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2.
J Virol ; 94(2)2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31666373

RESUMO

The low-pathogenic H7N9 influenza viruses that emerged in 2013 acquired an insertion of four amino acids in their hemagglutinin cleavage site and thereby became highly pathogenic to chickens in 2017. Previous studies indicated that these highly pathogenic H7N9 viruses are virulent in chickens but have distinct pathotypes in mice. A/chicken/Guangdong/SD098/2017 (CK/SD098) is avirulent, with a 50% mouse lethal dose (MLD50) of >7.5 log10 50% egg infectious dose (EID50), whereas A/chicken/Hunan/S1220/2017 (CK/S1220) is virulent in mice, with an MLD50 of 3.2 log10 EID50 In this study, we explored the genetic determinants that contribute to the difference in virulence between these two H7N9 viruses by generating a series of reassortants and mutants in the CK/S1220 virus background and testing their virulence in mice. We found that the reassortant CK/1220-SD098-NP, carrying the nucleoprotein (NP) of CK/SD098, was avirulent in mice, with an MLD50 of >107.5 EID50 The NPs of these two viruses differ by two amino acids, at positions 286 and 437. We further demonstrated that the amino acid mutations A286V and T437M of NP independently slowed the process of NP import to and export from the nucleus and thus jointly impaired the viral life cycle and attenuated the virulence of these H7N9 viruses in mice. Our study identified new virulence determinants in NP and provided novel targets for the development of live attenuated vaccines and antiviral drugs against influenza viruses.IMPORTANCE The H7N9 influenza viruses that emerged in China in 2013 have caused over 1,500 human infections, with a mortality rate of nearly 40%. The viruses were initially low pathogenic but became highly pathogenic in chickens at the beginning of 2017 and caused severe disease outbreaks in poultry. Several studies suggested that the highly pathogenic H7N9 viruses have increased virulence in mammals; however, the genetic basis of the virulence of H7N9 viruses in mammals is not fully understood. Here, we found that two amino acids, 286A and 437T, in NP are prerequisites for the virulence of H7N9 viruses in mice and the mutations A286V and T437M collectively eliminate the virulence of H7N9 viruses in mice. Our study further demonstrated that the virulence of influenza viruses is a polygenic trait, and the newly identified virulence-related residues in NP may provide new targets for attenuated influenza vaccine and antiviral drug development.

3.
J Virol ; 94(2)2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694949

RESUMO

Influenza A virus (IAV) coopts numerous host factors to complete its replication cycle. Here, we identify free fatty acid receptor 2 (FFAR2) as a cofactor for IAV entry into host cells. We found that downregulation of FFAR2 or Ffar2 expression significantly reduced the replication of IAV in A549 or RAW 264.7 cells. The treatment of A549 cells with small interfering RNA (siRNA) targeting FFAR2 or the FFAR2 pathway agonists 2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide (4-CMTB) and compound 58 (Cmp58) [(S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide] dramatically inhibited the nuclear accumulation of viral nucleoprotein (NP) at early time points postinfection, indicating that FFAR2 functions in the early stage of the IAV replication cycle. FFAR2 downregulation had no effect on the expression of sialic acid (SA) receptors on the cell membrane, the attachment of IAV to the SA receptors, or the activity of the viral ribonucleoprotein (vRNP) complex. Rather, the amount of internalized IAVs was significantly reduced in FFAR2-knocked-down or 4-CMTB- or Cmp58-treated A549 cells. Further studies showed that FFAR2 associated with ß-arrestin1 and that ß-arrestin1 interacted with the ß2-subunit of the AP-2 complex (AP2B1), the essential adaptor of the clathrin-mediated endocytosis pathway. Notably, siRNA knockdown of either ß-arrestin1 or AP2B1 dramatically impaired IAV replication, and AP2B1 knockdown or treatment with Barbadin, an inhibitor targeting the ß-arrestin1/AP2B1 complex, remarkably decreased the amount of internalized IAVs. Moreover, we found that FFAR2 interacted with three G protein-coupled receptor (GPCR) kinases (i.e., GRK2, GRK5, and GRK6) whose downregulation inhibited IAV replication. Together, our findings demonstrate that the FFAR2 signaling cascade is important for the efficient endocytosis of IAV into host cells.IMPORTANCE To complete its replication cycle, IAV hijacks the host endocytosis machinery to invade cells. However, the underlying mechanisms of how IAV is internalized into host cells remain poorly understood, emphasizing the need to elucidate the role of host factors in IAV entry into cells. In this study, we identified FFAR2 as an important host factor for the efficient replication of both low-pathogenic and highly pathogenic IAV. We revealed that FFAR2 facilitates the internalization of IAV into target cells during the early stage of infection. Upon further characterization of the role of FFAR2-associated proteins in virus replication, we found that the FFAR2-ß-arrestin1-AP2B1 signaling cascade is important for the efficient endocytosis of IAV. Our findings thus further our understanding of the biological details of IAV entry into host cells and establish FFAR2 as a potential target for antiviral drug development.

4.
BioData Min ; 12: 21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31807141

RESUMO

Background: The sequencing platform BGISEQ-500 is based on DNBSEQ technology and provides high throughput with low costs. This sequencer has been widely used in various areas of scientific and clinical research. A better understanding of the sequencing process and performance of this system is essential for stabilizing the sequencing process, accurately interpreting sequencing results and efficiently solving sequencing problems. To address these concerns, a comprehensive database, SEQdata-BEACON, was constructed to accumulate the run performance data in BGISEQ-500. Results: A total of 60 BGISEQ-500 instruments in the BGI-Wuhan lab were used to collect sequencing performance data. Lanes in paired-end 100 (PE100) sequencing using 10 bp barcode were chosen, and each lane was assigned a unique entry number as its identification number (ID). From November 2018 to April 2019, 2236 entries were recorded in the database containing 65 metrics about sample, yield, quality, machine state and supplies information. Using a correlation matrix, 52 numerical metrics were clustered into three groups signifying yield-quality, machine state and sequencing calibration. The distributions of the metrics also delivered information about patterns and rendered clues for further explanation or analysis of the sequencing process. Using the data of a total of 200 cycles, a linear regression model well simulated the final outputs. Moreover, the predicted final yield could be provided in the 15th cycle of the early stage of sequencing, and the corresponding R2 of the 200th and 15th cycle models were 0.97 and 0.81, respectively. The model was run with the test sets obtained from May 2019 to predict the yield, which resulted in an R2 of 0.96. These results indicate that our simulation model was reliable and effective. Conclusions: Data sources, statistical findings and application tools provide a constantly updated reference for BGISEQ-500 users to comprehensively understand DNBSEQ technology, solve sequencing problems and optimize run performance. These resources are available on our website http://seqBEACON.genomics.cn:443/home.html.

5.
Zhongguo Zhong Yao Za Zhi ; 44(16): 3399-3405, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31602901

RESUMO

Tripterygium wilfordii is widely used in the treatment of rheumatism with curative effect. However,its toxicity and adverse reactions,especially the hepatotoxicity,rank the first in the herbs induced liver injury,is the key factors hindering its clinical application. This paper reviewed the literatures related to the hepatotoxicity of T. wilfordii in recent 20 years,and summarized the characteristic of hepatotoxicity induced by T. wilfordii,the factors causing liver injury,the mechanism of toxicity,and the measures to reduce toxicity. In animal experiments,the T. wilfordii induced-hepatotoxicity in physiological state was more serious than pathological state. The T. wilfordii induced-hepatotoxicity is related to various toxic components contained in it,but alkaloids are the most toxic one.Overdose and cumulative overdose are the lead causing of hepatotoxicity induced by T. wilfordii. The theory of oxidative stress is still an important mechanism of T. wilfordii induced-hepatotoxicity,and Nrf2,as a key regulatory enzyme of oxidative stress,has become an important target for drugs to against T. wilfordii induced-hepatotoxicity. Mitochondrial autophagy and liver hypersensitivity are new mechanisms of liver injury induced by T. wilfordii. The measures such as dosage control,drug compatibility and dosage form variations can help to reduce the hepatotoxicity induced by T. wilfordii. This paper clarified the current situation and shortcomings of safety research on T. wilfordii,so as to propose new research strategies and provide ideas for rational evaluation of safety and clinical safe drug use of T. wilfordii.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Medicamentos de Ervas Chinesas/toxicidade , Tripterygium/toxicidade , Animais
6.
Zhongguo Zhong Yao Za Zhi ; 44(16): 3441-3447, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31602907

RESUMO

To observe the effect of Tripterygium Glycosides Tablets on angiogenesis of rats with type Ⅱ collagen-induced arthritis( CIA) and on the tube formation of human umbilical vein endothelial cells( HUVEC) in vitro. The HUVEC were induced by 20 µg·L-1 vascular endothelial growth factor( VEGF) in vitro,and were treated with 0. 1,1,10 mg·L-1 Tripterygium Glycosides Tablets continuously for 7 hours. The numbers of branches of tube formation were measured. SD rats were immunized to establish CIA. CIA rats were treated with 9,18,36 mg·kg-1·d-1 Tripterygium Glycosides Tablets for 42 days. Histopathological examination( HE) was performed to observe the vascular morphology and vascular density in the synovial membrane of the inflamed joints. Immunohistochemistry and immunofluorescence were performed to observe the expression of platelets-endothelial cell adhesion molecule( CD31) and αsmooth muscle actin( αSMA) in synovial membrane. Immunohistochemistry and Western blot were performed to observe the expression of hypoxia-inducible factors 1α( HIF1α) and angiotensin 1( Ang1) in the synovial tissue. The results showed that the numbers of branches of tube formation of HUVEC induced by VEGF were improved,and declined significantly after treated by Tripterygium Glycosides Tablets. Compared with the normal group,the vascular density,CD31 positive expression,CD31 +/αSMA-immature and total vascular positive expression in the synovial membrane of the model group were significantly increased,and so as HIF1α and Ang1 in the synovium. Tripterygium Glycosides Tablets reduced the synovial vascular density and inhibited the positive expression of CD31,CD31+/αSMA-immature blood vessels and total vascular,but has no effect on CD31+/αSMA+mature blood vessels. Tripterygium Glycosides Tablets also inhibited the expression of HIF1α and Ang1 in synovial membrane of inflammatory joints. Our results demonstrated that Tripterygium Glycosides Tablets could inhibit the angiogenesis of synovial tissue in CIA rats and the tube formation of HUVEC,which is related to the down-regulation of HIF1α/Ang1 signal axis.


Assuntos
Artrite Experimental/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Glicosídeos/farmacologia , Tripterygium/química , Inibidores da Angiogênese/farmacologia , Angiotensina I/metabolismo , Animais , Artrite Experimental/induzido quimicamente , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Membrana Sinovial/efeitos dos fármacos , Comprimidos , Fator A de Crescimento do Endotélio Vascular
7.
Zhongguo Zhong Yao Za Zhi ; 44(16): 3486-3493, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31602913

RESUMO

The aim of this paper was to observe the toxic effect of Tripterygium Glycosides Tablets( TG) on the reproductive system of Ⅱ type collagen induced arthritis( CIA) male rats,and to explore the toxic mechanism preliminarily. Fifty SD rats were randomly divided into normal control group( Con),model group( CIA),Tripterygium Glycosides Tablets clinical equivalent dose groups of 1,2,4 times( 9,18,36 mg·kg-1),10 rats in each group,and were given by gavage once a day for 42 days after the first immunization.The organ indexes of uterine and ovarian were calculated on days 21 and 42. Histopathological and morphological changes of uterine and ovarian were observed under optical microscope. The concentration of estradiol( E2),follicle-stimulating hormone( FSH),luteinizing hormone( LH),17α-hydroxylase( CYP17 A1) and cytochrome P450 19 A1( CYP19 A1) in serum were detected by ELISA. Immunohistochemistry was used to observe the expression of Bax and Bcl-2 related proteins in the apoptosis pathway of uterus and ovary. The results showed that compared with the Con group,CIA group could reduce the number of uterine glands( P<0.05),but no significant changes were observed in other groups. Compared with the CIA group,there were no significant changes in the coefficients of uterus and ovary in the Tripterygium Glycosides Tablets groups. The number of uterine glands,total follicles in the ovary,mature follicles and corpus luteum,the distribution of blood vessels and mitochondria had a certain inhibitory trend,and also slightly increased the number of atresia follicles,but the histopathological quantitative indicators were not statistically different. Except that 2 times clinical dose of Tripterygium Glycosides Tablets could significantly reduce the content of CYP19 A1( P<0. 05) after 42 d administration,there were no significant changes in serum estrogen E2,FSH,LH and estrogen synthesis key enzymes CYP17 A1 in each administration group. Medium and high doses of Tripterygium Glycosides Tablets could increase the expression of apoptotic protein Bax in uterine and ovarian tissues( P<0. 05,P<0. 01),and all the administration groups could inhibit the expression of apoptotic inhibiting protein Bcl-2( P <0. 05,P<0. 01,P<0.001),42 d was more obvious than 21 d. In conclusion,4 times and less than 4 times Tripterygium Glycosides Tablets did not cause obvious toxicity and histopathological changes in the reproductive organs of CIA rats,but it could reduce the level of serum estrogen synthesis key enzyme CYP19 A1 and affect the content of apoptosis-related proteins Bax and Bcl-2 in uterus and ovary tissues. The relevant mechanism needs further study.


Assuntos
Artrite Experimental/tratamento farmacológico , Medicamentos de Ervas Chinesas/toxicidade , Genitália Feminina/efeitos dos fármacos , Glicosídeos/toxicidade , Tripterygium/química , Animais , Apoptose , Aromatase/metabolismo , Artrite Experimental/induzido quimicamente , Medicamentos de Ervas Chinesas/farmacologia , Feminino , Glicosídeos/farmacologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Comprimidos
8.
Zhongguo Zhong Yao Za Zhi ; 44(16): 3502-3511, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31602915

RESUMO

The aim of this paper was to compare the properties of Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets from dose-effect-toxicity on type Ⅱ collagen-induced arthritis( CIA) in rats. SD rats were randomly divided into eight groups,including normal group,model group,Tripterygium Glycosides Tablets groups( 1 times equivalent dose 0.009 g·kg-1,4 times equivalent dose 0.036 g·kg-1,16 times equivalent dose 0.144 g·kg-1),Tripterygium wilfordii Tablets groups( 1 times equivalent dose 0.007 5 mg·kg-1,4 times equivalent dose 0.030 mg·kg-1,16 times equivalent dose 0.120 mg·kg-1). Beginning on the first immunization,Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets administered intraperitoneally once a day. After the second immunization,the symptoms such as redness and swelling of joints were observed,and the clinical score and incidence of arthritis were evaluated. HE and Masson staining were used to examine the histopathological changes of joints. The expression level of anti-type Ⅱ collagen antibody Ig G in serum was detected by ELISA,routine testing of blood components,the concentration of ALP( alkaline phosphatase),ALT( alanine aminotransferase),AST( aspartate aminotransferase),GGT( gamma-glutamyltransferase),TBi L( total bilirubin),CRE( creatinine) and UREA( urea) in serum were detected by enzymatic assay. The rate of sperm deformity in the epididymis was evaluated under light microscope. The extent of damage to the testis and ovarian tissue was assessed by HE staining. Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets attenuated the inflammation,redness,swelling and deformity of joints and reduced the clinical score and incidence of arthritis in CIA rats. Meanwhile,it also exhibited obvious reduction in all pathological features such as joint synovitis,pannus,cartilage erosion and bone destruction. Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets reduced Ig G in a dose-dependent manner,and Tripterygium Glycosides Tablets is better than Tripterygium wilfordii Tablets( P<0.05 or P<0.01). The high doses of Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets could significantly increase the organ coefficient of liver and spleen and reduced RBC and HGB in CIA rats( P<0.01),and severity leading to death. Gastric mucosal injury and morphological changes of liver and kidney were not observed in CIA rats of Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets treatment group. The 4 and 16 times doses of Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets could significantly increase serum ALT,GGT and decrease CRE( P<0.05 or P<0.01). Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets could increase the sperm deformity rate and damage the testicular seminiferous tubules of CIA male rats. Severity increased with dose and time increasing. The effect of Tripterygium Glycosides Tablets( 16 times) is more significant than Tripterygium wilfordii Tablets( 16 times). Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets significantly delayed onset of arthritis and inhibited the paw edema and arthritic score. Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets also caused male reproductive damage,high dose affected hematopoiesis,and maximum dose leading to death. Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets all depended on dose-effect-toxicity manner. Anti-arthritis effect of Tripterygium Glycosides Tablets is better than Tripterygium wilfordii Tablets,but the toxicity of Tripterygium Glycosides Tablets maximum dose is more obvious. The relevant conclusions of our study will provide experimental references for clinical rational use of drugs,and further clinical studies are needed to confirm our conclusions.


Assuntos
Artrite Experimental/tratamento farmacológico , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/toxicidade , Glicosídeos/administração & dosagem , Glicosídeos/toxicidade , Tripterygium/toxicidade , Animais , Relação Dose-Resposta a Droga , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Comprimidos
9.
Emerg Microbes Infect ; 8(1): 1280-1290, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31495283

RESUMO

The H3N2 influenza viruses became widespread in humans during the 1968 H3N2 pandemic and have been a major cause of influenza epidemics ever since. Different lineages of H3N2 influenza viruses are also commonly found in animals. If a different lineage of H3N2 virus jumps to humans, a human influenza pandemic could occur with devastating consequences. Here, we studied the genetics, receptor-binding properties, and replication and transmission in mammals of 15 H3N2 avian influenza viruses detected in live poultry markets in China. We found that the H3N2 avian influenza viruses are complicated reassortants with distinct replication phenotypes in mice. Five viruses replicated efficiently in mice and bound to both human-type and avian-type receptors. These viruses transmitted efficiently to direct-contact guinea pigs, and three of them also transmitted among guinea pigs and ferrets via respiratory droplets. Moreover, ferret antiserum induced by human H3N2 viruses did not react with any of the H3N2 avian influenza viruses. Our study demonstrates that the H3N2 avian influenza viruses pose a clear threat to human health and emphasizes the need for continued surveillance and evaluation of the H3N2 influenza viruses circulating in nature.


Assuntos
Transmissão de Doença Infecciosa , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/fisiologia , Influenza Aviária/virologia , Aves Domésticas/virologia , Ligação Viral , Animais , China , Modelos Animais de Doenças , Furões , Cobaias , Humanos , Vírus da Influenza A Subtipo H3N2/genética , Camundongos Endogâmicos BALB C , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação , Vírus Reordenados/fisiologia , Receptores Virais/metabolismo , Doenças dos Roedores/virologia , Replicação Viral
10.
Vaccine ; 37(40): 5925-5929, 2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31471151

RESUMO

Ducks play a key role in the maintenance and spread of avian influenza viruses (AIVs) in nature, and control of AIVs in ducks has important implications for AIV eradication from poultry. We previously constructed a recombinant duck enteritis virus (DEV), rDEVus78HA, that expresses the HA gene of an H5N1 AIV and showed that rDEVus78HA immunization provides complete protection against both DEV and H5N1 AIV challenge in specific-pathogen-free ducks. In this study, we performed a 60-week clinical trial and found that this rDEVus78HA vaccine can function as a bivalent vaccine in farmed ducks against lethal challenge with DEV and H5N1 virus. Moreover, we found that rDEVus78HA-vaccinated ducks were efficiently protected against challenges with recently isolated heterologous H5N6 and H5N8 viruses. Our results demonstrate that rDEVus78HA could be extremely valuable for the control of DEV and H5 AIVs in ducks.

11.
Cancer Res ; 79(19): 4828-4839, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31416841

RESUMO

Mutations in KEAP1 and NFE2L2 (encoding the protein Nrf2) are prevalent in both adeno and squamous subtypes of non-small cell lung cancer, as well as additional tumor indications. The consequence of these mutations is stabilized Nrf2 and chronic induction of a battery of Nrf2 target genes. We show that knockdown of Nrf2 caused modest growth inhibition of cells growing in two-dimension, which was more pronounced in cell lines expressing mutant KEAP1. In contrast, Nrf2 knockdown caused almost complete regression of established KEAP1-mutant tumors in mice, with little effect on wild-type (WT) KEAP1 tumors. The strong dependency on Nrf2 could be recapitulated in certain anchorage-independent growth environments and was not prevented by excess extracellular glutathione. A CRISPR screen was used to investigate the mechanism(s) underlying this dependence. We identified alternative pathways critical for Nrf2-dependent growth in KEAP1-mutant cell lines, including the redox proteins thioredoxin and peroxiredoxin, as well as the growth factor receptors IGF1R and ERBB3. IGF1R inhibition was effective in KEAP1-mutant cells compared with WT, especially under conditions of anchorage-independent growth. These results point to addiction of KEAP1-mutant tumor cells to Nrf2 and suggest that inhibition of Nrf2 or discrete druggable Nrf2 target genes such as IGF1R could be an effective therapeutic strategy for disabling these tumors. SIGNIFICANCE: This study identifies pathways activated by Nrf2 that are important for the proliferation and tumorigenicity of KEAP1-mutant non-small cell lung cancer.

12.
Biol Reprod ; 101(4): 664-674, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31291448

RESUMO

Evidence indicates that microRNAs (miRNAs) play essential roles in early embryonic development. The miRNA-518 family is a special biomarker of the placenta, and miRNA-518b is abnormally expressed in placental tissue in preeclampsia. Early growth response protein 1 (EGR1), a zinc finger transcriptional factor, plays an essential role in regulating cell differentiation, angiogenesis, and migration. Moreover, earlier studies have shown that EGR1 protein plays a key role in implantation. However, little is known about the role of miR-518b and EGR1 on early embryonic arrest (EEA) in humans. In our study, increased miR-518b along with decreased EGR1 was found in human villus tissues with EEA. Furthermore, we demonstrated by luciferase assay that miR-518b is a direct regulator of EGR1. After comparing the effect of silencing EGR1, vascular endothelial growth factor (VEGF) individually, and EGR1/VEGF in combination, we found that EGR1 can inhibit migration and angiogenesis of HTR-8 SVneo cells by decreasing the VEGF expression. Hypoxia plays an initial role in early embryonic development, and we found that hypoxia reduces the expression of miR-518b and increases the expression of EGR1 and VEGF to facilitate migration and angiogenesis in a hypoxic model of HTR-8/SVneo cell line. Our findings provide new insights into the role of miR-518b in EEA and implicate the potential application of miR-518b in the diagnosis and development of intervention for EEA.

13.
mBio ; 10(3)2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213560

RESUMO

Avian influenza viruses (AIVs) must acquire mammalian-adaptive mutations before they can efficiently replicate in and transmit among humans. The PB2 E627K mutation is known to play a prominent role in the mammalian adaptation of AIVs. The H7N9 AIVs that emerged in 2013 in China easily acquired the PB2 E627K mutation upon replication in humans. Here, we generate a series of reassortant or mutant H7N9 AIVs and test them in mice. We show that the low polymerase activity attributed to the viral PA protein is the intrinsic driving force behind the emergence of PB2 E627K during H7N9 AIV replication in mice. Four residues in the N-terminal region of PA are critical in mediating the PB2 E627K acquisition. Notably, due to the identity of viral PA protein, the polymerase activity and growth of H7N9 AIV are highly sensitive to changes in expression levels of human ANP32A protein. Furthermore, the impaired viral polymerase activity of H7N9 AIV caused by the depletion of ANP32A led to reduced virus replication in Anp32a-/- mice, abolishing the acquisition of the PB2 E627K mutation and instead driving the virus to acquire the alternative PB2 D701N mutation. Taken together, our findings show that the emergence of the PB2 E627K mutation of H7N9 AIV is driven by the intrinsic low polymerase activity conferred by the viral PA protein, which also involves the engagement of mammalian ANP32A.IMPORTANCE The emergence of the PB2 E627K substitution is critical in the mammalian adaptation and pathogenesis of AIV. H7N9 AIVs that emerged in 2013 possess a prominent ability in gaining the PB2 E627K mutation in humans. Here, we demonstrate that the acquisition of the H7N9 PB2 E627K mutation is driven by the low polymerase activity conferred by the viral PA protein in human cells, and four PA residues are collectively involved in this process. Notably, the H7N9 PA protein leads to significant dependence of viral polymerase function on human ANP32A protein, and Anp32a knockout abolishes PB2 E627K acquisition in mice. These findings reveal that viral PA and host ANP32A are crucial for the emergence of PB2 E627K during adaptation of H7N9 AIVs to humans.

14.
Drug Metab Dispos ; 47(10): 1156-1163, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31085544

RESUMO

In cells, catalytic disulfide cleavage is an essential mechanism in protein folding and synthesis. However, detailed enzymatic catalytic mechanism relating cleavage of disulfide bonds in xenobiotics is not well understood. This study reports an enzymatic mechanism of cleavage of disulfide bonds in xenobiotic small molecules and antibody conjugate (ADC) linkers. The chemically stable disulfide bonds in substituted disulfide-containing pyrrolobenzodiazepine (PBD, pyrrolo[2,1-c][1,4]benzodiazepine) monomer prodrugs in presence of glutathione or cysteine were found to be unstable in incubations in whole blood of humans and rats. It was shown the enzymes involved were thioredoxin (TRX) and glutaredoxin (GRX). For a diverse set of drug-linker conjugates, we determined that TRX in the presence of TRX-reductase and NADPH generated the cleaved products that are consistent with catalytic disulfide cleavage and linker immolation. GRX was less rigorously studied; in the set of compounds studied, its role in the catalytic cleavage was also confirmed. Collectively, these in vitro experiments demonstrate that TRX as well as GRX can catalyze the cleavage of disulfide bonds in both small molecules and linkers of ADCs.

16.
Gene ; 697: 19-25, 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-30776465

RESUMO

Early Embryonic Arrest (EEA) is one of the major causes of female infertility. Genetic factors including specific genes and miRNAs may play pivotal roles on EEA. However, it is not well defined what genes and micro RNAs participate the pathophysiological alterations of EEA. In this work, we compared the Transcriptome -Seq and microRNA profiles from three pairs of villi (three EEA patients and three normal pregnancy, NP). We first confirmed the array data by qPCR with ten randomly selected differentially expressed genes and ten differentially expressed miRNAs in villi from 20 EEA and 20 NP controls. We next applied Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis and found that these differentially expressed genes enriched in the PI3K-Akt signaling pathway, Jak-STAT signaling pathway, MAPK signaling pathway, Complement and coagulation cascades, Hypertrophic cardiomyopathy (HCM), Dilated cardiomyopathy (DCM). Interestingly, hsa-miR-6515-5p and its target genes NLRP3, UGP2 may regulate the Immune system and carbohydrate metabolism. Hsa-miRNA 518 and its target gene EGR1 may regulate cell proliferation, angiogenesis, and cell apoptosis to impact early embryonic development. Moreover, novel-m0045-5p and its target gene RMDN3 may regulate microtubule formation on the development of EEA. Our research provides novel biomarkers for EEA and establishes a foundation for further study of the mechanism of EEA.


Assuntos
Perda do Embrião/genética , Desenvolvimento Embrionário/genética , MicroRNAs/genética , Adulto , Grupo com Ancestrais do Continente Asiático/genética , Vilosidades Coriônicas/fisiologia , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Infertilidade Feminina/genética , Gravidez , Transdução de Sinais , Transcriptoma , Sequenciamento Completo do Exoma/métodos
17.
Sci China Life Sci ; 62(1): 76-83, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30515728

RESUMO

Antigenic drift forces us to frequently update influenza vaccines; however, the genetic basis for antigenic variation remains largely unknown. In this study, we used clade 7.2 H5 viruses as models to explore the molecular determinants of influenza virus antigenic variation. We generated eight monoclonal antibodies (MAbs) targeted to the hemagglutinin (HA) protein of the index virus A/chicken/Shanxi/2/2006 and found that two representative antigenically drifted clade 7.2 viruses did not react with six of the eight MAbs. The E131N mutation and insertion of leucine at position 134 in the HA protein of the antigenically drifted strains eliminated the reactivity of the virus with the MAbs. We also found that the amino acid N131 in the H5 HA protein is glycosylated. Our results provide experimental evidence that glycosylation and an amino acid insertion or deletion in HA influence antigenic variation.


Assuntos
Aminoácidos/imunologia , Antígenos Virais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Sequência de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Antígenos Virais/genética , Antígenos Virais/metabolismo , Galinhas/virologia , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/metabolismo , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/metabolismo , Influenza Aviária/genética , Influenza Aviária/imunologia , Influenza Aviária/virologia , Influenza Humana/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Homologia de Sequência de Aminoácidos
18.
Cell Host Microbe ; 24(4): 558-568.e7, 2018 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-30269969

RESUMO

H7N9 low pathogenic influenza viruses emerged in China in 2013 and mutated to highly pathogenic strains in 2017, resulting in human infections and disease in chickens. To control spread, a bivalent H5/H7 inactivated vaccine was introduced in poultry in September 2017. To monitor virus evolution and vaccine efficacy, we collected 53,884 poultry samples across China from February 2017 to January 2018. We isolated 252 H7N9 low pathogenic viruses, 69 H7N9 highly pathogenic viruses, and one H7N2 highly pathogenic virus, of which two low pathogenic and 14 highly pathogenic strains were collected after vaccine introduction. Genetic analysis of highly pathogenic strains revealed nine genotypes, one of which is predominant and widespread and contains strains exhibiting high virulence in mice. Additionally, some H7N9 and H7N2 viruses carrying duck virus genes are lethal in ducks. Thus, although vaccination reduced H7N9 infections, the increased virulence and expanded host range to ducks pose new challenges.


Assuntos
Doenças Transmissíveis Emergentes/virologia , Evolução Molecular , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Animais , Galinhas , China , Doenças Transmissíveis Emergentes/sangue , Doenças Transmissíveis Emergentes/imunologia , Doenças Transmissíveis Emergentes/mortalidade , Patos , Feminino , Humanos , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/sangue , Influenza Aviária/imunologia , Influenza Aviária/mortalidade , Influenza Humana/sangue , Influenza Humana/imunologia , Influenza Humana/mortalidade , Camundongos , Camundongos Endogâmicos BALB C , Organismos Livres de Patógenos Específicos , Vacinas de Produtos Inativados/imunologia , Virulência/genética
19.
Mol Pharm ; 15(11): 5103-5113, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30222362

RESUMO

Permeability assays are commonly conducted with Madin-Darby canine kidney (MDCK) cells to predict the intestinal absorption of small-molecule drug candidates. In addition, MDCK cells transfected to overexpress efflux transporters are often used to identify substrates. However, MDCK cells exhibit endogenous efflux activity for a significant proportion of experimental compounds, potentially leading to the underestimation of permeability and confounded findings in transport studies. The goal of this study was to evaluate canine Mdr1 knockout MDCK (gMDCKI) cells in permeability screening and human MDR1 substrate determination in a drug discovery setting. The gMDCKI cells were established by CRISPR-Cas9-mediated knockout of the canine Mdr1 gene in MDCKI wildtype (wt) cells. A comparison of efflux ratios (ER) between MDCKI wt and gMDCKI showed that out of 135 compounds tested, 38% showed efflux activity in MDCKI wt, while no significant efflux was observed in gMDCKI cells. Apparent permeability (Papp) from apical-to-basolateral (A-to-B) and basolateral-to-apical were near unity in gMDCKI cells, which approximated passive permeability, and 17% of compounds demonstrated increases in their Papp A-to-B values. Overexpression of human MDR1 in gMDCKI (gMDCKI-MDR1) cells enabled substrate determination without the contribution of endogenous efflux, and the assay was able to deconvolute ambiguous results from MDCKI-MDR1 and identify species differences in substrate specificity. An analysis of 395 and 474 compounds in gMDCKI and gMDCKI-MDR1, respectively, suggested physicochemical properties that were associated with low permeability correlated with MDR1 recognition. Poorly permeable compounds and MDR1 substrates were more likely to be large, flexible, and more capable of forming external hydrogen bonds. On the basis of our evaluation, we concluded that gMDCKI is a better cell line for permeability screening and efflux substrate determination than the MDCK wt cell line.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Cães , Avaliação Pré-Clínica de Medicamentos/métodos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Permeabilidade
20.
Pestic Biochem Physiol ; 149: 104-112, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30033006

RESUMO

Extracts from the aerial parts of Solidago canadensis L. were evaluated for molluscicidal activity against Pomacea canaliculata Lam. using an immersion bioassay method. The petroleum ether fraction of the ethanolic extract (PEEE) from S. canadensis exhibited strong molluscicidal activity. The PEEE mode of action in the hepatopancreas tissue of P. canaliculata was tested at several concentrations. Biochemical parameters, namely, soluble sugar content, protein, malondialdehyde (MDA), acetylcholinesterase (AChE) activity, alanine aminotransferase (ALT), and aspartate transaminase (AST) were significantly decreased or increased after exposure to PEEE for 48 h (p<0.05). Histological assessment results showed that hepatopancreas tissue structure was destroyed by exposure to PEEE. Gas chromatography-mass spectrometry analysis (GC-MS) was used to identify 15 compounds that could contribute to the molluscicidal efficacy of the PEEE. Molluscicidal assay, biochemical tests and histological assessments suggest that the PEEE from S. canadensis has potential utility as a molluscicide.


Assuntos
Moluscocidas/farmacologia , Extratos Vegetais/farmacologia , Caramujos/efeitos dos fármacos , Solidago/química , Acetilcolinesterase/metabolismo , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Hepatopâncreas/efeitos dos fármacos , Hepatopâncreas/enzimologia , Hepatopâncreas/metabolismo , Hepatopâncreas/patologia , Malondialdeído/metabolismo , Componentes Aéreos da Planta/química , Proteínas/análise , Açúcares/análise
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