bluuebunny/crossref_metadata_2025 huggingface dataset:
Timing Is Everything In Autumn – Coordination Of Senescence Onset By A Transcriptional Program In Response To Environmental And Phytohormone Signals
Authors: Lihavainen Jenna , Šimura Jan , Bag Pushan , Fataftah Nazeer , Robinson Kathryn Megan , Delhomme Nicolas , Novák Ondřej , Ljung Karin , Jansson Stefan
Published: April 2022

European aspen (Populus tremula L.) undergoes a coordinated senescence program during autumn; however, it is not known what exactly triggers it. To identify the cellular program leading to senescence, we utilized natural variation among Swedish aspen genotypes in a common garden to study senescence timing and the underlying changes in leaf phytohormone and transcriptome profiles. Apart from the patterns of major transcriptional cascade that was similar between the genotypes and closely associated with cytokinin and auxin metabolite levels and gradually decreasing air temperature during autumn, we detected patterns that consistently preceded or coincided with senescence onset in individual genotypes. Another cascade seemed to respond to short-term changes in weather conditions that re-wired the transcriptional network; the up-regulation of genes related to ethylene and abiotic stress, programmed cell death and translation occurred first in the early-senescing genotypes and later in the late one. Network analyses displayed a connection between the two cascades, metabolic stress and immunity responses mediated by salicylic acid (SA)-signalling pathway that was repressed along with SA levels at senescence onset. We propose that autumn senescence in aspen trees is affected by environmental variation that evokes stress and the timing is fine-tuned by their stress tolerance mechanism.

One sentence summary

Salicylic acid signalling pathway fine-tunes the timing of senescence onset under challenging environmental conditions in autumn.
Contributions of the N-terminal intrinsically disordered region of the SARS-CoV-2 nucleocapsid protein to RNA-induced phase separation
Authors: Zachrdla Milan , Savastano Adriana , de Opakua Alain Ibáñez , Cima-Omori Maria-Sol , Zweckstetter Markus
Published: April 2022

SARS-CoV-2 nucleocapsid protein is an essential structural component of mature virions, encapsulating the genomic RNA and modulating RNA transcription and replication. Several of its activities might be associated with the protein’s ability to undergo liquid-liquid phase separation. NSARS-CoV-2 contains an intrinsically disordered region at its N-terminus (NTE) that can be phosphorylated and is affected by disease-relevant mutations. Here we show that NTE deletion decreases the range of RNA concentrations that can induce phase separation of NSARS-CoV-2. In addition, deletion of the prion-like NTE allows NSARS-CoV-2 droplets to retain their liquid-like nature during incubation. We further demonstrate that RNA-binding engages multiple parts of the NTE and changes NTE’s structural properties. The results form the foundation to characterize the impact of N-terminal mutations and post-translational modifications on the molecular properties of the SARS-CoV-2 nucleocapsid protein.

Statement

The nucleocapsid protein of SARS-CoV-2 plays an important role in both genome packaging and viral replication upon host infection. Replication has been associated with RNA-induced liquid-liquid phase separation of the nucleocapsid protein. We present insights into the role of the N-terminal part of the nucleocapsid protein in the protein’s RNA-mediated liquid-liquid phase separation.
Electrophysiological signatures reveal spinal learning mechanisms for a lasting sensorimotor adaptation
Authors: Lavaud Simon , D’Andola Mattia , Bichara Charlotte , Takeoka Aya
Published: April 2022

Neurocircuits within the spinal cord are essential for movement automaticity. However, spinal mechanisms that underlie lasting sensorimotor adjustments remain unclear. Here, we establish a quantitative kinematic framework to characterize a conditioning behavior in which spinal circuits without brain input learn to adapt motor output upon multimodal sensory integration, undergo extinction, and reinforcement of learned behavior with repetitive training. In-vivo Neuropixel spinal cord recordings from awake behaving mice reveal learning phase-tuned single unit activities. In addition, optically identified unit recordings and a loss-of-function experiment demonstrate an essential function of a class of spinal inhibitory interneurons in this learning paradigm. Together, these data reveal neuronal underpinnings that shape lasting sensorimotor adaptation where stable sensory dissemination regulates learning and the existence of neuronal assembly that retains learned behavior.
Discussion-based DEI education to help create inclusive and open BME research lab environments
Authors: Naiknavare Ritika , Maisel Katharina
Published: April 2022

Diversity in teams has been shown to enhance creativity and innovation, particularly in teams where all members felt a sense of belonging. Creating an inclusive environment in a lab setting that provides a sense of belonging to all is challenging. This is particularly true in a field like Biomedical Engineering/Bioengineering where diversity is multifaceted and includes people’s diverse personal/cultural backgrounds and also diverse scientific backgrounds. In a research lab, there is additional diversity in training, as most labs contain trainees at different levels (undergrad, grad, postdoc, high school). To aid in creating a sense of belonging in a research lab, we have devised a novel initiative based upon open group discussions on diversity, equity, and inclusion topics. The initiative included a first presentation/discussion by the PI to set the stage for defining diversity, equity, and inclusion, and provided examples of existing DEI issues within our field, such as lack of racial diversity in awards like the NIH New Innovator Award and among conference speakers. After the initial presentation, trainee-led, bi-weekly-structured discussions were maintained. First, discussions focused broadly on any DEI related topic to enhance general knowledge on DEI issues and provide discussion comfort among the group. Second, there was a period to deepen knowledge of a specific topic (in our example, microaggressions), which was followed up with a period of discussions on potential solutions (such as how to react when observing a microaggression and what to do in response to realizing one’s own microaggression). Students reported that our discussions were the only ones they have had in their training thus far, and felt that these discussions made them feel like they belonged in the lab, made the lab more inclusive, enhanced their awareness of how to create inclusive spaces, and taught them about a variety of DEI topics. Overall, our DEI discussions have fostered open conversations within our lab group about DEI-related issues and topics at the lab, department, university, and countrywide levels and has established a space where students feel safe to voice their opinions and ask questions. We hope to ultimately use these DEI discussions to create actionable steps for addressing topics, e.g., microaggressions, in different scenarios that can be applied by group members in their future careers.
Unraveling axonal mechanisms of traumatic brain injury
Authors: Pozo Devoto Victorio M. , Lacovich Valentina , Feole Monica , Bhat Pratiksha , Chovan Jaroslav , Čarna Maria , Onyango Isaac G. , Dragišic Neda , Sűsserová Martina , Barrios-Llerena Martin E. , Stokin Gorazd B.
Published: April 2022

Axonal swellings (AS) are the neuropathological hallmark of axonal injury in several disorders from trauma to neurodegeneration. Current evidence proposes a role of perturbed Ca2+ homeostasis in AS formation, involving impaired axonal transport and focal distension of the axons. Mechanisms of AS formation, in particular moments following injury, however, remain unknown. Here we show that AS form independently from intra-axonal Ca2+ changes, which are required primarily for the persistence of AS in time. We further show that the majority of axonal proteins undergoing de/phosphorylation immediately following injury belong to the cytoskeleton. This correlates with an increase in the distance of the actin/spectrin periodic rings and with microtubule tracks remodeling within AS. Observed cytoskeletal rearrangements support axonal transport without major interruptions. Our results demonstrate that the earliest axonal response to injury consists in physiological adaptations of axonal structure to preserve function rather than in immediate pathological events signaling axonal destruction.
Methylene blue, Mycophenolic acid, Posaconazole, and Niclosamide inhibit SARS-CoV-2Omicron variant BA.1 infection of human airway epithelial explant cultures
Authors: Volle Romain , Murer Luca , Petkidis Anthony , Andriasyan Vardan , Savi Alessandro , Bircher Cornelia , Meili Nicole , Fischer Lucy , Sequeira Daniela Policarpo , Mark Daniela Katharina , Gomez-Gonzalez Alfonso , Greber Urs F.
Published: April 2022

Sublineages of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Omicron variants continue to amass mutations in the spike (S) glycoprotein, which leads to immune evasion and rapid spread of the virus across the human population. Here we demonstrate the susceptibility of the Omicron variant BA.1 (B.1.1.529.1) to four repurposable drugs, Methylene blue (MB), Mycophenolic acid (MPA), Posaconazole (POS), and Niclosamide (Niclo) in post-exposure treatments of primary human airway cell cultures. MB, MPA, POS, and Niclo are known to block infection of human nasal and bronchial airway epithelial explant cultures (HAEEC) with the Wuhan strain, and four variants of concern (VoC), Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28), Delta (B.1.617.2) (1, 2). Our results not only show broad anti-coronavirus effects of MB, MPA, POS and Niclo, but also demonstrate that the Omicron variant BA.1 (B.1.1.529.1) sheds infectious virus from HAEEC over at least 15 days, and maintains both intracellular and extracellular viral genomic RNA without overt toxicity, suggesting viral persistence. The data underscore the broad effects of MB, MPA, POS, and Niclo against SARS-CoV-2 and the currently circulating VoC, and reinforce the concept of repurposing drugs in clinical trials against COVID-19.
dartR v2: an accessible genetic analysis platform for conservation, ecology, and agriculture
Authors: Mijangos Jose Luis , Gruber Bernd , Berry Oliver , Pacioni Carlo , Georges Arthur
Published: April 2022

Innumerable approaches to analyse genetic data are now available to guide conservation, ecological and agricultural projects. However, streamlined and accessible tools are needed to bring these approaches within reach of a broader user base. dartR was released in 2018 to lessen the intrinsic complexity of analysing single nucleotide polymorphisms (SNPs) and dominant markers (presence/absence of amplified sequence tags) by providing user-friendly data quality control and marker selection functions. dartR users have grown steadily since its release and provided valuable feedback on their interaction with the package allowing us to enhance dartR capabilities.

Here, we present Version 2 of dartR. In this iteration, we substantially increased the number of available functions from 45 to 144. In addition to improved functionality, we focused on enhancing the user experience by extending plot customisation, function standardisation, increasing user support and function speed. dartR provides functions for various stages in analysing genetic data, from data manipulation to reporting.

dartR provides many functions for importing, exporting and linking to other packages, to provide an easy-to-navigate conduit between data generation and analysis options already available via other packages. We also implemented simulation functions whose results can be analysed seamlessly with several other dartR functions.

As more methods and approaches mature to inform conservation, we envision that accessible platforms to analyse genetic data will play a crucial role in translating science into practice.
The cardiopharyngeal mesoderm contributes to lymphatic vessel development
Authors: Maruyama Kazuaki , Miyagawa-Tomita Sachiko , Haneda Yuka , Kida Mayuko , Matsuzaki Fumio , Imanaka-Yoshida Kyoko , Kurihara Hiroki
Published: April 2022

Lymphatic vessels are crucial for tissue homeostasis and immune responses in vertebrates. Recent studies have demonstrated that lymphatic endothelial cells (LECs) arise from both venous sprouting (lymphangiogenesis) and de novo production from non-venous origins (lymphvasculogenesis), which is similar to blood vessel formation through angiogenesis and vasculogenesis. However, the contribution of LECs from non-venous origins to lymphatic networks is considered to be relatively small. Here, we identify the Islet1 (Isl1)-expressing cardiopharyngeal mesoderm (CPM) as a non-venous origin of craniofacial and cardiac LECs. Genetic lineage tracing with Isl1-Cre and Isl1-MerCreMer mice suggested that a subset of CPM cells gives rise to LECs. These CPM-derived LECs are distinct from venous-derived LECs in terms of their developmental processes and anatomical locations. Later, they form the craniofacial and cardiac lymphatic vascular networks in collaboration with venous-derived LECs. Collectively, our results demonstrate that there are two major sources of LECs, the cardinal vein and the CPM. As the CPM is evolutionarily conserved, these findings may improve our understanding of the evolution of lymphatic vessel development across species. Most importantly, our findings may provide clues to the pathogenesis of lymphatic malformations, which most often develop in the craniofacial and mediastinal regions.
Hierarchical architecture of dopaminergic circuits enables second-order conditioning in Drosophila
Authors: Yamada Daichi , Bushey Daniel , Feng Li , Hibbard Karen , Sammons Megan , Funke Jan , Litwin-Kumar Ashok , Hige Toshihide , Aso Yoshinori
Published: April 2022

Dopaminergic neurons with distinct projection patterns and physiological properties compose memory subsystems in a brain. However, it is poorly understood whether or how they interact during complex learning. Here, we identify a feedforward circuit formed between dopamine subsystems and show that it is essential for second-order conditioning, an ethologically important form of higher-order associative learning. The Drosophila mushroom body comprises a series of dopaminergic compartments, each of which exhibits distinct memory dynamics. We find that a slow and stable memory compartment can serve as an effective “teacher” by instructing other faster and transient memory compartments via a single key interneuron, which we identify by connectome analysis and neurotransmitter prediction. This excitatory interneuron acquires enhanced response to reward-predicting odor after first-order conditioning and, upon activation, evokes dopamine release in the “student” compartments. These hierarchical connections between dopamine subsystems explain distinct properties of first- and second-order memory long known by behavioral psychologists.
Monitoring promoter activity by RNA editing based reporter
Authors: Wang Jing , Zhao Yu-Ting , Hu Lu-Feng , Wang Yangming
Published: April 2022

Traditional methods monitoring the promoter activity require the insertion of reporter protein (e.g. fluorescent protein) downstream of a targeted promoter. These approaches suffer from low sensitivity and potential interference to endogenous transcripts especially when the targeted transcript is a noncoding RNA. Here, we develop a mechanistically different reporter system to monitor promoter activity based on ribozyme processed ADAR engaging RNA directed editing (REDDIT). We show that REDDIT can be used to monitor the promoter activity of protein coding, long noncoding RNA, and microRNA (miRNA) genes. Furthermore, REDDIT reporter can also be adapted to use bioluminescence imaging to monitor the promoter activity, which is more suitable for in vivo live imaging. Finally, the reporter sensitivity may be further increased by the circularization of ADAR recruiting RNA. REDDIT provides a powerful platform that is promising in a variety of applications such as monitoring the promoter activity, cell lineage tracing, and processing and manipulating genetic information for synthetic biology applications.
Screening the Pathogen Box Compounds for Activity Against Plasmodium falciparum Sporozoite Motility
Authors: Kanatani Sachie , Elahi Rubayet , Kanchanabhogin Sukanat , Vartek Natasha , Tripathi Abhai K. , Prigge Sean T. , Sinnis Photini
Published: April 2022

As the malaria parasite becomes resistant to every drug that we develop, identification and development of novel drug candidates is essential. Many studies have screened compounds designed to target the clinically important blood stages. However, if we are to shrink the malaria map, new drugs that block transmission of the parasite are needed. Sporozoites are the infective stage of the malaria parasite, transmitted to the mammalian host as mosquitoes probe for blood. Sporozoite motility is critical to their ability to exit the inoculation site and establish infection and drug-like compounds targeting motility are effective in blocking infection in the rodent malaria model. In this study, we established a moderate throughput motility assay for sporozoites of the human malaria parasite Plasmodium falciparum, enabling us to screen the 400 drug-like compounds from the Pathogen box provided by Medicines for Malaria Venture for their activity. Compounds exhibiting inhibitory effects on P. falciparum sporozoite motility were further assessed against transmission-blocking activity and asexual stage growth. Five compounds had a significant inhibitory effect on P. falciparum sporozoite motility at 1 μM concentration and four of these compounds also showed significant inhibition on transmission of P. falciparum gametocytes to the mosquito and of these four, three had previously been shown to have inhibitory activity on asexual blood stage parasites. Our findings provide new antimalarial drug candidates that have multi-stage activity.
Cross-talk between red blood cells and plasma influences blood flow and omics phenotypes in severe COVID-19
Authors: Recktenwald Steffen M. , Simionato Greta , Lopez Marcelle G. M. , Gamboni Fabia , Dzieciatkowska Monika , Meybohm Patrick , Zacharowski Kai , Knethen Andreas von , Wagner Christian , Kaestner Lars , D’Alessandro Angelo , Quint Stephan
Published: April 2022

Coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and can affect multiple organs, among which is the circulatory system. Inflammation and mortality risk markers were previously detected in COVID-19 plasma and red blood cells (RBCs) metabolic and proteomic profiles. Additionally, biophysical properties, such as deformability, were found to be changed during the infection. Based on such data, we aim to better characterize RBC functions in COVID-19. We evaluate the flow properties of RBCs in severe COVID-19 patients admitted to the intensive care unit by usingin vitromicrofluidic techniques and automated methods, including artificial neural networks, for an unbiased RBC analysis. We find strong flow and RBC shape impairment in COVID-19 samples and demonstrate that such changes are reversible upon suspension of COVID-19 RBCs in healthy plasma. Vice versa, healthy RBCs immediately resemble COVID-19 RBCs when suspended in COVID-19 plasma. Proteomics and metabolomics analyses allow us to detect the effect of plasma exchanges on both plasma and RBCs and demonstrate a new role of RBCs in maintaining plasma equilibria at the expense of their flow properties. Our findings provide a framework for further investigations of clinical relevance for therapies against COVID-19 and possibly other infectious diseases.
Identification of carcinogenesis and tumor progression processes in pancreatic ductal adenocarcinoma using high-throughput proteomics
Authors: Trilla-Fuertes Lucía , Gámez-Pozo Angelo , Lumbreras-Herrera María Isabel , López-Vacas Rocío , Heredia-Soto Victoria , Ghanem Ismael , López-Camacho Elena , Zapater-Moros Andrea , Miguel María , Peña-Burgos Eva M , Palacios Elena , de Uribe Marta , Guerra Laura , Dittmann Antje , Mendiola Marta , Ángel Fresno Vara Juan , Feliu Jaime
Published: April 2022

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with an overall 5 year-survival rate of just 5%. A better understanding of the carcinogenesis processes and the mechanisms of progression of PDAC is mandatory.

Fifty-two PDAC patients treated with surgery and adjuvant therapy, with available primary tumor, normal tissue, preneoplastic lesions (PanIN), and/or lymph node metastases, were selected for the study. Proteins were extracted from small punches and analyzed by LC-MS/MS using data-independent acquisition. Proteomics data was analyzed using probabilistic graphical models, allowing functional characterization. Comparisons between groups were done using linear mixed models. Three proteomics tumor subtypes were defined. T1 (32% of patients) was related to adhesion, T2 (34%) had metabolic features, and T3 (34%) presented high splicing and nucleoplasm activity. These proteomics subtypes were validated in the PDAC TCGA cohort. Relevant biological processes related to carcinogenesis and tumor progression were studied in each subtype. Carcinogenesis in T1 subtype seems to be related to an increase of adhesion and complement activation nodes activity, whereas tumor progression seems to be related to nucleoplasm and translation nodes. Regarding T2 subtype, it seems that metabolism and, especially, mitochondria act as the motor of cancer development. T3 analyses point out that nucleoplasm, mitochondria and metabolism, and extracellular matrix nodes could be involved in T3 tumors carcinogenesis. Identified processes were different among proteomics subtypes, suggesting that the molecular motor of the disease is different in each subtype. These differences can have implications in the development of future tailored therapeutic approaches for each PDAC proteomics subtype.
Size of societal volunteering predicts COVID-19 mortality
Authors: Schiltz Fritz , Van Remoortel Hans , Scheers Hans , Vandekerckhove Philippe
Published: April 2022

Introduction

Different countries responded differently to the COVID-19 pandemic in terms of timing and stringency of measures, and in types of policies adopted. Typically, policy makers tried to balance the capacity of healthcare systems to take care of the ill (as determined by ICU capacity, availability of nurses, etc.), with safeguarding economic output (preventing total lockdown of the labor force, etc.). Later on, also a broader array of considerations such as impact on schooling or the need for social contact were taken into account to varying degrees.

The broad and relatively fast availability of data on healthcare and economic capacity, together with the political estimate that these were the most critical determinants for maintaining societal structure and compliance with the measures taken, in many countries prioritized decision-making. What received far less attention, in part due to the difficulty of obtaining reliable data in a timely manner, was the opposite question: to what extent do societal structures – besides healthcare and economic systems - contribute to a country’s resilience during catastrophes such as the pandemic? While it is commonly understood that the impact of a pandemic goes beyond its death count, perhaps the death count itself is impacted by the way societies are structured.

One example of such societal structure is the contribution of volunteers during the COVID-19 response. Volunteers may contribute to well-functioning societies in different ways, both through practical actions (e.g. knitting face masks) as by strengthening societal cohesion (e.g. encouraging fellow citizens to comply with measures). This paper quantifies the association between COVID-19 mortality and the size of societal volunteering, using the unique context of the COVID-19 crisis with its intensity, sudden onset and global spread.
The genetic architecture of multiple mutualisms and mating system in Turnera ulmifolia
Authors: Laurich Jason R. , Reid Christopher G. , Biel Caroline , Wu Tianbu , Knox Christopher , Frederickson Megan E.
Published: April 2022

Plants often associate with multiple arthropod mutualists. These partners provide important services to their hosts, but multiple interactions can constrain a plant’s ability to respond to complex, multivariate selection. Here, we quantified patterns of genetic variance and covariance among rewards for pollination, biotic defence, and seed dispersal mutualisms in multiple populations of Turnera ulmifolia to better understand how the genetic architecture of multiple mutualisms might influence their evolution. We phenotyped plants cultivated from 17 Jamaican populations for several mutualism and mating system-related traits. We then fit genetic variance-covariance (G) matrices for the island metapopulation and the 5 largest individual populations. At the metapopulation level, we observed significant positive genetic correlations among stigma-anther separation, floral nectar production, and extrafloral nectar production. These correlations have the potential to significantly constrain or facilitate the evolution of multiple mutualisms in T. ulmifolia and suggest that pollination, seed dispersal, and dispersal mutualisms do not evolve independently. In particular, we found that positive genetic correlations between floral and extrafloral nectar production may help explain their stable co-existence in the face of physiological trade-offs and negative interactions between pollinators and ant bodyguards. Locally, we found only small differences in G among our T. ulmifolia populations, suggesting that geographic variation in G may not shape the evolution of multiple mutualisms.
Predicting brain functions from structural connectome using graph neural network
Authors: Hui Edward S. , Sun Yuxiang , Ko Ho , Chan Chetwyn C.H. , Cao Peng
Published: April 2022

Abstract

The relationship between brain structure and function remains elusive, amidst the tremendous advances in brain mapping techniques. In this work, we attempt to partially disentangle this relationship by connecting task–evoked functional MRI (fMRI) responses with the underlying structural connectome using graph neural network (GNN). MRI data (n = 1,063) were collected from the Human Connectome Project. We demonstrate that our GNN–based model predicts task–evoked fMRI responses with high fidelity. Using a graph attention mechanism, it is possible to infer the subsets of neighboring cortical regions whose structural connections are important for the prediction of the functional responses of individual cortical regions. Notably, for each cortical region, such subset of neighboring cortical regions is predominantly localized to the ipsilateral hemisphere and much smaller than that with direct structural connections. We found that the higher cognitive functions subserved by the cingulo–opercular, dorsal attention, frontoparietal and default mode clusters may depend on neighboring cortical regions across a wide range of functional brain clusters in the ipsilateral hemisphere, whilst the sensory functions subserved by the visual1 and auditory clusters on neighboring cortical regions across much fewer functional brain clusters.
Early Endosome Capture Proteomics and its Application to Amyloid Precursor Protein Intramembrane Processing by β and γ-Secretases
Authors: Park Hankum , Yu Qing , Hundley Frances V. , Overmyer Katherine , Serrano Lia , Paulo Joao A. , Paoli Julia C. , Swarup Sharan , Coon Joshua J. , Gygi Steven P. , Harper J. Wade
Published: April 2022

Degradation and recycling of plasma membrane-localized proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. The endolysosomal system also participates in intracellular membrane protein trafficking, signaling, and protein degradation via autophagy. Our understanding of the endolysosomal system has been significantly advanced through the development of methods for selective capture of lysosomes (termed Lyso-IP), but methods for analogous isolation of early intermediates in the endolysosomal system are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early endosome-associated protein EEA1 (Endo-IP) and provide proteomic and lipidomic snapshots of early endosomes in action. In addition to resident endosomal components, we identify numerous recycling, regulatory and membrane fusion complexes as well as candidate cargo, providing a proteomic landscape of early/sorting endosomes that we distinguished from lysosomes via comparative proteomics. The approach allows capture of internalized transferrin within minutes of its addition to cells, indicating the utility of the approach for dynamic analysis of early/sorting endosomes. Among the endocytic cargo identified by Endo-IP was the amyloid precursor protein (APP) genetically linked with Alzheimer’s disease. Processing of APP to amyloidogenic Aβ peptides by β- and γ-Secretases can occur within the endolysosomal system among other compartments, but methods for spatial quantification of Aβ products in individual organelles are lacking. We combined Endo- and Lyso-IP with targeted proteomics to provide a spatial digital snapshot of Aβ products. This analysis revealed that products of Aβ processing by β- and γ-Secretases, and alterations in the specificity of cleavage by small molecule γ-Secretase modulators, can be quantified in both early/sorting endosomes and lysosomes. We anticipate that the Endo-IP approach will facilitate systematic interrogation of the many processes that are coordinated on early endosomes.
Disease-associated mutations in WDR34 lead to diverse impacts on the assembly and function of dynein-2
Authors: Shak Caroline , Vuolo Laura , Uddin Borhan , Katoh Yohei , Brown Tom , Mukhopadhyay Aakash G. , Heesom Kate , Roberts Anthony J. , Stevenson Nicola , Nakayama Kazuhisa , Stephens David J.
Published: April 2022

The primary cilium is a sensory organelle, receiving signals from the external environment and relaying them into the cell. Mutations in proteins required for transport in the primary cilium result in ciliopathies, a group of genetic disorders that commonly lead to the malformation of organs such as the kidney, liver and eyes and skeletal dysplasias. Motor proteins dynein-2 and kinesin-2 mediate retrograde and anterograde transport respectively in the cilium. WDR34, a dynein-2 intermediate chain, is required for the maintenance of cilia function. Here, we investigated WDR34 mutations identified in Jeune syndrome, short-rib polydactyly syndrome or asphyxiating thoracic dysplasia patients. There is a poor correlation between genotype and phenotype in these cases making diagnosis and treatment highly complex. We set out to define the biological impacts on cilia formation and function of WDR34 mutations by stably expressing the mutant proteins in WDR34 knockout cells. WDR34 mutations led to different spectrums of phenotypes. Quantitative proteomics demonstrated changes in dynein-2 assembly, whereas initiation and extension of the axoneme, IFT-B protein localization, transition zone (TZ) integrity, and Hedgehog signalling were also affected.

Summary statement

Disease-associated mutations in WDR34 are found to have diverse impacts on ciliogenesis and cilia function following stable expression in a WDR34 knockout cell model.
RNA helicase DDX5 mediates adaptive response to multi-kinase inhibitors in liver cancer
Authors: Li Zhili , Sun Jiazeng , Rahman Naimur , Yan Bingyu , Utturkar Sagar , Lanman Nadia Atallah , Kazemian Majid , Caron de Fromentel Claude , Levrero Massimo , Andrisani Ourania
Published: April 2022

Objective: To determine the role of RNA helicase DDX5 in sorafenib/multi-tyrosine kinase inhibitor (mTKI) response. Sorafenib and mTKIs downregulate DDX5 in vitro and preclinical hepatocellular carcinoma (HCC) models. In turn, sorafenib-mediated DDX5 downregulation activates Wnt/β-catenin and non-canonical NF-κB signaling, resulting in ferroptosis escape and mTKI resistance.
Design and Results: Molecular, pharmacologic and bioinformatic approaches were employed in human HCC cell lines, preclinical HCC models, and HCCs from TCGA. Earlier studies linked sorafenib effectiveness to ferroptosis. Herein we demonstrate sorafenib/mTKIs downregulate DDX5 in vitro and in vivo. To understand the effect of DDX5 downregulation, we compared TCGA-derived HCCs expressing low vs. high DDX5 focusing on ferroptosis-related genes. Glutathione Peroxidase 4 (GPX4), a key ferroptosis regulator, was significantly overexpressed in DDX5LOW HCCs. Importantly, DDX5-knockdown (DDX5KD) HCC cell lines lacked lipid peroxidation by GPX4 inhibition, indicating DDX5 downregulation suppresses ferroptosis. RNAseq of wild type vs. DDX5KD cells untreated or treated with sorafenib, identified a unique set of genes repressed by DDX5 and upregulated by sorafenib. This set significantly overlaps genes from Wnt/β-catenin and non-canonical NF-κB pathways, including NF-κB inducing Kinase required for non-canonical NF-κB activation. Pharmacologic inhibition of these pathways in combination with sorafenib reduced DDX5KD cell viability. Mechanistically, sorafenib-mediated NIK expression induced NRF2 transcription, while DDX5KD extended NRF2 half/life by stabilizing p62/SQSTM1, enhancing GPX4 expression and ferroptosis escape. Conclusion: Sorafenib/mTKI-mediated DDX5 downregulation results in adaptive mTKI resistance by enhancing NRF2 expression, leading to ferroptosis escape. We propose inhibition of the pathways leading to NRF2 expression will enhance the therapeutic effectiveness of sorafenib/mTKIs.
Recurrent but short-lived duplications of centromeric proteins in holocentricCaenorhabditisspecies
Authors: Caro Lews , Raman Pravrutha , Steiner Florian A. , Ailion Michael , Malik Harmit S.
Published: April 2022

Centromeric histones (CenH3s) are essential for chromosome inheritance during cell division in most eukaryotes.CenH3genes have rapidly evolved and undergone repeated gene duplications and diversification in many plant and animal species. InCaenorhabditis,two independent duplications ofCenH3(namedhcp-3forHoloCentric chromosome-bindingProtein 3) have been previously identified: inC. elegansandC. remanei. Here, using phylogenomic analyses inCaenorhabditis,we find strict retention of the ancestralhcp-3gene and eight additional independenthcp-3duplications, most of which are only found in one or two species.hcp-3L(hcp-3-like) paralogs are expressed in both sexes (males and females/ hermaphrodites) and have a conserved histone fold domain. We identified novel N-terminal protein motifs, including putative kinetochore protein-interacting motifs and a potential separase cleavage site, which are well-conserved acrossCaenorhabditisHCP-3 proteins. Other N-terminal motifs vary in their retention across paralogs or species, revealing potential sub-functionalization or functional loss following duplication.C. afraencodes an unprecedented protein fusion, where thehcp-3paralog fused to duplicated segments fromhcp-4(nematode CENP-C). Extending our analyses beyond CenH3, we found gene duplications of six inner and outer kinetochore genes inCaenorhabditis, including co-retention of different kinetochore protein paralogs in a few species. Our findings suggest that centromeric protein duplications occur frequently inCaenorhabditisnematodes, are selectively retained under purifying selection but only for short evolutionary periods, then degenerate or are lost entirely. We hypothesize that unique challenges associated with holocentricity inCaenorhabditismay lead to this rapid ‘revolving door’ of kinetochore protein paralogs.
Performance comparison of systemic activity correction in functional near-infrared spectroscopy for methods with and without short distance channels
Authors: Klein Franziska , Lührs Michael , Benitez-Andonegui Amaia , Roehn Pauline , Kranczioch Cornelia
Published: April 2022

Significance

Functional near-infrared spectroscopy (fNIRS) is a promising tool for neurofeedback (NFB) or brain–computer interfaces (BCIs). However, fNIRS signals are typically highly contaminated by systemic activity (SA) artifacts, and, if not properly corrected, NFB or BCIs run the risk of being based on noise instead of brain activity. This risk can likely be reduced by correcting for SA, in particular when short-distance channels (SDCs) are available. Literature comparing correction methods with and without SDCs is still sparse, specifically comparisons considering single trials are lacking.

Aim

This study aimed at comparing the performance of SA correction methods with and without SDCs.

Approach

Semisimulated and real motor task data of healthy older adults were used. Correction methods without SDCs included a simple and a more advanced spatial filter. Correction methods with SDCs included a regression approach considering only the closest SDC and two GLM-based methods, one including all eight SDCs and one using only twoa prioriselected SDCs as regressors. All methods were compared with data uncorrected for SA and correction performance was assessed with quality measures quantifying signal improvement and spatial specificity at single trial level.

Results

All correction methods were found to improve signal quality and enhance spatial specificity as compared with the uncorrected data. Methods with SDCs usually outperformed methods without SDCs. Correction methods without SDCs tended to overcorrect the data. However, the exact pattern of results and the degree of differences observable between correction methods varied between semisimulated and real data, and also between quality measures.

Conclusions

Overall, results confirmed that both Δ[HbO] and Δ[HbR] are affected by SA and that correction methods with SDCs outperform methods without SDCs. Nonetheless, improvements in signal quality can also be achieved without SDCs and should therefore be given priority over not correcting for SA.
Conformational flexibility, lipid binding, and regulatory domains in CelTOS
Authors: Kumar Hirdesh , Jimah John R. , Ntumngia Francis B. , Misal Santosh A. , Barnes Samantha J. , Fried Michal , Adams John H. , Schlesinger Paul H. , Tolia Niraj H.
Published: April 2022

CelTOS is an essential Plasmodium traversal protein and conserved in apicomplexan parasites. We showed that CelTOS forms pores in cell membranes to enable traversal of parasites through cells (Jimah et al., 2016). Here, we establish roles for the distinct regions of CelTOS, examine the mechanism of pore formation and evaluate the immunogenicity of engineered CelTOS variants. CelTOS dimer dissociation is required for pore formation as disulfide bridging between monomers inhibits pore formation and this inhibition is rescued by disulfide-bridge reduction. A helix destabilizing Pro127 allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C-terminus of CelTOS is a negative regulator that limits pore formation. Lipid binding is a pre-requisite for pore assembly as mutation of a phospholipid binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. The disulfide-locked and N-terminal deletion mutants showed improved immunogenicity relative to wild-type CelTOS in mice. These findings have implications for pore-forming proteins that are essential for diverse functions, identify critical regions in CelTOS, and will guide the design of effective CelTOS-targeting vaccines to combat infection and transmission of malaria and apicomplexan parasites.
Gallbladder adenocarcinomas undergo subclonal diversification and selection from precancerous lesions to metastatic tumors
Authors: Kang Minsu , Na Hee Young , Ahn Soomin , Kim Ji-Won , Lee Sejoon , Ahn Soyeon , Lee Ju Hyun , Youk Jeonghwan , Kim Haesook T. , Kim Kui-Jin , Suh Koung Jin , Lee Jun Suh , Kim Se Hyun , Kim Jin Won , Kim Yu Jung , Lee Keun-Wook , Yoon Yoo-Seok , Kim Jee Hyun , Chung Jin-Haeng , Han Ho-Seong , Lee Jong Seok
Published: April 2022

We aimed to elucidate the evolutionary trajectories of gallbladder adenocarcinoma (GBAC) using multi-regional and longitudinal tumor samples. Using whole-exome sequencing data, we constructed phylogenetic trees in each patient, and analyzed mutational signatures. A total of 11 patients including 2 rapid autopsy cases were enrolled. The most frequently altered gene in primary tumors was ERBB2 (54.5%), followed by TP53 (45.5%), and FBXW7 (27.3%). Most mutations in frequently altered genes in primary tumors were detectable in concurrent precancerous lesions (biliary intraepithelial neoplasia, BilIN), but some of them were subclonal. Subclonal diversity was common in BilIN (n=4). However, among subclones in BilIN, a certain subclone commonly shrank in concurrent primary tumors. In addition, selected subclones underwent linear and branching evolution, maintaining subclonal diversity. In combined analysis with metastatic tumors (n=11), branching evolution was identified in 9 (81.8%) patients. Of these, 8 patients (88.9%) had a total of 11 subclones expanded at least 7-fold during metastasis. These subclones harbored putative metastasis-driving mutations in tumor suppressor genes such as SMAD4, ROBO1, and DICER1. In mutational signature analysis, 6 mutational signatures were identified: 1, 3, 7, 13, 22, and 24 (cosine similarity >0.9). Signatures 1 (age) and 13 (APOBEC) decreased during metastasis while signatures 22 (aristolochic acid) and 24 (aflatoxin) were relatively highlighted. Subclonal diversity arose early in precancerous lesions and the clonal selection was a common event during malignant transformation in GBAC. However, selected cancer clones continued to evolve and thus maintained subclonal diversity in metastatic tumors.
The role of clearance in neurodegenerative diseases
Authors: Brennan Georgia S. , Thompson Travis B. , Oliveri Hadrien , Rognes Marie E. , Goriely Alain
Published: April 2022

Alzheimer’s disease, the most common form of dementia, is a systemic neurological disorder associated with the formation of toxic, pathological aggregates of proteins within the brain that lead to severe cognitive decline, and eventually, death. In normal physiological conditions, the brain rids itself of toxic proteins using various clearance mechanisms. The efficacy of brain clearance can be adversely affected by the presence of toxic proteins and is also known to decline with age. Motivated by recent findings, such as the connection between brain cerebrospinal fluid clearance and sleep, we propose a mathematical model coupling the progression of toxic proteins over the brain’s structural network and protein clearance. The model is used to study the interplay between clearance in the brain, toxic seeding, brain network connectivity, aging, and progression in neurodegenerative diseases such as Alzheimer’s disease. Our findings provide a theoretical framework for the growing body of medical research showing that clearance plays an important role in the etiology, progression and treatment of Alzheimer’s disease.
Sensitive detection and structural characterisation of UV-induced cross-links in protein-RNA complexes using CLIR-MS
Authors: Sarnowski Chris P. , Knörlein Anna , de Vries Tebbe , Götze Michael , Beusch Irene , Aebersold Ruedi , Allain Frédéric H.-T. , Hall Jonathan , Leitner Alexander
Published: April 2022

Cross-linking coupled with mass spectrometry is an increasingly popular methodology for elucidating structural information from biological complexes. Whilst protein-protein cross-linking workflows are widely used and well characterised, adoption of protein-RNA cross-linking workflows for structural studies is less widespread, and data produced from such experiments remains less well understood. The cross-linking of stable isotope labelled RNA coupled to mass spectrometry (CLIR-MS) workflow uses isotope labelled RNA to simultaneously confirm that peptides are cross-linked to RNA and aid cross-link localisation in an RNA sequence. For broader application of CLIR-MS as part of the structural analysis of ribonucleoproteins, the method must be sensitive, robust, and its reaction products need to be well characterised. We enhanced our previously published workflow, improving coverage and sensitivity. We used it to infer common properties of protein-RNA cross-links such as cross-linking distance, and to assess the impact of substitution of uracil with 4-thio-uracil in structural proteomics experiments. We profiled the compositional diversity of RNA-derived peptide modifications, and subsequently defined a more inclusive data analysis approach which more than doubles the number of cross-link spectrum matches compared with our past work. We defined distance restraints from these cross-links, and with the aid of visualisation software, demonstrated that on their own they provide sufficient information to localise an RNA chain to the correct position on the surface of a protein. We applied our enhanced workflow and understanding to characterise the binding interface of several protein-RNA complexes containing classical and uncommon RNA binding domains. The enhanced sensitivity and understanding demonstrated here underpin a wider adoption of protein-RNA cross-linking in structural biology.
High predation risk decimates survival during the reproduction season
Authors: Smolinský Radovan , Hiadlovská Zuzana , Maršala Štěpán , Škrabánek Pavel , Škrobánek Michal , Martínková Natália
Published: April 2022

Predators attack conspicuous prey phenotypes that are present in the environment. Male display behaviour of conspicuous nuptial colouration becomes risky in the presence of a predator, and adult males face higher predation risk. High predation risk in one sex will lead to low survival and sex ratio bias in adult cohorts, unless the increased predation risk is compensated by higher escape rate.

Here, we tested the hypothesis that sand lizards (Lacerta agilis) have sex-specific predation risk and escape rate. We expected the differences to manifest in changes in sex ratio with age, differences in frequency of tail autotomy, and in sex-specific survival rate.

We developed a statistical model to estimate predation risk and escape rate, combining the observed sex ratio and frequency of tail autotomy with likelihood-based survival rate. Using Bayesian framework, we estimated the model parameters. We projected the date of the tail autotomy events from growth rates derived from capturerecapture data measurements.

We found statistically stable sex ratio in age groups, equal frequency of tail regenerates between sexes, and similar survival rate. Predation risk is similar between sexes, and escape rate increases survival by about 5%. We found low survival rate and a low number of tail autotomy events in females during months when sand lizards mate and lay eggs, indicating high predator pressure throughout reproduction. Our data show that gravid females fail to escape predation.

The risks of reproduction season in an ectotherm are a convolution of morphological changes (conspicuous colouration in males, body allometry changes in gravid females), behaviour (nuptial displays), and environmental conditions which challenge lizard thermal performance. Performance of endotherm predators in cold spring months endangers gravid females more than displaying males in bright nuptial colouration.