bluuebunny/crossref_metadata_2025_split huggingface dataset:
Machine learning-guided reconstruction of cytoskeleton network from Live-cell AFM Images
Authors: Ju Hanqiu , Skibbe Henrik , Fukui Masaya , Yoshimura Shige H. , Naoki Honda 
Published: March 2024

AbstractHow actin filaments (F-actins) are dynamically reorganized in motile cells at the level of individual filaments is an open question. To this end, we developed a high-speed atomic force microscopy (HS-AFM) to live-imagine intracellular dynamics of the individual F-actins. However, noise and low resolution made it difficult to fully recognize individual F-actins in the HS-AFM images. To tackle this problem, we developed a new machine learning method that quantitatively recognizes individual F-actins. The method estimates F-actin orientation from the image while improving the resolution. We found that F-actins were oriented at ±35° toward the membrane in the lamellipodia, which is consistent with Arp2/3 complex-induced branching. Furthermore, in the cell cortex our results showed non-random orientation at four specific angles, suggesting a new mechanism for F-actin organization demonstrating the potential of our newly developed method to fundamentally improve our understanding of the structural dynamics of F-actin networks.
34-parameter full spectrum immunophenotyping panel of human regulatory and effector lymphocytes
Authors: Georgieva Zoya , Coppard Valerie , Yang Jennie HM , Grenfell Richard , Jones Joanne 
Published: March 2024

AbstractThis 34-marker sentinel, intracellular, full-spectrum flow cytometry panel profiles regulatory and effector T, B and NK lymphocytes in human cryopreserved peripheral blood mononuclear cells. The panel focuses on cell trafficking, activation, exhaustion and proliferation, and permits easy customisation in two positions to accommodate other targets of the user’s interest. By combining breadth and depth of phenotyping, this panel is designed to maximise the information obtained from limited cell material and therefore will be particularly useful in mechanistic studies of immunomodulatory drugs for autoimmune disease, cancer and transplantation, where multiple immune populations may be affected.
Mx1-labeled pulp progenitor cells are main contributors to postnatal odontoblasts and pulp cells in murine molars
Authors: Yang Dongwook , Jeong YoungJae , Ortinau Laura , Solidum Jea , Park Dongsu 
Published: March 2024

SUMMARYRegeneration of dentin and odontoblasts from dental pulp stem cells (DPSCs) is essential for permanent tooth maintenance. However, thein vivoidentity and role of endogenous DPSCs in reparative dentinogenesis are elusive. Here, using pulp single-cell analysis before and after molar eruption, we revealed that endogenous DPSCs are enriched inCxcl12-GFP+coronal papilla-like cells withMx1-Cre labeling. TheseMx1+Cxcl12-GFP+cells are long-term repopulating cells that contribute to the majority of pulp cells and new odontoblasts after eruption. Upon molar injury,Mx1+DPSCs localize into the injury site and differentiate into new odontoblasts, formingscleraxis-GFP+andosteocalcin-GFP+dentinal tubules and reparative dentin. Single-cell and FACS analysis showed thatMx1+Cxcl12-GFP+DPSCs are the most primitive cells with stem cell marker expression and odontoblast differentiation. Taken together, our findings demonstrate thatMx1labels postnatal DSPCs, which are the main source of pulp cells and new odontoblasts with reparative dentinogenesisin vivo.
Hierarchal single-cell lineage tracing reveals differential fate commitment of CD8 T-cell clones in response to acute infection
Authors: Abdullah Leena , Emiliani Francesco E. , Vaidya Chinmay M. , Stuart Hannah , Kolling Fred W. , Ackerman Margaret E. , Song Li , McKenna Aaron , Huang Yina H. 
Published: March 2024

SummaryGenerating balanced populations of CD8 effector and memory T cells is necessary for immediate and durable immunity to infections and cancer. Yet, a definitive understanding of CD8 differentiation remains unclear. We used CARLIN, a processive lineage recording mouse model with single-cell RNA-seq and TCR-seq to track endogenous antigen-specific CD8 T cells during acute viral infection. We identified a diverse repertoire of expanded T-cell clones represented by seven transcriptional states. TCR enrichment analysis revealed differential memory- or effector-fate biases within clonal populations. Shared Vb segments and amino acid motifs were found within biased categories despite high TCR diversity. Using single-cell CARLIN barcode-seq we tracked multi-generational clones and found that unlike unbiased or memory-biased clones, which stably retain their fate profiles, effector-biased clones could adopt memory- or effector-bias within subclones. Collectively, our study demonstrates that a heterogenous T-cell repertoire specific for a shared antigen is composed of clones with distinct TCR-intrinsic fate-biases.
Ribosome biogenesis factor AtRRB1 confers pollen heat stress tolerance in Arabidopsis
Authors: Jin Chunlian , Schindfessel Cédric , Sun Limin , Keçeli Burcu Nur , Vanneste Steffen , Geelen Danny 
Published: March 2024

AbstractThe plant male reproductive system is very sensitive to high temperature stress leading to a reduction in fertility. Damage caused by heat stress is restored by the activation of transcription and the synthesis of chaperones that regulate the heat stress response. Here we report that AtRRB1 is a homolog of the yeast ribosome chaperone protein Rrb1p.AtRRB1is an essential gene and a T-DNA insertion in the coding region impairs male and female gametogenesis. The heterozygousrrb1-1mutant shows decreased expression of AtRRB1 and increased transcription of the 60S ribosome proteins RPL3B and RPL4, in line with a chaperone role of AtRRB1 in ribosome biogenesis. Embryo sac development across ovules of a single pistil occurs uncoordinated and about half of the ovules abort. Half ofrrb1-1pollen is substantially smaller and produce shorter pollen tubes than WT pollen. In contrast to the Col-0 pollen, smaller pollen is overly sensitivity to high temperature (24h at 32°C) treatment, specifically during the early bicellular microspore development stage. Heat stressedrrb1-1bicellular microspores accumulated excessively rough endoplasmic reticulum stacks, suggesting that loss of AtRRB1 activity causes an arrest in ER associated protein biosynthesis. These findings support a critical requirement for ribosome biogenesis and protein synthesis in bicellular microspores to recover from high temperature stress.
A 6-minute Limb Function Assessment for Therapeutic Testing in Experimental Peripheral Artery Disease Models
Authors: Palzkill Victoria R. , Tan Jianna , Tice Abigail L. , Ferriera Leonardo F. , Ryan Terence E. 
Published: March 2024

ABSTRACTBackgroundThe translation of promising therapies from pre-clinical models of hindlimb ischemia (HLI) to patients with peripheral artery disease (PAD) has been inadequate. While this failure is multifactorial, primary outcome measures in preclinical HLI models and clinical trials involving patients with PAD are not aligned well. For example, laser Doppler perfusion recovery measured under resting conditions is the most used outcome in HLI studies, whereas clinical trials involving patients with PAD primarily assess walking performance. Here, we sought to develop a 6-min limb function test for preclinical HLI models that assess muscular performance and hemodynamics congruently.MethodsWe developed anin situ6-min limb function test that involves repeated isotonic (shortening) contractions performed against a submaximal load. Continuous measurement of muscle blood flow was performed using laser Doppler flowmetry. Quantification of muscle power, work, and perfusion are obtained across the test. To assess the efficacy of this test, we performed HLI via femoral artery ligation on several mouse strains: C57BL6J, BALBc/J, and MCK-PGC1α (muscle-specific overexpression of PGC1α). Additional experiments were performed using an exercise intervention (voluntary wheel running) following HLI.ResultsThe 6-min limb function test was successful at detecting differences in limb function of C57BL6/J and BALBc/J mice subjected to HLI with effect sizes superior to laser Doppler perfusion recovery. C57BL6/J mice randomized to exercise therapy following HLI had smaller decline in muscle power, greater hyperemia, and performed more work across the 6-min limb function test compared to non-exercise controls with HLI. Mice with muscle-specific overexpression of PGC1α had no differences in perfusion recovery in resting conditions, but exhibited greater capillary density, increased muscle mass and absolute force levels, and performed more work across the 6-min limb function test compared to their wildtype littermates without the transgene.ConclusionThese results demonstrate the efficacy of the 6-min limb function test to detect differences in the response to HLI across several interventions including where traditional perfusion recovery, capillary density, and muscle strength measures were unable to detect therapeutic differences.
An inhibitory acetylcholine receptor gates context dependent mechanosensory processing inC. elegans
Authors: Kumar Sandeep , Sharma Anuj K. , Leifer Andrew M. 
Published: March 2024

SUMMARYAn animal’s current behavior influences its response to sensory stimuli, but the molecular and circuit-level mechanisms of this context-dependent decision-making is not well understood. In the nematodeC. elegans, inhibitory feedback from turning associated neurons alter downstream mechanosensory processing to gate the animal’s response to stimuli depending on whether the animal is turning or moving forward [1–3]. Until now, the specific neurons and receptors that mediate this inhibitory feedback were not known. We use genetic manipulations, single-cell rescue experiments and high-throughput closed-loop optogenetic perturbations during behavior to reveal the specific neuron and receptor responsible for receiving inhibition and altering sensorimotor processing. An inhibitory acetylcholine gated chloride channel comprised oflgc-47andacc-1expressed in neuron RIM receives inhibitory signals from turning neurons and performs the gating that disrupts the worm’s mechanosensory evoked reversal response.
Temporal constraints on enhancer usage shape the regulation of limb gene transcription
Authors: Rouco Raquel , Rauseo Antonella , Sapin Guillaume , Bompadre Olimpia , Darbellay Fabrice , Andrey Guillaume 
Published: March 2024

AbstractRepertoires of transcriptional enhancers orchestrate gene expression during embryonic development, thereby shaping the forms and functions of organs. Within these repertoires individual enhancers display spatially distinct or overlapping activities that collectively build up the expression domain of cognate genes. However, the temporal specificity of these enhancers - how their activities change over developmental time to dynamically influence gene expression - remains uncharacterized. Here, we observed that temporally restricted enhancer repertoires are embedded at numerous loci associated with mouse limb development. To monitor how such enhancer repertoires govern gene transcriptionin vivoacross extensive developmental periods, we introduce the regulatory trajectory framework. This paradigm conceptually involves transcriptional initiation, marking the beginning of gene expression, followed by its maintenance over time, and ultimately decommissioning, leading to gene repression. To track and sort cells undergoing these distinct phases, we devised a transgenic recorder approach at theShox2model locus. Through this method, we discovered that cells maintainingShox2transcription in early and late limb development relies on distinct, temporally restricted enhancer repertoires. We demonstrate that eliminating early-or late-acting enhancers only transiently affectsShox2expression indicating that these enhancer repertoires function independently. Additionally, we found that changes in the 3D topology of the locus associate with enhancer activities and that a rapid loss of enhancer-promoter contacts occurs during decommissioning. Finally, we show that the decommissioning of theShox2locus can be actively driven byHoxd13, a gene which expression is known to antagonizeShox2. Overall, our work uncovers the dependency of developmental genes on enhancers with temporally restricted activities to generate complex expression patterns over time and shed light on the dynamics of enhancer-promoter interactions.
Bacterial-tyramine-induced renal-gut countercurrent flow regulates gut immune response by promoting ROS accumulation and gut peristalsis in the flyBactrocera dorsalis
Authors: Liu Yanning , Luo Rengang , Lemaitre Bruno , Zhang Hongyu , Li Xiaoxue 
Published: March 2024

SummaryInsects increase gut peristalsis to purge ingested pathogens. They also rely on the Duox-ROS system to mount an efficient gut response. However, how these two host defense mechanisms are coordinated to clear pathogens remained unclear. We found that gut peristalsis is the major force for bacteria elimination, while Duox is necessary as a signaling molecule to activate gut peristalsis in a Tephritidae fruit flyBactrocera dorsalis. We found an insect-conserved renal-gut countercurrent flow formed upon recognizing bacteria-derived tyramine counteracts the gut peristalsis forced defecation and drives the accumulation of ROS. Furthermore, we demonstrated that the renal-gut countercurrent flow maintains proper microbiota composition. Our work provided a novel renal-gut interaction that ensures an efficient gut-immune response.
Extracellular vesicles promote proliferation in an animal model of regeneration
Authors: Avalos Priscilla N. , Wong Lily L. , Forsthoefel David J. 
Published: March 2024

AbstractExtracellular vesicles (EVs) are secreted nanoparticles composed of a lipid bilayer that carry lipid, protein, and nucleic acid cargo between cells as a mode of intercellular communication. Although EVs can promote tissue repair in mammals, their roles in animals with greater regenerative capacity are not well understood. Planarian flatworms are capable of whole body regeneration due to pluripotent somatic stem cells called neoblasts that proliferate in response to injury. Here, using transmission electron microscopy, nanoparticle tracking analysis, and protein content examination, we showed that EVs enriched from the tissues of the planarianSchmidtea mediterraneahad similar morphology and size as other eukaryotic EVs, and that these EVs carried orthologs of the conserved EV biogenesis regulators ALIX and TSG101. PKH67-labeled EVs were taken up more quickly by S/G2 neoblasts than G1 neoblasts/early progeny and differentiated cells. When injected into living planarians, EVs from regenerating tissue fragments enhanced upregulation of neoblast-associated transcripts. In addition, EV injection increased the number ofF-ara-EdU-labelled cells by 49% as compared to buffer injection only. Our findings demonstrate that regenerating planarians produce EVs that promote stem cell proliferation, and suggest the planarian as an amenablein vivomodel for the study of EV function during regeneration.
Establishing Primary and Stable Cell Lines from Frozen Wing Biopsies for Cellular, Physiological, and Genetic Studies in Bats
Authors: Deng Fengyan , Morales-Sosa Pedro , Bernal-Rivera Andrea , Wang Yan , Tsuchiya Dai , Javier Jose Emmanuel , Rohner Nicolas , Zhao Chongbei , Camacho Jasmin 
Published: March 2024

AbstractBats stand out among mammalian species for their exceptional traits, including the capacity to navigate through flight and echolocation, conserve energy through torpor/hibernation, harbor a multitude of viruses, exhibit resistance to disease, survive harsh environmental conditions, and demonstrate exceptional longevity compared to other mammals of similar size.In vivostudies of bats can be challenging for several reasons such as ability to locate and capture them in their natural environments, limited accessibility, low sample size, environmental variation, long lifespans, slow reproductive rates, zoonotic disease risks, species protection, and ethical concerns. Thus, establishing alternative laboratory models is crucial for investigating the diverse physiological adaptations observed in bats. Obtaining quality cells from tissues is a critical first step for successful primary cell derivation. However, it is often impractical to collect fresh tissue and process the samples immediately for cell culture due to the resources required for isolating and expanding cells. As a result, frozen tissue is typically the starting resource for bat primary cell derivation. Yet, cells in frozen tissue are usually damaged and represent low integrity and viability. As a result, isolating primary cells from frozen tissues poses a significant challenge. Herein, we present a successfully developed protocol for isolating primary dermal fibroblasts from frozen bat wing biopsies. This protocol marks a significant milestone, as this the first protocol specially focused on fibroblasts isolation from bat frozen tissue. We also describe methods for primary cell characterization, genetic manipulation of primary cells through lentivirus transduction, and the development of stable cell lines.Basic Protocol 1:Bat wing biopsy collection and preservationSupport Protocol 1:Blood collection from bat-venipunctureBasic Protocol 2:Isolation of primary fibroblasts from adult bat frozen wing biopsySupport Protocol 2:Maintenance of primary fibroblastsSupport Protocol 3:Cell banking and thawing of primary fibroblastsSupport Protocol 4:Growth curve and doubling timeSupport Protocol 5:Lentiviral transduction of bat primary fibroblastsBasic Protocol 3:Bat stable fibroblasts cell lines developmentSupport Protocol 6:Bat fibroblasts validation by immunofluorescence stainingSupport Protocol 7:Chromosome counting
Sediment-associated processes drive spatial variation in ecosystem respiration in the Yakima River basin
Authors: Kaufman Matthew , Garayburu-Caruso Vanessa A. , Forbes Brieanne , Lin Xinming , Hall Robert O. , Fulton Stephanie , Renteria Lupita , Fang Yilin , Son Kyongho , Stegen James C. 
Published: March 2024

AbstractAreas where groundwater and surface water mix (i.e., hyporheic zones, HZ) contribute substantially to stream ecosystem respiration (ERtot). We rely on reactive transport models to understand HZ respiration at large scales; however, model outputs have not been evaluated with field estimates of ERtot. Here we evaluate the degree to which spatial variation in model-predicted HZ respiration can explain spatial variation in field-estimated ERtot across 32 sites in the Yakima River basin (YRB). We find that predicted HZ respiration did not explain spatial variation in ERtot. We hypothesize that ERtot is influenced by processes that integrate contributions from sediments, such as benthic algae, submerged macrophytes, and shallow HZ. Our results indicate that sediment-associated processes hydrologically connected to the active channel are primary drivers of spatial variation in ERtot in the YRB. We encourage conceptual and physical models of stream ERtot to integrate shallow hyporheic exchange with sediment-associated primary production.
mm2-gb: GPU Accelerated Minimap2 for Long Read DNA Mapping
Authors: Dong Juechu , Liu Xueshen , Sadasivan Harisankar , Sitaraman Sriranjani , Narayanasamy Satish 
Published: March 2024

AbstractLong-read DNA sequencing is becoming increasingly popular for genetic diagnostics. Minimap2 is the state-of-the-art long-read aligner. However, Minimap2’s chaining step is slow on the CPU and takes 40-68% of the time especially for long DNA reads. Prior works in accelerating Minimap2 either lose mapping accuracy, are closed source (and not updated) or deliver inconsistent speedups for longer reads. We introducemm2-gbwhich accelerates the chaining step of Minimap2 on GPU without compromising mapping accuracy. In addition to intra- and inter-read parallelism exploited by prior works,mm2-gbexploits finer levels of parallelism by breaking down high latency large workloads into smaller independent segments that can be run in parallel and leverages several strategies for better workload balancing including split-kernels and prioritized scheduling of segments based on sorted size. We show thatmm2-gbon an AMD Instinct™ MI210 GPU achieves 2.57-5.33x performance improvement on long nanopore reads (10kb-100kb), and up to 1.87x performance gain on super long reads (100kb-300kb) compared to SIMD accelerated mm2-fast.mm2-gbis open-sourced and available athttps://github.com/Minimap2onGPU/mm2-gb.
Genome-wide analysis of mRNA decay in Arabidopsis shoot and root reveals the importance of co-translational mRNA decay in the general mRNA turnover
Authors: Marie-Christine Carpentier , Anne-Elodie Receveur , Alexandre Boubegtitene , Adrien Cadoudal , Cécile Bousquet-Antonelli , Rémy Merret 
Published: March 2024

ABSTRACTUntil recently, the general 5’-3’ mRNA decay was placed in the cytosol after the mRNA was released from ribosomes. However, the discovery of an additional 5’ to 3’ pathway, the Co-Translational mRNA Decay (CTRD), changed this paradigm. Up to date, defining the real contribution of CTRD in the general mRNA turnover has been hardly possible as the enzyme involved in this pathway is also involved in cytosolic decay. Here we overcame this obstacle and created an Arabidopsis line specifically impaired for CTRD called XRN4ΔCTRD. Through a genome-wide analysis of mRNA decay rate in shoot and root, we tested the importance of CTRD in mRNA turnover. First, we observed that mRNAs tend to be more stable in root than in shoot. Next, using XRN4ΔCTRD line, we demonstrated that CTRD is a major determinant in mRNA turnover. In shoot, the absence of CTRD leads to the stabilization of thousands of transcripts while in root its absence is highly compensated resulting in faster decay rates. We demonstrated that this faster decay rate is partially due to the XRN4-dependent cytosolic decay. Finally, we correlated this organ-specific effect with XRN4ΔCTRD line phenotypes revealing a crucial role of CTRD in mRNA homeostasis and proper organ development.
Development of a tightly regulated copper-inducible transient gene expression system inNicotiana benthamianaincorporating suicide exon and Cre recombinase
Authors: Chiang Bing-Jen , Lin Kuan-Yu , Chen Yi-Feng , Huang Ching-Yi , Goh Foong-Jing , Huang Lo-Ting , Chen Li-Hung , Wu Chih-Hang 
Published: March 2024

AbstractChemical-inducible gene expression systems have been frequently used to regulate gene expression for functional genomics in various plant species. However, a convenient chemical-inducible system that can tightly regulate transgene expression inNicotiana benthamianais still missing. In this study, we developed a tightly regulated copper-inducible system that can be used to regulate transgene expression and perform cell death assays inN. benthamiana. We tested several chemical-inducible systems usingAgrobacterium-mediated transient expression and found that the copper-inducible system showed the least concerns of leakiness issues. Using the MoClo-based synthetic biology approach, we optimized the design of the copper-inducible system and incorporated the use of the suicide exon HyP5SM/OsL5 and Cre/LoxP as additional regulatory elements to enhance the tightness of the regulation. This new design allowed us to tightly control the hypersensitive cell death induced by several tested NLRs and their matching AVRs, and it can also be easily applied to regulate the expression of other transgenes in transient expression assays. Our findings provide new approaches for both fundamental and translational studies in plant functional genomics.