RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells

Inhibins are members of the TGFβ superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse.     

Insect spiracle as the main penetration route of pyrethroids

The penetration route of adhered pyrethroids following direct aerosol spraying was studied in the German cockroach (Blattella germanica) by investigating the relationship between the application site of insecticide and knockdown efficacy. In direct spray, KT₅₀ was 26.4 s and the adhered amount of pyrethroid was 0.745 μg. On the contrary, required amount of pyrethroid to obtain the same KT₅₀ was one-eighth in topical application to the mesothoracic spiracle, while 2.6 times to the ventral mesothorax. KT₅₀ of cockroaches with blocked mesothoracic spiracles was greater than those with unblocked spiracles by 1.8-fold. The amount of directly sprayed pyrethroid penetrating through the inner wall of the mesothoracic trachea was significantly higher than the amount penetrating through the body wall of the ventral mesothorax. Therefore, the knockdown effect of the direct spray was believed to be caused by the flow of pyrethroids into the mesothoracic spiracles and its subsequent penetration through the inner wall of the mesothoracic trachea.     

The pyrethroid resistance status and mechanisms in Aedes aegypti from the Guerrero state,Mexico

Dengue is one of the most important vector-borne diseases worldwide and is a public health problem in Mexico. Most programs in dengue endemic countries rely on insecticides for Aedes control. In Mexico, pyrethroid insecticides (mainly permethrin and deltamethrin) have been extensively used over a decade as adulticides and represented a strong selection for insecticide resistance for dengue vectors in several parts of the country. We studied the type, frequency and distribution of insecticide resistance mechanisms in Aedes aegypti from six municipalities in the state of Guerrero selected on the basis of historically intense chemical control and a high risk for dengue transmission. Ae. aegypti eggs were collected from October 2009 to January 2010 using ovitraps. F₁ adults, emerged from these collections, were exposed to permethrin, deltamethrin and DDT in WHO diagnostic tests and showed high resistance levels to both pyrethroids and DDT. This was consistent with the presence of increased metabolic enzyme activities and target site insensitivity due to kdr mutations. Biochemical assays showed elevated esterase and glutathione S-transferase activities in the six municipalities. The V1016I kdr mutation on the IIS6 domain of the sodium channel gene was present in an overall frequency of 0.80. A second mutation, F1534C on the IIIS6 domain of the same gene was also detected, being the first report of this mutation in Guerrero. The multiple resistance mechanisms present in Ae. aegypti from Guerrero state represent a warning for the efficacy of the pyrethroid usage and consequently for the success of the dengue control program.     

Biochemical and molecular analyses to determine pyrethroid resistance in Aedes aegypti

Dengue fever is an important mosquito-borne viral disease in Taiwan. Insecticide resistance has been shown to significantly reduce the efficacy of vector control interventions. The detection of insecticide resistance is an important component in mosquito abatement programs. In this study, we used the insecticide-impregnated papers bioassay method to reveal high levels of resistance to permethrin in the LYPR and field strains of Aedes aegypti. We used the standard glass cylinder method to observe the knockdown effect of paralysis within 2 to 4 minutes after exposing mosquitoes to pyrethroid vapors. Biochemical assays showed elevated detoxification enzyme activities. Glutathione S-transferases, monooxygenases and β-esterases were the enzymes predominantly responsible for the permethrin resistance of Ae. aegypti in Taiwan. Molecular screening for common insecticide target-site mutations revealed the presence of V1023G and D1794Y mutations. Pearson’s correlation analysis was used to investigate the correlations between the allelic frequency of kdr mutation associated increase with the LC50 values of permethrin and the KT50 values of pyrethroid vaporizers. These findings will be used to assess resistance levels, estimate resistance potential, and formulate monitoring and resistance management strategies.     

RNAi-mediated Knockdown of Xist Does Not Rescue the Impaired Development of Female Cloned Mouse Embryos

In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of Xist during the preimplantation period in both sexes. We found that this aberrant Xist expression could be impeded by deletion of Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with Xist-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of Xist RNA signals remained within single embryos. After implantation, the dysregulated Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.     

Application of DNA-based genotyping techniques for the detection of kdr-like pyrethroid resistance in field populations of Colorado potato beetle

Permethrin resistance in 16 field populations of Colorado potato beetle was established by bioassay. Permethrin-resistant genotypes were detected using two genotyping techniques, bidirectional polymerase chain reaction amplification of specific alleles (bi-PASA) and single stranded conformational polymorphism (SSCP). The bi-PASA technique was 86% and SSCP was 95% reliable. In terms of accuracy, bi-PASA determined the correct genotype compared to the actual sequencing data 98% and SSCP 93% of the time. There was marked correlation between increasing survivorship (increasing LD₅₀values) and increasing proportions of resistant genotypes (increasing percentage of resistant allele). These techniques allowed the detection of a resistant allele 98% of the time when sampling only 384 beetles from an overwintering adult population (4995 beetles/ha) that possessed a 1% resistant allele frequency at a cost of $500/ha, substantiating the cost effectiveness of the genotyping approaches to monitoring.     

反义抑制miR-202引起斑马鱼早期胚胎发育异常研究

为了探讨miR-202在鱼类胚胎发育中的功能,采用实时定量反转录PCR技术和整体原位杂交技术检测了miR-202在斑马鱼胚胎发育阶段的表达。结果发现miR-202是母源性分子并在斑马鱼胚胎发育过程中持续表达,尤其在早期胚胎发育阶段表达水平较高。在此基础上,采用基因沉默技术在斑马鱼受精卵中显微注射miR-202的反义锁核苷酸,实时荧光定量反转录PCR技术和整体原位杂交技术结果显示miR-202反义锁核苷酸可以显著下调斑马鱼胚胎中miR-202表达水平,同时发现反义抑制miR-202后胚胎发育停滞在4 hpf时左右。共同注射miR-202前体可以部分挽救反义抑制miR-202后导致的胚胎发育停滞。本研究证明miR-202在斑马鱼胚胎发育过程中起着重要的调控作用,其功能是斑马鱼胚胎早期发育所必需的。为进一步探索miR-202在鱼类胚胎发育过程的功能奠定了基础。     

Frequency detection of pyrethroid resistance allele in Anopheles sinensis populations by real-time PCR amplification of specific allele (rtPASA)

To investigate the level of pyrethroid resistance in Anopheles sinensis Wiedemann 1828 (Diptera: Culicidae), a major malaria vector in Korea, we cloned and sequenced the IIS4-6 transmembrane segments of the sodium channel gene that encompass the most widely known kdr mutation sites. Sequence analysis revealed the presence of the major Leu-Phe mutation and a minor Leu-Cys mutation at the same position in permethrin-resistant field populations of An. sinensis. To establish a routine method for monitoring resistance, we developed a simple and accurate real-time PCR amplification of specific allele (rtPASA) protocol for the estimation of resistance allele frequencies on a population basis. The kdr allele frequency of a field population predicted by the rtPASA method (60.8%) agreed well with that determined by individual genotyping (61.7%), demonstrating the reliability and accuracy of rtPASA in predicting resistance allele frequency. Using the rtPASA method, the kdr allele frequencies in several field populations of An. sinensis were determined to range from 25.0 to 96.6%, suggestive of widespread pyrethroid resistance in Korea.     

Estimation of knockdown resistance in diamondback moth using real-time PASA

We have developed a simple and accurate real-time PASA (PCR amplification of specific allele) (rtPASA) protocol for the estimation of pyrethroid resistance allele frequency in pooled DNA samples using the T929I-mutated allele of sodium channel α-subunit gene from the diamondback moth, Plutella xylostella, as a model. Conditions for the rtPASA for the detection of the T929I mutation were optimized by adjusting annealing temperature, template, and primer concentrations. Using standard DNA mixtures of susceptible and resistant alleles in various ratios, a plot of allele frequency versus cycle threshold value (C_t value) was generated for the prediction of allele frequency. The semi log plot was linear within the 1-80% range of resistance allele frequencies with a high correlation coefficient (r²=0.997) when highly stringent conditions (67 °C annealing temperature and 2 ng template DNA) were used. For the detection of lower frequencies of the resistant allele (0.004-1%), another hyperbolic semi log plot was constructed (r²=0.983) using the C_t values obtained from the rtPASA with less stringent conditions (66 °C annealing temperature and 5 ng template DNA). The rtPASA was able to detect the resistant allele at frequency as low as 0.02%. The performance of the rtPASA was evaluated by comparing with the data generated from 50 individual genotypings. We demonstrated that the actual resistance allele frequency of a field population of P. xylostella precisely matched the predicted frequency deduced by our protocol. The rtPASA format is applicable for the detection of additional mutations associated with resistance in other insect pest species and will allow rapid and efficient monitoring of resistance in field populations in a high throughput format.