bluuebunny/crossref_metadata_2025 huggingface dataset:
Outcome Disparities by Insurance Plan and Educational Attainment in Patients with Atrial Fibrillation
Authors: Apiyasawat Sirin , Thongsri Tomon , Jongpiputvanich Kulyot , Krittayaphong Rungroj ,   for the COOL-AF Investigators
Published: April 2021

Objectives

Despite efforts to cut poverty, improve public health, and raise educational attainment, countries still suffer from disparities. Since 2002, Thailand has successfully implemented the Universal Health Coverage Scheme (UCS) to eradicate health care access inequity. Here, we explored the interlink between insurance plan, educational attainment, and adverse clinical outcomes in the national registry of patients with atrial fibrillation (AF) in Thailand.

Design

A nationwide prospective multicenter cohort of non-valvular 3402 AF.

Primary Outcomes

Patients were followed for 36 months for primary outcomes of all-cause mortality, ischemic stroke, and major bleeding. Survival analysis was performed using restricted mean survival time (RMST) and adjusted with multiple covariates.

Results

Data from 3026 AF patients (mean age 67, 59% male) were analyzed. The mean CHA2DS2VASc and HASBLED scores were 3.0 (SD 1.7) and 1.5 (SD 1.0) respectively. Most of the patients attained the elementary level of education (N=1739, 57.4%). The major health insurance plans were Civil Servant Medical Benefit Scheme (CSMBS; N=1397, 46.2%) and UCS (N=1333, 44.1%). After 36 months of follow-up, 248 patients died (8.2%), 95 suffered from ischemic stroke (3.1%), and 136 suffered from major bleeding (4.5%). AF patients with no formal education lost 1.78 months before they died (adjusted RMST difference -1.78; 95% CI, -3.25 to -0.30; P =.02) and 1.04 months before they developed ischemic stroke (adjusted RMST difference -1.04; 95% CI, -2.03 to -0.04; P =.04) compared to those with higher education. Educational attainment level was not associated with major bleeding. Across all types of insurance plan, RMSTs to all three clinical outcomes were essentially similar.

Conclusion

Education attainment level was independently associated with all-cause mortality and ischemic stroke in AF patients.

Health insurance plans were not associated with adverse clinical outcomes.

Clinical Trial Registration

Thai Clinical Trial Registration; Study ID: TCTR20160113002

Strengths and limitations of this study

The study links two of the most important of sociodemographic factors, education and health insurance plan, and clinical outcomes of atrial fibrillation (AF), one of the most common cardiovascular conditions.

Data is derived from a large prospective and nationwide cohort.

The study evaluates healthcare quality in universal health coverage system.

Causal relationship cannot be assumed from a cohort trial.

Enrollment took place in the medical center, thus, excluding patients who could not access the healthcare facilities.
Asymmetric effects of acute stress on cost and benefit learning
Authors: Voulgaropoulou Stella , Fauzani Fasya , Pfirrmann Janine , Vingerhoets Claudia , van Amelsvoort Thérèse , Hernaus Dennis
Published: April 2021

Stressful events trigger a complex physiological reaction – thefight-or-flightresponse – that can hamper flexible decision-making. Inspired by key neural and peripheral characteristics of the fight-or-flight response, here we ask whether acute stress changes how humans learn about costs and benefits. Participants were randomly exposed to an acute stress or no-stress control condition after which they completed a cost-benefit reinforcement learning task. Acute stress improved learning to maximize benefits (monetary rewards) relative to minimising energy expenditure (grip force). Using computational modelling, we demonstrate that costs and benefits can exert asymmetric effects on decisions when prediction errors that convey information about the reward value and cost of actions receive inappropriate importance; a process associated with distinct alterations in pupil size fluctuations. These results provide new insights into learning strategies under acute stress – which, depending on the context, may be maladaptive or beneficial - and candidate neuromodulatory mechanisms that could underlie such behaviour.
Deprivation and Exposure to Public Activities during the COVID-19 Pandemic in England and Wales
Authors: Beale Sarah , Braithwaite Isobel , Navaratnam Annalan M D , Hardelid Pia , Rodger Alison , Aryee Anna , Byrne Thomas , Erica Fong Wing Lam , Fragaszy Ellen , Geismar Cyril , Kovar Jana , Nguyen Vincent , Patel Parth , Shrotri Madhumita , Aldridge Robert W , Hayward Andrew , Michie Susan , Wijlaars Linda , Nastouli Eleni , Spyer Moira , Killingley Ben , Cox Ingemar , Lampos Vasileios , McKendry Rachel A , Cheng Tao , Liu Yunzhe , Johnson Anne M , Gibbs Jo , Gilson Richard
Published: April 2021

Background

Differential exposure to public activities and non-household contacts may contribute to stark deprivation-related inequalities in SARS-CoV-2 infection and outcomes, but has not been directly investigated. We set out to investigate whether participants in Virus Watch – a large community cohort study based in England and Wales – reported different levels of exposure to public activities and non-household contacts during the Autumn-Winter phase of the COVID-19 pandemic according to postcode-level socioeconomic deprivation.

Methods

Participants (n=20120-25228 across surveys) reported their daily activities during three weekly periods in late November 2020, late December 2020, and mid-February 2021. Deprivation was quantified based on participants’ postcode of residence using English or Welsh Indices of Multiple Deprivation quintiles. We used Poisson mixed effect models with robust standard errors to estimate the relationship between deprivation and risk of exposure to public activities during each survey period.

Results

Relative to participants in the least deprived areas, participants in the most deprived areas persistently exhibited elevated risk of exposure to vehicle sharing (aRR range across time points 1.73-8.52), public transport (aRR 3.13-5.73), work or education outside of the household (aRR 1.09-1.21), essential shops (aRR 1.09-1.13) and non-household contacts (aRR 1.15-1.19) across multiple survey periods.

Conclusion

Differential exposure to essential public activities in deprived communities is likely to contribute to inequalities in infection risk and outcomes during the COVID-19 pandemic. Public health interventions to reduce exposure during essential activities and financial and practical support to enable low-paid workers to stay at home during periods of intense transmission may reduce COVID-related inequalities.
Clinical validation of RCSMS: a rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva
Authors: del Prado Joaquín Abugattás Núñez , Reyes Angélica Quintana , La Torre Juan Blume , Gutiérrez Loli Renzo , Pinzón Olejua Alejandro , Chamorro Chirinos Elena Rocío , Loza Mauricio Félix Antonio , Maguiña Jorge L. , Leon Julio , Rodríguez Aliaga Piere , Málaga Trillo Edward
Published: April 2021

Early detection of SARS-CoV-2 using molecular techniques is paramount to the fight against COVID-19. Due to its high sensitivity and specificity, RT-qPCR is the “gold standard” method for this purpose. However, its technical requirements, processing time and elevated costs hamper its use towards massive and timely molecular testing for COVID-19 in rural and socioeconomically deprived areas of Latin America. The advent and rapid evolution of CRISPR-Cas technology has boosted the development of new pathogen detection methodologies. Recently, DETECTR -a combination of isothermal RT-LAMP amplification and Cas12a-mediated enzymatic detection-has been successfully validated in the Netherlands and the USA as a rapid and low-cost alternative to RT-qPCR for the detection of SARS-CoV-2 from nasopharyngeal swabs. Here, we evaluated the performance of RCSMS, a locally adapted variant of DETECTR, to ascertain the presence of SARS-CoV-2 in saliva samples from 276 patients in two hospitals in Lima, Perú (current status over a total of 350 samples). We show that a low-cost thermochemical treatment with TCEP/EDTA is sufficient to inactivate viral particles and cellular nucleases in saliva, eliminating the need to extract viral RNA with commercial kits, as well as the cumbersome nasopharyngeal swab procedure and the requirement of biosafety level 2 laboratories for molecular analyses. Our clinical validation shows that RCSMS detects up to 5 viral copies per reaction in 40 min, with sensitivity and specificity of 93.8% and 99.0% in the field, respectively, relative to RT-qPCR. Since CRISPR-Cas biosensors can be easily reprogrammed by using different guide RNA molecules, RCSMS has the potential to be quickly adapted for the detection of new SARS-CoV-2 variants. Notably, estimation of its negative and positive predictive values suggests that RCSMS can be confidently deployed in both high and low prevalence settings. Furthermore, our field study validates the use of lateral flow strips to easily visualize the presence of SARS-CoV-2, which paves the way to deploy RCSMS as a “point of care” test in environments with limited access to state-of-the-art diagnostic laboratories. In sum, RCSMS is a fast, efficient and inexpensive alternative to RT-qPCR for expanding COVID-19 testing capacity in low- and middle-income countries.
Building Back Better after COVID-19: a systematic scoping review of wicked problems affecting developed countries and implications for global governance
Authors: van Kessel Robin , Li Han Wong Brian
Published: April 2021

The COVID-19 pandemic is a textbook example of a ‘wicked problem’, one which is complex, open-ended, unpredictable, or intractable and seems resistant to any solution. This presents a window of opportunity to explore other wicked problems and their implications after the pandemic. A systematic scoping review was conducted to investigate the COVID-19 aftermath and identify public health topics which may be of great significance in the years to come. Through the adoption of three megadrivers as fundamental drivers of change (globalisation, demographic change, and digitalisation), it narratively explored how different wicked problems – and the driving mechanisms which sustain them – persist. It further explored the implications of these public health topics on global (health) governance. While the wicked problems mapped in this article show a large variance in where their apparent roots lie, they share one factor in common: health. These wicked problems must be first and foremost addressed if we as a globalised world are to successfully and sustainably build back better from COVID-19.

Summary Box

What is already known?

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COVID-19 is a textbook example of a wicked problem; a problem that is complex, open-ended, unpredictable, or intractable and seems resistant to any solution;

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Digitalisation, globalisation, and demographic change are seen as the three megadrivers of change and are theorised to create and sustain modern wicked problems;

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The megadrivers respectively have complex relationships with health; this includes both positive and negative associations.

What are the new findings?

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A series of wicked problems exist in multiple domains of society that continue to obstruct the progress of the 2030 Agenda for Sustainable Development;

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These wicked problems each have roots in the three megadrivers: digitalisation, globalisation, and demographic change;

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There exists a complex interrelation between the megadrivers, wicked problems, and health.

What do the new findings imply?

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It currently remains unclear to what extent wicked problems and megadrivers respectively affect health outcomes; therefore, further research is indicated;

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A ‘once in a generation’ opportunity has presented itself to build back better from the COVID-19 pandemic by addressing the existing wicked problems; therefore, governance mechanisms should follow and adapt accordingly;

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Wicked problems have bidirectional implications for modern policy; this has created an environment for wicked problems to manifest and sustain themselves, which in turn produce further policies that sustain wicked problems.
Detecting in-school transmission of SARS-CoV-2 from case ratios and documented clusters
Authors: Johnson Kaitlyn E. , Lachmann Michael , Stoddard Madison , Pasco Remy , Fox Spencer J. , Meyers Lauren Ancel , Chakravarty Arijit
Published: April 2021

Claims that in-person schooling has not amplified SARS-CoV-2 transmission are based on similar infection rates in schools and their surrounding communities and limited numbers of documented in-school transmission events. Simulations assuming high in-school transmission suggest that these metrics cannot exclude the possibility that transmission in schools exacerbated overall pandemic risks.
Response to Polygenic Risk: Results of the MyGeneRank Mobile Application-Based Coronary Artery Disease Study
Authors: Muse Evan D. , Chen Shang-Fu , Liu Shuchen , Fernandez Brianna , Schrader Brian , Molparia Bhuvan , León André Nicolás , Lee Raymond , Pubbi Neha , Mejia Nolan , Ren Christina , El-kalliny Ahmed , Montes de Oca Ernesto Prado , Aguilar Hector , Ghoshal Arjun , Dias Raquel , Evans Doug , Chen Kai-Yu , Zhang Paris , Wineinger Nathan E , Spencer Emily G. , Topol Eric J. , Torkamani Ali
Published: April 2021

The degree to which polygenic risk scores (PRS) influence preventive health is the subject of debate, with few prospective studies completed to date. We developed a smartphone application for the prospective and automated generation, communication, and electronic capture of response to a PRS for coronary artery disease (CAD). We evaluated self-reported actions taken in response to personal CAD PRS information, with special interest in the initiation of lipid lowering therapy (NCT03277365). 20% of high genetic risk (n=95) vs 7.9% of low genetic risk individuals (n=101) initiated lipid lowering therapy at follow-up (p-value = 0.002). The initiation of both statin and non-statin lipid lowering therapy was associated with degree of genetic risk – 15.2% (n=92) vs 6.0% (n=100) for statins (p-value = 0.018) and 6.8% (n=118) vs 1.6% (n=123) for non-statins (p-value = 0.022) in high vs low genetic risk, respectively. Overall, degree of genetic risk was associated with use of any lipid lowering therapy at follow-up - 42.4% (n=132) vs 28.5% (n=130) (p-value = 0.009). We also find that CAD PRS information is perceived to be understandable, actionable, and does not induce health anxiety.
Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes the hexameric complex
Authors: Hurdiss Daniel L. , El Kazzi Priscila , Bauer Lisa , Papageorgiou Nicolas , Ferron François P. , Donselaar Tim , van Vliet Arno L.W. , Canard Bruno , Decroly Etienne , Brancale Andrea , Zeev-Ben-Mordehai Tzviya , Förster Friedrich , van Kuppeveld Frank J.M , Coutard Bruno
Published: April 2021

The enterovirus genus encompasses many clinically important human pathogens such as poliovirus, coxsackieviruses, echoviruses, numbered enteroviruses and rhinoviruses. These viruses are the etiological agents of several human diseases, including hand-foot-and-mouth disease, neonatal sepsis, encephalitis, meningitis, paralysis and respiratory infections. There is an unmet need for antivirals to treat these diseases. The non-structural protein 2C is a AAA+ helicase and plays a key role in viral replication. As such, it is an attractive target for antiviral drug development. Several repurposing screens with FDA-approved drugs have identified 2C-targeting compounds such as fluoxetine and dibucaine, but the molecular basis of 2C inhibition has remained enigmatic. Here we present the 1.5 Å resolution crystal structure of the soluble fragment of coxsackievirus B3 2C protein in complex with (S)-fluoxetine (SFX), which reveals a conserved, hydrophobic drug-binding pocket which is distal to the ATP binding site. To decipher the molecular mechanism of inhibition by fluoxetine and other 2C-targeting compounds, we engineered a soluble, hexameric and ATPase competent 2C protein. Using this system, we show that SFX, dibucaine, HBB and guanidine hydrochloride inhibit 2C ATPase activity in a dose-dependent manner. Moreover, using cryo-EM analysis, we demonstrate that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition and allowing us to generate the first reconstruction of the oligomeric complex. Taken together, these results provide important structural and mechanistic insights into 2C inhibition and provide a robust engineering strategy which can be used for structural, functional and drug-screening analysis of 2C proteins from current or future enteroviruses.
Frontal cortex learns to add evidence across modalities
Authors: Coen Philip , Sit Timothy P.H. , Wells Miles J , Carandini Matteo , Harris Kenneth D
Published: April 2021

To make accurate perceptual decisions, the brain often combines information across sensory modalities. For instance, localizing objects by integrating their image and sound. However, the cortical substrates underlying this audiovisual integration remain uncertain. Here, we show that mouse frontal cortex combines auditory and visual evidence; that this combination is additive, mirroring behavior; and that it evolves with learning. Scanning optogenetic inactivation demonstrated that inactivating frontal cortex impaired choices based on either sensory modality. Recordings from >10,000 neurons indicated that after task learning, activity in frontal area MOs (secondary motor cortex) encodes an additive combination of visual and auditory signals, consistent with the mice’s behavioral strategy. An accumulator model applied to these sensory representations reproduced both the observed choices and reaction times. These results indicate that frontal cortex adapts through learning to combine evidence across sensory cortices, providing a signal that is transformed into a binary decision by a downstream accumulator.
SARS-CoV-2 Entry Protein TMPRSS2 and Its Homologue, TMPRSS4 Adopts Structural Fold Similar to Blood Coagulation and Complement Pathway Related Proteins
Authors: Muley Vijaykumar Yogesh , Singh Amit , Gruber Karl , Varela-Echavarría Alfredo
Published: April 2021

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes TMPRSS2 receptor to enter target human cells and subsequently causes coronavirus disease 19 (COVID-19). TMPRSS2 belongs to the type II serine proteases of subfamily TMPRSS, which is characterized by the presence of the serine-protease domain. TMPRSS4 is another TMPRSS member, which has a domain architecture similar to TMPRSS2. TMPRSS2 and TMPRSS4 have been shown to be involved in SARS-CoV-2 infection. However, their normal physiological roles have not been explored in detail. In this study, we analyzed the amino acid sequences and predicted 3D structures of TMPRSS2 and TMPRSS4 to understand their functional aspects at the protein domain level. Our results suggest that these proteins are likely to have common functions based on their conserved domain organization. Furthermore, we show that the predicted 3D structure of their serine protease domain has significant similarity to that of plasminogen which dissolves blood clot, and of other blood coagulation related proteins. Additionally, molecular docking analyses of inhibitors of four blood coagulation and anticoagulation factors show the same high specificity to TMPRSS2 and TMPRSS4 3D structures. Hence, our observations are consistent with the blood coagulopathy observed in COVID-19 patients and their predicted functions based on the sequence and structural analyses offer avenues to understand better and explore therapeutic approaches for this disease.
Sequential single cell transcriptional and protein marker profiling reveals TIGIT as a marker of CD19 CAR-T cell dysfunction in patients with non-Hodgkin’s lymphoma
Authors: Jackson Zachary , Hong Changjin , Schauner Robert , Dropulic Boro , Caimi Paolo F. , de Lima Marcos , Gupta Kalpana , Reese Jane S. , Hwang Tae Hyun , Wald David N.
Published: April 2021

Chimeric antigen receptor T cell (CAR-T cell) therapy is known to produce durable remissions in the treatment of CD19+relapsed/refractory B cell malignancies. Nonetheless, a significant portion of patients receiving the therapy experience poor outcomes in the acute response for unknown reasons. Given the decreased expansion and persistence of CD8 CAR-T cells in poor outcome groups, this failure may be attributed to CAR-T cell dysfunction. However, a comparison of the post-infusion transcriptional profiles and phenotypes between CAR-T cells of poor and favorable response groups has not been performed. Here, we employed single cell RNA sequencing and protein surface marker profiling of serial CAR-T cell blood samples from patients with CD19+relapsed/refractory non-Hodgkin’s lymphoma (NHL) to reveal CAR-T cell evolution, identify biomarkers of response, and test for evidence of exhaustion in CAR-T cells of poor responders. At the transcriptional and protein levels, we note the evolution of a majority of CAR-T cells toward a non-proliferative and highly-differentiated state. In poor outcome patients, we observed a more marked enrichment of an exhaustion profile as compared to favorable outcome patients. Lastly, we identified the checkpoint receptor TIGIT (T cell immunoreceptor with Ig and ITIM domains) as a novel prognostic biomarker and potential driver of CAR-T cell exhaustion. Altogether, we provide evidence of CAR-T cell dysfunction marked by TIGIT expression driving poor response in NHL patients.
Subcellular Organization of Viral Particles During Maturation of Nucleus-Forming Jumbo Phage
Authors: Chaikeeratisak Vorrapon , Khanna Kanika , Nguyen Katrina T. , Egan MacKennon E. , Enustun Eray , Armbruster Emily , Pogliano Kit , Villa Elizabeth , Pogliano Joe
Published: April 2021

Many eukaryotic viruses assemble mature particles within distinct subcellular compartments, but bacteriophages were long assumed to assemble randomly throughout the host cell cytoplasm. Here we visualized the subcellular location of viral particles formed during replication ofPseudomonasnucleus-forming jumbo phages and discovered that they assemble a unique structure inside cells we term phage bouquets. We show that after capsids complete DNA packaging at the surface of the phage nucleus, tails assemble and attach to the capsids, and these particles accumulate to form bouquets at specific subcellular locations. In these bouquets, the viral particles are arranged in a spherical pattern with tails oriented inward and the heads outwards. Localized at fixed distances on either side of the phage nucleus, bouquets grow in size and number over time as new phage particles are added. In the presence of mutations that cause the phage nucleus to be mispositioned away from its typical position at the midcell, bouquets still localize at the same fixed distance from the nucleus, suggesting an active mechanism for their formation and positioning. These results mark the discovery of a pathway for organizing mature viral particles inside bacteria and demonstrate that nucleus-forming jumbo phage, like most eukaryotic viruses, are highly spatially organized during all stages of their lytic cycle.
Mathematical model of the multi-amino acid multi-transporter system predicts uptake flux in CHO cells
Authors: Sreejan Ashley , Gadgil Mugdha , Gadgil Chetan J.
Published: April 2021

Chinese hamster ovary (CHO) cells express several amino acid (AA) transporters including uniporters and exchangers. Each transporter transports multiple AAs, making prediction of the effect of changed medium composition or transporter levels on individual AA transport rate challenging. A general kinetic model and a simplified analytical expression for the uptake rate is presented. A CHO cell-specific AA transport model, to our knowledge the first such network model for any cell type, is constructed. The model is validated by its prediction of reported uptake flux and amino acid inter-dependencies from experiments that were not used in model construction or parameter estimation. The model defines theoretical conditions for synergistic/repressive effect on the uptake rates of other AAs upon external addition of one AA. This model will help formulate testable hypotheses of the effect of process changes on AA initial uptake, and serve as the AA transport component of kinetic models for cellular metabolism.
Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages
Authors: Ullrich Sebastian , Arnan Carme , Pulido-Quetglas Carlos , Nurtdinov Ramil , Esteban Alexandre , Blanco-Fernandez Joan , Aparicio-Prat Estel , Johnson Rory , Pérez-Lluch Sílvia , Guigó Roderic
Published: April 2021

CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify both protein coding (pc) and non-coding genes playing a role along different processes. In particular, the usage of a nuclease active Cas9 coupled to a single gRNA has proven to efficiently impair the expression of pc-genes by generating deleterious frameshifts. Here, we first demonstrate that the usage of a second gRNA targeting the same gene synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next take advantage of our paired-guide (pgRNA) system to design a library to simultaneously target 874 pc-genes and 166 lncRNAs which are known to change expression during the transdifferentiation from pre-B cells to macrophages. We show that this system is able to identify known players in this process, and also predicts 26 potential novel ones, of which we select four for deeper characterization. Two of these,FURINandNFE2, code for proteins related to cell differentiation and macrophage function; the other two,LINC02432andMIR3945HG, are lncRNAs associated with cancerous and infectious diseases, respectively. The CRISPR-Cas9 coupled to pgRNAs system is, therefore, a suitable tool to target simultaneously pc-genes and lncRNAs for genomic perturbation assays.
JAFFAL: Detecting fusion genes with long read transcriptome sequencing
Authors: Davidson Nadia M. , Chen Ying , Sadras Teresa , Ryland Georgina L. , Blombery Piers , Ekert Paul G. , Göke Jonathan , Oshlack Alicia
Published: April 2021

Massively parallel short read transcriptome sequencing has greatly expanded our knowledge of fusion genes which are drivers of tumor initiation and progression. In cancer, many fusions are also important diagnostic markers and targets for therapy. Long read transcriptome sequencing allows the full length of fusion transcripts to be discovered, however, this data has a high rate of errors and fusion finding algorithms designed for short reads do not work. While numerous fusion finding algorithms now exist for short read RNA sequencing data, there are few methods to detect fusions using third generation or long read sequencing data. Fusion finding in long read sequencing will allow the discovery of the full isoform structure of fusion genes.

Here we present JAFFAL, a method to identify fusions from long-read transcriptome sequencing. We validated JAFFAL using simulation, cell line and patient data from Nanopore and PacBio. We show that fusions can be accurately detected in long read data with JAFFAL, providing better accuracy than other long read fusion finders and with similar performance as state-of-the-art methods applied to short read data. By comparing Nanopore transcriptome sequencing protocols we find that numerous chimeric molecules are generated during cDNA library preparation that are absent when RNA is sequenced directly. We demonstrate that JAFFAL enables fusions to be detected at the level of individual cells, when applied to long read single cell sequencing. Moreover, we demonstrate JAFFAL can identify fusions spanning three genes, highlighting the utility of long reads to characterise the transcriptional products of complex structural rearrangements with unprecedented resolution. JAFFAL is open source and available as part of the JAFFA package athttps://github.com/Oshlack/JAFFA/wiki.
Construction of a neural network energy function for protein physics
Authors: Yang Huan , Xiong Zhaoping , Zonta Francesco
Published: April 2021

Classical potentials are widely used to describe protein physics, due to their simplicity and accuracy, but they are continuously challenged as real applications become more demanding with time. Deep neural networks could help generating alternative ways of describing protein physics. Here we propose an unsupervised learning method to derive a neural network energy function for proteins. The energy function is a probability density model learned from plenty of 3D local structures which have been extensively explored by evolution. We tested this model on a few applications (assessment of protein structures, protein dynamics and protein sequence design), showing that the neural network can correctly recognize patterns in protein structures. In other words, the neural network learned some aspects of protein physics from experimental data.
Neutrophil transit time and localization within the megakaryocyte define morphologically distinct forms of emperipolesis
Authors: Huang Frank Y. , Cunin Pierre , Radtke Felix A. , Grieshaber-Bouyer Ricardo , Nigrovic Peter A.
Published: April 2021

In emperipolesis, neutrophils transit through megakaryocytes, but it is unknown whether this interaction represents a single type of cell-in-cell interaction or a set of distinct processes. Using anin vitromodel of murine emperipolesis, we characterized neutrophils entering megakaryocytes using live-cell spinning disk microscopy and electron microscopy. Approximately half of neutrophils exited the megakaryocyte rapidly, typically in 10 minutes or less, displaying ameboid morphology as they passed through the host cell (fast emperipolesis). The remaining neutrophils assumed a sessile morphology, most remaining within the megakaryocyte for at least 60 minutes (slow emperipolesis). These neutrophils typically localized near the megakaryocyte nucleus. By ultrastructural assessment, all internalized neutrophils remained morphologically intact. Most neutrophils resided within emperisomes, but some could be visualized exiting the emperisome into the cell cytoplasm. Neutrophils in the cytoplasm assumed close contact with the platelet-forming demarcation membrane system or with the perinuclear endoplasmic reticulum, as confirmed by immunofluorescence microscopy. Together, these findings reveal that megakaryocyte emperipolesis reflects at least two processes, fast and slow emperipolesis, each with its own characteristic transit time, morphology, and intracellular localization, suggesting distinct functions.

Key Points

Neutrophil passage through megakaryocytes, termed emperipolesis, diverges into fast and slow forms that differ in transit time, morphology, and intracellular localization

During emperipolesis, neutrophils can reside in vacuoles (emperisomes) or escape into the cell cytoplasm to assume positions near the megakaryocyte’s demarcation membrane system, endoplasmic reticulum, or nucleus.
Mitochondrial unfolded protein response transcription factor ATFS-1 increases resistance to exogenous stressors through upregulation of multiple stress response pathways
Authors: Soo Sonja K. , Traa Annika , Mistry Meeta , Van Raamsdonk Jeremy M.
Published: April 2021

The mitochondrial unfolded protein response (mitoUPR) is an evolutionarily conserved pathway that responds to various insults to the mitochondria through transcriptional changes that restore mitochondrial homeostasis in order to facilitate cell survival. Gene expression changes resulting from the activation of the mitoUPR are mediated by the transcription factor ATFS-1/ATF-5. To further define the mechanisms through which the mitoUPR protects the cell during mitochondrial dysfunction, we characterized the role of ATFS-1 in responding to organismal stress. We found that activation of ATFS-1 is sufficient to cause upregulation of genes involved in multiple stress response pathways, including the DAF-16-mediated stress response pathway, the SKN-1-mediated oxidative stress response pathway, the HIF-mediated hypoxia response pathway, the p38-mediated innate immune response pathway, and antioxidant genes. Moreover, ATFS-1 is required for the upregulation of stress response genes after exposure to exogenous stressors, especially oxidative stress and bacterial pathogens. Constitutive activation of ATFS-1 increases resistance to multiple acute exogenous stressors, while disruption ofatfs-1decreases stress resistance. Although ATFS-1-dependent genes are upregulated in multiple long-lived mutants, constitutive activation of ATFS-1 in wild-type animals results in decreased lifespan. Overall, our work demonstrates that ATFS-1 serves a vital role in organismal survival of acute stresses through its ability to activate multiple stress response pathways, but that chronic ATFS-1 activation is detrimental for longevity.
Partial resistance to HDAC inhibitors in FAPs of dystrophic muscles at late stages of disease is associated to epigenetic and transcriptional features of cellular senescence
Authors: Consalvi S. , Tucciarone L. , Macrì E. , De Bardi M. , Picozza M. , Salvatori I. , Renzini A. , Valente S. , Mai A. , Moresi V. , Puri P.L.
Published: April 2021

Pharmacological treatment of Duchenne Muscular Dystrophy (DMD) with histone deacetylase inhibitors (HDACi) is currently being tested in clinical trials. Pre-clinical studies performed in mdx mice - the mouse model of DMD - have shown that HDACi promote compensatory muscle regeneration, while inhibiting fibro-adipogenic degeneration, by targeting fibro-adipogenic progenitors (FAPs); however, these beneficial effects are restricted to early stages of disease progression. We show here that FAPs from late stage mdx mice exhibit epigenetic and transcriptional features of senescence that could not be fully reversed by HDACi. In particular, genome-wide increase in H3K9/14 acetylation at gene promoters of Senescence Associated Secretory Phenotype (SASP) genes was associated with their upregulation in late stage mdx FAPs. Treatment with the HDACi Trichostatin A (TSA) could inhibit SASP gene activation in FAPs, by decreasing H3K9/14 acetylation. Conversely, combinatorial decrease of H3K27 and/or H3K9/14 acetylation at promoters of genes required for cycle activation and progression was associated with their downregulation in FAPs from late stage mdx mice. However, these epigenetic and transcriptional alterations could not be reversed by TSA, due to a general resistance exhibited by FAPs from late stage mdx mice to HDACi-induced H3K9/14 hyperacetylation. Overall, this data reveal that disease-associated features of senescence develop in FAPs of DMD muscle through epigenetically distinct and pharmacologically dissociable events, and suggests that HDACi might at least retain anti- fibrotic and inflammatory activity at late stages of DMD, by repressing FAP-derived SASP.
De novo identification of universal cell mechanics gene signatures
Authors: Urbanska Marta , Ge Yan , Winzi Maria , Abuhattum Shada , Ali Syed Shafat , Herbig Maik , Kräter Martin , Toepfner Nicole , Durgan Joanne , Florey Oliver , Dori Martina , Calegari Federico , Lolo Fidel-Nicolás , del Pozo Miguel Á. , Taubenberger Anna , Cannistraci Carlo V. , Guck Jochen
Published: April 2021

Cell mechanical properties determine many physiological functions, such as cell fate specification, migration, or circulation through vasculature. Identifying factors that govern the mechanical properties is therefore a subject of great interest. Here we present a mechanomics approach for establishing links between single-cell mechanical phenotype changes and the genes involved in driving them. We combine mechanical characterization of cells across a variety of mouse and human systems with machine learning-based discriminative network analysis of associated transcriptomic profiles to infer a conserved network module of five genes with putative roles in cell mechanics regulation. We validate in silico that the identified gene markers are universal, trustworthy and specific to the mechanical phenotype across the studied mouse and human systems, and demonstrate experimentally that a selected target,CAV1, changes the mechanical phenotype of cells accordingly when silenced or overexpressed. Our data-driven approach paves the way towards engineering cell mechanical properties on demand to explore their impact on physiological and pathological cell functions.
Synthesising the multiple impacts of climatic variability on community responses to climate change
Authors: Terry J. Christopher D. , O’Sullivan Jacob D. , Rossberg Axel G.
Published: April 2021

Recent developments in understanding and predicting species responses to climate change have emphasised the importance of both environmental variability and consideration of the wider biotic community. However, to date, the interaction between the two has received less attention. Both theoretical and empirical results suggest that the combined effect of environmental variability and interspecific interactions can have strong impacts on existing range limits. Here we explore how competitive interactions and temporal variability can interact with the potential to strongly influence range shift dynamics. We highlight the need to understand these between-process interactions in order to predict how species will respond to global change. In particular, future research will need to move from evaluating possibilities to quantifying their impact. We emphasise the value and utility of empirically parameterised models to determine the direction and relative importance of these forces in natural systems.
Secondary Structure Prediction for RNA Sequences Including N6-methyladenosine
Authors: Kierzek Elzbieta , Zhang Xiaoju , Watson Richard M. , Kierzek Ryszard , Mathews David H.
Published: April 2021

There is increasing interest in the roles played by covalently modified nucleotides in mRNAs and non-coding RNAs. New high-throughput sequencing technologies localize these modifications to exact nucleotide positions. There has been, however, and inability to account for these modifications in secondary structure prediction because of a lack of software tools for handling modifications and a lack of thermodynamic parameters for modifications. Here, we report that we solved these issues for N6-methyladenosine (m6A), for the first time allowing secondary structure prediction for a nucleotide alphabet of A, C, G, U, and m6A. We revised the RNAstructure software package to work with any user-defined alphabet of nucleotides. We also developed a set of nearest neighbor parameters for helices and loops containing m6A, using a set of 45 optical melting experiments. Interestingly, N6-methylation decreases the folding stability of structures with adenosines in the middle of a helix, has little effect on the folding stability of adenosines at the ends of helices, and stabilizes the folding stability for structures with unpaired adenosines stacked on the end of a helix. The parameters were tested against an additional two melting experiments, including a consensus sequence for methylation and an m6A dangling end. The utility of the new software was tested using predictions of the structure of a molecular switch in the MALAT1 lncRNA, for which a conformation change is triggered by methylation. Additionally, human transcriptome-wide calculations for the effect of N6-methylation on the probability of an adenosine being buried in a helix compare favorably with PARS structure mapping data. Now users of RNAstructure are able to develop hypothesis for structure-function relationships for RNAs with m6A, including conformational switching triggered by methylation.
Characterization of methylation profiles in spontaneous preterm birth placental villous tissue
Authors: Brockway Heather M , Wilson Samantha L , Kallapur Suhas G , Buhimschi Catalin S , Muglia Louis J , Jones Helen N
Published: April 2021

Preterm birth is a global public health crisis which results in significant neonatal and maternal mortality. Yet little is known regarding the molecular mechanisms of idiopathic spontaneous preterm birth, and we have few diagnostic markers for adequate assessment of placental development and function. Previous studies of placental pathology and our transcriptomics studies suggest a role for placental maturity in idiopathic spontaneous preterm birth. It is known that placental DNA methylation changes over gestation. We hypothesized that if placental hypermaturity is present in our samples, we would observe a unique idiopathic spontaneous preterm birth DNA methylation profile potentially driving the gene expression differences we previously identified in our placental samples. Our results indicate the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern suggesting hypermaturity. Only seven significant differentially methylated regions fitting the idiopathic spontaneous preterm birth specific (relative to the controls) profile were identified, indicating unusually high similarity in DNA methylation between idiopathic spontaneous preterm birth and term birth samples. We identified an additional 1,718 significantly methylated regions in our gestational age matched controls were the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern, again indicating a striking level of similarity between the idiopathic spontaneous preterm birth and term birth samples. Pathway analysis of these regions revealed differences in genes within the WNT and Cadherin signaling pathways, both of which are essential in placental development and maturation. Taken together, these data demonstrate that the idiopathic spontaneous preterm birth samples are molecularly more mature than expected given their respective gestational age which likely impacts birth timing.
Determining clinically relevant features in cytometry data using persistent homology
Authors: Mukherjee Soham , Wethington Darren , Dey Tamal K. , Das Jayajit
Published: April 2021

Cytometry experiments yield high-dimensional point cloud data that is difficult to interpret manually. Boolean gating techniques coupled with comparisons of relative abundances of cellular subsets is the current standard for cytometry data analysis. However, this approach is unable to capture more subtle topological features hidden in data, especially if those features are further masked by data transforms or significant batch effects or donor-to-donor variations in clinical data. We present that persistent homology, a mathematical structure that summarizes the topological features, can distinguish different sources of data, such as from groups of healthy donors or patients, effectively. Analysis of publicly available cytometry data describing non-naïve CD8+ T cells in COVID-19 patients and healthy controls shows that systematic structural differences exist between single cell protein expressions in COVID-19 patients and healthy controls.

Our method identifies proteins of interest by a decision-tree based classifier and passes them to a kernel-density estimator (KDE) for sampling points from the density distribution. We then compute persistence diagrams from these sampled points. The resulting persistence diagrams identify regions in cytometry datasets of varying density and identify protruded structures such as ‘elbows’. We compute Wasserstein distances between these persistence diagrams for random pairs of healthy controls and COVID-19 patients and find that systematic structural differences exist between COVID-19 patients and healthy controls in the expression data for T-bet, Eomes, and Ki-67. Further analysis shows that expression of T-bet and Eomes are significantly downregulated in COVID-19 patient non-naïve CD8+ T cells compared to healthy controls. This counter-intuitive finding may indicate that canonical effector CD8+ T cells are less prevalent in COVID-19 patients than healthy controls. This method is applicable to any cytometry dataset for discovering novel insights throughtopological data analysiswhich may be difficult to ascertain otherwise with a standard gating strategy or in the presence of large batch effects.

Author summary

Identifying differences between cytometry data seen as a point cloud can be complicated by random variations in data collection and data sources. We applypersistent homologyused intopological data analysisto describe the shape and structure of the data representing immune cells in healthy donors and COVID-19 patients. By looking at how the shape and structure differ between healthy donors and COVID-19 patients, we are able to definitively conclude how these groups differ despite random variations in the data. Furthermore, these results are novel in their ability to capture shape and structure of cytometry data, something not described by other analyses.
Single-virus content mixing assay reveals cholesterol-enhanced influenza membrane fusion efficiency
Authors: Liu Katherine N. , Boxer Steven G.
Published: April 2021

In order to infect a cell, enveloped viruses must first undergo membrane fusion, which proceeds through a hemifusion intermediate, followed by the formation of a fusion pore through which the viral genome is transferred to a target cell. Single-virus fusion studies to elucidate the dynamics of content mixing typically require extensive fluorescent labeling of viral contents. The labeling process must be optimized depending on the virus identity and strain and can potentially be perturbative to viral fusion behavior. Here, we introduce a single-virus assay where content-labeled vesicles are bound to unlabeled influenza A virus (IAV) to eliminate the problematic step of content-labeling virions. We use fluorescence microscopy to observe individual, pH-triggered content mixing and content loss events between IAV and target vesicles of varying cholesterol compositions. We show that target membrane cholesterol increases the efficiency of IAV content mixing and decreases the fraction of content mixing events that result in content loss. These results are consistent with previous findings that cholesterol stabilizes pore formation in IAV entry and limits leakage following pore formation. We also show that content loss due to hemagglutinin fusion peptide engagement with the target membrane is independent of composition. This approach is a promising strategy for studying the single-virus content mixing kinetics of other enveloped viruses.

Statement of Significance

To replicate, enveloped viruses, like influenza A virus, must successfully deliver their contents to a host cell through viral membrane fusion. Most single-virus fusion assays require extensive fluorescent labeling of virions which can be perturbative to fusion kinetics. Here, we utilize content-labeled vesicles in a single-virus content mixing assay, which eliminates the need to fluorescently label virus contents. We use this assay to show that target membrane cholesterol increases the fraction of stable influenza virus content mixing events. This assay also enables the study of target membrane destabilization due to viral fusion peptide engagement.