Biology subjects

Zhang, M.

Publications and source records attributed to Zhang, M..

At least 685 records · Page 38Linked to original sources

bioRxiv2020

Identification of Lipid Droplets in Gut Microbiota

Recent research has accumulating evidence to support that gut microbiota can be a newly discovered organ/tissue for humans. Although the functions of gut microbiota have been linked to almost all aspects of human physiological, most findings so far are still either belonged to correlational research or at preliminary stage of causal study due to the complexity of gut microbiota as well as lack of proper tools and methods. Thus, development of new approaches is essential and necessary. Lipid droplet is a cellular organelle governing cell lipid homeostasis that is directly related to human metabolic syndromes. Previously we and other groups found lipid droplets in several bacterial strains. We wonder if gut bacteria have lipid droplets. If so, these bacteria may play key roles to regulate gut lipid content, remove unnecessary lipids and produce useful lipid molecules for host. Here we identified lipid droplet-like structures in freshly isolated bacteria from mouse small and large intestines, as well as mouse and human feces. We also purified and analyzed lipid droplets from a cloned human gut bacterium Streptomyces thermovulgaris. Together, we demonstrate the existence of lipid droplets in gut microbiota and established an approach to study them.

microbiology↗

bioRxiv2020

Autoantibody landscape of advanced prostate cancer

Although the importance of T-cell immune responses is well appreciated in cancer, autoantibody responses are less well-characterized. Nevertheless, autoantibody responses are of great interest, as they may be concordant with T-cell responses to cancer antigens or predictive of response to cancer immunotherapies. We performed serum epitope repertoire analysis (SERA) on a total of 1,229 serum samples obtained from a cohort of 72 men with metastatic castration-resistant prostate cancer (mCRPC) and 1,157 healthy control patients to characterize the autoantibody landscape of mCRPC. Using whole-genome sequencing results from paired solid-tumor metastasis biopsies and germline specimens, we identified tumor-specific epitopes in 29 mutant and 11 non-mutant proteins. Autoantibody enrichments for the top candidate autoantigen (NY-ESO-1) were validated using ELISA performed on the prostate cancer cohort and an independent cohort of 106 patients with melanoma. Our study recovers antigens of known importance and identifies novel tumor-specific epitopes of translational interest in advanced prostate cancer. Statement of significanceAutoantibodies have been shown to inform treatment response and candidate drug targets in various cancers. We present the first large-scale profiling of autoantibodies in advanced prostate cancer, utilizing a new next-generation sequencing-based approach to antibody profiling to reveal novel cancer-specific antigens and epitopes. Disclosure of Potential Conflicts of InterestJJA reports receiving consulting income from Janssen Biotech and Merck and honoraria from Astellas for speakers fees. MR reports receiving commercial research support from Novartis, Johnson & Johnson, Merck, Astellas, and Medivation, and is a consultant/advisory board member for Constellation Pharmaceuticals, Amgen, Ambrx, Johnson & Johnson, and Bayer. A.R. has received honoraria from consulting with Amgen, Bristol-Myers Squibb, Chugai, Dynavax, Genentech, Merck, Nektar, Novartis, Roche and Sanofi, is or has been a member of the scientific advisory board and holds stock in Advaxis, Arcus Biosciences, Bioncotech Therapeutics, Compugen, CytomX, Five Prime, RAPT, ImaginAb, Isoplexis, Kite-Gilead, Lutris Pharma, Merus, PACT Pharma, Rgenix and Tango Therapeutics. FYF serves on the advisory board for Dendreon, EMD Serono, Janssen Oncology, Ferring, Sanofi, Blue Earth Diagnostics, Celgene, consults for Bayer, Medivation/Astellas, Genetech, and Nutcracker Therapeutics, has honoraria from Clovis Oncology, and is a founder and has an ownership stake in PFS Genomics. SGZ and FYF have patent applications with Decipher Biosciences. SGZ and FYF have a patent application licensed to PFS Genomics. SGZ and FYF have patent applications with Celgene. WAH, RW, KK, PSD, and JCS have ownership of stocks or shares at Serimmune, paid employment at Serimmune, board membership at Serimmune, and patent applications on behalf of Serimmune.

cancer biology↗

bioRxiv2020

Optimized photochemistry and enzymology enable efficient analysis of RNA structures and interactions in cells and virus infections

Direct determination of RNA structures and interactions in living cells is critical for understanding their functions. Current crosslinking and proximity-ligation approaches are fundamentally limited due to inefficient RNA crosslinking, purification and high-level photochemical damages. Here we present PARIS2 (psoralen analysis of RNA interactions and structures, second generation), a re-invented method for capturing RNA duplexes in cells with three orders of magnitude improved efficiency. PARIS2 captures ribosome small subunit (SSU) binding sites on mRNAs, reporting translation status on a transcriptome wide scale, and captures spliceosomal snRNP binding sites on various RNA targets. We determine the RNA genome structure of enterovirus D68, a re-emerging viral pathogen associated with severe neurological symptoms, and discover alternative conformations in the internal ribosome entry site (IRES) that controls translation initiation. Together, these results reveal new aspects of RNA photochemistry and enzymology, and enable highly efficient interrogation of the RNA structurome and interactome in cells.

genomics↗

bioRxiv2020

Endocrine signaling mediates asymmetric motor deficits after unilateral brain injury

A paradigm in neurology is that brain injury-induced motor deficits (e.g. hemiparesis and hemiplegia) arise due to aberrant activity of descending neural pathways. We discovered that a unilateral injury of the hindlimb sensorimotor cortex of rats with completely transected thoracic spinal cord produces hindlimb postural asymmetry with contralateral flexion, and asymmetric changes in nociceptive hindlimb withdrawal reflexes and gene expression patterns in lumbar spinal cord. The injury-induced postural effects were abolished by prior hypophysectomy and were mimicked by transfusion of serum from animals with unilateral brain injury. Antagonists of the opioid and vasopressin receptors blocked formation of hindlimb postural asymmetry suggesting that these neurohormones mediate effects of brain injury on lateralized motor responses. Our data indicate that descending neural control of spinal circuits is complemented by a previously unknown humoral signaling from injured brain to the contra- and ipsilesional hindlimbs, and suggest the existence of a body side-specific neuroendocrine regulation in bilaterally symmetric animals.

neuroscience↗

bioRxiv2020

CNSA: a data repository for archiving omics data

With the application and development of high-throughput sequencing technology in life and health sciences, massive multi-dimensional biological data brings the problem of efficient management and utilization. Database development and biocuration are the prerequisites for the reuse of these big data. Here, relying on China National GeneBank (CNGB), we present CNGB Sequence Archive (CNSA) for archiving omics data, including raw sequencing data and its analytical data and related metadata which are organized into six objects, namely Project, Sample, Experiment, Run, Assembly, and Variation at present. Moreover, CNSA has created the correlation model of living samples, sample information, and analytical data on some projects, so that all data can be traced throughout the life cycle from the living sample to the sample information to the analytical data. Complying with the data standards commonly used in the life sciences, CNSA is committed to building a comprehensive and curated data repository for the storage, management and sharing of omics data, improving the data standards, and providing free access to open data resources for worldwide scientific communities to support academic research and the bio-industry. Database URL: https://db.cngb.org/cnsa/

genomics↗

bioRxiv2020

Partnership between epigenetic reader BRD4 and transcription factor CEBPD

Vascular smooth muscle cell (SMC) state/phenotype transitions underlie neointimal hyperplasia (IH) predisposing to cardiovascular diseases. Bromodomain protein BRD4 is a histone acetylation reader and enhancer mark that co-activates transcription elongation. CCAAT enhancer binding protein delta (CEBPD) is a transcription factor typically studied in adipogenesis and immune cell differentiation. Here we investigated the association between BRD4 and CEBPD in SMC state transition. Chromatin immunoprecipitation sequencing (ChIPseq) showed enrichment of BRD4 and histone acetylation (H3K27ac) at Cebpd and enhancer in rat carotid arteries undergoing IH. In vitro, BRD4 silencing with siRNA reduced SMC expression of CEBPD. Bromodomain-1 but not bromodoamin-2 accounted for this BRD4 function. Endogenous BRD4 co-IPed with CEBPD; Cebpd promoter and enhancer DNA fragments co-IPed with CEBPD or endogenous BRD4 (ChIP-qPCR). These co-IPs were abolished by the BRD4 bromodomain blocker JQ1. TNF upregulated both BRD4 and CEBPD. Silencing CEBPD averted TNF-induced inflammatory SMC state transition (heightened IL-1{beta}, IL6, and MCP-1 mRNA levels), so did JQ1. CEBPD overexpression increased PDGFR preferentially over PDGFR{beta}; so did TNF, and JQ1 abolished TNFs effect. Our data reveal a BRD4/CEBPD partnership that promotes CEBPDs own transcription and inflammatory SMC state transition, thus shedding new light on epigenetic reader and transcription factor cooperative actions in SMC pathobiology.

cell biology↗

bioRxiv2020

DeeReCT-APA: Prediction of Alternative Polyadenylation Site Usage Through Deep Learning

Alternative polyadenylation (APA) is a crucial step in post-transcriptional regulation. Previous bioinformatic works have mainly focused on the recognition of polyadenylation sites (PAS) in a given genomic sequence, which is a binary classification problem. Recently, computational methods for predicting the usage level of alternative PAS in a same gene have been proposed. However, all of them cast the problem as a non-quantitative pairwise comparison task and do not take the competition among multiple PAS into account. To address this, here we propose a deep learning architecture, DeeReCT-APA, to quantitatively predict the usage of all alternative PAS of a given gene. To accommodate different genes with potentially different numbers of PAS, DeeReCT-APA treats the problem as a regression task with a variable-length target. Based on a CNN-LSTM architecture, DeeReCT-APA extracts sequence features with CNN layers, uses bidirectional LSTM to explicitly model the interactions among competing PAS, and outputs percentage scores representing the usage levels of all PAS of a gene. In addition to the fact that only our method can predict quantitatively the usage of all the PAS within a gene, we show that our method consistently outperforms other existing methods on three different tasks for which they are trained: pairwise comparison task, highest usage prediction task and ranking task. Finally, we demonstrate that our method can be used to predict the effect of genetic variations on APA patterns and shed light on future mechanistic understanding in APA regulation. Our code and data are available at https://github.com/lzx325/DeeReCT-APA-repo.

bioinformatics↗

bioRxiv2020

Fibrinolytic niche is requested for alveolar type 2 cell-mediated alveologenesis and injury repair

COVID-19, SARS, and MERS are featured by fibrinolytic dysfunction. To test the role of the fibrinolytic niche in the regeneration of alveolar epithelium, we compared the self-renewing capacity of alveolar epithelial type 2 (AT2) cells and its differentiation to AT1 cells between wild type (wt) and fibrinolytic niche deficient mice (Plau-/- and Serpine1Tg). A significant reduction in both proliferation and differentiation of deficient AT2 cells was observed in vivo and in 3D organoid cultures. This decrease was mainly restored by uPA derived A6 peptide, a binding fragment to CD44 receptors. The proliferative and differential rate of CD44+ AT2 cells was greater than that of CD44- controls. There was a reduction in transepithelial ion transport in deficient monolayers compared to wt cells. Moreover, we found a marked suppression in total AT2 cells and CD44+ subpopulation in lungs from brain dead patients with acute respiratory distress syndrome (ARDS) and a mouse model infected by influenza viruses. Thus, we demonstrate that the fibrinolytic niche can regulate AT2-mediated homeostasis and regeneration via a novel uPA-A6-CD44+-ENaC cascade.

cell biology↗

bioRxiv2020

Iatrogenic Iron Promotes Neurodegeneration and Activates Self-protection of Neural Cells against Exogenous Iron Attacks

Metal implants are used worldwide, with millions of metal nails, plates and fixtures grafted during orthopaedic surgeries. Iron is the most common element of these metal implants. As time passes metal elements can be corroded and iron can be released from the implants in the form of ferric (Fe3+) or ferrous (Fe2+). These iron ions can permeate the surrounding tissues and enter circulation; importantly both Fe3+ and Fe2+ freely pass blood brain barrier (BBB). Can iron from implants represent a risk factor for neurological diseases? This remains an unanswered question. In this study, we discovered that the probability of metal implants delivered through orthopaedic surgeries was higher in patients of Parkinsons diseases (PD) or ischemic stroke than in healthy subjects. This finding instigated subsequent study of iron effects on neuronal cells. In experiments in vivo, we found that iron selectively decreased presence of divalent metal transporter 1 (DMT1) in neurones through increasing the expression of Ndfip1, which degrades DMT1 and rarely exists in glial cells. At the same time iron accumulation increased expression of DMT1 in astrocytes and microglial cells and triggered reactive astrogliosis and microglial activation. Facing the attack of excess iron, glial cells act as neuroprotectors to uptake more extracellular iron by up-regulating DMT1, whereas neurones limit iron uptake through decreasing DMT1 operation. Cerebral accumulation of iron was associated with impaired cognition, locomotion and mood. Excess iron thus affects neural cells and could increase the risk of neurodegeneration.

neuroscience↗

bioRxiv2020

In Vivo Efficacy and PK/PD Modeling of KBP-7072, An Aminomethylcycline Antibiotic, in Neutropenic Pneumonia and Thigh Infection Models

KBP-7072 is a novel aminomethylcycline with broad-spectrum activity against Gram-positive and Gram-negative multidrug resistant bacterial isolates and strains. Antibacterial activity and the PK/PD relationship were assessed using in vivo infection models. Six to 8-week-old female CD-1 mice were randomized to oral KBP-7072, minocycline and vehicle in a Klebsiella pneumoniae murine model, and KBP-7072, linezolid and vehicle for a Streptococcus pneumoniae murine model. Each animal was inoculated with K. pneumoniae or S. pneumoniae placed on the tip of the nares. KBP-7072 and antibiotics were started 3 hours post inoculation and continued for 3 days for K. pneumoniae, and were started 18 hours post inoculation and continued for 3 days for S. pneumoniae. Animals were euthanized at 0 (control group), 24, 48 or 72 hours post final dose. In vivo efficacy and PK/PD parameters were determined in Staphylococcus aureus isolate (6424MRSA-363), K. pneumoniae isolate (6680kpn-522), and E. coli isolate (6691eco-558) murine thigh infection models. In vivo efficacy and PK/PD parameters (fAUC/MIC, fCmax/MIC and %T>MICfree) were calculated. Respiratory infection occurred in all inoculated mice. KBP-7072 produced a significant (p<0.05 to <0.001) dose-dependent decrease in colony forming units (CFUs) at all doses and a dose-dependent increase in survival rate (p<0.001 vs. vehicle). The median survival in all KBP-7072-treated groups was significantly greater vs. comparators (p<0.001). These results demonstrate potent in vivo efficacy for KBP-7072 and determined that the AUC/MIC parameter was optimal for assessing bacteriostatic and bactericidal effects of KBP-7072.

pharmacology and toxicology↗

bioRxiv2020

Adaptive evolution of DNA methylation reshaped gene regulation in maize

DNA methylation is a ubiquitous chromatin feature -- in maize, more than 25% of cytosines in the genome are methylated. Recently, major progress has been made in describing the molecular mechanisms driving methylation, yet variation and evolution of the methylation landscape during maize domestication remain largely unknown. Here we leveraged whole-genome sequencing (WGS) and whole genome bisulfite sequencing (WGBS) on populations of modern maize, landrace, and teosinte (Zea mays ssp. parviglumis) to investigate the adaptive and phenotypic consequences of methylation variations in maize. By using a novel estimation approach, we inferred the methylome site frequency spectrum (mSFS) to estimate forward and backward epimutation rates and selection coefficients. We found weak evidence for direct selection on DNA methylation in any context, but thousands of differentially methylated regions (DMRs) were identified in population-wide that are correlated with recent selection. Further investigation revealed that DMRs are enriched in 5 untranslated regions, and that maize hypomethylated DMRs likely helped rewire distal gene regulation. For two trait-associated DMRs, vgt1-DMR and tb1-DMR, our HiChIP data indicated that the interactive loops between DMRs and respective downstream genes were present in B73, a modern maize line, but absent in teosinte. Functional analyses suggested that these DMRs likely served as cis-acting elements that modulated gene regulation after domestication. Our results enable a better understanding of the evolutionary forces acting on patterns of DNA methylation and suggest a role of methylation variation in adaptive evolution.

plant biology↗

bioRxiv2020

Angioplasty-induced epigenomic remodeling entails BRD4 and EZH2 hierarchical regulations

Atherosclerosis is commonly treated with angioplasty which, however, evokes neointimal hyperplasia (IH) and recurrent stenotic diseases. Epigenomic investigation was lacking on post-angioplasty IH. The histone acetylation reader BRD4 and H3K27me3 writer EZH2 are potent epigenetic factors; their relationship is little understood. Through genome-wide survey in the rat angioplasty model, we studied BRD4 and EZH2 functional regulations involved in IH. We performed chromatin immunoprecipitation sequencing (ChIPseq) using rat carotid arteries. While H3K27me3 ChIPseq signal prevalently intensified in balloon-injured (vs uninjured) arteries, BRD4 and H3K27ac became more enriched at Ezh2. Indeed, BRD4-siRNA or CRISPR-deletion of BRD4-associated enhancer abated the smooth muscle cell (SMC) expression of EZH2, and SMC-specific BRD4 knockout in BRD4fl/fl; Myh11CreERT2 mice reduced both H3K27me3 and IH in wire-injured femoral arteries. In accordance, post-angioplasty IH was exacerbated and mitigated, respectively, by lentiviral expression and pharmacological inhibition of EZH2/1; EZH2 (or EZH1) loss- and gain-of-function respectively attenuated and aggravated pro-IH SMC proliferative behaviors. Furthermore, while H3K27me3 ChIPseq signal magnified at P57 and ebbed at Ccnd1 and Uhrf1 after injury, silencing either EZH2 or EZH1 in SMCs up-regulated P57 and down-regulated Ccnd1 and Uhrf1. In summary, our results reveal an upsurge of EZH2/H3K27me3 after angioplasty, BRD4s control over EZH2 expression, and non-redundant EZH2/1 functions. As such, this study unravels angioplasty-induced loci-specific H3K27me3/ac redistribution in the epigenomic landscape rationalizing BRD4/EZH2-governed pro-IH regulations.

pathology↗

bioRxiv2020

A quasi-paired cohort strategy reveals the impaired detoxifying function of microbes in the gut of autistic children

Growing evidence suggests that autism spectrum disorder (ASD) is highly associated with dysbiosis in the gut microbiome. However, results of metagenome-based microbiome studies are not always consistent due to great individual diversity that overwhelms disease-associated alterations. Here, we proposed a novel analysis strategy--quasi-paired cohort and applied it to a metagenomic study of ASD microbiomes. By comparing the paired samples of ASD and neurotypical subjects, we identified significant deficiencies in ASD children in detoxifying enzymes and pathways, which showed strong correlations to mitochondrial damage. Diagnostic models with these detoxifying enzymes accurately discriminated ASD individuals from controls, and the dysfunction score inferred from the model increased with the clinical rating scores of ASD. Conclusively, our findings suggest a previously undiscovered mechanism in which impaired microbial detoxification leads to toxicant accumulation and mitochondrion damage contributes to the pathogenesis of ASD. This novel mechanism points to future therapeutic strategies of rebuilding microbial detoxification for ASD.

microbiology↗

bioRxiv2020

Establishment of Etv5 gene knockout mice as a recipient model for spermatogonial stem cell transplantation

Transplantation of spermatogonial stem cells (SSCs) is an alternative reproductive method to achieve conservation and production of elite animals in livestock production. Creating a recipient animal without endogenous germ cells is important for effective SSC transplantation. However, natural mutants with depletion of SSCs are difficult to obtain, and drug ablation of endogenous germ cells is arduous to perform for practical use. In this study, we used mouse models to study the preparation of recipients with congenital germ cell ablation. We knocked out (KO) Ets-variant gene 5 (Etv5) in mice using the CRISPR/Cas9 system. The testicular weight of Etv5-/- mice was significantly lower than that of wild-type (WT) mice. The germ cell layer of the seminiferous tubules gradually receded with age in Etv5-/- mice. At 12 weeks of age, the tubules of Etv5-/- mice lacked germ cells (Sertoli cell-only syndrome), and sperm were completely absent in the epididymis. We subsequently transplanted allogeneic SSCs with enhanced green fluorescent protein (EGFP) into 3-(immature) or 7-week-old (mature) Etv5-/- mice. Restoration of germ cell layers in the seminiferous tubules and spermatogenesis was observed in all immature testes but not in mature adult testes at 2 months post-transplantation. The presence of heterologous genes Etv5 and EGFP in recipient testicular tissue and epididymal sperm by PCR indicated that sperm originated from the transplanted donor cells. Our study demonstrates that, although Etv5-/- mice could accommodate and support foreign germ cell transplantation, this process occurs in a quite low efficiency to support a full spermatogenesis of transplanted SSCs. However, using Etv5-/- mice as a recipient model for SSC transplantation is feasible, and still needs further investigation to establish an optimized transplantation process.

bioengineering↗

bioRxiv2020

Structural basis underlying strong interactions between ankyrins and spectrins

Ankyrins (encoded by ANK1/2/3 corresponding to Ankyrin-R/B/G or AnkR/B/G), via binding to spectrins, connect plasma membranes with actin cytoskeleton to maintain mechanical strengths and to modulate excitabilities of diverse cells such as neurons, muscle cells, and erythrocytes. Cellular and genetic evidences suggest that each isoform of ankyrins pairs with a specific {beta}-spectrin in discrete subcellular membrane microdomains for distinct functions, though the molecular mechanisms underlying such ankyrin/{beta}-spectrin pairings are unknown. In this study, we discover that a conserved and short extension N-terminal to the ZU5N-ZU5C-UPA tandem (exZZU) is critical for each ankyrin to bind to {beta}-spectrins with high affinities. Structures of AnkB/G exZZU in complex with spectrin repeats13-15 of {beta}2/{beta}4-spectrins solved here reveal that the extension sequence of exZZU forms an additional {beta}-strand contributing to the structural stability and enhanced affinity of each ZU5N/spectrin repeat interaction. The junction site between the extension and ZU5N is exactly the position of a splicing-mediated miniexon insertion site of AnkB/G. The complex structures further reveal that the UPA domain of exZZU directly participates in spectrin binding. Formation of the exZZU supramodule juxtaposes the ZU5N and UPA domains for simultaneous interacting with spectrin repeats 14 and 15. However, our biochemical and structural investigations indicate that the direct and strong interactions between ankyrins and {beta}-spectrins do not appear to determine their pairing specificities. Therefore, there likely exists additional mechanism(s) for modulating functional pairings between ankyrins and {beta}-spectrins in cells.

biochemistry↗

bioRxiv2020

A Novel Non-hypermucoviscous ST11 Hypervirulent Carbapenem-resistant Klebsiella pneumoniae Lacking Classical Virulence Factors

BackgroundHypervirulent Klebsiella pneumoniae lacking classical virulence factors is uncommon, and the virulence mechanisms of this organism are not understood. MethodsFollowing a retrospective study of carbapenem-resistant K. pneumoniae based on core genome multilocus sequence typing (cgMLST), isolates that caused high mortality were investigated with a genome-wide association study (GWAS), proteome analysis and an animal model. ResultsThe sublineage of sequence type 11 (ST11) K. pneumoniae, which belongs to complex type 3176 (CT3176) and K-locus 47 (KL47), was highlighted due to the high mortality of infected patients. GWAS analysis showed that ampR was associated with the CT3176 isolates. In a mouse model, the mortality of ampR-carrying isolates was comparable to that of the typical hypervirulent isolate GM2. Even during the first 24 hours of infection, the bacterial load and pathological changes of the ampR-carrying isolates in the lungs were more severe than those of GM2. The ampR complement mutant was able to enhance the virulence of the KL47 isolate but not the virulence of KL1. Proteome analysis showed that the expression of WcaJ in the ampR+ isolates was significantly higher than that in the ampR- isolates, and this result was also confirmed by transcription tests and capsule staining. It is suggested that the enhancement of the initial stage of capsule synthesis may be the cause of the high virulence of these non-hypermucoviscous ST11 carbapenem-resistant K. pneumoniae isolates. ConclusionsNon-hypermucoviscous ST11 hypervirulent carbapenem-resistant K. pneumoniae warrants continued surveillance and investigation.

microbiology↗

bioRxiv2020

Oxidative Modifications of Parkin Underlie its Selective Neuroprotection in Adult Human Brain

The mechanisms by which Parkinson disease-linked parkin confers neuroprotection of human dopamine cells remain elusive. We hypothesized that its cysteines mediate multiple anti-oxidant effects in the midbrain. By studying >60 control specimens, we found that in adult human brain - but not in skeletal muscle- parkin is mostly aggregated and insoluble due to oxidative modifications, such as at C253. In vitro, parkins oxidation directly reduces hydrogen peroxide (H2O2) to water. In parkin-deficient human brain, H2O2 concentrations are elevated. In dopamine toxicity studies, wild-type parkin -but not disease-associated mutants-prevents neural death by lowering H2O2 and sequestering radicals within insoluble aggregates. Parkin conjugates dopamine metabolites at the human-specific residue C95 and augments melanin formation in vitro. Using epitope-mapped antibodies, we found that in adult Substantia nigra neurons parkin localizes to neuromelanin within LAMP-3/CD63-positive lysosomes. We conclude that parkins own oxidation, previously considered a loss-of-function event, underlies three neuroprotective effects in adult midbrain: its cysteines participate in H2O2 reduction, dopamine radical conjugation and the formation of neuromelanin.

neuroscience↗

bioRxiv2020

CoolMPS™: Advanced massively parallel sequencing using antibodies specific to each natural nucleobase

Massively parallel sequencing (MPS) on DNA nanoarrays provides billions of reads at relatively low cost and enables a multitude of genomic applications. Further improvement in read length, sequence quality and cost reduction will enable more affordable and accurate comprehensive health monitoring tests. Currently the most efficient MPS uses dye-labeled reversibly terminated nucleotides (RTs) that are expensive to make and challenging to incorporate. Furthermore, a part of the dye-linker (scar) remains on the nucleobase after cleavage and interferes with subsequent sequencing cycles. We describe here the development of a novel MPS chemistry (CoolMPS) utilizing unlabeled RTs and four natural nucleobase-specific fluorescently labeled antibodies with fast (30 sec) binding. We implemented CoolMPS on MGIs PCR-free DNBSEQ MPS platform using arrays of 200nm DNA nanoballs (DNBs) generated by rolling circle replication and demonstrate 3-fold improvement in signal intensity and elimination of scar interference. Single-end 100-400 base and pair-end 2x150 base reads with high quality were readily generated with low out-of-phase incorporation. Furthermore, DNBs with less than 50 template copies were successfully sequenced by strong-signal CoolMPS with 3-times higher accuracy than in standard MPS. CoolMPS chemistry based on natural nucleobases has potential to provide longer, more accurate and less expensive MPS reads, including highly accurate "4-color sequencing" on the most efficient dye-crosstalk-free 2-color imagers with an estimated sequencing error rate of 0.00058% (one error in 170,000 base calls) in a proof-of-concept demonstration.

genomics↗