Dissection of PIK3CA Aberration for Cervical Adenocarcinoma Outcomes.

Personalized treatment of genetically stratified subgroups has the potential to improve outcomes in many malignant tumors. This study distills clinically meaningful prognostic/predictive genomic marker for cervical adenocarcinoma using signature genomic aberrations and single-point nonsynonymous mutation-specific droplet digital PCR (ddPCR). Mutations in PIK3CA E542K, E545K, or H1047R were detected in 41.7% of tumors. PIK3CA mutation detected in the patient's circulating DNA collected before treatment or during follow-up was significantly associated with decreased progression-free survival or overall survival. PIK3CA mutation in the circulating DNA during follow-up after treatment predicted recurrence with 100% sensitivity and 64.29% specificity. It is the first indication of the predictive power of PIK3CA mutations in cervical adenocarcinoma. The work contributes to the development of liquid biopsies for follow up surveillance and a possibility of tailoring management of this particular women's cancer.

Risk of tuberculosis in rheumatoid arthritis patients on tumour necrosis factor-alpha inhibitor treatment in Taiwan.

OBJECTIVES: To quantify the incidence of tuberculosis (TB) in rheumatoid arthritis patients undergoing treatment with tumour necrosis factor-alpha inhibitors (TNFi). DESIGN: In a retrospective cohort study conducted using data from Taiwan's National Health Insurance claims databases, rheumatoid arthritis patients notified during the period 2006-2008 were recruited and classified based on types of TNFi treatment received. Active TB was the primary outcome. TB risk was estimated using Cox's proportional hazard model. The TB screening rate within 30 days of initiating treatment with TNFi was examined. RESULTS: Respectively 5079 and 829 patients were included in the non-TNFi and TNFi groups. Active TB rates were respectively 1411.3 and 679.5 events per 100,000 person-years in patients treated with adalimumab and etanercept. Significant TB risk was noted in patients treated with TNFi (aHR 4.87, 95%CI 2.14-11.06). No significant difference in active TB was observed between the TNFi subgroups (etanercept as reference, aHR 1.89, 95%CI 0.40-6.04). Only 8.7% (n = 9) of TNFi users underwent screening for TB before the first dose of TNFi. CONCLUSIONS: Patients on TNFi have a significantly greater risk of active TB than non-TNFi patients in the Taiwanese population. No difference in TB risk between the two available TNFi groups was noted. Screening for TB before initiating treatment with TNFi should be implemented.

BACTEC FX system as a tool for culturing gastric biopsies and Helicobacter pylori diagnosis.

Helicobacter pylori infection represents a key factor in the etiology of various gastrointestinal diseases. There are several acceptable methods to identify this microorganism. Some are invasive and some are noninvasive. This study demonstrates the use of BACTEC FX system for the growth and diagnosis of H. pylori isolated from gastric biopsy specimens, cut and placed in blood culture bottles, with subsequent incubation in the apparatus. Twenty-five positive and 15 negative biopsy samples tested using the quick urease technique, CUTest, were collected from 40 patients with confirmed chronic gastric inflammation. The biopsy samples were manually cut using a sterile scalpel and placed in tubes containing 5 ml of fetal bovine serum. The resulting suspensions were transferred using a syringe into anaerobic blood culture bottles. These bottles were incubated at 35 °C for a period of 7 days in the BACTEC FX system. All biopsy samples that reacted positive to the CUTest and one biopsy sample that reacted negative to the CUTest were confirmed as positive by the BACTEC FX system. In addition, there was a correlation between the positive culture and histology examination results. The use of BACTEC FX system significantly shortens the time needed for culturing, which makes the system more efficient in the identification of H. pylori. It should be emphasized that performing microbial culture testing has a significant role in monitoring antibiotic resistance, which cannot be done using other existing methods for H. pylori diagnosis.

Evidence-based recommendations for bowel cleansing before colonoscopy in children: a report from a national working group.

BACKGROUND: There are no current recommendations for bowel cleansing before colonoscopy in children. The Israeli Society of Pediatric Gastroenterology and Nutrition (ISPGAN) established an iterative working group to formulate evidence-based guidelines for bowel cleansing in children prior to colonoscopy. METHOD: Data were collected by systematic review of the literature and via a national-based survey of all endoscopy units in Israel. Based on the strength of evidence, the Committee reached consensus on six recommended protocols in children. Guidelines were finalized after an open audit of ISPGAN members. RESULTS: Data on 900 colonoscopies per year were accrued, which represents all annual pediatric colonoscopies performed in Israel. Based on the literature review, the national survey, and the open audit, several age-stratified pediatric cleansing protocols were proposed: two PEG-ELS protocols (polyethylene-glycol with electrolyte solution); Picolax-based protocol (sodium picosulphate with magnesium citrate); sodium phosphate protocol (only in children over the age of 12 years who are at low risk for renal damage); stimulant laxative-based protocol (e. g. bisacodyl); and a PEG 3350-based protocol. A population-based analysis estimated that the acute toxicity rate of oral sodium phosphate is at most 3/7320 colonoscopies (0.041 %). Recommendations on diet and enema use are provided in relation to each proposed protocol. CONCLUSION: There is no ideal bowel cleansing regimen and, thus, various protocols are in use. We propose several evidence-based protocols to optimize bowel cleansing in children prior to colonoscopy and minimize adverse events.

Pelargonium zonate spot virus is transmitted vertically via seed and pollen in tomato.

In autumn 2007, a new disease with unknown etiology was observed in open-field tomato (Solanum lycopersicum) in the Lachish region of Israel. The symptoms included mild mosaic, leaf malformation, and severe stunting of the plants. The causal agent was readily transmitted mechanically from the sap of infected plants to indicator plants. Viral particles were purified from infected plants and cDNA was synthesized from RNA isolated from the particles. Cloning and sequencing of the cDNA showed 95% identity to RNA 3 of Pelargonium zonate spot virus (PZSV). Using reverse-transcription polymerase chain reaction, PZSV was detected in both seed and pollen grains of infected tomato plants. Attempts to disinfect seed by using hydrochloric acid and trisodium phosphate failed to eliminate this PZSV detection. Seed from infected tomato plants gave rise to infected seedlings with a seed-transmission rate of PZSV of 11 to 29%. Pollen grains collected from flowers of infected plants were used to hand pollinate healthy mother tomato plants. Although none of the pollinated mother plants became infected with PZSV, 29% of the seedlings produced from seed harvested from these plants were found to be infected. This is the first demonstration that PZSV is transmitted vertically via both pollen and seed in tomato plants.

Electrophysiological assessment of sexual dysfunction in spinal cord injured patients.

STUDY DESIGN: Survey. OBJECTIVES: To determine associations between sexual dysfunctions and electrophysiological examinations of the genital system in spinal cord injured patients. SETTING: Ege University Hospital, Izmir, Turkey. METHODS: In total, 25 patients (17 men, eight women) who were out of the spinal shock period were examined. Neurological levels were determined according to the American Spinal Injury Association (ASIA) impairment scale. Data about erection, ejaculation and vaginal lubrication were obtained via inquiry forms. Bulbocavernosus reflex (BCR), pudendal somatosensorial evoked potentials (pSEP) and perineal sympathetic skin responses (pSSR) were recorded by an electromyographer unaware of the sexual state or neurological level of the patient. RESULTS: BCRs could be obtained from all patients with lesion levels above the sacral centre. A significant association was found between reflex erection and BCR positivity, while psychogenic erection was shown to have a significant association with the preservation of pSSR in men. Despite the lack of statistical significance due to the small sample size of the women examined, a similar association with lubrication was observed. Ejaculation and orgasm were not shown to be significantly associated with any electrophysiological examination. However, ejaculation was preserved in all men with a lesion level below T12 and with positive pSSR. There was no significant relationship between pSEP and sexual functions. The relationship between the existence of sacral sensation and pSEP positivity was statistically significant. CONCLUSION: This study has proved that BCR and pSSR have an important role in the estimation of the remaining sexual function in spinal cord injured patients. STATEMENT ON ETHICS: We certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during the course of this research.

Breakage of resistance to Cucumber mosaic virus by co-infection with Zucchini yellow mosaic virus: enhancement of CMV accumulation independent of symptom expression.

Resistance to the cucumovirus Cucumber mosaic virus (CMV) in cucumber cv. Delila was manifested as a very low level of accumulation of viral RNA and capsid protein, and an absence of CMV-induced symptoms. In addition, resistance was observed at the single cell level, with a reduction in accumulation of CMV RNAs, compared to accumulation in cells of the susceptible cucumber cv. Bet Alpha. Resistance to CMV in cv. Delila was broken by co-infection with the potyvirus Zucchini yellow mosaic virus (ZYMV). Resistance breakage in cv. Delila plants was manifested by an increase in the accumulation of (+) and (-) CMV RNA as well as CMV capsid protein, with no increase in the level of accumulation of ZYMV. Resistance breakage in the resistant cultivar by ZYMV also occurred at the single cell level. Thus, synergistic interactions known to occur between a potyvirus and a cucumovirus led to resistance breakage during a double infection. However, resistance breakage was not accompanied by an increase in disease symptoms beyond those induced by ZYMV itself. On co-inoculation with an asymptomatic variant of ZYMV-AG an enhancement of CMV infection occurred without disease manifestation. Consequently, intensification of viral RNA and capsid protein accumulation can occur without a corresponding increase in disease development, suggesting that different host genes regulate viral accumulation and disease development in the CMV-resistant cucumber plants.

Shoot production in squash (Cucurbita pepo) by in vitro organogenesis.

Seedling-derived cotyledon explants of squash ( Cucurbita pepo L.) of commercial cultivars True French, Ma'yan and Goldy were regenerated in vitro on Murashige and Skoog medium augmented with 1 mg/l benzyladenine. After 4 weeks in culture small shoots and buds regenerated only on the most proximal cotyledon edge. Culture on an elongation medium with a reduced cytokinin concentration (0.1 mg/l) with or without 1 mg/l gibberellic acid (GA(3)) facilitated the recovery of shoots. Fresh shoots could be recovered at each subculture of the regenerating mass. Peak productivity was during the third cycle of subculture, and shoot production ceased after the fifth subculture. Culture on elongation medium supplemented with GA(3) was 55% more effective with respect to overall shoot production than that on medium without GA(3), with 22 shoots recovered in total per explant from the former. Regeneration occurred under both light and dark conditions. All of the shoots tested were diploid. The shoots were rooted and transferred to the greenhouse where they grew and flowered normally.

Increase in Zucchini yellow mosaic virus Symptom Severity in Tolerant Zucchini Cultivars Is Related to a Point Mutation in P3 Protein and Is Associated with a Loss of Relative Fitness on Susceptible Plants.

ABSTRACT Zucchini yellow mosaic virus (ZYMV, Potyvirus) is a very damaging cucurbit virus worldwide. Interspecific crosses with resistant Cucurbita moschata have led to the release of "resistant" zucchini squash (C. pepo) F(1) hybrids. However, although the resistance is almost complete in C. moschata, the commercial C. pepo hybrids are only tolerant. ZYMV evolution toward increased aggressiveness on tolerant hybrids was observed in the field and was obtained experimentally. Sequence comparisons and recombination experiments revealed that a point mutation in the P3 protein of ZYMV was enough to induce tolerance breaking. Competition experiments were performed between quasi-isogenic wild-type, and aggressive variants of ZYMV distinguished by monoclonal antibodies. The aggressive mutants were more fit than wild-type strains in mixed infections of tolerant zucchini, but they presented a drastic fitness loss in mixed infections of susceptible zucchini or melon. Thus, the ability to induce severe symptoms in tolerant zucchini is related to a genetic load in susceptible zucchini, but also on other susceptible hosts. This represents the first quantitative study of the fitness cost associated with tolerance breaking for a plant virus. Thus, although easily broken, the tolerance might prove durable in some conditions if the aggressive variants are counterselected in susceptible crops.

A new approach for weed control in a cucurbit field employing an attenuated potyvirus-vector for herbicide resistance.

Expression of bar, a phosphinothricin acetyltransferase, in plant tissues, leads to resistance of these plants to glufosinate ammonium based herbicides. We have created a bar expressing, attenuated zucchini yellow mosaic potyvirus-vector, AGII-Bar, to enable herbicide use in cucurbit fields. The parental vector, ZYMV-AGII, has been rendered environmentally safe by both disease-symptom attenuation and aphid-assisted virus transmission abolishment. The recombinant AGII-Bar virus-encoding cDNA, when inoculated on diverse cucurbits was highly infectious, accumulated to similar levels as AGII, and elicited attenuated AGII-like symptoms. Potted cucurbits inoculated with AGII-Bar became herbicide resistant about a week post-inoculation. Herbicide resistance was sustained in squash over a period of at least 26 days and for at least 60 days in cucumber grown in a net-house under commercial conditions. To test the applicability of AGII-Bar use in a weed-infested field, a controlled experiment including more than 450 plants inoculated with this construct, was performed. Different dosages of glufosinate ammonium were sprayed, 2 weeks after planting, on the foliage of melons, cucumbers, squash, and watermelons. AGII-Bar provided protection to all inoculated plants, of every variety tested, at each dosage applied, including the highest doses that totally eradicated weeds. This study demonstrates that AGII-Bar can be utilized to facilitate weed control in cucurbits and exemplifies the practical potential of attenuated virus-vector use in agriculture.

The effect of head and neck positions on oropharyngeal swallowing: a clinical and electrophysiologic study.

OBJECTIVES: To determine the clinical usefulness of an electrophysiologic method for evaluating dysphagia and to identify the effects of various head and neck positions on oropharyngeal swallowing. DESIGN: Experimental, with control group. SETTING: An electromyography laboratory. PARTICIPANTS: Patients with neurogenic dysphagia (n = 51) and healthy controls (n = 24). Patients were divided into 2 groups: those patients with unilateral lower cranial lesions (n = 9) and those without laterality in the function of the oropharyngeal muscles (n = 42). INTERVENTIONS: Subjects were instructed to swallow doses of water, gradually increasing in quantity from 1 to 25 mL under 5 conditions: neutral, chin up, chin tucked, head rotated right, and head rotated left. MAIN OUTCOME MEASURES: Change in dysphagia limit through specified head and neck postures. Oropharyngeal swallowing was evaluated by laryngeal movements that were detected by a piezoelectric sensor and electromyography of the submental muscle complex. Laryngeal sensor signals occurring within 8 seconds of a swallow were accepted as a sign of the dysphagia limit. RESULTS: In the control group, dysphagia limit did not change significantly with changes in head and neck postures, except for the chin-up posture (p <.05) in which piecemeal deglutition occurred when subjects swallowed volumes less than 20 mL. Dysphagia limit improved significantly (p <.05) in 67% of the patients with unilateral lower cranial lesions when the head was rotated toward the paretic side. In dysphagic patients with bilateral symptoms, a significant (p <.01) improvement in dysphagia limit occurred in 50% of patients in chin-tuck position, but in the chin-up position, 55% of the patients experienced a significant (p <.01) decrease in dysphagia limit. CONCLUSION: The electrophysiologic method of measuring dysphagia limit confirms neurogenic dysphagia and its severity in the neutral head position. Changes in head and neck positions do not significantly alter dysphagia limit in unimpaired subjects except for the chin-up position. Although the results obtained were not compared with other techniques (eg, videofluoroscopy), this simple electrophysiologic method for describing dysphagia limit may have a place in the evaluation of dysphagia and its variability in various head and neck positions.

A nonviral peptide can replace the entire N terminus of zucchini yellow mosaic potyvirus coat protein and permits viral systemic infection.

Systematic deletion and peptide tagging of the amino-terminal domain (NT, ~43 amino acids) of an attenuated zucchini yellow mosaic potyvirus (ZYMV-AGII) coat protein (CP) were used to elucidate its role in viral systemic infection. Deletion mutants truncated by 8, 13, and 33 amino acid residues from the CP-NT 5' end were systemically infectious and produced symptoms similar to those of the AGII virus. Tagging these deletion mutants with either human c-Myc (Myc) or hexahistidine peptides maintained viral infectivity. Similarly, addition of these peptides to the intact AGII CP-NT did not affect viral life cycle. To determine which parts, if any, of the CP-NT are essential for viral systemic infection, a series of Myc-tagged mutants with 8 to 43 amino acids removed from the CP-NT were constructed. All Myc-tagged CP-NT deletion mutants, including those from which virtually all the viral CP-NT had been eliminated, were able to encapsidate and cause systemic infection. Furthermore, chimeric viruses with deletions of up to 33 amino acids from CP-NT produced symptoms indistinguishable from those caused by the parental AGII virus. In contrast to CP-NT Myc fusion, addition of the foot-and-mouth disease virus (FMDV) immunogenic epitope to AGII CP-NT did not permit systemic infection. However, fusion of the Myc peptide to the N terminus of the FMDV peptide restored the capability of the virus to spread systemically. We have demonstrated that all CP-NT fused peptides were exposed on the virion surface, masking natural CP immunogenic determinants. Our findings demonstrate that CP-NT is not essential for ZYMV spread and that it can be replaced by an appropriate foreign peptide while maintaining systemic infectivity.

Engineering zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits.

Plant virus vectors provide an attractive biotechnological tool for the transient expression of foreign genes in whole plants. As yet there has been no use of recombinant viruses for the improvement of commercial crops. This is mainly because the viruses used to create vectors usually cause significant yield loss and can be transmitted in the field. A novel attenuated zucchini yellow mosaic potyvirus (AG) was used for the development of an environmentally safe non-pathogenic virus vector. The suitability of AG as an expression vector in plants was tested by analysis of two infectious viral constructs, each containing a distinct gene insertion site. Introduction of a foreign viral coat protein gene into AG genome between the P1 and HC-Pro genes, resulted in no expression in planta. In contrast, the same gene was stably expressed when inserted between NIb and CP genes, suggesting that this site is more suitable for a gene vector. Virus-mediated expression of reporter genes was observed in squash and cucumber leaves, stems, roots and edible fruit. Furthermore, AG stably expressed human interferon-alpha 2, an important human anti-viral drug, without affecting plant development and yield. Interferon biological activity was measured in cucumber and squash fruit. Together, these data corroborate a biotechnological utility of AG as a non-pathogenic vector for the expression of a foreign gene, as a benefit trait, in cucurbits and their edible fruit.

Biological and molecular characterization of a new cucurbit-infecting tobamovirus.

ABSTRACT An uncharacterized virus was isolated from greenhouse-grown cucumber plants. Biological and serological data described in the present study indicated that the virus belonged in the genus Tobamovirus. The host range of the virus included several plant species within the family Cucurbitaceae. The virus designated Cucumber fruit mottle mosaic virus (CFMMV) causes severe mottling or mosaic on cucumber fruits, and its fast spread within greenhouses could lead to significant economic losses in cucumber crops. The genome of CFMMV has been completely sequenced and its genome organization was typical of a Tobamovirus. However, its sequence was distinct from other described viruses within the group of cucurbit-infecting Tobamoviruses. Comparisons of sequences and phylogenetic analysis suggested that the cucurbit-infecting Tobamoviruses be separated into two subgroups: subgroup I comprising the strains and isolates referred to in the literature as Cucumber green mottle mosaic virus (CGMMV) (CV3, CV4, CGMMV-W, CGMMV-SH, and CGMMV-Is) and subgroup II comprising CFMMV, Kyuri green mottle mosaic virus (KGMMV), and the Yodo strain of CGMMV, which is closely related to KGMMV and may be considered a strain of it.

Tourniquet paralysis after primary nerve repair.

Paralysis after use of a tourniquet during surgery is a well recognized complication. Based on the electrophysiologic findings, 35 cases referred to our electromyography laboratory have been diagnosed as tourniquet paralysis within a 3-yr period. This preliminary report suggests that tourniquet paralysis should be considered during clinical and electrophysiologic evaluations after primary nerve repairs.

Identification of a novel plant virus promoter using a potyvirus infectious clone.

A putative promoter from the strawberry vein banding caulimovirus (SVBV) genome was identified by its ability to drive infection with full-length cDNA of the zucchini yellow mosaic RNA potyvirus (ZYMV). A high rate of infection was obtained with the cDNA under control of the SVBV promoter using particle bombardment technology. The SVBV promoter shows 60% homology to the cauliflower mosaic virus 35S promoter in the domain spanning the conserved motifs of CCACT (at -83) and the TATA box (at -31), to the transcription start. The 3'-end one-third of the putative promoter (328 bp) was sufficient to invoke full infectivity with the ZYMV clone, and drove transient reporter gene expression in Solanaceae and Cucurbitaceae transformed with a binary plant transformation vector. Stable expression of a reporter gene (GUS) under control of the truncated SVBV promoter was shown in transformed tobacco shoots in roots, leaves and stems.

Characterisation of genetically modified cucumber mosaic virus expressing histidine-tagged 1a and 2a proteins.

Biological active cDNA clones of cucumber mosaic virus (CMV) RNAs 1 and 2 were modified by addition of sequences that encode hexahistidine (His-tag) at the amino- (N-) or carboxy- (C-) terminus of the 1a and 2a proteins. These proteins are essential components of the viral RNA-dependent RNA polymerase (RdRp). In all but one case, addition of the His-tag did not significantly affect the yields of the corresponding viruses and the His-tag-encoding sequences were maintained after mechanical passages. No differences were observed among the in vitro activities of the modified vs. wild-type viral RdRps. Subcellular fractionation showed that 2a protein was found both membrane-associated and in the 30,000 x g soluble fraction. Both termini of the native His-tag 2a protein could bind to a resin containing nickel-nitrilotriacetic acid (Ni(2+)-NTA). Detergent-treated RdRp containing C-terminal His-tagged 1a and 2a proteins was chromatographed on Ni(2+)-NTA resin. The activity of the eluted RdRp was template- dependent, in contrast to pre-chromatography fractions. However, only a small proportion of the viral RdRp as well as numerous host proteins bound to and eluted from the resin under non-denaturing conditions.

A Point Mutation in the FRNK Motif of the Potyvirus Helper Component-Protease Gene Alters Symptom Expression in Cucurbits and Elicits Protection Against the Severe Homologous Virus.

Sequence comparison had previously shown three amino acid changes in conserved motifs in the 455-amino acid sequence of the helper component-protease (HC-Pro) between a severe field strain of Zucchini yellow mosaic virus (ZYMV-NAT) and a mild field strain of ZYMV (ZYMV-WK). In this study, exchange of fragments and site-directed mutagenesis within the HC-Pro gene in an infectious clone of ZYMV enabled the effects of the mutations on symptom expression to be mapped. The substitution of Ile for Arg at position 180 in the conserved motif Phe-Arg-Asn-Lys (FRNK) of potyviruses was found to affect symptom expression. Infection of cucurbits with the engineered ZYMV (ZYMV-AG) that contained this mutation caused a dramatic symptom change from severe to mild in squash and to a symptom-free appearance in cucumber, melon, and watermelon. The Ile to Arg mutation was found to be stable, and no revertant virus was found after several passages through plants after long incubation periods. The AG strain was detected 4 days postinoculation and accumulated in cucurbits to a level and with kinetics similar to that of the wild-type ZYMV-AT strain. Cucurbit plants infected with the AG strain were protected against infection by the severe strain.

Normal range of fetal knee movements.

Fetal limb movement abnormalities have been described to date in the literature only in subjective terms. The aim of the present study was to define objectively the normal range of fetal knee motion. The study population consisted of 240 normal fetuses at gestational age 16-39 weeks. The range of active knee motion was determined on routine prenatal ultrasound scans by measuring the angles between the midtibia, midfemur, and midknee on maximum flexion and extension. From 16 to 32 weeks, average knee flexion was 129.4 degrees (range, 120-140 degrees) and average extension was 23.7 degrees (range, 15-40 degrees). From 33 to 39 weeks, there was a gradual decrease in knee extension owing to lack of space in uterus. Knowledge of the normal range of fetal knee motion will assist clinicians in the prenatal diagnosis of neuromusculoskeletal disease.