High air humidity dampens salicylic acid pathway and NPR1 function to promote plant disease.

The occurrence of plant disease is determined by interactions among host, pathogen, and environment. Air humidity shapes various aspects of plant physiology and high humidity has long been known to promote numerous phyllosphere diseases. However, the molecular basis of how high humidity interferes with plant immunity to favor disease has remained elusive. Here we show that high humidity is associated with an "immuno-compromised" status in Arabidopsis plants. Furthermore, accumulation and signaling of salicylic acid (SA), an important defense hormone, are significantly inhibited under high humidity. NPR1, an SA receptor and central transcriptional co-activator of SA-responsive genes, is less ubiquitinated and displays a lower promoter binding affinity under high humidity. The cellular ubiquitination machinery, particularly the Cullin 3-based E3 ubiquitin ligase mediating NPR1 protein ubiquitination, is downregulated under high humidity. Importantly, under low humidity the Cullin 3a/b mutant plants phenocopy the low SA gene expression and disease susceptibility that is normally observed under high humidity. Our study uncovers a mechanism by which high humidity dampens a major plant defense pathway and provides new insights into the long-observed air humidity influence on diseases.

Pattern-recognition receptors are required for NLR-mediated plant immunity.

The plant immune system is fundamental for plant survival in natural ecosystems and for productivity in crop fields. Substantial evidence supports the prevailing notion that plants possess a two-tiered innate immune system, called pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is triggered by microbial patterns via cell surface-localized pattern-recognition receptors (PRRs), whereas ETI is activated by pathogen effector proteins via predominantly intracellularly localized receptors called nucleotide-binding, leucine-rich repeat receptors (NLRs)1-4. PTI and ETI are initiated by distinct activation mechanisms and involve different early signalling cascades5,6. Here we show that Arabidopsis PRR and PRR co-receptor mutants-fls2 efr cerk1 and bak1 bkk1 cerk1 triple mutants-are markedly impaired in ETI responses when challenged with incompatible Pseudomonas syrinage bacteria. We further show that the production of reactive oxygen species by the NADPH oxidase RBOHD is a critical early signalling event connecting PRR- and NLR-mediated immunity, and that the receptor-like cytoplasmic kinase BIK1 is necessary for full activation of RBOHD, gene expression and bacterial resistance during ETI. Moreover, NLR signalling rapidly augments the transcript and/or protein levels of key PTI components. Our study supports a revised model in which potentiation of PTI is an indispensable component of ETI during bacterial infection. This revised model conceptually unites two major immune signalling cascades in plants and mechanistically explains some of the long-observed similarities in downstream defence outputs between PTI and ETI.

A plant genetic network for preventing dysbiosis in the phyllosphere.

The aboveground parts of terrestrial plants, collectively called the phyllosphere, have a key role in the global balance of atmospheric carbon dioxide and oxygen. The phyllosphere represents one of the most abundant habitats for microbiota colonization. Whether and how plants control phyllosphere microbiota to ensure plant health is not well understood. Here we show that the Arabidopsis quadruple mutant (min7 fls2 efr cerk1; hereafter, mfec)1, simultaneously defective in pattern-triggered immunity and the MIN7 vesicle-trafficking pathway, or a constitutively activated cell death1 (cad1) mutant, carrying a S205F mutation in a membrane-attack-complex/perforin (MACPF)-domain protein, harbour altered endophytic phyllosphere microbiota and display leaf-tissue damage associated with dysbiosis. The Shannon diversity index and the relative abundance of Firmicutes were markedly reduced, whereas Proteobacteria were enriched in the mfec and cad1S205F mutants, bearing cross-kingdom resemblance to some aspects of the dysbiosis that occurs in human inflammatory bowel disease. Bacterial community transplantation experiments demonstrated a causal role of a properly assembled leaf bacterial community in phyllosphere health. Pattern-triggered immune signalling, MIN7 and CAD1 are found in major land plant lineages and are probably key components of a genetic network through which terrestrial plants control the level and nurture the diversity of endophytic phyllosphere microbiota for survival and health in a microorganism-rich environment.

Ethylene signaling modulates air humidity responses in plants.

Air humidity significantly impacts plant physiology. However, the upstream elements that mediate humidity sensing and adaptive responses in plants remain largely unexplored. In this study, we define high humidity-induced cellular features of Arabidopsis plants and take a quantitative phosphoproteomics approach to obtain a high humidity-responsive landscape of membrane proteins, which we reason are likely the early checkpoints of humidity signaling. We found that a brief high humidity exposure (i.e., 0.5 h) is sufficient to trigger extensive changes in membrane protein abundance and phosphorylation. Enrichment analysis of differentially regulated proteins reveals high humidity-sensitive processes such as 'transmembrane transport', 'response to abscisic acid', and 'stomatal movement'. We further performed a targeted screen of mutants, in which high humidity-responsive pathways/proteins are disabled, to uncover genes mediating high humidity sensitivity. Interestingly, ethylene pathway mutants (i.e., ein2 and ein3eil1) display a range of altered responses, including hyponasty, reactive oxygen species level, and responsive gene expression, to high humidity. Furthermore, we observed a rapid induction of ethylene biosynthesis genes and ethylene evolution after high humidity treatment. Our study sheds light on the potential early signaling events in humidity perception, a fundamental but understudied question in plant biology, and reveals ethylene as a key modulator of high humidity responses in plants.

Ascorbate peroxidase 1 allows monitoring of cytosolic accumulation of effector-triggered reactive oxygen species using a luminol-based assay.

Biphasic production of reactive oxygen species (ROS) has been observed in plants treated with avirulent bacterial strains. The first transient peak corresponds to pattern-triggered immunity (PTI)-ROS, whereas the second long-lasting peak corresponds to effector-triggered immunity (ETI)-ROS. PTI-ROS are produced in the apoplast by plasma membrane-localized NADPH oxidases, and the recognition of an avirulent effector increases the PTI-ROS regulatory module, leading to ETI-ROS accumulation in the apoplast. However, how apoplastic ETI-ROS signaling is relayed to the cytosol is still unknown. Here, we found that in the absence of cytosolic ascorbate peroxidase 1 (APX1), the second phase of ETI-ROS accumulation was undetectable in Arabidopsis (Arabidopsis thaliana) using luminol-based assays. In addition to being a scavenger of cytosolic H2O2, we discovered that APX1 served as a catalyst in this chemiluminescence ROS assay by employing luminol as an electron donor. A horseradish peroxidase (HRP)-mimicking APX1 mutation (APX1W41F) further enhanced its catalytic activity toward luminol, whereas an HRP-dead APX1 mutation (APX1R38H) reduced its luminol oxidation activity. The cytosolic localization of APX1 implies that ETI-ROS might accumulate in the cytosol. When ROS were detected using a fluorescent dye, green fluorescence was observed in the cytosol 6 h after infiltration with an avirulent bacterial strain. Collectively, these results indicate that ETI-ROS eventually accumulate in the cytosol, and cytosolic APX1 catalyzes luminol oxidation and allows monitoring of the kinetics of ETI-ROS in the cytosol. Our study provides important insights into the spatial dynamics of ROS accumulation in plant immunity.

CURLY LEAF modulates apoplast liquid water status in Arabidopsis leaves.

The apoplast of plant leaves, the intercellular space between mesophyll cells, is normally largely filled with air with a minimal amount of liquid water in it, which is essential for key physiological processes such as gas exchange to occur. Phytopathogens exploit virulence factors to induce a water-rich environment, or "water-soaked" area, in the apoplast of the infected leaf tissue to promote disease. We propose that plants evolved a "water soaking" pathway, which normally keeps a nonflooded leaf apoplast for plant growth but is disturbed by microbial pathogens to facilitate infection. Investigation of the "water soaking" pathway and leaf water control mechanisms is a fundamental, yet previously overlooked, aspect of plant physiology. To identify key components in the "water soaking" pathway, we performed a genetic screen to isolate Arabidopsis (Arabidopsis thaliana) severe water soaking (sws) mutants that show liquid water overaccumulation in the leaf under high air humidity, a condition required for visible water soaking. Here, we report the sws1 mutant, which displays rapid water soaking upon high humidity treatment due to a loss-of-function mutation in CURLY LEAF (CLF), encoding a histone methyltransferase in the POLYCOMB REPRESSIVE COMPLEX 2 (PRC2). We found that the sws1 (clf) mutant exhibits enhanced abscisic acid (ABA) levels and stomatal closure, which are indispensable for its water soaking phenotype and mediated by CLF's epigenetic regulation of a group of ABA-associated NAM, ATAF, and CUC (NAC) transcription factor genes, NAC019/055/072. The clf mutant showed weakened immunity, which likely also contributes to the water soaking phenotype. In addition, the clf plant supports a substantially higher level of Pseudomonas syringae pathogen-induced water soaking and bacterial multiplication, in an ABA pathway and NAC019/055/072-dependent manner. Collectively, our study sheds light on an important question in plant biology and demonstrates CLF as a key modulator of leaf liquid water status via epigenetic regulation of the ABA pathway and stomatal movement.

Efficacy and Safety of Immune Checkpoint Inhibitors Combined With Chemotherapy as First-line Treatment for Recurrent or Metastatic Nasopharyngeal Carcinoma: A Network Meta-analysis of Randomized Controlled Trials.

BACKGROUND: Different clinical trials for recurrent or metastatic nasopharyngeal carcinoma have studied different combinations of immuno-oncology in first-line treatment, but the optimal choice has not been determined. OBJECTIVE: To systematically examine and compare the efficacy and safety of different immune checkpoint inhibitors (ICIs) combined with chemotherapy as first-line treatment for recurrent or metastatic nasopharyngeal carcinoma. METHODS: Several electronic databases were systematically searched up to February 2023. Articles meeting the inclusion criteria were included. RESULTS: Three RCTs were eligible in the study. Compared with placebo plus gemcitabine-cisplatin (GP), toripalimab plus GP (HR = 0.59, 95% CI: 0.37-0.95) was significantly associated with a better OS. Tislelizumab plus GP generated best progression-free survival (PFS) benefit (HR = 0.50, 95% CI: 0.37-0.67), greatest improvement in 1-year PFS rate (RR = 3.00, 95% CI: 1.84-5.22), and objective response rate (ORR) (RR = 1.26, 95% CI: 1.04-1.53) over the placebo plus GP. Furthermore, tislelizumab plus GP appeared to be safer than toripalimab plus GP and camrelizumab plus GP in terms of adverse events (AEs)-grade ≥3, treatment-related AEs (TRAEs)-grade ≥3, serious AEs (SAEs), treatment-related SAEs (TRSAEs), and AEs leading to discontinuation of treatment. CONCLUSION AND RELEVANCE: In recurrent or metastatic nasopharyngeal carcinoma, programmed death 1 (PD-1) inhibitors plus GP as first-line treatment have better survival outcomes than placebo plus GP with comparable toxicity. Toripalimab plus GP shows the best OS benefit over placebo plus GP, while tislelizumab plus GP generates the best PFS, 1-year PFS rate, ORR, and safety. Tislelizumab plus GP could be the best choice among the ICIs combined with chemotherapy regimens as first-line treatment in recurrent or metastatic nasopharyngeal carcinoma.

DGK5-mediated phosphatidic acid homeostasis interplays with reactive oxygen species in plant immune signaling.

Reactive oxygen species (ROS) and phosphatidic acid (PA) are important second messengers in plant immunity. PA binding to RBOHD, an NADPH oxidase responsible for ROS production, enhances RBOHD stability and promotes ROS production. Distinct phosphorylation of the lipid kinase DGK5 optimizes the PA burst in regulating ROS production.

Flip a coin: cell senescence at the maternal-fetal interface†.

During pregnancy, cell senescence at the maternal-fetal interface is required for maternal well-being, placental development, and fetal growth. However, recent reports have shown that aberrant cell senescence is associated with multiple pregnancy-associated abnormalities, such as preeclampsia, fetal growth restrictions, recurrent pregnancy loss, and preterm birth. Therefore, the role and impact of cell senescence during pregnancy requires further comprehension. In this review, we discuss the principal role of cell senescence at the maternal-fetal interface, emphasizing its "bright side" during decidualization, placentation, and parturition. In addition, we highlight the impact of its deregulation and how this "dark side" promotes pregnancy-associated abnormalities. Furthermore, we discuss novel and less invasive therapeutic practices associated with the modulation of cell senescence during pregnancy.

Bistability at the onset of neuronal oscillations.

The Hodgkin-Huxley (HH) model and squid axon (bathed in reduced Ca2+) fire repetitively for steady current injection. Moreover, for a current-range just suprathreshold, repetitive firing coexists with a stable steady state. Neuronal excitability, as such, shows bistability and hysteresis providing the opportunity for the system to perform as switchable between firing and non-firing states with transient input and providing the backbone as a dynamical mechanism for bursting oscillations. Some conditions for bistability can be derived by intricate analysis (bifurcation theory) and characterized by simulation, but conditions for emergence and robustness of such bistability do not typically follow from intuition. Here, we demonstrate with a semi-quantitative two-variable, V-w, reduction of the HH model features that promote/reduce bistability. Visualization of flow and trajectories in the V-w phase plane provides an intuitive grasp for bistability. The geometry of action potential recovery involves a late phase during which the dynamic negative feedback of [Formula: see text] inactivation and [Formula: see text] activation over/undershoot, respectively, their resting values, thereby leading to hyperexcitabilty and an intrinsically generated opportunity to by-pass the spiral-like stable rest state and initiate the next spike upstroke. We illustrate control of bistability and dependence of the degree of hysteresis on recovery timescales and gating properties. Our dynamical dissection reveals the strongly attracting depolarized phase of the spike, enabling approximations like the resetting feature of adapting integrate-and-fire models. We extend our insights and show that the Morris-Lecar model can also exhibit robust bistability.

[Application of high-speed counter current chromatography in extraction and separation of alkaloids in natural products].

Alkaloids, widespread in plants, have a series of pharmacological activities and have been widely used to treat various diseases. Because alkaloids are usually presented in multicomponent mixtures and are deeply low in content, they are very difficult to extract and separate by traditional methods. High-speed counter current chromatography(HSCCC) is a kind of liquid-liquid chromatography without solid support phase, which has the advantages of large injection volume, low cost, and no irreversible adsorption. Compared with the traditional methods of extraction and separation of alkaloids, HSCCC can ensure the separation of many different alkaloids at one time, with a high recovery and large amount. In this paper, the advantages and disadvantages of HSCCC compared with traditional separation methods were discussed and the solvent system and elution mode of HSCCC used to separate alkaloids in recent years were summarized by referring to the relevant literature to provide some references for the separation of alkaloids by HSCCC.

Effects of perfluoroundecanoic acid on the function of Leydig cells in adult male rats.

Perfluoroundecanoic acid (PFUnA), a perfluorinated compound, has environmental persistence, bioaccumulation, and potential toxicity. However, its effect on Leydig cell function remains unclear. Rats (age of 56 days) were gavaged with 0 (corn oil), 0.1, 0.5, 1, or 5 mg/kg/day PFUnA for 28 days. PFUnA significantly reduced serum testosterone levels as low as 0.5 mg/kg. PFUnA markedly decreased Leydig cell number as low as 0.1 mg/kg. PFUnA markedly reduced transcript levels of Star and Insl3 in the testes at 1 mg/kg after adjusting to Leydig cell number. It also reduced their protein levels. PFUnA significantly decreased the phosphorylation of AKT1 and mTOR as low as 0.1 mg/kg and the phosphorylation of ERK1/2 at 1 mg/kg and the phosphorylation of AKT1, AKT2, ERK1/2, and mTOR in Leydig cells at various concentrations (0.01-10 µM) after 24 h of in vitro treatment. In conclusion, PFUnA inhibits Leydig cell function possibly via AKT/ERK1/2/mTOR signaling pathways.

Bacteria establish an aqueous living space in plants crucial for virulence.

High humidity has a strong influence on the development of numerous diseases affecting the above-ground parts of plants (the phyllosphere) in crop fields and natural ecosystems, but the molecular basis of this humidity effect is not understood. Previous studies have emphasized immune suppression as a key step in bacterial pathogenesis. Here we show that humidity-dependent, pathogen-driven establishment of an aqueous intercellular space (apoplast) is another important step in bacterial infection of the phyllosphere. Bacterial effectors, such as Pseudomonas syringae HopM1, induce establishment of the aqueous apoplast and are sufficient to transform non-pathogenic P. syringae strains into virulent pathogens in immunodeficient Arabidopsis thaliana under high humidity. Arabidopsis quadruple mutants simultaneously defective in a host target (AtMIN7) of HopM1 and in pattern-triggered immunity could not only be used to reconstitute the basic features of bacterial infection, but also exhibited humidity-dependent dyshomeostasis of the endophytic commensal bacterial community in the phyllosphere. These results highlight a new conceptual framework for understanding diverse phyllosphere-bacterial interactions.

Bizarre giant cells in human angiosarcoma exhibit chemoresistance and contribute to poor survival outcomes.

Giant cells (GC) are a poorly understood subset of tumor cells that have been increasingly recognized as a potential contributor to tumor heterogeneity and treatment resistance. We aimed to characterize the biological and clinical significance of GC in angiosarcoma, an aggressive rare cancer of endothelial origin. Archival angiosarcoma samples were examined for the presence of GC and compared with clinicopathological as well as NanoString gene expression data. GC were examined in angiosarcoma cell lines MOLAS and ISOHAS using conventional and electron microscopy, single cell whole genome profiling, and other assays. In the cell lines, GC represented a rare population of mitotically active, non-senescent CD31+ cells, and shared similar genomic profiles with regular-sized cells, consistent with a malignant endothelial phenotype. GC remained viable and persisted in culture following exposure to paclitaxel and doxorubicin. In patient samples, GC were present in 24 of 58 (41.4%) cases. GC was correlated with poorer responses to chemotherapy (25.0% vs 73.3%, P = 0.0213) and independently contributed to worse overall survival outcomes (hazard ratio 2.20, 95% confidence interval 1.17-4.15, P = 0.0142). NanoString profiling revealed overexpression of genes, including COL11A1, STC1, and ERO1A, accompanied by upregulation of immune-related metabolic stress and metastasis/matrix remodeling pathways in GC-containing tumors. In conclusion, GC may contribute to chemoresistance and poor prognosis in angiosarcoma.

In utero exposure to dipentyl phthalate disrupts fetal and adult Leydig cell development.

Phthalates as plasticizers are widely used in many consumer products. Dipentyl phthalate (DPeP) is one of phthalates. However, there are currently few data on whether DPeP exposure affects rat Leydig cell development. In this study, we investigated the effects of in utero DPeP exposure on Leydig cell development in the testes of male newborn and adult rats. From gestational days 14 to 21, Sprague-Dawley pregnant rats were gavaged vehicle (corn oil, control) or DPeP (10, 50, 100, and 500 mg/kg body weight/day). Testosterone and the expression of Leydig cell genes and proteins in the testis at birth and at postnatal day 56 were examined. DPeP dose-dependently reduced serum testosterone levels of male offspring at birth and at postnatal day 56 at 100 and 500 mg/kg and lowered serum luteinizing hormone levels at adult males at ≥10 mg/kg when compared with the control. In addition, DPeP increased number of fetal Leydig cells by inducing their proliferation but down-regulated the expression of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, and Insl3 in fetal Leydig cells per se. DPeP reduced number of adult Leydig cells by inducing cell apoptosis and down-regulated the expression of Lhcgr and Star in adult Leydig cells at postnatal day 56. DPeP lowered SIRT1 and BCL2 levels in the testis of adult rats. In conclusion, DPeP adversely affects both fetal and adult Leydig cell development after in utero exposure.

Perfluoroundecanoic acid inhibits Leydig cell development in pubertal male rats via inducing oxidative stress and autophagy.

Perfluoroundecanoic acid (PFUnA) is one of long-chain perfluoroalkyl carboxylic acids. However, the effect of PFUnA on pubertal development of Leydig cells remains unclear. The goal of this study was to investigate the effect of PFUnA on Leydig cell development in pubertal male rats. We orally dosed male Sprague-Dawley rats (age 35 days) with PFUnA at doses of 0, 1, 5, and 10 mg/kg/day from postnatal day (PND) 35 to PND 56. Serum testosterone and luteinizing hormone levels were remarkably reduced by PFUnA at ≥1 mg/kg while serum follicle-stimulating hormone levels were lowered at 5 and 10 mg/kg. PFUnA down-regulated the expression of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, Insl3, Nr5a1, Fshr, Dhh, Sod1, and Sod2 and their proteins in the testis and the expression of Lhb and Fshb in the pituitary. PFUnA reduced Leydig cell number at 5 and 10 mg/kg. PFUnA induced oxidative stress and increased autophagy. These may result from the inhibition of phosphorylation of mTOR, AKT1, AKT2, and ERK1/2 in the testis. In conclusion, PFUnA exhibits inhibitory effects on pubertal Leydig cell development possibly via inducing oxidative stress and increasing autophagy.

Structural basis of JAZ repression of MYC transcription factors in jasmonate signalling.

The plant hormone jasmonate plays crucial roles in regulating plant responses to herbivorous insects and microbial pathogens and is an important regulator of plant growth and development. Key mediators of jasmonate signalling include MYC transcription factors, which are repressed by jasmonate ZIM-domain (JAZ) transcriptional repressors in the resting state. In the presence of active jasmonate, JAZ proteins function as jasmonate co-receptors by forming a hormone-dependent complex with COI1, the F-box subunit of an SCF-type ubiquitin E3 ligase. The hormone-dependent formation of the COI1-JAZ co-receptor complex leads to ubiquitination and proteasome-dependent degradation of JAZ repressors and release of MYC proteins from transcriptional repression. The mechanism by which JAZ proteins repress MYC transcription factors and how JAZ proteins switch between the repressor function in the absence of hormone and the co-receptor function in the presence of hormone remain enigmatic. Here we show that Arabidopsis MYC3 undergoes pronounced conformational changes when bound to the conserved Jas motif of the JAZ9 repressor. The Jas motif, previously shown to bind to hormone as a partly unwound helix, forms a complete α-helix that displaces the amino (N)-terminal helix of MYC3 and becomes an integral part of the MYC N-terminal fold. In this position, the Jas helix competitively inhibits MYC3 interaction with the MED25 subunit of the transcriptional Mediator complex. Our structural and functional studies elucidate a dynamic molecular switch mechanism that governs the repression and activation of a major plant hormone pathway.

Phyllosphere microbiota: Community dynamics and its interaction with plant hosts.

Plants are colonized by various microorganisms in natural environments. While many studies have demonstrated key roles of the rhizosphere microbiota in regulating biological processes such as nutrient acquisition and resistance against abiotic and biotic challenges, less is known about the role of the phyllosphere microbiota and how it is established and maintained. This review provides an update on current understanding of phyllosphere community assembly and the mechanisms by which plants and microbes establish the phyllosphere microbiota for plant health.

Neurotrophin-3 stimulates stem Leydig cell proliferation during regeneration in rats.

Neurotrophin-3 (NT-3) acts as an important growth factor to stimulate and control tissue development. The NT-3 receptor, TRKC, is expressed in rat testis. Its function in regulation of stem Leydig cell development and its underlying mechanism remain unknown. Here, we reported the role of NT-3 to regulate stem Leydig cell development in vivo and in vitro. Ethane dimethane sulphonate was used to kill all Leydig cells in adult testis, and NT-3 (10 and 100 ng/testis) was injected intratesticularly from the 14th day after ethane dimethane sulphonate injection for 14 days. NT-3 significantly reduced serum testosterone levels at doses of 10 and 100 ng/testis without affecting serum luteinizing hormone and follicle-stimulating hormone levels. NT-3 increased CYP11A1-positive Leydig cell number at 100 ng/testis and lowered Leydig cell size and cytoplasmic size at doses of 10 and 100 ng/testis. After adjustment by the Leydig cell number, NT-3 significantly down-regulated the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, Insl3, Trkc and Nr5a1) and the proteins. NT-3 increased the phosphorylation of AKT1 and mTOR, decreased the phosphorylation of 4EBP, thereby increasing ATP5O. In vitro study showed that NT-3 dose-dependently stimulated EdU incorporation into stem Leydig cells and inhibited stem Leydig cell differentiation into Leydig cells, thus leading to lower medium testosterone levels and lower expression of Lhcgr, Scarb1, Trkc and Nr5a1 and their protein levels. NT-3 antagonist Celitinib can antagonize NT-3 action in vitro. In conclusion, the present study demonstrates that NT-3 stimulates stem Leydig cell proliferation but blocks the differentiation via TRKC receptor.

Japanese Encephalitis Virus Induces Apoptosis and Encephalitis by Activating the PERK Pathway.

Accumulated evidence demonstrates that Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, the precise role of PERK in JEV-induced apoptosis and encephalitis remains unknown. Here, we report that JEV infection activates the PERK-ATF4-CHOP apoptosis pathway both in vitro and in vivo PERK activation also promotes the formation of stress granule, which in turn represses JEV-induced apoptosis. However, PERK inhibitor reduces apoptosis, indicating that JEV-activated PERK predominantly induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway. Among JEV proteins that have been reported to induce ER stress, only JEV NS4B can induce PERK activation. PERK has been reported to form an active molecule by dimerization. The coimmunoprecipitation assay shows that NS4B interacts with PERK. Moreover, glycerol gradient centrifugation shows that NS4B induces PERK dimerization. Both the LIG-FHA and the LIG-WD40 domains within NS4B are required to induce PERK dimerization, suggesting that JEV NS4B pulls two PERK molecules together by simultaneously interacting with them via different motifs. PERK deactivation reduces brain cell damage and encephalitis during JEV infection. Furthermore, expression of JEV NS4B is sufficient to induce encephalitis via PERK in mice, indicating that JEV activates PERK primarily via its NS4B to cause encephalitis. Taken together, our findings provide a novel insight into JEV-caused encephalitis.IMPORTANCE Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, whether the PERK pathway of ER stress response plays important roles in JEV-induced apoptosis and encephalitis remains unknown. Here, we found that JEV infection activates ER stress sensor PERK in neuronal cells and mouse brains. PERK activation induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway upon JEV infection. Among the JEV proteins prM, E, NS1, NS2A, NS2B, and NS4B, only NS4B activates PERK. Moreover, activated PERK participates in apoptosis and encephalitis induced by JEV and NS4B. These findings provide a novel therapeutic approach for JEV-caused encephalitis.