The Mycobacterium tuberculosis complex pangenome is small and shaped by sub-lineage-specific regions of difference

The Mycobacterium tuberculosis complex (MTBC) is a group of bacteria causing tuberculosis (TB) in humans and animals. Understanding MTBC genetic diversity is crucial for insights into its adaptation and traits related to survival, virulence, and antibiotic resistance. While it is known that within-MTBC diversity is characterised by large deletions found only in certain lineages (regions of difference [RDs]), a comprehensive pangenomic analysis incorporating both coding and non-coding regions remains unexplored. We utilised a curated dataset representing various MTBC genomes, including under-represented lineages, to quantify the full diversity of the MTBC pangenome. The MTBC was found to have a small, closed pangenome with distinct genomic features and RDs both between lineages (as previously known) and between sub-lineages. The accessory genome was identified to be a product of genome reduction, showing both divergent and convergent deletions. This variation has implications for traits like virulence, drug resistance, and metabolism. The study provides a comprehensive understanding of the MTBC pangenome, highlighting the importance of genome reduction in its evolution, and underlines the significance of genomic variations in determining the pathogenic traits of different MTBC lineages

Strigolactone analogs act as new anti-cancer agents in inhibition of breast cancer in xenograft model

Strigolactones (SLs) are a novel class of plant hormones. Previously, we found that analogs of SLs induce growth arrest and apoptosis in breast cancer cell lines. These compounds also inhibited the growth of breast cancer stem cell enriched-mammospheres with increased potency. Furthermore, strigolactone analogs inhibited growth and survival of colon, lung, prostate, melanoma, osteosarcoma and leukemia cancer cell lines. To further examine the anti-cancer activity of SLs in vivo, we have examined their effects on growth and viability of MDA-MB-231 tumor xenografts model either alone or in combination with paclitaxel. We show that strigolactone act as new anti-cancer agents in inhibition of breast cancer in xenograft model. In addition we show that SLs affect the integrity of the microtubule network and therefore may inhibit the migratory phenotype of the highly invasive breast cancer cell lines that were examined

A cluster randomized trial of a transition intervention for adolescents with congenital heart disease: rationale and design of the CHAPTER 2 study

BACKGROUND: The population of adolescents and young adults with congenital heart disease (CHD) is growing exponentially. These survivors are at risk of late cardiac complications and require lifelong cardiology care. However, there is a paucity of data on how to prepare adolescents to assume responsibility for their health and function within the adult health care system. Evidence-based transition strategies are required. METHODS: The Congenital Heart Adolescents Participating in Transition Evaluation Research (CHAPTER 2) Study is a two-site cluster randomized clinical trial designed to evaluate the efficacy of a nurse-led transition intervention for 16–17 year olds with moderate or complex CHD. The primary endpoint is excess time to adult CHD care, defined as the time interval between the final pediatric cardiology appointment and the first adult CHD appointment, minus the recommended time interval between these appointments. Secondary endpoints include the MyHeart score (CHD knowledge), Transition Readiness Assessment Questionnaire score, and need for catheter or surgical re-intervention. Participants are enrolled in clusters based on week of attendance in the pediatric cardiology clinic. The intervention consists of two one-hour individualized sessions between a cardiology nurse and study participant. Session One focuses on knowledge of the participant’s CHD, review of their cardiac anatomy and prior interventions, and potential late cardiac complications. Session Two focuses on self-management and communication skills through review and discussion of videos and role-play. The study will recruit 120 participants. DISCUSSION: Many adolescents and young adults experience a gap in care predisposing them to late cardiac complications. The CHAPTER 2 Study will investigate the impact of a nurse-led transition intervention among adolescents with CHD. Fidelity of the intervention is a major focus and priority. This study will build on our experience by (i) enrolling at two tertiary care programs, (ii) including a self-management intervention component, and (iii) evaluating the impact of the intervention on time to ACHD care, a clinically relevant outcome. The results of this study will inform pediatric cardiology programs, patients and policy makers in judging whether a structured intervention program provides clinically meaningful outcomes for adolescents and young adults living with CHD. TRIAL REGISTRATION: ClinicalTrials.gov ID NCT0172333

Bistability by multiple phosphorylation of regulatory proteins.

The activity of a protein can be reversibly modulated by post-translational, covalent modifications, such as phosphorylation and dephosphorylation. In many cases, the modulated protein may be phosphorylated by the same kinase on many different amino acid residues. Such multisite phosphorylations may occur progressively (during a single binding event of kinase to substrate) or distributively (the kinase dissociates from its substrate after each phosphorylation reaction). If a protein is phosphorylated by a distributive multisite mechanism, then the net activity of a population of these protein molecules can be a highly nonlinear function of the ratio of activities of the kinase and phosphatase enzymes. If the multiply phosphorylated protein is embedded in a positive feedback loop with its kinase and/or phosphatase, then the network may exhibit robust bistable behavior. Using numerical simulations and bifurcation theory, we study the properties of a particular bistable reaction network motivated by the antagonistic relationship between cyclin-dependent kinase and its multiply phosphorylated target, Cdh1, which is involved in the degradation of cyclin molecules. We characterize the bistable switch in terms of (i) the mechanism of distributive phosphorylation (ordered or disordered), (ii) the number of phosphorylation sites on the target protein, (iii) the effect of phosphorylation on the target protein (abrupt or progressive inactivation), and (iv) the effects of stochastic fluctuations in small cells with limited numbers of kinase, phosphatase and target proteins

Bistability by multiple phosphorylation of regulatory proteins.

The activity of a protein can be reversibly modulated by post-translational, covalent modifications, such as phosphorylation and dephosphorylation. In many cases, the modulated protein may be phosphorylated by the same kinase on many different amino acid residues. Such multisite phosphorylations may occur progressively (during a single binding event of kinase to substrate) or distributively (the kinase dissociates from its substrate after each phosphorylation reaction). If a protein is phosphorylated by a distributive multisite mechanism, then the net activity of a population of these protein molecules can be a highly nonlinear function of the ratio of activities of the kinase and phosphatase enzymes. If the multiply phosphorylated protein is embedded in a positive feedback loop with its kinase and/or phosphatase, then the network may exhibit robust bistable behavior. Using numerical simulations and bifurcation theory, we study the properties of a particular bistable reaction network motivated by the antagonistic relationship between cyclin-dependent kinase and its multiply phosphorylated target, Cdh1, which is involved in the degradation of cyclin molecules. We characterize the bistable switch in terms of (i) the mechanism of distributive phosphorylation (ordered or disordered), (ii) the number of phosphorylation sites on the target protein, (iii) the effect of phosphorylation on the target protein (abrupt or progressive inactivation), and (iv) the effects of stochastic fluctuations in small cells with limited numbers of kinase, phosphatase and target proteins

A multiplex culture system for the long-term growth of fission yeast cells: fission yeast long-term cultures

International audienceMaintenance of long-term cultures of yeast cells is central to a broad range of investigations, from metabolic studies to laboratory evolution assays. However, repeated dilutions of batch cultures lead to variations in medium composition, with implications for cell physiology. In Saccharomyces cerevisiae, powerful miniaturized chemostat setups, or ministat arrays, have been shown to allow for constant dilution of multiple independent cultures. Here we set out to adapt these arrays for continuous culture of a morphologically and physiologically distinct yeast, the fission yeast Schizosaccharomyces pombe, with the goal of maintaining constant population density over time. First, we demonstrated that the original ministats are incompatible with growing fission yeast for more than a few generations, prompting us to modify different aspects of the system design. Next, we identified critical parameters for sustaining unbiased vegetative growth in these conditions. This requires deletion of the gsf2 flocculin-encoding gene, along with addition of galactose to the medium and lowering of the culture temperature. Importantly, we improved the flexibility of the ministats by developing a piezo-pump module for the independent regulation of the dilution rate of each culture. This made it possible to easily grow strains that have different generation times in the same assay. Our system therefore allows for maintaining multiple fission yeast cultures in exponential growth, adapting the dilution of each culture over time to keep constant population density for hundreds of generations. These multiplex culture systems open the door to a new range of long-term experiments using this model organism. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd

From Diagnoses to Ongoing Journey: Parent Experiences Following Congenital Heart Disease Diagnoses

AbstractDespite improved survival among children with congenital heart disease (CHD), the risk of psychosocial difficulties remains largely unchanged with an increased emphasis of improving support for parents as a mechanism to optimize outcomes.ObjectiveUsing qualitative and quantitative methods, the current cross-sectional study examined parents' experiences at the time of their child's diagnosis, what they thought helped their child recover, barriers to support, and identified needs for future models of care.MethodThe sample included 26 parents (22 mothers, 3 fathers, and 1 mother/father pair) of children with CHD, ranging in age between 6 months and 4 years with a mean age of 2 years.ResultsQualitative results were organized around five themes: (a) They (medical team) saved my child's life, (b) My child is going to be okay, (c) Not out of the woods, (d) Optimizing support for my child and myself, and (e) What still gets in the way. Parents uniformly expressed a need for greater mental health support for their children as well as programs to improve parents' skill and confidence, with no difference between age groups (&lt; 2 years and &gt; 2 years of age). Common barriers to service included distance and time off work.ConclusionParents' experiences informed both acute and long term implications following CHD diagnoses, and highlight current gaps in mental health care. Direction for clinical care and improved intervention opportunities are discussed.</jats:sec

A KLK4 proteinase substrate capture approach to antagonize PAR1

AbstractProteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site. On the basis of in silico site-saturation mutagenesis, we then generated KLK4S207A,L185D, a first-of-a-kind ‘decoy’ PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This ‘substrate-capturing’ KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 ‘proteinase substrate capture’ approach to regulate proteinase-mediated PAR1 signaling.</jats:p

Protein phosphatase 2A controls the order and dynamics of cell-cycle transitions.

Bistability of the Cdk1-Wee1-Cdc25 mitotic control network underlies the switch-like transitions between interphase and mitosis. Here, we show by mathematical modeling and experiments in Xenopus egg extracts that protein phosphatase 2A (PP2A), which can dephosphorylate Cdk1 substrates, is essential for this bistability. PP2A inhibition in early interphase abolishes the switch-like response of the system to Cdk1 activity, promoting mitotic onset even with very low levels of Cyclin, Cdk1, and Cdc25, while simultaneously inhibiting DNA replication. Furthermore, even if replication has already initiated, it cannot continue in mitosis. Exclusivity of S and M phases does not depend on bistability only, since partial PP2A inhibition prevents replication without inducing mitotic onset. In these conditions, interphase-level mitotic kinases inhibit Cyclin E-Cdk2 chromatin loading, blocking initiation complex formation. Therefore, by counteracting both Cdk1 activation and activity of mitotic kinases, PP2A ensures robust separation of S phase and mitosis and dynamic transitions between the two states