A model for intracellular movement of Cauliflower mosaic virus: the concept of the mobile virion factory.

The genomes of many plant viruses have a coding capacity limited to <10 proteins, yet it is becoming increasingly clear that individual plant virus proteins may interact with several targets in the host for establishment of infection. As new functions are uncovered for individual viral proteins, virologists have realized that the apparent simplicity of the virus genome is an illusion that belies the true impact that plant viruses have on host physiology. In this review, we discuss our evolving understanding of the function of the P6 protein of Cauliflower mosaic virus (CaMV), a process that was initiated nearly 35 years ago when the CaMV P6 protein was first described as the 'major inclusion body protein' (IB) present in infected plants. P6 is now referred to in most articles as the transactivator (TAV)/viroplasmin protein, because the first viral function to be characterized for the Caulimovirus P6 protein beyond its role as an inclusion body protein (the viroplasmin) was its role in translational transactivation (the TAV function). This review will discuss the currently accepted functions for P6 and then present the evidence for an entirely new function for P6 in intracellular movement.

Association of the P6 protein of Cauliflower mosaic virus with plasmodesmata and plasmodesmal proteins.

The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata.

The P6 protein of Cauliflower mosaic virus interacts with CHUP1, a plant protein which moves chloroplasts on actin microfilaments.

The gene VI product, protein 6 (P6), of Cauliflower mosaic virus (CaMV) assembles into large, amorphous inclusion bodies (IBs) that are considered sites for viral protein synthesis and viral genome replication and encapsidation. P6 IBs align with microfilaments and require them for intracellular trafficking, a result implying that P6 IBs function to move virus complexes or virions within the cell to support virus physiology. Through a yeast two-hybrid screen we determined that CHUP1, a plant protein allowing chloroplast transport through an interaction with chloroplast and microfilament, interacts with P6. The interaction between CHUP1 and P6 was confirmed through colocalization in vivo and co-immunoprecipitation assays. A truncated CHUP1 fused with enhanced cyan fluorescent protein, unable to transport chloroplasts, inhibited intracellular movement of P6-Venus inclusions. Silencing of CHUP1 in N. edwardsonii impaired the ability of CaMV to infect plants. The findings suggest that CHUP1 supports CaMV infection through an interaction with P6.

A survey of resistance to Tomato bushy stunt virus in the genus Nicotiana reveals that the hypersensitive response is triggered by one of three different viral proteins.

In this study, we screened 22 Nicotiana spp. for resistance to the tombusviruses Tomato bushy stunt virus (TBSV), Cucumber necrosis virus, and Cymbidium ringspot virus. Eighteen species were resistant, and resistance was manifested in at least two different categories. In all, 13 species responded with a hypersensitive response (HR)-type resistance, whereas another five were resistant but either had no visible response or responded with chlorotic lesions rather than necrotic lesions. Three different TBSV proteins were found to trigger HR in Nicotiana spp. in an agroinfiltration assay. The most common avirulence (avr) determinant was the TBSV coat protein P41, a protein that had not been previously recognized as an avr determinant. A mutational analysis confirmed that the coat protein rather than the viral RNA sequence was responsible for triggering HR, and it triggered HR in six species in the Alatae section. The TBSV P22 movement protein triggered HR in two species in section Undulatae (Nicotiana glutinosa and N. edwardsonii) and one species in section Alatae (N. forgetiana). The TBSV P19 RNA silencing suppressor protein triggered HR in sections Sylvestres (N. sylvestris), Nicotiana (N. tabacum), and Alatae (N. bonariensis). In general, Nicotiana spp. were capable of recognizing only one tombusvirus avirulence determinant, with the exceptions of N. bonariensis and N. forgetiana, which were each able to recognize P41, as well as P19 and P22, respectively. Agroinfiltration failed to detect the TBSV avr determinants responsible for triggering HR in N. arentsii, N. undulata, and N. rustica. This study illustrates the breadth and variety of resistance responses to tombusviruses that exists in the Nicotiana genus.

Pyloromyotomy: randomized control trial of laparoscopic vs open technique.

PURPOSE: Open pyloromyotomy remains as the criterion standard treatment for hypertrophic pyloric stenosis with the laparoscopic approach rapidly gaining adoption. We present a prospective, randomized trial between the 2 approaches. METHODS: After institutional review board approval, 98 patients with hypertrophic pyloric stenosis were consecutively randomized to either open or laparoscopic pyloromyotomy. Postoperative and hospital course were evaluated by review of the hospital records and long-term follow-up with scripted telephone survey using Likert scales. The length of operating room time, surgical procedure, postoperative stay, time to refeeding, and complications were evaluated. Secondary outcomes of cosmetic results and parental satisfaction were determined. RESULTS: Ninety-eight patients were enrolled during a 4-year period. There were no significant differences between 2 groups on all primary outcomes. There were 3 complications in the open group-a wound dehiscence, a surgical site infection, and a gastric serosal tear-and 2 complications in the laparoscopic group-mucosal perforation and a suture granuloma. In long-term follow-up on 72 patients (56 months), parents described significant cosmetic results with laparoscopic approach. CONCLUSIONS: There was no difference in operating time, hospital stay, or refeeding patterns between open and laparoscopic pyloromyotomy. The complication rates were similar between the 2 methods. However, long-term cosmetic results were significantly superior in the laparoscopic group.

Comparative analysis of the capacity of tombusvirus P22 and P19 proteins to function as avirulence determinants in Nicotiana species.

We have used an agroinfiltration assay for a comparative study of the roles of tombusvirus P22 and P19 proteins in elicitation of hypersensitive response (HR)-like necrosis and the role of P19 in silencing suppression in Nicotiana species. The advantage of agroinfiltration rather than expression in plant virus vectors is that putative viral avirulence proteins can be evaluated in isolation, eliminating the possibility of synergistic effects with other viral proteins. We found that tombusvirus P22 and P19 proteins elicited HR-like necrosis in certain Nicotiana species but, also, that Nicotiana species could recognize subtle differences in sequence between these proteins. Furthermore, Nicotiana species that responded with systemic necrosis to virion inoculations responded to agroinfiltration of tombusvirus P19 with a very weak and delayed necrosis, indicating that the rapid HR-like necrosis was associated with putative resistance genes and a plant defense response that limited the spread of the virus. Tombusvirus P19 proteins also appeared to differ in their effectiveness as silencing suppressors; in our assay, the P19 proteins of Cymbidium ringspot virus and Tomato bushy stunt virus were stronger silencing suppressors than Cucumber necrosis virus P20. Finally, we show that agroinfiltration can be used to track the presence of putative plant resistance genes in Nicotiana species that target either tombusvirus P19 or P22.

Outcome of gastroschisis: a 20-year case review of infants with gastroschisis born in Galveston, Texas.

BACKGROUND/PURPOSE: In the past decade, the preferred method of closure of gastroschisis at our institution has been staged reduction using a silo with repair on an elective basis (SR) rather than primary surgical closure (PC). We performed a 20-year case review of infants with gastroschisis at a university hospital to compare these shifts in management and to determine factors affecting outcome. METHODS: Seventy-two cases were reviewed from 1983 to 2003. Times to first and full feeds were outcome variables for statistical analysis. RESULTS: The prevalence of gastroschisis increased from 0.03% to 0.1% since 1983. Patients had low birth weights (mean = 2294 g) and were borderline premature (mean = 35.8 weeks). Only 3% of the infants were African American. There was a high rate of cesarean deliveries (57%). Ten patients (15%) had gastroschisis complicated by liver herniation, intestinal atresia(s), and/or necrosis/perforation. Most patients were managed by SR (67%). Eight percent of the infants died, 9% developed necrotizing enterocolitis, and 50% had other gastrointestinal complications. Twenty-seven percent of the infants managed with SR did not need initial mechanical ventilation. However, the patients who underwent SR were ventilated longer after birth as compared with those who underwent PC (P < .08). Infants with a complicated gastroschisis had significantly longer times to first and full feeds (P < .001). Patients managed with SR took significantly longer to reach full feeds (P = .001), and there was a trend of starting feeds later (P = .06). When patients with a complicated gastroschisis were excluded, the differences between the SR and PC groups were even greater (P = .01; P < .001). CONCLUSIONS: In our patient population, the prevalence of gastroschisis increased by more than 400% since 1983. The defect was rare in African-American infants. Management by SR was associated with longer ventilation times and longer times to first and full feeds for both uncomplicated and complicated gastroschisis cases.

Imaging of acute appendicitis: US as the primary imaging modality.

BACKGROUND: The investigation of the acute abdomen in infants and children has evolved during the last two decades, placing imagers at the forefront of the evaluation and diagnosis of acute right lower quadrant abdominal problems. US and CT have recently been shown to be equally accurate in the diagnosis of acute appendicitis, but not everyone agrees. OBJECTIVE: To demonstrate the efficacy of triaging patients with acute abdominal problems that suggest appendicitis with US as the primary imaging modality. MATERIALS AND METHODS: We retrospectively reviewed the prospective imaging diagnoses in 622 children who presented to our emergency room (ER) and clinics with acute abdominal symptoms suggestive of appendicitis. We documented whether US or CT was performed and noted the diagnoses made. All of the patients had plain films. In addition, all patients undergoing surgical appendectomy during this time were also documented so as not to miss any cases of appendicitis. None was missed. RESULTS: There were 622 consecutive patients in our study. Three patients, diagnosed as normal, were eventually excluded because of lack of follow-up. In all, 152 patients were evaluated clinically and with plain films only. They were not subject to surgical exploration or further imaging. None returned with appendicitis. Eighty-one patients were directly subject to laparotomy after clinical and plain film evaluation. Of these patients, 20% had a normal appendix. Of the remaining 389 patients, 386 had US and three had CT alone. Four patients had both CT and US because of an inconclusive US examination. Three patients had CT alone because of their size. In total, 137 patients were diagnosed with appendicitis with US and/or CT. Four of these patients (3%) had normal appendices. Forty-two patients (less three lost to follow-up) were diagnosed as normal, and none returned with findings of appendicitis. Nine others had conditions other than acute appendicitis. Three had surgically proven, nonrelated conditions, and of the other six, one had pancreatitis and five nonsurgical adnexal problems. In all, 201 patients were diagnosed (with US) with mesenteric adenitis-enteritis, and none returned with findings of appendicitis. CONCLUSION: We attained a high degree of diagnostic accuracy in patients presenting with findings suggestive of appendicitis using US as the primary imaging modality. Our false-positive appendectomy rate was 3%. Therefore, triage of the acute abdomen with US supported by CT when required has considerable merit, especially when considering that US is noninvasive and does not use ionizing radiation.

Intestinal zygomycosis due to Absidia corymbifera mimicking necrotizing enterocolitis in a preterm neonate.

Zygomycosis is a rare fungal disease that occurs in compromised human hosts, including the preterm infant. The three clinical forms of zygomycosis are cellulitis, disseminated, and gastrointestinal, and the last often mimics necrotizing enterocolitis (NEC), complicating the diagnosis. This report details a case of primary gastrointestinal zygomycosis due to Absidia corymbifera, mimicking NEC, in a preterm infant, and emphasizes features that may lead to earlier diagnosis and treatment of future cases.

Severe ciprofloxacin-associated pseudomembranous colitis in an eight-year-old child.

Clostridium difficile is the principal cause of antibiotic-associated diarrhea and pseudomembranous enterocolitis in children. A case of severe pseudomembranous colitis developing in an 8-year-old child who had received oral ciprofloxacin therapy as part of an investigational protocol is presented. The safety and efficacy of fluoroquinolones in children has not yet been established. Use of these antibiotics in children outside investigational protocols ("off-label" use) as oral antipseudomonas agents is discouraged.

Validity of plain films in intussusception.

The aim of this study was to re-evaluate the specificity of plain film findings in intussusception. The plain film findings in 80 cases of proven intussusception were reviewed. Findings documented were: (1) presence or absence of small bowel obstruction, (2) paucity of right lower quadrant gas, (3) presence of an intracolonic mass, (4) presence of a rim or target sign, and (5) presence of the classic triad of intestinal obstruction, intracolonic mass, and paucity of right lower quadrant gas. Intestinal obstruction was present in 54% of patients. In 19 patients (24%) the abdominal films were completely normal. Paucity of right lower quadrant gas was seen in 10% of patients, while specific findings of a mass or a target (rim) sign were seen in 29% of patients. The classic triad of an intracolonic mass, obstruction, and paucity of gas in the right lower quadrant occurred in only 1 patient (1%). Plain films of the abdomen were diagnostic of intussusception in only 29% of cases. A completely normal gas pattern was seen in one-quarter of our patients. This being the case, most patients with suspected intussusception will require further imaging, either by ultrasound or contrast enema. In our institution we favor the ultrasound study.

Meconium obstruction in the very low birth weight premature infant.

Meconium obstruction of prematurity is a distinct clinical condition that occurs in very low birth weight infants, predisposing them to intestinal perforation and a prolonged hospitalization if not diagnosed and treated promptly. We report a series of 21 infants, including 2 detailed case reports, whose clinical course is indicative of meconium obstruction of prematurity. Specific risk factors are identified along with descriptions of clinical and radiologic findings, disease course, treatment, and outcome. Meconium obstruction of prematurity was more common in infants with a maternal history of pregnancy-induced or chronic hypertension, suggesting the possibility of decreased intestinal perfusion prenatally. Inspissated meconium was located most frequently in the distal ileum, making this disease process difficult to treat. Gastrografin enemas were safe, diagnostic, and therapeutic. Delay in diagnosis and treatment was associated with perforation and delay in institution of enteral feeds.