The NADPH oxidase enzyme complex, NOX2, is responsible for reactive oxygen species (ROS) production in neutrophils and has been recognized as a key mediator of inflammation. Here, we have performed rational design and in silico screen to identify a small molecule inhibitor, Phox-I1, targeting the interactive site of p67phox with Rac GTPase that is a necessary step of the signaling leading to NOX2 activation. Phox-I1 binds to p67phox with a submicromolar affinity and abrogates Rac1 binding, and is effective in inhibiting NOX2-mediated superoxide production dose-dependently in human and murine neutrophils without detectable toxicity. Medicinal chemistry characterizations have yielded promising analogs and initial information of the structure-activity relationship of Phox-I1. Our studies suggest the potential utility of Phox-I class inhibitors in NOX2 oxidase inhibition and present the first application of rational targeting of a small GTPase - effector interface.
Rac1; Rho GTPase; p67phox; NADPH oxidase; small molecule targeting; reactive oxygen species; neutrophil
Although it has been hypothesized that muscle metabolism and fatigability could be impaired in sickle cell patients, no study has addressed this issue.
We compared muscle metabolism and function (muscle microvascular oxygenation, microvascular blood flow, muscle oxygen consumption and muscle microvascular oxygenation variability, which reflects vasomotion activity, maximal muscle force and local muscle fatigability) and the hemorheological profile at rest between 16 healthy subjects (AA), 20 sickle cell-hemoglobin C disease (SC) patients and 16 sickle cell anemia (SS) patients.
Muscle microvascular oxygenation was reduced in SS patients compared to the SC and AA groups and this reduction was not related to hemorhelogical abnormalities. No difference was observed between the three groups for oxygen consumption and vasomotion activity. Muscle microvascular blood flow was higher in SS patients compared to the AA group, and tended to be higher compared to the SC group. Multivariate analysis revealed that muscle oxygen consumption was independently associated with muscle microvascular blood flow in the two sickle cell groups (SC and SS). Finally, despite reduced muscle force in sickle cell patients, their local muscle fatigability was similar to that of the healthy subjects.
Sickle cell patients have normal resting muscle oxygen consumption and fatigability despite hemorheological alterations and, for SS patients only, reduced muscle microvascular oxygenation and increased microvascular blood flow. Two alternative mechanisms can be proposed for SS patients: 1) the increased muscle microvascular blood flow is a way to compensate for the lower muscle microvascular oxygenation to maintain muscle oxygen consumption to normal values or 2) the reduced microvascular oxygenation coupled with a normal resting muscle oxygen consumption could indicate that there is slight hypoxia within the muscle which is not sufficient to limit mitochondrial respiration but increases muscle microvascular blood flow.
Patient evaluations are an important part of monitoring primary healthcare reforms, but there is little comparative information available to guide evaluators in the choice of instruments or to determine their relevance for Canada.
To compare values and the psychometric performances of validated instruments thought to be most pertinent to the Canadian context for evaluating core attributes of primary healthcare.
Among validated instruments in the public domain, we selected six: the Primary Care Assessment Survey (PCAS); the Primary Care Assessment Tool – Short Form (PCAT-S); the Components of Primary Care Index (CPCI); the first version of the EUROPEP (EUROPEP-I); the Interpersonal Processes of Care Survey, version II (IPC-II); and part of the Veterans Affairs National Outpatient Customer Satisfaction Survey (VANOCSS). We mapped subscales to operational definitions of attributes. All were administered to a sample of adult service users balanced by English/French language (in Nova Scotia and Quebec, respectively), urban/rural residency, high/low education and overall care experience. The sample was recruited from previous survey respondents, newspaper advertisements and community posters. We used common factor analysis to compare our factor resolution for each instrument to that of the developers.
Our sample of 645 respondents was approximately balanced by design variables, but considerable effort was required to recruit low-education and poor-experience respondents. Subscale scores are statistically different by excellent, average and poor overall experience, but interpersonal communication and respectfulness scores were the most discriminating of overall experience. We found fewer factors than did the developers, but when constrained to the number of expected factors, our item loadings were largely similar to those found by developers. Subscale reliability was equivalent to or higher than that reported by developers.
These instruments perform similarly in the Canadian context to their original development context, and can be used with confidence. Interpersonal and respectfulness scores are most discriminating of excellent, average or poor overall experience and are crucial dimensions of patient evaluations.
The operational definition of relational continuity is “a therapeutic relationship between a patient and one or more providers that spans various healthcare events and results in accumulated knowledge of the patient and care consistent with the patient's needs.”
To examine how well relational continuity is measured in validated instruments that evaluate primary healthcare from the patient's perspective.
645 adults with at least one healthcare contact in the previous 12 months responded to six instruments that evaluate primary healthcare. Five subscales map to relational continuity: the Primary Care Assessment Survey (PCAS, two subscales), the Primary Care Assessment Tool – Short Form (PCAT-S) and the Components of Primary Care Index (CPCI, two subscales). Scores were normalized for descriptive comparison. Exploratory and confirmatory (structural equation modelling) factor analysis examined fit to operational definition, and item response theory analysis examined item performance on common constructs.
All subscales load reasonably well on a single factor, presumed to be relational continuity, but the best model has two underlying factors corresponding to (1) accumulated knowledge of the patient and (2) relationship that spans healthcare events. Some items were problematic even in the best model. The PCAS Contextual Knowledge subscale discriminates best between different levels of accumulated knowledge, but this dimension is also captured well by the CPCI Accumulated Knowledge subscale and most items in the PCAT-S Ongoing Care subscale. For relationship-spanning events, the items' content captures concentration of care in one doctor; this is captured best by the CPCI Preference for Regular Provider subscale and, to a lesser extent, by the PCAS Visit-Based Continuity subscale and one relevant item in the PCAT-S Ongoing Care subscale. But this dimension correlates only modestly with percentage of reported visits to the personal doctor. The items function as yes/no rather than ordinal options, and are especially informative for poor concentration of care.
These subscales perform well for key elements of relational continuity, but do not capture consistency of care. They are more informative for poor relational continuity.
The operational definition of first-contact accessibility is “the ease with which a person can obtain needed care (including advice and support) from the practitioner of choice within a time frame appropriate to the urgency of the problem”; accommodation is “the way healthcare resources are organized to accommodate a wide range of patients' abilities to contact healthcare providers and reach healthcare services, that is to say telephone services, flexible appointment systems, hours of operation, and walk-in periods.”
To compare how well accessibility is measured in validated subscales that evaluate primary healthcare from the patient's perspective.
645 adults with at least one healthcare contact in the previous 12 months responded to six instruments that evaluate primary healthcare with four subscales that measure accessibility: the Primary Care Assessment Survey (PCAS), the Primary Care Assessment Tool – Short Form (PCAT-S, two subscales) and the first version of the EUROPEP (EUROPEP-I). Scores were normalized to a 0-to-10 scale for descriptive comparison. Exploratory and confirmatory (structural equation modelling) factor analysis examined fit to operational definition, and item response theory analysis examined item performance on common constructs.
The subscales demonstrate similar psychometric measures to those reported by developers. The PCAT-S First-Contact Utilization subscale does not fit the accessibility construct. The remaining three subscales load reasonably onto a single factor, presumed to be accessibility, but the best-fitting model has two factors: “timeliness of obtaining needed care” (PCAT-S First-Contact Access, some EUROPEP-I items) and “how resources are organized to accommodate clients” (PCAS Organizational Access and most of EUROPEP-I organization of care). Items in the PCAS and PCAT-S subscales have good discriminability.
Only three of the four subscales measure accessibility; all are appropriate for use in Canada. The PCAT-S First-Contact Access subscale is the best measure for first-contact accessibility, and PCAS Organizational Accessibility has good metric properties and measures for accommodation.
Respectfulness is one measurable and core element of healthcare responsiveness. The operational definition of respectfulness is “the extent to which health professionals and support staff meet users' expectations about interpersonal treatment, demonstrate respect for the dignity of patients and provide adequate privacy.”
To examine how well respectfulness is captured in validated instruments that evaluate primary healthcare from the patient's perspective, whether or not their developers had envisaged these as representing respectfulness.
645 adults with at least one healthcare contact with their own regular doctor or clinic in the previous 12 months responded to six instruments, two subscales that mapped to respectfulness: the Interpersonal Processes of Care, version II (IPC-II, two subscales) and the Primary Care Assessment Survey (PCAS). Additionally, there were individual respectfulness items in subscales measuring other attributes in the Components of Primary Care Index (CPCI) and the first version of the EUROPEP (EUROPEP-I). Scores were normalized for descriptive comparison. Exploratory and confirmatory (structural equation modelling) factor analyses examined fit to operational definition.
Respectfulness scales correlate highly with one another and with interpersonal communication. All items load adequately on a single factor, presumed to be respectfulness, but the best model has three underlying factors corresponding to (1) physician's interpersonal treatment (eigenvalue=13.99), (2) interpersonal treatment by office staff (eigenvalue=2.13) and (3) respect for the dignity of the person (eigenvalue=1.16). Most items capture physician's interpersonal treatment (IPC-II Compassionate, Respectful Interpersonal Style, IPC-II Hurried Communication and PCAS Interpersonal Treatment). The IPC-II Interpersonal Style (Disrespectful Office Staff) captures treatment by staff, but only three items capture dignity.
Various items or subscales seem to measure respectfulness among currently available validated instruments. However, many of these items related to other constructs, such as interpersonal communication. Further studies should aim at developing more refined measures – especially for privacy and dignity – and assess the relevance of the broader concept of responsiveness.
Comprehensiveness relates both to scope of services offered and to a whole-person clinical approach. Comprehensive services are defined as “the provision, either directly or indirectly, of a full range of services to meet most patients' healthcare needs”; whole-person care is “the extent to which a provider elicits and considers the physical, emotional and social aspects of a patient's health and considers the community context in their care.” Among instruments that evaluate primary healthcare, two had subscales that mapped to comprehensive services and to the community component of whole-person care: the Primary Care Assessment Tool – Short Form (PCAT-S) and the Components of Primary Care Index (CPCI, a limited measure of whole-person care).
To examine how well comprehensiveness is captured in validated instruments that evaluate primary healthcare from the patient's perspective.
645 adults with at least one healthcare contact in the previous 12 months responded to six instruments that evaluate primary healthcare. Scores were normalized for descriptive comparison. Exploratory and confirmatory (structural equation modelling) factor analysis examined fit to operational definition, and item response theory analysis examined item performance on common constructs.
Over one-quarter of respondents had missing responses on services offered or doctor's knowledge of the community. The subscales did not load on a single factor; comprehensive services and community orientation were examined separately. The community orientation subscales did not perform satisfactorily. The three comprehensive services subscales fit very modestly onto two factors: (1) most healthcare needs (from one provider) (CPCI Comprehensive Care, PCAT-S First-Contact Utilization) and (2) range of services (PCAT-S Comprehensive Services Available). Individual item performance revealed several problems.
Measurement of comprehensiveness is problematic, making this attribute a priority for measure development. Range of services offered is best obtained from providers. Whole-person care is not addressed as a separate construct, but some dimensions are covered by attributes such as interpersonal communication and relational continuity.
The operational definition of interpersonal communication is “the ability of the provider to elicit and understand patient concerns, to explain healthcare issues and to engage in shared decision-making if desired.”
To examine how well interpersonal communication is captured in validated instruments that evaluate primary healthcare from the patient's perspective.
645 adults with at least one healthcare contact in the previous 12 months responded to instruments that evaluate primary healthcare. Eight subscales measure interpersonal communication: the Primary Care Assessment Survey (PCAS, two subscales); the Components of Primary Care Index (CPCI, one subscale); the first version of the EUROPEP (EUROPEP-I); and the Interpersonal Processes of Care Survey, version II (IPC-II, four subscales). Scores were normalized for descriptive comparison. Exploratory and confirmatory (structural equation) factor analysis examined fit to operational definition, and item response theory analysis examined item performance.
Items not pertaining to interpersonal communication were removed from the EUROPEP-I. Most subscales are skewed positively. Normalized mean scores are similar across subscales except for IPC-II Patient-Centred Decision-Making and IPC-II Hurried Communication. All subscales load reasonably well on a single factor, presumed to be interpersonal communication. The best model has three underlying factors corresponding to eliciting (eigenvalue = 26.56), explaining (eigenvalue = 2.45) and decision-making (eigenvalue = 1.34). Both the PCAS Communication and the EUROPEP-I Clinical Behaviour subscales capture all three dimensions. Individual subscales within IPC-II measure each sub-dimension.
The operational definition is well reflected in the available measures, although shared decision-making is poorly represented. These subscales can be used with confidence in the Canadian context to measure this crucial aspect of patient-centred care.
Management continuity, operationally defined as “the extent to which services delivered by different providers are timely and complementary such that care is experienced as connected and coherent,” is a core attribute of primary healthcare. Continuity, as experienced by the patient, is the result of good care coordination or integration.
To provide insight into how well management continuity is measured in validated coordination or integration subscales of primary healthcare instruments.
Relevant subscales from the Primary Care Assessment Survey (PCAS), the Primary Care Assessment Tool – Short Form (PCAT-S), the Components of Primary Care Instrument (CPCI) and the Veterans Affairs National Outpatient Customer Satisfaction Survey (VANOCSS) were administered to 432 adult respondents who had at least one healthcare contact with a provider other than their family physician in the previous 12 months. Subscales were examined descriptively, by correlation and factor analysis and item response theory analysis. Because the VANOCSS elicits coordination problems and is scored dichotomously, we used logistic regression to examine how evaluative subscales relate to reported problems.
Most responses to the PCAS, PCAT-S and CPCI subscales were positive, yet 83% of respondents reported having one or more problems on the VANOCSS Overall Coordination subscale and 41% on the VANOCSS Specialist Access subscale. Exploratory factor analysis suggests two distinct factors. The first (eigenvalue=6.98) is coordination actions by the primary care physician in transitioning patient care to other providers (PCAS Integration subscale and most of the PCAT-S Coordination subscale). The second (eigenvalue=1.20) is efforts by the primary care physician to create coherence between different visits both within and outside the regular doctor's office (CPCI Coordination subscale). The PCAS Integration subscale was most strongly associated with lower likelihood of problems reported on the VANOCSS subscales.
Ratings of management continuity correspond only modestly to reporting of coordination problems, possibly because they rate only the primary care physician, whereas patients experience problems across the entire system. The subscales were developed as measures of integration and provider coordination and do not capture the patient's experience of connectedness and coherence.
This paper presents an overview of the analytic approaches that we used to assess the performance and structure of measures that evaluate primary healthcare; six instruments were administered concurrently to the same set of patients. The purpose is (a) to provide clinicians, researchers and policy makers with an overview of the psychometric methods used in this series of papers to assess instrument performance and (b) to articulate briefly the rationale, the criteria used and the ways in which results can be interpreted. For illustration, we use the case of instrument subscales evaluating accessibility. We discuss (1) distribution of items, including treatment of missing values, (2) exploratory and confirmatory factor analysis to identify how items from different subscales relate to a single underlying construct or sub-dimension and (3) item response theory analysis to examine whether items can discriminate differences between individuals with high and low scores, and whether the response options work well. Any conclusion about the relative performance of instruments or items will depend on the type of analytic technique used. Our study design and analytic methods allow us to compare instrument subscales, discern common constructs and identify potentially problematic items.
The aim of this study was to rate the importance of primary healthcare (PHC) attributes in evaluations of PHC organizational models in Canada.
Using the Delphi process, we conducted a consensus consultation with 20 persons recognized by peers as Canadian PHC experts, who rated the importance of PHC attributes within professional and community-oriented models of PHC.
Attributes rated as essential to all models were designated core attributes: first-contact accessibility, comprehensiveness of services, relational continuity, coordination (management) continuity, interpersonal communication, technical quality of clinical care and clinical information management. Overall, while all were important, non-core attributes – except efficiency/productivity – were rated as more important in community-oriented than in professional models. Attributes rated as essential for community-oriented models were equity, client/community participation, population orientation, cultural sensitivity and multidisciplinary teams.
Evaluation tools should address core attributes and be customized in accordance with the specific organizational models being evaluated to guide health reforms.
To evaluate chronic illness care delivery from the patient’s perspective and to examine its main correlates.
Cross-sectional, descriptive study using questionnaires and medical chart review.
Nine teaching family practices in Quebec.
A total of 364 patients with diabetes, hypertension, or chronic obstructive pulmonary disease.
Main outcomes measures
Score on the Patient Assessment of Chronic Illness Care (PACIC) questionnaire, which evaluates the patient’s perspective on the care received based on the chronic care model (CCM); patients characteristics (sex, level of education, number of chronic illnesses); patient-physician relationship (relational continuity, interpersonal communication assessed from the patient’s perspective); and interdisciplinary care and technical quality of care abstracted from patients’ medical charts.
The mean PACIC score obtained (2.8 out of 5) indicates that, on average, CCM-concordant care “generally did not occur” or occurred only “sometimes” in this network of teaching practices. However, with a mean technical quality-of-care score of nearly 80%, physicians in this network showed a high degree of adherence to clinical guidelines for the chronic illnesses under study. Patient education level lower than high school was negatively associated with PACIC scores, while positive associations were found with male sex, number of chronic illnesses, relational continuity, interpersonal communication, interdisciplinary care, and technical quality of care.
Patients with less education reported receiving less CCM-concordant care. The patient-physician relationship was the strongest correlate of PACIC scores, while interdisciplinary care and technical quality of care had modest contributions.
The objective of this study is to evaluate the feasibility, tolerance and efficacy of salvage external beam radiotherapy (EBRT) in persistent or recurrent prostate cancer after failed high intensity focused ultrasound (HIFU) therapy.
We reviewed data on tolerance and oncologic outcomes for all patients with biopsy-proven locally recurrent or persistent prostate cancer who underwent salvage EBRT in our department between April 2004 and June 2008. Minimum follow-up for inclusion was 2 years. Failure with EBRT was defined as biochemical relapse (Phoenix definition) or introduction of androgen deprivation therapy (ADT). Gastrointestinal and urinary toxicity and urinary stress incontinence were scored at 12 and 24 months (Radiation Therapy Oncology Group and Ingelman Sundberg rating, respectively).
The mean age of the patients was 68.8 years (range: 60–79). Mean prostate-specific antigen (PSA) before EBRT was 5.57 ng/mL (range: 2.5–14.8). Median follow-up was 36.5 ± 10.9 months (range: 24–54). No patient received adjunctive ADT. The EBRT course was well-tolerated and completed by all patients. The mean PSA nadir was 0.62 ng/mL (range: 0.03–2.4) and occurred after a median of 22 months (range: 12–36). One patient experienced biochemical failure and was prescribed ADT 30 months after EBRT. The disease-free survival rate was 83.3% at 36.5 months. There was no major EBRT-related toxicity at 12 or 24 months.
Our early clinical results confirm the feasibility and good tolerance of salvage radiotherapy after HIFU failure. Oncological outcomes were promising. A prospective study with longer follow-up is needed to identify factors predictive of success for salvage EBRT therapy after HIFU failure.
Prochlorococcus and Synechococcus, which numerically dominate vast oceanic areas, are the two most abundant oxygenic phototrophs on Earth. Although they require solar energy for photosynthesis, excess light and associated high UV radiations can induce high levels of oxidative stress that may have deleterious effects on their growth and productivity. Here, we compared the photophysiologies of the model strains Prochlorococcus marinus PCC 9511 and Synechococcus sp. WH7803 grown under a bell-shaped light/dark cycle of high visible light supplemented or not with UV. Prochlorococcus exhibited a higher sensitivity to photoinactivation than Synechococcus under both conditions, as shown by a larger drop of photosystem II (PSII) quantum yield at noon and different diel patterns of the D1 protein pool. In the presence of UV, the PSII repair rate was significantly depressed at noon in Prochlorococcus compared to Synechococcus. Additionally, Prochlorococcus was more sensitive than Synechococcus to oxidative stress, as shown by the different degrees of PSII photoinactivation after addition of hydrogen peroxide. A transcriptional analysis also revealed dramatic discrepancies between the two organisms in the diel expression patterns of several genes involved notably in the biosynthesis and/or repair of photosystems, light-harvesting complexes, CO2 fixation as well as protection mechanisms against light, UV, and oxidative stress, which likely translate profound differences in their light-controlled regulation. Altogether our results suggest that while Synechococcus has developed efficient ways to cope with light and UV stress, Prochlorococcus cells seemingly survive stressful hours of the day by launching a minimal set of protection mechanisms and by temporarily bringing down several key metabolic processes. This study provides unprecedented insights into understanding the distinct depth distributions and dynamics of these two picocyanobacteria in the field.
marine cyanobacteria; Synechococcus; Prochlorococcus; light/dark cycle; light stress; UV radiations; oxidative stress; photophysiology
During progression of melanoma, malignant melanocytes can be reprogrammed into mesenchymal-like cells through a process similar to epithelial-mesenchymal transition (EMT), which is associated with downregulation of the junctional protein E-cadherin and acquisition of a migratory phenotype. Recent evidence supports a role for SLUG, a transcriptional repressor of E-cadherin, as a melanocyte lineage transcription factor that predisposes to melanoma metastasis. However, the signals responsible for SLUG expression in melanoma are unclear and its role in the invasive phenotype is not fully elucidated. Here, we report that SLUG expression and activation is driven by SPARC (also known as osteonectin), a secreted extracellular matrix-associated factor that promotes EMT-like changes. Ectopic expression or knockdown of SPARC resulted in increased or reduced expression of SLUG, respectively. SLUG increase occurred concomitantly with SPARC-mediated downregulation of E-cadherin and P-cadherin, and induction of mesenchymal traits in human melanocytes and melanoma cells. Pharmacological blockade of PI3 kinase/AKT signaling impeded SPARC-induced SLUG levels and cell migration, whereas adenoviral introduction of constitutively active AKT allowed rescue of SLUG and migratory capabilities of SPARC knockdown cells. We also observed that pharmacological inhibition of oncogenic BRAFV600E using PLX4720 did not influence SLUG expression in melanoma cells harboring BRAFV600E. Furthermore, SLUG is a bona fide transcriptional repressor of E-cadherin as well as a regulator of P-cadherin in melanoma cells and its knockdown attenuated invasive behavior and blocked SPARC-enhanced cell migration. Notably, inhibition of cell migration in SPARC-depleted cells was rescued by expression of a SLUG transgene. In freshly isolated metastatic melanoma cells, a positive association between SPARC and SLUG mRNA levels was also found. These findings reveal that autocrine SPARC maintains heightened SLUG expression in melanoma cells and indicate that SPARC may promote EMT-associated tumor invasion by supporting AKT-dependent upregulation of SLUG.
In France, the proportion of MRSA has been over 25% since 2000. Prevention of hospital-acquired (HA) MRSA spread is based on isolation precautions and antibiotic stewardship. At our institution, before 2000, the Infection Disease and the Infection Control teams had failed to reduce HA-MRSA rates.
Objectives and methods
We implemented a multifaceted hospital-wide prevention program and measured the effects on HA-MRSA colonization and bacteremia rates between 2000 and 2009. From 2000 to 2003, active screening and decontamination of ICU patients, hospital wide alcohol based hand rubs (ABHR) use, control of specific classes of antibiotics, compliance audits, and feed-backs to the care providers were successively implemented. The efficacy of the program was assessed by HA-MRSA colonized and bacteremic patient rates per 1000 patient-days in patients hospitalized for more than twenty-four hours.
Compliance with the isolation practices increased between 2000 and 2009. Consumption of ABHR increased from 6.8 L to 27.5 L per 1000 patient-days. The use of antibiotic Defined Daily Doses (DDD) per 1000 patient-days decreased by 31%. HA-MRSA colonization decreased by 84% from 1.09 to 0.17 per 1000 patient-days and HA-MRSA bacteremia by 93%, from 0.15 to 0.01 per 1000 patient-days (p < 10−7 for each rate).
In an area highly endemic for MRSA, a multifaceted prevention program allows for sustainable reduction in HA-MRSA bacteremia rates.
MRSA; Bacteremia; Hospital-acquired; Isolation precaution; Alcohol based hand rub; Antibiotic stewardship
Quality control of drugs in formulations is still a major challenge in developing countries. For the quality control of artesunate and amodiaquine tablets in fixed-dose combination, only liquid chromatographic methods have been proposed in the literature. There are no capillary electrophoretic methods reported for the determination of these active substances, although this technique presents several advantages over liquid chromatography (long lifetime, low price of the capillary, low volumes of electrolyte consumption) in addition to simplicity. In this paper, a reliable capillary electrophoresis method has been developed and validated for the quality control of these drugs in commercial fixed-dose combination tablets.
Artesunate and amodiaquine hydrochloride in bilayer tablets were determined by micellar electrokinetic capillary chromatography (MEKC). Analytes were extracted from tablets by sonication with a solvent mixture phosphate buffer pH 7.0-acetonitrile containing benzoic acid as internal standard. Separation was carried out on Beckman capillary electrophoresis system equipped with fused silica capillary, 30 cm long (20 cm to detector) × 50 μm internal diameter, using a 25 mM borate buffer pH 9.2 containing 30 mM sodium dodecyl sulfate as background electrolyte, a 500 V cm−1 electric field and a detection wavelength of 214 nm.
Artesunate, amodiaquine and benzoic acid were separated in 6 min. The method was found to be reliable with respect to specificity,linearity of the calibration line (r2 > 0.995), recovery from synthetic tablets (in the range 98–102%), repeatability (RSD 2–3%, n = 7 analytical procedures). Application to four batches of commercial formulations with different dosages gave content in good agreement with the declared content.
The MEKC method proposed is reliable for the determination of artesunate and amodiaquine hydrochloride in fixed-dose combination tablets. The method is well-suited for drug quality control and detection of counterfeit or substandard medicines.
Anti-malarials; Amodiaquine; Artesunate; Fixed-dose combination; MEKC
Primary healthcare in developed countries is undergoing important reforms, and these require evaluation strategies to assess how well the population's expectations are being met. Although numerous instruments are available to evaluate primary healthcare (PHC) from the patient perspective, they do not all measure the same range of constructs. To analyze the extent to which important PHC attributes are covered in validated instruments measuring quality of care from the patient perspective.
We systematically identified validated instruments from the literature and by consulting experts. Using a Delphi consensus-building process, Canadian PHC experts identified and operationally defined 24 important PHC attributes. One team member mapped instrument subscales to these operational definitions; this mapping was then independently validated by members of the research team and conflicts were resolved by the PHC experts.
Of the 24 operational definitions, 13 were evaluated as being best measured by patients, 10 by providers, three by administrative databases and one by chart audits (some being best measured by more than one source). Our search retained 17 measurement tools containing 118 subscales. After eliminating redundancies, we mapped 13 unique measurement tools to the PHC attributes. Accessibility, relational continuity, interpersonal communication, management continuity, respectfulness and technical quality of clinical care were the attributes widely covered by available instruments. Advocacy, management of clinical information, comprehensiveness of services, cultural sensitivity, family-centred care, whole-person care and equity were poorly covered.
Validated instruments to evaluate PHC quality from the patient perspective leave many important attributes of PHC uncovered. A complete assessment of PHC quality will require adjusting existing tools and/or developing new instruments.
Primary healthcare; Quality of healthcare; Qualitative analysis; Measurement instruments
Subchondral bone modifications occur early in the development of osteoarthritis (OA). The level of bone resorption might impact cartilage remodeling. We therefore assessed the in vivo and in vitro effects of targeting bone resorption in OA and cartilage metabolism.
OA was induced by meniscectomy (MNX) in ovariectomized osteopenic mice (OP) treated with estradiol (E2), pamidronate (PAM), or phosphate buffered saline (PBS) for 6 weeks. We assessed the subchondral bone and cartilage structure and the expression of cartilage matrix proteases. To assess the involvement of bone soluble factors in cartilage metabolism, supernatant of human bone explants pre-treated with E2 or PAM were transferred to cartilage explants to assess proteoglycan release and aggrecan cleavage. OPG/RANKL mRNA expression was assessed in bone explants by real-time quantitative PCR. The role of osteoprotegerin (OPG) in the bone-cartilage crosstalk was tested using an OPG neutralizing antibody.
Bone mineral density of OP mice and osteoclast number were restored by E2 and PAM (p<0.05). In OP mice, E2 and PAM decreased ADAMTS-4 and -5 expression, while only PAM markedly reduced OA compared to PBS (2.0±0.63 vs 5.2±0.95; p<0.05). OPG/RANKL mRNA was increased in human bone explants treated with both drugs (2.2–3.7-fold). Moreover, supernatants from bone explants cultured with E2 or PAM reduced aggrecan cleavage and cartilage proteoglycan release (73±8.0% and 80±22% of control, respectively, p<0.05). This effect was reversed with osteoprotegerin blockade.
The inhibition of bone resorption by pamidronate in osteopenic mice alleviates the histological OA score with a reduction in the expression of aggrecanases. Bone soluble factors, such as osteoprotegerin, impact the cartilage response to catabolic factors. This study further highlights the importance of subchondral bone in the regulation of joint cartilage damage in OA.
Acute kidney injury (AKI) and acute lung injury (ALI) represent serious, complex clinical problems. The combination of AKI and ALI drastically decreases survival. However, detailed knowledge about the interactions between these two organs is scarce.
We used two different models of AKI together with P. aeruginosa inhalation to study kidney-lung cross-talk in mice during AKI and bacterial pneumonia. AKI was induced by folic-acid injections or by myohemoglobinuria following i.m. injection of glycerol. To characterize pneumonia, we measured O2-saturation, colony-forming units in lung homogenates, and neutrophil (PMN) recruitment. Plasma creatinine and cystatin C concentrations served to quantify AKI. We also examined lung and kidney histology as well as PMN transmigration and F-actin polymerization. Sub-groups of mice received anti-PMN-antibody or platelet-depleting serum to assess the role of PMN and platelets, respectively.
AKI by itself did not cause clinically-relevant ALI. P. aeruginosa-induced pneumonia was PMN-dependent, whereas pneumonia-induced AKI was platelet-dependent. AKI attenuated pulmonary PMN recruitment during pneumonia and worsened pneumonia. Mice with AKI had lower O2-saturations and greater bacterial load than mice without it. PMN from mice with FA-induced AKI also had impaired transmigration and F-actin polymerization in vitro.
Our data demonstrate clinically-relevant kidney-lung interactions during AKI and pneumonia, that depend both PMN and platelets.
An important function of all organisms is to ensure that their genetic material remains intact and unaltered through generations. This is an extremely challenging task since the cell's DNA is constantly under assault by endogenous and environmental agents. To protect against this, cells have evolved effective mechanisms to recognize DNA damage, signal its presence, and mediate its repair. While these responses are expected to be highly regulated because they are critical to avoid human diseases, very little is known about the regulation of the expression of genes involved in mediating their effects. The Nucleotide Excision Repair (NER) is the major DNA–repair process involved in the recognition and removal of UV-mediated DNA damage. Here we use a combination of in vitro and in vivo assays with an intermittent UV-irradiation protocol to investigate the regulation of key players in the DNA–damage recognition step of NER sub-pathways (TCR and GGR). We show an up-regulation in gene expression of CSA and HR23A, which are involved in TCR and GGR, respectively. Importantly, we show that this occurs through a p53 independent mechanism and that it is coordinated by the stress-responsive transcription factor USF-1. Furthermore, using a mouse model we show that the loss of USF-1 compromises DNA repair, which suggests that USF-1 plays an important role in maintaining genomic stability.
UV is responsible for DNA damage and genetic alterations of key players of the Nucleotide Excision Repair (NER) machinery promote the development of UV-induced skin cancers. The NER is the major DNA–repair process involved in the recognition and removal of UV-mediated DNA damage. Different factors participating in this DNA repair are essential, and their mutations are associated with severe genetic diseases such as Cockayne Syndrome and Xeroderma Pigmentosum. Here, we show for the first time that the specific regulation of expression in response to UV of two NER factors CSA and HR23A is required to efficiently remove DNA lesions and to maintain genomic stability. We also implicate the USF-1 transcription factor in the regulation of the expression of these factors using in vitro and in vivo models. This finding is particularly important because UV is the major cause of skin cancers and dramatically compromises patients with highly sensitive genetic diseases.
Relative to the cefotaxime-gentamicin combination, the moxifloxacin-cefotaxime combination significantly reduced microglial activation and immature oligodendrocyte cell death and delayed myelination in the developing white matter of neonatal rats with experimental Escherichia coli sepsis. These neuroprotective effects were not due to differences in in vivo bactericidal activities or in the systemic inflammatory responses and could be related to the intrinsic immunomodulatory properties of moxifloxacin. Molecular mechanisms underlying the neuroprotective effect of moxifloxacin remain to be elucidated.
Panton-Valentine Leucocidin (PVL), one of the β-barrel pore-forming staphylococcal leucotoxins, is known to be associated to furuncles and some severe community pneumonia. However, it is still uncertain how many other virulence factors are also associated to furuncles and what the risk factors of furuncles are in immuno-compromised status of patients, especially the HIV (+) patients. In this paper, we use antigen immunoprecipitation and multiplex PCR approach to determine the presence of 19 toxins, 8 adhesion factors and the PFGE profiles associated to furuncles in three independent patient study groups of S. aureus (SA) isolates collected from the Cayenne General Hospital (French Guiana). The patient groups were made of: 16 isolates from HIV (−) patients, 9 from HIV (+) patients suffering from furuncles, and 30 control isolates from patients with diverse secondary infected dermatitis. Our data reveals that the majority (96%) of SA strains isolated from HIV patient-derived furuncles significantly produced PVL (p<10−7), whereas only 10% of SA strains produced this toxin in secondary infected dermatosis. A high prevalence of LukE-LukD-producing isolates (56 to 78%) was recorded in patient groups. Genes encoding clumping factor B, collagen- and laminin-binding proteins (clfB, cna, lbp, respectively) were markedly frequent (30 to 55%), without being associated to a specific group. Pulse field gel electrophoresis evidenced 24 overall pulsotypes, whereas the 25 PVL-producing isolates were distributed into 15 non clonal fingerprints. These pulsotypes were not specific PVL-producing isolates. PVL appears to be the major virulence factor associated to furuncles in Europe and in South America regardless of the immune status of the HIV patients.