Biology subjects

Wu, C.

Publications and source records attributed to Wu, C..

At least 289 records · Page 16Linked to original sources

bioRxiv2020

An unexpected bifurcation in the Pointed transcriptional effector network contributes specificity and robustness to retinal cell fate acquisition

Spatiotemporally specific and robust cell fate transitions are fundamental to the development of appropriately patterned tissues. In the Drosophila retina, receptor tyrosine kinase / mitogen activated protein kinase (MAPK) signaling acts through the transcriptional effector Pointed (Pnt) to direct two distinct rounds of photoreceptor specification. A relay mechanism between two Pnt isoforms, a MAPK responsive form PntP2 and a constitutively active form PntP1, initiates and sustains the transcriptional response. Here, we report an unexpected bifurcation in the Pnt effector network. We show that PntP2 works redundantly with a closely related but previously uncharacterized isoform, PntP3, to activate pntP1 during specification of first round photoreceptors. Intrinsic activity differences between PntP2 and PntP3, combined with positive and negative transcriptional auto- and cross-regulation, buffer first-round fates against conditions of low signaling. In contrast, in a mechanism that may be adaptive to the stronger signaling environment used to specify second round fates, PntP2 uniquely activates pntP1. We propose that differences in expression patterns, transcriptional activities and regulatory interactions between Pnt isoforms together facilitate context-appropriate cell fate specification in different signaling environments.

developmental biology↗

bioRxiv2020

Early Postnatal Tobacco Smoke Exposure Aggravates Experimental Autoimmune Encephalomyelitis in Adult Rats

Although substantial evidence supports smoking as a risk factor for the development of multiple sclerosis (MS) in adulthood, it remains controversial as to whether early-life exposure to environmental tobacco smoke (ETS) increases the risk of MS later in life. Here, using experimental autoimmune encephalomyelitis (EAE) as an animal model for MS, we show that exposing neonatal rats during the 1st week (ETS1-EAE), but not the 2nd week (ETS2-EAE) and the 3rd week (ETS3-EAE) after birth, increased the severity of EAE in adulthood in comparison to pups exposed to filtered compressed air (AIR-EAE). The EST1-EAE rats showed a worse neurological deficit score and a significant increase in CD4+ cell infiltration, demyelination, and axonal injury in the spinal cord compared to AIR-EAE, ETS2-EAE, and ETS3-EAE groups. Flow cytometry analysis showed that the ETS1 group had decreased numbers of regulatory T (Treg) cells and increased effector T (Teff) cells in the brain and spinal cord. The expressions of Treg upstream regulator Foxp3 and downstream cytokines such as IL-10 were also altered accordingly. Together, these findings demonstrate that neonatal ETS exposure suppresses Treg functions and aggravates the severity of EAE, confirming early-life exposure to EST as a potential risk factor for multiple sclerosis in adulthood.

neuroscience↗

bioRxiv2020

barcodetrackR: an R package for the interrogation of clonal tracking data

Clonal tracking methods provide quantitative insights into the cellular output of genetically labelled progenitor cells across time and cellular compartments. In the context of gene and cell therapies, clonal tracking methods have enabled the tracking of progenitor cell output both in humans receiving cellular therapies and in corresponding animal models, providing valuable insight into lineage reconstitution, clonal dynamics, and vector genotoxicity. However, the absence of a toolbox by which to interrogate these data has precluded the development of standardized analytical frameworks within the field. Thus, we developed barcodetrackR, an R package that provides users with tools for the analysis and visualization of clonal dynamics across time and cellular compartments in clonal tracking experiments. Here, we demonstrate the utility of barcodetrackR in exploring longitudinal clonal patterns and lineage relationships in the context of a number of clonal tracking studies of hematopoietic stem and progenitor cells (HSPCs) in humans receiving HSPC gene therapy and in animals receiving lentivirally transduced HSPC transplants.

bioinformatics↗

bioRxiv2020

A gene-level methylome-wide association analysis identifies novel Alzheimer's disease genes

MotivationTranscriptome-wide association studies (TWAS) have successfully facilitated the discovery of novel genetic risk loci for many complex traits, including late-onset Alzheimers disease (AD). However, most existing TWAS methods rely only on gene expression and ignore epigenetic modification (i.e., DNA methylation) and functional regulatory information (i.e., enhancer-promoter interactions), both of which contribute significantly to the genetic basis of AD. ResultsThis motivates us to develop a novel gene-level association testing method that integrates genetically regulated DNA methylation and enhancer-target gene pairs with genome-wide association study (GWAS) summary results. Through simulations, we show that our approach, referred to as the CMO (cross methylome omnibus) test, yielded well controlled type I error rates and achieved much higher statistical power than competing methods under a wide range of scenarios. Furthermore, compared with TWAS, CMO identified an average of 124% more associations when analyzing several brain imaging-related GWAS results. By analyzing to date the largest AD GWAS of 71,880 cases and 383,378 controls, CMO identified six novel loci for AD, which have been ignored by competing methods. Availability and implementationSoftware: https://github.com/ChongWuLab/CMO Contactcwu3@fsu.edu Supplementary informationSupplementary data are available at Bioinformatics online.

genomics↗

bioRxiv2020

Accurate recognition of colorectal cancer with semi-supervised deep learning on pathological images

BackgroundMachine-assisted recognition of colorectal cancer (CRC) has been mainly focused on supervised deep learning that suffers from a significant bottleneck of requiring massive labeled data. We hypothesize that semi-supervised deep learning leveraging a small amount of labeled data with abundant available unlabeled data can provide a powerful alternative strategy. MethodWe proposed a semi-supervised model based on the mean teacher architecture that provides pathological predictions at both patch- and patient-levels. We demonstrated the general utility of the model utilizing 13,111 CRC whole slide images from 8,803 subjects gathered from 13 independent centers. We compared our proposed method with the prevailing supervised learning and six pathologists. Two extended evaluations on 15,000 lung and 294,912 lymph node images were also performed to confirm the generality of utility of semi-supervised learning for different cancers. ResultsWith a small amount of labeled training patches ([~]3,150 labeled, [~]40,950 unlabeled or [~]6,300 labeled, [~]37,800 unlabeled), the semi-supervised learning (SSL) performed significantly better than the supervised learning (SL, which only used the labeled data) (area under the curve, AUC: 0.90 {+/-} 0.06 vs. 0.84 {+/-} 0.07, P value = 0.02 or AUC: 0.98 {+/-} 0.01 vs. 0.92 {+/-} 0.04, P value = 0.0004). Moreover, we found no significant difference between SL using massive [~]44,100 labeled patches and SSL ([~]6,300 labeled, [~]37,800 unlabeled) at patch-level diagnoses (AUC:0.98 {+/-} 0.01 vs. 0.987 {+/-} 0.01, P value = 0.134) and patient-level diagnoses (average AUC: 0.974 vs. 0.980, P value = 0.117). SSL was close to human pathologists in diagnosis performance (average AUC: 0.972 vs. 0.969). This extended evaluation on lung and lymph node also confirmed when a small amount of labeled data were used, SSL was better than SL, and achieved similar performance as that of SL with massive labeling. ConclusionsWe reported that SSL can achieve excellent performance through a multi-center study. Because SSL dramatically reduces the need and cost of pathological image annotation, it has great potential to effectively build pathological artificial intelligence (AI) platforms in practice.

pathology↗

bioRxiv2020

Global proteome and phosphoproteome alterations in third generation EGFR TKI resistance reveal drug targets to circumvent resistance

Lung cancer is the leading cause of cancer mortality worldwide. The treatment of lung cancer patients harboring mutant EGFR with orally administered EGFR TKIs has been a paradigm shift. Osimertinib and rociletinib are 3rd generation irreversible EGFR TKIs targeting the EGFR T790M mutation. Osimertinib is the current standard care for patients with EGFR mutations due to increased efficacy, lower side effects, and enhanced brain penetrance. Unfortunately, all patients develop resistance. Genomic approaches have primarily been used to interrogate resistance mechanisms. Here, we have characterized the proteome and phosphoproteome of a series of isogenic EGFR mutant lung adenocarcinoma cell lines that are either sensitive or resistant to these drugs. To our knowledge, this is the most comprehensive proteomic dataset resource to date to investigate 3rd generation EGFR TKI resistance in lung adenocarcinoma. We have interrogated this unbiased global quantitative mass spectrometry dataset to uncover alterations in signaling pathways, reveal a proteomic signature of epithelial mesenchymal transition (EMT) and identify kinases and phosphatases with altered expression and phosphorylation in TKI resistant cells. Decreased tyrosine phosphorylation of key sites in the phosphatase SHP2 suggests its inhibition resulting in inhibition of RAS/MAPK and activation of PI3K/AKT pathways. Furthermore, we performed anticorrelation analyses of this phosphoproteomic dataset with the published drug-induced P100 phosphoproteomic datasets from the Library of Integrated Network-Based Cellular Signatures (LINCS) program to predict drugs with the potential to overcome EGFR TKI resistance. We identified dactolisib, a PI3K/mTOR inhibitor, which in combination with osimertinib overcomes resistance both in vitro and in vivo.One Sentence Summary Global quantitative proteome and phosphoproteome analyses to examine altered signaling pathways in isogenic 3rd generation EGFR TKI sensitive and resistant cells.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2020

Single-cell transcriptional atlas of the Chinese horseshoe bat (Rhinolophus sinicus) provides insight into the cellular mechanisms which enable bats to be viral reservoirs

Bats are a major "viral reservoir" in nature and there is a great interest in not only the cell biology of their innate and adaptive immune systems, but also in the expression patterns of receptors used for cellular entry by viruses with potential cross-species transmission. To address this and other questions, we created a single-cell transcriptomic atlas of the Chinese horseshoe bat (Rhinolophus sinicus) which comprises 82,924 cells from 19 organs and tissues. This atlas provides a molecular characterization of numerous cell types from a variety of anatomical sites, and we used it to identify clusters of transcription features that define cell types across all of the surveyed organs. Analysis of viral entry receptor genes for known zoonotic viruses showed cell distribution patterns similar to that of humans, with higher expression levels in bat intestine epithelial cells. In terms of the immune system, CD8+ T cells are in high proportion with tissue-resident memory T cells, and long-lived effector memory nature killer (NK) T-like cells (KLRG1, GZMA and ITGA4 genes) are broadly distributed across the organs. Isolated lung primary bat pulmonary fibroblast (BPF) cells were used to evaluate innate immunity, and they showed a weak response to interferon {beta} and tumor necrosis factor- compared to their human counterparts, consistent with our transcriptional analysis. This compendium of transcriptome data provides a molecular foundation for understanding the cell identities, functions and cellular receptor characteristics for viral reservoirs and zoonotic transmission.

cell biology↗

bioRxiv2020

Calcium ion chelation preserves platelet function during cold storage

ObjectivePlatelet transfusion is a life-saving therapy to prevent or treat bleeding in patients with thrombocytopenia or platelet dysfunction. However, for more than six decades, safe and effective strategies for platelet storage have been an impediment to widespread use of platelet transfusion. Refrigerated platelets are cleared rapidly from circulation, precluding cold storage of platelets for transfusion. Consequently, platelets are stored at room temperature (RT) with an upper limit of 5 days due to risks of bacterial contamination and loss of platelet function. This practice severely limits platelet availability for transfusion. This study is to identify the mechanism of platelet clearance after cold storage and develop a method for platelet cold storage. Approach and ResultsWe found that rapid clearance of cold-stored platelets was largely due to integrin activation and apoptosis. Deficiency of integrin {beta}3 or caspase-3 prolonged cold-stored platelets in circulation. Pre-treatment of platelets with EGTA, a cell impermeable calcium ion chelator, reversely inhibited cold storage-induced platelet activation and consequently prolonged circulation of cold-stored platelets. Moreover, transfusion of EGTA-treated, cold-stored platelets, but not RT-stored platelets, into the mice deficient in glycoprotein Ib significantly shortened tail-bleeding times and diminished blood loss. ConclusionIntegrin activation and apoptosis is the underlying mechanism of rapid clearance of platelets after cold storage. Addition of a cell impermeable calcium ion chelator to platelet products is potentially a simple and effective method to enable cold storage of platelets for transfusion.

cell biology↗

bioRxiv2020

Extracellular histones trigger disseminated intravascular coagulation by lytic cell death

Histones are cationic nuclear proteins that are essential for the structure and functions of eukaryotic chromatin. However, extracellular histones trigger inflammatory responses and contribute to death in sepsis by unknown mechanisms. We recently reported that inflammasome activation and pyroptosis trigger coagulation activation through a tissue factor (TF)-dependent mechanism. Here, we show that histones trigger coagulation activation in vivo as evidenced by coagulation parameters and fibrin deposition in tissues. However, histone-induced coagulopathy was neither dependent on caspase 1/11 and gasdermin D (GSDMD), nor on TLR2 and TLR4, as deficiency of these genes in mice did not protect against histone-induced coagulopathy. Incubation of histones with macrophages induced lytic cell death and phosphatidylserine (PS) exposure, which is required for TF activity, a key initiator of coagulation. Neutralization of TF diminished histone-induced coagulation. Our findings reveal lytic cell death as a novel mechanism of histone-induce coagulation activation and thrombosis. Key PointsO_LIHistones trigger DIC in a tissue factor dependent mechanism C_LIO_LIHistones induce tissue factor activation through lytic cell death C_LI

immunology↗

bioRxiv2020

Effects of Renin-Angiotensin Inhibition on ACE2 and TMPRSS2 Expression

Angiotensin-converting enzyme 2 (ACE2), a component of the renin-angiotensin system, is a receptor for SARS-CoV-2, the virus that causes COVID-19. To determine whether the renin-angiotensin inhibition regulates ACE2 expression, either enalapril (an angiotensin-converting enzyme inhibitor) or losartan (an AT1 receptor blocker) was infused subcutaneously to male C57BL/6J mice for two weeks. Neither enalapril nor losartan changed abundance of ACE2 mRNA in lung, ileum, kidney, and heart. Viral entry also depends on transmembrane protease serine 2 (TMPRSS2) to prime the S protein. TMPRSS2 mRNA was abundant in lungs and ileum, modest in kidney, but barely detectable in heart. TMPRSS2 mRNA abundance was not altered by either enalapril or losartan in any of the 4 tissues. Next, we determined whether depletion of angiotensinogen (AGT), the unique substrate of the renin-angiotensin system, changes ACE2 and TMPRSS2 mRNA abundance. AGT antisense oligonucleotides (ASO) were injected subcutaneously to male C57BL/6J mice for 3 weeks. Abundance of ACE2 mRNA was unchanged in any of the 4 tissues, but TMPRSS2 mRNA was significantly decreased in lungs. Our data support that the renin-angiotensin inhibition does not regulate ACE2 and hence are not likely to increase risk for COVID-19.

molecular biology↗

bioRxiv2020

An emerging role of Prevotella histicola on estrogen-deficiency induced bone loss through the gut microbiota-bone axis

Gut microbiota (GM)-bone axis has emerged as a crucial mediator in the maintenance of bone homeostasis. Estrogen-deficiency induced bone loss is closely associated with an altered GM. However, the underlying mechanisms remain not fully understood. To this end, feces samples collected from the postmenopausal patients with osteoporosis (PMO) and with normal bone mass (PMN) were examined by 16s rRNA gene sequencing. We found that GM in PMO group was featured by a significant decreased proportion of genus Prevotella in comparison with that in the PMN group. Next, Prevotella histicola (P.histicola), a typical specie of Prevotella, was orally given to the mice following the ovariectomy (OVX) procedure and it significantly prevented OVX induced bone loss. Mechanistically, the protective effects of P.histicola on bone mass were found to be associated with the inhibition of osteoclastic resorption by attenuating osteoclastogenic cytokines expression and ameliorating gut permeability. Thus, P.histicola may prevent estrogen-deficiency induced bone loss through the GM-bone axis.

microbiology↗

bioRxiv2020

Combination of favorable gene haplotypes plays an important role in rice hybrid grain quality traits based on genome-wide association analysis

Yield level and grain quality determine the commercial potential of rice (Oryza sativa L.) varieties. Mining and using genes that control important rice grain quality characteristics are major tasks for plant breeders. Here, a genome-wide association study was conducted to determine the genetic bases of 12 rice grain quality traits in 113 varieties and 565 testcross hybrids. A total of 56 significant SNPs were associated with 9 of the traits in variety phenotypic, general combining ability, testcross hybrid phenotypic and mid-parental heterotic datasets, from which 45 novel loci were identified. The cumulative effects of superior alleles or favorable haplotypes of genes closest to significant quality trait-associated loci were found in the four datasets. Additionally, the favorable gene haplotypes performed better than those of superior alleles in the variety and testcross hybrid datasets. Pyramiding the favorable haplotypes of five cloned rice grain quality genes resulted in a very low amylose content and high yield in the latter. These testcross hybrids had rice grain qualities similar to their parental lines but with much higher yields. The amylose content, grain width and grain length predictions for potential hybrids among the 113 varieties using genomic selection based on the BayesB method revealed a performance trend similar to those the testcross hybrids in our study. Thus, the selection of combination of favorable rice quality-related gene haplotypes is recommended to breed hybrids with high yields and elite grain qualities.

genetics↗

bioRxiv2020

Hypoxia Depletes Contaminating CD45+ Hematopoietic Cells from Bone Marrow Stromal Cell (BMSC) Cultures: Methods for BMSC Culture Purification

Culture expanded bone marrow stromal cells (BMSCs) are easily isolated, can be grown rapidly en masse, and contain both skeletal stem cells (SSCs) and multipotent mesenchymal progenitors (MMPs). Despite this functional heterogeneity, BMSC cultures continue to be utilized for many applications due to the lack of definitive and universally accepted markers to prospectively identify and purify SSCs. Isolation of BMSCs is widely based on their adherence to tissue culture plastic; however, this method poses unique challenges when working with murine models. In particular, hematopoietic cell contamination is a significant impediment during the isolation or murine BMSCs. Several strategies to reduce hematopoietic contamination have been developed with varying levels of success. In this study, we found that up to 60% of cells isolated in standard BMSC cultures expressed the hematopoietic marker CD45. Remarkably, when cultured at a physiological oxygen tension of 1% O2, there was a 10-fold reduction in hematopoietic cells associated with a concomitant increase in PDGFR+ expressing stromal cells. This finding was due, in part, to a differential response of the two populations to low oxygen tension, or hypoxia. Specifically, in standard tissue culture conditions of 21% O2, CD45+ hematopoietic cells showed increased proliferation coupled with no observable changes in cell death when compared to their counterparts grown at 1% O2. In contrast, PDGFR+ stromal cells responded to hypoxia by increasing proliferation and exhibiting a 10-fold decrease in cell death. In summary, we describe a simple and reliable method exploiting the divergent biological response of hematopoietic and stromal cells to hypoxia to significantly increase the PDGFR+ stromal cell population in murine BMSC cultures.

cell biology↗

bioRxiv2020

Mask, the Drosophila Ankyrin Repeat and KH domain-containing protein, regulates microtubule dynamics

Proper regulation of microtubule (MT) dynamics is vital for essential cellular processes and neuronal activities, including axonal transport as well as synaptic growth and remodeling. Here we demonstrate that Mask negatively regulates MT stability and maintains a balanced dynamics of MT length and architecture in both fly larval muscles and motor neurons. In larval muscles, loss of mask increases MT length, and genetically altering mask levels modifies the Tau-induced MT fragmentation. In motor neurons, loss of mask function reduces the number of End-Binding Protein 1 (EB1)-positive MT plus-end structures in the axons and also results in overexpansion of the presynaptic terminal at larval neuromuscular junctions (NMJs). mask shows strong genetic interaction with stathmin (stai), a neuronal modulator of MT dynamics, in regulation of axon transportation and synaptic terminal stability. Our structure/function analysis on Mask revealed that truncated Mask transgenes carrying only its N-terminal portion that contains the two Ankyrin repeats domains is able to rescue the MT-related mask loss-of-function defects in larval muscles and NMJs, suggesting an essential role of the Ankyrin repeats domains in mediating Masks MT stability-regulating function. Furthermore, we discovered that Mask negatively regulates the abundance of the microtubule-associated protein Jupiter in motor neuron axons, and that neuronal knocking down of Jupiter partially suppresses mask loss of function phenotypes at the larval NMJs. Together, our study identified Mask as a novel regulator for microtubule stability and dynamics. Author SummaryMicrotubules (MTs) are an essential part of the cellular cytoskeleton, providing the structural basis for critical cellular processes and functionality. A series of factors are required to orchestrate the assembly and disassembly of MTs. Here, we identified Mask as a novel regulator of MT dynamics in Drosophila. Mask shows prominent interplay with two important modulators of MT, Tau and Stathmin (Stai). These findings not only support the role of Mask as a novel microtubule regulator but also provide the foundation to explore future therapeutic strategies in mitigating deficits related to dysfunction of Tau and/or Stai, both of which are linked to human neurodegenerative disorders. Further analyses on Mask demonstrated that Jupiters localization to the MTs in the axons is negatively affected by Mask, and that reducing Jupiter level is able to partially suppress synaptic defects caused by mask mutant. Together, these data imply that Masks function in regulating MT dynamics requires Jupiter.

neuroscience↗

bioRxiv2020

Paxillin Promotes ATP-induced Activation of P2X7 Receptor and NLRP3 Inflammasome

The stimulation of P2X7 receptor by extracellular ATP leads to activation of NLRP3 inflammasome and release of pro-inflammatory cytokines. Here, we reveal a distinct mechanism by which Paxillin promotes ATP-induced activation of P2X7 receptor and NLRP3 inflammasome. Extracellular ATP induces Paxillin phosphorylation and facilitates Paxillin-NLRP3 interaction. Interestingly, Paxillin enhances NLRP3 deubiquitination and activates NLRP3 inflammasome upon ATP treatment and K+ efflux. Moreover, we reveal that UPS13 is a key enzyme for Paxillin-mediated NLRP3 deubiquitination upon ATP treatment. Notably, extracellular ATP promotes Paxillin and NLRP3 migration from cytosol to plasma membrane and facilitates P2X7-Paxillin interaction and Paxillin-NLRP3 association, resulting in the formation of P2X7-Paxillin-NLRP3 complex. Functionally, Paxillin is essential for ATP-induced NLRP3 inflammasome activation in mouse BMDMs and BMDCs as we as in human PBMCs and THP-1-differentiated macrophages. Thus, Paxillin plays key roles in ATP-induced activation of P2X7 receptor and NLRP3 inflammasome by facilitating the formation of the P2X7-Paxillin-NLRP3 complex.

immunology↗

bioRxiv2020

Long read assembly of the pandemic strain of Austropuccinia psidii (myrtle rust) reveals an unusually large (gigabase sized) and repetitive fungal genome.

Austropuccinia psidii, originating in South America, is a globally invasive fungal plant pathogen that causes rust disease on Myrtaceae. Several biotypes are recognized, with the most widely distributed pandemic biotype spreading throughout the Asia-Pacific and Oceania regions over the last decade. Austropuccinia psidii has a broad host range with more than 480 myrtaceous species. Since first detected in Australia in 2010, the pathogen has caused the near extinction of at least three species and negatively affected commercial production of several Myrtaceae. To enable molecular and evolutionary studies into A. psidii pathogenicity, we assembled a highly contiguous genome for the pandemic biotype. With an estimated haploid genome size of just over 1 Gb (gigabases), it is the largest assembled fungal genome to date. The genome has undergone massive expansion via distinct transposable element (TE) bursts. Over 90% of the genome is covered by TEs predominantly belonging to the Gypsy superfamily. These TE bursts have likely been followed by deamination events of methylated cytosines to silence the repetitive elements. This in turn led to the depletion of CpG sites in transposable elements and a very low overall GC content of 33.8%. The overall gene content is highly conserved, when compared to other closely related Pucciniales, yet the intergenic distances are increased by an order of magnitude indicating a general insertion of TEs between genes. Overall, we show how transposable elements shaped the genome evolution of A. psidii and provide a greatly needed resource for strategic approaches to combat disease spread.

genomics↗

bioRxiv2020

Automated design and optimization of multitarget schizophrenia drug candidates by deep learning

Complex neuropsychiatric diseases such as schizophrenia require drugs that can target multiple G protein-coupled receptors (GPCRs) to modulate complex neuropsychiatric functions. Here, we report an automated system comprising a deep recurrent neural network (RNN) and a multitask deep neural network (MTDNN) to design and optimize multitargeted antipsychotic drugs. The system successfully generates novel molecule structures with desired multiple target activities, among which high-ranking compound 3 was synthesized, and demonstrated potent activities against dopamine D2, serotonin 5-HT1A and 5-HT2A receptors. Hit expansion based on the MTDNN was performed, 6 analogs of compound 3 were evaluated experimentally, among which compound 8 not only exhibited specific polypharmacology profiles but also showed antipsychotic effect in animal models with low potential for sedation and catalepsy, highlighting their suitability for further preclinical studies. The approach can be an efficient tool for designing lead compounds with multitarget profiles to achieve the desired efficacy in the treatment of complex neuropsychiatric diseases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/999615v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@17046d8org.highwire.dtl.DTLVardef@3b4c2aorg.highwire.dtl.DTLVardef@10590e9org.highwire.dtl.DTLVardef@6cc609_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2020

Cell-free protein synthesis enables one-pot cascade biotransformation in an aqueous-organic biphasic system

Biocatalytic cascade reactions have become increasingly important and useful for chemical synthesis. However, biocatalysts are often incompatible with organic solvents, which prohibits many cascade reactions involving nonpolar substrates. In this work, we used cell-free protein synthesis (CFPS) to express enzymes in an aqueous-organic biphasic system for the construction of an artificial enzymatic pathway. CFPS-expressed enzymes without purification performed efficiently to convert styrene (below 20 mM) to (S)-1-phenyl-1,2-ethanediol (two steps in one pot) with 100% conversion. In addition, our CFPS system showed great tolerance to different organic solvents and, importantly, the entire biocatalytic system can be consistently scaled up without reduction of the substrate conversion rate. We therefore anticipate that our cell-free approach will make possible cost-effective, high-yielding synthesis of valuable chemicals.

synthetic biology↗