Virus-derived transgenes expressing hairpin RNA give immunity to Tobacco mosaic virus and Cucumber mosaic virus

<p>Abstract</p> <p>Background</p> <p>An effective method for obtaining resistant transgenic plants is to induce RNA silencing by expressing virus-derived dsRNA in plants and this method has been successfully implemented for the generation of different plant lines resistant to many plant viruses.</p> <p>Results</p> <p>Inverted repeats of the partial <it>Tobacco mosaic virus </it>(TMV) movement protein (MP) gene and the partial <it>Cucumber mosaic virus </it>(CMV) replication protein (Rep) gene were introduced into the plant expression vector and the recombinant plasmids were transformed into <it>Agrobacterium tumefaciens</it>. <it>Agrobacterium</it>-mediated transformation was carried out and three transgenic tobacco lines (MP16-17-3, MP16-17-29 and MP16-17-58) immune to TMV infection and three transgenic tobacco lines (Rep15-1-1, Rep15-1-7 and Rep15-1-32) immune to CMV infection were obtained. Virus inoculation assays showed that the resistance of these transgenic plants could inherit and keep stable in T₄progeny. The low temperature (15℃) did not influence the resistance of transgenic plants. There was no significant correlation between the resistance and the copy number of the transgene. CMV infection could not break the resistance to TMV in the transgenic tobacco plants expressing TMV hairpin MP RNA.</p> <p>Conclusions</p> <p>We have demonstrated that transgenic tobacco plants expressed partial TMV movement gene and partial CMV replicase gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to TMV or CMV infection.</p

From Tuberculosis Bedside to Bench: UBE2B Splicing as a Potential Biomarker and Its Regulatory Mechanism

Alternative splicing (AS) is an important approach for pathogens and hosts to remodel transcriptome. However, tuberculosis (TB)-related AS has not been sufficiently explored. Here we presented the first landscape of TB-related AS by long-read sequencing, and screened four AS events (S100A8-intron1-retention intron, RPS20-exon1-alternaitve promoter, KIF13B-exon4-skipping exon (SE) and UBE2B-exon7-SE) as potential biomarkers in an in-house cohort-1. The validations in an in-house cohort-2 (2274 samples) and public datasets (1557 samples) indicated that the latter three AS events are potential promising biomarkers for TB diagnosis, but not for TB progression and prognosis. The excellent performance of classifiers further underscored the diagnostic value of these three biomarkers. Subgroup analyses indicated that UBE2B-exon7-SE splicing was not affected by confounding factors and thus had relatively stable performance. The splicing of UBE2B-exon7-SE can be changed by heat-killed mycobacterium tuberculosis through inhibiting SRSF1 expression. After heat-killed mycobacterium tuberculosis stimulation, 231 ubiquitination proteins in macrophages were differentially expressed, and most of them are apoptosis-related proteins. Taken together, we depicted a global TB-associated splicing profile, developed TB-related AS biomarkers, demonstrated an optimal application scope of target biomarkers and preliminarily elucidated mycobacterium tuberculosis-host interaction from the perspective of splicing, offering a novel insight into the pathophysiology of TB

Multi-Vortex Regulation for Efficient Fluid and Particle Manipulation in Ultra-Low Aspect Ratio Curved Microchannels

Inertial microfluidics enables fluid and particle manipulation for biomedical and clinical applications. Herein, we developed a simple semicircular microchannel with an ultra-low aspect ratio to interrogate the unique formations of the helical vortex and Dean vortex by introducing order micro-obstacles. The purposeful and powerful regulation of dimensional confinement in the microchannel achieved significantly improved fluid mixing effects and fluid and particle manipulation in a high-throughput, highly efficient and easy-to-use way. Together, the results offer insights into the geometry-induced multi-vortex mechanism, which may contribute to simple, passive, continuous operations for biochemical and clinical applications, such as the detection and isolation of circulating tumor cells for cancer diagnostics

Numerical Study of Multivortex Regulation in Curved Microchannels with Ultra-Low-Aspect-Ratio

The field of inertial microfluidics has been significantly advanced in terms of application to fluid manipulation for biological analysis, materials synthesis, and chemical process control. Because of their superior benefits such as high-throughput, simplicity, and accurate manipulation, inertial microfluidics designs incorporating channel geometries generating Dean vortexes and helical vortexes have been studied extensively. However, existing technologies have not been studied by designing low-aspect-ratio microchannels to produce multi-vortexes. In this study, an inertial microfluidic device was developed, allowing the generation and regulation of the Dean vortex and helical vortex through the introduction of micro-obstacles in a semicircular microchannel with ultra-low aspect ratio. Multi-vortex formations in the vertical and horizontal planes of four dimension-confined curved channels were analyzed at different flow rates. Moreover, the regulation mechanisms of the multi-vortex were studied systematically by altering the micro-obstacle length and channel height. Through numerical simulation, the regulation of dimensional confinement in the microchannel is verified to induce the Dean vortex and helical vortex with different magnitudes and distributions. The results provide insights into the geometry-induced secondary flow mechanism, which can inspire simple and easily built planar 2D microchannel systems with low-aspect-ratio design with application in fluid manipulations for chemical engineering and bioengineering

Concentration Gradient Constructions Using Inertial Microfluidics for Studying Tumor Cell–Drug Interactions

With the continuous development of cancer therapy, conventional animal models have exposed a series of shortcomings such as ethical issues, being time consuming and having an expensive cost. As an alternative method, microfluidic devices have shown advantages in drug screening, which can effectively shorten experimental time, reduce costs, improve efficiency, and achieve a large-scale, high-throughput and accurate analysis. However, most of these microfluidic technologies are established for narrow-range drug-concentration screening based on sensitive but limited flow rates. More simple, easy-to operate and wide-ranging concentration-gradient constructions for studying tumor cell&ndash;drug interactions in real-time have remained largely out of reach. Here, we proposed a simple and compact device that can quickly construct efficient and reliable drug-concentration gradients with a wide range of flow rates. The dynamic study of concentration-gradient formation based on successive spiral mixer regulations was investigated systematically and quantitatively. Accurate, stable, and controllable dual drug-concentration gradients were produced to evaluate simultaneously the efficacy of the anticancer drug against two tumor cell lines (human breast adenocarcinoma cells and human cervical carcinoma cells). Results showed that paclitaxel had dose-dependent effects on the two tumor cell lines under the same conditions, respectively. We expect this device to contribute to the development of microfluidic chips as a portable and economical product in terms of the potential of concentration gradient-related biochemical research

Plants at population edges enjoy longer mating distances: Implications for conservation practice

Habitat fragmentation and population declines have led to increased population edges, exposing higher proportions of plants to ecotones. However, it remains a challenging question how edge plants perform compared with interior ones, as they usually experience dissimilar abiotic and biotic interactions. To address this question, we performed experiments on a monoecious herb (Sagittaria trifolia) focusing on the reproductive fitness and mating patterns of individuals at the edge of two common gardens established under varied light conditions (open vs. understory) with natural pollination. Plant female fitness was determined by seed production, whereas male fitness and mating patterns were resolved by paternity analysis employing eight microsatellite markers. Results showed that edge plants had lower pollinator visitation frequency than interior ones in both gardens. However, there was no significant reduction in female or male reproductive success of edge plants. Instead, edge plants enjoyed longer distances of effective pollen dispersal when reproduced as males, as well as longer distances of sires when reproduced as females. This pattern was consistent across habitats and years. Together, the results indicate that edge plants not only maintained a comparable level of reproduction as interior ones but also enjoyed greater mating distances, which would likely bring about higher genetic diversity in their offspring. This further implies that edge effects may play an important role in promoting gene exchange and encouraging population recovery

Monoclonal antibody-based colloid gold immunochromatographic strip for the rapid detection of Tomato zonate spot tospovirus

Abstract Background Tomato zonate spot virus (TZSV), a new species of genus Tospovirus, caused significant losses in yield and problems in quality of many important vegetables and ornamentals in Southwest China and posed a serious threat to important economic crops for the local farmers. A convenient and reliable method was urgently needed for rapid detection and surveillance of TZSV. Methods The nucleocapsid protein (N) of TZSV was expressed in Escherichia coli and purified, and was used as the antigen to immunize BALB/c mice. Three monoclonal antibodies (mAbs) 3A2, 5D2 and 5F7 against TZSV were obtained through the hybridoma technique. The mAb 3A2 was conjugated with colloid gold as detecting reagent; mAb 5D2 was coated on a porous nitrocellulose membrane as the detection line and protein A was coated as the control line respectively. The colloid gold immunochromatographic (GICA) strip was assembled. Results The analysis of Dot-ELISA and Western blot showed that the obtained three independent lines of mAbs 3A2, 5D2 and 5F7 specifically recognized TZSV N. Based on the assembly of GICA strip, the detection of TZSV was achieved by loading the infected sap onto the test strip for visual inspection. The analysis could be completed within 5–10 min. No cross-reaction occurred between TZSV and other tested viruses. The visual detection limit of the test strip for TZSV was 800 fold dilutions of TZSV-infected leaf samples. Conclusion The mAbs were specific and the colloidal GICA strip developed in this study was convenient, fast and reliable for the detection of TZSV. The method could be applied for the rapid diagnosis and surveillance of TZSV in the field

Molecular characterization of feline astrovirus in domestic cats from Northeast China.

Feline astrovirus (FeAstV) which belonged to the genus Mamastrovirus was first identified in the feces of kittens with diarrhea in the USA in 1981 by electron microscopy, and had been reported in many countries. Presently, there are no any reports of the circulation of FeAstV in mainland China. We performed this study to investigate the apparent prevalence and genetic variability of FeAstV infected in cats in mainland China for the first time. We tested fecal samples of 105 cats with diarrhea and 92 asymptomatic cats in five cities in northeast China by RT-PCR targeting RNA-dependent RNA polymerase (RdRp) gene of FeAstV, and analyzed sequences variability and phylogenetic evolution based on the complete capsid gene of FeAstV strains obtained from positive samples. The overall prevalence of FeAstV was 23.4% (46/197) of which 38 were tested in cats with diarrhea (36.2%, 38/105) and 8 were in asymptomatic cats (8.7%, 8/92). Mixed infection with other enteroviruses including feline parvovirus (FPV), feline bocavirus (FBoV) and feline kobuvirus (FeKoV) was found in 38 FeAstV-positive samples. Phylogenetic analysis based on the complete capsid gene revealed all FeAstV strains were divided into two different groups with a 0.454±0.016 of mean amino acid genetic distance between two groups, suggesting that FeAstVs should be classified into two different genotype species. This study provided the first molecular evidence that FeAstV with considerable genetic diversity was circulating in northeast China, and analyzed genetic variability and classification of FeAstVs for the first time