bluuebunny/crossref_metadata_2025_split huggingface dataset:
Large donor CRISPR for whole-CDS replacement of cell adhesion molecule LRRTM2
Authors: Pollitt Stephanie L , Levy Aaron D , Anderson Michael C , Blanpied Thomas A 
Published: May 2024

AbstractThe cell adhesion molecule LRRTM2 is crucial for synapse development and function. However, our understanding of its endogenous trafficking has been limited due to difficulties in manipulating its coding sequence (CDS) using standard genome editing techniques. We instead replaced the whole LRRTM2 CDS by adapting the recent CRISPR method Targeted Knock-in using Two Guides (TKIT), enabling complete control of LRRTM2. In primary rat hippocampal cultures, N-terminally tagged, endogenous LRRTM2 was found in 80% of synapses, and synaptic LRRTM2 content correlated with PSD-95 and AMPAR levels. LRRTM2 was also enriched with AMPARs outside synapses, demonstrating the sensitivity of this method to detect relevant new biology. Finally, we leveraged total genomic control to increase the synaptic levels of LRRTM2 via simultaneous mutation of its C-terminal domain, which did not correspondingly increase AMPAR enrichment. The coding region of thousands of genes span lengths suitable for whole-CDS replacement, suggesting this simple approach will enable straightforward structure-function analysis in diverse cellular systems.
Induced systemic resistance by the root colonization ofTrichoderma atrovirideis independent from the chitin-mediated signaling pathway in Arabidopsis
Authors: Sakai Ayae , Yamagata Hisako , Naito Keigo , Tominaga Takaya , Ifuku Shinsuke , Kaminaka Hironori 
Published: May 2024

AbstractBeneficial root endophytic fungi induce systemic responses, growth promotion, and induced systemic resistance (ISR) in the colonized host plants. Soil application of chitin, a main component of fungal cell walls, also systemically induces disease resistance. Thus, chitin recognition and its downstream signaling pathway are supposed to mediate ISR triggered by beneficial fungi colonizing the root. This study compared systemic disease resistance and transcriptional responses induced byTrichoderma, a representative beneficial root endophytic fungus, and chitin in Arabidopsis. A significant plant growth promotion was observed under root colonization by the three tested beneficial fungi,Trichoderma atroviride,Serendipita indica, andS. vermifera. Still, onlyT. atrovirideandS. indicatriggered ISR against the necrotrophic fungal pathogenAlternaria brassicicola. Induced systemic resistance triggered byT. atroviridewas compromised in the chitin-receptor mutant, while systemic resistance caused by soil application of chitin was not. Transcriptome analysis demonstrated that the chitin-regulated genes are mostly shared with those regulated byT. atroviride, but many of the latter were specific. However, the commonly enriched gene ontologies for those regulated genes indicated thatT. atrovirideinoculation and chitin application systemically control similar transcriptional responses, mainly associated with cell wall functions. Taken together,Trichodermacould trigger ISR primarily independently from the chitin-mediated signaling pathway; however, chitin andTrichodermawould systemically induce similar cellular functions in ISR aboveground.
Label-free Assessment of Complement-Dependent Cytotoxicity of Therapeutic Antibodies via a Whole-Cell MALDI Mass Spectrometry Bioassay
Authors: Schmidt Stefan , Geisel Alexander , Enzlein Thomas , Fröhlich Björn C. , Pritchett Louise , Verneret Melanie , Graf Christian , Hopf Carsten 
Published: May 2024

AbstractPotency assessment of monoclonal antibodies or corresponding biosimilars in cell-based assays is an essential prerequisite in biopharmaceutical research and development. However, cellular bioassays are still subject to limitations in sample throughput, speed, and often need costly reagents or labels as they are based on an indirect readout by luminescence or fluorescence. In contrast, whole-cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) has emerged as a direct, fast and label-free technology for functional drug screening being able to unravel the molecular complexity of cellular response to pharmaceutical reagents. However, this approach has not yet been used for cellular testing of biologicals. In this study, we have conceived, developed and bench-marked a label-free MALDI-MS based cell bioassay workflow for the functional assessment of complement-dependent cytotoxicity (CDC) of Rituximab antibody. By computational evaluation of response profiles followed by subsequentm/zfeature annotation via fragmentation analysis and trapped ion mobility MS, we identified adenosine triphosphate and glutathione as readily MS-assessable metabolite markers for CDC and demonstrate that robust concentration-response characteristics can be obtained by MALDI-TOF MS. Statistical assay performance indicators suggest that whole-cell MALDI-TOF MS could complement the toolbox for functional cellular testing of biopharmaceuticals.
Paralogue-selective degradation of the lysine acetyltransferase EP300
Authors: Chen Xuemin , Crawford McKenna C. , Xiong Ying , Shaik Anver Basha , Suazo Kiall F. , Penkalapati Manini S. , Williams Joycelyn H. , Andressen Thorkell , Swenson Rolf E. , Meier Jordan L. 
Published: May 2024

AbstractThe transcriptional coactivators EP300 and CREBBP are critical regulators of gene expression that share high sequence identity but exhibit non-redundant functions in basal and pathological contexts. Here, we report the development of a bifunctional small molecule, MC-1, capable of selectively degrading EP300 over CREBBP. Using a potent aminopyridine-based inhibitor of the EP300/CREBBP catalytic domain in combination with a VHL ligand, we demonstrate that MC-1 preferentially degrades EP300 in a proteasome-dependent manner. Mechanistic studies reveal that selective degradation cannot be predicted solely by target engagement or ternary complex formation, suggesting additional factors govern paralogue-specific degradation. MC-1 inhibits cell proliferation in a subset of cancer cell lines and provides a new tool to investigate the non-catalytic functions of EP300 and CREBBP. Our findings expand the repertoire of EP300/CREBBP-targeting chemical probes and offer insights into the determinants of selective degradation of highly homologous proteins.
A heterogeneous population code at the first synapse of vision
Authors: Herzog Tessa , Yoshimatsu Takeshi , Moya-Diaz Jose , James Ben , Lagnado Leon , Baden Tom 
Published: May 2024

SUMMARYVision begins when photoreceptors convert light fluctuations into temporal patterns of glutamate release that drive the retinal network. The input-output relation at this first stage has not been systematically measuredin vivoso it is not known how it operates across a photoreceptor population. Using kHz-rate glutamate imaging in zebrafish, we find that individual red cones encode visual stimuli with high reliability and time-precision, but routinely vary in sensitivity to luminance, contrast, and frequency across the population. Variations in input-output relations are generated by feedback from the horizontal cell network that effectively decorrelate feature representation. A model capturing how zebrafish sample their visual environment indicates that this heterogeneity expands the dynamic range of the retina to improve the coding of natural scenes. Moreover, we find that different kinetic release components are used to encode distinct stimulus features in parallel: sustained release linearly encodes low amplitude light and dark contrasts, but transient release encodes large amplitude dark contrasts. Together, this study reveals an unprecedented degree of functional heterogeneity within same-type photoreceptors and illustrates how separation of different visual features begins in the first synapse in vision.
A temperature-sensitive and interferon-silent Sendai virus vector for CRISPR-Cas9 delivery and gene editing in primary human cells
Authors: Stevens Christian S , Carmichael Jillian , Watkinson Ruth , Kowdle Shreyas , Reis Rebecca A , Hamane Kory , Jang Jason , Park Arnold , Pernet Olivier , Khamaikawin Wannisa , Hong Patrick , Thibault Patricia , Gowlikar Aditya , An Dong Sung , Lee Benhur 
Published: May 2024

ABSTRACTThe transformative potential of gene editing technologies hinges on the development of safe and effective delivery methods. In this study, we developed a temperature-sensitive and interferon-silent Sendai virus (ts SeV) as a novel delivery vector for CRISPR-Cas9 and for efficient gene editing in sensitive human cell types without inducing IFN responses. ts SeV demonstrates unprecedented transduction efficiency in human CD34+ hematopoietic stem and progenitor cells (HSPCs) including transduction of the CD34+/CD38-/CD45RA-/CD90+(Thy1+)/CD49fhighstem cell enriched subpopulation. The frequency ofCCR5editing exceeded 90% and bi-allelicCCR5editing exceeded 70% resulting in significant inhibition of HIV-1 infection in primary human CD14+ monocytes. These results demonstrate the potential of the ts SeV platform as a safe, efficient, and flexible addition to the current gene-editing tool delivery methods, which may help to further expand the possibilities in personalized medicine and the treatment of genetic disorders.
The Ccm3-GckIII signaling axis regulates Rab11-dependent recycling to the apical compartment
Authors: Burguete Alondra S. , Song Yanjun , Ghabrial Amin S. 
Published: May 2024

SummaryKinase cascades underlie many signaling pathways and are key regulators of development and morphogenesis. We have characterized a Hippo-like kinase cascade consisting of Thousand and One kinase (Tao), Germinal Center Kinase III (GckIII/Wheezy), and Tricornered (Trc) that plays an essential role in morphogenesis of tracheal terminal cell tubes inDrosophila. In this cascade, GckIII is the central kinase and is thought to act together with its binding partner, Cerebral Cavernous Malformations 3 (Ccm3). As suggested by its name,Drosophila Ccm3is the ortholog of a human vascular disease gene. As such, defining the Ccm3 pathway is critical to understanding both normal development and disease. Here we generate and characterize a null allele ofCcm3inDrosophila. We uncover a maternal contribution ofCcm3to embryonic development, show that maternal/zygotic null embryos have defective multicellular tracheal tubes, and that tracheal terminal cells derived from zygotic clones that also lack maternalCcm3, show fully penetrant tube dilation defects identical to those we previously described for other pathway genes. We show that wildtype Ccm3 colocalizes with p-GckIII during early embryogenesis, and that in larval terminal cells, is found in the nucleus as well as associated with the apical membrane. We further demonstrate thatMouse protein 25 (Mo25),known to encode a protein that binds and stabilizes GckIII proteins in the active conformation, is required to preventCcm3-like tube dilations, showing that Mo25 and Ccm3, together with Tao, are required to fully activate GckIII, which directly phosphorylates and activates Tricornered (Trc). We show that this Ccm3 signaling cassette operates in other epithelial tissues such as the wing, and in non-epithelial tissues such as motor neurons. Lastly, we define a role of Ccm3-GckIII signaling in the distribution of active Rab11, leading us to propose that persistent local Rab11 activity results in elevated recycling of membrane and apical determinants to the apical domain, and consequent dilation of tubes. We validate this model by showing that loss of Rab11 activity ameliorates the tube dilation defects of pathway mutants.
Elemental profiling and genome-wide association mapping reveal genomic variants modulating ionomic composition inPopulus trichocarpaleaves
Authors: Ployet Raphael , Feng Kai , Zhang Jin , Baxter Ivan , Glasgow David C. , Chen Jin-Gui , Tuskan Gerald A. , Tschaplinski Timothy J. , Weston David J. , Martin Madhavi Z. , Muchero Wellington 
Published: May 2024

AbstractThe ionome represents elemental composition in plant tissues and can be an indicator of nutrient status as well as overall plant performance. Thus, identifying genetic determinants governing elemental uptake and storage is an important goal in plant breeding and engineering. In this study, we coupled high-throughput ionome characterization with high-resolution genome-wide association studies (GWAS) to uncover genetic loci that modulate ionomic composition in leaves of 584 black cottonwood poplar (Populus trichocarpa) genotypes. Congruence of alternate ionomic profiling platforms, i.e., inductively coupled plasma-mass spectrometry (ICP-MS), neutron activation analysis (NAA) and laser-induced breakdown spectroscopy (LIBS), was performed on leaf samples from a subset of the population. Significant agreement was observed across the three platforms with some notable exceptions for individual elements. Subsequently, we used the ICP-MS platform to profile the 584 genotypes focusing on 20 elements. GWAS performed using a set of high-density (>8.2 million) single nucleotide polymorphisms (SNP), identified multiple loci significantly associated with variations in these mineral elements. The potential causal genes for variations in the ionome were significantly enriched in genes whose homologs were previously associated to ion homeostasis in other species. Notably, a polymorphic copy of the high-affinity molybdenum transporter MOT1 was found directly associated to molybdenum content in leaf tissues. The results of the GWAS also provided evidence of physiological and genetic interactions between mineral elements in poplar. The new candidate genes predicted to play a key role in cross-homeostasis of multiple elements are new targets for engineering a variety of traits of interest in tree species.
Novel PotentialCannabisPathogen: Discovery of Tobacco necrosis virus A in a Diseased ColombianCannabis sativaPlant
Authors: Lopez-Jimenez Juliana , Palacio-Torres Herman D. , Alzate Juan F. 
Published: May 2024

ABSTRACTPlant viral infections pose a significant threat to global crop productivity. Despite their profound impact on agriculture, plant viruses have been relatively understudied, primarily due to technological limitations associated with classical molecular methods. However, the advent of NGS RNA-seq analysis has revolutionized virus characterization in environmental settings, overcoming previous limitations and providing a powerful tool for studying plant viruses.In an RNA-seq experiment conducted on a diseased ColombianCannabis sativahemp plant, we identified a linear single-stranded RNA (ssRNA) genome belonging to Tobacco Necrosis Virus A (TNVA), a common cause of necrotic lesions in plants such as tobacco and tulipa. The affectedCannabis sativahemp plant exhibited severe symptoms, including alterations in pigmentation, leaf morphology such as chlorosis, necrotic tissue formation, and surface wear on the leaves. The complete genome sequence of theCannabis sativaTNVA was 3,656 nucleotides long, containing five putative ORFs, and was classified in the family Tombusviridae, genus Alphanecrovirus, and belonging to the Necro-like clade based on RdRp protein phylogenetic analysis. Our analysis revealed a well-conserved RdRp protein among the Alphanecroviruses, with 89% of the amino acid residues in the peptide being entirely conserved. In contrast, the coat protein exhibited significantly higher variability, with only 49.3% of the residues being 100% conserved. Regarding the viral genome expression ofCannabis sativaTNVA, we observed that the virus was highly abundant in the leaves of the diseased plant, ranking among the topmost abundant transcripts, occupying the percentile position of 3.06%. Overall, our study generated the first reference genome of TNVA virus in the tropical region and reported the first case of this virus infecting aCannabis sativaplant.
Controlling the frequency dynamics of homing gene drives for intermediate outcomes
Authors: Camm Benjamin J. , Fournier-Level Alexandre 
Published: May 2024

AbstractGene drives have enormous potential for solving biological issues by forcing the spread of desired alleles through populations. However, to safeguard from the potentially irreversible consequences on natural populations, gene drives with intermediate outcomes that neither fixate nor get removed from the population are of outstanding interest.To elucidate the conditions leading to intermediate gene drive frequency, a stochastic, individual allele-focused gene drive model accessible was developed to simulate the diffusion of a homing gene drive in a population. The frequencies of multiple alleles at a locus targeted by a gene drive were tracked under various scenarios. These explored the effect of gene drive conversion efficiency, strength and frequency of resistance alleles, presence and strength of a fitness cost for the gene drive, its dominance and the level of inbreeding.Four outcomes were consistently observed: Fixation, Loss, Temporary and Equilibrium. The latter two are defined by the frequency of the gene drive peaking then crashing or plateauing, respectively. No single variable determined the outcome of a drive, instead requiring a combination of variables. The difference between the conversion efficiency and resistance level differentiated the Temporary and Equilibrium outcomes. The frequency dynamics of the gene drive within outcomes varied extensively, with different variables driving this dynamics between outcomes.These simulation results highlight the possibility of fine-tuning gene drive outcomes and compensating through biotechnological design constraint imposed by population features. To that end, we provide a web application implementing our model which will guide the safer design of gene drives able to achieve a range of controllable outcome tailored to population management needs.
Changes in Cerebrospinal Fluid Proteins across the Spectrum of Untreated and Treated Chronic HIV-1 Infection
Authors: Hu Zicheng , Cinque Paola , Dravid Ameet , Hagberg Lars , Yilmaz Aylin , Zetterberg Henrik , Fuchs Dietmar , Gostner Johanna , Blennow Kaj , Spudich Serena S. , Kincer Laura , Zhou Shuntai , Joseph Sarah , Swanstrom Ronald , Price Richard W. , Gisslén Magnus 
Published: May 2024

ABSTRACTUsing theOlink Explore 1536platform, we measured 1,463 unique proteins in 303 cerebrospinal fluid (CSF) specimens from four clinical centers that included uninfected controls and 12 groups of people living with HIV-1 infection representing the spectrum of progressive untreated and treated chronic infection.We present three initial analyses of these measurements: an overview of the CSF protein features of the sample; correlations of the CSF proteins with CSF HIV-1 RNA and neurofilament light chain protein (NfL) concentrations; and comparison of the CSF proteins in HIV-associated dementia (HAD) and neurosymptomatic CSF escape (NSE). These reveal a complex but coherent picture of CSF protein changes that includes highest concentrations of many proteins during CNS injury in theHADandNSEgroups and variable protein changes across the course of neuroasymptomatic systemic HIV-1 progression, including two common patterns, designated aslymphoidandmyeloidpatterns, related to the principal involvement of their underlying inflammatory cell lineages. Antiretroviral therapy reduced CSF protein perturbations, though not always to control levels.The dataset of these CSF protein measurements, along with background clinical information, is posted online. Extended studies of this unique dataset will provide more detailed characterization of the dynamic impact of HIV-1 infection on the CSF proteome across the spectrum of HIV-1 infection, and further the mechanistic understanding of HIV-1-related CNS pathobiology.
Synchronization between chloroplastic and cytosolic protein synthesis for photosynthesis complex assembly
Authors: Fujita Tomoya , Wakigawa Taisei , Kawamoto Naohiro , Kurihara Yukio , Hirose Yuu , Hirayama Takashi , Toh Hirotaka , Kuriyama Tomoko , Hashimoto Atsushi , Matsuura-Suzuki Eriko , Mochida Keiichi , Yoshida Minoru , Matsui Minami , Iwasaki Shintaro 
Published: May 2024

SummaryThrough symbiosis, subunits of chloroplastic complexes are encoded in distinct genomes in the nucleus and organelles. For plant cells to maintain the stoichiometry of subunits and respond to environmental cues, orchestration of the nuclear and organellar gene expression systems is an essential task. However, the mechanism maintaining chloroplastic complexes remains largely enigmatic. Here, we simultaneously assessed the translatomes of the chloroplast and the cytoplasm via ribosome profiling and revealed the differential mechanisms employed by these two systems to cope with acute light/dark transitions: in chloroplasts, translational regulation is employed, whereas in the cytoplasm, control of the mRNA abundance is employed. This strategy is widely conserved in land plants (Arabidopsisand the grass plantBrachypodium) and green algae (Chlamydomonas). The translational control in chloroplasts may be established on the basis of organelle symbiosis; the primitive chloroplast in Glaucophyta (Cyanophora) was found to have already acquired translational control, whereas cyanobacteria (Synechocystis) employ control of the mRNA abundance. Moreover, treatment with photosynthetic electron transport inhibitors indicated that reduced plastoquinones may trigger translational activation in chloroplasts. Our work reveals an early origin of coordination of chloroplast and nuclear/cytoplasmic gene expression upon light exposure.
Docking interactions determine substrate specificity of members of a widespread family of protein phosphatases
Authors: Caban-Penix Suhaily , Ho Kristin , Yang Wendy , Baral Rishika , Bradshaw Niels 
Published: May 2024

AbstractHow protein phosphatases achieve specificity for their substrates is a major outstanding question. PPM family serine/threonine phosphatases are widespread in bacteria and eukaryotes, where they dephosphorylate target proteins with a high degree of specificity. In bacteria, PPM phosphatases control diverse transcriptional responses by dephosphorylating anti-anti-sigma factors of the STAS domain family, exemplified byB. subtilisphosphatases SpoIIE, which controls cell-fate during endospore formation, and RsbU, which initiates the General Stress Response. Using a combination of forward genetics, biochemical reconstitution, and AlphaFold2 structure prediction, we identified a conserved, tripartite substrate docking interface comprised of three variable loops on the surface of the PPM phosphatase domain that recognize the three-dimensional structure of the substrate protein. Non-conserved amino acids in these loops facilitate the accommodation of the cognate substrate and prevent dephosphorylation of the non-cognate substrate. Together, single-amino acid substitutions in these three elements cause an over five-hundred fold change in specificity. Our data additionally suggest that substrate-docking interactions regulate phosphatase specificity through a conserved allosteric switch element that controls the catalytic efficiency of the phosphatase by positioning the metal cofactor and substrate. We hypothesize that this is a generalizable mechanistic model for PPM family phosphatase substrate specificity. Importantly, the substrate docking interface with the phosphatase is only partially overlapping with the much more extensive interface with the upstream kinase, suggesting the possibility that kinase and phosphatase specificity evolved independently.