we have developed protocols for CRISPR/Cas9-based genetic modification. Until now, genome editing inD. citrihas been challenging due to the general fragility and size ofD.citrieggs. Here we present optimized methods for collecting and preparing eggs to introduce the Cas9 ribonucleoprotein (RNP) into early embryos and alternative methods (ReMOT Control) for injecting RNP into the hemocoel of adult females for ovarian transduction. Using these methods, we have generated visible somatic mutations, indicating their suitability for gene editing inD. citri. These methods represent the first steps towards advancingD. citriresearch in preparation for future genetic-based systems for controlling HLB.
B. subtilisMutS2 splits stalled ribosomes into subunits without mRNA cleavage
Authors: Park Esther , Mackens-Kiani Timur , Berhane Rebekah , Esser Hanna , Erdenebat Chimeg , Burroughs A. Maxwell , Berninghausen Otto , Aravind L. , Beckmann Roland , Green Rachel , Buskirk Allen R.
Published: May 2023

Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. InE. coli, these pathways are triggered by ribosome collisions through recruitment of SmrB, a nuclease that cleaves the mRNA. InB. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains and reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination ofin vivoandin vitroapproaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation by the ribosome quality control pathway. Notably, we see no evidence of mRNA cleavage by MutS2, nor does it promote ribosome rescue by tmRNA as SmrB cleavage does inE. coli. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue inB. subtilisand raise questions about how these pathways function differently in various bacteria.
Structural basis ofγ-chain family receptor sharing at the membrane level
Authors: Cai Tiantian , Lenoir Capello Rachel , Pi Xiong , Wu Hao , Chou James J.
Published: May 2023

The common γ-chain (γc) family of cytokine receptors, including interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, are activated upon engagement with the common γc receptor in ligand dependent manner. Sharing of γc by the IL receptors (ILRs) is thought to be achieved by concomitant binding of γc and ILR ectodomains to a cytokine. Here, we found that direct interactions between the transmembrane domain (TMD) of γc and those of the ILRs are also required for receptor activation, and remarkably, the same γc TMD can specifically recognize multiple ILR TMDs of diverse sequences. Heterodimer structures of γc TMD bound to the TMDs of IL-7R and IL-9R, determined in near lipid bilayer environment, reveal a conserved knob-into-hole mechanism of recognition that mediates receptor sharing within the membrane. Functional mutagenesis data indicate the requirement of the heterotypic interactions of TMDs in signaling, which could explain disease mutations within the receptor TMDs.

One-Sentence Summary

The transmembrane anchors of interleukin receptors of the gamma-chain family are critical for receptor sharing and activation.
Lipid modification of DNA nanocages enhances cellular uptake, migration, andin vivouptake
Authors: Kansara Krupa , Singh Ramesh , Yadav Pankaj , Kumar Ashutosh , Bhatia Dhiraj
Published: May 2023

The extraordinary self-assembling nature of DNA nanostructures and high functionality enables the formulation of DNA nanostructures with multiple chemical and biological molecules. How the whole organisms in native as well as modified; and how stable they are inside take up exactly these modified DNA nanostructures the organisms still remains to be explored. Here we report the fabrication and evaluation of a new conjugate of a cationic lipid, N-[one-(two, 3-dioleyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTMA) and DNA tetrahedron nanostructure (TdN) for the enhanced uptake, stability, bioimaging, and biotherapeutics in cells and zebrafish (Danio rerio) eleuthero embryos as a model organism. We summarise the enhanced uptake potential of TdN-DOTMA conjugate for futuristic biomedical applications such as drug delivery, bioimaging, biosensing, and therapeutics.
Hypoxia-induced CTCF mediates alternative splicing via coupling chromatin looping and RNA Pol II pause to promote EMT in breast cancer
Authors: Kakani Parik , Dhamdhere Shruti G , Pant Deepak , Mishra Jharna , Samaiya Atul , Shukla Sanjeev
Published: May 2023

Cancer cells experiencing hypoxic stress employ Epithelial-Mesenchymal Transition (EMT) to undergo metastasis through rewiring chromatin landscape, epigenetics, and importantly alternative splicing. Here, we investigated the role of CTCF, chromatin, epigenetic and alternative splicing modulator under hypoxia to promote EMT. Our result shows that hypoxia-induced epigenetic changes upregulate CTCF expression in breast cancer. We delineate that CTCF is a direct target of HIF1α and the hypoxia-induced HIF1α-CTCF axis functionally contributes to promoting EMT in breast cancer cells. Finally, our work uncovers COL5A1, an EMT gene, as a direct target of CTCF. We demonstrated that hypoxia-mediated CTCF enrichment on COL5A1 promoter regulates expression as well as alternative splicing events to promote EMT. Here, we put forward an intricate mechanism of alternative splicing where CTCF-mediated promoter-exon upstream looping regulates DNA de-methylation and CTCF-mediated RNA Pol II pausing at COL5A1 exon 64A. Our global analysis of CTCF-ChIP-seq data reveals that the hypoxia-induced differential CTCF occupancy possibly regulates gene expression and alternative splicing events of many genes that are enriched in the EMT pathway, cancer cell motility and invasion, angiogenesis, and stemness under hypoxia, similarly to the proposed model. Finally, we employed the epigenetic modulator dCas9-DNMT3A system to specifically disrupt HIF1α or CTCF binding under hypoxia and hence the HIF1α-CTCF-COL5A1exon64A axis that alleviates the EMT potential of breast cancer cells that may represent a novel therapeutic target in breast cancer.
Autoacetylation-mediated phase separation of TIP60 is critical for its functions
Authors: Dubey Shraddha , Gupta Himanshu , Gupta Ashish
Published: May 2023

TIP60 is an important lysine acetyl transferase protein that participates in various essential cellular activities by catalyzing the post-translational acetylation of lysine residues on histones and various non-histone protein substrates. TIP60 typically localizes to the nucleus in a punctate foci pattern, although defining factors and mechanisms regulating the assembly of TIP60 foci and their spatial distribution inside the nucleus are not understood. In the present study, we report that TIP60 can undergo phase separation to form liquid like droplets in the nuclear compartment, which is facilitated by the presence of an intrinsically disordered region (IDR) located between its chromodomain and catalytic domain. Importantly, we identified that autoacetylation on lysine 187, located within the IDR region of TIP60, is important for nuclear localization, oligomer formation and phase separation. Finally, we observed that the phase separation of TIP60 promotes its interaction with its partner proteins and actively contribute to its cellular functions.
Combinatorial quantification of 5mC and 5hmC at individual CpG dyads and the transcriptome in single cells reveals modulators of DNA methylation maintenance fidelity
Authors: Chialastri Alex , Sarkar Saumya , Schauer Elizabeth E. , Lamba Shyl , Dey Siddharth S.
Published: May 2023

Transmission of 5-methylcytosine (5mC) from one cell generation to the next plays a key role in regulating cellular identity in mammalian development and diseases. While recent work has shown that the activity of DNMT1, the protein responsible for the stable inheritance of 5mC from mother to daughter cells, is imprecise; it remains unclear how the fidelity of DNMT1 is tuned in different genomic and cell state contexts. Here we describe Dyad-seq, a method that combines enzymatic detection of modified cytosines with nucleobase conversion techniques to quantify the genome-wide methylation status of cytosines at the resolution of individual CpG dinucleotides. We find that the fidelity of DNMT1-mediated maintenance methylation is directly related to the local density of DNA methylation, and for genomic regions that are lowly methylated, histone modifications can dramatically alter the maintenance methylation activity. Further, to gain deeper insights into the methylation and demethylation turnover dynamics, we extended Dyad-seq to quantify all combinations of 5mC and 5-hydroxymethylcytosine (5hmC) at individual CpG dyads to show that TET proteins preferentially hydroxymethylate only one of the two 5mC sites in a symmetrically methylated CpG dyad rather than sequentially convert both 5mC to 5hmC. To understand how cell state transitions impact DNMT1-mediated maintenance methylation, we scaled the method down and combined it with the measurement of mRNA to simultaneously quantify genome-wide methylation levels, maintenance methylation fidelity and the transcriptome from the same cell (scDyad&T-seq). Applying scDyad&T-seq to mouse embryonic stem cells transitioning from serum to 2i conditions, we observe dramatic and heterogenous demethylation and the emergence of transcriptionally distinct subpopulations that are closely linked to the cell-to-cell variability in loss of DNMT1-mediated maintenance methylation activity, with regions of the genome that escape 5mC reprogramming retaining high levels of maintenance methylation fidelity. Overall, our results demonstrate that while distinct cell states can substantially impact the genome-wide activity of the DNA methylation maintenance machinery, locally there exists an intrinsic relationship between DNA methylation density, histone modifications and DNMT1-mediated maintenance methylation fidelity that is independent of cell state.