bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulus novelty uncovers coding diversity in survey of visual cortex
Authors: Garrett Marina , Groblewski Peter , Piet Alex , Ollerenshaw Doug , Najafi Farzaneh , Yavorska Iryna , Amster Adam , Bennett Corbett , Buice Michael , Caldejon Shiella , Casal Linzy , D’Orazi Florence , Daniel Scott , de Vries Saskia EJ , Kapner Daniel , Kiggins Justin , Lecoq Jerome , Ledochowitsch Peter , Manavi Sahar , Mei Nicholas , Morrison Christopher B. , Naylor Sarah , Orlova Natalia , Perkins Jed , Ponvert Nick , Roll Clark , Seid Sam , Williams Derric , Williford Allison , Ahmed Ruweida , Amine Daniel , Billeh Yazan , Bowman Chris , Cain Nicholas , Cho Andrew , Dawe Tim , Departee Max , Desoto Marie , Feng David , Gale Sam , Gelfand Emily , Gradis Nile , Grasso Conor , Hancock Nicole , Hu Brian , Hytnen Ross , Jia Xiaoxuan , Johnson Tye , Kato India , Kivikas Sara , Kuan Leonard , L’Heureux Quinn , Lambert Sophie , Leon Arielle , Liang Elizabeth , Long Fuhui , Mace Kyla , de Abril Ildefons Magrans , Mochizuki Chris , Nayan Chelsea , North Katherine , Ng Lydia , Ocker Gabriel Koch , Oliver Michael , Rhoads Paul , Ronellenfitch Kara , Schelonka Kathryn , Sevigny Josh , Sullivan David , Sutton Ben , Swapp Jackie , Nguyen Thuyanh K , Waughman Xana , Wilkes Joshua , Wang Michael , Farrell Colin , Wakeman Wayne , Zeng Hongkui , Phillips John , Mihalas Stefan , Arkhipov Anton , Koch Christof , Olsen Shawn R
Published: February 2023

Detecting novel stimuli in the environment is critical for learning and survival, yet the neural basis of novelty processing is not understood. To characterize cell type-specific novelty processing, we surveyed the activity of ∼15,000 excitatory and inhibitory neurons in mice performing a visual task with novel and familiar stimuli. Clustering revealed a dozen functional neuron types defined by experience-dependent encoding. Vasoactive-intestinal-peptide (Vip) expressing inhibitory neurons were diverse, encoding novel stimuli, omissions of familiar stimuli, or behavioral features. Distinct Somatostatin (Sst) expressing inhibitory neurons encoded either familiar or novel stimuli. Subsets of excitatory neurons co-clustered with specific Vip or Sst subpopulations, while Sst and Vip inhibitory clusters were non-overlapping. This study establishes that novelty processing is mediated by diverse functional neuron types in the visual cortex.
Nearly (?) sterile avian egg in a passerine bird
Authors: Těšický Martin , Schmiedová Lucie , Krajzingrová Tereza , Gomez Samblas Mercedes , Bauerová Petra , Kreisinger Jakub , Vinkler Michal
Published: February 2023

During early ontogeny, microbiome affects development of the gastrointestinal tract, immunity and survival in vertebrates. Bird egg has been suggested to be either (1) initially sterile (Sterile egg hypothesis) and (2) colonized throughhorizontal trans-shell migrationafter egg laying, or (3) initially seeded with bacteria throughvertical transferfrom mother’ ss oviduct. Little empirical data illuminate so far the contribution of these mechanisms to gut microbiota formation in avian embryos. We evaluated microbiome of the egg content (day 0; E0-egg), embryonic gut at day 13 (E13) and female faeces in a free-living passerine, the great tit (Parus major), using a methodologically advanced procedure combining16S rRNAgene sequencing and microbe-specific qPCR assays. Our metabarcoding revealed that avian egg is (nearly)sterile, barely distinguishable in microbial composition from negative controls. Of the three potentially pathogenic bacteria targeted by qPCR, onlyDietziawas found in E0-egg (yet also in controls), E13 gut and female samples, which might indicate its possiblevertical transfer. Unlike in poultry, we have shown in passerines that major bacterial colonisation of the gut does not occur before chick’s hatching. We stress that protocols carefully checking for environmental contamination are critically important in studies using samples with low bacterial biomass.
Long-term survival and induction of operational tolerance to murine islet allografts through the co-transplantation of cyclosporine A eluting microparticles
Authors: Kuppan Purushothaman , Wong Jordan , Kelly Sandra , Lin Jiaxin , Worton Jessica , Castro Chelsea , Paramor Joy , Seeberger Karen , Anderson Colin C. , Korbutt Gregory S. , Pepper Andrew R.
Published: February 2023

One strategy to prevent islet rejection, is to create a favorable immune-protective local environment at the transplant site. Herein, we utilize localized cyclosporine A (CsA) delivery to islet grafts via poly(lactic-co-glycolic acid) (PLGA) microparticles to attenuate allograft rejection. CsA microparticles alone significantly delayed islet allograft rejection compared to islets alone (p<0.05). Over 50% (6/11) of recipients receiving CsA microparticles and short-term cytotoxic T lymphocyte-associated antigen 4-Ig (CTLA4-Ig) therapy displayed prolonged allograft survival for 214 days, compared to 25% (2/8) receiving CTLA4-Ig alone (p>0.05). CsA microparticles + CTLA4-Ig islet allografts exhibited reduced T-cell (CD4+and CD8+cells) and macrophage (CD68+cells) infiltration compared to islets alone. We observed reduced mRNA expression of proinflammatory cytokines (IL-6, IL-10, INF-γ & TNF-α; p<0.05) and chemokines (CCL2, CCL5, CCL22, and CXCL10; p<0.05) in CsA microparticles + CTLA4-Ig allografts compared to islets alone. Long-term islet allografts contained insulin+and intra-graft FoxP3+T regulatory cells. Rapid rejection of third-party skin grafts (C3H) in islet allograft recipients suggested that CsA microparticles + CTLA4-Ig therapy induced donor specific operational tolerance. This study demonstrates that localized CsA drug delivery plus short-course systemic immunosuppression promotes an immune protective transplant niche for allogeneic islets.

Article Highlights

Systemic immunosuppression limits patient inclusion for beta cell replacement therapies

Localized islet graft immunosuppression may reduce drug toxicity and improve graft survival

Cyclosporine eluting microparticles + CTLA4-Ig therapy induced donor specific operational tolerance

Graft localized drug delivery can create an immune protective transplant niche
Human gene regulatory evolution is driven by the divergence of regulatory element function in bothcisandtrans
Authors: Hansen Tyler , Fong Sarah , Capra John A. , Hodges Emily
Published: February 2023

Gene regulatory divergence between species can result fromcis-acting local changes to regulatory element DNA sequences or globaltrans-acting changes to the regulatory environment. Understanding how these mechanisms drive regulatory evolution has been limited by challenges in identifyingtrans-acting changes. We present a comprehensive approach to directly identifycis-andtrans-divergent regulatory elements between human and rhesus macaque lymphoblastoid cells using ATAC-STARR-seq. In addition to thousands ofcischanges, we discover an unexpected number (~10,000) oftranschanges and show thatcisandtranselements exhibit distinct patterns of sequence divergence and function. We further identify differentially expressed transcription factors that underlie >50% oftransdifferences and trace howcischanges can produce cascades oftranschanges. Overall, we find that most divergent elements (67%) experienced changes in bothcisandtrans, revealing a substantial role fortransdivergence—alone and together withcischanges—to regulatory differences between species.
A Brighter picALuc Generated Through the Loss of a Salt Bridge Interaction
Authors: Biswas Kabir H
Published: February 2023

Recently, a miniaturized variant of an artificial luciferase (ALuc), named picALuc, with a molecular weight of 13 kDa and thus, the smallest luciferase, was reported. While picALuc was found to be as active as the ALuc, questions remained on the structural organization and residue-residue interactions in the protein. Here, combining structural modeling, molecular dynamics (MD) simulations and mutational analysis, we show that the loss of a salt bridge interaction formed by Glu50 (E50) residue results in an increased enzymatic activity of picALuc. Specifically, we generated a model of picALuc using the available structure of theGaussialuciferase (GLuc) and performed a 1 μs long Gaussian accelerated molecular dynamics (GaMD) simulation which revealed a general compaction of the protein structure as well as residue level interactions in the protein. Given that picALuc contains a number of charged residues, we focused our attention to salt bridge interactions and decided to mutate E10, E50 and D94 that were found to form a fluctuating, stable or a new salt bridge interaction, respectively. Live cell assays showed an enhanced bioluminescence in cells expressing the E50A mutant picALuc while in vitro assays revealed an increasedVmaxof the E50A mutant without affecting its thermal stability. Dynamic cross-correlation and principal component analyses of the GaMD simulation trajectories revealed altered collective dynamics in the protein, in which residue E50 contributed substantially. Finally, we developed a protein fragment complementation assay using picALuc that allows monitoring protein-protein interaction in live cells. We envisage that the brighter variant of picALuc and the protein fragment complementation assay reported here will find a general applicability in developing bioluminescence-based assays and the strategy developed here will pave the way for further engineering of brighter variants of picALuc.
Effects of a field-sprayed antibiotic on bee foraging behavior and pollination in pear orchards
Authors: Avila Laura , McCullough Christopher , Schiffer Annie , Moreno JoMari , Ganjur Neha , Ofenloch Zachary , DuPont Tianna , Nottingham Louis , Gerardo Nicole M. , Brosi Berry J.
Published: February 2023

Broadcast spraying of antibiotics in crops is widely used for controlling bacterial plant pathogens. The effects of antibiotics on non-target (and especially beneficial) organisms in cropping systems, however, are not well studied. Pollinators are of particular concern because in pear and apple crops, antibiotics for controlling fire blight (Erwinia amylovora) are sprayed during bloom, likely exposing pollinators. This is especially relevant as laboratory evidence suggests that antibiotics could have sublethal effects on bee foraging behavior and colony health. But to our knowledge these potential impacts have not been studied in field settings. Here, we compared the effects of two fire blight control methods, a single spray of an antibiotic (oxytetracycline) and a biological antagonist (Aureobasidium pullulans), on honey bee (Apis mellifera) foraging, pollination, and fruit set in pear orchards. Complementing these field assessments, we conducted laboratory experiments to examine the effects of these treatments on locomotion and foraging behavior of the bumble bee species,Bombus vosnesenskii. We found that honey bees visited fewer flowers and foraged longer on each flower in orchards sprayed with antibiotics than with biological product, but there were no differences in pollination and seed set. The pear cultivars we worked in, however, can self-pollinate. In the lab, we found that feeding on high doses of either the antibiotic or the biological antagonist reduced bumble bee foraging behavior relative to controls. The limited impact of antibiotics on pear pollination observed in this study suggest that antibiotics pose a low economic risk to pear growers, especially for self-compatible cultivars. Still, crops with higher pollinator dependence may be more affected by reductions in pollinator visitation. Future studies should examine the impacts of multiple antibiotic sprays within a season, which are common during warm springs, and their long-term health impacts on both individual bees and colonies.

Highlights

Antibiotics are sprayed on many crops to control plant bacterial pathogens.

The impacts of antibiotics on beneficial organisms in agriculture are unknown.

We studied antibiotic impacts on bee behavior and pollination function in pears.

Bees exposed to antibiotics visit fewer flowers and this could impact bee fitness.

Despite decreased bee visitation, we did not detect a reduction in crop pollination.

Graphical Abstract
The genome of the toxic invasive speciesHeracleum sosnowskyicarries an increased number of genes despite absence of recent whole-genome duplications
Authors: Schelkunov MI , Shtratnikova VYu , Klepikova AV , Makarenko MS , Omelchenko DO , Novikova LA , Obukhova EN , Bogdanov VP , Penin AA , Logacheva MD
Published: February 2023

Heracleum sosnowskyi, belonging to a group of giant hogweeds, is a plant with large effects on ecosystems and human health. It is an invasive species that contributes to the deterioration of grassland ecosystems. The ability ofH. sosnowskyito produce linear furanocoumarins (FCs), photosensitizing compounds, makes it very dangerous. At the same time, linear FCs are compounds with high pharmaceutical value that are used in skin disease therapies. Despite this high importance, it has not been the focus of genetic and genomic studies. Here, we report a chromosome-scale assembly of the Sosnowsky’s hogweed genome. Genomic analysis revealed an unusually high number of genes (55 206) in the hogweed genome, in contrast to the 25-35 thousand found in most plants. However, we did not find any traces of recent whole genome duplications not shared with its confamiliar,Daucus carota(carrot), which has approximately thirty thousand genes. The analysis of the genomic proximity of duplicated genes indicates tandem duplications as a main reason for this increase. We performed a genome-wide search of the genes of the FC biosynthesis pathway and their expression in aboveground plant parts. Using a combination of expression data and phylogenetic analysis, we found candidate genes for psoralen synthase and experimentally showed the activity of one of them using a heterologous yeast expression system. These findings expand our knowledge on the evolution of gene space in plants and lay a foundation for further analysis of hogweed as an invasive plant and as a source of FCs.
Sampling bias and the robustness of ecological metrics for plant–damage-type association networks: Comment
Authors: Schachat Sandra R.
Published: February 2023

Bipartite network metrics, which link taxa at two trophic levels, are notoriously biased when sampling is incomplete or uneven (Blüthgen et al., 2008; Dormann and Blüthgen, 2017; Fründ et al., 2016). Yet a new contribution (Swain et al., 2023, henceforth SEA) claims the opposite: that bipartite network metrics are minimally sensitive to incomplete sampling and, in fact, perform better at low sample sizes than traditional richness metrics. Here I show that SEA achieved this extraordinary finding by abandoning accepted practices, including practices from the authors’ previous papers.
Escherichia coliaggregates mediated by native or synthetic adhesins exhibit both core and adhesin-specific transcriptional responses
Authors: Chekli Yankel , Stevick Rebecca J. , Kornobis Etienne , Briolat Valérie , Ghigo Jean-Marc , Beloin Christophe
Published: February 2023

Bacteria can rapidly tune their physiology and metabolism to adapt to environmental fluctuations. In particular, they can adapt their lifestyle to the close proximity of other bacteria or presence of different surfaces. However, whether these interactions trigger transcriptomic responses is poorly understood. We used a specific set up ofE. colistrains expressing native or synthetic adhesins mediating bacterial aggregation to study the transcriptomic changes of aggregated compared to non-aggregated bacteria. Our results show that following aggregation, bacteria exhibit a core response independent of the adhesin type, with differential expression of 56.9% of the coding genome, including genes involved in stress response and anaerobic lifestyle. Moreover, when aggregates were formed via a naturally expressedE. coliadhesin (Antigen 43), the transcriptomic response of the bacteria was more exaggerated compared to aggregates formed via a synthetic adhesin. This suggests that the response to aggregation induced by nativeE. coliadhesins could have been finely tuned during bacterial evolution. Our study therefore provides insights on the effect of self-interaction in bacteria and allows a better understanding of why bacterial aggregates exhibit increased stress tolerance.

Importance

Formation of bacterial aggregates has an important role in both clinical and ecological contexts. Although these structures have been previously shown to be more resistant to stressful conditions, the genetic basis of this stress tolerance associated with the aggregate lifestyle is poorly understood. Surface sensing mediated by different adhesins can result in varying changes on bacterial physiology. However, whether adhesin-adhesin interactions as well as the type of adhesin mediating aggregation affects bacterial cell physiology is unknown. By sequencing the transcriptomes of aggregated and non-aggregated cells expressing native or synthetic adhesins, we characterized the effects of aggregation and adhesin type onE. coliphysiology.
Structural genomic variations and their effects on phenotypes inPopulus
Authors: Porth Ilga , Goessen Roos , Heinze Berthold
Published: February 2023

DNA copy numbers have recently emerged as an important new marker system. In the absence of a contiguous reference genome, alternative detection systems such as the comparative hybridization method have been used to detect copy number variations (CNVs). With the advent of chromosome-level resolved reference genomes based on the incorporation of long-read sequencing and powerful bioinformatics pipelines, comprehensive detection of all structural variations (SVs) in the poplar genome is now within reach. Gene CNVs and their inheritance are important because they can cause dosage effects in phenotypic variations. These are potent genetic markers that should be considered in complex trait variation such as growth and adaptation in poplar. SVs such as CNVs could be used in future genomic selection studies for poplar, especially in cases when heterosis increases hybrid performance (hybrid vigor). This Chapter reports recent findings on SVs in natural populations ofPopulusspp. as well as on artificially induced SVs in poplar to understand their potential importance in generating a considerable amount of phenotypic improvement. The Chapter concludes with an outlook on the future implementation of knowledge on SVs in poplar crop breeding.
CXCR6 promotes dermal CD8+T cell survival and transition to long-term tissue residence
Authors: Heim Taylor A. , Lin Ziyan , Steele Maria M. , Mudianto Tenny , Lund Amanda W.
Published: February 2023

Tissue resident memory T cells (TRM) provide important protection against infection, and yet the interstitial signals necessary for their formation and persistence remain incompletely understood. Here we show that antigen-dependent induction of the chemokine receptor, CXCR6, is a conserved requirement for TRMformation in peripheral tissue after viral infection. CXCR6 was dispensable for the early accumulation of antigen-specific CD8+T cells in skin and did not restrain their exit. Single cell sequencing indicated that CXCR6-/-CD8+T cells were also competent to acquire a transcriptional program of residence but exhibited deficiency in multiple pathways that converged on survival and metabolic signals necessary for memory. As such, CXCR6-/-CD8+T cells exhibited increased rates of apoptosis relative to controls in the dermis, leading to inefficient TRMformation. CXCR6 expression may therefore represent a common mechanism across peripheral non-lymphoid tissues and inflammatory states that increases the probability of long-term residence.

One Sentence Summary

CXCR6 promotes mechanisms of cellular adaptation to tissue infiltration that support local survival and the transition to tissue residence.
Epigenomic mapping in B-cell acute lymphoblastic leukemia identifies transcriptional regulators and noncoding variants promoting distinct chromatin architectures
Authors: Barnett Kelly R. , Mobley Robert J. , Diedrich Jonathan D. , Bergeron Brennan P. , Bhattarai Kashi Raj , Yang Wenjian , Crews Kristine R. , Manring Christopher S. , Jabbour Elias , Paietta Elisabeth , Litzow Mark R. , Kornblau Steven M. , Stock Wendy , Inaba Hiroto , Jeha Sima , Pui Ching-Hon , Mullighan Charles G. , Relling Mary V. , Yang Jun J. , Evans William E. , Savic Daniel
Published: February 2023

B-cell lineage acute lymphoblastic leukemia (B-ALL) is comprised of diverse molecular subtypes and while transcriptional and DNA methylation profiling of B-ALL subtypes has been extensively examined, the accompanying chromatin landscape is not well characterized for many subtypes. We therefore mapped chromatin accessibility using ATAC-seq for 10 B-ALL molecular subtypes in primary ALL cells from 154 patients. Comparisons with B-cell progenitors identified candidate B-ALL cell-of-origin and AP-1-associatedcis-regulatory rewiring in B-ALL.Cis-regulatory rewiring promoted B-ALL-specific gene regulatory networks impacting oncogenic signaling pathways that perturb normal B-cell development. We also identified that over 20% of B-ALL accessible chromatin sites exhibit strong subtype enrichment, with transcription factor (TF) footprint profiling identifying candidate TFs that maintain subtype-specific chromatin architectures. Over 9000 inherited genetic variants were further uncovered that contribute to variability in chromatin accessibility among individual patient samples. Overall, our data suggest that distinct chromatin architectures are driven by diverse TFs and inherited genetic variants which promote unique gene regulatory networks that contribute to transcriptional differences among B-ALL subtypes.

HIGHLIGHTS

Pro-B progenitor cells as the most common cell-of-origin for B-ALL

AP-1 TF-associatedcis-regulatory rewiring in B-ALL

Subtype-specific accessible chromatin signatures representing 20% of all B-ALL sites

Role for distinct TFs in promoting subtype-specific chromatin architectures

Thousands of inherited genetic variants identified impacting chromatin state
BorderlinerpoBmutations transmit at the same rate as commonrpoBmutations in a tuberculosis cohort in Bangladesh
Authors: Lempens Pauline , Van Deun Armand , Aung Kya J.M. , Hossain Mohammad A. , Behruznia Mahboobeh , Decroo Tom , Rigouts Leen , de Jong Bouke C. , Meehan Conor J.
Published: February 2023

The spread of multidrug-resistant tuberculosis (MDR-TB) is a growing problem in many countries worldwide. Resistance to one of the primary first-line drugs, rifampicin, is caused by mutations in theMycobacterium tuberculosis rpoBgene. While some of these infrequent mutations show lower fitnessin vitrothan more common mutations, theirin vivofitness is currently unknown.

We used a dataset of 394 whole genome sequenced MDR-TB isolates from Bangladesh, representing around 44% of notified MDR-TB cases over 6 years, to look at differences in transmission clustering between isolates with borderlinerpoBmutations and those with commonrpoBmutations. We found a relatively low percentage of transmission clustering in the dataset (34.8%) but no difference in clustering between different types ofrpoBmutations. Compensatory mutations inrpoA, rpoB, andrpoCwere associated with higher levels of transmission clustering as were lineages 2, 3, and 4 relative to lineage 1. Young people as well as patients with high sputum smear positive TB were more likely to be in a transmission cluster.

Our findings show that although borderlinerpoBmutations have lowerin vitrogrowth potential this does not translate into lower transmission potential orin vivofitness. Proper detection of these mutations is crucial to ensure they do not go unnoticed and spread MDR-TB within communities.

Data summary

WGS reads are available in the European Nucleotide Archive (PRJEB39569). In addition, WGS reads, as well as pDST and clinical data, are included in the ReSeqTB data platform and are accessible on registration athttps://platform.reseqtb.org/. Custom scripts for clustering are available athttps://github.com/conmeehan/pathophy.
Imaging the immune synapse: three-dimensional analysis of the immune synapse
Authors: Ruiz-Navarro Javier , Blázquez-Cucharero Sofía , Calvo Víctor , Izquierdo Manuel
Published: February 2023

T cell receptor (TCR) stimulation of T lymphocytes by antigen bound to the major histocompatibility complex (MHC) of an antigen-presenting cell (APC), together with the interaction of accessory molecules, induces the formation of the immunological synapse (IS), the convergence of secretion vesicles towards the centrosome, and the polarization of the centrosome to the IS. Upon IS formation, an initial increase in cortical filamentous actin (F-actin) at the IS takes place, followed by a decrease in F-actin density at the central region of the IS, which contains the secretory domain. These reversible, cortical actin cytoskeleton reorganization processes that characterize a mature IS occur during lytic granule secretion in cytotoxic T lymphocytes (CTL) and cytokine-containing vesicle secretion in T-helper (Th) lymphocytes. Besides, IS formation constitutes the basis of a signalling platform that integrates signals and coordinates molecular interactions that are necessary for an appropriate antigen-specific immune response. In this chapter we deal with the three-dimensional (3D) analysis of the synaptic interface architecture, as well as the analysis of the localization of different markers at the IS.
Metformin impairs trophoblast metabolism and differentiation in dose dependent manner
Authors: Nashif Sereen K. , Mahr Renee M. , Jena Snehalata , Jo Seokwon , Nelson Alisa B. , Sadowski Danielle , Crawford Peter A. , Puchalska Patrycja , Alejandro Emilyn U. , Gearhart Micah D. , Wernimont Sarah A.
Published: February 2023

Metformin is a widely prescribed medication whose mechanism of action is not completely defined and whose role in gestational diabetes management remains controversial. In addition to increasing risks of fetal growth abnormalities and preeclampsia, gestational diabetes is associated with abnormalities in placental development including impairments in trophoblast differentiation. Given that metformin impacts cellular differentiation events in other systems, we assessed metformin’s impact on trophoblast metabolism and differentiation. Using established cell culture models of trophoblast differentiation, oxygen consumption rates and relative metabolite abundance were determined following 200 μM (therapeutic range) and 2000 μM (supra-therapeutic range) metformin treatment using Seahorse and mass-spectrometry approaches. While no differences in oxygen consumption rates or relative metabolite abundance were detected between vehicle and 200 μM metformin treated cells, 2000 μM metformin impaired oxidative metabolism and increased abundance of lactate and TCA cycle intermediates, α-ketoglutarate, succinate, and malate. Examining differentiation, treatment with 2000 μM, but not 200 μM metformin, impaired HCG production and expression of multiple trophoblast differentiation markers. Overall, this work suggests that supra-therapeutic concentrations of metformin impairs trophoblast metabolism and differentiation whereas metformin concentrations in the therapeutic range do not strongly impact these processes.
CUR(E)ating a New Approach to Study Fungal Effectors and Enhance Undergraduate Education through Authentic Research
Authors: Li Gengtan , McWilliams Mai , Rodrigues Matheus , Mearkle Benjamin , Jaafar Nader , Golla Vivek , Yu Houlin , Yang He , Ayhan Dilay Hazal , Allen Kelly , Martínez-Soto Domingo , Springer Amy , Ma Li-Jun
Published: February 2023

Course-based Undergraduate Research Experiences (CUREs) integrate active, discovery-based learning into undergraduate curriculums, adding tremendous value to Biochemistry and Molecular Biology (BMB) education. There are multiple challenges in transforming a research project into a CURE, such as the readiness of students, the time commitment of the instructor, and the productivity of the research. In this article, we report a CURE course developed and offered in the University of Massachusetts Amherst BMB Department since 2018 that addresses these challenges. Our CURE focuses on fungal effectors which are proteins secreted by a destructive pathogenic fungusFusarium oxysporum, one of the top five most devastating plant pathogens. By studying this group of proteins, students are connected to real-world problems and participate in the search for potential solutions. A three-week “standard Bootcamp” is implemented to help students familiarize themselves with all basic techniques and boost their confidence. Next, molecular cloning, a versatile technique with modularity and repeatability, is used as the bedrock of the course. Our past five years of experience have confirmed that we have developed a novel and feasible CURE protocol. Measurable progress documented by students who took this course includes stimulated active learning and increased career trajectory to pursue hypothesis-based research to address societal needs. In addition, data generated through the course advance ongoing lab research. Collectively, we encourage the implementation of CURE among research-intensive faculty to provide a more inclusive research experience to all students, an important element in predicting career success.
Self-organizing actin networks drive sequential endocytic protein recruitment and vesicle release on synthetic lipid bilayers
Authors: Stoops Emily H. , Ferrin Michael A. , Jorgens Danielle M. , Drubin David G.
Published: February 2023

Forces generated by actin assembly assist membrane invagination during clathrin-mediated endocytosis (CME). The sequential recruitment of core endocytic proteins and regulatory proteins, and assembly of the actin network, are well documented in live cells and are highly conserved from yeasts to humans. However, understanding of CME protein self-organization, as well as the biochemical and mechanical principles that underlie actin’s role in CME, is lacking. Here, we show that supported lipid bilayers coated with purified yeast WASP, an endocytic actin assembly regulator, and incubated in cytoplasmic yeast extracts, recruit downstream endocytic proteins and assemble actin tails. Time-lapse imaging of WASP-coated bilayers revealed sequential recruitment of proteins from different endocytic modules, faithfully replicatingin vivobehavior. Reconstituted actin networks assemble in a WASP-dependent manner and deform lipid bilayers, as seen by electron microscopy. Time-lapse imaging revealed that vesicles are released from the lipid bilayers with a burst of actin assembly. Actin networks pushing on membranes have previously been reconstituted; here, we have reconstituted a biologically important variation of these actin networks that self-organize on bilayers and produce pulling forces sufficient to bud off membrane vesicles. We propose that actin-driven vesicle generation may represent an ancient evolutionary precursor to diverse vesicle forming processes adapted for a wide array of cellular environments and applications.

Significance Statement

Actin filament assembly participates in many vesicle-forming processes. However, the underlying principles for how assembly is initiated and organized to effectively harness assembly forces remain elusive. To address this gap, we report a novel reconstitution of actin-driven vesicle release from supported lipid bilayers. Using real-time imaging, we observe sequential recruitment of endocytic proteins and, following a burst of actin assembly, vesicle release from bilayers. Given the absence of cargo or upstream endocytic regulatory proteins on the bilayers, and the participation of actin in many vesicle-forming processes, we posit that this mode of vesicle formation represents an early evolutionary precursor for multiple trafficking pathways. We expect that this assay will be of great use for future investigations of actin-mediated vesicle-forming processes.
Distinct roles of α- and β-tubulin C-terminal tails for ciliary function as revealed by a CRISPR/Cas9 mediated gene editing inChlamydomonas
Authors: Kubo Tomohiro , Tani Yuma , Yanagisawa Haru-Aki , Kikkawa Masahide , Oda Toshiyuki
Published: February 2023

α- and β-tubulin have an unstructured glutamate-rich region at their C-terminal tails (CTT). The function of this region in cilia/flagella is still unclear, except that glutamates in CTT act as the sites for posttranslational modifications that affect ciliary motility. A unicellular algaChlamydomonaspossesses only two α-tubulin genes and two β-tubulin genes, each pair encoding an identical protein. This simple gene organization may enable a complete replacement of the wild-type tubulin with its mutated version. Here, using CRISPR/Cas9, we generated mutants expressing tubulins with modified CTTs. We found that the mutant whose four glutamate residues in the α-tubulin CTT have been replaced by alanine almost completely lacked polyglutamylated tubulin and displayed paralyzed cilia. In contrast, the mutant lacking the glutamate-rich region of the β-tubulin CTT assembled short cilia without the central apparatus. This phenotype is similar to the mutants harboring a mutation in a subunit of katanin, whose function has been shown to depend on the β-tubulin CTT. Therefore, our study reveals distinct and important roles of α- and β-tubulin CTT in the formation and function of cilia.

Summary statement

Chlamydomonasmutants were produced by CRISPR/Cas9 mediated gene editing to investigate ciliary function of tubulin C-terminal tails (CTTs). We found that α- and β-tubulin CTTs are essential for ciliary motility and assembly.
Astroglial exosome HepaCAM signaling and ApoE antagonization coordinates early postnatal cortical pyramidal neuronal axon growth and dendritic spine formation
Authors: Jin Shijie , Chen Xuan , Tian Yang , Jarvis Rachel , Promes Vanessa , Yang Yongjie
Published: February 2023

Developing astroglia play important roles in regulating synaptogenesis through secreted and contact signals. Whether they regulate postnatal axon growth is unknown. By selectively isolating exosomes using size-exclusion chromatography (SEC) and employing cell-type specific exosome reporter mice, our current results define a secreted astroglial exosome pathway that can spread long-rangein vivoand stimulate axon growth of cortical pyramidal neurons. Subsequent biochemical and genetic studies found that surface expression of glial HepaCAM protein essentially and sufficiently mediates the axon-stimulating effect of astroglial exosomes. Interestingly, apolipoprotein E (ApoE), a major astroglia-secreted cholesterol carrier to promote synaptogenesis, strongly inhibits the stimulatory effect of astroglial exosomes on axon growth. Developmental ApoE deficiency also significantly reduces spine density of cortical pyramidal neurons. Together, our study suggests a surface contact mechanism of astroglial exosomes in regulating axon growth and its antagonization by ApoE, which collectively coordinates early postnatal pyramidal neuronal axon growth and dendritic spine formation.
Physiological reprogrammingin vivomediated by Sox4 pioneer factor activity
Authors: Katsuda Takeshi , Sussman Jonathan , Ito Kenji , Katznelson Andrew , Yuan Salina , Li Jinyang , Merrell Allyson J. , Takenaka Naomi , Cure Hector , Li Qinglan , Rasool Reyaz Ur , Asangani Irfan A. , Zaret Kenneth S. , Stanger Ben Z.
Published: February 2023

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate “reprogramming” by opening new chromatin sites for expression that can attract transcription factors from the starting cell’s enhancers. Here we report that Sox4 is sufficient to initiate hepatobiliary metaplasia in the adult liver. In lineage-traced cells, we assessed the timing of Sox4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, Sox4 directly binds to and closes hepatocyte regulatory sequences via a motif it overlaps with Hnf4a, a hepatocyte master regulator. Subsequently, Sox4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals.
Whole-brain, gray and white matter time-locked functional signal changes with simple tasks and model-free analysis
Authors: Schilling Kurt G , Li Muwei , Rheault Francois , Gao Yurui , Cai Leon , Zhao Yu , Xu Lyuan , Ding Zhaohua , Anderson Adam W , Landman Bennett A , Gore John C
Published: February 2023

Recent studies have revealed the production of time-locked blood oxygenation-level dependent (BOLD) functional MRI (fMRI) signals throughout the entire brain in response to a task, challenging the idea of sparse and localized brain functions, and highlighting the pervasiveness of potential false negative fMRI findings. In these studies, ‘whole-brain’ refers to gray matter regions only, which is the only tissue traditionally studied with fMRI. However, recent reports have also demonstrated reliable detection and analyses of BOLD signals in white matter which have been largely ignored in previous reports. Here, using model-free analysis and simple tasks, we investigate BOLD signal changes in both white and gray matters. We aimed to evaluate whether white matter also displays time-locked BOLD signals across all structural pathways in response to a stimulus. We find that both white and gray matter show time-locked activations across the whole-brain, with a majority of both tissue types showing statistically significant signal changes for all task stimuli investigated. We observed a wide range of signal responses to tasks, with different regions showing very different BOLD signal changes to the same task. Moreover, we find that each region may display different BOLD responses to different stimuli. Overall, we present compelling evidence that the whole brain, including both white and gray matter, show time-locked activation to multiple stimuli, not only challenging the idea of sparse functional localization, but also the prevailing wisdom of treating white matter BOLD signals as artefacts to be removed.
Perinuclear damage from nuclear envelope deterioration elicits stress responses that contribute toLMNAcardiomyopathy
Authors: Sikder Kunal , Phillips Elizabeth , Zhong Zhijiu , Wang Nadan , Saunders Jasmine , Mothy David , Kossenkov Andrew , Schneider Timothy , Nichtova Zuzana , Csordas Gyorgy , Margulies Kenneth B. , Choi Jason C.
Published: February 2023

Mutations in theLMNAgene encoding nuclear lamins A/C cause a diverse array of tissue-selective diseases, with the heart being the most commonly affected organ. Despite progress in understanding the molecular perturbations emanating fromLMNAmutations, an integrative understanding of the pathogenesis leading to cardiac dysfunction remains elusive. Using a novel cell-type specificLmnadeletion mouse model capable of translatome profiling, we found that cardiomyocyte-specificLmnadeletion in adult mice led to rapid cardiomyopathy with pathological remodeling. Prior to the onset of cardiac dysfunction, lamin A/C-depleted cardiomyocytes displayed nuclear envelope deterioration, golgi dilation/fragmentation, and CREB3-mediated golgi stress activation. Translatome profiling identified upregulation of Med25, a transcriptional co-factor that can selectively dampen UPR axes. Autophagy is disrupted in the hearts of these mice, which can be recapitulated by disrupting the golgi or inducing nuclear damage by increased matrix stiffness. Systemic administration of pharmacological modulators of autophagy or ER stress significantly improved the cardiac function. These studies support a hypothesis wherein stress responses emanating from the perinuclear space contribute to the development ofLMNAcardiomyopathy.

Teaser

Interplay of stress responses underlying the development ofLMNAcardiomyopathy
SynEcoSys: a multifunctional platform of large-scale single-cell omics data analysis
Authors: Zhang Yan , Li Bingyu , Duan Jiachen , Chen Xuezhen , Zhang Xiaogang , Ye Jun , Veloso Ana , Fan Jue , Fang Nan
Published: February 2023

Next generation sequencing technologies enable the analysis of the transcriptomes of individual cells, providing a higher resolution of gene expression and function at the single cell level. Various single-cell data are continuously generated every year, covering fields from scientific research to clinical development. The fast-growing public datasets are collected by distinctive platforms, which are designed to facilitate biological discoveries, disease diagnosis, and new treatments. However, these platforms are hard to meet the urgency of having a unified data integration pipeline to improve comparability between datasets. Here we present SynEcoSys, an online multifunctional platform for single-cell transcriptomic data analysis, visualization, and exploration. SynEcoSys by Singleron Biotechnologies currently provides a massive collection of publicly available single-cell sequencing dataset, involving 46,326,175 cells from 731 datasets across multiple platforms and species. All datasets are generated with a strict and uniform data analysis pipeline and cell marker-based manual annotation, thus facilitating more comprehensive and reliable data mining. SynEcoSys is available at:https://www.synecosys.com.
Signalling pathway crosstalk stimulated by L-proline drives differentiation of mouse embryonic stem cells to primitive-ectoderm-like cells
Authors: Glover Hannah J. , Holliday Holly , Shparberg Rachel A. , Winkler David , Day Margot , Morris Michael B.
Published: February 2023

The amino acid L-proline exhibits novel growth factor-like properties during development - from improving blastocyst development to driving neurogenesisin vitro. Addition of 400 μM L-proline to self-renewal medium drives mouse embryonic stem cells (ESCs) to a transcriptionally distinct pluripotent cell population - early primitive ectoderm-like (EPL) cells - which lies between the naïve and primed states. EPL cells retain expression of pluripotency genes, upregulate primitive ectoderm markers, undergo a morphological change and have increased cell number.

These changes are facilitated by a complex signalling network hinging on the Mapk, Fgfr, Pi3k and mTor pathways. We use a factorial experimental design coupled with linear modelling and Bayesian regularised neural networks to understand which signalling pathways are involved in the transition between ESCs and EPL cells, and how they underpin changes in morphology, cell number, apoptosis, proliferation and gene expression. This approach allows for consideration of where pathways work antagonistically or synergistically.

Modelling showed that most properties were affected by more than one inhibitor, and each inhibitor blocked specific aspects of differentiation. These mechanisms underpin both progression of stem cells across thein vitropluripotency continuum and serve as a model for pre-, peri- and post-implantation embryogenesis.

Summary Statement

L-proline acts as growth factor to modulate phosphorylation of the Mapk, Pi3k, Fgf and mTor signalling pathways to drive embryonic stem cells to primitive ectoderm-like cells.
Deep Learning Reveals Endogenous Sterols as Allosteric Modulators of the GPCR-Gα Interface
Authors: Mohanty Sanjay Kumar , Mittal Aayushi , Namra  , Gaur Aakash , Duari Subhadeep , Solanki Saveena , Sharma Anmol Kumar , Arora Sakshi , Kumar Suvendu , Gautam Vishakha , Dixit Nilesh Kumar , Subramanian Karthika , Ghosh Tarini Shankar , Sengupta Debarka , Gupta Shashi Kumar , Murugan Natarajan Arul , Sharma Deepak , Ahuja Gaurav
Published: February 2023

Endogenous intracellular allosteric modulators of GPCRs remain largely unexplored, with limited binding and phenotype data available. This gap arises from the lack of robust computational methods for unbiased cavity identification, cavity-specific ligand design, synthesis, and validation across GPCR topology. Here, we developed Gcoupler, an AI-driven generalized computational toolkit that leverages an integrative approach combiningde novoligand design, statistical methods, Graph Neural Networks, and Bioactivity-based ligand prioritization for rationally predicting high-affinity ligands. Using Gcoupler, we interrogated intracellular metabolites that target and regulate the GPCR-Gα interface (Ste2p-Gpa1p), affecting pheromone-induced programmed cell death in yeast. Our computational analysis, complemented by experimental validations, including genetic screening, multi-omics, site-directed mutagenesis, biochemical assays, and physiological readouts, identified endogenous hydrophobic metabolites, notably sterols, as direct intracellular allosteric modulators of Ste2p. Molecular simulations coupled with biochemical signaling assessment in site-directed Ste2p mutants further confirmed metabolites binding to GPCR-Gα obstruct downstream signaling, possibly via cohesive effect. Finally, by utilizing isoproterenol-induced, GPCR-mediated human and neonatal rat cardiac hypertrophy models, we observed elevated metabolite levels attenuate hypertrophic response, reinforcing the evolutionary relevance of this mechanism.
In search of the Goldilocks zone for hybrid speciation II: hard times for hybrid speciation?
Authors: Blanckaert Alexandre , Sriram Vedanth , Bank Claudia
Published: February 2023

Hybridization opens a unique window for observing speciation mechanisms and is a potential engine of speciation. One controversially discussed outcome of hybridization is homoploid hybrid speciation by reciprocal sorting, where a hybrid population maintains a mixed combination of the parental genetic incompatibilities, preventing further gene exchange between the newly formed population and the two parental sources. Previous work showed that, for specific linkage architectures (i.e., the genomic location and order of hybrid incompatibilities), reciprocal sorting could reliably result in hybrid speciation. Yet, the sorting of incompatibilities creates a risk of population extinction. To understand how demographic consequences of the purging of incompatibilities interact with the formation of a hybrid species, we model an isolated hybrid population resulting from a single admixture event. We study how population size, linkage architecture and the strength of the incompatibility affect survival of the hybrid population, resolution/purging of the genetic incompatibilities and the probability of observing hybrid speciation. We demonstrate that the extinction risk is highest for intermediately strong hybrid incompatibilities. In addition, the linkage architecture displaying the highest hybrid speciation probabilities changes drastically with population size. Overall, this indicates that population dynamics can strongly affect the outcome of hybridization and the hybrid speciation probability.
Dibromomethyl- and bromomethyl- or bromo-substituted benzenes and naphthalenes: C—Br...Br interactions
Authors: Kuś Piotr , Jones Peter G. , Kusz Joachim , Książek Maria
Published: February 2023

The structures of six benzene and three naphthalene derivatives involving bromo, bromomethyl and dibromomethyl substituents, namely, 1,3-dibromo-5-(dibromomethyl)benzene, C7H4Br4, 1,4-dibromo-2,5-bis(bromomethyl)benzene, C8H4Br6, 1,4-dibromo-2-(dibromomethyl)benzene, C7H4Br4, 1,2-bis(dibromomethyl)benzene, C8H6Br4, 1-(bromomethyl)-2-(dibromomethyl)benzene, C8H7Br3, 2-(bromomethyl)-3-(dibromomethyl)naphthalene, C12H9Br3, 2,3-bis(dibromomethyl)naphthalene, C12H8Br4, 1-(bromomethyl)-2-(dibromomethyl)naphthalene, C12H9Br3, and 1,3-bis(dibromomethyl)benzene, C8H6Br4, are presented. The packing patterns of these compounds are dominated by Br...Br contacts and C—H...Br hydrogen bonds. The Br...Br contacts, shorter than twice the van der Waals radius of bromine (3.7 Å), seem to play a crucial role in the crystal packing of all these compounds. The occurrence of Type I and Type II interactions is also discussed briefly, considering the effective atomic radius of bromine, as is their impact on the packing of molecules in the individual structures.