Biology subjects

Qi, Y.

Publications and source records attributed to Qi, Y..

At least 127 records · Page 7Linked to original sources

bioRxiv2022

Identification of the Metaphyseal Skeletal Stem Cell

Identification of novel regional skeletal stem cells (SSCs) will provide a new cellular paradigm for bone physiology and dysfunction. Several populations of SSCs have been identified at distinct skeletal sites. However, a bona fide SSC population has not yet been formally characterized in the bone marrow. Here, we identify a metaphyseal SSCs (mpSSCs) population whose transcriptional landscape is distinct from other bone mesenchymal stromal cells (bMSCs) in the bone marrow. These mpSSCs emerge at the postnatal stage and reside just underneath the growth plate, consistent with the fact that these mpSSCs are exclusively derived from hypertrophic chondrocytes (HCs). These mpSSCs possess SSC properties such as self-renewal and multipotency in vitro and in vivo, stand at the top of the HC de-differentiation path, and produce most HC progeny. Genetic block of the conversion from HCs to mpSSCs significantly compromises trabecular bone formation and bone regeneration. Thus, metaphysis houses a unique HC-derived SSC population, which is a major source of osteoblasts and bMSCs supporting postnatal trabecular bone formation.

developmental biology↗

bioRxiv2022

Genome-wide investigation of multiplexed CRISPR-Cas12a-mediated editing in rice

We previously reported highly specific genome editing in rice by CRISPR-Cas9 and Cas12a with single DNA double strand break (DSB) (Tang et al., 2018). Two concurrent DSBs by CRISPR-Cas9 could generate defined deletions (Zhou et al., 2014), inversions (Schmidt et al., 2020), and translocations (Beying et al., 2020). Off-target effects of many simultaneous DSBs are unknown in plants. Here, we used whole-genome sequencing (WGS) to investigate off-target effects in rice plants edited by highly multiplexable CRISPR-Cas12a systems (Zhang et al., 2021). Our WGS study revealed highly specific multiplexed genome editing by CRISPR-Cas12a. We found low-frequency large chromosomal deletion and duplication events in plants that endured many (e.g., >50) simultaneous DSBs, but not in plants that endured a lower order DSBs (e.g., <10). While our short reads sequencing may not capture all chromosomal rearrangements, our results nevertheless shed important light on the analysis and regulation of engineered crops derived from multiplexed genome editing.

genomics↗

bioRxiv2022

Tumor landscape of epithelial ovarian cancer highlights that EGR1 drives tumor invasion at single-cell resolution

Identifying underlying molecular mechanisms and biomarkers of epithelial ovarian carcinoma (EOC) proliferation and metastasis remains challenging. Patients of EOC are usually diagnosed at an advanced stage and the availability of invasion-related targets is limited. Herein, we explored the single-cell RNA sequencing (scRNA-seq) dataset of EOC and defined tumor physiological reprograming compared to bulk RNA-seq. The energy metabolism and anti-apoptotic pathway was found as critical contributors to intratumor heterogeneity. Moreover, hypoxia, oxidative phosphorylation (OXPHOS) and glycolysis were positively correlated, which have biologically activity trajectories during epithelial mesenchymal transition (EMT). The HMGH1, EGR1 and RUNX1 were found to be critical inducers of the EMT process in EOC. Experimental validation revealed that suppressed EGR1 decreased the expression of FAS and HSPG2 and associating with EMT progression in EOC. In tumor microenvironment (TME), CAFs were found have significant contribution to tumor immune infiltration and metastasis and accumulation of CAFs was associated with poorer patient survival. In conclusion, physiological features and molecular mechanisms in the TME of EOC were revealed and provided effective targets for the suppression of tumor metastasis.

bioinformatics↗

bioRxiv2022

Identification of a multipotent lung progenitor for lung regeneration

We recently showed that intravenous infusion of mouse or human, fetal or adult lung cells following conditioning of recipient mice leads to lung chimerism within alveolar and bronchiolar lineages, in distinct patches containing both epithelial and endothelial cells. We show here, using R26R-Confetti mice as donors, that these multi-lineage patches are derived from a single lung progenitor. FACS of adult mouse lung cells revealed that the putative patch-forming progenitors co-express the endothelial marker CD31 (PECAM-1) and the epithelial marker CD326 (EPCAM). Transplantation of lung cells from transgenic Cre/lox mice expressing nuclear GFP under the VEcad promoter (VEcad-Cre-nTnG), led to GFP+ patches comprising both GFP+ endothelial and epithelial cells in vivo, and in ex-vivo culture of CD326+CD31+ progenitors. Single cell RNA sequencing of CD326+CD31+ lung cells revealed a subpopulation expressing canonical epithelial and endothelial genes. Such double positive GFP+NKX2.1+SOX17+ cells were also detected by immunohistological staining in lungs of VEcad-Cre-nTnG (expressing nuclear GFP) mice in proximity to blood vessels. These findings provide new insights on lung progenitors and lung development and suggest a potential novel approach for lung regeneration. SummaryWe show in the present study, that multi-lineage regenerative patches in our transplantation model are derived from a single lung progenitor, co-expressing the endothelial marker CD31 and the epithelial marker CD326. These findings provide new insights on lung progenitors and lung development.

cell biology↗

bioRxiv2022

Accurate and efficient protein sequence designthrough learning concise local environment of residues

Protein sequence design has been widely applied in rational protein engineering and increasing the design accuracy and efficiency is highly desired. Here we present ProDESIGN-LE, an accurate and efficient design approach, which adopts a concise but informative representation of residues local environment and trains a transformer to select an appropriate residue at a position from its local environment. ProDESIGN-LE iteratively applies the transformer on the positions in the target structure, eventually acquiring a designed sequence with all residues fitting well with their local environments. ProDESIGN-LE designed sequences for 68 naturally occurring and 129 hallucinated proteins within 20 seconds per protein on average, and the predicted structures from the designed sequences perfectly resemble the target structures with state-of-the-art average TM-score exceeding 0.80. We further experimentally validated ProDESIGN-LE by designing five sequences for an enzyme, chloramphenicol O-acetyltransferase type III (CAT III), and recombinantly expressing the proteins in E. coli. Of these proteins, three exhibited excellent solubility, and one yielded monomeric species with circular dichroism spectra consistent with the natural CAT III protein.

bioinformatics↗

bioRxiv2022

Species existence and coexistence under nutrient enrichment in the Park Grass

Numerous studies have shown that nutrient enrichment causes the loss of plant diversity in different grasslands across the globe. Thus far, three main hypotheses (niche dimension, competitive asymmetry, and soil acidification) have been proposed to account for this general phenomenon, but our knowledge of the underlying mechanisms remains rather vague. To reveal the cryptic mechanisms, we analyzed the famous long-term Park Grass Experiment (1856-) under modern coexistence theory by fitting Lotka-Volterra competition models with time-series data from the different treatments (15 different combinations of nutrient addition fully crossed with four levels of soil pH) to quantitatively test the three competing hypotheses. Supportive of the competitive asymmetry and soil acidification hypotheses, both nutrient addition and soil acidification overall decreased intrinsic population growth rates (r) and intensified competitive differences or asymmetries dramatically, which mostly favored grasses over forbs and legumes (and also forbs over legumes). These changes in r and competitive differences or asymmetries are generally consistent with the abundance changes of different functional groups following the various treatments. Moreover, the altered r (determining species existence) and competitive differences (affecting species coexistence) effectively explained the diversity loss and recovery (after nitrogen addition was withheld). However, while nutrient addition significantly decreased per-capita intra- and inter-specific competition (which indicates that belowground competition becomes less important when soil nutrients are more abundant), it did not decrease niche differences as predicted, poorly supporting the niche dimension hypothesis. These findings advance our understanding of fundamental mechanisms driving the response of plant communities to nutrient deposition in nature. Significance StatementAn unresolved fundamental scientific mystery with the crucial applied value in ecology is what causes the general loss of biodiversity following nutrient enrichment in the Anthropocene. In this study, we combined the recent advance in coexistence theory with the longest-running experiment existing in the world to wrestle with this conundrum. Our major results, which highlight the critical role of both existence and coexistence, are helpful in settling the debate between the three popular hypotheses and also, for the first time, provide quantitative explanations for the general findings in numerous nutrient-addition experiments. Our study shows the importance of applying modern coexistence theory to more quantitatively explain, predict and cope with the responses of ecological communities to global change factors in nature.

ecology↗

bioRxiv2022

Spatiotemporal heterogeneity and the long-term impact of meteorological , environmental and socio-economic factors of scrub typhus in China from 2012 to 2018

Large-scale outbreaks of scrub typhus combined with the emergence of this vector-borne rickettsiosis in new areas indicate that this disease remains seriously neglected. This study aimed to explore the long-term changes and regional leading factors of scrub typhus in China, so as to provide fresh insights for the prevention and control of this disease. In this study, a Bayesian space-time hierarchical model (BSTHM) was used to identify the long-term spatiotemporal heterogeneity of scrub typhus and quantify the association between meteorological factors and scrub typhus in southern and northern China from 2012 to 2018. GeoDetector model was used to quantify the dominant forces of environmental and socioeconomic factors in the Northern and the Southern China. Scrub typhus often appeared in summer and autumn (June to November), and epidemically peaked in October, with obvious temporal seasonality. Spatially, the hot spots (high-risk regions) were concentrated in the south, on the contrary the cold spots (low-risk regions) in the north. In addition, the main meteorological factor, average temperature, gave a significant impact in both areas. The average temperature increased by 1 {degrees}C, resulting in a decrease of 1.10% in southern China and an increase of 0.96% in northern China in the risk of scrub typhus. The determinant environmental and socio-economic factors of scrub typhus in the two areas were altitude and per capita GDP, with q-values of 0.91 and 0.87, respectively. Meteorological, environmental and socio-economic factors had a significant impact on the distribution of scrub typhus, with obvious seasonality and spatial heterogeneity. This study provides helpful suggestions and basis for reasonably allocating resources and controlling the occurrence of scrub typhus. Author summaryScrub typhus is a natural-focus disease caused by the bite of chigger mite larval. In this study, we use BSTHM to capture the overall temporal trend and spatial hot spots of scrub typhus, and quantify the relationship between the disease and major meteorological factors. Meanwhile, Geodetector model was used to quantify the influence of other potential risk factors and estimate the spatio-temporal heterogeneity of scrub typhus. The results showed that scrub typhus had significant seasonality, with a q value of 0.52, and spatial heterogeneity, with a q-value of 0.64. Scrub typhus mainly occurred in summer and autumn, and high-risk areas were mainly distributed in southern China (Yunnan, Hainan and Guangdong). These heterogeneity were closely related to the vector and host. Whether in the South or the north, scrub typhus was closely related to risk factors such as temperature, per capita GDP, NDVI, altitude and the percentage of children aged 0-14. These results suggest that the relevant departments should strengthen the monitoring of the ecological environment, the host and vector of Orientia tsutsugamushi, and strengthen the risk awareness, so as to prevent and control the possible increased risk of scrub typhus under these meteorological, environmental and socio-economic conditions. Considering the differences in different regions, resources should be allocated reasonably.

ecology↗

bioRxiv2022

Longitudinal white matter alterations in SIVmac239 infected rhesus monkeys with and without regular cART treatment

ObjectiveWe use the SIV-mac239 infected Chinese rhesus monkeys to longitudinally investigate white matters alterations with and without regular combined antiretroviral therapy (cART) treatment, and its relationship with clinical tests. Material and methodsDiffusion tensor imaging (DTI), CD4 T cell counts, and CD4/CD8 were obtained at baseline, 10 days, 4th week,12th week, 24th week, and 36th week post virus inoculation. postinfection (wpi). Microstructural properties were examined within 76 white matter defined by DTI-WM atlas for rhesus macaques. Corrections for multiple comparisons were performed using a false discovery rate (p < 0.05, FDR). Correlation analyses between imaging markers and clinical measures (CD4 T-cell counts, CD4/CD8 ratio) were determined using Pearsons correlations. ResultsIn our model, White matter alterations in SIV-infected macaques can be detected as soon as 4 weeks post inoculation in several brain regions. And with time proceeding, the cART can reverse, relieve, or even progressive effects. CD4 T-cell count is mainly associated with DTI metrics before the cART, whereas CD4/CD8 ratio was associated with white matter alteration with and without cART. ConclusionSIV-mac239 infection can be an idol modal to explore HIV induced HIV associated brain alterations, and the first group of white matter alterations was as soon as 4 weeks post inoculation in structure next to the periventricular area. As the time progressed, cART can bring different effect to each region, including reversed, relieved, and even progressive effects. In addition, these changes are closely linked to CD4/CD8 ratio even after cART. Importance

animal behavior and cognition↗

bioRxiv2022

Phase separation and correlated motions in motorized genome

The human genome is arranged in the cell nucleus non-randomly, and phase separation has been proposed as an important driving force for genome organization. However, the cell nucleus is an active system, and the contribution of non-equilibrium activities to phase separation and genome structure and dynamics remains to be explored. We simulated the genome using an energy function parameterized with chromosome conformation capture (Hi-C) data with the presence of active, nondirectional forces that break the detailed balance. We found that active forces that may arise from transcription and chromatin remodeling can dramatically impact the spatial localization of heterochromatin. When applied to euchromatin, active forces can drive heterochromatin to the nuclear envelope and compete with passive interactions among heterochromatin that tend to pull them in opposite directions. Furthermore, active forces induce long-range spatial correlations among genomic loci beyond single chromosome territories. We further showed that the impact of active forces could be understood from the effective temperature defined as the fluctuation-dissipation ratio. Our study suggests that non-equilibrium activities can significantly impact genome structure and dynamics, producing unexpected collective phenomena.

biophysics↗

bioRxiv2022

A unique epigenomic landscape defines CD8+ tissue-resident memory T cells

Memory T cells provide rapid and long-term protection against infection and tumors. The memory CD8+ T cell repertoire contains phenotypically and transcriptionally heterogeneous subsets with specialized functions and recirculation patterns. While these T cell populations have been well characterized in terms of differentiation potential and function, the epigenetic changes underlying memory T cell fate determination and tissue-residency remain largely unexplored. Here, we examined the single-cell chromatin landscape of CD8+ T cells over the course of acute viral infection. We reveal an early bifurcation of memory precursors displaying distinct chromatin accessibility and define epigenetic trajectories that lead to a circulating (TCIRC) or tissue-resident memory T (TRM) cell fate. While TRM cells displayed a conserved epigenetic signature across organs, we demonstrate that these cells exhibit tissue-specific signatures and identify transcription factors that regulate TRM cell populations in a site-specific manner. Moreover, we demonstrate that TRM cells and exhausted T (TEX) cells are distinct epigenetic lineages that are distinguishable early in their differentiation. Together, these findings show that TRM cell development is accompanied by dynamic alterations in chromatin accessibility that direct a unique transcriptional program resulting in a tissue-adapted and functionally distinct T cell state. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/490680v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@b03f1corg.highwire.dtl.DTLVardef@ff6871org.highwire.dtl.DTLVardef@220db2org.highwire.dtl.DTLVardef@1b15166_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIscATAC atlas reveals the epigenetic variance of memory CD8+ T cell subsets over the course of acute infection C_LIO_LIEarly bifurcation of memory precursors leads to circulating versus tissue-resident cell fates C_LIO_LIIntegrating transcriptional and epigenetic analyses identified organ-specific TRM cell regulators including HIC1 and BACH2 C_LIO_LIEpigenetic distinction of TRM cells and TEX cell subsets C_LI

immunology↗

bioRxiv2022

Glutamine is essential for overcoming theimmunosuppressive microenvironment in malignant salivary gland tumors

RationaleImmunosuppression in the tumor microenvironment (TME) is key to the pathogenesis of solid tumors. Tumor cell-intrinsic autophagy is critical for sustaining both tumor cell metabolism and survival. However, the role of autophagy in the host immune system that allows cancer cells to escape immune destruction remains poorly understood. Here, we determined if attenuated host autophagy is sufficient to induce tumor rejection through reinforced adaptive immunity. Furthermore, we determined whether dietary glutamine supplementation, mimicking attenuated host autophagy, is capable of promoting antitumor immunity. MethodsA syngeneic orthotopic tumor model in Atg5+/+ and Atg5flox/flox mice was established to determine the impact of host autophagy on the antitumor effects against mouse malignant salivary gland tumors (MSTs). Multiple cohorts of immunocompetent mice were used for oncoimmunology studies, including inflammatory cytokine levels, macrophage, CD4+, and CD8+ cells tumor infiltration at 14 days and 28 days after MST inoculation. In vitro differentiation and in vivo dietary glutamine supplementation were used to assess the effects of glutamine on Treg differentiation and tumor expansion. ResultsWe showed that mice deficient in the essential autophagy gene, Atg5, rejected orthotopic allografts of isogenic MST cells. An enhanced antitumor immune response evidenced by reduction of both M1 and M2 macrophages, increased infiltration of CD8+ T cells, elevated IFN-{gamma} production, as well as decreased inhibitory Tregs within TME and spleens of tumor-bearing Atg5flox/flox mice. Mechanistically, ATG5 deficiency increased glutamine level in tumors. We further demonstrated that dietary glutamine supplementation partially increased glutamine levels and restored potent antitumor responses in Atg5+/+ mice. ConclusionsDietary glutamine supplementation exposes a previously undefined difference in plasticity between cancer cells, cytotoxic CD8+ T cells and Tregs.

cancer biology↗

bioRxiv2022

Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat generated by CRISPR/Cas9

Staphylococcus aureus infections pose a potential threat to livestock production and public health. A novel strategy is needed to control S. aureus infection due to its adaptive evolution to antibiotics. Autophagy plays a key role in degrading bacteria for innate immune cells. In order to promote S. aureus clearance via TLR induced autophagy pathway, the domain fusion TLR2-4 with the extracellular domain of TLR2, specific recognizing S. aureus, and transmembrane and intracellular domains of TLR4 is assembled, then the goats expressing TLR2-4 is generated. TLR2-4 substantially augments the removal of S. aureus within macrophages by elevating autophagy level. Phosphorylated JNK/ERK1/2 promote LC3- puncta in TLR2-4 macrophages during S. aureus-induced autophagy via MyD88-mediated the TAK1 signaling cascade. Meantime, the TRIF-dependent TBK1-TFEB-OPTN signaling is involved in TLR2-4-triggered autophagy after S. aureus challenge. Moreover, the transcript of ATG5 and ATG12 is significantly increased via cAMP-PKA-NF-{kappa}B signaling, which facilitates S. aureus-induced autophagy in TLR2-4 macrophages. Overall, the novel receptor TLR2-4 enhances the autophagy-dependent clearance of S. aureus in macrophages via TAK1/TBK1-JNK/ERK, TBK1-TFEB-OPTN and cAMP-PKA-NF-{kappa}B-ATGs signaling pathways, which provide an alternative approach to resistant against S. aureus infection.

immunology↗

bioRxiv2022

Genome-wide analyses of PAM-relaxed Cas9 genome editors reveal substantial off-target effects by ABE8e in rice

PAM-relaxed Cas9 nucleases, cytosine base editors and adenine base editors are promising tools for precise genome editing in plants. However, their genome-wide off-target effects are largely undetermined. Here, we conduct whole-genome sequencing (WGS) analyses of transgenic plants edited by xCas9, Cas9-NGv1, Cas9-NG, SpRY, nCas9-NG-PmCDA1, nSpRY-PmCDA1 and nSpRY-ABE8e in rice. Our results reveal different guide RNA (gRNA)-dependent off-target effects with different editors. De novo generated new gRNAs by SpRY editors lead to additional but not substantial off-target mutations. Strikingly, ABE8e results in ~500 genome-wide A-to-G off-target mutations at TA motif sites per transgenic plant. The preference of the TA motif by ABE8e is also observed at the target sites. Finally, we investigate the timeline and mechanism of somaclonal variation due to tissue culture, which chiefly contributes to the background mutations. This study provides a comprehensive understanding on the scales and mechanisms of off-target and background mutations during PAM-relaxed genome editing in plants.

genetics↗

bioRxiv2022

HiDiver: A Suite of Methods to Merge Magnetic Resonance Histology, Light Sheet Microscopy, and Complete Brain Delineations

We have developed new imaging and computational workflows to produce accurately aligned multimodal 3D images of the mouse brain that exploit high resolution magnetic resonance histology (MRH) and light sheet microscopy (LSM) with fully rendered 3D reference delineations of brain structures. The suite of methods starts with the acquisition of geometrically accurate (in-skull) brain MRIs using multi-gradient echo (MGRE) and new diffusion tensor imaging (DTI) at an isotropic spatial resolution of 15 m. Whole brain connectomes are generated using over 100 diffusion weighted images acquired with gradients at uniformly spaced angles. Track density images are generated at a super-resolution of 5 m. Brains are dissected from the cranium, cleared with SHIELD, stained by immunohistochemistry, and imaged by LSM at 1.8 m/pixel. LSM channels are registered into the reference MRH space along with the Allen Brain Atlas (ABA) Common Coordinate Framework version 3 (CCFv3). The result is a high-dimensional integrated volume with registration (HiDiver) that has a global alignment accuracy of 10-50 m. HiDiver enables 3D quantitative and global analyses of cells, circuits, connectomes, and CNS regions of interest (ROIs). Throughput is sufficiently high that HiDiver is now being used in comprehensive quantitative studies of the impact of gene variants and aging on rodent brain cytoarchitecture.

neuroscience↗

bioRxiv2022

Study on gene knockout mice and human mutant individual reveals absence of CEP78 causes photoreceptor and sperm flagella impairments

BackgroundCone-rod dystrophy (CRD) is a genetically inherited retinal disease characterized by photoreceptor degeneration. In some rare cases, CRD and hearing loss can be associated with male fertility, while the underlying mechanism is not well known. MethodsUsing CRISPR/Cas9 system, we generated Cep78-/- mice. And electroretinogram (ERG), immunofluorescence staining and transmission electron microscopy (TEM) were used to analyze visual function and photoreceptor ciliary structure changes in Cep78-/- mouse. HE/PAS staining, scanning-electron microscopy (SEM) were conducted to Cep78-/- mice and human CRD patient with CEP78 protein loss to illustrate male infertility and multiple morphological abnormalities of the sperm flagella (MMAF) caused by CEP78 deficiency. TEM and immunofluorescence staining were performed to characterize morphological and molecular changes of sperm flagella microtubule arrangement, centriole development and spermatid head shaping in Cep78-/- mice. Mass-spectrometry analyses were conducted to identify protein abnormalities after Cep78 deletion and Cep78 interacting proteins in spermiogenesis. Co-immunoprecipitation was used to show the Cep78-Ift20-Ttc21a trimer. The role of Cep78-Ift20-Ttc21a trimer in cilliogenesis and centriole elongation was assessed by cilia induction assay. ResultsCep78 knockout mice exhibited impaired function and morphology of photoreceptors, typified by reduced electroretinogram amplitudes, disrupted translocation of cone arrestin, attenuated and disorganized photoreceptor outer segments (OS) disks and widen OS bases, as well as interrupted cilia elongations and structures. Cep78 deletion also caused male infertility and MMAF, with disordered "9 + 2" structure and triplet microtubules in sperm flagella. CEP78 forms a trimer with intraflagellar transport (IFT) proteins IFT20 and TTC21A essential for sperm flagella formation, is essential for their interaction and stability, and recruits IFT20 to centrosome. Insufficiency of any component in the trimer causes centriole elongation and cilia shortening. Additionally, absence of CEP78 protein in human leaded to similar phenotypes in vision and MMAF as Cep78-/- mice. ConclusionsWe found CEP78 as the causative gene of CRD with MMAF in human and mouse. Cep78 forms a trimer with Ift20 and Ttc21a, and regulate the interaction, stability and localization of the trimer proteins, which regulate cilliogenesis, centriole length, and sperm flagella formation. FundingThis work was supported by the National Key R&D Program (2021YFC2700200 to X.G); National Natural Science Foundation of China (82020108006, 81730025 to C.Z, 81971439, 81771641 to X.G, 82070974 to X.C, 82060183 to X.S); Shanghai Outstanding Academic Leaders (2017BR013 to C.Z); and Six Talent Peaks Project in Jiangsu Province (YY-019 to X.G). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

developmental biology↗

bioRxiv2022

Resolution and b value dependent Structural Connectome in ex vivo Mouse Brain

Diffusion magnetic resonance imaging has been widely used in both clinical and preclinical studies to characterize tissue microstructure and structural connectivity. The diffusion MRI protocol for the Human Connectome Project (HCP) has been developed and optimized to obtain high-quality, high-resolution diffusion MRI (dMRI) datasets. However, such efforts have not been fully explored in preclinical studies, especially for rodents. In this study, high quality dMRI datasets of mouse brains were acquired at 9.4T system from two vendors. In particular, we acquired a high-spatial resolution dMRI dataset (25 m isotropic with 126 diffusion encoding directions), which we believe to be the highest spatial resolution yet obtained; and a high-angular resolution dMRI dataset (50 m isotropic with 384 diffusion encoding directions), which we believe to be the highest angular resolution compared to the dMRI datasets at the microscopic resolution. We systematically investigated the effects of three important parameters that affect the final outcome of the connectome: b value (1000 s/mm2 to 8000 s/mm2), angular resolution (10 to 126), and spatial resolution (25 {micro}m to 200 {micro}m). The stability of tractography and connectome increase with the angular resolution, where more than 50 angles are necessary to achieve consistent results. The connectome and quantitative parameters derived from graph theory exhibit a linear relationship to the b value (R2 > 0.99); a single-shell acquisition with b value of 3000 s/mm2 shows comparable results to the multi-shell high angular resolution dataset. The dice coefficient decreases and both false positive rate and false negative rate gradually increase with coarser spatial resolution. Our study provides guidelines and foundations for exploration of tradeoffs among acquisition parameters for the structural connectome in ex vivo mouse brain.

neuroscience↗

bioRxiv2021

All-atom molecular dynamics simulations of synaptic vesicle fusion I: a glimpse at the primed state

Synaptic vesicles are primed into a state that is ready for fast neurotransmitter release upon Ca2+-binding to synaptotagmin-1. This state likely includes trans-SNARE complexes between the vesicle and plasma membranes that are bound to synaptotagmin-1 and complexins. However, the nature of this state and the steps leading to membrane fusion are unclear, in part because of the difficulty of studying this dynamic process experimentally. To shed light into these questions, we performed all-atom molecular dynamics simulations of systems containing trans-SNARE complexes between two flat bilayers or a vesicle and a flat bilayer with or without fragments of synaptotagmin-1 and/or complexin-1. Our results help visualize potential states of the release machinery en route to fusion, and suggest mechanistic features that may control the speed of release. In particular, the simulations suggest that the primed state contains almost fully assembled trans-SNARE complexes bound to the synaptotagmin-1 C2B domain and complexin-1 in a spring-loaded configuration where interactions of the C2B domain with the plasma membrane orient complexin-1 toward the vesicle, avoiding premature membrane merger but keeping the system ready for fast fusion upon Ca2+ influx.

biophysics↗

bioRxiv2021

Divergent clonal differentiation trajectories of T cell exhaustion

T cells activated by chronic antigen exposure in the setting of viral infections or cancer can adopt an exhausted T cell (Tex) state, characterized by reduced effector function and proliferative capacity, and the upregulation of inhibitory receptors. However, whether all antigen-specific T cell clones follow the same molecular and cellular Tex differentiation trajectory remains unclear. Here, we generate a single-cell multi-omic atlas of T cell exhaustion that redefines the phenotypic diversity and molecular regulation of Tex phenotypes. Longitudinal analysis during chronic viral infection identifies an early effector phenotype that is epigenetically primed for Tex differentiation and two late-stage Tex cell states with either a terminal exhaustion or a killer cell lectin-like receptor (KLR)-expressing cytotoxic gene signature. We define clonal trajectories of antigen-specific T cells using paired single-cell RNA and T cell receptor sequencing and reveal distinct differentiation trajectories resulting in terminal Tex-biased, KLR Tex-biased, or divergent clones that differentiate into both phenotypes. Comparison of Tex phenotypes among shared T cell clones that traffic to multiple organs reveals that clonal differentiation trajectories are maintained across tissues. Finally, we show that differences in clonal differentiation trajectory are driven by TCR signal strength, whereby high-affinity T cell clones preferentially adopt a terminal Tex fate, while low-affinity clones adopt an effector-like KLR Tex fate that is detectable long-term but depleted in high antigen settings. These findings reveal clonal heterogeneity in the T cell response to chronic antigen and genomic programs that underlie Tex fates and persistence. HighlightsO_LIA single-cell atlas of T cell exhaustion identifies novel early effector and KLR Tex states. C_LIO_LIClonal T cell analysis defines divergent differentiation trajectories during chronic viral infection leading to terminal and KLR Tex fates. C_LIO_LIThe heterogeneity of the Tex pool arises from three primary differentiation patterns and are differentially persistent in the setting of high antigen. C_LIO_LIClonal Tex differentiation patterns are conserved across organ sites and driven by TCR signal strength. C_LI

immunology↗