Biology subjects

Li, A.

Publications and source records attributed to Li, A..

At least 235 records · Page 13Linked to original sources

bioRxiv2020

Cellular Anatomy of the Mouse Primary Motor Cortex

An essential step toward understanding brain function is to establish a cellular-resolution structural framework upon which multi-scale and multi-modal information spanning molecules, cells, circuits and systems can be integrated and interpreted. Here, through a collaborative effort from the Brain Initiative Cell Census Network (BICCN), we derive a comprehensive cell type-based description of one brain structure - the primary motor cortex upper limb area (MOp-ul) of the mouse. Applying state-of-the-art labeling, imaging, computational, and neuroinformatics tools, we delineated the MOp-ul within the Mouse Brain 3D Common Coordinate Framework (CCF). We defined over two dozen MOp-ul projection neuron (PN) types by their anterograde targets; the spatial distribution of their somata defines 11 cortical sublayers, a significant refinement of the classic notion of cortical laminar organization. We further combine multiple complementary tracing methods (classic tract tracing, cell type-based anterograde, retrograde, and transsynaptic viral tracing, high-throughput BARseq, and complete single cell reconstruction) to systematically chart cell type-based MOp input-output streams. As PNs link distant brain regions at synapses as well as host cellular gene expression, our construction of a PN type resolution MOp-ul wiring diagram will facilitate an integrated analysis of motor control circuitry across the molecular, cellular, and systems levels. This work further provides a roadmap towards a cellular resolution description of mammalian brain architecture.

neuroscience↗

bioRxiv2020

The transmembrane domain of DWORF activates SERCA directly; P15 and W22 residues are essential.

The Ca-ATPase isoform 2a (SERCA2a) re-sequesters cytosolic Ca2+ into the sarcoplasmic reticulum (SR) of cardiac myocytes, enabling muscle relaxation during diastole. A central factor in heart failure is abnormally high cytosolic [Ca2+], resulting in pathophysiology and decreased cardiac performance. Therefore, augmentation of the SERCA2a Ca2+ transport activity is a promising therapeutic approach. A novel transmembrane peptide, dwarf open reading frame (DWORF), is proposed to enhance SR Ca2+ uptake and myocyte contractility by displacing the protein phospholamban (PLB) from its inhibitory site on SERCA2a. In the present study, we have developed several cell-based FRET biosensor systems for time-resolved FRET (TR-FRET) measurements of the protein-protein interactions and structural changes in SERCA2a complexes with PLB and/or DWORF. To test the hypothesis that DWORF competes with PLB to occupy the putative SERCA2a binding site, we transiently transfected DWORF into a stable cell line expressing SERCA2a labeled with green fluorescent protein (GFP, the FRET donor) and PLB labeled with red fluorescent protein (RFP, the FRET acceptor). We observed a significant decrease in FRET efficiency, consistent with a decrease in the fraction of SERCA2a bound to PLB. Functional analysis demonstrates that DWORF activates SERCA in both the presence and absence of PLB. Furthermore, using site-directed mutagenesis, we generated DWORF variants that do not activate SERCA, thus identifying residues that are necessary for functional SERCA2a-DWORF interactions. This work advances our mechanistic understanding of the regulation of SERCA2a by small transmembrane proteins and sets the stage for future therapeutic development in heart failure research.

biophysics↗

bioRxiv2020

Worldwide tracing of mutations and the evolutionary dynamics of SARS-CoV-2

Understanding the mutational and evolutionary dynamics of SARS-CoV-2 is essential for treating COVID-19 and the development of a vaccine. Here, we analyzed publicly available 15,818 assembled SARS-CoV-2 genome sequences, along with 2,350 raw sequence datasets sampled worldwide. We investigated the distribution of inter-host single nucleotide polymorphisms (inter-host SNPs) and intra-host single nucleotide variations (iSNVs). Mutations have been observed at 35.6% (10,649/29,903) of the bases in the genome. The substitution rate in some protein coding regions is higher than the average in SARS-CoV-2 viruses, and the high substitution rate in some regions might be driven to escape immune recognition by diversifying selection. Both recurrent mutations and human-to-human transmission are mechanisms that generate fitness advantageous mutations. Furthermore, the frequency of three mutations (S protein, F400L; ORF3a protein, T164I; and ORF1a protein, Q6383H) has gradual increased over time on lineages, which provides new clues for the early detection of fitness advantageous mutations. Our study provides theoretical support for vaccine development and the optimization of treatment for COVID-19. We call researchers to submit raw sequence data to public databases.

microbiology↗

bioRxiv2020

Enhanced specificity of high sensitivity somatic variant profiling in cell-free DNA via paired normal sequencing: design, validation, and clinical experience of the MSK-ACCESS liquid biopsy assay

Circulating cell-free DNA (cfDNA) from blood plasma of cancer patients can be used to interrogate somatic tumor alterations non-invasively or when adequate tissue is unavailable. We have developed and clinically implemented MSK-ACCESS (Analysis of Circulating cfDNA to Evaluate Somatic Status), an NGS assay for detection of very low frequency somatic alterations in select exons and introns of 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 98% for a priori mutation profiling. To evaluate the performance and utility of MSK-ACCESS, we report results from the first 681 prospective blood samples (617 patients) that underwent clinical analysis to guide patient management. Somatic mutations, copy number, and/or structural variants were detected in 73% of the samples, and 56% of these circulating-tumor DNA (ctDNA) positive samples had clinically actionable alterations. The utilization of matched white blood cell sequencing allowed retention of somatic alterations while filtering out over 10,000 germline and clonal hematopoiesis variants, thereby greatly enhancing the specificity of the assay. Taken together, our experience illustrates the importance of analyzing a matched normal sample when interpreting cfDNA results and highlights the potential of cfDNA profiling to guide treatment selection, monitor treatment response, and identify mechanisms of treatment resistance.

genomics↗

bioRxiv2020

Germline gene de-silencing by a transposon insertion is triggered by an altered landscape of local piRNA biogenesis

Transposable elements (TE) are selfish genetic elements that can cause harmful mutations. In Drosophila, it has been estimated that half of all spontaneous visible marker phenotypes are mutations caused by TE insertions. Because of the harm posed by TEs, eukaryotes have evolved systems of small RNA-based genome defense to limit transposition. However, as in all immune systems, there is a cost of autoimmunity and small RNA-based systems that silence TEs can inadvertently silence genes flanking TE insertions. In a screen for essential meiotic genes in Drosophila melanogaster, a truncated Doc retrotransposon within a neighboring gene was found to trigger the germline silencing of ald, the Drosophila Mps1 homolog, a gene essential for meiosis. A subsequent screen for modifiers of this silencing identified a new insertion of a Hobo DNA transposon in the same neighboring gene. Here we describe how the original Doc insertion triggers flanking piRNA biogenesis and local gene silencing and how the additional Hobo insertion leads to de-silencing by reducing flanking piRNA biogenesis triggered by the original Doc insertion. These results support a model of TE-mediated silencing by piRNA biogenesis in cis that depends on local determinants of transcription. This may explain complex patterns of off-target gene silencing triggered by TEs within populations and in the laboratory. It also provides a mechanism of sign epistasis among TE insertions. Author SummaryTransposable elements (TEs) are selfish DNA elements that can move through genomes and cause mutation. In some species, the vast majority of DNA is composed of this form of selfish DNA. Because TEs can be harmful, systems of genome immunity based on small RNA have evolved to limit the movement of TEs. However, like all systems of immunity, it can be challenging for the host to distinguish self from non-self. Thus, TE insertions occasionally cause the small RNA silencing machinery to turn off the expression of critical genes. The rules by which this inadvertent form of autoimmunity causes gene silencing are not well understood. In this article, we describe a phenomenon whereby a TE insertion, rather than silencing a nearby gene, rescues the silencing of a gene caused by another TE insertion. This reveals a mode of TE interaction via small RNA silencing that may be important for understanding how TEs exert their effects on gene expression in populations and across species.

genetics↗

bioRxiv2020

Visualizing dendritic characteristics of corticotropin-releasing hormone neurons at single-cell resolution in the whole mouse brain

Corticotropin-releasing hormone (CRH) is an important neuromodulator with wide distribution in the brain. Here, we screened the CRH-IRES-Cre;Ai32 mouse line to reveal the morphologies of individual CRH neurons throughout the mouse brain by using fluorescence micro-optical sectioning tomography (fMOST) system. Diverse dendritic morphologies and projection fibers were found in various brain regions. Reconstructions showed hypothalamic CRH neurons had the smallest somatic volumes and simplest dendritic branches, and CRH neurons in several regions shared a bipolar morphology. Further investigations in the medial prefrontal cortex unveiled somatic depth-dependent morphologies that exhibited three types of connections and CRH neurons in the anterior parvicellular area of hypothalamus had fewer and smaller Herring bodies whereas in the periventricular area had more and larger Herring bodies that were present within fibers projecting to the third ventricle. Our findings provide the most comprehensive intact morphologies of CRH neurons throughout the mouse brain that is currently available.

neuroscience↗

bioRxiv2020

Selective linkage of mitochondrial enzymes to intracellular calcium stores differs between human induced pluripotent stem cells, neural stem cells and neurons

The coupling of the endoplasmic reticulum (ER) with mitochondria modulates neuronal calcium signaling. Whether this link changes with neuronal development is unknown. The current study first determined whether ER calcium stores are similar during development of human neurons, and then tested if the ER/mitochondrial coupling varied with development. The release of ER calcium to the cytosol by the IP3 agonist bradykinin was determined in human induced-pluripotent stem cells (iPSC), neural stem cells (NSC) and neurons. The concentration dependence for the release of ER calcium was similar at different stages of development. Metabolism changes dramatically with development. Glycolysis is the main energy source in iPSC and NSC whereas mitochondrial metabolism is more prominent in neurons. To test whether the coupling of mitochondria and ER changed with development, bombesin or bradykinin releasable calcium stores (BRCS) were monitored after inhibiting either of two key mitochondrial enzyme complexes: the alpha-ketoglutarate dehydrogenase complex (KGDHC) or the pyruvate dehydrogenase complex (PDHC). Inhibition of KGDHC did not alter BRCS in either iPSC or NSC. Inhibition of PDHC in neurons diminished BRCS whereas decreased KGDHC activity exaggerated BRCS. The latter finding may help understand the pathology of Alzheimers disease (AD). BRCS is exaggerated in cells from AD patients and KGDHC is reduced in brains of patients with AD. In summary, a prominent ER/mitochondrial link in neurons is associated with selective mitochondrial enzymes. The ER/mitochondrial link changes with human neuronal development and plausibly links ER calcium changes to AD.

neuroscience↗

bioRxiv2020

Exaggerated postnatal surge of orexin and the effects of elimination of excess orexinon blood pressure in spontaneously hypertensive rats in postnatal development

It has been established that an overactive orexin (OX) system is associated with neurogenic hypertension in spontaneously hypertensive rats (SHRs). However, the chronology and mechanism of such association between orexin system and hypertension is unclear. We hypothesized that an aberrant surge of OX neurons in SHRs precedes the aberrant increase of arterial blood pressure (ABP) during postnatal development, which was primarily contributed by the exaggerated postnatal OX neurogenesis. We found that (1) SHRs experienced a greater surge in the number of orexin neurons than normotensive Wistar-Kyoto (WKY) rats before P16, which led to significantly more OX neurons than age-matched controls by P15-16 (3680{+/-}219 vs 2407{+/-}182, respectively, P=0.002). (2) Exaggerated OX neurogenesis, marked by bromodeoxyuridine (BrdU), was the primary contributor to excessive OX neurons in SHRs during development. (3) In contrast, SHRs and normotensive control rats have similar mean arterial blood pressure (ABP) at P15, and a significantly higher ABP in SHR than WKY emerges at P20 (74.8 {+/-} 2.5 vs 66.9 {+/-} 4.4 mmHg in wakefulness, respectively, P<0.05), a few days following the surge of OX activity. (4) Selectively eliminating excess ([~]30%) orexin neurons, via a targeted neurotoxin, in SHRs between P30 and P40 results in a significantly lowered ABP compared to non-lesioned SHRs at P40. We suggest that the postnatal surge of OX neurons, primarily attributed to the exaggerated postnatal OX neurogenesis, may be necessary for the development of higher ABP in SHRs, and modulation of the overactive OX system may have a preventative effect during the pre-hypertensive period. New FindingsO_ST_ABSWhat is the central question of this study?C_ST_ABSExcess orexin neurons have been associated with hypertension in spontaneously hypertensive rats, however, the association and mechanism between developing excess orexin neurons and high blood pressure are unknown. What is the main finding and its importance?Using spontaneously hypertensive rats in anatomical and physiological studies, we provided evidence showing that the excess OX neurons, primarily via exaggerated OX neurogenesis, may be necessary in developing a higher ABP in SHRs during development, and modulation of the overactive orexin system may be beneficial in treating hypertension.

neuroscience↗

bioRxiv2020

CHMP2B regulates TDP-43 phosphorylation and proteotoxicity via modulating CK1 turnover independent of the autophagy-lysosomal pathway

Protein inclusions containing phosphorylated TDP-43 are a shared pathology in several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, most ALS/FTD patients do not have a mutation in TDP-43 or the enzymes directly regulating its phosphorylation. It is intriguing how TDP-43 becomes hyperphosphorylated in each disease condition. In a genetic screen for novel TDP-43 modifiers, we found that knockdown (KD) of CHMP2B, a key component of the endosomal sorting complex required for transport (ESCRT) machinery, suppressed TDP-43-mediated neurodegeneration in Drosophila. Further investigation using mammalian cells indicated that CHMP2B KD decreased whereas its overexpression (OE) increased TDP-43 phosphorylation levels. Moreover, a known FTD-causing mutation CHMP2Bintron5 promoted hyperphosphorylation, insolubility and cytoplasmic accumulation of TDP-43. Interestingly, CHMP2B did not manifest these effects by its well-known function in the autophagy-lysosomal pathway. Instead, the kinase CK1 tightly regulated TDP-43 phosphorylation level in cells, and CHMP2B OE or CHMP2BIntron5 significantly decreased ubiquitination and the turnover of CK1 via the ubiquitin-proteasome (UPS) pathway. Finally, we showed that CHMP2B protein levels increased in the cerebral cortices of aged mice, which might underlie the age-associated TDP-43 pathology and disease onset. Together, our findings reveal a molecular link between the two ALS/FTD-pathogenic proteins CHMP2B and TDP-43, and provide an autophagy-independent mechanism for CHMP2B in pathogenesis. SIGNIFICANCE STATEMENTTDP-43 and CHMP2B are both ALS/FTD-associated proteins. Protein aggregations containing phosphorylated TDP-43 are a pathological hallmark of ALS/FTD; however, it is unclear how increased phosphorylation of TDP-43 occurs in diseases. The pathogenesis of CHMP2B has mainly been considered as a consequence of autophagy-lysosomal dysfunction. Here, we reveal that increase of CHMP2B levels (which occurs in aged mouse brains) or expression of the disease-causing mutation CHMP2BIntron5 promotes TDP-43 hyperphosphorylation, insolubility and cytoplasmic mislocalization. This effect is independent of the autophagy-lysosomal pathway but rather relies on the proteasome-mediated turnover of the kinase CK1 that phosphorylates TDP-43. Together, we provide a new molecular mechanism of CHMP2B pathogenesis by linking it to TDP-43 pathology via CK1.

neuroscience↗

bioRxiv2020

Single-cell Transcriptomics Identifies Gene Expression Networks Driving Differentiation and Tumorigenesis in the Human Fallopian Tube

The human fallopian tube harbors the cell-of-origin for the majority of high-grade serous ovarian cancers (HGSCs), but its cellular composition, particularly the epithelial component, is poorly characterized. We performed single-cell transcriptomic profiling in 12 primary fallopian specimens from 8 patients, analyzing around 53,000 individual cells to map the major immune, fibroblastic and epithelial cell types present in this organ. We identified 10 epithelial sub-populations, characterized by diverse transcriptional programs including SOX17 (enriched in secretory epithelial cells), TTF3 and RFX3 (enriched in ciliated cells) and NR2F2 (enriched in early, partially differentiated secretory cells). Based on transcriptional signatures, we reconstructed a trajectory whereby secretory cells differentiate into ciliated cells via a RUNX3high intermediate. Computational deconvolution of the cellular composition of advanced HGSCs based on epithelial subset signatures identified the early secretory population as a likely precursor state for the majority of HGSCs. The signature of this rare population of cells comprised both epithelial (EPCAM, KRT) and mesenchymal (THY1, ACTA2) features, and was enriched in mesenchymal-type HGSCs (P = 6.7 x 10-27), a group known to have particularly poor prognoses. This cellular and molecular compendium of the human fallopian tube in cancer-free women is expected to advance our understanding of the earliest stages of fallopian epithelial neoplasia.

genomics↗

bioRxiv2020

SRY: An Effective Method for Sorting Long Reads of Sex-limited Chromosome

Most of available reference genomes are lack of the sequence map of sex-limited chromosomes, that make the assemblies uncompleted. Recent advances on long reads sequencing and population sequencing raise the opportunity to assemble sex-limited chromosomes without the traditional complicated experimental efforts. We introduce a computational method that shows high efficiency on sorting and assembling long reads sequenced from sex-limited chromosomes. It will lead to the complete reference genomes and facilitate downstream research of sex-limited chromosomes.

bioinformatics↗

bioRxiv2020

Imaging ATP Consumption in Resting Skeletal Muscle: One Molecule at a Time

Muscle contraction is driven by sarcomere shortening and powered by cyclic hydrolysis of ATP by myosin molecular motors. However, myosin in relaxed muscle continues to slowly hydrolyze ATP, analogous to an idling engine. Utilizing super-resolution microscopy to directly image single molecule fluorescent ATP turnover in relaxed rat soleus skeletal muscle sarcomeres, we observed two rates of myosin ATP consumption that differed 5-fold. These distinct hydrolysis rates were spatially segregated, with the slower or "super relaxed" rate localized predominantly to the sarcomere C-zone, where Myosin Binding Protein-C (MyBP-C), a known modulator of muscle contractile function, exists. This super relaxed hydrolysis rate and its location suggest that MyBP-C can sequester myosin motors to regulate muscle metabolism and heat production in resting muscle and force generation upon activation. One Sentence SummarySuper relaxed skeletal muscle myosin is stabilized by Myosin-Binding Protein C as imaged by single ATP molecule consumption.

biophysics↗

bioRxiv2020

Single cell RNA sequencing reveals regulation of fetal ovary development in the monkey (Macaca fascicularis)

Germ cells are vital for reproduction and heredity. However, the mechanisms underlying female germ cell development in primates, especially in late embryonic stages, remain elusive. Here, we performed single-cell RNA sequencing of 12471 cells from whole fetal ovaries, and explored the communications between germ cells and niche cells. We depicted the two waves of oogenesis at single cell resolution and demonstrated that progenitor theca cells exhibit similar characteristics to Leydig cells in fetal monkey ovaries. Notably, we found that ZGLP1 displays differentially expressed patterns between mouse and monkey, which is not overlapped with NANOG in monkey germ cells, suggesting its role in meiosis entry but not in activating oogenic program in primates. Furthermore, the majority of germ cell clusters that highly expressed PRDM9 and SPO11 might undergo apoptosis after cyst breakdown, leading to germ cell attrition. Overall, our work provides new insights into the molecular and cellular basis of primate fetal ovary development at single-cell resolution.

developmental biology↗

bioRxiv2020

Polymerase Theta Inhibition Kills Homologous Recombination Deficient Tumors

PARP inhibitors (PARPi) have become a new line of therapy for Homologous Recombination (HR)-deficient cancers. However, resistance to PARPi has emerged as a major clinical problem. DNA polymerase theta (POL{theta}) is synthetic lethal with HR and a druggable target in HR-deficient cancers. Here, we identified the antibiotic Novobiocin (NVB) as a specific POL{theta} inhibitor that selectively kills HR-deficient tumor cells in vitro and in vivo. NVB directly binds to the POL{theta} ATPase domain, inhibits its ATPase activity, and phenocopies POL{theta} depletion. BRCA-deficient tumor cells and those with acquired PARPi resistance are sensitive to NVB in vitro and in vivo. Increased POL{theta} expression levels predict NVB sensitivity. The mechanism of NVB-mediated cell death in PARPi resistant cells is the accumulation of toxic RAD51 foci, which also provides a pharmacodynamic biomarker for NVB response. Our results demonstrate that NVB may be useful alone or in combination with PARPi in treating HR-deficient tumors, including those with acquired PARPi resistance. One Sentence SummaryWe identified Novobiocin as a specific POL{theta} inhibitor that selectively kills naive and PARPi resistance HR-deficient tumors in vitro and in vivo.

cancer biology↗

bioRxiv2020

Decoupling transcription factor expression and activity enables dimmer-switch gene regulation

Our mechanistic understanding of how gene regulatory networks can achieve complex biological responses is limited. We find that the galactose-responsive pathway in S. cerevisiae has a complex behavior - the decision to induce is controlled independently from the induction level, resembling a mechanical dimmer-switch. Surprisingly, this behavior is not achieved directly through chromatin regulation or combinatorial control at galactose-responsive promoters. Instead, this behavior is achieved by hierarchical regulation of the expression and catalytic activity of a single transcription factor. This genetic motif allows evolution to independently act on both properties, providing a means to tune both resource allocation strategies in dynamic multi-input environments on both physiological and evolutionary time-scales. Hierarchical regulation is ubiquitous and thus dimmer-switch regulation is likely a key feature of many biological systems.

systems biology↗

bioRxiv2020

Automated platform for high-throughput screening of base-modified aptamers for affinity and specificity

Aptamers incorporating chemically modified bases can achieve superior affinity and specificity compared to natural aptamers, but their characterization remains a labor-intensive, low-throughput task. Here we describe the non-natural aptamer array (N2A2) system, in which a minimally modified Illumina MiSeq instrument is used for the high-throughput generation and characterization of large libraries ([~]106) of base-modified DNA aptamer candidates on the basis of both target affinity and specificity. We first demonstrate the capability to screen multiple different base modifications to identify the optimal chemistry for high-affinity target binding. We next use N2A2 to generate aptamers that can maintain excellent specificity even in complex samples, with equally strong target affinity in both buffer and diluted human serum. Given that N2A2 requires only minor mechanical modifications to the MiSeq, we believe N2A2 offers a broadly accessible tool for generating high-quality affinity reagents for diverse applications.

bioengineering↗

bioRxiv2020

Development of a Novel Class of Self-Assembling dsRNA Cancer Therapeutics: a Proof of Concept Investigation

Cancer has proven to be an extremely difficult challenge to treat. Several fundamental issues currently underlie cancer treatment including differentiating self from non-self, functional coupling of the recognition and therapeutic components of various therapies, and the propensity of cancerous cells to develop resistance to common treatment modalities via evolutionary pressure. Given these limitations, there is an increasing need to develop an all-encompassing therapeutic that can uniquely target malignant cells, decouple recognition from treatment, and overcome evolutionarily driven cancer resistance. We describe herein, a new class of programmable self-assembling dsRNA-based cancer therapeutics, that uniquely targets aberrant genetic sequences, and in a functionally decoupled manner, undergoes oncogenic RNA activated displacement (ORAD), initiating a therapeutic cascade that induces apoptosis and immune activation. As a proof-of-concept, we show that RNA strands targeting the EWS/Fli1 fusion gene in Ewing Sarcoma cells that are end-blocked with phosphorothioate bonds and additionally sealed with a 2-U modified DNA protector can be used to induce specific and potent killing of cells containing the target oncogenic sequence, but not wildtype.

bioengineering↗

bioRxiv2020

Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics

Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. Yet such efforts are rare and focus only on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report the first multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We developed a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative sign and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and beta-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers lessons for engineering P450s.

evolutionary biology↗