Biology subjects

Sun, F.

Publications and source records attributed to Sun, F..

At least 109 records · Page 6Linked to original sources

bioRxiv2020

RND3/RhoE deficiency in preeclamptic women impairs placental mitochondrial function via regulation of the PPARgamma-UCP2 pathway

Preeclampsia (PE) is a life-threatening disease of pregnant women associated with severe hypertension, proteinuria, or multi-organ injuries. Mitochondrial-mediated placental oxidative stress plays a key role in the pathogenesis of PE. However, the underlying mechanism remains to be revealed. Here, we identify Rnd3, a small Rho GTPase, regulating placental mitochondrial reactive oxygen species (ROS). We showed that Rnd3 is down-regulated in primary trophoblasts isolated from PE patients. Loss of Rnd3 in trophoblasts resulted in excessive ROS generation, cell apoptosis, mitochondrial injury, and proton leakage from the respiratory chain. Moreover, Rnd3 overexpression partially rescues the mitochondrial defects and oxidative stress in human PE primary trophoblasts. Rnd3 physically interacts with the peroxisome proliferators-activated receptor {gamma} (PPAR{gamma}) and promotes the PPAR{gamma}-mitochondrial uncoupling protein 2 (UCP2) cascade. Forced expression of PPAR{gamma} rescues deficiency of Rnd3-mediated mitochondrial dysfunction. We conclude that Rnd3 acts as a novel protective factor in placental mitochondria through PPAR{gamma}-UCP2 signaling and highlight that downregulation of Rnd3 is a potential factor involved in PE pathogenesis.

pathology↗

bioRxiv2020

Cystic fibrosis transmembrane conductance regulator (CFTR) inhibition results in mucus accumulation in human airway epithelia Calu-3 cells: Experimental and Machine Learning Studies

Porosomes are cup-shaped lipoprotein structures at the cell plasma membrane involved in fractional release of intra-vesicular contents during secretion. At the base of the porosome facing the cell cytoplasm, secretory vesicles dock, fuse and swell, to release intra-vesicular content during secretion. Earlier studies demonstrate the cystic fibrosis trans-membrane conductance regulator (CFTR) associated with the porosome in human airways epithelial Calu-3 mucous-secreting cells, suggesting its possible involvement in porosome-mediated mucus secretion. The current study was undertaken to test this hypothesis. Electron microscopy followed by morphometric analysis using manual and computational machine learning approaches were used to assess changes in secretory vesicle size and content, following stimulation of secretion in the absence and presence of CFTR inhibitors. Results from the study demonstrate that stimulated Calu-3 cells pre-exposed to CFTR inhibitors, demonstrate attenuation of secretory vesicle swelling and the release of mucin. Consequently, accumulation of intracellular mucin is observed in cells exposed to CFTR inhibitors. These results further suggest that mucin secretion from Calu-3 cells involve CFTR both at the secretory vesicle membrane to regulate vesicle volume and hydration, and at the porosome to facilitate mucin hydration and secretion. These new findings progress our understanding of the involvement of CFTR on mucus hydration and secretion, providing critical insights into the etiology of CF disease.

cell biology↗

bioRxiv2020

Rapid Isothermal Amplification and Portable Detection System for SARS-CoV-2

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay details and primer sequences become widely known, many laboratories could perform diagnostic tests using methods such as RT-PCR or isothermal RT-LAMP amplification. A key advantage of RT-LAMP based approaches compared to RT-PCR is that RT-LAMP is known to be robust in detecting targets from unprocessed samples. In addition, RT-LAMP assays are performed at a constant temperature enabling speed, simplicity, and point-of-use testing. Here, we provide the details of an RT-LAMP isothermal assay for the detection of SARS-CoV-2 virus with performance comparable to currently approved tests using RT-PCR. We characterize the assay by introducing swabs in virus spiked synthetic nasal fluids, moving the swab to viral transport medium (VTM), and using a volume of that VTM for performing the amplification without an RNA extraction kit. The assay has a Limit-of-Detection (LOD) of 50 RNA copies/L in the VTM solution within 20 minutes, and LOD of 5000 RNA copies/L in the nasal solution. Additionally, we show the utility of this assay for real-time point-of-use testing by demonstrating detection of SARS-CoV-2 virus in less than 40 minutes using an additively manufactured cartridge and a smartphone-based reader. Finally, we explore the speed and cost advantages by comparing the required resources and workflows with RT-PCR. This work could accelerate the development and availability of SARS-CoV-2 diagnostics by proving alternatives to conventional laboratory benchtop tests. Significance StatementAn important limitation of the current assays for the detection of SARS-CoV-2 stem from their reliance on time- and labor-intensive and laboratory-based protocols for viral isolation, lysis, and removal of inhibiting materials. While RT-PCR remains the gold standard for performing clinical diagnostics to amplify the RNA sequences, there is an urgent need for alternative portable platforms that can provide rapid and accurate diagnosis, potentially at the point-of-use. Here, we present the details of an isothermal amplification-based detection of SARS-CoV-2, including the demonstration of a smartphone-based point-of-care device that can be used at the point of sample collection.

bioengineering↗

bioRxiv2020

Dynamic dopaminergic activity controls the timing of self-timed movement

Clues from human movement disorders have long suggested that the neurotransmitter dopamine plays a key role in motor control, but how the endogenous dopaminergic system regulates movement is unknown. Here we show dynamic dopaminergic signaling over seconds-long timescales controls movement timing in mice. Animals were trained to initiate licking after a self-timed interval following a start-timing cue. The movement time was variable from trial-to-trial, as expected from previous studies. Surprisingly, dopaminergic signals ramped-up over seconds between the start-timing cue and the self-timed movement, with variable dynamics that predicted the movement time on single trials. Steeply rising signals preceded early lick-initiation, whereas slowly rising signals preceded later initiation. Higher baseline signals also predicted earlier self-timed movements. Optogenetic activation of dopamine neurons during self-timing did not trigger immediate movements, but rather caused systematic early-shifting of movement initiation, whereas inhibition caused late-shifting, as if modulating the probability of movement. Consistent with this view, the dynamics of the endogenous dopaminergic signals quantitatively predicted the moment-by-moment probability of movement initiation on single trials. These results reveal a causal role for dynamic dopaminergic signaling unfolding over seconds in modulating the decision of when to move.

neuroscience↗

bioRxiv2020

New and improved GRAB fluorescent sensors for monitoring dopaminergic activity in vivo

The monoamine neuromodulator dopamine (DA) plays a critical role in the brain, and the ability to directly measure dopaminergic activity is essential for understanding its physiological functions. We therefore developed the first red fluorescent GPCR-activation-based DA (GRABDA) sensors and optimized versions of green fluorescent GRABDA sensors following our previous studies. In response to extracellular DA, both the red and green GRABDA sensors have a large increase in fluorescence ({Delta}F/F0 values of 150% and 340%, respectively), with subcellular resolution, subsecond kinetics, and nanomolar to submicromolar affinity. Moreover, both the red and green GRABDA sensors readily resolve evoked DA release in mouse brain slices, detect compartmental DA release in live flies with single-cell resolution, and report optogenetically elicited nigrostriatal DA release as well as mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. Importantly, co-expressing red GRABDA with either green GRABDA or the calcium indicator GCaMP6s provides a robust tool for simultaneously tracking neuronal activity and dopaminergic signaling in distinct circuits in vivo.

neuroscience↗

bioRxiv2020

Inhibition of SARS-CoV-2 infection (previously 2019-nCoV) by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion

The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be confirmed. Therefore, we herein used a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed plasma membrane fusion capacity superior to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. We then generated a series of lipopeptides and found that the EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than that of EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, potently inhibiting replication of 4 live human coronaviruses, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by currently circulating SARS-CoV-2 and emerging SARSr-CoVs.

microbiology↗

bioRxiv2020

Amorphous nickel titanium alloy film: a new choice for cryo electron microscopy sample preparation

Cryo-electron microscopy (cryoEM) has become one of the most important approach for structural biology. However, barriers are still there for an increase successful rate, a better resolution and improved efficiency from sample preparation, data collection to image processing. CryoEM sample preparation is one of the bottlenecks with many efforts made recently, including the optimization of supporting substrate (e.g. ultra-thin carbon, graphene, pure gold, 2d crystal of streptavidin, and affinity modification), which was aimed to solve air-water interface problem, or reduce beam induced motion (BIM), or change particle distribution in the grid hole. Here, we report another effort of developing a new supporting substrate, the amorphous nickel-titanium alloy (ANTA) film, for cryoEM sample preparation. Our investigations showed advantages of ANTA film in comparison with conventional carbon film, including superior electron conductivity and trace non-specific interaction with protein. These advantages yield less BIM and significantly improved particle distribution during cryoEM experiment of human apo-ferritn, thus resulting an improved reconstruction resolution from a reduced number of micrographs and particles. Unlike the pure gold film, the usage of the ANTA film is just same with the carbon film, compatible to conventional automatic cryoEM data collection procedure.

biophysics↗

bioRxiv2020

Cytosine methylation dynamics during post-testicular sperm maturation in mammals

Beyond the haploid genome, mammalian sperm contribute a payload of epigenetic information which can modulate offspring phenotypes. Recent studies have shown that the small RNA payload of sperm undergoes extensive remodeling during post-testicular maturation in the epididymis. Intriguingly, epididymal maturation has also been linked to changes in the sperm methylome, suggesting that the epididymis might play a broader role in remodeling the sperm epigenome. Here, we build on prior studies of the maturing sperm methylation landscape, further characterizing the genome-wide methylation landscape in seven germ cell populations collected from throughout the male reproductive tract. Overall, we find very few changes in the cytosine methylation landscape between testicular germ cell populations and cauda epididymal sperm, demonstrating that the sperm methylome is largely stable throughout post-testicular maturation. Intriguingly, although our sequencing data suggested that caput epididymal sperm exhibit a highly unusual methylome, follow-up studies revealed that this resulted from contamination of caput sperm by extracellular DNA. Extracellular DNA formed web-like structures that ensnared sperm, was present only in the caput epididymis of virgin males, where it was associated with citrullinated histone H3 and presumably resulted from a PAD-driven genome decondensation process. Taken together, our data emphasize the stability of the cytosine methylation landscape in mammalian sperm, and identify a surprising but transient period during which immature sperm are associated with extracellular DNA.

developmental biology↗

bioRxiv2019

In vitro induced floral reversion in switchgrass (Panicum Virgatum L.)

Switchgrass (Panicum Virgatum L.) is a warm-season perennial grass native to North America, it was used as forage and vegetative filter strips in early days, and have developed into a bioenergy crop in recent years. In this study, we found that the switchgrass cultivar Alamo at elongation stage 4 have developed inflorescences about 1 cm in length, and in vitro incubation of the shoot apexes harboring inflorescences on Murashige and Skoogs basal medium supplemented with 3 mg/L 6-benzylaminopurine generated multiple shoot clumps. Anatomical study showed that some of the regenerated shoots originated from axillary buds on the explants, some of them originated from adventurous buds and some of them originated from young florets. Further study of shoots originated from young florets found that the floral organs degenerated or developed into leaf-like organs, and the flower terminal transformed into a vegetative shoot apical meristem, thats to say these shoots arise from flower reversion. In vitro induction of floral reversion provided a novel protocol to manipulate flower development in switchgrass, which might contribute a fundamental for flower development study in switchgrass and other plants.

developmental biology↗

bioRxiv2019

A unified framework for dopamine signals across timescales

Rapid phasic activity of midbrain dopamine neurons are thought to signal reward prediction errors (RPEs), resembling temporal difference errors used in machine learning. Recent studies describing slowly increasing dopamine signals have instead proposed that they represent state values and arise independently from somatic spiking activity. Here, we developed novel experimental paradigms using virtual reality that disambiguate RPEs from values. We examined the dopamine circuit activity at various stages including somatic spiking, axonal calcium signals, and striatal dopamine concentrations. Our results demonstrate that ramping dopamine signals are consistent with RPEs rather than value, and this ramping is observed at all the stages examined. We further show that ramping dopamine signals can be driven by a dynamic stimulus that indicates a gradual approach to a reward. We provide a unified computational understanding of rapid phasic and slowly ramping dopamine signals: dopamine neurons perform a derivative-like computation over values on a moment-by-moment basis.

neuroscience↗

bioRxiv2019

VHUT-cryo-FIB, a method to fabricate frozen-hydrated lamella of tissue specimen for in situ cryo-electron tomography

ABSTACTCryo-electron tomography (cryo-ET) provides a promising technique to study high resolution structures of macromolecules in situ, opening a new era of structural biology. One major bottleneck of this technique is to prepare suitable cryo-lamellas of cell/tissue samples. The emergence of cryo-focused ion beam (cryo-FIB) milling technique provides a good solution of this bottleneck. However, there are still large limitations of using cryo-FIB to prepare cryo-lamella of tissue specimen because the thickness of tissue increases the difficulty of specimen freezing and cryo-FIB milling. Here we report a new workflow, VHUT-cryo-FIB (Vibratome - High pressure freezing - Ultramicrotome Trimming - cryo-FIB), aiming for efficient preparation of frozen hydrated tissue lamella for subsequent cryo-ET data collection. This workflow includes tissue slicing using vibratome, high pressure freezing, ultramicrotome cryo-trimming, cryo-FIB milling and the subsequent cryo-electron microscopy (cryo-EM). The modification of equipment in this workflow is highly eliminated. We developed two strategies with a special cryo-holder tip or carrier for loading cryo-lamella into side entry cryo-holder or Autoloader catridge. We tested this workflow using the tissue sample of rat skeleton muscle and spinach leaf and collected high quality cryo-ET tilt series, which enabled us to obtain an in situ structure of spinach ribosome by sub-tomogram averaging.

biophysics↗

bioRxiv2019

THOC1 deficiency leads to late-onset nonsyndromic hearing loss through p53-mediated hair cell apoptosis

Apoptosis of cochlear hair cells is a key step towards age-related hearing loss. Although numerous genes have been implicated in the genetic causes of late-onset, progressive hearing loss, few show direct links to the proapoptotic process. By genome-wide linkage analysis and whole exome sequencing, we identified a heterozygous p.L183V variant in THOC1 as the probable cause of the late-onset, progressive, non-syndromic hearing loss in a large dominant family. Thoc1, a member of the conserved multisubunit THO/TREX ribonucleoprotein complex, is highly expressed in mouse and zebrafish hair cells. The Thoc1 mutant zebrafish generated by gRNA-Cas9 system lacks the C-startle response, indicative of the hearing dysfunction. Both Thoc1 mutant and knockdown zebrafish have greatly reduced hair cell numbers, while the latter can be rescued by embryonic microinjection of human wild-type THOC1 mRNA but to significantly lesser degree by the p.L183V mutant mRNA. The Thoc1 deficiency resulted in marked apoptosis in zebrafish hair cells. Consistently, transcriptome sequencing of the mutants showed significantly increased gene expression in the p53-associated signaling pathway. Depletion of p53 or applying the p53 inhibitor Pifithrin- significantly rescued the hair cell loss in the Thoc1 knockdown zebrafish. Our results suggested that THOC1 deficiency lead to late-onset, progressive hearing loss through p53-mediated hair cell apoptosis. This is to our knowledge the first human disease associated with THOC1 mutations and may shed light on the molecular mechanism underlying the age-related hearing loss.\n\nSignificance StatementFor the first time, we found that THOC1 deficiency leads to late-onset nonsyndromic hearing loss. Furthermore, we revealed the hypomorphic THOC1 induced p53-mediated hair cell apoptosis. This is to our knowledge the first human disease associated with THOC1 mutations and may shed light on the molecular mechanism underlying the age-related hearing loss.

genetics↗

bioRxiv2019

Molecular mechanism of mitochondrial phosphatidate transfer by Ups1/Mdm35

Cardiolipin plays many important roles for mitochondrial physiological function and is synthesized from phosphatidic acid (PA) at inner mitochondrial membrane (IMM). PA synthesized from endoplasmic reticulum needs to transfer to IMM via outer mitochondrial membrane (OMM). The transfer of PA between IMM and OMM is mediated by Ups1/Mdm35 protein family. Although there are many structures of this family available, the detailed molecular mechanism of how PA is transferred between membranes is yet unknown. Here, we report another crystal structures of Ups1/Mdm35 in the authentic monomeric apo state and the DHPA bound state. By combining subsequent all-atom molecular dynamics simulations, extensive structural comparisons and biophysical assays, we discovered the conformational changes of Ups1/Mdm35, identified key structural elements and residues during membrane binding and PA entry. We found the monomeric Ups1 on membrane is an important transit for the success of PA transfer, and the equilibrium between monomeric Ups1 and Ups1/Mdm35 complex on membrane affects the PA transfer rate and can be regulated by many factors including environmental pH.

biochemistry↗

bioRxiv2019

Benchmarking of alignment-free sequence comparison methods

Alignment-free (AF) sequence comparison is attracting persistent interest driven by data-intensive applications. Hence, many AF procedures have been proposed in recent years, but a lack of a clearly defined benchmarking consensus hampers their performance assessment. Here, we present a community resource (http://afproject.org) to establish standards for comparing alignment-free approaches across different areas of sequence-based research. We characterize 74 AF methods available in 24 software tools for five research applications, namely, protein sequence classification, gene tree inference, regulatory element detection, genome-based phylogenetic inference and reconstruction of species trees under horizontal gene transfer and recombination events. The interactive web service allows researchers to explore the performance of alignment-free tools relevant to their data types and analytical goals. It also allows method developers to assess their own algorithms and compare them with current state-of-the-art tools, accelerating the development of new, more accurate AF solutions.

bioinformatics↗

bioRxiv2019

Three-dimensional genome reorganization during mouse spermatogenesis

Three-dimensional genome organization plays an important role in many biological processes. Yet, how the genome is packaged at the molecular level during mammalian spermatogenesis remains unclear. Here, we performed Hi-C in seven sequential stages during mouse spermatogenesis. We found that topological associating domains (TADs) and chromatin loops underwent highly dynamic reorganization. They displayed clear existence in primitive type A spermatogonia, disappearance at pachytene stage, and reestablishment in spermatozoa. Surprisingly, even in the absence of TADs and chromatin loops at pachytene stage, CTCF remained bound at TAD boundary regions (identified in primitive type A spermatogonia). Additionally, many enhancers and promoters exhibited features of open chromatin and transcription remained active at pachytene stage. A/B compartmentalization and segmentation ratio were conserved in different stages of spermatogenesis in autosomes, although there were A/B compartment switching events correlated with gene activity changes. Intriguingly, A/B compartment structure on the X chromosome disappeared during pacSC, rST and eST stages. Together, our work uncovered a dynamic three-dimensional chromatin organization during mouse spermatogenesis and suggested that transcriptional regulation could be independent of TADs and chromatin loops at specific developmental stages.

genomics↗

bioRxiv2019

Cryo-EM structures reveal a conformational change of SOPA1 during mitochondrial inner membrane fusion

Mammalian mitochondrial inner membrane fusion is mediated by OPA1(optic atrophy 1). Under physiological condition, OPA1 undergoes proteolytic processing to form a membrane-anchored long isoform (LOPA1) and a soluble short isoform (SOPA1). A combination of LOPA1 and SOPA1 are required for membrane fusion, however, the relevant mechanism is not well understood. In this study, we investigate the cryo-EM structures of SOPA1 coated liposome tube at nucleotide-free and GTP{gamma}S bound states. SOPA1 exhibits a general structure of dynamin family and can assemble onto membrane in a helical array with a building block of dimer and thus induce membrane tubulation. A predicted amphipathic helix is discovered to mediate the tubulation activity of SOPA1. The binding of GTP{gamma}S causes a conformational rotation between GTPase domain and stalk region, and then induces a rearrangement of the helical array and an expansion of the tube, which is opposite to the behavior of other dynamin proteins, suggesting a unique role of SOPA1 in the fusion of mitochondrial inner membrane.

biophysics↗

bioRxiv2018

A network-based integrated framework for predicting virus-host interactions with applications

Metagenomic sequencing has greatly enhanced the discovery of viral genomic sequences; however it remains challenging to identify the host(s) of these new viruses. We developed VirHostMatcher-Net, a flexible, network-based, Markov random field framework for predicting virus-host interactions using multiple, integrated features: CRISPR sequences, sequence homology, and alignment-free similarity measures ([Formula] and WIsH). Evaluation of this method on a benchmark set of 1,075 known viruses-host pairs yielded host prediction accuracy of 62% and 85% at the genus and phylum levels, representing 12-27% and 10-18% improvement respectively over previous single-feature prediction approaches. We applied our host-prediction tool to three metagenomic virus datasets: human gut crAss-like phages, marine viruses, and viruses recovered from globally-distributed, diverse habitats. Host predictions were frequently consistent with those of previous studies, but more importantly, this new tool made many more confident predictions than previous tools, up to 6-fold more (n>60,000), greatly expanding the diversity of known virus-host interactions.

bioinformatics↗

bioRxiv2018

Metagenomic unmapped reads provide important insights into human microbiota and disease associations

We developed a computational pipeline, MicroPro, for metagenomic data analyses that take into account all the reads from known and unknown microbial organisms and for associating viruses with complex diseases. We utilized MicroPro to analyze metagenomics data related to three diseases: colorectal cancer, type-2 diabetes and liver cirrhosis, and showed that including reads from unknown organisms will markedly increase the prediction accuracy of the disease status based on metagenomics data. We identified new microbial organisms associated with these diseases. Viruses were shown to play important roles in colorectal cancer and liver cirrhosis, but not in type-2 diabetes. MicroPro is available at https://github.com/zifanzhu/MicroPro.

bioinformatics↗