Biology subjects

He, K.

Publications and source records attributed to He, K..

66 records · Page 4Linked to original sources

bioRxiv2021

An ultrasensitive GRAB sensor for detecting extracellular ATP in vitro and in vivo

The purinergic transmitter ATP (adenosine 5-triphosphate) plays an essential role in both the central and peripheral nervous systems, and the ability to directly measure extracellular ATP in real time will increase our understanding of its physiological functions. We developed an ultrasensitive GPCR Activation-Based ATP sensor called GRABATP1.0, with a robust fluorescence response to extracellular ATP when expressed in several cell types. This sensor has sub-second kinetics, ATP affinity in the range of tens of nanomolar, and can be used to localize ATP release with subcellular resolution. Using this sensor, we monitored ATP release under a variety of in vitro and in vivo conditions, including primary hippocampal neurons, a zebrafish model of injury-induced ATP release, and LPS-induced ATP-release events in individual astrocytes in the mouse cortex measured using in vivo two-photon imaging. Thus, the GRABATP1.0 sensor is a sensitive, versatile tool for monitoring ATP release and dynamics under both physiological and pathophysiological conditions.

neuroscience↗

bioRxiv2021

Myoglobin primary structure reveals multiple convergent transitions to semi-aquatic life in the world smallest mammalian divers

Identifying the phylogenomic underpinnings of specialized phenotypes that fueled transitions into new adaptive zones is central to evolutionary biology but is often confounded by a fragmentary fossil record, morphological convergence, and unresolved phylogenetic relationships. The speciose mammalian order Eulipotyphla (e.g., moles, shrews, hedgehogs, solenodons) combines an unusual diversity of semi-aquatic, semi-fossorial, and fossorial forms that arose from terrestrial forbearers, yet the ecomorphological pathways leading to these lifestyles have been disputed for a century and more, calling for novel approaches. Here we resolve previously intractable eulipotyphlan intra-family relationships and establish the net surface charge of the oxygen-storing muscle protein myoglobin-readily determined from its primary structure-as a molecular signature to trace ancient lifestyle transitions based on protein sequence alone. Our analyses confidently resolve fossorial habits having evolved twice in talpid moles and reveal five independent origins of a semi-aquatic lifestyle in the order housing the worlds smallest endothermic divers.

evolutionary biology↗

bioRxiv2020

2-Arachidonoylglycerol mobilization following brief synaptic stimulation in the dorsal lateral striatum requires glutamatergic and cholinergic neurotransmission

Several forms of endocannabinoid (eCB) signaling have been described in the dorsal lateral striatum (DLS), however most experimental protocols used to induce plasticity do not recapitulate the firing patterns of striatal-projecting pyramidal neurons in the cortex or firing patterns of striatal medium spiny neurons. Therefore, it is unclear if current models of eCB signaling in the DLS provide a reliable description of mechanisms engaged under physiological conditions. To address this uncertainty, we investigated mechanisms of eCB mobilization following brief synaptic stimulation that mimics in vivo patterns of neural activity in the DLS. To monitor eCB mobilization, the novel genetically encoded fluorescent eCB biosensor, GRABeCB2.0, was expressed in corticostriatal afferents of C57BL6J mice and evoked eCB transients were measured in the DLS using a brain slice photometry technique. We found that brief bouts of synaptic stimulation induce long lasting eCB transients. Inhibition of monoacylglycerol lipase, prolonged the duration of the eCB transient, while inhibition of diacylglycerol lipase inhibited the peak amplitude, suggesting that 2-AG is the predominate eCB generated following brief synaptic stimulation. 2-AG transients were robustly inhibited by AMPA and NMDA receptor antagonists, DNQX and DL-AP5 respectively. Additionally, the 2-AG transient was inhibited by the muscarinic M1 receptor (M1R) antagonist, VU 0255035, and augmented by the M1R positive allosteric modulator, VU 0486846, indicating that acetylcholine (ACh) release is required for efficient 2-AG production. The dopamine D2 receptor (D2R) agonist, quinpirole, inhibited the 2-AG transient. However, in slices from mice lacking D2Rs on cholinergic interneurons (CINs), quinpirole did not inhibit the 2-AG transient, demonstrating that D2Rs on CINs can modulate 2-AG production. The AMPA receptor or NMDA receptor antagonists, DNQX or DL-AP5 respectively, occluded 2-AG augmentation by VU 0486846 suggesting that converging glutamatergic and cholinergic signals are required for efficient 2-AG production following brief synaptic stimulation. Collectively, these data uncover unrecognized mechanisms underlying 2-AG mobilization in the DLS.

neuroscience↗

bioRxiv2020

A fluorescent sensor for spatiotemporally resolved endocannabinoid dynamics in vitro and in vivo

Endocannabinoids (eCBs) are retrograde neuromodulators that play an important role in a wide range of physiological processes; however, the release and in vivo dynamics of eCBs remain largely unknown, due in part to a lack of suitable probes capable of detecting eCBs with sufficient spatiotemporal resolution. Here, we developed a new eCB sensor called GRABeCB2.0. This genetically encoded sensor consists of the human CB1 cannabinoid receptor fused to circular-permutated EGFP, providing cell membrane trafficking, second-resolution kinetics, high specificity for eCBs, and a robust fluorescence response at physiological eCB concentrations. Using the GRABeCB2.0 sensor, we monitored evoked changes in eCB dynamics in both cultured neurons and acute brain slices. Interestingly, in cultured neurons we also observed spontaneous compartmental eCB transients that spanned a distance of approximately 11 m, suggesting constrained, localized eCB signaling. Moreover, by expressing GRABeCB2.0 in the mouse brain, we readily observed foot shock-elicited and running-triggered eCB transients in the basolateral amygdala and hippocampus, respectively. Lastly, we used GRABeCB2.0 in a mouse seizure model and observed a spreading wave of eCB release that followed a Ca2+ wave through the hippocampus. Thus, GRABeCB2.0 is a robust new probe for measuring the dynamics of eCB release under both physiological and pathological conditions.

neuroscience↗

bioRxiv2020

PCV2 aggravated SS2 infection directly showed from differentially expressed proteome of PK15 cells.

PCV2 and SS2 were clinically two major pathogens in pigs and they were zoonotic pathogens. There was extensive cellular tropism for both PCV2 and SS2, as well as, PK15 cells was PCV2 infection mainly cell model. It was found that when PCV2 infected PK15 cells before at the MOI=0.1, SS2 could cause more damage to PK15 cells. ITRAQ labeling proteomic technology was used to explore the differentially expressed proteome of PCV2 and SS2 single infection and co-infection in PK15 cells. The results showed that there were total 4736 proteins distinct changed in this infection models for PK15 cells. PCV2 aggravated SS2 infection were showed directly in the big amount of differentially expressed proteins like AGO3, OSBPL1A, ALB, RIC8A, UBL4A, INTS5 and so on. For the KEGG pathway analysis, it indicated that PCV2 mainly induced the proteins lessen though a series of disease pathway like metabolic pathways, huntingtons disease, insulin signaling pathways, long-term depression, etc. to make cell at a state of compensation. PCV2 before infection made the cells was on the chopping block. Contemporary, SS2 induced the proteins of PK15 rapidly changing, including pathways like spliceosome, endoplasmic reticulum, tight junction, actin cytoskeleton and some involved in parasitic infections pathways like ECM-receptor interaction, Leishmaniasis, Toxoplasmosis and so on. Simultaneously, PCV2 and SS2 existed common ground that they influence PK15 cell by some virulence factor interacted with cytomembrane, influenced the function of ribosome and tRNA. The role of SS2 in the co-infection like a cook chopper.

microbiology↗

bioRxiv2019

Allergen-induced airway inflammation and Th2 cell development requires Blimp-1

A Th2 immune response is central to allergic airway inflammation, which afflicts millions worldwide. However, the mechanisms that augment GATA3 expression in an antigen-primed developing Th2 cell are not well understood. Here, we describe an unexpected role for Blimp-1, a transcriptional repressor that constrains autoimmunity, as an upstream promoter of GATA3 expression that is critical for Th2 cell development in the lung, but dispensable for TFH function and IgE production. Mechanistically, Blimp-1 acts through Bcl6, which is necessary to drive GATA3 expression. Surprisingly, the anti-inflammatory cytokine IL-10, but not the pro-inflammatory cytokines IL-6 or IL-21, is required via STAT3 activation to upregulate Blimp-1 and promote Th2 cell development. These data reveal a hitherto unappreciated role for an IL-10-STAT3-Blimp-1 circuit as an initiator of an inflammatory Th2 response in the lung to allergens. Thus, Blimp-1 in a context-dependent fashion can drive inflammation by promoting rather than terminating effector T cell responses. SummaryThe transcriptional repressor Blimp-1 acts via a pro-inflammatory IL-10-STAT3 axis as a critical positive regulator of Th2 cells in the lung in response to allergens driving pathophysiology associated with asthma disease.

immunology↗

bioRxiv2019

Dynamics of Auxilin1 and GAK in clathrin-1 mediated traffic

Clathrin coated vesicles formed at the plasma membrane lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 \"uncoating ATPase\". The J-domain containing proteins, auxilin1 (Aux1) and auxilin2/cyclin-G dependent kinase (GAK), recruit Hsc70. Aux1 and GAK are closely related homologs, each with a phosphatase- and tensin-like (PTEN-like) domain, a clathrin-binding region, and a C-terminal J-domain; GAK has an additional, N-terminal Ser/Thr kinase domain. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups. Aux1 and GAK appear on coated vesicles in successive transient bursts, immediately after dynamin mediated membrane scission has released the vesicle from the plasma membrane. We show here that these bursts represent recruitment of a very small number of auxilins such that even 4-6 molecules are sufficient to mediate uncoating. In contrast, we could not detect auxilins in abortive pits or at any time during coated-pit assembly. We have also shown previously that clathrin coated vesicles have a dynamic phosphoinositide landscape, and we have proposed that lipid head group recognition might determine the timing of Aux1 and GAK appearance. We now show that differential recruitment of Aux1 and GAK correlates with temporal variations in phosphoinositide composition, consistent with a lipid-switch timing mechanism.

cell biology↗

bioRxiv2019

A Novel Neoantigen Discovery Approach based on Chromatin High Order Conformation: Mapping the Neoantigen to 3D Genome

The high-throughput sequencing technology has yielded reliable and ultra-fast sequencing for DNA and RNA. For tumor cells of cancer patients, when combining the results of DNA and RNA sequencing, one can identify potential neoantigens that stimulate immune response of the T cells. However, when the somatic mutations are abundant, it is computationally challenging to efficiently prioritize the identified neoantigen candidates according to their ability of activating the T cell immuno-response. Numerous prioritization or prediction approaches have been proposed to address this issue but none of them considers the original DNA loci of the neoantigens from the perspective of 3D genome. Here we retrospect the DNA origins of the immune-positive and non-negative neoantigens in the context of 3D genome and discovered that 1) DNA loci of the immuno-positive neoantigens tend to cluster genome-wise. 2) DNA loci of the immuno-positive neoantigens tend to belong to active chromosomal compartment (compartment A) in some chromosomes. 3). DNA loci of the immuno-positive neoantigens tend to locate at specific regions in the 3D genome. We believe that the 3D genome information will help more precise neoantigen prioritization and discovery and eventually benefit precision and personalized medicine in cancer immunotherapy.

bioinformatics↗

bioRxiv2019

A Pipeline for Fecal Host DNA Analysis by Absolute Quantification of LINE-1 and Mitochondrial Genomic Elements Using ddPCR

Stool contains DNA shed from cells of the gastrointestinal (GI) tract and has great potential as a bio-specimen for non-invasive, nucleic acid-based detection of GI diseases. Whereas methods for studying faecal microbiome DNA are plentiful, there is a lack of well-characterised procedures for stabilisation, isolation, and quantitative analysis of faecal host DNA. We report an optimised pipeline for faecal host DNA analysis from the point-of-collection to droplet digital PCR (ddPCR) absolute quantification of host-specific gene targets. We evaluated multiple methods for preservation and isolation of host DNA from stool to identify the highest performing methods. To quantify host DNA even if present in partially degraded form, we developed sensitive, human-specific short-amplicon ddPCR assays targeting repetitive nuclear genomic elements (LINE-1) and mitochondrial genes. We validated the ability of these optimised methods to perform absolute quantification of host DNA in 200 stool DNA extracts from samples that were serially collected from three healthy individuals and three hospitalised patients. These specimens allowed assessment of host DNA day-to-day variability in stool specimens with widely varying physical characteristics (i.e., Bristol scores). We further extended this approach to mouse stool analysis, to enable faecal host DNA studies in animal disease models as well.

molecular biology↗

bioRxiv2018

Colistin resistance prevalence in Escherichia coli from domestic animals in intensive breeding farms of Jiangsu Province, China

The global dissemination of colistin resistance has received a great deal of attention. Recently, the plasmid-mediated colistin resistance encoded by mcr-1 and mcr-2 genes in Escherichia coli (E.coli) strains from animals, food, and patients in China have been reported continuously. To make clear the colisin resistance and mcr gene spread in domestic animals in Jiangsu Province, we collected fecael swabs from pigs, chicken and cattle at different age distributed in intensive feeding farms. The selected chromogenic agar and mcr-PCR were used to screen the colisin resistance and mcr gene carriage. Colistin resistant E.coli colonies were identified from 54.25 % (440/811) pig faecal swabs, from 35.96 % (443/1232) chicken faecal swabs, and 26.92 % (42/156) from cattle faecal swabs. Of all the colisin resistant E.coli colonies, the positive amplifications of mcr-1 were significantly higher than mcr-2. The mcr-1 prevalence was 68.86 % (303/440) in pigs, 87.58 % (388/443) in chicken, and 71.43 % (30/42), compared with 46.82 % (206/440) in pigs, 14.90 % (66/443) in chicken, and 19.05 % (8/42) in cattle of prevalence of mcr-2. Co-occurrence of mcr-1 and mcr-2 was identified in 20 % (88/440) in pigs, 7.22 % (32/443) in chickens, and in 9.52 % (4/42) cattle. These data indicate that mcr was the most important colistin resistance mechanism. Interventions and alternative options are necessary to minimise further dissemination of mcr between food-producing animals and human.\n\nIMPORTANCEColistin is recognized one of the last defence lines for the treatment of highly resistant bacteria, but the emergence of resistance that conferred by a transferable plasmid-mediated mcr genes to this vital antibiotic is extremely disturbing. Here, we used E. coli as an index to monitor drug resistance in domestic animals (pigs, chicken and cattle). It was found that the colistin resistance widely occurred at all ages of domestic animals and the mcr-dependent mechanism dominated in E.coli. We also found that the elder and adult animals were a reservoir of resistant strains, suggesting a potential food safety issue and greater public health problems.

epidemiology↗

bioRxiv2017

Cryptic phylogeographic history sheds light on the generation of species diversity in sky-island mountains

Biodiversity hotspots should be given high priority for conservation under the situation of global climate change. The sky islands in southwestern China are characterized by extraordinarily high species diversity and are among one of the worlds top biodiversity hotspots. However, neither the actual species diversity in this region or mechanisms generating this diversity are well explored. Here, we report on the phylogeographic analysis of the long-tailed mole (Scaptonyx fusicaudus), a semi-fossorial mammal that inhabits the montane cool forests across the Chinese sky islands and is considered to represent one species divided into two subspecies. Analyses using DNA sequence data from one mitochondrial and six nuclear genes revealed that populations inhabiting different mountains exhibited exceptionally strong geographic structure. The lowlands and large rivers act as \"soft\" and \"hard\" barriers to dispersal, respectively, isolating evolutionary lineages for up to 11 million years. Our results suggest that the mountain ranges act as interglacial refugia buffering populations from climate fluctuations, further facilitating allopatric diversification. Strikingly, species delimitation analyses suggests that the long-tailed mole may comprise 18 operational taxonomic units and 17 putative species. Our results suggest that for low-vagility species, the complex topography of the Chinese sky islands has shaped genetic diversity and structure and promoted exceptional diversification through a combination of eco-environmental stability as well as geographic fragmentation. The patterns observed in S. fusicaudus may be representative for other cold- adapted species, reflecting the generation of mammalian faunal diversity in the sky-island mountains of southwestern China.

zoology↗

bioRxiv2016

An improved de novo pipeline for enrichment of high diversity mitochondrial genomes from Amphibia to high-throughput sequencing

We present an improved de novo pipeline by combining long-range polymerase chain reaction (LR-PCR) and capture hybridization for enriching mitochondrial DNA to high-throughput sequencing. We test a new set of primers and hybridizing long-range library (LR-HY) with 112 mitochondrial genomes (MtG) representing three orders, 12 families, 54 genera, and 106 species of Amphibia. The primers are used for obtaining wide taxonomic MtG amplicons to sequence directly and/or make probes for closely related species. LR-HY is compared to standard hybridization. The primers successfully amplify 82 MtGs from all three order, all families, 92.6% (50/54) of the genera, and 74.5% (79/106) of the species, despite some DNA degradation and gene rearrangement. We observe a significantly negative correlation between sequence depth and gene variation. The pattern of highly variable regions is separately distributed in different regions within the length of < 4 kb in the 33-pooled sample. We demonstrate that using 2 kb libraries generate deeper sequence coverage in the highly variable loci than using 400 bp libraries. In total, the pipeline successfully recovers 83 complete and 14 almost complete MtGs from 53 of 54 genera, including 14 MtGs had rearranged protein-coding genes. This universal primers combined with LR-HY is an efficient way to enrich complete MtGs across the entire Amphibia.

genomics↗