Insect exclusion limits variation in bacterial microbiomes of tomato flowers and fruit.

AIMS: The effect of insect exclusion via netting on bacterial microbiota associated with field-grown tomato fruit and flowers was evaluated. METHODS AND RESULTS: Amplicon-based bacterial community profiling from insect-exposed plants and plants wrapped in nylon mosquito netting was conducted on total DNA extracted from tomato flower and mature unripe fruit washes. The V1-V3 region of the 16S rRNA gene was sequenced using Illumina MiSeq and analysed using qiime ver. 1.8. The carposphere supported significantly more phylogenetic diversity (PD) compared to the anthosphere, as measured by operational taxonomic unit richness (P = 0·001) and Faith's PD (P = 0·004). Flowers and fruit hosted distinct bacterial community structures (R2 = 0·27, P = 0·001), with specific taxonomic differences in taxa that included the Xanthomonadaceae (higher in flowers), and the Pseudomonadaceae, Methylobacteriaceae and Rhizobiales (higher in fruit) (FDR-P < 0·05). Bacterial community profiles of netted plants were overall statistically similar to non-netted plants for both flowers and fruit (P > 0·10). However, less variation between samples was observed among flowers (~50% less, P = 0·004) and green fruit (~10% less, P = 0·038) collected from netted than non-netted plants. CONCLUSION: Insects may introduce or augment variability in bacterial diversity associated with tomato flowers and potentially green fruit surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to knowledge on microbiome dynamics of the tomato holobiont. Deciphering drivers of bacterial diversity and community structure of fruit crops could reveal processes important to agricultural management, such as competitive exclusion of pathogens and priming of plant defense mechanisms.

Rational Clinical Pathology Assessment in the Intensive Care Unit.

Blood tests are ordered on a daily basis in intensive care units (ICU). There are no widely accepted guidelines for testing requirements. This study investigated the impact on ICU laboratory test costs of a multi-strategy change in practice involving routine blood testing. A single centre, prospective, interventional study using historical controls was undertaken to investigate the impact of ICU specialist authorisation of high-volume routine tests on ICU laboratory test costs. Prior to commencement of the study, ICU nursing and junior ICU doctors were able to order tests. During the six-month intervention period, the ICU specialists authorised routine blood tests. Adverse events related to not performing blood tests were also recorded. Overall ICU laboratory test costs decreased by 12.3% over the six months (P=0.0022 versus historical control) with a mean compliance of 51% with the test authorisation protocol. The costs of frequently ordered tests (classified as high-volume) decreased by 20% (P=0.0022 versus historical control). These accounted for an average of 54 ± 3% of the overall ICU blood test costs (blood gas analyses 17%, simple chemistry tests consisting of electrolytes, liver function, calcium, phosphate, magnesium 14%, coagulation 12% and full blood count 11%). Two protocol-related adverse events were recorded and judged as minor and were resolved by ordering tests during the day. No adverse patient outcomes resulted from these two events. Blood testing authorisation by an ICU specialist was associated with significant cost savings in ICU and no adverse patient outcomes.

An Escalation for Bivariate Binary Endpoints Controlling the Risk of Overtoxicity (EBE-CRO): Managing Efficacy and Toxicity in Early Oncology Clinical Trials.

For about a decade, early clinical development in oncology is facing new challenges. This is due to two main reasons. The first one is linked to the developed molecular targeted agents (MTA) themselves for which the maximum tolerated dose (MTD) is no longer the only dose of interest. The second reason is related to the fact that costs and attrition rates are huge. When selecting a dose, evidence of early activity signal becomes required for future engagements. This implies the need to handle both toxicity and activity endpoints in the analysis and also in the dose escalation design of early-phase trials. We propose a model-based design taking into account both safety and activity for dose escalation. The proposed model involves a bivariate Gaussian latent variable depending on a monotonic toxicity curve and a quadratic activity curve. This model is fitted under the Bayesian framework that allows the incorporation of prior information. The predictive distributions of dose-response curves are used to lead the dose recommendation. Uncertainty in the dose-response relationship is taken into account to calculate the probability of being an over-toxic or a target dose. The proposed design is compared to two other widely used methods.

Chemoprevention of breast cancer among women at elevated risk as defined by Gail Score.

The risk of an individual woman to develop breast cancer over a 5-year period can be estimated using the Gail Model. The risk factors included in this model effectively classify patients into two different subgroups. One subgroup comprises patients at increased risk because of increased exposure to estrogen. These women are more likely to benefit from endocrine chemopreventive therapies, namely selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs). The second subgroup comprises women who have inherited genetic mutations that predispose them to breast cancer. Chemoprevention in these patients is more likely to be achieved by novel agents, such as lapatinib, gefitinib, fenretinide, rexinoids and poly(ADP-ribose) polymerase (PARP)-inhibitors.

INS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells.

AIMS/HYPOTHESIS: We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS⁺) cells derived in vitro. METHODS: Homologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS⁺ cells. RESULTS: Differentiation of INS(GFP/w) hESCs using published protocols demonstrated that all GFP⁺ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP⁺ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS(GFP/w) hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP⁺ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP⁺ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS⁺ cells. CONCLUSIONS/INTERPRETATION: INS(GFP/w) hESCs are a valuable tool for investigating the nature of early INS⁺ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS⁺ cells from differentiating hESCs.

The functional and molecular characterisation of human embryonic stem cell-derived insulin-positive cells compared with adult pancreatic beta cells.

AIMS/HYPOTHESIS: Using a novel directed differentiation protocol, we recently generated up to 25% insulin-producing cells from human embryonic stem cells (hESCs) (insulin(+) cells). At this juncture, it was important to functionally and molecularly characterise these hESC-derived insulin(+) cells and identify key differences and similarities between them and primary beta cells. METHODS: We used a new reporter hESC line with green fluorescent protein (GFP) cDNA targeted to the INS locus by homologous recombination (INS (GFP/w)) and an untargeted hESC line (HES2). INS (GFP/w) allowed efficient identification and purification of GFP-producing (INS:GFP(+)) cells. Insulin(+) cells were examined for key features of adult beta cells using microarray, quantitative PCR, secretion assays, imaging and electrophysiology. RESULTS: Immunofluorescent staining showed complete co-localisation of insulin with GFP; however, cells were often multihormonal, many with granules containing insulin and glucagon. Electrophysiological recordings revealed variable K(ATP) and voltage-gated Ca(2+) channel activity, and reduced glucose-induced cytosolic Ca(2+) uptake. This translated into defective glucose-stimulated insulin secretion but, intriguingly, appropriate glucagon responses. Gene profiling revealed differences in global gene expression between INS:GFP(+) cells and adult human islets; however, INS:GFP(+) cells had remarkably similar expression of endocrine-lineage transcription factors and genes involved in glucose sensing and exocytosis. CONCLUSIONS/INTERPRETATION: INS:GFP(+) cells can be purified from differentiated hESCs, providing a superior source of insulin-producing cells. Genomic analyses revealed that INS:GFP(+) cells collectively resemble immature endocrine cells. However, insulin(+) cells were heterogeneous, a fact that translated into important functional differences within this population. The information gained from this study may now be used to generate new iterations of functioning beta cells that can be purified for transplant.

Defective stem cell factor expression in c-myb null fetal liver stroma.

High levels of c-Myb are observed in immature precursor myeloid and lymphoid cells, while downregulation of c-myb accompanies terminal differentiation to a mature phenotype. This has established c-Myb as a crucial transcription factor for hematopoiesis. Further evidence for this is the embryonic death of the c-myb homozygous mutant mouse at ED15 due to defective fetal liver erythropoiesis. Cells from fetal liver of wild-type and c-myb-/- embryos were examined in detail for their hematopoietic potential and the capacity of the stroma to support wild-type hematopoiesis. The c-myb-/- fetal liver was shown to harbor sevenfold fewer spleen focus-forming cells and a similarly lower number of cells with long-term repopulating capacity (high proliferative potential cells). However, shorter term repopulating cells were not substantially reduced. c-myb-/- stromal cells were unable to support the proliferation of wild-type bone marrow lineage-negative cells. This was found to be partly due to a decrease in stem cell factor (SCF) expression while partial rescue of the stromal cell cultures was achieved through the addition of exogenous SCF. DNA binding studies for two sites within the SCF promoter demonstrated an in vitro interaction between the SCF promoter and c-Myb and transient transfection studies demonstrated that c-Myb could substantially transactivate the SCF promoter in HEK293 cells. These data explain why the c-myb-/- embryos are so impaired in their ability to establish hematopoiesis.

An evaluation of the HPLC-Gel chromatographic method for analyzing metallothioneins in aquatic organisms.

The use of high-performance liquid chromatography to determine the concentrations of metal-binding proteins (MBP) in freshwater mussels has been evaluated. Initial use of dilute buffers (e.g., 1OmM TRIS, pH 7 or 8), to minimize competition between the buffer and the metal-cytosolic ligand complexes, proved unacceptable; high losses of low molecular weight marker proteins occurred during chromatographic separation, presumably as a result of adsorption to the gel-permeation column packing. Losses were more dependent on salt than pH; satisfactory recoveries were obtained in the pH range 6-8 with 1OmM buffer solutions and 100mM added electrolyte (NACl; KCl). Competition experiments performed with commercial metallothionein (pre-labelled with (109)Cd) and fresh mussel cytosol extract demonstrated that no appreciable metal exchange occurred during the 20-min pre-equilibration or the subsequent chromatographic separation step. With (203)Hg-labelled metallothionein occasional losses were noted, however, appreciable loss of the radioisotope ranging from 50 to 58% did occur with (65)Zn-labelled metallothionein during the chromatographic separation step. These results, particularly for Zn indicate that the recovery of metals after separation using this chromatographic method may vary, even for metals sharing similar chemical properties.

Gas chromatographic-mass spectrometric profiling with negative-ion chemical ionization detection of prostaglandins and their 15-keto and 15-keto-13,14-dihydro catabolites in rat blood.

A method was set up in which the primary prostaglandins (PGF2 alpha, PGD2, PGE2, thromboxane B2, 6-keto-PGF1 alpha and 6-keto-PGE1) and their catabolites (15-keto and 15-keto-13,14-dihydro) could be analyzed in the same sample at the same time. The method makes use of long capillary columns (60 m) to resolve the complex mixture during gas chromatography and mass fragmentography to provide the specificity of detection of these products. Selectivity and sensitivity is provided through use of appropriate derivatives (pentafluorobenzyl esters) which allow detection by negative-ion chemical ionization in which high-abundance fragments in the high end of the mass spectrum (M-pentafluorobenzyl) are observed. A purification procedure of whole blood is described involving diethyl ether extraction, C18 Sep-Pak chromatography, derivatization into the pentafluorobenzyl-O-methyloxime, C18 Sep-Pak and silicic acid chromatography followed by final derivatization into trimethylsilyl ethers for gas chromatographic-mass spectrometric analysis. Recovery of added [3H]PGF2 alpha was 73.8 +/- 2.2% (n = 10). Sample workup and analysis takes ten days for six samples. The method is sufficiently sensitive for the profiling of a 10-ml sample of whole blood (limit approximately 1 pg/ml; 1-pg injection on column).

Catabolism of 6-keto prostaglandin E1 by rat kidney homogenates.

6-Keto prostaglandin E1 (PGE1) was catabolized into two major products by homogenates of rat kidney in the presence of NAD. Rat brain was mostly deficient in this catabolic activity. A total profile of products from these experiments could be obtained using the pentafluorobenzyl esters, O-methyloxime, and trimethylsilyl either derivatives and analysis by capillary gas chromatography with electron capture detection. The same samples could be effectively analyzed by mass spectrometry using negative ion chemical ionization (NICI) detection. While 6-keto PGE1 showed spasmogenic activity on the rat stomach fundus as well as inhibition of the ADP-induced platelet aggregation, its catabolites lacked such activity. Analysis of rat whole blood by mass spectrometry using the NICI technique showed the presence of small amounts of 6-keto PGE1 and its 15-keto and 15-keto 13,14-dihydro catabolites in two of eight blood samples.