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Unexpected Favourites
Whining, pouting, crying and tantrums are all a part of a normal day when you have a toddler. Sometimes the toddlers even do those things too. Ha! If you're a parent of a toddler or have already gone through that phase, you know very well what it's like to have emotional whiplash on a daily basis. These are typically NOT the times when you'd likely be breaking out your camera to capture these special moments as digital pixels to save forever. Let's face it, we all want those minutes of madness to pass as quickly as possible. And when you're in the thick of it, holding on to those epic (and mild) emotions is not on the priority list.
I will admit that when I'm photographing families with toddlers I LOVE IT when they have their inevitable breakdowns. Why?! Because this shizer is R.E.A.L. folks! The crying tantrum is a universal sign that you are in the midst of Crazy Land and we ALL go through it to some degree. And better yet, when you come out of Crazy Land and everyone has survived the ride it's a pretty big deal that deserves a high five and some serious stickers on the reward board of life.
What surprises people the most from a documentary family photo session is just how much they like the photos of the hard moments. Because kids look like this a surprising amount when they are in their younger years and it's as important to treasure those super real + genuine moments as it is the happy, swinging, jumping and smiling ones.

subset: commoncrawl


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Carla A. Arena Ventura, PhD, MBA, MS
Carla A. A. Ventura is a Full Professor at the Department of Psychiatric Nursing and Human Sciences from the University of São Paulo at Ribeirão Preto College of Nursing. She coordinates an interdisciplinary Research Group on Global Health, Law and Development (GEPESADES), leading a team of undergraduate, Master, Doctoral and Post-Doctoral students. Since June 2017, she is the Director of the PAHO/WHO Collaborating Centre for Nursing Research Development at the University of São Paulo at Ribeirão Preto College of Nursing, Brazil. During 2017, she was a fellow of the Program Leaders for Health Equity, offered by George Washington University, Milken Institute School of Public Health, USA. Since May 2018, she is an Adjunct International Professor at the John Dossetor Health Ethics Centre, University of Alberta, Canada. She is a member of the Editorial Board of the Nursing Ethics Journal and is being nominated an Associate Editor for South America of the Primary Health Care Research and Development Journal. She develops research on human rights, mental health and drugs; health equity; stigma and human rights; health law; global health and development; human rights education, social participation and control.
Focus: Teaches human rights, right to health, and the social determinants of health to nursing students, and coordinates interdisciplinary and international research on nursing, global health, law and development.
Lives in: Riberão Preto, Brazil


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Mapped by Rob , Stewart Long, Rob Goodwin
Cartographer: Stewart Long
Published by gonzoearth
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Ground resolution: 22.0 cm/px
Capture date: 2010-06-02T00:00:00
Publication date: 2011-05-23T00:00:00
License: Public Domain
Mapping the property of Delbert Gunnoe, Dry Creek, WV.
While on this trip we created an improvised tether while in the field in order to fly high altitudes. In the somewhat rural area of Eastern Kentucky that we were in we had access to a farm supply store. Choosing from available parts at the farm supply store. The tether spool during construction.
Hi Stew--perhaps you could update the image links? And is this a mountain top removal site?
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Inspiring 'What Is Best in Nursing'Posted by Karen Haller | Academic Medicine, Collaboration, Health Care Leadership Development, Latin America, Medical Education, Nurse Development
The 1893 World's Fair in Chicago introduced Americans to hamburgers, the Ferris wheel—and the idea of higher education for nurses. Nurses hosted their first global meeting there, advocating for an educated workforce with professional standards of practice, rather than their traditional role as apprentices in hospitals. This meeting attracted notable and progressive nursing leaders, including(...)
Dec 20, 2017 No comments
Advancing Nursing in Saudi Arabia: A Case StudyPosted by Guest Author | Collaboration, Health Care Leadership Development, Medical Education, Middle East, Nurse Development
By Zeina Khouri-Stevens I was pleased when my colleague Karen Haller approached me to tag-team on a pair of posts about how Johns Hopkins Medicine International is evolving and empowering the nursing profession at its global affiliations. We decided to use Johns Hopkins Aramco Healthcare (JHAH) as a successful case study on how we are(...)
Dec 14, 2017 1 comment
Why I Can't Say Enough About the Importance of NursingPosted by Karen Haller | Collaboration, Health Care Leadership Development, Latin America, Medical Education, Middle East, Nurse Development
Countries worldwide are facing unprecedented challenges related to health and health care. This is made worse by a global shortage of 17.4 million qualified health care workers (2.6 million doctors and more than 9 million nurses), according to the World Health Organization. Half of all hospitals are operating with personnel deficits today. If current trends(...)
Dec 12, 2017 2 comments
Diet vs. Drugs in Treating High Blood PressurePosted by Charles Wiener | Academic Medicine, Health Innovation, Research
If you're one of the 1.13 billion people worldwide who are suffering from high blood pressure, Johns Hopkins researchers have some encouraging news. According to a new study our investigators published in the Nov. 12 issue of Journal of the American College of Cardiology, changes to your diet in conjunction with reduced salt intake may(...)
Dec 7, 2017 3 comments
Saving Lives by Lowering High Blood PressurePosted by oscarcingolani | Health Innovation, Medical Technology, Patient Care, Patient Safety, Precision Medicine, Research
By Dr. Oscar Cingolani We all know high blood pressure is a global threat, often called "the silent killer." In fact, hypertension is responsible for one in eight deaths worldwide, and it is the major culprit behind stroke, heart and kidney disease. In a new effort to curb this deadly impact, the American Heart Association(...)
Dec 5, 2017 No comments
Partners Forum 2017: Outcomes and Off-site CarePosted by Mohan Chellappa | Collaboration, Health Innovation, Health System Sustainability, Latin America, Medical Technology, Middle East, Patient Care, Patient Safety
Each year at Johns Hopkins Medicine International's Partners Forum, leaders from our global affiliates discuss ways to improve health and health care for the collective good. This year's theme, "The Evolution of Health Care: Predictions and Practices in a Changing Environment," helped us better understand the context and landscape of health care today. We wrestled(...)
Nov 30, 2017 No comments
Partners Forum 2017: Data vs. Delays in CarePosted by Mohan Chellappa | Collaboration, Health Innovation, Health System Sustainability, Medical Technology, Middle East, Patient Care, Patient Safety
We were thrilled to host the recent Johns Hopkins Medicine International Partners Forum, an annual meeting that brings together leaders, care providers and researchers from our nearly 20 affiliates in 15 countries. Looking at the past 20 years of my involvement with Johns Hopkins Medicine International when these relationships developed, this is an event I(...)
Nov 28, 2017 1 comment
Medical Education Across BordersPosted by Charles Wiener | Academic Medicine, Collaboration, Health Innovation, Health System Sustainability, Medical Education
As a Johns Hopkins faculty member, I have the privilege of teaching medical students and residents in the classroom and on the wards. I love participating in the learning process—helping students grapple with information, evaluate its potential for impact, and finally put it into play in their own careers and lives. Through my role as(...)
Nov 24, 2017 No comments
Brain Drain or Gain?Posted by Pamela Paulk | Academic Medicine, Asia Pacific, Collaboration, Health Care Leadership Development, Health Innovation, Health System Sustainability, Latin America, Medical Education, Nurse Development
Health care professionals have always been attracted to developed countries in search of better career and educational opportunities, political stability, and greater personal freedom, among myriad other factors. However, we've seen this trend go up significantly in recent years. The perceived need to relocate starts early for young people who dream of becoming nurses, physicians,(...)
Nov 22, 2017 2 comments


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Antimicrobial drug resistance affects broad changes in metabolomic phenotype in addition to secondary metabolism
Edited by Christopher T. Walsh, Harvard Medical School, Boston, MA, and approved December 20, 2012 (received for review October 23, 2012)
Bacteria develop resistance to many classes of antibiotics vertically, by engendering mutations in genes encoding transcriptional and translational apparatus. These severe adaptations affect global transcription, translation, and the correspondingly affected metabolism. Here, we characterize metabolome scale changes in transcriptional and translational mutants in a genomically characterized Nocardiopsis, a soil-derived actinomycete, in stationary phase. Analysis of ultra-performance liquid chromatography–ion mobility–mass spectrometry metabolomic features from a cohort of streptomycin- and rifampicin-resistant mutants grown in the absence of antibiotics exhibits clear metabolomic speciation, and loadings analysis catalogs a marked change in metabolic phenotype. Consistent with derepression, up to 311 features are observed in antibiotic-resistant mutants that are not detected in their progenitors. Mutants demonstrate changes in primary metabolism, such as modulation of fatty acid composition and the increased production of the osmoprotectant ectoine, in addition to the presence of abundant emergent potential secondary metabolites. Isolation of three of these metabolites followed by structure elucidation demonstrates them to be an unusual polyketide family with a previously uncharacterized xanthene framework resulting from sequential oxidative carbon skeletal rearrangements. Designated as "mutaxanthenes," this family can be correlated to a type II polyketide gene cluster in the producing organism. Taken together, these data suggest that biosynthetic pathway derepression is a general consequence of some antibiotic resistance mutations.
Increasing evidence is accumulating that a large fraction of antibiotic resistance may be elicited via horizontal transfer of resistance elements from the reservoir of microbial diversity found in soil (1, 2). Additionally, bacterial resistance toward a significant subset of antibiotics is rapidly generated vertically by mutations in central housekeeping genes encoding polymerases and ribosomal subunits. For example, bacterial resistance to rifampicin is afforded by mutations in the RNA polymerase β-subunit, encoded by rpoB, whereas resistance to streptomycin results from mutations in the ribosomal 30S subunit and/or ribosomal methyltransferases (3⇓–5). Notably in these adaptive strategies, a small number of mutations ultimately result in complex and significant global changes in gene expression and protein synthesis. For instance, rpoB mutations foster the up-regulation of proteins involved in central metabolism (including nucleoside and nucleotide, amino acid, carbohydrate, lipid, and phospholipid biosynthesis), detoxification, signal transduction, protein synthesis, and cell envelope processes, to name a few, whereas cell division proteins are generally down-regulated (6, 7). Streptomycin resistance, via rpsL mutation (encoding for ribosomal protein S12, a subunit of the 30S ribosome), leads to increased translational accuracy, slower overall translation, and has demonstrated late growth phase protein synthesis increases in comparison with the wild type (8). It follows causally that these changes are predicted to result in wholesale alterations in metabolism. Indeed, recent studies describe an apparent connection between vertically acquired antibiotic resistance and secondary metabolic fecundity in antibiotic-producing soil organisms such as the actinomycetes. The acquisition of resistance to antibiotics in these strains results in enhanced levels of antibiotic production for both known (8⇓–10) and previously unknown (11, 12) compounds. These data suggest that vertically selected antibiotic resistance may be a general strategy for eliciting secondary metabolism, but the scope of metabolic changes resulting from these mutations remains to be systematically described.
Herein, we describe an assessment of the consequences of antibiotic resistance mutations on a comprehensive metabolome scale. Comparative analysis of metabolomic data are greatly facilitated by using multivariate statistical analysis methods [e.g., principal component analysis (PCA), orthogonal projection to latent structures-discriminant analysis (OPLS-DA)], which convert multidimensional microbial extract ultra-performance liquid chromatography (UPLC)–MS data into lower-dimensional plots of maximally distinguishing eigenvectors (13). Critical loadings evaluation (14) efficiently validates the observed changes and identifies subsets of metabolites that are unique or shared within the datasets of a given antibiotic-resistant mutant and its progenitor strain. With these tools, we endeavor to describe and discriminate how antibiotic-induced mutations affect global metabolite pools and how PCA-based comparative analysis can identify secondary metabolite derepression in an unbiased fashion. Using a genomically characterized soil actinomycete of the Nocardiopsis genus as an example, we demonstrate the degree of metabolomic speciation afforded by antibiotic resistance mutations in the RNA polymerase β-subunit and the ribosome. Additionally, this analysis facilitates the rapid identification of up-regulated secondary metabolites. Isolations of several up-regulated features, followed by structure elucidation by 2D-NMR and isotopic incorporation experiments, reveal a polyketide carbon framework that may be correlated to a formerly unexpressed type II polyketide gene cluster present in the producing strain. Methods described herein will enable future studies to determine if this derepression is an adaptive or coincidental trait, in the context of the evolutionary benefits of antibiotic resistance.
Results and Discussion
Generation of a Cohort of Antibiotic-Resistant Mutants.
Nocardiopsis sp. FU40 ΔApoS8 was selected as a case study as it has been sequenced to high coverage, contains at least 19 identified secondary metabolic gene clusters, including several putative polyketides, and because the only secondary metabolite family identified from this organism to date are the proapoptotic macrolide polyketide apoptolidins A–H (15). We have previously disabled the biosynthetic genes for the production of apoptolidins via gene replacement of the terminal polyketide synthase ApoS8 with an apramycin resistance cassette, yielding a clean background for secondary metabolite analysis (16). Antibiotic-resistant mutants of this strain, Nocardiopsis sp. FU40 ΔApoS8, were generated by dilution plating of spore preparations on antibiotic-containing media followed by selection of several rifampicin (R1–R5)- or streptomycin (S1–S6)-resistant colonies with and without obvious morphological phenotypic differences.
Resistant and progenitor strains were then grown in an antibiotic-free vegetative medium and in production cultures from which total culture metabolite extracts were generated. Solid-phase extraction of combined broth and mycelium with Diaion HP-20 resin followed by extraction of mycelium and resin with increasingly nonpolar solvents captured a wide subset of moderate to highly hydrophobic excreted and cell-associated components. Genetic analyses of rpoB and ribosomal mutations were performed by PCR amplification and resequencing of the corresponding genes (summarized in SI Appendix, Table S1B). Sequencing of rpoB in resistant colonies demonstrated that all genes possessed a single G→A transition at position 3,280, a mutation not previously reported in antibiotic-resistant Streptomyces (9). Similarly, among streptomycin-resistant clones, a handful of resistance mutations were observed in S3,4,6, whereas in S1,2,5 no mutations were present in the amplified rpsL gene, suggesting that resistance to streptomycin in Nocardiopsis may occur outside of previously reported regions.
Analysis of Changes in Extracted Metabolomes in Antibiotic-Resistant Mutants.
The challenges to identifying mutation-induced changes in metabolism include detection/identification of discrete metabolites and ascertaining changes in generated metabolomes. We used unsupervised PCA of UPLC-ion mobility–mass spectrometry (IM-MS) data followed by loadings-plot evaluation, reasoning that linking metabolomics changes to a biological phenomenon would be facilitated by identifying metabolic species that are unique and/or shared to mutational phenotype. UPLC-IM-MS data were first manually inspected to verify reproducible peak retention times and intensities across samples and then peak deconvoluted and deisotoped to provide discrete features corresponding to a specific retention time and m/z pair. These data were normalized to total ion count, and each feature was used as a dimension for subsequent PCA. This results in relatively smaller changes being attenuated less than large-fold changes (17). Data compression occurs through the determination of the highest eigenvalue eigenvectors of the covariance matrix. The largest eigenvalue eigenvector is considered the principal component and describes the greatest variation in the data, with each subsequent eigenvector being orthogonal and describing progressively less variation. This provides sample grouping and separation based on patterns in the most significant detected features.
As shown in Fig. 1A, PCA effectively clusters technical replicates of UPLC-IM-MS–analyzed metabolomes from various antibiotic-resistance mutants selected by streptomycin and rifampicin plating, reflecting changes induced in the translational and transcriptional apparatus. Each data point represents 1,065 feature/intensities, generated by UPLC-IM-MS and peak identification using MarkerLynx (Waters). Plotting selected mutants in this manner reveals, without manual analysis of chromatograms or other visual phenotypic bias, which members are the most metabolically distinct from the progenitor strain. Interestingly, there is a marked metabolomic differentiation between antibiotic-resistant clones selected using the same antibiotic. In the case of rifampicin-induced mutations, for example, which possess identical mutations in rpoB, these data suggest that additional mutations are responsible for transcriptional variation. This likelihood is in accordance with observations from compensatory mutations found in streptomycin- and rifampicin-resistant organisms (18, 19), which have been demonstrated to reverse the fitness costs of ribosomal and polymerase mutations. The PCA plots also demonstrate that different antibiotic-induced mutations inducing transcriptional or translational variation can result in apparently similar global changes in metabolism.
Analysis of Metabolic Features.
Interpretation of the significance of groupings determined via PCA requires that the individual variance components be identified and, if possible, validated. Correspondingly, we endeavored to identify strain-specific metabolic features to develop an understanding of the lasting metabolic perturbations resulting from the acquisition of antibiotic resistance. Aiding in these identifications, the addition of IM (20) provided sufficient analyte separation to acquire untargeted fragmentation data of all detected species. Using both chromatography and mobility dimensions, high-energy fragmentation data for analytes of interest were extracted by correlating both retention and drift times of product ions to precursors. This depletion of coeluting species and chemical noise increases the confidence of identification. In-source fragmentation can be discerned from retention and drift time correlations.
Loadings analysis is a tool used for identifying the contribution of individual peaks to the PCA dispersion. The cosine of the angle between a component and a feature describes the weight of that particular feature for that component. For example, Fig. 1B shows three-way loadings analysis for selected strains shown in Fig. 1C. To interpret these plots, the features in Fig. 1B aligned along the vector of the strains in the inset PCA plot in Fig. 1C represent those species that contribute most to the dispersion in the PCA analysis. Features with greater magnitudes describe more group variation, through both uniqueness and intensity in the UPLC-IM-MS. The three-way loadings analysis is useful for illuminating clone-specific biosynthesis shared among cohorts. An analogous pairwise comparison (SI Appendix, Fig. S2) of a mutant to its progenitor stain is also useful for identifying new features in a single mutant (14). Fig. 1D demonstrates how loadings analysis successfully identifies and deconvolutes clone-specific metabolites from UPLC-IM-MS data without manual chromatogram analysis. Table 1 summarizes a selection of the most abundant metabolites unique to R4, S5, and the progenitor strains, respectively. We have included a complete list of the 280 up-regulated metabolites in this mutant pair, as well as extracted ion chromatograms (SI Appendix, Figs. S73–S99) and MS/MS analyses for all features in Table 1 (SI Appendix, Figs. S37–S72).
Fig. 2A depicts a summary of the global effects of acquiring antibiotic resistance on the metabolomes for R4, S5 mutants and WT via the tripartite analysis of Fig. 2B. These values were obtained by permuting the three groups in binary comparisons using OPLS-DA, a supervised multivariate statistical method that defines the first component (abscissa) as the eigenvector describing the greatest intergroup variation, with the second component (ordinate) defined as the eigenvector describing the greatest intragroup separation (21). A large portion of the features are present in all groups. In addition to metabolites specific to a clonal metabolome, this also includes unmetabolized media components and other sample background peaks. Of the 993 total features identified as exclusive to S5 and R4 mutants, and the wild-type Nocardiopsis sp. FU40 ΔApoS8, 56 features are exclusively expressed in the wild-type Nocardiopsis sp. FU40 ΔApoS8, 641 are shared between the wild type and at least one mutant, and 280 of all features are produced as a result of rpoB or rpsL mutation. The resulting model was used to generate a plot of loadings contribution vs. correlation value. To generate group-specific features, a correlation threshold of ≥0.9 was applied.
Half-matrix representations of these comparisons is displayed in Fig. 2 B and C. Fig. 2B can be interpreted as the number of unique features specific to the metabolomic extract of the organism indicated on the bottom row when compared specifically to the organism on the left column. Conversely, the half-matrix in Fig. 2C represents the number of unique features specific to the organisms indicated on the right column, when compared specifically to the organisms on the bottom row. For perspective, a similar comparison of Escherichia coli, Micrococcus luteus, and Bacillus subtilis yields feature differences on the order of as little as 13 specific features, and a maximum of 166 unique features, when compared in a binary fashion (SI Appendix, Fig. S1). This suggests that the metabolic phenotypes of the mutant organisms are on the scale of interphylogenic differences. Of note, the biosynthesis of new features not detected, or at low levels, in the progenitor strain is often found to be increased in multiple distinct mutants. The number of newly observed features found in the resistant mutants range from 100 to over 300 (237 on average) in comparison with the nonresistant Nocardiopsis progenitor, whereas the number of features unique to the progenitor strain in comparison with the mutants average 80. For example, an increase in feature 14, putatively identified as ectoine, is observed in multiple antibiotic resistance mutants. Likewise, features 11–15, in addition to many others, are shared by mutant groups. This includes the up-regulation of a putatively identified quorum-sensing molecule (feature 13), dipeptides (feature 11), and features with no known match (feature 10, for example). Interestingly, lipid-like features in the progenitor Nocardiopsis (e.g., features 23, 24, and 27) appear attenuated in all antibiotic-resistant strains. Together, these trends and specific analyses are consistent with generalized gene derepression as a consequence of mutations in the rpsL and rpoB genes. Normalized heat maps of features described as unique in Fig. 2 A–C are found in SI Appendix, Tables S10–S23 and Datasets S1–S4.
Isolation and Identification of Selected Secondary Metabolic Features.
Many of the metabolic features corresponding to up-regulated species in antibiotic-resistant mutants (207 of ∼650) possess high molecular weights (>400 Da) and are found to be up-regulated in more than one mutant. To investigate whether these species correspond to secondary metabolites, we isolated a subset of the most abundant features in rifampicin-resistant mutant R4.
Feature 1 (Fig. 3B, and SI Appendix, Table S2) was isolated with UV spectrum showing absorption maxima at 225, 252, 279, and 409 nm. The high-resolution mass spectrum yields an apparent m/z of 443.101 [M+H]+ and, along with UV and NMR data, yields a chemical formula of C22H18O10 that does not correlate with known entries in the aforementioned chemical databases. Challenges to structural elucidation of feature 1 include the large number of quaternary carbons, a high degree of unsaturation, and rapid dynamic chemical exchange properties of several resonances over the timescale of the NMR experiments. Briefly, three partial structures (I–III; Fig. 3A) were proposed on the basis correlation spectroscopy (COSY), heteronuclear single-quantum coherence (HSQC), heteronuclear multiple-bond correlation spectroscopy (HMBC), and adequate double-quantum transfer experiment (ADEQUATE) NMR correlations. HMBC correlations from C14 methylene protons to C3 quaternary carbon determine the relationship of fragment II with fragment III and crucial HMBC and 1,1-ADEQUATE correlations from protons H12 to carbon C11a and from proton C4a to carbon C5 determine the connectivity between fragments I and II and permit the closure of the last ring, which revealed that the compound contains an unusual xanthene scaffold (Fig. 3B). This compound, which we named mutaxanthene A 1, contained two minor variants, mutaxanthenes B 2 and C 3, which correlate to features 2 and 3 from the loadings analysis in Fig. 1B. The remaining four most abundant features in this plot correspond to adducts of the mutaxanthenes. As less abundant features, they further validate the utility of loadings analysis in comparing metabolomic perturbations in antibiotic-resistant mutants.
An advantageous property of mutaxanthenes A–C is their ready conversion in pH ∼6.6 ammonium acetate buffer into mutaxanthenes D (4) and E (5), which are tautomeric eneamine congeners of their parent compounds (Fig. 3B). Notably, substantial line broadening of 13C resonances of C1, C2, and C4 in mutaxanthenes A–C, ostensibly due to dynamic chemical exchange of the enol tautomers, appears as sharp singlets in D and E (SI Appendix, Fig. S35). This line width improvement in the amino congeners permits critical J-coupling values to be obtained for these atoms from 1,2-13C sodium acetate incorporation experiments (SI Appendix, Fig. S36). Correspondingly, the structures of mutaxanthenes A–E are supported by these incorporation experiments, which confirm the substructures and correct assembly of the xanthene scaffold and support the likely type II polyketide synthase origins of this family of compounds. A detailed description of NMR studies can be found in SI Appendix.
Analysis of Biosynthetic Gene Cluster and Proposed Biosynthetic Pathway.
The 12,12a-dihydro-1H-benzo[b]xanthene–based carbon framework of the mutaxanthenes has, to our knowledge, never before been reported in polyketide natural products. This unusual scaffold requires multiple skeletal rearrangements of an unreduced decaketide precursor. Although the process for generating the xanthene scaffold remains highly speculative, we can propose a pathway (Fig. 3C) consistent with the following features: (i) acetate incorporation reveals that the two carbons at positions C16 and C4a are uncoupled, suggesting that these carbons were once an intact acetate unit in the decaketide chain; (ii) the ether and carboxylate moieties together with the position of the uncoupled acetate-derived carbons formally imply oxidative cleavage, but not cleavage between the aforementioned acetate unit; (iii) the position of the acetate-derived carboxylate in the middle of the polyketide chain suggests an additional carbon skeletal rearrangement involving a ring contraction. In the proposed pathway, the intermediate precursor 6 is common to several polyketide families including polyketomycin (22) and mithramycin (23). The first oxidative cleavage resulting in 7 is a regiochemical variant of the Baeyer–Villiger oxidation found in mithramycin biosynthesis (24). The ring contraction rearrangement may occur in analogy to the Favorskii-like reaction proven in the biosynthesis of the enterocins (25) and implied in okadaic acids (26), in which an oxidative carbonylation sequence via 8 precedes the formation of a cyclopropanone intermediate 9, which upon hydration may rearrange to the internally situated carboxylate 11. An intramolecular Michael addition may be proposed to complete the xanthene framework. The previously reported genome sequence of Nocardiopsis sp. FU 40 ΔApoS8 contains one typical type II polyketide gene cluster. Analysis of this gene cluster (abbreviated Mux; SI Appendix, Table S7) reveals hypothetical polyketide synthase proteins consistent with the generation of the common precursor 6 and genes encoding translated sequence similarity to flavin-dependent oxidases (Mux O1, O2, O3) consistent with the proposed oxidative rearrangement biotransformations. Future studies will define the relationship of these oxidases to the skeletal rearrangements implied in the proposed biosynthesis mutaxanthenes A–C.
We are broadly interested in detecting and identifying metabolome-scale changes in microorganisms and understanding their roles in cellular processes. As the end product of the central dogma, metabolites and their dynamics may be used to complete our understanding of the molecular basis for biological phenomenon such as cell signaling, antibiotic resistance, pathogenicity, and metabolic fitness. The subject of this work, antibiotic resistance-induced changes in the transcriptional and translational apparatus of bacteria, has multiple aspects of interest. Investigating metabolome-scale changes resulting from these adaptations may provide insight into these widespread modes of vertically acquired bacterial resistance in the clinic. Additionally, due to the established connection between acquired resistance adaptations and secondary metabolism, unbiased analytical methods to assess new features engendered by resistance can potentially accelerate natural product discovery.
To address these subjects, we describe here metabolome-scale changes from resistance mutations in a secondary metabolite producing nonpathogenic soil organism. Notably, similarly drastic regulatory adaptations, engendered by mutations in the S12 ribosomal subunit and rpoB polymerase, are stably maintained in some, but not all clinically resistance strains (e.g., Mycobacterium tuberculosis, Streptococcus pyogenes, Staphylococcus aureus, Neisseria gonorrheae) (27⇓–29). However, despite the importance of antibiotic-selected mutations in both pathogenic and antibiotic-producing microorganisms, the downstream effects of a modified transcriptome and/or proteome on global metabolism have not been broadly investigated in a systematic manner. Here, we quantify the metabolic feature differences in stationary phase between cohorts of mutants revealing the scope of changes in metabolomic phenotype. Notably, the number of previously undetected features found in resistant mutants is, on average, threefold greater than the number of features identified as unique to wild type using the same comparison. Together with the results that these up-regulated features number in the hundreds and that the majority of the up-regulated features are not found in the progenitor Nocardiopsis are shared throughout the diverse mutant cohort, suggest that one means by which S12/rpoB mutations affect survival may be by a strategy of general derepression of biosynthetic processes.
In the case of secondary metabolite producing actinomycetes, antibiotic-induced S12 ribosomal subunit and rpoB polymerase mutants have been demonstrated to modulate the production of specific secondary metabolites resulting in increased production of poorly expressed or "silent" gene clusters (5, 8). However, it is not known whether secondary metabolite production is simply a symptom of broader transcriptional and translational derepression, or a specific adaptive response to antibiotic challenge. The results described here measure metabolomic changes in bacteria in the contexts of the phenomenon of vertically acquired antibiotic resistance and secondary metabolism. The observation that a large fraction of accumulated metabolites are of high molecular weight, are not identifiable in primary metabolism databases, and are demonstrated to be secondary metabolites, corroborates the hypothesis that generalized secondary metabolic derepression may be an important by-product of antibiotic resistance to transcription/translation targeting antibiotics. It is conceivable that metabolic plasticity may be an evolutionary "save all ships" response to antibiotic challenge. However, whether or not general metabolic derepression is an adaptive or coincident trait is unknown at this time. Future studies linking the quantification of identified accumulated metabolites to gene transcription and translation quantification data will define the relationship between primary and secondary metabolism, derepression, survival, and the mechanism of resistance in microorganisms.
Experimental Procedures
Preparation of Antibiotics-Resistant Mutants.
Spore suspensions of Nocardiopsis sp. FU40 ΔApoS8 were inoculated onto GYM agar plates containing 50 μg/mL apramycin, 400 μg/mL streptomycin, and 50 μg/mL apramycin, 100 μg/mL rifampicin. Agar plates were incubated for 10–14 d at 30 °C to select for antibiotic-resistant colonies. Individual streptomycin- and rifampicin-resistant colonies were selected and inoculated onto GYM agar containing the appropriate antibiotic for validation of resistance phenotype and to prepare glycerol stock solution for cryopreservation.
Mass Spectrometry-Based Feature Identification.
Features prioritized using the methods described herein were subjected to putative identification using both accurate mass (<10 ppm) and fragmentation spectra (accurate mass, <20 ppm). Liquid chromatography and IM retention times were used to isolate features in separations space. This provided enhanced subtraction of concomitant peaks present in the analyzed fragmentation spectra. These spectra were then subjected to manual interpretation to determine the type of parent ion (e.g., protonated, sodiated, in-source fragment, etc.). In source fragmentation can be determined by determining whether the ion in question occurs at multiple drift times. After determination of ion type, and isolation of fragmentation spectra, accurate mass was used for database searching from multiple sources. Databases queried include KEGG, Metlin, ChemSpider, PubChem, Human Metabolome Database, Dictionary of Natural Products, and MetaCyc. These databases were searched with a mass tolerance of 0.01 Da. If the accurate mass deviated by more than 20 ppm, the compound was excluded from further database investigation. Fragmentation spectra were subjected to both manual interpretation, and MetFrag evaluation, an in silico interpreter. High energy peak lists were imported into MetFrag using centroided data with an intensity cutoff of ∼1%. To use MetFrag with the Dictionary of Natural Products and MetaCyc, .sdf files were prepared with all matching compounds within a tolerance of 0.01 Da and scored accordingly. Congruency between retention time and ClogP was also considered when assigning identifications.
Data Processing and Multivariate Statistical Analysis.
Data were centroided postacquisition. Peaks were deisotoped, peak picked, and normalized using MarkerLynx data processing software (Waters). Peak detection was performed on low-energy data across the mass range of 100–2,000 Da with retention times between 0.0 and 20.0 min, peak widths automatically detected, an intensity threshold of 2,000, mass window of 0.08, retention time window of 0.60, and noise elimination of 4.00. Multivariate statistical analyses were performed using Umetrics extended statistics software EZinfo, version 220.127.116.11 (Umetrics). All data were pareto scaled.
We acknowledge support from National Institutes for Health Grants 1R01GM092218 and RC2DA028981, the Defense Threat Reduction Agency Grant HDTRA-09-1-0013, the Vanderbilt Institute of Chemical Biology, and the Vanderbilt Institute of Integrative Biosystems Research and Education.
↵1D.K.D. and C.R.G. contributed equally to this work.
- ↵2To whom correspondence may be addressed. E-mail: brian.bachmann{at}vanderbilt.edu or john.a.mclean{at}vanderbilt.edu.
Author contributions: D.K.D., C.R.G., J.A.M., and B.O.B. designed research; D.K.D. designed and executed experiments in microbiology, compound isolation, and NMR structure determination; C.R.G. designed and executed multivariate analyses methods and UPLC IM-MS methods and analyses; D.K.D., C.R.G., and C.R.M. performed research; D.K.D., C.R.G., and J.A.M. analyzed data; J.A.M. and B.O.B. supervised metabolomics analysis and microbiology/secondary metabolism; and D.K.D., C.R.G., J.A.M., and B.O.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218524110/-/DCSupplemental.
Freely available online through the PNAS open access option.
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Our story
Gasrec was founded in 2003 and is the UK's leading supplier of gas to the road transport sector.
As a pioneer in the supply of gas fuels, Gasrec has built nine refuelling stations across the UK to service gas-powered vehicles, including, in 2013, the first open-access facility at the Daventry International Rail Freight Terminal (DIRFT) logistics park, the largest LNG refuelling station in Europe.
Gasrec has developed customer relationships which enable the development of facilities in tandem with vehicle deployments. It is vital that infrastructure capacity is increased to meet growing demand working with operators and not in isolation. A strong management team is in place to best support development in a market which is growing sharply following the introduction of dedicated gas-powered vehicles in 2017/18.
Our People
Executive Leadership Team
Meet the Gasrec Executive Team who have more than 85 combined years experience in industrial gas, process plant management, sustainable project development and waste recycling logistics.
Rob Wood
Chief Executive Officer
Rob joined Gasrec in 2013 to lead the company through a key stage of growth.
Before joining Gasrec he held a variety of commercial roles in BP's downstream, chemical and upstream gas businesses over a 24-year career. His current focus is government and industry affairs promoting the use of gas and its contribution to decarbonising heavy transport.
Brian Cutler
Chief Financial Officer
Brian joined Gasrec in 2014 to redefine core financial systems and processes ensuring robustness and scalability to support growth. His current focus is fundraising and structuring to support growth and deliver economies of scale.
Prior to joining Gasrec he spent 9 years at Petrochem Carless, a chemical and fuel blending business, contributing to substantial growth, preparing the businesses for sale, providing due diligence support to shareholders, potential investors and banks.
Brian is a Chartered Management Accountant with 19 years PQE
Mark Siddle
Mark joined Gasrec in 2016 to develop the projects team and is now Projects Director with responsibility for delivery of all gas refuelling station projects, at customer sites and open access. His current focus is the efficienct project delivery in relation to customer timescales and reliability requirements.
Prior to joining Gasrec he worked for BP for 25 years in the chemicals and power and gas businesses, following which he acted as consultant on a number of gas-fired power developments.
James Westcott
Chief Commerical Officer
James joined Gasrec in 2012 to support the first stages of infrastructure development, he now has responsibility for new business development and strategic growth opportunities.
Prior to joining Gasrec James was Commercial Manager for a renewable energy company specialising in anaerobic digestion and the production of biomethane. His current focus is aligning industry requirements with future infrastructure developments, such that Gasrec delivers what is necessary for industry to make the leap to clean fuels.
Tony Robinson
Chief Technical Officer
Tony joined Gasrec in 2013 to build in house technical capability in operations and engineering and is now responsible for safety, operations and engineering across Gasrec.
Prior to joining Gasrec he worked at National Grid for 13 years initially in the LNG storage peak shave business followed by a period in the Pipeline Maintenance Centre with National Grid Transmission.
His current focus is on the efficiency of operations and engineering using emerging technology to improve the customers experience and confidence of using gas as a transport fuel, with zero tolerance and high focus on safety.


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5 Tips to Strike Marketing Gold with Landing Pages
Before you post on social media, create a new video, run a Google Ad, or blast an email to your list, define your purpose. And bundle it with a landing page to drive conversions. Make your post more than a mere company slogan, or feel-good quote. Promise to solve a problem or serve a need, and you'll find more people clicking to learn more. But, be careful: don't send them onto your general overview home page. Instead, take them to a focused landing page on your site, solely designed to deliver on that original promise, and guide them to the next steps.
Hi, I'm Perry Alexander, and today, we're going to get down to the purpose, value, and details of how to build a landing page that delivers. And, using landing pages is just as important for non-profits.
Be sure to subscribe to my channel and hit the bell icon to be notified when I share more videos about digital marketing.
But first, have you had success using landing pages? If so, leave a comment that says "Yes." But, if not, leave a comment that says "No," and let me know what's holding your back, so I can make future videos that address your needs.
Tip 1
Your initial contact—that is your post, video, ad, or email—AND your landing page must be a message-matched pair of elements. The original post, video, ad, or email is an invitation to consider your proposition. Will people find it relevant and trustworthy enough for further consideration? If so, include an obvious call-to-action (or CTA)—a clickable button that links to your landing page, which expands on and reinforces that initial message. The narrow purpose of the landing page is to further inform and compel the next step, which may be leaving their email address for something of value, filling out your contact form, or acting on their decision to move to a free trial or purchase of your product or service.
Tip 2
When considering what to post, what problem or pain point will you address? Does your audience understand how it's impacted by various other forces? Should you set the table with a few basics? Have you shown that you really get their pain or frustration, before jumping to an answer? How far and how fast should you get into the weeds to explain nuances and build trust in your authority?
Tip 3
Use landing page best practices, like those listed in this Unbounce.com blog post. See the link below. A few selected ones:
Ensure a consistent experience between your post, video, ad, or email and your landing page.
Repeat headlines and major concepts from your post to your landing page.
Make your first post and your landing page appear graphically related.
Keep the landing page simple and clutter free. Stick with a powerful, direct-to-the-point headline, simple bullet points, with the call-to-action button as high on the page as possible, once you've made your point.
Get rid of your normal website navigation bar on the landing page, to minimize distraction from the path you want them to stay on.
Display your phone number prominently, should they just want to call you.
Include social proof, if you have it—a testimonial or two, or how many others have already done what you're asking them to do.
Don't overstate or exaggerate your case. Be realistic and ditch the superlatives.
If your landing page turns out to be a corresponding page in your blog, incorporate landing page best practices into that blog post.
Tip 4
Make your call-to-action (CTA) specific. Go beyond simply, "Submit," "Go," or "Subscribe." Tell them what will happen if they click, such as "Get Your Free Trial," "Download Your Free Ebook," or "Schedule Your Free Consultation." And if you have a lot to explain on the landing page, repeat your call-to-action button every few paragraphs as they move down the page.
Tip 5
To increase landing page conversion rates by as much as 80%, include a video. Also include a thumbnail of the video in your first message, along with the word "video" in the headline or subject line. I've posted links to videos I've previously made about using and formatting videos.
For several great landing page examples, along with commentary about what makes them effective, check out the Hubspot post in the links below.
It's a Start
There's lots more to honing your landing page into a well-oiled conversion machine, but this is a solid start. Know your purpose before creating each social media post, new video, Google ad, or email blast, and then drive home conversions with smart landing pages.
If the idea of following up your posts with landing pages feels right, but also feels like a tall order without a little help, it's one of the many services I provide for my clients. Check out my digital marketing website and let's talk about how we might work together to improve your conversions through landing pages.
Thanks for watching. And, if you liked this video, please subscribe, hit the bell, tell others about it, like it, and leave a comment below or even a quick question, and I'll be sure to answer.
Resources and Links
Unbounce.com: Landing Page Best Practices
Hubspot: 19 Best Landing Page Designs
My Videos About Making Videos


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I've been looking for something to do lately.
So picked on a geeky item.
Been looking at modding my CM Stacker case, as its had something dribbled on it before I got it which has caused the grills etc to rust. Looking to sand it all back, and give it a new paint job with some LED lighting etc…..
Now, me being a geek and not handy at all with mechanical/DIY stuff really, I decided to do a trial run on an old PC case before modding my good one……. had a beat up CM Centurion 532 kicking around the garage, so picked on that.
Have now stripped that right down, removed everything from it I can from that that was glued/screwed, and started cleaning it up.
Have so far sanded back on side of the case (this is the non-window version of the case) – thank christ for electric sanders 😀
I've cut off the columns from the side (one was broken anyway) and started tidying up the top console (power, USB etc) to make it seem more "natural" for it being there now the columns are gone.
Also doing the same with the bottom part which is plastic where the columns were attached and act as feet for the front of the case.
The end goal, (and may change) is to sand it all back to bare metal, then spray it white inside and out, put the Overwatch logo (fav game of mine) on the sides, spray the front cover orange, and the grills will leave black and put orange LED's in behind them, and an orange LED fan at the bottom.
Thats the rough plan anyway…… Wont be a short project, as got other DIY things to get done around the house, so will be done over a few months I'm guessing.


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No one would be surprised to hear that standover tactics, bullying and violence are rife in prison. But you may be surprised to learn that prison nurses in New Zealand also engage in this kind of behaviour. In 2006, management at Corrections were so concerned about bullying by prison nurses, they asked the Health & Disability Commission to investigate.
The HDC dispatched two employees, Dr Elizabeth Finn and Mr David Webber to conduct a series of workshops in prison health centres. The first was held at the Waikeria prison in 2006 and workshops were subsequently held in another 12 prisons.
In 2008, a damning summary of the HDC's findings was released in a report titled "Team development workshops for prisons nursing services delivered by Dr Elizabeth Finn and Mr David Webber". In September 2009, a copy of the report was sent to the Ombudsman by HDC chief legal advisor, Nicola Sladden, describing a "culture of horizontal violence and bullying" within prison health centres. It identified the following problems:
Lack of leadership
Discouraged medication policy
Another failing identified by Finn and Webber concerned the prescribing of medication by prison doctors. The authors wrote:
"It appears that some doctors are not sufficiently aware of the context of the prison environment and the particular challenges it presents. For example, some medications are inappropriate in this environment because of their potential for use as 'currency'. Lack of national consistency with respect to provision of medications to patients who are prisoners may lead to stand-over tactics and abuse directed towards doctors in the first instance, and also towards nurses. A robust induction programme for doctors would be helpful."
From time to time, prisoners have to be taken to hospital with serious injuries or medical problems. Finn and Webber even reprimand doctors in Emergency Departments – apparently for not doing what they are told by prison nurses. The report says:
"(Doctors in) EDs may not be cooperating with advice from nurses that certain medications are not allowable in prisons, and may still be prescribing these (codeine, morphine)."
For severe pain, opiates like codeine and morphine are the medications of choice. But the HDC seems to believe that prisoners should not have their pain relieved, even in emergency situations. Apparently, they should continue to suffer - even when the pain is so bad they need to be taken to hospital.
This 'discouraged medication policy' extends well beyond opiate pain relief. All medications including antidepressants and antipsychotic drugs are taken away from prisoners - usually on their first day in prison. Although this practice varies from one prison to another, often such medications are never reinstated.
Breach of medical ethics and human rights
Denying patients clinically appropriate medication, especially when they are in severe pain or have mental health disorders, is inhumane and a breach of human rights. It contributes to depression and even to suicide. The policy also encourages prison doctors to breach their medical ethics which require them to "Consider the health and well-being of the patient to be your first priority"; and to "render medical service to that person without discrimination." When doctors are coerced into making prison policy their first priority, and discriminate against the patient because he or she is in prison, they risk being struck off for breaching their ethics.
The systematic denial of opiates and other clinically appropriate medication is a form of pharmacological or 'clean' torture; it causes serious harm and distress to the prisoner without leaving any visible evidence. But the Health and Disability Commission seems to condone this practice.
For an agency responsible for promoting safe medical practices, this is truly disturbing. It suggests the HDC is complicit in Corrections strategy to breach the human rights of prisoners and raises serious questions about the integrity of investigations into complaints by prisoners who need these medications.
The Commissioner's response
In July this year, I wrote to the HDC and asked if they still endorse this 'discouraged medication policy'. Mr Anthony Hill, the Commissioner, replied:
"It is not HDC's practice to endorse the policies or procedures of health care providers. This is because I must consider complaints to HDC fairly, with an open mind and free from bias. This may be called into question if I have previously endorsed a policy or procedure that is the subject of a complaint to HDC."
That's the whole point. The Finn Webber report is quite clearly an endorsement of Corrections discouraged medication policy and my letter therefore questions the integrity of all HDC investigations into prisoner healthcare in the last five years. Since Mr Hill refused to acknowledge the bias in the HDC's position, I have now written to the Ombudsman suggesting that if the HDC does not endorse this particular policy, then the Ombudsman should encourage the Commissioner to write to Corrections and tell them so. Otherwise, the Finn Webber report gives the Department every reason to continue removing clinically appropriate medication from prisoners, leaving them in pain and distress - and believing the HDC endorses this unethical practice.
I also suggested to the Ombudsman that he/she should persuade the Corrections Department to change this practice and remove it from their Medicines Policy. This is because New Zealand is party to the Convention against Torture and Cruel, Inhuman or Degrading Treatment or Punishment which requires Governments to take effective measures to prevent the torture and ill treatment of people who are detained by the State. And since 2007, when New Zealand signed the Optional Protocol to the Convention against Torture (OPCAT), the Ombudsman became one of the monitoring bodies with responsibility to ensure that New Zealand meets its obligations under the Convention. If he does not intervene, then by failing to act, the Ombudsman is also condoning and endorsing the torture and ill treatment of New Zealand prisoners.


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This is, by far, the cleanest my office has ever been. One moving box
full of stuff was taken home this weekend, two phones eliminated
(still need two), and I found out what color my desk is. Not pictured
in other parts of the office: 40 old voip phones removed (shipped to a
liquidator) and 35 boxes the new phones came in also removed (sent to the recycling bin).
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text: by Jamey Mann Often students and professionals find themselves at a plateau that can be difficult to work out of. Just like athletes need to change up their workouts to make gains in sports, musicians need to get out of comfort zones to make gains musically. At a certain point everyone's routine begins to get… Read more »


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If you want to sell more books an author platform is essential. Blogging your book is the perfect way to get your name out there.
You have a book that you have published, but have not exploited yet or needs a marketing boost?
Would you love to blog your book to raise your credibility and convert visitors to customers?
Do you need to build your author platform so that your book and other products and services are more visible?
The second part of this program takes you from outlined book to content mastery and building your author platform.
Blog Your Book will help you to discover the right content blogged, create your author platform and create greater awareness around your book and message
Discover what you want to create, what you want to be known for, blog your book, build your author platform, maximise your books reach.
There is no shortage of people to sell you the dream, what I do is make this real. I will set your expectations and be honest with you about how to make this work for you and what you have to do to make your book and blog work for you.
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Charles, At 13:37 2000-04-25 -0400, Charles McCathieNevile wrote: >Marjolein, > >this seems pretty clear (at least to me). The difference between your >understanding of prompt, and my understanding of the way it is defined and >used in the guidelines, is that where a checkpoint requires prompting, it is >required that there is a response, and that a prompt doesn't necessarily get >any data from the user. > >Note that the relevant checkpoint (3.1) says to prompt the author to provide >alternative content, not when, how often, or whether it has to be done all >the time for each piece of content that may have an alternative specified, or >can be done once in a prompt that covers everything (but requires some >response) and is more like what you are describing as an alert with a >required response. Not quite - since an alert (in my definition) gives information *to* the user but doesn't get information *from* the user. The way I understand the checkpoint (or its purpose), it would be more like a dialog, containing a prompt; where the dialog requires a response (OK or Cancel) but the prompt on that dialog doesn't necessarily require information (data to be filled in). >Charles > >On Tue, 25 Apr 2000, Marjolein Katsma wrote: > > It seems I understand the terms "Alert" and "Prompt" very differently > from the rest of the group. I also found this actually very hard to > explain on the phone. I'm not sure where this difference comes from (my > background as a software developer?), but of course I also missed the > start of the definition process in this group. > > So I'll attempt to write down my definition of these terms here: >[snip] Marjolein Katsma HomeSite Help - http://hshelp.com/ Bookstore for Webmasters - http://hshelp.com/bookstore/bookstore.htmlReceived on Tuesday, 25 April 2000 14:06:12 UTC
This archive was generated by hypermail 2.3.1 : Tuesday, 6 January 2015 21:39:44 UTC


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