Longitudinal profiling identifies co-occurring BRCA1/2 reversions, TP53BP1, RIF1 and PAXIP1 mutations in PARP inhibitor-resistant advanced breast cancer.

BACKGROUND: Resistance to therapies that target homologous recombination deficiency (HRD) in breast cancer limits their overall effectiveness. Multiple, preclinically validated, mechanisms of resistance have been proposed, but their existence and relative frequency in clinical disease are unclear, as is how to target resistance. PATIENTS AND METHODS: Longitudinal mutation and methylation profiling of circulating tumour (ct)DNA was carried out in 47 patients with metastatic BRCA1-, BRCA2- or PALB2-mutant breast cancer treated with HRD-targeted therapy who developed progressive disease-18 patients had primary resistance and 29 exhibited response followed by resistance. ctDNA isolated at multiple time points in the patient treatment course (before, on-treatment and at progression) was sequenced using a novel >750-gene intron/exon targeted sequencing panel. Where available, matched tumour biopsies were whole exome and RNA sequenced and also used to assess nuclear RAD51. RESULTS: BRCA1/2 reversion mutations were present in 60% of patients and were the most prevalent form of resistance. In 10 cases, reversions were detected in ctDNA before clinical progression. Two new reversion-based mechanisms were identified: (i) intragenic BRCA1/2 deletions with intronic breakpoints; and (ii) intragenic BRCA1/2 secondary mutations that formed novel splice acceptor sites, the latter being confirmed by in vitro minigene reporter assays. When seen before commencing subsequent treatment, reversions were associated with significantly shorter time to progression. Tumours with reversions retained HRD mutational signatures but had functional homologous recombination based on RAD51 status. Although less frequent than reversions, nonreversion mechanisms [loss-of-function (LoF) mutations in TP53BP1, RIF1 or PAXIP1] were evident in patients with acquired resistance and occasionally coexisted with reversions, challenging the notion that singular resistance mechanisms emerge in each patient. CONCLUSIONS: These observations map the prevalence of candidate drivers of resistance across time in a clinical setting, information with implications for clinical management and trial design in HRD breast cancers.

Tolerance's improvement of flexible bronchoscopy by Ericksonian hypnosis: The BREATH study.

AIM: The aim of this study was to determine whether the use of Ericksonian hypnosis may allow an improvement of the tolerance of flexible bronchoscopy. METHODS: A comparative, two parallel-group, prospective, randomized monocentric clinical trial was conducted. After randomization, patients were divided into two groups: a standard group, in which bronchoscopy was performed according to the official French good practice guidelines and a study group, in which bronchoscopy was performed under hypnosis. RESULTS: Sixty-seven patients were included, 7 patients were excluded and 60 patients were randomized. No significant differences in age, gender, examination indication and duration were observed between both groups. Two patients of the standard group removed the endoscope by themselves, resulting in a premature termination of bronchoscopy and they were excluded from the statistical analysis. In the standard group, the levels of anxiety, cough, dyspnoea and pain increased during the examination and the addition of local anaesthesia was more often required. In the hypnosis group, levels of anxiety, cough, dyspnoea decreased, whereas only the level of pain increased. There was a statistic significative difference in favour of hypnosis for all the other variables. Moreover, the behaviour score was higher in the standard group: 19.5±14.5 versus 7.3±4.7 (P<0.001), indicating a better tolerance in the hypnosis group. In the standard group, 14 patients refused a new examination under the same conditions versus 7 in the hypnosis group, and 12 patients asked for general anaesthesia in case of a new examination versus 7 in the hypnosis group. CONCLUSION: This randomised control trial is the first to test the faisability and the potential usefulness of Ericksonian hypnosis during flexible bronchoscopy. Our results indicates an improvement of tolerance and a positive effect on all studied parameters except pain. This method could be widely offered to all patients undergoing flexible bronchoscopy.

Comparative assessment of next-generation sequencing, denaturing gradient gel electrophoresis, clonal restriction fragment length polymorphism and cloning-sequencing as methods for characterizing commercial microbial consortia.

Characterization of commercial microbial consortia products for human and environmental health risk assessment is a major challenge for regulatory agencies. As a means to develop an approach to assess the potential environmental risk of these products, research was conducted to compare four genomics methods for characterizing bacterial communities; (i) Denaturing Gradient Gel Electrophoresis (DGGE), (ii) Clonal-Restriction Fragment Length Polymorphism (C/RFLP), (iii) partial 16S rDNA amplification, cloning followed by Sanger sequencing (PRACS) and (iv) Next-Generation Sequencing (NGS) based on Ion Torrent technology. A commercially available microbial consortium, marketed as a remediation agent for degrading petroleum hydrocarbon contamination in soil and water, was assessed. The bacterial composition of the commercial microbial product was characterized using the above four methods. PCR amplification of 16S rDNA was performed targeting the variable region V6 for DGGE, C/RFLP and PRACS and V5 for Ion Torrent sequencing. Ion Torrent technology was shown to be a promising tool for initial screening by detecting the majority of bacteria in the consortium that were also detected by DGGE, C/RFLP and PRACS. Additionally, Ion Torrent sequencing detected some of the bacteria that were claimed to be in the product, while three other methods failed to detect these specific bacteria. However, the relative proportions of the microbial composition detected by Ion Torrent were found to be different from DGGE, C/RFLP and PRACS, which gave comparable results across these three methods. The discrepancy of the Ion Torrent results may be due to the short read length generated by this technique and the targeting of different variable regions on the 16S rRNA gene used in this study. Arcobacter spp. a potential pathogenic bacteria was detected in the product by all methods, which was further confirmed using genus and species-specific PCR, RFLP and DNA-based sequence analyses. However, the viability of Arcobacter spp. was not confirmed. This study suggests that a combination of two or more methods may be required to ascertain the microbial constituents of a commercial microbial consortium reliably and for the presence of potentially human pathogenic contaminants.

[Possible responsibility of EDNRA gene triplication, coding for the endothelin 1 ET-A receptor in a case of congenital diaphragmatic hernia]. / Implication potentielle de la triplication du gène EDNRA codant pour le récepteur ET-A de l'endothéline 1 chez un fœtus porteur de hernie de coupole diaphragmatique.

Congenital diaphragmatic hernia has a physiopathology unfully understood, and is the cause of an important morbimortality. We report the case of a fetus suffering from a diaphragmatic hernia associated with a EDNRA gene triplication, coding for the endothelin 1 receptor. High-resolution genetic techniques were able to find the possible origin of this pathology, and showed that it was an isolated form with a good prognostic. ET-A receptor over-expression in lung vessels may cause a vascular remodeling and a lung arterial high blood pressure. This lung abnormality would have occurred before the diaphragmatic defect.

Quantum microbiology.

During his famous 1943 lecture series at Trinity College Dublin, the reknown physicist Erwin Schrodinger discussed the failure and challenges of interpreting life by classical physics alone and that a new approach, rooted in Quantum principles, must be involved. Quantum events are simply a level of organization below the molecular level. This includes the atomic and subatomic makeup of matter in microbial metabolism and structures, as well as the organic, genetic information code of DNA and RNA. Quantum events at this time do not elucidate, for example, how specific genetic instructions were first encoded in an organic genetic code in microbial cells capable of growth and division, and its subsequent evolution over 3.6 to 4 billion years. However, due to recent technological advances, biologists and physicists are starting to demonstrate linkages between various quantum principles like quantum tunneling, entanglement and coherence in biological processes illustrating that nature has exerted some level quantum control to optimize various processes in living organisms. In this article we explore the role of quantum events in microbial processes and endeavor to show that after nearly 67 years, Schrödinger was prophetic and visionary in his view of quantum theory and its connection with some of the fundamental mechanisms of life.

Development of a DNA microarray for enterococcal species, virulence, and antibiotic resistance gene determinations among isolates from poultry.

A DNA microarray (Enteroarray) was designed with probes targeting four species-specific taxonomic identifiers to discriminate among 18 different enterococcal species, while other probes were designed to identify 18 virulence factors and 174 antibiotic resistance genes. In total, 262 genes were utilized for rapid species identification of enterococcal isolates, while characterizing their virulence potential through the simultaneous identification of endogenous antibiotic resistance and virulence genes. Enterococcal isolates from broiler chicken farms were initially identified by using the API 20 Strep system, and the results were compared to those obtained with the taxonomic genes atpA, recA, pheS, and ddl represented on our microarray. Among the 171 isolates studied, five different enterococcal species were identified by using the API 20 Strep system: Enterococcus faecium, E. faecalis, E. durans, E. gallinarum, and E. avium. The Enteroarray detected the same species as API 20 Strep, as well as two more: E. casseliflavus and E. hirae. Species comparisons resulted in 15% (27 isolates) disagreement between the two methods among the five API 20 Strep identifiable species and 24% (42 isolates) disagreement when considering the seven Enteroarray identified species. The species specificity of key antibiotic and virulence genes identified by the Enteroarray were consistent with the literature adding further robustness to the redundant taxonomic probe data. Sequencing of the cpn60 gene further confirmed the complete accuracy of the microarray results. The new Enteroarray should prove to be a useful tool to accurately genotype strains of enterococci and assess their virulence potential.

How much cytoplasm can a bacterial genome control?

In this perspective we discuss that bacterial genomes have optimized during evolution to control a range of cytoplasm, from immediately after cell division to a maximum amount/volume present just prior to DNA replication and subsequent cell division. The genetic expansion of bacteria via evolution may be limited to a genome size:cytoplasm amount/volume ratios and energetics that have been selected for during 3.6-4 billion years of evolution on the Earth. The optimal genome size is one that is relatively constant, but also has some plasticity for evolutionary change (via gene transfer) and mutational events, and can control a range of cytoplasm during the cell cycle.

Use of a bacterial antimicrobial resistance gene microarray for the identification of resistant Staphylococcus aureus.

As diagnostic and surveillance activities are vital to determine measures needed to control antimicrobial resistance (AMR), new and rapid laboratory methods are necessary to facilitate this important effort. DNA microarray technology allows the detection of a large number of genes in a single reaction. This technology is simple, specific and high-throughput. We have developed a bacterial antimicrobial resistance gene DNA microarray that will allow rapid antimicrobial resistance gene screening for all Gram-positive and Gram-negative bacteria. A prototype microarray was designed using a 70-mer based oligonucleotide set targeting AMR genes of Gram-negative and Gram-positive bacteria. In the present version, the microarray consists of 182 oligonucleotides corresponding to 166 different acquired AMR gene targets, covering most of the resistance genes found in both Gram-negative and -positive bacteria. A test study was performed on a collection of Staphylococcus aureus isolates from milk samples from dairy farms in Québec, Canada. The reproducibility of the hybridizations was determined, and the microarray results were compared with those obtained by phenotypic resistance tests (either MIC or Kirby-Bauer). The microarray genotyping demonstrated a correlation between penicillin, tetracycline and erythromycin resistance phenotypes with the corresponding acquired resistance genes. The hybridizations showed that the 38 antimicrobial resistant S. aureus isolates possessed at least one AMR gene.

DNA technologies: what's next applied to microbiology research?

This perspective discusses current DNA technologies used in basic and applied microbiology research and speculates on possible new future technologies. DNA remains one of the most fascinating molecules known to humans and will continue to revolutionize many areas ranging from medicine, food and forensics to robotics and new industrial bioproducts/biofuel from waste materials. What's next with DNA is not always obvious, but history shows the international microbiology research community will readily adopt it.

Meal and snacking patterns of school-aged children in Scotland.

BACKGROUND: Current lifestyles and the choice and availability of foods may influence the eating patterns of children. The aim of this study was to investigate the meal and snacking patterns of school-aged children in Scotland. METHODS: A sub-sample of 156 children (5-17 years) from the national Survey of Sugar Intake among Children in Scotland completed a 4-day non-weighed diet diary. Meals and snacks were defined using a food-based classification system based on 'core' and 'non-core' foods. The first eating event containing a solid food item up to and including 0900 hours (1100 hours on weekend days) was defined as breakfast. Frequency of meal and snack consumption was compared between age, sex, body mass index (BMI) and socio-economic sub-groups, between term-time and school holidays and between weekdays and weekend days. Intakes of total fat, saturated fatty acids (SFA) and non-milk extrinsic sugars (NMES) on these days were also compared. RESULTS: Children ate a median of 3.3 meals plus 2.0 snacks per day, which did not vary between age and BMI groups. In all, 83% of children ate breakfast on all 4 days. Boys ate more meals than girls but the number of snacks was similar. Children from lower socio-economic groups tended to eat more meals and fewer snacks. Snacks accounted for 21% of the total daily energy intake, 22% of total fat, 24% of SFA and 39% of NMES intake. Daily intake of energy, total fat, SFA and NMES did not differ between term-time and holidays or weekdays and weekend days. CONCLUSIONS: Children tended to follow a traditional pattern of three meals a day, which was consistent between age and BMI subgroups and between school term-time and holidays.

Characterization of formulated microbial products by denaturing gradient gel electrophoresis, total cellular fatty acid analysis, and DNA microarray analysis.

Two commercial products, Biotize and Cycle, containing bacteria as an active ingredient were characterized for species identification and batch-to-batch variation by denaturing gradient gel electrophoresis (DGGE), total cellular fatty acid analysis (FAA), and a taxonomic DNA microarray. DGGE was useful at assessing the stability of consortia in different batches, and cluster analysis differentiated each batch even when only slight differences in species composition were observed. DGGE, FAA, and DNA microarray results indicated little batch-to-batch variation in Biotize and some batch variation in Cycle. The 3 methods agreed well with species identification in Biotize but generated conflicting results in the species composition of Cycle. This multi-method approach was useful in determining if the observed bacterial species present in the products matched the expected species composition.

Microarray analysis of Escherichia coli strains from interstitial beach waters of Lake Huron (Canada).

DNA microarray analyses revealed that clusters of repetitive extragenic palindromic PCR-related Escherichia coli isolates were isogenic only within interstitial Lake Huron beach water samples and not in surrounding waters. This suggested that adaptation and growth occurred within the interstitial water sites tested. All isolates were nonpathogenic, and three lake isolates possessed tetracycline resistance genes.

Lifestyle and socio-demographic factors associated with high-risk HPV infection in UK women.

The world age-standardised prevalence of high-risk HPV (hrHPV) infection among 5038 UK women aged 20-59 years, with a low-grade smear during 1999-2002, assessed for eligibility for TOMBOLA (Trial Of Management of Borderline and Other Low-grade Abnormal smears) was 34.2%. High-risk HPV prevalence decreased with increasing age, from 61% at ages 20-24 years to 14-15% in those over 50 years. The age-standardised prevalence was 15.1, 30.7 and 52.7%, respectively, in women with a current normal, borderline nuclear abnormalities (BNA) and mild smear. In overall multivariate analyses, tertiary education, previous pregnancy and childbirth were associated with reduced hrHPV infection risk. Risk of infection was increased in non-white women, women not married/cohabiting, hormonal contraceptives users and current smokers. In stratified analyses, current smear status and age remained associated with hrHPV infection. Data of this type are relevant to the debate on human papillomavirus (HPV) testing in screening and development of HPV vaccination programmes.

Altered gene expression in Choristoneura fumiferana and Manduca sexta in response to sublethal intoxication by Bacillus thuringiensis Cry1Ab toxin.

In order to understand how lepidopteran insects react physiologically to Bacillus thuringiensis crystal toxin ingestion, transcriptional profiling of Choristoneura fumiferana larvae exposed to sublethal doses of Cry1Ab protoxin were monitored using a C. fumiferana-specific cDNA microarray derived from a protoxin-specific subtractive library. Differential gene expression occurred primarily between 2 and 5 h postingestion. Metabolic enzymes such as lipases and proteases were generally repressed, whereas genes involved in detoxification, immune system regulation or general stress response were upregulated. A similar protoxin-specific transcriptional pattern was also observed with Manduca sexta larvae, using three upregulated genes (serpin, cytochrome P450 and carboxyl/cholinesterase) and one downregulated gene (beta-glucosidase), suggesting that a susceptible larval response to Cry toxin exposure might be universal among lepidopterous insects.

Development of a new integrated diagnostic test for identification and characterization of pathogens.

Animal diseases directly cause multi-million dollar losses world-wide. Therefore a rapid, highly specific, cost-effective diagnostic test for detecting a large set of bacterial virulence and antimicrobial resistance genes simultaneously is necessary. Hence, our group, the BCBG (Bacterial Chips Bacterial Genes) group, proposes developing a powerful molecular tool (DNA microarray) to detect a broad range of infectious agents, their endogenous main virulence factors and antibiotic resistance genes simultaneously. Effectively, a 70-mer oligonucleotide microarray capable of detecting the presence or absence of 169 Escherichia coli virulence genes or virulence marker genes as well as their variants, in addition to 30 principal antimicrobial resistance genes previously characterized in E. coli strains was developed by our group. This microarray was validated with a large collection of well characterized pathogenic and reference E. coli strains. Moreover, we are developing a new powerful clinical diagnostic microarray tool, to identify pathogenic bacteria of veterinary interest. The commercialization of this assay would allow same day diagnosis of infectious agents and their antibiotic resistance resulting in early treatment. In addition, this technology is also applicable to microbial quality control of food and water.

Transcriptional response of Choristoneura fumiferana to sublethal exposure of Cry1Ab protoxin from Bacillus thuringiensis.

Bacillus thuringiensis is a microbial control agent active against Choristoneura fumiferana, a lepidopteran defoliator of North American forests. Although the B. thuringiensis insecticidal crystal protoxins have a relatively narrow host range, there is concern about their impact on non-target species where intoxication effects may not be overt. Larval toxicity effects can be assessed at the molecular level by determining altered transcriptional profiles in response to sublethal protoxin exposure in sensitive insects. Subtraction hybridization libraries were created using two larval populations, control and protoxin-fed and were characterized by sequencing 1091 clones. Differential mRNA expression of selected clones, as measured by quantitative polymerase chain reaction, identified a number of metabolic and stress-related genes that were either transcriptionally enhanced or repressed after protoxin exposure.

Variation of milk citrate with stage of lactation and de novo fatty acid synthesis in dairy cows.

Citrate is a normal constituent of milk that affects milk-processing characteristics. It is an intermediate in the tricarboxylic acid cycle and plays an indirect role in fat synthesis by providing reducing equivalents in the form of NADPH. The objective of this study was to investigate variation in citrate with stage of lactation and de novo fatty acid synthesis, without confounding dietary effects. Twenty-four cows were fed the same diet, and milk citrate and fatty acids were determined over a 10-d period. Eight cows were in early lactation [13 +/- 1.8 d in milk (DIM; mean +/-standard error], 8 in midlactation (130 +/-4.6 DIM), and 8 in late lactation (283 +/-3.4 DIM). For cows in early, mid, and late lactation, milk yield was 34.4, 34.4, and 21.4 L/d [standard error of difference (SED) 1.78]; milk fat was 50.4, 40.3, and 41.4 g/L (3.68); milk citrate was 11.3, 9.7, and 10.1 mmol/L (0.64); the ratio of 4-14 C:18-20 C fatty acids was 0.9, 1.3, and 1.2 (0.07). Activity of the fatty acid synthase enzyme system (EC 2.3.1.85) was calculated as acetate used for chain elongation (ACE); ACE (mol/d) for cows in early, mid, and late lactation, was 7.3, 11.1, and 8.1 (SED 1.05). For individual cows, citrate (mmol/L) = 14.3 -0.44 xACE (r2 = 0.58). We propose that ACE provides a more accurate indication of synthase activity than do fatty acid ratios or yields. This study confirms the hypothesis that variation in milk citrate with stage of lactation is related to de novo synthesis of fatty acids and that the relationship is independent of diet and milk yield.

Psychological effects of a low-grade abnormal cervical smear test result: anxiety and associated factors.

Receipt of an abnormal cervical smear result often generates fear and confusion and can have a negative impact on a woman's well-being. Most previous studies have focussed on high-grade abnormal smears. This study describes the psychological and psychosocial effects, on women, of having received a low-grade abnormal smear result. Over 3500 women recruited to TOMBOLA (Trial Of Management of Borderline and Other Low-grade Abnormal smears) participated in this study. Anxiety was assessed using the Hospital Anxiety and Depression Scale (HADS) at recruitment. Socio-demographic and lifestyle factors, locus of control and factors associated with the psychosocial impact of the abnormal smear result were also assessed. Women reported anxiety levels consistent with those found in previous studies of women with high-grade smear results. Women at highest risk of anxiety were younger, had children, were current smokers, or had the highest levels of physical activity. Interventions that focus particularly on women's understanding of smear results and pre-cancer, and/or directly address their fears about cancer, treatment and fertility might provide the greatest opportunity to reduce the adverse psychosocial impact of receiving a low-grade abnormal cervical smear result.

Optimization of microbial DNA extraction and purification from raw wastewater samples for downstream pathogen detection by microarrays.

Numerous waterborne pathogens are difficult to detect and enumerate with accuracy due to methodological limitations and high costs of direct culturing. The purity of DNA extracted from wastewater samples is an important issue in the sensitivity and the usefulness of molecular methods such as polymerase chain reaction (PCR) and hybridizations on DNA microarrays. Ten different DNA extraction procedures, including physical and chemical extraction and purification steps, were examined to ascertain their relative effectiveness for extracting bacterial DNA from wastewater samples. The quality of the differentially extracted DNAs was subsequently assessed by PCR amplification and microarray hybridization. Our results showed that great differences existed among the ten procedures and only a few of the methods gave satisfactory results when applied to bacterial pathogens. This observation suggested that the extraction method needed to be carefully selected to produce significant and confident results in the detection of pathogens from environmental samples.

Cytochrome P-450 1A1 gene polymorphisms and risk of breast cancer: a HuGE review.

Cytochrome P-450 (CYP) 1A1 plays a key role in phase I metabolism of polycyclic aromatic hydrocarbons and in estrogen metabolism. It is expressed predominantly in extrahepatic tissues, including the breast. Four CYP1A1 gene polymorphisms (3801T --> C, Ile462Val, 3205T --> C, and Thr461Asp) have been studied in relation to breast cancer. The 3801C variant is more common than the Val variant. Both variants occur more frequently in Asians than in White populations. The 3205T --> C polymorphism has been observed in African Americans only. Little data are available on the geographic/ethnic distribution of the Thr461Asp polymorphism. The functional significance of the polymorphisms is unclear. In 17 studies, no consistent association between breast cancer and CYP1A1 genotype was found. Meta-analysis found no significant risk for the genotypes 1) 3801C/C (relative risk (RR) = 0.97, 95% confidence interval (CI): 0.52, 1.80) or 3801T/C (RR = 0.91, 95% CI: 0.70, 1.19) versus 3801T/T, 2) Val/Val (RR = 1.04, 95% CI: 0.63, 1.74) or Ile/Val (RR = 0.92, 95% CI: 0.76, 1.10) versus Ile/Ile, or 3) Asp/Asp (RR = 0.95, 95% CI: 0.20, 4.49) or Thr/Asp (RR = 1.12, 95% CI: 0.87, 1.43) versus Thr/Thr. Future studies should explore possible interactions between CYP1A1 and sources of polycyclic aromatic hydrocarbons, markers of estrogen exposure, other lifestyle factors influencing hormonal levels, and other genes involved in polycyclic aromatic hydrocarbon metabolism or hormonal biosynthesis.