Biology subjects

Ren, Q.

Publications and source records attributed to Ren, Q..

32 records · Page 2Linked to original sources

bioRxiv2022

Evidence for the role of music on the growth and signal response in duckweed

Sound vibration, an external mechanical force, has been proved to modulate plant growth and development like rain, wind, and vibration. However, the role of music on plants, especially on signal response, has been usually neglected in research. Herein, we investigated the growth state, gene expression, and signal response in duckweed treated with soft music. The protein content in duckweed after music treatment for 7 days was about 1.6 times that in duckweed without music treatment. Additionally, Fv/Fm in duckweed treated with music was 0.78, which was significantly higher in comparison with the control group (P < 0.01). Interestingly, music promoted the Glu and Ca signaling response. To further explore the global molecular mechanism, we performed transcriptome analysis. A total of 1296 DEGs were found for all these investigated genes in duckweed treated with music compared to the control group. Among these, up-regulation of the expression of metabolism-related genes related to glycolysis, cell wall biosynthesis, oxidative phosphorylation, and pentose phosphate pathways were found. Overall, these results provided a molecular basis to music-triggered signal response, transcriptomic, and growth changes in duckweed, which also highlighted the potential of music as an environmentally friendly stimulus to promote improved protein production in duckweed.

plant biology↗

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↗

bioRxiv2021

The framework of plant regeneration in duckweed (Lemna turonifera) comprises genetic transcript regulation and cyclohexane release

Regeneration is important for vegetative propagation of excellent variety, detoxification and the obtain of transgenic plant, but plant regeneration is time-consuming. Here, we found that duckweed regeneration could be enhanced by regenerating callus. Firstly, Genetic transcript regulation has been applied to study the molecular mechanism controlling regeneration. Auxin related genes have been significantly down-regulated in regenerating callus. Cytokinin signal pathway genes have been up-regulated in regenerating callus. Secondly, volatile organic compounds release has been analysised by gas chromatography/mass spectrum during the stage of plant regeneration, and 11 kinds of unique volatile organic compounds in the regenerating callus were increased. Among them, cyclohexane treatment enhanced duckweed regeneration by initiating root. Moreover, Auxin signal pathway genes were down-regulated in callus treated by cyclohexane. All together, these results provide novel mechanistic insights into how regenerating callus promotes duckweed regeneration. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/453434v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1c28166org.highwire.dtl.DTLVardef@bd86cforg.highwire.dtl.DTLVardef@dd9802org.highwire.dtl.DTLVardef@26ba1c_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2020

KCC2 is required for the survival of mature neurons but not for their development

The K+/Cl- co-transporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl- levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A {gamma}-aminobutyric acid receptors GABAARs. In accordance with this, compromised KCC2 activity results in seizures but whether such deficits directly contribute to the subsequent changes in neuronal viability that lead to epileptogenesis, remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression resulted in the rapid activation of the extrinsic apoptotic pathway, in mature hippocampal neurons. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization or dendritic structure. However ablating KCC2 expression in immature neurons was sufficient to prevent the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to loss of KCC2 function. In contrast KCC2 appears to play a minimal role in mediating neuronal development or architecture.

neuroscience↗

bioRxiv2020

MicroRNA-1 promotes the development of and prolongs engorgement time in Hyalomma anatolicum anatolicum (Acari: Ixodidae) ticks

MicroRNAs act as mRNA posttranscriptional regulators, playing important roles in cell differentiation, transcriptional regulation, growth and development. In this study, microRNA expression profiles of Hyalomma anatolicum anatolicum ticks at different developmental stages were detected by high-throughput sequencing and functionally assessed. In total, 2,585,169, 1,252,678, 1,558,217 and 1,155,283 unique reads were obtained from eggs, larvae, nymphs and adults, respectively, with 42, 46, 45 and 41 conserved microRNAs in these stages, respectively. Using eggs as a control, 48, 43 and 39 microRNAs were upregulated and 3, 10 and 9 downregulated in larvae, nymphs and adults, respectively. microRNA-1 (miR-1) was expressed in high abundance throughout Ha. anatolicum development, with an average of nearly one million transcripts, and it is highly conserved among tick species. Quantitative real-time PCR (qPCR) showed that miR-1 expression gradually increased with tick development, reaching the highest level at engorgement. Differential tissue expression was detected, with significantly higher levels in the salivary glands and epidermis than in the midgut. Inhibition assays showed no significant change in body weight or spawning time or amount between experimental and control groups, but there was a significant difference (p<0.01) in engorgement time. With miR-1 inhibition, ticks displayed obvious deformities during later development. To more fully explain the microRNA mechanism of action, the miR-1 family was analyzed regarding target gene; members acting on Hsp60 include miR-5, miR-994, miR-969, and miR-1011, which jointly play a role. Therefore, microRNAs are critical for normal tick development, and the primary structure of the mature sequence of miR-1 is highly conserved. Nonetheless, different developmental stages and tissues show different expression patterns, with a certain role in prolonging feeding. miR-1, together with other family members, regulates mRNA function and may be used as a molecular marker for species origin and evolution analysis and internal reference gene selection.

microbiology↗

bioRxiv2020

Isolation and characterization of multi-protein complexes enriched in the K-Cl co-transporter 2 from brain plasma membranes

Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanism neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of Kcc2 from the plasma membrane of murine forebrain. We resolved these using Blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS. Purified Kcc2 migrated as distinct molecular species of 300, 600 and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N and C-termini of Kcc2 was obtained. The 300kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. Intriguingly, lower levels of Kcc1 were also found in this species suggesting the existence of "mixed" Kcc2/Kcc1 heterodimers. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed association of Kcc2. These included spectrins, ankyrins, and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5 and Lmtk3. Finally, we used LC-MS/MS on highly purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity.

neuroscience↗

bioRxiv2020

A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation

The isocortex and hippocampal formation are two major structures in the mammalian brain that play critical roles in perception, cognition, emotion and learning. Both structures contain multiple regions, for many of which the cellular composition is still poorly understood. In this study, we used two complementary single-cell RNA-sequencing approaches, SMART-Seq and 10x, to profile [~]1.2 million cells covering all regions in the adult mouse isocortex and hippocampal formation, and derived a cell type taxonomy comprising 379 transcriptomic types. The completeness of coverage enabled us to define gene expression variations across the entire spatial landscape without significant gaps. We found that cell types are organized in a hierarchical manner and exhibit varying degrees of discrete or continuous relatedness with each other. Such molecular relationships correlate strongly with the spatial distribution patterns of the cell types, which can be region-specific, or shared across multiple regions, or part of one or more gradients along with other cell types. Glutamatergic neuron types have much greater diversity than GABAergic neuron types, both molecularly and spatially, and they define regional identities as well as inter-region relationships. For example, we found that glutamatergic cell types between the isocortex and hippocampal formation are highly distinct from each other yet possess shared molecular signatures and corresponding layer specificities, indicating their homologous relationships. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation for the first time, and begins to shed light on its underlying relationship with the development, evolution, connectivity and function of these two brain structures.

neuroscience↗

bioRxiv2020

The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products.

KCC2 plays a critical role in determining the efficacy of synaptic inhibition and deficits in its activity lead to epilepsy and neurodevelopmental delay. Here we use unbiased proteomic analyses to demonstrate that KCC2 forms stable protein complexes in the neuronal plasma membrane with 96 autism and/or epilepsy risk gene (ASD/Epi) products including ANKB, ANKG, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1, and SPTBN1. Many of these proteins are also targets of Fragile-X mental retardation protein (FMRP), the inactivation of which is the leading monogenic cause of autism. Accordingly, the expression of a subset of these KCC2-binding partners was decreased in Fmr1 knockout mice. Fmr1 knockout compromised KCC2 phosphorylation, a key regulatory mechanism for transporter activity and the postnatal development of GABAergic inhibition. Thus, KCC2 is a point of convergence for multiple ASD/Epi risk genes and therapies targeting this transporter may have broad utility in alleviating these heterogeneous disorders and their associated epilepsies.

neuroscience↗

bioRxiv2020

An integrated model system to gain mechanistic insights into biofilm formation and antimicrobial resistance development in Pseudomonas aeruginosa MPAO1

Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and missed genes by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq datasets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth. Experiments conducted across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated both known and novel genes involved in biofilm growth and antibiotic resistance identified in screens of the mutant collection. Differential protein expression data from planktonic cells versus biofilm confirmed upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type six secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance and resistance evolution.

systems biology↗

bioRxiv2019

Single-cell transcriptome profiling reveals neutrophil heterogeneity and orchestrated maturation during homeostasis and bacterial infection

The full neutrophil heterogeneity and differentiation landscape remains incompletely characterized. Here we profiled >25,000 differentiating and mature mouse neutrophils using single-cell RNA sequencing to provide a comprehensive transcriptional landscape of neutrophil maturation, function, and fate decision in their steady state and during bacterial infection. Eight neutrophil populations were defined by distinct molecular signatures. The three mature peripheral blood neutrophil subsets arise from distinct maturing bone marrow neutrophil subsets. Driven by both known and uncharacterized transcription factors, neutrophils gradually acquire microbicidal capability as they traverse the transcriptional landscape, representing an evolved mechanism for fine-tuned regulation of an effective but balanced neutrophil response. Bacterial infection reprograms the genetic architecture of neutrophil populations, alters dynamic transition between each subpopulation, and primes neutrophils for augmented functionality without affecting overall heterogeneity. In summary, these data establish a reference model and general framework for studying neutrophil-related disease mechanisms, biomarkers, and therapeutic targets at single-cell resolution.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC=\"FIGDIR/small/792200v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (28K):\norg.highwire.dtl.DTLVardef@18fed35org.highwire.dtl.DTLVardef@1e68b7org.highwire.dtl.DTLVardef@6cfbc8org.highwire.dtl.DTLVardef@1061a26_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA comprehensive single-cell resolution transcriptional landscape of mouse neutrophil maturation and fate decision under steady-state and bacterial infection conditions.\nC_LIO_LIThe pathogen clearance machinery in neutrophils is continuously and gradually built during neutrophil differentiation, maturation, and aging, driven by both known and uncharacterized transcription factors.\nC_LIO_LIThe three mature neutrophil subsets in peripheral blood, including a novel ISG-expressing subset, are derived from distinct bone marrow neutrophil precursors.\nC_LIO_LIBacterial infection reprograms the genetic architecture of neutrophil populations, alters dynamic transition between each subpopulation, and primes neutrophils for augmented functionality without affecting overall neutrophil heterogeneity.\nC_LIO_LIBacterial infection-induced emergency granulopoiesis is mediated by augmented proliferation of early stage neutrophil progenitors and accelerated post-mitotic maturation.\nC_LI

immunology↗

bioRxiv2019

Enhanced Golic+: Gene targeting with 100% recovery in Drosophila male germ cells

The efficiency of gene targeting can vary drastically. Even utilizing CRISPR/Cas9 does not ensure rapid, successful gene targeting. In Drosophila, we previously established Golic+ to augment gene-targeting productivity simply through fly pushing. This transgene-based system elicits gene targeting in germ cells. It further implements lethality selection to enrich for progeny with accurate gene targeting. However, limitations still remain. Here we deliver an improved Golic+ technique that we name Enhanced Golic+ (E-Golic+). E-Golic+ incorporates genetic modifications to eliminate false positives while simultaneously boosting efficiency. Strikingly, male germ cells are exceptionally susceptible to gene targeting using E-Golic+. With male founders, we easily achieve 100% recovery of correct gene targeting. Given the superior efficiency/specificity and relatively effortless scalability, E-Golic+ promises to triumph in any challenging and otherwise unattainable gene targeting projects in Drosophila.

genetics↗

bioRxiv2019

Conservation and Divergence of Related Neuronal Lineages in the Drosophila Central Brain

Wiring a complex brain requires enormous cell specificity. This specificity is laid out via a developmental process where neural stem cells produce countless diverse neurons. To help elucidate this process and resolve the considerable dynamic specificity, we need to observe the development of multiple neuronal lineages. Drosophila central brain lineages are predetermined, comprised of a fixed set of neurons born in pairs in a specific order. To reveal specific roles of lineage identity, Notch-dependent sister fate specification, and temporal patterning in morphological diversification, we mapped approximately one quarter of the Drosophila central brain lineages. While we found large aggregate differences, we also discovered similar patterns of morphological specification and diversification. Lineage identity plus Notch state govern primary neuronal trajectories, whereas temporal fates diversify terminal elaborations in target-specific manners. In addition, we identified related lineages of analogous neuron types produced in similar temporal patterns. Two stem cells even yield identical series of dopaminergic neuron types, but with completely disparate sister neurons. These phenomena suggest that large changes in morphological diversity can be the consequence of relatively small differences in lineage fating. Taken together, this large-scale lineage mapping study reveals that relatively simple rules drive incredible neuronal complexity.

developmental biology↗

bioRxiv2018

A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 nucleases in rice

Targeting specificity has been an essential issue for applying genome editing systems in functional genomics, precise medicine and plant breeding. Understanding the scope of off-target mutations in Cas9 or Cpf1-edited crops is critical for research and regulation. In plants, only limited studies had used whole-genome sequencing (WGS) to test off-target effects of Cas9. However, the cause of numerous discovered mutations is still controversial. Furthermore, WGS based off-target analysis of Cpf1 has not been reported in any higher organism to date. Here, we conducted a WGS analysis of 34 plants edited by Cas9 and 15 plants edited by Cpf1 in T0 and T1 generations along with 20 diverse control plants in rice, a major food crop with a genome size of ~380 Mb. The sequencing depth ranged from 45X to 105X with reads mapping rate above 96%. Our results clearly show that most mutations in edited plants were created by tissue culture process, which caused ~102 to 148 single nucleotide variations (SNVs) and ~32 to 83 insertions/deletions (indels) per plant. Among 12 Cas9 single guide RNAs (sgRNAs) and 3 Cpf1 CRISPR RNAs (crRNAs) assessed by WGS, only one Cas9 sgRNA resulted in off-target mutations in T0 lines at sites predicted by computer programs. Moreover, we cannot find evidence for bona fide off-target mutations due to continued expression of Cas9 or Cpf1 with guide RNAs in T1 generation. Taken together, our comprehensive and rigorous analysis of WGS big data across multiple sample types suggests both Cas9 and Cpf1 nucleases are very specific in generating targeted DNA modifications and off-targeting can be avoided by designing guide RNAs with high specificity.

molecular biology↗

bioRxiv2017

Stem cell intrinsic, Seven-up-triggered temporal factor gradients diversify intermediate neural progenitors

Drosophila type II neuroblasts produce numerous neurons and glia due to transiently amplifying, intermediate neural progenitors (INP). Consecutively born INPs produce morphologically distinct progeny, presumably due to temporal patterning in type II neuroblasts. We therefore profiled type II neuroblasts transcriptome across time. Our results reveal opposing temporal gradients of Imp and Syp RNA-binding proteins (descending and ascending, respectively). Maintaining Imp expression throughout brain development expands the number of neurons/glia with early temporal fate at the expense of cells with late fate. Conversely, precocious upregulation of Syp reduces the number of cells with early fate. Further, we reveal that the transcription factor, Seven-up initiates progression of the Imp/Syp gradients. Interestingly, genetic manipulations that fix Imp or Syp levels still yield progeny with a small range of early fates. We propose that the Seven-up-initiated Imp/Syp gradients create coarse temporal windows within type II neuroblasts to pattern INPs, which subsequently undergo fine-tuned subtemporal patterning.

neuroscience↗