Clinicopathological features of cytokeratin 5-positive pulmonary adenocarcinoma.

Cytokeratin 5 (CK5) is a marker for pulmonary squamous cell carcinoma; however, CK5 is sometimes present in pulmonary adenocarcinoma (ADC), and there is insufficient information regarding the clinicopathological features of CK5-positive ADC. We aimed to explore the clinicopathological characteristics of CK5-positive ADC using immunohistochemistry. We prepared the following two cohorts: a resected cohort containing 220 resected tumours for primarily studying the detailed morphological characteristics, and a tissue microarray (TMA) cohort containing 337 samples for investigating the associations of CK5 expression with other protein expressions, genetic and prognostic findings. CK5-positive ADC was defined to have ≥ 10% tumour cells and presence of CK5-positive tumour cells in the resected and TMA cohorts, respectively. CK5-positive ADCs were identified in 91 (16.3%) patients in the combined cohort. CK5-positive ADCs had male predominance (P = 0.012), smoking history (P = 0.001), higher stage (P < 0.001), histological high-grade components (P < 0.001), vascular invasion (P < 0.001), mucinous differentiation (P < 0.001), spread through airspaces (P < 0.001), EGFR wild-type (P < 0.001), KRAS mutations (P < 0.001), ALK rearrangement (P < 0.001) and ROS1 rearrangement (P = 0.002). In the resected cohort, more than half the CK5-positive ADCs (19 cases, 65.5%) showed mucinous differentiation; the remaining cases harboured high-grade components. In the TMA cohort, CK5-positive ADCs correlated with TTF-1 negativity (P = 0.002) and MUC5B, MUC5AC and HNF4alpha positivity (P < 0.001, 0.048, < 0.001). Further, CK5-positive ADCs had significantly lower disease-free and overall survival rates than CK5-negative ADCs (P < 0.001 for each). Additionally, multivariate analysis revealed that CK5 expression was an independent poor prognostic factor. CK5-positive ADCs showed aggressive clinical behaviour, with high-grade morphology and mucinous differentiation.

Protocol of a two arm randomised, multi-centre, 12-month controlled trial: evaluating the impact of a Cognitive Behavioural Therapy (CBT)-based intervention Supporting UPtake and Adherence to antiretrovirals (SUPA) in adults with HIV.

BACKGROUND: Delay to start antiretroviral therapy (ART) and nonadherence compromise the health and wellbeing of people living with HIV (PLWH), raise the cost of care and increase risk of transmission to sexual partners. To date, interventions to improve adherence to ART have had limited success, perhaps because they have failed to systematically elicit and address both perceptual and practical barriers to adherence. The primary aim of this study is to determine the efficacy of the Supporting UPtake and Adherence (SUPA) intervention. METHODS: This study comprises 2 phases. Phase 1 is an observational cohort study, in which PLWH who are ART naïve and recommended to take ART by their clinician complete a questionnaire assessing their beliefs about ART over 12 months. Phase 2 is a randomised controlled trial (RCT) nested within the observational cohort study to investigate the effectiveness of the SUPA intervention on adherence to ART. PLWH at risk of nonadherence (based on their beliefs about ART) will be recruited and randomised 1:1 to the intervention (SUPA intervention + usual care) and control (usual care) arms. The SUPA intervention involves 4 tailored treatment support sessions delivered by a Research Nurse utilising a collaborative Cognitive Behavioural Therapy (CBT) and Motivational Interviewing (MI) approach. Sessions are tailored to individual needs and preferences based on the individual patient's perceptions and practical barriers to ART. An animation series and intervention manual have been developed to communicate a rationale for the personal necessity for ART and illustrate concerns and potential solutions. The primary outcome is adherence to ART measured using Medication Event Monitoring System (MEMS). Three hundred seventy-two patients will be sufficient to detect a 15% difference in adherence with 80% power and an alpha of 0.05. Costs will be compared between intervention and control groups. Costs will be combined with the primary outcome in cost-effectiveness analyses. Quality adjusted life-years (QALYs) will also be estimated over the follow-up period and used in the analyses. DISCUSSION: The findings will enable patients, healthcare providers and policy makers to make informed decisions about the value of the SUPA intervention. TRIAL REGISTRATION: The trial was retrospectively registered 21/02/2014, ISRCTN35514212 .

Right and left inverted lobar lung transplantation.

Adult recipients frequently withdraw from living-donor lobar lung transplantation because of the small size of donor grafts. The right lower lobe is 120% larger than the left lower lobe. We developed a novel surgical technique in which an inverted right lower lobe graft can be transplanted into the left thorax. The first patient was a 43-year-old woman with end-stage idiopathic interstitial pneumonia. Her husband was the only eligible donor for living-donor lobar lung transplantation. His right lower lobe was estimated to provide 45% of the recipient's predicted forced vital capacity, which would provide the borderline function required for living-donor lobar lung transplantation. Since lung perfusion scintigraphy of the recipient showed a right-to-left ratio of 64:36, transplanting the right lower lobe graft into the left thorax and sparing the native right lung was considered the only treatment option. We simulated this procedure using three-dimensional models produced by a three-dimensional printer. In living-donor lobar lung transplantation, all anastomoses were performed smoothly as planned preoperatively. Because of the initial success, this procedure was performed successfully in two additional patients. This procedure enables larger grafts to be transplanted, potentially solving critical size matching problems in living-donor lobar lung transplantation.

Sparing Native Upper Lobes in Living-Donor Lobar Lung Transplantation: Five Cases From a Single Center.

Living-donor lobar lung transplantation (LDLLT) is indicated for rapidly deteriorating patients, and the total volume of two lower lobe grafts must be sufficient for the recipient. To rescue patients with small lobar grafts, we performed five LDLLTs sparing native upper lobes. This strategy was used when upper lobes or segments were preoperatively less impaired. There were no hospital deaths. Extracorporeal circulation time and operative time were similar to those of conventional LDLLTs. The length of intensive care unit stay was also similar. Late complications attributed to the spared lungs were airway infection in one recipient and pneumothorax in two but they were successfully managed. All recipients were discharged without supplemental oxygen. The spared lung volumes measured by volumetry did not change after LDLLT. Lung perfusion scintigraphy performed at 1 year showed remaining perfusion in the spared lungs, although much less than in the grafts. These results suggested that the spared lobes kept adequate space in the thoracic cavity and kept functioning to a limited extent. The new lobar-sparing strategy appears feasible and effective in LDLLT using small grafts for selected patients when the upper lobes or segments are less impaired.

Osteoprotegerin is secreted into the coronary circulation: a possible association with the renin-angiotensin system and cardiac hypertrophy.

The circulating osteoprotegerin (OPG) level reflects a series of cardiovascular diseases; however, the source(s) of circulating OPG remain(s) to be determined. This study explored whether OPG is released in the coronary circulation and whether it is associated with cardiac structure and function. Fifty-six patients (67±10 years old, male 57%, hypertension 73%, coronary artery disease 50%) were enrolled, and blood samples were collected simultaneously from the orifice of the left coronary artery (CA) and the coronary sinus (CS) after angiography. The concentration of OPG was higher in the CS than in the CA (7.7±4.1 vs. 6.7±3.6 pmol/l, p<0.001). The trans-cardiac OPG concentration was significantly (p=0.019) decreased in patients who have been prescribed either an angiotensin converting enzyme inhibitor or an angiotensin II type 1 receptor blocker (ACEI/ARB). In patients subgroup who did not take an ACEI/ARB (n=27), the trans-cardiac OPG level was positively correlated with age (r=0.396, p=0.041) and relative wall thickness of left ventricle (r=0.534, p=0.004). In multivariate linear regression analysis, relative wall thickness remained to be the independent variable for the trans-cardiac OPG level (p=0.004). Moreover, trans-cardiac OPG was significantly (p=0.021) increased in patients with relative wall thickness greater than 0.45 but it did not differ if the left ventricular mass index was increased (≥116 for males, or ≥ 104 for females, g/m(2)) or not (p=0.627). This study suggests that OPG is secreted into the coronary circulation and is associated with concentric remodeling/hypertrophy of LV, possibly in interactions with the renin-angiotensin system.

Itraconazole prophylaxis for invasive Aspergillus infection in lung transplantation.

Invasive Aspergillus infection (IA) is a significant cause of morbidity in lung transplantation (LT). However, its optimal prophylaxis is unclear. We routinely administer itraconazole (ITCZ) prophylaxis to all patients undergoing LT. In this study, we retrospectively evaluated the duration of prophylaxis and risk factors of IA. Among 30 adult patients who underwent LT, 5 patients developed IA. All patients with IA stopped ITCZ treatment within 1 year. At least 1 year of ITCZ prophylaxis is essential for the prevention of IA. Cytomegalovirus infection, renal replacement therapy, and tracheotomy were risk factors for IA.

Adaptation over a wide range of donor graft lung size discrepancies in living-donor lobar lung transplantation.

Living-donor lobar lung transplantation (LDLLT), unlike deceased donor lung transplantation, often involves a wide range of size discrepancies between donors and recipients. The aim of this study was to evaluate the function of donor lung grafts in the recipient thorax in 14 cases of bilateral LDLLT involving 28 successfully transplanted lower-lobe grafts. Pulmonary function tests and three-dimensional computed tomography (3D-CT) volumetry were performed perioperatively. According to 3D-CT size matching, donor graft volumes ranged from 40% to 161% of the hemilateral thoracic volumes of the recipients. Graft forced vital capacity (FVC) values increased over time, reaching 102 ± 39% of preoperatively estimated values at 12 months postoperatively. Graft volumes also increased over time, reaching 120 ± 38% of the original values at 12 months postoperatively. Undersized donor grafts expanded more after LDLLT than oversized donor grafts, producing greater FVC values than those estimated preoperatively, whereas oversized donor grafts became inflated to their original size and maintained FVC values that approached the preoperative estimates. Thus, donor grafts were found to overinflate or underinflate to the extent that they could preserve their native function in the new recipient's environment.

A prospective study analyzing one-year multidimensional outcomes in living lung transplant donors.

The success of living-donor lobar lung transplantation (LDLLT) largely depends on donor outcome; but to date, no authors have studied health-related quality of life (HRQOL) of donors. We prospectively evaluated multidimensional outcomes before and 1 year after donor lobectomies. Patient-reported HRQOL, dyspnea, psychological status and sleep quality, and physiological pulmonary function were determined. All donors were alive without any limitations in their activities of daily living after 1 year. Postoperative pulmonary function was better than the estimated preoperative values; but, with respect to HRQOL, four of the eight subscales of the Medical Outcomes Study 36-item short form (SF-36) deteriorated significantly after donation. In addition, dyspnea assessed by the modified Medical Research Council scale also worsened significantly. In contrast, postoperative anxiety assessed by the Hospital Anxiety and Depression Scale significantly improved from baseline. The donors whose recipients died reported lower SF-36 scores with worsening sleep quality measured by Pittsburgh Sleep Quality Index. Thus, although postoperative pulmonary functions in donors were preserved, their HRQOL and dyspnea deteriorated postoperatively. Moreover, HRQOL and sleep quality were impaired in recipients who experienced poor outcomes. To capture the comprehensive outcomes in LDLLT donors after donation, patient-reported outcomes should be analyzed separately from physiological outcomes.

Successful lung transplantation in a case with diffuse pulmonary arteriovenous malformations and hereditary hemorrhagic telangiectasia.

Diffuse pulmonary arteriovenous malformations (AVMs) are associated with a poor prognosis and the therapeutic strategy remains controversial. We describe a pediatric patient with diffuse pulmonary AVMs associated with hereditary hemorrhagic telangiectasia (HHT), who presented with two cerebral AVMs in the parietal and occipital lobes as well. Of note, successful bilateral lung transplantation not only improved the hypoxemia but also resulted in size reduction of the cerebral AVMs. Although it is essential to consider involvements other than pulmonary AVMs, especially brain AVMs, to decide the indication, lung transplantation can be a viable therapeutic option for patients with diffuse pulmonary AVMs and HHT.

Suppression of aggregate formation and apoptosis by transglutaminase inhibitors in cells expressing truncated DRPLA protein with an expanded polyglutamine stretch.

To elucidate the molecular mechanisms whereby expanded polyglutamine stretches elicit a gain of toxic function, we expressed full-length and truncated DRPLA (dentatorubral-pallidoluysian atrophy) cDNAs with or without expanded CAG repeats in COS-7 cells. We found that truncated DRPLA proteins containing an expanded polyglutamine stretch form filamentous peri- and intranuclear aggregates and undergo apoptosis. The apoptotic cell death was partially suppressed by the transglutaminase inhibitors cystamine and monodansyl cadaverine (but not putrescine), suggesting involvement of a transglutaminase reaction and providing a potential basis for the development of therapeutic measures for CAG-repeat expansion diseases.

Early-onset ataxia with ocular motor apraxia and hypoalbuminemia is caused by mutations in a new HIT superfamily gene.

Friedreich ataxia (FRDA), the most common autosomal recessive neurodegenerative disease among Europeans and people of European descent, is characterized by an early onset (usually before the age of 25), progressive ataxia, sensory loss, absence of tendon reflexes and pyramidal weakness of the legs. We have recently identified a unique group of patients whose clinical presentations are characterized by autosomal recessive inheritance, early age of onset, FRDA-like clinical presentations and hypoalbuminemia. Linkage to the FRDA locus, however, was excluded. Given the similarities of the clinical presentations to those of the recently described ataxia with oculomotor apraxia (AOA) linked to chromosome 9p13, we confirmed that the disorder of our patients is also linked to the same locus. We narrowed the candidate region and have identified a new gene encoding a member of the histidine triad (HIT) superfamily as the 'causative' gene. We have called its product aprataxin; the gene symbol is APTX. Although many HIT proteins have been identified, aprataxin is the first to be linked to a distinct phenotype.

Current status of organ transplantation in Japan.

To overcome severe donor shortage, Japanese doctors over the years have developed innovative strategies to maximize organs transplanted per brain death donor and expanded the donor pool using living donors. They also used living and marginal organs and drastically improved living donor lung, liver, pancreas and kidney transplantations. Moreover, they initiated ABO blood type incompatible liver transplantation advancements and succeeded in overcoming the blood type barrier in kidney and liver transplantations. Similar efforts are underway for pancreas transplantation. Furthermore, Japanese doctors have developed a nonaggressive step to achieve immunosuppression following organ transplantation by carefully monitoring donor-specific hyporesponsiveness and infectious immunostatus. However, the institution of amendments to allocation systems and the intensification of efforts to decrease living donor morbidity and to increase the number of brain death donors have remained important issues needing attention. Overall, the strategies Japan has adopted to overcome donor shortage can provide useful insights on how to increase organ transplantations.

[Surgical treatment of lung cancer in patients with pulmonary fibrosis].

Acute exacerbation( AE) of idiopathic pulmonary fibrosis( IPF) is the most common cause of postoperative mortality after lung cancer surgery. ostoperative mortality rate of lung cancer patients with IPF has been reported to range approximately from 3% to 15%. The degree of fibrosis on preoperative high-resolution computed tomography( CT), preoperative high serum levels of KL-6, lactate dehydrogenase(LDH), and C-reactive protein( CRP), and preoperative low %TLC, %VC, and %diffusing capacity of CO( DLco) in pulmonary function test have been reported as the predictive risk factors for postoperative AE, but the clinical relevance remains controversial. Most of reports regarding the 5-year survival rate after resection of lung cancer with IPF have been ranged between 40% and 60%, and significantly poorer than that without IPF. However, considering the high risk of AE following chemotherapy or radiotherapy, the poor prognosis after surgical treatment can be acceptable. A number of prophylactic strategies against AE have been reported in the literature, but none of them has been generally established. A nationwide survey concerning postoperative AE after resection of lung cancer with pulmonary fibrosis has been conducted in Japan, and the final results will be reported soon. Based on the outcomes of the survey, standard strategy for the treatment of lung cancer with IPF is expected to be established.6.

Less maintenance immunosuppression in lung transplantation following hematopoietic stem cell transplantation from the same living donor.

Living-donor lobar lung transplantation (LDLLT) is one of the final options for saving patients with pulmonary complications after hematopoietic stem cell transplantation (HSCT). We retrospectively investigated 19 patients who had undergone LDLLT after HSCT in Japan. Eight patients underwent LDLLT after HSCT in which one of the donors was the same living donor as in HSCT (SD group), while 11 received LDLLT from relatives who were not the HSCT donors (non-SD group). In the SD group, three patients underwent single LDLLT. The 5-year survival rate was 100% and 58% in the SD and non-SD groups, respectively. In the SD group, postoperative immunosuppression was significantly lower than in the non-SD group. Two patients died of infection and one died of post-transplant lymphoproliferative disease (PTLD) in the non-SD group, while only one patient died of PTLD 7 years after LDLLT in the SD group. Hematologic malignancy relapsed in two patients in the non-SD group. For the three single LDLLTs in the SD group, immunosuppression was carefully tapered. In our study, LDLLT involving the same donor as for HSCT appeared to have advantages related to lower immunosuppression compared to LDLLT from relatives who were not the HSCT donors.

Doctor-patient concordance during HIV treatment switching decision-making.

OBJECTIVES: The aim of the study was to explore levels of doctor-patient concordance during the making of decisions regarding HIV treatment switching and stopping in relation to patient health-related outcomes. METHODS: Adult patients attending five HIV clinics in the United Kingdom were requested to complete the study questionnaire, which included a Concordance Scale, and measures of symptoms [Memorial Symptom Assessment Short Form (MSAS) index], quality of life (EuroQol), satisfaction, adherence and sexual risk behaviour. Clinical health measures (HIV viral load and CD4 cell count) were also obtained. A total of 779 patients completed the questionnaire, giving a response rate of 86%; of these 779 patients, 430 had switched or stopped their HIV treatment and were thus eligible for inclusion. Of these patients, 217 (50.5%) fully completed the Concordance Scale. RESULTS: Concordance levels were high (88% scored between 30 and 40 on the scale; score range 10-40). Higher concordance was related to several patient outcomes, including: better quality of life (P=0.003), less severe and burdensome symptom experience (lower MSAS-physical score, P=0.001; lower MSAS-psychological score, P=0.008; lower MSAS-global distress index score, P=0.011; fewer symptoms reported, P=0.007), higher CD4 cell count (at baseline, P=0.019, and 6-12 months later, P=0.043) and greater adherence (P=0.029). CONCLUSIONS: High levels of doctor-patient concordance in HIV treatment decision-making are associated with greater adherence and better physical and psychological functioning. More research is needed to establish a causal relationship between concordance and these outcomes.

Dynamic instability of central airways and peripheral airspace in rat lungs perfused with cold preservation solutions.

BACKGROUND/AIMS: For lung preservation, one of two types of solutions is commonly employed: Euro-Collins (EC) or low potassium dextran glucose (LPDG). These two solutions have been compared regarding biological, morphometrical and physiological outcomes in many experiments. However, the dynamic mechanics of perfused lung are not well understood because the dynamic characteristics cannot be assessed under static conditions; hence, the primary goal of the present study was to assess this in perfused rat lungs during the preservation period, comparing EC with LPDG at 0 or 9 h at 4°C. METHODS: Lung impedance was measured using a forced oscillation technique. Lung resistance and elastance values were obtained by the fast Fourier transform algorithm. The instability of central airways and heterogeneity of ventilation were estimated. RESULTS: In the EC group, airway resistance and instability were high after perfusion, and the lung elastance was high and more heterogeneous after cold storage. In contrast, those parameters were stable in the LPDG group during cold storage. CONCLUSION: Such dynamic stability might facilitate the handling of lung grafts and eliminate injurious cyclic ventilation stress after reperfusion. Thus, we conclude that the impedance frequency characteristic represents a novel informative parameter for investigating lung preservation techniques.

Self-reported non-adherence to ART and virological outcome in a multiclinic UK study.

Adherence is of fundamental importance to ART success. We examined the association of self-reported non-adherence with demographic factors, health and behaviour issues, and virological outcome, in a multi-clinic study. Seven hundred and seventy-eight HIV patients in five clinics in London and Brighton completed a questionnaire on adherence and HIV/health issues at baseline in 2005/6. For 486 subjects taking ART, non-adherence in the past week was defined as: (A)>or=1 dose missed or taken incorrectly (wrong time/circumstances); (B)>or=1 dose missed; (C)>or=2 doses missed. Questionnaire data were matched with routine treatment and virology data for consenting subjects (61.4%). We assessed four virological outcomes in 307 of 486 patients: (i) VL>50c/mL using latest VL at the questionnaire and excluding patients starting HAART<24 weeks ago; (ii) VL>50c/mL using the first VL from 6 to 12 months post-questionnaire; (iii) any VL>50c/mL from 6 to 12 months post-questionnaire; (iv) among patients with VL<50c/mL at questionnaire, time to first subsequent VL>50c/mL over two years follow up. Non-adherence was reported by 278 (57.2%), 102 (21.0%) and 49 (10.1%) of 486 patients, for definitions A, B and C, respectively. Non-adherence declined markedly with older age, and tended to be more commonly reported by Black patients, those born outside the UK, those with greater psychological symptoms and those with suicidal thoughts. There was a weaker association with physical symptoms and no association with gender/sexuality, education, unemployment, or risk behaviour (p>0.1). In logistic regression analyses, younger age, non-UK birth and psychological variables were independent predictors of non-adherence [e.g., for non-adherence B: odds ratios (95% CI) were 0.95 (0.92, 0.98) for every year older age; 1.6 (1.0, 2.5) for non-UK born; 2.3 (1.5, 3.7) for suicidal thoughts]. Non-adherence was associated with poorer virological outcome; the most consistent association was for definition C. Among 255 patients with VL<50c/mL at baseline, non-adherence definition C was independently associated with subsequent VL>50c/mL [adjusted hazard ratio (95% CI) 3.2 (1.5, 7.2)]. Non-UK birth and psychological symptoms predicted non-adherence, but the most striking association was with younger age. Age should be an important consideration in clinical strategies to minimise non-adherence and in decisions regarding ART initiation. A simple measure of non-adherence can identify patients at risk of poorer virological outcome.

[Treatment strategy and outcomes of invading apical lung cancer].

Invading apical lung cancers are generally the non-small-cell lung cancers (NSCLCs) which involve the apex of the chest wall. These tumors should be classified into 2 types based on the main location of tumor because of the difference of involved surrounding structures ; (1) the superior sulcus tumor origi nally termed Pancoast tumor which involves posterior region of the apex and (2) the anterior apical tumor which involves anterior region of the apex. Previously, these NSCLCs were considered to be inoperable showing a dismal prognosis. With the development of combined modality therapies for locally advanced NSCLCs, the prognosis of invading apical NSCLCs has been improved, especially since intro duction of the neoadjuvant chemoradiotherapy. Surgical resection for invading apical NSCLCs is 1 of challenging procedures for thoracic surgeons. The point is the anatomical complication of the small apex surrounding vital structures. Several approaches have been developed such as the posterior Paul-son's approach or anterior Masaoka's approach. In particular, the approach from anterior chest has been modified or devised to achieve safe and complete resection of tumors invading anterior structures like subclavian vessels. In this article, we reviewed our 13 cases of invading apical NSCLCs, especially from the view point of surgical approach. Thoracic surgeons should understand the properties of each approach and master them for complete resection avoiding serious complications.

De novo synthesis of sphingolipids is required for cell survival by down-regulating c-Jun N-terminal kinase in Drosophila imaginal discs.

Mitogen-activated protein kinase (MAPK) is a conserved eukaryotic signaling factor that mediates various signals, cumulating in the activation of transcription factors. Extracellular signal-regulated kinase (ERK), a MAPK, is activated through phosphorylation by the kinase MAPK/ERK kinase (MEK). To elucidate the extent of the involvement of ERK in various aspects of animal development, we searched for a Drosophila mutant which responds to elevated MEK activity and herein identified a lace mutant. Mutants with mild lace alleles grow to become adults with multiple aberrant morphologies in the appendages, compound eye, and bristles. These aberrations were suppressed by elevated MEK activity. Structural and transgenic analyses of the lace cDNA have revealed that the lace gene product is a membrane protein similar to the yeast protein LCB2, a subunit of serine palmitoyltransferase (SPT), which catalyzes the first step of sphingolipid biosynthesis. In fact, SPT activity in the fly expressing epitope-tagged Lace was absorbed by epitope-specific antibody. The number of dead cells in various imaginal discs of a lace hypomorph was considerably increased, thereby ectopically activating c-Jun N-terminal kinase (JNK), another MAPK. These results account for the adult phenotypes of the lace mutant and suppression of the phenotypes by elevated MEK activity: we hypothesize that mutation of lace causes decreased de novo synthesis of sphingolipid metabolites, some of which are signaling molecules, and one or more of these changes activates JNK to elicit apoptosis. The ERK pathway may be antagonistic to the JNK pathway in the control of cell survival.

Molecular Characterization and Diagnosis of QoI Resistance in Cucumber and Eggplant Fungal Pathogens.

ABSTRACT The molecular mechanism of QoI fungicide resistance was studied using isolates of cucumber Corynespora leaf spot fungus (Corynespora cassiicola) and the eggplant leaf mold (Mycovellosiella nattrassii). In both pathogens, a mutation at position 143 from glycine to alanine (G143A) was detected in the cytochrome b gene that encodes for the fungicide-targeted protein. Moreover, the nucleotide sequence at amino acid position 143 was converted from GGT or GGA in sensitive (wild-type) to GCT or GCA in resistant (mutant-type) isolates. The methods of polymerase chain reaction restriction fragment length polymorphism commonly used for QoI resistance monitoring were employed successfully, leading to the amplified gene fragment from resistant isolates being cut with the restriction enzyme ItaI. However, heteroplasmy (the coexistence of wild-type and mutated alleles) was found when the resistant isolates of C. cassiicola, M. nattrassii, and Colletotrichum gloeosporioides (strawberry anthracnose fungus) were subcultured in the presence or absence of QoI fungicides. QoI resistance of cucumber powdery and downy mildew isolates persisted for a few years following the removal of the selection pressure imposed by the fungicide under both laboratory and commercial greenhouse conditions. The proportion of mutated sequences in cytochrome b gene decreased over time in the pathogen population. The protective efficacy of the full dose of azoxystrobin decreased when the populations of powdery and downy mildews contained resistant isolates at 10%. Using FMBIO, a fluorescence bio-imaging analyzer, the mutant allele from the QoI-resistant isolates could be detected at the level of 1%, whereas the detection sensitivity of ethidium-bromide-stained gels was approximately 10 times lower.