Indexing the Pseudomonas specialized metabolome enabled the discovery of poaeamide B and the bananamides

Pseudomonads are cosmopolitan microorganisms able to produce a wide array of specialized metabolites. These molecules allow Pseudomonas to scavenge nutrients, sense population density and enhance or inhibit growth of competing microorganisms. However, these valuable metabolites are typically characterized one-molecule–one-microbe at a time, instead of being inventoried in large numbers. To index and map the diversity of molecules detected from these organisms, 260 strains of ecologically diverse origins were subjected to mass-spectrometry-based molecular networking. Molecular networking not only enables dereplication of molecules, but also sheds light on their structural relationships. Moreover, it accelerates the discovery of new molecules. Here, by indexing the Pseudomonas specialized metabolome, we report the molecular-networking-based discovery of four molecules and their evolutionary relationships: a poaeamide analogue and a molecular subfamily of cyclic lipopeptides, bananamides 1, 2 and 3. Analysis of their biosynthetic gene cluster shows that it constitutes a distinct evolutionary branch of the Pseudomonas cyclic lipopeptides. Through analysis of an additional 370 extracts of wheat-associated Pseudomonas, we demonstrate how the detailed knowledge from our reference index can be efficiently propagated to annotate complex metabolomic data from other studies, akin to the way in which newly generated genomic information can be compared to data from public databases

Fortified breakfast cereal consumed daily for 12 wk leads to a significant improvement in micronutrient intake and micronutrient status in adolescent girls: a randomised controlled trial

Background: Poor micronutrient status is reported among adolescents across Europe and USA. This may be related to the well-documented decline in the regular consumption of breakfast by this group. The regular consumption of a breakfast cereal offers a possible means to improve micronutrient status; fortified cereal is likely to have enhanced benefit. A study was conducted to determine the efficacy of the regular consumption of a fortified cereal with milk, compared with unfortified cereal, consumed either as a breakfast or a supper, in improving micronutrient intake and micronutrient status of adolescent girls. Methods: A randomised, double-blind, placebo-controlled intervention trial was conducted in girls recruited at ages 16–19 years, from schools and colleges in Sheffield, UK. Girls were randomised to receive 50 g fortified or unfortified cereal, with 150 ml semi-skimmed milk, daily, for 12 weeks, as a breakfast or as a supper. Dietary intake was estimated using a 4-d food diary and blood collected for the assessment of nutritional status. Within-group changes were tested using a paired sample t test; two-way ANOVA was used to analyse effects of the intervention, with cereal type and time of consumption as factors, correcting for baseline values. The analysis was conducted on 71 girls who completed the study. Results: Consumption of unfortified cereal elicited an increase in the intake of vitamins B1, B2 and B6; consumption of fortified cereal elicited increases in vitamins B1, B2, B6, B12, folate and iron (P < 0.001) and of vitamin D (P = 0.007), all increases were significantly greater than for unfortified cereal. Consumption of the fortified cereal also led to a significant improvement in biomarkers of status for vitamins B2, B12, folate and of iron, compared with girls receiving the unfortified cereal, and maintained vitamin D status, in contrast with the girls receiving the unfortified cereal (P < 0.001). Conclusions: The daily consumption of cereal with milk for 12 weeks by adolescent girls, increased intakes of micronutrients. The consumption of fortified cereal elicited greater increases than for unfortified cereal and improved biomarkers of micronutrient status. The findings justify strategies to encourage the consumption of fortified cereal with milk by adolescents, either as a breakfast or a supper

The effect of iterative model reconstruction on coronary artery calcium quantification

Coronary artery calcium (CAC) scoring with computed tomography (CT) is an established tool for quantifying calcified atherosclerotic plaque burden. Despite the widespread use of novel image reconstruction techniques in CT, the effect of iterative model reconstruction on CAC score remains unclear. We sought to assess the impact of iterative model based reconstruction (IMR) on coronary artery calcium quantification as compared to the standard filtered back projection (FBP) algorithm and hybrid iterative reconstruction (HIR). In addition, we aimed to simulate the impact of iterative reconstruction techniques on calcium scoring based risk stratification of a larger asymptomatic population. We studied 63 individuals who underwent CAC scoring. Images were reconstructed with FBP, HIR and IMR and CAC scores were measured. We estimated the cardiovascular risk reclassification rate of IMR versus HIR and FBP in a larger asymptomatic population (n = 504). The median CAC scores were 147.7 (IQR 9.6-582.9), 107.0 (IQR 5.9-526.6) and 115.1 (IQR 9.3-508.3) for FBP, HIR and IMR, respectively. The HIR and IMR resulted in lower CAC scores as compared to FBP (both p < 0.001), however there was no difference between HIR and IMR (p = 0.855). The CAC score decreased by 7.2 % in HIR and 7.3 % in IMR as compared to FBP, resulting in a risk reclassification rate of 2.4 % for both HIR and IMR. The utilization of IMR for CAC scoring reduces the measured calcium quantity. However, the CAC score based risk stratification demonstrated modest reclassification in IMR and HIR versus FBP

CD34-related coexpression of MDR1 and BCRP indicates a clinically resistant phenotype in patients with acute myeloid leukemia (AML) of older age

Clinical resistance to chemotherapy in acute myeloid leukemia (AML) is associated with the expression of the multidrug resistance (MDR) proteins P-glycoprotein, encoded by the MDR1/ABCB1 gene, multidrug resistant-related protein (MRP/ABCC1), the lung resistance-related protein (LRP), or major vault protein (MVP), and the breast cancer resistance protein (BCRP/ABCG2). The clinical value of MDR1, MRP1, LRP/MVP, and BCRP messenger RNA (mRNA) expression was prospectively studied in 154 newly diagnosed AML patients ≥60 years who were treated in a multicenter, randomized phase 3 trial. Expression of MDR1 and BCRP showed a negative whereas MRP1 and LRP showed a positive correlation with high white blood cell count (respectively, p < 0.05, p < 0.001, p < 0.001 and p < 0.001). Higher BCRP mRNA was associated with secondary AML (p < 0.05). MDR1 and BCRP mRNA were highly significantly associated (p < 0.001), as were MRP1 and LRP mRNA (p < 0.001) expression. Univariate regression analyses revealed that CD34 expression, increasing MDR1 mRNA as well as MDR1/BCRP coexpression, were associated with a lower complete response (CR) rate and with worse event-free survival and overall survival. When adjusted for other prognostic actors, only CD34-related MDR1/BCRP coexpression remained significantly associated with a lower CR rate (p = 0.03), thereby identifying a clinically resistant subgroup of elderly AML patients

CD133 expression in chemo-resistant Ewing sarcoma cells

<p>Abstract</p> <p>Background</p> <p>Some human cancers demonstrate cellular hierarchies in which tumor-initiating cancer stem cells generate progeny cells with reduced tumorigenic potential. This cancer stem cell population is proposed to be a source of therapy-resistant and recurrent disease. Ewing sarcoma family tumors (ESFT) are highly aggressive cancers in which drug-resistant, relapsed disease remains a significant clinical problem. Recently, the cell surface protein CD133 was identified as a putative marker of tumor-initiating cells in ESFT. We evaluated ESFT tumors and cell lines to determine if high levels of CD133 are associated with drug resistance.</p> <p>Methods</p> <p>Expression of the CD133-encoding <it>PROM1 </it>gene was determined by RT-PCR in ESFT tumors and cell lines. CD133 protein expression was assessed by western blot, FACS and/or immunostaining. Cell lines were FACS-sorted into CD133+ and CD133- fractions and proliferation, colony formation in soft agar, and <it>in vivo </it>tumorigenicity compared. Chemosensitivity was measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays.</p> <p>Results</p> <p><it>PROM1 </it>expression was either absent or extremely low in most tumors. However, <it>PROM1 </it>was highly over-expressed in 4 of 48 cases. Two of the 4 patients with <it>PROM1 </it>over-expressing tumors rapidly succumbed to primary drug-resistant disease and two are long-term, event-free survivors. The expression of <it>PROM1 </it>in ESFT cell lines was similarly heterogeneous. The frequency of CD133+ cells ranged from 2-99% and, with one exception, no differences in the chemoresistance or tumorigenicity of CD133+ and CD133- cell fractions were detected. Importantly, however, the STA-ET-8.2 cell line was found to retain a cellular hierarchy in which relatively chemo-resistant, tumorigenic CD133+ cells gave rise to relatively chemo-sensitive, less tumorigenic, CD133- progeny.</p> <p>Conclusions</p> <p>Up to 10% of ESFT express high levels of <it>PROM1</it>. In some tumors and cell lines the CD133+ fraction is relatively more drug-resistant, while in others there is no apparent difference between CD133+ and CD133- cells. These studies reveal heterogeneity in <it>PROM1</it>/CD133 expression in ESFT tumors and cell lines and confirm that high levels of <it>PROM1 </it>expression are, in at least some cases, associated with chemo-resistant disease. Further studies are required to elucidate the contribution of <it>PROM1/</it>CD133 expressing cells to therapeutic resistance in a large, prospective cohort of primary ESFT.</p

Chronic Myeloid Leukemia Stem Cell Biology

Leukemia progression and relapse is fueled by leukemia stem cells (LSC) that are resistant to current treatments. In the progression of chronic myeloid leukemia (CML), blast crisis progenitors are capable of adopting more primitive but deregulated stem cell features with acquired resistance to targeted therapies. This in turn promotes LSC behavior characterized by aberrant self-renewal, differentiation, and survival capacity. Multiple reports suggest that cell cycle alterations, activation of critical signaling pathways, aberrant microenvironmental cues from the hematopoietic niche, and aberrant epigenetic events and deregulation of RNA processing may facilitate the enhanced survival and malignant transformation of CML progenitors. Here we review the molecular evolution of CML LSC that promotes CML progression and relapse. Recent advances in these areas have identified novel targets that represent important avenues for future therapeutic approaches aimed at selectively eradicating the LSC population while sparing normal hematopoietic progenitors in patients suffering from chronic myeloid malignancies

Processing of Hand-Related Verbs Specifically Affects the Planning and Execution of Arm Reaching Movements

Even though a growing body of research has shown that the processing of action language affects the planning and execution of motor acts, several aspects of this interaction are still hotly debated. The directionality (i.e. does understanding action-related language induce a facilitation or an interference with the corresponding action?), the time course, and the nature of the interaction (i.e. under what conditions does the phenomenon occur?) are largely unclear. To further explore this topic we exploited a go/no-go paradigm in which healthy participants were required to perform arm reaching movements toward a target when verbs expressing either hand or foot actions were shown, and to refrain from moving when abstract verbs were presented. We found that reaction times (RT) and percentages of errors increased when the verb involved the same effector used to give the response. This interference occurred very early, when the interval between verb presentation and the delivery of the go signal was 50 ms, and could be elicited until this delay was about 600 ms. In addition, RTs were faster when subjects used the right arm than when they used the left arm, suggesting that action–verb understanding is left-lateralized. Furthermore, when the color of the printed verb and not its meaning was the cue for movement execution the differences between RTs and error percentages between verb categories disappeared, unequivocally indicating that the phenomenon occurs only when the semantic content of a verb has to be retrieved. These results are compatible with the theory of embodied language, which hypothesizes that comprehending verbal descriptions of actions relies on an internal simulation of the sensory–motor experience of the action, and provide a new and detailed view of the interplay between action language and motor acts

Immunoregulation of bovine macrophages by factors in the salivary glands of Rhipicephalus microplus

<p>Abstract</p> <p>Background</p> <p>Alternative strategies are required to control the southern cattle tick, <it>Rhipicephalus microplus</it>, due to evolving resistance to commercially available acaricides. This invasive ectoparasite is a vector of economically important diseases of cattle such as bovine babesiosis and anaplasmosis. An understanding of the biological intricacies underlying vector-host-pathogen interactions is required to innovate sustainable tick management strategies that can ultimately mitigate the impact of animal and zoonotic tick-borne diseases. Tick saliva contains molecules evolved to impair host innate and adaptive immune responses, which facilitates blood feeding and pathogen transmission. Antigen presenting cells are central to the development of robust T cell responses including Th1 and Th2 determination. In this study we examined changes in co-stimulatory molecule expression and cytokine response of bovine macrophages exposed to salivary gland extracts (SGE) obtained from 2-3 day fed, pathogen-free adult <it>R. microplus</it>.</p> <p>Methods</p> <p>Peripheral blood-derived macrophages were treated for 1 hr with 1, 5, or 10 μg/mL of SGE followed by 1, 6, 24 hr of 1 μg/mL of lipopolysaccharide (LPS). Real-time PCR and cytokine ELISA were used to measure changes in co-stimulatory molecule expression and cytokine response.</p> <p>Results</p> <p>Changes were observed in co-stimulatory molecule expression of bovine macrophages in response to <it>R</it>. <it>microplus </it>SGE exposure. After 6 hrs, CD86, but not CD80, was preferentially up-regulated on bovine macrophages when treated with 1 μg/ml SGE and then LPS, but not SGE alone. At 24 hrs CD80, CD86, and CD69 expression was increased with LPS, but was inhibited by the addition of SGE. SGE also inhibited LPS induced upregulation of TNFα, IFNγ and IL-12 cytokines, but did not alter IL-4 or CD40 mRNA expression.</p> <p>Conclusions</p> <p>Molecules from the salivary glands of adult <it>R. microplus </it>showed bimodal concentration-, and time-dependent effects on differential up-regulation of CD86 in bovine macrophages activated by the TLR4-ligand, LPS. Up regulation of proinflammatory cytokines and IL-12, a Th1 promoting cytokine, were inhibited in a dose-dependent manner. The co-stimulatory molecules CD80, as well as the cell activation marker, CD69, were also suppressed in macrophages exposed to SGE. Continued investigation of the immunomodulatory factors will provide the knowledge base to research and develop therapeutic or prophylactic interventions targeting <it>R. microplus</it>-cattle interactions at the blood-feeding interface.</p

An Information Theoretic, Microfluidic-Based Single Cell Analysis Permits Identification of Subpopulations among Putatively Homogeneous Stem Cells

An incomplete understanding of the nature of heterogeneity within stem cell populations remains a major impediment to the development of clinically effective cell-based therapies. Transcriptional events within a single cell are inherently stochastic and can produce tremendous variability, even among genetically identical cells. It remains unclear how mammalian cellular systems overcome this intrinsic noisiness of gene expression to produce consequential variations in function, and what impact this has on the biologic and clinical relevance of highly ‘purified’ cell subgroups. To address these questions, we have developed a novel method combining microfluidic-based single cell analysis and information theory to characterize and predict transcriptional programs across hundreds of individual cells. Using this technique, we demonstrate that multiple subpopulations exist within a well-studied and putatively homogeneous stem cell population, murine long-term hematopoietic stem cells (LT-HSCs). These subgroups are defined by nonrandom patterns that are distinguishable from noise and are consistent with known functional properties of these cells. We anticipate that this analytic framework can also be applied to other cell types to elucidate the relationship between transcriptional and phenotypic variation

Live cell dynamics of production, explosive release and killing activity of phage tail-like weapons for Pseudomonas kin exclusion.

Interference competition among bacteria requires a highly specialized, narrow-spectrum weaponry when targeting closely-related competitors while sparing individuals from the same clonal population. Here we investigated mechanisms by which environmentally important Pseudomonas bacteria with plant-beneficial activity perform kin interference competition. We show that killing between phylogenetically closely-related strains involves contractile phage tail-like devices called R-tailocins that puncture target cell membranes. Using live-cell imaging, we evidence that R-tailocins are produced at the cell center, transported to the cell poles and ejected by explosive cell lysis. This enables their dispersal over several tens of micrometers to reach targeted cells. We visualize R-tailocin-mediated competition dynamics between closely-related Pseudomonas strains at the single-cell level, both in non-induced condition and upon artificial induction. We document the fatal impact of cellular self-sacrifice coupled to deployment of phage tail-like weaponry in the microenvironment of kin bacterial competitors, emphasizing the necessity for microscale assessment of microbial competitions