bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct RNA-RNA interaction between Neat1 and RNA targets, as a mechanism for RNAs paraspeckle retention
Authors: Jacq Audrey , Becquet Denis , Guillen Séverine , Boyer Bénédicte , Bello-Goutierrez Maria-Montserrat , Franc Jean-Louis , François-Bellan Anne-Marie
Published: October 2020

Paraspeckles are nuclear ribonucleic complex formed of a long non-coding RNA, nuclear-enriched abundant transcript one (Neat1) and associated RNA-binding proteins (RBP) whose cellular known functions are to sequester in the nucleus both proteins and RNAs. However, how RNAs are bound to paraspeckles is largely unknown. It is highly likely that binding of RNAs may occur via interactions with RBPs and accordingly, two structures present in the 3’UTR of some RNAs have been shown to allow their association to paraspeckles via protein binding. However, Neat1 could also be involved in the targeting of RNAs through direct RNA-RNA interactions. Using a RNA pull-down procedure adapted to select only RNAs engaged in direct RNA-RNA interactions and followed by RNA-seq we showed that in a rat pituitary cell line, GH4C1 cells, 1791 RNAs were associated with paraspeckles by direct interaction with Neat1. Neat1 was actually found able to bind more than 30% of the total transcripts targeted by the paraspeckles, we have identified in this cell line in a previous study. Furthermore, given the biological processes in which direct RNAs targets of Neat1 were involved as determined by gene ontology analysis, it was proposed that Neat1 played a major role in paraspeckle functions such as circadian rhythms, mRNA processing, RNA splicing and regulation of cell cycle. Finally, we provided evidence that direct RNA targets of Neat1 were preferentially bound to the 5’ end of Neat1 demonstrating that they are located in the shell region of paraspeckles.
Tracking cytosine depletion in SARS-CoV-2
Authors: Luo Ruibang , Wong Yat-Sing , Lam Tak-Wah
Published: October 2020

Motivation

Danchin et al. have pointed out that cytosine drives the evolution of SARS-CoV-2. A depletion of cytosine might lead to the attenuation of SARS-CoV-2.

Results

We built a website to track the composition change of mono-, di-, and tri-nucleotide of SARS-CoV-2 over time. The website downloads new strains available from GISAID and updates its results daily. Our analysis suggests that the composition of cytosine in coronaviruses is related to their reported mortality. Using 137,315 SARS-CoV-2 strains collected in ten months, we observed cytosine depletion at a rate of about one cytosine loss per month from the whole genome.

Availability

The website is available athttp://www.bio8.cs.hku.hk/sarscov2/.

Contact

rbluo@cs.hku.hk

Supplementary information

Supplementary data are available atBioinformaticsonline.
Which are the central aspects of infant sleep? The dynamic of sleep composites across infancy
Authors: Schoch Sarah F. , Huber Reto , Kohler Malcolm , Kurth Salome
Published: October 2020

Sleep is ubiquitous during infancy and important for the well-being of both infant and parent. Therefore, there is large interest to characterize infant sleep with reliable tools, for example by means of combining actigraphy with 24-h-diaries. However, it is critical to select the right variables to characterize sleep. With a principal component analysis, we identified 5 underlying sleep composites from 48 commonly used sleep variables:Sleep Night, Sleep Day, Sleep Activity, Sleep Timing and Sleep Variability.These composites accurately reflect the known changes of sleep throughout infancy asSleep Day(representing naps), Sleep Activity(representing sleep efficiency and consolidation) andSleep Variability(representing day-to-day stability) decrease across infancy, whileSleep Night(representing nighttime sleep) slightly increases andSleep Timingbecomes earlier with increasing age. Additionally, we uncover interesting dynamics between the sleep composites and demonstrate that infant sleep is not only highly variable between infants but also considerably dynamic within infants across time. Interestingly,Sleep Dayis associated with behavioral development and therefore a potential marker for maturation. We recommend the use of sleep composites or of those specific single variables, which are solid representatives of the sleep composites for more reliable research.
No impact of functional connectivity of the motor system on the resting motor threshold: A replication study
Authors: Engelhardt Melina , Komnenić Darko , Roth Fabia , Kawelke Leona , Finke Carsten , Picht Thomas
Published: October 2020

The physiological mechanisms of corticospinal excitability and factors influencing its measurement with transcranial magnetic stimulation are still poorly understood. A recent study reported an impact of functional connectivity between the primary motor cortex and dorsal premotor cortex on the resting motor threshold of the dominant hemisphere. We aimed to replicate these findings in a larger sample of 38 healthy right-handed subjects with data from both hemispheres. Resting-state functional connectivity was assessed between the primary motor cortex and five a-priori defined motor-relevant regions on each hemisphere as well as interhemispherically between both primary motor cortices. Following the procedure by the original authors, we included age, the cortical grey matter volume and coil to cortex distance as further predictors in the analysis. We report replication models for the dominant hemisphere as well as an extension to data from both hemispheres and support the results with Bayes factors. Functional connectivity between the primary motor cortex and dorsal premotor cortex did not explain variability in the resting motor threshold and we obtained moderate evidence for the absence of this effect. In contrast, coil to cortex distance could be confirmed as an important predictor with strong evidence. These findings contradict the previously proposed effect, thus questioning the notion of the dorsal premotor cortex playing a major role in modifying corticospinal excitability.
SETD2 regulates the methylation of translation elongation factor eEF1A1 in clear cell renal cell carcinoma
Authors: Hapke Robert , Venton Lindsay , Rose Kristie Lindsay , Sheng Quanhu , Reddy Anupama , Jones Angela , Rathmell W. Kimryn , Haake Scott
Published: October 2020

SET domain-containing protein 2 (SETD2) is commonly mutated in renal cell carcinoma. SETD2 methylates histone H3 as well as a growing list of non-histone proteins. To explore SETD2-dependent regulation of the kidney cancer proteome, we performed a systems-wide analysis of protein lysine-methylation and expression in wild type (WT) andSETD2-knock out (KO) kidney cells. We observed decreased lysine methylation of the translation elongation factor eEF1A1.EEF1AKMT2andEEF1AKMT3are known to methylate eEF1A1, and we show here that their expression is dependent on SET-domain function ofSETD2. Globally, we observe differential expression of hundreds of proteins in WT versusSETD2-KO cells, including increased expression of many involved in protein translation. Finally, we observe decreased progression free survival and loss of EEF1AKMT2 gene expression inSETD2-mutated tumors. Overall, these data suggest thatSETD2-mutated ccRCC, via loss of enzymetic function of the SET domain, displays dysregulation of protein translation as a potentially important component of the transformed phenotype.
Invasion byFusobacterium nucleatumCauses Upregulation ofdll4andklf4Expression in Caco2 Cells
Authors: Cochrane Kyla , Robinson Avery V. , Powers Jacqueline , Brown Scott D. , Holt Robert A. , Allen-Vercoe Emma
Published: October 2020

Fusobacterium nucleatumis an emerging microbe of importance in the pathogenesis of colorectal cancer. Strains of this enigmatic bacterial species vary in their capacity to invade human epithelial cells, a virulence determinant which has important implications in disease. Here, we infected human colorectal epithelial (Caco-2) cellsin vitrowith a known, highly invasive strain ofF. nucleatumisolated from a Crohn’s Disease patient, as well as a further invasive isolate ofF. nucleatumderived from a colorectal cancer tumour. We used transcriptional profiling to determine the human genes upregulated during the invasion process compared to exposure to a non-invasiveE.colicontrol strain. Infection withF. nucleatumstrains resulted in the upregulation of several host genes, including two associated with tumorigenesis:dll4andklf4.
Set1-dependent H3K4 methylation becomes critical for DNA replication fork progression in response to changes in S phase dynamics inSaccharomyces cerevisiae
Authors: de La Roche Saint-André Christophe , Géli Vincent
Published: October 2020

DNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. InSaccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth oforc5-1 set1Δ. Interestingly,orc5-1 set1Δis sensitive to the lack of RNase H activity while a reduction of histone levels is able to counterbalance the loss of Set1. We propose that the recently described Set1-dependent mitigation of transcription-replication conflicts becomes critical for growth when the replication forks accelerate due to decreased origin firing in theorc5-1background. Furthermore, we show that an increase of reactive oxygen species (ROS) levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality inorc5-1 set1Δ.

Author summary

DNA replication, that ensures the duplication of the genetic material, starts at discrete sites, termed origins, before proceeding at replication forks whose progression is carefully controlled in order to avoid conflicts with the transcription of genes. In eukaryotes, DNA replication occurs in the context of chromatin, a structure in which DNA is wrapped around proteins, called histones, that are subjected to various chemical modifications. Among them, the methylation of the lysine 4 of histone H3 (H3K4) is carried out by Set1 inSaccharomyces cerevisiae, specifically at transcribed genes. We report that, when the replication fork accelerates in response to a reduction of active origins, the absence of Set1 leads to accumulation of DNA damage. Because H3K4 methylation was recently shown to slow down replication at transcribed genes, we propose that the Set1-dependent becomes crucial to limit the occurrence of conflicts between replication and transcription caused by replication fork acceleration. In agreement with this model, stabilization of transcription-dependent structures or reduction histone levels, to limit replication fork velocity, respectively exacerbates or moderates the effect of Set1 loss. Last, but not least, we show that the oxidative stress associated to DNA damage is partly responsible for cell lethality.
Darwin was right, in any given field, the spatial variability of earthworm communities in pastures isn’t driven by measurable soil properties
Authors: Hodson Mark E , Corstanje Ron , Jones David T , Witton Jo , Burton Victoria J , Sloan Tom , Eggleton Paul
Published: October 2020

Abundance and distribution of earthworms in agricultural fields is frequently proposed as a measure of soil quality assuming that observed patterns of abundance are in response to improved or degraded environmental conditions. However, it is not clear that earthworm abundances can be directly related to their edaphic environment, as noted in Darwin’s final publication, perhaps limiting or restricting their value as indicators of ecological quality in any given field.

We present results from a spatially explicit intensive survey of pastures within United Kingdom farms, looking for the main drivers of earthworm density at a range of scales. When describing spatial variability of earthworm abundance within any given field, the best predictor was earthworm abundance itself within 20 – 30 m of the sampling point; there were no consistent environmental correlates with earthworm numbers, suggesting that biological factors (e.g. colonisation rate, competition, predation, parasitism) drive or at least significantly modify earthworm distributions at this spatial level. However, at the national scale, earthworm abundance is well predicted by soil nitrate levels, density, temperature and moisture content, albeit not in a simple linear fashion. This suggests that although land can be managed at the farm scale to promote earthworm abundance and the resulting soil processes that deliver ecosystem services, within a field, earthworm distributions will remain patchy. The divergence in the interpretative value of earthworm abundance as an ecological indicator is a function of spatial scale, corresponding to species specific biological factors as well as a response to environmental pressures. Species abundance can effectively be used as ecological indicators, even if, at first, distributions seem random. However, care must be exercised, in the sampling design for the indicator species, if its abundance is to be used as a proxy for environmental quality at a particular scale (e.g. a management scale such as field scale).
Seipin traps triacylglycerols to facilitate their nanoscale clustering in the ER membrane
Authors: Prasanna Xavier , Salo Veijo T. , Li Shiqian , Ven Katharina , Vihinen Helena , Jokitalo Eija , Vattulainen Ilpo , Ikonen Elina
Published: October 2020

Seipin is a disk-like oligomeric ER protein important for lipid droplet (LD) biogenesis and triacylglycerol (TAG) delivery to growing LDs. Here we show through biomolecular simulations bridged to experiments that seipin can trap TAGs in the ER bilayer via the luminal hydrophobic helices of the protomers delineating the inner opening of the seipin disk. This promotes the nanoscale sequestration of TAGs at a concentration that by itself is insufficient to induce TAG clustering in a lipid membrane. We identify Ser166 in the α3 helix as a favored TAG occupancy site and show that mutating it compromises the ability of seipin complexes to sequester TAGin silicoand to promote TAG transfer to LDs in cells. While seipin-S166D mutant colocalizes poorly with promethin, the association of nascent wild-type seipin complexes with promethin is promoted by TAGs. Together, these results suggest that seipin traps TAGs via its luminal hydrophobic helices, serving as a catalyst for seeding the TAG cluster from dissolved monomers inside the seipin ring, thereby generating a favorable promethin binding interface.
Atomic-level evolutionary information improves protein-protein interface scoring
Authors: Quignot Chloé , Granger Pierre , Chacón Pablo , Guerois Raphael , Andreani Jessica
Published: October 2020

The crucial role of protein interactions and the difficulty in characterising them experimentally strongly motivates the development of computational approaches for structural prediction. Even when protein-protein docking samples correct models, current scoring functions struggle to discriminate them from incorrect decoys. The previous incorporation of conservation and coevolution information has shown promise for improving protein-protein scoring. Here, we present a novel strategy to integrate atomic-level evolutionary information into different types of scoring functions to improve their docking discrimination.

We applied this general strategy to our residue-level statistical potential from InterEvScore and to two atomic-level scores, SOAP-PP and Rosetta interface score (ISC). Including evolutionary information from as few as ten homologous sequences improves the top 10 success rates of these individual scores by respectively 6.5, 6 and 13.5 percentage points, on a large benchmark of 752 docking cases. The best individual homology-enriched score reaches a top 10 success rate of 34.4%. A consensus approach based on the complementarity between different homology-enriched scores further increases the top 10 success rate to 40%.

All data used for benchmarking and scoring results, as well as pipelining scripts, are available athttp://biodev.cea.fr/interevol/interevdata/
Nitric Oxide Regulates theRalstonia solanacearumType 3 Secretion System
Authors: Hendrich Connor G. , Truchon Alicia N. , Dalsing Beth L. , Allen Caitilyn
Published: October 2020

Ralstonia solancearumcauses bacterial wilt disease on diverse plant hosts.R. solanacearumcells enter a host from soil or infested water through the roots, then multiply and spread in the water-transporting xylem vessels. Despite the low nutrient content of xylem sap,R. solanacearumgrows very well inside the host, using denitrification to respire in this hypoxic environment.R. solanacearumgrowthin plantaalso depends on the successful deployment of protein effectors into host cells via a Type III Secretion System (T3SS). The T3SS is absolutely required forR. solanacearumvirulence, but it is metabolically costly and can trigger host defenses. Thus, the pathogen’s success depends on optimized regulation of the T3SS. We found that a byproduct of denitrification, the toxic free-radical nitric oxide (NO), positively regulates theR. solanacearumT3SS bothin vitroandin planta. Using chemical treatments andR. solanacearummutants with altered NO levels, we show that the expression of a key T3SS regulator is induced by NO in culture. Analyzing the transcriptome ofR. solanacearumresponding to varying levels of NO both in culture andin plantarevealed that the T3SS and effectors were broadly upregulated with increasing levels of NO. This regulation was specific to the T3SS and was not shared by other stressors. Our results suggest thatR. solanacearumexperiences an NO-rich environment in the plant host and may use this NO as a signal to activate T3SS during infection.
Associations between childhood family emotional health, fronto-limbic grey matter volume, and saliva 5mC in young adulthood
Authors: Pfeiffer JR , Bustamante Angela C. , Kim Grace S. , Armstrong Don , Knodt Annchen R. , Koenen Karestan C. , Hariri Ahmad R. , Uddin Monica
Published: October 2020

Background

Poor family emotional health (FEH) during childhood is prevalent and impactful, and likely confers similar neurodevelopmental risks as other adverse social environments. Pointed FEH study efforts are underdeveloped, and the mechanisms by which poor FEH are biologically embedded are unclear. The current exploratory study examined whether variability in DNA methylation (DNAm) and fronto-limbic grey matter volume may represent pathways through which FEH may become biologically embedded.

Results

Self-reported childhood FEH was nominally associated with right hemisphere hippocampus (b=10.4, p=0.005), left hemisphere amygdala (b=5.3, p=0.009), and right hemisphere amygdala (b=5.8, p=0.016) volumes. Childhood FEH was also nominally associated with 49 DNAm MEs (prange=3×10−6to 0.047). After limiting analyses to probes correlated between saliva and brain, saliva-derived DNAm MEs partially mediated the association between FEH and right hippocampal volume (Burlywood ME indirect effect b=-111, p=0.014), and fully mediated the FEH and right amygdala volume relationship (Pink4 ME indirect effect b=-48, p=0.026). Modules were enriched with probes falling in genes with immune, CNS, and metabolic functions.

Conclusions

Findings extend work highlighting neurodevelopmental variability associated with adverse social environment exposure during childhood by specifically implicating poor FEH, while informing a mechanism of biological embedding. FEH-associated epigenetic signatures could function as proxies of altered fronto-limbic grey matter volume associated with poor childhood FEH and inform further investigation into primarily affected tissues such as endocrine, immune, and CNS cell types.
Self-similar synchronization of calcium and membrane potential transitions during AP cycles predict HR across species
Authors: Sirenko Syevda Tagirova , Tsutsui Kenta , Tarasov Kirill , Yang Dongmei , Wirth Ashley N , Maltsev Victor A. , Ziman Bruce D. , Yaniv Yael , Lakatta Edward G.
Published: October 2020

Background

Translation of knowledge of sinoatrial nodal “SAN” automaticity gleaned from animal studies to human dysrhythmias, e.g. “Sick Sinus” Syndrome (SSS) requiring electronic pacemaker insertion has been sub-optimal, largely because heart rate (HR) varies widely across species.

Objectives

To discover regulatory universal mechanisms of normal automaticity in SAN pacemaker cells that are self-similar across species.

Method

Sub-cellular Ca2+releases, whole cell AP-induced Ca2+transients and APs were recorded in isolated mouse, guinea-pig, rabbit and human SAN cells. Parametric Ca2+and Vm Kinetic Transitions (PCVKT) during phases of AP cycles from their ignition to recovery were quantified.

Results

Although both action potential cycle lengths (APCL) and PCVKT during AP cycles differed across species by ten-fold, trans-species scaling of PCVKT during AP cycles and scaling, of PCVKT to APCL in cellsin vitro, EKG RR intervalsin vivo, and BM were self-similar (obeyed power laws) across species. Thus, APCLin vitro, HRin vivo, and BM ofanyspecies can be predicted by PCVKT during AP cycles in SAN cells measured in any single speciesin vitro.

Conclusions

In designing optimal HR to match widely different BM and energy requirements from mice to humans, nature did not “reinvent pacemaker cell wheels”, but differentially scaled kinetics of gears that regulate the rates at which the “wheels spin”. This discovery will facilitate the development of novel pharmalogic therapies and biologic pacemakers featuring a normal, wide-range rate regulation in animal models and the translation of these to humans to target recalcitrant human SSS.

Condensed Abstract

Studies in animal models are an important facet of cardiac arrhythmia research. Because HR differs by over ten-fold between some animals and humans, translation of knowledge about regulatory mechanisms of SAN normal automaticity gleaned from studies in animal models to target human SSS has been sub-optimal. Our findings demonstrating that trans-species self-similarity of sub-cellular and cellular mechanisms that couple Ca2+to Vm during AP cycles can predict heart ratein vivofrom mice to humans will inform on the design of novel studies in animal models and facilitate translation of this knowledge to target human disease.
Fatty acid binding protein 5 forms higher order assemblies with FLAP or COX-2 in LPS-stimulated macrophages
Authors: Elder Shelby E. , Bauer Nicholas C. , Soberman Roy J. , Schmider Angela B.
Published: October 2020

Immune cells must integrate multiple extracellular signals to produce an appropriate inflammatory response, including production of a dynamic mix of eicosanoids and related bioactive lipids. Synthesis of these lipids is initiated on the membrane surface of the nuclear envelope and endoplasmic reticulum. One critical question is how the precursor arachidonic acid (AA) is distributed between the initial biosynthetic enzymes of the arachidonate 5-lipoxygenase (5-LO) and prostaglandin-endoperoxidase synthase-1/2 (COX-1/2) related pathways. To understand these balancing mechanisms, we hypothesized that fatty acid binding proteins mediate this process. We employed a multi-modal imaging approach by combining direct stochastic optical reconstruction microscopy (dSTORM) with computational analyses and fluorescence lifetime imaging microscopy (FLIM) to delineate the relationships of fatty acid binding proteins 3, 4, and 5 (FABP3–5) with 5-LO activating protein (FLAP), COX-1, and COX-2 in the presence of a stimulus (lipopolysaccharide, LPS) that triggers the synthesis of prostaglandin E2(PGE2). LPS triggers a redistribution of FABP5 to higher order assemblies of COX-2 or FLAP. This was evidenced by a decrease in lifetime determined by FLIM. Colocalization between FABP3 and FLAP decreased, but no other changes in distribution were observed for FABP3 and FABP4. In contrast, assemblies of FABP5 with COX-1 were smaller and showed an increase in lifetime. The data indicate that FABP5 is a member of higher order assemblies of eicosanoid biosynthetic enzymes and that FABP5 may play a key role in regulating the organization of these structures. FABP5 is positioned to distribute AA to both the 5-LO and COX-2 pathways.
Melanocytes determine angiogenesis gene expression across human tissues
Authors: Freilikhman Shirly , Halasi Marianna , Eran Alal , Adini Irit
Published: October 2020

Several angiogenesis-dependent diseases, including age-related macular degeneration and infantile hemangioma, display differential prevalence among Black, as compared to White individuals. Although socioeconomic status and genetic architecture have been suggested as explaining these differences, we have recently shown that pigment productionper semight be involved. For example, we have shown that the extracellular protein fibromodulin is a pro-angiogenic factor highly secreted by melanocytes in White but not Black individuals. Still, additional pigment-dependent angiogenic factors and their molecular mechanisms remain to be identified. Understanding the contribution of pigmentation to angiogenesis in health and disease is essential for precision medicine of angiogenesis-dependent diseases with racial disparity. Toward that goal, we compared the transcriptomes of Black and White individuals in three tissues with angiogenic activity, namely artery, whole blood, and skin. We identified several differentially expressed angiogenesis pathways, including artery morphogenesis, regulation of endothelial cell chemotaxis, and cellular response to vascular endothelial growth factor stimulus. We then demonstrated that the expression of key genes in these pathways is directly modulated by the degree of pigmentation. We further identified the precise pigment production pathway controlling the expression of these genes, namely melanocortin 1 receptor (MC1R) signaling. These results demonstrate pigment-mediated regulation of angiogenesis-related pathways and their driver genes across human tissues.
Disparate Bone Anabolic Cues Activate Bone Formation by Regulating the Rapid Lysosomal Degradation of Sclerostin Protein
Authors: Gould Nicole R. , Williams Katrina M. , Joca Humberto C. , Torre Olivia M. , Lyons James S. , Leser Jenna M. , Srikanth Manasa P. , Hughes Marcus , Khairallah Ramzi J. , Feldman Ricardo A. , Ward Christopher W. , Stains Joseph P.
Published: October 2020

The down regulation of sclerostin mediates bone formation in response to mechanical cues and parathyroid hormone (PTH). To date, the regulation of sclerostin has been attributed exclusively to the transcriptional downregulation that occurs hours after stimulation. Here, we describe, for the first time, the rapid post-translational degradation of sclerostin protein by the lysosome following mechanical load or PTH. We present a unifying model, integrating both new and established mechanically- and hormonally-activated effectors into the regulated degradation of sclerostin by lysosomes. Using anin vivomechanical loading model, we find transient inhibition of lysosomal degradation or the upstream mechano-signaling pathway controlling sclerostin abundance impairs subsequent load-induced bone formation. We also link dysfunctional lysosomes to aberrant sclerostin regulation using Gaucher disease iPSCs. These results inform a paradigm shift in how bone anabolic cues post-translationally regulate sclerostin and expands our understanding of how osteocytes regulate this fundamentally important protein to regulate bone formation.
Ten complete mitochondrial genomes of Gymnocharacini (Stethaprioninae, Characiformes): evolutionary relationships and a repetitive element in the Control Region (D-loop)
Authors: Pasa Rubens , Menegídio Fabiano Bezerra , Rodrigues-Oliveira Igor Henrique , da Silva Iuri Batista , de Campos Matheus Lewi Cruz Bonaccorsi , Rocha-Reis Dinaíza Abadia , Heslop-Harrison John Seymour (Pat) , Schwarzacher Trude , Kavalco Karine Frehner
Published: October 2020

We are presenting the complete mitogenomes of eight fish species/cytotypes from Neotropical region belonging to theAstyanaxandPsalidodongenus:A. aeneus, A. altiparanae, P. fasciatus(from two locations - Upper Paraná and São Francisco river basins),A. lacustris, P. rivularis(two cytotypes) andP. rioparanaibano. We perform the whole-genome sequencing for six of these species in a Novaseq 6000 - by Illumina, meanwhile two genomes were assembled from raw data available in databases. Plus, we reassembled and annotated the mitochondrial genomes forA. mexicanusandP. paranae, both already described and with raw data available online. All the genomes presented the same organization, with 13 protein-coding genes, 22 tRNA genes and two rRNA genes. Aiming to contribute to the understanding of the several cryptic species complexes and phylogeny of the genus, we perform Bayesian analysis using the 13 protein-coding genes from these species, plusDeuterodon gitonand using aBryconspecies as outgroup.
Chromatin accessibility profiles of castration-resistant prostate cancers reveal novel subtypes and therapeutic vulnerabilities
Authors: Tang Fanying , Wang Shangqian , Wong Chen Khuan , Lee Cindy J. , Cohen Sandra , Park Jane , Hill Corinne E. , Eng Kenneth , Bareja Rohan , Han Teng , Liu Eric Minwei , Palladino Ann , Di Wei , Gao Dong , Abida Wassim , Beg Shaham , Puca Loredana , Berger Michael F. , Gopalan Anuradha , Dow Lukas E. , Mosquera Juan Miguel , Beltran Himisha , Sternberg Cora N. , Chi Ping , Scher Howard I. , Sboner Andrea , Chen Yu , Khurana Ekta
Published: October 2020

In castration-resistant prostate cancer (CRPC), the loss of androgen receptor (AR)-dependence due to lineage plasticity, which has become more prevalent, leads to clinically highly aggressive tumors with few therapeutic options and is mechanistically poorly defined. To identify the master transcription factors (TFs) of CRPC in a subtype-specific manner, we derived and collected 29 metastatic human prostate cancer organoids and cell lines, and generated ATAC-seq, RNA-seq and DNA sequencing data. We identified four subtypes and their master TFs using novel computational algorithms: AR-dependent; Wnt-dependent, driven by TCF; neuroendocrine, driven by ASCL1 and NEUROD1 and stem cell-like (SCL), driven by the AP-1 family. The transcriptomic signatures of these four subtypes enabled the classification of 370 patients. We find that AP-1 co-operates with the inhibitable YAP/TAZ/TEAD pathway in the SCL subtype, the second most common group of CRPC tumors after AR-dependent. Together, this molecular classification reveals new drug targets and can potentially guide therapeutic decisions.
A toolbox for imaging RIPK1, RIPK3 and MLKL in mouse and human cells
Authors: Samson André L. , Fitzgibbon Cheree , Patel Komal M. , Hildebrand Joanne M. , Whitehead Lachlan W. , Rimes Joel S. , Jacobsen Annette V. , Horne Christopher R. , Gavin Xavier J. , Young Samuel N. , Rogers Kelly L. , Hawkins Edwin D. , Murphy James M.
Published: October 2020

Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3 and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol-is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3 and MLKL signals.
LACCASE is Necessary for Root Development inSalvia miltiorrhiza
Authors: Zhou Zheng , Li Qing , Wang Yun , Xiao Liang , Bu Qitao , Hao Kai , Guo Meili , Chen Wansheng , Zhang Lei
Published: October 2020

Laccases are multicopper-containing glycoproteins related to monolignol oxidation and polymerization. These properties indicate that laccases are involved in the formation of important medicinal phenolic acid compounds inSalvia miltiorrhizasuch as salvianolic acid B (SAB), which is used for cardiovascular disease treatment. To date, 29 laccases have been found inS. miltiorrhiza, some of which influence the synthesis of phenolic acids. Because of the functional redundancy of laccase genes, their roles inS. miltiorrhizaare poorly understood. In this study, the CRISPR/Cas9 system was first used for dual gene locus targeting inS. miltiorrhizato knock out multiple laccase family genes. The development of the roots was retarded, and root microstructure was abnormal in laccase mutant lines. Additionally, the accumulation of phenolic acid compounds as well as lignin was dramatically reduced. This study suggests that SmLACs are necessary for root development and phenolic acid compound metabolism inS. miltiorrhiza.
Development and analytical validation of a finite element model of fluid transport through osteochondral tissue
Authors: Hislop Brady D. , Heveran Chelsea M. , June Ronald K.
Published: October 2020

Fluid transport between cartilage and bone is critical to joint health. The objective of this study was to develop and analytically validate a finite element model of osteochondral tissue capable of modeling cartilage-bone fluid transport. A biphasic viscoelastic model using an ellipsoidal fiber distribution was created with three distinct layers of cartilage (superficial zone, middle zone, and deep zone) along with a layer of subchondral bone. For stress-relaxation in unconfined compression, our results for compressive stress, radial stress, effective fluid pressure, and elastic recoil were compared with established biphasic analytical solutions. Our model also shows the development of fluid pressure gradients at the cartilage-bone interface during loading. Fluid pressure gradients developed at the cartilage-bone interface with consistently higher pressures in cartilage following initial loading to 10% strain, followed by convergence towards equal pressures in cartilage and bone during the 400s relaxation period. These results provide additional evidence that fluid is transported between cartilage and bone during loading and improves upon estimates of the magnitude of this effect through incorporating a realistic distribution and estimate of the collagen ultrastructure. Understanding fluid transport between cartilage and bone may be key to new insights about the mechanical and biological environment of both tissues in health and disease.
Somatic CAG expansion in Huntington’s disease is dependent on the MLH3 endonuclease domain, which can be excluded viaMLH3splice redirection to suppress expansion
Authors: Roy Jennie C. L. , Vitalo Antonia , Andrew Marissa A. , Mota-Silva Eduarda , Kovalenko Marina , Burch Zoe , Nhu Anh M. , Cohen Paula E. , Grabczyk Ed , Wheeler Vanessa C. , Pinto Ricardo Mouro
Published: October 2020

Somatic expansion of the CAG repeat tract that causes Huntington’s disease (HD) is thought to contribute to the rate of disease pathogenesis. Therefore, factors influencing repeat expansion are potential therapeutic targets. Genes in the DNA mismatch repair pathway are critical drivers of somatic expansion in HD mouse models. Here, we have tested, using genetic and pharmacological approaches, the role of the endonuclease domain of the mismatch repair protein MLH3 in somatic CAG expansion in HD mice and patient cells. A point mutation in the MLH3 endonuclease domain completely eliminated CAG expansion in the brain and peripheral tissues of a HD knock-in mouse model (HttQ111). To test whether the MLH3 endonuclease could be manipulated pharmacologically, we delivered splice switching oligonucleotides in mice to redirectMlh3splicing to exclude the endonuclease domain. Splice redirection to an isoform lacking the endonuclease domain was associated with reduced CAG expansion. Finally, CAG expansion in HD patient-derived primary fibroblasts was also significantly reduced by redirectingMLH3splicing to the endogenous endonuclease domain-lacking isoform. These data indicate the potential of targeting the MLH3 endonuclease domain to slow somatic CAG repeat expansion in HD, a therapeutic strategy that may be applicable across multiple repeat expansion disorders.
Large-Scale Labeling and Assessment of Sex Bias in Publicly Available Expression Data
Authors: Flynn Emily , Chang Annie , Altman Russ B.
Published: October 2020

Women are at more than 1.5-fold higher risk for clinically relevant adverse drug events. While this higher prevalence is partially due to gender-related effects, biological sex differences likely also impact drug response. Publicly available gene expression databases provide a unique opportunity for examining drug response at a cellular level. However, missingness and heterogeneity of metadata prevent large-scale identification of drug exposure studies and limit assessments of sex bias. To address this, we trained organism-specific models to infer sample sex from gene expression data, and used entity normalization to map metadata cell line and drug mentions to existing ontologies. Using this method, we infer sex labels for 450,371 human and 245,107 mouse microarray and RNA-seq samples from refine.bio. Overall, we find slight female bias (52.1%) in human samples and (62.5%) male bias in mouse samples; this corresponds to a majority of single sex studies, split between female-only and male-only (33.3% vs 18.4% in human and 31.0% vs 30.4% in mouse respectively). In drug studies, we find limited evidence for sex-sampling bias overall; however, specific categories of drugs, including human cancer and mouse nervous system drugs, are enriched in female-only and male-only studies respectively. Our expression-based sex labels allow us to further examine the complexity of cell line sex and assess the frequency of metadata sex label misannotations (2-5%). We make our inferred and normalized labels, along with flags for misannotated samples, publicly available to catalyze the routine use of sex as a study variable in future analyses.
A β-catenin-driven switch in TCF/LEF transcription factor binding to DNA target sites promotes commitment of mammalian nephron progenitor cells
Authors: Guo Qiuyu , Kim Albert , Li Bin , Ransick Andrew , Bugacov Helena , Chen Xi , Lindstrom Nils , Brown Aaron , Oxburgh Leif , Ren Bing , McMahon Andrew P.
Published: October 2020

The canonical Wnt pathway transcriptional co-activator β-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated β-catenin levels in NPC cultures using the GSK3 inhibitor CHIR9902 (CHIR) to examine opposing developmental actions of β-catenin. Low CHIR-mediated maintenance and expansion of NPCs is independent of direct engagement of TCF/LEF/ β-catenin transcriptional complexes at low-CHIR dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/β-catenin complexes replaced TCF7L1/TCF7L2 binding on enhancers of differentiation-promoting target genes. Chromosome confirmation studies showed pre-established promoter-enhancer connections to these target genes in NPCs. High CHIR-associatedde novolooping was observed in positive transcriptional feedback regulation to the canonical Wnt pathway. Thus, β-catenin’s direct transcriptional role is restricted to the induction of NPCs where rising β-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program.
Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking and synchronized extracellular vesicle release of CD9 and CD63
Authors: Mathieu Mathilde , Névo Nathalie , Jouve Mabel , Valenzuela José Ignacio , Maurin Mathieu , Verweij Frederik , Palmulli Roberta , Lankar Danielle , Dingli Florent , Loew Damarys , Rubinstein Eric , Boncompain Gaёlle , Perez Franck , Théry Clotilde
Published: October 2020

Despite their important and multiple roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized yet. In particular, how and to what extent EVs form either as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (ectosomes) remains unclear. We followed in HeLa cells the intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment and identified transient co-localization both at the plasma membrane (PM) and in endosomes, before they finally segregate. CD9 was more abundantly released in EVs than CD63. However, when forcing expression of CD63 at the PM, by mutating its lysosome-addressing motive, its secretion in EVs was increased. Thus, in HeLa cells, small ectosomes are more prominently released than exosomes. By comparative proteomic analysis, we identified a few surface proteins likely specific of either exosomes (e.g. LAMP1) or ectosomes (e.g. BSG, SLC3A2), based on their known intracellular location in lysosomes or the PM, and on the different effects on their release of Bafilomycin A1, a drug that neutralizes endosomal pH. Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.
Deep ultraviolet light based wide-field fluorescence microscope for brain imaging
Authors: Kasaragod Deepa , Zhu Meina , Terai Haruhi , Kawakami Koichi , Aizawa Hidenori
Published: October 2020

Three-dimensional (3D) cellular scale imaging techniques that improve our understanding of the brain architecture is crucial for structural and functional integration and analysis of normal and pathological conditions in the brain. We have developed a wide-field fluorescent microscope using deep ultra violet (DUV) light emitting diode as the illumination source. This microscope employs oblique illumination of deep UV light and the optical sectioning is obtained on the tissue surface over a few micron thickness; largely attributed to the large absorption and hence low tissue penetration of the DUV light. Fluorescence emissions in the visible range that are spectrally separates allows for multiple channel of fluorophore detection using single or a combination of dyes. The fluorescence signal is captured using water immersion objectives and a color camera. Arduino Mega 2560 controlled 3-axis motorised microscope stage is developed for obtaining the wide-field imaging. To enable 3D imaging, the microscope setup is integrated with vibrating microtome that can slice thin sections for serial block-face imaging. In this paper, we show the versatility of the DUV microscope as a microscopy tool for use in neuroscience labs for a range of 2D brain imaging applications. We also present its applicability for 3D imaging of rodent brain in combination with whole brain staining protocol.
A curated dataset of modern and ancient high-coverage shotgun human genomes
Authors: Delser Pierpaolo Maisano , Jones Eppie R. , Hovhannisyan Anahit , Cassidy Lara , Pinhasi Ron , Manica Andrea
Published: October 2020

Over the last few years, genome-wide data for a large number of ancient human samples have been collected. Whilst datasets of capture SNPs have been collated, high coverage shotgun genomes (which are relatively few but allow certain type of analyses not possible with ascertained captured SNPs) have to be reprocessed by individual groups from raw reads. This task is computationally intensive. Here, we release a dataset including 34 whole-genome sequenced samples, previously published and distributed worldwide, together with the genetic pipeline used to process them. The dataset contains 73,435,604 sites called across 18 ancient and 16 modern individuals and includes sequence data from four previously published ancient samples which we sequenced to higher coverage (10-18x). Such a resource will allow researchers to analyse their new samples with the same genetic pipeline and directly compare them to the reference dataset without re-processing published samples. Moreover, this dataset can be easily expanded to increase the sample distribution both across time and space.
Deep-learning based denoising and reconstruction of super-resolution structured illumination microscopy images
Authors: Shah Zafran Hussain , Müller Marcel , Wang Tung-Cheng , Scheidig Philip Maurice , Schneider Axel , Schüttpelz Mark , Huser Thomas , Schenck Wolfram
Published: October 2020

Super-resolution structured illumination microscopy (SR-SIM) provides an up to two-fold enhanced spatial resolution of fluorescently labeled samples. The reconstruction of high quality SR-SIM images critically depends on patterned illumination with high modulation contrast. Noisy raw image data, e.g. as a result of low excitation power or low exposure times, result in reconstruction artifacts. Here, we demonstrate deep-learning based SR-SIM image denoising that results in high quality reconstructed images. A residual encoding-decoding convolution neural network (RED-Net) was used to successfully denoise computationally reconstructed noisy SR-SIM images. We also demonstrate the entirely deep-learning based denoising and reconstruction of raw SIM images into high-resolution SR-SIM images. Both image reconstruction methods prove to be very robust against image reconstruction artifacts and generalize very well over various noise levels. The combination of computational reconstruction and subsequent denoising via RED-Net shows very robust performance during inference after training even if the microscope settings change.
Refining physico-chemical rules for herbicides using an antimalarial library
Authors: Sukhoverkov Kirill V. , Corral Maxime G. , Leroux Julie , Haywood Joel , Johnen Philipp , Newton Trevor , Stubbs Keith A. , Mylne Joshua S.
Published: October 2020

Successful herbicides, like drugs, have physico-chemical properties that usually fall within certain limits. A recent analysis of 334 herbicides showed similar properties to the ‘rule of five’ for human orally-delivered drugs, but herbicides diverged from this for proton donors, partition coefficients and molecular weight. To refine rules for herbicides, we exploited the close evolutionary relationship betweenP. falciparumand plants by screening the Malaria Box, a 400-compound library composed of novel chemical scaffolds with activity against blood stage malaria parasitePlasmodium falciparum. A high proportion (52 of 400) were herbicidal toArabidopsis thalianaon agar plates. Thirty-nine of these 52 herbicidal compounds were tested on soil and 16 compounds were herbicidal. These data were used to predict whether a herbicidal hit found on agar will work on soil-grown plants. The physico-chemical parameters were weighted to logP and formal charge and used to generate weighted scores to a large chemical library of liver-stage effective antimalarial leads. Of the six top-scoring compounds, one had a potency comparable to commercial herbicides. This novel compound MMV1206386 had no close structural matches among commercial herbicides. Physiological profiling suggested that MMV1206386 has a new mode of action and overall demonstrates how weighted rules can help during herbicide discovery programs.