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Biology subjects

Dong, L.

Publications and source records attributed to Dong, L..

At least 109 records · Page 6Linked to original sources

Cell minor-axis length is a critical feature for breast cancer cell migration on straight, wavy, loop and grid microfibre patterns

Cell migration plays an important role in physiological and pathological processes where the fibrillar morphology of extracellular matrice (ECM) could regulate the migration dynamics. To mimic the morphological characteristics of fibrillar matrix structures, low-voltage continuous electrospinning was adapted to construct straight, wavy, looped and gridded fibre patterns made of polystyrene (of fibre diameter ca. 3 m). With microfibres deposited onto non-passivated surfaces, cells were permitted to explore their different shapes in response to the directly-adhered fibre, as well as to the neighbouring patterns. For all the patterns studied, analysing cellular migration dynamics of MDA-MB-231 (a highly migratory breast cancer cell line) demonstrated a switch in behaviour when the pattern features approach the upper limit of the cell minor axis. Our findings suggest that, although cells dynamically adjust their shapes in response to different fibrillar environments during migration, their ability to divert from an existing fibre track is limited by the size along the cell minor axis. We therefore conclude that the upper limit of cell minor axis might act as a guide for the design of microfibre patterns for different purposes of cell migration.

bioengineering↗

The Genome Sequence Archive Family: Towards Explosive Data Growth and Diverse Data Types

The Genome Sequence Archive (GSA) is a data repository for archiving raw sequence data, which provides data storing and sharing services for worldwide scientific communities. Considering explosive data growth with diverse data types, here we present the GSA family by expanding into a set of resources for raw data archive with different purposes, namely, GSA (https://ngdc.cncb.ac.cn/gsa/), GSA for Human (GSA-Human, https://ngdc.cncb.ac.cn/gsa-human/), and Open Archive for Miscellaneous Data (OMIX, https://ngdc.cncb.ac.cn/omix/). Compared with the 2017 version, GSA has been significantly updated in data model, online functionalities, and web interfaces. GSA-Human, as a new partner of GSA, is a data repository specialized in human genetics-related data with controlled access and security. OMIX, as a critical complement to the two resources mentioned above, is an open archive for miscellaneous data. Together, all these resources form a family of resources dedicated to archiving explosive data with diverse types, accept data submissions from all over the world and provide free open access to all publicly available data in support of worldwide research activities.

bioinformatics↗

The effect of histone deacetylase inhibitor Trichostatin A on IL-17A-induced transformation of MRC5 into myofibroblasts and its mechanism

ObjectiveTo elucidate potential IL-17A- and TSA-mediated regulation of fibroblasts transformation. MethodsMTT assay, HDAC1 activity assay, cell immunofluorescence and Western blot were employed to detect the expression of related indicators. ResultsMRC5 cells expressed only a small amount of Vimentin. IL-17A treatment upregulated MRC5 cell proliferation, in a concentration-dependent manner. TSA treatment, however, suppressed MRC5 cell proliferation. IL-17A treatment also upregulated HDAC1 activity in MRC5 cells, in a concentration-dependent manner. Using immunofluorescence, we demonstrated that IL-17A-treated MRC5 cells had markedly elevated Vimentin, Collagen-I and a-SMA levels, compared to controls. However, a combined treatment of IL-17A and TSA resulted in markedly reduced levels of the Vimentin, Collagen-I and a-SMA, compared to IL-17A alone, yet the amount was higher than controls. Using western blot analysis, we also revealed that the IL-17A-treated MRC5 cells had markedly elevated levels of Vimentin, a-SMA, HDAC1, p-Smad2, and p-Smad3, and markedly reduced level of Smad7, compared to controls. In TSA intervention group, the expression effect of the above protein was opposite. Moreover, no discernible difference was observed in the levels of Smad2 and Smad3 among the treated and un-treated groups. ConclusionIL-17A stimulates proliferation of MRC5 cells and increases HDAC1 activity and protein expression. It also transforms MRC5 cells into myofibroblasts via activation of the TGF -{beta}1/ Smads signaling network. TSA, on the other hand, strongly suppresses TGF -{beta}1/ Smads pathway-mediated fibrosis by ceasing HDAC1 activity and protein expression.

cell biology↗

Easy multiple sequential CRISPR/Cas9 knockouts in cell lines using a Cre/LoxP re-cyclable vector

To easily generate cell lines lacking multiple proteins, we inserted Cre/Lox sites flanking the guide RNA, Cas9, and mCherry fluorescent protein coding regions of a CRISPR/Cas9 lentiviral vector. Cells bearing an inducible Cre recombinase construct can be transfected with the CRISPR/Cas9 lentiviral vector, and mCherry positive cells sorted by flow cytometry. Induction of Cre causes deletion of the guide RNA, Cas9, and mCherry genes, so that mCherry negative cells can be isolated. After confirming successful targeting of the gene, the cells can be re-infected with the same vector bearing a different guide RNA, and mCherry-positive cells sorted once more. In this way, multiple genes can be mutated sequentially using the same vector and selection marker, without persistent expression of the guide RNA or Cas9. We used this system to sequentially mutate two candidate genes, Bak1 and Bcl2, and generated lines that lacked expression of both proteins.

molecular biology↗

Natural variation in GmRAV confers ecological adaptation through photoperiod control of flowering time and maturity in soybean

Photoperiod strictly controlled vegetative and reproductive growth stages in soybean. A soybean GmRAV transcription factor containing both AP2 and B3 domains was shown to be a key component of this process. We identified six polymorphisms in GmRAV promoter that showed significant association with flowering time and maturity of soybean in one or multiple environments. Soybean varieties with minor polymorphism exhibited longer growth period contributing to soybean adaptation to lower latitudes. The cis-acting element GT1CONSENSUS motif of GmRAV promoter controlled the growth period and shortened R5-R7 by reducing the expression level of GmRAV in soybean. Three GmRAV-overexpressing (GmRAV-ox) transgenic lines displayed later flowering time and maturity, shorter height and fewer numbers of leaves compared with control plants, and transgenic inhibition of GmRAV (GmRAV-i) soybean displayed earlier flowering time and maturity, and increased plant heights. 163 GmRAV-target genes were determined to be putatively directly bound and transcriptionally regulated by GmRAV by combining the results from the DAP-seq and RNA-seq analyses. Two GmRAV binding motifs [C(A/G/T)A(C)ACAA(G/T)A(C/T)A(G/T)] and [C(T/A)A(C/T) C(T/G)CTG] were identified. GmRAV acting downstream of E3E4 delayed soybean growth period by repressing GmFT5a transcriptional activity to guaranteed both vegetative and reproductive phase long enough to allow necessary energy reserved to be accumulated.

plant biology↗

Integrated characterization of SARS-CoV-2 genome, microbiome, antibiotic resistance and host response from single throat swabs

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, poses a severe threat to humanity. Rapid and comprehensive analysis of both pathogen and host sequencing data is critical to track infection and inform therapies. In this study, we performed unbiased metatranscriptomic analysis of clinical samples from COVID-19 patients using a newly-developed RNA-seq library construction method (TRACE-seq), which utilizes tagmentation activity of Tn5 on RNA/DNA hybrids. This approach avoids the laborious and time-consuming steps in traditional RNA-seq procedure, and hence is fast, sensitive and convenient. We demonstrated that TRACE-seq allowed integrated characterization of full genome information of SARS-CoV-2, putative pathogens causing coinfection, antibiotic resistance and host response from single throat swabs. We believe that the integrated information will deepen our understanding of pathogenesis and improve diagnostic accuracy for infectious diseases.

microbiology↗

Co-infection of influenza A virus enhances SARS-CoV-2 infectivity

The upcoming flu season in the northern hemisphere merging with the current COVID-19 pandemic raises a potentially severe threat to public health. Through experimental co-infection of IAV with either pseudotyped or SARS-CoV-2 live virus, we found that IAV pre-infection significantly promoted the infectivity of SARS-CoV-2 in a broad range of cell types. Remarkably, increased SARS-CoV-2 viral load and more severe lung damage were observed in mice co-infected with IAV in vivo. Moreover, such enhancement of SARS-CoV-2 infectivity was not seen with several other viruses probably due to a unique IAV segment as an inducer to elevate ACE2 expression. This study illustrates that IAV has a special nature to aggravate SARS-CoV-2 infection, and prevention of IAV is of great significance during the COVID-19 pandemic.

microbiology↗

Supraphysiological testosterone induces ferroptosis and activates NF-kappaB mediated immune pathways in prostate cancer through nucleophagy

The discovery that androgens play an important role in the progression of prostate cancer (PCa) has led to the development of androgen deprivation therapy as a first line of treatment against PCa. However, paradoxical growth inhibition has been observed, both experimentally and clinically, in a subset of PCa upon administration of supraphysiological levels of testosterone (SupraT). Here we report that SupraT activates cytoplasmic nucleic acid sensors and induces growth inhibition of SupraT-sensitive PCa cells. This is initiated by induction of two parallel autophagy-mediated processes, namely, ferritinophagy and nucleophagy. Consequently, autophagosomal DNA activates nucleic acid sensors that converge on NF-kappaB to drive immune signaling pathways. Chemokines and cytokines secreted by the tumor cells in response to SupraT results in increased migration of cytotoxic immune cells to tumor beds of animal xenografts and patient tumors. Collectively, our findings indicate that SupraT may inhibit a subset of PCa by activating nucleic acid sensors and downstream immune signaling.

cancer biology↗

Characterization of extracellular vesicles and artificial nanoparticles with four orthogonal single-particle analysis platforms

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

cell biology↗

In Vitro efficacy comparison of linezolid, tedizolid, sutezolid and delpazolid against rapid growing Mycobacteria isolated in Beijing, China

The natural resistance of rapid growth Mycobacterium (RGM) against multiple antibiotics renders the treatment of caused infections less successful and time consuming. Therefore, new effective agents are urgently needed. The aim of this study was to evaluate the in vitro susceptibility of 115 isolates, constituting different RGM species, against four oxazolidinones i.e. delpazolid, sutezolid, tedizolid and linezolid. Additionally, 32 reference strains of different RGM species were also tested. The four oxazolidinones exhibited potent in vitro activity against the recruited RGM reference strains, 24 out of 32 RGM species had MICs [≤] 8 g/mL against all four oxazolidinones whereas tedizolid and delpazolid generally presented lower MICs than linezolid or sutezolid. Tedizolid showed the strongest activity against clinical isolates of M. abscessus with MIC50=1g/mL and MIC90=2g/mL. MIC values for tedizolid were usually 4- to 8-fold less than the MICs of linezolid for M. abscessus subsp. abscessus. The MIC distributions of sutezolid and linezolid were similar, while delpazolid showed 2-fold lower MIC as compared with linezolid. Linezolid was not active against most of the tested M. fortuitum isolates, 22 out of the 25 M. fortuitum were resistant against linezolid. However, delpazolid exhibited better antimicrobial activity against these isolates with 4-fold lower MIC values, in contrast with linezolid. In addition, the protein alignment of RplC and RplD and structure based analysis showed that there may be no correlation between oxazolidinones resistance and mutations in rplC, rplD and 23 srRNAgenes in tested RGM. This study showed tedizolid harbors the strongest inhibitory activity against M. abscessus in vitro, while delpazolid presented the best activity against M. fortuitum, which provided important insights on the potential clinical application of oxazolidinones to treat RGM infections.

microbiology↗

Identification of Retinal Ganglion Cell Types and Brain Nuclei expressing the transcription factor Brn3c/Pou4f3 using a Cre recombinase knock-in allele.

Members of the POU4F/Brn3 transcription factor family have an established role in the development of retinal ganglion cell types (RGCs), the projection sensory neuron conveying visual information from the mammalian eye to the brain. Our previous work using sparse random recombination of a conditional knock-in reporter allele expressing Alkaline Phosphatase (AP) and intersectional genetics had identified three types of Pou4f3/Brn3c positive (Brn3c+) RGCs. Here, we describe a novel Brn3cCre mouse allele generated by serial Dre to Cre recombination. We use this allele to explore the expression overlap of Brn3c with Brn3a and Brn3b and the dendritic arbor morphologies and visual stimulus properties of Brn3c+ RGC types. Furthermore, we explore Brn3c-expressing brain nuclei. Our analysis reveals a much larger number of Brn3c+ RGCs and more diverse set of RGC types than previously reported. The majority of RGCs having expressed Brn3c during development are still Brn3c positive in the adult, and all of them express Brn3a while only about half express Brn3b. Intersection of Brn3b and Brn3c expression highlights an area of increased RGC density, similar to an area centralis, corresponding to part of the binocular field of view of the mouse. Brn3c+ neurons and projections are present in multiple brain nuclei. Brn3c+ RGC projections can be detected in the Lateral Geniculate Nucleus (LGN), Pretectal Area (PTA) and Superior Colliculus (SC) but also in the thalamic reticular nucleus (TRN), a visual circuit station that was not previously described to receive retinal input. Most Brn3c+ neurons of the brain are confined to the pretectum and the dorsal midbrain. Amongst theses we identify a previously unknown Brn3c+ subdivision of the deep mesencephalic nucleus (DpMe). Thus, our newly generated allele provides novel biological insights into RGC type classification, brain connectivity and midbrain cytoarchitectonic, and opens the avenue for specific characterization and manipulation of these structures.

neuroscience↗

Identification and verification of 3 key genes associated with survival and prognosis of patients with colon adenocarcinoma via integrated bioinformatics analysis

BackgroundColorectal cancer (CRC) is the third most lethal malignancy in the world, wherein colon adenocarcinoma (COAD) is the most prevalent type of CRC. Exploring biomarkers is important for the diagnosis, treatment, and prevention of COAD. MethodsWe used GEO2R and Venn online software for differential gene screening analysis. Hub genes were screened via STRING and Cytoscape, following Gene Ontology and KEGG enrichment analysis. Finally, survival analysis and expression validation were performed via UALCAN online software, real-time PCR and immunohistochemistry. ResultsIn this study, we screened 323 common differentially expressed genes from four GSE datasets. Furthermore, four hub genes were selected for survival correlation analysis and expression level verification, three of which were shown to be statistically significant. ConclusionOur study suggests that SERPINE1, SPP1 and TIMP1 may be biomarkers closely related to the prognosis of CRC patients.

bioinformatics↗

Optimizing nucleic acid extraction and transcriptome evaluation from low-input, fixed clinical samples

Tumor biopsies are commonly formalin-fixed and paraffin-embedded (FFPE) for long-term and efficient storage. However, FFPE preservation can greatly compromise the quality of samples, the extraction of nucleic acids, and feasibility of downstream studies, including RNA sequencing. These challenges are especially evident in the studies of clinical trial samples, where the sizes of biopsies often limit the amount of material available for study. Here, we evaluate two nucleic acid extraction kits (Covaris truXTRAC FFPE tNA Plus, QIAGEN miRNeasy FFPE) and three hybridized-capture-based RNA sequencing library preparations (Agilent SureSelect XT RNA Direct, Agilent SureSelect XT HS, and Illumina TruSeq RNA Exome) to evaluate the impact of these sample processing steps on transcriptome evaluation in a melanoma biopsy procured in a clinical setting and preserved by FFPE. While there exist many options for extraction and library preparation that are appropriate for RNA sequencing from FFPE samples, we observed that combinations of experimental approaches may have subtle impacts on downstream analysis, including gene expression quantification and fusion detection.

genomics↗

True S-cones are concentrated in the ventral mouse retina for color detection in the upper visual field

Color, an important visual cue for survival, is encoded by comparing signals from photoreceptors with different spectral sensitivities. The mouse retina expresses a short wavelength-sensitive and a middle/long wavelength-sensitive opsin (S- and M-opsin), forming opposing, overlapping gradients along the dorsal-ventral axis. Here, we analyzed the distribution of all cone types across the entire retina for two commonly used mouse strains. We found, unexpectedly, that true S-cones (S-opsin only) are highly concentrated (up to 30% of cones) in ventral retina. Moreover, S-cone bipolar cells (SCBCs) are also skewed towards ventral retina, with wiring patterns matching the distribution of true S-cones. In addition, true S-cones in the ventral retina form clusters, which may augment synaptic input to SCBCs. Such a unique true S-cone pattern forms a basis for mouse color vision, likely reflecting evolutionary adaption to enhance color coding for the upper visual field suitable for mices habitat and behavior.

evolutionary biology↗

Transposase assisted tagmentation of RNA/DNA hybrid duplexes

Tn5-mediated transposition of double-strand DNA has been widely utilized in various high-throughput sequencing applications. Here, we report that the Tn5 transposase is also capable of direct tagmentation of RNA/DNA hybrids in vitro. As a proof-of-concept application, we utilized this activity to replace the traditional library construction procedure of RNA sequencing, which contains many laborious and time-consuming processes. Results of activity of transposase assisted RNA/DNA hybrids co-tagmentation (termed "ATRAC-seq") are comparable to traditional RNA-seq methods in terms of gene number, gene body coverage and gene expression analysis; at the meantime, ATRAC-seq enables a one-tube library construction protocol and hence is more rapid (within 8 h) and convenient. We expect this tagmentation activity on RNA/DNA hybrids to have broad potentials on RNA biology and chromatin research.

genomics↗

Strigolactone defective mutants of Arabidopsis exhibit delayed sepal senescence

Flower sepals are critical for flower development and vary greatly in lifespan depending on their function postpollination. However, very little is known on what controls sepal longevity. Using a sepal senescence mutant screen, we directly connected strigolactones (SL) with sepal longevity. We identified two Arabidopsis mutants that harbour novel mutations in the SL biosynthetic gene MORE AXILLARY GROWTH1 (MAX1) and receptor DWARF14 (AtD14). The mutation in AtD14 caused a substitution of the catalytic Ser-97 to Phe in the enzyme active site. The lesion in MAX1 changed a highly conserved Gly-469 to Arg in the haem-iron ligand signature of the cytochrome P450 protein, which caused loss-of-function of MAX1. nCounter-based transcriptional analysis suggested an interaction between SL and sugar signalling in controlling dark-induced inflorescence senescence. The results uncover an important function for SL in regulating floral organ senescence in addition to its other diverse functions in plant development and stress response. One-sentence summaryTwo novel mutants in the strigolactone pathway demonstrate a role for the hormone in sepal senescence, and transcriptional analysis highlights interaction between strigolactones and sugar signalling.

plant biology↗

Single-Cell Map of Diverse Immune Phenotypes in the Metastatic Brain Tumor Microenvironment of Non Small Cell Lung Cancer

Cancer immunotherapies have shown sustained clinical success in treating primary non-small-cell lung cancer (NSCLC). However, patients with brain metastasis are excluded from the trials because the brain is viewed traditionally as an immune-privileged organ. The composition and properties of tumor-infiltrating myeloid cells in metastatic brain tumors are mostly unknown. To depict the baseline landscape of the composition, gene signature, and functional states of these immune cells, we performed - single-cell RNA sequencing (scRNAseq) for 12,196cellsafter data preprocessing, including 2,241 immunecells from three surgically removed brain lesions of treatment-naive NSCLC patients. We found a lack of T lymphocyte infiltration and activation, as well as the vast expansion of tumor-associated macrophage(TAM) in the brain lesions of NSCLC patients. By comparing our scRNAseq dataset with published data from early and late-stage primary NSCLC tumors, we showed that this compromised T cell response is unique to brain lesions. We identified a unique alternative activation (M2) gene expression pattern of the TAM in the brain metastasis and a lack of known T cell co-stimulator expression. Accumulation of M2 polarized TAM may, therefore, cause the comprised anti-tumor T cell response in metastatic brain lesions. These findings can contribute to the design of new immunotherapy strategies for NSCLC patients with brain metastasis.

cancer biology↗

Correlation of p53 and DNA repair gene mutation patterns in human malignancies indicates different tumour suppression mechanisms of p53

P53 suppresses tumorigenesis through multiple cellular functions/mechanisms. Recently, Janic A, et al. reported that DNA repair pathways are critical mediators of p53-dependent tumor suppression. We showed, by mining cBioPortal data of a range of human cancers, that the tendency of mutual exclusivity of mutations in p53 and DNA repair genes only exist in very limited human cancer types. In the majority of human cancers, p53 mutations are equally distributed between DNA repair gene mutation positive and negative cases and in a number of human cancers, p53 and DNA repair gene mutations have a tendency of co-occurrence. These different correlation patterns of p53 and DNA repair gene mutations in human malignancies may reflect different critical molecular/cellular pathways activated by p53 in different organs or cell types to suppress tumorigenesis.

cancer biology↗