Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth

While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E. histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E. histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E. histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual E. histolytica RNAi pathway. These studies dramatically improve the tractability of E. histolytica and open up the possibility of applying genetics to improve understanding of this important pathogen.

Root-to-Shoot Long-Distance Mobile miRNAs Identified from Nicotiana Rootstocks

Root-derived mobile signals play critical roles in coordinating a shoot’s response to underground conditions. However, the identification of root-to-shoot long-distance mobile signals has been scant. In this study, we aimed to characterize root-to-shoot endogenous mobile miRNAs by using an Arabidopsis/Nicotiana interfamilial heterograft in which these two taxonomically distant species with clear genetic backgrounds had sufficient diversity in differentiating miRNA sources. Small RNA deep sequencing analysis revealed that 82 miRNAs from the Arabidopsis scion could travel through the graft union to reach the rootstock, whereas only a very small subset of miRNA (6 miRNAs) preferred the root-to-shoot movement. We demonstrated in an ex vivo RNA imaging experiment that the root-to-shoot mobile Nb-miR164, Nb-miR395 and Nb-miR397 were targeted to plasmodesmata using the bacteriophage coat protein MS2 system. Furthermore, the Nb-miR164 was shown to move from the roots to the shoots to induce phenotypic changes when its overexpressing line was used as rootstock, strongly supporting that root-derived Nb-miR164 was able to modify the scion trait via its long-distance movement.

Myrislignan Induces Redox Imbalance and Activates Autophagy in Toxoplasma gondii

Toxoplasma gondii (T. gondii) is an important health problem in human and animals, and the highlighting side effects of launched therapeutic chemicals cannot be ignored. Thus, it is urgent to develop new drugs to against the infection. Myrislignan originated from nutmeg exhibited excellent anti-T. gondii activity in vitro and in vivo, and was able to destroy mitochondrial function. However, the exact mechanism of action is still unknown. In this study, combining RNAs deep-sequencing analysis and surface plasmon resonance (SPR) analysis, the differentially expressed genes (DEGs) and high affinity proteins suggested that myrislignan may affect the oxidation-reduction process of T. gondii. Furthermore, the upregulating ROS activity after myrislignan incubation verified that myrislignan destroyed the oxidant-antioxidant homeostasis of tachyzoites. Transmission electron microscopy (TEM) indicated that myrislignan induced the formation of autophagosome-like double-membrane structure. Moreover, monodansyl cadaverine (MDC) staining and western blot further illustrated autophagosome formation. Myrislignan treatment induced a significant reduction in T. gondii by flow cytometry analysis. Together, these findings demonstrated that myrislignan can induce the oxidation-reduction in T. gondii, lead to the autophagy, and cause the death of T. gondii.

Zn2+-dependent DNAzymes that cleave all combinations of ribonucleotides

Although several DNAzymes are known, their utility is limited by a narrow range of substrate specificity. Here, we report the isolation of two zinc-dependent DNAzymes, ZincDz1 and ZincDz2, which exhibit compact catalytic core sequences with highly versatile hydrolysis activity. They were selected through in vitro selection followed by deep sequencing analysis. Despite their sequence similarity, each DNAzyme showed different Zn2+-concentration and pH-dependent reaction profiles, and cleaved the target RNA sequences at different sites. Using various substrate RNA sequences, we found that the cleavage sequence specificity of ZincDz2 and its highly active mutant ZincDz2-v2 to be 5′-rN↓rNrPu-3′. Furthermore, we demonstrated that the designed ZincDz2 could cut microRNA miR-155 at three different sites. These DNAzymes could be useful in a broad range of applications in the fields of medicine and biotechnology.

Failure to maintain full-term pregnancies in pig carrying klotho monoallelic knockout fetuses

Background Small animals that show a deficiency in klotho exhibit extremely shortened life span with multiple aging-like phenotypes. However, limited information is available on the function of klotho in large animals such as pigs. Results In an attempt to produce klotho knockout pigs, an sgRNA specific for klotho (targeting exon 3) was designed and Cas9-sgRNA ribonucleoproteins were transfected into porcine fibroblasts. Transfected fibroblasts were cultured for one to 2 days and then directly used for nuclear transfer without selection. The cloned embryos were cultured in vitro for 7 days and analyzed to detect modifications of the klotho gene by both T7E1 and deep sequencing analysis. Modification succeeded in 13 of 20 blastocysts (65%), 8 of which (40.0%) were monoallelic modifications and 5 (25.0%) were biallelic modifications. Based on high mutation rates in blastocysts, we transferred the cloned embryos to 5 recipient pigs; 1 recipient was pregnant and 16 fetuses were recovered at Day 28 post transfer. Of the 16 fetuses, 9 were resorbing and 7 were viable. Four of 9 (44.4%) resorbing fetuses and 3 of the 7 (42.9%) viable fetuses had monoallelic modifications. Thus, 3 klotho monoallelic knockout cell lines were established by primary culture. A total of 2088 cloned embryos reconstructed with 2 frame-shifted cell lines were transferred to 11 synchronized recipients. Of the recipients, 7 of 11 eleven (63.6%) became pregnant. However, none of the pregnancies was maintained to term. To discover why klotho monoallelic knockout fetuses were aborted, expression of aging- and apoptosis-related genes and klotho protein in placentas from klotho monoallelic knockout and wild-type fetuses was investigated. Placentas from klotho monoallelic knockout fetuses showed negatively changed expression of aging- and apoptosis-related genes with lower relative expression of klotho protein. These results indicated that the reason why klotho monoallelic knockout fetuses were not maintained to term was possibly due to decreased klotho expression in placentas, negatively affecting aging- and apoptosis-related genes. Conclusions Klotho monoallelic knockout porcine fetal fibroblasts were successfully established. However, pigs carrying klotho monoallelic knockout fetuses failed to maintain full-term pregnancy and a decrease in klotho expression in placenta likely leads to pregnancy loss.

Replication Dynamics, Pathogenicity, and Evolution of Influenza Viruses in Intestinal Caco-2 Cells

Background: Influenza virus is a major cause of respiratory infections worldwide. Besides the common respiratory symptoms, namouras cases with gastrointestinal symptoms have been reported. Moreover, influenza virus has been detected in feces of up to 20.6 % of influenza-infected patients. Therefore, direct infection of intestinal cells with influenza virus is suspected; however, the mechanism of this infection has not been explored. AIM: To investigate influenza virus replication, cellular responses to infection, and virus evolution following serial infection in human Caucasian colon adenocarcinoma cells (Caco-2 cells). Method: Two influenza A subtypes (A/H3N2 and A/H1N1pdm 09) and one influenza B virus (B/Yamagata) were serially passaged in Caco-2. Quantitative PCR was used to study hormones and cytokines expression following infection. Deep sequencing analysis of viral genome was used to assess the virus evolution. Results: The replication capacity of the three viruses was maintained throughout 12 passages, with H3N2 virus being the fastest in adaptation. The expression of hormone and cytokines in Caco-2 cells was considerably different between the viruses and among the passages, however, a pattern of induction was observed at the late phase of infection. Deep sequencing analysis revealed a few amino acid substitutions in the HA protein of H3N2 and H1N1 viruses, mostly in the antigenic site. Moreover, virus evolution at the quasispecies level based on HA protein revealed that H3N2 and H1N1 harbored more diverse virus populations when compared to IBV, indicating their higher evolution within Caco-2 cells. Conclusion: The findings of this study indicate the possibility of influenza virus replication in intestinal cells. To further explain the gastrointestinal complications of influenza infections in-vivo experiments with different influenza viruses are needed.