Biology subjects

Liu, L.

Publications and source records attributed to Liu, L..

At least 541 records · Page 30Linked to original sources

bioRxiv2022

Longitudinal investigation of changes in resting-state co-activation patterns and their predictive ability in the zQ175 DN mouse model of Huntington's disease

Huntingtons disease (HD) is a neurodegenerative disorder caused by expanded ([≥]40) glutamine-encoding CAG repeats in the huntingtin gene, which leads to dysfunction and death of predominantly striatal and cortical neurons. While the genetic profile and behavioural signs of the disease are better known, changes in the functional architecture of the brain, especially before the behavioural symptoms become apparent, are not fully and consistently characterized. In this study, we sought markers at pre, early and late manifest states of phenotypic progression in the heterozygous (HET) zQ175 delta-neo (DN) mouse model, using resting-state functional magnetic resonance imaging (RS-fMRI). This mouse model shows molecular, cellular and circuitry alterations that resemble those seen in HD in humans. Specifically, we investigated, longitudinally, changes in co-activation patterns (CAPs) that are the transient states of brain activity constituting the resting-state networks (RSNs). Most robust changes in the temporal properties of CAPs occurred at the late manifest state; the durations of two anti-correlated CAPs, characterized by simultaneous co-activation of default-mode like network (DMLN) and co-deactivation of lateral-cortical network (LCN) and vice-versa, were reduced in the zQ175 DN HET animals compared to the wild-type mice. Changes in the spatial properties, measured in terms of activation levels of different brain regions, during CAPs were found at all three states and became progressively more pronounced at the manifest states. We then assessed the cross-validated predictive power of CAP metrics to distinguish HET animals from controls. Spatial properties of CAPs performed significantly better than the chance level at all three states with 80% classification accuracy at the early and late manifest states.

neuroscience↗

bioRxiv2022

Brevicidine, a bacterial non-ribosomally produced cyclic antimicrobial lipopeptide with a unique modus operandi

Due to the accelerated appearance of antibiotic-resistant (AMR) pathogens in clinical infections, new first-in-class antibiotics, operating via novel modes of action, are desperately-needed. Brevicidine, a bacterial non-ribosomally produced cyclic lipopeptide, has shown potent and selective antimicrobial activity against Gram-negative pathogens. However, before our investigations, little was known about how brevicidine exerts its potent bactericidal effect against Gram-negative pathogens. In this study, we find that brevicidine has potent antimicrobial activity against AMR Enterobacteriaceae pathogens, with a MIC value ranging between 0.5M (0.8mg/L) and 2M (3.0mg/L). In addition, brevicidine showed potent anti-biofilm activity against the Enterobacteriaceae pathogens, with same 100% inhibition and 100% eradication concentration of 4M (6.1mg/L). Further mechanistic studies showed that brevicidine exerts its potent bactericidal activity via interacting with lipopolysaccharide in the outer membrane, targeting phosphatidylglycerol and cardiolipin in the inner membrane, and dissipating the proton motive force of bacteria. This results in metabolic perturbation, including inhibition of adenosine triphosphate synthesis, inhibits the dehydrogenation of nicotinamide adenine dinucleotides, accumulation of reactive oxygen species in bacteria, and inhibition of protein synthesis. Lastly, brevicidine showed a good therapeutic effect in a mouse peritonitis-sepsis model. Our findings pave the way for further research on clinical applications of brevicidine, to combat the prevalent infections caused by AMR Gram-negative pathogens worldwide.

microbiology↗

bioRxiv2022

JGI Plant Gene Atlas: An updateable transcriptome resource to improve structural annotations and functional gene descriptions across the plant kingdom

Gene functional descriptions, which are typically derived from sequence similarity to experimentally validated genes in a handful of model species, offer a crucial line of evidence when searching for candidate genes that underlie trait variation. Plant responses to environmental cues, including gene expression regulatory variation, represent important resources for understanding gene function and crucial targets for plant improvement through gene editing and other biotechnologies. However, even after years of effort and numerous large-scale functional characterization studies, biological roles of large proportions of protein coding genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, a public and updateable data resource consisting of transcript abundance assays from 2,090 samples derived from 604 tissues or conditions across 18 diverse species. We integrated across these diverse conditions and genotypes by analyzing expression profiles, building gene clusters that exhibited tissue/condition specific expression, and testing for transcriptional modulation in response to environmental queues. For example, we discovered extensive phylogenetically constrained and condition-specific expression profiles across many gene families and genes without any functional annotation. Such conserved expression patterns and other tightly co-expressed gene clusters let us assign expression derived functional descriptions to 64,620 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome-next.jgi.doe.gov), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

plant biology↗

bioRxiv2022

Structural conservation of Lassa virus glycoproteins and recognition by neutralizing antibodies

SummaryLassa fever is an acute hemorrhagic fever caused by the zoonotic Lassa virus (LASV). The LASV glycoprotein complex (GPC) mediates viral entry and is the sole target for neutralizing antibodies. Immunogen design is complicated by the metastable nature of recombinant GPCs and the antigenic differences amongst LASV lineages. Despite the sequence diversity of GPC, structures of most lineages are lacking. We present the development and characterization of prefusion-stabilized, trimeric GPCs of LASV lineages II, V, and VI, revealing structural conservation despite sequence diversity. High-resolution structures and biophysical characterization of GPC in complex with GP1-A antibodies reveal their neutralization mechanisms. Finally, we present the isolation and characterization of a novel trimer-preferring neutralizing antibody belonging to the GPC-B competition group with an epitope that spans adjacent protomers and includes the fusion peptide. Our work provides molecular detail information on LASV antigenic diversity and will guide efforts to design pan-LASV vaccines. HighlightsO_LIStructural characterization of soluble glycoproteins from four Lassa virus lineages. C_LIO_LIMAb 12.1F, belonging to the GP1-A cluster, inhibits matriglycan and LAMP-1 binding. C_LIO_LIGP1-A mAbs show glycan-dependence with 19.7E demonstrating lineage-dependent binding. C_LIO_LIA novel trimer-preferring NAb S370.7 targets the GPC-B epitope. C_LI

immunology↗

bioRxiv2022

RGA1 alleviates low-light-repressed pollen tube growth by improving the metabolism and allocation of sugars and energy

Low-light stress compromises photosynthetic and energy efficiency and leads to spikelet sterility; however, the effect of low-light stress on pollen tube elongation in the pistil remains poorly understood. The gene RGA1, which encodes a G subunit of the heterotrimeric G protein, enhanced low-light tolerance in rice plants at anthesis by preventing the cessation of pollen tube elongation in the pistil. The levels of reactive oxygen species were higher and the content of ATP and ATPase was lower in RGA1 mutant (d1) plants compared with wild-type and RGA1-overexpressing (OE-1) plants under low-light conditions. Energy deficits, rather than interference with signaling transduction pathways, were the main contributors to the inhibition of pollen tube elongation in the pistil by low-light stress. In this process, marked increases in the activities of acid invertase (INV), sucrose synthase (SUS), and mitochondrial respiratory electron transport chain complexes, as well as the relative expression levels of SUTs, SWEETs, SUSs, INVs, CINs, SnRK1A, and SnRk1B, were observed in OE-1 plants. INV and ATPase activators (sucrose and Na2SO3, respectively) increased spikelet fertility by improving the energy status in the pistil under low-light conditions, and the ATPase inhibitor Na2VO4 induced spikelet sterility and decreased ATPase activity. Therefore, RGA1 could alleviate the low-light stress-induced impairment of pollen tube elongation to increase spikelet fertility by promoting sucrose unloading in the pistil and improving the metabolism and allocation of energy.

plant biology↗

bioRxiv2022

Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis

Obesity has always been considered a significant risk factor in OA progression, but the underlying mechanism of obesity-related inflammation in OA synovitis remains unclear. The present study found that synovial macrophages infiltrated and polarized in the obesity microenvironment and identified the essential role of M1 macrophages in impaired macrophage efferocytosis using pathology analysis of obesity-associated OA. The present study revealed that obese OA patients and ApoE-/- mice showed a more pronounced synovitis and enhanced macrophage infiltration in synovial tissue, accompanied by dominant M1 macrophage polarization. Obese OA mice had a more severe cartilage destruction and increased levels of synovial apoptotic cells than OA mice in the control group. Enhanced M1-polarized macrophages in obese synovium decreased growth arrest-specific 6 (GAS6) secretion, resulting in impaired macrophage efferocytosis in synovial apoptotic cells. Intracellular contents released by accumulated apoptotic cells further triggered an immune response and lead to a release of inflammatory factors, such as TNF-, IL-1{beta}, and IL-6, which induce chondrocyte homeostasis dysfunction in obese OA patients. Intra-articular injection of GAS6 restored the phagocytic capacity of macrophages, reduced the accumulation of local apoptotic cells, and decreased the levels of TUNEL- and caspase-3-positive cells, preserving cartilage thickness and preventing the progression of obesity-associated OA. Therefore, blocking M1 macrophage polarization or intra-articular injection of GAS6 is a potential therapeutic strategy for obesity-associated OA.

immunology↗

bioRxiv2022

LINC02428, a liver-specific long noncoding RNA suppresses hepatocellular carcinoma progression

There is growing evidence that lncRNAs play an important role in the progression of HCC, especially liver-specific expression of lncRNAs. LINC02428 is a liver-specific labeled lncRNA, but its role in HCC is unclear. We investigated the expression characteristics of LINC02428 in hepatocellular carcinoma tissues and analyzed its function in HCC. Using TCGA public data we found that the expression level of LINC02428 was significantly reduced in hepatocellular carcinoma tissues, compared to normal tissues. LINC02428 was also significantly reduced in hepatocellular carcinoma tissues in our cDNA microarray data from HCC patients. Survival analysis suggests that HCC patients with lower LINC02428 have a lower overall survival rate. In an in vitro analysis, overexpression of LINC02428 inhibited HCC cell proliferation, migration, and induced apoptosis. In conclusion, we report for the first time the expression profile of a liver-specific lncRNA, LINC02428, in HCC and confirmed its ability to inhibit the progression of HCC. LINC02428 may become a new target for the diagnosis and treatment of HCC.

cancer biology↗

bioRxiv2022

Large scale production of human blastoids amenable to modeling blastocyst development and maternal-fetal crosstalk

Recent advances in human blastoids generated from naive pluripotent stem cells have opened a new avenue for modelling early human development and implantation. Despite the success, however, existing protocols have several limitations, e.g., the use of custom-built microwell arrays impedes wide adoption by the research community, and mass production of human blastoids is hampered by low-output or low-efficiency methods. To address these issues, here we developed an optimized protocol based on commercially available microwell plates, which enabled efficient generation of high-fidelity human blastoids at a large scale. Leveraging on the improved protocol, we identified MAPK. PI3K/AKT and mTOR signaling pathways were activated in both blastoids and blastocyst, and discovered endometrial stromal effects in promoting trophoblast cell survival, proliferation and syncytialization during extended co-culture with blastoids. Our optimized protocol will facilitate broader use of human blastoids as an accessible, perturbable, scalable, tractable, and ethical model for human blastocysts.

developmental biology↗

bioRxiv2022

Droplet microfluidics forward for tracing target cells at single-cell transcriptome resolution

The rapid promotion of single-cell omics in various fields has begun to help solve many problems encountered in research including precision medicine, prenatal diagnosis, and embryo development. Meanwhile, single-cell techniques are also constantly updated with increasing demand. For some specific target cells, the workflow from droplet screening to single-cell sequencing is a preferred option, which also should reduce the impact of operation steps such as demulsification and cell recovery. We developed an all-in-droplet method integrating cell encapsulation, target sorting, droplet picoinjection, and single-cell transcriptome profiling on chips to achieve a labor-saving monitor of TCR-T cells. As a proof of concept, in this research, TCR-T cells were encapsulated, sorted, and performed single-cell transcriptome sequencing (scRNA-seq) by injecting reagents into droplet. It avoided the tedious operation of droplet breakage and re-encapsulation between droplet sorting and scRNA-seq. Moreover, convenient device operation will accelerate the progress of chip marketization. The strategy achieved an excellent recovery performance of single cell transcriptome with a median gene number over 4000 and a cross-contamination rate of 8.2 {+/-} 2%. Furthermore, this strategy allows us to develop a device with high integrability to monitor infused TCR-T cells, which will promote the development of adoptive T cell immunotherapy and their clinical application.

cell biology↗

bioRxiv2022

Optimization and redevelopment of single-cell data analysis workflow based on deep generative models

The present single-cell RNA sequencing(scRNA-seq) analysis pipelines require a combination of appropriate normalization, dimension reduction, clustering, and specific-gene analysis algorithms, but the rationale for the choice of these algorithms is relatively subjective because of the lack of ground truth assessment conclusions. As the number of captured single-cells increases, the number of different types of noise cells also increases, which can strongly affect the analysis efficiency. For scRNA-seq, a technology that generates data through multi-process operations, the deep generative model should be a good choice for this type of data analysis, allowing simultaneous estimation of multiple unobservable parameters assumed in the data generation process. Hence, in our study, we sequenced a pool of pre-labeled single cells to obtain a batch of scRNA-seq data with main and fine labels, which was then used to evaluate the clustering and specific-gene analysis methods. Afterward, we applied two deep generative models to infer the probabilities of pseudo and impurity cells. And by stepwise removing the inferred noise cells, the clustering performance and the consistency of different specific-gene analysis methods are both greatly improved. After that, we applied Deep-LDA (a latent Dirichlet allocation-based deep generative model) to scRNA-seq data analysis. And this model takes the count matrix as input, and makes the classification and specific gene optimization process mutually dependent, which has more practical sense and simplifies the analysis workflow. At last, we successfully implemented the model with transferred knowledge to make single-cell annotation and verified its superior performance.

bioinformatics↗

bioRxiv2022

The SARS-CoV-2 spike N-terminal domain engages 9-O-acetylated α2-8-linked sialic acids

SARS-CoV-2 viruses engage ACE2 as a functional receptor with their spike protein. The S1 domain of the spike protein contains a C-terminal receptor-binding domain (RBD) and an N-terminal domain (NTD). The NTD of other coronaviruses includes a glycan-binding cleft. However, for the SARS-CoV-2 NTD protein-glycan binding was only observed weakly for sialic acids with highly sensitive methods. Amino acid changes in the NTD of Variants of Concern (VoC) shows antigenic pressure, which can be an indication of NTD-mediated receptor binding. Trimeric NTD proteins of SARS-CoV-2, Alpha, Beta, Delta, and Omicron did not reveal a receptor binding capability. Unexpectedly, the SARS-CoV-2 Beta subvariant strain (501Y.V2-1) NTD binding to Vero E6 cells was sensitive to sialidase pretreatment. Glycan microarray analyses identified a putative 9-O-acetylated sialic acid as a ligand, which was confirmed by catch-and-release ESI-MS, STD-NMR analyses, and a graphene-based electrochemical sensor. The Beta (501Y.V2-1) variant attained an enhanced glycan binding modality in the NTD with specificity towards 9-O-acetylated structures, suggesting a dual-receptor functionality of the SARS-CoV-2 S1 domain, which was quickly selected against. These results indicate that SARS-CoV-2 can probe additional evolutionary space, allowing binding to glycan receptors on the surface of target cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=76 HEIGHT=200 SRC="FIGDIR/small/507904v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1f98a70org.highwire.dtl.DTLVardef@1efc119org.highwire.dtl.DTLVardef@16e8bc6org.highwire.dtl.DTLVardef@9a74e6_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisCoronaviruses utilize their N-terminal domain (NTD) for initial reversible low-affinity interaction to (sialylated) glycans. This initial low-affinity/high-avidity engagement enables viral surfing on the target membrane, potentially followed by a stronger secondary receptor interaction. Several coronaviruses, such as HKU1 and OC43, possess a hemagglutinin-esterase for viral release after sialic acid interaction, thus allowing viral dissemination. Other coronaviruses, such as MERS-CoV, do not possess a hemagglutinin-esterase, but interact reversibly to sialic acids allowing for viral surfing and dissemination. The early 501Y.V2-1 subvariant of the Beta SARS-CoV-2 Variant of Concern has attained a receptor-binding functionality towards 9-O-acetylated sialic acid using its NTD. This binding functionality was selected against rapidly, most likely due to poor dissemination. Ablation of sialic acid binding in more recent SARS-CoV-2 Variants of Concern suggests a fine balance of sialic acid interaction of SARS-CoV-2 is required for infection and/or transmission.

molecular biology↗

bioRxiv2022

Regulatory and evolutionary annotation identifies KAN1, MYB44 and PIF4 as regulators of Arabidopsis lincRNAs expressed in root

Thousands of long intergenic noncoding RNAs (lincRNAs) have been identified in plant genomes. While some lincRNAs have been characterized as important regulators in different biological processes, little is known about the transcriptional regulation for most plant lincRNAs. Through the integration of eight annotation resources, we defined 6,599 high-confidence lincRNA loci in Arabidopsis thaliana. For lincRNAs belonging to different evolutionary age categories, we identified major differences in sequence and chromatin features, as well as in the level of conservation and purifying selection acting during evolution. Spatiotemporal gene expression profiles combined with transcription factor (TF) chromatin immunoprecipitation data were used to construct a TF- lincRNA regulatory network containing 2,659 lincRNAs and 15,686 interactions. We found that properties characterizing lincRNA expression, conservation and regulation differ between plants and animals. Experimental validation confirmed the role of three TFs, KAN1, MYB44, and PIF4, as key regulators controlling root- specific lincRNA expression, demonstrating the predictive power of our network. Furthermore, we identified 58 lincRNAs, regulated by these TFs, showing strong root cell-type specific expression or chromatin accessibility, which are linked with GWAS genetic associations related to root system development and growth. The multi-level genome-wide characterization covering chromatin state information, promoter conservation, and ChIP-based TF binding, for all detectable lincRNAs across 769 expression samples, permits to rapidly define the biological context and relevance of lincRNAs in Arabidopsis regulatory networks. One-line sentenceA multi-level Arabidopsis gene regulatory network identifies novel regulators controlling root-specific lincRNA expression, offering a promising strategy to identify lincRNAs involved in plant biology.

plant biology↗

bioRxiv2022

Resistance of SARS-CoV-2 Omicron Subvariant BA.4.6 to Antibody Neutralization

SARS-CoV-2 Omicron subvariants BA.4.6, BA.4.7, and BA.5.9 have recently emerged, and BA.4.6 appears to be expanding even in the presence of BA.5 that is globally dominant. Compared to BA.5, these new subvariants harbor a mutation at R346 residue in the spike glycoprotein, raising concerns for further antibody evasion. We compared the viral receptor binding affinity of the new Omicron subvariants with BA.5 by surface plasmon resonance. We also performed VSV-based pseudovirus neutralization assays to evaluate their antigenic properties using sera from individuals who received three doses of a COVID-19 mRNA vaccine (boosted) and patients with BA.1 or BA.2 breakthrough infection, as well as using a panel of 23 monoclonal antibodies (mAbs). Compared to the BA.5 subvariant, BA.4.6, BA.4.7, and BA.5.9 showed similar binding affinities to hACE2 and exhibited similar resistance profiles to boosted and BA.1 breakthrough sera, but BA.4.6 was slightly but significantly more resistant than BA.5 to BA.2 breakthrough sera. Moreover, BA.4.6, BA.4.7, and BA.5.9 showed heightened resistance over to a class of mAbs due to R346T/S/I mutation. Notably, the authorized combination of tixagevimab and cilgavimab completely lost neutralizing activity against these three subvariants. The loss of activity of tixagevimab and cilgavimab against BA.4.6 leaves us with bebtelovimab as the only therapeutic mAb that has retained potent activity against all circulating forms of SARS-CoV-2. As the virus continues to evolve, our arsenal of authorized mAbs may soon be depleted, thereby jeopardizing the wellbeing of millions of immunocompromised persons who cannot robustly respond to COVID-19 vaccines.

microbiology↗

bioRxiv2022

Heterologous boost with mRNA vaccines against SARS-CoV-2 Delta/Omicron variants following an inactivated whole-virus vaccine

The coronavirus SARS-CoV-2 has mutated quickly and caused significant global damage. This study characterizes two mRNA vaccines ZSVG-02 (Delta) and ZSVG-02-O (Omicron BA.1), and associating heterologous prime-boost strategy following the prime of a most widely administrated inactivated whole-virus vaccine (BBIBP-CorV). The ZSVG-02-O induces neutralizing antibodies that effectively cross-react with Omicron subvariants following an order of BA.1>BA.2>BA.4/5. In naive animals, ZSVG-02 or ZSVG-02-O induce humoral responses skewed to the vaccines targeting strains, but cellular immune responses cross-react to all variants of concern (VOCs) tested. Following heterologous prime-boost regimes, animals present comparable neutralizing antibody levels and superior protection across all VOCs. Single-boost only generated ancestral and omicron dual-responsive antibodies, probably by "recall" and "reshape" the prime immunity. New Omicron-specific antibody populations, however, appeared only following the second boost with ZSVG-02-O. Overall, our results support a heterologous boost with ZSVG-02-O, providing the best protection against current VOCs in inactivated virus vaccine- primed populations.

molecular biology↗

bioRxiv2022

IL-33 Involved in the Progression of Liver Fibrosis Through Innate Immunity Cells (MΦ) Regulated by ICOS/ICOSL Signaling in Early Stage of Mice Schistosomiasis

Schistosomiasis liver fibrosis is characterized by the foci of a classical type 2 granulomatous inflammation in response to egg soluble antigen. The inducible costimulator (ICOS)/ICOSL signaling can mediate Th2 polarization and regulate the disease process of chronic schistosomiasis by adaptive immune response. ICOS/ICOSL signaling could regulate innate immune cells, including B cells and macrophage (M{varphi}), and participate in pulmonary fibrosis. The purpose of the study was to identify whether ICOS/ICOSL signaling could regulate the polarization of M{varphi}, and to explore the potential role of IL-33 in ICOSL knockout (ICOSL-KO) mice with schistosomiasis. Firstly, the expression level of CD86, CD206 and IL-33 was checked respectively in ICOSL-KO and WT mice infected with Schistosoma japonicum (S. japonicum). Then recombinant IL-33 (rIL-33) was injected into ICOSL-KO mice infected with S. japonicum. Our experiments showed that the liver M{varphi} was successfully polarized toward to classical activated M{varphi} (M1) and the expression of IL-33 was inhibited in ICOSL-KO mice. Furthermore, the injection of rIL-33 successfully aggravated liver fibrosis in ICOSL-KO mice, increased the numbers of lymphocyte antigen 6C (Ly6C)hi, enhanced the expression of C-C chemokine ligand (CCL)2,, CCL5 and C-X-C motif chemokine 2 (CXCL2), and promoted polarization of T helper (Th) cells to Th2 cells, as well as induced the autophagy and apoptosis of hepatic stellate cells (HSCs). Overall, the liver fibrosis was alleviated in ICOSL-KO mice along with the reduced expression of IL-33, which could skew the polarization of M{varphi} toward to M1, induce Th cells activation, HSCs apoptosis and autophagy through Smad2/3 and TGF-{beta} signaling pathway, thus participated in the homeostasis of liver fibrosis of schistosomiasis.

immunology↗

bioRxiv2022

An outer nuclear membrane protein promotes a polarized nuclear position and the future division plane during asymmetric cell division

Asymmetric cell division generates new cell types and is a feature of development in multicellular organisms. Prior to asymmetric cell division, cell polarity is established. Zea mays stomatal development serves as an excellent plant model system for asymmetric cell division, especially the asymmetric division of the subsidiary mother cell (SMC). In SMCs, the nucleus migrates to a polar location after the accumulation of polarly localized proteins, but before the appearance of the preprophase band. We examined a mutant of the outer nuclear membrane protein, which is part of the LINC (linker of nucleoskeleton and cytoskeleton) complex that localizes to the nuclear envelope in interphase cells. Previously, mlks2 (maize linc kash sine-like2) was observed to have abnormal stomata. We confirmed and identified the precise defects that lead to abnormal asymmetric divisions. Proteins that are polarly localized in SMCs prior to division polarize normally in mlks2. However, polar localization of the nucleus is sometimes impaired, even in cells that have otherwise normal polarity. This leads to a misplaced preprophase band and atypical division planes. MLKS2 is localized to mitotic structures, however the structure of the preprophase band, spindle and phragmoplast appeared normal in mlks2. Timelapse imaging revealed that mlks2 has defects in pre-mitotic nuclear migration towards the polarized site, and unstable position at the division site after formation of the preprophase band. We show that nuclear envelope proteins promote pre-mitotic nuclear migration and stable nuclear position, and that the position of the nucleus influences division plane establishment in asymmetrically dividing cells. One-sentence summaryNuclear movement and positioning prior to asymmetric cell division is required for deciding the future division site.

plant biology↗

bioRxiv2022

Dissecting the co-segregation probability from genome architecture mapping

The genome architecture mapping (GAM) is a recently developed methodology that offers the co-segregation probability of two genomic segments from an ensemble of thinly sliced nuclear profiles, enabling to probe and decipher the 3D chromatin organization. The co-segregation probability from GAM, which typically probes the length scale associated with the genomic separation greater than 1 MB, is, however, not identical to the contact probability obtained in Hi-C, and its correlation with inter-locus distance measured with FISH is not so good as the contact probability. In this study, by using a polymer-based model of chromatins, we derive a theoretical expression of the co-segregation probability as well as that of the contact probability, and carry out quantitative analyses of how they differ from each other. The results from our study, validated with in-silico GAM analysis on 3D genome structures from FISH, suggest that to attain strong correlation with the inter-locus distance, a properly normalized version of co-segregation probability needs to be calculated based on a large number of nuclear slices (n > 103). SIGNIFICANCEBy leveraging a polymer model of chromatin, we critically assess the utility of co-segregation probability captured from GAM analysis. Our polymer model, which offers analytical expressions for the co-segregation probability as well as for the contact probability and inter-locus distance, enables quantitative comparison between the data from GAM, Hi-C, and FISH. Although the plain co-segregation probabilities from GAM are not well correlated with inter-locus distances measured from FISH, properly normalized versions of the probability calculated from a large number of nuclear profiles can still reasonably represent the inter-locus distance. Our study offers instructions of how to take full advantage of GAM analysis in deciphering 3D genome organization.

biophysics↗

bioRxiv2022

Combinatorial perturbation sequencing on single cells using microwell-based droplet random pairing

Combinatorial drug therapy reduces drug resistance and disease relapse, but informed drug combinations are lacking due to the high scale of possible combinations and the relatively simple phenotyping strategies. Here we report combinatorial perturbation sequencing (CP-seq) on single cells using microwell-base droplet random pairing. CP-seq uses oligonucleotides to barcode drugs, encapsulates drugs and cells in separate droplets, and pairs cell droplets with two drug droplets randomly on a microwell array chip to complete combinatorial drug treatment and barcode-tagging on cells. The subsequent single-cell RNA sequencing simultaneously detects the single-cell transcriptomes and drug barcodes to demultiplex the corresponding drug treatment. The microfluidic droplet operations had robust performance, with overall success rate among the microwells being up to 83%. We then progressively validated the CP-seq by performing single drug treatment and then combinatorial drug treatment. Leveraging the advantage of droplet microfluidics in massive multiplexing, the CP-seq can test thousands of drug combinations in a single experiment and represents a great technology for combinatorial perturbation screening with high throughput and comprehensive profiling.

bioengineering↗