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1.  Evaluation of an ethidium monoazide–enhanced 16S rDNA real-time polymerase chain reaction assay for bacterial screening of platelet concentrates and comparison with automated culture 
Transfusion  2013;54(3 Pt 2):870-878.
Culture-based systems are currently the preferred means for bacterial screening of platelet (PLT) concentrates. Alternative bacterial detection techniques based on nucleic acid amplification have also been developed but these have yet to be fully evaluated. In this study we evaluate a novel 16S rDNA polymerase chain reaction (PCR) assay and compare its performance with automated culture.
A total of 2050 time-expired, 176 fresh, and 400 initial-reactive PLT packs were tested by real-time PCR using broadly reactive 16S primers and a “universal” probe (TaqMan, Invitrogen). PLTs were also tested using a microbial detection system (BacT/ALERT, bioMérieux) under aerobic and anaerobic conditions.
Seven of 2050 (0.34%) time-expired PLTs were found repeat reactive by PCR on the initial nucleic acid extract but none of these was confirmed positive on testing frozen second aliquots. BacT/ALERT testing also failed to confirm any time-expired PLTs positive on repeat testing, although 0.24% were reactive on the first test. Three of the 400 “initial-reactive” PLT packs were found by both PCR and BacT/ALERT to be contaminated (Escherichia coli, Listeria monocytogenes, and Streptococcus vestibularis identified) and 14 additional packs were confirmed positive by BacT/ALERT only. In 13 of these cases the contaminating organisms were identified as anaerobic skin or oral commensals and the remaining pack was contaminated with Streptococcus pneumoniae.
These results demonstrate that the 16S PCR assay is less sensitive than BacT/ALERT and inappropriate for early testing of concentrates. However, rapid PCR assays such as this may be suitable for a strategy of late or prerelease testing.
PMCID: PMC4282358  PMID: 23701338
2.  High-throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label 
Quantifying the concentration and purity of a target protein is essential for high-throughput protein expression test and rapid screening of highly soluble proteins. However, conventional methods such as PAGE and dot blot assay generally involve multiple time-consuming tasks requiring hours or do not allow instant quantification. Here, we demonstrate a new method based on the Photoactive yellow protein turn Off/On Label (POOL) system that can instantly quantify the concentration and purity of a target protein. The main idea of POOL is to use Photoactive Yellow Protein (PYP), or its miniaturized version, as a fusion partner of the target protein. The characteristic blue light absorption and the consequent yellow color of PYP is absent when initially expressed without its chromophore, but can be turned on by binding its chromophore, p-coumaric acid. The appearance of yellow color upon adding a precursor of chromophore to the co-expressed PYP can be used to check the expression amount of the target protein via visual inspection within a few seconds as well as to quantify its concentration and purity with the aid of a spectrometer within a few minutes. The concentrations measured by the POOL method, which usually takes a few minutes, show excellent agreement with those by the BCA Kit, which usually takes ∼1 h. We demonstrate the applicability of POOL in E. coli, insect, and mammalian cells, and for high-throughput protein expression screening.
PMCID: PMC3810716  PMID: 23740751
protein expression test; high-throughput protein expression screening; protein purification; color tag; protein quantification; protein purity estimation
3.  High-throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label 
Quantifying the concentration and purity of a target protein is essential for high-throughput protein expression test and rapid screening of highly soluble proteins. However, conventional methods such as PAGE and dot blot assay generally involve multiple time-consuming tasks requiring hours or do not allow instant quantification. Here, we demonstrate a new method based on the Photoactive yellow protein turn Off/On Label (POOL) system that can instantly quantify the concentration and purity of a target protein. The main idea of POOL is to use Photoactive Yellow Protein (PYP), or its miniaturized version, as a fusion partner of the target protein. The characteristic blue light absorption and the consequent yellow color of PYP is absent when initially expressed without its chromophore, but can be turned on by binding its chromophore, p-coumaric acid. The appearance of yellow color upon adding a precursor of chromophore to the co-expressed PYP can be used to check the expression amount of the target protein via visual inspection within a few seconds as well as to quantify its concentration and purity with the aid of a spectrometer within a few minutes. The concentrations measured by the POOL method, which usually takes a few minutes, show excellent agreement with those by the BCA Kit, which usually takes ∼1 h. We demonstrate the applicability of POOL in E. coli, insect, and mammalian cells, and for high-throughput protein expression screening.
PMCID: PMC3810716  PMID: 23740751
protein expression test; high-throughput protein expression screening; protein purification; color tag; protein quantification; protein purity estimation
4.  Transparency in Nigeria's public pharmaceutical sector: perceptions from policy makers 
Pharmaceuticals are an integral component of health care systems worldwide, thus, regulatory weaknesses in governance of the pharmaceutical system negatively impact health outcomes especially in developing countries [1]. Nigeria is one of a number of countries whose pharmaceutical system has been impacted by corruption and has struggled to curtail the production and trafficking of substandard drugs. In 2001, the National Agency for Food and Drug Administration and Control (NAFDAC) underwent an organizational restructuring resulting in reforms to reduce counterfeit drugs and better regulate pharmaceuticals [2]. Despite these changes, there is still room for improvement. This study assessed the perceived level of transparency and potential vulnerability to corruption that exists in four essential areas of Nigeria's pharmaceutical sector: registration, procurement, inspection (divided into inspection of ports and of establishments), and distribution.
Standardized questionnaires were adapted from the World Health Organization assessment tool and used in semi-structured interviews with key stakeholders in the public and private pharmaceutical system. The responses to the questions were tallied and converted to scores on a numerical scale where lower scores suggested greater vulnerability to corruption and higher scores suggested lower vulnerability.
The overall score for Nigeria's pharmaceutical system was 7.4 out of 10, indicating a system that is marginally vulnerable to corruption. The weakest links were the areas of drug registration and inspection of ports. Analysis of the qualitative results revealed that the perceived level of corruption did not always match the qualitative evidence.
Despite the many reported reforms instituted by NAFDAC, the study findings suggest that facets of the pharmaceutical system in Nigeria remain fairly vulnerable to corruption. The most glaring deficiency seems to be the absence of conflict of interest guidelines which, if present and consistently administered, limit the promulgation of corrupt practices. Other major contributing factors are the inconsistency in documentation of procedures, lack of public availability of such documentation, and inadequacies in monitoring and evaluation. What is most critical from this study is the identification of areas that still remain permeable to corruption and, perhaps, where more appropriate checks and balances are needed from the Nigerian government and the international community.
PMCID: PMC2775729  PMID: 19874613
5.  Validity of visual inspection of diagnostic peritoneal lavage fluid 
Canadian Journal of Surgery  1996;39(2):114-119.
To determine the positive and negative predictive values of visual inspection of peritoneal lavage fluid and the threshold concentration of erythrocytes for diagnosing significant hemoperitoneum by this method.
Nineteen residents in surgery and 21 staff surgeons were asked to inspect mock peritoneal lavage fluid and state whether they would proceed with urgent laparotomy.
Main Results
The overall positive and negative predictive values for visual inspection were 52.0% and 98.9%, respectively. The threshold for diagnosing significant hemoperitoneum by visual inspection was between 10 000 and 20 000 erythrocytes/mL for most subjects. There were no significant differences between residents and staff surgeons.
Visual inspection of peritoneal lavage fluid has good negative but poor positive predictive value, and the threshold for diagnosing significant hemoperitoneum by visual inspection is less than 100 000 erythrocytes/mL. Therefore, patients whose condition is stable and for whom visual inspection of lavage fluid indicates apparently significant hemoperitoneum should not undergo laparotomy without confirmation by laboratory testing.
PMCID: PMC3949848  PMID: 8769921
6.  Patient-Centered Design of an Information Management Module for a Personally Controlled Health Record 
The development of health information technologies should be informed by iterative experiments in which qualitative and quantitative methodologies provide a deeper understanding of the abilities, needs, and goals of the target audience for a personal health application.
Our objective was to create an interface for parents of children with attention-deficit hyperactivity/disorder (ADHD) to enter disease-specific information to facilitate data entry with minimal task burden.
We developed an ADHD-specific personal health application to support data entry into a personally controlled health record (PCHR) using a three-step, iterative process: (1) a needs analysis by conducting focus groups with parents of children with ADHD and an heuristic evaluation of a prerelease version of a PCHR, (2) usability testing of an initial prototype personal health application following a “think aloud” protocol, (3) performance testing of a revised prototype, and (4) finalizing the design and functionality of the ADHD personal health application. Study populations for the three studies (focus groups and two usability testing studies) were recruited from organizations in the greater Boston area. Study eligibility included being an English- or Spanish-speaking parent who was the primary caretaker of a school-age child with ADHD. We determined subjects’ health literacy using the Test of Functional Health Literacy in Adults (TOFHLA). We assessed subjects’ task burden using the National Aeronautics and Space Administration (NASA) Task Load Index. To assess the impact of factors associated with the time spent entering data, we calculated Pearson correlation coefficients (r) between time on task and both task burden and subject characteristics. We conducted t tests to determine if time on task was associated with successful task completion.
The focus groups included three cohorts: 4 Spanish-speaking parents with diverse health literacy, 4 English-speaking parents with lower health literacy, and 7 English-speaking parents with higher health literacy. Both the initial usability testing cohort (n = 10) and the performance-testing cohort (n = 7) included parents of diverse health literacy and ethnicity. In performance testing, the prototype PCHRs captured patient-specific data with a mean time on task of 11.9 minutes (SD 6.5). Task burden experienced during data entry was not associated with successful task completion (P = .92). Subjects’ past computer experience was highly correlated with time on task (r = .86, P = .01), but not with task burden (r = .18, P = .69). The ADHD personal health application was finalized in response to these results by (1) simplifying the visual environment, (2) including items to support users limited by health literacy or technology experience, and (3) populating the application’s welcome screen with pictures of culturally diverse families to establish a personal family-oriented look and feel.
Our patient-centered design process produced a usable ADHD-specific personal health application that minimizes the burden of data entry.
PMCID: PMC2956329  PMID: 20805091
Attention deficit disorder with hyperactivity; patient-centered care; personal health record; computerized medical record systems; user-computer interface; comprehension; questionnaires; software design
7.  Integrated inspection of services for people with learning disabilities in Scotland: the way forward? 
The article summarises the process and the results of the first, integrated inspection of managed care services for people with learning disabilities in Scotland. The multi-agency model used was developed to be congruent with the existing performance inspection models, used by single agency inspection. The inspection activities and main outcomes are described, and suggestions are made for improvements.
Context of case
In 2006 an inspection model was devised to assess the quality of health, social services and education services for people with learning disabilities in one geographical area of Scotland, as a precursor to a programme of inspections nationally. The first joint, integrated inspection of all services for people with learning disabilities in Scotland took place in June 2006, and the report was published in March 2007. This was the first multi-agency inspection of its kind in the UK, and the first to involve carers and people with learning disabilities on the inspection team.
Data sources
A number of data sources were used to check existing practice against agreed Quality Outcome indicators. Primary sources of data were social work records, health records, education records, staff surveys, carer surveys, interviews with staff, family carers and people with learning disabilities, and self evaluations completed by the services being inspected. Eleven different domains, each with sub-indicators were investigated.
Case description
This paper summarises the process of an integrated, multi-agency inspection, how the inspection activities were conducted and the main findings of this inspection. Practical improvements to the process are suggested, and these may be of use to other services and inspectorates.
Conclusions and discussion
The integrated inspection was a qualified success. Most major objectives were achieved. The sharing of data amongst inspection agencies, establishing the level of commitment to integrated inspection and conducting multiple, integrated inspections nationally in a reasonable timescale are the main barriers remaining. The data were collected in an innovative way during this inspection, to make the analysis directly relevant to services, by providing domain specific and area specific details about how well local needs are being met.
The lessons from this integrated inspection may be of interest to other practitioners in the UK and beyond, both in terms of process and outcomes.
PMCID: PMC2092399  PMID: 18043724
integrated inspection; quality of care; clinical efficiency; client perspective
8.  A service-oriented architecture for integrating the modeling and formal verification of genetic regulatory networks 
BMC Bioinformatics  2009;10:450.
The study of biological networks has led to the development of increasingly large and detailed models. Computer tools are essential for the simulation of the dynamical behavior of the networks from the model. However, as the size of the models grows, it becomes infeasible to manually verify the predictions against experimental data or identify interesting features in a large number of simulation traces. Formal verification based on temporal logic and model checking provides promising methods to automate and scale the analysis of the models. However, a framework that tightly integrates modeling and simulation tools with model checkers is currently missing, on both the conceptual and the implementational level.
We have developed a generic and modular web service, based on a service-oriented architecture, for integrating the modeling and formal verification of genetic regulatory networks. The architecture has been implemented in the context of the qualitative modeling and simulation tool GNA and the model checkers NUSMV and CADP. GNA has been extended with a verification module for the specification and checking of biological properties. The verification module also allows the display and visual inspection of the verification results.
The practical use of the proposed web service is illustrated by means of a scenario involving the analysis of a qualitative model of the carbon starvation response in E. coli. The service-oriented architecture allows modelers to define the model and proceed with the specification and formal verification of the biological properties by means of a unified graphical user interface. This guarantees a transparent access to formal verification technology for modelers of genetic regulatory networks.
PMCID: PMC2813247  PMID: 20042075
9.  Population pharmacokinetic and pharmacodynamic modeling of transformed binary effect data of triflusal in healthy Korean male volunteers: a randomized, open-label, multiple dose, crossover study 
Triflusal is a drug that inhibits platelet aggregation. In this study we investigated the dose-exposure-response relationship of a triflusal formulation by population pharmacokinetic (PK) and pharmacodynamic (PD) modeling of its main active metabolite, hydroxy-4-(trifluoromethyl) benzoic acid (HTB).
This study was a randomized, open-label, multiple-dose, two-period, two-treatment, comparative crossover design. All volunteers received a single oral loading dose of 900 mg of triflusal on Day 1, followed by a dose of 600 mg/day from Day 2 to 9. Using data from 34 healthy volunteers, 476 HTB plasma concentration data points and 340 platelet aggregation data points were used to construct PK and PD models respectively using NONMEM (version 6.2). As the PD endpoint was qualitative, we implemented binary analysis of ‘inhibition’ and ‘non-inhibition’ rather than using the actual value of the test. The final PK-PD model was evaluated using a visual predictive check (VPC) and bootstrap.
The time-concentration profile of HTB over the entire dosing period was described by a one-compartment model with a first-order formation rate constant for HTB. Weight was selected as a covariate for clearance and volume of triflusal, respectively. The structure and the population estimates for triflusal PK were as follows: oral clearance (CL/F) = 0.2 · (weight/71.65)0.845 L/h, oral volume of distribution (V/F) = 8.3 · (weight/71.65) L, and kf = 0.341 h-1. A sigmoid relationship between triflusal concentration and the probability of significant inhibition with shape factor was chosen as the final PD model. No time delay between concentration and response was identified. The final structure between predicted concentration and the probability of inhibition of platelet aggregation (IPA) relationship was as follows: Probability of . Thus, we concluded this relationship is more like quantal concentration-response relationship. The current dosing regimen was considered to be efficacious based on the EC50 estimate of 84.9 μg/mL obtained in this study.
A PK and binary probability PD model of triflusal was successfully developed for Korean healthy volunteers. The model may be used to further prediction inhibition of platelet aggregation by triflusal.
Trial registration
Clinical Research Information Service (CRIS), KCT0001299 (Registered December 5, 2014)
PMCID: PMC4416384  PMID: 25534747
HTB; Transformed binary data; Platelet aggregation; Binary probability model; NONMEM; Population pharmacokinetics and pharmacodynamics
10.  Diagnostic efficiency of abattoir meat inspection service in Ethiopia to detect carcasses infected with Mycobacterium bovis: Implications for public health 
BMC Public Health  2010;10:462.
Bovine Tuberculosis (BTB) is a widespread and endemic disease of cattle in Ethiopia posing a significant threat to public health. Regular surveillance by skin test, bacteriology and molecular methods is not feasible due to lack of resource. Thus, routine abattoir (RA) inspection will continue to play a key role for national surveillance. We evaluated efficiency of RA inspection for diagnosis of Mycobacterium bovis infection and discussed its public health implications in light of a high risk of human exposure.
The study was conducted in five abattoirs: Addis Ababa, Adama, Hawassa, Yabello and Melge-Wondo abattoirs. The efficiency of routine abattoir (RA) inspection was validated in comparison to detailed abattoir (DA) inspection, followed by culture and microscopy (CM) and region of difference (RD) deletion analysis. Diagnostic accuracies (with corresponding measures of statistical uncertainty) were determined by computing test property statistics (sensitivity and specificity) and likelihood estimations using web-based SISA diagnostic statistics software. Post-test probability of detecting TB infected carcasses was estimated using nomograms. Agreement between RA and DA inspections was measured using kappa statistics. The study was conducted and reported in accordance with standards for reporting of diagnostic accuracy (STARD) requirements. Both routine and detailed meat inspection protocols were performed on a subpopulation of 3322 cattle selected randomly from among 78,269 cattle slaughtered during the study period. Three hundred thirty seven carcasses identified through detailed meat inspection protocols were subjected to culture and microscopy; of the 337, a subset of 105 specimens for culture and microscopy were subjected to further molecular testing.
There was a substantial agreement between RA and DA inspections in Addis Ababa (Kappa = 0.7) and Melge-Wondo abattoirs (Kappa = 0.67). In Adama, Hawassa and Yabello abattoirs, the agreement was however poor (Kappa ≤ 0.2). RA inspection was able to detect only 117 of the total 3322 carcasses inspected (3.5%). The sensitivity (Sn) and specificity (Sp) of RA inspection were 28.2% (95/337) [95%CI: 23.4-33.0] and 99.3% (2963/2985) [95%CI: 99.0-99.6], respectively, when DA inspection was considered as reference test. When culture and microscopy (CM) was considered as reference test, the Sn and Sp of RA were 55.2% (58/105) [95%CI: 45.7-64.7] and 84.1% (195/232) [95%CI: 79.3-88.8]. RA inspection failed to detect 71.8% (242/337) and 44.8% (47/105) of TB infected carcasses as judged by DA inspection and CM, respectively. On the other hand, a much higher sensitivity of DA was obtained when CM and RD deletion analysis were considered as reference tests (96.3% (105/109) and 100.0% (24/24), respectively).
The study results indicate that meat inspection protocols currently utilized in abattoirs are insufficient to detect the majority of TB lesions at the gross level. DA inspection protocols were demonstrated to improve the detection level by approximately 3-fold. The failure of current inspection techniques to detect approximately 70% of carcasses presented with grossly-visible lesions of TB at the slaughter-plants indicates the magnitude of meat-borne zoonotic TB as an on-going risk to public health. Standardization of abattoir inspection protocols (in line with international sanitary requirements), enhanced training and proficiency testing of meat inspections, and raising public awareness are recommended as essential and cost-effective interventions to improve meat inspection service in Ethiopia, with subsequent protection of consumers' health.
PMCID: PMC2924289  PMID: 20691081
11.  Use of modelling and simulation techniques to support decision making on the progression of PF-04878691, a TLR7 agonist being developed for hepatitis C 
To use non-linear mixed effects modelling and simulation techniques to predict whether PF-04878691, a toll-like receptor 7 (TLR7) agonist, would produce sufficient antiviral efficacy while maintaining an acceptable side effect profile in a ‘proof of concept’ (POC) study in chronic hepatitis C (HCV) patients.
A population pharmacokinetic–pharmacodynamic (PKPD) model was developed using available ‘proof of pharmacology’ (POP) clinical data to describe PF-04878691 pharmacokinetics (PK) and its relationship to 2′,5′-oligoadenylate synthetase (OAS; marker of pharmacology) and lymphocyte levels (marker of safety) following multiple doses in healthy subjects. A second model was developed to describe the relationship between change from baseline OAS expressed as fold change and HCV viral RNA concentrations using clinical data available in HCV patients for a separate compound, CPG-10101 (ACTILON™), a TLR9 agonist. Using these models the antiviral efficacy and safety profiles of PF-04878691 were predicted in HCV patients.
The population PKPD models described well the clinical data as assessed by visual inspection of diagnostic plots, visual predictive checks and precision of the parameter estimates. Using these relationships, PF-04878691 exposure and HCV viral RNA concentration was simulated in HCV patients receiving twice weekly administration for 4 weeks over a range of doses. The simulations indicated that significant reductions in HCV viral RNA concentrations would be expected at doses >6 mg. However at these doses grade ≥3 lymphopenia was also predicted.
The model simulations indicate that PF-04878691 is unlikely to achieve POC criteria and support the discontinuation of this compound for the treatment of HCV.
PMCID: PMC3248258  PMID: 21689140
PKPD; population modelling; TLR7
12.  A genome-scale metabolic flux model of Escherichia coli K–12 derived from the EcoCyc database 
BMC Systems Biology  2014;8:79.
Constraint-based models of Escherichia coli metabolic flux have played a key role in computational studies of cellular metabolism at the genome scale. We sought to develop a next-generation constraint-based E. coli model that achieved improved phenotypic prediction accuracy while being frequently updated and easy to use. We also sought to compare model predictions with experimental data to highlight open questions in E. coli biology.
We present EcoCyc–18.0–GEM, a genome-scale model of the E. coli K–12 MG1655 metabolic network. The model is automatically generated from the current state of EcoCyc using the MetaFlux software, enabling the release of multiple model updates per year. EcoCyc–18.0–GEM encompasses 1445 genes, 2286 unique metabolic reactions, and 1453 unique metabolites. We demonstrate a three-part validation of the model that breaks new ground in breadth and accuracy: (i) Comparison of simulated growth in aerobic and anaerobic glucose culture with experimental results from chemostat culture and simulation results from the E. coli modeling literature. (ii) Essentiality prediction for the 1445 genes represented in the model, in which EcoCyc–18.0–GEM achieves an improved accuracy of 95.2% in predicting the growth phenotype of experimental gene knockouts. (iii) Nutrient utilization predictions under 431 different media conditions, for which the model achieves an overall accuracy of 80.7%. The model’s derivation from EcoCyc enables query and visualization via the EcoCyc website, facilitating model reuse and validation by inspection. We present an extensive investigation of disagreements between EcoCyc–18.0–GEM predictions and experimental data to highlight areas of interest to E. coli modelers and experimentalists, including 70 incorrect predictions of gene essentiality on glucose, 80 incorrect predictions of gene essentiality on glycerol, and 83 incorrect predictions of nutrient utilization.
Significant advantages can be derived from the combination of model organism databases and flux balance modeling represented by MetaFlux. Interpretation of the EcoCyc database as a flux balance model results in a highly accurate metabolic model and provides a rigorous consistency check for information stored in the database.
PMCID: PMC4086706  PMID: 24974895
Escherichia coli; Flux balance analysis; Constraint-based modeling; Metabolic network reconstruction; Metabolic modeling; Genome-scale model; Gene essentiality; Systems biology; EcoCyc; Pathway Tools
13.  Differential Proteomic Analysis of Platelets Suggested Possible Signal Cascades Network in Platelets Treated with Salvianolic Acid B 
PLoS ONE  2011;6(2):e14692.
Salvianolic acid B (SB) is an active component isolated from Danshen, a traditional Chinese medicine widely used for the treatment of cardiovascular disorders. Previous study suggested that SB might inhibit adhesion as well as aggregation of platelets by a mechanism involving the integrin α2β1. But, the signal cascades in platelets after SB binding are still not clear.
Methodology/Principal Findings
In the present study, a differential proteomic analysis (two-dimensional electrophoresis) was conducted to check the protein expression profiles of rat platelets with or without treatment of SB. Proteins altered in level after SB exposure were identified by MALDI-TOF MS/MS. Treatment of SB caused regulation of 20 proteins such as heat shock-related 70 kDa protein 2 (hsp70), LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C. The regulation of SB on protein levels was confirmed by Western blotting. The signal cascades network induced by SB after its binding with integrin α2β1 was predicted. To certify the predicted network, binding affinity of SB to integrin α2β1 was checked in vitro and ex vivo in platelets. Furthermore, the effects of SB on protein levels of hsp70, coronin-1B and intracellular levels of Ca(2+) and reactive oxygen species (ROS) were checked with or without pre-treatment of platelets using antibody against integrin α2β1. Electron microscopy study confirmed that SB affected cytoskeleton structure of platelets.
Integrin α2β1 might be one of the direct target proteins of SB in platelets. The signal cascades network of SB after binding with integrin α2β1 might include regulation of intracellular Ca(2+) level, cytoskeleton-related proteins such as coronin-1B and cytoskeleton structure of platelets.
PMCID: PMC3040754  PMID: 21379382
14.  Screening for childhood strabismus by primary care physicians. 
Canadian Family Physician  1998;44:337-343.
OBJECTIVE: To review the clinical classification of strabismus, to describe the timing and method of strabismus screening examinations, and to discuss the principles of treatment. QUALITY OF EVIDENCE: Current literature (1983 to 1995) was searched via MEDLINE using the MeSH headings strabismus, ocular motility disorders, and amblyopia. Articles were selected based on their date of publication, clinical relevance, and availability. Preference was given to more recent articles, articles with large numbers of subjects, and well-designed cohort studies. Official recommendations from academic groups were analyzed. Descriptions of clinical tests and their illustrations are based on classic texts. MAIN FINDINGS: Primary care physicians should screen all low-risk children. High-risk children (low birth weight, family history of strabismus, congenital ocular abnormality, or systemic conditions with vision-threatening ocular manifestations) should be referred to an ophthalmologist for screening. Screening should be performed in the neonatal period, at 6 months, and at 3 years (Grade A recommendation), as well as at 5 to 6 years (Grade B recommendation). Screening examination includes inspection, examining visual acuity, determining pupillary reactions, checking ocular alignment, testing eye movements, and ophthalmoscopy. CONCLUSIONS: Primary care physicians are essential to early detection of strabismus and amblyopia. Early detection can help minimize visual dysfunction, allow for normal development of binocular vision and depth perception, and prevent psychosocial dysfunction.
PMCID: PMC2277602  PMID: 9512837
15.  Isolation and characterization of actin and actin-binding protein from human platelets 
The Journal of Cell Biology  1981;91(1):201-211.
Human blood platelets, which are highly motile cells essential for the maintenance of hemostasis, contain large quantities of actin and other contractile proteins. We have previously introduced a method (Lucas, R. C., T. C. Detwiler, and A. Stracher, J. Cell Biol., 1976, 70(2, Pt. 2):259 a) for the quantitative recovery of the platelets' cytoskeleton using a solution containing 1% Triton X-100 and 10 mM EGTA. This cytoskeleton contains most of the platelets' actin, actin-binding protein (ABP, subunit molecular weight = 260,000), and a 105,000-dalton protein. Negative staining of this Triton-insoluble residue on an EM grid shows it to consist of branched cables of actin filaments aligned in parallel. When this cytoskeletal structure is dissolved in high-salt solutions, the actin and ABP dissociate and can subsequently be separated. Here we will present simple and rapid methods for the individual purifications of platelet actin and platelet ABP. When purified actin and ABP are recombined in vitro, they are shown to be both necessary and sufficient for the reformation of the cytoskeletal complex. The reformed structure is visualized as a complex array of fibers, which at the EM level are seen to be bundles of actin filaments. The reformation of the cytoskeleton requires only that the actin be in the filamentous form--no accessory proteins, chelating agents, divalent cations, or energy sources are necessary. In vivo, however, the state of assembly of the platelets' cytoskeleton appears to be under the control of the intracellular concentration of free calcium. Under conditions where proteolysis is inhibited and EGTA is omitted from the Triton-solubilization step, no cytoskeleton can be isolated. The ability of Ca+2 to control the assembly and disassembly of the platelets' cytoskeleton provides a mechanism for cytoskeletal involvement in shape change and pseudopod formation during platelet activation.
PMCID: PMC2111948  PMID: 7197680
16.  Iterative Development of Visual Control Systems in a Research Vivarium 
PLoS ONE  2014;9(4):e90076.
The goal of this study was to test the hypothesis that reintroduction of Continuous Performance Improvement (CPI) methodology, a lean approach to management at Seattle Children’s (Hospital, Research Institute, Foundation), would facilitate engagement of vivarium employees in the development and sustainment of a daily management system and a work-in-process board. Such engagement was implemented through reintroduction of aspects of the Toyota Production System. Iterations of a Work-In-Process Board were generated using Shewhart’s Plan-Do-Check-Act process improvement cycle. Specific attention was given to the importance of detecting and preventing errors through assessment of the following 5 levels of quality: Level 1, customer inspects; Level 2, company inspects; Level 3, work unit inspects; Level 4, self-inspection; Level 5, mistake proofing. A functioning iteration of a Mouse Cage Work-In-Process Board was eventually established using electronic data entry, an improvement that increased the quality level from 1 to 3 while reducing wasteful steps, handoffs and queues. A visual workplace was realized via a daily management system that included a Work-In-Process Board, a problem solving board and two Heijunka boards. One Heijunka board tracked cage changing as a function of a biological kanban, which was validated via ammonia levels. A 17% reduction in cage changing frequency provided vivarium staff with additional time to support Institute researchers in their mutual goal of advancing cures for pediatric diseases. Cage washing metrics demonstrated an improvement in the flow continuum in which a traditional batch and queue push system was replaced with a supermarket-type pull system. Staff engagement during the improvement process was challenging and is discussed. The collective data indicate that the hypothesis was found to be true. The reintroduction of CPI into daily work in the vivarium is consistent with the 4P Model of the Toyota Way and selected Principles that guide implementation of the Toyota Production System.
PMCID: PMC3987998  PMID: 24736460
17.  Accuracy of direct digital radiography for detecting occlusal caries in primary teeth compared with conventional radiography and visual inspection: an in vitro study 
Dentomaxillofacial Radiology  2010;39(6):362-367.
The diagnosis of caries lesions is still a matter of concern in dentistry. The diagnosis of dental caries by digital radiography has a number of advantages over conventional radiography; however, this method has not been explored fully in the field of paediatric dentistry. This in vitro research evaluated the accuracy of direct digital radiography compared with visual inspection and conventional radiography in the diagnosis of occlusal caries lesions in primary molars.
50 molars were selected and evaluated under standardized conditions by 2 previously calibrated examiners according to 3 diagnostic methods (visual inspection, conventional radiography and direct digital radiography). Direct digital radiographs were obtained with the Dixi3 system (Planmeca, Helsinki, Finland) and the conventional radiographs with InSight film (Kodak Eastman Co., Rochester, NY). The images were scored and a reference standard was obtained histologically. The interexaminer reliability was calculated using Cohen's kappa test and the specificity, sensitivity and accuracy of the methods were calculated.
Examiner reliability was good. For lesions limited to the enamel, visual inspection showed significantly higher sensitivity and accuracy than both radiographic methods, but no significant difference was found in specificity. For teeth with dentinal caries, no significant differences were found for any parameter when comparing visual and radiographic evaluation.
Although less accurate than the visual method for detecting caries lesions confined to the enamel, the direct digital radiographic method is as effective as conventional radiographic examination and visual inspection of primary teeth with occlusal caries when the dentine is involved.
PMCID: PMC3520238  PMID: 20729186
direct digital radiography; conventional radiography; dental caries; primary teeth
18.  Real-time imaging required for optimal echocardiographic assessment of aortic valve calcification 
Aortic valve calcification (AVC), even without haemodynamic significance, may be prognostically import as an expression of generalized atherosclerosis, but techniques for echocardiographic assessment are essentially unexplored.
Two-dimensional (2D) echocardiographic recordings (Philips IE33) of the aortic valve in short-axis and long-axis views were performed in 185 consecutive patients within 1 week before surgery for aortic stenosis (n = 109, AS), aortic regurgitation (n = 61, AR), their combination (n = 8) or dilation of the ascending aorta (n = 7). The grey scale mean (GSMn) of the aortic valve in an end-diastolic short-axis still frame was measured. The same frame was scored visually 1–5 as indicating that the aortic valve was normal, thick, or had mild, moderate or severe calcification. The visual echodensity of each leaflet was determined real time applying the same 5-grade scoring system for each leaflet, and the average for the whole valve was calculated. Finally, a similar calcification score for the whole valve based on inspection and palpation by the surgeon was noted.
Visual assessment of real-time images using the proposed scoring system showed better correlation with the surgical evaluation of the degree of valve calcification (r = 0·83, P<0·001) compared to evaluation of stop frames by visual assessment (r = 0·66, P<0·001) or the GSMn score (r = 0·64, P<0·001). High inter- and intra-observer correlations were observed for real-time visual score (both intraclass correlation coefficient = 0·93).
Real-time evaluation of the level of AVC is superior to using stop frames assessed either visually or by dedicated computer grey scale measurement software.
PMCID: PMC3549484  PMID: 23031068
aortic regurgitation; aortic stenosis; grey scale; sclerosis; ultrasound
19.  Platelet gel in the treatment of cutaneous ulcers: the experience of the Immunohaematology and Transfusion Centre of Parma 
Blood Transfusion  2010;8(4):237-247.
Platelet gel is being ever more frequently used to promote healing of cutaneous ulcers. However, the factors that determine the often variable clinical outcome of this procedure are still incompletely understood.
The aims of this study were to demonstrate that platelet gel, even when obtained under strictly controlled conditions, produces highly variable outcomes in patients with cutaneous ulcers and to propose a method for in vitro standardisation of the biological properties of platelet gel.
Material and methods.
Patients were enrolled on the basis of a pre-defined protocol. Platelet concentrate was produced with standard methods, with a variability in platelet count among the different samples of less than 10%. The platelet gel for clinical use was obtained, under strictly standardized conditions, by adding thrombin and calcium gluconate to the concentrates. For in vitro studies, platelet gel, obtained from platelet-rich plasma from four donors, was frozen and thawed twice so as to increase gel contraction. The supernatant was used to modify cell proliferation, protein synthesis, and the expression of selected genes in cultures of human diploid fibroblasts.
Seventeen patients (aged 44–78 years) with ulcers (4 diabetic, 11 vascular, 1 post-traumatic, 1 decubitus) were treated with platelet gel (4 autologous, 13 homologous). Complete re-epithelialisation of four ulcers (1 diabetic, 1 post-traumatic, 2 vascular) was obtained after applications of platelet gel (2 autologous, 2 homologous); in 11 other cases there was a greater than 50% reduction in the size of the ulcer. Two patients had no benefit. The supernatant of the platelet gel was able to promote dose-dependent proliferation and changes in gene expression as well as in metabolic activities related to protein synthesis.
Although the use of platelet gel in the treatment of cutaneous ulcers is increasing, and conditions for its production are better standardised, very considerable variability of clinical outcomes is still observed, even within single centres, suggesting that there are differences in biological properties of platelet concentrates from individual patients which cannot be readily controlled with current techniques. The biological effects of the platelet gel supernatant described in this article may provide the basis for a simple biological validation of platelet preparations before their clinical use, so as to reduce this potentially important source of variability.
PMCID: PMC2957488  PMID: 20967164
platelet gel; fibroblasts; ulcers; cell proliferation
20.  Optimized Preparation Method of Platelet-Concentrated Plasma and Noncoagulating Platelet-Derived Factor Concentrates: Maximization of Platelet Concentration and Removal of Fibrinogen 
Platelet-rich plasma (PRP) has been clinically used as an easily prepared growth factor cocktail that can promote wound healing, angiogenesis, and tissue remodeling. However, the therapeutic effects of PRP are still controversial, due partly to the lack of optimized and standardized preparation protocols. We used whole blood (WB) samples to optimize the preparation protocols for PRP, white blood cell-containing (W-PRP), platelet-concentrated plasma (PCP), and noncoagulating platelet-derived factor concentrate (PFC). PRP and W-PRP were most efficiently collected by 10 min centrifugation in a 15-mL conical tube at 230–270 g and 70 g, respectively. To prepare PCP, platelets were precipitated by centrifugation of PRP at >2300 g, 90% of supernatant plasma was removed, and the platelets were resuspended. For preparation of noncoagulating PFC, the supernatant was replaced with one-tenth volume of saline, followed by platelet activation with thrombin. Platelet (before activation) and platelet-derived growth factor (PDGF)-BB (after activation) concentrations in PCP were approximately 20 times greater than those in WB, whereas PFC contained a 20-times greater concentration of platelets before platelet activation and a 50-times greater concentration of PDGF-BB without formation of a fibrin gel after platelet activation than WB. Surprisingly, total PDGF-BB content in the PFC was twice that of activated WB, which suggested that a substantial portion of the PDGF-BB became trapped in the fibrin glue, and replacement of plasma with saline is crucial for maximization of platelet-derived factors. As an anticoagulant, ethylene di-amine tetra-acetic acid disodium inhibited platelet aggregation more efficiently than acid citrate dextrose solution, resulting in higher nonaggregated platelet yield and final PDGF-BB content. These results increase our understanding of how to optimize and standardize preparation of platelet-derived factors at maximum concentrations.
PMCID: PMC3285602  PMID: 21951067
21.  Reduced Haemodynamic Response in the Ageing Visual Cortex Measured by Absolute fNIRS 
PLoS ONE  2015;10(4):e0125012.
The effect of healthy ageing on visual cortical activation is still to be fully explored. This study aimed to elucidate whether the haemodynamic response (HDR) of the visual cortex altered as a result of ageing. Visually normal (healthy) participants were presented with a simple visual stimulus (reversing checkerboard). Full optometric screening was implemented to identify two age groups: younger adults (n = 12, mean age 21) and older adults (n = 13, mean age 71). Frequency-domain Multi-distance (FD-MD) functional Near-Infrared Spectroscopy (fNIRS) was used to measure absolute changes in oxygenated [HbO] and deoxygenated [HbR] haemoglobin concentrations in the occipital cortices. Utilising a slow event-related design, subjects viewed a full field reversing checkerboard with contrast and check size manipulations (15 and 30 minutes of arc, 50% and 100% contrast). Both groups showed the characteristic response of increased [HbO] and decreased [HbR] during stimulus presentation. However, older adults produced a more varied HDR and often had comparable levels of [HbO] and [HbR] during both stimulus presentation and baseline resting state. Younger adults had significantly greater concentrations of both [HbO] and [HbR] in every investigation regardless of the type of stimulus displayed (p<0.05). The average variance associated with this age-related effect for [HbO] was 88% and [HbR] 91%. Passive viewing of a visual stimulus, without any cognitive input, showed a marked age-related decline in the cortical HDR. Moreover, regardless of stimulus parameters such as check size, the HDR was characterised by age. In concurrence with present neuroimaging literature, we conclude that the visual HDR decreases as healthy ageing proceeds.
PMCID: PMC4409147  PMID: 25909849
22.  Pharmacodynamics of RP 59500 (quinupristin-dalfopristin) administered by intermittent versus continuous infusion against Staphylococcus aureus-infected fibrin-platelet clots in an in vitro infection model. 
We evaluated the bactericidal activity of RP 59500 (quinupristin-dalfopristin) against fibrin-platelet clots (FPC) infected with two clinical isolates of Staphylococcus aureus, one constitutively erythromycin and methicillin resistant (S. aureus AW7) and one erythromycin and methicillin susceptible (S. aureus 1199), in an in vitro pharmacodynamic infection model. RP 59500 was administered by continuous infusion (peak steady-state concentration of 6 microg/ml) or intermittent infusion (simulated regimens of 7.5 mg/kg of body weight every 6 h (q6h) q8h, and q12h. FPCs were infected with S. aureus to achieve an initial bacterial density of 10(9) CFU/g. Model experiments were run in duplicate over 72 h. Two FPCs were removed from each model at 0, 12, 24, 36, 48, and 72 h, and the bacterial densities (in CFU per gram) were determined and compared to those of growth control experiments. Additional samples were also removed from the model over the 72-h period for pharmacokinetic evaluation. All regimens significantly (P < or = 0.01) decreased bacterial densities in the infected FPCs for both isolates compared to growth controls. This occurred even though MBCs were equal to or greater than the RP 59500 concentrations achieved in the models. There were no significant differences found between the dosing frequencies and levels of killing when examining each isolate separately. However, examination of the residual bacterial densities (CFU per gram at 72 h) and visual inspection of the overall killing effect (killing curve plots over 72 h) clearly demonstrated a more favorable bactericidal activity against 1199 than against the AW7 isolate. This was most apparent when the q8h and the q12h AW7 regimens were compared to all 1199 treatment regimens by measuring the 72-h bacterial densities (P < or = 0.01). Killing (99.9%) was not achieved against the AW7 isolate. However, a 99.9% kill was demonstrated for all dosing regimens against the 1199 isolate. The area under the concentration-time curve from 0 to 24 h was found to be significantly correlated with reduction in bacterial density for the AW7 isolate (r = 0.74, P = 0.04). No resistance was detected during any experiment for either isolate. RP 59500 efficacy against constitutively erythromycin- and methicillin-resistant S. aureus may be improved by increasing organism exposure to RP 59500 as a function of dosing frequency.
PMCID: PMC163915  PMID: 9174199
23.  Vaccine Storage Practices and the Effects of Education in Some Private Medical Institutions 
Although vaccination rates have increased, problems still remain in the storage and handling of vaccines. This study focused on inspecting actual vaccine storage status and awareness, and comparing them before and after education was provided.
In the primary inspection, a status survey checklist was completed by visual inspection. A questionnaire on the awareness of proper vaccine storage and handling was also administered to vaccine administrators in private medical institutions in 4 regions in Gyeongsangbuk-province. One-on-one education was then carried out, and our self-produced manual on safe vaccine storage and management methods was provided. In the secondary inspection, the investigators visited the same medical institutions and used the same questionnaire and checklist used during the primary inspection. The results before and after education were compared, by treating each appropriate answer as 1 point.
The average checklists score was 9.74 (out of 15 points), which increased significantly after education was provided (by 0.84, p<0.001). The participants demonstrated improved practices in recording storage temperatures (p=0.016), storing vaccines in the center of the refrigerator (p=0.004), storing vaccines with other medication and non-medical items (p=0.031) after education. The average score calculated from the questionnaires was 10.48 (out of 14 points), which increased after education (by 1.03, p<0.001).
This study suggests that vaccine storage practices and awareness are inadequate, but can be partially improved by providing relevant education. Repetitive education and policy-making are required to store vaccines safely because one-off education and unenforced guidelines offer limited efficacy.
PMCID: PMC3324719  PMID: 22509448
Awareness; Checklist; Education; Questionnaires; Vaccines
24.  The Use of Spinning-Disk Confocal Microscopy for the Intravital Analysis of Platelet Dynamics in Response to Systemic and Local Inflammation 
PLoS ONE  2011;6(9):e25109.
Platelets are central players in inflammation and are an important component of the innate immune response. The ability to visualize platelets within the live host is essential to understanding their role in these processes. Past approaches have involved adoptive transfer of labelled platelets, non-specific dyes, or the use of fluorescent antibodies to tag platelets in vivo. Often, these techniques result in either the activation of the platelet, or blockade of specific platelet receptors. In this report, we describe two new methods for intravital visualization of platelet biology, intravenous administration of labelled anti-CD49b, which labels all platelets, and CD41-YFP transgenic mice, in which a percentage of platelets express YFP. Both approaches label endogenous platelets and allow for their visualization using spinning-disk confocal fluorescent microscopy. Following LPS-induced inflammation, we were able to measure a significant increase in both the number and size of platelet aggregates observed within the vasculature of a number of different tissues. Real-time observation of these platelet aggregates reveals them to be large, dynamic structures that are continually expanding and sloughing-off into circulation. Using these techniques, we describe for the first time, platelet recruitment to, and behaviour within numerous tissues of the mouse, both under control conditions and following LPS induced inflammation.
PMCID: PMC3176312  PMID: 21949865
25.  Platelet Activation Test in Unprocessed Blood (Pac-t-UB) to Monitor Platelet Concentrates and Whole Blood of Thrombocytopenic Patients 
Platelet concentrate transfusion is the standard treatment for hemato-oncology patients to compensate for thrombocytopenia. We have developed a novel platelet activation test in anticoagulated unprocessed blood (pac-t-UB) to determine platelet function in platelet concentrates and in blood of thrombocytopenic patients.
We have measured platelet activity in a platelet concentrate and in anticoagulated unprocessed blood of a post-transfusion thrombocytopenic patient.
Our data show time-dependent platelet activation by GPVI agonist (collagen related peptide; CRP), PAR-1 agonist (SFLLRN), P2Y12 agonist (ADP), and thromboxane receptor agonist (U46619) in a platelet concentrate. Furthermore, pac-t-UB showed time-dependent platelet activation in unprocessed blood of a post-transfusion patient with thrombocytopenia. Testing platelet function by different agonists in relation to storage show that 3-day-old platelet concentrates are still reactive to the studied agonists. This reactivity rapidly drops for each agonists during longer storage.
Pac-t-UB is a novel tool to estimate platelet function by different agonists in platelet concentrates and in unprocessed blood of thrombocytopenic patients. In the near future, we will validate whether pac-t-UB is an adequate test to monitor the quality of platelet concentrates and whether pac-t-UB predicts the bleeding risk of transfused thrombocytopenic patients.
PMCID: PMC3638932  PMID: 23652405
Flow cytometry; Platelet, Platelet activation; Platelet concentrates; Platelet function; Platelet storage; Platelet transfusion; Platelets; Thrombocytopenia

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