Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Functional verification”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,243 records · Page 69Linked to original sources
PubMed1996

Double-lung transplantation in the rat: an acute, syngeneic in situ model.

BACKGROUND: The high rate of reperfusion injury in clinical lung transplantation mandates significant improvements in lung preservation. Innovations should be validated using standardized and low-cost experimental models. METHODS: The model introduced here is analyzed by comparing global lung function after varying ischemic times (2, 4, 8, 16, and 24 hours). A rat double-lung block is flush-perfused, and the main pulmonary artery and left atrium are connected to the left pulmonary artery and vein of a syngeneic recipient using a T-shaped stent. With pressure side ports and incorporated flow crystals, measurement of vascular resistance and graft oxygenation can be performed. The transplant is ventilated separately, and compliance and resistance are determined. RESULTS: The increase in the ischemic interval from 2 to 24 hours caused an increase in the alveolar arterial oxygen difference from 220 +/- 20 to 600 +/- 34 mm Hg, pulmonary vascular resistance from 198 +/- 76 to 638 +/- 212 mm Hg.mL-1.min-1, and resistance to airflow from 274 +/- 50 to 712 +/- 30 cm H2O/L H2O, and a decrease in pulmonary compliance from 0.4 +/- 0.05 to 0.12 +/- 0.06 mL/cm H2O. CONCLUSIONS: This in situ, syngeneic rat lung transplantation model offers an alternative to large animal models for verification of lung preservation solutions and for modification of donor or recipient treatment regimens.

Airway Resistance↗
PubMed1988

Changes in blood platelet function, coagulation, and fibrinolytic activity in response to moderate, exhaustive, and prolonged exercise.

In a group of 47 healthy men aged 25-55, the influence of moderate, exhaustive, and prolonged exercise on blood platelet function, coagulation, and fibrinolytic activity was evaluated. Static exertion of short duration and moderate intensity did not cause significant alterations in any of the indices analyzed. Prolonged exercise (60 min) performed on a bicycle ergometer in aerobic conditions led to strong activation of the fibrinolytic system [euglobulin lysis time (ELT) fell from 208 to 88 min] and a slight increase in platelet count (PC) but did not cause significant changes in platelet factor 4 (PF 4) and platelet aggregate ratio, recalcification (RT), and prothrombin time (PT). Repeated bouts of maximum exercise leading to considerable acidosis (pH = 7.22; BE = -13.3) caused a significant increase in blood platelet count and an increase in PF 4 release. These changes were accompanied by strong intensification in fibrinolytic activity (over 63% reduction of ELT and a marked increase of fibrin/fibrinogen degradation products). In some individuals, unfavorable hemostatic changes were observed following intensive exercise. It is concluded that more attention should be given in future to the problem of verification of preventive and rehabilitative training programs from the point of view of exercise-induced hemostatic changes.

Adult↗
PubMed2003

[Arterial hypertension in glomerulonephritis].

Numerous factors causing the disturbances of pressure natriuresis mechanism may be involved in the development of arterial hypertension in kidney diseases. In particular patients with parenchymatous kidney disease, the relative effect of the impaired renal sodium excretion causing extracellular fluid volume expansion and the disequilibrium of vasoconstricting and vasorelaxating factors may differ, and in addition, these effects are influenced by the type of kidney disease, the degree of the kidney function impairment and sodium intake. In our studies on the hypertension in kidney disease we have choosen the evaluation of incidence of hypertension in the early stage of primary glomerulonephritis (GN) and the comparison of distribution of genotypes and alleles depending on insertion/deletion polymorphism of angiotensin converting enzyme (ACE) gene in patients with early stage of GN and healthy subjects. Arterial hypertension was diagnosed according to actual criteria in 46% out of 74 patients with early stage of GN. It was also found that hypertension was more common in patients with nephrotic syndrome (63%) than in patients with lower proteinuria (36% of patients). Among 50 patients with early stage of GN the distribution of genotypes and alleles depending on the polymorphism ACE gene was significantly different from 100 healthy controls. These results indicate that hypertension is often present in early stage of GN, particularly in patients with nephrotic syndrome and depends on the morphological type of glomerular disease. Further studies are needed for verification of the role of ACE gene polymorphism in the predisposition to development of GN and associated arterial hypertension.

Adult↗
PubMed2004

Analysis of genomic and expressed major histocompatibility class Ia and class II genes in a hexaploid Lake Tana African 'large' barb individual (Barbus intermedius).

Expression of too many co-dominant major histocompatibility complex (MHC) alleles is thought to be detrimental to proper functioning of the immune system. Polyploidy of the genome will increase the number of expressed MHC genes unless they are prone to a silencing mechanism. In polyploid Xenopus species, the number of MHC class I and II genes has been physically reduced, as it does not increase with higher ploidy genomes. In the zebrafish some class II B loci have been silenced, as only two genomically bona fide loci, DAA/DAB and DEA/DEB, have been described. Earlier studies indicated a reduction in the number of genomic and expressed class II MHC genes in a hexaploid African 'large' barb. This prompted us to study the number of MHC genes present in the genome of an African 'large' barb individual (Barbus intermedius) in relation to those expressed, adopting the following strategy. Full-length cDNA sequences were generated from mRNA and compared with partial genomic class Ia and II sequences generated by PCR using the same primer set. In addition, we performed Southern hybridizations to obtain a verification of the number of class I and II B genes. Our study revealed three beta2-microglobulin, five class Ia, four class II A, and four class II B genes at the genomic level, which were shown to be expressed in the hexaploid barb individual. The class Ia and class II data indicate that the ploidy status does not correlate with the presence and expression of these MHC genes.

Amino Acid Sequence↗
PubMed2005

Injection of glutamate into the pedunculopontine tegmental nuclei of anesthetized rat causes respiratory dysrhythmia and alters EEG and EMG power.

The pedunculopontine tegmental nucleus (PPT) has been shown to have important functions relevant to the regulation of behavioral states and various motor control systems, including breathing control. Our previous work has shown that the activation of neurons within the PPT, a structure that is typically active during rapid eye movement (REM) sleep, can produce respiratory disturbances in freely moving and anesthetized rats. The aim of this study was to test the hypothesis that respiratory modulation by the PPT in anesthetized rats can be evoked in the absence of other signs of an REM-sleep-like state. We characterized electroencephalogram (EEG) and electromyogram (EMG) changes during respiratory disturbances induced by glutamatergic stimulation of the PPT in spontaneously breathing, adult male Sprague-Dawley rats anesthetized with a ketamine/xylazine combination or with nembutal. Respiratory movements were monitored by a piezoelectric strain gauge. Two-barrel glass pipettes were used to pressure inject glutamate, to probe for respiratory effective sites within the PPT, and to inject oil red dye at the end of the experiments for histological verification of the injection sites. The EEGs were recorded from the sensorimotor cortex, hippocampus, and from the pons contralateral from the injection site. The EMGs were recorded from the genioglossus muscle. The initial response to glutamate injection into the respiratory modulating region of the PPT was always a respiratory pattern disturbance. Subsequent activation of EMG and EEG often occurred in ketamine/xylazine-anesthetized rats, but REM-sleep-like patterns were not observed. Respiratory pattern and EMG power changes in nembutal-anesthetized rats were similar, but EEG activation was never observed. Thus, we conclude that respiratory suppression produced by the local activation of PPT neurons may not necessarily be accompanied by an REM-sleep-like cortical state in this anesthetized model.

Anesthesia↗
PubMed2004

Strengthening early warning function of surveillance in the Republic of Serbia: lessons learned after a year of implementation.

The Republic of Serbia, with WHO support, has implemented an early warning system (ALERT) for priority communicable diseases, to complement the routine surveillance system which notifies individual confirmed cases. The results of its evaluation, conducted one year after implementation is presented here. ALERT relies on notification of 11 syndromes by primary care facilities. Data is analysed weekly at district level and transmitted to national epidemiologists. ALERT is perceived to be a simple and flexible tool. Acceptability is higher at national level than at district level. Some districts perceive ALERT as a parallel system poorly connected to control measures. Sensitivity of ALERT in detecting cases of meningitis is 93%, and 37% for cases of hepatitis. Retrospective analysis of ALERT data identified 9 outbreaks, 5 of which had been recognized by epidemiologists. ALERT was the timeliest system for detecting 4 outbreaks identified by both systems. ALERT was useful for triggering timely investigation and control of outbreaks of hantavirus and salmonellosis and for detecting the start of the influenza season. However, ALERT did not detect clusters of brucellosis and tularaemia targeted by the unexplained fever syndrome. This evaluation underlined the need for a global review of surveillance activities when implementing new components such as ALERT. While control measures based on notification of individual confirmed cases are well understood and implemented, the investigation and verification process that should result from an increase in ALERT syndromes is not fully understood. Field epidemiology training programmes, such as the EPIET programme, are best suited to bring about this change of perspective.

Communicable Disease Control↗
PubMed2006

Transport and fate of radionuclides in aquatic environments--the use of ecosystem modelling for exposure assessments of nuclear facilities.

In safety assessments of nuclear facilities, a wide range of radioactive isotopes and their potential hazard to a large assortment of organisms and ecosystem types over long time scales need to be considered. Models used for these purposes have typically employed approaches based on generic reference organisms, stylised environments and transfer functions for biological uptake exclusively based on bioconcentration factors (BCFs). These models are of non-mechanistic nature and involve no understanding of uptake and transport processes in the environment, which is a severe limitation when assessing real ecosystems. In this paper, ecosystem models are suggested as a method to include site-specific data and to facilitate the modelling of dynamic systems. An aquatic ecosystem model for the environmental transport of radionuclides is presented and discussed. With this model, driven and constrained by site-specific carbon dynamics and three radionuclide specific mechanisms: (i) radionuclide uptake by plants, (ii) excretion by animals, and (iii) adsorption to organic surfaces, it was possible to estimate the radionuclide concentrations in all components of the modelled ecosystem with only two radionuclide specific input parameters (BCF for plants and Kd). The importance of radionuclide specific mechanisms for the exposure to organisms was examined, and probabilistic and sensitivity analyses to assess the uncertainties related to ecosystem input parameters were performed. Verification of the model suggests that this model produces analogous results to empirically derived data for more than 20 different radionuclides.

Adsorption↗
PubMedDate not supplied

[Tinnitus Handicap Inventory: cross-cultural adaptation to Brazilian Portuguese].

BACKGROUND: Tinnitus can cause damage to the life quality of patients. The assessment of to what extent a patient's quality of life has been affected by tinnitus can be useful to understand the limitations imposed by this symptom. Also it can be useful for therapeutic programming and for the verification of therapy effectiveness (pre and post treatment evaluation). AIM: To culturally adapt the Tinnitus Handicap Inventory (THI), to use it with the Brazilian population and to evaluate its reproducibility. METHOD: The cultural adaptation of the THI (Newman et al., 1996) followed the steps indicated by Guillemin et al., (1993), which include the translation from English to Portuguese, linguistic adaptation and revision of the grammatical and idiomatic equivalences. Assessment of inter and intra-researcher reproducibility of results was also made. Participants of this study were thirty patients (19 females, 63,3% and 11 males, 36,7%) with tinnitus from an otoneurology specialized ambulatory, with ages between 39 and 79 years. RESULTS: The THI was adapted in order to be used with the Brazilian population and was named Brazilian Tinnitus Handicap Inventory. There was no statistically significant difference regarding inter and intra-researcher reproducibility of the results when using the Brazilian version of the questionnaire. A decline in the quality of life regarding emotional, functional and/or catastrophic aspects was observed for all of the assessed patients. CONCLUSION: The THI was translated and culturally adapted in order to be used with the Brazilian population, demonstrating to be a reliable instrument to verify the damage caused by tinnitus in the quality of life of individuals.

Adult↗
PubMed1989

Mechanisms that improve referential access.

Two mechanisms, suppression and enhancement, are proposed to improve referential access. Enhancement improves the accessibility of previously mentioned concepts by increasing or boosting their activation; suppression improves concepts' accessibility by decreasing or dampening the activation of other concepts. Presumably, these mechanisms are triggered by the informational content of anaphors. Six experiments investigated this proposal by manipulating whether an anaphoric reference was made with a very explicit, repeated name anaphor or a less explicit pronoun. Subjects read sentences that introduced two participants in their first clauses, for example, "Ann predicted that Pam would lose the track race," and the sentences referred to one of the two participants in their second clauses, "but Pam/she came in first very easily." While subjects read each sentence, the activation level of the two participants was measured by a probe verification task. The first two experiments demonstrated that explicit, repeated name anaphors immediately trigger the enhancement of their own antecedents and immediately trigger the suppression of other (nonantecedent) participants. The third experiment demonstrated that less explicit, pronoun anaphors also trigger the suppression of other nonantecedents, but they do so less quickly--even when, as in the fourth experiment, the semantic information to identify their antecedents occurs prior to the pronouns (e.g., "Ann predicted that Pam would lose the track race. But after winning the race, she..."). The fifth experiment demonstrated that more explicit pronouns--pronouns that match the gender of only one participant-trigger suppression more powerfully. A final experiment demonstrated that it is not only rementioned participants who improve their referential access by triggering the suppression of other participants; newly introduced participants do so too (e.g., "Ann predicted that Pam would lose the track race, but Kim..."). Thus, both suppression and enhancement improve referential access, and the contribution of these two mechanisms is a function of explicitness. The role of these two mechanisms in mediating other referential access phenomena is also discussed.

Cognition↗
PubMed1998

Partial status epilepticus associated with asomatognosia and alien hand-like behaviors [corrected].

BACKGROUND: Paroxysmal alien hand syndrome (AHS) has been reported in association with ictal phenomena, but simultaneous electroencephalographic verification has not been made. OBSERVATION: A 61-year-old woman with a right hemisphere glioblastoma multiforme developed movements of the left hand that she claimed were not under her control, and she denied ownership of the affected limb. Simultaneous electroencephalograms documented continuous spikes in the right frontotemporal region. Intravenous diazepam therapy (2.5 mg) reversed both the abnormal movements and the spike activity on the electroencephalograms. CONCLUSIONS: Our case demonstrates that partial seizures can produce AHS along with asomatognosia. This variety of AHS appears to be different, both etiologically and phenomenologically, from other forms of AHS.

Aged↗
PubMed1999

Bilateral reductions of hippocampal volume, glucose metabolism, and wada hemispheric memory performance are related to the duration of mesial temporal lobe epilepsy.

In refractory temporal lobe epilepsy (TLE) temporal lobe structures and functions are continuously or intermittently affected by abnormal brain electrical events, noxious neurochemical agents, and metabolic disturbances. There is conflicting evidence regarding the relationship between the duration of refractory mesial TLE and quantitative measures of temporal lobe functions and volumes of the hippocampi. Twenty patients (aged 28 +/- 7 years, 14 males) with an initial precipitating injury before the age of 5 years were subjected to high-resolution magnetic resonance imaging, fluoro-2-deoxy-d-glucose positron-emission tomography (PET), and the Wada test. We investigated whether the duration of unilateral refractory TLE (12 left, 8 right) affects hippocampal volume, glucose metabolism, or Wada hemispheric memory performance. Ipsilateral to the epileptogenic zone the hippocampal volume, metabolism, and Wada hemispheric memory performance were reduced compared to the corresponding contralateral measures. The duration of epilepsy controlled for age at investigation, side of seizure origin, underlying cause, and sex were negatively correlated with ipsi- and contralateral hippocampal volume, hippocampal metabolism, and Wada hemispheric memory performance. Moreover, ipsilateral Wada hemispheric memory performance and contralateral hippocampal glucose metabolism were correlated with the frequency of habitual seizures. Refractory TLE seems to be associated with a slow but ongoing bilateral temporal lobe damage. These cross-sectional results require verification by longitudinal studies carried out over a period of more than two decades.

Adolescent↗
PubMed2019

Network pharmacology exploration reveals the bioactive compounds and molecular mechanisms of Li-Ru-Kang against hyperplasia of mammary gland.

Li-Ru-Kang (LRK) has been commonly used in the treatment of hyperplasia of mammary gland (HMG) as a cipher prescription and achieved obvious therapeutic effects. However, the bioactive compounds and underlying pharmacological mechanisms remain unclear. This study aims to decipher the bioactive compounds and potential action mechanisms of LRK in the treatment of HMG using an integrated pharmacology approach. The ingredients of LRK and the corresponding drug targets were retrieved through drug target databases and were used to construct the "compound-target-disease" network and function-pathway network. Ultimately, 89 compounds and 2150 drug targets were collected. Gene ontology enrichment analysis revealed that mammary gland alveolus development and mammary gland lobule development were the key biological processes and were regulated simultaneously by three direct targets, including androgen receptor (AR), estrogen receptor (ER) and cyclin-D1. Moreover, 14 compounds of LRK were directly involved in the regulation of the three aforementioned targets. KEGG pathway enrichment analysis found that five signaling pathways and seven direct targets were closely related with HMG treatment by LRK. The results of animal experiments showed that LRK significantly improved the histopathological status of HMG in rats. Additionally, LRK markedly regulated the protein expressions of AR, cyclin-D1, MMP2, MMP3 and MMP9. But interestingly, the effect of LRK on ER was not obvious. This study demonstrated that LRK exerted its therapeutic efficacy based on multi-components, multi-targets and multi-pathways. This research confirms the advantages of network pharmacology analyses and the necessity for experimental verification.

Animals↗
PubMed2005

Evaluation and improvement of the efficiency of the Seidel humeral nail by numerical-experimental analysis of the bone-implant contact.

Seidel intramedullary humeral nail is locked distally by a spreading device and proximally by transverse cross locking screws. The main problems found in its use are: the loosening of the distal anchorage, even after X-ray verification of the correct expansion of the device; the formation of distal fractures, occurring even 1 month after the operation; the system's low stability. The problems noted can be ascribed to the behaviour of the spreading device for distal fixing. The present study, therefore, was directed at analysing the contact between the spreading mechanism and the medullary canal in relation to the geometric parameters of the mechanism itself. The main objective was to define the correct regulation of the expansion in the operating theatre, and to delimit the more appropriate conditions of use, allowing the surgeon to evaluate the suitability of this particular device for the type of fracture under treatment. Numerical and experimental techniques were used to perform an analysis of the implant behaviour. This investigation, referred to immediate post-operative condition, allowed to evaluate the stability to traction of the system, to define the typology and properties of the bone-implant contact zone and to quantify the stresses produced, all as a function of the parameter on which the surgeon intervenes in the operating theatre: the number of turns used to tighten the spreading screw. The results obtained confirm and explain the disadvantages associated with the distal expansion system: the bone-implant contact turns out to be inefficient, as revealed by the distribution of the pressure on the inner wall of the medullary canal. On the basis of the results, it is possible to define the optimal conditions of use of the nail, and to formulate a simple solution for the improvement of its performance.

Biomechanical Phenomena↗
PubMed1999

Semiempirical and Raman spectroscopic studies of carotenoids.

Semiempirical AM1 calculations have been carried out for beta-carotene and the three xanthophylls (zeaxanthin, canthaxanthin, and astaxanthin) containing oxygen functions (hydroxy/keto groups) found in the majority of natural pigment. The fully optimized geometries correspond well with the X-ray structures of beta-carotene and canthaxanthin and indicate that substitutions on the terminal rings have a minimal effect on the conformation of the chromophore. Twisting along the polyenic chain results from steric interaction involving methyl substituents, and a Ci point group can be proposed for the four investigated carotenoids. AM1 calculated excitation energies for the strongly allowed excited states can be compared to with the experimental absorption band in the visible region, considering solvent effect. Resonance Raman (RR) and Fourier transform (FT) Raman spectra of natural astaxanthin as well as astaxanthins specifically 13C labeled at the positions 12,12'; 13,13'; 14,14'; 15,15'; 15, and 20,20' were recorded. Furthermore the RR and FT Raman spectra of the asymmetric carotenoid 20-norastaxanthin are presented. The data reveal a substantial amount of information about the coupling between the different vibrations, and enabled an extensive experimental verification of the theoretical normal-coordinate analysis previously performed on polyenic molecules [J Raman Spectrosc 1983, 14, 310-321; Advances in Infrared and Raman Spectroscopy, Vol. 12, 1985, pp. 115-178; Spectrochim Acta 1996, 53, 381-392; Biochim Biophys Acta 1994, 1185, 188-196]. The results make up a very interesting dataset which allowed the interpretation and/or observation of several, hitherto never observed or not well understood, effects in the Raman spectra of the differently labeled astaxanthins.

Absorption↗
PubMed1992

Comparative techniques for determining cellular iron distribution in brain tissues.

Iron is essential for a number of normal brain functions and accumulates in high concentrations in specific regions of the brain. In pathological states, it may further accumulate in these and other areas that are typically low in iron content. The contribution of excess iron to potential central nervous system damage through its ability to donate an electron and to promote oxygen free radical formation has made the nature, location, extent, and process of iron deposition in the brain important areas of investigation. Nevertheless, there is relatively little information currently available on the cellular and subcellular distribution of iron in the central nervous system in either normal or diseased states. We describe and compare a number of the currently available techniques by which iron can be detected within the cellular components of the brain. Histochemical approaches, primarily in the form of the Perls' stain, yields information only on iron in its ferric state and is a relatively insensitive technique. Electron microscopy with x-ray spectrometry can provide positive identification of iron but has limitations regarding morphological verification of the specific cells being probed and also has a relatively high lowest detection limit. Secondary ion mass spectrometry and proton-induced x-ray spectrometry are both expensive, highly complex techniques with greater detection sensitivity, but they have problems identifying the cellular components being analyzed. Finally, laser microprobe mass analysis combines histological localization and identification of probe sites in plastic-embedded histological sections with detection limits in the single part per million range.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

Organization and transcription of the division cell wall (dcw) cluster in Neisseria gonorrhoeae.

A cluster of genes involved in cell division and cell wall (dcw) biosynthesis was identified in Neisseria gonorrhoeae using genomic analysis and through verification of gene order by polymerase chain reaction (PCR) analysis. The gonococcal dcw cluster consists of 17 genes, in the order 5'-mraZ-mraW-ftsI-murE-hyp1-murF- mraY-hyp2-murD-ftsW-murG-murC-ddl -ft sQ-ftsA-ftsZ-hyp3-3'. The gene organization of the dcw cluster of N. gonorrhoeae is more similar to that observed in Gram-negative rods such as Escherichia coli and Haemophilus influenzae than in Gram-positive bacteria. The cluster is characterized by several intergenic spaces. Compared with E. coli, two genes, ftsL and envA, are absent in the gonococcal dcw cluster and three hypothetical genes are novel to the cluster. The cluster is flanked by two transcriptional terminators consisting of paired neisserial uptake sequences and also includes four internal terminators, three of which are paired neisserial uptake sequences. We also found that a repeated sequence on the gonococcal genome, commonly called a Correia element, acts as the fourth transcriptional terminator. All termination sequences were shown to be fully functional by using reverse transcription PCR experiments. Transcriptional start sites upstream of ftsQ, ftsA and ftsZ were determined by primer extension and six promoters were identified; three promoters were located upstream of ftsZ in the intergenic space, two were upstream of ftsA within ftsQ and one was upstream of ftsQ within ddl. Some of these promoters were preferentially used under anaerobic conditions. The location of these promoters differed from those described in E. coli indicating dissimilar transcriptional regulation.

Base Sequence↗
PubMedDate not supplied

A comprehensive cardiovascular waveform analysis program for IBM-compatible personal computers.

Computerized cardiovascular waveform processing has become a necessary and fundamental tool in the analysis of physiologic data. The availability of numerous commercial data-analysis programs has significantly enhanced the efficiency of waveform analysis. Many such programs come with their own programming language, which enables the researcher to create an application program suited to a specific series of calculations. Once written, an application program can significantly increase the efficiency with which data from a specific experiment can be analyzed. However, creating or modifying a program for each new experimental protocol can be time-consuming, especially in the error-detection and verification stages. Single-purpose programs also prove somewhat inflexible to unexpected changes in experimental formats. These problems suggested the need for a more flexible program, but one that is nevertheless specifically suited to the analysis of cardiovascular signals. This need led to the development of a program designed in collaboration with surgeons and physiologists. This program addresses some important analysis problems in cardiovascular research, and allows the user to survey and manipulate cardiovascular waveforms in an intuitive and spontaneous manner.

Algorithms↗
PubMedDate not supplied

A novel protocol to identify mutations in patients with wiskott-Aldrich syndrome.

Mutations of WASP (Wiskott-Aldrich syndrome protein) underlie the severe immunodeficiency/platelet disorder Wiskott-Aldrich syndrome (WAS) and its milder variant X-linked thrombocytopenia (XLT). The affected gene, a 12-exon structure on the X-chromosome, is expressed exclusively in blood cells. The encoded product WASP is a 502-amino-acid scaffolding protein that functions in stimulus-induced nucleation of actin filaments to form dynamic cell surface projections. To date, more than 150 mutations have been identified in 300 WAS/XLT kindred worldwide, generally through methodologies that include sophisticated exon screening steps such as single-strand conformation analysis. We report here a simpler protocol, which was designed for use in clinical settings to identify the mutations of newly diagnosed patients. The approach relies on directly sequencing amplified exons according to a staggered schedule based on statistical evaluation of previous cases. In a 2 1/2-year trial, samples from 28 consecutive patients were analyzed; these included 3 "blindly labeled" previously studied cases. The mutations that were identified include a broad spectrum (8 missense, 3 nonsense, 5 splice site mutations, 11 small insertion/deletions, 1 large deletion) and were broadly distributed (in 10 of the 12 exons). All mutations were verified and no discrepancies were encountered. Per patient, a mean of six DNA sequencing reactions and 6-7 h of staff effort sufficed for mutation identification and verification, indicating that the protocol is cost-effective. This cumulative experience demonstrates the suitability, reliability, and versatility of the new protocol.

Child, Preschool↗