RESUMO
Vitellogenesis-inhibiting hormone (VIH) negatively regulates reproduction in shrimp and other decapod crustaceans. In order to assess the effects of transcriptional silencing by multiple VIH subtype I sinus gland peptides (SGPs) on ovarian maturation in female whiteleg shrimp, Litopenaeus vannamei, we synthesized five dsRNAs targeting Liv-SGP-A, -B, -C, -F, and -G and injected them into subadults. The following treatments were employed: sgpG-dsRNA (targeting Liv-SGP-G), sgpC-dsRNA (targeting Liv-SGP-C), and mixed-dsRNA (targeting Liv-SGP-A, -B, and -F). The expression of Liv-SGP-G in eyestalks was significantly decreased at 10, 20, and 30 days after the injection of sgpG-dsRNA In addition, it was significantly decreased at 10 and 30 days after the injection of mixed-dsRNA. The expression of vitellogenin (Vg) gene expression in the ovaries, and concentrations of Vg protein in the hemolymph, were not changed by the administration of any dsRNA treatment (the ovaries remained immature in all treated individuals and contained mostly oogonia and previtellogenic oocytes). Although the administration of dsRNAs corresponding to multiple VIHs did not promote ovarian maturation, this is the first report of the co-transcriptional repression of Liv-SGP-G by the injection of dsRNA for homologous genes (Liv-SGP-A, -B, and -F). These results indicate that subadults can respond to the techniques of transcriptional silencing.
Assuntos
Hormônios de Invertebrado/genética , Penaeidae/genética , Peptídeos/metabolismo , RNA de Cadeia Dupla/genética , Animais , Feminino , Hemolinfa/metabolismo , Hormônios de Invertebrado/metabolismo , Penaeidae/metabolismo , Peptídeos/genética , RNA de Cadeia Dupla/metabolismo , Vitelogênese , Vitelogeninas/genética , Vitelogeninas/metabolismoRESUMO
Immune responses counteract infections but also cause collateral damage to hosts. Oligoadenylate synthetase 1 (OAS1) binds double-stranded RNA from invading viruses and produces 2'-5' linked oligoadenylate (2-5A) to activate ribonuclease L (RNase L), which cleaves RNA to inhibit virus replication. OAS1 can also undergo autoactivation by host RNAs, a potential trade-off to antiviral activity. We investigated functional variation in primate OAS1 as a model for how immune pathways evolve to mitigate costs and observed a surprising frequency of loss-of-function variation. In gorillas, we identified a polymorphism that severely decreases catalytic function, mirroring a common variant in humans that impairs 2-5A synthesis through alternative splicing. OAS1 loss-of-function variation is also common in monkeys, including complete loss of 2-5A synthesis in tamarins. The frequency of loss-of-function alleles suggests that costs associated with OAS1 activation can be so detrimental to host fitness that pathogen-protective effects are repeatedly forfeited.
Assuntos
2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/farmacologia , Antivirais/farmacologia , Mutação , Primatas/imunologia , 2',5'-Oligoadenilato Sintetase/metabolismo , Nucleotídeos de Adenina/metabolismo , Sequência de Aminoácidos , Animais , Endorribonucleases/metabolismo , Evolução Molecular , Variação Genética , Haplorrinos , Humanos , Modelos Moleculares , Oligorribonucleotídeos/metabolismo , Conformação Proteica , RNA de Cadeia Dupla/metabolismo , Análise de Sequência de Proteína , Replicação Viral/efeitos dos fármacos , Vírus/efeitos dos fármacosRESUMO
The cattle fever tick, Rhipicephalus microplus (Canestrini) (Acari: Ixodidae), is a one-host tick that infests primarily cattle in tropical and sub-tropical regions of the world. This species transmits deadly cattle pathogens, especially Babesia spp., for which a recombinant vaccine is not available. Therefore, disease control depends on tick vector control. Although R. microplus was eradicated in the USA, tick populations in Mexico and South America have acquired resistance to many of the applied acaricides. Recent acaricide-resistant tick reintroductions detected in the U.S. underscore the need for novel tick control methods. The octopamine and tyramine/octopamine receptors, both G protein-coupled receptors (GPCR), are believed to be the main molecular targets of the acaricide amitraz. This provides the proof of principle that investigating tick GPCRs, especially those that are invertebrate-specific, may be a feasible strategy for discovering novel targets and subsequently new anti-tick compounds. The R. microplus leucokinin-like peptide receptor (LKR), also known as the myokinin- or kinin receptor, is such a GPCR. While the receptor was previously characterized in vitro, the function of the leucokinin signaling system in ticks remains unknown. In this work, the LKR was immunolocalized to the periphery of the female midgut and silenced through RNA interference (RNAi) in females. To optimize RNAi experiments, a dual-luciferase system was developed to determine the silencing efficiency of LKR-double stranded RNA (dsRNA) constructs prior to testing those in ticks placed on cattle. This assay identified two effective dsRNAs. Silencing of the LKR with these two validated dsRNA constructs was verified by quantitative real time PCR (qRT-PCR) of female tick dissected tissues. Silencing was significant in midguts and carcasses. Silencing caused decreases in weights of egg masses and in the percentages of eggs hatched per egg mass, as well as delays in time to oviposition and egg hatching. A role of the kinin receptor in tick reproduction is apparent.
Assuntos
Proteínas de Artrópodes/análise , Proteínas de Artrópodes/metabolismo , Trato Gastrointestinal/química , Aptidão Genética , Receptores de Neuropeptídeos/análise , Receptores de Neuropeptídeos/metabolismo , Rhipicephalus/química , Rhipicephalus/fisiologia , Animais , Proteínas de Artrópodes/antagonistas & inibidores , Proteínas de Artrópodes/genética , Feminino , Inativação Gênica , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/genéticaRESUMO
We present an algorithm to calculate free energies and rates from molecular simulations on biased potential energy surfaces. As input, it uses the accumulated times spent in each state or bin of a histogram and counts of transitions between them. Optimal unbiased equilibrium free energies for each of the states/bins are then obtained by maximizing the likelihood of a master equation (i.e., first-order kinetic rate model). The resulting free energies also determine the optimal rate coefficients for transitions between the states or bins on the biased potentials. Unbiased rates can be estimated, e.g., by imposing a linear free energy condition in the likelihood maximization. The resulting "dynamic histogram analysis method extended to detailed balance" (DHAMed) builds on the DHAM method. It is also closely related to the transition-based reweighting analysis method (TRAM) and the discrete TRAM (dTRAM). However, in the continuous-time formulation of DHAMed, the detailed balance constraints are more easily accounted for, resulting in compact expressions amenable to efficient numerical treatment. DHAMed produces accurate free energies in cases where the common weighted-histogram analysis method (WHAM) for umbrella sampling fails because of slow dynamics within the windows. Even in the limit of completely uncorrelated data, where WHAM is optimal in the maximum-likelihood sense, DHAMed results are nearly indistinguishable. We illustrate DHAMed with applications to ion channel conduction, RNA duplex formation, α-helix folding, and rate calculations from accelerated molecular dynamics. DHAMed can also be used to construct Markov state models from biased or replica-exchange molecular dynamics simulations. By using binless WHAM formulated as a numerical minimization problem, the bias factors for the individual states can be determined efficiently in a preprocessing step and, if needed, optimized globally afterward.
Assuntos
Simulação de Dinâmica Molecular , Algoritmos , Ligação de Hidrogênio , Canais Iônicos/química , Canais Iônicos/metabolismo , Cadeias de Markov , Peptídeos/química , Estrutura Secundária de Proteína , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , TermodinâmicaRESUMO
Over a decade has passed since the first description of RNAi in animals--the fundamental endogenous process by which small dsRNAs mediate sequence-specific gene silencing. This discovery has radically transformed our understanding of gene regulation and function and spawned a whole new biotechnology industry focused on developing RNAi-based therapeutic approaches to a variety of human diseases that have otherwise proved challenging to conventional therapies. While RNAi technologies hold great promise as a powerful medical tool, successful delivery of RNAi agents and effective measurement of their uptake are major challenges in translating RNAi therapies to the clinic. Exciting developments in the field have also been tempered by safety concerns surrounding the immunogenic potential of this gene silencing technology and the potential side effects associated with exploiting a crucial biological pathway for therapeutic benefit. This article examines the progress of RNAi therapeutics including advances in delivery and safety, and recent findings from several Phase I-III clinical trials. The emergence of a novel application of RNAi in enhancing the delivery of low-molecular weight drugs to neuronal tissues will also be presented to provide an outlook on the future of RNAi technologies.
Assuntos
Inativação Gênica , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Animais , Biotecnologia/métodos , Ensaios Clínicos como Assunto , Sistemas de Liberação de Medicamentos , Indústria Farmacêutica , Humanos , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/química , RNA de Cadeia Dupla/metabolismoRESUMO
The advent of flow cytometry-based applications has significantly impacted the study of cellular apoptosis. Propidium iodide (PI) is a commonly used viability stain in these studies. Unfortunately, we find that conventional Annexin V/PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment. Both primary cells and cell lines are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence. This distribution spans a wide range of animal models including mice, swine, avian, and teleost fish and potentially affects up to 1016 out of 1019 of peer-reviewed papers published in this area since 1995. We show that the primary ramifications from these findings relate to cells experiencing changes in RNA content. Virally infected cells, for example, are qualified as undergoing apoptosis in response to infection based on conventional staining protocols; in fact, these cells are alive and actively producing viral RNA that can serve to produce additional infectious viral particles. Based on our observations we propose a modified protocol, show that it overcomes previous drawbacks for this technique, and that it will allow for more accurate assessment of cell death across various platforms.
Assuntos
Apoptose , Propídio/metabolismo , Coloração e Rotulagem/métodos , Animais , Antraquinonas/metabolismo , Morte Celular , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Embrião de Galinha , Chlorocebus aethiops , Citoplasma/metabolismo , DNA/metabolismo , Reações Falso-Positivas , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Formaldeído/metabolismo , Carpa Dourada , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Ribonucleases/metabolismo , Sus scrofa , Fixação de Tecidos , Células Vero , Replicação Viral/genéticaRESUMO
Large-scale production of long dsRNA is needed if antiviral applications of RNAi are to succeed in shrimp farm operations. A novel hairpin-RNA expression vector was developed based on the RNA-dependent RNA polymerase (RdRp) gene of yellow head virus (YHV), the cause of a lethal shrimp disease. Using transformed RNase-deficient Escherichia coli, large amounts (approximately 5 mg dsRNA from 130 ml bacterial culture) of long dsRNA (>300 nt) were produced. Large-scale in vivo dsRNA production was approximately one-fourth the cost of production of a commercial in vitro transcription kit. The hairpin-RNA consisted of the target RdRp sequence ("forward") and a 100-base shortened version of its inverted repeat ("reverse") to introduce a loop and bypass the difficulty of including a small "loop" connector into the "carrier" vector. A test group of whiteleg shrimp Penaeus (Litopenaeus) vannamei (approximately 10-15 g) was injected with 25 microg of this dsRNA 1-day prior to YHV challenge while control groups were injected with NaCl solution or similarly prepared dsGFP-RNA. The group injected with YHV-specific dsRNA did not develop yellow head disease during 14-day of observation after YHV challenge, whereas the control groups injected with NaCl and dsGFP-RNA developed gross signs of yellow head disease and died within 7-10 days after challenge. Quantitative RT-PCR and immunohistochemistry revealed that both viral mRNA and viral proteins were suppressed in the protected shrimp.
Assuntos
Biotecnologia , Penaeidae/imunologia , Penaeidae/virologia , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Polimerase Dependente de RNA , Roniviridae/patogenicidade , Animais , Biotecnologia/economia , Biotecnologia/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Imuno-Histoquímica , Penaeidae/enzimologia , Penaeidae/genética , Interferência de RNA/imunologia , RNA de Cadeia Dupla/administração & dosagem , RNA de Cadeia Dupla/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Polimerase Dependente de RNA/administração & dosagem , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Roniviridae/enzimologia , Roniviridae/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Gene expression analysis by cDNA-AFLP in barley ( Hordeum vulgare L.) after powdery mildew ( Blumeria graminis f.sp. hordei , Bgh ) inoculation revealed 615 (3.7%) of 16 500 screened cDNA fragments being differentially regulated 4 and/or 12 h after inoculation. Of these transcript derived fragments (TDFs), 120 were sequenced, and for 28 out of 29 tested, induction was confirmed via RT-PCR. Most TDFs did not show any homology to sequences with known functions, others showed homology to genes involved in primary and secondary metabolism, pathogen response, redox regulation, and signal transduction. TDFs with homology to a MAP kinase ( PWMK1 ), a WRKY transcription factor, a heparanase, an immunophilin, a cytochrome P450, and a receptor-like protein kinase were isolated as full length cDNAs. Knockdown by RNA interference via biolistic delivery of sequence specific double stranded RNA to leaf segments tagged two of these genes as possible candidates being causally involved in the outcome of the barley- Bgh interaction. Knockdown of the receptor-like protein kinase and the WRKY transcription factor increased resistance to the fungus, while knockdown of PWMK1 only led to a slightly enhanced susceptibility of epidermal cells to Bgh . This suggests that the receptor-like protein kinase and the WRKY protein are candidates for negative regulators of powdery mildew resistance. Based on expression analyses, PWMK1 appears to be more generally involved in stress response.