Cooperative recruitment of RDR6 by SGS3 and SDE5 during small interfering RNA amplification in Arabidopsis.

Small interfering RNAs (siRNAs) are often amplified from transcripts cleaved by RNA-induced silencing complexes (RISCs) containing a small RNA (sRNA) and an Argonaute protein. Amplified siRNAs, termed secondary siRNAs, are important for reinforcement of target repression. In plants, target cleavage by RISCs containing 22-nucleotide (nt) sRNA and Argonaute 1 (AGO1) triggers siRNA amplification. In this pathway, the cleavage fragment is converted into double-stranded RNA (dsRNA) by RNA-dependent RNA polymerase 6 (RDR6), and the dsRNA is processed into siRNAs by Dicer-like proteins. Because nonspecific RDR6 recruitment causes nontarget siRNA production, it is critical that RDR6 is specifically recruited to the target RNA that serves as a template for dsRNA formation. Previous studies showed that Suppressor of Gene Silencing 3 (SGS3) binds and stabilizes 22-nt sRNA-containing AGO1 RISCs associated with cleaved target, but how RDR6 is recruited to targets cleaved by 22-nt sRNA-containing AGO1 RISCs remains unknown. Here, using cell-free extracts prepared from suspension-cultured Arabidopsis thaliana cells, we established an in vitro system for secondary siRNA production in which 22-nt siRNA-containing AGO1-RISCs but not 21-nt siRNA-containing AGO1-RISCs induce secondary siRNA production. In this system, addition of recombinant Silencing Defective 5 (SDE5) protein remarkably enhances secondary siRNA production. We show that RDR6 is recruited to a cleavage fragment by 22-nt siRNA-containing AGO1-RISCs in coordination with SGS3 and SDE5. The SGS3-SDE5-RDR6 multicomponent recognition system and the poly(A) tail inhibition may contribute to securing specificity of siRNA amplification.

Tomato brown rugose fruit virus resistance generated by quadruple knockout of homologs of TOBAMOVIRUS MULTIPLICATION1 in tomato.

Tomato brown rugose fruit virus (ToBRFV) is an emerging virus of the genus Tobamovirus. ToBRFV overcomes the tobamovirus resistance gene Tm-22 and is rapidly spreading worldwide. Genetic resources for ToBRFV resistance are urgently needed. Here, we show that clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9)-mediated targeted mutagenesis of four tomato (Solanum lycopersicum) homologs of TOBAMOVIRUS MULTIPLICATION1 (TOM1), an Arabidopsis (Arabidopsis thaliana) gene essential for tobamovirus multiplication, confers resistance to ToBRFV in tomato plants. Quadruple-mutant plants did not show detectable ToBRFV coat protein (CP) accumulation or obvious defects in growth or fruit production. When any three of the four TOM1 homologs were disrupted, ToBRFV CP accumulation was detectable but greatly reduced. In the triple mutant, in which ToBRFV CP accumulation was most strongly suppressed, mutant viruses capable of more efficient multiplication in the mutant plants emerged. However, these mutant viruses did not infect the quadruple-mutant plants, suggesting that the resistance of the quadruple-mutant plants is highly durable. The quadruple-mutant plants also showed resistance to three other tobamovirus species. Therefore, tomato plants with strong resistance to tobamoviruses, including ToBRFV, can be generated by CRISPR/Cas9-mediated multiplexed genome editing. The genome-edited plants could facilitate ToBRFV-resistant tomato breeding.

Cytoplasmic assembly and selective nuclear import of Arabidopsis Argonaute4/siRNA complexes.

In plants, DNA methylation can be mediated by a class of Argonaute4 (AGO4)-associated heterochromatic siRNAs (hc-siRNAs), through a pathway termed RNA-directed DNA methylation (RdDM). It has been thought that RdDM is solely a nuclear process, as both the biogenesis and functioning of hc-siRNAs take place in the nucleus. In this study, we unexpectedly found that hc-siRNAs are predominantly present in the cytoplasm. We demonstrated that AGO4 is loaded with hc-siRNAs in the cytoplasm and the formation of mature AGO4/siRNA complexes requires HSP90 and the cleavage activity of AGO4. Intriguingly, siRNA binding facilitates the redistribution of AGO4 into the nucleus, likely through inducing conformational change that leads to the exposure of the nuclear localization signal (NLS). Our findings reveal an unsuspected cytoplasmic step in the RdDM pathway. We propose that selective nuclear import of mature AGO4/siRNA complexes is a key regulatory point prior to the effector stage of RdDM.

Purification and functional characterization of tomato mosaic virus 130K protein expressed in silkworm pupae using a baculovirus vector.

Tomato mosaic virus (ToMV; genus, Tobamovirus) is a member of the alpha-like virus superfamily of positive-strand RNA viruses, which includes many plant and animal viruses of agronomical and clinical importance. The genomes of alpha-like viruses encode replication-associated proteins that contain methyltransferase, helicase and/or polymerase domains. The three-dimensional structure of the helicase domain fragment of ToMV has been determined, but the structures of the other domains of alpha-like virus replication proteins are not available. In this study, we expressed full-length ToMV replication-associated protein 130 K, which contains the methyltransferase and helicase domains, using the baculovirus-silkworm expression system and purified the recombinant protein to near homogeneity. Purified 130 K, which was stable in phosphate buffer containing magnesium ions and ATP, formed a dimer in solution and hydrolyzed nucleoside 5'-triphosphates.

Correlation between vancomycin penetration into cerebrospinal fluid and protein concentration in cerebrospinal fluid/serum albumin ratio.

Bacterial meningitis is a life-threatening condition. Vancomycin (VCM) is one of the antibiotics used as empirical therapy for bacterial meningitis. It is essential to maintain an adequate concentration of VCM in cerebrospinal fluid (CSF) to treat bacterial meningitis effectively. VCM administered intravenously must pass the blood-brain barrier (BBB) to enter the CSF and the extent of VCM penetration into CSF varies widely among patients. Previous report indicated that CSF albumin level is useful for estimation of VCM CSF penetration. However, CSF albumin level is not measured in routine practice. We focused on CSF protein concentration that is generally examined at the beginning of diagnosis and treatment of bacterial meningitis. We examined the relationship between CSF protein concentration/serum albumin ratio and the extent of VCM penetration into CSF. This retrospective study involved 7 patients admitted to our hospital who were treated with VCM for suspected bacterial meningitis. The VCM concentrations in serum and CSF were 17.6 ± 7.2 µg/mL and 3.31 ± 3.14 µg/mL, respectively. The serum VCM concentrations showed no significant correlation with CSF VCM concentrations. On the other hand, the protein concentration in CSF/serum albumin ratio showed a strong positive correlation with the VCM CSF/serum ratio (r = 0.877, p < 0.005). Our study indicates that the ratio of CSF protein concentration/serum albumin is likely useful for estimating the approximate VCM CSF/serum ratio. This could contribute to an improvement in the treatment of bacterial meningitis.

Viruses roll the dice: the stochastic behavior of viral genome molecules accelerates viral adaptation at the cell and tissue levels.

Recent studies on evolutionarily distant viral groups have shown that the number of viral genomes that establish cell infection after cell-to-cell transmission is unexpectedly small (1-20 genomes). This aspect of viral infection appears to be important for the adaptation and survival of viruses. To clarify how the number of viral genomes that establish cell infection is determined, we developed a simulation model of cell infection for tomato mosaic virus (ToMV), a positive-strand RNA virus. The model showed that stochastic processes that govern the replication or degradation of individual genomes result in the infection by a small number of genomes, while a large number of infectious genomes are introduced in the cell. It also predicted two interesting characteristics regarding cell infection patterns: stochastic variation among cells in the number of viral genomes that establish infection and stochastic inequality in the accumulation of their progenies in each cell. Both characteristics were validated experimentally by inoculating tobacco cells with a library of nucleotide sequence-tagged ToMV and analyzing the viral genomes that accumulated in each cell using a high-throughput sequencer. An additional simulation model revealed that these two characteristics enhance selection during tissue infection. The cell infection model also predicted a mechanism that enhances selection at the cellular level: a small difference in the replication abilities of coinfected variants results in a large difference in individual accumulation via the multiple-round formation of the replication complex (i.e., the replication machinery). Importantly, this predicted effect was observed in vivo. The cell infection model was robust to changes in the parameter values, suggesting that other viruses could adopt similar adaptation mechanisms. Taken together, these data reveal a comprehensive picture of viral infection processes including replication, cell-to-cell transmission, and evolution, which are based on the stochastic behavior of the viral genome molecules in each cell.

In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90.

RNA-induced silencing complexes (RISCs) play central roles in posttranscriptional gene silencing. In plants, the mechanism of RISC assembly has remained elusive due to the lack of cell-free systems that recapitulate the process. In this report, we demonstrate that plant AGO1 protein synthesized by in vitro translation using an extract of evacuolated tobacco protoplasts incorporates synthetic small interfering RNA (siRNA) and microRNA (miRNA) duplexes to form RISCs that sequester the single-stranded siRNA guide strand and miRNA strand, respectively. The formed RISCs were able to recognize and cleave the complementary target RNAs. In this system, the siRNA duplex was incorporated into HSP90-bound AGO1, and subsequent removal of the passenger strand was triggered by ATP hydrolysis by HSP90. Removal of the siRNA passenger strand required the ribonuclease activity of AGO1, while that of the miRNA star strand did not. Based on these results, the mechanism of plant RISC formation is discussed.

[Surgical Technique for Lumbar Spinal Extraforaminal Schwannoma].

INTRODUCTION: Spinal neurinomas are frequently observed in the cauda equina, particularly within the dura mater. However, extraforaminal schwannomas(EFS), which are neither intradural nor dumbbell type, are relatively rare; hence, different surgical procedures have been employed. Here, we report 12 cases of lumbar EFS with total resection that were safely performed. METHODS AND SUBJECTS: This study was comprised of 12 patients with lumbar EFS(excluding those with neurofibromas)who were previously treated at our hospital. Using the Wiltse paraspinal approach, we inserted the microscope until it reached the tumor surface. After confirming the tumor surface, we inserted a scalpel to make an incision in a portion of the outer capsule. Then, we confirmed that the surfaces of the outer capsule and the tumor within the capsule could be dissected. Next, a thread was attached to the outer capsule and pulled upwards so that there was enough space to perform the required procedures within the capsule. Using a CUSA®, we removed the intracapsular tumor. After its removal, the outer capsule was treated carefully to ensure that the origin nerve was not damaged. RESULTS: In all cases, we successfully performed a total removal of the intracapsular tumor;in the mean postoperative observation period of 8 years, no recurrence was observed. Although temporary paralysis was observed in three patients, this improved over time. CONCLUSION: We report our experience with a surgical technique that ensured the safe removal of lumbar EFS. Satisfactory results were achieved, with no recurrences observed during the mean 8-year postoperative observation period.

[Virus resistance genes in plants].

Plants defend themselves from virus infection by RNA silencing and resistance (R) gene-mediated mechanisms. Many dominant R genes encode nucleotide-binding site and leucine-rich repeat (NB-LRR)-containing proteins. NB-LRR proteins are also encoded by R genes against bacteria or fungi, suggesting a similar mechanism underlies defense systems to diverse pathogens. In contrast, several non-NB-LRR-type R genes have recently been cloned, each of which differs from others in sequences and functions. In this review, we introduce a diversity of R gene-mediated plant defense systems against viruses. Tm-1, JAX1, and Scmv1, resistance genes against tomato mosaic virus, potexviruses, and sugarcane mosaic virus, respectively, inhibit virus multiplication at a single cell level. The RTM1, RTM2, RTM3 genes of Arabidopsis thaliana inhibit systemic transport of potyviruses through the phloem. STV11 of rice against rice stripe virus and Ty-1 and Ty-3 genes of tomato against tomato yellow leaf curl virus allow low level virus multiplication and confer tolerance. The wide diversity of plant defense systems against viruses implies their recent emergence. We suggest that plants evolved new defense systems to counter infection by viruses that had overcome pre-existing defense systems (RNA silencing and NB-LRR-type R gene-mediated systems).

A Short Open Reading Frame Encompassing the MicroRNA173 Target Site Plays a Role in trans-Acting Small Interfering RNA Biogenesis.

trans-Acting small interfering RNAs (tasiRNAs) participate in the regulation of organ morphogenesis and determination of developmental timing in plants by down-regulating target genes through mRNA cleavage. The production of tasiRNAs is triggered by microRNA173 (miR173) and other specific microRNA-mediated cleavage of 5'-capped and 3'-polyadenylated primary TAS transcripts (pri-TASs). Although pri-TASs are not thought to encode functional proteins, they contain multiple short open reading frames (ORFs). For example, the primary TAS2 transcript (pri-TAS2) contains 11 short ORFs, and the third ORF from the 5' terminus (ORF3) encompasses the miR173 target site. Here, we show that nonsense mutations in ORF3 of pri-TAS2 upstream of the miR173 recognition site suppress tasiRNA accumulation and that ORF3 is translated in vitro. Glycerol gradient centrifugation analysis of Arabidopsis (Arabidopsis thaliana) plant extracts revealed that pri-TAS2 and its miR173-cleaved 5' and 3' fragments are fractionated together in the polysome fractions. These and previous results suggest that the 3' fragment of pri-TAS2, which is a source of tasiRNAs, forms a huge complex containing SGS3, miR173-programmed AGO1 RNA-induced silencing complex, the 5' fragment, and ribosomes. This complex overaccumulated, moderately accumulated, and did not accumulate in rdr6, sde5, and sgs3 mutants, respectively. The sgs3 sde5 and rdr6 sde5 double mutants showed phenotypes similar to those of sgs3 and sde5 single mutants, respectively, with regard to the TAS2-related RNA accumulation, suggesting that the complex is formed in an SGS3-dependent manner, somehow modified and stabilized by SDE5, and becomes competent for RDR6 action. Ribosomes in this complex likely play an important role in this process.

An extremely high bioavailability of orally administered vancomycin in a patient with severe colitis and renal insufficiency.

Because there is little absorption of orally administered vancomycin hydrochloride (VCM) through the normal intestinal microvillus membrane, the pharmacokinetics of VCM absorbed from the digestive tract are mostly unknown. Here we report a case of severe colitis and renal insufficiency in which the serum concentration of VCM reached the supratherapeutic range after oral administration. A 54-year-old man receiving outpatient chemotherapy for rectal cancer was admitted to our hospital for severe sepsis and acute renal failure. Multimodal therapy including continuous renal replacement therapy (CRRT) and mechanical ventilation was initiated, and oral VCM administration (0.5 g every 6 h) was begun for suspected severe pseudomembranous colitis with large amounts of watery stool. Despite continued CRRT, the serum VCM concentration increased to 30.6 µg/mL after 4 days. Based on pharmacokinetic analysis, the bioavailability of VCM was estimated to be over 54.5%. Colonoscopy showed that the mucosa was severely damaged throughout the large intestine, resulting in considerable exudation of plasma and blood. This case indicates the need for careful and early monitoring during high-dose oral VCM administration to patients with severe mucosal injury and renal insufficiency.

Replication protein of tobacco mosaic virus cotranslationally binds the 5' untranslated region of genomic RNA to enable viral replication.

Genomic RNA of positive-strand RNA viruses replicate via complementary (i.e., negative-strand) RNA in membrane-bound replication complexes. Before replication complex formation, virus-encoded replication proteins specifically recognize genomic RNA molecules and recruit them to sites of replication. Moreover, in many of these viruses, selection of replication templates by the replication proteins occurs preferentially in cis. This property is advantageous to the viruses in several aspects of viral replication and evolution, but the underlying molecular mechanisms have not been characterized. Here, we used an in vitro translation system to show that a 126-kDa replication protein of tobacco mosaic virus (TMV), a positive-strand RNA virus, binds a 5'-terminal ∼70-nucleotide region of TMV RNA cotranslationally, but not posttranslationally. TMV mutants that carried nucleotide changes in the 5'-terminal region and showed a defect in the binding were unable to synthesize negative-strand RNA, indicating that this binding is essential for template selection. A C-terminally truncated 126-kDa protein, but not the full-length 126-kDa protein, was able to posttranslationally bind TMV RNA in vitro, suggesting that binding of the 126-kDa protein to the 70-nucleotide region occurs during translation and before synthesis of the C-terminal inhibitory domain. We also show that binding of the 126-kDa protein prevents further translation of the bound TMV RNA. These data provide a mechanistic explanation of how the 126-kDa protein selects replication templates in cis and how fatal collision between translating ribosomes and negative-strand RNA-synthesizing polymerases on the genomic RNA is avoided.

Structural basis for the recognition-evasion arms race between Tomato mosaic virus and the resistance gene Tm-1.

The tomato mosaic virus (ToMV) resistance gene Tm-1 encodes a protein that shows no sequence homology to functionally characterized proteins. Tm-1 binds ToMV replication proteins and thereby inhibits replication complex formation. ToMV mutants that overcome this resistance have amino acid substitutions in the helicase domain of the replication proteins (ToMV-Hel). A small region of Tm-1 in the genome of the wild tomato Solanum habrochaites has been under positive selection during its antagonistic coevolution with ToMV. Here we report crystal structures for the N-terminal inhibitory domains of Tm-1 and a natural Tm-1 variant with an I91-to-T substitution that has a greater ability to inhibit ToMV RNA replication and their complexes with ToMV-Hel. Each complex contains a Tm-1 dimer and two ToMV-Hel monomers with the interfaces between Tm-1 and ToMV-Hel bridged by ATP. Residues in ToMV-Hel and Tm-1 involved in antagonistic coevolution are found at the interface. The structural differences between ToMV-Hel in its free form and in complex with Tm-1 suggest that Tm-1 affects nucleoside triphosphatase activity of ToMV-Hel, and this effect was confirmed experimentally. Molecular dynamics simulations of complexes formed by Tm-1 with ToMV-Hel variants showed how the amino acid changes in ToMV-Hel impair the interaction with Tm-1 to overcome the resistance. With these findings, together with the biochemical properties of the interactions between ToMV-Hel and Tm-1 variants and effects of the mutations in the polymorphic residues of Tm-1, an atomic view of a step-by-step coevolutionary arms race between a plant resistance protein and a viral protein emerges.

[A case of Evans syndrome in a long-term hemodialysis patient].

A 75-year-old woman, who had been on maintenance hemodialysis (HD) from 2000, was receiving erythro- poiesis stimulating agent (ESA) for renal anemia. In November 2013, although still continuing ESA, she was admitted to our hospital due to worsening anemia. Since blood tests suggested the possibility of hemolytic anemia, we consulted,with hematologists, and she was transferred to another hospital. Differential diagnosis for anemia revealed that she had newly developed Evans syndrome, which is the complication of autoimmune hemolytic ane- mia (AIHA) and idiopathic thrombocytopenic purpura(ITP). She was successfully treated for AIHA with blood transfusion and administration of steroids, and for ITP by eradicating Melicobacter pylort. Anemia is commonly seen in HD patients, and the majority of anemia cases are diagnosed as renal anemia; however, hemolytic anemia should be considered in order to make a differential diagnosis. There are few reports of Evans syndrome in HD, and the pathogenesis of Evans syndrome is largely unknown. Further accumulation of clinical reports is needed to clarify its etiology.

Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants.

Posttranscriptional gene silencing is mediated by RNA-induced silencing complexes (RISCs) that contain AGO proteins and single-stranded small RNAs. The assembly of plant AGO1-containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90-dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex-bound AGO1. Moreover, CYP40 that was synthesized by in-vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin-binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90-AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants.

3' fragment of miR173-programmed RISC-cleaved RNA is protected from degradation in a complex with RISC and SGS3.

trans-acting small interfering RNAs (tasiRNAs) are plant-specific endogenous siRNAs produced via a unique pathway whose first step is the microRNA (miRNA)-programmed RNA-induced silencing complex (RISC)-mediated cleavage of tasiRNA gene (TAS) transcripts. One of the products is subsequently transformed into tasiRNAs by a pathway that requires several factors including SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6. Here, using in vitro assembled ARGONAUTE (AGO)1-RISCs, we show that SGS3 is recruited onto RISCs only when they bind target RNA. Following cleavage by miRNA173 (miR173)-programmed RISC, SGS3 was found in complexes containing cleaved TAS2 RNA and RISC. The 3' cleavage fragment (the source of tasiRNAs) was protected from degradation in this complex. Depletion of SGS3 did not affect TAS2 RNA cleavage by miR173-programmed RISC, but did affect the stability of the 3' cleavage fragment. When the 3' nucleotide of 22-nt miR173 was deleted or the corresponding nucleotide in TAS2 RNA was mutated, the complex was not observed and the 3' cleavage fragment was degraded. Importantly, these changes in miR173 or TAS2 RNA are known to lead to a loss of tasiRNA production in vivo. These results suggest that (i) SGS3 associates with AGO1-RISC via the double-stranded RNA formed by the 3'-terminal nucleotides of 22-nt miR173 and corresponding target RNA, which probably protrudes from the AGO1-RISC molecular surface, (ii) SGS3 protects the 3' cleavage fragment of TAS2 RNA from degradation, and (iii) the observed SGS3-dependent stabilization of the 3' fragment of TAS2 RNA is key to tasiRNA production.

Cortical bone trajectory and traditional trajectory--a radiological evaluation of screw-bone contact.

BACKGROUND: Cortical bone trajectory (CBT), a relatively new technique for pedicle screw insertion in the lumbar spine, is believed to have equivalent pullout and toggle characteristics compared with the traditional trajectory (TT). It has been hypothesized that the new trajectory offers higher cortical bone contact with the pedicle screws and therefore has an improved anchoring property over the traditional trajectory where the screws are inserted into the vertebral body trabecular space. The aim of this study is to evaluate the pedicle screw-cortical bone contact between the two trajectories from a radiological standpoint. METHODS: Two hundred twenty-two patients with degenerative lumbar spine disease underwent computed tomography scanning. For each patient, axial slices of the L4 and L5 vertebra were cut in two planes, one horizontal to the pedicle representing the plane at which pedicle screws are inserted using the TT and another in a more caudo-cranial plane representing the plane at which pedicle screws are inserted using CBT. For each trajectory, a region of interest (ROI) was selected within the area in which the screws are inserted. A CT number (Hounsfield scale) was then calculated within each ROI to compare the bone density. RESULTS: The CT numbers within the ROI for CBT were constantly almost over four times higher than that for the TT, and there was a significant difference between the values (p < 0.0001). CONCLUSIONS: This study has demonstrated that, with the cortical bone trajectory, the pedicle screws penetrate a region that is richer in cortical bone compared to when using the traditional trajectory. This is in keeping with previous hypotheses that the new trajectory offers higher cortical bone contact.

Postoperative behavior of thoracolumbar/lumbar curve and coronal balance after posterior thoracic fusion for Lenke 1C and 2C adolescent idiopathic scoliosis.

BACKGROUND: Controversy still exists around surgical strategies for Lenke type 1C and 2C curves with primary thoracic and compensatory lumbar curves in adolescent idiopathic scoliosis (AIS). The benefit of selective thoracic fusion (STF) for these curve types is spontaneous lumbar curve correction while saving more mobile lumbar segments. However, a risk of postoperative coronal decompensation after STF has also been reported. This multicenter retrospective study was conducted to evaluate postoperative behavior of thoracolumbar/lumbar (TLL) curve and coronal balance after posterior thoracic fusion for Lenke 1C and 2C AIS. METHODS: Twenty-four Lenke 1C and 2C AIS patients who underwent posterior thoracic fusion were included. The mean age of patients was 15.7 years old at time of surgery. Constructs used for surgery in all cases were pedicle screw constructs ending at L3 or above. Radiographic measurements were performed on Cobb angles of the main thoracic and TLL curves and coronal balance. Factors related to final Cobb angle of TLL curve and postoperative change of coronal balance were investigated. RESULTS: Mean Cobb angles for main thoracic and TLL curves were 59.0° and 43.9° preoperatively, and were corrected to 21.5° and 22.0° at final follow-up, respectively. Mean coronal balance was -5.6 mm preoperatively and was corrected to -14.6 mm at final follow-up. Final Cobb angle of TLL curve was significantly correlated with immediate postoperative Cobb angle of main thoracic curve and tilt of lowest instrumented vertebra (LIV). Postoperative change of coronal balance was significantly correlated with selection of LIV relative to stable vertebra. CONCLUSION: Spontaneous correction of TLL curve occurred consistently by correcting the main thoracic curve and making the LIV more horizontal after posterior thoracic fusion for Lenke 1C and 2C AIS. The more distal fixation to stable vertebra resulted in coronal balance shifting more to the left postoperatively.

The resistance protein Tm-1 inhibits formation of a Tomato mosaic virus replication protein-host membrane protein complex.

The Tm-1 gene of tomato confers resistance to Tomato mosaic virus (ToMV). Tm-1 encodes a protein that binds ToMV replication proteins and inhibits the RNA-dependent RNA replication of ToMV. The replication proteins of resistance-breaking mutants of ToMV do not bind Tm-1, indicating that the binding is important for inhibition. In this study, we analyzed how Tm-1 inhibits ToMV RNA replication in a cell-free system using evacuolated tobacco protoplast extracts. In this system, ToMV RNA replication is catalyzed by replication proteins bound to membranes, and the RNA polymerase activity is unaffected by treatment with 0.5 M NaCl-containing buffer and remains associated with membranes. We show that in the presence of Tm-1, negative-strand RNA synthesis is inhibited; the replication proteins associate with membranes with binding that is sensitive to 0.5 M NaCl; the viral genomic RNA used as a translation template is not protected from nuclease digestion; and host membrane proteins TOM1, TOM2A, and ARL8 are not copurified with the membrane-bound 130K replication protein. Deletion of the polymerase read-through domain or of the 3' untranslated region (UTR) of the genome did not prevent the formation of complexes between the 130K protein and the host membrane proteins, the 0.5 M NaCl-resistant binding of the replication proteins to membranes, and the protection of the genomic RNA from nucleases. These results indicate that Tm-1 binds ToMV replication proteins to inhibit key events in replication complex formation on membranes that precede negative-strand RNA synthesis.

Coevolution and hierarchical interactions of Tomato mosaic virus and the resistance gene Tm-1.

During antagonistic coevolution between viruses and their hosts, viruses have a major advantage by evolving more rapidly. Nevertheless, viruses and their hosts coexist and have coevolved, although the processes remain largely unknown. We previously identified Tm-1 that confers resistance to Tomato mosaic virus (ToMV), and revealed that it encodes a protein that binds ToMV replication proteins and inhibits RNA replication. Tm-1 was introgressed from a wild tomato species Solanum habrochaites into the cultivated tomato species Solanum lycopersicum. In this study, we analyzed Tm-1 alleles in S. habrochaites. Although most part of this gene was under purifying selection, a cluster of nonsynonymous substitutions in a small region important for inhibitory activity was identified, suggesting that the region is under positive selection. We then examined the resistance of S. habrochaites plants to ToMV. Approximately 60% of 149 individuals from 24 accessions were resistant to ToMV, while the others accumulated detectable levels of coat protein after inoculation. Unexpectedly, many S. habrochaites plants were observed in which even multiplication of the Tm-1-resistance-breaking ToMV mutant LT1 was inhibited. An amino acid change in the positively selected region of the Tm-1 protein was responsible for the inhibition of LT1 multiplication. This amino acid change allowed Tm-1 to bind LT1 replication proteins without losing the ability to bind replication proteins of wild-type ToMV. The antiviral spectra and biochemical properties suggest that Tm-1 has evolved by changing the strengths of its inhibitory activity rather than diversifying the recognition spectra. In the LT1-resistant S. habrochaites plants inoculated with LT1, mutant viruses emerged whose multiplication was not inhibited by the Tm-1 allele that confers resistance to LT1. However, the resistance-breaking mutants were less competitive than the parental strains in the absence of Tm-1. Based on these results, we discuss possible coevolutionary processes of ToMV and Tm-1.