Silk-based biomaterials in combination with extracellular matrix (ECM) coatings were assessed as templates for cell-seeded bladder tissue engineering approaches. Two structurally diverse groups of silk scaffolds were produced by a gel spinning process and consisted of either smooth, compact multi-laminates (Group 1) or rough, porous lamellar-like sheets (Group 2). Scaffolds alone or coated with collagen types I or IV or fibronectin were assessed independently for their ability to support attachment, proliferation, and differentiation of primary cell lines including human bladder smooth muscle cells (SMC) and urothelial cells as well as pluripotent cell populations, such as murine embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells. AlamarBlue evaluations revealed that fibronectin-coated Group 2 scaffolds promoted the highest degree of primary SMC and urothelial cell attachment in comparison to uncoated Group 2 controls and all Group 1 scaffold variants. Real time RT-PCR and immunohistochemical (IHC) analyses demonstrated that both fibronectin-coated silk groups were permissive for SMC contractile differentiation as determined by significant upregulation of α-actin and SM22α mRNA and protein expression levels following TGFβ1 stimulation. Prominent expression of epithelial differentiation markers, cytokeratins, was observed in urothelial cells cultured on both control and fibronectin-coated groups following IHC analysis. Evaluation of silk matrices for ESC and iPS cell attachment by alamarBlue showed that fibronectin-coated Group 2 scaffolds promoted the highest levels in comparison to all other scaffold formulations. In addition, real time RT-PCR and IHC analyses showed that fibronectin-coated Group 2 scaffolds facilitated ESC and iPS cell differentiation toward both urothelial and smooth muscle lineages in response to all trans retinoic acid as assessed by induction of uroplakin and contractile gene and protein expression. These results demonstrate that silk scaffolds support primary and pluripotent cell responses pertinent to bladder tissue engineering and that scaffold morphology and fibronectin coatings influence these processes.
The performance of scoring tools to select clinical vignettes for presentation at academic meetings has never been assessed.
To measure the psychometric properties of two scoring tools used to select clinical vignettes and to determine which elements are most helpful.
Prospective observational study.
Participants submitting clinical vignette abstracts, Society of General Internal Medicine annual meetings (2006–2007).
The 2006 scoring tool had three criteria (clarity, significance, and relevance) with brief general descriptors. The 2007 modified tool had five criteria (clarity, significance, relevance, teaching value, and overall assessment) with more detailed descriptors.
A total of 938 clinical vignette abstracts were submitted (484 in 2006; 454 in 2007); 59.5% (n = 288) were accepted for presentation. Cronbach’s alpha was 0.81 for the 2006 three-item tool and 0.95 for the 2007 modified five-item tool. Simplifying the five-item 2007 tool to three items (relevance, teaching value, overall assessment) yielded a Cronbach’s alpha of 0.95. The agreement between the number of clinical vignettes accepted for presentation (2007) using the average score of the five items with the number that would have been accepted using the simplified three items (relevance, teaching value, overall assessment) was almost perfect, with kappa 0.89 (95% confidence interval, 0.85 to 0.93).
Both scoring tools performed well, but a simplified tool with three items (relevance, teaching value, and overall assessment) and detailed descriptors was optimal; the simplified tool could improve the reviewer efficiency and quality of clinical vignettes presented at national meetings.
professional competence; internship and residency; education medical/methods; educational measurement/methods/standards; congresses as topic; reproducibility of results
The macrocyclic polyketide tacrolimus (FK506) is a potent immunosuppressant that prevents T-cell proliferation produced solely by Streptomyces species. We report here the first draft genome sequence of a true FK506 producer, Streptomyces tsukubaensis NRRL 18488, the first tacrolimus-producing strain that was isolated and that contains the full tacrolimus biosynthesis gene cluster.
Little is known about how faculty, residents, and fellows practice for oral presentations at academic meetings. We sought to categorize presenters' practice styles and the impact of feedback.
We surveyed oral presenters at 5 annual academic general internal medicine meetings between 2008 and 2010, using a cross-sectional design. Main measures were frequency and settings of practice, most helpful practice setting, changes made in response to feedback, impact of feedback, and perceived quality of presentation.
The response rate was 63% (333/525 responders). Respondents represented 59 academic medical centers. Presenters reported practicing in a mean ± SD of 2.3 (±1.3) of 5 different settings. Of the 46% of presenters (152/333) who practiced in front of a group of more experienced colleagues, 80% of presenters (122/152) reported it was the most helpful setting. Eighty-one percent of presenters (268/333) practiced alone, and 25% of presenters (82/333) reported practicing alone was the most helpful setting. The mean numbers of change types reported by faculty were fewer than those reported by residents and fellows (mean 2.3 ± 1.8, and 3.1 ± 2.0, respectively; P < .001). Practicing alone was not associated with changes in content (P = .30), visual aids (P = .12), or delivery style (P = .53).
Practicing in front of a group of experienced colleagues was the most helpful setting in which to prepare for an oral academic meeting presentation, but it was not universally utilized. Feedback given at these sessions was more likely to result in changes made to the presentation; however, broader implementation of such sessions 5 require institutional support.
To determine the effectiveness of a provider-based education and implementation intervention for improving diabetes control.
Cluster-randomized trial with baseline and follow-up cross sections of diabetes patients in each participating physician's practice.
Eleven US Southeastern states, 2006–08.
Two hundred and five rural primary care physicians.
Multi-component interactive intervention including Web-based continuing medical education, performance feedback and quality improvement tools.
Primary Outcome Measures
‘Acceptable control’ [hemoglobin A1c ≤9%, blood pressure (BP) <140/90 mmHg, low-density lipoprotein cholesterol (LDL) <130 mg/dl] and ‘optimal control’ (A1c <7%, BP <130/80 mmHg, LDL <100 mg/dl).
Of 364 physicians attempting to register, 205 were randomized to the intervention (n= 102) or control arms (n= 103). Baseline and follow-up data were provided by 95 physicians (2127 patients). The proportion of patients with A1c ≤9% was similar at baseline and follow-up in both the control [adjusted odds ratio (AOR): 0.94; 95% confidence interval (CI): 0.61, 1.47] and intervention arms [AOR: 1.16 (95% CI: 0.80, 1.69)]; BP <140/90 mmHg and LDL <130 mg/dl were also similar at both measurement points (P= 0.66, P= 0.46; respectively). We observed no significant effect on diabetes control attributable to the intervention for any of the primary outcome measures. Intervention physicians engaged with the Website over a median of 64.7 weeks [interquartile range (IQR): 45.4–81.8) for a median total of 37 min (IQR: 16–66).
A wide-reach, low-intensity, Web-based interactive multi-component intervention did not improve control of glucose, BP or lipids for patients with diabetes of physicians practicing in the rural Southeastern US.
internet; translational research; diabetes mellitus; rural health services; education; medical; continuing process assessment (Health Care)
CME providers may be interested in identifying effective marketing strategies to direct users to specific content. The use of online advertisements to recruit participants for clinical trials, public health programs, and Continuing Medical Education (CME) has been shown to be effective in some but not all studies. The purpose of this study was to compare the impact of two marketing strategies in the context of an online CME cultural competence curriculum (www.c-comp.org).
In an interrupted time-series quasi-experimental design, two marketing strategies were tested: a) wide dissemination to relevant organizations over a period of approximately four months, and b) Internet paid search using Google Ads (five consecutive eight-week periods--control 1, cultural/ CME advertisement, control 2, hypertension/ content advertisement, control 3). Outcome measures were CME credit requests, Web traffic (visits per day, page views, pages viewed per visit), and cost.
Overall, the site was visited 19,156 times and 78,160 pages were viewed. During the wide dissemination phase, the proportion of visits requesting CME credit decreased between the first (5.3%) and second halves (3.3%) of this phase (p= .04). During the Internet paid search phase, the proportion of visits requesting CME credit was highest during the cultural/ CME advertisement period (control 1, 1.4%; cultural/CME ad, 4.3%; control 2, 1.5%; hypertension/content ad, 0.6%; control 3, 0.8%; p<.001). All measures of Web traffic changed during the Internet paid search phase (p<.01); however, changes were independent of the advertisement periods. The incremental cost for the cultural advertisement per CME credit requested was $0.64US.
Internet advertisement focusing on cultural competence and CME was associated with about a three-fold increase in requests for CME credit at an incremental cost of under $1; however, Web traffic changes were independent of the advertisement strategy.
Marketing; Continuing Medical Education; Internet; Web; Online; Cultural Competence
Chemokines and their receptors play a role in the innate immune response as well as in the disruption of the balance between pro-inflammatory Th17 cells and regulatory T cells (Treg), underlying the pathogenesis of coronary vasculitis in Kawasaki disease (KD).
Here we show that genetic inactivation of chemokine receptor (CCR)-2 is protective against the induction of aortic and coronary vasculitis following injection of Candida albicans water-soluble cell wall extracts (CAWS). Mechanistically, both T and B cells were required for the induction of vasculitis, a role that was directly modulated by CCR2. CAWS administration promoted mobilization of CCR2-dependent inflammatory monocytes (iMo) from the bone marrow (BM) to the periphery as well as production of IL-6. IL-6 was likely to contribute to the depletion of Treg and expansion of Th17 cells in CAWS-injected Ccr2+/+ mice, processes that were ameliorated following the genetic inactivation of CCR2.
Collectively, our findings provide novel insights into the role of CCR2 in the pathogenesis of vasculitis as seen in KD and highlight novel therapeutic targets, specifically for individuals resistant to first-line treatments.
CCR2; Coronary vasculitis; Treg; Treg/Th17 imbalance
Autologous gastrointestinal segments are utilized as the primary option for bladder reconstructive procedures despite their inherent morbidity and significant complication rate. Multi-laminate biomaterials derived from Bombyx mori silk fibroin and prepared from a gel spinning process may serve as a superior alternative for bladder tissue engineering due to their robust mechanical properties, biocompatibility, and processing plasticity. In the present study, we sought to determine the impact of variations in winding (axial slew rate: 2 and 40 mm/sec) and post-winding (methanol and lyophilization) fabrication parameters on the in vivo performance of gel spun silk scaffolds in a murine model of bladder augmentation. Three silk matrix groups with distinct structural and mechanical properties were investigated following 10 weeks of implantation including our original prototype previously shown to support bladder regeneration, Group 1 (2mm/sec, methanol) as well as Group 2 (40mm/sec, methanol) and Group 3 (40mm/sec, lyophilization) configurations. Non surgical animals were assessed in parallel as controls. Quantification of residual scaffold area demonstrated that while Group 1 and 2 scaffolds were largely intact, processing parameters utilized for Group 3 led to significantly higher degrees of scaffold degradation in comparison to Group 1. Histological (hematoxylin and eosin, masson’s trichrome) and immunohistochemical (IHC) analyses showed comparable extents of smooth muscle regeneration and contractile protein (α-smooth muscle actin and SM22α) expression within the original defect site throughout all matrix groups similar to controls. Parallel evaluations demonstrated transitional urothelial formation with prominent uroplakin and p63 protein expression supported by Group 1 and 3 scaffolds, while Group 2 variants supported a thin, immature epithelium composed primarily of uroplakin-negative, p63-positive basal cells. Voided stain on paper analysis revealed similar voiding patterns between all matrix groups; however Group 2 animals displayed substantially lower voided volumes with increased frequency in comparison to controls. In addition, cystometric assessments revealed all matrix groups supported comparable degrees of bladder compliance similar to control levels. The results of this study demonstrate that selective alterations in winding and post-winding fabrication parameters can enhance the degradation rate of gel spun silk scaffolds in vivo while preserving their ability to support bladder tissue regeneration and function.
silk; bladder tissue engineering; smooth muscle cell; epithelium; urinary tract
To determine the effectiveness of a provider-based intervention to improve medication intensification among patients with diabetes.
Effectiveness cluster-randomised trial. Baseline and follow-up cross-sections of diabetes physicians’ patients.
Eleven U.S. Southeastern states, 2006–2008.
205 Rural primary care physicians, 95 completed the study.
Multicomponent interactive intervention including web-based continuing medical education (CME), performance feedback and quality improvement tools.
Primary outcome measures
Medication intensification, a dose increase of an existing medication or the addition of a new class of medication for glucose, blood pressure and lipids control on any of the three most recent office visits.
Of 364 physicians attempting to register, 102 were randomised to the intervention and 103 to the control arms; 95 physicians (intervention, n=48; control, n=47) provided data on their 1182 of their patients at baseline (intervention, n=715; control, n=467) and 945 patients at follow-up (intervention, n=479; control, n=466). For A1c control, medication intensification increased in both groups (intervention, pre 26.4% vs post 32.6%, p=0.022; control, pre 24.8% vs post 31.1%, p=0.033) (intervention, adjusted OR (AOR) 1.37; 95% CI 1.06 to 1.76; control, AOR 1.41 (95% CI 1.06 to 1.89)); however, we observed no incremental benefit solely due to the intervention (group-by-time interaction, p=0.948). Among patients with the worst glucose control (A1c >9%), intensification increased in both groups (intervention, pre 34.8% vs post 62.5%, p=0.002; control, pre 35.7% vs post 61.4%, p=0.008).
A wide-reach, low-intensity, web-based interactive multicomponent intervention had no significant incremental effect on medication intensification for control of glucose, blood pressure or lipids for patients with diabetes of physicians practising in the rural Southeastern USA.
Diabetes & Endocrinology; General Medicine (see Internal Medicine); Primary Care
Dendritic cells (DCs) have long been recognized as potential therapeutic targets of rheumatoid arthritis (RA). Increasing evidence has showed that DCs are capable of suppressing autoimmunity by expanding FoxP3+ regulatory T cells (Treg), which in turn exert immunosuppression by increasing TGFβ-1. In the SKG mice, activated DC prime autoreactive T cells causing autoantibody production and an inflammatory arthritic response. Recently, we reported that CC-chemokine receptor-2 deficient (Ccr2−/−) mice had impaired DCs migration and reduced CD8α+ DCs in the C57Bl/6J mice strain and that these mice were more susceptible to collagen antibody-induced arthritis (CAIA), compared to wild type mice. To examine the mechanism by which DCs contribute to the increased susceptibility of arthritis in Ccr2−/− mice, we tested the hypothesis that CD8α+ DCs are protective (tolerogenic) against autoimmune arthritis by examining the role of CD8α+ DCs in Ccr2−/− and SKG mice.
To examine the mechanism by which DCs defects lead to the development of arthritis, we used two murine models of experimental arthritis: collagen-induced arthritis (CIA) in DBA1/J mice and zymosan-induced arthritis in SKG mice. DBA1/J mice received recombinant Flt3L-injections to expand endogenous DCs populations or adoptive transfers of CD8α+ DCs.
Flt3L-mediated expansion of endogenous CD8α+ DCs resulted in heightened susceptibility of CIA. In contrast, supplementation with exogenous CD8α+ DCs ameliorated arthritis in Ccr2−/− mice and enhanced TGFβ1 production by T cells. Furthermore, SKG mice with genetic inactivation of CCR2 did not affect the numbers of DCs nor improve the arthritis phenotype.
CD8α+ DCs were tolerogenic to the development of arthritis. CD8α+ DCs deficiency heightened the sensitivity to arthritis in Ccr2−/− mice. Ccr2 deficiency did not alter the arthritic phenotype in SKG mice suggesting the arthritis in Ccr2−/− mice was T cell-independent.
CCR2; dendritic cells; CD8α+ DC; arthritis; SKG
Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.
Bioengineering; Issue 66; Medicine; Biomedical Engineering; Physiology; Silk; bladder tissue engineering; biomaterial; scaffold; matrix; augmentation; cystometry
It is widely accepted that, when feasible, nephron-sparing surgery (NSS) is preferable to radical nephrectomy (RN) for treatment of renal tumors in adults. However, RN is more frequently used in children. We sought to compare in-hospital outcomes after NSS and RN for malignant pediatric renal tumors.
Patients & Methods
The Pediatric Health Information System (PHIS) combines data from over 40 North American pediatric hospitals. We queried PHIS to identify children with malignant renal tumors who underwent surgery from 2003 to 2009. We examined whether outcomes (complication rates, cost and length of stay) differed by procedure type. Multivariate regression models were used to adjust for confounding and generalized estimating equations were used to adjust for hospital clustering.
We identified 1,235 children with renal tumors who underwent RN (91%) or NSS (9%). Patients undergoing RN and NSS had similar median co-morbidity scores (p=0.98), hospital length of stays (each 6.0 days, p=0.54), in-hospital charges, ($25,700 v. $37,000, p=0.11), and surgical complication rates (16.4 v. 20.5%, p=0.24). These outcomes remained similar after adjusting for other patient and hospital factors.
The majority of children with malignant renal tumors treated at children’s hospitals undergo RN. RN and NSS use were not significantly different in terms of their length of hospital stay, in-hospital charges, and complication rates. While oncologic outcomes are lacking, these data suggest that NSS may be performed in selected children with malignant renal tumors without significantly increasing their hospital charges, length of stay, or surgical complication rates.
Kidney Neoplasms; Wilms Tumor; Renal Cell Carcinoma; Pediatrics
To assess whether a novel evaluation tool could guide curricular change in an internal medicine residency program.
The authors developed an 8-item Ecological Momentary Assessment tool and collected daily evaluations from residents of the relative educational value of 3 differing ambulatory morning report formats (scale: 8 = best, 0 = worst). From the evaluations, they made a targeted curricular change and used the tool to assess its impact.
Residents completed 1388 evaluation cards for 223 sessions over 32 months, with a response rate of 75.3%. At baseline, there was a decline in perceived educational value with advancing postgraduate (PGY) year for the overall mean score (PGY-1, 7.4; PGY-2, 7.2; PGY-3, 7.0; P < .01) and for percentage reporting greater than 2 new things learned (PGY-1, 77%; PGY-2, 66%; PGY-3, 50%; P < .001). The authors replaced the format of a lower scoring session with one of higher cognitive content to target upper-level residents. The new session's mean score improved (7.1 to 7.4; P = .03); the adjusted odds ratios before and after the change for percentage answering, “Yes, definitely” to “Area I need to improve” was 2.53 (95% confidence interval [CI], 1.45–4.42; P = .001) and to “Would recommend to others,” it was 2.08 (95% CI, 1.12–3.89; P = .05).
The Ecological Momentary Assessment tool successfully guided ambulatory morning report curricular changes and confirmed successful curricular impact. Ecological Momentary Assessment concepts of multiple, frequent, timely evaluations can be successfully applied in residency curriculum redesign.
To identify, prioritize, and organize components of a cultural competence curriculum to address disparities in cardiovascular disease.
In 2006, four separate nominal group technique sessions were conducted with medical students, residents, community physicians, and academic physicians to generate and prioritize a list of concepts (i.e., ideas) to include in a curriculum. Afterward, 45 educators and researchers organized and prioritized the concepts using a card-sorting exercise. Multidimensional scaling (MDS) and hierarchical cluster analysis produced homogeneous groupings of related concepts and generated a cognitive map. The main outcome measures were the number of cultural competence concepts, their relative ranks, and the cognitive map.
Thirty participants generated 61 concepts, 29 were identified by at least 2 participants. The cognitive map organized concepts into four clusters, interpreted as: (1) patient’s cultural background (e.g.,, information on cultures, habits, values); (2) provider and health care (e.g., clinical skills, awareness of one’s bias, patient-centeredness, and professionalism), communication skills (e.g., history, stereotype avoidance, and health disparities epidemiology); (3) cross-culture (e.g., idiomatic expressions, examples of effective communication); and (4) resources to manage cultural diversity (e.g., translator guides, instructions and community resources). The MDS two-dimensional solution demonstrated a good fit (stress=0.07; R2=0.97).
A novel, combined approach allowed stakeholders’ inputs to identify and cognitively organize critical domains used to guide development of a cultural competence curriculum. Educators may use this approach to develop and organize educational content for their target audiences, especially in ill-defined areas like cultural competence.
Even though pay-for-performance programs are being rapidly implemented, little is known about how patient complexity affects practice-level performance assessment in rural settings. We sought to determine the association between patient complexity and practice-level performance in the rural United States.
Using baseline data from a trial aimed at improving diabetes care, we determined factors associated with a practice’s proportion of patients having controlled diabetes (hemoglobin A1c ≤7%): patient socioeconomic factors, clinical factors, difficulty with self-testing of blood glucose, and difficulty with keeping appointments. We used linear regression to adjust the practice-level proportion with A1c controlled for these factors. We compared practice rankings using observed and expected performance and classified practices into hypothetical pay-for-performance categories.
Rural primary care practices (n = 135) in 11 southeastern states provided information for 1641 patients with diabetes. For practices in the best quartile of observed control, 76.1% of patients had controlled diabetes vs 19.3% of patients in the worst quartile. After controlling for other variables, proportions of diabetes control were 10% lower in those practices whose patients had the greatest difficulty with either self testing or appointment keeping (p < .05 for both). Practice rankings based on observed and expected proportion of A1c control showed only moderate agreement in pay-for-performance categories (κ = 0.47; 95% confidence interval, 0.32–0.56; p < .001).
Basing public reporting and resource allocation on quality assessment that does not account for patient characteristics may further harm this vulnerable group of patients and physicians.
diabetes; quality of care; primary care
Filamentous fungi produce an impressive variety of secondary metabolites; many of them have important biological activities. The biosynthesis of these secondary metabolites is frequently induced by plant-derived external elicitors and appears to also be regulated by internal inducers, which may work in a way similar to that of bacterial autoinducers. The biosynthesis of penicillin in Penicillium chrysogenum is an excellent model for studying the molecular mechanisms of control of gene expression due to a good knowledge of the biochemistry and molecular genetics of β-lactam antibiotics and to the availability of its genome sequence and proteome. In this work, we first developed a plate bioassay that allows direct testing of inducers of penicillin biosynthesis using single colonies of P. chrysogenum. Using this bioassay, we have found an inducer substance in the conditioned culture broths of P. chrysogenum and Acremonium chrysogenum. No inducing effect was exerted by γ-butyrolactones, jasmonic acid, or the penicillin precursor δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine. The conditioned broth induced penicillin biosynthesis and transcription of the pcbAB, pcbC, and penDE genes when added at inoculation time, but its effect was smaller if added at 12 h and it had no effect when added at 24 h, as shown by Northern analysis and lacZ reporter studies. The inducer molecule was purified and identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR) as 1,3-diaminopropane. Addition of pure 1,3-diaminopropane stimulated the production of penicillin by about 100% compared to results for the control cultures. Genes for the biosynthesis of 1,3-diaminopropane have been identified in the P. chrysogenum genome.
Fungi comprise a vast group of microorganisms including the Ascomycota (majority of all described fungi), the Basidiomycota (mushrooms or higher fungi), and the Zygomycota and Chytridiomycota (basal or lower fungi) that produce industrially interesting secondary metabolites, such as β-lactam antibiotics. These compounds are one of the most commonly prescribed drugs world-wide. Since Fleming's initial discovery of Penicillium notatum 80 years ago, the role of Penicillium as an antimicrobial source became patent. After the isolation of Penicillium chrysogenum NRRL 1951 six decades ago, classical mutagenesis and screening programs led to the development of industrial strains with increased productivity (at least three orders of magnitude). The new “omics” era has provided the key to understand the underlying mechanisms of the industrial strain improvement process. The review of different proteomics methods applied to P. chrysogenum has revealed that industrial modification of this microorganism was a consequence of a careful rebalancing of several metabolic pathways. In addition, the secretome analysis of P. chrysogenum has opened the door to new industrial applications for this versatile filamentous fungus.
The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway.
Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells.
In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.
secretory pathways; chymosin; filamentous fungi; casein phosphopeptides; vesicles; extracellular proteins
To investigate the association between physician participants’ levels of engagement in a Web-based educational intervention and their patients’ baseline diabetes measures.
The authors conducted a randomized trial of online CME activities designed to improve diabetes care provided by family, general, and internal medicine physicians in rural areas of 11 southeastern states between September 2006 and July 2008. Using incidence rate ratios derived from negative binomial models, the relationship between physicians’ engagement with the study Web site and baseline proportion of their patients having controlled diabetes (hemoglobin A1c ≤7%) was explored.
One hundred thirty-three participants (intervention = 64; control = 69) provided information for 1,637 patients with diabetes. In the intervention group, physicians in practices in the worst quartiles of A1c control were least engaged with the study Web site in nearly all dimensions. Total number of pages viewed decreased as quartile of A1c control worsened (137, 73, 68, 57; P = .007); similarly, for a given 10% increase in proportion of patients with controlled A1c, participants viewed 1.13 times more pages (95% CI: 1.02–1.26, P = .02). In the control group, engagement was neither correlated with A1c control nor different across quartiles of A1c control.
Engagement in Web-based interventions is measurable and has important implications for research and education. Because physicians of patients with the greatest need for improvement in A1c control may not use online educational resources as intensely as others, other strategies may be necessary to engage these physicians in professional development activities.
Physician use of the Internet for practice improvement has increased dramatically over the last decade, but research shows that many physicians choose not to participate. The current study investigated the association of specific physician characteristics with enrollment rates and intensity of participation in a specific Internet-delivered educational intervention to improve care to post–myocardial infarction (MI) patients.
Primary-care physicians were recruited for participation in a randomized controlled trial designed to compare effectiveness of an intervention Web site versus a control Web site in the management of adult chronic disease. Physicians were informed that the intervention focused on ambulatory post–myocardial infarction patients. Physician characteristics were obtained from a commercial vendor with data merged from the American Medical Association and Alabama State Licensing Board. Enrollment and Web use were tracked electronically.
Out of a sample of 1337 eligible physicians, 177 (13.2%) enrolled in the study. Enrollment was higher for physicians with more post-MI patients (≥20 vs < 20 patients, 15.3% vs 9.3%, P = .002) and for those practicing in rural compared to urban areas (16.3% vs 12.1%, P = .046). Intensity of use of the Internet courses after initial enrollment was not predicted by physician characteristics in the current sample.
Physicians with more post-MI patients and rural practice location were found to predict enrollment in an Internet-delivered continuing medical education (CME) intervention designed to improve care for post-MI patients. These factors predicted program interest but not program use. More research is needed to replicate these findings to investigate variables that determine physician engagement in Internet CME.
education; medical; continuing; evidenced-based practice; Internet use; myocardial-infarction care; diffusion of innovation
This paper highlights a descriptive study of the challenges and lessons learned in the recruitment of rural primary care physicians into a randomized clinical trial using an Internet-based approach.
A multidisciplinary/multi-institutional research team used a multilayered recruitment approach, including generalized mailings and personalized strategies such as personal office visits, letters, and faxes to specific contacts. Continuous assessment of recruitment strategies was used throughout study in order to readjust strategies that were not successful.
We recruited 205 primary care physicians from 11 states. The 205 lead physicians who enrolled in the study were randomized, and the overall recruitment yield was 1.8% (205/11 231). In addition, 8 physicians from the same practices participated and 12 nonphysicians participated. The earlier participants logged on to the study Web site, the greater yield of participation. Most of the study participants had logged on within 10 weeks of the study.
Despite successful recruitment, the 2 major challenges in recruitment in this study included defining a standardized definition of rurality and the high cost of chart abstractions. Because many of the patients of study recruits were African American, the potential implications of this study on the field of health disparities in diabetes are important.
recruitment; physicians; diabetes; Internet intervention
Rationale, aims and objectives
Efforts to implement evidence-based medicine (EBM) training in developing countries are limited. We describe the results of an international effort to improve research capacity in a developing country; we conducted a course aimed at improving basic EBM attitudes and identified challenges.
Between 2005 and 2009, we conducted an annual 3-day course in Perú consisting of interactive lectures and case-based workshops. We assessed self-reported competence and importance in EBM using a Likert scale (1 = low, 5 = high).
Totally 220 clinicians participated. For phase I (2005–2007), self-reported EBM competence increased from a median of 2 to 3 (P < 0.001) and the perceived importance of EBM did not change (median = 5). For phase II (2008–2009), before the course, 8–72% graded their competence very low (score of 1–2). After the course, 67–92% of subjects graded their increase in knowledge very high (score of 4–5). The challenges included limited availability of studies relevant to the local reality written in Spanish, participants’ limited time and lack of long-term follow-up on practice change. Informal discussion and written evaluation from participants were universally in agreement that more training in EBM is needed.
In an EBM course in a resource-poor country, the baseline self-reported competence and experience on EBM were low, and the course had measurable improvements of self-reported competence, perceived utility and readiness to incorporate EBM into their practices. Similar to developed countries, translational research and building the research capacity in developing countries is critical for translating best available evidence into practice.
developing countries; education; evidence-based medicine; evidence-based practice; international cooperation
The phylum Apicomplexa includes a large group of protozoan parasites responsible for a wide range of animal and human diseases. Destructive pathogens, such as Plasmodium falciparum and Plasmodium vivax, causative agents of human malaria, Cryptosporidium parvum, responsible of childhood diarrhoea, and Toxoplasma gondii, responsible for miscarriages and abortions in humans, are frequently associated with HIV immunosuppression in AIDS patients. The lack of effective vaccines, along with years of increasing pressure to eradicate outbreaks with the use of drugs, has favoured the formation of multi-drug resistant strains in endemic areas. Almost all apicomplexan of medical interest contain two endosymbiotic organelles that contain their own mitochondrial and apicoplast DNA. Apicoplast is an attractive target for drug testing because in addition to harbouring singular metabolic pathways absent in the host, it also has its own transcription and translation machinery of bacterial origin. Accordingly, apicomplexan protozoa contain an interesting mixture of enzymes to unwind DNA from eukaryotic and prokaryotic origins. On the one hand, the main mechanism of DNA unwinding includes the scission of one—type I—or both DNA strands—type II eukaryotic topoisomerases, establishing transient covalent bonds with the scissile end. These enzymes are targeted by camptothecin and etoposide, respectively, two natural drugs whose semisynthetic derivatives are currently used in cancer chemotherapy. On the other hand, DNA gyrase is a bacterial-borne type II DNA topoisomerase that operates within the apicoplast and is effectively targeted by bacterial antibiotics like fluoroquinolones and aminocoumarins. The present review is an update on the new findings concerning topoisomerases in apicomplexan parasites and the role of these enzymes as targets for therapeutic agents.
DNA topoisomerases; chemotherapy; apicomplexan protozoa; tropical diseases