Biology subjects

Shi, Y.

Publications and source records attributed to Shi, Y..

At least 289 records · Page 16Linked to original sources

bioRxiv2022

Identification of RNA-based cell-type markers for the stem cell manufacture system with a statistical scoring function

Cell-type biomarkers are useful in stem cell manufacture to monitor cell purification, cell quantity, and quality. However, the study on cell-type markers, specifically for stem cell manufacture, is limited. The emerging questions are which RNA transcripts can serve as biomarkers during stem cell culture, and what method can efficiently and accurately discover these biomarkers. We developed a scoring function system to identify RNA biomarkers with RNA-seq data. We applied the method to two data sets, one for extracellular RNAs (ex-RNAs) and the other for intracellular microRNAs (miRNAs). The data set have RNA-seq data of ex-RNAs from cell culture media for six different types of cells, including human embryonic stem cells. To get the RNA-seq data from intracellular miRNAs, we cultured three types of cells: human embryonic stem cells (H9), neural stem cells (NSC), hESC-derived endothelial cells (EC) and conducted small RNA-seq to their intracellular miRNAs. Using these data, we identified a set of ex-RNAs/smRNAs as candidates of biomarkers for different types of cells for cell manufacture. We also used deep-learning based prediction methods and simulated data to validate these discovered biomarkers.

bioinformatics↗

bioRxiv2022

METTL7A and METTL7B are responsible for S-thiol methyl transferase activity in liver

S-Methylation of drugs containing thiol-moieties often alters their activity and results in detoxification. Historically, scientists attributed methylation of exogenous aliphatic and phenolic thiols to a putative S-adenosyl-L-methionine dependent membrane-associated phase II enzyme known as thiol methyltransferase (TMT). TMT has a broad substrate specificity and methylates the thiol metabolite of spironolactone, mertansine, ziprasidone, captopril, and the active metabolites of the thienopyridine pro-drugs, clopidogrel, and prasugrel. Despite TMTs role in the S-methylation of clinically relevant drugs, the enzyme(s) responsible for this activity remained unknown. We recently identified methyltransferase-like protein 7B (METTL7B) as an alkyl thiol-methyltransferase. METTL7B is an endoplasmic-reticulum-associated protein with similar biochemical properties and substrate specificity to TMT. Yet, the historic TMT inhibitor, 2,3-dichloro--methylbenzylamine (DCMB), has no effect on the activity of METTL7B, indicating that multiple enzymes contribute to TMT activity. Here we report that methyltransferase-like protein 7A (METTL7A), an uncharacterized member of the METTL7 family, also acts as a thiolmethyltransferase. METTL7A exhibits similar biochemical properties to TMT, including inhibition by DCMB (IC50 1.2 {micro}M). Applying quantitative proteomics to human liver microsomes and gene modulation experiments in HepG2 and HeLa cells, we determined that TMT activity correlates closely with METTL7A and METTL7B protein levels. Furthermore, purification of a novel His-GST-tagged recombinant protein and subsequent activity experiments prove that METTL7A can selectively methylate exogenous thiol-containing substrates, including 7-thiospironolactone, dithiothreitol, 4-chlorothiophenol, and mertansine. We conclude that the METTL7 family encodes for two enzymes, METTL7A and METTL7B, which we have renamed TMT1A1 and TMT1B1, respectively, that are responsible for TMT activity in liver microsomes. Significance StatementWe identified METTL7A (TMT1A1) and METTL7B (TMT1B1) as the enzymes responsible for the microsomal alkyl thiol methyltransferase activity. These are the first two enzymes directly associated with microsomal TMT activity. S-Methylation of many commonly prescribed thiol-containing drugs alters their pharmacological activity and/or toxicity, and identifying the enzymes responsible, will improve our understanding of the DMPK properties of alkyl- or phenolic-thiol-containing therapeutics. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/511968v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@2419f9org.highwire.dtl.DTLVardef@254c5borg.highwire.dtl.DTLVardef@b81378org.highwire.dtl.DTLVardef@1064c79_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Efficient and rapid fluorescent protein knock-in with universal donors in mammalian stem cells

Fluorescent protein (FP) tagging is a foundational approach in cell biology because it allows observation of protein distribution, dynamics, and interaction with other proteins in living cells. However, the typical approach using overexpression of tagged proteins can perturb cell behavior and introduce localization artifacts. To preserve native expression, fluorescent proteins can be inserted directly into endogenous genes. This approach has been standard in yeast for decades, and more recently in invertebrate model organisms with the advent of CRISPR/Cas9. However, endogenous fluorescent protein tagging has not been widely used in mammalian cells due to inefficient homology-directed repair (HDR). Here, we describe a streamlined method for efficient and fast integration of FP tags into native loci via non-homologous end joining (NHEJ) in mouse embryonic stem cells. Our protocol minimizes cloning with universal donors, allows for N or C-terminal tagging of endogenous proteins, and requires less than 2 weeks from transfection to imaging, thereby improving the applicability of FP knock-in in mammalian cells.

cell biology↗

bioRxiv2022

Pathway elucidation of pharmaceutical cucurbitacin IIa in Hemsleya chinensis and high-level production of precursor cucurbitadienol in engineered Saccharomyces cerevisiae and Nicotiana benthamiana

Cucurbitacin IIa is a triterpene isolated exclusively from Hemsleya plants, which is non-steroidal anti-inflammatory drug that function as the main ingredient of Hemslecin capsules and Supplemental Tablets in China. In this study, the biosynthetic pathway of cucurbitacin IIa was elucidated by characterization of squalene epoxidases (HcSE1, HcSE2), cucurbitenol synthases (HcOSC6) and acyltransferases (HcAT1) in Hemsleya chinensis. Meanwhile, cycloartenol synthases (HcOSC1), isomultiflorenol synthases (HcOSC5) and {beta}-amyrin synthase (HcOSC2-4) involved in sterol and triterpenes biosynthesis were functionally illustrated. The high-level production of yeast the key cucurbitacin precursor, cucurbitadienol, was constructed to produce 296.37 mg/L cucurbitadienol and 722.99 mg/L total triterpenoid which is the highest yield cucurbitadienol from known engineered microbes. Moreover, production of cucurbitenol in transient expression of tobacco was employed to achieve 94.8 mg/g dry weight (dw) cucurbitenol from leaves. In this study, the key genes involved in cucurbitacin IIa biosynthesis were identified to facilitate its medical applications via biosynthetic strategy. Meanwhile, the high-level production of cucurbitadienol chassis yeast and tobacco transient expression offered a robust and sSupplemental Table substrate for pharmaceutical cucurbitacin production and screening platform for candidate genes involved in cucurbitacin biosynthesis.

synthetic biology↗

bioRxiv2022

A Purinergic Mechanism Underlying Metformin Regulation of Hyperglycemia

Metformin, created in 1922, has been the first-line medication treating type 2 diabetes mellitus for almost 70 years; however, its mechanism of action has been heavily debated, partly because most prior studies used supratherapeutic concentrations exceeding 1mM. Here we report that at a clinically relevant concentration of 10 M, metformin blocks high glucose-stimulated ATP secretion from hepatocytes mediating its antihyperglycemic action. Following glucose administration, mice demonstrate increased circulating ATP concentrations, which are prevented by metformin. Extracellular ATP through P2Y2 receptors (P2Y2R) compromises insulin-induced AKT activation and increases hepatic glucose production. In addition, metformin-dependent improvement in glucose tolerance is abolished in P2Y2R-null mice. Thus, removing the target of extracellular ATP, P2Y2R, mimics the effects of metformin, revealing a novel purinergic antidiabetic mechanism for metformin. One-Sentence SummaryMetformin abolishes high glucose-stimulated ATP secretion, preventing purinergic mechanism-mediated hepatic insulin resistance and glucose production.

pharmacology and toxicology↗

bioRxiv2022

Conserved allosteric inhibition mechanism in SLC1 transporters

Excitatory Amino Acid Transporter 1 (EAAT1) is a plasma-membrane glutamate transporter belonging to the SLC1 family of solute carriers. It plays a key role in neurotransmitter transport and contributes to the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined using cryo-EM, in complex with UCPH-101, a highly potent and non-competitive inhibitor of EAAT1. Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid transporter, which regulates pools of amino acids such as glutamine, serine and alanine between intracellular and extracellular compartments in a Na+ dependent manner. ASCT2 also belongs to the SLC1 family and shares 58% sequence similarity with EAAT1. However, allosteric modulation of ASCT2 via non-competitive inhibitors is unknown. Here we explore the UCPH-101 inhibitory mechanisms of EAAT1 and ASCT2 by using rapid kinetic experiments. Our results show that UCPH-101 slows substrate translocation rather than substrate or Na+ binding, confirming a non-competitive inhibitory mechanism, but only partially inhibits wild-type ASCT2 with relatively low affinity. Guided by computational modeling using ligand docking and molecular dynamics (MD) simulations, we selected two residues involved in UCPH-101/EAAT1 interaction, which were mutated in ASCT2 (F136Y, I237M, F136Y/I237M) in the corresponding positions. We show that in the F136Y/I237M double mutant transporter, 100% of the inhibitory effect of UCPH-101 on anion current could be restored, and the apparent affinity was increased (Ki = 9.3 M), much closer to the EAAT1 value of 0.6 M. Finally, we identify a novel non-competitive ASCT2 inhibitor, identified through virtual screening and experimental testing against the allosteric site, further supporting its localization. Together, these data indicate that the mechanism of allosteric modulation is conserved between EAAT1 and ASCT2. Due to the difference in binding site residues between ASCT2 and EAAT1, these results raise the possibility that more potent, and potentially selective inhibitors can be designed that target the ASCT2 allosteric binding site.

biophysics↗

bioRxiv2022

Illuminating histidine phosphorylation in the pancreatic tumor microenvironment

Development of phosphohistidine (pHis) antibodies has significantly advanced our understanding of pHis contributions to tumor biology, including a tumor suppressive role for a pHis phosphatase, a metastasis suppressive role for His kinases, and pHis regulation of T cell receptor signaling. Using these antibodies, we investigated pHis pathway regulation in the mouse pancreatic tumor microenvironment. We identified deregulated expression of pHis and pHis phosphatases that correlated with mouse pancreatic tumor progression. We developed a protocol to circumvent the acid and heat-sensitivity of pHis signals, enabling their co-staining with other proteins in FFPE tissue, identifying a significant enrichment of 1-pHis and a subtype of 3-pHis signals (Gly-3-pHis) in the stroma. We discovered increased Gly-3-pHis levels in tumor-associated myeloid cells mainly resulting from elevated ATP citrate lyase 3-pHis levels and predicted the existence of pHis in cell-cell adhesion proteins. We provide evidence that mitochondrial delocalization of PGAM5, a pHis phosphatase with increased expression during pancreatic tumorigenesis, occurs in tumor cells as compared to stromal cells, enabling access to PGAM5s known cytoplasmic substrate, pHis-NME (Non-MEtastatic), and two potential Gly-3-pHis substrates, SCS (Succinyl CoA Synthetase) and {beta}-catenin. Overall, we introduce a new method and possible targets for future studies of pHis pathway deregulation during tumorigenesis.

cancer biology↗

bioRxiv2022

A machine learning liver-on-a-chip system for safer drug formulation

Drug metabolism leads to biotransformations of pharmaceutical substances that alter drug efficacy, toxicity, as well as drug interactions. Modeling these processes ex vivo stands to greatly accelerate our capacity to develop safe and efficacious drugs and formulations. Recognizing the liver as the primary site of drug metabolism, here we report a novel whole-tissue ex vivo liver screening platform that enabled modeling of hepatic metabolism and tracking of hepatotoxic drug metabolites. We applied the system for the characterization of acetaminophen (APAP) metabolism and identified interactions that can mitigate the generation of toxic metabolites ex vivo. Combining our experimental platform with state-of-the-art machine learning, we validated two novel functional excipients that can prevent APAP hepatotoxicity in vivo in mice. To assess translational potential, we prototyped a novel solid dosage form with controlled release of both APAP and our functional excipients. Our this platform provides innovative potential access to actionable data on drug metabolism to support the development of new therapeutic approaches.

bioengineering↗

bioRxiv2022

Pharmacogenetic allele variant frequencies: An analysis of the VAs Million Veteran Program (MVP) as a representation of the diversity in US population.

We present allele frequencies of pharmacogenomics relevant variants across multiple ancestry in a sample representative of the US population. We analyzed 658,582 individuals with genotype data and extracted pharmacogenomics relevant single nucleotide variant (SNV) alleles, human leukocyte antigens (HLA) 4-digit alleles and an important copy number variant (CNV), the full deletion/duplication of CYP2D6. We compiled distinct allele frequency tables for European, African American, Hispanic, and Asian ancestry individuals. In addition, we compiled allele frequencies based on local ancestry reconstruction in the African-American (2-way deconvolution) and Hispanic (3-way deconvolution) cohorts.

genomics↗

bioRxiv2022

Modeling and Simulation of Rat Non-Barrel Somatosensory Cortex. Part I: Modeling Anatomy

The function of the neocortex is fundamentally determined by its repeating microcircuit motif, but also by its rich, interregional connectivity. We present a data-driven computational model of the anatomy of non-barrel primary somatosensory cortex of juvenile rat, integrating whole-brain scale data while providing cellular and subcellular specificity. The model consists of 4.2 million morphologically detailed neurons, placed in a digital brain atlas. They are connected by 14.2 billion synapses, comprising local, mid-range and extrinsic connectivity. We delineated the limits of determining connectivity from neuron morphology and placement, finding that it reproduces targeting by Sst+ neurons, but requires additional specificity to reproduce targeting by PV+ and VIP+ interneurons. Globally, connectivity was characterized by local clusters tied together through hub neurons in layer 5, demonstrating how local and interegional connectivity are complicit, inseparable networks. The model is suitable for simulation-based studies, and a 211,712 neuron subvolume is made openly available to the community.

neuroscience↗

bioRxiv2022

Inhibition inputs from hippocampal CA1 to retrosplenial agranular cortex gate social behavior

Retrosplenial cortex has been implicated in processing sensory information and spatial learning, with abnormal neural activity observed in association with psychedelics and in mouse and non-human primate models of autism spectrum disorders (ASD). The direct role of the retrosplenial cortex in regulating social behaviors remains unclear. This work reveals that the neural activity of retrosplenial agranular cortex (RSA), a subregion of retrosplenial cortex, is initially activated, then quickly suppressed upon social contact. The up-down phase of RSA neurons is crucial for normal social behaviors. PV-positive GABAergic neurons in the hippocampal CA1 region were found to send inhibitory projections to RSA. Blocking these CA1-RSA inhibitory inputs significantly impaired social behavior. Notably, enhancing the CA1-RSA inhibitory input could rescue social behavior defects in an ASD mouse model. This work suggests a neural mechanism for salience processing of social behavior and identifies a potential target for ASD intervention using neural modulation approaches.

neuroscience↗

bioRxiv2022

Coronaviruses using different strategies to antagonize antiviral responses and pyroptosis

Viral infection triggers inflammasome-mediated caspase-1 activation. Nevertheless, limited understanding exists regarding how viruses use the active caspase-1 to evade host immune response. Here, we use porcine epidemic diarrhea virus (PEDV) as a model of coronaviruses (CoVs) to illustrate the intricate regulation of CoVs to combat IFN-I signaling and pyroptosis. Our findings demonstrate that PEDV infection stabilizes caspase-1 expression via papain-like protease PLP2s deubiquitinase activity and the enhanced stabilization of caspase-1 disrupts IFN-I signaling by cleaving RIG-I at D189 residue. 6-Thioguanine (6TG), a PLP2 inhibitor, plays a critical role in reversing the inhibitory effect of IFN-I via PLP2 and also inhibiting PEDV replication. Meanwhile, PLP2 can degrade GSDMD-p30 by removing its K27-linked ubiquitin chain at K275 to restrain pyroptosis. Papain-like proteases from other genera of CoVs (PDCoV and SARS-CoV-2) have the similar activity to degrade GSDMD-p30. We further demonstrate that SARS-CoV-2 N protein induced NLRP3 inflammasome activation also uses the active caspase-1 to counter IFN-I signaling by cleaving RIG-I. Therefore, our work unravels a novel antagonistic mechanism employed by CoVs to evade host antiviral response.

immunology↗

bioRxiv2022

Development of Equine Polyclonal Antibodies as a Broad-Spectrum Therapy Against SARS-CoV-2 Variants

The Coronavirus disease 19 (COVID-19) pandemic has accumulated over 550 million confirmed cases and more than 6.34 million deaths worldwide. Although vaccinations has largely protected the population through the last two years, the effect of vaccination has been increasingly challenged by the emerging SARS-CoV-2 variants. Although several therapeutics including both monoclonal antibodies and small molecule drugs have been used clinically, high cost, viral escape mutations, and potential side effects have reduced their efficacy. There is an urgent need to develop a low cost treatment with wide-spectrum effect against the novel variants of SARS-CoV-2. Here we report a product of equine polyclonal antibodies that showed potential broad spectrum neutralization effect against the major variants of SARS-CoV-2. The equine polyclonal antibodies were generated by horse immunization with the receptor binding domain (RBD) of SARS-CoV-2 spike protein and purified from equine serum. A high binding affinity between the generated equine antibodies and the RBD was observed. Although designed against the RBD of the early wild type strain sequenced in 2020, the equine antibodies also showed a highly efficient neutralization capacity against the major variants of SARS-CoV-2, including the recent BA.2 Omicron variant (IC50 =1.867g/ml) in viral neutralization assay in Vero E6 cells using live virus cultured. The broad-spectrum neutralization capacity of the equine antibodies was further confirmed using pseudovirus neutralization assay covering the major SARS-CoV-2 variants including wild type, alpha, beta, delta, and omicron, showing effective neutralization against all the tested strains. Ex vivo reconstructed human respiratory organoids representing nasal, bronchial, and lung epitheliums were employed to test the treatment efficacy of the equine antibodies. Antibody treatment protected the human nasal, bronchial, and lung epithelial organoids against infection of the novel SARS-CoV-2 variants challenging public health, the Delta and Omicron BA.2 isolates, by reducing >95% of the viral load. The equine antibodies were further tested for potential side effects in a mouse model by inhalation and no significant pathological feature was observed. Equine antibodies, as a mature medical product, have been widely applied in the treatment of infectious diseases for more than a century, which limits the potential side effects and are capable of large scale production at a low cost. A cost-effective, wide-spectrum equine antibody therapy effective against the major SARS-CoV-2 variants can contribute as an affordable therapy to cover a large portion of the world population, and thus potentially reduce the transmission and mutation of SARS-CoV-2.

immunology↗

bioRxiv2022

Metabolic functional redundancy of the CYP9A subfamily members leads to P450-mediated lambda-cyhalothrin resistance in Cydia pomonella

BACKGROUNDThe evolution of insect resistance to pesticides poses a continuing threat to sustainable pest management. While much is known about the molecular mechanisms that confer resistance in model insects and few agricultural pests, far less is known about fruit pests. RESULTSHere we found that functional redundancy and preference of metabolism by cytochrome P450 monooxygenases (P450s) genes in the CYP9A subfamily confer resistance to lambda-cyhalothrin in Cydia pomonella, a major invasive pest of pome fruit. A total of four CYP9A genes, including CYP9A61, CYP9A120, CYP9A121, and CYP9A122, were identified from C. pomonella. Among these, CYP9A120, CYP9A121, and CYP9A122 were predominantly expressed in the midgut of larvae. The expression levels of these P450 genes were significantly induced by LD10 of lambda-cyhalothrin and were overexpressed in a field-evolved lambda-cyhalothrin resistant population. Knockdown of CYP9A120 and CYP9A121 by RNA-mediated interference (RNAi) increased the susceptibility of larvae to lambda-cyhalothrin. In vitro assays demonstrated that recombinant P450s expressed in Sf9 cells can metabolize lambda-cyhalothrin, but with functional redundancy and divergence through regioselectivity of metabolism. CYP9A121 preferred to convert lambda-cyhalothrin to 2'-hydroxy-lambda-cyhalothrin, whereas CYP9A122 only generated 4'-hydroxy metabolite of lambda-cyhalothrin. Although possesses a relatively low metabolic capability, CYP9A120 balanced catalytic competence to generate both 2'- and 4'-metabolites. CONCLUSIONCollectively, these results reveal that metabolic functional redundancy of three members of the CYP9A subfamily leads to P450-mediated lambda-cyhalothrin resistance in C. pomonella, thus representing a potential adaptive evolutionary strategy during its worldwide expansion.

molecular biology↗

bioRxiv2022

Functional characterization of multidomain protein Vip3Aa from Bacillus thuringiensis reveals a strategy to increase its insecticidal potency

Microbially derived, protein-based biopesticides offer a more sustainable pest management alternative to synthetic pesticides. Vegetative insecticidal proteins (Vip3), multidomain proteins secreted by Bacillus thuringiensis, represent a second-generation insecticidal toxin that have been used in transgenic crops. However, the molecular mechanism underlying Vip3s toxicity is poorly understood. Here, we determine the distinct functions and contributions of the domains of the Vip3Aa protein to its toxicity against Spodoptera frugiperda larvae. Vip3Aa domains II and III (DII-DIII) bind the midgut epithelium, while DI maintains the tetrameric state of the Vip3Aa protoxin, which is essential for its stability and toxicity. DI-DIII can be activated by midgut proteases, and exhibits ex vivo cytotoxicity similar to full-length Vip3Aa. We also determine that DV binds the peritrophic matrix via its glycan-binding activity, which is essential for Vip3Aa insecticidal activity. We further show that Vip3Aa has multiple protease activation sites and that introducing additional cleavage sites between DI and DII can increase the proteolysis efficiency and boost Vip3Aa insecticidal potency. This study provides insights into Vip3Aas mode-of-action and demonstrates a proof-of-concept strategy to enhance the insecticidal potency of Vip3Aa, which should significantly improve its application and development as a biopesticide.

microbiology↗

bioRxiv2022

Selective Regulation of Tuft Cell-Like Small Cell Lung Cancer by Novel Transcriptional Co-activators C11orf53 and COLCA2

The DepMap project has generated a huge resource for investigating selectively essential genes, which represent potential cancer therapeutic targets. However, manually sorting out which of the hundreds of selectively essential genes is understudied and warrants investigations is time-consuming and probably not practical. To efficiently identify uncharacterized, selectively essential genes, we collected and ranked the 347 selectively essential genes from the DepMap dataset by their PubMed publication counts, based on the assumption that genes with low publication counts are un-studied or under-studied. We successfully validated two of the top candidates in our ranking system, C11orf53 and COLCA2, as new vulnerabilities that are selectively essential in the class II POU domain transcription factor POU2F3-dependent tuft cell-like small cell lung cancer (SCLC) cell lines. Importantly, we found that the sequence motif, which mediates physical interactions of the transcriptional co-activator POU2AF1 with two of the class II POU domain-containing family of transcription factors (POU2F1 and POU2F2), is also present in the N-terminal regions of C11orf53 and COLCA2. We further confirmed that 1) COLCA2 physically interacts with POU2F3 through this conserved sequence motif; 2) this interaction is important for COLCA2 to regulate tuft cell-like SCLC cell growth, and 3) both C11orf53 and COLCA2 contain transcriptional co-activator domains. Consistently, we find similar transcriptomic changes in response to the loss of COLCA2 or POU2F3 in SCLC cells. In summary, our analysis pipeline enables identification and prioritization of understudied but important, selectively essential genes, leading to the identification of two new transcriptional co-activators for the class II POU domain transcription factors. Disruption of this important physical interaction is predicted to be a potential therapeutic strategy to selectively inhibit tuft cell-like SCLCs.

molecular biology↗

bioRxiv2022

Cryo-EM structures of α-synuclein filaments from Parkinson's disease and dementia with Lewy bodies

Parkinsons disease (PD) is the most common movement disorder, with resting tremor, rigidity, bradykinesia and postural instability being major symptoms (1). Neuropathologically, it is characterised by the presence of abundant filamentous inclusions of -synuclein in the form of Lewy bodies and Lewy neurites in some brain cells, including dopaminergic nerve cells of the substantia nigra (2). PD is increasingly recognised as a multisystem disorder, with cognitive decline being one of its most common non-motor symptoms. Many patients with PD develop dementia more than 10 years after diagnosis (3). PD dementia (PDD) is clinically and neuropathologically similar to dementia with Lewy bodies (DLB), which is diagnosed when cognitive impairment precedes parkinsonian motor signs or begins within one year from their onset (4). In PDD, cognitive impairment develops in the setting of well-established PD. Besides PD and DLB, multiple system atrophy (MSA) is the third major synucleinopathy (5). It is characterised by the presence of abundant filamentous -synuclein inclusions in brain cells, especially oligodendrocytes (Papp-Lantos bodies). We previously reported the electron cryo-microscopy (cryo-EM) structures of two types of -synuclein filaments extracted from the brains of individuals with MSA (6). Each filament type is made of two different protofilaments. Here we report that the cryo-EM structures of -synuclein filaments from the brains of individuals with PD, PDD and DLB are made of a single protofilament (Lewy fold) that is markedly different from the protofilaments of MSA. These findings establish the existence of distinct molecular conformers of assembled -synuclein in neurodegenerative disease.

neuroscience↗

bioRxiv2022

Tumor subclones, where are you?

IntroductionTumor clonal structure is closely related to future progression, which has been mainly investigated via mutation abundance clustering in bulk sample. With limited studies at single-cell resolution, a systematic comparison of the two approaches is still lacking. MethodsHere, using bulk and single-cell mutational data from liver and colorectal cancers, we would like to check the possibility of obtaining accurate tumor clonal structures from bulk-level analysis. We checked whether co-mutations determined by single-cell analysis had corresponding bulk variant allele frequency (VAF) peaks. We examined VAF ranges for different groups of co-mutations, and also the possibility of discriminating them. ResultsWhile bulk analysis suggested absence of subclonal peaks and possibly neutral evolution in some cases, single-cell analysis identified co-existing subclones. The overlaps of bulk VAF ranges for co-mutations from different subclones made it difficult to separate them, even with other parameter introduced. The difference between mutation cluster and tumor subclone is accountable for the challenge in bulk clonal deconvolution, especially in case of branched evolution as shown in colorectal cancer. ConclusionComplex subclonal structures and dynamic evolution are hidden under the seemingly clonal neutral pattern at bulk level, suggesting single-cell analysis will be needed to avoid under-estimation of tumor heterogeneity. Research HighlightsO_LIBulk-level mutation abundance clusters are not equal to tumor subclones. C_LIO_LIDifferent groups of co-mutations could not be discriminated at bulk-level. C_LIO_LISingle-cell mutational analysis can identify rather than infer tumor subclones. C_LIO_LICo-existing tumor subclones may have clonal neutral appearance at bulk-level. C_LI Lay summarySystematic comparison of tumor clonal structure differences between bulk and single-cell mutational analysis is lacking. Here we performed such as study and found that complex subclonal structures and dynamic evolution are hidden under clonal neutral appearance at bulk level in liver and colorectal cancers, suggesting single-cell analysis will be needed to avoid under-estimation of tumor heterogeneity.

cancer biology↗