Marvin Ryou

Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor

Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome.

Evaluation of a manually driven, multitasking platform for complex endoluminal and natural orifice transluminal endoscopic surgery applications (with video)

BACKGROUND The Direct Drive Endoscopic System (DDES) is a multitasking platform developed to overcome the limitations of the currently available rigid and flexible endoscopic systems in application to natural orifice transluminal endoscopic surgery (NOTES), single-port laparoscopy, and advanced endoluminal procedures. The system consists of a 3-channel, steerable guide sheath accepting a 6-mm endoscope and two 4-mm articulating instruments. The systems overall design enables the interventionalist to operate instruments bimanually from a stable platform, conveying a laparoscopic paradigm to the functional working space at the distal end of the flexible guide sheath. OBJECTIVE To assess the basic functionality of the DDES device in a series of defined exercises by using ex vivo porcine stomachs and 1 in vivo animal model. DESIGN Ex vivo calibration and training exercises, including EMR, full-thickness suturing, and knot tying. SETTING Animal laboratory. INTERVENTIONS EMR, full-thickness suturing, and knot tying. MAIN OUTCOME MEASUREMENTS Successful completion of specified tasks. RESULTS Independent instrument movement with a wide range of motion allowed the interventionalist to perform several complex tasks efficiently. The DDES was able to (1) grasp tissue and hold it under tension, (2) cut through layers of porcine stomach in a controlled fashion, (3) suture, and (4) tie knots. LIMITATION Ex vivo study. CONCLUSIONS This novel multitasking platform demonstrated surgical functionality including triangulation, cutting, grasping, suturing, and knot tying. Preliminary results suggest that the DDES can perform complex endosurgical tasks that have traditionally been challenging or impossible with the standard endoscopic paradigm, and may enable NOTES, single-port laparoscopy, and complex endoluminal procedures.

Transluminal closure for NOTES: an ex vivo study comparing leak pressures of various gastrotomy and colotomy closure modalities.

BACKGROUND AND STUDY AIMS Transluminal closure is fundamental to the safe introduction of natural orifice transluminal endoscopic surgery (NOTES) into humans. Suture, staples, and clips have been used. We aimed to evaluate the acute strength of various gastrotomy and colotomy closure techniques in an ex vivo porcine model by assessing air leak pressures. PATIENTS AND METHODS The following closure modalities were assessed with at least five samples per arm: conventional open/laparoscopic suturing techniques including full-thickness interrupted sutures, double-layer sutures, and purse-string sutures, as well as endoscopic clips and endoscopic staples. Historical values for transgastric closures with hand-sewn interrupted sutures, endoscopic clips, and a prototype endoscopic suture device were used from our laboratorys prior study. RESULTS Using Kruskal-Wallis analysis, the overall comparisons were significant ( P = 0.0038 for gastrotomy closure; P = 0.0018 for colotomy closure). Post hoc paired comparisons revealed that the difference between all closure arms versus negative control were significant. Significance could not be established among the various closure arms. However, trends suggested hand-sewn double-layer sutures, endoscopic staples, and both hand-sewn and endoscopically-placed purse-string sutures produced the strongest closures. Furthermore, endoscopic clips appeared sufficient for colotomy closure when ideally placed. CONCLUSIONS Suture (both hand-sewn and endoscopically deployed) appears to produce the strongest closures in both stomach and colon, with the important caveats that (1) a continuous through-thickness suture track be avoided, such as in the full-thickness closure, or (2) suture holes be buried, such as in the purse-string configuration. When suture tracks are full-thickness, they can serve as leak sites. Staples and clips can produce comparable closures, but only under ideal conditions.

Evaluation of a novel access and closure device for NOTES applications: a transcolonic survival study in the porcine model (with video)

BACKGROUND Natural orifice transluminal endoscopic surgery (NOTES) is a novel, potentially less invasive alternative to laparoscopic surgery. However, the problems of transluminal access and closure represent significant obstacles to its successful introduction in humans. OBJECTIVE Our purpose was to evaluate the feasibility and safety of a novel device designed for transluminal access and closure. DESIGN Experimental endoscopic study of transcolonic incision and closure with use of a prototype device in a survival porcine model. SUBJECTS Four adult female Yorkshire pigs were used in the study. INTERVENTIONS While under general anesthesia, the animals were prepped with multiple tap water enemas followed by instillation of an antibiotic suspension and povidone-iodine lavage. At a distance of 15 to 20 cm from the anus, the prototype device deployed a circumscribing purse-string suture around the planned incision site and subsequently used a blade mechanism to create a 2.5-cm linear incision. The peritoneum was then accessed with a standard double-channel enodoscope. The transcolonic incision was then closed by cinching and securing the purse-string suture with a titanium knot by use of a separate hand-activated suture-locking device. All animals were allowed to eat immediately after recovering from general anesthesia. MAIN OUTCOME MEASUREMENTS The animals were monitored daily for signs of peritonitis and sepsis and were electively killed on day 14. The peritoneal cavity was examined for peritonitis, and the colonic incision site was examined for wound dehiscence, pericolic abscess formation, and gross adhesions. Tissue samples from both incisional and random peritoneal sites were obtained for histologic examination. RESULTS Transcolonic incision and closure were successful in all 4 animals. The device performed in a rapid and reproducible fashion. All animals recovered without septic complications. At necropsy, there was no evidence of peritonitis, abscesses, or wound dehiscence. Salpingocolonic and colovesicular adhesions were noted in 3 of 4 animals. Histologic examination revealed microabscesses at the incision site in all animals. CONCLUSIONS The prototype incision and closure device represents a promising solution to the problems of transluminal access for NOTES. The presence of incision-related adhesions and microabscesses signal the need for further refinement in aseptic technique.

Current status of endoluminal bariatric procedures for primary and revision indications.

Endoscopic bariatric procedures are gaining traction as possible minimally invasive treatment modalities for obesity. This article focuses on the various endoscopic devices and procedures that pertain to primary and revisional treatments. Additionally, the article discusses the potential for applying these various devices and procedures to other points of intervention, including early intervention, bridge to surgery, and primary metabolic treatment (eg, diabetes management). Devices that are currently in human use are preferentially discussed, followed by references to devices that may see clinical use in the near future.

Percutaneous Gastrostomy Device for the Treatment of Class II and Class III Obesity: Results of a Randomized Controlled Trial

Objectives:The AspireAssist System (AspireAssist) is an endoscopic weight loss device that is comprised of an endoscopically placed percutaneous gastrostomy tube and an external device to facilitate drainage of about 30% of the calories consumed in a meal, in conjunction with lifestyle (diet and exercise) counseling.Methods:In this 52-week clinical trial, 207 participants with a body-mass index (BMI) of 35.0–55.0 kg/m2 were randomly assigned in a 2:1 ratio to treatment with AspireAssist plus Lifestyle Counseling (n=137; mean BMI was 42.2±5.1 kg/m2) or Lifestyle Counseling alone (n=70; mean BMI was 40.9±3.9 kg/m2). The co-primary end points were mean percent excess weight loss and the proportion of participants who achieved at least a 25% excess weight loss.Results:At 52 weeks, participants in the AspireAssist group, on a modified intent-to-treat basis, had lost a mean (±s.d.) of 31.5±26.7% of their excess body weight (12.1±9.6% total body weight), whereas those in the Lifestyle Counseling group had lost a mean of 9.8±15.5% of their excess body weight (3.5±6.0% total body weight) (P<0.001). A total of 58.6% of participants in the AspireAssist group and 15.3% of participants in the Lifestyle Counseling group lost at least 25% of their excess body weight (P<0.001). The most frequently reported adverse events were abdominal pain and discomfort in the perioperative period and peristomal granulation tissue and peristomal irritation in the postoperative period. Serious adverse events were reported in 3.6% of participants in the AspireAssist group.Conclusions:The AspireAssist System was associated with greater weight loss than Lifestyle Counseling alone.

Smart Self-Assembling MagnetS for ENdoscopy (SAMSEN) for transoral endoscopic creation of immediate gastrojejunostomy (with video)

BACKGROUND Gastrojejunostomy is important for palliation of malignant gastric outlet obstruction and surgical obesity procedures. A less-invasive endoscopic technique for gastrojejunostomy creation is conceptually attractive. Our group has developed a compression anastomosis technology based on endoscopically delivered self-assembling magnets for endoscopy (SAMSEN) to create an instant, large-caliber gastrojejunostomy. OBJECTIVE To develop and evaluate an endoscopic means of gastrojejunostomy creation by using SAMSEN. SETTING Developmental laboratory and animal facility. DESIGN Animal study and human cadaveric study. SUBJECTS Yorkshire pigs (7 cadaver, 5 acute); human (1 cadaver). INTERVENTIONS A transoral procedure for SAMSEN delivery was developed in porcine and human cadaver models. Subsequently, gastrojejunostomy creation by using SAMSEN was performed in 5 acute pigs. The endoscope was advanced into the peritoneal cavity through the gastrotomy, and a segment of the small bowel was grasped and pulled closer to the stomach. An enterotomy was created, and a custom overtube was advanced into the small bowel for deployment of the first magnetic assembly. Next, a reciprocal magnetic assembly was deployed in the stomach. The 2 magnetic systems were mated under fluoroscopic and endoscopic guidance. Contrast studies assessed for gastrojejunostomy leak. Immediate necropsies were performed. MAIN OUTCOME MEASUREMENTS Technical feasibility and complications. RESULTS Gastrojejunostomy creation by using SAMSEN was successful in all 5 animals. Deep enteroscopy was performed through the stoma without difficulty. No leaks were identified on contrast evaluation. At necropsy, the magnets were properly deployed and robustly coupled together, resistant to vigorous tissue manipulation. LIMITATIONS Acute animal study. CONCLUSIONS Endoscopic creation of immediate gastrojejunostomy by using SAMSEN is technically feasible.

Magnetic retraction in natural-orifice transluminal endoscopic surgery (NOTES): addressing the problem of traction and countertraction

BACKGROUND AND STUDY AIMS Because of their reliance on the flexible endoscope, most current procedures in natural-orifice transluminal endoscopic surgery (NOTES) suffer from the inability to vigorously grasp and move tissue or to retract organs. We aimed to assess the use of internal and external magnets that might allow the vigorous multiaxial traction/countertraction required in more complicated NOTES procedures. METHODS Ex vivo and in vivo porcine model. Study components were: (1) Evaluation of force-distance relationship of this magnetic retraction system using a digital tensiometer. (2) Application of this magnetic retraction system to two procedures in the porcine model: (a) Liver retraction during transcolonic cholecystectomy in five nonsurvival pigs. Procedure time was recorded and compared to historical controls. (b) Mesh positioning for implantation into the anterior abdominal wall for ventral hernia repair in three survival pigs. RESULTS Over a distance of 5 cm to 0.25 cm, the magnetic force of our system increased from 3 to 90 gramforce (29.42 to 882.60 mN.) In vivo, the magnet system provided robust liver retraction, shortening the procedure time of NOTES cholecystectomy from a historical mean of 68 minutes (range 42 - 90 minutes; n = 5) to 49.6 minutes (range 33 - 61 minutes; n = 3). The magnetic system also greatly enhanced mesh positioning and stability, and these animals survived for 2 weeks without complications. CONCLUSIONS Our basic system provided critical liver retraction during NOTES cholecystectomy and was also instrumental in moving and stabilizing mesh for implantation during NOTES ventral hernia repair. Magnets can potentially provide the vigorous traction and countertraction required to advance NOTES procedures.

Randomized sham-controlled trial evaluating efficacy and safety of endoscopic gastric plication for primary obesity: The ESSENTIAL trial

Evaluate safety and efficacy of the pose™ procedure for obesity treatment.

Endoscopic foreign body removal for treatment of chronic abdominal pain in patients after Roux-en-Y gastric bypass

BACKGROUND Common endoscopic findings in patients who have undergone Roux-en-Y gastric bypass (RYGB) with chronic abdominal pain have included marginal ulceration, gastrogastric fistula, and jejunal erosion. However, suture or staples eroding into the gastric pouch can also contribute to abdominal pain. Redundant suture is typically regarded as a normal part of the postoperative anatomy. The objectives of the present study were to assess the effects of endoscopic foreign body removal of partially exposed sutures and staples in post-RYGB patients with chronic abdominal pain at a university hospital in the United States. METHODS We performed a retrospective study of consecutive patients from January 2006 to July 2007. Post-RYGB patients with chronic abdominal pain underwent endoscopic foreign body removal of exposed sutures/staples. Pain scores were obtained before the procedure, immediately after the procedure, and at the telephone follow-up (median 7.2 months). RESULTS Of 21 patients, 15 (71%) reported immediate symptomatic improvement. Specific endoscopic accessories were found to be more useful than others in managing the various foreign materials. Of the 21 patients, 15 (71%) were available for telephone follow-up. Of these 15 patients, 13 (87%) reported continued symptomatic improvement, with 9 (60%) reporting complete pain resolution and 4 (27%) reporting partial improvement. Eroded foreign material was seen in association with marginal ulcers in 3 patients (14%), gastritis in 7 patients (33%), and an inflammatory polyp in 1 patient (5%). CONCLUSIONS Eroded suture and staples can cause chronic abdominal pain in post-RYGB patients. In symptomatic patients, visible suture or staples should be considered a potential etiology of chronic pain, instead of normal postoperative findings. Endoscopic foreign body removal might be of therapeutic benefit in these patients.