Real-world analysis of clinical and economic impact of 21-gene recurrence score (RS) testing in early-stage breast cancer (ESBC) in Ireland.

PURPOSE: Results from TAILOR-X suggest that up to 70% of hormone receptor-positive (HR+) node-negative (N0) ESBC patients (pts) may avoid chemotherapy (CT) with RS ≤ 25. We assess clinical and economic impacts of RS testing on treatment using real-world data. METHODS: From October 2011 to February 2019, a retrospective, cross-sectional observational study was conducted of HR+ N0 ESBC pts who had RS testing in Ireland. Pts were classified low risk (RS ≤ 25) and high risk (RS > 25). Clinical risk was calculated. Data were collected via electronic patient records. Cost data were supplied by the National Healthcare Pricing Regulatory Authority. RESULTS: 963 pts. Mean age is 56 years. Mean tumour size is 1.7 cm. 114 (11.8%), 635 (66%), 211 (22%), 3 (0.2%) pts had G1, G2, G3 and unknown G, respectively. 796 pts (82.8%) low RS, 159 (16.5%) high RS and 8 pts (0.7%) unknown RS. 263 pts (26%) were aged ≤ 50 at diagnosis; 117 (45%) had RS 0-15, 63 (24.5%) 16-20, 39 (15.3%) 21-25 and 40 (15.2%) RS 26-100. 4 pts (1.5%) had unknown RS. Post-RS testing, 602 pts (62.5%) had a change in CT decision; 593 changed to hormone therapy (HT) alone. In total, 262 pts received CT. Of pts receiving CT; 138 (53%) had RS > 25, 124 (47%) had RS ≤ 25. Of pts aged ≤ 50, 153 (58%) had high clinical risk, of whom 28 had RS 16-20. Assay use achieved a 62.5% change in treatment with 73% of pts avoiding CT. This resulted in savings of €4 million in treatment costs. Deducting assay costs, savings of €1.9 million were achieved. CONCLUSION: Over the 8 years of the study, a 62.5% reduction in CT use was achieved with savings of over €1,900,000.

The MarR family regulator OsbR controls oxidative stress response, anaerobic nitrate respiration, and biofilm formation in Chromobacterium violaceum.

BACKGROUND: Chromobacterium violaceum is an environmental opportunistic pathogen that causes rare but deadly infections in humans. The transcriptional regulators that C. violaceum uses to sense and respond to environmental cues remain largely unknown. RESULTS: Here, we described a novel transcriptional regulator in C. violaceum belonging to the MarR family that we named OsbR (oxidative stress response and biofilm formation regulator). Transcriptome profiling by DNA microarray using strains with deletion or overexpression of osbR showed that OsbR exerts a global regulatory role in C. violaceum, regulating genes involved in oxidative stress response, nitrate reduction, biofilm formation, and several metabolic pathways. EMSA assays showed that OsbR binds to the promoter regions of several OsbR-regulated genes, and the in vitro DNA binding activity was inhibited by oxidants. We demonstrated that the overexpression of osbR caused activation of ohrA even in the presence of the repressor OhrR, which resulted in improved growth under organic hydroperoxide treatment, as seem by growth curve assays. We showed that the proper regulation of the nar genes by OsbR ensures optimal growth of C. violaceum under anaerobic conditions by tuning the reduction of nitrate to nitrite. Finally, the osbR overexpressing strain showed a reduction in biofilm formation, and this phenotype correlated with the OsbR-mediated repression of two gene clusters encoding putative adhesins. CONCLUSIONS: Together, our data indicated that OsbR is a MarR-type regulator that controls the expression of a large number of genes in C. violaceum, thereby contributing to oxidative stress defense (ohrA/ohrR), anaerobic respiration (narK1K2 and narGHJI), and biofilm formation (putative RTX adhesins).

Trastuzumab emtansine (T-DM1) in patients with HER2-positive metastatic breast cancer and brain metastases: exploratory final analysis of cohort 1 from KAMILLA, a single-arm phase IIIb clinical trial☆.

BACKGROUND: Patients with brain metastases (BM) from human epidermal growth factor receptor 2 (HER2)-positive breast cancer represent a difficult-to-treat population. Trastuzumab emtansine (T-DM1) has shown potential activity in this subset of patients in small clinical series. PATIENTS AND METHODS: KAMILLA is an ongoing, phase IIIb study of T-DM1 in patients with HER2-positive locally advanced/metastatic breast cancer with prior HER2-targeted therapy and chemotherapy. Patients received T-DM1 3.6 mg/kg every 3 weeks (intravenously) until unacceptable toxicity, withdrawal of consent, or disease progression. Tumor response and clinical outcomes in patients with baseline BM were evaluated in this post hoc, exploratory analysis. The main outcome measures were best overall response rate (complete response + partial response) and clinical benefit rate (complete response + partial response + stable disease lasting ≥6 months) by RECIST v1.1 criteria, progression-free survival, overall survival, and safety. RESULTS: Of 2002 treated patients, 398 had baseline BM. In 126 patients with measurable BM, the best overall response rate and clinical benefit rate were 21.4% [95% confidence interval (CI) 14.6-29.6] and 42.9% (95% CI 34.1-52.0), respectively. A reduction in the sum of the major diameters of BM ≥30% occurred in 42.9% (95% CI 34.1-52.0), including 49.3% (95% CI 36.9-61.8) of 67 patients without prior radiotherapy to BM. In the 398 patients with baseline BM, median progression-free survival and overall survival were 5.5 (95% CI 5.3-5.6) months and 18.9 (95% CI 17.1-21.3) months, respectively. The adverse event profile was broadly similar in patients with and without baseline BM, although nervous system adverse events were more common in patients with [208 (52.3%)] versus without [701 (43.7%)] baseline BM. CONCLUSION: This exploratory analysis of patients with HER2-positive metastatic breast cancer and BM enrolled in a prospective clinical trial shows that T-DM1 is active and well-tolerated in this population. T-DM1 should be explored further in this setting. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01702571.

Development of guidelines for family and non-professional helpers on assisting an older person who is developing cognitive impairment or has dementia: a Delphi expert consensus study.

BACKGROUND: Assisting a person with dementia can lead to significant carer burden and possible negative outcomes for the person. Using the Delphi method, this study developed expert consensus guidelines for how family and non-professional carers should assist a person who is developing cognitive impairment, or has dementia or delirium. METHODS: A systematic search of websites, books and journal articles was conducted to develop a questionnaire containing items about the knowledge, skills and actions needed for assisting a person who is developing cognitive impairment, or has dementia or delirium. These items were rated over three rounds by two international expert panels comprising professionals specialising in research or treatment of dementia, and dementia carer advocates. RESULTS: A total of 65 participants (43 in the professional panel and 22 in the carer advocate panel) completed all three survey rounds. Of the 656 survey items that were rated, a total of 389 items were endorsed by at least 80 % of each panel. The endorsed items formed the basis of a guidelines document that explains what family and non-professional carers need to know and do when assisting a person who is developing cognitive impairment, or has dementia or delirium. CONCLUSIONS: The two groups of experts were able to reach substantial consensus on how to assist a person who is developing cognitive impairment, or has dementia or delirium.

Vismodegib in the treatment of advanced BCC.

Basal-cell carcinoma (BCC) is the most commonly diagnosed malignancy, comprising over 80 per thousand of non-melanoma skin cancers. Surgical excision is adequate treatment for most BCC's. Options are however limited for the minority of patients presenting with locally advanced inoperable or metastatic BCC. The Hedgehog signalling pathway is a critical driver in the pathogenesis of both sporadic and hereditary BCC. On 31st January 2012, based on a phase II clinical trial the US Food and Drug Administration approved Vismodegib (Erivedge, Roche) a first-in-class, small-molecule oral Hedgehog-inhibitor for the treatment of locally advanced inoperable and metastatic BCC. We present our experience treating the first Irish patient with this agent.

Development and characterisation of mgTHP-1, a novel in vitro model for neural macrophages with microglial characteristics.

Neuroinflammation is primarily characterised by activation of the brain's resident macrophages - the microglia. However, other central nervous system (CNS) cells also contribute to this response, including the astrocytes and endothelial cells. In addition, there is infiltration into the CNS of peripherally derived immune cells. Together these cells mediate inflammation by the production of cytokines, chemokines, reactive oxygen species, and secondary messengers, and enacting of the appropriate response to those signals. However, deciphering the specific contributions of each cell type has been challenging. Studying CNS cell biology is often challenging, as the isolation of primary cells is not always feasible, and differentiation towards microglia-like cells is complex. Here, we demonstrate a novel method whereby THP-1 monocytic cells are differentiated into neural macrophage cells with microglia-like cell characteristics. The cells, designated mgTHP-1, show typical morphological and gene expression patterns of resident CNS macrophages and functionally respond to inflammatory stimuli by producing inflammatory cytokines. Furthermore, with the addition of Vicenin-2 (an anti-inflammatory flavonoid) such responses can be reversed. This novel cell model will allow further investigations, and hence insights, into the neuroinflammatory mechanisms associated with CNS diseases.

Variability in feed intake the first days following weaning impacts gastrointestinal tract development, feeding patterns, and growth performance in nursery pigs.

The present study investigated the effects of voluntary feed intake (FI) the first days after weaning on gastrointestinal development and protein fermentation the first week after weaning and growth performance and feeding patterns during the nursery phase. A total of 144 mixed-sex weaned pigs (24 ±â€…2 d old; 7.2 ±â€…0.8 kg body weight [BW]) were allocated to 12 pens with 12 pigs/pen. Each pen was equipped with an electronic feeding station for monitoring individual FI during a 40-d study. Pigs were classified based on their cumulative FI over the initial 3 d after weaning (FId1-3) being above or below their pen median FId1-3 (high = 919 ±â€…244 g or low = 507 ±â€…222 g FId1-3). Similarly, weaning BW classes (BW0; high = 7.72 ±â€…0.59 kg or low = 6.62 ±â€…0.88 kg BW) were created to study interactions with FId1-3. Two female pigs with either a high or a low FId1-3 per pen (n = 24) were selected for sampling at d6 and were used to study gastrointestinal development and fermentation products in the small intestine. Feeding patterns per day, FI, and growth performance were measured individually. Low FId1-3 pigs had lower (P < 0.05) daily FI during d0 to d8, d8 to d15, and d22 to d28, BW on d15, d22, d29, and d40, and average daily gain during d0 to d8, d22 to d29, and d29 to d40 compared to high FId1-3. High FId1-3 pigs increased (P < 0.05) the number of visits to the feeder between d1 to d13 and d31 to d35, and the time spent per visit only for d1 to d4 (P < 0.05). The daily rate of FI (g/min) was higher (P < 0.05) for High FId1-3 pigs on d6, d8, d9, and d10, and again several days later (d20 to d39). In addition, the high FId1-3 × high BW0 interaction improved daily FI during d18 to d40 compared to low FId1-3 × low BW0 class (P < 0.05). For the sampling on d6, low FId1-3 pigs had a lighter small intestine, colon, and pancreas, and reduced villi length, smaller villi surface area, and a lower number of goblet cells size in jejunum (P < 0.05), while concentrations of lactic acid, histamine, and cadaverine in small intestinal content were increased (P < 0.05). In conclusion, pigs with high FId1-3 became faster eaters with higher FI and growth rates toward the second half of the nursery, which was similar and additive for pigs with higher weaning BW. High FId1-3 was also associated with greater development of the gastrointestinal tract and a reduced protein fermentation 1-wk after weaning.

Poor adaptation to solid feed after weaning is often associated with a reduced digestive function and growth in nursery pigs. The reasons driving an early acceptance of feed and its consequences are still largely unknown. We investigated the effects of high and low feed intake between d1-3 after weaning on gastrointestinal development and morphometrics 1-wk after weaning and growth performance and feeding patterns in the nursery phase. The results showed that pigs with a high initial feed intake (increased number of visits to the feeder and time spent per visit early after weaning), consumed feed faster throughout the nursery resulting in higher intakes early and late in the nursery but not for the intermediate period. Higher weaning body weight was also associated with improved feed intake and growth from d17 onwards, which was an additional but independent effect of the early feed intake effect. Besides, pigs with high feed intake between d1 and d3 after weaning had heavier empty gastrointestinal organs, improved intestinal wall morphometrics, and reduced protein fermentation in the small intestine 1-wk after weaning.

Palbociclib combined with endocrine treatment in hormone receptor-positive, HER2-negative breast cancer patients with high relapse risk after neoadjuvant chemotherapy: subgroup analyses of premenopausal patients in PENELOPE-B.

BACKGROUND: The PENELOPE-B study demonstrated that the addition of 1-year post-neoadjuvant palbociclib to endocrine therapy (ET) in patients with high-risk early breast cancer (BC) did not improve invasive disease-free survival (iDFS) compared to placebo. Here, we report results for premenopausal women. PATIENTS AND METHODS: Patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative BC at high risk of relapse [defined as no pathological complete response after neoadjuvant chemotherapy and a clinical, pathological stage, estrogen receptor, grading (CPS-EG) score ≥3 or 2/ypN+] were randomized to receive 13 cycles of palbociclib or placebo + standard ET. Ovarian function (OF) was evaluated by centrally assessed estradiol, follicle-stimulating hormone and anti-Müllerian hormone serum levels. RESULTS: Overall, 616 of 1250 randomized patients were premenopausal; of these, 30.0% were <40 years of age, 47.4% had four or more metastatic lymph nodes, and 58.2% had a CPS-EG score ≥3. 66.1% of patients were treated with tamoxifen alone, and 32.9% received ovarian function suppression (OFS) in addition to either tamoxifen or aromatase inhibitor (AI). After a median follow-up of 42.8 months (97.2% completeness) no difference in iDFS between palbociclib and placebo was observed [hazard ratio = 0.95, 95% confidence interval (CI) 0.69-1.30, P = 0.737]. The estimated 3-year iDFS rate was marginally higher in the palbociclib arm (80.6% versus 78.3%). Three year iDFS was higher in patients receiving AI than tamoxifen plus OFS or tamoxifen alone (86.0% versus 78.6% versus 78.0%). Patients receiving tamoxifen plus OFS showed a favorable iDFS with palbociclib (83.0% versus 74.1%, hazard ratio = 0.52, 95% CI 0.27-1.02, P = 0.057). Hematologic adverse events were more frequent with palbociclib (76.1% versus 1.9% grade 3-4, P < 0.001). Palbociclib seems not to negatively impact the OF throughout the treatment period. CONCLUSIONS: In premenopausal women, who received tamoxifen plus OFS as ET, the addition of palbociclib to ET results in a favorable iDFS. The safety profile seems favorable and in contrast to chemotherapy palbociclib does not impact OF throughout the treatment period.

Reproducibility of positive results for the detection of serum galactomannan by Platelia™ aspergillus EIA.

Galactomannan (GM) was recently included in consensus guidelines as an indirect mycological criterion for the diagnosis of invasive aspergillosis. Currently, there is an enzyme immunoassay available to detect GM in biological samples, the Platelia™ Aspergillus EIA. In this study, the reproducibility of positive results obtained using this assay was evaluated using serum samples from neutropenic patients. A trend toward lower values was observed, and 55 %(27/49) of positive results were negative after retesting. A low reproducibility of positive results for the detection of GM in serum was observed.

In vitro acetylcholinesterase activity of peptide derivatives isolated from two species of Leguminosae.

CONTEXT: Cratylia mollis Martius ex Benth. and Cenostigma macrophyllum Tul. (Leguminosae) are both endemic Brazilian plants and they are used by the natives as medicinal plants, and the leaves of C. mollis are also employed as forage for cattle during the dry season of region. OBJECTIVE: Isolation of the compounds responsible for the acetylcholinesterase (AChE) inhibition from the CHCl3 active extract. MATERIALS AND METHODS: Two peptidic compounds were isolated by chromatographic techniques from the CHCl3 extract of the leaves of C. mollis and C. macrophyllum. They were identified by spectrometric data analysis (MS and NMR) and they were subjected to AChE inhibition employing Ellman's test. RESULTS: The peptides were identified as N-benzoylphenylalaninoyl-phenlyalaninolacetate (aurentiamide acetate) (1) and N-benzoylphenylalaninyl-N-benzoylphenylalaninate (2). Both peptides 1 and 2 exhibit AChE inhibition, with IC50 values equal to 111.34 µM and 137.6 µM, respectively. DISCUSSION AND CONCLUSION: Compound 1 (aurentiamide acetate) has rarely been isolated from the Leguminosae family, and N-benzoylphenylalaninyl-N-benzoylphenylalaninate (2) is a compound that has never previously been isolated from this family. Compound 1 is shown to be a potent inhibitor of AChE, with IC50 values similar to the physostigmine control (141.51 µM).

Bayesian variable selection in quantile regression with random effects: an application to Municipal Human Development Index.

According to the Atlas of Human Development in Brazil, the income dimension of Municipal Human Development Index (MHDI-I) is an indicator that shows the population's ability in a municipality to ensure a minimum standard of living to provide their basic needs, such as water, food and shelter. In public policy, one of the research objectives is to identify social and economic variables that are associated with this index. Due to the income inequality, evaluate these associations in quantiles, instead of the mean, could be more interest. Thus, in this paper, we develop a Bayesian variable selection in quantile regression models with hierarchical random effects. In particular, we assume a likelihood function based on the Generalized Asymmetric Laplace distribution, and a spike-and-slab prior is used to perform variable selection. The Generalized Asymmetric Laplace distribution is a more general alternative than the Asymmetric Laplace one, which is a common approach used in quantile regression under the Bayesian paradigm. The performance of the proposed method is evaluated via a comprehensive simulation study, and it is applied to the MHDI-I from municipalities located in the state of Rio de Janeiro.

Investigating a clinically actionable BRAF mutation for monitoring low-grade serous ovarian cancer: A case report.

Low-grade serous ovarian cancer (LGSOC) poses a specific clinical challenge due to advanced presentation at diagnosis and the lack of effective systemic treatments. The aim of this study was to use a precision medicine approach to identify clinically actionable mutations in a patient with recurrent LGSOC. Primary, metastatic and recurrence tissue, and blood samples were collected from a stage IV LGSOC patient. Single-gene testing for clinically actionable mutations (BRAF V600, KRAS and NRAS) and subsequent whole-exome sequencing (WES) were performed. Droplet digital PCR was used to evaluate the presence of an identified BRAF D594G mutation in the matched plasma cell-free DNA (cfDNA). No clinically actionable mutations were identified using single-gene testing. WES identified a BRAF D594G mutation in six of seven tumor samples. The patient was commenced on a MEK inhibitor, trametinib, but with minimal clinical response. A newly designed ddPCR assay detected the BRAF alteration in the matched tissues and liquid biopsy cfDNA. The identification and sensitive plasma detection of a common "druggable" target emphasises the impact of precision medicine on the management of rare tumors and its potential contribution to novel monitoring regimens in this field.

Coping with uncertainty: T1a,bN0M0 HER2-positive breast cancer, do we have a treatment threshold?

BACKGROUND: Recent retrospective studies have suggested that patients with T1a,bN0M0 human epidermal growth factor receptor 2 (HER2)-positive breast cancer are at a higher risk for recurrence and might benefit from adjuvant trastuzumab. The absolute benefits associated with treating this subgroup are uncertain. DESIGN: We reviewed recent studies examining the prognostic value of HER2 in patients with node-negative T1a,b HER2-positive breast cancer. We calculated the number needed to treat (NNT) using baseline risk estimates for untreated T1a,bN0M0 breast cancer and the number needed to harm (NNH) using the incidence of cardiac events in each of the adjuvant trastuzumab clinical trials. RESULTS: Several studies were identified, each with limitations inherent to retrospective database analyses: small cohort sizes, lack of systematic HER2 testing in older specimens, variations in the use of adjuvant therapy and definitions of study end points, and lack of information relating to comorbidities. The 5-year disease-free survival in the pre-trastuzumab era ranged from 77% to 95%. Comparisons between small HER2 -positive and small HER2 -negative cancers showed numerically worse outcome for the HER2-positive cohort in some but not all studies. In many instances, the NNH was larger (26-250) than the NNT (13-35); however, in a subset of patients, the NNH was lower (6) than the NNT (13-35). CONCLUSIONS: Better prediction tools to estimate more precisely the risk for death due to comorbid illness versus breast cancer are needed. In some patients, the risks of therapy could outweigh the benefits. Treatment selection for T1a,bN0 HER2-positive cancers remains in the transition area between evidence- and subjective judgment-based medicine.

Acupuncture at "Zusanli" (St.36) and "Sanyinjiao" (SP.6) Points on the Gastrointestinal Tract: A Study of the Bioavailability of (99m)Tc-Sodium Pertechnetate in Rats.

The objective of this study is to investigate the differences of acupuncture effect between the Zusanli (St.36) and Sanyinjiao (SP.6) points on the gastrointestinal-tract (GIT) segment performed by the bioavailability of (99m)Tc-sodium-pertechnetate (Na(99m)TcO(4)) in rats. Male Wistar rats (n = 21) were allocated into three groups of seven each. Group 1 was treated by acupuncture bilaterally at St.36; Group 2 at SP.6; and Group 3 was untreated (control). After 10 min of needle insertion in anesthetized rats, 0.3 mL of Na(99m)TcO(4) (7.4 MBq) was injected via ocular-plexus. After 20 min, the exitus of animals was induced by cervical-dislocation and GIT organs isolated. However, immediately before the exitus procedure, blood was collected by cardiac-puncture for blood radio-labeling (BRL). The radioactivity uptake of the blood constituents was calculated together with the GIT organs by a well gamma counter. The percentage of injected dose per gram of tissue (%ID/g) of Na(99m)TcO(4) was calculated for each GIT organs, while BRL was calculated in %ID. According to the one-way ANOVA, the stomach, jejunum, ileum from the treated groups (Group 1 and Group 2) had significant differences compared to the controls (Group 3). However, between the treated groups (Group 1 and Group 2), there were significant differences (P < .05) in the stomach, jejunum, ileum, cecum, transverse and rectum. In BRL analysis, Group 2 showed significant increase and decrease of the insoluble and soluble fractions of the blood cells, respectively (P < .0001). The authors suggest that St.36 may have a tendency of up-regulation effect on GIT, whereas SP.6, down-regulation effect. However, further rigorous experimental studies to examine the effectiveness of acupuncture in either acupuncture points need to be carried out.

Bayesian factor models for multivariate categorical data obtained from questionnaires.

Factor analysis is a flexible technique for assessment of multivariate dependence and codependence. Besides being an exploratory tool used to reduce the dimensionality of multivariate data, it allows estimation of common factors that often have an interesting theoretical interpretation in real problems. However, standard factor analysis is only applicable when the variables are scaled, which is often inappropriate, for example, in data obtained from questionnaires in the field of psychology, where the variables are often categorical. In this framework, we propose a factor model for the analysis of multivariate ordered and non-ordered polychotomous data. The inference procedure is done under the Bayesian approach via Markov chain Monte Carlo methods. Two Monte Carlo simulation studies are presented to investigate the performance of this approach in terms of estimation bias, precision and assessment of the number of factors. We also illustrate the proposed method to analyze participants' responses to the Motivational State Questionnaire dataset, developed to study emotions in laboratory and field settings.

Collagen expression, ultrastructural assembly, and mineralization in cultures of chicken embryo osteoblasts.

A newly defined chick calvariae osteoblast culture system that undergoes a temporal sequence of differentiation of the osteoblast phenotype with subsequent mineralization (Gerstenfeld, L. C., S. Chipman, J. Glowacki, and J. B. Lian. 1987. Dev. Biol. 122:49-60) has been examined for the regulation of collagen synthesis, ultrastructural organization of collagen fibrils, and extracellular matrix mineralization. Collagen gene expression, protein synthesis, processing, and accumulation were studied in this system over a 30-d period. Steady state mRNA levels for pro alpha 1(I) and pro alpha 2 collagen and total collagen synthesis increased 1.2- and 1.8-fold, respectively, between days 3 and 12. Thereafter, total collagen synthesis decreased 10-fold while mRNA levels decreased 2.5-fold. In contrast to the decreasing protein synthesis after day 12, total accumulated collagen in the cell layers increased sixfold from day 12 to 30. Examination of the kinetics of procollagen processing demonstrated that there was a sixfold increase in the rate of procollagen conversion to alpha chains from days 3 to 30 and the newly synthesized collagen was more efficiently incorporated into the extracellular matrix at later culture times. The macrostructural assembly of collagen and its relationship to culture mineralization were also examined. High voltage electron microscopy demonstrated that culture cell layers were three to four cells thick. Each cell layer was associated with a layer of well developed collagen fibrils orthogonally arranged with respect to adjacent layers. Fibrils had distinct 64-70-nm periodicity typical of type I collagen. Electron opaque areas found principally associated with the deepest layers of the fibrils consisted of calcium and phosphorus determined by electron probe microanalysis and were identified by electron diffraction as a very poorly crystalline hydroxyapatite mineral phase. These data demonstrate for the first time that cultured osteoblasts are capable of assembling their collagen fibrils into a bone-specific macrostructure which mineralizes in a manner similar to that characterized in vivo. Further, this matrix maturation may influence the processing kinetics of the collagen molecule.

Membrane permeability coefficients of murine primary neural brain cells in the presence of cryoprotectant.

Neural cells isolated from the brain have a number of research and clinical applications, including transplantation to patients with neurodegenerative conditions. Tissue supply is one of the major limiting factors to clinical transplantation. Cryopreservation of primary neural cells would improve supply, aid in organisation of transplantation surgery and facilitate research. To date, cryopreservation using standard methods has resulted in reduced yield and/or viability of primary neural tissue. In order to optimise freezing protocols specifically for such cells, the non-osmotic volume (V(b)), water permeability (L(p)) and permeability to cryoprotectant (P(cpa)) were determined. Murine foetal brain tissue from the ganglionic eminence (GE), ventral mesencephalon (VM), or neocortical mantle (Ctx) was trypsinised to a single cell suspension. To determine V(b,) cell volume was measured after exposure to anisotonic solutions of sucrose (150-1500 mOsmol/kg). L(p) (mum/min.atm) and P(cpa) (mum/s) were determined for GE cells by measuring cell volume during exposure to 1.5 mol/l cryoprotectant. Cell volume was determined using an electronic particle counting method. V(b) was 27% for Ctx and GE, and 30% for VM. The osmotic response of GE cells was similar in the presence of propane-1,2-diol and dimethyl sulphoxide. In the presence of ethylene glycol, cell volume decrease was greater on initial exposure to cryoprotectant and recovery slower. Differences in L(p,) but not P(cpa), were found between cryoprotectants. The present results provide key parameters for optimisation of freezing protocols for cryopreservation of primary foetal brain tissues for application in neural cell transplantation.

EmrR-Dependent Upregulation of the Efflux Pump EmrCAB Contributes to Antibiotic Resistance in Chromobacterium violaceum.

Chromobacterium violaceum is an environmental Gram-negative bacterium that causes infections in humans. Treatment of C. violaceum infections is difficult and little is known about the mechanisms of antibiotic resistance in this bacterium. In this work, we identified mutations in the MarR family transcription factor EmrR and in the protein GyrA as key determinants of quinolone resistance in C. violaceum, and we defined EmrR as a repressor of the MFS-type efflux pump EmrCAB. Null deletion of emrR caused increased resistance to nalidixic acid, but not to other quinolones or antibiotics of different classes. Moreover, the ΔemrR mutant showed decreased production of the purple pigment violacein. Importantly, we isolated C. violaceum spontaneous nalidixic acid-resistant mutants with a point mutation in the DNA-binding domain of EmrR (R92H), with antibiotic resistance profile similar to that of the ΔemrR mutant. Other spontaneous mutants with high MIC values for nalidixic acid and increased resistance to fluoroquinolones presented point mutations in the gene gyrA. Using DNA microarray, Northern blot and EMSA assays, we demonstrated that EmrR represses directly a few dozen genes, including the emrCAB operon and other genes related to transport, oxidative stress and virulence. This EmrR repression on emrCAB was relieved by salicylate. Although mutation of the C. violaceum emrCAB operon had no effect in antibiotic susceptibility or violacein production, deletion of emrCAB in an emrR mutant background restored antibiotic susceptibility and violacein production in the ΔemrR mutant. Using a biosensor reporter strain, we demonstrated that the lack of pigment production in ΔemrR correlates with the accumulation of quorum-sensing molecules in the cell supernatant of this mutant strain. Therefore, our data revealed that overexpression of the efflux pump EmrCAB via mutation and/or derepression of EmrR confers quinolone resistance and alters quorum-sensing signaling in C. violaceum, and that point mutation in emrR can contribute to emergence of antibiotic resistance in bacteria.

Oral cancer: exploring the stories in United Kingdom newspaper articles.

Objective Reports suggest that patients with oral cancer delay seeking help because they are unaware of the symptoms. The majority of adults (95%) engage with news reports and 40% read newspapers. Newspaper oral cancer stories may influence awareness and health-seeking behaviour. The aim of this study was to explore how oral cancer is portrayed in UK newspaper print media.Design Qualitative content analysis of articles from ten newspapers with the widest UK print circulation. All articles using the terms 'mouth cancer' and 'oral cancer' over a three year period were retrieved. Duplicates, non-cancer and non-human articles were excluded.Results 239 articles were analysed. Common topics included 'recent research', 'survivor stories', 'health information' and 'celebrity linkage'. Articles were often emotive, featuring smoking, alcohol, sex and celebrity. Articles lacked a proper evidence base and often failed to provide accurate information about signs and symptoms, information about prevention and signposting to treatment.Conclusions Opportunities to save lives are being missed. Further work to improve social responsibility in the media and develop guidance to enhance the quality of information, health reporting and signposting to help are indicated.