Reprint of “GELFrEE fractionation combined with mass spectrometry for proteome analysis of secreted toxins from Enteropathogenic Escherichia coli (EPEC)”

Enteropathogenic Escherichia coli, or EPEC, is a human pathogen associated with gastroenteritis and diarrheal disease whose pathogenicity is related to the secretion of effector proteins (exotoxins). Determining exotoxin expression level is of considerable interest to those studying toxin function and pathological phenotypes. Mass spectrometry (MS) provides an ideal platform for detection and quantification of proteins from complex mixtures. Here, we apply a solution-phase electrophoretic platform (GELFrEE) followed by MS to characterize the secreted proteome of a wild type and mutant strain of EPEC (ΔsepD), exhibiting enhanced secretion of effector proteins. Through peptide-level analysis, a total of 363 and 155 proteins were identified from the wild type and mutant strains, respectively. Semi-quantitative analysis of the MS data reveals the effector proteins EspB, EspC, and EspD appear in a relatively greater abundance from wild type EPEC, while two major virulence factors in EPEC, Tir and NleA appear in greater abundance from the secreted proteome of the mutant strain. Additionally, intact proteins may further be characterized following GELFrEE with MS to improve throughput of analysis. This study demonstrates the application of GELFrEE-MS to differentiate wild type and mutant strains of EPEC. This platform is therefore a powerful tool to study exotoxin secretion from pathogenic bacteria.     

Reprint of “Assessment of marker proteins identified in whole cell extracts for bacterial speciation using liquid chromatography electrospray ionization tandem mass spectrometry”

Staphylococcal strains (CoNS) were speciated in this study. Digests of proteins released from whole cells were converted to tryptic peptides for analysis. Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS/MS, Orbitrap) was employed for peptide analysis. Data analysis was performed employing the open-source software X!Tandem which uses sequenced genomes to generate a virtual peptide database for comparison to experimental data. The search database was modified to include the genomes of the 11 Staphylococcus species most commonly isolated from man. The number of total peptides matching each protein along with the number of peptides specifically matching to the homologue (or homologues) for strains of the same species were assessed. Any peptides not matching to the species examined were considered conflict peptides. The proteins typically identified with the largest percentage of sequence coverage, number of matched peptides and number of peptides corresponding to only the correct species were elongation factor Tu (EF Tu) and enolase (Enol). Additional proteins with consistently observed peptides as well as peptides matching only homologues from the same species were citrate synthase (CS) and 1-pyrroline-5-carboxylate dehydrogenase (1P5CD).Protein markers, previously identified from gel slices, (aconitate hydratase and oxoglutarate dehydrogenase) were found to provide low confidence scores when employing whole cell digests. The methodological approach described here provides a simple yet elegant way of identification of staphylococci. However, perhaps more importantly the technology should be applicable universally for identification of any bacterial species.     

Plasma estrone sulfate assay in men: Comparison of radioimmunoassay,mass spectrometry coupled to gas chromatography (GC–MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS)

BackgroundEstrogens are involved in the natural history of the prostate cancer and estrone sulfate, the quantitatively main circulating plasma estrogen in men, has been associated with an aggressive form of this cancer. A convenient and accurate plasma assay of this steroid has become important.MethodsWe simultaneously assayed estrone sulfate in the plasma of one hundred men aged 30–50 years, according to LC-MS/MS, GC–MS after solvolysis of E₁S, radioimmunoassay after a chromatographic purification step, and a direct RIA commercial kit.ResultsEstrone sulfate plasma levels obtained with the first three methods were not significantly different. However, estrone sulfate levels measured by the direct RIA were three-fold higher than those obtained by the first three methods. We showed that the excessively high estrone sulfate levels obtained with the direct RIA kit had two origins: interference by high dehydroepiandrosterone sulfate plasma levels in men, and estrone sulfate inaccurate low concentrations in the standards.ConclusionThe LC-MS/MS method can be considered as an optimum option for clinical laboratory. The GC–MS method requires solvolysis to estrone, but allows simultaneous unconjugated steroid measurement. RIA method, with chromatographic purification, is cumbersome, but less expensive. DSL-5400 kit yielded estrone sulfate plasma levels that were too high.     

Role of liquid chromatography–high-resolution mass spectrometry (LC-HR/MS) in clinical toxicology

Background. Gas chromatography (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS) are widely used to confirm drug screening results and for urine screening in presumed intoxicated patients. These techniques are better suited to targeted analysis than to general unknown screening and, due to the complexity of testing, results are seldom available rapidly enough to contribute to the immediate care of the patient. High resolution (HR)/MS with time-of-flight (TOF) or orbitrap instruments offer potential advantages in clinical toxicology. Comparison of GC-MS, LC-MS/MS and LC-HR/MS. For unknown analyses, GC-MS and LC-MS/MS require comparison of full-scan spectra against preestablished libraries. Operation in full-scan mode greatly reduces sensitivity and some drugs present in low but significant concentrations may be missed. Selected ion monitoring (SIM) in GC/MS and selected reaction monitoring (SRM) in LC-MS/MS, where only targeted ions are monitored, increase sensitivity but require prior knowledge of what compound is to be measured. LC-HR/MS offers mass assignment with an accuracy of 0.001 atomic mass units (amu) compared with 1 amu in conventional MS. Tentative identification is thus directed to a very limited set of compounds (or even one unique compound) based on the exact molecular formula rather than a fragmentation pattern, since HR/MS can discriminate between compounds with the same nominal molecular mass. LC-MS/MS has clear advantages over GC/MS in ease and speed of sample preparation and the opportunities for its automation. LC-HR/MS is more suitable to clinical toxicology because the drugs present in a sample are rarely known a priori, and tentative identifications of unknowns can be made without the availability of a reference standard or a library spectrum. Blood can be used in preference to urine which is more relevant to the patient's current clinical situation. Methods. A literature search was conducted using PUBMED for clinical toxicology, adulterants in illicit drugs and herbal supplements, and case reports using LC-TOF/MS and LC-HR/MS. Only 42 papers in English were identified in these searches. LC-HR/MS in clinical toxicology. LC-HR/MS has been used to detect designer drugs, doping agents, (neurosteroids) and adulterants such as levamisole, a veterinary antihelmitic found in street cocaine, and pharmaceuticals in herbal medications marketed to contain only natural ingredients. LC-HR/MS has proved useful for cases where existing tests were unable to identify the cause of the intoxication. One patient suffered a drug-induced seizure which was originally thought to be caused by an herbal medication, but diphenhydramine was determined to be the culprit. In another, 5-oxoproline was identified as the cause of metabolic acidosis seen in chronic acetaminophen (paracetamol) use. LC-HR/MS has successfully identified medications that were mislabeled or misrepresented street drugs. In one case, medications sold as diazepam were determined to be glyburide instead. The identification of novel designer amines, stimulants found in “bath salts”, and synthetic cannabinoids are well suited to LC-HR/MS. Dozens or even hundreds of possible compounds cannot realistically be tested on an individual basis by targeted LC-MS/MS or GC/MS analysis. Conclusions. LC-HR/MS offers unique opportunities for time-sensitive clinical analysis of blood samples from intoxicated patients and for comprehensive screening in a wide range of situations and materials. While the identification is not as definitive as that obtained by conventional fragmentation MS, the presumptive identification can be confirmed later with standards and spectral library matches. Optimum utilization of the presumptive diagnosis requires close collaboration between the laboratory analysts and their clinical counterparts.     

Identification of the “Minimal Triangle” and Other Common Event-to-Event Transitions in Conflict and Containment Incidents

Although individual conflict and containment events among acute psychiatric inpatients have been studied in some detail, the relationship of these events to each other has not. In particular, little is known about the temporal order of events for individual patients. This study aimed to identify the most common pathways from event to event. A sample of 522 patients was recruited from 84 acute psychiatric wards in 31 hospital locations in London and the surrounding areas during 2009–2010. Data on the order of conflict and containment events were collected for the first two weeks of admission from patients’ case notes. Event-to-event transitions were tabulated and depicted diagrammatically. Event types were tested for their most common temporal placing in sequences of events. Most conflict and containment occurs within and between events of the minimal triangle (verbal aggression, de-escalation, and PRN medication), and the majority of these event sequences conclude in no further events; a minority transition to other, more severe, events. Verbal abuse and medication refusal were more likely to start sequences of disturbed behaviour. Training in the prevention and management of violence needs to acknowledge that a gradual escalation of patient behaviour does not always occur. Verbal aggression is a critical initiator of conflict events, and requires more detailed and sustained research on optimal management and prevention strategies. Similar research is required into medication refusal by inpatients.     

Clinical Characteristics and Outcomes of “Silent” and “Non-Silent” Peritonitis in Patients on Peritoneal Dialysis

♦ Objectives: We compared the clinical characteristics and outcomes of “silent” peritonitis (meaning episodes without fever and abdominal pain) and “non-silent” peritonitis in patients on peritoneal dialysis (PD).♦ Methods: Our cohort study collected data about all peritonitis episodes occurring between January 2008 and April 2010. Disease severity score, demographics, and biochemistry and nutrition data were recorded at baseline. Effluent cell counts were examined at regular intervals, and the organisms cultured were examined. Treatment failure was defined as peritonitis-associated death or transfer to hemodialysis.♦ Results: Of 248 episodes of peritonitis occurring in 161 PD patients, 20.9% led to treatment failure. Of the 248 episodes, 51 (20.6%) were not accompanied by fever and abdominal pain. Patients with these silent peritonitis episodes tended to be older (p = 0.003). The baseline values for body mass index, triglycerides, and daily energy intake were significantly lower before silent peritonitis episodes than before non-silent episodes (p = 0.01, 0.003, and 0.001 respectively). Although silent peritonitis episodes were more often culture-negative and less often caused by gram-negative organisms, and although they presented with low effluent white cell counts on days 1 and 3, the risk for treatment failure in those episodes was not lower (adjusted odds ratio: 1.33; 95% confidence interval: 0.75 to 2.36; p = 0.33).♦ Conclusions: Silent peritonitis is not a rare phenomenon, especially in older patients on PD. Although these episodes were more often culture-negative, silent presentation was not associated with a better outcome.Key words: Peritonitis, outcomesPeritonitis, a common complication of peritoneal dialysis (PD), contributes to treatment failure, hospitalization, and death. It is important to diagnose PD-associated peritonitis and to initiate empiric antibiotic therapy as early as possible to prevent those serious consequences (1). Peritonitis is defined based on the presentation of symptoms, peritoneal effluent cell count, and culture of micro-organisms. Patients have to recognize their symptoms—including cloudy effluent, fever, and abdominal pain—early in the onset of peritonitis. Awareness of the symptoms of peritonitis is a key precondition for obtaining an effluent cell count and culture to promptly make the diagnosis, especially for patients who live far from the hospital and who cannot easily send dialysate samples to a lab.In fact, the symptoms of peritonitis vary in severity across the PD population. In pediatric PD patients, disease severity scores (DSSs) at the onset of peritonitis are calculated as a points total for pain and fever (2-5). The range shows that some patients might present with cloudy effluent alone, and some might complain of severe abdominal pain or high fever, or both.In most PD centers, DSS is not a routine measurement tool for PD-related peritonitis in adults. The DSS range for the adult PD population is not known, but we recognized that abdominal pain and fever might be absent at the onset of some peritonitis episodes. Here, we refer to those peritonitis episodes as “silent” peritonitis episodes. We coined the term silent peritonitis for this phenomenon because patients do not complain of fever or abdominal pain, and yet they have peritonitis. By contrast, we refer to peritonitis episodes with varying extents of fever and abdominal pain as “non-silent”peritonitis.Because previous studies reported that an absence of fever in bacteremic community-acquired pneumonia predicts a worse outcome in elderly individuals (6,7), comparisons of the clinical characteristics and outcomes in silent and non-silent peritonitis in the PD population are of interest. In the present study, we addressed that issue in a single-center cohort of patients presenting with incident episodes of peritonitis. Specifically, we set out to determine the clinical characteristics (including demographics, biochemical and nutrition indices, effluent cell counts, and causative organisms) and final outcomes associated with these two types of peritonitis.     

Do two oxidants (ferric-peroxo and ferryl-oxo species) act in the biosynthesis of estrogens? A DFT calculation

Density functional theory calculations were performed in order to reveal the mysterious catalytic step of the biosynthesis of estrogens. The results indicated two reactive oxidants, ferric-peroxo and ferryl-oxo (compound I) species, to participate in the conversion of androgens to estrogens. The ferric-peroxo species was determined, according to our derived mechanism, to act in the oxidation of 19-OH androgen to yield the 19,19-gem-diol intermediate and generate the ferryl-oxo (compound I) species. This species was then modeled to effect, in the final step, an abstraction of H from an O–H group of 19,19-gem-diol to give the experimentally observed products. We considered our new mechanistic scenario to reasonably explain the latest experimental observations and to provide deep insight complementing the newly accepted compound I (Cpd I) mechanism.

Density functional theory calculations were performed in order to reveal the mysterious catalytic step of the biosynthesis of estrogens.     

The prediction of total skeletal muscle mass in a Caucasian population – comparison of Magnetic resonance imaging (MRI) and Dual-energy X-ray absorptiometry (DXA)

Dual-energy X-ray (DXA) is an alternative to magnetic resonance imaging (MRI) to measure skeletal muscle mass. DXA assesses lean body mass (LBM), and MRI measures skeletal muscle mass (SMM). Kim et al. (Am J Clin Nutr 2002; 76 : 378; J Appl Physiol (1985) 2004; 97 : 655) developed MRI-based algorithms to estimate whole-body SMM by DXA. These algorithms were based on an ethnically mixed study population (Kim et al., Am J Clin Nutr 2002; 76 : 378; J Appl Physiol (1985) 2004; 97 : 655). It is unclear whether Kim's algorithms are accurate in an exclusive Caucasian population. The aim of our study was to validate Kim's equation in a Caucasian population of 346 subjects. SMM_MRI was assessed using MRI, and LBM and BMC_DXA were measured by DXA and fat mass (FM_ADP) by air-displacement plethysmographie (ADP). SMM_MRI and predicted SMM were highly correlated (r = 0·944; P<0·05). The standard error of estimate of the regression equation was 2·4 kg. However, Bland–Altman plots showed a significant (P<0·001) systematic bias between SMM_MRI (median 25·1 kg; IQ 20·2–31·1 kg) and predicted SMM (median 26·3 kg; IQ 22·6–33·0 kg), overestimating SMM by 9·8%. Multiple regression analyses showed that weight explained 4·4% of the variance in the differences between SMM_MRI and predicted SMM with the major part unexplained. Kim's algorithm has a systematic unexplained bias and is not recommended in Caucasians.     

Identification of NDM-1 in a Putatively Novel Acinetobacter Species (“NB14”) Closely Related to Acinetobacter pittii

In this study, we describe the molecular characterization of a plasmid-located bla_NDM-1 harbored by an Acinetobacter clinical isolate recovered from a patient in Turkey that putatively constitutes a novel Acinetobacter species, as shown by its distinct ARDRA (amplified 16S ribosomal DNA restriction analysis) profile and molecular sequencing techniques. bla_NDM-1 was carried by a conjugative plasmid widespread among non-baumannii Acinetobacter isolates, suggesting its potential for dissemination before reaching more clinically relevant Acinetobacter species.     

The development and implementation of a training package for dietitians on cow’s milk protein allergy in infants and children based on UK RCPCH competencies for food allergies – a pilot study

Background

Many food allergy guidelines have been published worldwide over recent years. The United Kingdom National Institute of Health and Clinical Excellence guidelines and The Royal College of Paediatrics and Child Health food allergy care pathways require dietitians to assist with the diagnosis and management of food allergies, which highlighted the need for further education of dietitians to meet these competencies. The aim of this study was to design a competence based one day education course for dietitians on the diagnosis and management of cow’s milk protein allergy in infants and children.

Methods

A one day training course was developed. Dietitians’ knowledge was assessed via multiple choice questions before and on the day of the course and retention of knowledge was assessed one month after the course. Pre course reading was given once the first assessment was completed.

Results

Thirty seven dietitians attended the course and 32 completed all three assessments. A significant improvement in assessment scores was seen between the pre course and on the day assessments of 7.2% (p < 0.001) and between pre course and post course assessments of 8.9% (p < 0.001). In delegates who rated their perceived level of knowledge as high, a significant increase was seen between pre course and on the day and between pre course and post course (both p < 0.001). Actual increase in knowledge was seen alongside a significant increase in high rating of perceived level of confidence between pre course and on the day and between pre course and post course (both p < 0.001).

Conclusions

Educating dietitians using the format of one day teaching with pre and post course assessment has improved both knowledge and competencies in the diagnosis and management of cow’s milk protein allergy. Further courses in other areas of food allergy could be developed using this approach within the UK and worldwide.     

Effects of galanin(1–16) on pancreatic secretion in anesthetized and conscious rats

Galanin, a 29-amino acid peptide, has been demonstrated in pancreatic nerve endings and found to inhibit insulin release in the rat. However, the data available concerning its effects on exocrine pancreatic secretion are contradictory. The aim of the present study was to evaluate the effects of a synthetic porcine galanin sequence, Gal(1–16), on stimulated pancreatic secretion in hyperglycemic anesthetized and conscious rats. Male Wistar rats were anesthetized and surgically prepared with pancreatic and femoral vein catheters. In anesthetized animals, the pancreatic secretion was continuously stimulated with 150 ng cholecystokinin octapeptide (CCK-8)/kg body weight per 30 min, dissolved in saline or 10% glucose. Synthetic Gal(1–16) (0.3 or 1 nmol/kg per h) was infused over a 60-min period. In conscious rats, 1, 3, or 10 nmol Gal(1–16)/kg per h was administered in a continuous saline or 10% glucose infusion over a 30-min period. The pancreatic secretory volume and protein output were determined in 30-min samples in both models. In anesthetized rats, 0.3 nmol Gal(1–16)/kg per h did not modify pancreatic secretion during CCK-8 stimulation. However, both the pancreatic secretory volume and the protein output were significantly inhibited compared with the basal levels by 1 nmol Gal(1–16)/kg per h. The inhibitory effect of Gal(1–16) on pancreatic secretion was more marked with CCK-8/glucose (53.9%) than with CCK-8/saline stimulation (20.1%). In conscious rats, significant inhibitory effects of 1 nmol Gal(1–16)/kg per h in saline were observed (18%). During glucose infusion, a dose-dependent inhibition of 1, 3, and 10 nmol Gal(1–16)/kg per h on pancreatic secretory volume and protein output (35% inhibition at 1 nmol/kg per h) was observed. In conclusion, the inhibitory effect of Gal(1–16) on exogenous and endogenous CCK-stimulated pancreatic secretion was found to be more potent in the presence of glucose both in anesthetized and in conscious rats. These results may suggest an indirect (insulin-mediated) inhibitory effect of porcine Gal(1–16) on pancreatic secretion in the rat.     

Patients’ view on health-related aspects of functioning and disability of joint contractures: a qualitative interview study based on the International Classification of Functioning,Disability and Health (ICF)

Abstract

Purpose: The aim of this study was to identify health-relevant aspects of functioning and disability of persons aged 65 years or older with joint contractures, to link the findings to corresponding ICF categories and to describe the patients’ perspective. Methods: We conducted 43 qualitative, semi-structured, face-to-face interviews with affected persons in two different locations (Witten, Munich) and in three different settings. Data were analyzed using the “meaning condensation procedure” and then linked to ICF categories. Results: From all interviews a total of 2499 single meaning-concepts were extracted which were linked to 324 different ICF categories. The participants in all settings mainly reported problems related to “Mobility of a single joint (b710)”, “Sensation of pain (b280)” and problems related to “Walking (d450)”. Almost all participants reported “Products and technology for personal indoor and outdoor mobility and transportation (e120)” as a relevant environmental factor. Conclusions: From the patients’ perspective, joint contractures have an impact on multifaceted aspects of functioning and disability, mainly body functions, environmental factors and activities and participation. The results of this study will contribute to the development of a standard instrument for measuring functioning, disability and health-relevant aspects for patients with joint contractures.

• Implications for Rehabilitation

• Joint contractures are a major cause for the development of disability in older people.

• Patients’ perspectives and their personal experiences have to be considered when assessing the impact of joint contractures.

• The International Classification of Functioning, Disability and Health (ICF) is an appropriate framework for describing the patients’ multifaceted experience of joint contractures.

     

Comparison of the Impact of “Fast Decline” in Residual Renal Function and “Initial Anuria” on Long-Term Outcomes in CAPD Patients

♦ Background: Residual renal function (RRF) is pivotal to long-term outcomes, while rapid RRF decline (RRFD) is associated with mortality risk for continuous ambulatory peritoneal dialysis (CAPD) patients. This study was conducted to compare the impact of “initial anuria” and rapid RRFD on the long-term prognosis of CAPD patients.♦ Method: According to the timing of anuria and the slope of RRFD, a total of 255 incident CAPD patients were divided into 3 groups. For the “anuria” group, anuria was detected from CAPD initiation and persisted for > 6 months (n = 27). Based on the median of the RRFD slope, the other 228 non-anuric patients were divided into a “slow decliner” group (n = 114), and a “rapid decliner” group (n = 114). The maximal observation period was 120 months.♦ Results: Logistic regression tests indicated that the “anuria” group was associated with previous hemodialysis > 3 months (odds ratio [OR]: 8.52, 95% confidence interval [CI]: 3.12 – 23.28), and female (OR: 0.29, 95% CI: 0.09 – 0.90), while the “fast decliner” group with higher Davies co-morbidity scores (DCS) (OR: 1.52; 95% CI: 1.08 – 2.14), body mass index (BMI) (OR: 1.12; 95% CI: 1.04 – 1.21), and male (OR: 1.12; 95% CI: 1.04 – 1.21). After adjusting for DCS, the “fast decliner” group (hazard ratio [HR]: 0.37; 95% CI: 0.17 – 0.80) showed a better outcome than that of the “anuria” group (reference = 1). Both baseline RRF (β = -0.24; p < 0.001) and DCS (β = -3.76; p < 0.001) showed inverse linear correlations to the slope of RRFD. From the Cox proportional analyses, higher baseline RRF (HR: 0.92; 95% CI: 0.88 –.97) and higher slope of RRFD (slower decline in RRF) (HR: 0.90; 95% CI: 0.85 – 0.96) were independent factors for less mortality risk in patients with DCS = 0. However, only a higher slope of RRFD (HR: 0.97; 95% CI: 0.94 – 0.99) was significant for better survival in CAPD patients with DCS > 0.♦ Conclusion: Compared to the baseline RRF, CAPD patients with co-morbidities that rapidly deteriorate RRFD are more crucially associated with long-term mortality risk.     

Impact of Kendall,Hollon, Beck,Hammen, and Ingram (1987) on treatment of the continuity issue in “depression” research

The article, Issues and Recommendations Regarding Use of the Beck Depression Inventory (Kendall, Hollon, Beck, Hammen, & Ingram, 1987), has had a major impact on depression research. A majority of studies using only the BDI in nonclinical samples now refer to the construct measured as dysphoria rather than depression. This word change, however, is not always accompanied by other changes in research design and interpretation that would seem warranted by the concerns that initially prompted the dysphoria recommendation, such as the nonspecificity of high BDI scores to major depression. Researchers typically continue to derive hypotheses from depression theory, use only the BDI to measure dysphoria rather than purer markers of negative affectivity, cite as a limitation of their findings the danger of assuming continuity between subclinical and clinical depression, and sometimes lapse into depression terminology. Alternative suggestions are made for considering how the particular goals of a study might lead to various ways of handling the continuity issue.We are grateful to Tony Ahrens, Diane Arnkoff, and anonymous reviewers for feedback on earlier drafts.     

Toxicologists in public health – Following the path of Louis Roche (based on the Louis Roche lecture “An accidental toxicologist in public health”, Bordeaux, 2010)

Background.?The global burden of clinical toxicology suggests a natural partnership with public health. This article reflects the content of a Louis Roche lecture given in 2010.

Historical context.?Our practice and research in clinical toxicology has evolved from clinical cases to toxico-epidemiology to public health. This evolution in practice was initially unplanned but gained momentum and impact as we placed it more formally in a public health framework. This perspective is implicit in Louis Roche's call to “examine all aspects of the poisoning problem” and still provides a valuable starting point for any clinical toxicologist.

Discussion.?Clinical toxicology has always had a patient centered focus but its greatest successes have been related to public health interventions. Our early failures and later success in pubic health toxicology correlated with our understanding of the importance of partnerships outside our field. The most rapid dissemination and implementation of information derived from research occur through apriori partnerships with other agencies and international partners.

Conclusion.?Addressing both local and global need has a number of bilateral synergies. Repositioning clinical toxicology into a public health framework increases access to strategic partnerships, research funds, and policy implementation while still addressing questions that are important to clinical practice.