High-throughput microfluidic single-cell digital polymerase chain reaction.

Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells. This device allows for the parallel processing of single cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis. The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning. The high density of this dPCR format (118,900 chambers/cm(2)) allows the analysis of 200 single cells per run, for a total of 204,000 PCR reactions using a device footprint of 10 cm(2). Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 10(4). We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods. We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells. High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity. We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing.

Method of preparing a decellularized porcine tendon using tributyl phosphate.

Extracellular matrix (ECM) materials are currently utilized for soft tissue repair applications such as vascular grafts, tendon reconstruction, and hernia repair. These materials are derived from tissues such as human dermis and porcine small intestine submucosa, which must be rendered acellular to prevent disease transmission and decrease the risk of an immune response. The ideal decellularization technique removes cells and cellular remnants, but leaves the original collagen architecture intact. The tissue utilized in this study was the central tendon of the porcine diaphragm, which had not been previously investigated for soft tissue repair. Several treatments were investigated during this study including peracetic acid, TritonX-100, sodium dodecyl sulfate, and tri(n-butyl) phosphate (TnBP). Of the decellularization treatments investigated, only 1% TnBP was effective in removing cell nuclei while leaving the structure and composition of the tissue intact. Overall, the resulting acellular tissue scaffold retained the ECM composition, strength, resistance to enzymatic degradation, and biocompatibility of the original tissue, making 1% TnBP an acceptable decellularization treatment for porcine diaphragm tendon.

Purification and characterization of a novel phosphorus-oxidizing enzyme from Pseudomonas stutzeri WM88.

The ptxD gene from Pseudomonas stutzeri WM88 encoding the novel phosphorus oxidizing enzyme NAD:phosphite oxidoreductase (trivial name phosphite dehydrogenase, PtxD) was cloned into an expression vector and overproduced in Escherichia coli. The heterologously produced enzyme is indistinguishable from the native enzyme based on mass spectrometry, amino-terminal sequencing, and specific activity analyses. Recombinant PtxD was purified to homogeneity via a two-step affinity protocol and characterized. The enzyme stoichiometrically produces NADH and phosphate from NAD and phosphite. The reverse reaction was not observed. Gel filtration analysis of the purified protein is consistent with PtxD acting as a homodimer. PtxD has a high affinity for its substrates with Km values of 53.1 +/- 6.7 microm and 54.6 +/- 6.7 microm, for phosphite and NAD, respectively. Vmax and kcat were determined to be 12.2 +/- 0.3 micromol x min(-1) x mg(-1) and 440 min(-1). NADP can substitute poorly for NAD; however, none of the numerous compounds examined were able to substitute for phosphite. Initial rate studies in the absence or presence of products and in the presence of the dead end inhibitor sulfite are most consistent with a sequential ordered mechanism for the PtxD reaction, with NAD binding first and NADH being released last. Amino acid sequence comparisons place PtxD as a new member of the d-2-hydroxyacid NAD-dependent dehydrogenases, the only one to have an inorganic substrate. To our knowledge, this is the first detailed biochemical study on an enzyme capable of direct oxidation of a reduced phosphorus compound.

Men making sense of their chest pain--niggles, doubts and denials.

Participant observation was undertaken of the early admission period of 25 men admitted to hospital with acute chest pain, followed by in-depth interviews of 10 of the men after discharge. Grounded theory methods were used in the analysis to develop a model of how the men came to interpret their experiences. An emerging feature of the men's experiences was that, although they had suffered intense pain prior to admission, there had been a series of delays whilst they tried to rationalize their symptoms. We relate our discussion to literature on men and masculinity and the notion of Foucault (1975) of self-surveillance, to offer an insight into the men's self concept and social situation. Our conclusions suggest that men's self concept as 'healthy' may inhibit a speedy response to the signs and symptoms of acute coronary occlusion, increasing the risk of cardiac arrest without nearby life support.

Molecular and immunologic characterization of group A bovine rotavirus field isolates with P8[11] spike protein.

Bovine rotavirus strain B223 is the North American prototype for group A P8[11] serotype/genotype rotaviruses. Rotaviruses with this serotype/genotype are a prevalent cause of neonatal calf diarrhea and have also been isolated from asymptomatic human infants. On the basis of deduced amino acid sequence of the outer capsid viral protein 4 (VP4), strain B223 lacks a cysteine at position 318 that is conserved among all other rotavirus strains. It has been speculated that this may result in the loss of disulfide bond and a change in the structure of the VP4 that may affect the infectivity and antigenicity of the virus. This paper describes partial sequences of the VP4 gene (nucleotides 613 to 1016 coding for amino acid positions 202 to 335) of 16 bovine rotavirus field isolates with P8[11] that were obtained from calves in Nebraska and Indiana. All the isolates lack a cystein at position 203 and had a conserved valine residue at position 318, thus indicating that the prototype strain B223 is representative of group A rotaviruses with P8[11] serotype/genotype.

Cecal spirochetosis associated with Serpulina pilosicoli in captive juvenile ring-necked pheasants.

Spirochetes similar to those described in the ceca of broilers with diarrhea and in laying hens with decreased egg production and growth were identified in the ceca of captive-raised juvenile ring-necked pheasants (Phasianus colchicus). The birds were submitted for diagnostic investigation of an illness characterized by a seromucoid ocular discharge, sneezing, swollen infraorbital sinuses, and weight loss. In addition to cecal spirochetosis, the birds had mild enteric coccidiosis, trichomoniasis, and nematodiasis (Heterakis spp.); esophageal capillariasis; and respiratory mycoplasmosis. Weakly beta-hemolytic spirochetes isolated from the ceca of one pheasant were identified as Serpulina pilosicoli with the use of a 16S rRNA sequence-specific polymerase chain reaction amplification assay. Diffuse cecal enterocyte attachment was reproduced in a 1-day-old chick challenged with the pheasant S. pilosicoli isolate. Immunohistochemical staining of sections of ceca from the pheasant and challenged chick with a Serpulina spp. flagellar antigen-specific monoclonal antibody confirmed spirochetal attachment to cecal enterocytes. The etiologic significance of the spirochete infection is unknown because respiratory signs and multiple gastroenteric pathogens dominated the clinicopathologic manifestations and an intestinal disorder was not a clinical complaint.

Efficacy of a temperature-sensitive modified-live bovine herpesvirus type-1 vaccine against abortion and stillbirth in pregnant heifers.

OBJECTIVE: To evaluate the efficacy of a commercially available temperature-sensitive modified-live bovine herpesvirus type-1 (BHV-1) vaccine against BHV-1 challenge-induced abortion and stillbirth. DESIGN: Prospective randomized control trial. ANIMALS: 20 cycling, nonpregnant, BHV-1 seronegative heifers of various breeds and weights, 12 to 15 months old. PROCEDURE: Heifers were randomly assigned to a vaccinate (n = 10) or nonvaccinate control (n = 10) group. Seventeen to 26 days after members of the vaccinate group received a second dose of vaccine, all heifers were artificially inseminated. Heifers were challenged intravenously with Cooper strain BHV-1 between days 177 and 187 of gestation. Aborted fetuses and stillborn calves were necropsied, and tissues collected for histologic examination and virus isolation. Heifers, calves, and fetuses were tested for BHV-1 antibody throughout the study. RESULTS: The difference in number of abortions or stillbirths between vaccinated heifers (1/10) and control heifers (10/10) was significant (P < 0.003). Seven of 10 control heifers had a virus neutralization antibody titer to BHV-1 at abortion or stillbirth that declined or remained unchanged from their titer at a previous serologic evaluation (7 to 66 days earlier). CLINICAL IMPLICATIONS: Prebreeding vaccination of replacement heifers with modified-live BHV-1 vaccine provides fetal protection at 6 months of gestation (7 months after vaccination) and appears to be a reasonable precaution to control economic losses associated with BHV-1 infection. Abortions induced by BHV-1 are not necessarily associated with rising or markedly high virus neutralization antibody titers. These titers should be used cautiously when assessing the role of BHV-1 in bovine abortion and stillbirth.

Comparison of the abilities of serologic tests to detect pseudorabies-infected pigs during the latent phase of infection.

OBJECTIVE: To compare the sensitivities of all available serologic tests in detecting pseudorabies virus (PRV) antibodies in pigs during long-term latent pseudorabies. DESIGN: Pigs experimentally infected with a virulent strain of PRV were maintained for 2 to 27 months after inoculation. At the time of necropsy of each pig, blood was collected for serologic evaluation, and tissues were obtained for polymerase chain reaction (PCR) verification of latency. ANIMALS: 65 crossbred pigs each weighing approximately 18 kg at the start of the study. PROCEDURE: Serum samples from each pig were analyzed by serum neutralization, latex agglutination, screening ELISA, particle concentration fluorescence immunoassay, automated latex agglutination, and differential ELISA for glycoproteins I, III, and X. DNA was extracted from the trigeminal ganglia and tonsils of each pig and was analyzed by PCR for PRV genomic sequences. RESULTS: PCR analysis of trigeminal ganglia and tonsils indicated that all pigs were latently infected with PRV at the time of necropsy, and serologic testing verified that all pigs had PRV-specific antibodies, regardless of duration of infection. The screening tests were virtually equivalent in sensitivity for detection of PRV antibodies. Of the differential serologic tests, the glycoprotein-I and -III marker systems, which performed with similar sensitivity as screening tests, were superior to the glycoprotein-X marker system in detecting PRV antibodies in latently infected pigs. CONCLUSION: Serologic testing consistently detects pigs in the latent phase of PRV infection, provided that the test detects the antibody response to the whole virus or to a reliable PRV-marker glycoprotein.

Tracheoesophageal fistula: a case report.

Tracheoesophageal fistula without atresia (H-type) is a known but uncommon cause of respiratory distress in the newborn. We present a rare case of a high cervical tracheoesophageal fistula unassociated with a laryngeal cleft or esophageal atresia. The diagnosis, associated anomalies, and surgical repair of this previously undescribed entity are discussed.

Salivary gland disease in infancy and childhood: non-malignant lesions.

The differential diagnosis for benign lesions of the salivary glands in infancy and childhood can be somewhat overwhelming, especially in light of their infrequent occurrence in a general otolaryngologic practice. The purpose of this review is to discuss these conditions under the headings of inflammatory conditions, immunologic disorders, granulomatous disease, trauma, cysts, systemic disorders and benign neoplasms. Basic techniques of diagnosis and treatment as they apply to children will be outlined.

Evaluation of neonatal subglottic stenosis: a 3-year prospective study.

Subglottic stenosis is the most common cause of chronic airway obstruction. It results in prolonged tracheal cannulation of infants and children. Following the widespread adoption over the past 20 years of prolonged intubation for respiratory support in neonates, the incidence of acquired subglottic stenosis increased dramatically. On January 1, 1987, we began a 3-year prospective study to delineate potential etiologic factors involved in the development of subglottic stenosis in neonates. The present study analyzes data from 289 infants. Relationships between birth weight, gestational age, endotracheal tube size, duration of intubation and ventilation, number and difficulty of intubations, and the subsequent need for medical and surgical therapy are discussed. Whole organ larynges from autopsy specimens provide histological correlation.

Treatment of tracheocutaneous fistulae in children.

Tracheocutaneous fistula is a frequent sequel of long-term tracheotomy in children and adults. Persistence of the tracheotomy stoma can lead to increased morbidity and mortality, especially in patients with limited pulmonary reserve. Recommendations for treatment have included both primary closure as well as excision of the fistulous tract with healing by secondary intention. Operative management and outcome of tracheocutaneous fistula performed at The Hospital For Sick Children between January 1978 and January 1988 was reviewed retrospectively. Complications of primary closure included pneumothorax, pneumomediastinum, and poor wound healing, often leading to significant patient morbidity. Excision of the fistulous tract and temporary replacement of the tracheotomy tube with secondary wound healing resulted in no complications and successful closure in all cases. In our experience, tracheocutaneous fistula in the pediatric population are best managed in this way.

Intradiploic epidermoid cyst of the sphenoid wing.

Epidermoids are blastomatous malformations of abnormal epithelial proliferation. Intradiploic epidermoids are very rare lesions that may occur in all flat bones of the calvarium and in the paranasal sinuses, maxilla, temporal bone, and sphenoid bone. This article describes the unique case of an intradiploic epidermoid tumor located in the greater sphenoid wing. Theories of pathogenesis, clinical behavior, diagnosis, and treatment of diploic epidermoids are reviewed.

Thyroid nodules in children.

Thyroid nodules are infrequently encountered in children. During the era of low-dose therapeutic irradiation, the incidence of malignancy in these lesions was 40% to 70%. Recent studies suggest that this incidence is declining, resulting in a concomitant relative increase in the proportion of benign nodular conditions. There is also a heightened awareness that secondary thyroid neoplasms may occur in children surviving primary malignant diseases. Records of 38 children treated surgically at Texas Children's Hospital between 1972 and 1984 have been reviewed to determine the incidence of benign vs. malignant pathosis and to study the role of prior irradiation or chemotherapy in the pathogenesis of thyroid disease. Benign conditions were diagnosed in 27 children (71%), with diffuse hyperplasia (10 children, 26%) and follicular adenoma (8 children, 21%) occurring most frequently. Thyroid carcinoma was diagnosed in the remaining 11 children (29%). All of these patients were euthyroid at presentation, none had received multimodal therapy for a prior malignant condition, and only one had a history of head and neck irradiation in infancy for a treatment of a benign condition (a congenital hemangioma). The clinical presentation, diagnostic evaluation, and surgical management of these patients are reviewed and closely parallel those of patients in other recently published series. No conclusions can be drawn regarding the development of thyroid neoplasia following multimodal therapy for primary disease; however, this incidence must be very low.

Head and neck manifestations of neurofibromatosis.

Neurofibromatosis is a neurocutaneous systemic disease that occurs in 1:2500 to 3300 live births. Prevalence figures have shown it to be as common as cystic fibrosis or Down's syndrome and more than twice as common as muscular dystrophy. In this study, our experience with 257 cases of neurofibromatosis seen since 1972 is reviewed. Intracranial, bony, and extracranial anomalies are described in the 223 patients (87%) who presented with, or ultimately developed, head and neck manifestations of the disease. The most common intracranial tumor was optic glioma, found in 35 patients (14%), 19 younger than 10 years of age. Acoustic neuromas were diagnosed in eight individuals (3%) and were bilateral in three. The most common skull anomaly was macrocephaly, noted 78 times (30%). Absence of the sphenoid wing occurred in 11 patients (4%) and 19 others (7%) had facial asymmetry due to other skull abnormalities. Extracranial manifestations included neurofibromas of the plexiform and nonplexiform type, Lisch nodules, and cafe-au-lait spots.

Malignant salivary gland tumors of the paranasal sinuses and nasal cavity.

Of a total of 122 patients with minor salivary gland tumors of the nose and paranasal sinuses, 66 patients were identified who had had their therapy at the University of Texas M.D. Anderson Hospital and Tumor Institute, Houston. Adenoid cystic carcinomas and adenocarcinomas were the most frequent tumors. Eighty percent of the patients were treated for relatively advanced (T3 and T4) cancers. Low-grade and high-grade carcinomas could be identified in the two groups studied, and the pathologic grading influenced prognosis in addition to the amount of local extent and degree of invasion of the neoplasm. The adequate preoperative documentation of tumor extension is mandatory, and a team effort in the surgical resection is worthwhile. Radiation therapy remains an important adjuvant in this treatment and occasionally has been used effectively as the only modality of treatment.