Biology subjects

Xu, J.

Publications and source records attributed to Xu, J..

At least 469 records · Page 26Linked to original sources

bioRxiv2023

Mice can learn a cognitive map based on a stable start location and self-motion signals

Animals navigate by learning the spatial layout of their environment. We investigated spatial learning of mice in an open maze where food was hidden in one of a hundred holes. Mice leaving from a stable entrance learned to efficiently navigate to the food without the need for landmarks. We developed a quantitative framework to reveal how the mice estimate the food location based on analyses of trajectories and active hole checks. After learning, the computed "target estimation vector" (TEV) closely approximated the mices route and its hole check distribution. The TEV required learning both the direction and distance of the start to food vector, and our data suggests that different learning dynamics underlie these estimates. We propose that the TEV can be precisely connected to the properties of hippocampal place cells. Finally, we provide the first demonstration that, after learning the location of two food sites, the mice took a shortcut between the sites, demonstrating that they had generated a cognitive map.

neuroscience↗

bioRxiv2023

The unfolded protein response of the endoplasmic reticulum protects Caenorhabditis elegans against DNA damage caused by stalled replication forks

All animals must maintain genome and proteome integrity, especially when experiencing endogenous or exogenous stress. To cope, organisms have evolved sophisticated and conserved response systems: unfolded protein responses (UPRs) ensure proteostasis while DNA damage responses (DDRs) maintains genome integrity. Emerging evidence suggests that UPRs and DDRs crosstalk, but this remains poorly understood. Here, we demonstrate that depletion of the DNA primases pri-1 or pri-2, which synthesize RNA primers at replication forks and whose inactivation causes DNA damage, activates the UPR of the endoplasmic reticulum (UPR-ER) in Caenorhabditis elegans, with especially strong activation in the germline. We observed activation of both the inositol-requiring-enzyme 1 (ire-1) and the protein kinase RNA-like ER kinase (pek-1) branches of the UPR-ER. Interestingly, activation of the UPR-ER output gene heat shock protein 4 (hsp-4) was partially independent of its canonical activators, ire-1 and X-box binding protein (xbp-1), and instead required the third branch of the UPR-ER, activating transcription factor 6 (atf-6), suggesting functional redundancy. We further found that primase depletion specifically induces the UPR-ER, but not the distinct cytosolic or mitochondrial UPRs, suggesting that primase inactivation causes compartment-specific rather than global stress. Functionally, loss of ire-1 or pek-1 sensitized animals to replication stress caused by hydroxyurea. Finally, transcriptome analysis of pri-1 embryos revealed several deregulated processes that could cause UPR-ER activation, including protein glycosylation, calcium signaling, and fatty acid desaturation. Together, our data show that the UPR-ER, but not other UPRs, responds to replication fork stress and that the UPR-ER is required to alleviate this stress.

genetics↗

bioRxiv2023

Reconstruction of TrkB complex assemblies and localizing an-tidepressant targets using Artificial Intelligence

Since Major Depressive Disorder (MDD) represents a neurological pathology caused by inter-synaptic messaging errors, membrane receptors, the source of signal cascades, constitute appealing drugs targets. G protein-coupled receptors (GPCRs) and ion channel receptors chelated antidepressants (ADs) high-resolution architectures were reported to realize receptors physical mechanism and design prototype compounds with minimal side effects. Tyrosine kinase receptor 2 (TrkB), a receptor that directly modulates synaptic plasticity, has a finite three-dimensional chart due to its high molecular mass and intrinsically disordered regions (IDRs). Leveraging breakthroughs in deep learning, the meticulous architecture of TrkB was projected employing Alphfold 2 (AF2). Furthermore, the Alphafold Multimer algorithm (AF-M) models the coupling of intra- and extra-membrane topologies to chaperones: mBDNF, SHP2, Etc. Conjugating firmly dimeric transmembrane helix with novel compounds like 2R,6R-hydroxynorketamine (2R,6R-HNK) expands scopes of drug screening to encompass all coding sequences throughout genomes. The operational implementation of TrkB kinase-SHP2, PLC{gamma}1, and SHC1 ensembles has paved the path for machine learning in which it can forecast structural transitions in the self-assembly and self-dissociation of molecules during trillions of cellular mechanisms. In silicon, the cornerstone of the alteration will be artificial intelligence (AI), empowering signal networks to operate at the atomic level and picosecond timescales.

bioinformatics↗

bioRxiv2023

Positive coping supports children's emotional wellness: Behavioral evidence and neuroendocrine mechanisms

A positive coping style is recognized as a stable disposition to foster emotional wellness and resilience, enabling an adaptive process of assessing and dealing with environmental challenges. Such an adaptive process is believed to rely on a nuanced interplay of the hippocampal system and the primary stress hormone cortisol activity. As a hallmark of diurnal cortisol rhythm, cortisol awakening response (CAR) is sensitive to upcoming stress and subserves the preparation of the hippocampal system for rapid behavioral adaption. Yet, little is known about how the hippocampal system and CAR contribute to the merit of positive coping on emotional wellness. By two studies, we investigate the effects of positive coping on childrens emotional wellness and CAR, as well as longitudinal changes in hippocampal-neocortical functional systems involved in emotional processing. Behaviorally, positive coping predicted better emotional regulation ability, but lower anxiety and lower response caution in emotional decision-making. At the endocrine and neurocognitive level, positive coping was associated with greater CAR, which further predicted higher connectivity of the hippocampus with ventrolateral prefrontal cortex (vlPFC) and stimulus-sensitive neocortex one year later. Furthermore, CAR mediated an indirect association between positive coping and longitudinal increases in hippocampal-neocortical connectivity. Positive coping and CAR together could account for the maturity of vlPFC through longitudinal changes in hippocampal-neocortical connectivity. Overall, our findings suggest a cognitive-neuroendocrinal framework in which positive coping shapes hippocampal-neocortical maturation via stress hormone response to support emotional wellness. SignificanceThe role of the hippocampal system in regulating stress response is well recognized, but its contribution to emotional well-being is not yet understood. Here we show that the protective effects of positive coping on emotional well-being are contingent on two factors: the cortisol awakening response (CAR), which is sensitive to upcoming stress, and hippocampal development. We found that positive coping practices promoted emotional wellness, enhanced emotional decision-making and increased CAR in young children. Longitudinal neuroimaging analysis revealed that positive coping-related CAR predicted greater hippocampal connectivity with stimulus-sensitive neocortex one year later. Importantly, CAR acted as a mediator of the promotive influence of positive coping on the longitudinal development of hippocampal-neocortical connectivity, which contributed to the maturity of prefrontal control systems. Our findings emphasize the importance of hippocampal-neocortical development in resilient coping and emotional wellness.

neuroscience↗

bioRxiv2023

Cerebellar Morphological Differences in Bipolar Disorder Type I

BackgroundThe neural underpinnings of bipolar disorder (BD) remain poorly understood. The cerebellum is ideally positioned to modulate emotional regulation circuitry yet has been understudied in BD. Previous studies have suggested differences in cerebellar activity and metabolism in BD, however findings on cerebellar structural differences remain contradictory. MethodsWe collected 3T anatomical MRI scans from participants with (N = 131) and without (N = 81) BD type I. Differences in cerebellar volumes were assessed along with factors that influence the results. ResultsThe cerebellar cortex was smaller bilaterally in participants with BD. Polygenic propensity score (bipolar N = 103, control N = 64) did not predict any cerebellar volumes, suggesting that non-genetic factors may have greater influence on the cerebellar volume difference we observed in BD. Cerebellar white matter volumes increased with more adverse childhood events, but we did not observe any associations with parental psychiatric illness. We also evaluated time from onset and symptom burden and found no associations with cerebellar volumes, suggesting neurodevelopment may differ prior to onset. Finally, we found taking sedatives was associated with larger cerebellar white matter and non-significantly larger cortical volume. LimitationsThis study was cross-sectional, limiting interpretation of possible mechanisms. Most of our participants were White, which could limit the generalizability. Additionally, we did not account for potential polypharmacy interactions. ConclusionsThese findings suggest that external influences, such as medications, may influence cerebellum structure in BD and may mask underlying differences. Accounting for medication may be critical for consistent findings in future studies.

neuroscience↗

bioRxiv2023

A burns and COVID-19 shared stress responding gene network deciphers CD1C-CD141- DCs as the key cellular components in septic prognosis

Differential body responses to various stresses, infectious or noninfectious, govern clinical outcomes ranging from asymptoma to death. However, the common molecular and cellular nature of the stress responsome across different stimuli is not described. In this study, we compared the expression behaviors between burns and COVID-19 infection by choosing the transcriptome of peripheral blood from related patients as the analytic target since the blood cells reflect the systemic landscape of immune homeostasis. We identified an immune co-stimulator (CD86)-centered network, named stress-response core (SRC), which coordinated multiple immune processes and was robust in membership and highly related to the clinical traits in both burns and COVID-19. An independent whole blood single-cell RNA sequencing of COVID-19 patients demonstrated that the monocyte-dendritic cell (Mono-DC) wing was the major cellular source of the SRC, among which the higher expression of the SRC in the monocyte was associated with the asymptomatic COVID-19 patients, while the quantity-restricted and function-defected CD1C-CD141-DCs were recognized as the key signature which linked to bad consequences in COVID-19. Specifically, the proportion of the CD1C-CD141-DCs and their SRC expression levels were step-wise reduced along with worse clinic conditions while the sub-cluster of CD1C-CD141-DCs of the critical COVID-19 patients was characterized of IFN signaling quiescence, high mitochondrial metabolism and immune-communication inactivation. Thus, our study identified an expression-synchronized and function-focused gene network which was decreased under burns and COVID-19 stress and argued the CD1C-CD141-DC as the prognosis-related cell population which might serve as a new target of diagnosis and therapy.

immunology↗

bioRxiv2023

Efficient generation of transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein

Citrus is one of the top three tree crops in the world and its production faces many devastating disease challenges such as citrus canker caused by Xanthomonas citri subsp. citri (Xcc). Genetic improvement of citrus via traditional approaches is a lengthy (approximately 20 years) and laborious process. Biotechnological approaches including CRISPR genome editing technologies have shown promise. However, none of the citrus plants generated by biotechnological approaches have been commercialized, which primarily resulted from the transgenic nature of the genetically modified plants. Here, we successfully developed transgene-free canker-resistant Citrus sinensis lines in the T0 generation within 10 months through transformation of embryogenic citrus protoplasts with Cas12a/crRNA ribonucleoprotein (RNP) to edit the canker susceptibility gene CsLOB1. Among the 39 regenerated lines, 38 are biallelic/homozygous mutants based on Sanger sequencing analysis of targeting sites and whole genome sequencing, demonstrating a 97.4% biallelic/homozygous mutation rate. The edited lines do not contain off-target mutations. The CsLOB1 edited C. sinensis lines demonstrate no differences from wild type plants except canker resistance. Importantly, the transgene-free canker-resistant C. sinensis lines have received regulatory approval by USDA APHIS. This study presents an efficient genome editing approach for citrus using Cas12a/crRNA RNP, which has a broad impact on genetic improvement of elite citrus varieties and potentially other tree crops and their genetic study. This study represents a breakthrough by generating the first transgene-free canker-resistant C. sinensis lines that provide a sustainable and efficient solution to control citrus canker.

bioengineering↗

bioRxiv2023

PNMA2 forms non-enveloped virus-like capsids that trigger paraneoplastic neurological syndrome

The paraneoplastic Ma antigen (PNMA) genes are associated with cancer-induced paraneoplastic syndromes that present with neurological symptoms and autoantibody production. How PNMA proteins trigger a severe autoimmune disease is unclear. PNMA genes are predominately expressed in the central nervous system with little known functions but are ectopically expressed in some tumors. Here, we show that PNMA2 is derived from a Ty3 retrotransposon that encodes a protein which forms virus-like capsids released from cells as non-enveloped particles. Recombinant PNMA2 capsids injected into mice induce a robust autoimmune reaction with significant generation of autoantibodies that preferentially bind external "spike" PNMA2 capsid epitopes, while capsid-assembly-defective PNMA2 protein is not immunogenic. PNMA2 autoantibodies present in cerebrospinal fluid of patients with anti-Ma2 paraneoplastic neurologic disease show similar preferential binding to PNMA2 "spike" capsid epitopes. These observations suggest that PNMA2 capsids released from tumors trigger an autoimmune response that underlies Ma2 paraneoplastic neurological syndrome.

neuroscience↗

bioRxiv2023

An Unexpected Role of Neutrophils in Clearing Apoptotic Hepatocytes In Vivo

Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis, and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.

physiology↗

bioRxiv2023

Brainstem Dbh+ Neurons Control Chronic Allergen-Induced Airway Hyperreactivity

Chronic exposure of the lung to irritants such as allergen is a primary cause of asthma characterized by exaggerated airway constriction, also called hyperreactivity, which can be life-threatening. Aside from immune cells, vagal sensory neurons are important for airway hyperreactivity 1-4. However, the identity and signature of the downstream nodes of this adaptive circuit remains poorly understood. Here we show that a single population of Dbh+neurons in the nucleus of the solitary tract (nTS) of the brainstem, and downstream neurons in the nucleus ambiguous (NA), are both necessary and sufficient for chronic allergen-induced airway hyperreactivity. We found that repeated exposures of mice to inhaled allergen activates nTS neurons in a mast cell-, interleukin 4 (IL-4)-and vagal nerve-dependent manner. Single-nucleus RNA-seq of the nTS at baseline and following allergen challenges reveals that a Dbh+ population is preferentially activated. Ablation or chemogenetic inactivation of Dbh+ nTS neurons blunted, while chemogenetic activation promoted hyperreactivity. Viral tracing indicates that Dbh+nTS neurons, capable of producing norepinephrine, project to the NA, and NA neurons are necessary and sufficient to relay allergen signals to postganglionic neurons that then directly drive airway constriction. Focusing on transmitters, delivery of norepinephrine antagonists to the NA blunted allergen-induced hyperreactivity. Together, these findings provide molecular, anatomical and functional definitions of key nodes of a canonical allergen response circuit. The knowledge opens the possibility of targeted neural modulation as an approach to control refractory allergen-induced airway constriction.

cell biology↗

bioRxiv2023

Thermal Stimulus Task fMRI in the Cervical Spinal Cord at 7 Tesla

PurposeAlthough functional MRI is widely applied in the brain, fMRI of the spinal cord is more technically demanding. Proximity to the vertebral column and lungs results in strong spatial inhomogeneity and temporal fluctuations in B0. Increasing field strength enables higher spatial resolution and improved sensitivity to BOLD signal, but amplifies the effects of B0 inhomogeneity. In this work, we present the first task fMRI in the spinal cord at 7 T. Further, we compare the performance of single-shot and multi-shot 2D EPI protocols, which differ in sensitivity to spatial and temporal B0 inhomogeneity. MethodsThe cervical spinal cords of 11 healthy volunteers were scanned at 7 T using single-shot 2D EPI at 0.75 mm in-plane resolution and multi-shot 2D EPI at 0.75 and 0.6 mm in-plane resolutions. All protocols used 3 mm slice thickness. For each protocol, the BOLD response to thirteen 10-second noxious thermal stimuli applied to the right thumb was acquired in a 10-minute fMRI run. Image quality, temporal SNR, and BOLD activation (percent signal change and z-stat) at both individual-and group-level were evaluated between the protocols. ResultsTemporal SNR was highest in single-shot and multi-shot 0.75 mm protocols. In group-level analyses, activation clusters appeared in all protocols in the ipsilateral dorsal quadrant at the expected C6 neurological level. In individual-level analyses, activation clusters at the expected level were detected in some, but not all subjects and protocols. Single-shot 0.75 mm generally produced the highest mean z-statistic, while multi-shot 0.60 mm produced the best-localized activation clusters and the least geometric distortion. Larger than expected within-subject segmental variation of BOLD activation along the cord was observed. ConclusionGroup-level sensory task fMRI of the cervical spinal cord is feasible at 7 T with single-shot or multi-shot EPI. The best choice of protocol will likely depend on the relative importance of sensitivity to activation versus spatial localization of activation for a given experiment. HighlightsFirst stimulus task fMRI results in the spinal cord at 7 T Single-shot 0.75 mm 2D EPI produced the highest mean z-statistic Multi-shot 0.60 mm 2D EPI provided the best-localized activation and least distortion

bioengineering↗

bioRxiv2023

A synthetic signalling network imitating the action of immune cells in response to bacterial metabolism

State-of-the-art bottom-up synthetic biology allows us to replicate many basic biological functions in artificial cell-like devices. To mimic more complex behaviours, however, artificial cells would need to perform many of these functions in a synergistic and coordinated fashion, which remains elusive. Here we considered a sophisticated biological response, namely the capture and deactivation of pathogens by neutrophil immune cells, through the process of netosis. We designed a consortium consisting of two synthetic agents - responsive DNA-based particles and antibiotic-loaded lipid vesicles - whose coordinated action mimics the sought immune-like response when triggered by bacterial metabolism. The artificial netosis-like response emerges from a series of interlinked sensing and communication pathways between the live and synthetic agents, and translates into both physical and chemical antimicrobial actions, namely bacteria immobilisation and exposure to antibiotics. Our results demonstrate how advanced life-like responses can be prescribed with a relatively small number of synthetic molecular components, and outlines a new strategy for artificial-cell-based antimicrobial solutions.

synthetic biology↗

bioRxiv2023

Functional Connectivity of the Cerebellar Vermis in Bipolar Disorder and Associations with Mood

PurposeStudies of the neural underpinnings of bipolar type I disorder have focused on the emotional control network. However, there is also growing evidence for cerebellar involvement, including abnormal structure, function, and metabolism. Here, we sought to assess functional connectivity of the cerebellum with the cerebrum in bipolar disorder and to assess whether any effects might depend on mood. MethodsThis cross-sectional study enrolled 128 participants with bipolar type I disorder and 83 control comparison participants who completed a 3T MRI scan, which included anatomical imaging as well as resting state BOLD imaging. Functional connectivity of the cerebellar vermis to all other brain regions was assessed. Based on quality control metrics of the fMRI data, 109 participants with bipolar disorder and 79 controls were used to in the statistical analysis comparing connectivity of the vermis as well as associations with mood. Potential impacts of medications were also explored. ResultsFunctional connectivity of the cerebellar vermis in bipolar disorder was found to differ significantly between brain regions known to be involved in the control of emotion, motor function, and language. While connections with emotion and motor control areas were significantly stronger in bipolar disorder, connection to a region associated language production was significantly weaker. In the participants with bipolar disorder, ratings of depression and mania were inversely associated with vermis functional connectivity. No effect of medications on these connections were observed. ConclusionTogether the findings suggest cerebellum may play a compensatory role in bipolar disorder and when it can no longer fulfill this role, depression and mania develop. The proximity of the cerebellar vermis to the skull may make this region a potential target for treatment with transcranial magnetic stimulation.

neuroscience↗

bioRxiv2023

A deep learning approach for the discovery of tumor-targeting small organic ligands from DNA-Encoded Chemical Libraries

DNA-Encoded Chemical Libraries (DELs) emerged as efficient and cost-effective ligand discovery tools, which enable the generation of protein-ligand interaction data of unprecedented size. In this article, we present an approach that combines DEL screening and instance-level deep learning modeling to identify tumor-targeting ligands against Carbonic Anhydrase IX (CAIX), a clinically validated marker of hypoxia and clear cell Renal Cell Carcinoma. We present a new ligand identification and HIT-to-LEAD strategy driven by Machine Learning (ML) models trained on DELs, which expand the scope of DEL-derived chemical motifs. CAIX screening datasets obtained from three different DELs were used to train ML models for generating novel HITs, dissimilar to elements present in the original DELs. Out of the 152 novel potential HITs that were identified with our approach and screened in an in vitro enzymatic inhibition assay, 70% displayed submicromolar activities (IC50 < 1 M). Based on the first HIT set, the model was further used to prioritize and generate LEAD compounds with nanomolar affinity for in vivo tumor-targeting applications. Three LEAD candidates showed accumulation on the surface of CAIX-expressing tumor cells in cellular binding assays. The best compound displayed in vitro KD of 5.7 nM and selectively targeted tumors in mice bearing human Renal Cell Carcinoma lesions. Our results demonstrate the synergy between DEL and machine learning for the identification of novel HITs and for the successful translation of LEAD candidates for in vivo targeting applications. Graphical Abstracts O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/525453v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@105913corg.highwire.dtl.DTLVardef@823522org.highwire.dtl.DTLVardef@6e85e9org.highwire.dtl.DTLVardef@19a611_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

Histone acetylation insulator SET orchestrates PP2A inhibition and super-enhancer activation

Wide-spread growth-essential genes are hyper-transcribed in the pancreatic cancer cells. Searching for the factors that reprogram this abnormal transcription, we identified the nuclear oncogene SET that supported CDK9-induced and Pol II-mediated transcription. SET disrupted PP2A-A/C interaction via its C-terminal domains. Through blocking PP2A activity, SET assisted CDK9 to maintain Pol II CTD phosphorylation and activated mRNA transcription. Meanwhile, as a histone acetylation insulator, SET mainly suppressed histone acetylation in the gene promoters but evaded enhancers. Massive super-enhancer associated genes, including the oncogene MET, were hence permitted to be transcribed by SET over-expression. Our findings position SET as a key factor that bridges histone acetylation and PP2A related transcription in cancer cells.

molecular biology↗

bioRxiv2023

The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partial preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, these findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies. Brief SummaryA mouse model of human DNAAF5 primary ciliary dyskinesia variants reveals gene dosage effects of mutant alleles and tissue-specific molecular requirements for cilia motor assembly.

cell biology↗

bioRxiv2023

Myricetin protected against Aβ oligomer-induced synaptic impairment, mitochondrial function and oxidative stress in SH-SY5Y cells via ERK1/2/GSK-3β pathways

Alzheimers disease is characterized by abnormal {beta}-amyloid (A{beta}) plaque accumulation, tau hyperphosphorylation, reactive oxidative stress, mitochondrial dysfunction and synaptic loss. Myricetin, a dietary flavonoid, has been shown to have neuroprotective effects in vitro and in vivo. Here, we aimed to elucidate the mechanism and pathways involved in myricetins protective effect on the toxicity induced by the A{beta}42 oligomer. Neuronal SH-SY5Y cells were pretreated with myricetin before incubation with A{beta}42 oligomer. The levels of pre- and postsynaptic proteins, mitochondrial division and fusion proteins, glycogen synthase kinase-3 {beta} (GSK-3{beta}) and extracellular regulated kinase (ERK) 1/2 were assessed by Western blotting. Flow cytometry assays for mitochondrial membrane potential (JC1) and reactive oxidative stress, as well immunofluorescence staining for lipid peroxidation (4-HNE) and DNA oxidation (8-OHdG), were performed. We found that myricetin prevented A{beta}42 oligomer-induced tau phosphorylation and the reduction in pre/postsynaptic proteins. In addition, myricetin reduced reactive oxygen species generation, lipid peroxidation, and DNA oxidation induced by the A{beta}42 oligomer. Moreover, myricetin prevented the A{beta}42 oligomer-induced reduction in mitochondrial fusion proteins (mitofusin-1, mitofusin-2), fission protein (dynamin-related protein 1) phosphorylation, and mitochondrial membrane potential via the associated GSK-3{beta} and ERK 1/2 signaling pathways. In conclusion, this study provides new insight into the neuroprotective mechanism of myricetin against A{beta}42 oligomer-induced toxicity.

neuroscience↗

bioRxiv2023

Structural insights into transcription activation mechanism of the global regulator GlnR from actinobacteria

GlnR, an OmpR/PhoB subfamily protein, is an orphan response regulator that globally coordinates the expression of genes responsible for nitrogen, carbon and phosphate metabolism in actinobacteria. Although much efforts at biochemical and genetic analyses have been made on the mechanism of GlnR-dependent transcription activation, it still remains unclear owing to lacking the structure of GlnR-dependent transcription activation complex (GlnR-TAC). Here, we report a crystal structure of a binary complex including a C terminal DNA binding domain of GlnR (GlnR_DBD) and its regulatory cis-element DNA, and a cryo-EM structure of GlnR-TAC comprising of Mycobacterium tuberculosis RNA polymerase, GlnR, and a promoter containing four well-characterized conserved GlnR binding sites. These structures show four GlnR protomers coordinately engage promoter DNA in a head-to-tail manner, with two N-terminal receiver domains of GlnR (GlnR-RECs) jointly act as a bridge to connect RNAP NTD with the upstream GlnR_DBD. GlnR-TAC is stabilized by complex protein-protein interactions between GlnR and the conserved {beta} flap, {sigma}AR4, CTD, NTD domains of RNAP. These are in good agreement with our mutational and kinetic single-molecule fluorescence assays. Altogether, our results reveal a general transcription activation mechanism for the global regulator GlnR and other OmpR/PhoB subfamily proteins, and present a unique mode of bacterial transcription regulation. Significance statementIn low-GC gram-negative bacteria, the typical two component system NtrB/NtrC accounts for the expression of genes related to nitrogen metabolism. In high-GC gram-positive actinobacteria, GlnR, an atypical and orphan response regulator (RR) of the OmpR/PhoB subfamily proteins, globally coordinates transcription of genes involved in nitrogen, carbon and phosphate metabolism. Here, using crystallography, cryo-electron microscopy, and single-molecule fluorescence assays, we show that GlnR activates transcription by contacting DNA between the -10 and -35 elements and further upstream through contacting {sigma} region 4 and RNAP {beta} flap subunit. We also identify a previously unobserved cooperative engagement of four GlnR protomers to the promoter DNA, which not only makes the transcription initiation complex (RPo) more stable, but also provides better transcription activities.

biochemistry↗