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KEY MESSAGE: MdERF023 is a transcription factor that can reduce salt tolerance by inhibiting ABA signaling and Na+/H+ homeostasis. Salt stress is one of the principal environmental stresses limiting the growth and productivity of apple (Malus × domestica). The APETALA2/ethylene response factor (AP2/ERF) family plays key roles in plant growth and various stress responses; however, the regulatory mechanism involved has not been fully elucidated. In the present study, we identified an AP2/ERF transcription factor (TF), MdERF023, which plays a negative role in apple salt tolerance. Stable overexpression of MdERF023 in apple plants and calli significantly decreased salt tolerance. Biochemical and molecular analyses revealed that MdERF023 directly binds to the promoter of MdMYB44-like, a positive modulator of ABA signaling-mediated salt tolerance, and suppresses its transcription. In addition, MdERF023 downregulated the transcription of MdSOS2 and MdAKT1, thereby reducing the Na+ expulsion, K+ absorption, and salt tolerance of apple plants. Taken together, these results suggest that MdERF023 reduces apple salt tolerance by inhibiting ABA signaling and ion transport, and that it could be used as a potential target for breeding new varieties of salt-tolerant apple plants via genetic engineering.
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Regulação da Expressão Gênica de Plantas , Malus , Proteínas de Plantas , Tolerância ao Sal , Transdução de Sinais , Fatores de Transcrição , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Malus/genética , Malus/metabolismo , Malus/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Tolerância ao Sal/genética , Sódio/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Activating transcription factor 6 (ATF6), an important signaling molecule in unfolded protein response (UPR), plays a role in the pathogenesis of several diseases, including diseases such as congenital retinal disease, liver fibrosis and ankylosing spondylitis. After endoplasmic reticulum stress (ERS), ATF6 is activated after separation from binding immunoglobulin protein (GRP78/BiP) in the endoplasmic reticulum (ER) and transported to the Golgi apparatus to be hydrolyzed by site 1 and site 2 proteases into ATF6 fragments, which localize to the nucleus and regulate the transcription and expression of ERS-related genes. In these diseases, ERS leads to the activation of UPR, which ultimately lead to the occurrence and development of diseases by regulating the physiological state of cells through the ATF6 signaling pathway. Here, we discuss the evidence for the pathogenic importance of ATF6 signaling in different diseases and discuss preclinical results.
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Background: Natural killer (NK) cells play important immunoregulatory roles in the immune pathogenesis of severe aplastic anemia (SAA). Our previous research showed that SAA caused a decrease in T cell immunoglobulin mucin-3 (TIM3) expression on NK cells. Here we investigated the expression of surface receptors, and the cytotoxicity of peripheral TIM3+ NK and TIM3- NK cells in patients with SAA. Methods: The expressions of surface receptors and cytoplasmic protein of TIM3+ NK and TIM3- NK cells from peripheral blood were detected by FCM. The functions of mDCs, and apoptosis rate of K562 cells after co-culture with TIM3+ NK and TIM3- NK cells were maesured by FCM. Westren-blot was used to detect the changes of TIM3+ NK and TIM3- NK signaling pathway proteins (AKT, P-AKT) and compare the functional activity of the two groups. Results: Activating receptors NKG2D and Granzyme B were higher, while inhibiting receptors NKG2A, CD158a and CD158b were lower on TIM3- NK cells compared with TIM3+ NK cells in patients with SAA. In SAA, the expression of CD80 and CD86 on mDCs (Myeloid dendritic cells) was significantly decreased after incubation with TIM3- NK cells. The apoptosis rate (AR) of K562 cells was significantly increased after being incubated with TIM3- NK cells in SAA. The level of signal pathway protein AKT of TIM3- NK cells in SAA was similar to that of TIM3+ NK cells, and the levels of P-AKT and P-AKT/AKT ratio of TIM3- NK cells were significantly higher than those of TIM3+ NK cells. Conclusions: Therefore, TIM3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA. Low expression of TIM3 contributes to the enhancement of NK cell activity which in turn inhibits the immune activation state of SAA and improves the disease state. Our research may aid the development of new therapeutic strategies based on TIM3-NK cells infusion for the treatment of SAA.
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MHY1485 is an mTOR activator that inhibits the autophagy process by inhibiting the fusion between autophagosomes and lysosomes. This study aimed to explore the role and mechanism of MHY1485 in hepatocellular carcinoma (HCC) and to provide an in-depth understanding of the mechanisms of autophagy regulation in relation to adriamycin (ADM) resistance, as well as the development of a molecularly targeted autophagy-modulating approach. Here, ADM was used to treat HepG2 cells and construct an ADM-resistant cell model. The HepG2/ADM cell line and HepG2 cells were treated with MHY1485 and ADM, respectively, and the proliferation and apoptosis of HCC cells were detected using CCK8, clone formation, flow cytometry, and 5-ethynyl-2'-deoxyuridine staining assays. Ki-67, mTOR phosphorylation, and LC3A expression were detected by IF staining; the expression or phosphorylation levels of autophagy-related proteins (i.e., GLUT1, PGI, PFK, END, and MTHFD2) and apoptosis-related proteins (caspase-3, caspase-8, and caspase-9) were detected by qPCR and western blotting. The number of autophagosomes was determined by monodansylcadaverine staining. Our results showed that MHY1485 can inhibit the proliferation and growth of liver cancer cells, and that MHY1485 combined with ADM can effectively inhibit the tolerance of HepG2/ADM cells to ADM and enhance the efficacy of ADM. The results of the detection of the autophagy-related protein LC3A also indicated that MHY1485 activates mTOR and can affect the phosphorylation level of ULK1, inhibit autophagy, and enhance the sensitivity of liver cancer cells to adriamycin. In summary, MHY1485 can enhance the sensitivity of adriamycin-resistant cells to adriamycin by activating mTOR and blocking the autophagy process in cells; therefore, mTOR may become a potential target for the treatment of liver cancer.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Morfolinas , Triazinas , Humanos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Doxorrubicina/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Células Hep G2 , Apoptose , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Autofagia , Proliferação de Células , Linhagem Celular TumoralRESUMO
To retrospectively analyze the efficacy and safety of venetoclax combined with azacitidine (VEN + AZA) in the treatment of elderly patients with acute myeloid leukemia. The clinical data for 57 AML patients treated with the VEN + AZA regimen from December 2019 to November 2022 in the Department of Hematology, General Hospital of Tianjin Medical University, were collected. Of the 57 patients included in this study, the mean age of onset was 69.89 (±8.88) years. The median follow-up time was 8.57 months, and the median OS time was 11.50 months. The ORR, CR rate, and MRD (<0.1%) negativity rate were 87.5%, 68.8%, and 58.3%, respectively. The median OS was longer in patients who achieved CR/CRi and who were MRD-negative than in those who did not. MRD negativity was less likely to be achieved in patients aged ≥75 years and with ECOG scores of ≥3. Compared to traditional intensive chemotherapy, MRD negative was achieved more quickly with VEN + AZA regimens in patients with newly diagnosed AML. Advanced age and ECOG score were risk factors for negative MRD. The dominant adverse reactions were hematological adverse events. VEN + AZA regimens in elderly unfit patients with previously untreated newly diagnosed AML have sufficient efficacy and safety.
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Azacitidina , Leucemia Mieloide Aguda , Sulfonamidas , Idoso , Humanos , Pessoa de Meia-Idade , Azacitidina/uso terapêutico , Estudos Retrospectivos , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêuticoRESUMO
Abscisic acid (ABA) is involved in salt and drought stress responses, but the underlying molecular mechanism remains unclear. Here, we demonstrated that the overexpression of MdMYB44-like, an R2R3-MYB transcription factor, significantly increases the salt and drought tolerance of transgenic apples and Arabidopsis. MdMYB44-like inhibits the transcription of MdPP2CA, which encodes a type 2C protein phosphatase that acts as a negative regulator in the ABA response, thereby enhancing ABA signaling-mediated salt and drought tolerance. Furthermore, we found that MdMYB44-like and MdPYL8, an ABA receptor, form a protein complex that further enhances the transcriptional inhibition of the MdPP2CA promoter by MdMYB44-like. Significantly, we discovered that MdPP2CA can interfere with the physical association between MdMYB44-like and MdPYL8 in the presence of ABA, partially blocking the inhibitory effect of the MdMYB44-like-MdPYL8 complex on the MdPP2CA promoter. Thus, MdMYB44-like, MdPYL8, and MdPP2CA form a regulatory loop that tightly modulates ABA signaling homeostasis under salt and drought stress. Our data reveal that MdMYB44-like precisely modulates ABA-mediated salt and drought tolerance in apples through the MdPYL8-MdPP2CA module.
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Arabidopsis , Malus , Malus/genética , Malus/metabolismo , Resistência à Seca , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Cloreto de Sódio/farmacologia , Arabidopsis/metabolismo , Ácido Abscísico/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Estresse FisiológicoRESUMO
BACKGROUND: It is difficult to definitively determine the degree of ischemia in the bowel in which an incarcerated groin hernia is embedded. Failure to diagnose and intervene promptly and accurately increases the rate of bowel resection and patient mortality. The aim of this study is to investigate the risk factors for incarcerated inguinal hernia complicating bowel necrosis with resection and to establish a predictive model as a reference for clinical work. METHODS: Patients with incarcerated groin hernia who were admitted to our hospital were retrospectively analyzed. They were divided into bowel resection and non-bowel resection groups based on whether bowel resection was performed in the surgical record and postoperative pathological results. Risk factors for the development of bowel resection in incarcerated groin hernia were analyzed by univariate analysis and multivariate logistic regression, respectively. The screened independent risk factors were used to establish a prediction model, and finally, the predictive ability and accuracy of the model were validated and the clinical benefit was analyzed. RESULTS: A total of 345 patients with incarcerated groin hernia were included, of whom 58 underwent bowel resection for bowel necrosis and 287 did not. Multifactorial logistic regression analysis identified bowel obstruction (OR, 7.285 [95% CI, 2.254-23.542], P = 0.001), peritonitis (OR, 16.786 [95% CI, 5.436-51.838], P = 0.000), duration of incarcerated groin hernia (OR, 1.009 [95% CI, 1. 001-1.018], P = 0.034), heart rate (OR, 1.109 [95% CI, 1.021-1.205], P = 0.014), and preoperative total protein (OR, 0.900 [95% CI, 0.836-0.969], P = 0.005) were independent risk factors for bowel resection in incarcerated groin hernia. The predictive value of the established prediction model was basically in agreement with the measured value with a consistency index of 0.938 (0.901-0.974) and had a good clinical benefit. CONCLUSION: Clinical screening and management of independent risk factors for bowel resection in patients with incarcerated groin hernia should be strengthened. The predictive model developed in this study has high diagnostic efficacy for bowel resection associated with incarcerated inguinal hernia, with the aim of reducing the incidence of bowel resection and unplanned secondary surgery.
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Hérnia Inguinal , Adulto , Humanos , Hérnia Inguinal/complicações , Hérnia Inguinal/cirurgia , Estudos Retrospectivos , Virilha/cirurgia , Intestinos/cirurgia , Herniorrafia/métodos , NecroseRESUMO
The human stress hormones catecholamines play a critical role in communication between human microbiota and their hosts and influence the outcomes of bacterial infections. However, it is unclear how M. tuberculosis senses and responds to certain types of human stress hormones. In this study, we screened several human catecholamine stress hormones (epinephrine, norepinephrine, and dopamine) for their effects on Mycobacterium growth. Our results showed that epinephrine significantly stimulated the growth of M. tuberculosis in the serum-based medium as well as macrophages. In silico analysis and molecular docking suggested that the extra-cytoplasmic domain of the MprB might be the putative adrenergic sensor. Furthermore, we showed that epinephrine significantly enhances M. tuberculosis biofilm formation, which has distinct texture composition, antibiotic resistance, and stress tolerance. Together, our data revealed the effect and mechanism of epinephrine on the growth and biofilm formation of M. tuberculosis, which contributes to the understanding of the environmental perception and antibiotic resistance of M. tuberculosis and provides important clues for the understanding of bacterial pathogenesis and the development of novel antibacterial therapeutics.
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Mycobacterium tuberculosis , Tuberculose , Humanos , Simulação de Acoplamento Molecular , Epinefrina/farmacologia , Catecolaminas , Biofilmes , Hormônios , Mycobacterium smegmatis , Proteínas de BactériasRESUMO
IMPORTANCE: African swine fever (ASF), caused by African swine fever virus (ASFV), has become a major crisis for the pork industry in recent years. The mechanism for ASFV pathology and the clinical symptoms difference of ASF between domestic pigs and reservoir hosts remain to be elucidated. We deciphered the comprehensive protein-protein interaction (PPI) network between ASFV and host immune pathways. The intensive PPI network contained both ASFV-host immune pathway PPI and ASFV-ASFV PPI information, providing a comprehensive ASFV-host interaction landscape. Furthermore, the ASFV-host PPI difference between domestic pigs and warthogs was explored, which will be instructive for exploring essential candidates involved in ASFV pathology. Moreover, we screened the inhibitory effect of ASFV proteins in the PPI with cGAS-STING pathway on IFN-I and NF-κB, further providing possible functions of ASFV-host PPI network in innate immune regulation.
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Vírus da Febre Suína Africana , Febre Suína Africana , Interferon Tipo I , Suínos , Animais , Febre Suína Africana/metabolismo , Sus scrofa , NF-kappa B/metabolismo , Interferon Tipo I/metabolismoRESUMO
Tetracycline (TC) is an effective antibiotic used to treat humans and livestock, but its inappropriate use imposes toxic effects, including pollution, on environmental ecology and food. Currently, sensitive, accurate, and cost-effective methods that can detect lower concentrations of TC residues in environmental and food samples are needed. In this study, a novel indirect competitive assay-based aptamer method was developed for detecting TC residues through signal amplification by real-time fluorescence-based quantitative polymerase chain reaction. The response surface methodology was introduced to optimize the optimal concentrations (influencing factors) of the three types of single-stranded DNA in the competitive assay process. The optimal conditions for the three types of ssDNA were 112 nM for the specific aptamer of TC (Apt40), 115 nM for the signal DNA, and 83 nM for the DNA catcher. As expected, under optimal conditions, the Ct value was linearly related to the logarithm of TC concentration. The calibration curve equation was Ct = -0.34516 log[TC] + 9.9345 (R2 = 0.998) in the range of 10-3-103 ng mL-1, and the limit of detection was 7.02 × 10-5 ng mL-1. The new method was effectively applied to detect TC residues in wastewater, honey, and milk samples. It achieved an average recovery rate of 101.19% with a small variation of 5.16%. The validation was carried out using an enzyme-linked immunosorbent assay. This approach demonstrates high sensitivity and selectivity, making it well suited for detecting leftover antibiotics in food when using suitable aptamers.
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Leaves and flowers are crucial for the growth and development of higher plants. In this study we identified a mutant with narrow leaflets and early flowering (nlef) in an ethyl methanesulfonate-mutagenized population of woodland strawberry (Fragaria vesca) and aimed to identify the candidate gene. Genetic analysis revealed that a single recessive gene, nlef, controlled the mutant phenotype. We found that FvH4_1g25470, which encodes a putative DNA polymerase α with a polymerase and histidinol phosphatase domain (PHP), might be the candidate gene, using bulked segregant analysis with whole-genome sequencing, molecular markers, and cloning analyses. A splice donor site mutation (C to T) at the 5' end of the second intron led to an erroneous splice event that reduced the expression level of the full-length transcript of FvePHP in mutant plants. FvePHP was localized in the nucleus and was highly expressed in leaves. Silencing of FvePHP using the virus-induced gene silencing method resulted in partial developmental defects in strawberry leaves. Overexpression of the FvePHP gene can largely restore the mutant phenotype. The expression levels of FveSEP1, FveSEP3, FveAP1, FveFUL, and FveFT were higher in the mutants than those in 'Yellow Wonder' plants, probably contributing to the early flowering phenotype in mutant plants. Our results indicate that mutation in FvePHP is associated with multiple developmental pathways. These results aid in understanding the role of DNA polymerase in strawberry development.
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Alternaria alternata (A. alternata) is a common pathogen that greatly influences apples' quantity and quality. However, chemical treatments produce increased health risks along with decreased food and environmental safety. Advancements in plant molecular biology, such as transgenic technology, have increased apple trees' resistance to pathogens and have therefore attracted widespread attention. WRKY transcription factors are involved in abiotic and biotic stress regulation; however, their biological role in non-model plants such as apple, is still unknown. In this investigation, MdWRKY120 was isolated from the 'GL-3' apple to determine its function during Alternaria alternate infection. The MdWRKY120-GFP fusion protein was located in the nucleus. MdWRKY120 in yeast cells exhibited activating transcriptional activity, meaning it is a transcription activator. MdWRKY120 overexpression transgenic plants were more sensitive to A. alternata, while RNAi transgenic plants showed increased resistance to A. alternata. This investigation demonstrates that MdWRKY120 enhances the susceptibility of apples to A. alternata.
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The nervous necrosis virus (NNV) mainly attacks the central nervous system of fish to cause viral nervous necrosis, which is an acute and serious prevalent disease in fish. Among different genotypes of NNV, red-spotted grouper nervous necrosis virus (RGNNV) is the most widely reported, with the highest number of susceptible species. To better understand the pathogenicity of RGNNV, we first developed a reverse genetic system for recombinant RGNNV rescue using B7GG and striped snakehead (SSN-1) cells. Furthermore, we constructed attenuated RGNNV strains rRGNNV-B2-M1 and rRGNNV-B2-M2 with the loss of B2 protein expression, which grew slower and induced less Mx1 expression than that of wild-type RGNNV. Moreover, rRGNNV-B2-M1 and rRGNNV-B2-M2 were less virulent than the wild-type RGNNV. Our study provides a potential tool for further research on the viral protein function, virulence pathogenesis, and vaccine development of RGNNV, which is also a template for the rescue of other fish viruses.
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Bass , Doenças dos Peixes , Nodaviridae , Infecções por Vírus de RNA , Animais , Bass/genética , Necrose , Nodaviridae/genética , Infecções por Vírus de RNA/veterinária , Genética ReversaRESUMO
Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.
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Mycobacterium tuberculosis , Tuberculose , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Edição de Genes , Interferência de RNA , Tuberculose/prevenção & controle , Tuberculose/genética , Tuberculose/microbiologia , Antituberculosos/metabolismo , Sistemas CRISPR-CasRESUMO
Mycobacterium tuberculosis can invade different cells with distinct persistence fates because cells are equipped with different host restriction factors. However, the underlying mechanisms remain elusive. Here, we infected THP1 and Raw264.7 macrophages cell lines, A549 epithelial cell line, and hBMEC and bEnd.3 endothelial cell lines with M. tuberculosis and demonstrated that M. tuberculosis significantly inhibited lysosome acidification in THP1, hBMEC, A549, and Raw264.7 cells, while, in bEnd.3 cells, M. tuberculosis was mainly delivered into acidified phagolysosomes and auto-lysosomes. The systematic gene profile analysis of different cells and intracellular M. tuberculosis showed that the phagosome autophagy-pathway-related genes itgb3 and atg3 were highly expressed in bEnd.3 cells. Knockdown of these genes significantly increased the number of viable intracellular M. tuberculosis bacilli by altering phagosomal trafficking in bEnd.3 cells. Treatment with itgb3 agonist significantly decreased M. tuberculosis survival in vivo. These findings could facilitate the identification of anti-M. tuberculosis host genes and guide M. tuberculosis-resistant livestock breeding. IMPORTANCE As an intracellular pathogen, Mycobacterium tuberculosis could avoid host cell immune clearance using multiple strategies for its long-term survival. Understanding these processes could facilitate the development of new approaches to restrict intracellular M. tuberculosis survival. Here, we characterized the detailed molecular events occurring during intracellular trafficking of M. tuberculosis in macrophage, epithelial, and endothelial cell lines and found that ITGB3 facilitates M. tuberculosis clearance in endothelial cells through altering phagosomal trafficking. Meanwhile, the treatment with ITGB3 agonist could reduce bacterial load in vivo. Our results identified new anti-M. tuberculosis restriction factors and illuminated a new anti-M. tuberculosis defense mechanism.
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Tuberculosis (TB) is a debilitating infectious disease responsible for more than one million deaths per year. The emergence of drug-resistant TB poses an urgent need for the development of new anti-TB drugs. In this study, we screened a library of over 4,000 small molecules and found that orbifloxacin and the peptide AK15 possess significant bactericidal activity against Mycobacterium tuberculosis (Mtb) in vitro. Orbifloxacin also showed an effective ability on the clearance of intracellular Mtb and protect mice from a strong inflammatory response but not AK15. Moreover, we identified 17 nucleotide mutations responsible for orbifloxacin resistance by whole-genome sequencing. A critical point mutation (D94G) of the DNA gyrase (gyrA) gene was found to be the key role of resistance to orbifloxacin. The computational docking revealed that GyrA D94G point mutation can disrupt the orbifloxacin-protein gyrase interactions mediated by magnesium ion bridge. Overall, this study indicated the potential ability of orbifloxacin as an anti-tuberculosis drug, which can be used either alone or in combination with first-line antibiotics to achieve more effective therapy on TB.
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Mycobacterium tuberculosis (M.tb) secretes numerous proteins to interfere with host immune response for its long-term survival. As one of the top abundant M.tb secreted proteins, Rv3722c was found to be essential for bacilli growth. However, it remains elusive how this protein interferes with the host immune response and regulates M.tb survival. Here, we confirmed that Rv3722c interacted with host TRAF3 to promote M.tb replication in macrophages. Knock-down of TRAF3 attenuated the effect of Rv3722c on the intracellular M.tb survival. The interaction between Rv3722c and TRAF3 hampered MAPK and NF-κB pathways, resulting in a significant increase of IFN-ß expression and decrease of IL-1ß, IL-6, IL-12p40, and TNF-α expression. Our study revealed that Rv3722c interacted with TRAF3 and interrupted its downstream pathways to promote M.tb survival in macrophages. These findings facilitate further understanding of the mechanism of M.tb secreted proteins in regulating the host cell immune response and promoting its intracellular survival.
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Mycobacterium tuberculosis , Tuberculose , Humanos , Macrófagos , Transdução de Sinais , Fator 3 Associado a Receptor de TNFRESUMO
Herpesvirus based multivalent vaccines have been extensively studied, whereas few of them have been successfully used in clinic and animal husbandry industry due to the low expression of foreign immunogens in herpesvirus. In this study, we developed a new strategy to construct herpesvirus based bivalent vaccine with high-level expression of foreign immunogen, by which the ORF2 gene encoding the major antigen protein Cap of porcine circovirus type 2 (PCV2), was highly expressed in pseudorabies virus (PRV). To obtain the high expression of PCV2 immunogen, tandem repeats of PCV2 ORF2 gene were firstly linked by protein quantitation ratioing (PQR) linker to reach equal expression of each ORF2 gene. Then, the multiple copies of ORF2 gene were respectively inserted into the gE and gG sites of PRV using CRISPR/Cas9 system, in which the expression of ORF2 gene was driven by endogenous strong promoters of PRV. Through this way, the highest yield of Cap protein was achieved in two copies of quadruple ORF2 gene insertion. Finally, in mice and pigs immunized with the bivalent vaccine candidate, we detected high titer of specific antibodies for PRV and neutralized antibodies for PCV2, and observed protective effect of the bivalent vaccine candidate against PRV challenge in immunized pigs, suggesting a potential clinical application of the bivalent vaccine candidate we constructed. Together, our strategy could be extensively applied to the generation of other multivalent vaccines, and will pave the way to construct herpesvirus based multivalent vaccines to effectively reduce the cost of vaccine.
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Circovirus/imunologia , Herpesvirus Suídeo 1 , Vacinas contra Pseudorraiva/imunologia , Pseudorraiva/prevenção & controle , Doenças dos Suínos/prevenção & controle , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Suínos , Doenças dos Suínos/sangue , Proteínas Virais/imunologiaRESUMO
The WRKY proteins are a large family of transcription factors that play important roles in stress responses and plant development. However, the roles of most WRKYs in strawberry are not well known. In this study, FvWRKY71 was isolated from the woodland strawberry 'Ruegen'. FvWRKY71 was highly expressed in the shoot apex and red fruit. Subcellular localization analysis showed that FvWRKY71 was located in the nucleus. Transactivation analysis showed that FvWRKY71 presented transcriptional activation activity in yeast. Overexpression of FvWRKY71 in Arabidopsis and woodland strawberry revealed early flowering in the transgenic plants compared with the wild-type control. Gene expression analysis indicated that the transcript levels of the flowering time and development integrator genes AP1, LFY, FT, AGL42, FUL, FPF1, SEP1, SEP2, and SEP3 were increased in FvWRKY71-overexpressing Arabidopsis and strawberry plants compared with the wild-type controls, which may result in accelerated flowering in transgenic plants. Furthermore, FvWRKY71 was proven to directly bind to the W-boxes (TTGACT/C) of the FvFUL, FvSEP1, FvAGL42, FvLFY, and FvFPF1 promoters in vitro and in vivo. Taken together, our results reveal a transcriptional regulatory cascade of FvWRKY71 involved in promoting flowering in woodland strawberry.
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Fragaria nilgerrensis is a wild diploid strawberry species endemic to east and southeast region in Asia and provides a rich source of genetic variations for strawberry improvement. Here, we present a chromosome-scale assembly of F. nilgerrensis using single-molecule real-time (SMRT) Pacific Biosciences sequencing and chromosome conformation capture (Hi-C) genome scaffolding. The genome assembly size was 270.3 Mb, with a contig N50 of â¼8.5 Mb. A total of 28 780 genes and 117.2 Mb of transposable elements were annotated for this genome. Next, detailed comparative genomics with the high-quality F. vesca reference genome was conducted to obtain the difference among transposable elements, SNPs, Indels, and so on. The genome size of F. nilgerrensis was enhanced by around 50 Mb relatively to F. vesca, which is mainly due to expansion of transposable elements. In comparison with the F. vesca genome, we identified 4 561 825 SNPs, 846 301 Indels, 4243 inversions, 35 498 translocations and 10 099 relocations. We also found a marked expansion of genes involved in phenylpropanoid biosynthesis, starch and sucrose metabolism, cyanoamino acid metabolism, plant-pathogen interaction, brassinosteroid biosynthesis and plant hormone signal transduction in F. nilgerrensis, which may account for its specific phenotypes and considerable environmental adaptability. Interestingly, we found sequence variations in the upstream regulatory region of FnMYB10, a core transcriptional activator of anthocyanin biosynthesis, resulted in the low expression level of the FnMYB10 gene, which is likely responsible for white fruit phenotype of F. nilgerrensis. The high-quality F. nilgerrensis genome will be a valuable resource for biological research and comparative genomics research.