A fluorogenic cyclic peptide for imaging and quantification of drug-induced apoptosis

Programmed cell death or apoptosis is a central biological process that is dysregulated in many diseases, including inflammatory conditions and cancer. The detection and quantification of apoptotic cells in vivo is hampered by the need for fixatives or washing steps for non-fluorogenic reagents, and by the low levels of free calcium in diseased tissues that restrict the use of annexins. In this manuscript, we report the rational design of a highly stable fluorogenic peptide (termed Apo-15) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. Furthermore, using a combination of chemical and biophysical methods, we identify phosphatidylserine as a molecular target of Apo-15. We demonstrate that Apo-15 can be used for the quantification and imaging of drug-induced apoptosis in preclinical mouse models, thus creating opportunities for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Maximising learning dialogues between workplace mentors and students undertaking professional field-based experiences

Field‐based experience is an integral component of many pre-service professional preparation programmes. In these practicum placements, students are paired with a mentor who is usually an experienced practitioner. While placements are regarded as a highly significant contributor to the overall programme, research suggests that student experience and the resultant learning can be varied. Sanders’ 2008 doctoral research focused on the use of intentional interventions within practicum experiences in initial primary teacher training as a means of enriching learning dialogue, which is when the conversation between a supervisor/mentor and learner is characterised by genuine professional co‐enquiry. This study takes that work and extends it into degree programmes preparing students for early childhood education and for counselling

Quorum sensing negatively regulates multinucleate cell formation during intracellular growth of Burkholderia pseudomallei in macrophage-like cells.

Burkholderia pseudomallei is a Gram-negative environmental bacterium and the causative agent of melioidosis, a potentially fatal, acute or chronic disease endemic in the tropics. Acyl homoserine lactone (AHL)-mediated quorum sensing and signalling have been associated with virulence and biofilm formation in numerous bacterial pathogens. In the canonical acyl-homoserine lactone signalling paradigm, AHLs are detected by a response regulator. B. pseudomallei encodes three AHL synthases, encoded by bpsI1, bpsI2 and bpsI3, and five regulator genes. In this study, we mutated the B. pseudomallei AHL synthases individually and in double and triple combination. Five AHLs were detected and quantified by tandem liquid chromatography-mass spectroscopy. The major AHLs produced were N-octanoylhomoserine lactone and N-(3-hydroxy-decanoyl)homoserine lactone, the expression of which depended on bpsI1 and bpsI2, respectively. B. pseudomallei infection of macrophage cells causes cell fusion, leading to multinucleated cells (3 or more nuclei per cell). A triple mutant defective in production of all three AHL synthases was associated with a striking phenotype of massively enhanced host cellular fusion in macrophages. However, neither abrogation of host cell fusion, achieved by mutation of bimA or hcp1, nor enhancement of fusion altered intracellular replication of B. pseudomallei. Furthermore, when tested in murine models of acute melioidosis the AHL synthase mutants were not attenuated for virulence. Collectively, this study identifies important new aspects of the genetic basis of AHL synthesis in B. pseudomallei and the roles of these AHLs in systemic infection and in cell fusion in macrophages for this important human pathogen

AHLs produced by <i>B. pseudomallei</i> MSHR520 and its derived mutants.

<p>Mutant strains are deleted in each AHL synthase gene indicated, either singly or in double or triple combination. Each value is presented as the mean ± SD. ND, not detected.</p

Infection of RAW264.7 cells by <i>B. pseudomallei</i> MSHR520, MSHR520Δ<i>bimA</i>, and MSHR520Δ<i>hcp</i>1.

<p>Infections were carried out over a period of 12 h and fixed for Giemsa staining (a) or lysed to release internalised bacteria, which were then plated for enumeration of cfu per well (b). (a) Formation of MNCs in cells infected with Δ<i>bimA</i> and Δ<i>hcp</i>1 mutants as compared with wt (MSHR520). Comparisons between mutants and wt were statistically significant (P<0.001). >1000 nuclei per well were counted and are expressed as number of nuclei in each category. Columns show the mean values with SEM. (b) Intracellular bacteria in cells infected with wt, Δ<i>bimA</i> and Δ<i>hcp</i>1 mutants as compared with wt (MSHR520). No significant difference (P = 0.46) is observed between wt and Δ<i>bimA</i> or wt and Δ<i>hcp</i>1. Scatter plots show each well as an individual point with the mean value represented by the horizontal bar. Data are derived from 4 separate experiments, each including duplicate or triplicate wells.</p

Representative views of <i>B. pseudomallei</i> infected RAW264.7 cells at 12 h post infection.

<p>A) & B) MSHR520 (wt) infected; C) MSHR520 Δ<i>bimA</i>; D) MSHR520 Δ<i>hcp</i>1; E) & F) MSHR520 Δ<i>bpsI</i>123. Nuclei are stained blue with DAPI; bacteria are labelled green with rabbit anti-<i>B. pseudomallei</i> IgG and anti-rabbit IgG-alexa 568; and actin is labelled red with phalloidin-alexa 488. Red actin tails, at the poles of bacteria, are visible in all panels with the exception of C. Scale bar represents 25 µm (A, C, D, E), 12.5 µm (B), 250 µm (F).</p