Maintenance of cardiorespiratory fitness, body composition, and a physically active lifestyle after structured exercise interventions in individuals with overweight and obesity: A mixed-method follow-up study.

Objectives: The aim of this mixed-method study was to explore maintenance of physical activity and health effects one year after completion of exercise interventions in transport and leisure-time domains of everyday life. We hypothesised that routinisation of active commuting would lead to better maintenance of physical activity and health effects compared with leisure-time exercise. Study design: Mixed-methods follow-up study. Methods: Individuals with overweight/obesity, who completed a 6-month exercise intervention (active commuting by bike (BIKE), moderate (MOD) or vigorous intensity leisure-time exercise (VIG)), were after one year invited to participate in a follow-up visit which included measurements of cardiorespiratory fitness during an incremental bicycle test and body composition using dual-energy X-ray absorptiometry. Variability in maintenance practices was assessed in a sub-sample of participants who experienced the greatest improvements ('VO2peak improvers') and reductions ('VO2peak reducers'), respectively, in cardiorespiratory fitness. Semi-structured interviews were conducted (15-30 min) and analysed using systematic text condensation to identify barriers and facilitators associated with maintenance of physical activity. Results: Out of the 74 participants completing an exercise intervention, 46 (62%) completed follow-up (BIKE: n = 14; MOD: n = 14; VIG: n = 18). Improvements in VO2peak and reductions in fat mass were maintained in BIKE and VIG. Body weight decreased in BIKE and fat free mass increased in VIG. Changes in VO2peak and anthropometry at follow-up did not differ between BIKE and MOD + VIG. Fat mass decreased and recreational physical activity increased in 'VO2peak improvers'. Findings from the interviews suggested that self-monitoring, collective exercising, and new personal exercise challenges facilitate maintenance of a physically active lifestyle. Conclusion: Completion of a structured exercise intervention consisting of 6 months of active commuting or vigorous intensity leisure-time exercise was associated with long-term maintenance of improvements in VO2peak and body composition, whereas moderate intensity leisure-time exercise was not. In contrast to our hypothesis, active commuting was not associated with better maintenance of physical activity and health effects after the intervention compared with leisure-time exercise.

Assessment of structural chromosomal instability phenotypes as biomarkers of carboplatin response in triple negative breast cancer: the TNT trial.

BACKGROUND: In the TNT trial of triple negative breast cancer (NCT00532727), germline BRCA1/2 mutations were present in 28% of carboplatin responders. We assessed quantitative measures of structural chromosomal instability (CIN) to identify a wider patient subgroup within TNT with preferential benefit from carboplatin over docetaxel. PATIENTS AND METHODS: Copy number aberrations (CNAs) were established from 135 formalin-fixed paraffin-embedded primary carcinomas using Illumina OmniExpress SNP-arrays. Seven published [allelic imbalanced CNA (AiCNA); allelic balanced CNA (AbCNA); copy number neutral loss of heterozygosity (CnLOH); number of telomeric allelic imbalances (NtAI); BRCA1-like status; percentage of genome altered (PGA); homologous recombination deficiency (HRD) scores] and two novel [Shannon diversity index (SI); high-level amplifications (HLAMP)] CIN-measurements were derived. HLAMP was defined based on the presence of at least one of the top 5% amplified cytobands located on 1q, 8q and 10p. Continuous CIN-measurements were divided into tertiles. All nine CIN-measurements were used to analyse objective response rate (ORR) and progression-free survival (PFS). RESULTS: Patients with tumours without HLAMP had a numerically higher ORR and significantly longer PFS in the carboplatin (C) than in the docetaxel (D) arm [56% (C) versus 29% (D), PHLAMP,quiet = 0.085; PFS 6.1 months (C) versus 4.1 months (D), Pinteraction/HLAMP = 0.047]. In the carboplatin arm, patients with tumours showing intermediate telomeric NtAI and AiCNA had higher ORR [54% (C) versus 20% (D), PNtAI,intermediate = 0.03; 62% (C) versus 33% (D), PAiCNA,intermediate = 0.076]. Patients with high AiCNA and PGA had shorter PFS in the carboplatin arm [3.4 months (high) versus 5.7 months (low/intermediate); and 3.8 months (high) versus 5.6 months (low/intermediate), respectively; Pinteraction/AiCNA = 0.027, Padj.interaction/AiCNA = 0.125 and Pinteraction/PGA = 0.053, Padj.interaction/PGA = 0.176], whilst no difference was observed in the docetaxel arm. CONCLUSIONS: Patients with tumours lacking HLAMP and demonstrating intermediate CIN-measurements formed a subgroup benefitting from carboplatin relative to docetaxel treatment within the TNT trial. This suggests a complex and paradoxical relationship between the extent of genomic instability in primary tumours and treatment response in the metastatic setting.

Prospective association between late evening food consumption and risk of prediabetes and diabetes: the Whitehall II cohort study.

AIMS: We examined whether late evening food consumption was prospectively associated with the risk of developing prediabetes or diabetes in a large observational study of individuals with normoglycaemia. METHODS: Participants were 2642 men and women with normoglycaemia (HbA1c < 39 mmol/mol; < 5.7%) from the Whitehall II study. Time of last eating episode (TLEE) before the examination day was assessed at baseline. We studied the associations of TLEE with 5-year changes in HbA1c and risk of developing prediabetes or diabetes (HbA1c ≥ 39 mmol/mol; ≥ 5.7%). Potential heterogeneity in the association between TLEE and prediabetes or diabetes was examined using recursive partitioning modelling for time-to-event outcomes. RESULTS: There was a tendency of an overall association of TLEE with change in HbA1c but with little effect size [ß per 1-h increase in TLEE = 0.2 mmol/mol, 95% CI -0.0 to 0.3 (0.01%, -0.00 to 0.03); P = 0.055] and no association with the risk of developing prediabetes/diabetes (risk ratio per 1-h increase in TLEE = 1.03, 95% CI 0.94 to 1.13; P = 0.511). According to the recursive partitioning modelling, women with HbA1c ≤ 36 mmol/mol and TLEE after 21:00 had a 1.51 times (95% CI 1.16 to 1.93) higher 5-year risk of developing prediabetes or diabetes than those having their TLEE between 16:00 and 21:00 (35.4% vs. 23.5%; P = 0.003). CONCLUSIONS: There was no overall association of TLEE with the development of prediabetes or diabetes in the Whitehall II population. However, explorative analyses suggested that eating late in the evening was associated with increased risk of developing prediabetes/diabetes among women with good glycaemic control. Whether restricting late evening food consumption is effective and feasible for the prevention of Type 2 diabetes needs testing in randomized controlled trials.

Effects of active commuting and leisure-time exercise on fat loss in women and men with overweight and obesity: a randomized controlled trial.

BACKGROUND: Aerobic exercise is recommended for weight management but energy balance is often less negative than predicted from exercise energy expenditure (ExEE). OBJECTIVE: To examine effects of active commuting and leisure-time exercise on fat loss in women and men with overweight and obesity. METHODS: We randomized 130 younger, physically inactive women and men with overweight and obesity (body mass index: 25-35 kg m-2) to 6 months of habitual lifestyle (control; CON, n=18), active commuting (BIKE, n=35) or leisure-time exercise of moderate (MOD, 50% VO2peak reserve, n=39) or vigorous intensity (VIG, 70% VO2peak reserve, n=38). The primary outcome was change in fat mass measured by dual-energy X-ray absorptiometry, which was analyzed intention-to-treat. Accumulated energy balance was calculated based on changes in body composition, and ExEE was calculated based on heart rate monitoring during exercise. RESULTS: Testing at 3 and 6 months was completed by 95 and 90 participants, respectively. Fat mass was reduced after 3 and 6 months in BIKE (3 months: -3.6 (-5.5; -1.7) kg (mean (95% CI)); 6 months: -4.2 (-6.6; -1.9) kg; both: P<0.001), MOD (3 months: -2.2 (-3.9; -0.4) kg; 6 months: -2.6 (-4.8; -0.5) kg, both: P<0.02) and VIG (3 months: -3.4 (-5.2; -1.7) kg; 6 months: -4.5 (-6.6; -2.3) kg; both: P<0.001) compared with CON. Furthermore, fat loss was greater in VIG compared with MOD (6 months: -1.8 (-3.6; -0.1) kg, P=0.043). Based on the ExEE and the accumulated energy balance MOD compensated for the ExEE (77 (48; 106) %) but not BIKE (38 (-18; 95) %) and VIG (21 (-14; 55) %). CONCLUSIONS: A meaningful fat loss was obtained by 6 months of active commuting and leisure-time exercise, but fat loss was greater with vigorous compared with moderate intensity exercise. Active commuting is an alternative to leisure-time exercise in the management of overweight and obesity. The trial was registered at clinicaltrials.gov as NCT01962259 (main trial) and NCT01973686 (energy metabolism sub-study).

Dietary shifts have consequences for the repertoire of allergens produced by the European house dust mite.

Products manufactured from mass-cultured house dust mites, currently commercialized for the diagnosis and immunotherapy of allergy, are heterogeneous in terms of allergen composition and thus present concerns to regulatory authorities. The most abundant species, Dermatophagoides pteronyssinus (Trouessart) (Astigmata: Pyroglyphidae), produces 19 allergenic proteins. Many of these are putatively involved in mite digestive physiology and metabolism. This study aimed to evaluate the effects of mite-rearing media on allergen production. Mites were adapted to feed on culture media supplemented with proteins, lipids, carbohydrates or beard shavings, and collected to quantify major allergens (Der p 1 and 2) by immunodetection, transcription of allergen genes by real-time quantitative polymerase chain reaction, and allergen-related enzymatic activities. All culture media significantly affected the content of major allergens. Modification of macronutrients in the diet produced minor effects on the transcription of allergen genes, but significantly altered mite allergen-related activities. The most remarkable impacts were detected in mites feeding on beard shavings and were reflected in reductions in the content of major allergens, alterations in the transcription of nine allergen genes, and changes in eight allergen-related activities. These results demonstrate the importance of culture media to the quality and consistency of mite extracts used for pharmaceuticals, and highlight the need to further elucidate allergen production by mites in the laboratory and in domestic environments.

Functional redundancy between Apc and Apc2 regulates tissue homeostasis and prevents tumorigenesis in murine mammary epithelium.

Aberrant Wnt signaling within breast cancer is associated with poor prognosis, but regulation of this pathway in breast tissue remains poorly understood and the consequences of immediate or long-term dysregulation remain elusive. The exact contribution of the Wnt-regulating proteins adenomatous polyposis coli (APC) and APC2 in the pathogenesis of human breast cancer are ill-defined, but our analysis of publically available array data sets indicates that tumors with concomitant low expression of both proteins occurs more frequently in the 'triple negative' phenotype, which is a subtype of breast cancer with particularly poor prognosis. We have used mouse transgenics to delete Apc and/or Apc2 from mouse mammary epithelium to elucidate the significance of these proteins in mammary homeostasis and delineate their influences on Wnt signaling and tumorigenesis. Loss of either protein alone failed to affect Wnt signaling levels or tissue homeostasis. Strikingly, concomitant loss led to local disruption of ß-catenin status, disruption in epithelial integrity, cohesion and polarity, increased cell division and a distinctive form of ductal hyperplasia with 'squamoid' ghost cell nodules in young animals. Upon aging, the development of Wnt activated mammary carcinomas with squamous differentiation was accompanied by a significantly reduced survival. This novel Wnt-driven mammary tumor model highlights the importance of functional redundancies existing between the Apc proteins both in normal homeostasis and in tumorigenesis.

Effects of domestic chemical stressors on expression of allergen genes in the European house dust mite.

The expression of allergen genes in house dust mites is influenced by temperature and relative humidity, but little is known of the impacts of other environmental factors that may alter the repertoire of allergens released by mites in home microhabitats. Bioassays were conducted in concave microscope slides in combination with real-time quantitative polymerase chain reaction (RT-qPCR) to analyse gene expression of 17 allergens of Dermatophagoides pteronyssinus (Acariformes: Pyroglyphidae) exposed to three chemical stressors that can be present in domestic environments. Short-term exposure (5-12 days) to diesel exhaust particles (DEPs) (1 µg/cm2 ), bacterial lipopolysaccharide endotoxin (0.1 µg/cm2 ) and benzyl benzoate (3.2 µg/cm2 ), at concentrations exceeding those expected in homes, had no significant effect on allergen transcription. A significant increase in the transcription of allergens Der p 3, Der p 8 and Der p 21 was observed only after exposing mites to a higher concentration of DEPs (10 µg/cm2 ) over a whole generation. In combination, the present results suggest that the analysed factors have low impact on allergen production. The methodology described here offers a sound and rapid approach to the broad-spectrum study of factors affecting allergen-related mite physiology, and allows the simultaneous screening of different factors in a relatively short period with consideration of the full spectrum of allergen genes.

Quality control of house dust mite extracts by broad-spectrum profiling of allergen-related enzymatic activities.

BACKGROUND: Diagnosis and immunotherapy of allergy against mites is based on complex extracts from large-scale cultures. However, the analysis of their composition using specific antibodies is limited. By taking advantage of the prevailing enzymatic nature of mite allergens, we have developed a broad-spectrum biochemical method for the standardization of native mite products. METHODS: Microplate-based assays have been implemented for thirteen Dermatophagoides pteronyssinus enzymatic activities, associated with Der p 1, 3, 4, 6, 8, 9, 15 and 20 allergens. The dynamics of these activities along culture growth, and their profile in purified fractions (bodies and faeces) and international reference standards (WHO/IUIS, two CBER/FDA), have been characterized. The stability of enzymatic activities and major allergens under stress conditions (40°C) has been assessed in the presence/absence of specific protease inhibitors. RESULTS: The analysis of enzymatic activities revealed distinct profiles along culture growth and between fractions (bodies and faeces). Remarkable differences were found when comparing international reference standards, being consistent with their source material (purified bodies or whole cultures). After 72 h at 40°C, only trypsin and alpha-amylase maintained high activity. Notably, the prominent role of trypsins in the hydrolytic degradation of major allergens is demonstrated by the use of inhibitors. CONCLUSIONS: Our method offers a robust approach to assess the complexity of mite extracts and highlights the critical importance of source materials for the composition and stability of finished products. The implementation of this approach in industry-based quality control procedures would contribute to the standardization of allergenic extracts used for diagnosis and immunotherapy.

Pharmacokinetic profile of methotrexate in psoriatic skin via the oral or subcutaneous route using dermal microdialysis showing higher methotrexate bioavailability in psoriasis plaques than in non-lesional skin.

AIMS: The aim of this pilot study was to use microdialysis to evaluate levels of Methotrexate (MTX) directly in psoriatic skin following oral or subcutaneous administration of MTX to elaborate a complete pharmacokinetic profile within the dermal skin. METHODS: Six patients with chronic plaque psoriasis on the arm undergoing treatment with MTX were included in a mono-centre clinical trial. Patients were under treatment with p.o. or s.c. MTX (7.5 and 15 mg) for at least 3 months. Interstitial fluid was collected ex vivo via dermal microdialysis from lesional or non-lesional skin and via intravenous microdialysis as well as blood serum every hour up to 10 h after methotrexate administration every hour. MTX was analysed via liquid chromatography. RESULTS: The area under the curve (AUC) of methotrexate from peripheral blood was up to four times higher than from microdiaylsis, which detection of free unbound MTX. The AUC from dialysates in psoriatic lesional skin was higher than in non-lesional psoriatic skin, and the AUC levels from i.v. microdialysis were non-significantly higher than those from lesional psoriatic skin. Pharmacokinetic profiles were individually quite different and did not primarily depend on the dose or the means (p.o. vs. s.c.) in which it was administered. CONCLUSION: Dermal microdialysis is a valid tool to evaluate levels of methotrexate in the skin of psoriasis patients. Drug levels and bioavailability of methotrexate were higher in lesional than non-lesional psoriatic skin. The individual AUC of MTX was not primarily dependent on the route or dose of administration.

Allergen expression in the European house dust mite Dermatophagoides pteronyssinus throughout development and response to environmental conditions.

House dust mites are a major source of allergy worldwide. While diagnosis and treatment based on mite extracts have remarkably advanced, little information exists on the expression of allergens in mites. We have studied gene expression of eight Dermatophagoides pteronyssinus (Trouessart) (Acari: Pyroglyphidae) allergens (Der p 1, 2, 3, 4, 5, 7, 10 and 21). All allergens showed higher transcription in nymphs compared with larvae or adults, with the only exception of Der p 10. The transcription of Der p 4 and Der p 10, together with the transcription and protein ratios Der p 1 to Der p 2, were higher in males than in females. One-week exposure of mite cultures to 16 or 35 °C (versus 24 °C) or low RH (44% versus 76%) significantly influenced the allergen gene transcription profile. Our results demonstrate that allergen expression is quantitatively and/or qualitatively influenced by mite development and sex, as well as by the environment. We suggest that monitoring allergen gene expression may be a useful tool to assist the optimization of mite cultures in the production of standardized allergenic extracts for clinical use.

Change in sleep duration and proposed dietary risk factors for obesity in Danish school children.

BACKGROUND: Recent cross-sectional studies found higher consumption of energy-dense foods among children with short sleep duration; however, longitudinal studies examining changes in sleep and diet over time are needed. OBJECTIVE: This study aimed to investigate prospective associations between changes in objectively measured sleep duration and alterations in proposed dietary risk factors for obesity in 8-11-year-old Danish children. METHODS: Four hundred forty-one children recorded dietary intake during seven consecutive days, along with accelerometer measurements estimating sleep duration at baseline and after ∼200 days. RESULTS: Baseline sleep duration did not predict changes in dietary intake or vice versa (all P ≥ 0.69). However, 1-h lower sleep duration was associated with higher intake of added sugar (1.59 E%; P = 0.001) and sugar-sweetened beverages (0.90 E%; P = 0.002) after 200 days with no change in energy density of the diet (P = 0.78). CONCLUSION: Our results suggest that a negative change in sleep duration is associated with higher intakes of sugar containing foods/beverages.

Arabidopsis thaliana and Pisum sativum models demonstrate that root colonization is an intrinsic trait of Burkholderia cepacia complex bacteria.

Burkholderia cepacia complex (Bcc) bacteria possess biotechnologically useful properties that contrast with their opportunistic pathogenicity. The rhizosphere fitness of Bcc bacteria is central to their biocontrol and bioremediation activities. However, it is not known whether this differs between species or between environmental and clinical strains. We investigated the ability of 26 Bcc strains representing nine different species to colonize the roots of Arabidopsis thaliana and Pisum sativum (pea). Viable counts, scanning electron microscopy and bioluminescence imaging were used to assess root colonization, with Bcc bacteria achieving mean (±sem) levels of 2.49±0.23×10(6) and 5.16±1.87×10(6) c.f.u. per centimetre of root on the A. thaliana and P. sativum models, respectively. The A. thaliana rhizocompetence model was able to reveal loss of colonization phenotypes in Burkholderia vietnamiensis G4 transposon mutants that had only previously been observed in competition experiments on the P. sativum model. Different Bcc species colonized each plant model at different rates, and no statistical difference in root colonization was observed between isolates of clinical or environmental origin. Loss of the virulence-associated third chromosomal replicon (>1 Mb DNA) did not alter Bcc root colonization on A. thaliana. In summary, Bcc bacteria possess intrinsic root colonization abilities irrespective of their species or source. As Bcc rhizocompetence does not require their third chromosomal replicon, the possibility of using synthetic biology approaches to engineer virulence-attenuated biotechnological strains is tractable.

Bacillus thuringiensis colonises plant roots in a phylogeny-dependent manner.

Although much is known about the pathology of Bacillus thuringiensis against invertebrates, current understanding of its natural ecology is limited. This study evaluated the biodiversity of B. thuringiensis in relation to its interaction with plants. Phylogenetic relationships between 44 reference and field-collected strains, determined using 16S rRNA and gyrB gene sequences, revealed a high degree of variability, similar to that found in databases. An Arabidopsis thaliana in vitro inoculation model was developed to screen the ability of B. thuringiensis to colonise roots. Significant colonisation differences up to 91-fold were observed between strains, and correlation between strain phylogeny and colonisation was found. The genetics and biochemistry of auxin production; presence of the gene encoding indole pyruvate decarboxylase; and the abilities of Bt strains to swarm, grow in rich/minimal media and affect root growth differed between the strains, but only auxin production correlated significantly with ability to colonise roots. Co-inoculation with Burkholderia phytofirmans PsJN or Pseudomonas fluorescens SBW25 produced no effect on B. thuringiensis colonisation levels, regardless of the co-inoculant. Similarly, root colonisation of A. thaliana mutants impaired in plant defences was not significantly higher compared with controls. This is the first systematic and phylogenetic evaluation of B. thuringiensis interaction with plants.

Occurrence, characterization and insecticidal activity of Bacillus thuringiensis strains isolated from argan fields in Morocco.

Soils collected from five locations in the argan forest (an endemic plant) in Morocco were used to form the first collection of Bacillus thuringiensis (Bt) strains from this area (58 strains). Here we found that the argan forest is a major source of Bt, as 90.62% of the samples contained Bt strains. These strains produced mainly spherical or irregular crystals that in some cases remained adhered to the spore after cell lysis. There was no strain producing bipyramidal crystals, suggesting the absence of strains bearing crv1 genes. This was confirmed by PCR analysis using eight primer pairs that can potentially detect 13 different groups of cry and cyt genes. Strains containing cry7/8 were the most abundant (25.53%), followed by strains harbouring cry9A (14.89%), cry11 (8.51%) and cry4 (4.25%). The mixtures of spores and crystals as well as culture supernatants were assayed for toxicity towards Ceratitis capitata (Medfly), showing up to 30% mortality. Our findings suggest that the argan region is a suitable target for future and wider screening programmes looking for strains bearing toxins or combinations of them to develop more efficient Bt-based formulates.

Cyt1Aa protein from Bacillus thuringiensis (Berliner) serovar israelensis is active against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann).

BACKGROUND: Ceratitis capitata (Wiedemann) is one of the world's most destructive fruit pests. The aim of this study was to ascertain insecticidal activity of Bacillus thuringiensis (Berliner) delta-endotoxins to C. capitata. RESULTS: Among 42 selected Bacillus strains, only B. thuringiensis serovar israelensis (Bti) solubilised protoxins showed biological activity against C. capitata neonate larvae, whereas Bti spore and crystal mixture was inactive. Insecticidal activity of Bti protoxins was significantly enhanced by incubation with Culex pipiens L. gut extracts. Overdigestion of Bti protoxins with Sesamia nonagrioides (Lefebvre) gut extracts suppressed biological activity against C. capitata, and this correlated with degradation of Cyt toxins. Cyt1Aa solubilised protoxin showed the highest toxicity, LC(50) after 7 days of 4.93 microg cm(-2), while proteolytical processing of Cyt1Aa protoxins by larval gut extracts did not enhance insecticidal activity. CONCLUSION: The present study provides evidence for the first time of the insecticidal activity of a B. thuringiensis strain against C. capitata and identifies a single delta-endotoxin with potential for controlling this pest.

Diversity of Bacillus thuringiensis strains isolated from citrus orchards in spain and evaluation of their insecticidal activity against Ceratitis capitata.

A survey of Bacillus thuringiensis (Berliner) strains isolated from Spanish citrus orchards has been performed, and the strains were tested for insecticidal activity against the Mediterranean fruit fly Ceratitis capitata (Wiedemann), a key citrus pest in Spain. From a total of 150 environmental samples, 376 isolates were selected, recording a total B. thuringiensis index of 0.52. The collection was characterized by means of phase-contrast microscopy, SDS-PAGE, and PCR analysis with primer pairs detecting toxin genes cry1, cry2, cry3, cry4, cry5, cry7, cry8, cry9, cry10, cry11, cry12, cry14, cry17, cry19, cry21, cry27, cry39, cry44, cyt1, and cyt2. Diverse crystal inclusion morphologies were identified: bipyramidal (45%), round (40%), adhered to the spore (7%), small (5%), and irregular (3%). SDSPAGE of spore-crystal preparations revealed 39 different electrophoresis patterns. All primer pairs used in PCR tests gave positive amplifications in strains of our collection, except for primers for detection of cry3, cry19, cry39, or cry44 genes. Strains containing cry1, cry2, cry4, and cry27 genes were the most abundant (48.7%, 46%, 11.2%, and 8.2% of the strains, respectively). Ten different genetic profiles were found, although a total of 109 strains did not amplify with the set of primers used. Screening for toxicity against C. capitata adults was performed using both spore-crystal and soluble fractions. Mortality levels were less than 30%. We have developed a large and diverse B. thuringiensis strain collection with huge potential to control several agricultural pests; however, further research is needed to find out Bt strains active against C. capitata.

Simple and rapid method for PCR characterization of large Bacillus thuringiensis strain collections.

Polymerase chain reaction (PCR)-based identification of Bacillus thuringiensis toxin genes has become a routine step in most B. thuringiensis isolation and characterization initiatives. In the present study we propose a simplified method for extensive PCR analysis of B. thuringiensis cry and cyt genes of particular interest in large-scale screening programs. Fifty B. thuringiensis strains were screened for the presence of genes of the cry1 subfamily. Identical results were obtained when our method was used in comparison to other methodology based on a standard alkaline lysis preparation of plasmid DNA. Additional tests evidenced the fitness of our method in a particular multiplex-PCR analysis. The main advantages of the proposed methodology are that it is simpler and quicker than commonly used protocols (as DNA template preparation is substituted by direct addition of small amounts of B. thuringiensis liquid cell suspension to the reaction mixture) and that it allows simultaneous handling (bacterial growth and PCR) of up to 96 strains per round.