bluuebunny/crossref_metadata_2025 huggingface dataset:
Combining ancestral reconstruction with folding-landscape simulations to engineer heterologous protein expression
Authors: Gamiz-Arco Gloria , Risso Valeria A. , Gaucher Eric A. , Gavira Jose A. , Naganathan Athi N. , Ibarra-Molero Beatriz , Sanchez-Ruiz Jose M.
Published: July 2021

Obligate symbionts exhibit high evolutionary rates and extensive sequence divergence. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its refolding in vitro is very slow and its expression in E. coli leads to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro refolding rate and that the ancestral expression efficiency can be achieved with only 1-2 back-to-ancestor replacements. These results demonstrate a sequence-engineering approach to rescue inefficient heterologous expression, a major biotechnological bottleneck.
Complex population history affects admixture analyses in nine-spined sticklebacks
Authors: Feng Xueyun , Merilä Juha , Löytynoja Ari
Published: July 2021

Introgressive hybridization is an important process in evolution but challenging to identify, undermining the efforts to understand its role and significance. On the other hand, many analytical methods assume direct descent from a single common ancestor, and admixture among populations can violate their assumptions and lead to seriously biased results. A detailed analysis of 888 whole genome sequences of nine-spined sticklebacks (Pungitius pungitius) revealed a complex pattern of population ancestry involving multiple waves of gene flow and introgression across northern Europe. The two recognized lineages were found to have drastically different histories and their secondary contact zone was wider than anticipated, displaying a smooth gradient of foreign ancestry with some curious deviations from the expected pattern. Interestingly, the freshwater isolates provided peeks into the past and helped to understand the intermediate states of evolutionary processes. Our analyses and findings paint a detailed picture of the complex colonization history of northern Europe and provide back-drop against which introgression and its role in evolution can be investigated. However, they also expose the challenges in analyses of admixed populations and demonstrate how hidden admixture and colonization history misleads the estimation of admixture proportions and population split times.
Creating detailed metadata for an R Shiny analysis of circadian behavior sequence data
Authors: Colomb Julien , Winter York
Published: July 2021

Automated mouse phenotyping through the high-throughput analysis of home cage behavior has brought hope of a more effective and efficient method for testing rodent models of diseases. Advanced video analysis software is able to derive behavioral sequence data sets from multiple-day recordings. However, no dedicated mechanisms exist for sharing or analyzing these types of data. In this article, we present a free, open-source software actionable through a web browser (an R Shiny application), which can perform state-of-the-art multidimensional analysis of home cage behavioral sequence data. The software aligns time-series data to the light/dark cycle, and then uses different time windows to produce up to 162 behavior variables per animal. It prevents p-hacking by providing an analysis that uses a principal component analysis strategy, while also representing the behavior graphically for further explorative analysis. A machine-learning approach was implemented, but it proved ineffective at separating the experimental groups.

The software requires spreadsheets that provide information about the experiment (i.e., metadata), thus promoting a data management strategy that leads to FAIR data production. This encourages the publication of some metadata even when the data are kept private. We tested our software by comparing the behavior of female mice in videos recorded twice at 3 and 7 months in a home cage monitoring system. This study demonstrated that combining data management with data analysis leads to a more efficient and effective research process.
Lower cognitive set shifting ability is associated with stiffer balance recovery behavior and larger perturbation-evoked cortical responses in older adults
Authors: Payne Aiden M. , Palmer Jacqueline A. , McKay J. Lucas , Ting Lena H.
Published: July 2021

1.

The mechanisms underlying associations between cognitive set shifting impairments and balance dysfunction are unclear. Cognitive set shifting refers to the ability to flexibly adjust behavior to changes in task rules or contexts, which could be involved in flexibly adjusting balance recovery behavior to different contexts, such as the direction the body is falling. Prior studies found associations between cognitive set shifting impairments and severe balance dysfunction in populations experiencing frequent falls. The objective of this study was to test whether cognitive set shifting ability is expressed in successful balance recovery behavior in older adults with high clinical balance ability (N=19, 71 ± 7 years, 6 female). We measured cognitive set shifting ability using the Trail Making Test and clinical balance ability using the miniBESTest. For most participants, cognitive set shifting performance (Trail Making Test B-A = 37 ± 20s) was faster than normative averages (46s for comparable age and education levels), and balance ability scores (miniBESTest = 25 ± 2 / 28) were above the threshold for fall risk (23 for people between 70-80 years). Reactive balance recovery in response to support-surface translations in anterior and posterior directions was assessed in terms of body motion, muscle activity, and brain activity. Across participants, lower cognitive set shifting ability was associated with smaller peak center of mass displacement during balance recovery, lower directional specificity of late phase balance-correcting muscle activity (i.e., greater antagonist muscle activity 200-300ms after perturbation onset), and larger cortical N1 responses (100-200ms). None of these measures were associated with clinical balance ability. Our results suggest that cognitive set shifting ability is expressed in balance recovery behavior even in the absence of profound clinical balance disability. Specifically, our results suggest that lower flexibility in cognitive task performance is associated with lower ability to incorporate the directional context into the cortically-mediated later phase of the motor response. The resulting antagonist activity and stiffer balance behavior may help explain associations between cognitive set shifting impairments and frequent falls.
Active antithrombin glycoforms are selectively physiosorbed on plasma extracellular vesicles
Authors: Radeghieri Annalisa , Alacqua Silvia , Zendrini Andrea , Previcini Vanessa , Todaro Francesca , Martini Giuliana , Ricotta Doris , Bergese Paolo
Published: July 2021

Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clotting proteases. A deficit in AT function leads to AT qualitative deficiency, challenging to diagnose. Here we report that active AT may travel physiosorbed on the surface of plasma extracellular vesicles (EVs), contributing to form the “EV-protein corona”. The corona is enriched in specific AT glycoforms, thus suggesting glycosylation to play a key role in AT partitioning between EVs and plasma. Differences in AT glycoform composition of the corona of EVs separated from plasma of healthy and AT qualitative deficiency-affected subjects were also noticed. This suggests deconstructing the plasma into its nanostructured components, as extracellular vesicles, could help to unravel pathophysiological mechanisms otherwise undiscovered.
Transposable element activity in the transcriptomic analysis of mouse pancreatic tumors
Authors: Lerat Emmanuelle , Burlet Nelly , Navratil Vincent , Noûs Camilles
Published: July 2021

Transposable elements (TEs) are middle-repeated DNA sequences that can move along chromosomes using internal coding and regulatory regions. By their ability to move and because they are repeated, TEs can promote mutations. Especially they can alter the expression pattern of neighboring genes and have been shown to be involved in the mammalian regulatory network evolution. Human and mouse share more than 95% of their genomes and are affected by comparable diseases, which makes the mouse a perfect model in cancer research. However not much investigation concerning the mouse TE content has been made on this topics. In human cancer condition, a global activation of TEs can been observed which may ask the question of their impact on neighboring gene functioning. In this work, we used RNA sequences of highly aggressive pancreatic tumors from mouse to analyze the gene and TE deregulation happening in this condition compared to pancreas from healthy animals. Our results show that several TE families are deregulated and that the presence of TEs is associated with the expression divergence of genes in the tumor condition. These results illustrate the potential role of TEs in the global deregulation at work in the cancer cells.
A synthetic lethal screen identifies HDAC4 as a potential target in MELK overexpressing cancers
Authors: Zhou Lin , Zheng Siqi , Rosas Bringas Fernando R. , Bakker Bjorn , Simon Judith E. , Bakker Petra L. , Kazemier Hinke G. , Schubert Michael , Roorda Maurits , van Vugt Marcel , Chang Michael , Foijer Floris
Published: July 2021

Maternal embryonic leucine zipper kinase (MELK) is frequently overexpressed in cancer, but the role of MELK in cancer is still poorly understood. MELK was shown to have roles in many cancer-associated processes including tumor growth, chemotherapy resistance, and tumor recurrence. To determine whether the frequent overexpression of MELK can be exploited in therapy, we performed a high-throughput screen using a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential when MELK is overexpressed. We identified two such genes: LAG2 and HDA3. LAG2 encodes an inhibitor of the SCF ubiquitin-ligase complex, while HDA3 encodes a subunit of the HDA1 histone deacetylase complex. We find that one of these synthetic lethal interactions is conserved in mammalian cells, as inhibition of a human homolog of HDA3 (HDAC4) is synthetically toxic in MELK overexpression cells. Altogether, our work might provide a new angle of how to exploit MELK overexpression in cancers and might thus lead to novel intervention strategies.
Classification of Free-Living Body Posture with ECG Patch Accelerometers: Application to the Multicenter AIDS Cohort Study
Authors: Etzkorn Lacey H. , Heravi Amir S. , Wu Katherine C. , Post Wendy S. , Urbanek Jacek , Crainiceanu Ciprian
Published: July 2021

Summary

As health studies increasingly monitor free-living heart performance via ECG patches with accelerometers, researchers will seek to investigate cardio-electrical responses to physical activity and sedentary behavior, increasing demand for fast, scalable methods to process accelerometer data. We provide the first published analysis of tri-axial accelerometry data from Zio XT patch and introduce an extension of posture classification algorithms for use with ECG patches worn in the free-living environment. Our novel extensions to posture classification include (1) estimation of an upright posture for each individual without the reference measurements used by existing posture classification algorithms; (2) correction for device removal and re-positioning using novel spherical change-point detection; and (3) classification of upright and recumbent periods using a clustering and voting process rather than a simple inclination threshold used in other algorithms. Methods were built using data from 14 participants from the Multicenter AIDS Cohort Study (MACS), and applied to 1, 250 MACS participants. As no posture labels exist in the free-living environment, we evaluate the algorithm against labelled data from the Towson Accelerometer Study and against data labelled by hand from the MACS study.
Global and Gene-specific Transcriptional Responses to Acute Stress
Authors: Fischl Harry , Brown Thomas , Angel Andrew , Mellor Jane
Published: July 2021

Nucleosomes may regulate transcription by controlling access to promoters by transcription factors and RNA polymerase II (Pol2). Potentially active genes display nucleosome depleted regions flanked by positioned -1 and +1 nucleosomes. On yeast genes, the transcription start site (TSS) is on the upstream face of the +1 nucleosome, but whether precise +1 nucleosome positioning controls Pol2 access to the TSS remains unclear. Here, using acute nutrient starvation to rapidly reprogramme the genome, we show highly dynamic upstream or downstream shifts in the position of +1 nucleosomes, coincident with levels of transcriptionally engaged Pol2 at 58% of genes. Transcript level changes broadly reflect Pol2 occupancy changes with a delay but can be further influenced by Pub1 or Puf3 dependent changes in transcript degradation rates. The response to acute stress has a second component as we also observed genome-wide changes in Pol2 distribution on genes, independent of changes in Pol2 occupancy, with Pol2 accumulating upstream of a +170 nt stalling site. Mathematical modelling supports a global increase in promoter-proximal early transcription termination as a major component of the global stress response. Thus, we uncover a two-component transcriptional response to stress, one focused on the +1 nucleosome, the second on Pol2 itself.

A two-component responses to acute stress involving a gene-specific response and a global response

Dynamic shifting of +1 nucleosome position with transcriptional activation or repression.

Global targeting of Pol2 leading to early transcription termination on acute stress
A study of the correlation between phenotypic antimicrobial susceptibility testing methods and the associated genotypes determined by whole genome sequencing for a collection of Escherichia coli of bovine origin
Authors: Maunsell Thomas J , Nguyen Scott , Garach Farid El , Miossec Christine , Cuinet Emmanuel , Woehrlé Frédérique , Fanning Séamus , Niedziela Dagmara A.
Published: July 2021

Antimicrobial resistance (AMR) has increased at an alarming pace in the recent years. Molecular-based methods such as whole genome sequencing (WGS) offer a potential alternative to the conventional labour-intensive methods traditionally used to characterise AMR phenotypes. The aim of this study was to investigate whether WGS could be used as a predictor of AMR in Escherichia coli isolates of bovine origin.

Genomes of 143 E. coli cultured from cattle presenting with diarrhoea or mastitis were sequenced on an Illumina MiSeq platform. AMR genes were identified using the ResFinder and AMRFinder databases. Antimicrobial susceptibility testing by disk diffusion was performed on a panel of 10 antibiotics, covering 7 antimicrobial classes. Minimum inhibitory concentration (MIC) measurements were made using the Sensititre plate with 6 antibiotics, covering 5 antimicrobial classes. Correlation between genotype and phenotype was assessed statistically by means of a two-by-two table analysis and Cohen’s kappa (κ) test.

The overall κ correlation between WGS and disk diffusion was 0.81, indicating a near perfect agreement, and the average positive predicted value was 77.4 %. Correlation for individual antimicrobial compounds varied, with five yielding near perfect agreement (κ = 0.81–1.00; amoxicillin, florfenicol, gentamicin, tetracycline and trimethoprim-sulfamethoxazole), one showing substantial agreement (κ = 0.65; nalidixic acid), and four showing moderate agreement (κ = 0.41– 0.60). The overall κ correlation between WGS and MIC was 0.55 indicating moderate agreement, and the average positive predicted value was 68.6 %. Three antibiotics yielded near perfect agreement (gentamicin, tetracycline and trimethoprim-sulfamethoxazole) and a further three showed fair agreement (κ = 0.21–0.40).

WGS is a useful tool that can be used for the prediction of AMR phenotypes, and correlates well with disk diffusion results. MIC measurements may be necessary for antimicrobial compounds with a high proportion of intermediately resistant isolates recorded, such as cephalothin.

Highlights

Culture based antimicrobial susceptibility testing is used to identify therapeutics in the treatment of clinical veterinary isolates

Whole genome sequencing is increasingly adopted for surveillance, epidemiological traceback investigations, and detection of antimicrobial resistance genes

Little is known in correlations between antimicrobial resistance genotypes and disk diffusion antimicrobial susceptibility testing

This study finds that whole genome sequencing is a useful predictor for antimicrobial susceptibility however, minimum inhibitory concentration measurements may still be needed for intermediately resistant isolates
Genetic instability from a single S-phase after whole genome duplication
Authors: Gemble Simon , Wardenaar René , Keuper Kristina , Srivastava Nishit , Nano Maddalena , Macé Anne-Sophie , Tijhuis Andréa E. , Bernhard Sara Vanessa , Spierings Diana C.J. , Simon Anthony , Goundiam Oumou , Hochegger Helfrid , Piel Matthieu , Foijer Floris , Storchová Zuzana , Basto Renata
Published: July 2021

Diploid and stable karyotypes are associated with health and fitness in animals. In contrast, whole genome duplications (WGDs) - doubling full chromosome content - are linked to genetic instability (GIN) and frequently found in human cancers 1–3. It has been established that WGDs fuel chromosome instability through abnormal mitosis 4–8, however, the immediate consequences of tetraploidy in the first interphase are not known. This is an essential question because single WGD events such as cytokinesis failure can promote tumorigenesis 9. Here, we found that newly born tetraploid human cells undergo high rates of DNA damage during DNA replication in the first S-phase. Using DNA combing and single cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we found that these defects result from the lack of protein mass scaling up at the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the GIN landscape that favors tumorigenesis after tetraploidization.
The context of experienced sensory discrepancies shapes multisensory integration and recalibration differently
Authors: Park Hame , Kayser Christoph
Published: July 2021

Whether two sensory cues interact during perceptual judgments depends on their immediate properties, but as suggested by Bayesian models, also on the observer’s a priori belief that these originate from a common source. While in many experiments this a priori belief is considered fixed, in real life it must adapt to the momentary context or environment. To understand the adaptive nature of human multisensory perception we investigated the context-sensitivity of spatial judgements in a ventriloquism paradigm. We exposed observers to audio-visual stimuli whose discrepancy either varied over a wider (± 46°) or a narrower range (± 26°) and hypothesized that exposure to a wider range of discrepancies would facilitate multisensory binding by increasing participants a priori belief about a common source for a given discrepancy. Our data support this hypothesis by revealing an enhanced integration (ventriloquism) bias in the wider context, which was echoed in Bayesian causal inference models fit to participants’ data, which assigned a stronger a priori integration tendency during the wider context. Interestingly, the immediate ventriloquism aftereffect, a multisensory response bias obtained following a multisensory test trial, was not affected by the contextual manipulation, although participant’s confidence in their spatial judgments differed between contexts for both integration and recalibration trials. These results highlight the context-sensitivity of multisensory binding and suggest that the immediate ventriloquism aftereffect is not a purely sensory-level consequence of the multisensory integration process.
Epigenetic regulator genes direct lineage switching in MLL-AF4 leukaemia
Authors: Tirtakusuma Ricky , Szoltysek Katarzyna , Milne Paul , Grinev Vasily V , Ptasinska Anetta , Meyer Claus , Nakjang Sirintra , Hehir-Kwa Jayne Y , Williamson Daniel , Cauchy Pierre , Assi Salam A , Imperato Maria R , Vogiatzi Fotini , Lin Shan , Wunderlich Mark , Stutterheim Janine , Komkov Alexander , Zerkalenkova Elena , Evans Paul , McNeill Hesta , Elder Alex , Martinez-Soria Natalia , Fordham Sarah E , Shi Yuzhe , Russell Lisa J , Pal Deepali , Smith Alex , Kingsbury Zoya , Becq Jennifer , Eckert Cornelia , Haas Oskar A , Carey Peter , Bailey Simon , Skinner Roderick , Miakova Natalia , Collin Matthew , Bigley Venetia , Haniffa Muzlifah , Marschalek Rolf , Harrison Christine J , Cargo Catherine A , Schewe Denis , Olshanskaya Yulia , Thirman Michael J , Cockerill Peter N , Mulloy James C , Blair Helen J , Vormoor Josef , Allan James M , Bonifer Constanze , Heidenreich Olaf , Bomken Simon
Published: July 2021

The fusion gene MLL-AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukaemia. However, relapse can be associated with a switch from acute lymphoblastic to acute myeloid leukaemia. Here we show that these myeloid relapses share oncogene fusion breakpoints with their matched lymphoid presentations and can originate in either early, multipotent progenitors or committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programmes indicating that the execution and maintenance of lymphoid lineage differentiation is impaired. We show that this subversion is recurrently associated with the dysregulation of repressive chromatin modifiers, notably the nucleosome remodelling and deacetylation complex, NuRD. In addition to mutations, we show differential expression or alternative splicing of NuRD members and other genes is able to reprogram the B lymphoid into a myeloid gene regulatory network. Lineage switching in MLL-AF4 leukaemia is therefore driven and maintained by defunct epigenetic regulation.

Statement of Significance

We demonstrate diverse cellular origins of lineage switched relapse within MLL-AF4 pro-B acute leukaemia. Irrespective of the developmental origin of relapse, dysregulation of NuRD and/or other epigenetic machinery underpins fundamental lineage reprogramming with profound implications for the increasing use of epitope directed therapies in this high-risk leukaemia.
Phenotypic determinism and stochasticity in antibody repertoires of clonally expanded plasma cells
Authors: Neumeier Daniel , Yermanos Alexander , Agrafiotis Andreas , Csepregi Lucia , Chowdhury Tasnia , Ehling Roy A , Kuhn Raphael , Roberto Raphaël Brisset-Di , Di Tacchio Mariangela , Antonialli Renan , Starkie Dale , Lightwood Daniel J , Oxenius Annette , Reddy Sai T
Published: July 2021

The capacity of humoral B cell-mediated immunity to effectively respond to and protect against pathogenic infections is largely driven by the presence of a diverse repertoire of polyclonal antibodies in the serum, which are produced by plasma cells (PCs).1,2 Recent studies have started to reveal the balance between deterministic mechanisms and stochasticity of antibody repertoires on a genotypic level (i.e., clonal diversity, somatic hypermutation, germline gene usage).3–8 However, it remains unclear if clonal selection and expansion of PCs follows any deterministic rules or is stochastic with regards to phenotypic antibody properties (i.e., antigen-binding, affinity, epitope specificity). Here we report on the in-depth genotypic and phenotypic characterization of clonally expanded PC antibody repertoires following protein immunization. We find that there is only a strong correlation with antigen-specificity among the most expanded clones (top ~ 10), whereas among the rest of the clonal repertoire antigen-specificity is stochastic. Furthermore, we report both on a polyclonal repertoire and clonal lineage level that antibody-antigen binding affinity does not correlate with clonal expansion or somatic hypermutation. Lastly, we provide evidence for convergence towards dominant epitopes despite clonal sequence diversity among the most expanded clones. Our results highlight the extent to which clonal expansion can be ascribed to antigen binding, affinity and epitope specificity and they have implications for the assessment of effective vaccines.

Graphical abstract
Neurotoxins subvert the allosteric activation mechanism of SARM1 to induce neuronal loss
Authors: Wu Tong , Zhu Jian , Strickland Amy , Ko Kwang Woo , Sasaki Yo , Dingwall Caitlin , Yamada Yurie , Figley Matthew D , Mao Xianrong , Neiner Alicia , Bloom Joseph , DiAntonio Aaron , Milbrandt Jeffrey
Published: July 2021

SARM1 is an inducible TIR-domain NAD+ hydrolase that mediates pathological axon degeneration. SARM1 is activated by an increased ratio of NMN to NAD+, which competes for binding to an allosteric activating site. When NMN binds, the TIR domain is released from autoinhibition, activating its NAD+ hydrolase activity. The discovery of this allosteric activating site led us to hypothesize that other NAD+-related metabolites might also activate SARM1. Here we show that the nicotinamide analogue 3-acetylpyridine (3-AP), first identified as a neurotoxin in the 1940s, is converted to 3-APMN which activates SARM1 and induces SARM1-dependent NAD+ depletion, axon degeneration and neuronal death. Systemic treatment with 3-AP causes rapid SARM1-dependent death, while local application to peripheral nerve induces SARM1-dependent axon degeneration. We also identify a related pyridine derivative, 2-aminopyridine, as another SARM1-dependent neurotoxin. These findings identify SARM1 as a candidate mediator of environmental neurotoxicity, and furthermore, suggest that SARM1 agonists could be developed into selective agents for neurolytic therapy.
Cellular and genetic drivers of RNA editing variation in the human brain
Authors: Cuddleston Ryn , Li Junhao , Fan Xuanjia , Kozenkov Alexey , Lalli Matthew , Khalique Shahrukh , Dracheva Stella , Mukamel Eran A. , Breen Michael S.
Published: July 2021

Posttranscriptional adenosine-to-inosine modifications amplify the functionality of RNA molecules in the brain, yet the cellular and genetic regulation of RNA editing is poorly described. We quantified base-specific RNA editing across three major cell populations from the human prefrontal cortex: glutamatergic neurons, medial ganglionic eminence GABAergic neurons, and oligodendrocytes. We found more selective editing and RNA hyper-editing in neurons relative to oligodendrocytes. The pattern of RNA editing was highly cell type-specific, with 189,229 cell type-associated sites. The cellular specificity for thousands of sites was confirmed by single nucleus RNA-sequencing. Importantly, cell type-associated sites were enriched in GTEx RNA-sequencing data, edited ∼twentyfold higher than all other sites, and variation in RNA editing was predominantly explained by neuronal proportions in bulk brain tissue. Finally, we discovered 661,791 cis-editing quantitative trait loci across thirteen brain regions, including hundreds with cell type-associated features. These data reveal an expansive repertoire of highly regulated RNA editing sites across human brain cell types and provide a resolved atlas linking cell types to editing variation and genetic regulatory effects.
Modeling lung cell development using human pluripotent stem cells
Authors: Ngan Shuk Yee , Quach Henry , Dierolf Joshua , Laselva Onofrio , Lee Jin-A , Huang Elena , Mangos Maria , Xia Sunny , Bear Christine E. , Wong Amy P.
Published: July 2021

Human pluripotent stem cells (hPSC) differentiations can capture developmental phenotypes and processes. They are useful for studying fundamental biological mechanisms driving tissue morphogenesis and cell lineage development. Here, we show temporal development of lung cell lineages using hPSC that recapitulate developmental milestones observed in primary tissue, the generation of renewable fetal lung epithelial spheroids, and the functional utility of the lung models at different differentiation stages for cystic fibrosis disease modeling. We first show the presence of hPSC-derived lung progenitor cells reminiscent of early trimester lung development and containing basal stem cells that generate renewable airway spheroids. Maturation and polarization in air liquid interface (ALI) generates additional epithelial cell lineages found in adult airways, including pulmonary neuroendocrine, brush, mature basal, ciliated and secretory cell types. Finally, pseudotime and RNA velocity analyses of the integrated datasets from fetal and ALI stages reveal both previously identified and new cell lineage relationships. Overall, hPSC differentiation can capture aspects of human lung development and potentially provide important insight into congenital causes of diseases.
Rescue of stalled clathrin-mediated endocytosis by asymmetric Arp2/3-mediated actin assembly
Authors: Jin Meiyan , Shirazinejad Cyna , Wang Bowen , Yan Amy , Schöneberg Johannes , Upadhyayula Srigokul , Xu Ke , Drubin David G.
Published: July 2021

Actin assembly facilitates vesicle formation in several trafficking pathways. Clathrin-mediated endocytosis (CME) shows elevated actin assembly dependence under high membrane tension. Why actin assembly at CME sites occurs heterogeneously even within the same cell, and how assembly forces are harnessed, are not fully understood. Here, endocytic dynamics, actin presence, and geometry of CME proteins from three different functional modules, were analyzed using three-dimensional (3D) super-resolution microscopy, live-cell imaging, and machine-learning-based computation. When hundreds of CME events were compared, sites with actin assembly showed a distinct signature, a delay between completion of coat expansion and vesicle scission, indicating that actin assembly occurs preferentially at stalled CME sites. N-WASP is recruited to one side of CME sites where it is positioned to stimulate asymmetric actin assembly. We propose that asymmetric actin assembly rescues stalled CME sites by pulling vesicles into the cell much like a bottle opener pulls off a bottle cap.
Autophagosomes fuse to phagosomes and facilitate the degradation of apoptotic cells inCaenorhabditis elegans
Authors: Peña-Ramos Omar , Chiao Lucia , Liu Xianghua , Yu Xiaomeng , Yao Tianyou , He Henry , Zhou Zheng
Published: July 2021

Autophagosomes are double-membrane intracellular vesicles that degrade protein aggregates, intracellular organelles, and other cellular components. During the development of the nematodeCaenorhabditis elegans, many somatic and germ cells undergo apoptosis. These cells are engulfed and degraded by their neighboring cells. We discovered a novel role of autophagosomes in facilitating the degradation of apoptotic cells using a real-time imaging technique. Specifically, the double-membrane autophagosomes in engulfing cells are recruited to the surfaces of phagosomes containing apoptotic cells and subsequently fuse to phagosomes, allowing the inner vesicle to enter the phagosomal lumen. Mutants defective in the production of autophagosomes display significant defects in the degradation of apoptotic cells, demonstrating the importance of autophagosomes to this process. The signaling pathway led by the phagocytic receptor CED-1, the adaptor protein CED-6, and the large GTPase dynamin (DYN-1) promotes the recruitment of autophagosomes to phagosomes. Moreover, the subsequent fusion of autophagosomes with phagosomes requires the functions of the small GTPase RAB-7 and the HOPS complex components. Further observations suggest that autophagosomes provide apoptotic cell-degradation activities in addition to and in parallel of lysosomes. Our findings reveal that, unlike the single-membrane,LC3-associatedphagocytosis (LAP) vesicles reported for mammalian phagocytes, the canonical double-membrane autophagosomes facilitate the clearance ofC. elegansapoptotic cells. These findings add autophagosomes to the collection of intracellular organelles that contribute to phagosome maturation, identify novel crosstalk between the autophagy and phagosome maturation pathways, and discover the upstream signaling molecules that initiate this crosstalk.
TIGAR-V2: Efficient TWAS Tool with Nonparametric Bayesian eQTL Weights of 49 Tissue Types from GTEx V8
Authors: Parrish Randy L. , Gibson Greg C. , Epstein Michael P. , Yang Jingjing
Published: July 2021

Standard Transcriptome-Wide Association Study (TWAS) methods first train gene expression prediction models using reference transcriptomic data, and then test the association between the predicted genetically regulated gene expression and phenotype of interest. Most existing TWAS tools require cumbersome preparation of genotype input files and extra coding to enable parallel computation. To improve the efficiency of TWAS tools, we develop TIGAR-V2, which directly reads VCF files, enables parallel computation, and reduces up to 90% computation cost (mainly due to loading genotype data) compared to the original version. TIGAR-V2 can train gene expression imputation models using either nonparametric Bayesian Dirichlet Process Regression (DPR) or Elastic-Net (as used by PrediXcan), perform TWAS using either individual-level or summary-level GWAS data, and implements both burden and variance-component statistics for gene-based association tests. We trained gene expression prediction models by DPR for 49 tissues using GTEx V8 by TIGAR-V2 and illustrated the usefulness of these Bayesian cis-eQTL weights through TWAS of breast and ovarian cancer utilizing public GWAS summary statistics. We identified 88 and 37 risk genes respectively for breast and ovarian cancer, most of which are either known or near previously identified GWAS (~95%) or TWAS (~40%) risk genes and three novel independent TWAS risk genes with known functions in carcinogenesis. These findings suggest that TWAS can provide biological insight into the transcriptional regulation of complex diseases. TIGAR-V2 tool, trained Bayesian cis-eQTL weights, and LD information from GTEx V8 are publicly available, providing a useful resource for mapping risk genes of complex diseases.
GAIT-GM integrative cross-omics analyses reveal cholinergic defects in a C. elegans model of Parkinson’s disease
Authors: Mor Danielle E. , Huertas Francisco , Morse Alison M. , Kaletsky Rachel , Murphy Coleen T. , Kalia Vrinda , Miller Gary W. , Moskalenko Olexander , Conesa Ana , McIntyre Lauren M.
Published: July 2021

Background

Parkinson’s disease (PD) is a disabling neurodegenerative disorder in which multiple cell types, including dopaminergic and cholinergic neurons, are affected. The mechanisms of neurodegeneration in PD are unknown, limiting the development of therapies directed at disease-relevant molecular targets. C. elegans is a genetically tractable model system that can be used to disentangle disease mechanisms in complex diseases such as PD. Such mechanisms can be studied combining high-throughput molecular profiling technologies such as transcriptomics and metabolomics. However, the integrative analysis of multi-omics data in order to unravel disease mechanisms is a challenging task without advanced bioinformatics training. Galaxy, a widely-used resource for enabling bioinformatics analysis by the broad scientific community, has poor representation of multi-omics integration pipelines.

Results

We present the integrative analysis of gene expression and metabolite levels of a C. elegans PD model using GAIT-GM, a new Galaxy tool for multi-omics data analysis. Using GAIT-GM, we discovered an association between branched-chain amino acid metabolism and cholinergic neurons in the C. elegans PD model. An independent follow-up experiment uncovered cholinergic neurodegeneration in the C. elegans model that is consistent with cholinergic cell loss observed in PD.

Conclusion

GAIT-GM is an easy to use Galaxy-based tool for generating novel testable hypotheses of disease mechanisms involving gene-metabolite relationships.
Mapping the vocal circuitry of Alston’s singing mouse with pseudorabies virus
Authors: Zheng Da-Jiang , Okobi Daniel E. , Shu Ryan , Agrawal Rania , Smith Samantha K. , Long Michael A. , Phelps Steven M.
Published: July 2021

Vocalizations, like many social displays, are often elaborate, rhythmically structured behaviors that are modulated by a complex combination of cues. Vocal motor patterns require close coordination of neural circuits governing the muscles of the larynx, jaw, and respiratory system. In the elaborate vocalization of Alston’s singing mouse (Scotinomys teguina), for example, each note of its rapid, frequency-modulated trill is accompanied by equally rapid modulation of breath and gape. To elucidate the neural circuitry underlying this behavior, we introduced the polysynaptic retrograde neuronal tracer pseudorabies virus (PRV) into the cricothyroid and digastricus muscles, which control frequency modulation and jaw opening respectively. Each virus singly labels ipsilateral motoneurons (nucleus ambiguous for cricothyroid, and motor trigeminal nucleus for digastricus). We find that the two isogenic viruses heavily and bilaterally co-label neurons in the gigantocellular reticular formation, a putative central pattern generator. The viruses also show strong co-labeling in compartments of the midbrain including the ventrolateral periaqueductal grey and the parabrachial nucleus, two structures strongly implicated in vocalizations. In the forebrain, regions important to social cognition and energy balance both exhibit extensive co-labeling. This includes the paraventricular and arcuate nuclei of the hypothalamus, the lateral hypothalamus, preoptic area, extended amygdala, central amygdala, and the bed nucleus of the stria terminalis. Finally, we find doubly labeled neurons in M1 motor cortex previously described as laryngeal, as well as in the prelimbic cortex, which indicate these cortical regions play a role in vocal production. Although we observe some novel patterns of double-labelling, the progress of both viruses is broadly consistent with vertebrate-general patterns of vocal circuitry, as well as with circuit models derived from primate literature.
The impact of whole genome duplications on the human gene regulatory networks
Authors: Mottes Francesco , Villa Chiara , Osella Matteo , Caselle Michele
Published: July 2021

This work studies the effects of the two rounds of Whole Genome Duplication (WGD) at the origin of the vertebrate lineage on the architecture of the human gene regulatory networks. We integrate information on transcriptional regulation, miRNA regulation, and protein-protein interactions to comparatively analyse the role of WGD and Small Scale Duplications (SSD) in the structural properties of the resulting multilayer network. We show that complex network motifs, such as combinations of feed-forward loops and bifan arrays, deriving from WGD events are specifically enriched in the network. Pairs of WGD-derived proteins display a strong tendency to interact both with each other and with common partners and WGD-derived transcription factors play a prominent role in the retention of a strong regulatory redundancy. Combinatorial regulation and synergy between different regulatory layers are in general enhanced by duplication events, but the two types of duplications contribute in different ways. Overall, our findings suggest that the two WGD events played a substantial role in increasing the multi-layer complexity of the vertebrate regulatory network by enhancing its combinatorial organization, with potential consequences on its overall robustness and ability to perform high-level functions like signal integration and noise control.
Agonism and grooming behavior explain social status effects on physiology and gene regulation in rhesus macaques
Authors: Simons Noah D. , Michopoulos Vasiliki , Wilson Mark , Barreiro Luis B. , Tung Jenny
Published: July 2021

Variation in social status predicts molecular, physiological, and life history outcomes across a broad range of species, including our own. Experimental studies indicate that some of these relationships persist even when the physical environment is held constant. Here, we draw on data sets from one such study—experimental manipulation of dominance rank in captive female rhesus macaques—to investigate how social status shapes the lived experience of these animals to alter gene regulation, glucocorticoid physiology, and mitochondrial DNA phenotypes. We focus specifically on dominance rank-associated dimensions of the social environment, including both competitive and affiliative interactions. Our results show that simple summaries of rank-associated behavioral interactions are often better predictors of molecular and physiological outcomes than dominance rank itself. However, while measures of immune function are best explained by agonism rates, glucocorticoid-related phenotypes tend to be more closely linked to affiliative behavior. We conclude that dominance rank serves as a useful summary for investigating social environmental effects on downstream outcomes. Nevertheless, the behavioral interactions that define an individual’s daily experiences reveal the proximate drivers of social status-related differences, and are especially relevant for understanding why individuals who share the same social status sometimes appear physiologically distinct.
Allosteric control of Ubp6 and the proteasome via a bidirectional switch
Authors: Hung Ka Ying Sharon , Klumpe Sven , Eisele Markus R. , Elsasser Suzanne , Tian Geng , Sun Shuangwu , Moroco Jamie A. , Cheng Tat Cheung , Joshi Tapan , Seibel Timo , Van Dalen Duco , Feng Xin-Hua , Lu Ying , Ovaa Huib , Engen John R. , Lee Byung-Hoon , Rudack Till , Sakata Eri , Finley Daniel
Published: July 2021

The proteasome is the principal cellular protease, and recognizes target proteins that have been covalently marked by ubiquitin chains. The ubiquitin signal is subject to rapid editing at the proteasome, allowing it to reject substrates based on topological features of their attached ubiquitin chains. Editing is mediated by a key regulator of the proteasome, deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome–both by deubiquitinating proteasome-bound ubiquitin conjugates, and through a noncatalytic effect that does not involve deubiquitination. We report mutants in both Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations fall within its ILR element, defined here, which is conserved from yeast to mammals. The ILR is a component of the BL1 blocking loop, other parts of which obstruct ubiquitin access to the catalytic groove in free Ubp6. Rpt1 docking at the ILR opens the catalytic groove by rearranging not only BL1 but also a novel network of three directly interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition by Ubp6 are linked in that they were eliminated by the same Ubp6 and Rpt1 mutations. Ubp6 and ubiquitin together drive the proteasome into a unique conformational state associated with proteasome inhibition. Our results identify a multicomponent allosteric switch that exerts simultaneous control over the activity of both Ubp6 and the proteasome, and suggest that their active states are in general mutually exclusive. The findings lead to a new paradigm for allosteric control of deubiquitinating enzymes.
The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions
Authors: Kumar Charanya , Batra Sahil , Griffith Jack D. , Remus Dirk
Published: July 2021

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.
Existence of positive solutions for p-Laplacian systems involving left and right fractional derivatives
Authors: Ramdane Samira , Guezane-Lakoud Assia
Published: July 2021

Purpose

The paper deals with the existence of positive solutions for a coupled system of nonlinear fractional differential equations with p-Laplacian operator and involving both right Riemann–Liouville and left Caputo-type fractional derivatives. The existence results are obtained by the help of Guo–Krasnosel'skii fixed-point theorem on a cone in the sublinear case. In addition, an example is included to illustrate the main results.

Design/methodology/approach

Fixed-point theorems.

Findings

No finding.

Originality/value

The obtained results are original.
Developing trauma-informed care: using psychodynamic concepts to help staff respond to the attachment needs of survivors of trauma
Authors: Rye Emma , Anderson Joanna , Pickard Max
Published: July 2021

Purpose

Many referrals to our mental health of learning disability service focus on concerns about behaviours that present risks to the referred person and to those around them, including support workers. If the referred person has good verbal ability, psychological therapy may be requested and offered, but the person may find it too difficult to engage for a number of reasons. Even when they do engage in therapy, the authors recognise the importance of helping staff better understand their attachment needs. This paper aims to demonstrate an innovative approach to helping staff provide Trauma-Informed Care (TIC).

Design/methodology/approach

The authors developed a training programme for support workers using psychodynamic concepts, such as splitting, transference and counter-transference, to help them develop insight into the processes that get enacted during their work. In this paper the authors have generally used the term “care staff”, recognising that an important aspect of the role of those staff is to provide care within an attachment framework; as well as support to maximise independence.

Findings

The feedback from staff who have attended has been very positive, both at the time and later. Staff who have attended have talked to multidisciplinary colleagues about the impact their learning has had on their ability to work with service users who present great challenges, in the context of their trauma histories.

Originality/value

The importance of providing TIC is gaining traction across varied settings. The authors are in the process of developing both qualitative and quantitative research programmes to evaluate this approach to increasing TIC for adults with learning disabilities, reducing staff burn out and placement breakdown.