Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

Heart rate response to a caudal block in children anesthetized with sevoflurane after ultrasound confirmation of placement.

INTRODUCTION: Previous studies identified decreasing heart rate (HR) as a predictor of successful caudal placement in children using halothane and isoflurane. No changes were found in HR in the one study using sevoflurane. We documented HR changes in children following a caudal block during sevoflurane anesthesia utilizing ultrasound to confirm successful caudal placement. METHODS: Seventy-one children (1-82 months) were anesthetized with sevoflurane. A caudal block was placed with confirmation by ultrasound. Four aliquots of bupivacaine 0.2% with epinephrine 5 µg · cc(-1) were administered for a total volume of 1 cc · kg(-1) with HR recorded for 4 min. The outcomes measured were HR changes from the initial baseline and during each 1-min interval. The age-related differences in HR were also analyzed. RESULTS: Heart rate change from the initial baseline after placing the caudal needle and allowing for equilibration ranged from -10.2% to +8.9% and the HR change from the baseline at the start of each aliquot injection ranged from -9.5% to +8.9%. Most participants (n = 60, 84.5%) experienced at least one HR reduction over the observation period. For patients < 36 months, the HR change ranged from -11 to +12 b · min(-1) (mean -0.3); for patients aged ≥ 36 months, the HR change ranged from -10 to +6 b · min(-1) (mean -1.1). CONCLUSIONS: Heart rate changes following a caudal block in children ≤ 82 months of age anesthetized with sevoflurane is not a reliable indicator of a successful block. Despite 100% caudal success, many children had no decrease in HR, and in those that did, the decline was of a magnitude indeterminate from beat-to-beat variability.

Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

Therapeutic ultrasound for glaucoma: clinical use of a low-frequency low-power ultrasound device for lowering intraocular pressure.

BACKGROUND: This is a first-in-human study to determine the efficacy and tolerability of a new method of treating glaucoma using a low-power, low-frequency, focused therapeutic ultrasound for glaucoma (TUG) device designed to trigger an inflammatory reaction in the anterior chamber angle and trabecular meshwork to enhance outflow. The use of the device is anticipated for mild or moderate open-angle glaucoma as an enhancement to outflow. METHODS: In a two-branch clinical trial, a total of 26 primary open-angle glaucoma patients underwent a procedure consisting of the external application of the TUG device. In branch 1, nine of these patients were naïve to pharmaceutical treatment or had been off of medication for over 6 months. In branch 2, 17 patients were treated after a medication washout period. All patients in the study were followed for 12 months. RESULTS: In branch 1, there was a decrease in intraocular pressure averaging over 20% lasting at least a year in 74% of the eyes with non-normotensive open-angle glaucoma. In branch 2, an average of two visits while on medication provided the comparison intraocular pressure (IOP) to the effect of the TUG treatment after washout. It was seen that the intraocular pressure over the year post-treatment was equal to or better than the pharmaceutical control in close to 80% of measurements. CONCLUSION: A novel device for lowering intraocular pressure is described with a potential for adding to our armamentarium for treating glaucoma. This is a small cohort study which indicates beneficial trends. TRIAL REGISTRATION NUMBER: The study was a registered clinical trial, #ISRCTN50904302.

Laryngeal mask airway placement in children prior to an intravenous line utilizing heart rate as an indicator of anesthetic depth.

INTRODUCTION: The usual practice in pediatric anesthesia cases requiring a laryngeal mask airway is to place an intravenous line (IV) prior to laryngeal mask airway placement. A different approach that has several clinical advantages is to place the laryngeal mask airway prior to the IV. We describe our experience with this technique, using heart rate as an indicator of adequate anesthetic depth. In addition, we analyzed heart rate data in children undergoing sevoflurane inductions, looking for age-related differences. METHODS: Following a sevoflurane induction, heart rates were recorded every 12 s for 3 min in 127 ASA I-II children under age 7. Laryngeal mask airway placement occurred when the heart rate dropped at least 10% from its maximum level or at 3 min. Ease of laryngeal mask airway placement was graded using a scale from 0 to 3. Endtidal sevoflurane concentration, occurrence of laryngospasm and blood pressure at laryngeal mask airway placement were also recorded. RESULTS: The laryngeal mask airway was successfully placed on the first attempt in all 127 children. Easy placement was noted in 98.4%. The youngest children's heart rates peaked earlier than the oldest (P < 0.001), while time to laryngeal mask airway placement increased with increasing age (P < 0.0001). CONCLUSIONS: Laryngeal mask airway placement before an IV is a safe alternative to the usual mask-IV-laryngeal mask airway sequence. Our data compare favorably to other studies where ease of laryngeal mask airway placement was reported. This technique has several advantages including securing the airway first for an anticipated difficult IV placement. Heart rate changes during a sevoflurane induction appear to be age-dependent.

Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli.

BACKGROUND: Isobutanol is a promising next-generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titer. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases of adaptation to exogenous isobutanol stress. RESULTS: The adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in marC, hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced RpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodeling the cell envelope and surprisingly, stress response attenuation. CONCLUSIONS: We have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to further efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis targets for future global phenotype engineering.

Microarrays for protease detection in tissues and cells.

Expression of a given protease and of the endogenous inhibitors that regulate protease activity can be readily determined at the transcript level by using whole genome microarray chips. In the case of proteases and protease inhibitors, however, determining which cells are expressing them is often critical to understanding the functional roles of the proteases. For example, in cancer many of the proteases are derived from cells that are found in the microenvironment surrounding the tumor, e.g., fibroblasts and inflammatory cells. Proteases from both fibroblasts and inflammatory cells have been implicated in malignant progression. Therefore, it is important to recognize the origin of these molecules if one is to develop effective therapies. In this regard, mouse transgenic models and xenograft models in which human tumor cells are implanted in mice are useful tools. To profile human and mouse proteases, protease inhibitors, and protease interactors, we have developed in partnership with Affymetrix a custom, single platform, dual species chip: the Hu/Mu ProtIn chip. The Hu/Mu ProtIn chip has been validated for its ability to identify human and mouse transcripts in single species specimens and to identify and distinguish between human and mouse transcripts in dual species specimens such as xenografts. In the latter specimens, the Hu/Mu ProtIn chip has enabled us to identify host (mouse) proteases that play a protective role in development of lung tumors. Here we outline a protocol for using the Hu/Mu ProtIn chip to profile proteases, protease inhibitors, and protease interactors in tissues and cells.

Determining the accuracy of caudal needle placement in children: a comparison of the swoosh test and ultrasonography.

BACKGROUND: The aim of the present study was to compare two confirmatory tests - the 'swoosh' test (auscultation during caudal injection) and real time ultrasound imaging (both transverse 2D imaging and color flow Doppler imaging) in pediatric patients receiving a caudal epidural block. METHODS/MATERIALS: This was a retrospective observational study of caudal injections administered to 83 pediatric patients (0-11 years) presenting for elective surgery over a 4 month time period. While injecting small aliquots of local anesthetic, a standard stethoscope was placed over the lower lumbar spine to auscultate for the 'swoosh' test. An ultrasound machine (Sonosite Titan, Sonosite Inc., Bothell, WA, USA) was then utilized for real-time visualization of caudal injectate. Each test performed during the caudal injection (swoosh, turbulence on 2D imaging, or color flow on Doppler imaging) was recorded as positive, negative or equivocal. RESULTS: Eighty out of 83 patients (96.4%) had a successful caudal block based on minimal or no perioperative narcotic use, minimal or no response to surgical stimulation, the presence of motor blockade and patient comfort in the PACU. Ultrasound was significantly superior to 'swoosh' for sensitivity (96.3% vs 57.5%), negative predictive (40% vs 5.6 value) % and likelihood ratio (2.89 vs 1.73). Specificity and positive predictive value were not different between 'swoosh' and ultrasound. Of the ultrasound tests, turbulence was more sensitive than color flow Doppler (95.0% vs 78.8%). CONCLUSION: Ultrasonography is superior to the 'swoosh' test as an objective confirmatory technique during caudal block placement in children. We found the presence or absence of turbulence during injection within the caudal space to be the best single indicator of caudal success. We think ultrasonography should be used, if available, when teaching this technique.

Ultrasonography and pediatric caudals.

Ultrasound is an important tool for performing pediatric regional blocks, including caudal blocks. We present a case in which the availability of ultrasound allowed us to proceed with a successful caudal block which we otherwise might have abandoned in an infant with difficult anatomy.