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1.
Gene ; 766: 145154, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32949699

RESUMO

CircRNA serves a crucial role in the development of heart failure (HF). Nevertheless, the regulatory mechanisms of circ_0062389 in HF are unknown. This study aims to examine the effect and mechanism of circ_0062389 on cardiomyocyte apoptosis in HF rats and H9C2 cells. Rats were divided into 5 groups (n = 8/group): the Control group, Sham group, HF group, HF + si-NC group, and HF + si-circRNA group. The echocardiography was used to examine the cardiac function, including LVIDd, LVIDs, IVSd, and IVSs. The apoptosis of myocardial tissue was detected through TUNEL method. H9C2 cells were randomly assigned into Control group (untransfected H9C2 cells), H/R group (untransfected H/R H9C2 cells), H/R + si-NC group (transfected si-NC) and H/R + si-circRNA group (transfect si-circ_0062389). Cell apoptosis was assessed through flow cytometry. The expression of circ_0062389 in myocardial tissues of HF rats was significantly higher than that of Control group and Sham group. Silencing circ_0062389 significantly reduced the levels of LVIDd, LVIDs, IVSd, and IVSs. Additionally, silencing circ_0062389 could significantly reduce the apoptosis rate of rat cardiomyocytes. Besides, silencing circ_0062389 significantly reduced the expression of TGF-ß1 and Smad3 protein. Silencing circ_0062389 could alleviate cardiomyocyte apoptosis in HF rats via modulating TGF-ß1/Smad3 signaling pathway, which might be a promising target for the treatment of HF.


Assuntos
Apoptose/genética , Insuficiência Cardíaca/genética , Miócitos Cardíacos/patologia , Interferência de RNA/fisiologia , RNA Circular/genética , Transdução de Sinais/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta1/genética , Animais , Linhagem Celular , Coração/fisiologia , Insuficiência Cardíaca/patologia , Masculino , Miocárdio/patologia , Ratos , Ratos Sprague-Dawley
2.
Exp Parasitol ; 218: 108003, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980317

RESUMO

Dermatophagoides farinae, an important pathogen, has multiple allergens. However, their expression under physiological conditions are not understood. Our previous RNA-seq showed that allergens of D. farinae were up-regulated under temperature stress, implying that they may be involved in stress response. Here, we performed a comprehensive study. qRT-PCR detection indicated that 26 of the 34 allergens showed differential expression. Der f1 had the most abundant basic expression quantity. Der f 28.0201 (HSP70) and Der f3 had the same regulation pattern in 9 highly expressed transcripts, which only up-regulated at 41 °C and 43 °C, but Der f 28.0201 showed stronger regulation than Der f 3 (19.88-fold vs 6.02-fold). Whereas Der f 1, 2, 7, 21, 22, 27, and 30 were up-regulated under both heat and cold stress, and Der f 27 showed the strongest regulation ability among them. Der f 27 showed more significant up-regulation than Der f 28.0201 under heat stress (23.59-fold vs 19.88-fold), and Der f27 had more obvious up-regulation under cold than heat stress (30.70-fold vs 23.59-fold). The expression of Der f 27, 28.0201 and 1, and D. farinae survival rates significantly decreased following RNAi, indicating the upregulation of these allergens under temperature stress conferred thermo-tolerance or cold-tolerance to D. farinae. In this study, we described for the first time that these allergens have temperature-stress response functions. This new scientific discovery has important clinical value for revealing the more frequent and serious allergic diseases caused by D. farinae during the change of seasons.


Assuntos
Antígenos de Dermatophagoides/fisiologia , Resposta ao Choque Frio/fisiologia , Dermatophagoides farinae/fisiologia , Resposta ao Choque Térmico/fisiologia , Estresse Fisiológico/fisiologia , Animais , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/metabolismo , Antígenos de Dermatophagoides/farmacologia , Sequência de Bases , Dermatophagoides farinae/genética , Feminino , Inativação Gênica , Anotação de Sequência Molecular , RNA/química , RNA/isolamento & purificação , Interferência de RNA/fisiologia , RNA de Cadeia Dupla/química , Reação em Cadeia da Polimerase em Tempo Real , Estações do Ano , Estresse Fisiológico/genética , Regulação para Cima
3.
Proc Natl Acad Sci U S A ; 117(35): 21504-21511, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817556

RESUMO

In fission yeast, the inverted repeats IR-L and IR-R function as boundary elements at the edges of a 20-kb silent heterochromatic domain where nucleosomes are methylated at histone H3K9. Each repeat contains a series of B-box motifs physically associated with the architectural TFIIIC complex and with other factors including the replication regulator Sap1 and the Rix1 complex (RIXC). We demonstrate here the activity of these repeats in heterochromatin formation and maintenance. Deletion of the entire IR-R repeat or, to a lesser degree, deletion of just the B boxes impaired the de novo establishment of the heterochromatic domain. Nucleation proceeded normally at the RNA interference (RNAi)-dependent element cenH but subsequent propagation to the rest of the region occurred at reduced rates in the mutants. Once established, heterochromatin was unstable in the mutants. These defects resulted in bistable populations of cells occupying alternate "on" and "off" epigenetic states. Deleting IR-L in combination with IR-R synergistically tipped the balance toward the derepressed state, revealing a concerted action of the two boundaries at a distance. The nuclear rim protein Amo1 has been proposed to tether the mating-type region and its boundaries to the nuclear envelope, where Amo1 mutants displayed milder phenotypes than boundary mutants. Thus, the boundaries might facilitate heterochromatin propagation and maintenance in ways other than just through Amo1, perhaps by constraining a looped domain through pairing.


Assuntos
Proteínas de Ligação a DNA/genética , Heterocromatina/metabolismo , Sequências Repetidas Invertidas/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica/fisiologia , Heterocromatina/genética , Histonas/metabolismo , Metilação , Proteínas Nucleares/metabolismo , Interferência de RNA/fisiologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/metabolismo
4.
J Biosci ; 452020.
Artigo em Inglês | MEDLINE | ID: mdl-32713862

RESUMO

The two biological evidences to endorse the antiviral activity of RNA interference (RNAi) are biogenesis of viral-siRNA (v-siRNA) by the host and encoding of RNAi-suppressor protein by viral genome. It has been recently established that mammals and mammalian cell lines mount antiviral RNAi to defend themselves against the invading viruses. The large part of viral pathogenicity is also due to the RNAi suppressor proteins. In this context it is only natural to ask what kinds of RNAi suppressors are encoded by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the central character of the present pandemic. The following mini review addresses this question.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Interferência de RNA/fisiologia , RNA Interferente Pequeno/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Imunidade Inata/imunologia , Pandemias , Células Vero
5.
J Vis Exp ; (159)2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32538906

RESUMO

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.


Assuntos
Besouros/genética , Expressão Gênica/genética , Interferência de RNA/fisiologia , Animais
6.
Gene ; 755: 144900, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32554046

RESUMO

Atherosclerosis (AS) is a serious threat to the cardiovascular system. Circular RNA circ_0003645 was found to be differentially expressed in the process of AS. Our study tried to unravel the effect and underlying mechanism of circ_0003645 in endothelial cells treated with oxidized low-density lipoprotein (oxLDL). Si-RNAs and over-circ0003645 were transfected into human umbilical vein endothelial cells (HUVECs), and the expression levels of circ_0003645 and NF-κB mRNA were measured. The protein level of NF-κB, lactate dehydrogenase leakage (LDH leakage), cell viability, and apoptosis were detected. Further, the expression of interleukin (IL)-6, tumor necrosis factor (TNF)-α, ICAM-1, and VCAM-1 were measured. Circ_0003645 was found up-regulated in AS patients and in HUVECs treated with oxLDL. The LDH leakage, cell apoptosis, and expression levels of IL-6, TNF-α, ICAM-1, VCAM-1, NF-κB mRNA, NF-κB protein were all inhibited by circ_0003645 silencing, while cell viability was promoted, and the opposite effects were observed by the overexpression of circ_0003645. In conclusion, circ_0003645 silencing alleviated inflammation and apoptosis, while promoted the viability in oxLDL-induced endothelial cells by the NF-κB pathway.


Assuntos
Aterosclerose/genética , Lipoproteínas LDL/farmacologia , NF-kappa B/metabolismo , RNA Circular/genética , Apoptose/fisiologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Estudos de Casos e Controles , Linhagem Celular , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interferência de RNA/fisiologia , RNA Circular/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
7.
Nat Commun ; 11(1): 2243, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32382029

RESUMO

Cells rely on a diverse repertoire of genes for maintaining homeostasis, but the transcriptional networks underlying their expression remain poorly understood. The MOF acetyltransferase-containing Non-Specific Lethal (NSL) complex is a broad transcription regulator. It is essential in Drosophila, and haploinsufficiency of the human KANSL1 subunit results in the Koolen-de Vries syndrome. Here, we perform a genome-wide RNAi screen and identify the BET protein BRD4 as an evolutionary conserved co-factor of the NSL complex. Using Drosophila and mouse embryonic stem cells, we characterise a recruitment hierarchy, where NSL-deposited histone acetylation enables BRD4 recruitment for transcription of constitutively active genes. Transcriptome analyses in Koolen-de Vries patient-derived fibroblasts reveals perturbations with a cellular homeostasis signature that are evoked by the NSL complex/BRD4 axis. We propose that BRD4 represents a conserved bridge between the NSL complex and transcription activation, and provide a new perspective in the understanding of their functions in healthy and diseased states.


Assuntos
Histonas/metabolismo , Ativação Transcricional/fisiologia , Acetilação , Animais , Células Cultivadas , Cromatina/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Epigenômica , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Gravidez , Regiões Promotoras Genéticas/genética , Interferência de RNA/fisiologia , Ativação Transcricional/genética
8.
Acta Diabetol ; 57(9): 1111-1116, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32447557

RESUMO

AIMS: Long non-coding RNA (lncRNA) VIM Antisense RNA 1 (VIM-AS1) has been reported to be correlated with type 2 diabetes (T2D) susceptibility, while the roles of this lncRNA in T2D and its complications remain unclear. This study aimed to explore the role of VIM-AS1 in diabetic retinopathy (DR). METHODS: Gene expression levels in both human specimens and in vitro cultivated cells were determined by qPCR and western blot. Overexpression experiments were performed to analyze gene interactions. Cell apoptosis after transfections was detected by cell apoptosis assay. RESULTS: We found that VIM-AS1 was significantly downregulated in T2D patients in comparison with that in healthy controls. Specifically, the expression levels of VIM-AS1 were lowest among T2D patients complicated with DR. Bioinformatics analysis showed that VIM-AS1 can interact with microRNA 29 (miR-29), which is a critical player in high glucose-induced apoptosis of human retinal pigment epithelial cells (RPEs). Dual-luciferase assay also revealed the direct interaction between them. High glucose treatment led to upregulated miR-29 and downregulated VIM-AS1. However, overexpression of VIM-AS1 and miR-29 did not affect the expression of each other. Cell apoptosis analysis showed that overexpression of VIM-AS1 reduced the enhancing effects of miR-29 overexpression on RPEs cell proliferation. CONCLUSIONS: Therefore, VIM-AS1 may sponge miR-29 to participate in DR.


Assuntos
Retinopatia Diabética/genética , MicroRNAs/genética , Interferência de RNA/fisiologia , RNA Longo não Codificante/fisiologia , Adulto , Idoso , Apoptose/genética , Estudos de Casos e Controles , Proliferação de Células/genética , Células Cultivadas , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Retinopatia Diabética/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
9.
Gene ; 752: 144783, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32428699

RESUMO

RNA interference (RNAi), which employs double-strand RNA (dsRNA) or small interference RNA (siRNA), is a popular reverse genetic manipulation tool to study gene function. Presently, there is few reports on the implementation of RNAi on the insulin-like androgenic gland gene (IAG) in red swamp crayfish Procambarus clarkii. In this study, the effective sequence of siRNA and optimal injection dose were determined, and the effects of RNAi using dsRNA, siRNA, and long-term RNAi were investigated. The results showed that the doses of 0.5 and 1 µg/g of body weight of IAG-siRNA3 produced significantly better inhibition than 0.1 µg/g. qPCR assays showed that both dsRNA and siRNA silenced the IAG expression in five tissues (brain, ventral nerve cord, androgenic gland, testis, and vas deferens) in adult P. clarkii, with the effectiveness decreasing over time, inhibiting the production of spermatid. dsRNA exhibited a longer interference effect than siRNA in adults. For long-term interference (P. clarkii juveniles were injected 7 times with 1 µg/g of body weight of IAG-dsRNA), and found that the secondary sexual characteristics of juveniles were affected, while the control group developed normally. The results of this study could lay the foundation for crayfish sex reversal with IAG RNAi, and provide the reference for those studies in which the technique of RNAi was used.


Assuntos
Astacoidea/genética , Hormônios Gonadais/genética , Androgênios/metabolismo , Animais , Astacoidea/metabolismo , Hormônios Gonadais/metabolismo , Masculino , Interferência de RNA/fisiologia , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Diferenciação Sexual/genética , Testículo/metabolismo
10.
Nat Commun ; 11(1): 1851, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32296040

RESUMO

Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. With a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs, we identify numerous lncRNAs involved in key steps of cell division such as chromosome segregation, mitotic duration and cytokinesis. Here, we provide evidence that the chromatin-associated lncRNA, linc00899, leads to robust mitotic delay upon its depletion in multiple cell types. We perform transcriptome analysis of linc00899-depleted cells and identify the neuronal microtubule-binding protein, TPPP/p25, as a target of linc00899. We further show that linc00899 binds TPPP/p25 and suppresses its transcription. In cells depleted of linc00899, upregulation of TPPP/p25 alters microtubule dynamics and delays mitosis. Overall, our comprehensive screen uncovers several lncRNAs involved in genome stability and reveals a lncRNA that controls microtubule behaviour with functional implications beyond cell division.


Assuntos
Divisão Celular/genética , Divisão Celular/fisiologia , RNA Longo não Codificante/genética , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Mitose/genética , Mitose/fisiologia , Proteínas/genética , Interferência de RNA/fisiologia
11.
Nat Cell Biol ; 22(5): 579-590, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32251399

RESUMO

In fission yeast and plants, RNA processing and degradation contribute to heterochromatin silencing, alongside conserved pathways of transcriptional repression. It has not been known whether similar pathways exist in metazoans. Here, we describe a pathway of silencing in Caenorhabditis elegans somatic cells, in which the highly conserved RNA-binding complex LSM2-8 contributes selectively to the repression of heterochromatic reporters and endogenous genes bearing the Polycomb mark, histone H3K27me3. This acts by degrading selected transcripts through the XRN-2 exoribonuclease. Disruption of the LSM2-8 pathway leads to mRNA stabilization. Unlike previously described pathways of heterochromatic RNA degradation, LSM2-8-mediated RNA degradation does not target nor require H3K9 methylation. Intriguingly, loss of this pathway coincides with a localized reduction in H3K27me3 at lsm-8-sensitive loci. We have thus uncovered a mechanism of RNA degradation that selectively contributes to the silencing of a subset of H3K27me3-marked genes, revealing a previously unrecognized layer of post-transcriptional control in metazoan heterochromatin.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Exorribonucleases/genética , Histonas/genética , Estabilidade de RNA/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Animais , Caenorhabditis elegans/genética , Inativação Gênica/fisiologia , Heterocromatina/genética , Metilação , Proteínas do Grupo Polycomb/genética , Interferência de RNA/fisiologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética
12.
Mol Cell ; 78(5): 862-875.e8, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32348780

RESUMO

Nuclear RNA interference (RNAi) pathways work together with histone modifications to regulate gene expression and enact an adaptive response to transposable RNA elements. In the germline, nuclear RNAi can lead to trans-generational epigenetic inheritance (TEI) of gene silencing. We identified and characterized a family of nuclear Argonaute-interacting proteins (ENRIs) that control the strength and target specificity of nuclear RNAi in C. elegans, ensuring faithful inheritance of epigenetic memories. ENRI-1/2 prevent misloading of the nuclear Argonaute NRDE-3 with small RNAs that normally effect maternal piRNAs, which prevents precocious nuclear translocation of NRDE-3 in the early embryo. Additionally, they are negative regulators of nuclear RNAi triggered from exogenous sources. Loss of ENRI-3, an unstable protein expressed mostly in the male germline, misdirects the RNAi response to transposable elements and impairs TEI. The ENRIs determine the potency and specificity of nuclear RNAi responses by gating small RNAs into specific nuclear Argonautes.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Inativação Gênica/fisiologia , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Células Germinativas/metabolismo , Proteínas Nucleares/metabolismo , Interferência de RNA/fisiologia , RNA de Cadeia Dupla/metabolismo , RNA Nuclear/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética
13.
Plant Sci ; 294: 110443, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32234229

RESUMO

High temperature (HT) is a main environmental restraint that affects rice yield and grain quality. In this study, SSIIIa-RNAi and its wild-type (WT) were used to investigate the effect of HT exposure on the isozyme-specific variation of several key starch biosynthesis enzymes in developing endosperms and its relation to starch properties. SSIIIa-RNAi had minimal impact on grain chalky occurrence under normal temperature growth, but it could up-grade the susceptibility of grain chalky occurrence to HT exposure, due to the relatively sensitive response of AGPase and SSI to HT exposure. Different from WT, SSIIIa-RNAi had the relatively enriched proportion of chains with DP 13-16 under HT, and HT-induced decline in the proportion of DP < 12 became much larger for SSIIIa-RNAi relative to WT. SSIIIa-RNAi significantly enhanced the expression of SSI isozyme and total SS activity, whereas SSI-RNAi deficiency had little impact on the expression of SSIIIa isozyme. In this regard, the compensatory increase in SSI isozyme as a result of SSIIIa deficiency occurred only in a one-way manner. SSIIIa-RNAi caused a striking elevation in BEIIa expression, and the effect of SSIIIa deficiency on the chain length distribution in relation to HT exposure was closely associated with the participation of BEIIa, SSI, and their interaction in amylopectin biosynthesis.


Assuntos
Oryza/metabolismo , Amido/metabolismo , Amilopectina/genética , Amilopectina/metabolismo , Temperatura Alta , Oryza/genética , Interferência de RNA/fisiologia , Amido/genética , Sintase do Amido/genética , Sintase do Amido/metabolismo , Temperatura
14.
Parasit Vectors ; 13(1): 202, 2020 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-32307003

RESUMO

BACKGROUND: Malaria vector mosquitoes acquire midgut microbiota primarily from their habitat. The homeostasis of these microbial communities plays an essential role in the mosquito longevity, the most essential factor in the mosquito vectorial capacity. Our recent study revealed that silencing genes involved in regulation of the midgut homeostasis including FN3D1, FN3D3 and GPRGr9 reduced the survival of female adult Anopheles arabiensis mosquitoes. In the present study, we investigate the stability of the gene silencing efficiency of mosquitoes reared in three different breeding conditions representing distinct larval habitat types: town brick pits in Jimma, flood pools in the rural land of Asendabo and roadside pools in Wolkite. METHODS: First-instar larvae of An. arabiensis mosquitoes were reared separately using water collected from the three breeding sites. The resulting adult females were micro-injected with dsRNA targeting the FN3D1 gene (AARA003032) and their survival was monitored. Control mosquitoes were injected with dsRNA Lacz. In addition, the load of midgut microbiota of these mosquitoes was determined using flow cytometry. RESULTS: Survival of naïve adult female mosquitoes differed between the three sites. Mosquitoes reared using water collected from brick pits and flood pools survived longer than mosquitoes reared using water collected from roadside. However, the FN3D1 gene silencing effect on survival did not differ between the three sites. CONCLUSIONS: The present study revealed that the efficacy of FN3D1 gene silencing is not affected by variation in the larval habitat. Thus, silencing this gene has potential for application throughout sub-Saharan Africa.


Assuntos
Anopheles/genética , Domínio de Fibronectina Tipo III/genética , Interferência de RNA/fisiologia , Animais , Anopheles/fisiologia , Cruzamento , Ecossistema , Larva/genética , Larva/fisiologia , Controle de Mosquitos/métodos , Mosquitos Vetores/genética , Mosquitos Vetores/fisiologia
15.
Sci Rep ; 10(1): 1604, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005880

RESUMO

Aphids are important agricultural pests causing major yield losses worldwide. Since aphids can rapidly develop resistance to chemical insecticides there is an urgent need to find alternative aphid pest management strategies. Despite the economic importance of bluegreen aphid (Acyrthosiphon kondoi), very few genetic resources are available to expand our current understanding and help find viable control solutions. An artificial diet is a desirable non-invasive tool to enable the functional characterisation of genes in bluegreen aphid and discover candidate target genes for future use in RNA interference (RNAi) mediated crop protection against aphids. To date no artificial diet has been developed for bluegreen aphid, so we set out to develop a suitable diet by testing and optimising existing diets. Here, we describe an artificial diet for rearing bluegreen aphid and also provide a proof of concept for the supplementation of the diet with RNAi molecules targeting the salivary gland transcript C002 and gap gene hunchback, resulting in bluegreen aphid mortality which has not yet been documented in this species. Managing this pest, for example via RNAi delivery through artificial feeding will be a major improvement to test bluegreen aphid candidate target genes for future pest control and gain significant insights into bluegreen aphid gene function.


Assuntos
Afídeos/genética , Suplementos Nutricionais , Fabaceae/parasitologia , Interferência de RNA/fisiologia , Animais , Dieta/métodos , Medicago truncatula/parasitologia , Fenótipo , Doenças das Plantas/parasitologia , Genética Reversa/métodos , Glândulas Salivares/parasitologia
16.
PLoS One ; 15(1): e0227685, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935250

RESUMO

The acyl-CoA-binding proteins (ACBP) act by regulating the availability of acyl-CoA in the cytoplasm and must have essential functions in lipid metabolism. The genome of the kissing-bug Rhodnius prolixus encodes five proteins of this family, but little is known about them. In this study we investigated the expression and function of RpACBP-5. Feeding induced RpACBP-5 gene expression in the posterior midgut, and an increase of about four times was observed two days after the blood meal. However, the amount of protein, which was only detected in this organ, did not change during digestion. The RpACBP-5 gene was also highly expressed in pre-vitellogenic and vitellogenic oocytes. Recombinant RpACBP-5 was shown to bind to acyl-CoA of different lengths, and it exhibited nanomolar affinity to lauroyl-CoA in an isothermal titration assay, indicating that RpACBP-5 is a functional ACBP. RpACBP-5 knockdown by RNA interference did not affect digestion, egg laying and hatching, survival, or accumulation of triacylglycerol in the fat body and oocytes. Similarly, double knockdown of RpACBP-1 and RpACBP-5 did not alter egg laying and hatching, survival, accumulation of triacylglycerol in the fat body and oocytes, or the neutral lipid composition of the posterior midgut or hemolymph. These results show that RpACBP-5 is a functional ACBP but indicate that the lack of a detectable phenotype in the knockdown insects may be a consequence of functional overlap of the proteins of the ACBP family found in the insect.


Assuntos
Inibidor da Ligação a Diazepam/genética , Inibidor da Ligação a Diazepam/metabolismo , Rhodnius/genética , Acil Coenzima A/metabolismo , Animais , Proteínas de Transporte/metabolismo , Corpo Adiposo/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Hemolinfa/metabolismo , Proteínas de Insetos/genética , Metabolismo dos Lipídeos/genética , Oócitos/metabolismo , Oviposição , Interferência de RNA/fisiologia , Rhodnius/metabolismo , Triglicerídeos/metabolismo
17.
Infect Immun ; 88(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31740529

RESUMO

Listeria monocytogenes is a foodborne bacterium that causes gastroenteritis, meningitis, or abortion. Listeria induces its internalization (entry) into some human cells through interaction of the bacterial surface protein InlB with its host receptor, the Met tyrosine kinase. InlB and Met promote entry through stimulation of localized actin polymerization and exocytosis. How actin cytoskeletal changes and exocytosis are controlled during entry is not well understood. Here, we demonstrate important roles for the host GTPase Arf1 and its effectors AP1 and PICK1 in actin polymerization and exocytosis during InlB-dependent uptake. Depletion of Arf1 by RNA interference (RNAi) or inhibition of Arf1 activity using a dominant-negative allele impaired InlB-dependent internalization, indicating an important role for Arf1 in this process. InlB stimulated an increase in the GTP-bound form of Arf1, demonstrating that this bacterial protein activates Arf1. RNAi and immunolocalization studies indicated that Arf1 controls exocytosis and actin polymerization during entry by recruiting the effectors AP1 and PICK1 to the plasma membrane. In turn, AP1 and PICK1 promoted plasma membrane translocation of both Filamin A (FlnA) and Exo70, two host proteins previously found to mediate exocytosis during InlB-dependent internalization (M. Bhalla, H. Van Ngo, G. C. Gyanwali, and K. Ireton, Infect Immun 87:e00689-18, 2018, https://doi.org/10.1128/IAI.00689-18). PICK1 mediated recruitment of Exo70 but not FlnA. Collectively, these results indicate that Arf1, AP1, and PICK1 stimulate exocytosis by redistributing FlnA and Exo70 to the plasma membrane. We propose that Arf1, AP1, and PICK1 are key coordinators of actin polymerization and exocytosis during infection of host cells by Listeria.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Exocitose/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Listeria monocytogenes/patogenicidade , Proteínas Nucleares/metabolismo , Fator de Transcrição AP-1/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Listeriose/metabolismo , Listeriose/microbiologia , Polimerização , Interferência de RNA/fisiologia , Transdução de Sinais/fisiologia
18.
Gene ; 729: 144300, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31884102

RESUMO

West Nile virus (WNV) has been found to be a common cause of neuroinvasive arboviral disease worldwide in human and horses. The process of RNA interference induced by small RNA molecules, like small interfering RNA (siRNA) and microRNA (miRNA), proved to be a novel approach for preventing viral infections. So far there is no published data for inhibition of West Nile virus by vector delivered artificial miRNA which believed to have more inhibitory potential than small interfering (siRNA). In the present study, we designed two artificial miRNA (amiRNAs) targeting the conserved NS5 and NS2A genomic regions of West Nile virus. These amiRNAs oligos were cloned in to miRNA based vector having murine miR-155 backbone which allows the high expression of amiRNAs in green fluorescent protein (GFP) tagged form. Vero cells were transiently transfected by cytomegalovirus (CMV) promoter derived vector expressing amiRNAs transcribed by RNA Pol II. Efficacy of amiRNA targeting the NS5 and NS2A regions of WNV was determined in highly virulent WNV Eg101 strain in Vero cells. The result indicated that both amiRNA effectively inhibit West Nile virus replication. The concatenated amiRNA having dual pre-amiRNA expression cassette showed better efficacy. amiRNA targeting NS5 showed best protection against WNV infection and percentage reduction of WNV titer was observed at 96 hpi is 97.11%. Further study for inhibition of WNV replication was assessed by plaque assay, quantitative reverse transcriptase PCR (qRT-PCR) assay, Immunofluorescence assay and Western blot analysis. Present study concludes that amiRNA (NS5) targeting conserved region of gene significantly reduced the virus replication as determined by plaque assay. Similarly, reduction was also observed at RNA and protein level through real-time RT-PCR and Western blot analysis directly correlate with the inhibition of WNV replication. Here, we describe our current understanding of the role of miRNAs in host defense response against West Nile virus, as well as their potential as new therapeutic approaches.


Assuntos
Replicação Viral/genética , Febre do Nilo Ocidental/prevenção & controle , Vírus do Nilo Ocidental/genética , Animais , Antivirais/metabolismo , Chlorocebus aethiops , Engenharia Genética/métodos , MicroRNAs/biossíntese , MicroRNAs/genética , MicroRNAs/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno/genética , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Células Vero , Febre do Nilo Ocidental/genética , Vírus do Nilo Ocidental/patogenicidade
19.
PLoS Pathog ; 15(12): e1008110, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790500

RESUMO

Viroids are small, non-protein-coding RNAs which can induce disease symptoms in a variety of plant species. Potato (Solanum tuberosum L.) is the natural host of Potato spindle tuber viroid (PSTVd) where infection results in stunting, distortion of leaves and tubers and yield loss. Replication of PSTVd is accompanied by the accumulation of viroid-derived small RNAs (sRNAs) proposed to play a central role in disease symptom development. Here we report that PSTVd sRNAs direct RNA silencing in potato against StTCP23, a member of the TCP (teosinte branched1/Cycloidea/Proliferating cell factor) transcription factor family genes that play an important role in plant growth and development as well as hormonal regulation, especially in responses to gibberellic acid (GA). The StTCP23 transcript has 21-nucleotide sequence complementarity in its 3' untranslated region with the virulence-modulating region (VMR) of PSTVd strain RG1, and was downregulated in PSTVd-infected potato plants. Analysis using 3' RNA ligase-mediated rapid amplification of cDNA ends (3' RLM RACE) confirmed cleavage of StTCP23 transcript at the expected sites within the complementarity with VMR-derived sRNAs. Expression of these VMR sRNA sequences as artificial miRNAs (amiRNAs) in transgenic potato plants resulted in phenotypes reminiscent of PSTVd-RG1-infected plants. Furthermore, the severity of the phenotypes displayed was correlated with the level of amiRNA accumulation and the degree of amiRNA-directed down-regulation of StTCP23. In addition, virus-induced gene silencing (VIGS) of StTCP23 in potato also resulted in PSTVd-like phenotypes. Consistent with the function of TCP family genes, amiRNA lines in which StTCP23 expression was silenced showed a decrease in GA levels as well as alterations to the expression of GA biosynthesis and signaling genes previously implicated in tuber development. Application of GA to the amiRNA plants minimized the PSTVd-like phenotypes. Taken together, our results indicate that sRNAs derived from the VMR of PSTVd-RG1 direct silencing of StTCP23 expression, thereby disrupting the signaling pathways regulating GA metabolism and leading to plant stunting and formation of small and spindle-shaped tubers.


Assuntos
Genes de Plantas , Doenças das Plantas/virologia , Solanum tuberosum/virologia , Viroides/patogenicidade , Virulência/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Interferência de RNA/fisiologia , Vírus de RNA , RNA Viral , Solanum tuberosum/genética , Fatores de Transcrição
20.
Sci Rep ; 9(1): 19926, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31882941

RESUMO

We have previously developed efficient peptide-based nucleic acid delivery vectors PF14 and NF55, where we have shown that these vectors preferentially transfect lung tissue upon systemic administration with the nucleic acid. In the current work, we have explored the utilization and potential of these vectors for the lung-targeted gene therapy. Accordingly, we assessed the efficacy of these peptides in (i) two different lung disease models - acute lung inflammation and asthma in mice and (ii) using two different nucleic acid cargos - siRNA and pDNA encoding shRNA. Using RNAi against cytokine TNFα, we showed efficient anti-inflammatory effects in both disease models and observed decreased disease symptoms. Our results highlight the potential of our transfection vectors for lung gene therapy.


Assuntos
Asma/metabolismo , Inflamação/metabolismo , Pulmão/metabolismo , Ácidos Nucleicos/metabolismo , Interferência de RNA/fisiologia , Animais , Asma/imunologia , Asma/terapia , Feminino , Terapia Genética , Inflamação/imunologia , Inflamação/terapia , Masculino , Camundongos , Reação em Cadeia da Polimerase
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