bluuebunny/crossref_metadata_2025 huggingface dataset:
Construction of a 3-color prism-based TIRF microscope to study the interactions and dynamics of macromolecules
Authors: Fairlamb Max S. , Whitaker Amy M. , Bain Fletcher E. , Spies Maria , Freudenthal Bret D.
Published: May 2021

Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for real-time visualization of macromolecular dynamics and complex assembly. Prism-based TIRF microscopes (prismTIRF) are relatively simple to operate and can be easily modulated to fit the needs of a wide variety of experimental applications. While building a prismTIRF microscope without expert assistance can pose a significant challenge, the components needed to build a prismTIRF microscope are relatively affordable and, with some guidance, the assembly can be completed by a determined novice. Here, we provide an easy-to-follow guide for the design, assembly, and operation of a 3-color prismTIRF microscope which can be utilized for the study macromolecular complexes, including the multi-component protein-DNA complexes responsible for DNA repair, replication, and transcription. Our hope is that this article can assist laboratories that aspire to implement single-molecule TIRF techniques, and consequently expand the application of this technology to a broader spectrum of scientific questions.
Fly seizure EEG: field potential activity in the Drosophila brain
Authors: Iyengar Atulya , Wu Chun-Fang
Published: May 2021

Hypersynchronous neural activity is a characteristic feature of seizures. Although many Drosophila mutants of epilepsy-related genes display clear behavioral spasms and motor unit hyperexcitability, field potential measurements of aberrant hypersynchronous activity across brain regions during seizures have yet to be described. Here, we report a straightforward method to observe local field potentials (LFPs) from the Drosophila brain to monitor ensemble neural activity during seizures in behaving tethered flies. High frequency stimulation across the brain reliably triggers a stereotypic sequence of electroconvulsive seizure (ECS) spike discharges readily detectable in the dorsal longitudinal muscle (DLM) and coupled with behavioral spasms. During seizure episodes, the LFP signal displayed characteristic large-amplitude oscillations with a stereotypic temporal correlation to DLM flight muscle spiking. ECS-related LFP events were clearly distinct from rest- and flight-associated LFP patterns. We further characterized the LFP activity during different types of seizures originating from genetic and pharmacological manipulations. In the ‘bang-sensitive’ sodium channel mutant bangsenseless (bss), the LFP pattern was prolonged, and the temporal correlation between LFP oscillations and DLM discharges was altered. Following administration of the pro-convulsant GABAA blocker picrotoxin, we uncovered a qualitatively different LFP activity pattern, which consisted of a slow (1-Hz), repetitive, waveform, closely coupled with DLM bursting and behavioral spasms. Our approach to record brain LFPs presents an initial framework for electrophysiological analysis of the complex brain-wide activity patterns in the large collection of Drosophila excitability mutants.
AGL16 regulates genome-wide gene expression and flowering time with partial dependency on SOC1 in Arabidopsis
Authors: Dong Xue , Zhang Li-Ping , Yu Dong-Mei , Cheng Fang , Dong Yin-Xin , Jiang Xiao-Dong , Qian Fu-Ming , Turck Franziska , Hu Jin-Yong
Published: May 2021

Flowering transition is pivotal and tightly regulated by complex gene-regulatory-networks, in which AGL16 plays important roles. But the molecular function and binding property of AGL16 is not fully explored in vivo. With ChIP-seq and comparative transcriptomics approaches, we characterized the AGL16 targets spectrum and tested its close molecular and genetic interactions with SOC1, the key flowering integrator. AGL16 bound to promoters of more than 2000 genes via CArG-box motifs that were highly similar to that of SOC1. Being consistent with this, AGL16 formed protein complex and shared a common set of targets with SOC1. However, only very few genes showed differential expression in the agl16-1 loss-of-function mutant, whereas in the soc1-2 knockout background, AGL16 repressed and activated the expression of 375 and 182 genes, respectively, with more than a quarter of the DEGs were also bound by AGL16. AGL16 targeted potentially to about seventy flowering time genes involved in multiple pathways. Corroborating with these, AGL16 repressed the flowering time stronger in soc1-2 than in Col-0 background. These data reveals that AGL16 regulates gene expression and flowering time with a partial dependency on SOC1 activity. Moreover, AGL16 participated in the regulation of water loss and seed dormancy. Our study thus defines the AGL16 molecular spectrum and provides insights underlining the molecular coordination of flowering and environmental adaptation.
Meiotic DSB-independent role of protein phosphatase 4 in Hop1 assembly to promote meiotic chromosome axis formation in budding yeast
Authors: Li Ke , Shinohara Miki
Published: May 2021

Dynamic changes in chromosomal structure that occur during meiotic prophase play an important role in the progression of meiosis. Among these structures, meiosis-specific chromosomal axis-loop structures are important as a scaffold for integrated control between the meiotic recombination reaction and the associated checkpoint system to ensure accurate chromosome segregation. However, the molecular mechanism of the initial step of chromosome axis-loop construction is not well understood. Here, we showed that in budding yeast, protein phosphatase 4 (PP4) that primarily counteracts Mec1/Tel1 phosphorylation is required to promote the assembly of chromosomal axis components Hop1 and Red1 onto meiotic chromatin via interaction with Hop1. PP4, in contrast, did not affect Rec8 assembly. Notably, unlike the previously known function of PP4, this novel function of PP4 was independent of meiotic DSB-dependent Tel1/Mec1 kinase activities. The defect in Hop1/Red1 assembly in the absence of PP4 function was not suppressed by dysfunction of Pch2, which removes Hop1 protein from the chromosome axis. This suggests that PP4 is required for the initial step of chromatin loading of Hop1 rather than stabilization of Hop1 on axes. These results indicate that phosphorylation/dephosphorylation-mediated regulation of Hop1 recruitment onto chromatin occurs during chromosome axis construction before meiotic double-strand break formation.
Therapeutic reversal of prenatal pontine ID1 signaling in DIPG
Authors: Yadav Viveka Nand , Harris Micah K. , Messinger Dana , Thomas Chase , Cummings Jessica R. , Yang Tao , Woo Rinette , Siddaway Robert , Burkert Martin , Stallard Stefanie , Qin Tingting , Mullan Brendan , Siada Ruby , Ravindran Ramya , Niculcea Michael , Ginn Kevin F. , Gener Melissa A.H. , Dorris Kathleen , Vitanza Nicholas A. , Schmidt Susanne V. , Spitzer Jasper , Li Jiang , Filbin Mariella G. , Cao Xuhong , Castro Maria G. , Lowenstein Pedro R. , Mody Rajen , Chinnaiyan Arul , Desprez Pierre-Yves , McAllister Sean , Hawkins Cynthia , Waszak Sebastian M. , Venneti Sriram , Koschmann Carl
Published: May 2021

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive brain tumor with rare survival beyond two years. This poor prognosis is largely due to the tumor’s highly infiltrative and invasive nature. Previous reports demonstrate upregulation of the transcription factor ID1 with H3K27M and ACVR1 mutations, but this has not been confirmed in human tumors or therapeutically targeted. We developed an in utero electroporation (IUE) murine H3K27M-driven tumor model, which demonstrates increased ID1 expression in H3K27M- and ACVR1-mutated tumor cells. In human tumors, elevated ID1 expression is associated with H3K27M/ACVR1-mutation, brainstem location, and reduced survival. The ID1 promoter demonstrates a similar active epigenetic state in H3K27M tumor cells and murine prenatal hindbrain cells. In the developing human brain, ID1 is expressed highest in oligo/astrocyte-precursor cells (OAPCs). These ID1+/SPARCL1+ cells share a transcriptional program with astrocyte-like (AC-like) DIPG cells, and demonstrate upregulation of gene sets involved with regulation of cell migration. Both genetic and pharmacologic [cannabidiol (CBD)] suppression of ID1 results in decreased DIPG cell invasion/migration in vitro and invasion/tumor growth in multiple in vivo models. CBD reduces proliferation through reactive oxygen species (ROS) production at low micromolar concentrations, which we found to be achievable in the murine brainstem. Further, pediatric high-grade glioma patients treated off-trial with CBD (n=15) demonstrate tumor ID1 reduction and improved overall survival compared to historical controls. Our study identifies that ID1 is upregulated in DIPG through reactivation of a developmental OAPC transcriptional state, and ID1-driven invasiveness of DIPG is therapeutically targetable with CBD.

One Sentence Summary

The transcription factor ID1 is upregulated in a subset of DIPG tumor cells, and ID1-driven invasiveness is therapeutically targetable with CBD.
The polar flagellar transcriptional regulatory network in Vibrio campbellii deviates from canonical Vibrio species
Authors: Petersen Blake D. , Liu Michael S. , Podicheti Ram , Yang Albert Ying-Po , Simpson Chelsea , Hemmerich Chris , Rusch Douglas B. , van Kessel Julia C.
Published: May 2021

Vibrio campbellii is a Gram-negative bacterium that is free-living and ubiquitous in marine environments, and it is a pathogen of fish and shellfish. Swimming motility via a single polar flagellum is a critical virulence factor in V. campbellii pathogenesis, and disruption of the flagellar motor significantly decreases host mortality. To examine V. campbellii flagellar gene regulation, we identified homologs of flagellar and chemotaxis genes conserved in other members of the Vibrionaceae and determined the transcriptional profile of these loci using differential RNA-seq. We systematically deleted all 63 predicted flagellar and chemotaxis genes in V. campbellii and examined their effects on motility and flagellum production. We specifically focused on the core flagellar regulators of the flagellar regulatory hierarchy established in other Vibrios: RpoN (σ54), FlrA, FlrC, and FliA. Our results show that V. campbellii transcription of flagellar and chemotaxis genes is governed by a multi-tiered regulatory hierarchy similar to other motile Vibrio species but with two critical differences: the σ54-dependent regulator FlrA is dispensable for motility, and Class II gene expression is independent of σ54 regulation. Our genetic and phenotypic dissection of the V. campbellii flagellar regulatory network highlights the differences that have evolved in flagellar regulation across the Vibrionaceae.
Fungi hijack a plant apoplastic endoglucanase to release a ROS scavenging β-glucan decasaccharide to subvert immune responses
Authors: Chandrasekar Balakumaran , Wanke Alan , Wawra Stephan , Saake Pia , Mahdi Lisa , Charura Nyasha , Neidert Miriam , Malisic Milena , Thiele Meik , Dama Murali , Pauly Markus , Zuccaro Alga
Published: May 2021

Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner, insoluble cell wall (CW) layer and an outer, soluble extracellular polysaccharide (EPS) matrix. Here we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, implicating different biological functions. The barley (Hordeum vulgare) β-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved β-1,3;1,6-glucan decasaccharide (β-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight β-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by β-1,3-endoglucanases due to the presence of three β-1,6-linked glucose branches and can scavenge reactive oxygen species. Additionally, exogenous application of β-GD leads to enhanced fungal colonization in barley. Our data highlights the hitherto undescribed capacity of this often overseen fungal EPS layer to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant-microbe interface.

Significance

Here we identify and characterize a conserved β-1,3;1,6-glucan decasaccharide with antioxidant activity released from the fungal extracellular polysaccharide (EPS) matrix by the activity of a plant apoplastic endoglucanase. In addition, we provide a quantitative proteomic analysis of the fungal EPS and cell wall (CW) layers.

HIGHLIGHTS

The fungal extracellular polysaccharide (EPS) matrix and the cell wall (CW) are specific layers with distinct protein and carbohydrate signatures

A conserved β-1,3;1,6-glucan decasaccharide (β-GD) is released from the EPS matrices of different fungi by the activity of the barley β-1,3-endoglucanase BGLUII, a member of the widely distributed apoplastic GH17 family

The β-GD efficiently scavenges reactive oxygen species (ROS) and enhances fungal colonization

The immunomodulatory potential as microbe-associated molecular pattern (MAMP) as well as the biochemical activity as ROS scavenger of soluble low molecular weight β-glucans are defined by the presence of β-1,6-glucose branches
Subspace based Multiple Constrained Minimum Variance (SMCMV) Beamformers
Authors: Moiseev Alexander , Herdman Anthony T. , Ribary Urs
Published: May 2021

In MEG and EEG brain imaging research two popular approaches are often used for spatial localization of focal task- or stimuli-related brain activations. One is a so called MUSIC approach applied in the form of RAP or TRAP MUSIC algorithms. Another one is the beamformer approach, specifically multiple constrained minimum variance (MCMV) beamformer when dealing with significantly correlated activations. Either method is using its own source localizer functions. Considering simplicity, accuracy and computational efficiency both approaches have their advantages and disadvantages. In this study we introduce a novel set of so called Subspace MCMV (or SMCMV) beamformers whose localizer functions combine MUSIC and MCMV localizers. We show that in ideal situations where forward modeling, data recording and noise measurements are error-free, SMCMV localizers allow precise identification of n arbitrarily correlated sources irrespective to their strength in just n scans of the brain volume using RAP MUSIC type algorithm. We also demonstrate by extensive computer simulations that with respect to source localization errors and the total number of identified sources SMCMV outperforms both the TRAP MUSIC and MIA MCMV (which is the most accurate MCMV algorithm to our knowledge) in non-ideal practical situations, specifically when the noise covariance cannot be estimated precisely, signal to noise ratios are small, source correlations are significant and larger numbers of sources are involved.
A burst of transposon expression accompanies the activation of Y chromosome fertility genes during Drosophila spermatogenesis
Authors: Lawlor Matthew A. , Cao Weihuan , Ellison Christopher E.
Published: May 2021

Transposable elements (TEs) must replicate in germline cells to pass novel insertions to offspring. In Drosophila melanogaster ovaries, TEs can exploit specific developmental windows of opportunity to evade host silencing and increase their copy numbers. However, TE activity and host silencing in the distinct cell types of the Drosophila melanogaster testis are not well understood. We reanalyzed publicly available single-cell RNA-seq datasets to quantify TE expression in the distinct cell types of the Drosophila testis. We developed a novel method for identification of TE and host gene expression programs and find that a distinct population of early spermatocytes expresses a large number of TEs at much higher levels than other germline and somatic components of the testes. This burst of TE expression coincides with the activation of Y chromosome fertility factors and spermatocyte-specific transcriptional regulators, as well as downregulation of many components of the piRNA pathway. The TEs expressed by this cell population are enriched on the Y chromosome and depleted on the X chromosome relative to other active TEs. These data suggest that some TEs may achieve high insertional activity in males by exploiting a window of opportunity for mobilization created by the activation of spermatocyte-specific and Y-chromosome-specific transcriptional programs.
Binding of SARS-CoV-2 fusion peptide to host membranes
Authors: Schaefer Stefan L. , Jung Hendrik , Hummer Gerhard
Published: May 2021

During infection the SARS-CoV-2 virus fuses its viral envelope with cellular membranes of its human host. Initial contact with the host cell and membrane fusion are both mediated by the viral spike (S) protein. Proteolytic cleavage of S at the S2′ site exposes its 40 amino acid long fusion peptide (FP). Binding of the FP to the host membrane anchors the S2 domain of S in both the viral and the host membrane. The reorganization of S2 then pulls the two membranes together. Here we use molecular dynamics (MD) simulations to study the two core functions of the SARS-CoV-2 FP: to attach quickly to cellular membranes and to form an anchor strong enough to withstand the mechanical force during membrane fusion. In eight 10 μs-long MD simulations of FP in proximity to endosomal and plasma membranes, we find that FP binds spontaneously to the membranes and that binding proceeds predominantly by insertion of two short amphipathic helices into the membrane interface. Connected via a flexible linker, the two helices can bind the membrane independently, yet binding of one promotes the binding of the other by tethering it close to the target membrane. By simulating mechanical pulling forces acting on the C-terminus of the FP we then show that the bound FP can bear forces up to 250 pN before detaching from the membrane. This detachment force is more than ten-fold higher than an estimate of the force required to pull host and viral membranes together for fusion. We identify a fully conserved disulfide bridge in the FP as a major factor for the high mechanical stability of the FP membrane anchor. We conclude, first, that the sequential binding of two short amphipathic helices allows the SARS-CoV-2 FP to insert quickly into the target membrane, before the virion is swept away after shedding the S1 domain connecting it to the host cell receptor. Second, we conclude that the double attachment and the conserved disulfide bridge establish the strong anchoring required for subsequent membrane fusion. Multiple distinct membrane-anchoring elements ensure high avidity and high mechanical strength of FP-membrane binding.
Convergent use of phosphatidic acid for Hepatitis C virus and SARS-CoV-2 replication organelle formation
Authors: Tabata Keisuke , Prasad Vibhu , Paul David , Lee Ji-Young , Pham Minh-Tu , Twu Woan-Ing , Neufeldt Christopher J. , Cortese Mirko , Cerikan Berati , Si Tran Cong , Lüchtenborg Christian , V’kovski Philip , Hörmann Katrin , Müller André C. , Zitzmann Carolin , Haselmann Uta , Beneke Jürgen , Kaderali Lars , Erfle Holger , Thiel Volker , Lohmann Volker , Superti-Furga Giulio , Brügger Britta , Bartenschlager Ralf
Published: May 2021

Double membrane vesicles (DMVs) are used as replication organelles by phylogenetically and biologically distant pathogenic RNA viruses such as hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Viral DMVs are morphologically analogous to DMVs formed during autophagy, and although the proteins required for DMV formation are extensively studied, the lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV replication and DMV formation. AGPAT1/2 double knockout also impaired SARS-CoV-2 replication and the formation of autophagosome-like structures. By using correlative light and electron microscopy, we observed the relocalization of AGPAT proteins to HCV and SARS-CoV-2 induced DMVs. In addition, an intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways via phosphotidylcholine (PC) and diacylglycerol (DAG). Pharmacological inhibition of these synthesis pathways also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as an important lipid used for replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. In addition, our data argue that host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses.

One Sentence Summary

Phosphatidic acid is important for the formation of double membrane vesicles, serving as replication organelles of hepatitis C virus and SARS-CoV-2, and offering a possible host-targeting strategy to treat SARS-CoV-2 infection.
A dual sequence and culture-based survey of maize rhizosphere protists reveals dominant, plant-enriched, and culturable community members
Authors: Taerum Stephen J. , Micciulla Jamie , Corso Gabrielle , Steven Blaire , Gage Daniel J. , Triplett Lindsay R.
Published: May 2021

Protists play important roles in shaping the microbial community of the rhizosphere. However, there is still a limited understanding of how plants shape the protist community, and how well protist isolate collections might represent rhizosphere protist composition and function in downstream studies. We sought to determine whether maize roots select for a distinct protist community in the field, and whether the common or dominant members of that community are readily culturable using standard protist isolation methods. We sequenced 18S and 16S rRNA genes from the rhizospheres of maize grown in two sites, and isolated 103 protists into culture from the same roots. While field site had the greatest effect, rhizospheres in both sites had distinct protist composition from the bulk soils, and certain taxa were enriched in both sites. Enriched taxa were correlated to bacterial abundance patterns. The isolated protists represented six supergroups, and the majority corresponded to taxa found in the sequencing survey. Twenty-six isolates matched eight of the 89 core rhizosphere taxa. This study demonstrates that maize roots select for a distinct protist community, but also illustrate the potential challenges in understanding the function of the dominant protist groups in the rhizosphere.

Originality-Significance Statement

This is the first study comparing cultivation-dependent and independent methods for studying the protist community of plant roots, and the first untargeted analysis of the maize rhizosphere’s effect on protist communities. We show that maize in different sites select for distinct communities and overlapping enriched taxa, but that isolating the most important plant-associated protists may be a challenge for researchers.
CRISPR screens for lipid regulators reveal a role for ER-bound SNX13 in lysosomal cholesterol export
Authors: Lu Albert , Hsieh Frank , Enrich Carlos , Pfeffer Suzanne R.
Published: May 2021

We report here two genome-wide CRISPR screens carried out to identify genes that when knocked out, alter levels of lysosomal cholesterol or bis(monoacylglycero)phosphate. In addition, these screens were also carried out under conditions of NPC1 inhibition to identify modifiers of NPC1 function in lysosomal cholesterol export. The screens confirm tight co- regulation of cholesterol and bis(monoacylglycero)phosphate levels in cells, and reveal an unexpected role for the ER-localized, SNX13 protein as a negative regulator of lysosomal cholesterol export. In the absence of NPC1 function, SNX13 knockout decreases lysosomal cholesterol, and is accompanied by triacylglycerol-rich lipid droplet accumulation and increased lysosomal bis(monoacylglycero)phosphate. These experiments provide unexpected insight into the regulation of lysosomal lipids and modification of these processes by novel gene products.

Genome-wide CRISPR screens carried out with and without NPC1 function identify shared pathways that coordinately control lysosomal cholesterol and bis(monoacylglycero)phosphate. ER-localized SNX13 protein plays an unexpected regulatory role in modifying NPC1 function to regulate cellular cholesterol localization.
Nanopore dwell time analysis permits sequencing and conformational assignment of pseudouridine in SARS-CoV-2
Authors: Fleming Aaron M. , Mathewson Nicole J. , Burrows Cynthia J.
Published: May 2021

Nanopore devices can directly sequence RNA, and the method has the potential to determine locations of epitranscriptomic modifications that have grown in significance because of their roles in cell regulation and stress response. Pseudouridine (Ψ), the most common modification in RNA, was sequenced with a nanopore system using a protein sensor with a helicase brake in synthetic RNAs with 100% modification at 18 known human pseudouridinylation sites. The new signals were compared to native uridine (U) control strands to characterize base calling and associated errors as well as ion current and dwell time changes. The data point to strong sequence context effects in which Ψ can easily be detected in some contexts while in others Ψ yields signals similar to U that would be false negatives in an unknown sample. We identified that the passage of Ψ through the helicase brake slowed the translocation kinetics compared to U and showed a smaller sequence bias that could permit detection of this modification in RNA. The unique signals from Ψ relative to U are proposed to reflect the syn-anti conformational flexibility of Ψ not found in U, and the difference in π stacking between these bases. This observation permitted analysis of SARS-CoV-2 nanopore sequencing data to identify five conserved Ψ sites on the 3’ end of the viral sub-genomic RNAs, and other less conserved Ψ sites. Using the helicase as a sensor protein in nanopore sequencing experiments enables detection of this modification in a greater number of relevant sequence contexts. The data are discussed concerning their analytical and biological significance.
Receptor binding may directly activate the fusion machinery in coronavirus spike glycoproteins
Authors: Wang Yuzhang , Fallon Lucy , Raguette Lauren , Budhan Stephanie , Belfon Kellon , Stepanenko Darya , Helbock Stephanie , Varghese Sarah , Simmerling Carlos
Published: May 2021

SARS-CoV-2, the causative agent of the COVID-19 pandemic, is an enveloped RNA virus. Trimeric spike glycoproteins extend outward from the virion; these class I viral membrane fusion proteins mediate entry of the virus into a host cell and are the dominant antigen for immune response. Cryo-EM studies have generated a large number of structures for the spike either alone, or bound to the cognate receptor ACE2 or antibodies, with the three receptor binding domains (RBDs) seen closed, open, or in various combinations. Binding to ACE2 requires an open RBD, and is believed to trigger the series of dramatic conformational changes in the spike that lead to the shedding of the S1 subunit and transition of the spring-loaded S2 subunit to the experimentally observed post-fusion structure. The steps following ACE2 binding are poorly understood despite extensive characterization of the spike through X-ray, cryo-EM, and computation. Here, we use all-atom simulations, guided by analysis of 81 existing experimental structures, to develop a model for the structural and energetic coupling that connects receptor binding to activation of the membrane fusion machinery.
Accuracies of genomic predictions for disease resistance of striped catfish to Edwardsiella ictaluri using artificial intelligence algorithms
Authors: Vu Nguyen Thanh , Phuc Tran Huu , Oanh Kim Thi Phuong , Van Sang Nguyen , Trang Trinh Thi , Nguyen Nguyen Hong
Published: May 2021

Assessments of genomic prediction accuracies using artificial intelligence (AI) algorithms (i.e., machine and deep learning methods) are currently not available or very limited in aquaculture species. The principal aim of this study was to examine the predictive performance of these new methods for disease resistance to Edwardsiella ictaluri in a population of striped catfish Pangasianodon hypophthalmus and to make comparisons with four common methods, i.e., pedigree-based best linear unbiased prediction (PBLUP), genomic-based best linear unbiased prediction (GBLUP), single-step GBLUP (ssGBLUP) and a non-linear Bayesian approach (notably BayesR). Our analyses using machine learning (i.e., ML-KAML) and deep learning (i.e., DL-MLP and DL-CNN) together with the four common methods (PBLUP, GBLUP, ssGBLUP and BayesR) were conducted for two main disease resistance traits (i.e., survival status coded as 0 and 1 and survival time, i.e., days that the animals were still alive after the challenge test) in a pedigree consisting of 560 individual animals (490 offspring and 70 parents) genotyped for 14,154 Single Nucleotide Polymorphism (SNPs). The results using 6470 SNPs after quality control showed that AI methods outperformed PBLUP, GBLUP and ssGBLUP, with the increases in the prediction accuracies for both traits by 9.1 – 15.4%. However, the prediction accuracies obtained from AI methods were comparable to those estimated using BayesR. Imputation of missing genotypes using AlphaFamImpute increased the prediction accuracies by 5.3 – 19.2% in all the methods and data used. On the other hand, there were insignificant decreases (0.3 – 5.6%) in the prediction accuracies for both survival status and survival time when multivariate models were used in comparison to univariate analyses. Interestingly, the genomic prediction accuracies based on only highly significant SNPs (P < 0.00001, 318 - 400 SNPs for survival status and 1362 – 1589 SNPs for survival time) were somewhat lower (0.3 to 15.6%) than those obtained from the whole set of 6,470 SNPs. In most of our analyses, the accuracies of genomic prediction were somewhat higher for survival time than survival status (0/1 data). It is concluded that there are prospects for the application of genomic selection to increase disease resistance to Edwardsiella ictaluri in striped catfish breeding programs.
Single cell transcriptomics and developmental trajectories of murine cranial neural crest cell fate determination and cell cycle progression
Authors: Ji Yu , Zhang Shuwen , Reynolds Kurt , Gu Ran , McMahon Moira , Islam Mohammad , Liu Yue , Imai Taylor , Donham Rebecca , Zhao Huan , Xu Ying , Burkart-Waco Diana , Zhou Chengji J.
Published: May 2021

Cranial neural crest (NC) cells migrate long distances to populate the future craniofacial regions and give rise to various tissues, including facial cartilage, bones, connective tissues, and cranial nerves. However, the mechanism that drives the fate determination of cranial NC cells remains unclear. Using single-cell RNA sequencing combined genetic fate mapping, we reconstructed developmental trajectories of cranial NC cells, and traced their differentiation in mouse embryos. We identified four major cranial NC cell lineages at different status: pre-epithelial-mesenchymal transition, early migration, NC-derived mesenchymal cells, and neural lineage cells from embryonic days 9.5 to 12.5. During migration, the first cell fate determination separates cranial sensory ganglia, the second generates mesenchymal progenitors, and the third separates other neural lineage cells. We then focused on the early facial prominences that appear to be built by undifferentiated, fast-dividing NC cells that possess similar transcriptomic landscapes, which could be the drive for the facial developmental robustness. The post-migratory cranial NC cells exit the cell cycle around embryonic day 11.5 after facial shaping is completed and initiates further fate determination and differentiation processes. Our results demonstrate the transcriptomic landscapes during dynamic cell fate determination and cell cycle progression of cranial NC lineage cells and also suggest that the transcriptomic regulation of the balance between proliferation and differentiation of the post-migratory cranial NC cells can be a key for building up unique facial structures in vertebrates.
Non-Transgenic Functional Rescue of Neuropeptides
Authors: DiLoreto Elizabeth M. , Reilly Douglas K. , Srinivasan Jagan
Published: May 2021

Animals constantly respond to changes in their environment and internal states via neuromodulation. Neuropeptide genes modulate neural circuits by encoding either multiple copies of the same neuropeptide or different neuropeptides. This architectural complexity makes it difficult to determine the function of discrete and active neuropeptides. Here, we present a novel genetic tool that facilitates functional analysis of individual peptides. We engineeredEscherichia colibacteria to express active peptides and fed loss-of-functionCaenorhabditis elegansto rescue gene activity. Using this approach, we rescued the activity of different neuropeptide genes with varying lengths and functions:trh-1, ins-6, andpdf-1. While some peptides are functionally redundant, others exhibited unique and previously uncharacterized functions. The mechanism of peptide uptake is reminiscent of RNA interference, suggesting convergent mechanisms of gene regulation in organisms. Our rescue-by-feeding paradigm provides a high-throughput screening strategy to elucidate the functional landscape of neuropeptide genes regulating different behavioral and physiological processes.
Repeated origins, gene flow, and allelic interactions of herbicide resistance mutations in a widespread agricultural weed
Authors: Kreiner J.M. , Sandler G. , Stern A.J. , Tranel P.J. , Weigel D , Stinchcombe J.R. , Wright S.I.
Published: May 2021

Causal mutations and their frequency in agricultural fields are well-characterized for herbicide resistance. However, we still lack an understanding of their evolutionary history: the extent of parallelism in the origins of target-site resistance (TSR), how long these mutations persist, how quickly they spread, and allelic interactions that mediate their selective advantage. We addressed these questions with genomic data from 18 agricultural populations of common waterhemp (Amaranthus tuberculatus), which we show to have undergone a massive expansion over the past century with a contemporary effective population size estimate of 8 × 107. We found variation at seven characterized TSR loci, two of which had multiple amino acid substitutions, and three of which were common. These three common resistance variants show parallelism in their mutational origins, with gene flow having shaped their distribution across the landscape. Allele age estimates supported a strong role of adaptation from de novo mutations, with a median allele age of 30 suggesting that most resistance alleles arose soon after the onset of herbicide use. However, resistant lineages varied in both their age and evidence for selection over two different timescales, implying considerable heterogeneity in the forces that govern their persistence. The evolutionary history of TSR has also been shaped by both intra- and inter-locus allelic interactions. We report a signal of extended haplotype competition between two common TSR alleles, and extreme linkage with genome-wide alleles with known functions in resistance adaptation. Together, this work reveals a remarkable example of spatial parallel evolution in a metapopulation, with important implications for the management of herbicide resistance.
Web-based Psychoacoustics: Hearing Screening, Infrastructure, and Validation
Authors: Mok Brittany A. , Viswanathan Vibha , Borjigin Agudemu , Singh Ravinderjit , Kafi Homeira , Bharadwaj Hari M.
Published: May 2021

Anonymous web-based experiments are increasingly and successfully used in many domains of behavioral research. However, online studies of auditory perception, especially of psychoacoustic phenomena pertaining to low-level sensory processing, are challenging because of limited available control of the acoustics, and the unknown hearing status of participants. Here, we outline our approach to mitigate these challenges and validate our procedures by comparing web-based measurements to labbased data on a range of classic psychoacoustic tasks. Individual tasks were created using jsPsych, an open-source javascript front-end library. Dynamic sequences of psychoacoustic tasks were implemented using Django, an open-source library for web applications, and combined with consent pages, questionnaires, and debriefing pages. Subjects were recruited via Prolific, a web-based human-subject marketplace. Guided by a meta-analysis of normative data, we developed and validated a screening procedure to select participants for (putative) normal-hearing status; this procedure combined thresholding of scores in a suprathreshold cocktail-party task with filtering based on survey responses. Headphone use was standardized by supplementing procedures from prior literature with a binaural hearing task. Individuals meeting all criteria were re-invited to complete a range of classic psychoacoustic tasks. Performance trends observed in re-invited participants were in excellent agreement with lab-based data for fundamental frequency discrimination, gap detection, sensitivity to interaural time delay and level difference, comodulation masking release, word identification, and consonant confusions. Our results suggest that web-based psychoacoustics is a viable complement to lab-based research. Source code for our infrastructure is also provided.
Veillonella parvula: a strictly anaerobic bacterium with high efficacy for safe and specific tumor targeting and colonization
Authors: Kefayat Amirhosein , Ghahremani Fatemeh , Rostami Soodabeh
Published: May 2021

Bacterial cancer therapy has gained lots of attention in the past decade and is now considering a reliable option for the future. However, some concerns have limited its application into clinic settings like insufficient colonization of tumors and infectious origin of the currently used bacteria like Clostridium and Salmonella species, especially in cancer patients which exhibit different levels of immunocompromising. In the present study, Veillonella parvula (V. parvula) as a strictly anaerobic bacterium which has rarely identified as a pathogen in human, was administrated into 4T1 breast tumor-bearing mice. At first, 4T1 breast tumor-bearing BALB/c mice were injected with 107 bacteria intravenously, intraperitoneally, orally, or intratumorally. The best administration route according to tumor colonization and safety was selected. Then, the therapeutic effect of V. parvula administration was investigated according to the 4T1 breast tumor’s growth, metastasis, and tumor-bearing mice survival. Besides, histopathological evaluations were done to estimate microscopic changes at the inner of the tumor. V. parvula exhibited significant tumor-targeting and colonization efficacy, 24 h after intravenous administration and formed clustered colonies at the central region of the tumors. Although a negligible number of the bacteria were localized at normal organs, these organs became clear from the bacteria after 72 h, and no side effects or death were observed at the animals after intravenous administration of V. parvula. Although mean tumor volumes in the V. parvula treated group was lower than the control (~ 25.4%), their difference wasn’t statistically significant (P > 0.05). Despite significant tumor colonization (5500000:1 in comparison with normal organs after 72 h), V. parvula didn’t cause a significant therapeutic effect on the metastasis or survival time of tumor-bearing mice. Taking together, V. parvula is a completely safe and tumor-specific agent per se, without any genetic manipulation. Also, it exhibits high tumor penetration and colonization at the deep regions of the tumor.
Precursor abundance influences divergent antigen specific CD8+ T cell responses after Yersinia pseudotuberculosis foodborne infection
Authors: Zhang Yue , Qiu Zhijuan , Sheridan Brian S. , Bliska James B.
Published: May 2021

Primary infection of C57BL/6 mice with the bacterial pathogen Yersinia pseudotuberculosis elicits an unusually large H-2Kb-restricted CD8+ T cell response to the endogenous and protective bacterial epitope YopE69-77. To better understand the basis for this large response, the model OVA257-264 epitope was inserted into YopE in Y. pseudotuberculosis and antigen specific CD8+ T cells in mice were characterized after foodborne infection with the resulting strain. The epitope YopE69-77 elicited significantly larger CD8+ T cell populations in the small intestine, mesenteric lymph nodes (MLNs), spleen and liver between 7- and 30-days post infection, despite residing in the same protein and having a similar affinity for H-2Kb as OVA257-264. YopE-specific CD8+ T cell precursors were ~4.6 times as abundant as OVA-specific precursors in the MLNs, spleen and other lymph nodes of naïve mice, explaining the dominance of YopE69-77 over OVA257-264 at early infection times. However, other factors contributed to this dominance as the ratio of YopE-specific to OVA-specific CD8+ T cells increased between 7- and 30-days post infection. We also compared the YopE-specific and OVA-specific CD8+ T cells generated during infection for effector and memory phenotypes. Significantly higher percentages of YopE-specific cells were characterized as short short-lived effectors while higher percentages of OVA-specific cells were memory precursor effectors at day 30 post infection in spleen and liver. Our results suggest that a large precursor number contributes to the dominance and effector and memory functions of CD8+ T cells generated to the protective YopE69-77 epitope during Y. pseudotuberculosis infection of C57BL/6 mice.
Kinesin-3 mediated axonal delivery of presynaptic neurexin stabilizes dendritic spines and postsynaptic components
Authors: Oliver Devyn , Ramachandran Shankar , Philbrook Alison , Lambert Christopher M. , Nguyen Ken C. Q. , Hall David H. , Francis Michael M.
Published: May 2021

The functional properties of neural circuits are defined by the patterns of synaptic connections between their partnering neurons, but the mechanisms that stabilize circuit connectivity are poorly understood. We systemically examined this question at synapses onto newly characterized dendritic spines of C. elegans GABAergic motor neurons. We show that the presynaptic adhesion protein, neurexin/NRX-1, is required for stabilization of postsynaptic structure. We find that early postsynaptic developmental events proceed without a strict requirement for synaptic activity and are not disrupted by deletion of neurexin/nrx-1. However, in the absence of presynaptic NRX-1, dendritic spines and receptor clusters become destabilized and collapse prior to adulthood. We demonstrate that kinesin-3/UNC-104 delivers NRX-1 to presynaptic terminals and show that ongoing delivery is required for postsynaptic maintenance in mature animals. By defining the dynamics and temporal order of synapse formation events in vivo, we describe a mechanism for stabilizing mature circuit connectivity through neurexin-based adhesion.
Viral shedding and transmission after natural infection and vaccination in an animal model of SARS-CoV-2 propagation
Authors: Zeiss Caroline J. , Asher Jennifer L. , Wyk Brent Vander , Allore Heather G. , Compton Susan
Published: May 2021

At present, global immunity to SARS-CoV-2 resides within a heterogeneous combination of susceptible, naturally infected and vaccinated individuals. The extent to which viral shedding and transmission occurs on re-exposure to SARS-CoV-2 after prior natural exposure or vaccination is an emerging area of understanding. We used Sialodacryoadenitis Virus (SDAV) in rats to model the extent to which immune protection afforded by prior natural infection via high risk (inoculation; direct contact) or low risk (fomite) exposure, or by vaccination, influenced viral shedding and transmission on re-exposure. On initial infection, we confirmed that amount, duration and consistency of viral shedding were correlated with exposure risk. Animals were reinfected after 3.7-5.5 months using the same exposure paradigm. Amount and duration of viral shedding were correlated with re-exposure type and serologic status. 59% of seropositive animals shed virus. Previously exposed seropositive reinfected animals were able to transmit virus to 25% of naive recipient rats after 24-hour exposure by direct contact. Rats vaccinated intranasally with a related virus (Parkers Rat Coronavirus) were able to transmit SDAV to only 4.7% of naive animals after a 7-day direct contact exposure, despite comparable viral shedding. Observed cycle threshold values associated with transmission in both groups ranged from 29-36 cycles, however observed shedding was not a prerequisite for transmission. Results indicate that low-level shedding in both naturally infected and vaccinated seropositive animals can propagate infection in susceptible individuals. Extrapolated to COVID-19, our results suggest that continued propagation of SARS-CoV-2 by seropositive previously infected or vaccinated individuals is possible.
Crystal structure and Hirshfeld surface analysis of 5,7-diphenyl-1,2,3,5,6,7-hexahydroimidazo[1,2-a]pyridine-6,6,8-tricarbonitrile methanol monosolvate
Authors: Naghiyev Farid N. , Mammadova Gunay Z. , Khalilov Ali N. , Atioğlu Zeliha , Akkurt Mehmet , Akobirshoeva Anzurat A. , Mamedov İbrahim G.
Published: May 2021

In the title compound, C22H17N5·CH4O, the imidazolidine ring of the 1,2,3,5,6,7-hexahydroimidazo[1,2-a]pyridine ring system is a twisted envelope, while the 1,2,3,4-tetrahydropyridine ring adopts a twisted boat conformation. In the crystal, pairs of molecules are linked by O—H...N and N—H...O hydrogen bonds via two methanol molecules, forming a centrosymmetric R
4
4(16) ring motif. These motifs are connected to each other by C—H...N hydrogen bonds and form columns along the a axis. The columns form a stable molecular packing, being connected to each other by van der Waals interactions. A Hirshfeld surface analysis indicates that the most significant contributions to the crystal packing are from H...H (43.8%), N...H/H...N (31.7%) and C...H/H...C (18.4%) contacts.
Crystal structure of potassium hydrogen bis((E)-2-{4-[3-(thiophen-3-yl)acryloyl]phenoxy}acetate)
Authors: Vu Quoc Trung , Phan Thuy Linh , Do Ba Dai , Tran Thi Thuy Duong , Nguyen Ngoc Linh , Nguyen Thuy Chinh , Duong Khanh Linh , Ha Manh Hung , Thai Hoang , Le Van Khoe , Van Meervelt Luc
Published: May 2021

The synthesis and spectroscopic data of (E)-2-{4-[3-(thiophen-3-yl)acryloyl]phenoxy}acetic acid are described. Crystallization from an ethanol–water mixture resulted in the title compound, C30H23KO8S2 or [K(C15H11O4S)(C15H12O4S)]
n
, containing one molecule of the acid and one molecule of the potassium salt in the asymmetric unit. Both molecules share the H atom between their carboxyl groups and a potassium ion. The C=C bonds display an E configuration. The thiophene and phenyl rings in the two molecules are inclined by 43.3 (2) and 22.7 (2)°. The potassium ion is octahedrally coordinated by six O atoms. This distorted octahedron shares on opposite sides two oxygen atoms with inversion-related octahedra, resulting in chains of octahedra running in the [010] direction, which form ladder-like chains by C—H...π interactions. A Hirshfeld surface analysis indicates that the highest contributions to the surface contacts arise from interactions in which H atoms are involved, with the most important contribution being from H...H (31.6 and 31.9% for the two molecules) interactions.