Trade-off between local replication and long-distance dissemination during experimental evolution of a satellite RNA.

Satellite RNAs (satRNAs) are molecular parasites that depend on their non-homologous helper viruses (HVs) for essential biological functions. While there are multiple molecular and phylogenetic studies on satRNAs, there is no experimental evolution study on how satRNAs may evolve in common infection conditions. In this study, we serially passaged the Bamboo mosaic virus (BaMV) associated-satRNA (satBaMV) under conditions in which satBaMV either coinfects an uninfected host plant, Nicotiana benthamiana, with BaMV or superinfects a transgenic N. benthamiana expressing the full-length BaMV genome. Single-nucleotide polymorphisms (SNPs) of satBaMV populations were analyzed by deep sequencing. Forty-eight SNPs were identified across four different experimental treatments. Most SNPs are treatment-specific, and some are also ephemeral. However, mutations at positions 30, 34, 63, and 82, all located at the 5' untranslated region (UTR), are universal in all treatments. These universal SNPs are configured into several haplotypes and follow different population dynamics. We constructed isogenic satBaMV strains only differing at positions 30 and 82 and conducted competition experiments in protoplasts and host plants. We found that the haplotype that reached high frequency in protoplasts and inoculation leaves also exhibited poor dissemination to systemic leaves and vice versa, thus suggesting an apparent trade-off between local replication and long-distance dissemination. We posit that the trade-off is likely caused by antagonistic pleiotropy at the 5' UTR. Our findings revealed a hitherto under-explored connection between satRNA genome replication and movement within a host plant. The significance of such a connection during satRNA evolution warrants a more thorough investigation.

Exploring the Multifunctional Roles of Odontoglossum Ringspot Virus P126 in Facilitating Cymbidium Mosaic Virus Cell-to-Cell Movement during Mixed Infection.

Synergistic interactions among viruses, hosts and/or transmission vectors during mixed infection can alter viral titers, symptom severity or host range. Viral suppressors of RNA silencing (VSRs) are considered one of such factors contributing to synergistic responses. Odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV), which are two of the most significant orchid viruses, exhibit synergistic symptom intensification in Phalaenopsis orchids with unilaterally enhanced CymMV movement by ORSV. In order to reveal the underlying mechanisms, we generated infectious cDNA clones of ORSV and CymMV isolated from Phalaenopsis that exerted similar unilateral synergism in both Phalaenopsis orchid and Nicotiana benthamiana. Moreover, we show that the ORSV replicase P126 is a VSR. Mutagenesis analysis revealed that mutation of the methionine in the carboxyl terminus of ORSV P126 abolished ORSV replication even though some P126 mutants preserved VSR activity, indicating that the VSR function of P126 alone is not sufficient for viral replication. Thus, P126 functions in both ORSV replication and as a VSR. Furthermore, P126 expression enhanced cell-to-cell movement and viral titers of CymMV in infected Phalaenopsis flowers and N. benthamiana leaves. Taking together, both the VSR and protein function of P126 might be prerequisites for unilaterally enhancing CymMV cell-to-cell movement by ORSV.

Argonaute 5 family proteins play crucial roles in the defence against Cymbidium mosaic virus and Odontoglossum ringspot virus in Phalaenopsis aphrodite subsp. formosana.

The orchid industry faces severe threats from diseases caused by viruses. Argonaute proteins (AGOs) have been shown to be the major components in the antiviral defence systems through RNA silencing in many model plants. However, the roles of AGOs in orchids against viral infections have not been analysed comprehensively. In this study, Phalaenopsis aphrodite subsp. formosana was chosen as the representative to analyse the AGOs (PaAGOs) involved in the defence against two major viruses of orchids, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). A total of 11 PaAGOs were identified from the expression profile analyses of these PaAGOs in P. aphrodite subsp. formosana singly or doubly infected with CymMV and/or ORSV. PaAGO5b was found to be the only one highly induced. Results from overexpression of individual PaAGO5 family genes revealed that PaAGO5a and PaAGO5b play central roles in the antiviral defence mechanisms of P. aphrodite subsp. formosana. Furthermore, a virus-induced gene silencing vector based on Foxtail mosaic virus was developed to corroborate the function of PaAGO5s. The results confirmed their importance in the defences against CymMV and ORSV. Our findings may provide useful information for the breeding of traits for resistance or tolerance to CymMV or ORSV infections in Phalaenopsis orchids.

Dual resistance of transgenic plants against Cymbidium mosaic virus and Odontoglossum ringspot virus.

Taxonomically distinct Cymbidium mosaic potexvirus (CymMV) and Odontoglossum ringspot tobamovirus (ORSV) are two of the most prevalent viruses worldwide; when co-infecting orchids, they cause synergistic symptoms. Because of the huge economic loss in quality and quantity in the orchid industry with virus-infected orchids, virus-resistant orchids are urgently needed. To date, no transgenic resistant lines against these two viruses have been reported. In this study, we generated transgenic Nicotiana benthamiana expressing various constructs of partial CymMV and ORSV genomes. Several transgenic lines grew normally and remained symptomless after mixed inoculation with CymMV and ORSV. The replication of CymMV and ORSV was approximately 70-90% lower in protoplasts of transgenic lines than wild-type (WT) plants. Of note, we detected extremely low or no viral RNA or capsid protein of CymMV and ORSV in systemic leaves of transgenic lines after co-infection. Grafting experiments further revealed that CymMV and ORSV trafficked extremely inefficiently from co-infected WT stocks to transgenic scions, presumably due to RNA-mediated interference. This study reports the first successful creation of dual resistant transgenic lines against CymMV and ORSV. Our studies shed light on the commercial development of transgenic orchid production to combat the global viral threat.

microRPM: a microRNA prediction model based only on plant small RNA sequencing data.

Motivation: MicroRNAs (miRNAs) are endogenous non-coding small RNAs (of about 22 nucleotides), which play an important role in the post-transcriptional regulation of gene expression via either mRNA cleavage or translation inhibition. Several machine learning-based approaches have been developed to identify novel miRNAs from next generation sequencing (NGS) data. Typically, precursor/genomic sequences are required as references for most methods. However, the non-availability of genomic sequences is often a limitation in miRNA discovery in non-model plants. A systematic approach to determine novel miRNAs without reference sequences is thus necessary. Results: In this study, an effective method was developed to identify miRNAs from non-model plants based only on NGS datasets. The miRNA prediction model was trained with several duplex structure-related features of mature miRNAs and their passenger strands using a support vector machine algorithm. The accuracy of the independent test reached 96.61% and 93.04% for dicots (Arabidopsis) and monocots (rice), respectively. Furthermore, true small RNA sequencing data from orchids was tested in this study. Twenty-one predicted orchid miRNAs were selected and experimentally validated. Significantly, 18 of them were confirmed in the qRT-PCR experiment. This novel approach was also compiled as a user-friendly program called microRPM (miRNA Prediction Model). Availability and implementation: This resource is freely available at http://microRPM.itps.ncku.edu.tw. Contact: nslin@sinica.edu.tw or sarah321@mail.ncku.edu.tw. Supplementary information: Supplementary data are available at Bioinformatics online.

The Nucleolar Fibrillarin Protein Is Required for Helper Virus-Independent Long-Distance Trafficking of a Subviral Satellite RNA in Plants.

RNA trafficking plays pivotal roles in regulating plant development, gene silencing, and adaptation to environmental stress. Satellite RNAs (satRNAs), parasites of viruses, depend on their helper viruses (HVs) for replication, encapsidation, and efficient spread. However, it remains largely unknown how satRNAs interact with viruses and the cellular machinery to undergo trafficking. Here, we show that the P20 protein of Bamboo mosaic potexvirus satRNA (satBaMV) can functionally complement in trans the systemic trafficking of P20-defective satBaMV in infected Nicotiana benthamiana The transgene-derived satBaMV, uncoupled from HV replication, was able to move autonomously across a graft union identified by RT-qPCR, RNA gel blot, and in situ RT-PCR analyses. Coimmunoprecipitation experiments revealed that the major nucleolar protein fibrillarin is coprecipitated in the P20 protein complex. Notably, silencing fibrillarin suppressed satBaMV-, but not HV-, phloem-based movement following grafting or coinoculation with HV Confocal microscopy revealed that the P20 protein colocalized with fibrillarin in the nucleoli and formed punctate structures associated with plasmodesmata. The mobile satBaMV RNA appears to exist as ribonucleoprotein (RNP) complex composed of P20 and fibrillarin, whereas BaMV movement proteins, capsid protein, and BaMV RNA are recruited with HV coinfection. Taken together, our findings provide insight into movement of satBaMV via the fibrillarin-satBaMV-P20 RNP complex in phloem-mediated systemic trafficking.

Application of an Integrated Omics Approach for Identifying Host Proteins That Interact With Odontoglossum ringspot virus Capsid Protein.

The glutamic acid at position 100 (E(100)) in the capsid protein (CP) of Odontoglossum ringspot virus (ORSV) plays an important role in long-distance viral movement in Nicotiana benthamiana. The ORSV(E100A) mutant, which has a glutamic acid to alanine substitution, shows a loss of systemic infectivity in N. benthamiana. Transmission electron microscopy and size-exclusion chromatography assays showed that E(100) is essential for CP-CP interaction and viral particle assembly. To identify the ORSV triggering or response genes and CP-interacting proteins (CP-IP), an integrated omics approach based on next-generation sequencing and proteomics profiling was used in this study. The whole-transcriptomes of healthy and ORSV-infected leaves of N. benthamiana were analyzed, and the gene information was used to create a N. benthamiana protein database that was used for protein identification following mass spectrometry analysis. The integrated omics approach identified several putative host proteins that interact with ORSV CP(WT) and were categorized as photosystem subunits, defense-associated proteins, and cell division components. The expression pattern and CP interaction of these CP-IP were examined by semiquantitative reverse transcription polymerase chain reaction and an in vitro binding assay, respectively, to verify the in silico data. Among these proteins, a proteinase inhibitor of N. benthamiana (NbPI2) was highly associated with CP(E100A) as compared with CP(WT), and NbPI1 and NbPI2 were highly induced in ORSV-infected plants. NbPI1- and NbPI2-silenced plants (via a Tobacco rattle virus-induced gene-silencing system) did not exhibit a difference in ORSV infection. Thus, whether NbPI1 and NbPI2 play a role in plant immunity requires further investigation. In summary, the integrated omics approach provides massive and valuable information to identify the ORSV CP-IP and these CP-IP will help us to understand the movement of this virus and plant-virus interaction.

The ubiquitin-like (UBX)-domain-containing protein Ubx2/Ubxd8 regulates lipid droplet homeostasis.

Lipid droplets (LDs) are central organelles for maintaining lipid homeostasis. However, how cells control the size and number of LDs remains largely unknown. Herein, we report that Ubx2, a UBX-domain-containing protein involved in endoplasmic reticulum (ER)-associated degradation, is crucial for LD maintenance. Ubx2 redistributes from the ER to LDs when LDs start to form and enlarge during diauxic shift and in the stationary phase. ubx2Δ cells contain abnormal numbers of LDs that are smaller than normal, and their triacylglycerol (TAG) is reduced to 50% of the normal level. Deletion of either the UBX or UBA domain in Ubx2 has no effect, but deletion of both causes LD phenotypes similar to that in ubx2Δ. The reduced level of TAG in ubx2Δ is probably the result of mislocalization of phospholipid:diacylglycerol acyltransferase (Lro1), one of the two TAG-synthesizing enzymes in yeast, which moves along the ER and distributes dynamically to the putative LD assembly sites abutting LDs. Thus, Ubx2 is important for the maintenance of cellular TAG homeostasis probably through Lro1. The mammalian Ubxd8 (also known as FAF2), when expressed in yeast, complements the defect of ubx2Δ, implying a functional conservation for these UBX-domain-containing proteins in lipid homeostasis.

Viral protein targeting to the cortical endoplasmic reticulum is required for cell-cell spreading in plants.

Many plant RNA viruses use their nonstructural proteins to target and move through the cortical endoplasmic reticulum (ER) tubules within the plant intercellular junction for cell-to-cell spreading. Most of these proteins, including the triple-gene-block 3 protein (TGBp3) of Potexvirus, are ER membrane proteins. We previously showed that TGBp3 of the Bamboo mosaic potexvirus partitions into tubular subdomains of the ER in both yeast and plants, but the mechanism and physiological significance of this localization is unclear. Here, we demonstrate that a sorting signal present in TGBp3 is necessary and sufficient for its oligomerization and for targeting integral membrane proteins into puncta within curved ER tubules. Mutations in the TGBp3 sorting signal impair viral spread, and plants infected with viruses harboring these mutants were either asymptomatic or had reduced symptoms. Thus, we propose that Potexvirus use the sorting signal in TGBp3 to target infectious viral derivatives to cortical ER tubules for transmission through the intercellular junctions in plants.

Traffic of a viral movement protein complex to the highly curved tubules of the cortical endoplasmic reticulum.

Intracellular trafficking of the nonstructural movement proteins of plant viruses plays a crucial role in sequestering and targeting viral macromolecules in and between cells. Many of the movement proteins traffic in unconventional, yet mechanistically unknown, pathways to localize to the cell periphery. Here we study trafficking strategies associated with two integral membrane movement proteins TGBp2 and TGBp3 of Potexvirus in yeast. We demonstrate that this simple eukaryote recapitulates the targeting of TGBp2 to the peripheral bodies at the cell cortex by TGBp3. We found that these viral movement proteins traffic as an approximately 1:1 stoichiometric protein complex that further polymerizes to form punctate structures. Many punctate structures depart from the perinuclear endoplasmic reticulum (ER) and move along the tubular ER to the cortical ER, supporting that it involves a lateral sorting event via the ER network. Furthermore, the peripheral bodies are associated with cortical ER tubules that are marked by the ER shaping protein reticulon in both yeast and plants. Thus, our data support a model in which the peripheral bodies partition into and/or stabilize at highly curved membrane environments.

Left internal iliac artery and vein tear during microendoscopic lumbar discectomy - a case report.

As minimally invasive techniques have gained popularity across surgical specialties, microendoscopic discectomy has been hailed as one of the newest and best methods for disc removal. Although problems are unusual and infrequent, the complications that can be associated with this procedure should be realized. Iatrogenic major vascular injury is a rare but serious complication during microendoscopic discectomy, and early detection is difficult due to the surgical positions, the specific anatomy of the spine, and the subtle changes of signs and symptoms the patient manifests. Here, we present a female patient who suffered from left internal iliac artery and vein tear during microendoscopic lumbar discectomy. We conclude that both surgeons and anesthesiologists should be aware of the possibility of this complication during surgery. When this complication occurs, it is vitally important that the correct crisis resolution protocols, such as proper massive transfusion algorithm and successful surgical interventions, be applied immediately during the critical period.

Depth of the thoracic epidural space in paramedian approach.

STUDY OBJECTIVE: To define the depth of the thoracic epidural space in the paramedian axis. DESIGN: Retrospective study. SETTING: Operating room of a tertiary care medical center. PATIENTS: Nine hundred ninety-eight consecutive adults scheduled for elective major cardiothoracic/abdominal surgery and postoperative thoracic epidural pain control. INTERVENTIONS: The thoracic epidural pain control was accomplished via paramedian approach at indicated levels in 977 of 998 patients with uniform and well-standardized technique routinely performed in this institute. MEASUREMENTS: The depth of the epidural space, defined as the distance from the needle tip just penetrating the epidural layer to the overlying skin, was measured by directly checking the length markers displayed on the needle. Association between demographic variables and epidural depths at different thoracic levels was analyzed. MAIN RESULTS: The mean thoracic epidural depth was (mean+/-SD) 5.11+/-0.94 cm, which was positively correlated with the body weight (regression coefficient=0.039, P<.001) and body mass index but was unrelated to sex, age, or body height. On stepwise linear multivariate regression analysis, each 10 kg of increase in body weight would result in a 0.39-cm increase in the depth. Besides, this paramedian depth was 0.34 cm longer at upper thoracic levels (T9 and upper) than that at lower levels (T10 and lower, P<.001). CONCLUSIONS: Body weight, body mass index, and anatomical levels determine the paramedian thoracic epidural depth. The greater the patient's weight and the higher the puncture level, the deeper the thoracic epidural space from the body surface.

Complications of fluid overloading with different distention media in hysteroscopy--a report of two cases.

Complications arising from hysteroscopy are relatively rare events. They occur more frequently with operative hysteroscopy than with diagnostic hysteroscopy. Hysteroscopic procedures, which are alternatives to hysterectomy for surgical treatment of menorrhagia and uterine fibroids, place women to run the risk of intravasation of uterine distention fluid. Excessive intravasation can entail fluid overload, pulmonary edema, congestive heart failure, and electrolyte imbalances. The prerequisite for treatment of hysteroscopic fluid overload is knowing the nature of the intravasation fluid and it should be promptly treated to prevent neurological sequelae. Almost all serious complications of operative hysteroscopy can be avoided if proper precautions are taken and close communication is maintained between the gynecologic surgeon, the anesthesiologist, and nursing staff. Here, we present two cases of fluid overload with acute pulmonary edema and electrolyte imbalance from hysteroscopy with different distention media.

Accidental subdural catheterization due to complication of epidural anesthesia--a case report.

Although accidental subdural injection is a well-recognized complication of epidural block, only a mere handful cases have been substantially proven by radiological evidence. Here we report a case of subdural catheterization during the attempt of epidural anesthesia for a gynecological procedure. Its clinical course and radiological findings are compared with those of the cases previously reported in literature. Whenever there is the occurrence of widespread of sensory block together with respiratory distress and hemodynamic unstability following epidural injection of local anesthetic, a subdural injection should be considered in spite of a negative confirmation. Repeated subdural injection of a local anesthetic at the same site may predispose patients to serious morbidity. Therefore, we recommend that when a subdural injection is evident or suspected, reinsertion of the catheter in the epidural space via another entry or contemplation of a switch to another anesthetic technique is mandatory.