Biology subjects

Fu, T.

Publications and source records attributed to Fu, T..

47 records · Page 3Linked to original sources

bioRxiv2020

Umbilical cord blood derived microglia-like cells to model COVID-19 exposure

Microglia, the resident brain immune cells, play a critical role in normal brain development, and are impacted by the intrauterine environment, including maternal immune activation and inflammatory exposures. The COVID-19 pandemic presents a potential developmental immune challenge to the fetal brain, in the setting of maternal SARS-CoV-2 infection with its attendant potential for cytokine production and, in severe cases, cytokine storming. There is currently no biomarker or model for in utero microglial priming and function that might aid in identifying the neonates and children most vulnerable to neurodevelopmental morbidity, as microglia remain inaccessible in fetal life and after birth. This study aimed to generate patient-derived microglial-like cell models unique to each neonate from reprogrammed umbilical cord blood mononuclear cells, adapting and extending a novel methodology previously validated for adult peripheral blood mononuclear cells. We demonstrate that umbilical cord blood mononuclear cells can be used to create microglial-like cell models morphologically and functionally similar to microglia observed in vivo. We illustrate the application of this approach by generating microglia from cells exposed and unexposed to maternal SARS-CoV-2 infection. Our ability to create personalized neonatal models of fetal brain immune programming enables non-invasive insights into fetal brain development and potential childhood neurodevelopmental vulnerabilities for a range of maternal exposures, including COVID-19.

neuroscience↗

bioRxiv2020

Co-evolution of Eukaryotic-like Vps4 and ESCRT-III Subunits in the Asgard Archaea

The emergence of the endomembrane system is a key step in the evolution of cellular complexity during eukaryogenesis. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery is essential and required for the endomembrane system functions in eukaryotic cells. Recently, genes encoding eukaryote-like ESCRT protein components have been identified in the genomes of Asgard archaea, a newly proposed archaeal superphylum that is thought to include the closest extant prokaryotic relatives of eukaryotes. However, structural and functional features of Asgard ESCRT remain uncharacterized. Here we show that Vps4, Vps2/24/46, and Vps20/32/60, the core functional components of the Asgard ESCRT, co-evolved eukaryote-like structural and functional features. Phylogenetic analysis shows that Asgard Vps4, Vps2/24/46, and Vps20/32/60 are closely related to their eukaryotic counterparts. Molecular dynamic simulation and biochemical assays indicate that Asgard Vps4 contains a eukaryote-like Microtubule Interacting and Transport (MIT) domain that binds the distinct type-1 MIT Interacting Motif and type-2 MIT Interacting Motif in Vps2/24/46, and Vps20/32/60, respectively. The Asgard Vps4 partly, but much more efficiently than homologs from other archaea, complements the vps4 null mutant of Saccharomyces cerevisiae, further supporting the functional similarity between the membrane remodeling machineries of Asgard archaea and eukaryotes. Thus, this work provides evidence that the ESCRT complexes from Asgard archaea and eukaryotes are evolutionarily related and functionally similar. Thus, despite the apparent absence of endomembranes in Asgard archaea, the eukaryotic ESCRT seems to have been directly inherited from an Asgard ancestor, to become a key component of the emerging endomembrane system. IMPORTANCEThe discovery of Asgard archaea has changed the exiting ideas on the origins of Eukaryotes. Researchers propose that eukaryotic cells evolve from Asgard archaea. This hypothesis partly stems from the presence of multiple eukaryotic signature proteins in Asgard archaea, including homologues of ESCRT proteins that are essential components of the endomembrane system in eukaryotes. However, structural and functional features of Asgard ESCRT remain unknown. Our study provides evidence that Asgard ESCRT is functionally comparable to the eukaryotic counterparts suggesting that, despite the apparent absence of endomembranes in archaea, eukaryotic ESCRT was inherited from an Asgard archaeal ancestor, alongside the emergence of endomembrane system during eukaryogenesis.

microbiology↗

bioRxiv2020

Navigating the translational roadblock: Towards highly specific and effective all-optical interrogations of neural circuits

Two-photon (2-P) all-optical approaches combine in vivo 2-P calcium imaging and 2-P optogenetic modulations and have the potential to build a framework for network-based therapies, e.g. for rebalancing maladaptive activity patterns in preclinical models of neurological disorders. Here, our goal was to tailor these approaches for this purpose: Firstly, we combined in vivo juxtacellular recordings and GCaMP6f-based 2-P calcium imaging in layer II/III of mouse visual cortex to tune our detection algorithm towards a 100 % specific identification of AP-related calcium transients. False-positive-free detection was achieved at a sensitivity of approximately 73 %. To further increase specificity, secondly, we minimized photostimulation artifacts as a potential source for false-positives by using extended-wavelength-spectrum laser sources for optogenetic stimulation of the excitatory opsin C1V1. We achieved artifact-free all-optical experiments performing photostimulations at 1100 nm or higher and simultaneous calcium imaging at 920 nm in mouse visual cortex in vivo. Thirdly, we determined the spectral range for maximizing efficacy of optogenetic control by performing 2-P photostimulations of individual neurons with wavelengths up to 1300 nm. The rate of evoked transients in GCaMP6f/C1V1-co-expressing cortical neurons peaked already at 1100 nm. By refining spike detection and defining 1100 nm as the optimal wavelength for artifact-free and effective stimulations of C1V1 in GCaMP-based all-optical interrogations, we increased the translational value of these approaches, e.g. for the use in preclinical applications of network-based therapies. One Sentence SummaryWe maximize translational relevance of 2-P all-optical physiology by increasing specificity, minimizing artifacts and optimizing stimulation efficacy.

neuroscience↗

bioRxiv2020

Differential RNA editing between epithelial and mesenchymal tumors impacts mRNA abundance in immune response pathways

Recent studies revealed global shifts in RNA editing, the modification of RNA sequences, across many cancers. Besides a few sites implicated in tumorigenesis or metastasis, most tumor-associated sites, predominantly in noncoding regions, have unknown function. Here, we characterize editing profiles between epithelial (E) and mesenchymal (M) phenotypes in seven cancer types, as epithelial-mesenchymal transition (EMT) is a key paradigm for metastasis. We observe distinct editing patterns between E and M tumors and EMT induction upon loss of ADAR enzymes in cultured cells. E-M differential sites are highly enriched in genes involved in immune and viral processes, some of which regulate mRNA abundance of their respective genes. We identify a novel mechanism in which ILF3 preferentially stabilizes edited transcripts. Among editing-dependent ILF3 targets is the transcript encoding PKR, a crucial player in immune response. Our study demonstrates the broad impact of RNA editing in cancer and relevance of editing to cancer-related immune pathways.

cancer biology↗

bioRxiv2020

LanD-like Flavoprotein-Catalyzed Aminovinyl-Cysteine Formation through Oxidative Decarboxylation and Cyclization of a Peptide at the C-Terminus

Aminovinyl-cysteine residues arise from processing the C-terminal O_SCPLOWLC_SCPLOW-Cys and an internal O_SCPLOWLC_SCPLOW-Ser/O_SCPLOWLC_SCPLOW-Thr or O_SCPLOWLC_SCPLOW-Cys of a peptide. Formation of these nonproteinogenic amino acids, which occur in a macrocyclic ring of diverse ribosomally synthesized lanthipeptides and non-lanthipeptides, remains poorly understood. Here, we report that LanD-like flavoproteins in the biosynthesis of distinct non-lanthipeptides share an unexpected dual activity for aminovinyl-cysteine formation. Each flavoprotein catalyzes oxidative decarboxylation of the C-terminal O_SCPLOWLC_SCPLOW-Cys and couples the resulting enethiol nucleophile with the internal residue to afford a thioether linkage for peptide cyclization. The cyclization step, which largely depends on proximity effect by positioning the enethiol intermediate with a bent conformation at the active site, can be substrate-dependent, proceeding inefficiently through nucleophilic substitution for an unmodified peptide or efficiently through Michael addition for a dehydrated/dethiolated peptide. Uncovering this unusual flavin-dependent paradigm for thioether residue formation advances the understanding in the biosynthesis of aminovinyl-cysteine-containing RiPPs and renews interest in flavoproteins, particularly those involved in non-redox transformations. LanD-like flavoproteins activity, which is flexible in peptide substrate and amenable for evolution by engineering, can be combined with different post-translational modifications for structural diversity, thereby holding promise for peptide macrocyclization/functionalization in drug development by chemoenzymatic or synthetic biology approaches.

biochemistry↗

bioRxiv2019

Neuronal activity triggers uptake of hematopoietic extracellular vesicles in vivo

Communication with the hematopoietic system is a vital component of regulating brain function in health and disease. Traditionally, the major routes considered for this neuroimmune communication are either by individual molecules such as cytokines carried by blood, by neural transmission, or in more severe pathologies, by entry of peripheral immune cells into the brain. In addition, functional mRNA from peripheral blood can be directly transferred to neurons via extracellular vesicles (EVs) but the parameters that determine their uptake are unknown. We show that transfer of EVs from blood is triggered by neuronal activity in vivo. Importantly, this transfer occurs not only in pathological stimulation but also by neuronal activation caused by the physiological stimulus of novel object recognition. This discovery suggests a continuous role of EVs under pathological conditions as well as during routine cognitive tasks in the healthy brain.

neuroscience↗

bioRxiv2018

A low-cost DIY device for high resolution, continuous measurement of microbial growth dynamics

High-resolution data on microbial growth dynamics allow characterisation of microbial physiology, as well as optimisation of genetic alterations thereof. Such data are routinely collected using bench-top spectrophotometers or so-called plate readers. These equipments present several drawbacks: (i) measurements from different devices cannot be compared directly, (ii) proprietary nature of devices makes it difficult for standardisation methods to be developed across devices, and (iii) high costs limit access to devices, which can become a bottleneck for researchers, especially for those working with anaerobic organisms or at higher containment level laboratories. These limitations could be lifted, and data reproducibility improved, if the scientific community could adopt standardised, low-cost and open-source devices that can be built in-house. Here, we present such a device, MicrobeMeter, which is a do-it-yourself (DIY), simple, yet robust photometer with continuous data-logging capability. It is built using 3D-printing and open-source Arduino platform, combined with purpose-built electronic circuits. We show that MicrobeMeter displays linear relation between culture density and turbidity measurement for microbes from different phylogenetic domains. In addition, culture density estimated from MicrobeMeter measurements produced less variance compared against three commercial bench-top spectrophotometers, indicating that its measurements are less affected by the differences in cell types. We show the utility of MicrobeMeter, as a programmable wireless continuous measurement device, by collecting long-term growth dynamics up to 458 hours from aerobic and anaerobic cultures. We provide a full open-source description of MicrobeMeter and its implementation for faster adaptation and future development by the scientific community. The blueprints of the device, as well as ready-to-assemble kit versions are also made available through www.humanetechnologies.co.uk.

microbiology↗

bioRxiv2018

Allele-specific binding of RNA-binding proteins reveals functional genetic variants in the RNA

Allele-specific protein-RNA binding is an essential aspect that may reveal functional genetic variants influencing RNA processing and gene expression phenotypes. Recently, genome-wide detection of in vivo binding sites of RNA binding proteins (RBPs) is greatly facilitated by the enhanced UV crosslinking and immunoprecipitation (eCLIP) protocol. Hundreds of eCLIP-Seq data sets were generated from HepG2 and K562 cells during the ENCODE3 phase. These data afford a valuable opportunity to examine allele-specific binding (ASB) of RBPs. To this end, we developed a new computational algorithm, called BEAPR (Binding Estimation of Allele-specific Protein-RNA interaction). In identifying statistically significant ASB sites, BEAPR takes into account UV cross-linking induced sequence propensity and technical variations between replicated experiments. Using simulated data and actual eCLIP-Seq data, we show that BEAPR largely outperforms often-used methods Chi-Squared test and Fishers Exact test. Importantly, BEAPR overcomes the inherent over-dispersion problem of the other methods. Complemented by experimental validations, we demonstrate that ASB events are significantly associated with genetic regulation of splicing and mRNA abundance, supporting the usage of this method to pinpoint functional genetic variants in post-transcriptional gene regulation. Many variants with ASB patterns of RBPs were found as genetic variants with cancer or other disease relevance. About 38% of ASB variants were in linkage disequilibrium with single nucleotide polymorphisms from genome-wide association studies. Overall, our results suggest that BEAPR is an effective method to reveal ASB patterns in eCLIP and can inform functional interpretation of disease-related genetic variants.

bioinformatics↗

bioRxiv2018

Condensin-dependent chromatin condensation represses transcription globally during quiescence

Quiescence is a stress-resistant state in which cells reversibly exit the mitotic cell cycle and suspend most cellular processes. Quiescence is essential for stem cell maintenance and its misregulation is implicated in tumor formation. One of the conserved hallmarks of quiescent cells, from Saccharomyces cerevisiae to humans, is highly condensed chromatin. Here, we use Micro-C XL to map chromatin contacts at single-nucleosome resolution genome-wide to elucidate mechanisms and functions of condensed chromatin in quiescent S. cerevisiae cells. We describe previously uncharacterized chromatin domains on the order of 10-60 kilobases that in quiescent cells are formed by condensin-mediated chromatin loops. Conditional depletion of condensin prevents chromatin condensation during quiescence entry and leads to widespread transcriptional de-repression. We further demonstrate that condensin-dependent chromatin compaction is conserved in quiescent human fibroblasts. We propose that condensin-dependent condensation of chromatin represses transcription throughout the quiescent cell genome.

molecular biology↗

bioRxiv2018

Mechanistic insight into the interactions of NAP1 with NDP52 and TAX1BP1 for the recruitment of TBK1

NDP52 and TAX1BP1, two SKICH domain-containing autophagy recetpors, play crucial roles in selective autophagy. The autophagic functions of NDP52 and TAX1BP1 are regulated by TBK1, which can indirectly associate with them through the adaptor protein NAP1. However, the molecular mechanism governing the interactions of NAP1 with NDP52 and TAX1BP1 as well as the effects induced by TBK1-mediated phosphorylation of NDP52 and TAX1BP1 remain elusive. Here, we reported the first atomic structures of the SKICH regions of NDP52 and TAX1BP1 in complex with NAP1, which not only uncover the mechanismtic basis underpinning the specific interactions of NAP1 with NDP52 and TAX1BP1, but also reveal the first binding mode of a SKICH domain. Moreover, we demonstrated that the phosphorylation of TAX1BP1 SKICH mediated by TBK1 may regulate the interaction between TAX1BP1 and NAP1. In all, our findings provide mechanistic insights into the NAP1-mediated recruitments of TBK1 to NDP52 and TAX1BP1, and are valuable for further understanding the functions of these proteins in selective autophagy.

biophysics↗

bioRxiv2017

Increased microglial synapse elimination in patient-specific models of schizophrenia

Schizophrenia patients display decreased synaptic density in postmortem studies, suggesting aberrant microglial synapse elimination during neurodevelopment. Here, we use cellular reprogramming to create patient-specific in vitro models of microglia-mediated synapse engulfment that demonstrate increased synapse elimination in schizophrenia-derived models compared to healthy controls. We show that excessive synaptic pruning in schizophrenia reflects abnormalities in microglia-like cells as well as synaptic structures. Further, we find that schizophrenia risk-associated variants within the complement component 4 locus contribute to the increased uptake in schizophrenia models. Finally, we demonstrate that the antibiotic minocycline reduces microglia-mediated synapse uptake and show that minocycline treatment for acne is associated with a reduction in incident schizophrenia risk compared to other treatments in a cohort of more than 9,000 young adults drawn from health records. Specific pharmacological interventions targeting excessive pruning merit further study for their capacity to delay or prevent the onset of schizophrenia in high-risk individuals.

neuroscience↗