Metabolic labeling and protein linearization technology allow the study of proteins secreted by cultured cells in serum-containing media.

Supernatants from cell cultures (also called conditioned media, CMs) are commonly analyzed to study the pool of secreted proteins (secretome). To reduce the exogenous protein background, serum-free media are often used to obtain CMs. Serum deprivation, however, can severely affect cell viability and phenotype, including protein secretion. We present a strategy to analyze the proteins secreted by cells in fetal bovine serum-containing CMs, which combines the advantage of metabolic labeling and protein concentration linearization techniques. Incubation of CMs with a hexapeptide ligand library was used to reduce the dynamic range of the samples and led to the identification of 3 times more proteins than in untreated CM samples. Labeling with a deuterated amino acid was used to distinguish between cellular proteins and homologous bovine proteins contained in the medium. Application of the strategy to two breast cancer cell lines led to the identification of proteins secreted in different amounts and which could correlate with their varying degree of aggressiveness. Selected reaction monitoring (SRM)-based quantitation of three proteins of interest in the crude samples yielded data in good agreement with the results from concentration-equalized samples.

Quantitative comparison of simulations of seismic wave propagation in heterogeneous poro-elastic media involving fluid-solid interfaces and in equivalent visco-elastic solids

There is increasing evidence to suggest that the presence of mesoscopic heterogeneities constitutes the predominant attenuation mechanism at seismic frequencies. As a consequence, centimeter-scale perturbations of the subsurface physical properties should be taken into account for seismic modeling whenever detailed and accurate responses of the target structures are desired. This is, however, computationally prohibitive since extremely small grid spacings would be necessary. A convenient way to circumvent this problem is to use an upscaling procedure to replace the heterogeneous porous media by equivalent visco-elastic solids. In this work, we solve Biot's equations of motion to perform numerical simulations of seismic wave propagation through porous media containing mesoscopic heterogeneities. We then use an upscaling procedure to replace the heterogeneous poro-elastic regions by homogeneous equivalent visco-elastic solids and repeat the simulations using visco-elastic equations of motion. We find that, despite the equivalent attenuation behavior of the heterogeneous poro-elastic medium and the equivalent visco-elastic solid, the seismograms may differ due to diverging boundary conditions at fluid-solid interfaces, where there exist additional options for the poro-elastic case. In particular, we observe that the seismograms agree for closed-pore boundary conditions, but differ significantly for open-pore boundary conditions. This is an interesting result, which has potentially important implications for wave-equation-based algorithms in exploration geophysics involving fluid-solid interfaces, such as, for example, wave field decomposition.

How many diagnosis fields are needed to capture safety events in administrative data? Findings and recommendations from the WHO ICD-11 Topic Advisory Group on Quality and Safety

OBJECTIVE: As part of the WHO ICD-11 development initiative, the Topic Advisory Group on Quality and Safety explores meta-features of morbidity data sets, such as the optimal number of secondary diagnosis fields. DESIGN: The Health Care Quality Indicators Project of the Organization for Economic Co-Operation and Development collected Patient Safety Indicator (PSI) information from administrative hospital data of 19-20 countries in 2009 and 2011. We investigated whether three countries that expanded their data systems to include more secondary diagnosis fields showed increased PSI rates compared with six countries that did not. Furthermore, administrative hospital data from six of these countries and two American states, California (2011) and Florida (2010), were analysed for distributions of coded patient safety events across diagnosis fields. RESULTS: Among the participating countries, increasing the number of diagnosis fields was not associated with any overall increase in PSI rates. However, high proportions of PSI-related diagnoses appeared beyond the sixth secondary diagnosis field. The distribution of three PSI-related ICD codes was similar in California and Florida: 89-90% of central venous catheter infections and 97-99% of retained foreign bodies and accidental punctures or lacerations were captured within 15 secondary diagnosis fields. CONCLUSIONS: Six to nine secondary diagnosis fields are inadequate for comparing complication rates using hospital administrative data; at least 15 (and perhaps more with ICD-11) are recommended to fully characterize clinical outcomes. Increasing the number of fields should improve the international and intra-national comparability of data for epidemiologic and health services research, utilization analyses and quality of care assessment.

Study Regarding Implementation of a Uniform Statewide System to Allow for Electronic Transactions for the Registration and Titling of Motor Vehicles

LEGISLATIVE STUDY – The 83rd General Assembly of the Iowa Legislature, in Senate File 2273, directed the Iowa Department of Transportation (DOT) to conduct a study of how to implement a uniform statewide system to allow for electronic transactions for the registration and titling of motor vehicles. PARTICIPANTS IN STUDY – As directed by Senate File 2273, the DOT formed a working group to conduct the study that included representatives from the Consumer Protection Division of the Office of the Attorney General, the Department of Public Safety, the Department of Revenue, the Iowa State County Treasurer’s Association, the Iowa Automobile Dealers Association, and the Iowa Independent Automobile Dealers Association. CONDUCT OF THE STUDY – The working group met eight times between June 17, 2010, and October 1, 2010. The group discussed the costs and benefits of electronic titling from the perspectives of new and used motor vehicle dealers, county treasurers, the DOT, lending institutions, consumers and consumer protection, and law enforcement. Security concerns, legislative implications, and implementation timelines were also considered. In the course of the meetings the group: 1. Reviewed the specific goals of S.F. 2273, and viewed a demonstration of Iowa’s current vehicle registration and titling system so participants that were not users of the system could gain an understanding of its current functionality and capabilities. 2. Reviewed the results of a survey of county treasurers conducted by the DOT to determine the extent to which county treasurers had processing backlogs and the extent to which county treasurers limited the number of dealer registration and titling transactions that they would process in a single day and while the dealer waited. Only eight reported placing a limit on the number of dealer transactions that would be processed while the dealer waited (with the number ranging from one to four), and only 11 reported a backlog in processing registration and titling transactions as of June 11, 2010, with most backlogs being reported in the range of one to three days. 3. Conducted conference calls with representatives of the American Association of Motor Vehicle Administrators (AAMVA) and representatives of three states -- Kansas, which has an electronic lien and titling (ELT) program, and Wisconsin and Florida, each of which have both an ELT program and an electronic registration and titling (ERT) program – to assess current and best practices for electronic transactions. In addition, the DOT (through AAMVA) submitted a survey to all U.S. jurisdictions to determine how, if at all, other states implemented electronic transactions for the registration and titling of motor vehicles. Twenty-eight states responded to the survey; of the 28 states that responded, only 13 allowed liens to be added or released electronically, and only five indicated allowing applications for registration and titling to be submitted electronically. DOT staff also heard a presentation from South Dakota on its ERT system at an AAMVA regional meeting. ELT information that emerged suggests a multi-vendor approach, in which vendors that meet state specifications for participation are authorized to interface with the state’s system to serve as a portal between lenders and the state system, will facilitate electronic lien releases and additions by offering lenders more choices and the opportunity to use the same vendor in multiple states. The ERT information that emerged indicates a multi-interface approach that offers an interface with existing dealer management software (DMS) systems and through a separate internet site will facilitate ERT by offering access that meets a variety of business needs and models. In both instances, information that emerged indicates that, in the long-term, adoption rates are positively affected by making participation above a certain minimum threshold mandatory. 4. To assess and compare functions or services that might be offered by or through a vendor, the group heard presentations from vendors that offer products or services that facilitate some aspect of ELT or ERT. 5. To assess the concerns, needs and interest of Iowa motor vehicle dealers, the group surveyed dealers to assess registration and titling difficulties experienced by dealers, the types of DMS systems (if any) used by dealers, and the dealers’ interest and preference in using an electronic interface to submit applications for registration and titling. Overall, 40% of the dealers that responded indicated interest and 57% indicated no interest, but interest was pronounced among new car dealers (75% were interested) and dealers with a high number of monthly transactions (85% of dealers averaging more than 50 sales per month were interested). The majority of dealers responding to the dealer survey ranked delays in processing and problems with daily limits on transaction as ―minor difficulty or ―no difficulty. RECOMMENDATIONS -- At the conclusion of the meetings, the working group discussed possible approaches for implementation of electronic transactions in Iowa and reached a consensus that a phased implementation of electronic titling that addressed first electronic lien and title transactions (ELT) and electronic fund transfers (EFT), and then electronic applications for registration and titling (ERT) is recommended. The recommendation of a phased implementation is based upon recognition that aspects of ELT and EFT are foundational to ERT, and that ELT and EFT solutions are more readily and easily attained than the ERT solution, which will take longer and be somewhat more difficult to develop and will require federal approval of an electronic odometer statement to fully implement. ELT – A multi-vendor approach is proposed for ELT. No direct costs to the state, counties, consumers, or dealers are anticipated under this approach. The vendor charges participating lenders user or transaction fees for the service, and it appears the lenders typically absorb those costs due to the savings offered by ELT. Existing staff can complete the programming necessary to interface the state system with vendors’ systems. The estimated time to implement ELT is six to nine months. Mandatory participation is not recommended initially, but should be considered after ELT has been implemented and a suitable number of vendors have enrolled to provide a fair assessment of participation rates and opportunities. EFT – A previous attempt to implement ELT and EFT was terminated due to concern that it would negatively impact county revenues by reducing interest income earned on state funds collected by the county and held until the monthly transfer to the state. To avoid that problem in this implementation, the EFT solution should remain revenue neutral to the counties, by allowing fees submitted by EFT to be immediately directed to the proper county account. Because ARTS was designed and has the capacity to accommodate EFT, a vendor is not needed to implement EFT. The estimated time to implement EFT is six to nine months. It is expected that EFT development will overlap ELT development. ERT – ERT itself must be developed in phases. It will not be possible to quickly implement a fully functioning, paperless ERT system, because federal law requires that transfer of title be accompanied by a written odometer statement unless approval for an alternate electronic statement is granted by the National Highway Traffic Safety Administration (NHTSA). It is expected that it will take as much as a year or more to obtain NHTSA approval, and that NHTSA approval will require design of a system that requires the seller to electronically confirm the seller’s identity, make the required disclosure to the buyer, and then transfer the disclosure to the buyer, who must also electronically confirm the buyer’s identity and electronically review and accept the disclosure to complete and submit the transaction. Given the time that it will take to develop and gain approval for this solution, initial ERT implementation will focus on completing and submitting applications and issuing registration applied for cards electronically, with the understanding that this process will still require submission of paper documents until an electronic odometer solution is developed. Because continued submission of paper documents undermines the efficiencies sought, ―full‖ ERT – that is, all documents necessary for registration and titling should be capable of approval and/or acceptance by all parties, and should be capable of submission without transmittal or delivery of duplicate paper documents .– should remain the ultimate goal. ERT is not recommended as a means to eliminate review and approval of registration and titling transactions by the county treasurers, or to place registration and titling approval in the hands of the dealers, as county treasurers perform an important role in deterring fraud and promoting accuracy by determining the genuineness and regularity of each application. Authorizing dealers to act as registration agents that approve registration and title applications, issue registration receipts, and maintain and deliver permanent metal license plates is not recommended. Although distribution of permanent plates by dealers is not recommended, it is recommended that dealers participating in ERT generate and print registration applied for cards electronically. Unlike the manually-issued cards currently in use, cards issued in this fashion may be queried by law enforcement and are less susceptible to misuse by customers and dealers. The estimated time to implement the electronic application and registration applied for cards is 12 to 18 months, to begin after ELT and EFT have been implemented. It is recommended that focus during this time be on facilitating transfers through motor vehicle dealers, with initial deployment focused on higher-volume dealers that use DMS systems. In the long term an internet option for access to ERT must also be developed and maintained to allow participation for lower-volume dealers that do not use a DMS system. This option will also lay the ground work for an ERT option for sales between private individuals. Mandatory participation in Iowa is not recommended initially. As with ELT, it is recommended that mandatory participation be considered after at least an initial phase of ERT has been implemented and a suitable number of dealers have enrolled to provide a fair assessment of participation rates and opportunities. The use of vendors to facilitate ERT is not initially proposed because 1) DOT IT support staff is capable of developing a system that will interact with DMS systems and will still have to develop a dealer and public interface regardless of whether a vendor acts as intermediary between the DMS systems, and 2) there is concern that the cost of the vendor-based system, which is funded by transaction-based payments from the dealer to the vendor, will be passed to the consumer in the form of additional documentation or conveyance fees. However, the DOT recommends flexibility on this point, as development and pilot of the system may indicate that a multi-vendor approach similar to that recommended for ELT may increase the adoption rate by larger dealers and may ultimately decrease the user management to be exercised by DOT staff. If vendors are used in the process, additional legislation or administrative rules may be needed to control the fees that may be passed to the consumer. No direct cost to the DOT or county treasurers is expected, as the DOT expects that it may complete necessary programming with existing staff. Use of vendors to facilitate ERT transactions by dealers using DMS systems would result in transaction fees that may ultimately be passed to consumers. LEGISLATION – As a result of the changes implemented in 2004 under Senate File 2070, the only changes to Iowa statutes proposed are to section 321.69 of the Iowa Code, ―Damage disclosure statement,and section 321.71, ―Odometer requirements.‖ In each instance, authority to execute these statements by electronic means would be clarified by authorizing language similar to that used in section 321.20, subsections ―2‖ and ―3,‖ which allows for electronic applications and directs the department to ―adopt rules on the method for providing signatures for applications made by electronic means.‖ In these sections, the authorizing language might read as follows: Notwithstanding contrary provisions of this section, the department may develop and implement a program to allow for any statement required by this section to be made electronically. The department shall adopt rules on the method for providing signatures for statements made by electronic means. Some changes to DOT administrative rules will be useful but only to enable changes to work processes that would be desirable in the long term. Examples of long term work processes that would be enabled by rule changes include allowing for signatures created through electronic means and electronic odometer certifications. The DOT rules, as currently written, do not hinder the ability to proceed with ELT, EFT, and ERT.

Systemic and pulmonary vascular dysfunction in children conceived by assisted reproductive technologies.

BACKGROUND: Assisted reproductive technology (ART) involves the manipulation of early embryos at a time when they may be particularly vulnerable to external disturbances. Environmental influences during the embryonic and fetal development influence the individual's susceptibility to cardiovascular disease, raising concerns about the potential consequences of ART on the long-term health of the offspring. METHODS AND RESULTS: We assessed systemic (flow-mediated dilation of the brachial artery, pulse-wave velocity, and carotid intima-media thickness) and pulmonary (pulmonary artery pressure at high altitude by Doppler echocardiography) vascular function in 65 healthy children born after ART and 57 control children. Flow-mediated dilation of the brachial artery was 25% smaller in ART than in control children (6.7±1.6% versus 8.6±1.7%; P<0.0001), whereas endothelium-independent vasodilation was similar in the 2 groups. Carotid-femoral pulse-wave velocity was significantly (P<0.001) faster and carotid intima-media thickness was significantly (P<0.0001) greater in children conceived by ART than in control children. The systolic pulmonary artery pressure at high altitude (3450 m) was 30% higher (P<0.001) in ART than in control children. Vascular function was normal in children conceived naturally during hormonal stimulation of ovulation and in siblings of ART children who were conceived naturally. CONCLUSIONS: Healthy children conceived by ART display generalized vascular dysfunction. This problem does not appear to be related to parental factors but to the ART procedure itself. CLINICAL TRIAL REGISTRATION: URL: www.clinicaltrials.gov. Unique identifier: NCT00837642.

Physicochemical and biological characterization of single-walled and double-walled carbon nanotubes in biological media

To study the toxicity of nanoparticles under relevant conditions, it is important to reproducibly disperse nanoparticles in biological media in in vitro and in vivo studies. Here, single-walled nanotubes (SWNTs) and double-walled nanotubes (DWNTs) were physicochemically and biologically characterized when dispersed in phosphate-buffered saline (PBS) and bovine serum albumin (BSA). BSA-SWNT/DWNT interaction resulted in a reduction of aggregation and an increase in particle stabilization. Based on the protein sequence coverage and protein binding results, DWNTs exhibited higher protein binding than SWNTs. SWNT and DWNT suspensions in the presence of BSA increased interleukin-6 (IL-6) levels and reduced tumor necrosis factor-alpha (TNF-α) levels in A549 cells as compared to corresponding samples in the absence of BSA. We next determined the effects of SWNTs and DWNTs on pulmonary protein modification using bronchoalveolar lavage fluid (BALF) as a surrogate collected form BALB/c mice. The BALF proteins bound to SWNTs (13 proteins) and DWNTs (11 proteins), suggesting that these proteins were associated with blood coagulation pathways. Lastly, we demonstrated the importance of physicochemical and biological alterations of SWNTs and DWNTs when dispersed in biological media, since protein binding may result in the misinterpretation of in vitro results and the activation of protein-regulated biological responses.

Modified Blalock Taussig shunt: a not-so-simple palliative procedure.

OBJECTIVES: Thirty-two consecutive isolated modified Blalock Taussig (BT) shunts performed in infancy since 2004 were reviewed and analysed to identify the risk factors for shunt intervention and mortality. METHODS: Sternotomy was the only approach used. Median age and weight were 10.5 (range 1-74) days and 2.9 (1.9-4.4) kg, respectively. Shunt palliation was performed for biventricular hearts (Tetralogy of Fallot/double outlet right ventricle/transposition of great arteries_ventricular septal defect_pulmonary stenosis/pulmonary atresia_ventricular septal defect/others) in 21, and univentricular hearts in 11, patients. Hypoplastic left heart syndrome patients were excluded. Two procedures required cardiopulmonary bypass. Median shunt size was 3.5 (3-4) mm and median shunt size/kg body weight was 1.2 (0.9-1.7) mm/kg. Reduction in shunt size was necessary in 5 of 32 (16%) patients. RESULTS: Three of 32 (9%) patients died after 3 (1-15) days due to cardiorespiratory decompensation. Lower body weight (P = 0.04) and bigger shunt size/kg of body weight (P = 0.004) were significant risk factors for mortality. Acute shunt thrombosis was observed in 3 of 32 (9%), none leading to death. Need for cardiac decongestive therapy was associated with univentricular hearts (P < 0.001), bigger shunt size (P = 0.054) and longer hospital stay (P = 0.005). Twenty-eight patients have undergone a successful shunt takedown at a median age of 5.5 (0.5-11.9) months, without late mortality. CONCLUSIONS: Palliation with a modified BT shunt continues to be indicated despite increased thrust on primary corrective surgery. Though seemingly simple, it is associated with significant morbidity and mortality. Effective over-shunting and acute shunt thrombosis are the lingering problems of shunt therapy.

Procedure for human saphenous veins ex vivo perfusion and external reinforcement.

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Profiling safety of intravitreal injections for retinoblastoma using an anti-reflux procedure and sterilisation of the needle track.

BACKGROUND: The preservation of globe integrity has always been a major concern during the treatment of retinoblastoma for fear of extraocular or metastatic spread. Intravitreal chemotherapy has been attempted as a desperate salvage therapy only for eyes with refractory retinoblastoma. Published data on the safety and efficacy of this route are, however, limited. METHODS: A modified technique of intravitreal injection in eyes with retinoblastoma is described. All children with retinoblastoma who received one or more intravitreal injections using this technique were retrospectively reviewed concerning ocular complications of the injection procedure as well as clinical or histopathological evidence of tumour spread. RESULTS: 30 eyes of 30 children with retinoblastoma received a total of 135 intravitreal injections, with a median follw-up duration of 13.5 months. No extraocular spread was seen on clinical follow-up in any patients and there was no tumour contamination of the retrieved entry sites histopathologically analysed among the five enucleated eyes. No significant ocular side effects were observed except transient localised vitreous haemorrhage (3/135). CONCLUSION: This technique is potentially safe and effective at a low cost and may play a promising role, especially in the treatment of recurrent and/or resistant vitreous disease in retinoblastoma, as an alternative to enucleation and/or external beam radiotherapy. However, this treatment should not replace the primary standard of care of retinoblastoma and should not be considered in group E eyes. Its application should be approved by an ophthalmological-oncological team and it should be performed by an experienced eye surgeon in a tertiary referral centre after careful selection of a tumour-free injection site.

New microfluidic-based sampling procedure for overcoming the hematocrit problem associated with dried blood spot analysis.

Hematocrit (Hct) is one of the most critical issues associated with the bioanalytical methods used for dried blood spot (DBS) sample analysis. Because Hct determines the viscosity of blood, it may affect the spreading of blood onto the filter paper. Hence, accurate quantitative data can only be obtained if the size of the paper filter extracted contains a fixed blood volume. We describe for the first time a microfluidic-based sampling procedure to enable accurate blood volume collection on commercially available DBS cards. The system allows the collection of a controlled volume of blood (e.g., 5 or 10 μL) within several seconds. Reproducibility of the sampling volume was examined in vivo on capillary blood by quantifying caffeine and paraxanthine on 5 different extracted DBS spots at two different time points and in vitro with a test compound, Mavoglurant, on 10 different spots at two Hct levels. Entire spots were extracted. In addition, the accuracy and precision (n = 3) data for the Mavoglurant quantitation in blood with Hct levels between 26% and 62% were evaluated. The interspot precision data were below 9.0%, which was equivalent to that of a manually spotted volume with a pipet. No Hct effect was observed in the quantitative results obtained for Hct levels from 26% to 62%. These data indicate that our microfluidic-based sampling procedure is accurate and precise and that the analysis of Mavoglurant is not affected by the Hct values. This provides a simple procedure for DBS sampling with a fixed volume of capillary blood, which could eliminate the recurrent Hct issue linked to DBS sample analysis.

General unknown screening procedure for the characterization of human drug metabolites: application to loratadine phase I metabolism.

This article describes the application of a recently developed general unknown screening (GUS) strategy based on LC coupled to a hybrid linear IT-triple quadrupole mass spectrometer (LC-MS/MS-LIT) for the simultaneous detection and identification of drug metabolites following in vitro incubation with human liver microsomes. The histamine H1 receptor antagonist loratadine was chosen as a model compound to demonstrate the interest of such approach, because of its previously described complex and extensive metabolism. Detection and mass spectral characterization were based on data-dependent acquisition, switching between a survey scan acquired in the ion-trapping Q3 scan mode with dynamic subtraction of background noise, and a dependent scan in the ion-trapping product ion scan mode of automatically selected parent ions. In addition, the MS(3) mode was used in a second step to confirm the structure of a few fragment ions. The sensitivity of the ion-trapping modes combined with the selectivity of the triple quadrupole modes allowed, with only one injection, the detection and identification of 17 phase I metabolites of loratadine. The GUS procedure used in this study may be applicable as a generic technique for the characterization of drug metabolites after in vitro incubation, as well as probably in vivo experiments.

Quasi-Nonparametric Blind Inversion of Wiener Systems

An e cient procedure for the blind inversion of a nonlinear Wiener system is proposed. We proved that the problem can be expressed as a problem of blind source separation in nonlinear mixtures, for which a solution has been recently proposed. Based on a quasi-nonparametric relative gradient descent, the proposed algorithm can perform e ciently even in the presence of hard distortions.