Bruno J. Salena

Minimizing postcolonoscopy abdominal pain by using CO2 insufflation: A prospective, randomized, double blind, controlled trial evaluating a new commercially available CO2 delivery system

BACKGROUND Abdominal pain after colonoscopy is a common, distressing symptom resulting from bowel distension by insufflated gas. CO(2), unlike air, is rapidly cleared from the colon by passive absorption. A commercially available CO(2) delivery system has only recently become available. The effects of CO(2) and air insufflation on residual bowel gas and postprocedure pain were compared. METHODS One hundred patients were randomized to undergo colonoscopy with insufflation of air (n = 51) or CO(2) (n = 49) by means of a regulator; 97 patients completed the study. Patients with active GI bleeding, inflammatory bowel disease, or previous colectomy were excluded. Pain scores (ordinal scale: 0 = none, to 5 = extreme) were recorded immediately after colonoscopy and at 1, 6, and 24 hours. Residual colonic gas was evaluated on abdominal radiographs at 1 hour. RESULTS Residual colonic gas and postprocedural pain at 1 and 6 hours were significantly less in the CO(2) group. 71% of patients insufflated with room air had colonic distension in excess of 6 cm versus 4% for those in the CO(2) group. 94% of patients insufflated with CO(2) had minimal colonic gas versus 2% in whom air was used (p < 0.0001). Of patients insufflated with air, 45% and 31% had pain at, respectively, 1 hour and 6 hours, versus 7% and 9%, respectively, for those insufflated with CO(2) (respectively, p < 0.0001 and p < O.02). No complications resulted from use of the CO(2) delivery system. CONCLUSIONS Insufflation of CO(2) rather than air significantly reduces abdominal pain and bowel distension after colonoscopy. CO(2) may be insufflated safely and effectively with the new CO(2) delivery system.

Translating Bacterial Detection by DNAzymes into a Litmus Test

Microbial pathogens pose serious threats to public health and safety, and results in millions of illnesses and deaths as well as huge economic losses annually. Laborious and expensive pathogen tests often represent a significant hindrance to implementing effective front-line preventative care, particularly in resource-limited regions. Thus, there is a significant need to develop low-cost and easy-to-use methods for pathogen detection. Herein, we present a simple and inexpensive litmus test for bacterial detection. The method takes advantage of a bacteria-specific RNA-cleaving DNAzyme probe as the molecular recognition element and the ability of urease to hydrolyze urea and elevate the pH value of the test solution. By coupling urease to the DNAzyme on magnetic beads, the detection of bacteria is translated into a pH increase, which can be readily detected using a litmus dye or pH paper. The simplicity, low cost, and broad adaptability make this litmus test attractive for field applications, particularly in the developing world.

A Catalytic DNA Activated by a Specific Strain of Bacterial Pathogen

Abstract Pathogenic strains of bacteria are known to cause various infectious diseases and there is a growing demand for molecular probes that can selectively recognize them. Here we report a special DNAzyme (catalytic DNA), RFD‐CD1, that shows exquisite specificity for a pathogenic strain of Clostridium difficile (C. difficile). RFD‐CD1 was derived by an in vitro selection approach where a random‐sequence DNA library was allowed to react with an unpurified molecular mixture derived from this strain of C. difficle, coupled with a subtractive selection strategy to eliminate cross‐reactivities to unintended C. difficile strains and other bacteria species. RFD‐CD1 is activated by a truncated version of TcdC, a transcription factor, that is unique to the targeted strain of C. difficle. Our study demonstrates for the first time that in vitro selection offers an effective approach for deriving functional nucleic acid probes that are capable of achieving strain‐specific recognition of bacterial pathogens.

A Sensitive DNA Enzyme-Based Fluorescent Assay for Bacterial Detection

Bacterial detection plays an important role in protecting public health and safety, and thus, substantial research efforts have been directed at developing bacterial sensing methods that are sensitive, specific, inexpensive, and easy to use. We have recently reported a novel “mix-and-read” assay where a fluorogenic DNAzyme probe was used to detect model bacterium E. coli. In this work, we carried out a series of optimization experiments in order to improve the performance of this assay. The optimized assay can achieve a detection limit of 1000 colony-forming units (CFU) without a culturing step and is able to detect 1 CFU following as short as 4 h of bacterial culturing in a growth medium. Overall, our effort has led to the development of a highly sensitive and easy-to-use fluorescent bacterial detection assay that employs a catalytic DNA.

Optimal DNA templates for rolling circle amplification revealed by in vitro selection.

Rolling circle amplification (RCA) has been widely used as an isothermal DNA amplification technique for diagnostic and bioanalytical applications. Because RCA involves repeated copying of the same circular DNA template by a DNA polymerase thousands of times, we hypothesized there exist DNA sequences that can function as optimal templates and produce more DNA amplicons within an allocated time. Herein we describe an in vitro selection effort conducted to search from a random sequence DNA pool for such templates for phi29 DNA polymerase, a frequently used polymerase for RCA. Diverse DNA molecules were isolated and they were characterized by richness in adenosine (A) and cytidine (C) nucleotides. The top ranked sequences exhibit superior RCA efficiency and the use of these templates for RCA results in significantly improved detection sensitivity. AC-rich sequences are expected to find useful applications for setting up effective RCA assays for biological sensing.

Relapse of duodenal ulcer.

which was concerned with different approaches to the medical treatment of duodenal ulcer. No physician I know believes that medical treatment cures duodenal ulcer disease. Prolonged medical treatment is required for hypertension, epilepsy, diabetes, and myxoedema so why not in principle for duodenal ulcer too? The difference between Dr Wormsley and myself concerns the nature of that treatment. When should medical treatment be abandoned and surgery be advised? There is little argument about the patient whose symptoms cannot be controlled medically or who has major haemorrhage. But what about the young patient whose symptoms are well controlled without complication? Many such patients do not want surgery and one has to be very confident to advise a young, largely asymptomatic patient to undergo elective surgery on the grounds that it might save his life some decades later. The mortality from elective gastric surgery is very low but finite, and ifthe number ofoperations increases so will the number of perioperative deaths. How many deaths at the age of 30 are acceptable to prevent 100 deaths at the age of 70? The 100/o mortality from upper gastrointestinal haemorrhage is largely confined to the elderly, many ofwhomhave other major medical disorders. What would their prognosis otherwise have been and how much has the bleed curtailed life? Only a few elderly patients with bleeding may have a history of peptic ulceration, and a third to a half are likely to have been taking anti-inflammatory drugs.I2 They may not have had longstanding ulcers amenable to surgery many years earlier. The recurrence rate after truncal vagotomy is low but the few patients who develop intractable diarrhoea or dumping are a major problem with little hope of satisfactory medical or surgical treatment, leading to the suggestion that the operation should be abandoned.3 Nevertheless, it remains the commonest operation for duodenal ulcer in the United Kingdom. Why has proximal gastric vagotomy not been universally adopted? Presumably because it is technically more demanding and frequently carries a higher relapse rate. It is unreasonable to compare relapse rates after gastric surgery with those during or after medical treatment, when repeating or changing the drug treatment will usually heal the ulcer again. Mr McCloy refers to the substantial relapse rate even on maintenance treatment but omits to mention that such relapses are rarely accompanied by complications.4 The surgical patient who relapses has to revert to medical treatment or undergo a more radical operation associated with higher morbidity. No mention has been made of the risk of cancer after gastric surgery.5 This may be small but it has to be considered. We do not yet know the optimal time to recommend surgery to the patient well controlled on medical treatment but Mr McCloys letter and the thoughtful article from his colleague6 are welcome contributions to the debate.

Detection of DNA Amplicons of Polymerase Chain Reaction Using Litmus Test

We report on a new colorimetric DNA detection method that takes advantage of the power of polymerase chain reaction (PCR) and the simplicity of the classic litmus test. The strategy makes use of a modified set of primers for PCR to facilitate ensuing manipulations of resultant DNA amplicons: their tagging with urease and immobilization onto magnetic beads. The amplicon/urease-laden beads are then used to hydrolyze urea, resulting in the increase of pH that can be conveniently reported by a pH-sensitive dye. We have successfully applied this strategy for the detection of two hypervirulent strains of the bacterium Clostridium difficile that are responsible for the recent increase in the global incidence and severity of C. difficile infections. Furthermore, the viability of this test for diagnostic applications is demonstrated using clinically validated stool samples from C. difficile infected patients.

Colorimetric Detection of Bacteria Using Litmus Test.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

Serendipitous Discovery of a Guanine-rich DNA Molecule with a Highly Stable Structure in Urea

We have made an accidental discovery of an unusual, single-stranded, guanine-rich DNA molecule that is capable of adopting a folded structure in 7 M urea (7MU) known to denature nucleic acid structures. The folding of this molecule requires Na+ and Mg2+ and the folded structure remains stable when subjected to denaturing (7MU) polyacrylamide gel electrophoresis. Results from sequence mutagenesis, DNA methylation, and circular dichroism spectroscopy studies suggest that this molecule adopts an intramolecular guanine-quadruplex structure with 5 layers of guanine tetrads. Our finding indicates that DNA has the ability to create extremely stable structural folds despite its limited chemical repertoire, making it possible to develop DNA-based systems for unconventional applications.

Target-Induced Catalytic Assembly of Y-Shaped DNA and Its Application for In Situ Imaging of MicroRNAs

DNA is a highly programmable material that can be configured into unique high-order structures, such as DNA branched junctions containing multiple helical arms converging at a center. Herein we show that DNA programmability can deliver in situ growth of a 3-way junction-based DNA structure (denoted Y-shaped DNA) with the use of three hairpin-shaped DNA molecules as precursors, a specific microRNA target as a recyclable trigger, and a DNA polymerase as a driver. We demonstrate that the Y-shaped configuration comes with the benefit of restricted freedom of movement in confined cellular environment, which makes the approach ideally suited for in situ imaging of small RNA targets, such as microRNAs. Comparative analysis illustrates that the proposed imaging technique is superior to both the classic fluorescence in situ hybridization (FISH) method and an analogous amplified imaging method via programmed growth of a double-stranded DNA (rather than Y-shaped DNA) product.