bluuebunny/crossref_metadata_2025 huggingface dataset:
A whole-genome sequenced control population in northern Sweden reveals subregional genetic differences
Authors: Svensson Daniel , Rentoft Matilda , Dahlin Anna M. , Lundholm Emma , Olason Pall I. , Sjödin Andreas , Nylander Carin , Melin Beatrice S. , Trygg Johan , Johansson Erik
Published: February 2020

The number of national reference populations that are whole-genome sequenced are rapidly increasing. Partly driving this development is the fact that genetic disease studies benefit from knowing the genetic variation typical for the geographical area of interest. A whole-genome sequenced Swedish national reference population (n=1000) has been recently published but with few samples from northern Sweden. In the present study we have whole-genome sequenced a control population (n=300) (ACpop) from Västerbotten County, a sparsely populated region in northern Sweden previously shown to be genetically different from southern Sweden. The aggregated variant frequencies within ACpop are publicly available (DOI 10.17044/NBIS/G000005) to function as a basic resource in clinical genetics and for genetic studies. Our analysis of ACpop, representing approximately 0.11% of the population in Västerbotten, indicates the presence of a genetic substructure within the county. Furthermore, a demographic analysis showed that the population from which samples were drawn was to a large extent geographically stationary, a finding that was corroborated in the genetic analysis down to the level of municipalities. Including ACpop in the reference population when imputing unknown variants in a Västerbotten cohort resulted in a strong increase in the number of high-confidence imputed variants (up to 81% for variants with minor allele frequency < 5%). ACpop was initially designed for cancer disease studies, but the genetic structure within the cohort will be of general interest for all genetic disease studies in northern Sweden.
Gut eukaryotic communities in pigs: diversity, composition and host genetics contribution
Authors: Ramayo-Caldas Yuliaxis , Prenafeta Francesc , Zingaretti Laura M , Gonzales Olga , Dalmau Antoni , Quintanilla Raquel , Ballester Maria
Published: February 2020

This study aims to characterize commensal fungi and protists inhabiting the gut of healthy pigs, and explore the putative host genetic control over diversity and composition of pig gut eukaryotes. Fecal fungi and protists communities from 514 Duroc pigs of two sexes and two different ages were characterized by 18S and ITS ribosomal RNA gene sequencing. The gut mycobiota was dominated by yeasts, with a high prevalence ofKazachstaniaspp. Regarding protists, representatives of four genera (Blastocystis, Neobalantidium, Tetratrichomonas and Trichomitus) persisted through more than the 80% of the pigs. Heritabilities for the diversity and abundance of gut eukaryotic communities were estimated with the subset of 60 days aged piglets (N=405). Obtained heritabilities ranged from 0.15 to 0.28, indicating a rather limited host-genetic control. A genome wide association study reported genetic variants associated with the fungal α-diversity (SSC6) and with the abundance ofBlastocystisspp. (SSC6, SSC17 and SSC18). Annotated candidate genes (IL23R, IL12RB2, PIK3C3, PIK3CD,HNF4AandTNFRSF9) were mainly related to immunity, gut homeostasis and metabolic processes. Our results point towards a minor and taxa specific genetic control over the diversity and composition of the pig gut eukaryotic communities.
Extensive genomic and transcriptomic variation defines the chromosome-scale assembly ofHaemonchus contortus, a model gastrointestinal worm
Authors: Doyle Stephen R. , Tracey Alan , Laing Roz , Holroyd Nancy , Bartley David , Bazant Wojtek , Beasley Helen , Beech Robin , Britton Collette , Brooks Karen , Chaudhry Umer , Maitland Kirsty , Martinelli Axel , Noonan Jennifer D. , Paulini Michael , Quail Michael A. , Redman Elizabeth , Rodgers Faye H. , Sallé Guillaume , Shabbir Muhammad Zubair , Sankaranarayanan Geetha , Wit Janneke , Howe Kevin L. , Sargison Neil , Devaney Eileen , Berriman Matthew , Gilleard John S. , Cotton James A.
Published: February 2020

Background

Haemonchus contortusis a globally distributed and economically important gastrointestinal pathogen of small ruminants, and has become the key nematode model for studying anthelmintic resistance and other parasite-specific traits among a wider group of parasites including major human pathogens. Two draft genome assemblies forH. contortuswere reported in 2013, however, both were highly fragmented, incomplete, and differed from one another in important respects. While the introduction of long-read sequencing has significantly increased the rate of production and contiguity ofde novogenome assemblies broadly, achieving high quality genome assemblies for small, genetically diverse, outcrossing eukaryotic organisms such asH. contortusremains a significant challenge.

Results

Here, we report using PacBio long read and OpGen and 10X Genomics long-molecule methods to generate a highly contiguous 283.4 Mbp chromosome-scale genome assembly including a resolved sex chromosome. We show a remarkable pattern of almost complete conservation of chromosome content (synteny) withCaenorhabditis elegans, but almost no conservation of gene order. Long-read transcriptome sequence data has allowed us to define coordinated transcriptional regulation throughout the life cycle of the parasite, and refine our understanding ofcis- andtrans-splicing relative to that observed inC. elegans. Finally, we use this assembly to give a comprehensive picture of chromosome-wide genetic diversity both within a single isolate and globally.

Conclusions

TheH. contortusMHco3(ISE).N1 genome assembly presented here represents the most contiguous and resolved nematode assembly outside of theCaenorhabditisgenus to date, together with one of the highest-quality set of predicted gene features. These data provide a high-quality comparison for understanding the evolution and genomics ofCaenorhabditisand other nematodes, and extends the experimental tractability of this model parasitic nematode in understanding pathogen biology, drug discovery and vaccine development, and important adaptive traits such as drug resistance.
Cystin gene mutations cause autosomal recessive polycystic kidney disease associated with alteredMycexpression
Authors: Yang Chaozhe , O’Connor Amber K. , Kesterson Robert A. , Watts Jacob A. , Majmundar Amar J. , Braun Daniela A. , Lek Monkol , Laricchia Kristen M. , Fathy Hanan M. , Shril Shirlee , Hildebrandt Friedhelm , Guay-Woodford Lisa M.
Published: February 2020

Mutation of theCys1gene underlies the renal cystic disease in theCys1cpk/cpk(cpk)mouse that phenocopies human autosomal recessive polycystic kidney disease (ARPKD). Cystin, the protein product ofCys1, is expressed in the primary apical cilia of renal ductal epithelial cells. In previous studies, we showed that cystin regulatesMycexpression via interaction with the tumor suppressor, necdin. Here, we demonstrate rescue of thecpkrenal phenotype by kidney-specific expression of a cystin-GFP fusion protein encoded by a transgene integrated into theRosa26locus. In addition, we show that expression of the cystin-GFP fusion protein in collecting duct cells down-regulates expression ofMycincpkkidneys. Finally, we report the first human patient with an ARPKD phenotype due to homozygosity for a predicted deleterious splicing defect inCYS1. These findings suggest that mutations in theCys1mouse andCYS1human orthologues cause an ARPKD phenotype that is driven by overexpression of theMycproto-oncogene.

Translational Statement

The cystin-deficientcpkmouse is a model for the study of autosomal recessive polycystic kidney disease (ARPKD). We show that thecpkmouse phenotype is associated with alteredMycexpression. To date, the clinical relevance of cystin deficiency to human disease was unclear, due to the absence of ARPKD cases associated withCYS1mutations. We report the first case of ARPKD linked to aCYS1mutation disrupting normal splicing. These findings confirm the relevance of cystin deficiency to human ARPKD, implicateMycin disease initiation or progression, and validate thecpkmouse as a translationally relevant disease model.
Love at first flight: Wing interference patterns are species-specific and sexually dimorphic in blowflies (Diptera: Calliphoridae)
Authors: Butterworth Nathan J. , White Thomas E. , Byrne Phillip G. , Wallman James F.
Published: February 2020

Wing interference patterns (WIPs) are stable structural colours displayed on insect wings which are only visible at specific viewing geometries and against certain backgrounds. These patterns are widespread among flies and wasps, and growing evidence suggests that they may function as species- and sex-specific mating cues in a range of taxa. As such, it is expected that WIPs should differ between species and show clear sexual dimorphisms. However, the true extent to which WIPs vary between species, sexes, and individuals is currently unclear, as previous studies have only taken a qualitative approach, without considering how WIPs might be perceived by the insect. Here, we perform the first quantitative analysis of inter- and intra-specific variation in WIPs across seven Australian species of the blowfly genusChrysomya. Using multispectral digital imaging and a tentative model of blowfly colour vision, we provide quantitative evidence that WIPs are species-specific, highlight that the extent of divergence is greater in males than in females, and demonstrate sexual dimorphisms in several species. These data provide evidence that WIPs have diversified substantially in blowflies and suggests that sexual selection may have played a role in this process.
Exon probe sets and bioinformatics pipelines for all levels of fish phylogenomics
Authors: Hughes Lily C. , Ortí Guillermo , Saad Hadeel , Li Chenhong , White William T. , Baldwin Carole C. , Crandall Keith A. , Arcila Dahiana , Betancur-R. Ricardo
Published: February 2020

Exon markers have a long history of use in phylogenetics of ray-finned fishes, the most diverse clade of vertebrates with more than 35,000 species. As the number of published genomes increases, it has become easier to test exons and other genetic markers for signals of ancient duplication events and filter out paralogs that can mislead phylogenetic analysis. We present seven new probe sets for current target-capture phylogenomic protocols that capture 1,104 exons explicitly filtered for paralogs using gene trees. These seven probe sets span the diversity of teleost fishes, including four sets that target five hyper-diverse percomorph clades which together compriseca. 17,000 species (Carangaria, Ovalentaria, Eupercaria, and Syngnatharia + Pelagiaria combined). We additionally included probes to capture exon markers that have been commonly used in fish phylogenetics, despite some being flagged for paralogs, to facilitate integration of old and new molecular phylogenetic matrices. We tested these probes and merged new exon-capture sequence data into an existing data matrix of 1,105 exons and 300 ray-finned fish species. We provide an optimized bioinformatics pipeline to assemble exon capture data from raw reads to alignments for downstream analysis. We show that loci with known paralogs are at risk of assembling duplicated sequences with target-capture, but we also assembled many useful orthologous sequences. These probe sets are a valuable resource for advancing fish phylogenomics because they can be easily extracted from increasingly available whole genome and transcriptome datasets, and also may be integrated with existing PCR-based exon and mitochondrial datasets.
COMPOSITUM 1 (COM1) contributes to the architectural simplification of barley inflorescence via meristem identity signals
Authors: Poursarebani N. , Trautewig C. , Melzer M. , Nussbaumer T. , Lundqvist U. , Rutten T. , Schmutzer T. , Brandt R. , Himmelbach A. , Altschmied L. , Koppolu R. , Youssef H. M. , Sibout R. , Dalmais M. , Bendahmane A. , Stein N. , Xin Z. , Schnurbusch T.
Published: February 2020

Grasses have varying inflorescence shapes; however, little is known about the genetic mechanisms specifying such shapes among tribes. We identified the grass-specific TCP transcription factor COMPOSITUM 1 (COM1) expressed in inflorescence meristematic boundaries of different grasses. COM1 specifies branch-inhibition in Triticeae (barley) versus branch-formation in non-Triticeae grasses. Analyses of cell size, cell walls and transcripts revealed barley COM1 regulates cell growth, affecting cell wall properties and signaling specifically in meristematic boundaries to establish identity of adjacent meristems.COM1acts upstream of the boundary geneLiguleless1and confers meristem identity partially independent of theCOM2pathway. Furthermore, COM1 is subject to purifying natural selection, thereby contributing to specification of the spike inflorescence shape. This meristem identity pathway has conceptual implications for both inflorescence evolution and molecular breeding in Triticeae.
Distinct representations of finger movement and force in human motor and premotor cortices
Authors: Flint Robert D. , Tate Matthew C. , Li Kejun , Templer Jessica W. , Rosenow Joshua M. , Pandarinath Chethan , Slutzky Marc W.
Published: February 2020

The ability to grasp and manipulate objects requires controlling both finger movement kinematics and isometric force. Previous work suggests that these behavioral modes are controlled separately, but it is unknown whether the cerebral cortex represents them differently. Here, we investigated this question by recording high-density electrocorticography from the motor and premotor cortices of seven human subjects performing a sequential movement-force motor task. We decoded finger movement (0.7±0.3 fractional variance account for; FVAF) and force (0.7±0.2 FVAF) with high accuracy, yet found different spatial representations. We also found clear distinctions in electrocorticographic activity by using deep learning methods to uncover state-space representations, and by developing a new metric, the neural vector angle. Thus, state-space techniques can help to investigate broad cortical networks. Finally, we were able to classify the behavioral mode from neural signals with high accuracy (90±6%). Thus, finger movement and force have distinct representations in motor/premotor cortices. This will inform our understanding of the neural control of movement as well as the design of grasp brain-machine interfaces.
Using computational modeling to teach metabolism as a dynamic system improves student performance
Authors: Booth Christine S. , Song Changsoo , Howell Michelle E. , Rasquinha Achilles , Saska Aleš , Helikar Resa , Sikich Sharmin M. , Couch Brian A. , Dijk Karin van , Roston Rebecca L. , Helikar Tomáš
Published: February 2020

Understanding metabolic function requires knowledge of the dynamics, interdependence, and regulation of biochemical networks. However, current approaches are not optimal to develop the needed mechanistic understanding, and misconceptions about biological processes persist even after graduation. To address these issues, we developed a computational modeling and simulation approach that employs scaffolded learning to teach biochemistry students about the regulation of metabolism. The power of the approach lies in students’ abilities to alter any component or connection in a modeled system and instantly observe the effects of their changes. We find that students who use our approach perform better on biochemistry metabolism questions compared to students in a course that did not use this approach. We also investigated performance by gender and found that our modules may have the potential to increase equity in education. We noted that students are generally positive about the approach and appreciate its benefits. Our modules provide life science instructors with a dynamic and systems-driven approach to teach metabolic regulation and control that improves learning and also equips students with important technical skills.
Towards domain-general predictive coding: Expected TMS excites the motor system less effectively than unexpected stimulation
Authors: Tran Dominic M. D. , McNair Nicolas A. , Harris Justin A. , Livesey Evan J.
Published: February 2020

The brain’s response to sensory input is modulated by prediction. For example, sounds that are produced by one’s own actions, or those that are strongly predicted by environmental cues, are perceived as less salient and elicit an attenuated N1 component in the auditory evoked potential. Here we examined whether the neural response to direct stimulation of the brain is attenuated by prediction in a similar manner. Transcranial magnetic stimulation (TMS) applied over primary motor cortex can be used to gauge the excitability of the motor system. Motor-evoked potentials (MEPs), elicited by TMS and measured in peripheral muscles, are larger when actions are being prepared and smaller when actions are voluntarily suppressed. We tested whether the amplitude of MEPs was attenuated under circumstances where the TMS pulse can be reliably predicted, even though control of the relevant motor effector was never required. Self-initiation of the TMS pulse and reliable cuing of the TMS pulse both attenuated MEP amplitudes, compared to MEPs generated programmatically in an unpredictable manner. These results suggest that predictive coding may be governed by domain-general mechanisms responsible for all forms predictive learning.
Specific Inhibition of GSK-3β by Tideglusib: Potential Therapeutic Target for Neuroblastoma Cancer Stem Cells
Authors: Bahmad Hisham F. , Chalhoub Reda M. , Harati Hayat , Bou-Gharios Jolie , Ballout Farah , Monzer Alissar , Msheik Hiba , Assi Sahar , Araji Tarek , Elajami Mohamad K. , Ghanem Paola , Chamaa Farah , Kadara Humam , Abou-Antoun Tamara , Daoud Georges , Fares Youssef , Abou-Kheir Wassim
Published: February 2020

Neuroblastoma is an embryonic tumor that represents the most common extracranial solid tumor in children. Resistance to therapy is attributed, in part, to the persistence of a subpopulation of slowly dividing cancer stem cells (CSCs) within those tumors. Glycogen synthase kinase (GSK)-3β is an active proline-directed serine/threonine kinase, well-known to be involved in different signaling pathways entangled in the pathophysiology of neuroblastoma. This study aims to assess the potency of an irreversible GSK-3β inhibitor drug, Tideglusib (TDG), in suppressing proliferation, viability, and migration of human neuroblastoma cell lines, as well as its effects on their CSCs subpopulationin vitroandin vivo. Our results showed that treatment with TDG significantly reduced cell proliferation, viability, and migration of SK-N-SH and SH-SY5Y cells. TDG also significantly inhibited neurospheres formation capability in both cell lines, eradicating the self-renewal ability of highly resistant CSCs. Importantly, TDG potently inhibited neuroblastoma tumor growth and progressionin vivo. In conclusion, TDG proved to be an effectivein vitroandin vivotreatment for neuroblastoma cell lines and may hence serve as a potential adjuvant therapeutic agent for this aggressive nervous system tumor.
Transcription-associated topoisomerase activities control DNA-breaks production by G-quadruplex ligands
Authors: Pipier A. , Bossaert M. , Riou J.F. , Noirot C. , Nguyễn L-T. , Serre R-F. , Bouchez O. , Defrancq E. , Calsou P. , Britton S. , Gomez D.
Published: February 2020

G-quadruplexes (G4), non-canonical DNA structures, are involved in several essential processes. Stabilization of G4 structures by small compounds (G4 ligands) affects almost all DNA transactions, including telomere maintenance and genomic stability. Here, thanks to a powerful and unbiased genetic approach, we identify topoisomerase 2-alpha (TOP2A) as the main effector of cell cytotoxicity induced by CX5461, a G4 ligand currently undergoing phase I/II clinical trials. This approach also allowed to identify new point mutations affecting TOP2A activity without compromising cell viability. Moreover, based on cross-resistance studies and siRNA-based protein depletion we report that TOP2A plays a major role in cell cytotoxicity induced by two unrelated clastogenic G4 ligands, CX5461 and pyridostatin (PDS). We also report that cytotoxic effects induced by both compounds are associated with topoisomerase 2-mediated DNA breaks production. Finally, we show that TOP2-mediated DNA breaks production is strongly associated with RNA Pol II-dependent transcription and is countered by topoisomerase 1 (TOP1). Altogether our results indicate that clastogenic G4 ligands act as DNA structure-driven TOP2-poisons at transcribed regions bearing G-quadruplex structures.
The stress-induced protein NUPR1 orchestrates protein translation during ER-stress by interacting with eIF2α
Authors: Borrello Maria Teresa , Santofimia-Castaño Patricia , Bocchio Marco , Listi Angela , Fraunhoffer Nicolas , Soubeyran Philippe , Chevet Eric , Pin Christopher , Iovanna Juan
Published: February 2020

NUPR1 is a stress response protein overexpressed upon cell injury in virtually all organs including the exocrine pancreas. Despite NUPR1’s well established role in the response to cell stress, the molecular and structural machineries triggered by NUPR1 activation remain largely unknown. In this study, we uncover an important role for NUPR1 in participating in the unfolded protein response pathway and the endoplasmic reticulum stress response. Biochemical results, confirmed by ultrastructural morphological observation, revealed alterations in the UPR in acinar cells of germline-deleted NUPR1 murine models, consistent with the inability to restore general protein translation. Bioinformatical analysis of NUPR1 interacting partners showed significant enrichment in translation initiation factors, including eukaryotic initiation factor (eIF) 2α. Co-immunoprecipitation and proximity ligation assays both confirmed interaction between NUPR1 and eIF2α and its phosphorylated form (p-eIF2α). Our. Moreover, our data also suggest loss of NUPR1 in cells results in maintained eIF2α phosphorylation and evaluation of nascent proteins by (peIF2α), and click chemistry revealed that NUPR1-depleted PANC-1 cells displayed a slower post stress protein translational recovery compared to wild-type. Combined, this data proposes a novel role for NUPR1 in the integrated stress response pathway, at least partially through promoting efficient PERK-branch activity and resolution through a unique interaction with eIF2α.

Significance

In the pancreas, NUPR1 is required for a resolution of the ER stress response. During ER stress response, NUPR1 binds both eIF2α allowing for its dephosphorylation and restoration of new protein synthesis.

Highlights

Biochemical analysis revealed a general reduction in the protein expression of downstream mediators of the unfolded protein response in the pancreas of mice lackingNupr1. This finding suggests a novel role for NUPR1 in the UPR/ER stress response.

Ultrastructural analysis of pancreata revealed reduced morphological alterations in tunicamycin-treatedNupr1-/-mice compared toNupr1+/+mice consistent with a maintained block in general protein translation.

Co-immunoprecipitation of tagged NUPR1 confirmed a novel interaction with eIF2α. Depletion of NUPR1 prolonged phosphorylation of eIF2α, suggesting it may be involved in attenuation of the PERK branch of the UPR.

NUPR1-depleted PANC-1 cells displayed a slower recovery of protein translation following UPR activation
A supernumerary designer chromosome for modularin vivopathway assembly inSaccharomyces cerevisiae
Authors: Postma Eline D. , Dashko Sofia , van Breemen Lars , Taylor Parkins Shannara K. , van den Broek Marcel , Daran Jean-Marc , Daran-Lapujade Pascale
Published: February 2020

The construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. UsingSaccharomyces cerevisiae, this study proposes Synthetic Chromosomes (SynChs) as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities. Capitalizing the powerful homologous recombination capability ofS. cerevisiae, modular SynChs of 50 and 100 Kb were fully assembledde novofrom up to 44 transcriptional-unit-sized fragments in a single transformation. These assemblies were remarkably efficient and faithful to theirin silicodesign. SynChs made of non-coding DNA were stably replicated and segregated irrespective of their size without affecting the physiology of their host. These non-coding SynChs were successfully used as landing pad and as exclusive expression platform for the essential glycolytic pathway. This work pushes the limit of DNA assembly inS. cerevisiaeand paves the way forde novodesigner chromosomes as modular genome engineering platforms inS. cerevisiae.
Bile Acid pool composition and Gallbladder function are controlled by TGR5 to protect the liver against Bile Acid overload
Authors: Bidault-Jourdainne Valeska , Merlen Grégory , Glénisson Mathilde , Doignon Isabelle , Garcin Isabelle , Péan Noémie , Boisgard Raphael , Ursic-Bedoya José , Serino Matteo , Ullmer Christoph , Humbert Lydie , Abdelrafee Ahmed , Vibert Eric , Duclos-Vallée Jean-Charles , Rainteau Dominique , Tordjmann Thierry
Published: February 2020

Backgrounds & Aims

As the bile acid (BA) pool composition is of major impact on liver pathophysiology, we studied its regulation by the BA receptor TGR5, promoting hepatoprotection against BA overload.

Methods

WT, total and hepato-specific TGR5-KO, and TGR5-overexpressing mice were used in: partial and 90% extended hepatectomies (EH) upon normal, ursodeoxycholic acid (UDCA)- or cholestyramine (CT)-enriched diet, bile duct ligation (BDL), cholic acid (1%)-enriched diet, and TGR5 agonist (RO) treatments. We thereby studied TGR5 impact on: BA pool composition, liver injury, regeneration and survival. Particular focus was made on gut microbiota (GM) and gallbladder (GB) function analysis. BA pool composition was analyzed in patients undergoing major hepatectomy.

Results

The TGR5-KO hyperhydrophobic BA pool was not related to BA synthesis alteration, nor to the TGR5-KO GM dysbiosis, as supported by hepatocyte-specific KO mice and cohousing experiments. The TGR5-dependent control of GB dilatation was crucial for BA pool composition, as determined by experiments including RO treatment +/− cholecystectomy. The poor TGR5-KO post-EH survival rate, related with exacerbated peribiliary necrosis and BA overload, was improved by shifting the BA pool towards a more hydrophilic composition (CT and UDCA treatments). After either BDL or CA-enriched diet +/− cholecystectomy, we found that GB dilatation had strong TGR5-dependent hepatoprotective properties. In patients, a more hydrophobic BA pool was correlated with an unfavorable outcome after hepatectomy.

Conclusion

BA pool composition is crucial for hepatoprotection in mice and humans. We point TGR5 as a key regulator of BA profile and thereby as a potential hepatoprotective target under BA overload conditions.

Lay summary

Through multiple in vivo experimental approaches in mice, together with a patients study, this work brings some new light on the relationships between biliary homeostasis, gallbladder function and liver protection. We showed that the bile acid pool composition is crucial for optimal liver repair, not only in mice but also in human patients undergoing major hepatectomy.
Variance-adjusted Mahalanobis (VAM): a fast and accurate method for cell-specific gene set scoring
Authors: Frost H. Robert
Published: February 2020

Single cell RNA sequencing (scRNA-seq) is a powerful tool for analyzing complex tissues with recent advances enabling the transcriptomic profiling of thousands to tens-of-thousands of individual cells. Although scRNA-seq provides unprecedented insights into the biology of heterogeneous cell populations, analyzing such data on a gene-by-gene basis is challenging due to the large number of tested hypotheses, high level of technical noise and inflated zero counts. One promising approach for addressing these challenges is gene set testing, or pathway analysis. By combining the expression data for all genes in a pathway, gene set testing can mitigate the impacts of sparsity and noise and improve interpretation, replication and statistical power. Unfortunately, statistical and biological differences between single cell and bulk expression measurements make it challenging to use gene set testing methods originally developed for bulk tissue on scRNA-seq data and progress on single cell-specific methods has been limited. To address this challenge, we have developed a new gene set testing method, variance-adjusted Mahalanobis (VAM), that seamlessly integrates with the Seurat framework and is designed to accommodate the technical noise, sparsity and large sample sizes characteristic of scRNA-seq data. The VAM method computes cell-specific pathway scores to transform a cell-by-gene matrix into a cell-by-pathway matrix that can be used for both exploratory data visualization and statistical gene set enrichment analysis. Because the distribution of these scores under the null of uncorrelated technical noise has an accurate gamma approximation, inference can be performed at both the population and single cell levels. As we demonstrate using both simulation studies and real data analyses, the VAM method provides superior classification accuracy at a lower computation cost relative to existing single sample gene set testing approaches.
Arabidopsis REI-LIKE proteins activate ribosome biogenesis during cold acclimation
Authors: Cheong Bo Eng , Beine-Golovchuk Olga , Gorka Michal , Ho William Wing Ho , Martinez-Seidel Federico , Firmino Alexandre Augusto Pereira , Skirycz Aleksandra , Roessner Ute , Kopka Joachim
Published: February 2020

Arabidopsis REIL proteins are cytosolic ribosomal 60S-biogenesis factors. After shift to 10°C,reilmutants deplete and slowly replenish non-translating eukaryotic ribosome complexes of root tissue, while tightly controlling the balance of non-translating 40S- and 60S-subunits.Reilmutations compensate by hyper-accumulation of non-translating subunits at steady-state temperature; after cold-shift, a KCl-sensitive 80S sub-fraction remains depleted. We infer that Arabidopsis buffers fluctuating translation by pre-existing non-translating ribosomes beforede novosynthesis meets temperature-induced demands.Reil1 reil2double mutants accumulate 43S-preinitiation and pre-60S-maturation complexes and have altered paralog composition of ribosomal proteins in non-translating complexes. With few exceptions, e.g. RPL3B and RPL24C, these changes are not under transcriptional control. Our study suggests requirement ofde novosynthesis of eukaryotic ribosomes for long-term cold acclimation, feedback control ofNUC2andeIF3C2transcription and links new proteins, AT1G03250, AT5G60530, to plant ribosome biogenesis. We propose that Arabidopsis requires biosynthesis of specialized ribosomes for cold acclimation.

Highlight of this study

REIL proteins affect paralog composition of eukaryotic ribosomes and suppress accumulation of 43S-preinitiation and pre-60S-maturation complexes, suggesting functions of ribosome heterogeneity and biogenesis in plant cold acclimation.
Constitutive activation of leucine-rich repeat receptor kinase signaling pathways by BAK1-interacting receptor-like kinase 3 chimera
Authors: Hohmann Ulrich , Ramakrishna Priya , Wang Kai , Lorenzo-Orts Laura , Nicolet Joel , Henschen Agnes , Barberon Marie , Bayer Martin , Hothorn Michael
Published: February 2020

Receptor kinases with extracellular leucine-rich repeat domains (LRR-RKs) form the largest group of membrane signaling proteins in plants. LRR-RKs can sense small molecule, peptide or protein ligands, and may be activated by ligand-induced interaction with a shape complementary SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) co-receptor kinase. We have previously shown that SERKs can also form constitutive, ligand-independent complexes with the LRR ectodomains of BAK1-interacting receptor-like kinase 3 (BIR3) receptor pseudokinases, negative regulators of LRR-RK signaling. Here we report that receptor chimaera in which the extracellular LRR domain of BIR3 is fused to the cytoplasmic kinase domains of the SERK-dependent LRR-RKs BRASSINOSTEROID INSENSITIVE1, HAESA and ERECTA form tight complexes with endogenous SERK co-receptors in the absence of ligand stimulus. Expression of these chimaera under the control of the endogenous promoter of the respective LRR-RK leads to strong gain-of-function brassinosteroid, floral abscission and stomatal patterning phenotypes, respectively. Importantly, a BIR3-GSO1/SGN3 chimera can partially complementsgn3Casparian strip formation phenotypes, suggesting that GSO1/SGN3 receptor activation is also mediated by SERK proteins. Collectively, our protein engineering approach may be used to elucidate the physiological functions of orphan LRR-RKs and to identify their receptor activation mechanism in single transgenic lines.
Single-cell chromatin profiling reveals demethylation-dependent metabolic vulnerabilities of breast cancer epigenome
Authors: Kusi Meena , Zand Maryam , Lin Chun-Lin , Wang Chiou-Miin , Lucio Nicholas D. , Kirma Nameer B. , Ruan Jianhua , Huang Tim H.-M. , Mitsuya Kohzoh
Published: February 2020

Metabolic reprogramming in cancer cells not only sustains bioenergetic and biosynthetic needs but also influences transcriptional programs, yet how chromatin regulatory networks are rewired by altered metabolism remains elusive. Here we investigate genome-scale chromatin remodeling in response to 2-hydroxyglutarate (2HG) oncometabolite using single-cell assay for transposase accessible chromatin with sequencing (scATAC-seq). We find that 2HG enantiomers differentially disrupt exquisite control of epigenome integrity by limiting α-ketoglutarate (αKG)-dependent DNA and histone demethylation, while enhanced cell-to-cell variability in the chromatin regulatory landscape is most evident upon exposure to L2HG enantiomer. Despite the highly heterogeneous responses, 2HG largely recapitulates two prominent hallmarks of the breast cancer epigenome, i.e., global loss of 5-hydroxymethylcytosine (5hmC) and promoter hypermethylation, particularly at tumor suppressor genes involved in DNA damage repair and checkpoint control. Single-cell mass cytometry further demonstrates downregulation of BRCA1, MSH2 and MLH1 in 2HG-responsive subpopulations, along with acute reversal of chromatin remodeling upon withdrawal. Collectively, this study provides a molecular basis for metabolism-epigenome coupling and identifies metabolic vulnerabilities imposed on the breast cancer epigenome.
A Superfamily of T6SS Antibacterial Effectors Displaying L,D-carboxypeptidase Activity Towards Peptidoglycan
Authors: de Sousa Stephanie Sibinelli , Hespanhol Julia Takuno , Nicastro Gianlucca Gonçalves , Matsuyama Bruno Yasui , Mesnage Stephane , Patel Ankur , de Souza Robson Francisco , Guzzo Cristiane Rodrigues , Bayer-Santos Ethel
Published: February 2020

Type VI secretion systems (T6SSs) are contractile nanomachines used by bacteria to inject toxic effectors into competitors. The identity and mechanism of many effectors remain unknown. We characterized aSalmonellaSPI-6 T6SS antibacterial effector called Tae5STM(type VIamidaseeffector 5). Tae5STMis toxic in target-cell periplasm and is neutralized by its cognate immunity protein (Tai5STM). Microscopy analysis revealed that cells expressing the effector stop dividing and lose cell envelope integrity. Bioinformatic analysis uncovered similarities between Tae5STMand the catalytic domain of L,D-transpeptidase. Point mutations on conserved catalytic histidine and cysteine residues abrogated toxicity. Biochemical assays revealed that Tae5STMdisplays L,D-carboxypeptidase activity, cleaving peptidoglycan tetrapeptides betweenmeso-diaminopimelic acid3and D-alanine4. Phylogenetic analysis showed that Tae5STMhomologs constitutes a new superfamily of T6SS-associated amidase effectors distributed among α-, β- and γ-proteobacteria. This work expands our current knowledge about bacterial effectors used in interbacterial competition.
Chronic Chemogenetic Manipulation of Ventral Pallidum Targeted Neurons in Rats Fed an Obesogenic Diet
Authors: Doucette Wilder T. , Smedley Elizabeth B. , Ruiz-Jaquez Metztli , Khokhar Jibran Y. , Smith Kyle
Published: February 2020

Current treatments for obesity are unable to reliably reduce weight over time and alternative treatments need to address this challenge. New interventions that target the nervous system could manipulate brain networks underlying reward, metabolic rate and behavior. Here, the ventral pallidum was evaluated as a target of manipulation due to its established role in the networks underlying motivation, pleasure and behavioral output. Chronic inhibitory or excitatory chemogenetic activation was used to modulate the activity of ventral pallidum (VP) targeted neurons in rats on an obesogenic diet. We hypothesized that inhibition of VP activity would decrease the salience of the rats’ high-sugar, high-fat diet and lead to reduced food consumption and weight gain over time. Paradoxically, measurements of weight, water and food consumption revealed significantly increased weight gain in both groups receiving VP targeted manipulation that was not readily explained by food or water consumption. We theorize that the complex reciprocal feedback between ventral striatal structures (e.g., VP) and metabolic centers of the hypothalamus and brainstem, demonstrated by prior research, likely underpin our findings. This study suggests that the treatment of appetitive disorders (e.g., obesity) with chronic neuromodulation-based interventions could be burdened by the delayed onset of outcomes that are difficult to predict from related prior studies that used acute interventions.
Molecular mechanism for direct actin force-sensing by α-catenin
Authors: Mei Lin , de los Reyes Santiago Espinosa , Reynolds Matthew J. , Liu Shixin , Alushin Gregory M.
Published: February 2020

The actin cytoskeleton mediates mechanical coupling between cells and their tissue microenvironments. The architecture and composition of actin networks are modulated by force, but it is unclear how interactions between actin filaments (F-actin) and associated proteins are mechanically regulated. Here, we employ both optical trapping and biochemical reconstitution with myosin motor proteins to show force greater than one piconewton applied solely to F-actin enhances binding by the essential cell-cell adhesion protein αE-catenin, but not its homolog vinculin. Near atomic-resolution cryo-electron microscopy structures of both proteins bound to F-actin reveal unique rearrangements that facilitate their flexible C-termini refolding to engage distinct interfaces. Truncating α-catenin’s C-terminus eliminates force-activated F-actin binding, and addition of this motif to vinculin confers force-activated binding, demonstrating that α-catenin’s C-terminus is a modular detector of F-actin tension. Our studies establish that piconewton force on F-actin can enhance partner binding, which we propose mechanically regulates cellular adhesion throughα-catenin.
The endometrial transcription landscape of MRKH syndrome
Authors: Hentrich T , Koch A , Weber N , Kilzheimer A , Burkhardt S , Rall K , Casadei N , Kohlbacher O , Riess O , Schulze-Hentrich JM , Brucker SY
Published: February 2020

The Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome (OMIM 277000) is characterized by agenesis of the uterus and upper part of the vagina in females with normal ovarian function. While genetic causes have been identified for a small subset of patients and epigenetic mechanisms presumably contribute to the pathogenic unfolding, too, the etiology of the syndrome has remained largely enigmatic. A comprehensive understanding of gene activity in the context of the disease is crucial to identify etiological components and their potential interplay. So far, this understanding is lacking, primarily due to the scarcity of samples and suitable tissue.

In order to close this gap, we profiled endometrial tissue of uterus rudiments in a large cohort of MRKH patients using RNA-seq and thereby provide a genome-wide view on the altered transcription landscape of the MRKH syndrome. Differential and co-expression analyses of the data identified cellular processes and candidate genes that converge on a core network of interconnected regulators that emerge as pivotal for the perturbed expression space. With these results and browsable access to the rich data through an online tool we seek to accelerate research to unravel the underlying biology of this syndrome.
Cerebral Atrophy in Amyotrophic Lateral Sclerosis Parallels the Pathological Distribution of TDP43
Authors: Dadar Mahsa , Manera Ana Laura , Zinman Lorne , Korngut Lawrence , Genge Angela , Graham Simon J. , Frayne Richard , Collins D. Louis , Kalra Sanjay
Published: February 2020

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a preferential involvement of both upper and lower motor neurons. Evidence from neuroimaging and post-mortem studies confirms additional involvement of brain regions extending beyond the motor cortex. The aim of this study was to assess the extent of cerebral disease in ALS cross-sectionally and longitudinally, and to compare the findings with a recently proposed disease-staging model of ALS pathology. Deformation-based morphometry (DBM) was used to identify the patterns of brain atrophy associated with ALS and to assess their relationship with clinical symptoms. Longitudinal T1-weighted MRI data and clinical measures were acquired at baseline, 4 months, and 8 months, from 66 ALS patients and 43 age-matched controls who participated in the Canadian ALS Neuroimaging Consortium (CALSNIC) study. Whole brain voxel-wise mixed-effects modelling analysis showed extensive atrophy patterns differentiating ALS patients from the normal controls. Cerebral atrophy was present in the motor cortex and corticospinal tract, involving both GM and WM, and to a lesser extent in non-motor regions. More specifically, the results showed significant bilateral atrophy in the motor cortex, the corticospinal tract including the internal capsule and brainstem, with an overall pattern of ventricular enlargement; along with significant progressive longitudinal atrophy in the precentral gyrus, frontal and parietal white matter, accompanied by ventricular and sulcal enlargement. Atrophy in the precentral gyrus was significantly associated with greater disability as quantified with the ALS Functional Rating Scale-Revised (ALSFRS-R) (p<0.0001). The pattern of atrophy observed using DBM was consistent with the Brettschneider’s four stage pathological model of the disease. Deformation based morphometry provides a sensitive indicator of atrophy in ALS, and has potential as a biomarker of disease burden, in both gray and white matter.
Allosteric regulation of Thioesterase Superfamily Member 1 by free fatty acids and lysophosphatidylcholine
Authors: Tillman Matthew C. , Imai Norihiro , Li Yue , Khadka Manoj , Okafor C. Denise , Juneja Puneet , Adhiyaman Akshitha , Hagen Susan J. , Cohen David E. , Ortlund Eric A.
Published: February 2020

Non-shivering thermogenesis occurs in brown adipose tissue to generate heat in response to cold temperatures. Thioesterase superfamily member 1 (Them1) is transcriptionally upregulated in brown adipose tissue upon cold exposure and suppresses thermogenesis to conserve energy reserves. Them1 hydrolyzes long-chain fatty acyl-CoAs, preventing their use as fuel for thermogenesis. Them1 contains a C-terminal StAR-related lipid transfer domain (StarD) with unknown ligand or function. By complementary biophysical approaches, we show that StarD binds to long-chain fatty acids, products of Them1’s enzymatic reaction, as well lysophosphatidylcholine (LPC), which activate thermogenesis in brown adipocytes. Certain fatty acids stabilize the StarD and allosterically enhance Them1 catalysis of acyl-CoA, whereas 18:1 LPC destabilizes and inhibits activity, which we verify in cell culture. Additionally, we demonstrate that the StarD functions to localize Them1 near lipid droplets. These findings define the role of the StarD as a lipid sensor that allosterically regulates Them1 activity and localization.
Development of a honey bee RNA virus vector based on the genome of Deformed wing virus
Authors: Ryabov Eugene V. , Christmon Krisztina , Heerman Matthew C. , Posada-Florez Francisco , Harrison Robert L. , Chen Yanping , Evans Jay D.
Published: February 2020

We developed a honey bee RNA-virus vector based on the genome of a picorna-like Deformed wing virus (DWV), the main viral pathogen of the honey bee (Apis mellifera). To test the potential of DWV to be utilized as a vector, the 717 nt sequence coding for the enhanced green fluorescent protein (eGFP), flanked by the peptides targeted by viral protease, was inserted into an infectious cDNA clone of DWV in-frame between the leader protein and the virus structural protein VP2 genes. Thein vitroRNA transcripts fromegfp-tagged DWV cDNA clones were infectious when injected into honey bee pupae. Stable DWV particles containing genomic RNA of the recovered DWV withegfpinserts were produced, as evidenced by cesium chloride density gradient centrifugation. These particles were infectious to honey bee pupae when injected intra-abdominally. Fluorescent microscopy showed GFP expression in the infected cells and Western blot analysis demonstrated accumulation of free eGFP rather than its fusions with DWV LP and/or VP2 proteins. Analysis of the progenyegfp-tagged DWV showed gradual accumulation of genome deletions foregfp, providing estimates for the rate of loss of a non-essential gene an insect RNA virus genome during natural infection.
Acoustogenetic Control of CAR T Cells via Focused Ultrasound
Authors: Wu Yiqian , Liu Yahan , Huang Ziliang , Wang Xin , Jin Zhen , Li Jiayi , Limsakul Praopim , Zhu Linshan , Allen Molly , Pan Yijia , Bussell Robert , Jacobson Aaron , Liu Thomas , Chien Shu , Wang Yingxiao
Published: February 2020

Optogenetics can control specific molecular events in living systems, but the penetration depth of light is typically limited at hundreds of micrometers. Focused ultrasound (FUS), on the other hand, can deliver energy safely and noninvasively into tissues at depths of centimeters. Here we have developed an acoustogenetic approach using short-pulsed FUS to remotely and directly control the genetics and cellular functions of engineered mammalian cells for therapeutic purposes. We applied this acoustogenetic approach to control chimeric antigen receptor (CAR) T cells with high spatiotemporal precision, aiming to mitigate the potentially lethal “on-target off-tumor” effects of CAR T cell therapy. We first verified the controllability of our acoustogenetic CAR T cells in recognizing and killing tumor cellsin vitro, and then applied this approachin vivoto suppress tumor growth of both lymphoma and prostate cancers. The results indicate that FUS-based acoustogenetics can allow the noninvasive and remote activation, without any exogenous cofactor, of different types of CAR T cells for cancer therapeutics.