210 Citations
The ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states However current RNA-seq methods are unable to simultaneously monitor both short and long poly A and poly A -transcripts at the single-cell level and thus deliver only a partial snapshot of the cellular RNAome Here we describe Smart-seq-total a method capable of assaying a broad spectrum of coding and non-coding RNA from a single cell Built upon the template-switch mechanism Smart-seq-total bears the key feature of its predecessor Smart-seq namely the ability to capture full-length transcripts ... More
The ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states. However, current RNA-seq methods are unable to simultaneously monitor both short and long, poly(A)+ and poly(A)-transcripts at the single-cell level, and thus deliver only a partial snapshot of the cellular RNAome. Here, we describe Smart-seq-total, a method capable of assaying a broad spectrum of coding and non-coding RNA from a single cell. Built upon the template-switch mechanism, Smart-seq-total bears the key feature of its predecessor, Smart-seq2, namely, the ability to capture full-length transcripts with high yield and quality. It also outperforms current poly(A)–independent total RNA-seq protocols by capturing transcripts of a broad size range, thus, allowing us to simultaneously analyze protein-coding, long non-coding, microRNA and other non-coding RNA transcripts from single cells. We used Smart-seq-total to analyze the total RNAome of human primary fibroblasts, HEK293T and MCF7 cells as well as that of induced murine embryonic stem cells differentiated into embryoid bodies. We show that simultaneous measurement of non-coding RNA and mRNA from the same cell enables elucidation of new roles of non-coding RNA throughout essential processes such as cell cycle or lineage commitment. Moreover, we show that cell types can be distinguished based on the abundance of non-coding transcripts alone. Less
N -Methyladenosine m A is the most widespread internal messenger RNA modification in humans Despite recent progress in understanding the biological roles of m A the inability to install m A site specifically in individual transcripts has hampered efforts to elucidate causal relationships between the presence of a specific m A and phenotypic outcomes In the present study we demonstrate that nucleus-localized dCas fusions with a truncated METTL methyltransferase domain and cytoplasm-localized fusions with a modified METTL METTL methyltransferase complex can direct site-specific m A incorporation in distinct cellular compartments with the former fusion protein having particularly low off-target activity ... More
N6-Methyladenosine (m6A) is the most widespread internal messenger RNA modification in humans. Despite recent progress in understanding the biological roles of m6A, the inability to install m6A site specifically in individual transcripts has hampered efforts to elucidate causal relationships between the presence of a specific m6A and phenotypic outcomes. In the present study, we demonstrate that nucleus-localized dCas13 fusions with a truncated METTL3 methyltransferase domain and cytoplasm-localized fusions with a modified METTL3:METTL14 methyltransferase complex can direct site-specific m6A incorporation in distinct cellular compartments, with the former fusion protein having particularly low off-target activity. Independent cellular assays across multiple sites confirm that this targeted RNA methylation (TRM) system mediates efficient m6A installation in endogenous RNA transcripts with high specificity. Finally, we show that TRM can induce m6A-mediated changes to transcript abundance and alternative splicing. These findings establish TRM as a tool for targeted epitranscriptome engineering that can reveal the effect of individual m6A modifications and dissect their functional roles. Less
ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes This posttranslational modification is carried out by ADP-ribosyltransferases converting -NAD into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR The reverse reaction release of ADP-ribose from the acceptor molecule is catalyzed by ADP-ribosylhydrolases Several hydrolases contain a macrodomain fold and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation Macrodomains have been linked to diseases such as cancer making them potential drug targets Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases and here we describe ... More
ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes. This posttranslational modification is carried out by ADP-ribosyltransferases converting β-NAD+ into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR. The reverse reaction, release of ADP-ribose from the acceptor molecule, is catalyzed by ADP-ribosylhydrolases. Several hydrolases contain a macrodomain fold, and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation. Macrodomains have been linked to diseases such as cancer, making them potential drug targets. Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases, and here we describe an inhibitor screening assay against mono-ADP-ribosylhydrolyzing enzymes. The activity-based assay uses an α-NAD+, anomer of β-NAD+, which is accepted as a substrate by MacroD1, MacroD2, and ARH3 due to its resemblance to the protein-linked ADP-ribose. The amount of α-NAD+ present after hydrolysis is measured by chemically converting it on a microtiter plate to a fluorescent compound. We optimized the assay for MacroD2 and performed a proof-of-concept compound screening. Three compounds were identified as screening hits with micromolar potency. However, further characterization of the compounds identified them as protein destabilizers, excluding further follow-up studies. Validation and screening demonstrated the usability of the in vitro assay for MacroD2, and we also demonstrate the applicability of the assay as a tool for other human ADP-ribosylhydrolases. Less
The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology these effectors are known as ROPs and GRAs respectively To examine the individual impacts of ROPs and GRAs on host gene expression we developed a robust novel protocol to enrich for ultrapure populations of a naturally occurring and reproducible population of host cells called uninfected-injected U-I cells which Toxoplasma injects with ROPs but subsequently fails to invade We then performed single-cell transcriptomic analysis at to h postinfection on U-I cells as well as on uninfected and ... More
The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultrapure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single-cell transcriptomic analysis at 1 to 3 h postinfection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild-type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host’s earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent proinflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggest that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes. Less
The pharmaceutical industry is continuing to face high research and development R D costs and low overall success rates of clinical compounds during drug development There is an increasing demand for development and validation of healthy or disease-relevant and physiological human cellular models that can be implemented in early-stage discovery thereby shifting attrition of future therapeutics to a point in discovery at which the costs are significantly lower There needs to be a paradigm shift in the early drug discovery phase which is lengthy and costly away from simplistic cellular models that show an inability to effectively and efficiently reproduce ... More
The pharmaceutical industry is continuing to face high research and development (R&D) costs and low overall success rates of clinical compounds during drug development. There is an increasing demand for development and validation of healthy or disease-relevant and physiological human cellular models that can be implemented in early-stage discovery, thereby shifting attrition of future therapeutics to a point in discovery at which the costs are significantly lower. There needs to be a paradigm shift in the early drug discovery phase (which is lengthy and costly), away from simplistic cellular models that show an inability to effectively and efficiently reproduce healthy or human disease-relevant states to steer target and compound selection for safety, pharmacology, and efficacy questions. This perspective article covers the various stages of early drug discovery from target identification (ID) and validation to the hit/lead discovery phase, lead optimization, and preclinical safety. We outline key aspects that should be considered when developing, qualifying, and implementing complex in vitro models (CIVMs) during these phases, because criteria such as cell types (e.g., cell lines, primary cells, stem cells, and tissue), platform (e.g., spheroids, scaffolds or hydrogels, organoids, microphysiological systems, and bioprinting), throughput, automation, and single and multiplexing endpoints will vary. The article emphasizes the need to adequately qualify these CIVMs such that they are suitable for various applications (e.g., context of use) of drug discovery and translational research. The article ends looking to the future, in which there is an increase in combining computational modeling, artificial intelligence and machine learning (AI/ML), and CIVMs. Less
Synthetic lethal screens have the potential to identify new vulnerabilities incurred by specific cancer mutations but have been hindered by lack of agreement between studies In the case of KRAS we identify that published synthetic lethal screen hits significantly overlap at the pathway rather than gene level Analysis of pathways encoded as protein networks could identify synthetic lethal candidates that are more reproducible than those previously reported Lack of overlap likely stems from biological rather than technical limitations as most synthetic lethal phenotypes are strongly modulated by changes in cellular conditions or genetic context the latter determined using a pairwise ... More
Synthetic lethal screens have the potential to identify new vulnerabilities incurred by specific cancer mutations but have been hindered by lack of agreement between studies. In the case of KRAS, we identify that published synthetic lethal screen hits significantly overlap at the pathway rather than gene level. Analysis of pathways encoded as protein networks could identify synthetic lethal candidates that are more reproducible than those previously reported. Lack of overlap likely stems from biological rather than technical limitations as most synthetic lethal phenotypes are strongly modulated by changes in cellular conditions or genetic context, the latter determined using a pairwise genetic interaction map that identifies numerous interactions that suppress synthetic lethal effects. Accounting for pathway, cellular and genetic context nominates a DNA repair dependency in KRAS-mutant cells, mediated by a network containing BRCA1. We provide evidence for why most reported synthetic lethals are not reproducible which is addressable using a multi-faceted testing framework. Less
We engineered a concatenated fluorescent biosensor and dual-wavelength fluorescence lifetime FLT detection to perform high-throughput screening HTS in living cells for discovery of potential heart-failure drugs Heart failure is correlated with insufficient activity of the sarcoplasmic reticulum Ca-pump SERCA a often due to excessive inhibition by phospholamban PLB a small transmembrane protein We sought to discover small molecules that restore SERCA a activity by disrupting this inhibitory interaction between PLB and SERCA a Our approach was to fluorescently tag the two proteins and measure fluorescence resonance energy transfer FRET to detect changes in binding or structure of the complex To ... More
We engineered a concatenated fluorescent biosensor and dual-wavelength fluorescence lifetime (FLT) detection, to perform high-throughput screening (HTS) in living cells for discovery of potential heart-failure drugs. Heart failure is correlated with insufficient activity of the sarcoplasmic reticulum Ca-pump (SERCA2a), often due to excessive inhibition by phospholamban (PLB), a small transmembrane protein. We sought to discover small molecules that restore SERCA2a activity by disrupting this inhibitory interaction between PLB and SERCA2a. Our approach was to fluorescently tag the two proteins and measure fluorescence resonance energy transfer (FRET) to detect changes in binding or structure of the complex. To optimize sensitivity to these changes, we engineered a biosensor that concatenates the two fluorescently labeled proteins on a single polypeptide chain. This SERCA2a-PLB FRET biosensor construct is functionally active and effective for HTS. By implementing 2-wavelength FLT detection at extremely high speed during primary HTS, we culled fluorescent compounds as false-positive Hits. In pilot screens, we identified Hits that alter the SERCA2a-PLB interaction, and a newly developed secondary calcium uptake assay revealed both activators and inhibitors of Ca-transport. We are implementing this approach for large-scale screens to discover new drug-like modulators of SERCA2a-PLB interactions for heart failure therapeutic development. Less
We present a deep learning-based framework to design and quantify point-of-care sensors As a use-case we demonstrated a low-cost and rapid paper-based vertical flow assay VFA for high sensitivity C-Reactive Protein hsCRP testing commonly used for assessing risk of cardio-vascular disease CVD A machine learning-based framework was developed to determine an optimal configuration of immunoreaction spots and conditions spatially-multiplexed on a sensing membrane and to accurately infer target analyte concentration Using a custom-designed handheld VFA reader a clinical study with human samples showed a competitive coefficient-of-variation of and linearity of R among blindly-tested VFAs in the hsCRP range i e ... More
We present a deep learning-based framework to design and quantify point-of-care sensors. As a use-case, we demonstrated a low-cost and rapid paper-based vertical flow assay (VFA) for high sensitivity C-Reactive Protein (hsCRP) testing, commonly used for assessing risk of cardio-vascular disease (CVD). A machine learning-based framework was developed to (1) determine an optimal configuration of immunoreaction spots and conditions, spatially-multiplexed on a sensing membrane, and (2) to accurately infer target analyte concentration. Using a custom-designed handheld VFA reader, a clinical study with 85 human samples showed a competitive coefficient-of-variation of 11.2% and linearity of R2 = 0.95 among blindly-tested VFAs in the hsCRP range (i.e., 0–10 mg/L). We also demonstrated a mitigation of the hook-effect due to the multiplexed immunoreactions on the sensing membrane. This paper-based computational VFA could expand access to CVD testing, and the presented framework can be broadly used to design cost-effective and mobile point-of-care sensors. Less
Treatments for cognitive deficits associated with central nervous system CNS disorders such as Alzheimer disease and schizophrenia remain significant unmet medical needs that incur substantial pressure on the health care system The nicotinic acetylcholine receptor nAChR has garnered substantial attention as a target for cognitive deficits based on receptor localization robust preclinical effects genetics implicating its involvement in cognitive disorders and encouraging albeit mixed clinical data with nAChR orthosteric agonists Importantly previous orthosteric agonists at this receptor suffered from off-target activity receptor desensitization and an inverted U-shaped dose-effect curve in preclinical assays that limit their clinical utility To overcome the ... More
Treatments for cognitive deficits associated with central nervous system (CNS) disorders such as Alzheimer disease and schizophrenia remain significant unmet medical needs that incur substantial pressure on the health care system. The α7 nicotinic acetylcholine receptor (nAChR) has garnered substantial attention as a target for cognitive deficits based on receptor localization, robust preclinical effects, genetics implicating its involvement in cognitive disorders, and encouraging, albeit mixed, clinical data with α7 nAChR orthosteric agonists. Importantly, previous orthosteric agonists at this receptor suffered from off-target activity, receptor desensitization, and an inverted U-shaped dose-effect curve in preclinical assays that limit their clinical utility. To overcome the challenges with orthosteric agonists, we have identified a novel selective α7 positive allosteric modulator (PAM), BNC375. This compound is selective over related receptors and potentiates acetylcholine-evoked α7 currents with only marginal effect on the receptor desensitization kinetics. In addition, BNC375 enhances long-term potentiation of electrically evoked synaptic responses in rat hippocampal slices and in vivo. Systemic administration of BNC375 reverses scopolamine-induced cognitive deficits in rat novel object recognition and rhesus monkey object retrieval detour (ORD) task over a wide range of exposures, showing no evidence of an inverted U-shaped dose-effect curve. The compound also improves performance in the ORD task in aged African green monkeys. Moreover, ex vivo 13C-NMR analysis indicates that BNC375 treatment can enhance neurotransmitter release in rat medial prefrontal cortex. These findings suggest that α7 nAChR PAMs have multiple advantages over orthosteric α7 nAChR agonists for the treatment of cognitive dysfunction associated with CNS diseases. Less
Single-cell RNA sequencing scRNA-seq is the leading technique for characterizing the transcriptomes of individual cells in a sample The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues organs and organisms However the protocols differ substantially with respect to their RNA capture efficiency bias scale and costs and their relative advantages for different applications are unclear In the present study we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states We performed a multicenter study comparing commonly used scRNA-seq and single-nucleus ... More
Single-cell RNA sequencing (scRNA-seq) is the leading technique for characterizing the transcriptomes of individual cells in a sample. The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues, organs and organisms. However, the protocols differ substantially with respect to their RNA capture efficiency, bias, scale and costs, and their relative advantages for different applications are unclear. In the present study, we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states. We performed a multicenter study comparing 13 commonly used scRNA-seq and single-nucleus RNA-seq protocols applied to a heterogeneous reference sample resource. Comparative analysis revealed marked differences in protocol performance. The protocols differed in library complexity and their ability to detect cell-type markers, impacting their predictive value and suitability for integration into reference cell atlases. These results provide guidance both for individual researchers and for consortium projects such as the Human Cell Atlas. Less
Brain endothelial cells BECs are key constituents of the blood-brain barrier BBB protecting the brain from pathogens and restricting access of circulatory factors Yet because circulatory proteins have prominent age-related effects on adult neurogenesis neuroinflammation and cognitive function in mice we wondered whether BECs receive and potentially relay signals between the blood and brain Using single-cell RNA sequencing of hippocampal BECs we discover that capillary BECs compared with arterial and venous BECs undergo the greatest transcriptional changes in normal aging upregulating innate immunity and oxidative stress response pathways Short-term infusions of aged plasma into young mice recapitulate key aspects of ... More
Brain endothelial cells (BECs) are key constituents of the blood-brain barrier (BBB), protecting the brain from pathogens and restricting access of circulatory factors. Yet, because circulatory proteins have prominent age-related effects on adult neurogenesis, neuroinflammation, and cognitive function in mice, we wondered whether BECs receive and potentially relay signals between the blood and brain. Using single-cell RNA sequencing of hippocampal BECs, we discover that capillary BECs—compared with arterial and venous BECs—undergo the greatest transcriptional changes in normal aging, upregulating innate immunity and oxidative stress response pathways. Short-term infusions of aged plasma into young mice recapitulate key aspects of this aging transcriptome, and remarkably, infusions of young plasma into aged mice exert rejuvenation effects on the capillary transcriptome. Together, these findings suggest that the transcriptional age of BECs is exquisitely sensitive to age-related circulatory cues and pinpoint the BBB itself as a promising therapeutic target to treat brain disease. Less
Next generation sequencing is in the process of evolving from a technology used for research purposes to one which is applied in clinical diagnostics Recently introduced high throughput and benchtop instruments offer fully automated sequencing runs at a lower cost per base and faster assay times In turn the complex and cumbersome library preparation starting with isolated nucleic acids and resulting in amplified and barcoded DNA with sequencing adapters has been identified as a significant bottleneck Library preparation protocols usually consist of a multistep process and require costly reagents and substantial hands-on-time Considerable emphasis will need to be placed on ... More
Next generation sequencing is in the process of evolving from a technology used for research purposes to one which is applied in clinical diagnostics. Recently introduced high throughput and benchtop instruments offer fully automated sequencing runs at a lower cost per base and faster assay times. In turn, the complex and cumbersome library preparation, starting with isolated nucleic acids and resulting in amplified and barcoded DNA with sequencing adapters, has been identified as a significant bottleneck. Library preparation protocols usually consist of a multistep process and require costly reagents and substantial hands-on-time. Considerable emphasis will need to be placed on standardisation to ensure robustness and reproducibility. This review presents an overview of the current state of automation of library preparation for next generation sequencing. Major challenges associated with library preparation are outlined and different automation strategies are classified according to their functional principle. Pipetting workstations allow high-throughput processing yet offer limited flexibility, whereas microfluidic solutions offer great potential due to miniaturisation and decreased investment costs. For the emerging field of single cell transcriptomics for example, microfluidics enable singularisation of tens of thousands of cells in nanolitre droplets and barcoding of the RNA to assign each nucleic acid sequence to its cell of origin. Finally, two applications, the characterisation of bacterial pathogens and the sequencing within human immunogenetics, are outlined and benefits of automation are discussed. Less
Histone lysine specific demethylase LSD has become a potential therapeutic target for the treatment of cancer Discovery and develop novel and potent LSD inhibitors is a challenge although several of them have already entered into clinical trials Herein for the first time we reported the discovery of a series of -cyano- -phenylpyrimidine derivatives as LSD inhibitors using flavin adenine dinucleotide FAD similarity-based designing strategy of which compound q was finally identified to repress LSD with IC nmol L Docking analysis suggested that compound q fitted well into the FAD-binding pocket Further mechanism studies showed that compound q may inhibit LSD ... More
Histone lysine specific demethylase 1 (LSD1) has become a potential therapeutic target for the treatment of cancer. Discovery and develop novel and potent LSD1 inhibitors is a challenge, although several of them have already entered into clinical trials. Herein, for the first time, we reported the discovery of a series of 5-cyano-6-phenylpyrimidine derivatives as LSD1 inhibitors using flavin adenine dinucleotide (FAD) similarity-based designing strategy, of which compound 14q was finally identified to repress LSD1 with IC50 = 183 nmol/L. Docking analysis suggested that compound 14q fitted well into the FAD-binding pocket. Further mechanism studies showed that compound 14q may inhibit LSD1 activity competitively by occupying the FAD binding sites of LSD1 and inhibit cell migration and invasion by reversing epithelial to mesenchymal transition (EMT). Overall, these findings showed that compound 14q is a suitable candidate for further development of novel FAD similarity-based LSD1 inhibitors. Less
Toxoplasma gondii a protozoan parasite undergoes a complex and poorly understood developmental process that is critical for establishing a chronic infection in its intermediate hosts Here we applied single-cell RNA-sequencing scRNA-seq on Toxoplasma in both tachyzoite and bradyzoite stages using three widely studied strains to construct a comprehensive atlas of cell-cycle and asexual development revealing hidden states and transcriptional factors associated with each developmental stage Analysis of SAG -related sequence SRS antigenic repertoire reveals a highly heterogeneous sporadic expression pattern unexplained by measurement noise cell cycle or asexual development Furthermore we identified AP IX- as a transcription factor that controls ... More
Toxoplasma gondii, a protozoan parasite, undergoes a complex and poorly understood developmental process that is critical for establishing a chronic infection in its intermediate hosts. Here, we applied single-cell RNA-sequencing (scRNA-seq) on >5,400 Toxoplasma in both tachyzoite and bradyzoite stages using three widely studied strains to construct a comprehensive atlas of cell-cycle and asexual development, revealing hidden states and transcriptional factors associated with each developmental stage. Analysis of SAG1-related sequence (SRS) antigenic repertoire reveals a highly heterogeneous, sporadic expression pattern unexplained by measurement noise, cell cycle, or asexual development. Furthermore, we identified AP2IX-1 as a transcription factor that controls the switching from the ubiquitous SAG1 to rare surface antigens not previously observed in tachyzoites. In addition, comparative analysis between Toxoplasma and Plasmodium scRNA-seq results reveals concerted expression of gene sets, despite fundamental differences in cell division. Lastly, we built an interactive data-browser for visualization of our atlas resource. Less
Carbapenem-resistant Enterobacteriaceae CRE represent an urgent threat to human health Here we report the application of several complementary whole-genome sequencing WGS technologies to characterise a hospital outbreak of blaIMP- carbapenemase-producing E hormaechei Using Illumina sequencing we determined that all outbreak strains were sequence type ST and near-identical Comparison to publicly available data linked all outbreak isolates to a isolate from the same ward suggesting an environmental source in the hospital Using Pacific Biosciences sequencing we resolved the complete context of the blaIMP- gene on a large IncHI plasmid carried by all IMP- -producing strains across different hospitals Shotgun metagenomic sequencing ... More
Carbapenem-resistant Enterobacteriaceae (CRE) represent an urgent threat to human health. Here we report the application of several complementary whole-genome sequencing (WGS) technologies to characterise a hospital outbreak of blaIMP-4 carbapenemase-producing E. hormaechei. Using Illumina sequencing, we determined that all outbreak strains were sequence type 90 (ST90) and near-identical. Comparison to publicly available data linked all outbreak isolates to a 2013 isolate from the same ward, suggesting an environmental source in the hospital. Using Pacific Biosciences sequencing, we resolved the complete context of the blaIMP-4 gene on a large IncHI2 plasmid carried by all IMP-4-producing strains across different hospitals. Shotgun metagenomic sequencing of environmental samples also found evidence of ST90 E. hormaechei and the IncHI2 plasmid within the hospital plumbing. Finally, Oxford Nanopore sequencing rapidly resolved the true relationship of subsequent isolates to the initial outbreak. Overall, our strategic application of three WGS technologies provided an in-depth analysis of the outbreak. Less
Sex is a key modifier of neurological disease outcomes Microglia are implicated in neurological diseases and modulated by microRNAs but it is unknown whether microglial microRNAs have sex-specific influences on disease We show in mice that microglial microRNA expression differs in males and females and that loss of microRNAs leads to sex-specific changes in the microglial transcriptome and tau pathology These findings suggest that microglial microRNAs influence tau pathogenesis in a sex-specific manner
In tropical iron ore regions biologically mediated reduction of crystalline iron oxides drives ongoing iron cycling that contributes to the stability of surface duricrusts This represents a biotechnological opportunity with respect to post-mining rehabilitation attempts requiring re-formation of these duricrusts However cultivated dissimilatory iron reducing bacteria typically reduce crystalline iron oxides quite poorly A glucose-fermenting microbial consortium capable of reducing at least mmol L goethite was enriched from an iron duricrust region Metagenome analysis led to the recovery of a metagenome assembled genome MAG of an iron reducer belonging to the alphaproteobacterial genus Telmatospirillum This is the first report of ... More
In tropical iron ore regions, biologically mediated reduction of crystalline iron oxides drives ongoing iron cycling that contributes to the stability of surface duricrusts. This represents a biotechnological opportunity with respect to post-mining rehabilitation attempts, requiring re-formation of these duricrusts. However, cultivated dissimilatory iron reducing bacteria typically reduce crystalline iron oxides quite poorly. A glucose-fermenting microbial consortium capable of reducing at least 27 mmol/L goethite was enriched from an iron duricrust region. Metagenome analysis led to the recovery of a metagenome assembled genome (MAG) of an iron reducer belonging to the alphaproteobacterial genus Telmatospirillum. This is the first report of iron reduction within the Telmatospirillum and the first reported genome of an iron-reducing, neutrophilic member of the Alphaproteobacteria. The Telmatospirillum MAG encodes putative metal transfer reductases (MtrA, MtrB) and a novel, multi-heme outer membrane cytochrome for extracellular electron transfer. In the presence of goethite, short chain fatty acid production shifted significantly in favor of acetate rather than propionate, indicating goethite is a hydrogen sink in the culture. Therefore, the presence of fermentative bacteria likely promotes iron reduction via hydrogen production. Stimulating microbial fermentation has potential to drive reduction of crystalline iron oxides, the rate limiting step for iron duricrust re-formation. Less
The over-expression and aggregation of -synuclein Syn are linked to the onset and pathology of Parkinson s disease Native monomeric Syn exists in an intrinsically disordered ensemble of interconverting conformations which has made its therapeutic targeting by small molecules highly challenging Nonetheless here we successfully target the monomeric structural ensemble of Syn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes Using a surface plasmon resonance high-throughput screen in which monomeric Syn is incubated with microchips arrayed with tethered compounds we identified novel Syn interacting drug-like compounds Because these small molecules could impact a variety of Syn ... More
The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson’s disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson’s disease. Less
In vitro models cell culture models microtissue organ-on-a-chip microfabrication micropumps membrane manufacturing filtration vascularization perfusion microfluidics channels cleanroom silicon glass polydimethylsiloxane additive manufacturing bioprinting bioprinter three-dimensional printing bioreactor stimulation biointerfaces scaffold barrier junction sterilization biodegradable polymers functionalization hydrogels coatings etching photolithography microinjection fluid control automation disposable autoclavable media exchange sampling pipetting viscous drag pump peristaltic pump capillary pump valves bubble traps flow sensor pressure sensor noninvasive monitoring oxygen sensor pH sensor mechanotransduction shear stress compression tensile stress gauge sensor TEER electrochemical sensor biosensor calibration immunosensor ion-sensitive field-effect transistor electrical impedance spectroscopy label-free sensor lactate sensor glucose sensor temperature sensor
Microfluidic devices have been increasingly used for low-volume liquid handling operations However laboratory automation of such delicate devices has lagged behind due to the lack of world-to-chip macro-to-micro interfaces In this paper we have presented the first pipette-free robotic microfluidic interface using a microfluidic-embedded container cap referred to as a microfluidic cap-to-dispense CD to achieve a seamless integration of liquid handling and robotic automation without any traditional pipetting steps The CD liquid handling platform offers a generic and modular way to connect the robotic device to standard liquid containers It utilizes the high accuracy and high flexibility of the robotic ... More
Microfluidic devices have been increasingly used for low-volume liquid handling operations. However, laboratory automation of such delicate devices has lagged behind due to the lack of world-to-chip (macro-to-micro) interfaces. In this paper, we have presented the first pipette-free robotic–microfluidic interface using a microfluidic-embedded container cap, referred to as a microfluidic cap-to-dispense (μCD), to achieve a seamless integration of liquid handling and robotic automation without any traditional pipetting steps. The μCD liquid handling platform offers a generic and modular way to connect the robotic device to standard liquid containers. It utilizes the high accuracy and high flexibility of the robotic system to recognize, capture and position; and then using microfluidic adaptive printing it can achieve high-precision on-demand volume distribution. With its modular connectivity, nanoliter processability, high adaptability, and multitask capacity, μCD shows great potential as a generic robotic–microfluidic interface for complete pipette-free liquid handling automation. Less
Human leukocyte antigen HLA alleles have been implicated as risk factors for immune-mediated adverse drug reactions The authors recently reported a strong association between HLA-A and vancomycin-induced drug reaction with eosinophilia and systemic symptoms Identification of individuals with the risk allele before or shortly after the initiation of vancomycin therapy is of great clinical importance to prevent morbidity and mortality and improve drug safety and antibiotic treatment options A prerequisite to the success of pharmacogenetic screening tests is the development of simple robust cost-effective single HLA allele test that can be implemented in routine diagnostic laboratories In this study the ... More
Human leukocyte antigen (HLA) alleles have been implicated as risk factors for immune-mediated adverse drug reactions. The authors recently reported a strong association between HLA-A*32:01 and vancomycin-induced drug reaction with eosinophilia and systemic symptoms. Identification of individuals with the risk allele before or shortly after the initiation of vancomycin therapy is of great clinical importance to prevent morbidity and mortality, and improve drug safety and antibiotic treatment options. A prerequisite to the success of pharmacogenetic screening tests is the development of simple, robust, cost-effective single HLA allele test that can be implemented in routine diagnostic laboratories. In this study, the authors developed a simple, real-time allele-specific PCR for typing the HLA-A*32:01 allele. Four-hundred and fifty-eight DNA samples including 30 HLA-A*32:01–positive samples were typed by allele-specific PCR. Compared with American Society for Histocompatibility and Immunogenetics–accredited, sequence-based, high-resolution, full-allelic HLA typing, this assay demonstrates 100% accuracy, 100% sensitivity (95% CI, 88.43% to 100%), and 100% specificity (95% CI, 99.14% to 100%). The lowest limit of detection of this assay using PowerUp SYBR Green is 10 ng of template DNA. The assay demonstrates a sensitivity and specificity to differentiate the HLA-A*32:01 allele from closely related non–HLA-A*32 alleles and may be used in clinical settings to identify individuals with the risk allele before or during the course of vancomycin therapy. Less
Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease Here we used single-nucleus RNA-sequencing analysis to perform a comprehensive study of cell types in the middle temporal gyrus of human cortex We identified a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse with excitatory types being less layer-restricted than expected Comparison to similar mouse cortex single-cell RNA-sequencing datasets revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of properties of human cell types Despite this general conservation we also ... More
Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease. Here we used single-nucleus RNA-sequencing analysis to perform a comprehensive study of cell types in the middle temporal gyrus of human cortex. We identified a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse, with excitatory types being less layer-restricted than expected. Comparison to similar mouse cortex single-cell RNA-sequencing datasets revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of properties of human cell types. Despite this general conservation, we also found extensive differences between homologous human and mouse cell types, including marked alterations in proportions, laminar distributions, gene expression and morphology. These species-specific features emphasize the importance of directly studying human brain. Less
Compositions devices methods and systems are provided for differential functionalization of a surface of a structure to support biopolymer synthesis Provided herein are processes which include use of lamps lasers and or microcontact printing to add functional groups to surfaces for the efficient and uniform synthesis of oligonucleic acids
Viral discovery is accelerating at an unprecedented rate due to continuing advances in culture-independent sequence-based analyses One important facet of this discovery is identification of the hosts of these recently characterized uncultured viruses To this end we have adapted the viral tagging approach which bypasses the need for culture-based methods to identify host phage pairings Fluorescently labelled anonymous virions adsorb to unlabelled anonymous bacterial host cells which are then individually sorted as host phage pairs followed by genome amplification and high-throughput sequencing to establish the identities of both the host and the attached virus es We demonstrate single-cell viral tagging ... More
Viral discovery is accelerating at an unprecedented rate due to continuing advances in culture-independent sequence-based analyses. One important facet of this discovery is identification of the hosts of these recently characterized uncultured viruses. To this end, we have adapted the viral tagging approach, which bypasses the need for culture-based methods to identify host–phage pairings. Fluorescently labelled anonymous virions adsorb to unlabelled anonymous bacterial host cells, which are then individually sorted as host–phage pairs, followed by genome amplification and high-throughput sequencing to establish the identities of both the host and the attached virus(es). We demonstrate single-cell viral tagging using the faecal microbiome, including cross-tagging of viruses and bacteria between human subjects. A total of 363 unique host–phage pairings were predicted, most of which were subject-specific and involved previously uncharacterized viruses despite the majority of their bacterial hosts having known taxonomy. One-fifth of these pairs were confirmed by multiple individual tagged cells. Viruses targeting more than one bacterial species were conspicuously absent in the host–phage network, suggesting that phages are not major vectors of inter-species horizontal gene transfer in the human gut. A high level of cross-reactivity between phages and bacteria from different subjects was noted despite subject-specific viral profiles, which has implications for faecal microbiota transplant therapy. Less
RNase H dependent PCR-enabled T-cell receptor sequencing rhTCRseq can be used to determine paired alpha beta T-cell receptor TCR clonotypes in single cells or perform alpha and beta TCR repertoire analysis in bulk RNA samples With the enhanced specificity of RNase H dependent PCR rhPCR it achieves TCR-specific amplification and addition of dual-index barcodes in a single PCR step For single cells the protocol includes sorting of single cells into plates generation of cDNA libraries a TCR-specific amplification step a second PCR on pooled sample to generate a sequencing library and sequencing In the bulk method sorting and cDNA library ... More
RNase H–dependent PCR-enabled T-cell receptor sequencing (rhTCRseq) can be used to determine paired alpha/beta T-cell receptor (TCR) clonotypes in single cells or perform alpha and beta TCR repertoire analysis in bulk RNA samples. With the enhanced specificity of RNase H–dependent PCR (rhPCR), it achieves TCR-specific amplification and addition of dual-index barcodes in a single PCR step. For single cells, the protocol includes sorting of single cells into plates, generation of cDNA libraries, a TCR-specific amplification step, a second PCR on pooled sample to generate a sequencing library, and sequencing. In the bulk method, sorting and cDNA library steps are replaced with a reverse-transcriptase (RT) reaction that adds a unique molecular identifier (UMI) to each cDNA molecule to improve the accuracy of repertoire-frequency measurements. Compared to other methods for TCR sequencing, rhTCRseq has a streamlined workflow and the ability to analyze single cells in 384-well plates. Compared to TCR reconstruction from single-cell transcriptome sequencing data, it improves the success rate for obtaining paired alpha/beta information and ensures recovery of complete complementarity-determining region 3 (CDR3) sequences, a prerequisite for cloning/expression of discovered TCRs. Although it has lower throughput than droplet-based methods, rhTCRseq is well-suited to analysis of small sorted populations, especially when analysis of 96 or 384 single cells is sufficient to identify predominant T-cell clones. For single cells, sorting typically requires 2–4 h and can be performed days, or even months, before library construction and data processing, which takes ~4 d; the bulk RNA protocol takes ~3 d. Less
Discovery of novel Mnk inhibitors using mutation-based induced-fit virtual high-throughput screening
Mnk kinases Mnk and are downstream effectors of Map kinase pathways and regulate phosphorylation of eukaryotic initiation factor E Engagement of the Mnk pathway is critical in acute myeloid leukemia AML leukemogenesis and Mnk inhibitors have potent antileukemic properties in vitro and in vivo suggesting that targeting Mnk kinases may provide a novel approach for treating AML Here we report the development and application of a mutation-based induced-fit in silico screen to identify novel Mnk inhibitors The Mnk structure was modeled by temporarily mutating an amino acid that obstructs the ATP-binding site in the Mnk crystal structure while carrying out ... More
Mnk kinases (Mnk1 and 2) are downstream effectors of Map kinase pathways and regulate phosphorylation of eukaryotic initiation factor 4E. Engagement of the Mnk pathway is critical in acute myeloid leukemia (AML) leukemogenesis and Mnk inhibitors have potent antileukemic properties in vitro and in vivo, suggesting that targeting Mnk kinases may provide a novel approach for treating AML. Here, we report the development and application of a mutation-based induced-fit in silico screen to identify novel Mnk inhibitors. The Mnk1 structure was modeled by temporarily mutating an amino acid that obstructs the ATP-binding site in the Mnk1 crystal structure while carrying out docking simulations of known inhibitors. The hit compounds display activity in Mnk biochemical and cellular assays, including acute myeloid leukemia progenitors. This approach will enable further rational structure-based drug design of new Mnk inhibitors and potentially novel ways of therapeutically targeting this kinase. Less
Objective To optimize post-differentiation freeze thaw and culture conditions for induced pluripotent stem cell-derived brain microvascular endothelial cell iPSC-BMEC maturation functionality and reproducibility Methods Human iPSCs were spontaneously differentiated into BMECs protocol based on Lippmann et al Nat Biotech Dissociation and sub-culture conditions were investigated using Design of Experiments DoE an unbiased method based on structured statistical analysis of variance ANOVA DoE efficiently identifies interactions amongst experimental variables and predicts responses Our design focused on maximizing trans-endothelial electrical resistance TEER We incorporated a Mantis liquid handler to execute precise culture conditions in -well transwell systems and measured TEER over time ... More
Objective: To optimize post-differentiation freeze, thaw, and culture conditions for induced pluripotent stem cell-derived brain microvascular endothelial cell (iPSC-BMEC) maturation, functionality, and reproducibility. Methods: Human iPSCs were spontaneously differentiated into BMECs (protocol based on Lippmann et al. Nat Biotech 2012). Dissociation and sub-culture conditions were investigated using Design of Experiments (DoE), an unbiased method based on structured statistical analysis of variance (ANOVA). DoE efficiently identifies interactions amongst experimental variables and predicts responses. Our design focused on maximizing trans-endothelial electrical resistance (TEER). We incorporated a Mantis liquid handler to execute precise culture conditions in 96-well transwell systems and measured TEER over time. Top hit conditions were validated on multiple batches of iPSC-BMECs, these included post-thaw cell viability, hourly TEER reads, and immunocytochemistry (ICC). Results: We have identified a robust cryopreservation method, time course and medium formulation for sub-culturing spontaneously differentiated iPSC-BMEC. The optimized cryopreservation protocol yields high cell viability recovery allowing the ability to produce bulk batches of iPSC-BMECs, thereby minimizing interexperimental variability. Surprisingly, we found that the removal of retinoic acid and the extension to at least 7 days in culture reproducibly resulted in prolonged high TEER, approximately 500 ohms * cm2 greater than TEER peak at 48 h, in iPSC-BMEC mono-culture transwells. Our sub-culture method generates iPSC-BMECs that express the endothelial surface marker PECAM1, relevant blood–brain barrier (BBB) tight junction proteins (claudin-5, ZO-1, occludin), and transporters enriched on brain microvessels (Glut-1, transferrin receptor, insulin receptor). Conclusion: We demonstrate the power of applying DoE to fine-tune complex culture conditions to maximize cell performance. Our cryopreservation and sub-culture protocols robustly produce functional endothelial cells of the BBB that are suited to address basic cerebral vascular biology questions, study the vascular phenotype of neurological disorders, and enable high-throughput screening for drug discovery. Less
To enable rapid selection of traits in marker-assisted breeding markers must be technically simple low-cost high-throughput and randomly distributed in a genome We developed such a technology designated as Multiplex Restriction Amplicon Sequencing MRASeq which reduces genome complexity by polymerase chain reaction PCR amplification of amplicons flanked by restriction sites The first PCR primers contain restriction site sequences at -ends preceded by - bases of specific or degenerate nucleotide sequences and then by a unique M -tail sequence which serves as a binding site for a second PCR that adds sequencing primers and barcodes to allow sample multiplexing for sequencing ... More
To enable rapid selection of traits in marker-assisted breeding, markers must be technically simple, low-cost, high-throughput and randomly distributed in a genome. We developed such a technology, designated as Multiplex Restriction Amplicon Sequencing (MRASeq), which reduces genome complexity by polymerase chain reaction (PCR) amplification of amplicons flanked by restriction sites. The first PCR primers contain restriction site sequences at 3’-ends, preceded by 6-10 bases of specific or degenerate nucleotide sequences and then by a unique M13-tail sequence which serves as a binding site for a second PCR that adds sequencing primers and barcodes to allow sample multiplexing for sequencing. The sequences of restriction sites and adjacent nucleotides can be altered to suit different species. Physical mapping of MRASeq SNPs from a biparental population of allohexaploid wheat (Triticum aestivum L.) showed a random distribution of SNPs across the genome. MRASeq generated thousands of SNPs from a wheat biparental population and natural populations of wheat and barley (Hordeum vulgare L.). This novel, next-generation sequencing-based genotyping platform can be used for linkage mapping to screen quantitative trait loci (QTL), background selection in breeding and many other genetics and breeding applications of various species. Less
DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications Here we describe a de novo enzymatic synthesis strategy designed for data storage which harnesses the template-independent polymerase terminal deoxynucleotidyl transferase TdT in kinetically controlled conditions Information is stored in transitions between non-identical nucleotides of DNA strands To produce strands representing user-defined content nucleotide substrates are added iteratively yielding short homopolymeric extensions whose lengths are controlled by apyrase-mediated substrate degradation With this scheme we synthesize DNA strands carrying bits including addressing and demonstrate retrieval with streaming nanopore sequencing We ... More
DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications. Here, we describe a de novo enzymatic synthesis strategy designed for data storage which harnesses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) in kinetically controlled conditions. Information is stored in transitions between non-identical nucleotides of DNA strands. To produce strands representing user-defined content, nucleotide substrates are added iteratively, yielding short homopolymeric extensions whose lengths are controlled by apyrase-mediated substrate degradation. With this scheme, we synthesize DNA strands carrying 144 bits, including addressing, and demonstrate retrieval with streaming nanopore sequencing. We further devise a digital codec to reduce requirements for synthesis accuracy and sequencing coverage, and experimentally show robust data retrieval from imperfectly synthesized strands. This work provides distributive enzymatic synthesis and information-theoretic approaches to advance digital information storage in DNA. Less
Human NimA-related kinases Neks have multiple mitotic and non-mitotic functions but few substrates are known We systematically determined the phosphorylation-site motifs for the entire Nek kinase family except for Nek While all Nek kinases strongly select for hydrophobic residues in the position the family separates into four distinct groups based on specificity for a serine versus threonine phospho-acceptor and preference for basic or acidic residues in other positions Unlike Nek -Nek Nek is a dual-specificity kinase that efficiently phosphorylates itself and peptide substrates on serine and tyrosine and its activity is enhanced by tyrosine auto-phosphorylation Nek dual-specificity depends on residues ... More
Human NimA-related kinases (Neks) have multiple mitotic and non-mitotic functions, but few substrates are known. We systematically determined the phosphorylation-site motifs for the entire Nek kinase family, except for Nek11. While all Nek kinases strongly select for hydrophobic residues in the −3 position, the family separates into four distinct groups based on specificity for a serine versus threonine phospho-acceptor, and preference for basic or acidic residues in other positions. Unlike Nek1-Nek9, Nek10 is a dual-specificity kinase that efficiently phosphorylates itself and peptide substrates on serine and tyrosine, and its activity is enhanced by tyrosine auto-phosphorylation. Nek10 dual-specificity depends on residues in the HRD+2 and APE-4 positions that are uncommon in either serine/threonine or tyrosine kinases. Finally, we show that the phosphorylation-site motifs for the mitotic kinases Nek6, Nek7 and Nek9 are essentially identical to that of their upstream activator Plk1, suggesting that Nek6/7/9 function as phospho-motif amplifiers of Plk1 signaling. Less
An aged circulatory environment can activate microglia reduce neural precursor cell activity and impair cognition in mice We hypothesized that brain endothelial cells BECs mediate at least some of these effects We observe that BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of vascular cell adhesion molecule VCAM a protein that facilitates vascular immune cell interactions Concomitantly levels of the shed soluble form of VCAM are prominently increased in the plasma of aged humans and mice and their plasma is sufficient to increase VCAM expression in cultured BECs and the hippocampi of young mice ... More
An aged circulatory environment can activate microglia, reduce neural precursor cell activity and impair cognition in mice. We hypothesized that brain endothelial cells (BECs) mediate at least some of these effects. We observe that BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of vascular cell adhesion molecule 1 (VCAM1), a protein that facilitates vascular–immune cell interactions. Concomitantly, levels of the shed, soluble form of VCAM1 are prominently increased in the plasma of aged humans and mice, and their plasma is sufficient to increase VCAM1 expression in cultured BECs and the hippocampi of young mice. Systemic administration of anti-VCAM1 antibody or genetic ablation of Vcam1 in BECs counteracts the detrimental effects of plasma from aged individuals on young brains and reverses aging aspects, including microglial reactivity and cognitive deficits, in the brains of aged mice. Together, these findings establish brain endothelial VCAM1 at the blood–brain barrier as a possible target to treat age-related neurodegeneration. Less
Neurological diseases such as Alzheimer s disease AD Parkinson s disease PD Epilepsy and Multiple Sclerosis are included in the Global burden of disease study as these disorders have a high impact on public health Lack of effective treatment has motivated the researchers to perform early diagnostics by identifying new gene mutations which can improve the therapies The aim of this thesis was a genetic analysis of PD using next-generation sequencing data In this thesis whole genome sequencing WGS and whole exome sequencing WES using DNA from familial PD patients and healthy individuals was performed in order to identify the ... More
Neurological diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Epilepsy and Multiple Sclerosis are included in the Global burden of disease study as these disorders have a high impact on public health. Lack of effective treatment has motivated the researchers to perform early diagnostics, by identifying new gene mutations, which can improve the therapies. The aim of this thesis was a genetic analysis of PD using next-generation sequencing data. In this thesis, whole genome sequencing (WGS) and whole exome sequencing (WES) using DNA from familial PD patients and healthy individuals was performed in order to identify the PD causal genes. A large repository of sporadic PD WES data and a genotyping array was used to replicate our findings. The PD patients from Germany were stratified for clinical trials on the basis of mitochondrial endo-phenotype by performing risk profiling of associated Single Nucleotide Polymorphisms (SNPs) using exome genotyping array. The sporadic PD WES and genotyping array data from International Parkinson’s disease Genomics Consortium was used to perform association tests, to determine the burden of rare variants in candidate genes of interest. Furthermore, mRNA sequencing of all the genes under the PD GWAS loci after knockdown with short hairpin RNAs was performed, to identify the actual genes contributing to PD risk and the novel pathways involved in PD. Finally, an epistatic interaction of a Mendelian PD gene and associated locus was performed to understand the joint contribution to PD risk. Taking everything into account, we identified pathogenic variants in known and some novel genes causing PD in families. On the basis of risk profiling some of the German PD patients will undergo clinical trials with coenzyme Q10 and vitamin K2. The association tests using sporadic PD data helped to identify some novel genes significantly associated with PD risk. The knockdown experiments facilitated the identification of genes contributing to PD risk in some of the PD GWAS loci. Less
Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates While the impact of localization and recruitment on kinase substrate targeting can be readily assessed evaluating the relative importance of direct phosphorylation site interactions remains challenging In this study we examine the STE family of protein serine threonine kinases to investigate basic mechanisms of substrate targeting We used peptide arrays to define the phosphorylation site specificity for the majority of STE kinases and categorized them into four distinct groups Using structure-guided mutagenesis we identified key specificity-determining residues within the kinase catalytic cleft ... More
Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates. While the impact of localization and recruitment on kinase–substrate targeting can be readily assessed, evaluating the relative importance of direct phosphorylation site interactions remains challenging. In this study, we examine the STE20 family of protein serine–threonine kinases to investigate basic mechanisms of substrate targeting. We used peptide arrays to define the phosphorylation site specificity for the majority of STE20 kinases and categorized them into four distinct groups. Using structure-guided mutagenesis, we identified key specificity-determining residues within the kinase catalytic cleft, including an unappreciated role for the kinase β3–αC loop region in controlling specificity. Exchanging key residues between the STE20 kinases p21-activated kinase 4 (PAK4) and Mammalian sterile 20 kinase 4 (MST4) largely interconverted their phosphorylation site preferences. In cells, a reprogrammed PAK4 mutant, engineered to recognize MST substrates, failed to phosphorylate PAK4 substrates or to mediate remodeling of the actin cytoskeleton. In contrast, this mutant could rescue signaling through the Hippo pathway in cells lacking multiple MST kinases. These observations formally demonstrate the importance of catalytic site specificity for directing protein kinase signal transduction pathways. Our findings further suggest that phosphorylation site specificity is both necessary and sufficient to mediate distinct signaling outputs of STE20 kinases and imply broad applicability to other kinase signaling systems. Less
Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease It is currently unknown whether these functions are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions Here we performed deep single-cell RNA sequencing scRNA-seq of microglia and related myeloid cells sorted from various regions of embryonic early postnatal and adult mouse brains We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes regardless of brain region By contrast early postnatal microglia are more heterogeneous We discovered a proliferative-region-associated microglia PAM ... More
Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease. It is currently unknown whether these functions are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions. Here, we performed deep single-cell RNA sequencing (scRNA-seq) of microglia and related myeloid cells sorted from various regions of embryonic, early postnatal, and adult mouse brains. We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes, regardless of brain region. By contrast, early postnatal microglia are more heterogeneous. We discovered a proliferative-region-associated microglia (PAM) subset, mainly found in developing white matter, that shares a characteristic gene signature with degenerative disease-associated microglia (DAM). Such PAM have amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease. Less
Here we present a compendium of single-cell transcriptomic data from the model organism Mus musculus that comprises more than cells from organs and tissues These data represent a new resource for cell biology reveal gene expression in poorly characterized cell populations and enable the direct and controlled comparison of gene expression in cell types that are shared between tissues such as T lymphocytes and endothelial cells from different anatomical locations Two distinct technical approaches were used for most organs one approach microfluidic droplet-based -end counting enabled the survey of thousands of cells at relatively low coverage whereas the other full-length ... More
Here we present a compendium of single-cell transcriptomic data from the model organism Mus musculus that comprises more than 100,000 cells from 20 organs and tissues. These data represent a new resource for cell biology, reveal gene expression in poorly characterized cell populations and enable the direct and controlled comparison of gene expression in cell types that are shared between tissues, such as T lymphocytes and endothelial cells from different anatomical locations. Two distinct technical approaches were used for most organs: one approach, microfluidic droplet-based 3′-end counting, enabled the survey of thousands of cells at relatively low coverage, whereas the other, full-length transcript analysis based on fluorescence-activated cell sorting, enabled the characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology. Less
Automation provides a number of benefits to clinical industrial and research laboratories including elimination of human errors improvement in consistency minimization of contamination increased throughput decreased costs and reduced hands-on time At the same time multiplex assays offer the benefits of more data per sample faster reaction times reduced sample consumption enhanced scalability and a number of additional benefits due to elimination of repetitive steps Integrating laboratory automation with multiplex assays provides significant efficiencies to workflow processes especially when a large number of samples are being analyzed or when a highly multiplexed assay is being developed Automation can be partially ... More
Automation provides a number of benefits to clinical, industrial, and research laboratories, including elimination of human errors, improvement in consistency, minimization of contamination, increased throughput, decreased costs, and reduced hands-on time. At the same time, multiplex assays offer the benefits of more data per sample, faster reaction times, reduced sample consumption, enhanced scalability, and a number of additional benefits due to elimination of repetitive steps. Integrating laboratory automation with multiplex assays provides significant efficiencies to workflow processes, especially when a large number of samples are being analyzed, or when a highly multiplexed assay is being developed. Automation can be partially or fully implemented. Less
Structure-based drug design SBDD heavily relies on the production of high-resolution three-dimensional D structures of the drug target in the presence or absence of the drug candidate X-ray crystallography is the predominant technique accounting for higher than of the structures in Protein Data Bank PDB considering the complexes involving protein and inhibitors Macromolecular X-ray crystallography provides an important and powerful technique in studying the specific interactions of a particular drug with its protein target at the atomic level that can help improve the drug design process It is the main technique used to obtain D information for binary complexes involving ... More
Structure-based drug design (SBDD) heavily relies on the production of high-resolution (<2Å) three-dimensional (3D) structures of the drug target in the presence or absence of the drug candidate [1]. X-ray crystallography is the predominant technique accounting for higher than 94% of the structures in Protein Data Bank (PDB), considering the complexes involving protein and inhibitors [2–4]. Macromolecular X-ray crystallography provides an important and powerful technique in studying the specific interactions of a particular drug with its protein target at the atomic level that can help improve the drug design process [5]. It is the main technique used to obtain 3D information for binary complexes involving protein and drugs [6]. One of the major ‘bottlenecks’ in X-ray crystallography is the lack of generalized methods for high quality crystal production. Since protein crystallization mechanism details remain unknown, protein crystallization is a complicated and time-consuming process and requires performing a significant number of trial-and-error experiments involving systematic testing of variable chemical and physical parameters [7]. High-throughput (HT) structural biology coincided with the dawn of the genomic era in biology requires the automation, miniaturization, and parallelization of protein crystallization in order to reach the capacity necessary for large-scale structure determination efforts [8]. HT protein crystallization screening (HTPCS) technologies appeared on the protein crystallization scene more than three decades ago and have since allowed accessing hundreds to thousands of protein crystallization conditions, thereby greatly impacting HT structural biology [9]. HTPCS has helped to identify critical components required for HT crystallization efforts [7]. In recent years, the rapid developments of manipulation techniques and devices provide effective and reliable solutions for protein crystallization screening with HT and low consumption. In spite of their advances, HTPCS has suffered from two main handicaps, namely, poor hit rate in protein crystallization screening and lack of predictive power of the scoring functions. To overcome these handicaps, several projects have recently been initiated to construct ‘smart systems’ that are not only capable of rapidly performing a large number of crystallization trials, but also scripting and triggering certain events based on the collected data used in predict the outcome of a protein x-ray crystallization trial [10]. In this contest, we will outline recent efforts in HTPCS that could improve the success rate of the structural pipeline. We will discuss the challenge and some of the possible avenues in that direction. Less
High-throughput single-cell RNA-seq methods assign limited unique molecular identifier UMI counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts We thus developed a high-throughput single-cell RNA-seq method Quartz-Seq to overcome these issues Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts at a rate of and detect more genes To demonstrate the power of Quartz-Seq we analyzed approximately transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads
Methods are provided for analyzing one or more genetic samples comprising procuring one or more genetic samples comprising genetic material from one or more individuals and sequencing the genetic material using non-targeted ultra-low coverage sequencing to obtain genetic information for individual associated with the one or more genetic samples Personal and genetic information associated with the individuals is stored in a database for retrieval and manipulation
Cells require some metals such as zinc and manganese but excess levels of these metals can be toxic As a result cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication Here we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity together with transcriptomics data for wild-type or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation Altogether we identified genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment and putative zinc-responsive genes Analysis of the omics data points to ... More
Cells require some metals, such as zinc and manganese, but excess levels of these metals can be toxic. As a result, cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication. Here, we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity, together with transcriptomics data for wild-type or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation. Altogether, we identified 47 genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment, and >1800 putative zinc-responsive genes. Analysis of the ‘omics data points to the relevance of ion transporters, glutathione (GSH)-related factors, and conserved disease-associated genes in zinc detoxification. Specific genes identified in the zinc screen include orthologs of human disease-associated genes CTNS, PTPRN (also known as IA-2), and ATP13A2 (also known as PARK9). We show that knockdown of red dog mine (rdog; CG11897), a candidate zinc detoxification gene encoding an ABCC-type transporter family protein related to yeast cadmium factor (YCF1), confers sensitivity to zinc intoxication in cultured cells, and that rdog is transcriptionally upregulated in response to zinc stress. As there are many links between the biology of zinc and other metals and human health, the ‘omics data sets presented here provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes. Less
In recent years highly detailed characterization of adult bone marrow BM myeloid progenitors has been achieved and as a result the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined Fetal liver FL hematopoietic progenitor cells HPCs are poorly characterized in comparison potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis Numerous disorders for example infant acute leukemias have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets We previously demonstrated that a Runx distal promoter P -GFP proximal promoter P -hCD ... More
In recent years, highly detailed characterization of adult bone marrow (BM) myeloid progenitors has been achieved and, as a result, the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined. Fetal liver (FL) hematopoietic progenitor cells (HPCs) are poorly characterized in comparison, potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis. Numerous disorders, for example infant acute leukemias, have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets. We previously demonstrated that a Runx1 distal promoter (P1)-GFP::proximal promoter (P2)-hCD4 dual-reporter mouse (Mus musculus) model can be used to identify adult BM progenitor subsets with distinct lineage preferences. In this study, we undertook the characterization of the expression of Runx1-P1-GFP and P2-hCD4 in FL. Expression of P2-hCD4 in the FL immunophenotypic Megakaryocyte-Erythroid Progenitor (MEP) and Common Myeloid Progenitor (CMP) compartments corresponded to increased granulocytic/monocytic/megakaryocytic and decreased erythroid specification. Moreover, Runx1-P2-hCD4 expression correlated with several endogenous cell surface markers’ expression, including CD31 and CD45, providing a new strategy for prospective identification of highly purified fetal myeloid progenitors in transgenic mouse models. We utilized this methodology to compare the impact of the deletion of either total RUNX1 or RUNX1C alone and to determine the fetal HPCs lineages most substantially affected. This new prospective identification of FL progenitors therefore raises the prospect of identifying the underlying gene networks responsible with greater precision than previously possible. Less
There is a pressing need to improve approaches for drug discovery related to neuropsychiatric disorders NSDs Therapeutic discovery in neuropsychiatric disorders would benefit from screening assays that can measure changes in complex phenotypes linked to disease mechanisms However traditional assays that track complex neuronal phenotypes such as neuronal connectivity exhibit poor scalability and are not compatible with high-throughput screening HTS procedures Therefore we created a neuronal phenotypic assay platform that focused on improving the scalability and affordability of neuron-based assays capable of tracking disease-relevant phenotypes First using inexpensive laboratory-level automation we industrialized primary neuronal culture production which enabled the creation ... More
There is a pressing need to improve approaches for drug discovery related to neuropsychiatric disorders (NSDs). Therapeutic discovery in neuropsychiatric disorders would benefit from screening assays that can measure changes in complex phenotypes linked to disease mechanisms. However, traditional assays that track complex neuronal phenotypes, such as neuronal connectivity, exhibit poor scalability and are not compatible with high-throughput screening (HTS) procedures. Therefore, we created a neuronal phenotypic assay platform that focused on improving the scalability and affordability of neuron-based assays capable of tracking disease-relevant phenotypes. First, using inexpensive laboratory-level automation, we industrialized primary neuronal culture production, which enabled the creation of scalable assays within functioning neural networks. We then developed a panel of phenotypic assays based on culturing of primary neurons from genetically modified mice expressing HTS-compatible reporters that capture disease-relevant phenotypes. We demonstrated that a library of 1,280 compounds was quickly screened against both assays using only a few litters of mice in a typical academic laboratory setting. Finally, we implemented one assay in a fully automated high-throughput academic screening facility, illustrating the scalability of assays designed using this platform. These methodological improvements simplify the creation of highly scalable neuron-based phenotypic assays designed to improve drug discovery in CNS disorders. Less
This review provides an overview of the science of high-throughput screening HTS within the pharmaceutical industry and the greater drug discovery community From its origins in the early s to the current state of the art key aspects of the process are introduced and described in increasing levels of detail Examples of technologies employed in the automation of HTS are provided together with an evaluation of their applicability and limitations The various detection modalities typically encountered and their suitability to high-density screening formats or well are described The increasing importance of informatics in screen design data interpretation quality control and ... More
This review provides an overview of the science of high-throughput screening (HTS) within the pharmaceutical industry and the greater drug discovery community. From its origins in the early 1990s to the current state of the art, key aspects of the process are introduced and described in increasing levels of detail. Examples of technologies employed in the automation of HTS are provided, together with an evaluation of their applicability and limitations. The various detection modalities typically encountered, and their suitability to high-density screening formats (96, 386, 1536, or 3456 well) are described. The increasing importance of informatics in screen design, data interpretation, quality control, and its contribution to the development of future HTS strategies is introduced. Current and future trends of how HTS is employed to meet the changing needs for new drug discovery are explored, including the parallel use of complementary screening modalities to sample diverse chemical matter and identify the best starting points for drug discovery programs. Less
The growing demand for fast highly sensitive and low cost diagnostic devices has stimulated efforts to generate simplified analytical systems capable of being applied in the field This scientific work involved the study of different approaches to enable the qualitative and quantitative detection of biomarkers for clinical diagnosis in paper-based platforms The first strategy consisted of understanding the operating principles of the lateral flow immunoassay device allowing the assessment of critical steps and consequently providing the most appropriate experimental conditions for the early diagnosis of malaria caused by Plasmodium falciparum by identifying the HRP biomarker The best device performance was ... More
The growing demand for fast, highly sensitive and low cost diagnostic devices has stimulated efforts to generate simplified analytical systems capable of being applied in the field. This scientific work involved the study of different approaches to enable the qualitative and quantitative detection of biomarkers for clinical diagnosis in paper-based platforms. The first strategy consisted of understanding the operating principles of the lateral flow immunoassay device, allowing the assessment of critical steps and, consequently, providing the most appropriate experimental conditions for the early diagnosis of malaria caused by Plasmodium falciparum, by identifying the HRP2 biomarker. The best device performance was achieved by using 0,05 µg (1 µL / 50 µg mL-1) of the capture antibody and incubation for 5 minutes for its adsorption; blocking of nitrocellulose with 1.5% BSA (m/v) containing 0.1% of the surfactant Tween-20 (v/v) by immersion and incubation for 10 minutes; 0.04 µg (20 µL/2 µg mL-1) detection antibody conjugated to the enzyme peroxidase; washing with 0.01 mol L-1 Tris-HCl buffer solution pH 7.4 containing 0.15 mol L-1 NaCl and 0.1% Tween 20 (v/v); addition of 5 µL of TMB chromogenic substrate and reading within 5-20 minutes after this addition. The colorimetric detection system had a visual detection limit of 5 ng mL-1 (135 pmol L-1), which value should be sufficient to identify malaria contamination on the first day of symptom onset.The developed platform was then applied to blood samples from patients infected by the disease, demonstrating efficiency in the qualitative discrimination of a positive and negative result and the generation of reliable results. In another study, with the aim of providing a second generation of the device, a paper-based 3D detection system capable of being coupled to a lateral flow immunoassay platform was built. This system enables quantitative, automated diagnosis with signal amplification through the incorporation of a new polymeric material of the class of poly(benzyl ethers) that selectively responds to hydrogen peroxide, in addition to requiring only color and color visualization. a timer for obtaining data and later analyzing the results.The delimitation of hydrophobic barriers on the paper was carried out by means of wax printing and the orientation processes and type of paper to be used in the layer containing the polymer were evaluated. The material deposition process on the paper surface demonstrated greater repeatability when performed automatically by means of a liquid handling robot. The highest sensitivity condition for the 3D detection system was achieved using a polymer concentration of 4.0 mg mL-1 with the achievement of a detection limit of 0.02 mmol L-1 of hydrogen peroxide. This system was then coupled to a lateral flow immunoassay platform and used in initial studies to detect the creatine kinase MB isoenzyme, one of the biomarkers indicated for the diagnosis of acute myocardial infarction (AU)one of the biomarkers indicated for the diagnosis of acute myocardial infarction (AU) Less
The androgen receptor AR NR C is a nuclear receptor whose main function is acting as a transcription factor regulating gene expression for male sexual development and maintaining accessory sexual organ function It is also a necessary component of female fertility by affecting the functionality of ovarian follicles and ovulation Pathological processes involving AR include Kennedy s disease and Klinefelter s syndrome as well as prostate ovarian and testicular cancer Strict regulation of sex hormone signaling is required for normal reproductive organ development and function Therefore testing small molecules for their ability to modulate AR is a first step in ... More
The androgen receptor (AR, NR3C4) is a nuclear receptor whose main function is acting as a transcription factor regulating gene expression for male sexual development and maintaining accessory sexual organ function. It is also a necessary component of female fertility by affecting the functionality of ovarian follicles and ovulation. Pathological processes involving AR include Kennedy’s disease and Klinefelter’s syndrome, as well as prostate, ovarian, and testicular cancer. Strict regulation of sex hormone signaling is required for normal reproductive organ development and function. Therefore, testing small molecules for their ability to modulate AR is a first step in identifying potential endocrine disruptors. We screened the Tox21 10K compound library in a quantitative high-throughput format to identify activators of AR using two reporter gene cell lines, AR β-lactamase (AR-bla) and AR-luciferase (AR-luc). Seventy-five compounds identified through the primary assay were characterized as potential agonists or inactives through confirmation screens and secondary assays. Biochemical binding and AR nuclear translocation assays were performed to confirm direct binding and activation of AR from these compounds. The top seventeen compounds identified were found to bind to AR, and sixteen of them translocated AR from the cytoplasm into the nucleus. Five potentially novel or not well-characterized AR agonists were discovered through primary and follow-up studies. We have identified multiple AR activators, including known AR agonists such as testosterone, as well as novel/not well-known compounds such as prulifloxacin. The information gained from the current study can be directly used to prioritize compounds for further in-depth toxicological evaluations, as well as their potential to disrupt the endocrine system via AR activation. Less
Antibiotic-resistant bacterial infections are increasingly prevalent worldwide and there is an urgent need for novel classes of antibiotics capable of overcoming existing resistance mechanisms One potential antibiotic target is the bacterial single-stranded DNA binding protein SSB which serves as a hub for DNA repair recombination and replication Eight highly conserved residues at the C-terminus of SSB use direct protein protein interactions PPIs to recruit more than a dozen important genome maintenance proteins to single-stranded DNA Mutations that disrupt PPIs with the C-terminal tail of SSB are lethal suggesting that small-molecule inhibitors of these critical SSB PPIs could be effective antibacterial ... More
Antibiotic-resistant bacterial infections are increasingly prevalent worldwide, and there is an urgent need for novel classes of antibiotics capable of overcoming existing resistance mechanisms. One potential antibiotic target is the bacterial single-stranded DNA binding protein (SSB), which serves as a hub for DNA repair, recombination, and replication. Eight highly conserved residues at the C-terminus of SSB use direct protein–protein interactions (PPIs) to recruit more than a dozen important genome maintenance proteins to single-stranded DNA. Mutations that disrupt PPIs with the C-terminal tail of SSB are lethal, suggesting that small-molecule inhibitors of these critical SSB PPIs could be effective antibacterial agents. As a first step toward implementing this strategy, we have developed orthogonal high-throughput screening assays to identify small-molecule inhibitors of the Klebsiella pneumonia SSB-PriA interaction. Hits were identified from an initial screen of 72,474 compounds using an AlphaScreen (AS) primary screen, and their activity was subsequently confirmed in an orthogonal fluorescence polarization (FP) assay. As an additional control, an FP assay targeted against an unrelated eukaryotic PPI was used to confirm specificity for the SSB-PriA interaction. Nine potent and selective inhibitors produced concentration–response curves with IC50 values of <40 μM, and two compounds were observed to directly bind to PriA, demonstrating the success of this screen strategy. Less
Evaluating the ligandability of a protein target is a key component when defining hit-finding strategies or when prioritize among drug targets Computational as well as biophysical approaches based on nuclear magnetic resonance NMR fragment screening are powerful approaches but suffer from specific constraints that limit their usage Here we demonstrate the applicability of high-throughput thermal scanning HTTS as a simple and generic biophysical fragment screening method to reproduce assessments from NMR-based screening By applying this method to a large set of proteins we can furthermore show that the assessment is predictive of the success of high-throughput screening HTS The few ... More
Evaluating the ligandability of a protein target is a key component when defining hit-finding strategies or when prioritize among drug targets. Computational as well as biophysical approaches based on nuclear magnetic resonance (NMR) fragment screening are powerful approaches but suffer from specific constraints that limit their usage. Here, we demonstrate the applicability of high-throughput thermal scanning (HTTS) as a simple and generic biophysical fragment screening method to reproduce assessments from NMR-based screening. By applying this method to a large set of proteins we can furthermore show that the assessment is predictive of the success of high-throughput screening (HTS). The few divergences for targets of low ligandability originate from the sensitivity differences of the orthogonal biophysical methods. We thus applied a new strategy making use of modulations in the solvent structure to improve assay sensitivity. This novel approach enables improved ligandability assessments in accordance with NMR-based assessments and more importantly positions the methodology as a valuable option for biophysical fragment screening. Less
Paper microzone plates in combination with a noncontact liquid handling robot were demonstrated as tools for studying the stability of enzymes stored on paper The effect of trehalose and SU- epoxy novolac resin SU- on the stability of horseradish peroxidase HRP was studied in both a short-term experiment where the activity of various concentrations of HRP dried on paper were measured after h and a long-term experiment where the activity of a single concentration of HRP dried and stored on paper was monitored for days SU- was found to stabilize HRP up to times more than trehalose in the short-term ... More
Paper microzone plates in combination with a noncontact liquid handling robot were demonstrated as tools for studying the stability of enzymes stored on paper. The effect of trehalose and SU-8 epoxy novolac resin (SU-8) on the stability of horseradish peroxidase (HRP) was studied in both a short-term experiment, where the activity of various concentrations of HRP dried on paper were measured after 1 h, and a long-term experiment, where the activity of a single concentration of HRP dried and stored on paper was monitored for 61 days. SU-8 was found to stabilize HRP up to 35 times more than trehalose in the short-term experiment for comparable concentrations of the two reagents, and a 1% SU-8 solution was found to stabilize HRP approximately 2 times more than a 34% trehalose solution in both short- and long-term experiments. The results suggest that SU-8 is a promising candidate for use as an enzyme-stabilizing reagent for paper-based diagnostic devices and that the short-term experiment could be used to quickly evaluate the capacity of various reagents for stabilizing enzymes to identify and characterize new enzyme-stabilizing reagents. Less
DNA has been proposed as a highly desirable medium for storage of digital information The barrier to such use of DNA is the low efficiency and speed as well as the high cost of current synthesis methods In the current state of the art DNA is synthesized using phosphoramidite precursors in organic solvents These chemical synthesis methods result in errors of approximately and take approximately minutes per addition step Furthermore the reagents that are used in this synthesis process are expensive Some of these same reagents also damage DNA a problem that precludes the possibility of synthesizing DNA strands that ... More
DNA has been proposed as a highly desirable medium for storage of digital information. The barrier to such use of DNA is the low efficiency and speed as well as the high cost of current synthesis methods. In the current state of the art, DNA is synthesized using phosphoramidite precursors in organic solvents. These chemical synthesis methods result in errors of approximately 1% and take approximately 10 minutes per addition step. Furthermore, the reagents that are used in this synthesis process are expensive. Some of these same reagents also damage DNA, a problem that precludes the possibility of synthesizing DNA strands that are longer than ˜200 bases, further hampering the efficiency of this chemical process. Despite multiple efforts, a feasible method for synthesis of custom nucleic acid sequences using terminal deoxynucleotidyl transferase (TdT) has not been described before. TdT is currently used in batch reactions for the addition of variable lengths of singular nucleotides or uncontrolled sequence of nucleotide mixtures to the 3′ end of a nucleic acid sequence. A method to control the number and nature of nucleotides that TdT incorporates to generate user-defined nucleic acid sequences is a significant challenge which has not been addressed. There thus remains a need for the development of faster and cheaper enzymatic oligonucleotide synthesis methods than the existing chemical oligonucleotide synthesis methods. Less
MEK is an upstream kinase in MAPK signaling pathways where it phosphorylates p MAPK and JNK in response to mitogenic and cellular stress queues MEK is overexpressed and induces metastasis in advanced prostate cancer lesions However the value of MEK as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK have not been developed Despite a high level of sequence homology in the ATP-binding site most reported MEK inhibitors are selective for MEK and display reduced potency toward other MEKs Here we present the first functional and binding selectivity-profiling platform of the MEK family We ... More
MEK4 is an upstream kinase in MAPK signaling pathways where it phosphorylates p38 MAPK and JNK in response to mitogenic and cellular stress queues. MEK4 is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. Despite a high level of sequence homology in the ATP-binding site, most reported MEK inhibitors are selective for MEK1/2 and display reduced potency toward other MEKs. Here, we present the first functional and binding selectivity-profiling platform of the MEK family. We applied the platform to profile a set of known kinase inhibitors and used the results to develop an in silico approach for small molecule docking against MEK proteins. The docking studies identified molecular features of the ligands and corresponding amino acids in MEK proteins responsible for high affinity binding versus those driving selectivity. WaterLOGSY and saturation transfer difference (STD) NMR spectroscopy techniques were utilized to understand the binding modes of active compounds. Further minor synthetic manipulations provide a proof of concept by showing how information gained through this platform can be utilized to perturb selectivity across the MEK family. This inhibitor-based approach pinpoints key features governing MEK family selectivity and clarifies empirical selectivity profiles for a set of kinase inhibitors. Going forward, the platform provides a rationale for facilitating the development of MEK-selective inhibitors, particularly MEK4 selective inhibitors, and repurposing of kinase inhibitors for probing the structural selectivity of isoforms. Less