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Long, H.

Publications and source records attributed to Long, H..

At least 37 records · Page 2Linked to original sources

One-Step PCR Amplicon Library Construction (OSPALC, version 1)

High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures, greatly promoting biological studies involving large amounts of complex samples, especially those involving environmental and pathogen-monitoring ones. However, commercial library preparation kits for amplicon sequencing, which generally require multiple steps, including adapter ligation and indexing, are expensive and time-consuming, especially for applications at a large scale. Here, to overcome this technical hurdle, we present a one-step PCR amplicon library construction (OSPALC) protocol for amplicon library preparations in the lab along with a QIIME2-based amplicon analysis pipeline. High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples. With this protocol, the amplicon library construction is completed through one regular PCR with long primers, and the total cost per DNA/cDNA sample decreases to just 1/15 of the typical cost via service providers. Empirically tested primers to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study. Criteria to design primers targeting at any regions of prokaryotic and eukaryotic genomes are also suggested. In principle, OSPALC can be conveniently used in amplicon library constructions of any target gene of both prokaryotes and eukaryotes at the DNA or RNA levels, and will facilitate research in numerous fields.

genomics↗

Resting fMRI-guided TMS evokes subgenual anterior cingulate response in depression

Depression alleviation following treatment with repetitive transcranial magnetic stimulation (rTMS) tends to be more effective when TMS is targeted to cortical areas with high resting state functional connectivity (rsFC) with the subgenual anterior cingulate cortex (sgACC). However, it has not yet been confirmed that rsFC-guided TMS coil placement leads to TMS modulation of the sgACC. For each participant (N=115, 34 depressed patients), a peak rsFC cortical hotspot for the sgACC and control targets were prospectively identified. Single pulses of TMS interleaved with fMRI readouts were then administered to these targets and established significant downstream fMRI BOLD responses in the sgACC. We then marked an association between TMS-evoked BOLD responses in the sgACC and rsFC between the stimulation site and sgACC. This effect was qualified by a difference between healthy and patient participants: only in depressed patients, positively connected sites of stimulation led to the strongest evoked responses in the sgACC. Our results highlight rsFC-based targeting as a viable strategy to causally modulate sgACC subcortical targets and further suggest that cortical sites with high positive rsFC to the sgACC might represent an alternative target for the treatment of depression.

neuroscience↗

A nuclear receptor facilitates differentiation of human PSCs into more mature hepatocytes

The capacity to generate functional hepatocytes from renewable human pluripotent stem cells (hPSCs) could address limited supplies of primary human hepatocytes. However, hepatocytes differentiated from hPSCs in vitro are functionally immature. To understand mechanisms regulating maturation of in vitro derived hepatocytes, we developed a 3D spheroid differentiation system and compared gene regulatory elements in uncultured human primary hepatocytes with those in hepatocytes that were differentiated in 2D or 3D conditions from human PSCs by RNA-seq, ATAC-seq, and H3K27Ac ChIP-seq. Three-dimensional differentiation improved enhancer activity and expression of transcription factor ONECUT1, but was insufficient to upregulate human-specific mature hepatocytes marker gene CYP3A4 or super-enhancer regulated transcription factor gene NFIC. Regulome comparisons showed reduced enrichment of thyroid receptor THRB motifs in accessible chromatin and in active enhancers without reduced transcription of THRB, suggesting the regulation at the level of THRB ligands in PSC-differentiated hepatocytes. Addition of thyroid hormone T3 to the PSC-differentiated hepatocytes increased CYP3A4 expression. T3 increased binding of THRB to the CYP3A4 proximal enhancer and restored the super-enhancer status and gene expression of NFIC and reduced expression of AFP. The resultant hPSC-hepatocytes showed gene expression, epigenetic status and super-enhancer landscape closer to primary hepatocytes and activated regulatory regions including non-coding SNPs associated with liver-related diseases. Transplanting the 3D PSC-hepatocytes into immunocompromised mice resulted in engraftment of human hepatocytes in the mouse liver parenchyma without disrupting normal liver histology at 6 months after transplantation. This work provides insights into the functions of nuclear receptor THRB and highlights the importance of the environmental factors-nuclear receptors axis in regulating maturation of human PSC-differentiated cell types.

developmental biology↗

MIRA: Joint regulatory modeling of multimodal expression and chromatin accessibility in single cells

Rigorously comparing gene expression and chromatin accessibility in the same single cells could illuminate the logic of how coupling or decoupling of these mechanisms regulates fate commitment. Here, we present MIRA: Probabilistic Multimodal Models for Integrated Regulatory Analysis, a comprehensive methodology that systematically contrasts transcription and accessibility to infer the regulatory circuitry driving cells along developmental trajectories. MIRA leverages topic modeling of cell states and regulatory potential modeling of individual gene loci. MIRA thereby represents cell states in an efficient and interpretable latent space, infers high fidelity lineage trees, determines key regulators of fate decisions at branch points, and exposes the variable influence of local accessibility on transcription at distinct loci. Applied to epidermal maintenance differentiation and embryonic brain development from two different multimodal platforms, MIRA revealed that early developmental genes were tightly regulated by local chromatin landscape whereas terminal fate genes were titrated without requiring extensive chromatin remodeling.

bioinformatics↗

Cortical-subcortical structural connections support transcranial magnetic stimulation engagement of the amygdala

The amygdala processes valenced stimuli, influences affective states, and exhibits aberrant activity across anxiety disorders, depression, and PTSD. Interventions that modulate amygdala activity hold promise for treating transdiagnostic affective symptoms. We investigated (N=45) whether transcranial magnetic stimulation (TMS) elicits indirect changes in amygdala activity when applied to ventrolateral prefrontal cortex (vlPFC), a region important for affect regulation. Harnessing in-scanner interleaved TMS/functional MRI (fMRI), we reveal that vlPFC neurostimulation evoked acute, dose-dependent modulations of amygdala fMRI BOLD signal. Larger TMS-evoked changes in amygdala fMRI signal were associated with higher fiber density in a vlPFC-amygdala white matter pathway, suggesting this pathway facilitated stimulation-induced communication between cortex and subcortex. This work provides evidence of amygdala engagement by TMS, highlighting stimulation of vlPFC-amygdala circuits as a candidate treatment for affective psychopathology. More broadly, it indicates that targeting cortical-subcortical connections may enhance the impact of TMS on subcortical neural activity and, by extension, subcortex-subserved behaviors. TeaserIndividualized, connectivity-guided transcranial magnetic stimulation modulates the amygdala, demonstrating therapeutic potential.

neuroscience↗

Robust SARS-CoV-2-specific T-cell immunity is maintained at 6 months following primary infection

The immune response to SARS-CoV-2 is critical in both controlling primary infection and preventing re-infection. However, there is concern that immune responses following natural infection may not be sustained and that this may predispose to recurrent infection. We analysed the magnitude and phenotype of the SARS-CoV-2 cellular immune response in 100 donors at six months following primary infection and related this to the profile of antibody level against spike, nucleoprotein and RBD over the previous six months. T-cell immune responses to SARS-CoV-2 were present by ELISPOT and/or ICS analysis in all donors and are characterised by predominant CD4+ T cell responses with strong IL-2 cytokine expression. Median T-cell responses were 50% higher in donors who had experienced an initial symptomatic infection indicating that the severity of primary infection establishes a setpoint for cellular immunity that lasts for at least 6 months. The T-cell responses to both spike and nucleoprotein/membrane proteins were strongly correlated with the peak antibody level against each protein. The rate of decline in antibody level varied between individuals and higher levels of nucleoprotein-specific T cells were associated with preservation of NP-specific antibody level although no such correlation was observed in relation to spike-specific responses. In conclusion, our data are reassuring that functional SARS-CoV-2-specific T-cell responses are retained at six months following infection although the magnitude of this response is related to the clinical features of primary infection.

immunology↗

A non-canonical EZH2 function sensitizes solid tumors to genotoxic stress

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring gain-of-function EZH2 mutations that enhance its polycomb repressive function. In contrast, in castration-resistant prostate cancer (CRPC) we have previously reported that EZH2 plays a non-canonical role as a transcriptional activator. In this setting, we now show that EZH2 inhibitors can also block the non-canonical activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomic profiling of cells treated with EZH2 inhibitors demonstrated that rather than de-repressing tumor suppressor genes silenced by PRC2, EZH2 inhibitors downregulate a set of DNA repair genes that are directly regulated by EZH2. In addition, genome-wide CRISPR/Cas9-mediated loss-of-function screens in the presence of EZH2 inhibitors identified these DNA repair genes to underlie the growth-inhibitory function of these compounds. Interrogation of public data from diverse solid tumor types expressing wild-type EZH2 showed that expression of DNA damage repair genes is significantly correlated with cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhanced their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential new combination cancer therapies.

cancer biology↗

Transcription-coupled structural dynamics of topologically associating domains regulate replication origin efficiency

Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Despite various genetic and epigenetic signatures are found to be related with active origins, it remains elusive how the selection of origins is determined. The classic Rosette model proposes that the origins clustered in a chromatin domain are preferentially and simultaneously fired, but direct imaging evidence has been lacking due to insufficient spatial resolution. Here, we applied dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We found that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase, in contrary to the Rosette model. Intriguingly, while both active and dormant origins are distributed homogeneously in the TAD during the G1 phase, active origins relocate to the TAD periphery before entering the S phase. We proved that such origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observed that the replication machinery protein PCNA forms immobile clusters around the TADs at the G1/S transition, which explains why origins at the TAD periphery are preferentially fired. Thus, we propose a "Chromatin Re-organization Induced Selective Initiation" (CRISI) model that the transcription-coupled chromatin structural re-organization determines the selection of replication origins, which transcends the scope of specific genetic and epigenetic signatures for origin efficiency. Our in situ super-resolution imaging unveiled coordination among DNA replication, transcription, and chromatin organization inside individual TADs, providing new insights into the biological functions of sub-domain chromatin structural dynamics.

genomics↗

Susceptibility of tree shrew to SARS-CoV-2 infection

Since SARS-CoV-2 became a pandemic event in the world, it has not only caused huge economic losses, but also a serious threat to global public health. Many scientific questions about SARS-CoV-2 and COVID-19 were raised and urgently need to be answered, including the susceptibility of animals to SARS-CoV-2 infection. Here we tested whether tree shrew, an emerging experimental animal domesticated from wild animal, is susceptible to SARS-CoV-2 infection. No clinical signs were observed in SARS-CoV-2 inoculated tree shrews during this experiment except the increasing body temperature (above 39{degrees} C) particular in female animals during infection. Low levels of virus shedding and replication in tissues occurred in all three age groups, each of which showed his own characteristics. Histopathological examine revealed that pulmonary abnormalities were mild but the main changes although slight lesions were also observed in other tissues. In summary, tree shrew is not susceptible to SARS-CoV-2 infection and may not be a suitable animal for COVID-19 related researches.

microbiology↗

Parkinson's disease-related phosphorylation at Tyr39 rearranges α-synuclein amyloid fibril structure revealed by cryo-EM

Post-translational modifications (PTMs) of -synuclein (-syn), e.g. phosphorylation, play an important role in modulating -syn pathology in Parkinsons disease (PD) and -synucleinopathies. Accumulation of phosphorylated -syn fibrils in Lewy bodies and Lewy neurites is the histological hallmark of these diseases. However, it is unclear how phosphorylation relates to -syn pathology. Here, by combining chemical synthesis and bacterial expression, we obtained homogeneous -syn fibrils with site-specific phosphorylation at Y39, which exhibits enhanced neuronal pathology in rat primary cortical neurons. We determined the cryo-EM structure of pY39 -syn fibril, which reveals a new fold of -syn with pY39 in the center of the fibril core forming electrostatic interaction network with eight charged residues in the N-terminal region of -syn. This structure composed of residues 1-100 represents the largest -syn fibril core determined so far. This work provides structural understanding on the pathology of pY39 -syn fibril, and highlights the importance of PTMs in defining the polymorphism and pathology of amyloid fibrils in neurodegenerative diseases.

biophysics↗

Comparison of SARS-CoV-2 infections among 3 species of non-human primates

COVID-19, caused by SARS-CoV-2 infection, has recently been announced as a pandemic all over the world. Plenty of diagnostic, preventive and therapeutic knowledges have been enriched from clinical studies since December 2019. However, animal models, particularly non-human primate models, are urgently needed for critical questions that could not be answered in clinical patients, evaluations of anti-viral drugs and vaccines. In this study, two families of non-human primates, Old world monkeys (12 Macaca mulatta, 6 Macaca fascicularis) and New world monkeys (6 Callithrix jacchus), were experimentally inoculated with SARS-CoV-2. Clinical signs were recorded. Samples were collected for analysis of viral shedding, viremia and histopathological examination. Increased body temperature was observed in 100% (12/12) M. mulatta, 33.3% (2/6) M. fascicularis and none (0/6) of C. jacchus post inoculation of SARS-CoV-2. All of M. mulatta and M. fascicularis showed chest radiographic abnormality. Viral genomes were detected in nasal swabs, throat swabs, anal swabs and blood from all 3 species of monkeys. Viral shedding from upper respiratory samples reached the peak between day 6 and day 8 post inoculation. From necropsied M. mulatta and M. fascicularis, the tissues showing virus positive were mainly lung, weasand, bronchus and spleen. No viral genome was seen in any of tissues from 2 necropsied C. jacchus. Severe gross lesions and histopathological changes were observed in lung, heart and stomach of SARS-CoV-2 infected animals. In summary, we have established a NHP model for COVID-19, which could be used to evaluate drugs and vaccines, and investigate viral pathogenesis. M. mulatta is the most susceptible to SARS-CoV-2 infection, followed by M. fascicularis and C. jacchus. One Sentence SummaryM. mulatta is the most susceptible to SARS-CoV-2 infection as compared to M. fascicularis and C. jacchus.

microbiology↗

Mechanism of monoallelic expression and allelic rheostat role of DNA methylation

In diploid cells, maternal and paternal copies of genes usually have similar transcriptional activity. Mammalian allele-specific epigenetic mechanisms such as X-chromosome inactivation (XCI) and imprinting were historically viewed as rare exceptions to this rule. The discovery of mitotically stable monoallelic autosomal expression (MAE) a decade ago revealed an additional allele-specific mode regulating thousands of mammalian genes. However, despite its prevalence, the mechanistic basis of MAE remains unknown. To uncover the mechanism of MAE maintenance, we devised a small-molecule screen for reactivation of silenced alleles across multiple loci using targeted RNA sequencing. Contrary to previous reports, we identified DNA methylation as a key mechanism of MAE mitotic maintenance. In contrast with the binary choice of the active allele in XCI, stringent transcriptome-wide analysis revealed MAE as a regulatory mode with tunable control of allele-specific expression, dependent on the extent of DNA methylation. In a subset of MAE genes, allelic imbalance was insensitive to changes in DNA methylation, implicating additional mechanisms in MAE maintenance in these loci. Our findings identify a key mechanism of MAE maintenance, reveal tunability of this mode of gene regulation, and provide the essential platform for probing the biological role of MAE in development and disease.

genomics↗

The Tung Tree (Vernicia Fordii) Genome Provides A Resource for Understanding Genome Evolution and Oil Improvement

Tung tree (Vernicia fordii) is an economically important woody oil plant that produces tung oil containing a high proportion of eleostearic acid ([~]80%). Here we report a high-quality, chromosome-scale tung tree genome sequence of 1.12 Gb with 28,422 predicted genes and over 73% repeat sequences. Tung tree genome was assembled by combining Illumina short reads, PacBio single-molecule real-time long reads and Hi-C sequencing data. Insertion time analysis revealed that the repeat-driven tung tree genome expansion might be due to long standing long terminal repeat (LTR) retrotransposon bursts and lack of efficient DNA deletion mechanisms. An electronic fluorescent pictographic (eFP) browser was generated based on genomic and RNA-seq data from 17 various tissues and developmental stages. We identified 88 nucleotide-binding site (NBS)-encoding resistance genes, of which 17 genes may help the tung tree resist the Fusarium wilt shortly after infection. A total of 651 oil-related genes were identified and 88 of them were predicted to be directly involved in tung oil biosynthesis. The fewer phosphoenolpyruvate carboxykinase (PEPC) genes, and synergistic effects between transcription factors and oil biosynthesis-related genes may contribute to high oil content in tung seeds. The tung tree genome should provide valuable resources for molecular breeding and genetic improvement.

bioinformatics↗

Parkinson's disease associated mutation E46K of α-synuclein triggers the formation of a novel fibril structure

-Synuclein (-syn) amyloid fibril, as the major component of Lewy bodies and pathological entity spreading in human brain, is closely associated with Parkinsons disease (PD) and other synucleinopathies. Several single amino-acid mutations (e.g. E46K) of -syn have been identified causative to the early onset of familial PD. Here, we determined the cryo-EM structure of a full-length -syn fibril formed by N-terminal acetylated E46K mutant -syn (Ac-E46K). The fibril structure represents a new fold of -syn, which demonstrates that the E46K mutation breaks the electrostatic interactions in the wild type (WT) -syn fibril and thus triggers the rearrangement of the overall structure. Furthermore, we show that the Ac-E46K fibril is less resistant to harsh conditions and protease cleavage, and more prone to be fragmented with a higher capability of seeding fibril formation than that of the WT fibril. Our work provides a structural view to the severe pathology of the PD familial mutation E46K of -syn and highlights the importance of electrostatic interactions in defining the fibril polymorphs.

biophysics↗

Predicting master transcription factors from pan-cancer expression data

The function of critical developmental regulators can be subverted by cancer cells to control expression of oncogenic transcriptional programs. These "master transcription factors" (MTFs) are often essential for cancer cell survival and represent vulnerabilities that can be exploited therapeutically. The current approaches to identify candidate MTFs examine super-enhancer associated transcription factor-encoding genes with high connectivity in network models. This relies on chromatin immunoprecipitation-sequencing (ChIP-seq) data, which is technically challenging to obtain from primary tumors, and is currently unavailable for many cancer types and clinically relevant subtypes. In contrast, gene expression data are more widely available, especially for rare tumors and subtypes where MTFs have yet to be discovered. We have developed a predictive algorithm called CaCTS (Cancer Core Transcription factor Specificity) to identify candidate MTFs using pan-cancer RNA-sequencing data from The Cancer Genome Atlas. The algorithm identified 273 candidate MTFs across 34 tumor types and recovered known tumor MTFs. We also made novel predictions, including for cancer types and subtypes for which MTFs have not yet been characterized. Clustering based on MTF predictions reproduced anatomic groupings of tumors that share 1-2 lineage-specific candidates, but also dictated functional groupings, such as a squamous group that comprised five tumor subtypes sharing 3 common MTFs. PAX8, SOX17, and MECOM were candidate factors in high-grade serous ovarian cancer (HGSOC), an aggressive tumor type where the core regulatory circuit is currently uncharacterized. PAX8, SOX17, and MECOM are required for cell viability and lie proximal to super-enhancers in HGSOC cells. ChIP-seq revealed that these factors co-occupy HGSOC regulatory elements globally and co-bind at critical gene loci including MUC16 (CA-125). Addiction to these factors was confirmed in studies using THZ1 to inhibit transcription in HGSOC cells, suggesting early down-regulation of these genes may be responsible for cytotoxic effects of THZ1 on HGSOC models. Identification of MTFs across 34 tumor types and 140 subtypes, especially for those with limited understanding of transcriptional drivers paves the way to therapeutic targeting of MTFs in a broad spectrum of cancers.

cancer biology↗

Social and non-social functions of infant vocalizations

Research on infant vocal development has provided notable insights into vocal interaction with caregivers, elucidating growth in foundations for language through parental elicitation and reaction to vocalizations. A role for infant vocalizations produced endogenously, potentially providing raw material for interaction and a basis for growth in the vocal capacity itself, has received less attention. We report that in laboratory recordings of infants and their parents, the bulk of infant speech-like vocalizations, or "protophones", were directed toward no one and instead appeared to be generated endogenously, mostly in exploration of vocal abilities. The tendency to predominantly produce protophones without directing them to others occurred both during periods when parents were instructed to interact with their infants and during periods when parents were occupied with an interviewer, with the infants in the room. The results emphasize the infant as an agent in vocal learning, even when not interacting socially and suggest an enhanced perspective on foundations for vocal language.

developmental biology↗

Mechanistic insights into NAD synthase NMNAT chaperoning phosphorylated Tau from pathological aggregation

Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimers disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model. By combining NMR spectroscopy, crystallography, single-molecule and computational approaches, we revealed that NMNAT adopts its enzymatic pocket to specifically bind the phosphorylated sites of pTau, which can be competitively disrupted by the enzymatic substrates of NMNAT. Moreover, we found that NMNAT serves as a co-chaperone of Hsp90 for the specific recognition of pTau over Tau. Our work uncovers a dedicated chaperone of pTau and suggests NMNAT as a key node between NAD metabolism and Tau homeostasis in aging and neurodegeneration.

biochemistry↗

An abscisic acid (ABA) homeostasis regulated by its production, catabolism and transport in peanut leaves in response to drought stress

ABA is an important messenger that acts as the signaling mediator for regulating the adaptive response of plants to drought stress. Two production pathways, de novo biosynthesis and hydrolysis of glucose-conjugated ABA by {beta}-glucosidase (BG), increase cellular ABA levels in plants. ABA catabolism via hydroxylation by 8-hydroxylase (CYP707A), or conjugation by uridine diphosphate glucosyltransferase (UGT), decreases cellular ABA levels. The transport of ABA through ATP-binding cassette (ABC)-containing transporter proteins, members of ABC transporter G family (ABCG), across plasma membrane (PM) is another important pathway to regulate cellular ABA levels. In this study, based on our previously constructed transcriptome of peanut leaves in response to drought stress, fourteen candidate genes involved in ABA production (including AhZEP, AhNCED1 and AhNCED3, AhABA2, AhAAO1 and AhAAO2, AhABA3, AhBG11 and AhBG24), catabolism (including AhCYP707A3, AhUGT71K1 and AhUGT73B4) and transport (including AhABCG22-1 and AhABCG22-2), were identified homologously and phylogenetically, and further analyzed at the transcriptional level by real-time RT-PCR, simultaneously determining ABA levels in peanut leaves in response to drought. The high sequence identity and very similar subcellular localization of the proteins deduced from 14 identified genes involved in ABA production, catabolism and transport with the reported corresponding enzymes in databases suggest their similar roles in regulating cellular ABA levels. In response to drought stress, ABA accumulation levels in peanut leaves agree very well with the up-regulated expressions of ABA-producing genes (AhZEP, AhNCED1, AhAAO2, AhABA3, AhBG11 and AhBG24) and PM-localized ABA importer genes (AhABCG22-1 and AhABCG22-2), although the expression of ABA catabolic genes (AhCYP707A3 and AhUGT71K1) was also up-regulated. It is likely that drought-responsive induction of catabolic genes helps not only to maintain ABA levels within a permissible range, but also to prepare the plant for degradation of ABA after removal of the stress. These results suggest that ABA homeostasis in peanut leaves in response to drought may be coordinated by a master regulatory circuit that involves production, catabolism, and as well as transport.

plant biology↗