Several fungi in two different families––the Clavicipitaceae and the Trichocomaceae––produce different profiles of ergot alkaloids, many of which are important in agriculture and medicine. All ergot alkaloid producers share early steps before their pathways diverge to produce different end products. EasA, an oxidoreductase of the old yellow enzyme class, has alternate activities in different fungi resulting in branching of the pathway. Enzymes beyond the branch point differ among lineages. In the Clavicipitaceae, diversity is generated by the presence or absence and activities of lysergyl peptide synthetases, which interact to make lysergic acid amides and ergopeptines. The range of ergopeptines in a fungus may be controlled by the presence of multiple peptide synthetases as well as by the specificity of individual peptide synthetase domains. In the Trichocomaceae, diversity is generated by the presence or absence of the prenyl transferase encoded by easL (also called fgaPT1). Moreover, relaxed specificity of EasL appears to contribute to ergot alkaloid diversification. The profile of ergot alkaloids observed within a fungus also is affected by a delayed flux of intermediates through the pathway, which results in an accumulation of intermediates or early pathway byproducts to concentrations comparable to that of the pathway end product.
ergot alkaloids; Claviceps; Epichloë, Neosartorya fumigata, old yellow enzyme; peptide synthetase; prenyl transferase
Children with chronic otitis media (OM) often have conductive hearing loss which results in communication difficulties and requires surgical treatment. Recent studies have provided clinical evidence that there is a one-to-one correspondence between chronic OM and the presence of a bacterial biofilm behind the tympanic membrane (TM). Here we investigate the acoustic effects of bacterial biofilms, confirmed using optical coherence tomography (OCT), in adult ears. Non-invasive OCT images are collected to visualize the cross-sectional structure of the middle ear, verifying the presence of a biofilm behind the TM. Wideband measurements of acoustic reflectance and impedance (0.2 to 6 [kHz]) are used to study the acoustic properties of ears with confirmed bacterial biofilms. Compared to known acoustic properties of normal middle ears, each of the ears with a bacterial biofilm has an elevated power reflectance in the 1 to 3 [kHz] range, corresponding to an abnormally small resistance (real part of the impedance). These results provide assistance for the clinical diagnosis of a bacterial biofilm, which could lead to improved treatment of chronic middle ear infection and further understanding of the impact of chronic OM on conductive hearing loss.
bacterial biofilm; optical coherence tomography; reflectance; impedance; middle ear; otitis media; tympanic membrane
This study characterizes middle ear complex acoustic reflectance (CAR) and impedance by fitting poles and zeros to real-ear measurements. The goal of this work is to establish a quantitative connection between pole-zero locations and the underlying physical properties of CAR data. Most previous studies have analyzed CAR magnitude; while the magnitude accounts for reflected power, it does not encode latency information. Thus, an analysis that studies the real and imaginary parts of the data together could be more powerful. Pole-zero fitting of CAR data is examined using data compiled from various studies, dating back to Voss and Allen (1994). Recent CAR measurements were taken using a middle ear acoustic power analyzer (MEPA) system (HearID, Mimosa Acoustics), which makes complex acoustic impedance and reflectance measurements in the ear canal over the 0.2 to 6.0 kHz frequency range. Pole-zero fits to measurements over this range are achieved with an average RMS relative error of less than 3% using 12 poles. Factoring the reflectance fit into its all-pass and minimum-phase components approximates the effect of the ear canal, allowing for comparison across measurements. It was found that individual CAR magnitude variations for normal middle ears in the 1 to 4 kHz range often give rise to closely-placed pole-zero pairs, and that the locations of the poles and zeros in the s-plane may differ between normal and pathological middle ears. This study establishes a methodology for examining the physical and mathematical properties of CAR using a concise parametric model. Pole-zero modeling shows promise for precise parameterization of CAR data and for identification of middle ear pathologies.
middle ear reflectance; poles; zeros
It is well-known that ultraviolet (UV) light exposure and a sun sensitive phenotype are risk factors for the development of non-melanoma skin cancer (NMSC), including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). In this New Hampshire population-based case-control study, we collected data from 5,072 individuals, including histologically-confirmed cases of BCC and SCC, and controls via a personal interview to investigate possible associations between photosensitizing medication use and NMSC. After adjustment for potentially confounding factors (e.g. lifetime number of painful sunburns), we found a modest increase in risk of SCC (OR = 1.2, 95% CI = 1.0–1.4) and BCC (OR = 1.2, 95% CI = 0.9–1.5), in particular early-onset BCC, (≤ 50 years of age) (OR = 1.5, 95% CI = 1.1–2.1) associated with photosensitizing medication use. For SCC the association was strongest amongst those with tendency to sunburn rather than tan. We also specifically found associations with BCC, and especially early-onset BCC, and photosensitizing antimicrobials. In conclusion, certain commonly prescribed photosensitizing medications may enhance the risk of developing SCC, especially in individuals with a sun sensitive phenotype, and may increase the risk of developing BCC and incidence of BCC at a younger age.
Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a “protein-first” method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes using mass spectrometry-based proteomics for the targeted detection of expressed nonribosomal peptide synthetases or polyketide synthases. Improvements to the original PrISM screening approach (Nature Biotechnology, 2009, 27, 951 – 956), e.g. improved de novo peptide sequencing, have enabled the discovery of ten NRPS/PKS gene clusters from six strains. Taking advantage of the concurrence of biosynthetic enzymes and the secondary metabolites they generate, two natural products were associated with their previously ‘orphan’ gene clusters. This work has demonstrated the feasibility of a proteomics-based strategy for use in screening for NRP/PK production in actinomycetes (often >8 Mbp, high GC genomes) versus the bacilli (2–4 Mbp genomes) used previously.
Nonribosomal peptide; polyketide; natural product biosynthesis; Actinobacteria; de novo sequencing; Fourier-Transform Mass Spectrometry
Background & Aims
Early fluid resuscitation is recommended to reduce morbidity and mortality among patients with acute pancreatitis (AP), although the impact of this intervention has not been quantified. We investigated the association between early fluid resuscitation and outcome of patients admitted to the hospital with AP.
Non-transfer patients admitted to our center with AP, from 1985 to 2009, were identified retrospectively. Patients were stratified into groups based on early (n=340) or late resuscitation (n=94). Early resuscitation was defined as receiving ≥ 1/3 of the total 72 h fluid volume within 24 hours of presentation, whereas late resuscitation was defined as receiving ≤ 1/3 of the total 72 h fluid volume within 24 hours of presentation. The primary outcomes were frequency of the systemic inflammatory response syndrome (SIRS), organ failure, and death.
Early resuscitation was associated with decreased SIRS, compared with late resuscitation, at 24 h (15% vs. 32% P=0.001), 48 h (14% vs. 33%, P =0.001), and 72 h (10% vs. 23%, P =0.01), as well as reduced organ failure at 72 h (5% vs. 10%, P <0.05), a lower rate of admission to the intensive-care unit (6% vs. 17%, P< 0.001), and a reduced length of hospital stay (8 vs. 11 days, P=0.01). Subgroup analysis demonstrated that these benefits were more pronounced in patients with interstitial, rather than severe, pancreatitis at admission.
In patients with AP, early fluid resuscitation was associated with reduced incidence of SIRS and organ failure at 72 hours. These effects were most pronounced in patients admitted with interstitial, rather than severe, disease.
pancreas; inflammation; treatment; efficacy
The mechanisms that generate dynamic spatial patterns within proliferating tissues are poorly understood, largely because of difficulties in unravelling interactions between cell specification, polarity, asymmetric division, rearrangements and growth. Here we address this problem for stomatal spacing in plants, which offer the simplifying advantage that cells do not rearrange. By tracking lineages and gene activities over extended periods, we show that limited stem cell behavior of stomatal precursors depends on maintenance of the SPEECHLESS (SPCH) transcription factor in single daughter cells. Modelling shows how this property can lead to observed stereotypical stomata lineages through a post-mitotic polarity switching mechanism. The model predicts the location of a polarity determinant BASL over multiple divisions, which we validate experimentally. Our results highlight the dynamic two-way interactions between stem cells and their neighborhood during developmental patterning.
Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are large enzymes responsible for the biosynthesis of medically and ecologically important secondary metabolites. In a previous report we described a proteomics approach to screen for expressed NRPSs or PKSs from bacteria with or without sequenced genomes. Here we use this proteome mining approach to discover a novel natural product arising from rare adenylation (A) and reductase (Red) domains in its biosynthetic machinery. We also clone the entire gene cluster and elucidate the biosynthesis of the new compound produced by an unsequenced Bacillus sp. isolated from soil collected in Koran, Louisiana.
Nonribosomal peptide synthetase; natural products; proteomics; Fourier-transform mass spectrometry
The cyclodepsipeptide jasplakinolide (1) (a.k.a. jaspamide), isolated previously from the marine sponge Jaspis splendens, is a unique cytotoxin and molecular probe that operates through stabilization of filamentous actin (F-actin). We have recently disclosed that two analogues of 1, jasplakinolides B (3) and E, were referred to the National Cancer Institute's (NCI) Biological Evaluation Committee and the objective of this study was to re-investigate a Fijian collection of J. splendens in an effort to find jasplakinolide congeners with similar biological properties. The current efforts have afforded six known jasplakinolide analogues (4 - 7, 9 - 10), two structures requiring revision (8 and 14) and four new congeners of 1 (11 - 13, 15) including open chain derivatives and structures with modified β-tyrosine residues. Compounds were evaluated for biological activity in the NCI's 60 cell line screen and in a microfilament disruption assay in both HCT-116 and HeLa cells. These two phenotypic screens provide evidence that each cytotoxic analogue, including jasplakinolide B (3), operates by modification of microfilaments. The new structure jasplakinolide V (13) has also been selected for study by the NCI's Biological Evaluation Committee. In addition, the results of a clonogenic dose response study on jasplakinolide are presented.
With many bioactive non-ribosomal peptides and polyketides produced in fungi, studies of their biosyntheses are an active area of research. Practical limitations of working with mega-dalton synthetases including cell lysis and protein extraction to recombinant gene and pathway expression has slowed understanding of many secondary metabolic processes relative to bacterial counterparts. Recent advances in accessing fungal biosynthetic machinery are beginning to change this. Here we describe the successes of some studies of thiotemplate biosynthesis in fungal systems, along with very recent advances in chemical tagging and mass spectrometric strategies to selectively study biosynthetic conveyer belts in isolation, and within a few years, in endogenous fungal proteomes.
fungal metabolism; nonribosomal peptide synthetases; polyketide synthases; thiotemplate biosynthesis; secondary metabolism; proteomics; mass spectrometry; Fourier-Transform mass spectrometry
The goal of this study was to isolate and study additional jasplakinolide analogues from two taxonomically distinct marine sponges including two Auletta spp. and one Jaspis splendens. This led to the isolation of jasplakinolide (1) and eleven jasplakinolide analogues (3 – 13) including seven new analogues (6 – 10, 12, and 13). Structure elucidation of the new compounds was based on a combination of 1D and 2D NMR analysis, optical rotation, circular dichroism, and preparation of Mosher’s esters. Five of the new compounds are oxidized tryptophan derivatives of 1, including a unique quinazoline derivative (9). Compounds 1, 3, 5 – 8, and 11 were evaluated in the NCI 60 cell line screen and all compounds were tested in a microfilament disruption assay. Jasplakinolide B (11) exhibited potent cytotoxicity (GI50 < 1 nM vs. human colorectal adenocarcinoma (HCT-116) cells) but did not exhibit microfilament-disrupting activity at 80 nM.
Enzyme screening of crude sponge extracts prioritized a 2005 Papua New Guinea collection of Hyrtios sp. for further study. The MeOH extract contained puupehenone and four puupehenone analogs (1, 2, 3, 5, and 7) along with a new diastereomer, 20-epi-hydroxyhaterumadienone (4) and a new analog 15-oxo-puupehenoic acid (6). The drimane terpene core of 4 and 6 was rapidly dereplicated, and modified Mosher’s method identified 4 while 1D and 2D NMR techniques were used to solve 6. These compounds plus noteworthy repository natural products and standards were tested against three lipoxygenase isozymes, human 5-, 12- and 15-lipoxygenases. Significant potency and selectivity profiles were exhibited in the human 5-lipoxygenase assay by puupehenone (1) and jaspaquinol (9) and structural factors responsible for activity identified.
Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications—914 in parotid and 917 in submandibular/sublingual saliva—were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.
Human saliva; parotid; submandibular; sublingual; ductal secretion; proteomics; mass spectrometry
To determine whether the knowledge specified in the specialty‐specific section of the College of Emergency Medicine curriculum covered the diagnoses presenting to a UK teaching hospital emergency department.
An audit of 1000 sets of notes was undertaken, the diagnosis abstracted and mapped to the curriculum.
1076 diagnoses were derived and all were covered by the curriculum. The three most common diagnostic categories were musculoskeletal, wound management and cardiology.
The curriculum covered all the diagnoses in this sample. Knowing the frequency of a diagnosis could be used to inform training and assessment.
The dramatic biogeographical variations in the secondary metabolites from Psammocinia aff. bulbosa have complicated our efforts to re-isolate the two most cytotoxic of its metabolites, (+)-psymberin and (+)-cyclocinamide A. Reported now are the results of a new study that demonstrates our ability to repeatedly isolate these two compounds through targeted collection efforts. Additional study of the new sample of (+)-cyclocinamide A has enabled finalizing its biological activity and absolute stereochemistry as 4S, 7S, 11S, 14S.
Tobacco use remains the leading cause of death and disability in Canada. Insufficient research capacity can inhibit evidence-informed decision making for tobacco control. This paper outlines a Canadian project to build research capacity, defined as a community's ability to produce research that adequately informs practice, policy, and future research in a timely, practical manner. A key component is that individuals and teams within the community must mutually engage around common, collectively negotiated goals to address specific practices, policies or programs of research. An organizing framework, a set of activities to build strategic recruitment, productivity tools, and procedures for enhancing social capital are described. Actions are intended to facilitate better alignment between research and the priorities of policy developers and service providers, enhance the external validity of the work performed, and reduce the time required to inform policy and practice.
To assess the relationship between low-grade inflammation, measured as basal high sensitivity (hs)-CRP, and IVF outcome.
We recruited a total of 220 women undergoing infertility work up prior to IVF. Patients were selected for a BMI < 30 kg/m2 with an upper age limit of 40 years. Serum hs-CRP levels were measured on day 3 of a spontaneous menstrual cycle preceding ovarian stimulation. A sensitive two-site ELISA was used for analysis. Dose of gonadotrophins required, follicles days 8 and 10, number of oocytes collected, number of oocytes fertilised and pregnancy outcome were recorded.
Median hs-CRP was 1.08 mg/L (0.43–3.00 mg/L). The hs-CRP was significantly related to BMI (r = 0.386, P < .001) but not to age and smoking habit. There were no significant relationships between basal hs-CRP and any of the measured IVF outcomes.
These findings demonstrate that serum hs-CRP concentration is not a predictive marker of cycle or pregnancy outcome in women undergoing IVF treatment.
Controlled ovarian hyperstimulation; hs-CRP; Inflammation; IVF; Pregnancy rate
The theory of planned behaviour states that attitudinal variables (e.g. job satisfaction) only have an indirect effect on retention whereas intentions have a direct effect. This study uses secondary data from a longitudinal cohort of newly qualified nurses to test for the direct and indirect effects of job satisfaction (client care, staffing, development, relationships, education, work-life interface, resources, pay) and intentions to nurse on working as a nurse during the 3 years after qualification.
A national sample (England) of newly qualified (1997/98) nurses (n = 3669) were surveyed at 6 months, 18 months and 3 years. ANOVA and MANOVA were used for comparison of mean job satisfaction scores between groups; intentions to nurse (very likely, likely vs. unlikely, very unlikely and unable to say at this stage); working (or not working as a nurse) at each time-point. Indirect and direct effects were tested using structural equation and logistic regression models.
Intentions expressed at 6 months to nurse at 18 months were associated with higher scores on pay and relationships, and intentions at 3 years were associated with higher scores on care, development, relationships, work-life interface, resources, pay respectively. Intentions expressed at 18 months to nurse at 3 years were associated with higher scores on development, relationships, education and work-life interface. Associations with actual nursing were fewer. Those working as a nurse had higher satisfaction scores for development (18 months) and relationships (3 years). Regression models found significant associations between the pay and staffing factors and intentions expressed at 6 months to nurse at 18 months, and between pay and intentions to nurse at 3 years. Many of the associations between intentions and working as a nurse were significant. Development was the only job satisfaction factor significantly associated with working as a nurse and just at 18 months.
Results partially support the theory of planned behaviour. Intentions expressed by nurses are stronger predictors of working as a nurse than job satisfaction. Retention strategies should focus on identifying nurses showing early signs of departure with emphasis on developmental aspects, mentoring and support.
Job satisfaction is an important component of nurses' lives that can impact on patient safety, productivity and performance, quality of care, retention and turnover, commitment to the organisation and the profession. Little is known about job satisfaction in early career and how it varies for different groups of nurses. This paper investigates how the components of job satisfaction vary during early career in newly qualified UK nurses.
Nurses were sampled using a combined census and multi-stage approach (n = 3962). Data were collected by questionnaire at 6 months, 18 months and 3 years after qualification between 1998 and 2001. Scores were calculated for seven job satisfaction components and a single item that measured satisfaction with pay. Scores were compared longitudinally and between nursing speciality (general, children's, mental health) using a mixed model approach.
No single pattern across time emerged. Trends varied by branch and job satisfaction component. Rank order of job satisfaction components, from high to low scores, was very similar for adult and child branch nurses and different for mental health. Nurses were least satisfied with pay and most satisfied with relationships at 6 and 18 months and with resources (adult and child) and relationships (mental health) at 3 years. Trends were typically upwards for adult branch nurses, varied for children's nurses and downwards for mental health nurses.
The impact of time on job satisfaction in early career is highly dependent on specialism. Different contexts, settings and organisational settings lead to varying experiences. Future research should focus on understanding the relationships between job characteristics and the components of job satisfaction rather than job satisfaction as a unitary construct. Research that further investigates the benefits of a formal one year preceptorship or probationary period is needed.
Determination of seabird diet usually relies on the analysis of stomach-content remains obtained through stomach flushing; this technique is both invasive and logistically difficult. We evaluate the usefulness of DNA-based faecal analysis in a dietary study on chick-rearing macaroni penguins (Eudyptes chrysolophus) at Heard Island. Conventional stomach-content data was also collected, allowing comparison of the approaches.
Prey-specific PCR tests were used to detect dietary DNA in faecal samples and amplified prey DNA was cloned and sequenced. Of the 88 faecal samples collected, 39 contained detectable DNA from one or more of the prey groups targeted with PCR tests. Euphausiid DNA was most commonly detected in the early (guard) stage of chick-rearing, and detection of DNA from the myctophid fish Krefftichthys anderssoni and amphipods became more common in samples collected in the later (crèche) stage. These trends followed those observed in the penguins' stomach contents. In euphausiid-specific clone libraries the proportion of sequences from the two dominant euphausiid prey species (Euphausia vallentini and Thysanoessa macrura) changed over the sampling period; again, this reflected the trend in the stomach content data. Analysis of prey sequences in universal clone libraries revealed a higher diversity of fish prey than identified in the stomachs, but non-fish prey were not well represented.
The present study is one of the first to examine the full breadth of a predator's diet using DNA-based faecal analysis. We discuss methodological difficulties encountered and suggest possible refinements. Overall, the ability of the DNA-based approach to detect temporal variation in the diet of macaroni penguins indicates this non-invasive method will be generally useful for monitoring population-level dietary trends in seabirds.