RESUMO
Immune dysfunction is a primary culprit behind spontaneous miscarriage (SM). To address this, immunosuppressive agents have emerged as a novel class of tocolytic drugs, modulating the maternal immune system's tolerance towards the embryo. Rapamycin (PubChem CID:5284616), a dual-purpose compound, functions as an immunosuppressive agent and triggers autophagy by targeting the mTOR pathway. Its efficacy in treating SM has garnered significant research interest in recent times. Autophagy, the cellular process of self-degradation and recycling, plays a pivotal role in numerous health conditions. Research indicates that autophagy is integral to endometrial decidualization, trophoblast invasion, and the proper functioning of decidual immune cells during a healthy pregnancy. Yet, in cases of SM, there is a dysregulation of the mTOR/autophagy axis in decidual stromal cells or immune cells at the maternal-fetal interface. Both in vitro and in vivo studies have highlighted the potential benefits of low-dose rapamycin in managing SM. However, given mTOR's critical role in energy metabolism, inhibiting it could potentially harm the pregnancy. Moreover, while low-dose rapamycin has been deemed safe for treating recurrent implant failure, its potential teratogenic effects remain uncertain due to insufficient data. In summary, rapamycin represents a double-edged sword in the treatment of SM, balancing its impact on autophagy and immune regulation. Further investigation is warranted to fully understand its implications.
Assuntos
Aborto Espontâneo , Autofagia , Sirolimo , Serina-Treonina Quinases TOR , Humanos , Autofagia/efeitos dos fármacos , Feminino , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Gravidez , Animais , Transdução de Sinais/efeitos dos fármacos , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Inibidores de MTOR/farmacologia , Inibidores de MTOR/uso terapêuticoRESUMO
In an effort to develop safe and innovative in vitro models for Ebola virus (EBOV) research, we generated a recombinant Ebola virus where the glycoprotein (GP) gene was substituted with the Cre recombinase (Cre) gene by reverse genetics. This defective virus could multiply itself in a complementary permissive cell line, which could express GP and reporter protein upon exogenous Cre existence. The main features of this novel model for Ebola virus are intact viral life cycle, robust virus multiplication and normal virions morphology. The design of this model ensures its safety, excellent stability and maneuverability as a tool for virology research as well as for antiviral agent screening and drug discovery, and such a design could be further adapted to other viruses.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Humanos , Ebolavirus/genética , Ebolavirus/metabolismo , Linhagem Celular , Glicoproteínas/genética , Replicação Viral , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
The ongoing coronavirus disease 2019 (COVID-19) pandemic caused more than 96 million infections and over 2 million deaths worldwide so far. However, there is no approved vaccine available for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the disease causative agent. Vaccine is the most effective approach to eradicate a pathogen. The tests of safety and efficacy in animals are pivotal for developing a vaccine and before the vaccine is applied to human populations. Here we evaluated the safety, immunogenicity, and efficacy of an inactivated vaccine based on the whole viral particles in human ACE2 transgenic mouse and in non-human primates. Our data showed that the inactivated vaccine successfully induced SARS-CoV-2-specific neutralizing antibodies in mice and non-human primates, and subsequently provided partial (in low dose) or full (in high dose) protection of challenge in the tested animals. In addition, passive serum transferred from vaccine-immunized mice could also provide full protection from SARS-CoV-2 infection in mice. These results warranted positive outcomes in future clinical trials in humans.
Assuntos
Vacinas contra COVID-19/imunologia , COVID-19 , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/prevenção & controle , Camundongos , Camundongos Transgênicos , Primatas , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Vacinas de Produtos Inativados/imunologiaRESUMO
Long ncRNAs regulate a complex array of fundamental biological processes, while its molecular regulatory mechanism in Leydig cells (LCs) remains unclear. In the present study, we established the lncRNA LOC102176306/miR-1197-3p/peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) regulatory network by bioinformatic prediction, and investigated its roles in goat LCs. We found that lncRNA LOC102176306 could efficiently bind to miR-1197-3p and regulate PPARGC1A expression in goat LCs. Downregulation of lncRNA LOC102176306 significantly supressed testosterone (T) synthesis and ATP production, decreased the activities of antioxidant enzymes and mitochondrial complex I and complex III, caused the loss of mitochondrial membrane potential, and inhibited the proliferation of goat LCs by decreasing PPARGC1A expression, while these effects could be restored by miR-1197-3p inhibitor treatment. In addition, miR-1197-3p mimics treatment significantly alleviated the positive effects of lncRNA LOC102176306 overexpression on T and ATP production, antioxidant capacity and proliferation of goat LCs. Taken together, lncRNA LOC102176306 functioned as a sponge for miR-1197-3p to maintain PPARGC1A expression, thereby affecting the steroidogenesis, cell proliferation and oxidative stress of goat LCs. These findings extend our understanding of the molecular mechanisms of T synthesis, cell proliferation and oxidative stress of LCs.
Assuntos
Células Intersticiais do Testículo/citologia , MicroRNAs/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , RNA Longo não Codificante/genética , Testículo/citologia , Animais , Apoptose , Proliferação de Células , Cabras , Células Intersticiais do Testículo/metabolismo , Masculino , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Testículo/metabolismo , Testosterona/metabolismoRESUMO
The ongoing COVID-19 pandemic is causing huge impact on health, life, and global economy, which is characterized by rapid spreading of SARS-CoV-2, high number of confirmed cases and a fatality/case rate worldwide reported by WHO. The most effective intervention measure will be to develop safe and effective vaccines to protect the population from the disease and limit the spread of the virus. An inactivated, whole virus vaccine candidate of SARS-CoV-2 has been developed by Wuhan Institute of Biological Products and Wuhan Institute of Virology. The low toxicity, immunogenicity, and immune persistence were investigated in preclinical studies using seven different species of animals. The results showed that the vaccine candidate was well tolerated and stimulated high levels of specific IgG and neutralizing antibodies. Low or no toxicity in three species of animals was also demonstrated in preclinical study of the vaccine candidate. Biochemical analysis of structural proteins and purity analysis were performed. The inactivated, whole virion vaccine was characterized with safe double-inactivation, no use of DNases and high purity. Dosages, boosting times, adjuvants, and immunization schedules were shown to be important for stimulating a strong humoral immune response in animals tested. Preliminary observation in ongoing phase I and II clinical trials of the vaccine candidate in Wuzhi County, Henan Province, showed that the vaccine is well tolerant. The results were characterized by very low proportion and low degree of side effects, high levels of neutralizing antibodies, and seroconversion. These results consistent with the results obtained from preclinical data on the safety.
Assuntos
Vacinas contra COVID-19/imunologia , SARS-CoV-2 , Animais , Anticorpos Antivirais , Vacinas contra COVID-19/efeitos adversos , Feminino , Imunidade Humoral , Masculino , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/imunologiaRESUMO
The aim of this study was to investigate the molecular mechanisms of arginine (Arg) on follicular development of acute feed-restricted ewes during the luteal phase. From day 6 of the estrous cycle, 24 multiparous Hu sheep were randomly assigned into three groups: control group (a maintenance diet; n = 6), feed restriction group (0.5 maintenance diet, saline infusion; n = 9) and Arg treatment group (0.5 maintenance diet, infusion with 155 µmol of Arg-HCl/kg body weight; n = 9). The intravenous administrations were performed three times per day from day 6 to day 15 of the estrous cycle. At the end of treatment, the hypothalamus and pituitary were collected, as well as the follicular fluid (FF) and granulose cells (GCs) in the ≥2.5 mm follicles. The transcription level of NPVF was significantly increased, and the expression level of GNRH was significantly decreased in the hypothalamus with feed restriction. In addition, feed restriction significantly decreased the number of ≥2.5 mm follicles in the ovaries. In the ≥2.5 mm follicles, feed restriction significantly increased estradiol (E2) level in FF and the expression levels of steroidogenesis related genes (STAR, 3BHSD and CYP19A1) in GCs, while significantly decreased the expressions of FSHR and cell proliferation related genes (YAP1, CCND1 and PCNA) in GCs. Moreover, the activities of glucose metabolism enzymes (PFKP and G6PDH) were significantly decreased in GCs of the ≥2.5 mm follicles with feed restriction. Interestingly, as a precursor of nitric oxide, Arg supplementation can rescue the effects of feed restriction on follicular development by enhancing glucose metabolism and cell proliferation of GCs, and alleviating the abnormal E2 secretion in the ≥2.5 mm follicles, accompanied with recovering the expressions of NPVF and GNRH in the hypothalamus. These findings will be helpful for understanding the role of nutrition and Arg in sheep follicular development.
Assuntos
Arginina , Fase Luteal , Animais , Dieta , Estradiol , Ciclo Estral , Feminino , Líquido Folicular , OvinosRESUMO
The 2019 novel coronavirus (SARS-CoV-2) outbreak is a major challenge for public health. SARS-CoV-2 infection in human has a broad clinical spectrum ranging from mild to severe cases, with a mortality rate of ~6.4% worldwide (based on World Health Organization daily situation report). However, the dynamics of viral infection, replication and shedding are poorly understood. Here, we show that Rhesus macaques are susceptible to the infection by SARS-CoV-2. After intratracheal inoculation, the first peak of viral RNA was observed in oropharyngeal swabs one day post infection (1 d.p.i.), mainly from the input of the inoculation, while the second peak occurred at 5 d.p.i., which reflected on-site replication in the respiratory tract. Histopathological observation shows that SARS-CoV-2 infection can cause interstitial pneumonia in animals, characterized by hyperemia and edema, and infiltration of monocytes and lymphocytes in alveoli. We also identified SARS-CoV-2 RNA in respiratory tract tissues, including trachea, bronchus and lung; and viruses were also re-isolated from oropharyngeal swabs, bronchus and lung, respectively. Furthermore, we demonstrated that neutralizing antibodies generated from the primary infection could protect the Rhesus macaques from a second-round challenge by SARS-CoV-2. The non-human primate model that we established here provides a valuable platform to study SARS-CoV-2 pathogenesis and to evaluate candidate vaccines and therapeutics.
Assuntos
Betacoronavirus/genética , Betacoronavirus/imunologia , Infecções por Coronavirus/patologia , Modelos Animais de Doenças , Macaca mulatta/virologia , Pneumonia Viral/patologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Betacoronavirus/isolamento & purificação , COVID-19 , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico por imagem , Infecções por Coronavirus/virologia , Feminino , Imuno-Histoquímica , Masculino , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico por imagem , Pneumonia Viral/virologia , RNA Viral/genética , Radiografia Torácica , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2 , Carga Viral , Replicação ViralRESUMO
Yes-associated protein 1 (YAP1) transcription regulator of the Hippo protein kinase pathway, serves as a key regulator of tissue growth and organ size by regulating cell proliferation and apoptosis. Effects of YAP1 on proliferation and apoptosis of sheep endometrial epithelial cells (EEC) as a result of estradiol-17ß (E2) treatment, however, remain unclear. In the present study, the abundance of YAP1 protein in the uterine horn was greater than that in the uterine body or cervix. The YAP1 protein was primarily localized in the endometrial luminal and glandular epithelial cells of the uterine horn of ewes on day 2 of the estrous cycle. Compared with control samples, there was a lesser abundance of YAP1 mRNA transcript that was associated with a lesser proliferation and greater apoptosis of EEC. There were also lesser concentrations of epidermal growth factor and insulin-like growth factor 1 in the spent culture medium when there was a lesser abundance of YAP1 mRNA in EEC compared with those in the control group. When there was a greater abundance of YAP1 mRNA transcript, there were greater concentrations of epidermal growth factor and insulin-like growth factor 1 in the spent media. Furthermore, with estradiol-17ß treatment the abundance of YAP1 mRNA transcript was similar to that of the control samples. Taken together, estradiol-17ß may function as an essential regulator of EEC proliferation and apoptosis by modulation of concentrations of YAP1 protein in the sheep uterus. These results indicate there are molecular mechanisms of estradiol-17ß and YAP1 in EEC proliferation and apoptosis of ewes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células/efeitos dos fármacos , Endométrio/citologia , Células Epiteliais/efeitos dos fármacos , Estradiol/farmacologia , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Ovinos , Fatores de Transcrição/genética , Regulação para Cima , Útero/metabolismoRESUMO
This study aimed to determine the effects of l-arginine (L-Arg) supplementation on steroid hormone receptors in non-pregnant ovine endometrium. All experimental ewes were randomly assigned to either a control group (nâ¯=â¯6), a nutrient-restricted group (nâ¯=â¯6), or an L-Arg supplemented nutrient-restricted group (nâ¯=â¯6). The effects of L-Arg on estrogen receptor α/ß (ERα/ß) and progesterone receptor (PGR) expression in the ovine endometrium were assessed. Our results showed that levels of ERß and PGR expression were significantly increased by nutrient restriction, but L-Arg counteracted the effect of nutrient restriction on ERß and PGR expression (pâ¯<â¯0.05). Also, expression of endometrial ERα was substantially increased (pâ¯<â¯0.05) by L-Arg supplementation. Furthermore, ERα/ß and PGR were mainly detected in the endometrial luminal epithelium and glandular epithelium. Therefore, we isolated and identified endometrial epithelial cells (EECs) from sheep. Different concentrations of L-Arg were added to investigate the effects on ERα/ß and PGR in EECs. The expression levels of endothelial nitric oxide synthase, ERß, and PGR were significantly increased in response to low-concentration (200⯵mol) L-Arg supplementation, which subsequently decreased with a high concentration (800⯵mol) (pâ¯<â¯0.05). Otherwise, ERα expression was remarkably increased at both L-Arg concentrations in EECs (pâ¯<â¯0.05). Overall, the results indicated that L-Arg performed crucial roles in the regulation of ovine steroid hormone receptor expression in the endometrium. The results of this study provide a theoretical basis and technical means for the normal function of endometrium in response to low nutrient levels.
Assuntos
Arginina/farmacologia , Restrição Calórica , Endométrio/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Receptores de Progesterona/genética , Ovinos , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Animais , Restrição Calórica/veterinária , Células Cultivadas , Endométrio/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Nutrientes , Gravidez , Receptores de Progesterona/metabolismo , Ovinos/genética , Ovinos/metabolismo , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
Nutrient deficiency in ruminants can lead to estrus cycle disorders, a decreased pregnancy rate, and reduce birth weight. l-arginine (L-Arg), an important amino acid, can improve uterine homeostasis in pregnant sheep and prevent intrauterine growth restriction (IUGR). However, most studies of L-Arg have been conducted on pregnant sheep and few have reported the effects of L-Arg on microvessel density (MVD) in the non-pregnant ovine endometrium. The processes of normal uterine cyclical development and implantation are dependent on a balanced of endometrial MVD. In this study, female Hu sheep were randomly assigned to either a control group (nâ¯=â¯6), a nutrient-restricted group (nâ¯=â¯6), or an L-Arg supplemented nutrient-restricted group (nâ¯=â¯6). The effects of L-Arg on MVD in ovine endometrium were then studied. Our results showed that ovine endometrial MVD was significantly increased by nutrient restriction, but L-Arg counteracted the effect of nutrient restriction on MVD (Pâ¯<â¯0.05). Levels of angiogenic growth factors (including VEGFA, VEGFR2, and FGF2) had significant increases (Pâ¯<â¯0.05) in endometrium of nutrient restriction on sheep, but that L-Arg supplementation substantially decreased (Pâ¯<â¯0.05) their expressions in nutrient restriction sheep. Furthermore, oxidative stress caused by nutrient restriction was also alleviated by L-Arg supplementation in the ovine endometrium. Overall, the results suggested that L-Arg has crucial roles in maintaining the balance of endometrial MVD and angiogenic growth factors, and increasing anti-oxidation capability in the endometrium of nutrient-restricted sheep. This study will provide a theoretical basis and technical means for the normal development of endometrial microvessels in low nutrition level.
Assuntos
Arginina/farmacologia , Endométrio/irrigação sanguínea , Privação de Alimentos , Ovinos , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacosRESUMO
Rapamycin (RAPA), an inhibitor of mammalian target of rapamycin (mTOR), exhibits a high neuroprotective action against neurodegenerative diseases in mouse models. Since neuroinflammation has been shown to be involved in Alzheimer's disease (AD) development and progression, the aim of this study was to examine the anti-inflammatory role of RAPA in AD in vivo and in vitro, and investigate the underlying mechanisms. We found that amyloid-ß (Aß) induced neuronal inflammation and a remarkable increase in mTOR activity in in-vivo and in-vitro models of inflammation, suggesting the critical role of mTOR signaling in neuronal inflammation. In addition, administration of RAPA was found to down-regulate mTOR, p-mTOR, Nuclear factor kappa B (NF-κB) p65, p-p65, TNF-α, IL-1ß and Bax protein expression in Aß25-35- or lipopolysaccharides (LPS)-treated mice and cultured Neuro-2a (N2a) cells. Moreover, RAPA disrupted Aß25-35-induced nuclear translocation of mTOR and NF-κB. Our findings indicate that RAPA inhibits Aß25-35- or LPS-induced neuronal inflammation through suppressing mTOR signaling and reducing nuclear import of NF-κB.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Anti-Inflamatórios/uso terapêutico , Inflamação/tratamento farmacológico , Lipopolissacarídeos/toxicidade , NF-kappa B/metabolismo , Fragmentos de Peptídeos/toxicidade , Transdução de Sinais/efeitos dos fármacos , Sirolimo/uso terapêutico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação/induzido quimicamente , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Neuroblastoma/patologia , Neuroglia/efeitos dos fármacos , TransfecçãoRESUMO
Mammalian target of rapamycin (mTOR) is a key regulator of metabolism, cell growth, and protein synthesis. Since decreased mTOR activity has been found to slow aging in many species, the aim of this study was to examine the activity of mTOR and its phosphorylated form in in vitro and in vivo models mimicking Alzheimer's disease (AD), and investigate the potential pathway of PGC-1ß in regulating mTOR expression. Primary neurons and N2a cells were treated with Aß25-35, while untreated cells served as controls. The expression of mTOR, p-mTOR (Ser2448), and PGC-1ß was determined with Western blotting and RT-PCR assay, and the translocation of mTOR was detected using confocal microscopy. Aß25-35 treatment stimulated the translocation of mTOR from cytoplasm to nucleus, and resulted in elevated expression of mTOR and p-mTOR (Ser2448) and reduced PGC-1ß expression. In addition, overexpression of PGC-1ß was found to decrease mTOR expression. The results of this study demonstrate that Aß increases the expression of mTOR and p-mTOR at the site of Ser2448, and the stimulation of Aß is likely to depend on sirtuin 1, PPARγ, and PGC-1ß pathway in regulating mTOR expression.
Assuntos
Neurônios/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Sirtuína 1/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Acetilação , Peptídeos beta-Amiloides , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Camundongos Transgênicos , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Fragmentos de Peptídeos , Fosforilação , Transporte Proteico/efeitos dos fármacos , RatosRESUMO
The specific binding between viral attachment proteins (VAPs) of a virus and its cellular receptors on host cells mediates virus entry into host cells, which triggers subsequent viral infections. Previous studies indicate that F1 ATP synthase ß subunit (named BP53), is found on the surface of shrimp cells and involved in white spot syndrome virus (WSSV) infection by functioning as a potential viral receptor. Herein, in a far-western blotting assay, three WSSV proteins with molecular weights of 28 kDa, 37 kDa, and >50 kDa were found to interact with BP53. The 28 kDa and 37 kDa proteins were identified as the envelope protein VP28 and VP37 of WSSV respectively, which could be recognized by the polyclonal antibodies. Enzyme-linked immunosorbent binding assays revealed that VP37 contributed to almost 80% of the binding capability for BP53 compared with the same amount of total WSSV protein. The relationship between BP53 and its complementary interacting protein, VP37, was visualized using a co-localization assay. Bound VP37 on the cell surface co-localized with BP53 and shared a similar subcellular location on the outer surface of shrimp cells. Pearson's correlation coefficients reached to 0.67 ± 0.05 and the Mander's overlap coefficients reached 0.70 ± 0.05, which indicated a strong relationship between the localization of BP53 and bound rVP37. This provides evidence for an interaction between BP53 and VP37 obtained at the molecular and cellular levels, supporting the hypothesis that BP53 serves as a receptor for WSSV by binding to VP37. The identification of the viral binding proteins of shrimp BP53 is helpful for better understanding the pathogenic mechanisms of WSSV to infect shrimp at the cellular level.
Assuntos
Complexos de ATP Sintetase/fisiologia , Interações Hospedeiro-Patógeno , Penaeidae/virologia , Receptores Virais/química , Vírus da Síndrome da Mancha Branca 1/patogenicidade , Complexos de ATP Sintetase/química , Complexos de ATP Sintetase/metabolismo , Animais , Ensaio de Imunoadsorção Enzimática , Penaeidae/metabolismo , Receptores Virais/fisiologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/fisiologia , Ligação Viral , Internalização do VírusRESUMO
Manchurian walnut (Juglans mandshurica Maxim.) is a vulnerable, temperate deciduous tree valued for its wood and nut, but transcriptomic and genomic data for the species are very limited. Next generation sequencing (NGS) has made it possible to develop molecular markers for this species rapidly and efficiently. Our goal is to use transcriptome information from RNA-Seq to understand development in J. mandshurica and develop polymorphic simple sequence repeats (SSRs, microsatellites) to understand the species' population genetics. In this study, more than 47.7 million clean reads were generated using Illumina sequencing technology. De novo assembly yielded 99,869 unigenes with an average length of 747 bp. Based on sequence similarity search with known proteins, a total of 39,708 (42.32 %) genes were identified. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) identified 15,903 (16.9 %) unigenes. Further, we identified and characterized 63 new transcriptome-derived microsatellite markers. By testing the markers on 4 to 14 individuals from four populations, we found that 20 were polymorphic and easily amplified. The number of alleles per locus ranged from 2 to 8. The observed and expected heterozygosity per locus ranged from 0.209 to 0.813 and 0.335 to 0.842, respectively. These twenty microsatellite markers will be useful for studies of population genetics, diversity, and genetic structure, and they will undoubtedly benefit future breeding studies of this walnut species. Moreover, the information uncovered in this research will also serve as a useful genetic resource for understanding the transcriptome and development of J. mandshurica and other Juglans species.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Juglans/genética , Repetições de Microssatélites/genética , Transcriptoma/genética , Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Frutas/genética , Frutas/crescimento & desenvolvimento , Juglans/crescimento & desenvolvimento , Anotação de Sequência Molecular , Filogenia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimentoRESUMO
BACKGROUND: Over the past a few years, evidences indicate that adenosine triphosphate (ATP) is an energy source for the binding, maturation, assembly, and budding process of many enveloped viruses. Our previous studies suggest that the F1-ATP synthase beta subunit (ATPsyn ß, BP53) of the shrimp Litopenaeus vannamei (L. vannamei) might serve as a potential receptor for white spot syndrome virus (WSSV)'s infection. METHODS: BP53 was localized on the surface of shrimp hemocytes and gill epithelial cells by immunofluorescence assay and immunogold labeling technique. Cell surface ATP synthesis was demonstrated by an in vitro bioluminescent luciferase assay. Furthermore, the expression of bp53 after WSSV infection was investigated by RT-PCR test. In addition, RNAi was developed to knock down endogenous bp53. RESULTS: BP53 is present on shrimp cell surface of hemocytes and gill epithelia. The synthesized ATP was detectable in the extracellular supernatant by using a bioluminescence assay, and the production declined post WSSV binding and infection. Knocking down endogenous bp53 resulted in a 50% mortality of L. vannamei. CONCLUSION: These results suggested that BP53, presenting on cell surface, likely served as one of the receptors for WSSV infection in shrimp. Correspondingly, WSSV appears to disturb the host energy metabolism through interacting with host ATPsyn ß during infection. This work firstly showed that host ATP production is required and consumed by the WSSV for binding and proceeds with infection process.