Genotype-by-genotype interactions between an insect and its pathogen.

Genotype-by-genotype (G×G) interactions are an essential requirement for the coevolution of hosts and parasites, but have only been documented in a small number of animal model systems. G×G effects arise from interactions between host and pathogen genotypes, such that some pathogen strains are more infectious in certain hosts and some hosts are more susceptible to certain pathogen strains. We tested for G×G interactions in the gypsy moth (Lymantria dispar) and its baculovirus. We infected 21 full-sib families of gypsy moths with each of 16 isolates of baculovirus and measured the between-isolate correlations of infection rate across host families for all pairwise combinations of isolates. Mean infectiousness varied among isolates and disease susceptibility varied among host families. Between-isolate correlations of infection rate were generally less than one, indicating nonadditive effects of host and pathogen type consistent with G×G interactions. Our results support the presence of G×G effects in the gypsy moth-baculovirus interaction and provide empirical evidence that correlations in infection rates between field-collected isolates are consistent with values that mathematical models have previously shown to increase the likelihood of pathogen polymorphism.

Effects of pathogen exposure on life-history variation in the gypsy moth (Lymantria dispar).

Investment in host defences against pathogens may lead to trade-offs with host fecundity. When such trade-offs arise from genetic correlations, rates of phenotypic change by natural selection may be affected. However, genetic correlations between host survival and fecundity are rarely quantified. To understand trade-offs between immune responses to baculovirus exposure and fecundity in the gypsy moth (Lymantria dispar), we estimated genetic correlations between survival probability and traits related to fecundity, such as pupal weight. In addition, we tested whether different virus isolates have different effects on male and female pupal weight. To estimate genetic correlations, we exposed individuals of known relatedness to a single baculovirus isolate. To then evaluate the effect of virus isolate on pupal weight, we exposed a single gypsy moth strain to 16 baculovirus isolates. We found a negative genetic correlation between survival and pupal weight. In addition, virus exposure caused late-pupating females to be identical in weight to males, whereas unexposed females were 2-3 times as large as unexposed males. Finally, we found that female pupal weight is a quadratic function of host mortality across virus isolates, which is likely due to trade-offs and compensatory growth processes acting at high and low mortality levels, respectively. Overall, our results suggest that fecundity costs may strongly affect the response to selection for disease resistance. In nature, baculoviruses contribute to the regulation of gypsy moth outbreaks, as pathogens often do in forest-defoliating insects. We therefore argue that trade-offs between host life-history traits may help explain outbreak dynamics.

Quantum Dots as Reporters in Multiplexed Immunoassays for Biomarkers of Exposure to Agrochemicals.

The application of quantum dots (QDs) as labels in immunoassay microarrays for the multiplex detection of 3-phenoxybenzoic acid (PBA) and atrazine-mercapturate (AM) has been demonstrated. PBA and AM are biomarkers of exposure to the pyrethroid insecticides and to the herbicide atrazine, respectively. Microarrays were fabricated by microcontact printing of the coating antigens in line patterns onto glass substrates. Competitive immunoassays were successfully performed using QDs (QD560 and QD620) as reporters. The multiplexed immunoassays were characterized by fluorescence microscopy and SEM. The application of QD fluorophores facilitates multiplex assays and therefore can contribute to enhanced throughput in biomonitoring.

Safety and efficacy of using the LigaSure vessel sealing system for securing the pedicles in vaginal hysterectomy: randomised controlled trial.

OBJECTIVE: To assess the safety and efficacy of using the LigaSure vessel sealing system for securing the pedicles during vaginal hysterectomy in comparison with the conventional method of securing the pedicles by suture ligation. DESIGN: Randomised controlled trial. SETTING: Gynaecology Department, Benenden Hospital, Kent. POPULATION: One hundred and sixteen women undergoing vaginal hysterectomy were prospectively randomised to either LigaSure (Group I) or suture ligation (Group II) for securing the pedicles. METHODS: Data of patients were collected prospectively. Statistical analysis was performed using the Mann-Whitney U test, chi(2) and Fisher's exact test as appropriate. MAIN OUTCOME MEASURES: Operating time, operative blood loss and peri-operative complications. RESULTS: The operating time was significantly shorter in the LigaSure group compared with the control group (P < 0.04). There was no statistical significant difference between the two groups in operative blood loss (P= 0.433), but peri-operative haemorrhagic complications were less frequent in the LigaSure group (0%vs 6.8%, P= 0.057). Four patients in the control group required either conversion to laparotomy because of bleeding, return to theatre for immediate post-operative haemorrhage or readmission for vault haematoma, whereas none in the LigaSure group had bleeding from unsecured pedicles. CONCLUSION: The LigaSure vessel sealing system is a safe alternative for securing pedicles in vaginal hysterectomy when compared with conventional suture ligation. Larger studies are required to determine its place in gynaecological surgery.

The value of human papillomavirus testing in the colposcopy clinic.

The aim of the study was to investigate if human papillomavirus (HPV) testing can help in treatment and management decision-making in patients referred to the colposcopy clinic with mildly dyskaryotic or borderline smears. Fifty-five patients referred to the colposcopy clinic in Benenden Hospital with mildly dyskaryotic or borderline smears had HPV testing in addition to colposcopy. Twenty-eight patients had a negative HPV test and normal colposcopy and were discharged back to their general practitioners for cytological surveillance. The most recent smear results of these 28 patients were obtained from their general practitioners to assess if any patient had progressed to a higher-grade smear. Twenty-one (75%) replies were obtained and in 17 (80.9%) patients smear results had regressed to normal. Three continued to have borderline or mildly dyskaryotic smear and one had progressed to moderate dyskaryosis. Our conclusion was that in the presence of normal colposcopic findings in patients with mildly dyskaryotic or borderline smears, negative HPV status justifies referral back to the general practitioner for cytological surveillance, thus reducing workload and cost.

Sexual function after using tension-free vaginal tape for the surgical treatment of genuine stress incontinence.

OBJECTIVE: To determine whether the use of the tension-free vaginal polypropylene tape (TVT) procedure for the treatment of genuine stress incontinence (GSI) affects sexual activity. PATIENTS AND METHODS: Sixty-seven women treated by insertion of a TVT between September 1998 and March 2001 for GSI were sent questionnaires 6-36 months after surgery to determine any urinary symptoms, sexual activity, patient satisfaction and the use of hormone-replacement therapy. RESULTS: The questionnaire was returned by 57 patients (87%); 43 (76%) reported being sexually active and 14 (25%) were not. Of the former, 31 (72%) reported no change in sexual function after surgery and only two reported an improvement; six (14%) reported that sexual function was worse and four did not reply to the questions. The patients reporting that sexual function was worse cited loss of libido as the main reason. No patients complained of dyspareunia. CONCLUSIONS: There was no significant change in sexual function or activity after the TVT procedure and patients can thus be reassured that this operation will not affect their sex life.

The efficacy of laparoscopic mesh colposuspension: results of a prospective controlled study.

OBJECTIVE: To investigate the efficacy of laparoscopic mesh colposuspension as an equivalent approach to the 'gold standard' open Burch colposuspension. PATIENTS AND METHODS: A prospective controlled study of laparoscopic mesh colposuspension was conducted over 2 years; 87 patients with genuine stress incontinence (GSI) were recruited. The preoperative evaluation included a history, examination, midstream urine analysis, urinary voiding diary, a Urilos pad test, and twin-channel subtracted cystometry, including urethral profilometry and measurement of the postvoid residual volume. The study included patients who had undergone previous incontinence surgery, but those with detrusor instability or neurogenic bladder were excluded. The patients were assessed at 6 weeks, 6 months and 1 year after surgery and then yearly thereafter. The urodynamic assessment was repeated 3 months after surgery. RESULTS: Forty-nine patients underwent laparoscopic colposuspension using Prolene mesh and titanium tacks to elevate the bladder neck, while 38 patients had open Burch colposuspension. There was no difference between the groups in age, parity, body mass index, menopausal status, medical history, previous bladder neck surgery and prolapse. At 6 weeks the cure rate was similarly high in the two groups (91% laparoscopic and 94% open). After a mean follow-up of 32 months, both groups showed a decline in efficacy, which was more marked in the laparoscopic group. Cure rates were 62% for laparoscopy and 79% for open surgery, and the improvement rates were 77% and 89%, respectively (P < 0.05). CONCLUSION: Laparoscopic colposuspension using a mesh and tacker technique reduces the technical difficulty and operating time of the endoscopic procedure, but the long-term cure rates are inferior to open Burch colposuspension.

Cold knife cone biopsy--a valid diagnostic tool and treatment option for lesions of the cervix.

We present a case series of cold knife cone biopsy with emphasis on validation of the technique for ongoing and future use. This involved: analysis of 100 cold knife cone biopsies performed between 1987 and 1997. Data were gathered relating to indications, technique, postoperative morbidity, histological findings and results of long-term follow-up. Mean age was 41.8 years, and mean parity 2. Smear abnormalities of moderate or severe dyskaryosis were present in 74% of cases, and glandular abnormalities in 8%. Colposcopic diagnosis of CIN II-III was made in 55% of cases, and invasion suspected in 12%. The main indications for conisation were inability to visualise the entire squamocolumnar junction (64%), disparity in cytological and colposcopic findings (26%), and colposcopic suspicion of invasion (12%). Mean blood loss was 90 ml. One patient (1%) required hysterectomy and blood transfusion to control secondary haemorrhage. Postoperative infection occurred in 5%, and cervical stenosis in 3%. All specimens were adequate for histopathological evaluation, including the margin, CIN was histologically proven in 67% of specimens, microinvasion in 4%, adenocarcinoma in-situ in 3%, and invasion in 3%. During a mean follow-up of 4.5 years (range 1.5-11 years), cure rate after complete excision was 97% and after incomplete excision was 85% (P > 0.05) Despite the advent of alternative methods of treatment, cold knife cone biopsy remains an acceptable option in the management of CIN and microinvasion of the cervix. There are valid indications for the technique. When properly performed, it gives accurate representation of the disease process, has low risk of complications and is curative in most cases.

Burch colposuspension: a 10 to 12 years follow up.

Burch colposuspension is considered to be the gold standard for the surgical treatment of genuine stress incontinence in women. Short-term cure rates are as high as 90%. (Jarvis, 1994). The long-term (10 or more years) cure rate of 62% in this study confirms the time dependent decline in efficacy.

Assessment of a new bone anchor system for the treatment of female genuine stress incontinence.

OBJECTIVE: To evaluate the efficacy of the In-Tac bone-anchor system (using shape-memory metal bone anchors, Influence Medical Technologies, Lancs, UK) as a vaginal procedure for the treatment of female genuine stress incontinence (GSI). PATIENTS AND METHODS: Between January 1997 and April 1998, 30 patients with GSI were recruited into the study (age range 36-74 years); patients who had undergone previous failed continence surgery were not excluded. All patients underwent a urodynamic assessment before and 3 months after surgery. All In-Tac bone-anchor procedures were performed under general anaesthesia. Patients were reviewed after surgery at 6 weeks, 3, 6 and 12 months, and yearly thereafter. RESULTS: The mean (range) operative duration was 42 (20-75) min and the blood loss 60 (10-200) mL (median 30). There were no intraoperative complications and minimal analgesia was required postoperatively. At 6 weeks, 27 patients (90%) were subjectively cured. The urodynamic assessment at 3 months revealed that 22 patients (73%) were objectively cured; at 6 months and one year the subjective cure rate was 80%. CONCLUSION: The In-Tac bone-anchor system is simple and safe, the procedure easily learned and the operation brief. It offers promise as an incision-less vaginal procedure that may have wide application for the treatment of women with GSI. A longer follow-up is needed to fully confirm its durability and effectiveness.

Vaginal endoscopic oophorectomy with vaginal hysterectomy: a simple minimal access surgery technique.

The main advantage of laparoscopic assisted vaginal hysterectomy (LAVH) is ease of access to the ovaries; despite this, LAVH is infrequently performed due to the inherent difficulty of the technique. Lower morbidity, shorter length of surgery, reduced hospital stay and more rapid recovery are the main advantages of vaginal hysterectomy, but one of its limitations is the accessibility of the ovaries. Three methods of vaginal endoscopic oophorectomy following vaginal hysterectomy are described in this paper. Endoscopic vaginal oophorectomy was successfully performed during 82 cases of vaginal hysterectomy and the results show that the technique is simple, safe and easy to learn, and an alternative to laparotomy and LAVH in the absence of pelvic adhesions.

The cytochrome bc1 complex but not CycM is necessary for symbiotic nitrogen fixation by Rhizobium leguminosarum.

Following Tn5 mutagenesis of Rhizobium leguminosarum biovar viciae, two mutants in one complementation group were identified as being unable to fix nitrogen in pea nodules. Spectroscopic analysis revealed that the mutants had lowered levels of c-type cytochromes and cytochromes aa3, but increased levels of cytochrome d. Cells of the mutants were greatly reduced in their ability to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine but membranes prepared from them had increased levels of succinate- and NADH-dependent respiration. NADH oxidation by the mutants was insensitive to the respiratory inhibitor antimycin A, that targets the cytochrome bc1 complex. Molecular analysis of the mutants revealed that they were affected in the cytochrome bc1 complex. One of the mutants contained Tn5 in a gene homologous to that encoding cytochrome c1, and in the other the Tn5 was in DNA homologous to that encoding the cytochrome b component of the cytochrome bc1 complex. Haem staining revealed that haem proteins of M(r)31,000 and M(r)23,000 were absent from membranes from the mutants whereas an additional soluble c-type cytochrome protein of M(r)23,000 was present. We conclude that the larger of these two haem proteins corresponds to cytochrome c1 and, in its absence, the protein of M(r)23,000 does not remain associated with the membrane. Formation of this M(r)23,000 component was specifically blocked in a third respiratory-defective mutant which contained Tn5 in a region of DNA showing homology to a Bradyrhizobium Japonicum gene previously shown to encode the membrane-bound c-type cytochrome CycM. Although the cytochrome bc1 complex is essential for symbiotic nitrogen fixation, the other membrane-bound c-type cytochrome (CycM) is not.

Characterization of the cycHJKL genes involved in cytochrome c biogenesis and symbiotic nitrogen fixation in Rhizobium leguminosarum.

Mutants of Rhizobium leguminosarum bv. viciae unable to respire via the cytochrome aa3 pathway were identified by the inability to oxidize N,N'-dimethyl-p-phenylenediamine. Two mutants which were complemented by cosmid pIJ1942 from an R. leguminosarum clone bank were identified. Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected. Heme staining of cellular proteins revealed that all cytochrome c-type heme proteins were absent. These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa3 and d were present, the latter at a higher-than-normal level. DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli nrfE and nrfF gene products involved in the assembly of c-type cytochromes. The absence of cytochrome c heme proteins in these R. leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme to apocytochrome c.

Pharyngeal sensation and gag reflex in healthy subjects.

The gag reflex is often used in the assessment of swallowing, yet its absence does not predict aspiration in acute stroke. Disordered pharyngeal sensation has been found to be a sensitive predictor. The occurrence of gag reflex and pharyngeal sensation in healthy people is unknown. We studied these tests in 140 healthy subjects (half elderly and half young). Gag reflex was absent in 37% of subjects whereas pharyngeal sensation was absent in only 1. The results largely explain the low predictive value of gag reflex in the assessment of aspiration in acute stroke. Testing pharyngeal sensation would be more likely to be useful in these circumstances.

Rhizobium leguminosarum nodulation gene (nod) expression is lowered by an allele-specific mutation in the dicarboxylate transport gene dctB.

To identify host genes that might influence nod (nodulation) gene expression in Rhizobium leguminosarum, a nodC-phoA reporter plasmid (carrying nodD) was introduced into a chemically mutagenized population of a R. leguminosarum strain lacking a symbiotic plasmid. The transconjugants were screened for expression of alkaline phosphatase (PhoA) on plates containing hesperetin, an inducer of nod genes, and a mutant with reduced expression was identified. When the nodC-phoA plasmid was cured from the mutant and the symbiotic plasmid pRL1Jl introduced, the mutant formed nodules, but symbiotic nitrogen fixation was less than 20% of normal. When the nodC-phoA allele was introduced on pRL1Jl a low level of nod gene induction was found. The reduced nodC expression appeared to be caused by a decrease in expression of the regulatory gene nodD, since expression of a nodD-lacZ fusion was also lower in the mutant than in the control. These mutant phenotypes and the low nitrogen fixation were complemented with a plasmid (plJ1848) from a R. leguminosarum cosmid library. DNA hybridization confirmed that plJ1848 was not from the symbiotic plasmid and showed that a DNA insertion was present in the mutant. The complementing region of plJ1848 was defined by transposon mutagenesis; DNA sequencing revealed that it carried the dicarboxylic acid transport (dct) genes. However, the mutant grew well with succinate as sole C-source. Genetic analysis revealed that the mutant appeared to contain IS50 in the regulatory gene dctB and that this mutation caused the reduction in nod gene expression. The effect was allele-specific since other mutations in dctB did not influence nod gene expression. Surprisingly, the mutant had a constitutive high level of succinate transport, indicating that the mutation caused unregulated expression of dctA the structural gene for dicarboxylic acid transport. This in some way appears to have lowered the expression of nodD, indicating that the nodD promoter may be influenced by the metabolic status of the cells or by expression of dctD in the absence of dctB.

Identification of a gene encoding a thioredoxin-like product necessary for cytochrome c biosynthesis and symbiotic nitrogen fixation in Rhizobium leguminosarum.

A Tn5-induced mutant of Rhizobium leguminosarum bv. viciae could not form nitrogen-fixing nodules on pea or vetch because of a lesion in electron transport to oxygen. The mutant lacked spectroscopically detectable cytochromes c and aa3. No proteins containing c-type cytochrome could be identified in the mutant by heme staining of proteins fractionated on polyacrylamide gels, indicating that the mutant was defective in maturation of all c-type cytochromes. The Tn5 mutation was determined to be located in a gene that was called cycY. The cycY gene product is homologous to the thioredoxin-like protein HelX involved in the assembly of c-type cytochromes in Rhodobacter capsulatus and to an open reading frame from a Bradyrhizobium japonicum gene cluster containing other genes involved in cytochrome c biogenesis. Our observations are consistent with CycY functioning as a thioredoxin that reduces cysteine residues in apocytochromes c before heme attachment.

Resistance to nodulation of cv. Afghanistan peas is overcome by nodX, which mediates an O-acetylation of the Rhizobium leguminosarum lipo-oligosaccharide nodulation factor.

Only some strains of Rhizobium leguminosarum biovar viciae can efficiently nodulate varieties of peas such as cv. Afghanistan, which carry a recessive allele that blocks efficient nodulation by most western isolates of R.I. viciae. One strain (TOM) which can nodulate cv. Afghanistan peas has a gene (nodX) that is required to overcome the nodulation resistance. Strain TOM makes significantly lower amounts of lipo-oligosaccharide nodulation factors than other strains of R.I. viciae and this effect appears to be due to lower levels of nod gene induction. These nodulation factors are similar to those from other R.I. viciae strains in that they consist of an oligomer of four or five beta 1-4-linked N-acetylglucosamine residues in which the terminal non-reducing glucosamine carries an O-acetyl group and a C18:4 or C18:1 N-acyl group. However, one of the nodulation factors made by strain TOM differs from the factors made by other strains of R.I. viciae in that it carries an O-acetyl group on the C-6 of the reducing N-acetylglucosamine residue. This acetylation is NodX-dependent and the pentameric nodulation factor is acetylated on the reducing N-acetylglucosamine residue whereas the tetrameric nodulation factor is not. Although the nodL gene product is also an O-acetyl transferase (it O-acetylates the C-6 of the terminal non-reducing glucosamine), there is very little similarity between the amino acid sequences of these two acetyl transferases.