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Chen, A.

Publications and source records attributed to Chen, A..

At least 217 records · Page 12Linked to original sources

Multiplex base editing to convert TAG into TAA codons in the human genome

Large-scale recoding has been shown to enable novel amino acids, biocontainment and viral resistance in bacteria only so far. Here we extend this to human cells demonstrating exceptional base editing to convert TAG to TAA for 33 essential genes via a single transfection, and examine base-editing genome-wide (observing ~ 40 C-to-T off-target events in essential gene exons). We also introduce GRIT, a computational tool for recoding. This demonstrates the feasibility of recoding, and multiplex editing in mammalian cells.

synthetic biology↗

Rapid cross-sensory adaptation of self-motion perception

Perceptual adaptation is often studied within a single sense. However, our experience of the world is naturally multisensory. Here, we investigated cross-sensory (visual-vestibular) adaptation of self-motion perception. It was previously found that relatively long visual self-motion stimuli ({gtrsim} 15s) are required to adapt subsequent vestibular perception, and that shorter duration stimuli do not elicit cross-sensory (visual{leftrightarrow}vestibular) adaptation. However, it is not known whether several discrete short-duration stimuli may lead to cross-sensory adaptation (even when their sum, if presented together, would be too short to elicit cross-sensory adaptation). This would suggest that the brain monitors and adapts to supra-modal statistics of events in the environment. Here we investigated whether cross-sensory (visual{leftrightarrow}vestibular) adaptation occurs after experiencing several short (1s) self-motion stimuli. Forty-five participants discriminated the headings of a series of self-motion stimuli. To expose adaptation effects, the trials were grouped in 140 batches, each comprising three prior trials, with headings biased to the right or left, followed by a single unbiased test trial. Right, and left-biased batches were interleaved pseudo-randomly. We found significant adaptation in both cross-sensory conditions (visual prior and vestibular test trials, and vice versa), as well as both unisensory conditions (when prior and test trials were of the same modality - either visual or vestibular). Fitting the data with a logistic regression model revealed that adaptation was elicited by the prior stimuli (not prior choices). These results suggest that the brain monitors supra-modal statistics of events in the environment, even for short-duration stimuli, leading to functional (supra-modal) adaptation of perception.

neuroscience↗

Mediobasal hypothalamic FKBP51 acts as a molecular switch linking autophagy to whole-body metabolism

The mediobasal hypothalamus (MBH) is the central region in the physiological response to metabolic stress. The FK506-binding protein 51 (FKBP51) is a major modulator of the stress response and has recently emerged as a scaffolder regulating metabolic and autophagy pathways. However, the detailed protein-protein interactions linking FKBP51 to autophagy upon metabolic challenges remain elusive. We performed mass spectrometry-based metabolomics of FKBP51 knockout (KO) cells revealing an increased amino acid and polyamine metabolism. We identified FKBP51 as a central nexus for the recruitment of the LKB1/AMPK complex to WIPI4 and TSC2 to WIPI3, thereby regulating the balance between autophagy and mTOR signaling in response to metabolic challenges. Furthermore, we demonstrated that MBH FKBP51 deletion strongly induces obesity, while its overexpression protects against high-fat diet (HFD) induced obesity. Our study provides an important novel regulatory function of MBH FKBP51 within the stress-adapted autophagy response to metabolic challenges.

neuroscience↗

Stem cell quiescence requires PRC2/PRC1-mediated mitochondrial checkpoint

Both normal and tumorous stem cells can arrest cell division, avoid apoptosis, and then regenerate lost daughter cells following acute genotoxic insult. This protective, reversible proliferative arrest, known as "quiescence," is still poorly understood. Here, we show that mTOR-regulated mitophagy is required for radiation insult-induced quiescence in Drosophila germline stem cells (GSCs). In GSCs, depletion of mito-fission (Drp1) or mitophagy (Pink1 and Parkin) eliminates entry into quiescence, while depletion of mitochondrial biogenesis (PGC1) or fusion (Mfn2) eliminates exit from quiescence. We also find that mitophagy-dependent quiescence is under epigenetic control; knockdown of Jarid2 (PRC2) or Pc or Sce (PRC1) stabilizes the mitochondria and locks GSCs out of quiescence, while knockdown of PRC2-specific demethylase, Utx, prevents re-accumulation of the mitochondria and locks GSCs in quiescence. These data suggest that mitochondrial number coordinates reversible quiescence. We further identify that the mechanism of quiescence in both GSCs and human induced pluripotent stem cells (iPSCs) relies on mitophagy to deplete the mitochondrial pool of CycE and limit cell cycle progression. This alternative method of G1/S regulation may present new opportunities for therapeutic purposes.

biochemistry↗

Genome-wide CRISPR activation screen identifies novel receptors for SARS-CoV-2 entry

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been endangering worldwide public health and economy. SARS-CoV-2 infects a variety of tissues where the known receptor ACE2 is low or almost absent, suggesting the existence of alternative pathways for virus entry. Here, we performed a genome-wide barcoded-CRISPRa screen to identify novel host factors that enable SARS-CoV-2 infection. In addition to known host proteins, i.e. ACE2, TMPRSS2 and NRP1, we identified multiple host components, among which LDLRAD3, TMEM30A and CLEC4G were confirmed as functional receptors for SARS-CoV-2. All these membrane proteins bind directly to spikes N-terminal domain (NTD). Their essential and physiological roles have all been confirmed in either neuron or liver cells. In particular, LDLRAD3 and CLEC4G mediate SARS-CoV-2 entry and infection in a fashion independent of ACE2. The identification of the novel receptors and entry mechanisms could advance our understanding of the multiorgan tropism of SARS-CoV-2, and may shed light on the development of the therapeutic countermeasures against COVID-19.

microbiology↗

SARS-CoV-2 immune evasion by variant B.1.427/B.1.429

SARS-CoV-2 entry is mediated by the spike (S) glycoprotein which contains the receptor-binding domain (RBD) and the N-terminal domain (NTD) as the two main targets of neutralizing antibodies (Abs). A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429) was originally detected in California and is currently spreading throughout the US and 29 additional countries. It is unclear whether antibody responses to SARS-CoV-2 infection or to the prototypic Wuhan-1 isolate-based vaccines will be impacted by the three B.1.427/B.1.429 S mutations: S13I, W152C and L452R. Here, we assessed neutralizing Ab responses following natural infection or mRNA vaccination using pseudoviruses expressing the wildtype or the B.1.427/B.1.429 S protein. Plasma from vaccinated or convalescent individuals exhibited neutralizing titers, which were reduced 3-6 fold against the B.1.427/B.1.429 variant relative to wildtype pseudoviruses. The RBD L452R mutation reduced or abolished neutralizing activity of 14 out of 35 RBD-specific monoclonal antibodies (mAbs), including three clinical-stage mAbs. Furthermore, we observed a complete loss of B.1.427/B.1.429 neutralization for a panel of mAbs targeting the N-terminal domain due to a large structural rearrangement of the NTD antigenic supersite involving an S13I-mediated shift of the signal peptide cleavage site. These data warrant closer monitoring of signal peptide variants and their involvement in immune evasion and show that Abs directed to the NTD impose a selection pressure driving SARS-CoV-2 viral evolution through conventional and unconventional escape mechanisms.

immunology↗

High-resolution single-molecule long-fragment rRNA gene amplicon sequencing for uncultured bacterial and fungal communities

Although several large-scale environmental microbial projects have been initiated in the past two decades, understanding of the role of complex microbiotas is still constrained by problems of detecting and identifying unknown microorganisms1-6.Currently, hypervariable regions of rRNA genes as well as internal transcribed spacer regions are broadly used to identify bacteria and fungi within complex communities7,8, but taxonomic and phylogenetic resolution is hampered by insufficient sequencing length9-11. Direct sequencing of full length rRNA genes is currently limited by read length using second generation sequencing or sacrificed quality and throughput by using single molecule sequencing. We developed a novel method to sequence and assemble nearly full length rRNA genes using second generation sequencing.Benchmarking was performed on mock bacterial and fungal communities as well as two forest soil samples. The majority of rRNA gene sequences of all species in the mock community samples were successfully recovered with identities above 99.5% compared to the reference sequences. For soil samples we obtained exquisite coverage with identification of a large number of putative new species, as well as high abundance correlation between replicates. This approach provides a cost-effective method for obtaining extensive and accurate information on complex environmental microbial communities.

microbiology↗

Structure and mechanistic analyses of the gating mechanism of elongating ketosynthases

Ketosynthases (KSs) catalyze carbon-carbon bond forming reactions in fatty acid synthases (FASs) and polyketide synthases (PKSs). KSs utilize a two-step ping pong kinetic mechanism to carry out an overall decarboxylative thio-Claisen condensation that can be separated into the transacylation and condensation reactions. In both steps, an acyl carrier protein (ACP) delivers thioester tethered substrates to the active sites of KSs. Therefore, protein-protein interactions (PPIs) and KS-mediated substrate recognition events are required for catalysis. Recently, crystal structures of Escherichia coli elongating type II FAS KSs, FabF and FabB, in complex with E. coli ACP, AcpP, revealed distinct conformational states of two active site KS loops. These loops were proposed to operate via a gating mechanism to coordinate substrate recognition and delivery followed by catalysis. Here we interrogate this proposed gating mechanism by solving two additional high-resolution structures of substrate engaged AcpP-FabF complexes, one of which provides the missing AcpP-FabF gate-closed conformation. Clearly defined interactions of one of these active site loops with AcpP are present in both the open and closed conformations, suggesting AcpP binding triggers or stabilizes gating transitions, further implicating PPIs in carrier protein-dependent catalysis. We functionally demonstrate the importance of gating in the overall KS condensation reaction and provide experimental evidence for its role in the transacylation reaction. Furthermore, we evaluate the catalytic importance of these loops using alanine scanning mutagenesis and also investigate chimeric FabF constructs carrying elements found in type I PKS KS domains. These findings broaden our understanding of the KS mechanism which advances future engineering efforts in both FASs and evolutionarily related PKSs.

biochemistry↗

Large field of view-spatially resolved transcriptomics at nanoscale resolution

Spatially resolved transcriptomic technologies are promising tools to study cell fate decisions in a physical microenvironment, which is fundamental for enhancing our knowledge of mammalian development. However, the imbalance between resolution, transcript capture and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation mammalian embryos. Here, we combined DNA nanoball (DNB) patterned arrays and tissue RNA capture to create SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq). This approach allows transcriptomic profiling of large histological sections with high resolution and sensitivity. We have applied Stereo-seq to study the kinetics and directionality of transcriptional variation in a time course of mouse organogenesis. We used this information to gain insight into the molecular basis of regional specification, neuronal migration and differentiation in the developing brain. Furthermore, we mapped the expression of a panel of developmental disease-related loci on our global transcriptomic maps to define the spatiotemporal windows of tissue vulnerability. Our panoramic atlas constitutes an essential resource to investigate longstanding questions concerning normal and abnormal mammalian development.

cell biology↗

N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2

SARS-CoV-2 entry into host cells is orchestrated by the spike (S) glycoprotein that contains an immunodominant receptor-binding domain (RBD) targeted by the largest fraction of neutralizing antibodies (Abs) in COVID-19 patient plasma. Little is known about neutralizing Abs binding to epitopes outside the RBD and their contribution to protection. Here, we describe 41 human monoclonal Abs (mAbs) derived from memory B cells, which recognize the SARS-CoV-2 S N-terminal domain (NTD) and show that a subset of them neutralize SARS-CoV-2 ultrapotently. We define an antigenic map of the SARS-CoV-2 NTD and identify a supersite recognized by all known NTD-specific neutralizing mAbs. These mAbs inhibit cell-to-cell fusion, activate effector functions, and protect Syrian hamsters from SARS-CoV-2 challenge. SARS-CoV-2 variants, including the 501Y.V2 and B.1.1.7 lineages, harbor frequent mutations localized in the NTD supersite suggesting ongoing selective pressure and the importance of NTD-specific neutralizing mAbs to protective immunity.

immunology↗

Stem cell derived astrocytes with POLG mutations and mitochondrial dysfunction including abnormal NAD+ metabolism is toxic for neurons

The inability to reliably replicate mitochondrial DNA (mtDNA) by mitochondrial DNA polymerase gamma (POLG) leads to a subset of common mitochondrial diseases associated with neuronal death and depletion of neuronal mtDNA. Defining disease mechanisms remains difficult due to the limited access to human tissue. Astrocytes are highly abundant in the brain, playing a crucial role in the support and modulation of neuronal function. Astrocytes also respond to insults affecting the brain. Following damage to the center neural system, which can be hypoxia, inflammation or neurodegeneration, astrocytes become activated and lose their supportive role and gain toxic functions that induce rapid death of neurons and oligodendrocytes. The role of astrocyte reactivation and the consequences this has for neuronal homeostasis in mitochondrial diseases has not been explored. Here, using patient cells carrying POLG mutations, we generated iPSCs and then differentiated into astrocytes. We demonstrated that POLG-astrocytes exhibited both mitochondrial dysfunctions, including loss of mitochondrial membrane potential, energy failure, complex I and IV defects, disturbed NAD+/NADH metabolism, and mtDNA depletion. Further, POLG derived astrocytes presented an A1-like reactive phenotype with increased proliferation, invasion, upregulation of pathways involved in response to stimulus, immune system process, cell proliferation and cell killing. Under direct and indirect co-culture with neurons, POLG-astrocytes exhibited a toxic effect leading to the death of neurons. Our findings demonstrate that mitochondrial dysfunction caused by POLG mutations leads not only to intrinsic defects in energy metabolism affecting both neurons and astrocytes, but also to neurotoxic damage driven by astrocytes. Our studies provide a robust astroglia-neuronal interaction model for future investigation of mitochondrial involvement in neurogenesis and neurodegenerative diseases. Highlights{blacksquare} Patient-specific astrocytes harbouring a POLG mutation showed lower mitochondrial membrane potential and mtDNA depletion. {blacksquare}POLG-astrocytes generated elevated L-lactate as the end glycolytic product. {blacksquare}Patient-specific astrocytes with POLG mutations exhibited mitochondrial respiratory chain disruption accompanied with abnormal UCP2/SirT1/SirT3 mediated NAD+ metabolism. {blacksquare}Suppressed complex I and IV-driven respiration contributed to the pathological mechanisms in POLG-related disease. {blacksquare}POLG-astrocytes exhibit A1-reactive phenotype and neurotoxic potential. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/423652v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@18995fdorg.highwire.dtl.DTLVardef@1d615d4org.highwire.dtl.DTLVardef@334aa8org.highwire.dtl.DTLVardef@16a84ba_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

In vitro analysis of colistin and ciprofloxacin antagonism of Pseudomonas aeruginosa phage PEV2 infection activities

Phage therapy is a century-old technique employing viruses (phages) to treat bacterial infections. In the clinic, phage therapy often is used in combination with antibiotics. Antibiotics, however, interfere with critical bacterial activities, such as DNA and protein synthesis, which also are required for phage infection processes. Resulting antagonistic impacts of antibiotics on phages nevertheless are not commonly determined in association with phage therapy studies using standard, planktonic approaches. Here we assess the antagonistic impact of two antibiotics, colistin and ciprofloxacin, on the bactericidal, bacteriolytic, and new virion production activities of Pseudomonas aeruginosa podovirus PEV2, using a broth culture, optical density-based lysis profile assay. Though phage-antibiotic combinations were more potent in reducing cell viability than phages or antibiotics alone, colistin substantially interfered with phage PEV2 bacteriolytic and virion-production activities at minimum inhibitory concentration (MIC). Ciprofloxacin, by contrast, had no such impact at 1x MIC or 3x MIC. At higher but still clinically relevant concentrations (9x MIC) burst sizes were still significant (~30 phages/infected bacterium). We corroborated these lysis profile results by more traditional measurements (colony forming units, plaque forming units, one-step growth experiments) and two other P. aeruginosa phages. To our knowledge this is the first study in which detailed antibiotic impact on P. aeruginosa phage infection activities has been determined under conditions similar to those used to determine antibiotic MICs and could point especially to ciprofloxacin as a minimally antagonistic phage therapy co-treatment of P. aeruginosa infections.

microbiology↗

Hypothalamic glucocorticoid receptor in CRF neurons is essential for HPA axis habituation to repeated stressor

Short abstractHabituation of the hypothalamic-pituitary-adrenal (HPA) axis to repeated homotypic stressors is crucial for the organisms well-being. Many physiological and psychological disorders are associated with HPA axis dysfunction. Here, we show that glucocorticoid receptors in CRF neurons of the hypothalamic paraventricular nucleus are essential for HPA habituation. By increasing inhibitory tone onto CRF neurons, glucocorticoid receptors led to essential cellular modulation and hypothalamic-pituitary-adrenal axis activation dampening, when re-exposed to the same stressor.

neuroscience↗

Aberrant structural synaptic dynamics precede disorganization of temporal and spatial coding in the hippocampus upon repeated stress exposure

Stress exposure strongly contributes to the pathophysiology of psychiatric diseases such as depression, schizophrenia, anxiety, and post-traumatic stress disorder. It also affects both function and structure of hippocampal CA1, leading to episodic memory impairment. Here, we used deep-brain optical imaging to elucidate the effects of stress on CA1 pyramidal neuron structural connectivity and activity. We tracked dynamics of dendritic spines during repeated stress and found decrease in spinogenesis followed by decrease in spine stability. In contrast to repeated stress, acute stress led to stabilization of the spines born in temporal proximity to the stressful event. To investigate the link between structural plasticity and activity patterns upon repeated stress, we studied the activity of thousands of CA1 pyramidal neurons in freely-moving. We found an increase in activity and loss of temporal organization followed by a disruption in temporal and spatial coding. Our data suggest that stress-induced sustained increase in activity leads to loss of structural connectivity and subsequent temporal and spatial coding impairments.

neuroscience↗

THE TRPV CHANNEL OSM-9 MEDIATES OLFACTORY LONG- TERM MEMORY CONSOLIDATION INDEPENDENT OF SLEEP

Memory, defined as an alteration in behavior towards a stimulus that follows as a consequence of experience, arises when a sensory stimulus is encountered at the same time that the animal experiences a negative or positive internal state. How this coincident detection of external and internal stimuli stably alters responses to the external stimulus is still not fully understood, especially in the context of an intact animal. One barrier to understanding how an intact biological circuit changes is knowing what molecular processes are required to establish and maintain the memory. The optically accessible and compact nervous system of C. elegans provides a unique opportunity to examine these processes. C. elegans can remember an odor such as butanone when it is paired with a single negative experience and the transient receptor potential (TRP) OSM-9/TRPV5/TRPV6 channel is known to be required for this memory. The multiple gating mechanisms of TRPV channels give them the potential to be the coincidence detectors required to integrate internal state and external stimuli. Here, we report that this TRPV channel is also required for acquisition and possibly consolidation of sleep-dependent, long-term memory of butanone. We find that in the anterior ganglion, endogenous GFP-tagged OSM-9 is expressed in the paired AWA olfactory neurons, the ASH nociceptive neuron pair, the mechanosensory OLQ tetrad, and the paired ADF and ADL sensory neurons. In these cells, OSM-9 protein is concentrated in the sensory endings, dendrites, and cell bodies, but excluded from the neurites in the nerve ring. In the tail, OSM-9 is expressed in the nociceptive phasmid neurons PHA and PHB, possibly PQR as well as PVP. In the midbody, it is possibly expressed in the mechanosensitive PVD neuron. It is notably absent from the AWC pair that are required for butanone attraction. Chronic loss of OSM-9 in a subset of ciliated neurons that do not include AWA interferes with consolidation but not learning. Because OSM-9 is expressed and required in sensory neurons that are not needed for butanone chemosensory behavior, two interpretations are possible. The first, is that OSM-9 loss leads to gain of function or neomorphic behavior of these cells that are extrinsic to the primary sensory circuit and their new activity interferes with acquisition and consolidation of memory. The second is that loss of OSM-9 leads to a loss of function phenotype in which the wild type function of these cells is diminished and this function is required for memory consolidation. Author summaryHow organisms learn from their environment and keep these memories for the long term ensures their survival. There is much known about the regions of the brain and the various proteins that are essential for memory, yet the exact molecular mechanisms and dynamics required are not known. We aimed to understand the genetics that underlie memory formation. We tested a gene that encodes a transient potential receptor channel vanilloid channel, which is similar to the channels we have that sense spicy foods and other harmful cues. Our studies have shown that this gene is required for the animal to be able to acquire and perhaps consolidate olfactory memory. This protein is not expressed in the sensory neurons that respond to the odor that is memorized or in other downstream interneurons in the odor-sensation circuit, but it is expressed in a distinct set of sensory neurons. This indicates that long-term memory involves wild type behavior of a wider array of sensory neurons than is required for the primary sensation. These channels are also implicated in neurological disorders where memory is affected, including Alzheimers disease. Understanding how memory formation is affected by cells outside the memory circuit might provide testable hypothesis about what goes awry in Alzheimers disease.

neuroscience↗

An adaptive optics module for deep tissue multiphoton imaging in vivo

Understanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at unprecedented depths in vivo.

bioengineering↗

A robust and versatile nanobody platform for drug delivery

Therapeutic and diagnostic efficacies of numerous small biomolecules and chemical compounds are hampered by the short half-lives. Here we report the development of a repertoire of diverse, high-affinity albumin-nanobodies (NbHSA) to facilitate drug delivery. By integrating biophysics, and hybrid structural biology, we have systematically characterized the NbHSA for albumin binding, mapped the epitopes, and resolved the architecture of a tetrameric Nb-albumin complex. We employed quantitative proteomics for accurate, multiplex Nb pharmacokinetic analysis. Using a humanized albumin mouse model, we found that the NbHSA has outstanding pharmacokinetics; the most stable NbHSA has a 771-fold T1/2 improvement compared with a control Nb. Interestingly, the pharmacokinetics of NbHSA is related to their biophysical and structural properties. To demonstrate the utility of NbHSA, we developed a highly stable NbHSA-hIL-2 cytokine conjugate "Duraleukin" and confirmed its improved anticancer properties than hIL-2 alone. We envision that this high-quality Nb resource will advance research into novel biotherapeutics.

bioengineering↗

Dynamic encoding of saccade sequences in primate frontal eye field

AO_SCPLOWBSTRACTC_SCPLOWFrontal eye field (FEF) is a key part of oculomotor system, with dominant responses to the direction of single saccades. However, whether and how FEF contributes to sequential saccades remain largely unknown. Here by training rhesus monkeys to perform sequential saccades and recording the neuronal activities in FEF, we found that the sequence-related activities are clearly represented in FEF, and many neurons selectivity to saccade direction undergoes dynamic changes during sequential task. In addition, the sequence-related activities are context-dependent, with different firing activities during memory- versus visually-guided sequence. Supra-threshold microstimulation in FEF evokes saccade without altering the overall sequence structure. Pharmacological inactivation of FEF severely impaired the monkeys performance of sequential saccades, with different effects on the same actions at different positions within the sequence. These results reveal the context-dependent dynamic encoding of saccade direction in FEF, and underscore a critical role of FEF in planning and execution of sequential saccades. IO_SCPLOWNC_SCPLOW BO_SCPLOWRIEFC_SCPLOWJia, Puyang et al. employed in vivo recording to reveal the dynamic encoding of sequential saccades in primate frontal eye field (FEF), then used electric microstimulation and reversible inactivation to demonstrate the causal role of FEF in controlling saccade sequences. HO_SCPLOWIGHLIGHTSC_SCPLOWO_LIFEF neurons respond differently during sequential vs. single saccades C_LIO_LISequence-related FEF activity is context-dependent C_LIO_LIFEF microstimulation induced saccade without altering sequence structure C_LIO_LIFEF inactivation severely impaired the performance of sequential saccades C_LI

neuroscience↗