Regeneration of transgenic tamarillo plants

Media were developed to regenerate shoots from leaf pieces of tamarillo (Cyphomandra betacea (Cav.) Sendtner). Shoots were derived via organogenesis and could be easily rooted and transferred to the growth chamber. Transgenic tamarillo plants were produced using the binary vector pKIWI110 in the avirulent Agrobacterium strain LBA4404. All transgenic plants were kanamycin resistant and some plants expressed the D-glucuronidase (gusA) reporter gene and were chlorsulfuron resistant. Molecular evidence for transformation was obtained using PCR (polymerase chain reaction) and Southern hybridization. Inheritance of the transgenic phenotypes was demonstrated in seedling progeny.     

Enhanced transformation of tomato co-cultivated with Agrobacterium tumefaciens C58C1Rifr::pGSFR1161 in the presence of acetosyringone

Summary Explants of tomato (Lycopersicon esculentum Mill cv. Ailsa Craig) were co-cultivated with Agrobacterium tumefaciens C58C1Rif^r::pGSFR1161 in the presence of 20 (M acetosyringone). Transformed root clones were selected on kanamycin medium and the presence of the nptII gene in the plant DNA confirmed by the polymerase chain reaction. Root clones derived from acetosyringone treatment grew more vigorously in the presence of kanamycin and synthesized a greater amount of NPT-II enzyme. The conclusion is that acetosyringone treatment enhances the transformation process, possibly by stimulating multiple insertions of the T-DNA into the host genome.Abbreviations AS acetosyringone - CTAB hexadecyltrimethylammonium bromide - EDTA ethylenediaminetetraacetic acid - ELISA enzyme-linked immunosorbant assay - MS Murashige and Skoog (1962) medium - PCR polymerase chain reaction     

Semiparametric estimation of the transformation model by leveraging external aggregate data in the presence of population heterogeneity

Leveraging information in aggregate data from external sources to improve estimation efficiency and prediction accuracy with smaller scale studies has drawn a great deal of attention in recent years. Yet, conventional methods often either ignore uncertainty in the external information or fail to account for the heterogeneity between internal and external studies. This article proposes an empirical likelihood-based framework to improve the estimation of the semiparametric transformation models by incorporating information about the t-year subgroup survival probability from external sources. The proposed estimation procedure incorporates an additional likelihood component to account for uncertainty in the external information and employs a density ratio model to characterize population heterogeneity. We establish the consistency and asymptotic normality of the proposed estimator and show that it is more efficient than the conventional pseudopartial likelihood estimator without combining information. Simulation studies show that the proposed estimator yields little bias and outperforms the conventional approach even in the presence of information uncertainty and heterogeneity. The proposed methodologies are illustrated with an analysis of a pancreatic cancer study.     

Frog call transmission in forestry monocultures: Are there drawbacks?

Anuran males emit advertisement calls for the purpose of attracting females and repelling conspecific males. Call transmission is influenced by the acoustics of the propagation environment, including vegetation. Thus, forestry monocultures of non-native trees represent artificial environments that could modify call transmission. These monocultures have substituted large areas of Atlantic Forest in southern Brazil, representing a conservation challenge. Considering this context, we hypothesized that anurans have calls that are less attenuated in their native environment than in forest plantations. To test it, we performed sound transmission experiments using calls of three anuran species native to southern Brazil: Boana bischoffi, B. leptolineata, and Hylodes meridionalis. We compared sound attenuation between the native forest and forestry monocultures (Eucalyptus sp. and Pinus sp. forests), and included distance from the sound source, air temperature, humidity, and vegetation density as cofactors in linear mixed models. Our results show that the advertisement calls of the two tree frogs (Boana) attenuate less in tree plantations than in native forests, while the third species shows either no differences or less attenuation in native forests. Our results can be understood according to differences in natural history and microhabitat use among the studied species. The two tree frog species vocalize perched from vegetation so their calls become better transmitted in forestry monocultures, which have fewer trees than the native forest. On the other hand, H. meridionalis calls from rocks on streams and the propagation is finely tuned to this peculiar microhabitat. We discuss the conservation implications of our findings for anurans and for the Atlantic Forest. Abstract in Portuguese and Spanish are available with online material.     

Influence of HIV-1 Genomic RNA on the Formation of Gag Biomolecular Condensates

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55^Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4⁺ T cell nuclear lysates led to the formation of larger BMCs compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggest that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.     

植物多基因干扰载体体系构建与效用分析

多数重要的功能基因属于多基因家族,这些家族成员间存在功能冗余,高效的多基因干扰体系对研究多基因家族成员的生物学功能及其分子调控机制具有重要意义。对pCAMBIA1301载体改造,构建了适用于植物的多基因干扰体系pCAMBIA1301m和pCAMBIA1301s。使用该多基因干扰体系构建了四基因的干扰载体pCAMBIA1301m：35S∷SlPP2C1-2-3-4,4个目标基因为来源于番茄PP2C家族A组的PP2C1、PP2C2、PP2C3和PP2C4,并通过遗传转化导入番茄,用GUS染色和PCR检测转基因阳性植株,再利用RT-qPCR技术检测T₁和T₂代转基因植株中目标基因的干扰效率,用T₂代种子分析转基因番茄对ABA敏感性。结果表明,应用该干扰体系成功获得了四基因干扰的转基因植株35S∷SlPP2C1-2-3-4。在转基因番茄中4个目标基因的表达量显著低于野生型,其干扰效率均高于70%,转基因番茄种子萌发具有强烈的ABA不敏感性。多基因干扰体系能高效地同时沉默多个目标基因。     

人上皮细胞角蛋白纤维结构转化的分析

用0℃冷冻处理2—3 h,一些PcaSE-1和BEL-7404细胞的角蛋白纤维能部分地转化成凝聚颗粒,但在HeLa 和CNE 细胞中不发生这种角蛋白纤维结构转化。当回复温度到37℃15—30 min 时,PcaSE-1 和BEL-7404细胞的这种结构转化能快速回复。相反,在HeLa 和CNE 细胞有丝分裂时,角蛋白纤维能转化成凝聚颗粒,但PcaSE-1细胞和BEL-7404细胞的角蛋白纤维网始终维持纤维状态,且围绕纺锤体分布。上述结果表明:两类上皮细胞角蛋白纤维结构的转化似由不同因子所引起。我们的结果还指出:(1)单用秋水仙素或用秋水仙素和细胞松弛素D 合并处理PcaSE-1细胞不能引起角蛋白纤维凝聚。但经秋水仙素解聚微管后,会增强细胞对冷处理的凝聚反应。(2)冷处理时角蛋白纤维凝聚的形成与细胞是否具有两套不同的中间纤维无关。(3)予先用TritonX-100抽提细胞,角蛋白纤维在冷冻后不能转化成凝聚颗粒。(4)冷冻处理引起的结构转化可能是某些上皮细胞系的角蛋白纤维的一种特殊性质。     

The restricted distribution of potato leafroll luteovirus antigen in potato plants with transgenic resistance resembles that in clones with one type of host genemediated resistance

The distribution of virus-infected cells was examined, by fluorescence microscopy, within plants of a range of potato clones infected with potato leafroll luteovirus (PLRV). This range included nine PLRV-resistant clones, of which four were transgenic lines carrying the PLRV coat protein gene and five were conventionally bred. Plants of these clones were resistant to PLRV multiplication and accumulated less virus antigen in leaf tissue than did susceptible clones. Indirect fluorescent antibody staining of thin sections from carbodiimide-fixed petiole tissue revealed that in plants of PLRV-susceptible clones, virus-infected cells were abundant within both external (abaxial) and internal (adaxial) phloem bundles. In plants of the PLRV-resistant conventionally bred clones and in resistant transgenic lines of cv. Pentland Squire, virus-infected cells were much fewer in number and largely restricted to internal phloem bundles. In resistant transgenic lines of cv. Désirée, this restricted distribution of PLRV antigen was only detected in petioles of young leaves. The results suggest that the transgenic and a host-mediated type of resistance that restricts virtis multiplication have underlying similarities.