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Apoptosis is a critical host antiviral defense mechanism. But many viruses have evolved multiple strategies to manipulate apoptosis and escape host antiviral immune responses. Herpesvirus infection regulated apoptosis; however, the underlying molecular mechanisms have not yet been fully elucidated. Hence, the present study aimed to study the relationship between herpesvirus infection and apoptosis in vitro and in vivo using the pseudorabies virus (PRV) as the model virus. We found that mitochondria-dependent apoptosis was induced by PRV gM, a late protein encoded by PRV UL10, a virulence-related gene involved in enhancing PRV pathogenicity. Mechanistically, gM competitively combines with BCL-XL to disrupt the BCL-XL-BAK complex, resulting in BCL-2-antagonistic killer (BAK) oligomerization and BCL-2-associated X (BAX) activation, which destroys the mitochondrial membrane potential and activates caspase-3/7 to trigger apoptosis. Interestingly, similar apoptotic mechanisms were observed in other herpesviruses (Herpes Simplex Virus-1 [HSV-1], human cytomegalovirus [HCMV], Equine herpesvirus-1 [EHV-1], and varicella-zoster virus [VZV]) driven by PRV gM homologs. Compared with their parental viruses, the pathogenicity of PRV-ΔUL10 or HSV-1-ΔUL10 in mice was reduced with lower apoptosis and viral replication, illustrating that UL10 is a key virulence-related gene in PRV and HSV-1. Consistently, caspase-3 deletion also diminished the replication and pathogenicity of PRV and HSV-1 in vitro and in mice, suggesting that caspase-3-mediated apoptosis is closely related to the replication and pathogenicity of PRV and HSV-1. Overall, our findings firstly reveal the mechanism by which PRV gM and its homologs in several herpesviruses regulate apoptosis to enhance the viral replication and pathogenicity, and the relationship between gM-mediated apoptosis and herpesvirus pathogenicity suggests a promising approach for developing attenuated live vaccines and therapy for herpesvirus-related diseases.

African swine fever (ASF) is an acute, hemorrhagic, and severe infectious disease caused by the ASF virus (ASFV). ASFV has evolved multiple strategies to escape host antiviral immune responses. Here, we reported that ASFV pB318L, a trans-geranylgeranyl-diphosphate synthase, reduced the expression of type I interferon (IFN-I) and IFN-stimulated genes (ISGs). Mechanically, pB318L not only interacted with STING to reduce the translocation of STING from the endoplasmic reticulum to the Golgi apparatus but also interacted with IFN receptors to reduce the interaction of IFNAR1/TYK2 and IFNAR2/JAK1. Of note, ASFV with interruption of B318L gene (ASFV-intB318L) infected PAMs produces more IFN-I and ISGs than that in PAMs infected with its parental ASFV HLJ/18 at the late stage of infection. Consistently, the pathogenicity of ASFV-intB318L is attenuated in piglets compared with its parental virus. Taken together, our data reveal that B318L gene may partially affect ASFV pathogenicity by reducing the production of IFN-I and ISGs. This study provides a clue to design antiviral agents or live attenuated vaccines to prevent and control ASF.

Calcium (Ca2+), a second messenger, plays a crucial role in plant growth and development as well as in responding to biotic and abiotic stresses. In this work, we explored the role of exogenous calcium in alleviating cold stress and examined the relationship between calcium chloride (CaCl2) and calcium channel blockers, lanthanum chloride (LaCl3), in tea plants under cold stress at the physiological and transcriptional levels. Exogenous Ca2+ partially offsets the negative impacts of cold stress which increased the tolerance of tea plants by significantly raising the photochemical efficiency of PSII, protective enzyme activities, and the ABA content, which reduced the relative electrical conductivity (REC) level and the malondialdehyde (MDA) concentration. At the transcriptome level, exogenous Ca2+ significantly enhanced the expression of key genes involved in cold response pathways. Nevertheless, LaCl3 treatment not only significantly inhibited the activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), but also increased cold damage. This study aims to provide essential insight into the role of exogenous Ca2+ in tea plants responding to cold stress, and to better understand the molecular mechanisms that facilitate Ca-mediated cold tolerance.

Abstract Background Osteosarcoma (OS) is a malignant bone tumor that commonly occurs in adolescents with a high mortality rate and frequent pulmonary metastasis. Emerging evidence has suggested that circular RNAs (circRNAs) are important regulators in multiple biological activities of carcinomas. Nevertheless, the role of circRNAs derived from forkhead box M1 (FOXM1), a well-accepted modulator of OS progression, has not been discussed in OS. Methods Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to test circ-FOXM1 (hsa_circ_0025033) expression in OS cell lines. Cell counting kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), transwell assays and western blot analysis of epithelial-mesenchymal transition (EMT) markers were conducted to evaluate cell proliferation, apoptosis, migration, and EMT process. Luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay were utilized to detect the interaction of circ-FOXM1 and RNAs. Results High expression of circ-FOXM1 was detected in OS cell lines. Functionally, circ-FOXM1 knockdown inhibited the proliferation, migration and EMT process, whereas induced the apoptosis of OS cells. From the aspect of molecular mechanism, circ-FOXM1 was discovered to upregulate FOXM1 expression via sponging miR-320a and miR-320b, therefore activating Wnt signaling pathway. Besides, rescue experiments elucidated that circ-FOXM1 regulated cellular activities of OS cells via FOXM1. Further, in vivo assays supported that loss of circ-FOXM1 restrained OS tumor growth. Conclusion Circ-FOXM1 facilitated the malignant phenotypes of OS cells through FOXM1-mediated Wnt pathway activation, revealing circ-FOXM1 as a potential biomarker for OS treatment.

Forest wood biomass can be used as a renewable resource for the sustainable production of fuels and chemicals. In this study, the methanol, methanol/ethanol, and ethanol/benzene solvent extracts of Platanus orientalis L. bark were analyzed using FTIR, IH NMR, 13C NMR, 2D-HSQC NMR, GC-MS, and TOF-LC-MS. The results revealed that the bark of Planus orientalis contained a wide variety of chemical compounds, such as 30-triacontanol, 1-Hexanol, hexadecanoic acid, methyl ester, 2-ethyl-, γ-Sitosterol, and 3,4,5-tri methoxy-Phenol. In addition, the fast pyrolysis of P. orientalis L. bark (POL-B) with nano-catalysts (Co3O4, Fe2O3, and Co3O4/Fe2O3) was investigated using pyrolysis/gas chromatography/mass spectrometry (Py-GC/MS) and a thermogravimetric analyzer coupled with an FTIR spectrophotometer (TG-FTIR). The TG results revealed that the nano-catalysts significantly affected the pyrolysis of P. orientalis bark. The nano-Fe2O3 catalyst was shown to increase acid and ketone compound production during the catalytic pyrolysis of cellulose. According to the Py-GC-MS results, the pyrolytic products contained several value-added chemicals and high-quality bio-oil. The nano-catalysts promoted the production of aromatics, phenols, ketones, olefins, furans and alkane compounds. These natural-product active molecules and bio-oil, as high-grade raw materials, could be used in many industrial and agricultural fields for the production of wetting agents, stabilizers, plasticizers and resins. In addition, a number of active molecules could be used as drugs and biomedical active ingredients for anti-cancer and anti-inflammatory purposes.

Wood adhesive is very important to the quality of wood-based panels in the forest product industry. Researchers are currently focused on developing green and environmentally friendly adhesives using biomass materials. Soybean meal (SM), a cheap and high-protein byproduct from soybean processing, is considered a green biomaterial resource for adhesive production. In this study, SM was modified using urea and sodium hydroxide as denaturants, and two cross-linking agents were prepared to incorporate into the soybean meal adhesive for bonding reactions. Sodium hydroxide added to soybean protein caused structural damage to the protein structure, which allowed the peptide chains to unfold extensively, forming a low-molecular-weight peptide mixture with a good size distribution and exposing numerous active functional groups for cross-linking with the tannin–phenol–formaldehyde resin (TR) and wood. Adding the epoxy resin CA and TR enabled the cross-linking agents to react with the active functional groups on proteins, forming a cross-linked network structure. As a result, the adhesive’s strength, residual rate, and solid content were improved.

We previously reported that dysregulation of histone deacetylase 2 (Hdac2) was associated with the prognosis of sudden sensorineural hearing loss. However, the underlying molecular mechanisms are poorly understood. In the present study, we developed an acute hearing loss animal model in guinea pigs by infusing lipopolysaccharides (LPS) into the cochlea and measured the expression of Hdac2 in the sensory epithelium. We observed that the level of Hdac2 was significantly decreased in the LPS-infused cochleae. The levels of apoptosis-inhibition genes Bcl-2 and Bcl-xl were also decreased in the cochlea and correlated positively with the levels of Hdac2. Caspase3 or TUNEL-positive spiral ganglion neurons, hair cells, and supporting cells were observed in the LPS-infused cochleae. These in vivo observations were recapitulated in cell culture experiments. Based on bioinformatics analysis, we found miR-204-5p was engaged in the regulation of Hdac2 on Bcl-2. Molecular mechanism experiments displayed that miR-204-5p could be regulated by Hdac2 through interacting with transcription factor Sp1. Taken together, these results indicated that the Hdac2/Sp1/miR-204-5p/Bcl-2 regulatory axis mediated apoptosis in the cochlea, providing potential insights into the progression of acute hearing loss. To our knowledge, the study describes a miRNA-related mechanism for Hdac2-mediated regulation in the cochlea for the first time.

Abstract The human leukocyte antigen F-associated transcript 10 (FAT10) is a member of the small ubiquitin-like protein family that binds to its target proteins and subjects them to degradation by the ubiquitin–proteasome system (UPS). In the heart, FAT10 plays a cardioprotective role and affects predisposition to cardiac arrhythmias after myocardial ischemia (MI). However, whether and how FAT10 influences cardiac arrhythmias is unknown. We investigated the role of FAT10 in regulating the sodium channel Nav1.5, a major regulator of cardiac arrhythmias. Fat10 was conditionally deleted in cardiac myocytes using Myh6-Cre and Fat10 F/F mice (cFat10 −/− ). Compared with their wild-type littermates, cFat10 −/− mice showed prolonged RR, PR, and corrected QT (QTc) intervals, were more likely to develop ventricular arrhythmia, and had increased mortality after MI. Patch-clamp studies showed that the peak Na+ current was reduced, and the late Na+ current was significantly augmented, resulting in a decreased action potential amplitude and delayed depolarization. Immunoblot and immunofluorescence analyses showed that the expression of the membrane protein Nav1.5 was decreased. Coimmunoprecipitation experiments demonstrated that FAT10 stabilized Nav1.5 expression by antagonizing Nav1.5 ubiquitination and degradation. Specifically, FAT10 bound to the lysine residues in the C-terminal fragments of Nav1.5 and decreased the binding of Nav1.5 to the Nedd4-2 protein, a ubiquitin E3 ligase, preventing degradation of the Nav1.5 protein. Collectively, our findings showed that deletion of the Fat10 in cardiac myocytes led to increased cardiac arrhythmias and increased mortality after MI. Thus, FAT10 protects against ischemia-induced ventricular arrhythmia by binding to Nav1.5 and preventing its Neddylation and degradation by the UPS after MI.

The molecular basis for the unique taste and aroma of tea cultivars is largely unknown, but is critical for breeding new cultivars. Here the authors use transcriptomics and metabolomics to study the relationship among phylogenetic groups and specialized metabolites from 136 tea accessions in China.

Abstract With the steadily increasing amount of leadless pacemaker implantations performed worldwide, it has called attention to the delivery difficulty in patients with severe large right heart. Nevertheless, limited studies have reported leadless pacemaker implantation in patients with tricuspid stenosis. Here, we report the successful implantation of leadless pacemaker in a 60‐year‐old female patient with giant right atrium and tricuspid valve stenosis. It is highlighted that leadless pacemaker should not be discouraged even if there are tricuspid valve stenosis and giant right atrium.

Abstract Carboxylesterases, historically referred as non-specific esterases, are ubiquitous hydrolases with high catalytic efficiency. Without exceptions, all mammalian species studied contain multiple forms of carboxylesterases. While having been widely studied in humans and experimental animals, these enzymes remain to be characterized in farm animals. In this study, we showed that pig liver esterase 1 (PLE1) and pig liver esterase 6 (PLE6) were highly active toward amoxicillin (AMO) and ampicillin (AMP), two major antibiotics that are widely used in food-supplements. Mass-spectrometric analysis established that the hydrolysis occurred at the β-lactam amide bond and the hydrolysis drastically decreased or completely eliminated the antibacterial activity. Furthermore, hydrolytic activity and proteomic analysis suggested that trace PLEs existed in pig plasma and contributed little to the hydrolysis of AMO and AMP. These results suggested that carboxylesterases-based hydrolysis determines the therapeutic intensity of these and related antibiotics and the magnitude of the determination occurs in a species-dependent manner.

As an important Ca2+ sensor, calmodulin (CaM) and calmodulin-like protein (CML) play core roles in plant growth, development, and response to environmental stimuli. The CaM/CML gene family has been well characterized in various plant species, such as Arabidopsis thaliana, rice, and tomato; however, in the tea plant, the CaM/CML gene family has not been systematically and comprehensively characterized. In the present study, a total of 5 CsCaM and 60 CsCML proteins were identified from the tea plant genome, which were unevenly distributed on the 14 chromosomes of the tea plant. All the proteins contained two to four EF-hand domains. Meanwhile, an integrated analysis of physicochemical properties, sequence structure, motif identification, phylogeny, gene duplication, promoter cis-elements, and RNA-seq expression profiles in the CsCaM/CML gene family was performed. Transcriptome analysis revealed that CsCaM/CMLs were differentially expressed in different tissues of the tea plant, suggesting their potential roles in plant growth and development. The expression profiles associated with various stress treatments revealed that CsCaM/CML genes were involved in a wide range of abiotic factors, including cold and drought stress. Quantitative real-time PCR (qRT-PCR) was also used to validate the differences in expression under abiotic stress. Overall, these findings enhanced our understanding of CsCaM/CML genes and provided useful information for further research into their molecular functions in abiotic stress response, and in multiple physiological processes in the tea plant.

Endocannabinoids are endogenous ligands of cannabinoid receptors and activation of these receptors has strong physiological and pathological significance. Structurally, endocannabinoids are esters (e.g., 2-arachidonoylglycerol, 2-AG) or amides (e.g., N-arachidonoylethanolamine, AEA). Hydrolysis of these compounds yields arachidonic acid (AA), a major precursor of proinflammatory mediators such as prostaglandin E2. Carboxylesterases are known to hydrolyze esters and amides with high efficiency. CES1, a human carboxylesterase, has been shown to hydrolyze 2-AG, and shares a high sequence identity with pig carboxylesterases: PLE1 and PLE6 (pig liver esterase). The present study was designed to test the hypothesis that PLE1 and PLE6 hydrolyze endocannabinoids and promote inflammatory response. Consistent with the hypothesis, purified PLE1 and PLE6 efficaciously hydrolyzed 2-AG and AEA. PLE6 was 40-fold and 3-fold as active as PLE1 towards 2-AG and AEA, respectively. In addition, both PLE1 and PLE6 were highly sensitive to bis(4-nitrophenyl) phosphate (BNPP), an aryl phosphodiester known to predominately inhibit carboxylesterases. Based on the study with BNPP, PLEs contributed to the hydrolysis of 2-AG by 53.4 to 88.4% among various organs and cells. Critically, exogenous addition or transfection of PLE6 increased the expression and secretion of proinflammatory cytokines in response to the immunostimulant lipopolysaccharide (LPS). This increase was recapitulated in cocultured alveolar macrophages and PLE6 transfected cells in transwells. Finally, BNPP reduced inflammation trigged by LPS accompanied by reduced formation of AA and proinflammatory mediators. These findings define an innovative connection: PLE-endocannabinoid-inflammation. This mechanistic connection signifies critical roles of carboxylesterases in pathophysiological processes related to the metabolism of endocannabinoids.

Tea plants: Controlling color and flavor Researchers have identified genes and related metabolic processes in tea plants that control the lack of green color known as albinism, which gives the tea a savory “umami” taste. The scientists, led by Xinghui Li at Nanjing Agricultural University in China, investigated genes associated with color changes in tender shoots of a novel albino variety of tea plant known as Huabai 1. The transition from white to green shoots is a temperature-dependent process associated with differences in the activity of 483 identified genes. Control of the color change is also linked to alterations in chemical reaction pathways involving a variety of plant compounds, including phenylpropanoids, flavonoids and the green chlorophylls most commonly associated with plant color. The insights may help plant breeders to create new varieties of teas with highly prized desirable flavors

Pseudorabies, caused by the pseudorabies virus (PRV), is an acute fatal disease, which can infect rodents, mammals, and other livestock and wild animals across species. Recently, the emergence of PRV virulent isolates indicates a high risk of a variant PRV epidemic and the need for continuous surveillance. In this study, PRV-GD and PRV-JM, two fatal PRV variants, were isolated and their pathogenicity as well as their effects on host natural immune responses were assessed. PRV-GD and PRV-JM were genetically closest to PRV variants currently circulating in Heilongjiang (HLJ8) and Jiangxi (JX/CH/2016), which belong to genotype 2.2. Consistently, antisera from sows immunized with PRV-Ea classical vaccination showed much lower neutralization ability to PRV-GD and PRV-JM. However, the antisera from the pigs infected with PRV-JM had an extremely higher neutralization ability to PRV-TJ (as a positive control), PRV-GD and PRV-JM. In vivo, PRV-GD and PRV-JM infections caused 100% death in mice and piglets and induced extensive tissue damage, cell death, and inflammatory cytokine release. Our analysis of the emergence of PRV variants indicate that pigs immunized with the classical PRV vaccine are incapable of providing sufficient protection against these PRV isolates, and there is a risk of continuous evolution and virulence enhancement. Efforts are still needed to conduct epidemiological monitoring for the PRV and to develop novel vaccines against this emerging and reemerging infectious disease.

The non-specific innate immunity can initiate host antiviral innate immune responses within minutes to hours after the invasion of pathogenic microorganisms. Therefore, the natural immune response is the first line of defense for the host to resist the invaders, including viruses, bacteria, fungi. Host pattern recognition receptors (PRRs) in the infected cells or bystander cells recognize pathogen-associated molecular patterns (PAMPs) of invading pathogens and initiate a series of signal cascades, resulting in the expression of type I interferons (IFN-I) and inflammatory cytokines to antagonize the infection of microorganisms. In contrast, the invading pathogens take a variety of mechanisms to inhibit the induction of IFN-I production from avoiding being cleared. Pseudorabies virus (PRV) belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus. PRV is the causative agent of Aujeszky’s disease (AD, pseudorabies). Although the natural host of PRV is swine, it can infect a wide variety of mammals, such as cattle, sheep, cats, and dogs. The disease is usually fatal to these hosts. PRV mainly infects the peripheral nervous system (PNS) in swine. For other species, PRV mainly invades the PNS first and then progresses to the central nervous system (CNS), which leads to acute death of the host with serious clinical and neurological symptoms. In recent years, new PRV variant strains have appeared in some areas, and sporadic cases of PRV infection in humans have also been reported, suggesting that PRV is still an important emerging and re-emerging infectious disease. This review summarizes the strategies of PRV evading host innate immunity and new targets for inhibition of PRV replication, which will provide more information for the development of effective inactivated vaccines and drugs for PRV.

Abstract Background Self-incompatibility (SI) is a major barrier that obstructs the breeding process in most horticultural plants including tea plants (Camellia sinensis). The aim of this study was to elucidate the molecular mechanism of SI in tea plants through a high throughput transcriptome analysis. Results In this study, the transcriptomes of self- and cross-pollinated pistils of two tea cultivars ‘Fudingdabai’ and ‘Yulv’ were compared to elucidate the SI mechanism of tea plants. In addition, the ion components and pollen tube growth in self- and cross-pollinated pistils were investigated. Our results revealed that both cultivars had similar pollen activities and cross-pollination could promote the pollen tube growth. In tea pistils, the highest ion content was potassium (K+), followed by calcium (Ca2+), magnesium (Mg2+) and phosphorus (P5+). Ca2+ content increased after self-pollination but decreased after cross-pollination, while K+ showed reverse trend with Ca2+. A total of 990 and 3 common differentially expressed genes (DEGs) were identified in un-pollinated vs. pollinated pistils and self- vs. cross-pollinated groups after 48 h, respectively. Function annotation indicated that three genes encoding UDP-glycosyltransferase 74B1 (UGT74B1), Mitochondrial calcium uniporter protein 2 (MCU2) and G-type lectin S-receptor-like serine/threonine-protein kinase (G-type RLK) might play important roles during SI process in tea plants. Conclusion Ca2+ and K+ are important signal for SI in tea plants, and three genes including UGT74B1, MCU2 and G-type RLK play essential roles during SI signal transduction.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-22150-y

Temperature and humidity are important parameters in the operation of proton exchange membrane fuel cell (PEMFC), which have an important impact on the performance of fuel cell. Fuel cell test platform is an important tool to study the performance of fuel cells, and its temperature and humidity control module is also the key in the research process of the test platform, so that it can provide the gas with precise temperature and humidity control during the test process of the fuel cell. In this paper, a humidifier combined with bubbling and spraying is adopted for the application of test platform, and the numerical simulation model of the humidifier is established. According to the model, the influence of operating conditions of humidifier on humidification performance is verified, such as inlet air velocity and the humidifying water temperature. The results indicate that the inlet air velocity and the humidifying water temperature have great influence on the humidifying performance of the humidifier. The humidifying performance decreases with the increase of the inlet air velocity and increases with the increase of the humidifying water temperature respectively. In addition, the humidification performance of the humidifier is verified.

Tea products made from purple leaves are highly preferred by consumers due to the health benefits. This study developed a proteome reference map related to color changes during leaf growth in tea (Camellia sinensis) plant with purple young shoots using two-dimensional electrophoresis (2-DE). Forty-six differentially expressed proteins were detected in the gel and successfully identified by using MALDI-TOF/TOF-MS. The pronounced changes in the proteomic profile between tender purple leaves (TPL) and mature green leaves (MGL) included: 1) the lower activity of proteins associated with CO2 assimilation, energy metabolism and photo flux efficiency and higher content of anthocyanins in TPL than those in MGL may protect tender leaves against photo-damage; 2) the higher abundance of chalcone synthase (CHS), chalcone isomerase (CHI) and flavonol synthase (FLS) likely contributes to the synthesis of anthocyanins, catechins and flavonols in TPL tissues; 3) higher abundance of stress response proteins, such as glutathione S-transferases (GST) and phospholipid hydroperoxide glutathione peroxidase (PHGPx), could enhance the tolerance of TPL tissues to adverse condition in; and 4) the increased abundance of proteins related to protein synthesis, nucleic acids and cell wall proteins should be beneficial for the proliferation and expansion of leaf cell in TPL tissues. qPCR analysis showed that the expression of differentially abundant proteins was regulated at the transcriptional level. Therefore, the results indicated that higher abundance of CHI and CHS may account for the production of the purple-shoot phenotype in Wuyiqizhong 18 and thereby, enhancing the anthocyanin biosynthesis. The higher abundance of glutamine synthetase (GS) proteins related to the theanine biosynthesis may improve the flavor of tea products from TPL materials. Thus, this work should help to understand the molecular mechanisms underlying the changes in leaf color alteration.

The proton exchange membrane fuel cell (PEMFC) is taken to be the ultimate technical direction of vehicle power. Cooling system is a key component which directly affects the fuel cell performance, reliability and durability. For the purpose of keeping accurate temperature control under dynamic loads and achieving rapid warm-up control during cold-start, a 35 kW PEMFC’s cooling system dynamic model is established and validated by experiments firstly. According to the simulation results, the model can well be fitted to the actual system. Then an integrate separate PID (Proportional-Integral-Derivative) algorithm and cooling fan prestart strategy is proposed. The result shows that it can effectively reduce the temperature overshoot under dynamic loads. In view of the thermostat mechanical characteristics tend to cause large temperature fluctuation during warm-up process, a thermostat control strategy is proposed to reduce the temperature fluctuation from 7.5 °C to 0.4 °C.

AIM:Pig liver carboxylesterase (PLE) gene sequences in GenBank are incomplete, which has led to difficulties in studying the genetic structure and regulation mechanisms of gene expression of PLE family genes. The aim of this study was to obtain and analysis of complete gene sequences of PLE family by screening from a Rongchang pig BAC library and third-generation PacBio gene sequencing. METHODS:After a number of existing incomplete PLE isoform gene sequences were analysed, primers were designed based on conserved regions in PLE exons, and the whole pig genome used as a template for Polymerase chain reaction (PCR) amplification. Specific primers were then selected based on the PCR amplification results. A three-step PCR screening method was used to identify PLE-positive clones by screening a Rongchang pig BAC library and PacBio third-generation sequencing was performed. BLAST comparisons and other bioinformatics methods were applied for sequence analysis. RESULTS:Five PLE-positive BAC clones, designated BAC-10, BAC-70, BAC-75, BAC-119 and BAC-206, were identified. Sequence analysis yielded the complete sequences of four PLE genes, PLE1, PLE-B9, PLE-C4, and PLE-G2. Complete PLE gene sequences were defined as those containing regulatory sequences, exons, and introns. It was found that, not only did the PLE exon sequences of the four genes show a high degree of homology, but also that the intron sequences were highly similar. Additionally, the regulatory region of the genes contained two 720bps reverse complement sequences that may have an important function in the regulation of PLE gene expression. SIGNIFICANCE:This is the first report to confirm the complete sequences of four PLE genes. In addition, the study demonstrates that each PLE isoform is encoded by a single gene and that the various genes exhibit a high degree of sequence homology, suggesting that the PLE family evolved from a single ancestral gene. Obtaining the complete sequences of these PLE genes provides the necessary foundation for investigation of the genetic structure, function, and regulatory mechanisms of the PLE gene family.

Mitochondria are major cellular sources of hydrogen peroxide (H(2)O(2)), the production of which is modulated by oxygen availability and the mitochondrial energy state. An increase of steady-state cell H(2)O(2) concentration is able to control the transition from proliferating to quiescent phenotypes and to signal the end of proliferation; in tumor cells thereby, low H(2)O(2) due to defective mitochondrial metabolism can contribute to sustain proliferation. Mitogen-activated protein kinases (MAPKs) orchestrate signal transduction and recent data indicate that are present in mitochondria and regulated by the redox state. On these bases, we investigated the mechanistic connection of tumor mitochondrial dysfunction, H(2)O(2) yield, and activation of MAPKs in LP07 murine tumor cells with confocal microscopy, in vivo imaging and directed mutagenesis. Two redox conditions were examined: low 1 microM H(2)O(2) increased cell proliferation in ERK1/2-dependent manner whereas high 50 microM H(2)O(2) arrested cell cycle by p38 and JNK1/2 activation. Regarding the experimental conditions as a three-compartment model (mitochondria, cytosol, and nuclei), the different responses depended on MAPKs preferential traffic to mitochondria, where a selective activation of either ERK1/2 or p38-JNK1/2 by co-localized upstream kinases (MAPKKs) facilitated their further passage to nuclei. As assessed by mass spectra, MAPKs activation and efficient binding to cognate MAPKKs resulted from oxidation of conserved ERK1/2 or p38-JNK1/2 cysteine domains to sulfinic and sulfonic acids at a definite H(2)O(2) level. Like this, high H(2)O(2) or directed mutation of redox-sensitive ERK2 Cys(214) impeded binding to MEK1/2, caused ERK2 retention in mitochondria and restricted shuttle to nuclei. It is surmised that selective cysteine oxidations adjust the electrostatic forces that participate in a particular MAPK-MAPKK interaction. Considering that tumor mitochondria are dysfunctional, their inability to increase H(2)O(2) yield should disrupt synchronized MAPK oxidations and the regulation of cell cycle leading cells to remain in a proliferating phenotype.

Pseudorabies virus (PRV) infection activates inflammatory responses to release robust proinflammatory cytokines, which are critical for controlling viral infection and clearance of PRV. However, the innate sensors and inflammasomes involved in the production and secretion of proinflammatory cytokines during PRV infection remain poorly studied. In this study, we report that the transcription and expression levels of some proinflammatory cytokines, including interleukin 1 b (IL-1 b), IL-6, and tumor necrosis factor alpha (TNF- a), are upregulated in primary peritoneal macrophages and in mice during PRV infection. Mechanistically, Toll-like receptor 2 (TLR2), TLR3, TLR4, and TLR5 were induced by the PRV infection to enhance the transcription levels of pro-IL-1 b, pro-IL-18, and gasdermin D (GSDMD). Additionally, we found that PRV infection and transfection of its genomic DNA triggered AIM2 inflammasome activation, apoptosis-related speckle-like protein (ASC) oligomerization, and caspase-1 activation to enhance the secretion of IL-1 b and IL-18, which was mainly dependent on GSDMD, but not GSDME, in vitro and in vivo. Taken together, our findings reveal that the activation of the TLR2-TLR3-TRL4-TLR5-NF- kB axis and AIM2 inflammasome, as well as GSDMD, is required for proinflammatory cytokine release, which resists the PRV replication and plays a critical role in host defense against PRV infection. Our findings provide novel clues to prevent and control PRV infection. IMPORTANCE PRV can infect several mammals, including pigs, other livestock, rodents, and wild animals, causing huge economic losses. As an emerging and reemerging infectious disease, the emergence of PRV virulent isolates and increasing human PRV infection cases indicate that PRV is still a high risk to public health. It has been reported that PRV infection leads to robust release of proinflammatory cytokines through activating inflammatory responses. However, the innate sensor that activates IL-1 b expression and the inflammasome involved in the maturation and secretion of proinflammatory cytokines during PRV infection remain poorly studied. In this study, our findings reveal that, in mice, activation of the TLR2-TLR3-TRL4- TLR5-NF- kB axis and AIM2 inflammasome, as well as GSDMD, is required for proin- flammatory cytokine release during PRV infection, and it resists PRV replication and plays a critical role in host defense against PRV infection. Our findings provide novel clues to prevent and control PRV infection. [ABSTRACT FROM AUTHOR]