Biology subjects

Gao, L.

Publications and source records attributed to Gao, L..

At least 181 records · Page 10Linked to original sources

bioRxiv2020

Yellow-Fruited Phenotype is Caused by an Insertion Event at 5' UTR of YFT1 Allele in yft1 Mutant Tomato

The yft1 tomato mutant has a yellow-fruited phenotype controlled by a recessive gene (YFT1), which has been shown by map-based cloning to be a homolog of ETHYLENE INSENSITIVE 2 (EIN2). The genetic lesion of the YFT1 allele of yft1 is attributed to a 573 bp DNA fragment (IF573) insertion at 1,200 bp downstream of the transcription start site (TSS). Transcriptome analysis revealed that the mutation resulted in 5,053 differentially expressed genes (DEGs) in the fruit pericarp compared with the M82 wild type cultivar. These were annotated as being involved in ethylene synthesis, chromoplast development, and carotenoid synthesis. Genetic lesion in YFT1 caused a reduction in its own transcript levels in yft1 and impaired ethylene emission and signal transduction, delayed chromoplast development and decreased carotenoid accumulation. The molecular mechanism underlying the reduced expression of YFT1 in yft1 was examined at both the RNA and DNA levels. The IF573 event was shown to introduce two negative regulatory sequences located at -272 to -173 bp and -172 to -73 bp in the YFT1 allele promoter, causing alterative splicing due to aberrant splicing sites, and also altering the structure of the open reading frame in the 5-UTR. This study contributes to the understanding of color formation in tomato fruit. One-sentence summaryLesion happened in regulatory region impairs expression of a key gene of ethylene signal pathway, and alters fruit color in tomato due to effect of carotenoids accumulation and ethylene synthesis.

plant biology↗

bioRxiv2020

Bottom-Illuminated Orbital Shaker for Microalgae Cultivation

A bottom-illuminated orbital shaker designed for the cultivation of microalgae suspensions is described in this open-source hardware report. The instrument agitates and illuminates microalgae suspensions grown inside flasks. It was optimized for low production cost, simplicity, low power consumption, design flexibility, consistent, and controllable growth light intensity. The illuminated orbital shaker is especially well suited for low-resource research laboratories and education. It is an alternative to commercial instruments for microalgae cultivation. It improves on typical do-it-yourself microalgae growth systems by offering consistent and well characterized illumination light intensity. The illuminated growth area is 20 cm x 15 cm, which is suitable for three T75 tissue culture flasks or six 100 ml Erlenmeyer flasks. The photosynthetic photon flux density, is variable in eight steps (26 - 800 mol {middle dot} m-2 {middle dot} s-1) and programmable in a 24-hour light/dark cycle. The agitation speed is variable (0 - 210 RPM). The overall material cost is around {pound}300, including an entry-level orbital shaker. The build takes two days, requiring electronics and mechanical assembly capabilities. The instrument build is documented in a set of open-source protocols, design files, and source code. The design can be readily modified, scaled, and adapted for other orbital shakers and specific experimental requirements. The instrument function was validated by growing fresh-water microalgae Desmodesmus quadricauda and Chlorella vulgaris. The cultivation protocols, microalgae growth curves, and doubling times are included in this report. Specifications table O_TBL View this table: org.highwire.dtl.DTLVardef@23c347org.highwire.dtl.DTLVardef@1e96f84org.highwire.dtl.DTLVardef@985cb5org.highwire.dtl.DTLVardef@ee9a76org.highwire.dtl.DTLVardef@dee9ab_HPS_FORMAT_FIGEXP M_TBL C_TBL

cell biology↗

bioRxiv2020

Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms

Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterization of DNMT3A and DNMT3B, we here report a multi-layered substrate-recognition mechanism underpinning their divergent genomic methylation activities. A hydrogen bond in the catalytic loop of DNMT3B causes a lower CpG specificity than DNMT3A, while the interplay of target recognition domain and homodimeric interface fine-tunes the distinct target selection between the two enzymes, with Lysine 777 of DNMT3B acting as a unique sensor of the +1 flanking base. The divergent substrate preference between DNMT3A and DNMT3B provides an explanation for site-specific epigenomic alterations seen in ICF syndrome with DNMT3B mutations. Together, this study reveals crucial and distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis.

genetics↗

bioRxiv2020

Direct readout of heterochromatic H3K9me3 regulates DNMT1-mediated maintenance DNA methylation

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.

genetics↗

bioRxiv2020

Systemic analysis of tissue cells potentially vulnerable to SARS-CoV-2 infection by the protein-proofed single-cell RNA profiling of ACE2, TMPRSS2 and Furin proteases

Single-cell RNA profiling of ACE2, the SARS-CoV-2 receptor, had proposed multiple tissue cells as the potential targets of SARS-CoV-2, the novel coronavirus causing the COVID-19 pandemic. However, most were not echoed by the patients clinical manifestations, largely due to the lack of protein expression information of ACE2 and co-factors. Here, we incorporated the protein information to analyse the expression of ACE2, together with TMPRSS2 and Furin, two proteases assisting SARS-CoV-2 infection, at single cell level in situ, which we called protein-proofed single-cell RNA (pscRNA) profiling. Systemic analysis across 36 tissues revealed a rank list of candidate cells potentially vulnerable to SARS-CoV-2. The top targets are lung AT2 cells and macrophages, then cardiomyocytes and adrenal gland stromal cells, followed by stromal cells in testis, ovary and thyroid. Whereas, the polarized kidney proximal tubule cells, liver cholangiocytes and intestinal enterocytes are less likely to be the primary SARS-CoV-2 targets as ACE2 localizes at the apical region of cells, where the viruses may not readily reach. Actually, the stomach may constitute a physical barrier against SARS-CoV-2 as the acidic environment in normal stomach (pH < 2.0) could completely inactivate SARS-CoV-2 pseudo-viruses. These findings are in concert with the clinical characteristics of prominent lung symptoms, frequent heart injury, and uncommon intestinal symptoms and acute kidney injury. Together, we provide a comprehensive view on the potential SARS-CoV-2 targets by pscRNA profiling, and propose that, in addition to acute respiratory distress syndrome, attentions should also be paid to the potential injuries in cardiovascular, endocrine and reproductive systems during the treatment of COVID-19 patients.

cell biology↗

bioRxiv2020

Collapse of the hepatic gene regulatory network in the absence of FoxA factors

The FoxA transcription factors are critical for liver development through their pioneering activity, which initiates a highly complex regulatory network thought to become progressively resistant to the loss of any individual hepatic transcription factor via mutual redundancy. To investigate the dispensability of FoxA factors for maintaining this regulatory network, we ablated all FoxA genes in the adult mouse liver. Remarkably, loss of FoxA caused rapid hepatocyte dedifferentiation manifested by a massive reduction in the expression of key liver genes. Interestingly, expression of these genes was reduced back to the low levels of the fetal prehepatic endoderm stage, leading to necrosis and lethality within days. Mechanistically, we found FoxA proteins to be required for maintaining enhancer activity, chromatin accessibility, nucleosome positioning and binding by HNF4. Thus, the FoxA factors act continuously, guarding hepatic enhancer activity throughout life.

genetics↗

bioRxiv2020

CytoTalk: De novo construction of signal transduction networks using single-cell RNA-Seq data

Single-cell technology has opened the door for studying signal transduction in a complex tissue at unprecedented resolution. However, there is a lack of analytical methods for de novo construction of signal transduction pathways using single-cell omics data. Here we present CytoTalk, a computational method for de novo constructing cell type-specific signal transduction networks using single-cell RNA-Seq data. CytoTalk first constructs intracellular and intercellular gene-gene interaction networks using an information-theoretic measure between two cell types. Candidate signal transduction pathways in the integrated network are identified using the prize-collecting Steiner forest algorithm. We applied CytoTalk to a single-cell RNA-Seq data set on mouse visual cortex and evaluated predictions using high-throughput spatial transcriptomics data generated from the same tissue. Compared to published methods, genes in our inferred signaling pathways have significantly higher spatial expression correlation only in cells that are spatially closer to each other, suggesting improved accuracy of CytoTalk. Furthermore, using single-cell RNA-Seq data with receptor gene perturbation, we found that predicted pathways are enriched for differentially expressed genes between the receptor knockout and wild type cells, further validating the accuracy of CytoTalk. In summary, CytoTalk enables de novo construction of signal transduction pathways and facilitates comparative analysis of these pathways across tissues and conditions.

bioinformatics↗

bioRxiv2020

The mouse cortico-tectal projectome

The superior colliculus (SC) is a midbrain structure that receives diverse and robust cortical inputs to drive a range of cognitive and sensorimotor behaviors. However, it remains unclear how descending cortical inputs arising from higher-order associative areas coordinate with SC sensorimotor networks to influence its outputs. In this study, we constructed a comprehensive map of all cortico-tectal projections and identified four collicular zones with differential cortical inputs: medial (SC.m), centromedial (SC.cm), centrolateral (SC.cl) and lateral (SC.l). Computational analyses revealed that cortico-tectal projections are organized as multiple subnetworks that are consistent with previously identified cortico-cortical and cortico-striatal subnetworks. Furthermore, we delineated the brain-wide input/output organization of each collicular zone and described a subset of their constituent neuronal cell types based on distinct connectional and morphological features. Altogether, this work provides a novel structural foundation for the integrative role of the SC in controlling cognition, orientation, and other sensorimotor behaviors.

neuroscience↗

bioRxiv2020

AfRip3, a RIP3-like kinase, is identified as a key modulator of necroptotic death in Aspergillus fumigatus

Aspergillus fumigatus exhibits autophagic and necroptotic process when its GPI anchor synthesis is suppressed. A putative kinase (AFUA_6G02590) is found to be overexpressed in response to GPI anchor suppression and identified as a RIP3-like protein, namely AfRip3. To elucidate its function, in this study a Afrip3-overexpressing strain OE-Afrip3 was constructed. Although OE-Afrip3 strain exhibited an increased cell death, neither apoptotic nor autophagic process was activated. Our evidences demonstrated that overexpression of Afrip3 gene in A. fumigatus only led to necroptosis, while the Afrip3-knockout mutant was unable to activate necroptotic process. Further analysis revealed that both JNK and SMase pathways were activated in OE-Afrip3 strain, by which an increase of reactive oxygen species (ROS) was induced. We also showed that expression of Afrip3 gene was induced by Ca2+. In addition, eEF1B{gamma} and adenylylsulfate kinase (ASK) were identified as potential candidates to interact with AfRip3. These results indicate that AfRip3 is a key modulator that activates necroptotic process in A. fumigatus, which can be induced by Ca2+ and in turn activate JNK (c-Jun NH2-terminal kinase) and SMase (sphingomyelinase) pathway. Our findings suggest that necroptotic pathway in A. fumigatus is distinct from that in mammalian cell and may provide a new strategy for development of anti-fungal drug. Author summaryAspergillus fumigatus is a human fungal pathogen and causes invasive aspergillosis (IA) in immunocompromised patients with high mortality (30-95%). Development of novel therapies is urgently needed. In this study, we confirm AfRip3 (AFUA_6G02590), a RIP3-like protein, is a key modulator that activates necroptotic process in A. fumigatus. We also find that cytosolic Ca2+ can induce the expression of Afrip3 and activated AfRip3 in turn activate JNK (c-Jun NH2-terminal kinase) and SMase (sphingomyelinase) pathway. Our findings suggest that necroptotic pathway in A. fumigatus is distinct from that in mammalian cell and may provide a new strategy for development of anti-fungal drug.

microbiology↗

bioRxiv2020

Glucose-mediated expansion of a gut commensal bacterium promotes Plasmodium infection through alkalizing mosquito midgut

SUMMARYDietary sugar is the major energy source for mosquitoes, but its influence on mosquitoes capability to transmit malaria parasite remains unclear. Here we show that Plasmodium berghei infection changes global metabolism of Anopheles stephensi with the most significant impact on glucose metabolism. Supplementation of glucose or trehalose (the main hemolymph sugar) to mosquito increases Plasmodium infection by alkalizing the mosquito midgut. The glucose/trehalose diets promote rapid growth of a commensal bacterium, Asaia bogorensis, which remodels glucose metabolism and consequently increases midgut pH. The pH increase in turn promotes Plasmodium gametogenesis. We also demonstrate the sugar composition from different natural plants influences A. bogorensis growth and Plasmodium infection is associated with their capability to expand A. bogorensis. Altogether, our results demonstrate that dietary glucose is an important factor that determines mosquitos competency to transmit Plasmodium and further highlight a key role for mosquito-microbiota metabolic interactions in regulating development of malaria parasite.

microbiology↗

bioRxiv2020

The Intergenic Small Non-Coding RNA ittA is Required for Optimal Infectivity and Tissue Tropism in Borrelia burgdorferi

Post-transcriptional regulation via small regulatory RNAs (sRNAs) has been implicated in diverse regulatory processes in bacteria, including virulence. One class of sRNAs, termed trans-acting sRNAs, can affect the stability and/or the translational efficiency of regulated transcripts. In this study, we utilized a collaborative approach that employed data from infection with the Borrelia burgdorferi Tn library, coupled with Tn-seq, together with borrelial sRNA and total RNA transcriptomes, to identify an intergenic trans-acting sRNA, which we designate here as ittA for infectivity-associated and tissue-tropic sRNA locus A. The genetic inactivation of ittA resulted in a significant attenuation in infectivity, with decreased spirochetal load in ear, heart, skin and joint tissues. In addition, the ittA mutant did not disseminate to peripheral skin sites or heart tissue, suggesting a role for ittA in regulating a tissue-tropic response. RNA-Seq analysis determined that 19 transcripts were differentially expressed in the ittA mutant relative to its genetic parent, including vraA, bba66, ospD and oms28 (bba74). Subsequent proteomic analyses also showed a significant decrease of OspD and Oms28 (BBA74) proteins. To our knowledge this is the first documented intergenic sRNA that alters the infectivity potential of B. burgdorferi. AUTHOR SUMMARYLyme disease is a tick-borne infection mediated by the spirochetal bacterium, Borrelia burgdorferi, that is responsible for greater than 300,000 infections in the United States per year. As such, additional knowledge regarding how this pathogen modulates its regulatory armamentarium is needed to understand how B. burgdorferi establishes and maintains infection. The identification and characterization of small, non-coding RNA molecules in living systems, designated as sRNAs, has recalibrated how we view post-transcriptional regulation. Recently, over 1,000 sRNAs were identified in B. burgdorferi. Despite the identification of these sRNAs, we do not understand how they affect infectivity or B. burgdorferi pathogenesis related outcomes. Here, we characterize the ittA B. burgdorferi sRNA and show that it is essential for optimal infection using murine experimental infection as our readout. We also track the effect of this sRNA on the transcriptional and proteomic profile as the first step in providing mechanistic insight into how this important sRNA mediates its regulatory effect.

microbiology↗

bioRxiv2020

Counteracting Genome Instability by p53-dependent Mintosis

Entosis was proposed to promote aneuploidy and genome instability by cell-in-cell mediated engulfment in tumor cells. We reported here, in epithelial cells, that entosis coupled with mitotic arrest functions to counteract genome instability by targeting aneuploid mitotic progenies for engulfment and elimination. We found that the formation of cell-in-cell structures associated with prolonged mitosis, which was sufficient to induce entosis. This process was controlled by the tumor suppressor p53 (wild type) that upregulates Rnd3 expression in response to DNA damages associated with prolonged metaphase. Rnd3 compartmentalized RhoA activities accumulated during prolonged metaphase to drive cell-in-cell formation. Remarkably, this prolonged mitosis-induced entosis (mintosis) selectively targets non-diploid progenies for internalization, blockade of which increased aneuploidy. Thus, our work uncovered a heretofore unrecognized mechanism of mitotic surveillance for entosis, which eliminates newly-born abnormal daughter cells in a p53-depedent way to maintain genome integrity.

cell biology↗

bioRxiv2020

Loss of the Er81 Transcription Factor in Cholinergic Cells Alters Striatal Activity and Habit Formation

The finely-tuned activity of cholinergic interneurons (CINs) in the striatum is key for motor control, learning, and habit formation. Yet, the molecular mechanisms that determine their unique functional properties remain poorly explored. Using a combination of genetic and biochemical assays, in vitro and in vivo physiological characterisation, we report that selective ablation of the Er81 transcription factor leads to prominent changes in CIN molecular, morphological and electrophysiological features. In particular, the lack of Er81 amplifies intrinsic delayed-rectifier and hyperpolarization-activated currents, which subsequently alters the tonic and phasic activity of CINs. We further demonstrate that these alterations enhance their pause and time-locked responses to sensorimotor inputs in awake mice. Finally, this study reveals an Er81-dependent developmental mechanism in CINs essential for habit formation in adult mice. Highlights- The Er81 transcription factor is expressed in striatal cholinergic interneurons (CINs) - Conditional deletion of Er81 alters key molecular, morphological and electrophysiological properties of CINs in adult mice - Deletion of Er81 reduces the intrinsic excitability of CINs by upregulating delayed rectifier and hyperpolarization-activated currents - Deletion of Er81 alters in vivo striatal activity and habit formation

neuroscience↗

bioRxiv2019

Pharmacological inhibition of acid sensing ion channel 1a protects the heart from ischemia-reperfusion injury

The proton-gated acid-sensing ion channel 1a (ASIC1a) is implicated in the injury response to cerebral ischemia but little is known about its role in cardiac ischemia. We provide genetic evidence that ASIC1a is involved in myocardial ischemia-reperfusion injury (IRI) and show that pharmacological inhibition of ASIC1a yields robust cardioprotection in rodent and human models of cardiac ischemia, resulting in improved post-IRI cardiac viability and function. Consistent with a key role for ASIC1a in cardiac ischemia, we show that polymorphisms in the ASIC1 genetic locus are strongly associated with myocardial infarction. Collectively, our data provide compelling evidence that ASIC1a is a key target for cardioprotective drugs to reduce the burden of disease associated with myocardial ischemia.

physiology↗

bioRxiv2019

Transcriptional regulatory network controlling the ontogeny of hematopoietic stem cells

Hematopoietic stem cell (HSC) ontogeny is accompanied by dynamic changes in gene regulatory networks. We performed RNA-Seq and histone mark ChIP-Seq to define the transcriptomes and epigenomes of cells representing key developmental stages of HSC ontogeny in the mouse. The five populations analyzed were embryonic day 10.5 (E10.5) endothelium and hemogenic endothelium from the major arteries (dorsal aorta, umbilical and vitelline), an enriched population of pre-hematopoietic stem cells (pre-HSCs), fetal liver HSCs, and adult bone marrow HSCs. We observed dynamic and combinatorial epigenetic changes that mark regulatory DNA sequences including gene promoters and enhancers. Using epigenetic signatures, we identified enhancers for each developmental stage. Only 12% of enhancers are primed, and 78% are active, suggesting the vast majority of enhancers are established de novo at the developmental stages where they are required to control their target genes, without prior priming in earlier stages. We constructed developmental-stage-specific transcriptional regulatory networks during HSC ontogeny by linking enhancers and predicted bound transcription factors to their target promoters using a novel computational algorithm. Our computational analyses predicted known transcriptional regulators for the endothelial-to-hematopoietic transition, validating our overall approach, and identified putative novel transcription factors whose regulon activities correlate with the emergence of pre-HSCs. We validated roles for the broadly expressed transcription factors SP3 and MAZ in arterial hemogenic endothelium. Our data and computational analyses provide a useful resource for uncovering regulators of HSC formation.

developmental biology↗

bioRxiv2019

Nova proteins direct synaptic integration of somatostatin interneurons through activity-dependent alternative splicing

Somatostatin interneurons are the earliest born population of cortical inhibitory cells. They are crucial to support normal brain development and function; however, the mechanisms underlying their integration into nascent cortical circuitry are not well understood. In this study, we begin by demonstrating that the maturation of somatostatin interneurons is activity dependent. We then investigated the relationship between activity, alternative splicing and synapse formation within this population. Specifically, we discovered that the Nova family of RNA-binding proteins are activity-dependent and are essential for the maturation of somatostatin interneurons, as well as their afferent and efferent connectivity. Within this population, Nova2 preferentially mediates the alternative splicing of genes required for axonal formation and synaptic function independently from its effect on gene expression. Hence, our work demonstrates that the Nova family of proteins through alternative splicing are centrally involved in coupling developmental neuronal activity to cortical circuit formation.

neuroscience↗

bioRxiv2019

The histone modification reader ZCWPW1 links histone methylation to repair of PRDM9-induced meiotic double stand breaks

The histone modification writer PRDM9 has been shown to deposit H3K4me3 and H3K36me3 at future double-strand break (DSB) sites during the very early stages of meiosis, but the reader of these marks remains unclear. Here, we demonstrate that ZCWPW1 is an H3K4me3 reader that is required for DSB repair and synapsis in mouse testes. We generated H3K4me3 reader-dead ZCWPW1 mutant mice and found that their spermatocytes were arrested at the pachytene-like stage, which phenocopies the Zcwpw1 knock-out mice. Based on various ChIP-seq and immunofluorescence analyses using several mutants, we found that ZCWPW1s occupancy on chromatin is strongly promoted by the histone-modification activity of PRDM9. ZCWPW1 localizes to DMC1-labelled hotspots in a largely PRDM9-dependent manner, where it facilitates completion of synapsis by mediating the DSB repair process. In sum, our study demonstrates the function of ZCWPW1 that acts as part of the selection system for epigenetics-based recombination hotspots in mammals.

developmental biology↗

bioRxiv2019

A versatile tiling light sheet microscope for cleared tissue imaging

We present a tiling light sheet microscope compatible with all tissue clearing methods for rapid multicolor 3D imaging of centimeter-scale cleared tissues with micron-scale (4x4x10 m3) to nanometer-scale (70x70x200 nm3) spatial resolution. The microscope uses tiling light sheets to achieve a more advanced multicolor 3D imaging ability than conventional light sheet microscopes. The illumination light is phase modulated to adjust the 3D imaging ability of the microscope based on the tissue size and the desired spatial resolution and imaging speed, and also to keep the microscope aligned. The ability of the microscope to align via phase modulation alone also makes the microscope reliable and easy to operate. Here we describe the working principle and design of the microscope. We demonstrate its imaging ability by imaging various cleared tissues prepared by different tissue clearing and tissue expansion techniques.

bioengineering↗