bluuebunny/crossref_metadata_2025 huggingface dataset:
A catalytic-independent function of human DNA polymerase Kappa controls the pool of the Checkpoint Kinase 1
Authors: Dall’Osto Marina , Pierini Laura , Valery Nicolas , Hoffmann Jean-Sébastien , Pillaire Marie-jeanne
Published: February 2021

DNA polymerase kappa (Pol κ) has been well documented thus far for its specialized DNA synthesis activity during translesion replication, progression of replication forks through regions difficult to replicate and replication checkpoint at stalled forks.

Here we unveiled an unexpected role for Pol κ in controlling the stability and abundance of Chk1, the major mediator of the replication checkpoint. We found that loss of Pol κ decreased the Chk1 protein level in the nucleus of four human cell lines. Pol κ and not the other Y‐family polymerase members is required to maintain the Chk1 protein pool all along the cell cycle. We showed that Pol κ depletion affected the protein stability of Chk1 and protected it from proteasome degradation and the replication recovery defects observed in Pol κ-depleted cells could be overcome by the re-expression of Chk1. Importantly, this new function of Pol κ does not require its catalytic activity, revealing that in addition to its known roles in the replication process, Pol κ can contribute to the maintenance of genome stability independently of its DNA synthesis activity.
Host tropism determination by convergent evolution of immunological evasion in the Lyme disease system
Authors: Hart Thomas M. , Dupuis Alan P. , Tufts Danielle M. , Blom Anna M. , Starkey Simon , Rego Ryan O. M. , Ram Sanjay , Kraiczy Peter , Kramer Laura D. , Diuk-Wasser Maria A. , Kolokotronis Sergios-Orestis , Lin Yi-Pin
Published: February 2021

Microparasites selectively adapt in some hosts, known as host tropism. Transmitted through ticks and carried mainly by mammals and birds, the Lyme disease (LD) bacterium is a well-suited model to study such tropism. LD bacteria species vary in host ranges through mechanisms eluding characterization. By feeding ticks infected with different LD bacteria species, utilizing feeding chambers and live mice and quail, we found species-level differences of bacterial transmission. These differences localize on the tick blood meal, and complement, a defense in vertebrate blood, and a bacterial polymorphic protein, CspA, which inactivates complement by binding to a host complement inhibitor, FH. CspA selectively confers bacterial transmission to vertebrates that produce FH capable of allele-specific recognition. Phylogenetic analyses revealed convergent evolution as the driver of such findings, which likely emerged during the last glacial maximum. Our results identify LD bacterial determinants of host tropism, defining an evolutionary mechanism that shapes host-microparasite associations.
Tandem mass spectrum similarity-based network analysis using13C-labeled and non-labeled metabolome data to identify the biosynthesis pathway of the blood pressure-lowering asparagus metabolite asparaptine A
Authors: Nakabayashi Ryo , Yamada Yutaka , Nishizawa Tomoko , Mori Tetsuya , Asano Takashi , Kuwabara Masanari , Saito Kazuki
Published: February 2021

Asparaptine, a conjugate of arginine and asparagusic acid, was found in asparagus (Asparagus officinalis) as a naturally occurring inhibitor of angiotensin-converting enzyme (ACE)in vitro.The biosynthetic pathway to asparaptine is largely unknown; however, it is suggested that asparagusic acid may be biosynthesized from valine. To determine which metabolites are involved in the asparaptine biosynthetic pathway, we performed tandem mass spectrometry similarity-based metabolome network analysis using13C labeled and non-labeled valine-fed asparagus calluses. We determined that valine is used as a starting material,S(2-carboxy-n-propyl)-cysteine as an intermediate, and two new metabolites as asparaptine analogs, lysine- and histidine-type conjugates, are involved in the pathway. Asparaptine was therefore renamed asparaptine A (arginine type), and the two analogs were named asparaptines B (lysine type) and C (histidine type). Oral feeding of asparaptine A to a hypertensive mouse species showed that this metabolite lowers both blood pressure and heart rate within two hours and both of which were back to normal two days later. These results suggest that asparaptine A may not only have effects as an ACE inhibitor, but also has β-antagonistic effects, which are well-known to be preventive for cardiovascular diseases.
An integrated strategy reveals complex glycosylation of erythropoietin using top-down and bottom-up mass spectrometry
Authors: Guan Yudong , Zhang Min , Gaikwad Manasi , Voss Hannah , Fazel Ramin , Ansari Samira , Shen Huali , Wang Jigang , Schlüter Hartmut
Published: February 2021

The characterization of glycoproteins, like erythropoietin, is challenging due to the structural micro- and macro-heterogeneity of the protein glycosylation. This study presents an in-depth strategy for glycosylation analysis of a first-generation erythropoietin (epoetin beta), including a developed top-down mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). Extending the coverage of our newly developed Python script to phosphorylated N-glycans enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin. The site-specificity of N-glycans was revealed at glycopeptide level by pGlyco software using different proteases. In total, 215 N-glycan compositions were identified from N-glycan and glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two different O-glycan compositions, based on the mass shifts between non-O-glycosylated and O-glycosylated species. This integrated strategy allows the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.
Specialization of actin isoforms derived from the loss of key interactions with regulatory factors
Authors: Sanders Micaela Boiero , Toret Christopher P. , Antkowiak Adrien , Robinson Robert C. , Michelot Alphée
Published: February 2021

A paradox of eukaryotic cells is that while some species assemble a complex actin cytoskeleton from a single ortholog, other species utilize a greater diversity of actin isoforms. The physiological consequences of using different actin isoforms, and the molecular mechanisms by which highly conserved actin isoforms are segregated into distinct networks, are poorly known. Here, we sought to understand how a simple biological system, composed of a unique actin and a limited set of actin-binding proteins, reacts to a switch to heterologous actin expression. Using yeast as a model system and biomimetic assays, we show that such perturbation causes drastic reorganization of the actin cytoskeleton. Our results indicate that defective interaction of a heterologous actin for important regulators of actin assembly limits certain actin assembly pathways while reinforcing others. Expression of two heterologous actin variants, each specialized in assembling a different network, rescues cytoskeletal organization and confers resistance to external perturbation. Hence, while species using a unique actin have homeostatic actin networks, actin assembly pathways in species using several actin isoforms may act more independently.
Capsule protects against intracellular killing and enables vascular endothelial cell translocation during invasive pneumococcal disease
Authors: Brissac Terry , Martínez Eriel , Kruckow Katherine L. , Riegler Ashleigh N. , Ganaie Feroze , Im Hansol , Bakshi Sayan , Arroyo-Diaz Nicole M. , Spencer Brady L. , Saad Jamil S. , Nahm Moon H. , Orihuela Carlos J.
Published: February 2021

Streptococcus pneumoniae(Spn) is a leading cause of invasive disease. Chief among its virulence determinants is capsular polysaccharide which protects the bacterium from phagocytosis. While 100 antigenically distinct capsule types are produced bySpn, i.e. serotypes, only 20-30 are commonly associated with invasive disease. A frequency that suggests serotypespecific properties of the capsule influence virulence. Herein, we show capsule has strong antioxidant properties. Moreover, that this property promotes invasive disease by protectingSpntaken up by vascular endothelial cells during bacteremia from endosome-killing and enhancing the translocation rate into organs. Crucially, isogenic capsule-switch mutants ofSpnvaried considerably in their resistance to H2O2-killing in culture and measured levels correlated positively with intracellular survival ratesin vitro, organ invasion ratesin vivo, and epidemiologically-established human attack rates for the corresponding serotype. The amount of capsule produced and specific biochemical features of a serotype, such as acetylation, also influencedSpnresistance to oxidative stress. Autolysin-mediated shedding was also found to be necessary, indicating that capsule worked as a distal sink for reactive oxygen species. Our results outline a new role for capsular polysaccharide, as an intracellular antioxidant. They help to explain why certain serotypes ofSpnhave greater propensity for human disease.
The climate benefits of yield increases in genetically engineered crops
Authors: Kovak Emma , Qaim Matin , Blaustein-Rejto Dan
Published: February 2021

The benefits of genetically engineered (GE) crops are systematically underestimated because previous studies did not incorporate the reduction in greenhouse gas (GHG) emissions associated with yield increases. We estimate this impact using the carbon opportunity cost of land use. Our results suggest that the GHG emissions reductions from the yield increases in GE crops are substantial and should be included in future analyses.
Bifurcated monocyte states are predictive of mortality in severe COVID-19
Authors: Cillo Anthony R. , Somasundaram Ashwin , Shan Feng , Cardello Carly , Workman Creg J. , Kitsios Georgios D. , Ruffin Ayana , Kunning Sheryl , Lampenfeld Caleb , Onkar Sayali , Grebinoski Stephanie , Deshmukh Gaurav , Methe Barbara , Liu Chang , Nambulli Sham , Andrews Lawrence , Duprex W. Paul , Joglekar Alok V. , Benos Panayiotis V. , Ray Prabir , Ray Anuradha , McVerry Bryan J. , Zhang Yingze , Lee Janet S. , Das Jishnu , Singh Harinder , Morris Alison , Bruno Tullia C. , Vignali Dario A.A.
Published: February 2021

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection presents with varied clinical manifestations1, ranging from mild symptoms to acute respiratory distress syndrome (ARDS) with high mortality2,3. Despite extensive analyses, there remains an urgent need to delineate immune cell states that contribute to mortality in severe COVID-19. We performed high-dimensional cellular and molecular profiling of blood and respiratory samples from critically ill COVID-19 patients to define immune cell genomic states that are predictive of outcome in severe COVID-19 disease. Critically ill patients admitted to the intensive care unit (ICU) manifested increased frequencies of inflammatory monocytes and plasmablasts that were also associated with ARDS not due to COVID-19. Single-cell RNAseq (scRNAseq)-based deconvolution of genomic states of peripheral immune cells revealed distinct gene modules that were associated with COVID-19 outcome. Notably, monocytes exhibited bifurcated genomic states, with expression of a cytokine gene module exemplified byCCL4(MIP-1β) associated with survival and an interferon signaling module associated with death. These gene modules were correlated with higher levels of MIP-1β and CXCL10 levels in plasma, respectively. Monocytes expressing genes reflective of these divergent modules were also detectable in endotracheal aspirates. Machine learning algorithms identified the distinctive monocyte modules as part of a multivariate peripheral immune system state that was predictive of COVID-19 mortality. Follow-up analysis of the monocyte modules on ICU day 5 was consistent with bifurcated states that correlated with distinct inflammatory cytokines. Our data suggests a pivotal role for monocytes and their specific inflammatory genomic states in contributing to mortality in life-threatening COVID-19 disease and may facilitate discovery of new diagnostics and therapeutics.
SPARC Data Structure: Rationale and Design of a FAIR Standard for Biomedical Research Data
Authors: Bandrowski Anita , Grethe Jeffrey S. , Pilko Anna , Gillespie Tom , Pine Gabi , Patel Bhavesh , Surles-Zeigler Monique , Martone Maryann E.
Published: February 2021

The NIH Common Fund’s Stimulating Peripheral Activity to Relieve Conditions (SPARC) initiative is a large-scale program that seeks to accelerate the development of therapeutic devices that modulate electrical activity in nerves to improve organ function. Integral to the SPARC program are the rich anatomical and functional datasets produced by investigators across the SPARC consortium that provide key details about organ-specific circuitry, including structural and functional connectivity, mapping of cell types and molecular profiling. These datasets are provided to the research community through an open data platform, the SPARC Portal. To ensure SPARC datasets are Findable, Accessible, Interoperable and Reusable (FAIR), they are all submitted to the SPARC portal following a standard scheme established by the SPARC Curation Team, called the SPARC Data Structure (SDS). Inspired by the Brain Imaging Data Structure (BIDS), the SDS has been designed to capture the large variety of data generated by SPARC investigators who are coming from all fields of biomedical research. Here we present the rationale and design of the SDS, including a description of the SPARC curation process and the automated tools for complying with the SDS, including the SDS validator and Software to Organize Data Automatically (SODA) for SPARC. The objective is to provide detailed guidelines for anyone desiring to comply with the SDS. Since the SDS are suitable for any type of biomedical research data, it can be adopted by any group desiring to follow the FAIR data principles for managing their data, even outside of the SPARC consortium. Finally, this manuscript provides a foundational framework that can be used by any organization desiring to either adapt the SDS to suit the specific needs of their data or simply desiring to design their own FAIR data sharing scheme from scratch.
Dung beetles as vertebrate samplers – a test of high throughput analysis of dung beetle iDNA
Authors: Drinkwater Rosie , Clare Elizabeth L. , Chung Arthur Y. C. , Rossiter Stephen J. , Slade Eleanor M.
Published: February 2021

The application of environmental DNA (eDNA) sampling in biodiversity surveys has gained widespread acceptance, especially in aquatic systems where free eDNA can be readily collected by filtering water. In terrestrial systems, eDNA-based approaches for assaying vertebrate biodiversity have tended to rely on blood-feeding invertebrates, including leeches and mosquitoes (termed invertebrate-derived DNA or iDNA). However, a key limitation of using blood-feeding taxa as samplers is that they are difficult to trap, and, in the case of leeches, are highly restricted to humid forest ecosystems. Dung beetles (superfamily Scarabaeoidea) feed on the faecal matter of terrestrial vertebrates and offer several potential benefits over blood-feeding invertebrates as samplers of vertebrate DNA. Importantly, these beetles can be easily captured in large numbers using simple, inexpensive baited traps; are globally distributed; and also occur in a wide range of biomes, allowing mammal diversity to be compared across habitats. In this exploratory study, we test the potential utility of dung beetles as vertebrate samplers by sequencing the mammal DNA contained within their guts. First, using a controlled feeding experiment, we show that mammalian DNA can be retrieved from the guts of large dung beetles (Catharsius renaudpauliani) for up to 10 hours after feeding. Second, by combining high-throughput sequencing of a multi-species assemblage of dung beetles with PCR replicates, we show that multiple mammal taxa can be identified with high confidence. By providing preliminary evidence that dung beetles can be used as a source of mammal DNA, our study highlights the potential for this widespread group to be used in future biodiversity monitoring surveys.
The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method
Authors: Marçal Repolês Bruno , Gorospe Choco Michael , Tran Phong , Nilsson Anna Karin , Wanrooij Paulina H.
Published: February 2021

The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues.
Change in protein, oil and fatty acid contents in soybeans (Glycine max (L.) Merr.) of different seed coat colors and seed weight
Authors: Choi Yu-Mi , Yoon Hyemyeong , Shin Myoung-Jae , Lee Yoonjung , Hur On Sook , Lee Bong Choon , Ha Bo-Keun , Wang XiaoHan , Desta Kebede Taye
Published: February 2021

Soybean seeds are one of the best sources of plant-based high-quality proteins and oils. The contents of these metabolites are affected by both environmental and genetic factors. In this study, 49 soybean germplasms were cultivated in Korea, the contents of total protein, total oil and five fatty acids were determined, and the influences of seed coat color and seed weight on each were assessed. The total protein and total oil contents were evaluated using Kjeldahl and Soxhlet methods and were in the ranges of 36.28-44.19% and 13.45-19.20%, respectively. Moreover, the contents of individual fatty acids were determined as area percentage from acquired gas-chromatography peaks. The contents of palmitic, stearic, oleic, linoleic and linolenic acids were in the ranges of 9.90-12.55, 2.45-4.00, 14.97-38.74, 43.22-60.26, and 5.37-12.33%, respectively and each significantly varied between the soybean germplasms. Unlike total oil and fatty acid contents, total protein content was not significantly affected by both seed coat color and seed weight. Cluster analysis grouped the soybeans into two classes with notable content differences. Fatty acids were the main factors for the variabilities seen between the soybean germplasms as observed in the principal component analysis. Correlation analysis revealed a significant but negative association between total oil and total protein contents (r = -0.714, p < 0.0001). Besides, a trade-off relationship was observed between oleic acid and linoleic acid (r = -0.936, p < 0.0001) which was reflected with respect to both seed coat color and seed weight. Among all colored soybeans, pale-yellow soybeans had the highest and the lowest levels of oleic acid and linoleic acid, respectively each being significantly different from the rest of colored soybeans (p < 0.05). Likewise, oleic acid content increased with seed weight while that of linoleic acid decreased with seed weight (p < 0.05). In general, this study showed the significance of seed coat color and seed weight to discriminate soybean genotypes, mainly in terms of their fatty acid contents. Moreover, the soybean germplasms with distinct characters and fatty acid contents identified in this study could be important genetic resources for cultivar development.
Electrical Unfolding of Cytochrome c During Translocation Through a Nanopore Constriction
Authors: Tripathi Prabhat , Benabbas Abdelkrim , Mehrafrooz Behzad , Yamazaki Hirohito , Aksimentiev Aleksei , Champion Paul M. , Wanunu Meni
Published: February 2021

Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field-driven translocation behavior of cytochrome c (cyt c) through ultrathin silicon nitride (SiNx) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5 nm diameter pore we find that, in a threshold electric field regime of ∼30-100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5 nm and 2.0 nm diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (∼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens new avenues for exploring protein folding structures, internal contacts, and electric field-induced deformability.

Significance Statement

Can localized electric fields drive the complete unfolding of a protein molecule? Protein unfolding prior to its translocation through a nanopore constriction is an important step in protein transport across biological membranes and also an important step in nanopore-based protein sequencing. We studied here the electric-field-driven translocation behavior of a model protein (cyt c) through nanopores of diameters ranging from 1.5 to 5.5 nm. These single molecule measurements show that electric fields at the nanopore constriction can select both partially and fully unfolded protein conformations. Zero-field free energy gaps between these conformations, found using a simple thermodynamic model, are in remarkable agreement with previously reported studies of cyt c unfolding energetics.
Cytogenetic events in the endosperm of amphiploidAvena maroccana×A. longiglumis
Authors: Tomaszewska Paulina , Kosina Romuald
Published: February 2021

This study analysed cytogenetic events occurring in the syncytial endosperm of theAvena maroccanaGand. ×Avena longiglumisDur. amphiploid, which is a product of two wild species having different genomes. Selection through the elimination of chromosomes and their fragments, including those translocated, decreased the level of ploidy in the endosperm below the expected 3n, leading to the modal number close to 2n. During intergenomic translocations, fragments of the heterochromatin-rich C-genome were transferred to the A and Al genomes. Terminal and non-reciprocal exchanges dominated, whereas other types of translocations, including microexchanges, were less common. Using two probes and by counterstaining with DAPI, theA. longiglumisand the rare exchanges between the A and Al genomes were detected by GISH. The large discontinuity in the probe labelling in the C chromosomes demonstrated inequality in the distribution of repetitive sequences along the chromosome and probable intragenomic rearrangements. In the nucleus, the spatial arrangement of genomes was non-random and showed a sectorial-concentric pattern, which can vary during the cell cycle, especially in the less stable tissue like the hybrid endosperm.
Fixational drift is driven by diffusive dynamics in central neural circuitry
Authors: Ben-Shushan Nadav , Shaham Nimrod , Joshua Mati , Burak Yoram
Published: February 2021

During fixation and between saccades, our eyes undergo diffusive random motion called fixational drift [1]. The role of fixational drift in visual coding and inference has been debated in the past few decades, but the mechanisms that underlie this motion remained unknown. In particular, it has been unclear whether fixational drift arises from peripheral sources, or from central sources within the brain. Here we show that fixational drift is correlated with neural activity, and identify its origin in central neural circuitry within the oculomotor system. We analyzed a large data set of ocular motoneuron (OMN) recordings in the rhesus monkey, alongside precise measurements of eye position [2, 3], and found that most of the variance of fixational eye drifts must arise upstream of the OMNs. The diffusive statistics of the motion points to the oculomotor integrator, a memory circuit responsible for holding the eyes still between saccades, as a likely source of the motion. Theoretical modeling, constrained by the parameters of the primate oculomotor system, supports this hypothesis by accounting for the amplitude as well as the statistics of the motion. Thus, we propose that fixational ocular drift provides a direct observation of diffusive dynamics in a neural circuit responsible for storage of continuous parameter memory in persistent neural activity. The identification of a mechanistic origin for fixational drift is likely to advance the understanding of its role in visual processing and inference.
Computational Simulations of the 4-D Micro-Circulatory Network in Zebrafish Tail Amputation and Regeneration
Authors: Roustaei Mehrdad , Baek Kyung In , Wang Zhaoqiang , Cavallero Susana , Satta Sandro , Lai Angela , O’Donnell Ryan , Vedula Vijay , Ding Yichen , Marsden Alison Lesley , Hsiai Tzung
Published: February 2021

Wall shear stress (WSS) in the micro-vasculature contributes to biomechanical cues that regulate mechanotransduction underlying vascular development, regeneration, and homeostasis. We hereby elucidate the interplay between hemodynamic shear forces and luminal remodeling in response to vascular injury and regeneration in the zebrafish model of tail amputation. Using the transgenicTg(fli1:eGFP; Gata1:ds-red) line, we were able to track the enhanced green-fluorescent protein (eGFP)-labeled endothelial lining of the 3-D microvasculature for post-image segmentation and reconstruction of fluid domain for computational fluid dynamics (CFD) simulation. At 1 day post amputation (dpa), dorsal aorta (DA) and posterior cardinal vein (PCV) were severed, and vasoconstriction developed in the dorsal longitudinal anastomotic vessel (DLAV) with a concomitant increase in WSS in the segmental vessels (SV) proximal to the amputation site and a decrease in WSS in SVs distal to amputation. Simultaneously, we observed angiogenesis commencing at the tips of the amputated DLAV and PCV where WSS was minimal in the absence of blood flow. At 2 dpa, vasodilation occurred in a pair of SVs proximal to amputation, resulting in increased flow rate and WSS, whereas in the SVs distal to amputation, WSS normalized to the baseline. At 3 dpa, the flow rate in the arterial SV proximal to amputation continued to rise and merged with DLAV that formed a new loop with PCV. Thus, our CFD modeling uncovered a well-coordinated micro-vascular adaptation process following tail amputation, accompanied by the rise and fall of WSS and dynamic changes in flow rate during vascular regeneration.
Relatively Shorter Muscle Lengths Increase the Metabolic Rate of Cyclic Force Production
Authors: Beck Owen N. , Schroeder Jordyn N. , Trejo. Lindsey H. , Franz Jason R. , Sawicki Gregory S.
Published: February 2021

During animal locomotion, force-producing leg muscles are almost exclusively responsible for the whole-body’s metabolic energy expenditure. Animals can change the length of these leg muscles by altering body posture (e.g.,joint angles), kinetics (e.g.,body weight), or the structural properties of their biological tissues (e.g.,tendon stiffness). Currently, it is uncertain whether relative muscle fascicle operating length has a measurable effect on the metabolic energy expenditure of cyclic locomotion-like contractions. To address this uncertainty, we measured the metabolic energy expenditure of human participants as they cyclically produce two distinct ankle moments at three separate ankle angles (90°, 105°, 120°) on a fixed-position dynamometer exclusively using their soleus. Overall, increasing participant ankle angle from 90° to 120° (more plantar flexion) reduced minimum soleus fascicle length by 17% (both moment levels, p<0.001) and increased metabolic energy expenditure by an average of 208% (both p<0.001). Across both moment levels, the increased metabolic energy expenditure was not driven by greater fascicle positive mechanical work (higher moment level, p=0.591), fascicle force rate (both p≥0.235), or active muscle volume (both p≥0.122); but it was correlated with average relative soleus fascicle length (r=-179, p=0.002) and activation (r=0.51, p<0.001). Therefore, the metabolic energy expended during locomotion can likely be reduced by lengthening active muscles that operate on the ascending-limb of their force-length relationship.
Cued Naming and the left inferior frontal cortex
Authors: Holland Rachel , Crinion Jennifer T.
Published: February 2021

Clinical studies have shown that naming can be behaviorally facilitated by priming, e.g., phonemic cues reduce anomia. Rehabilitation of language is argued to rely upon the same processes of priming in healthy speakers. Here we show, in healthy older adults, the immediate facilitatory behavioral and neural priming elicited by phonemic cues presented during an fMRI experiment of overt naming; thus, bridging the gap between lesion and neuroimaging studies. Four types of auditory cues were presented concurrently with an object picture (e.g., cat): (i) word (i.e., the target name (/kat/), (ii) initial phoneme segment (e.g., /ka/), (iii) final phoneme segment (/at/), or (iv) acoustic (noise) control cue. Naming was significantly faster with word, initial and final phonemic cues compared to noise; and word and initial cues compared to final cues, with no difference between word and initial cues. A neural priming effect – a significant decrease in neural activity – was observed in the left inferior frontal cortex (LIFC, pars triangularis, BA45) and the anterior insula bilaterally consistent with theories of primed articulatory encoding and post-lexical selection. The reverse contrast revealed increased activation in left posterior dorsal supramarginal gyrus for word cues that, we argue, may reflect integration of semantic and phonology processing during word rather than phonemic conditions. Taken together, these data from unimpaired speakers identified nodes within the naming network affected by phonemic cues. Activity within these regions may act as a possible biomarker to index anomic individuals’ responsiveness to phonemically cued anomia treatment.
Differential roles of RIG-I-like receptors in SARS-CoV-2 infection
Authors: Yang Duomeng , Geng Tingting , Harrison Andrew G. , Wang Penghua
Published: February 2021

The retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) are the major viral RNA sensors that are essential for activation of antiviral immune responses. However, their roles in severe acute respiratory syndrome (SARS)-causing coronavirus (CoV) infection are largely unknown. Herein we investigate their functions in human epithelial cells, the primary and initial target of SARS-CoV-2, and the first line of host defense. A deficiency in MDA5 (MDA5−/−), RIG-I or mitochondrial antiviral signaling protein (MAVS) greatly enhanced viral replication. Expression of the type I/III interferons (IFN) was upregulated following infection in wild-type cells, while this upregulation was severely abolished inMDA5−/−andMAVS−/−, but not inRIG-I−/−cells. Of note, ACE2 expression was ~2.5 fold higher inRIG-I−/−than WT cells. These data demonstrate a dominant role of MDA5 in activating the type I/III IFN response to SARS-CoV-2, and an IFN-independent anti-SARS-CoV-2 role of RIG-I.
Human retinal organoids release extracellular vesicles that regulate gene expression in target human retinal progenitor cells
Authors: Zhou Jing , Flores-Bellver Miguel , Pan Jianbo , Benito-Martin Alberto , Shi Cui , Onwumere Onyekwere , Mighty Jason , Qian Jiang , Zhong Xiufeng , Hogue Tasmim , Amponsah-Antwi Baffour , Einbond Linda , Gharbaran Rajendra , Wu Hao , Chen Bo-Juen , Zheng Zhiliang , Tchaikovskaya Tatyana , Zhang Xusheng , Peinado Hector , Canto-Soler Maria Valeria , Redenti Stephen
Published: February 2021

The mechanisms underlying retinal development have not been completely elucidated. Extracellular vesicles (EVs) are novel essential mediators of cell-to-cell communication with emerging roles in developmental processes. Nevertheless, the identification of EVs in human retinal tissue, characterization of their cargo, and analysis of their potential role in retina development has not been accomplished. Three-dimensional retinal tissue derived from human induced pluripotent stem cells (hiPSC) provide an ideal developmental system to achieve this goal. Here we report that hiPSC-derived retinal organoids release exosomes and microvesicles with small noncoding RNA cargo. EV miRNA cargo-predicted targetome correlates with GO pathways involved in mechanisms of retinogenesis relevant to specific developmental stages corresponding to hallmarks of native human retina development. Furthermore, uptake of EVs by human retinal progenitor cells leads to changes in gene expression correlated with EV miRNA cargo predicted gene targets, and mechanisms involved in retinal development, ganglion cell and photoreceptor differentiation and function.
Expression of a tetracycline-controlled transactivator (Tet-On/Off system) in beta cells reduces insulin expression and secretion in mice
Authors: Jouvet Nathalie , Bouyakdan Khalil , Baldwin Cindy , Marcinkiewicz Jadwiga , Alquier Thierry , Estall Jennifer L.
Published: February 2021

Controllable genetic manipulation is an indispensable tool in research, greatly advancing our understanding of cell biology and physiology. However, in beta cells, transgene silencing, low inducibility, ectopic expression and off-targets effects on cell function and glucose homeostasis are a persistent challenge. In this study, we investigated whether an inducible, Tet-Off system with beta-cell specific MIP-itTA driven expression of TetO-CreJaw/Jcould circumvent previous issues of specificity, efficacy and toxicity. Following assessment of tissue-specific gene recombination; beta cell architecture;in vitroandin vivoglucose-stimulated insulin secretion (GSIS); and whole-body glucose homeostasis, we discovered that expression of any tetracycline-controlled transactivator (e.g. itTA, rtTA or tTA) in beta cells significantly reducedInsulingene expression and decreased insulin content. This translated into lower pancreatic insulin levels and reduced insulin secretion in mice carrying a MIP-itTA transgene, independent of Cre-recombinase expression or doxycycline treatment. These results raise significant concern regarding the use of Tet-On or Tet-Off systems for genome editing in beta cells and emphasize the need to control for effects of transactivator expression. Our study echoes ongoing challenges faced by fundamental researchers focused on beta cells and highlights the need for consistent and careful control of experiments using these research tools.
A system for continuous and automated measurement of mouse home-cage drinking with automated control of liquid access
Authors: Bagley Jared R. , Kashinsky Wayne M. , Jentsch James D.
Published: February 2021

Measurement of drinking behavior in laboratory animals is an often utilized method in many areas of scientific research, including the study of ingestive behaviors and addictions. We have designed a system that measures drinking by continuously tracking fluid-filled bottle weights with load cells and calculating change in fluid weight per drinking bout. The load cells serve both as a contact sensor that detects mouse-spout contact, as well as a monitor of fluid weight change per contact bout. The design described here fulfills several key criteria, including automated and continuous recording of drinking in the home-cage, automated control of liquid access, and inexpensive/reproducible fabrication. These features may allow researchers to generate high-resolution, detailed information on drinking behavior in high-throughput experimental designs. Here, we provide an overview of the design and present results from tests to validate the system. C57BL/6J mice were offered water and ethanol concurrently, using this system. Consumption weights were determined by the system and independently by conventional approaches. The results indicated a near-perfect correlation between the two methods, indicating the system returned valid consumption weights. This system functions as a valid drinking monitor that provides temporally precise data with a low cost design.
The role of gene segment interactions in driving the emergence of dominant gene constellations during influenza virus reassortment
Authors: Trifkovic Sanja , Gilbertson Brad , Fairmaid Emily , Cobbin Joanna , Rockman Steven , Brown Lorena E.
Published: February 2021

A segmented genome enables influenza virus to undergo reassortment when two viruses infect the same cell. Resulting reassorted progeny have a spectrum of gene constellations and potentially different phenotypes. Although reassortment is involved in the creation of pandemic influenza strains and is routinely used to produce influenza vaccines, our understanding of the factors that drive the emergence of dominant gene constellations during this process is incomplete. Using an influenza vaccine seed production model, reassortant genotypes were tracked through the reassortment process under antibody selective pressure. We discovered that certain gene constellations conferring low replicative fitness were selected at the expense of more fit progeny. Nevertheless, relatively unfit reassortants likely provide high hemagglutinin antigen yields through co-production of non-infectious particles and/or by more hemagglutinin molecules per virion. Our data illustrate the dynamics and complexity of reassortment and highlight how gene segment interactions formed during packaging, in addition to antibody pressure, restrict the final viruses that dominate.
VIA: Generalized and scalable trajectory inference in single-cell omics data
Authors: Stassen Shobana V. , Yip Gwinky G. K. , Wong Kenneth K. Y. , Ho Joshua W. K. , Tsia Kevin K.
Published: February 2021

Inferring cellular trajectories using a variety of omic data is a critical task in single-cell data science. However, accurate prediction of cell fates, and thereby biologically meaningful discovery, is challenged by the sheer size of single-cell data, the diversity of omic data types, and the complexity of their topologies. We present VIA, a scalable trajectory inference algorithm that overcomes these limitations by using lazy-teleporting random walks to accurately reconstruct complex cellular trajectories beyond tree-like pathways (e.g. cyclic or disconnected structures). We show that VIA robustly and efficiently unravels the fine-grained sub-trajectories in a 1.3-million-cell transcriptomic mouse atlas without losing the global connectivity at such a high cell count. We further apply VIA to discovering elusive lineages and less populous cell fates missed by other methods across a variety of data types, including single-cell proteomic, epigenomic, multi-omics datasets, and a new in-house single-cell morphological dataset.
A strategy to address dissociation-induced compositional and transcriptional bias for single-cell analysis of the human mammary gland
Authors: Engelbrecht Lisa K. , Twigger Alecia-Jane , Ganz Hilary M. , Gabka Christian J. , Bausch Andreas R. , Lickert Heiko , Sterr Michael , Kunze Ines , Khaled Walid T. , Scheel Christina H.
Published: February 2021

Single-cell transcriptomics provide insights into cellular heterogeneity and lineage dynamics that are key to better understanding normal mammary gland function as well as breast cancer initiation and progression. In contrast to murine tissue, human mammary glands require laborious dissociation protocols to isolate single cells. This leads to unavoidable procedure-induced compositional and transcriptional bias. Here, we present a new strategy on how to identify and minimize systematic error by combining different tissue dissociation strategies and then directly comparing composition and transcriptome of isolated cells using single-cell RNA sequencing and flow cytometry. Depending on the tissue isolation strategy, we found dramatic differences in abundance and heterogeneity of certain stromal cells types. Moreover, we identified lineage-specific dissociation-induced gene expression changes that, if left unchecked, could lead to misinterpretation of cellular heterogeneity and, since the basal epithelial population is particularly affected by this, wrongful assignment of putative stem cell populations.
CONTEXT-INDEPENDENT FUNCTION OF A CHROMATIN BOUNDARYIN VIVO
Authors: Willemin Andréa , Lopez-Delisle Lucille , Bolt Christopher Chase , Gadolini Marie-Laure , Duboule Denis , Rodríguez-Carballo Eddie
Published: February 2021

Mammalian genomes are partitioned into sub-megabase to megabase-sized units of preferential interactions called topologically associating domains or TADs, which are likely important for the proper implementation of gene regulatory processes. These domains provide structural scaffolds for distantcisregulatory elements to interact with their target genes within the three-dimensional nuclear space and architectural proteins such as CTCF as well as the cohesin complex participate in the formation of the boundaries between them. However, the importance of the genomic context in providing a given DNA sequence the capacity to act as a boundary element remains to be fully investigated. To address this question, we randomly relocated a topological boundary functionally associated with the mouseHoxDgene cluster and show that it can indeed act similarly outside its initial genomic context. In particular, the relocated DNA segment recruited the required architectural proteins and induced a significant depletion of contacts between genomic regions located across the integration site. The host chromatin landscape was re-organized, with the splitting of the TAD wherein the boundary had integrated. These results provide evidence that topological boundaries can function independently of their site of origin, under physiological conditions during mouse development.

AUTHOR SUMMARY

During development, enhancer sequences tightly regulate the spatio-temporal expression of target genes often located hundreds of kilobases away. This complex process is made possible by the folding of chromatin into domains, which are separated from one another by specific genomic regions referred to as boundaries. In order to understand whether such boundary sequences require their particular genomic contexts to achieve their isolating effect, we analyzed the impact of introducing one such boundary, taken from theHoxDgene cluster, into a distinct topological domain. We show that this ectopic boundary splits the host domain into two sub-domains and affects the expression levels of a neighboring gene. We conclude that this sequence can work independently from its genomic context and thus carries all the information necessary to act as a boundary element.
Lattice defects induced by microtubule-stabilizing agents exert a long-range effect on microtubule growth by promoting catastrophes
Authors: Rai Ankit , Liu Tianyang , Katrukha Eugene A. , Estévez-Gallego Juan , Paterson Ian , Díaz J. Fernando , Kapitein Lukas C. , Moores Carolyn A. , Akhmanova Anna
Published: February 2021

Microtubules are dynamic cytoskeletal polymers that spontaneously switch between phases of growth and shrinkage. The probability of transitioning from growth to shrinkage, termed catastrophe, increases with microtubule age, but the underlying mechanisms are poorly understood. Here, we set out to test whether microtubule lattice defects formed during polymerization can affect growth at the plus end. To generate microtubules with lattice defects, we used microtubule-stabilizing agents that promote formation of polymers with different protofilament numbers. By employing different agents during nucleation of stable microtubule seeds and subsequent polymerization phase, we could reproducibly induce switches in protofilament number and induce stable lattice defects. Such drug-induced defects led to frequent catastrophes, which were not observed when microtubules were grown in the same conditions but without a protofilament number mismatch. Microtubule severing at the site of the defect was sufficient to suppress catastrophes. We conclude that structural defects within microtubule lattice can exert effects that can propagate over long distances and affect the dynamic state of the microtubule end.
Topologically associating domains and their role in the evolution of genome structure and function inDrosophila
Authors: Liao Yi , Zhang Xinwen , Chakraborty Mahul , Emerson J.J.
Published: February 2021

Topologically associating domains (TADs) were recently identified as fundamental units of three-dimensional eukaryotic genomic organization, although our knowledge of the influence of TADs on genome evolution remains preliminary. To study the molecular evolution of TADs inDrosophilaspecies, we constructed a new reference-grade genome assembly and accompanying high-resolution TAD map forD. pseudoobscura. Comparison ofD. pseudoobscuraandD. melanogaster, which are separated by ∼49 million years of divergence, showed that ∼30%–40% of their genomes retain conserved TADs. Comparative genomic analysis of 17Drosophilaspecies revealed that chromosomal rearrangement breakpoints are enriched at TAD boundaries but depleted within TADs. Additionally, genes within conserved TADs show lower expression divergence than those located in nonconserved TADs. Furthermore, we found that a substantial proportion of long genes (>50 kbp) inD. melanogaster(42%) andD. pseudoobscura(26%) constitute their own TADs, implying transcript structure may be one of the deterministic factors for TAD formation. By using structural variants (SVs) identified from 14D. melanogasterstrains, its three closest sibling species from theD. simulansspecies complex, and two obscura clade species, we uncovered evidence of selection acting on SVs at TAD boundaries, but with the nature of selection differing between SV types. Deletions are depleted at TAD boundaries in both divergent and polymorphic SVs, suggesting purifying selection, whereas divergent tandem duplications are enriched at TAD boundaries relative to polymorphism, suggesting they are adaptive. Our findings highlight how important TADs are in shaping the acquisition and retention of structural mutations that fundamentally alter genome organization.