Evaluation of an endoscopic suturing device for transoral outlet reduction in patients with weight regain following Roux-en-Y gastric bypass.

BACKGROUND AND STUDY AIMS: A dilated gastrojejunal anastomosis (GJA) is thought to be associated with weight regain in patients with Roux-en-Y gastric bypass (RYGB). Due to a high rate of perioperative morbidity, surgical revision is not generally performed. The aim of this study was to assess the technical feasibility, safety, and early outcomes of a procedure using a commercially available endoscopic suturing device to reduce the diameter of the GJA. PATIENTS AND METHODS: This was a retrospective analysis of 25 consecutive patients who underwent transoral outlet reduction (TORe) for dilated GJA and weight regain. An endoscopic suturing device was used to place sutures at the margin of the GJA in order to reduce its aperture. On chart review, clinical data were available at 3, 6, and 12 months. RESULTS: Patients had regained a mean of 24 kg from their weight loss nadir and had a mean body mass index of 43 kg/m2 at the time of endoscopic revision. Average anastomosis diameter was 26.4 mm. Technical success was achieved in all patients (100 %) with a mean reduction in anastomosis diameter to 6 mm (range 3 - 10 mm), representing a 77.3 % reduction. The mean weight loss in successful cases was 11.5 kg, 11.7 kg, and 10.8 kg at 3, 6, and 12 months, respectively. There were no major complications. CONCLUSION: This case series demonstrated the technical feasibility, safety, and efficacy of performing gastrojejunostomy reduction using a commercially available endoscopic suturing device. This technique may represent an effective and minimally invasive option for the management of weight regain in patients with RYGB.

Real-time computed tomography-based augmented reality for natural orifice transluminal endoscopic surgery navigation.

BACKGROUND: Natural orifice transluminal endoscopic surgery (NOTES) is technically challenging owing to endoscopic short-sighted visualization, excessive scope flexibility and lack of adequate instrumentation. Augmented reality may overcome these difficulties. This study tested whether an image registration system for NOTES procedures (IR-NOTES) can facilitate navigation. METHODS: In three human cadavers 15 intra-abdominal organs were targeted endoscopically with and without IR-NOTES via both transgastric and transcolonic routes, by three endoscopists with different levels of expertise. Ease of navigation was evaluated objectively by kinematic analysis, and navigation complexity was determined by creating an organ access complexity score based on the same data. RESULTS: Without IR-NOTES, 21 (11·7 per cent) of 180 targets were not reached (expert endoscopist 3, advanced 7, intermediate 11), compared with one (1 per cent) of 90 with IR-NOTES (intermediate endoscopist) (P = 0·002). Endoscope movements were significantly less complex in eight of the 15 listed organs when using IR-NOTES. The most complex areas to access were the pelvis and left upper quadrant, independently of the access route. The most difficult organs to access were the spleen (5 failed attempts; 3 of 7 kinematic variables significantly improved) and rectum (4 failed attempts; 5 of 7 kinematic variables significantly improved). The time needed to access the rectum through a transgastric approach was 206·3 s without and 54·9 s with IR-NOTES (P = 0·027). CONCLUSION: The IR-NOTES system enhanced both navigation efficacy and ease of intra-abdominal NOTES exploration for operators of all levels. The system rendered some organs accessible to non-expert operators, thereby reducing one impediment to NOTES procedures.

Marginal ulceration after Roux-en-Y gastric bypass surgery: characteristics, risk factors, treatment, and outcomes.

BACKGROUND AND STUDY AIMS: Marginal ulcers are one of the most common complications after gastric bypass. Reported incidence varies widely (0.6-16 %) and pathogenesis is unclear. The aim of the present study was to describe characteristics, risk factors, management, and outcomes of endoscopically documented ulcers. PATIENTS AND METHODS: Data from all patients diagnosed with marginal ulcers at endoscopy between 2003 and 2010 were retrospectively reviewed. RESULTS: A total of 103 patients with marginal ulcers presented with pain (63 %) and/or bleeding (24 %), a median of 22 months after surgery. Ulcers were located on the anastomosis (50 %) or the jejunum (40 %); sutures were visible in 35 %, and gastrogastric fistulae in 8 %. The mean pouch length was 5.6 cm. Diabetes (odds ratio [OR] 2.5; P = 0.03), smoking (OR 2.5; P = 0.02), and gastric pouch length (OR 1.2; P = 0.02) were significantly associated with marginal ulcer formation on univariate analysis; diabetes was significantly associated on multivariate analysis (OR 5.6; P = 0.003). The risk of developing a marginal ulcer decreased with time (OR 0.8; P < 0.01) and was not associated with the use of nonsteroidal anti-inflammatory drugs. At first endoscopic follow-up, 67 % of ulcers had healed. Recurrence occurred in four patients and nine patients required surgical revision. CONCLUSIONS: The vast majority of marginal ulcers had a favorable outcome after medical treatment. However, 9 % of patients eventually required surgical revision. Therefore, endoscopic follow-up is essential. Diabetes, smoking, and long gastric pouches were significant risk factors for marginal ulcer formation, suggesting increased acid exposure and mucosal ischemia are both involved in marginal ulcer pathogenesis. Management of these factors may prove effective in managing marginal ulcers, and tailoring postoperative proton pump inhibitor therapy to patients with multiple risk factors could be effective.

NOTES spin-off for the therapeutic gastroenterologist: natural orifice surgery.

Natural orifice transluminal endoscopic surgery (NOTES) has ushered in a new era in flexible endoscopy. Over the past decade, modest advances have been made in addressing the fundamental challenges of NOTES surgery including transluminal access and closure techniques, and advancement of NOTES-specific technology. Despite these encouraging initial efforts significant obstacles to widespread acceptance of NOTES as a surgical option persist. Moreover, due to the well-documented safety and efficacy of laparoscopic techniques, the question remains as to the best candidate NOTES procedure. Presently, interest has shifted from true NOTES to hybrid procedures and single incision laparoscopic surgery, due to the lure of more immediate success. Additionally, there is also a growing awareness of the potential applications of natural orifice surgery techniques to the present field of therapeutic endoscopy. Research into transluminal access and closure has born several techniques and devices that are now being explored in endoscopic procedures such as full-thickness resection, endoscopic myotomy, direct endoscopic pancreatic necrosectomy and bariatric endoscopy. Such NOTES "spin-off" procedures are expanding the armamentarium of today's therapeutic endoscopists, and a growing body of literature suggests that they will play a significant role in the evolution of therapeutic endoscopy in the future.

Image Registered Gastroscopic Ultrasound (IRGUS) in human subjects: a pilot study to assess feasibility.

BACKGROUND AND STUDY AIMS: Endoscopic ultrasound (EUS) is a complex procedure due to the subtleties of ultrasound interpretation, the small field of observation, and the uncertainty of probe position and orientation. Animal studies demonstrated that Image Registered Gastroscopic Ultrasound (IRGUS) is feasible and may be superior to conventional EUS in efficiency and image interpretation. This study explores whether these attributes of IRGUS will be evident in human subjects, with the aim of assessing the feasibility, effectiveness, and efficiency of IRGUS in patients with suspected pancreatic lesions. PATIENTS AND METHODS: This was a prospective feasibility study at a tertiary care academic medical center in human patients with pancreatic lesions on computed tomography (CT) scan. Patients who were scheduled to undergo conventional EUS were randomly chosen to undergo their procedure with IRGUS. Main outcome measures included feasibility, ease of use, system function, validated task load (TLX) assessment instrument, and IRGUS experience questionnaire. RESULTS: Five patients underwent IRGUS without complication. Localization of pancreatic lesions was accomplished efficiently and accurately (TLX temporal demand 3.7 %; TLX effort 8.6 %). Image synchronization and registration was accomplished in real time without procedure delay. The mean assessment score for endoscopist experience with IRGUS was positive (66.6 ± 29.4). Real-time display of CT images in the EUS plane and echoendoscope orientation were the most beneficial characteristics. CONCLUSIONS: IRGUS appears feasible and safe in human subjects, and efficient and accurate at identification of probe position and image interpretation. IRGUS has the potential to broaden the adoption of EUS techniques and shorten EUS learning curves. Clinical studies comparing IRGUS with conventional EUS are ongoing.

The role of a computed tomography-based image registered navigation system for natural orifice transluminal endoscopic surgery: a comparative study in a porcine model.

BACKGROUND AND STUDY AIMS: Most natural orifice transluminal endoscopic surgery (NOTES) procedures have been performed in animal models through the anterior stomach wall, but this approach does not provide efficient access to all anatomic areas of interest. Moreover, injury of the adjacent structures has been reported when using a blind access. The aim of the current study was to assess the utility of a CT-based (CT: computed tomography) image registered navigation system in identifying safe gastrointestinal access sites for NOTES and identifying intraperitoneal structures. METHODS: A total of 30 access procedures were performed in 30 pigs: anterior gastric wall (n = 10), posterior gastric wall (n = 10), and anterior rectal wall (n = 10). Of these, 15 procedures used image registered guidance (IR-NOTES) and 15 procedures used a blind access (NOTES only). Timed abdominal exploration was performed with identification of 11 organs. The location of the endoscopic tip was tracked using an electromagnetic tracking system and was recorded for each case. Necropsy was performed immediately after the procedure. The primary outcome was the rate of complications; secondary outcome variables were number of organs identified and kinematic measurements. RESULTS: A total of 30 animals weighting a mean (± SD) of 30.2 ± 6.8 kg were included in the study. The incision point was correctly placed in 11 out of 15 animals in each group (73.3 %). The mean peritoneoscopy time and the number of properly identified organs were equivalent in the two groups. There were eight minor complications (26.7 %), two (13.3 %) in the IR-NOTES group and six (40.0 %) in the NOTES only group ( P = n. s.). Characteristics of the endoscope tip path showed a statistically significant improvement in trajectory smoothness of motion for all organs in the IR-NOTES group. CONCLUSION: The image registered system appears to be feasible in NOTES procedures and results from this study suggest that image registered guidance might be useful for supporting navigation with an increased smoothness of motion.

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.

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 laboratory's 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.

Transcolonic ventral wall hernia mesh fixation in a porcine model.

BACKGROUND AND STUDY AIMS: A natural orifice transluminal endoscopic surgery (NOTES) approach to ventral wall hernia repair may represent a potentially less invasive alternative to current transabdominal surgical techniques. We aimed to investigate the feasibility of using transcolonic NOTES to deliver hernia repair mesh into the peritoneal cavity, as well as the ability to manipulate composite mesh and secure it to the abdominal wall. METHODS: Five pigs weighing 20 to 25 kg were used in this feasibility study involving two acute and three survival experiments. A prototype mesh delivery system was used to transfer 1.5 - 2-cm x 2.5 - 3-cm pieces of composite hernia mesh into the peritoneal cavity. Neodymium magnets on a prototype control arm were used to help position the mesh by magnetically engaging previously placed endoscopic clips. Transfascial fixation of the mesh with 3-0 monofilament sutures was achieved using a 19-gauge hollow needle, pusher wire, and a suture tag system. RESULTS: Composite hernia mesh was successfully transferred and secured in 5/5 attempts. All three survival animals thrived for 14 days prior to elective sacrifice. At necropsy, the mesh sites were found to be well peritonealized without adhesions. Suture placement through the posterior fascia was confirmed in 10/12 sutures. Of these, four sutures were within the abdominal musculature, and two sutures were through the anterior fascia (transfascial). CONCLUSIONS: Transcolonic delivery, transcutaneous magnetic manipulation, and fixation of composite hernia mesh are technically feasible in a porcine model with animal survival. An effective suturing method that allows consistent anchoring through the anterior fascia would be preferred and may require the development of novel devices.

Dual-port distal pancreatectomy using a prototype endoscope and endoscopic stapler: a natural orifice transluminal endoscopic surgery (NOTES) survival study in a porcine model.

BACKGROUND AND STUDY AIMS: Natural orifice transluminal endoscopic surgery (NOTES) is a potentially less invasive alternative to laparoscopic surgery that may be applicable to distal pancreatectomy. We aimed to demonstrate the technical feasibility of a NOTES distal pancreatectomy in an in vivo porcine model via a combined transvaginal-transcolonic approach. MATERIAL AND METHODS: The procedure was performed in five female Yorkshire pigs weighing approximately 30 kg each. A prototype endoscope ("R-scope"), advanced into the peritoneal cavity through an anterior colotomy, and a computer-assisted linear stapler, introduced transvaginally, were used in dissection and resection of the distal pancreas. Prone positioning was used to enhance retroperitoneal exposure. Pneumodissection was used for blunt dissection. The colotomies were closed with endoloops. Necropsies were done immediately after the procedure in the first three animals, and after 2 weeks' survival in the final two animals. RESULTS: Distal pancreatectomy was successful in all five animals. Prone positioning was critically important for proper exposure of retroperitoneal and pelvic structures. Pneumodissection was effective for blunt dissection, and both the linear stapler and R-scope functioned smoothly. Transvaginal and transcolonic access provided similar intraperitoneal views, and the dual-lumen approach enhanced triangulation. Both survival animals thrived postoperatively. Necropsies revealed clean staple lines; closed transcolonic and transvaginal incisions; and absence of infection, hemorrhage, or fluid collections. CONCLUSIONS: NOTES distal pancreatectomy is technically feasible in the porcine model. The transvaginal approach provides a vantage point very similar to that of the transcolonic route and holds promise as a NOTES access site, either singly or as part of a dual-lumen approach. The endoscopic linear stapler and R-scope both advance NOTES capabilities. The novel concepts of fully prone positioning, pneumodissection, and endoloop colotomy closures are introduced. Considering anatomical differences and that healthy animals were used, transferring this technique to patients with pancreatic disease might be difficult and further modifications would likely be needed.

Pancreatic cancer cells overexpress gelsolin family-capping proteins, which contribute to their cell motility.

BACKGROUND: Previously, proteomic methods were applied to characterise differentially expressed proteins in microdissected pancreatic ductal adenocarcinoma cells. AIMS: To report that CapG and a related protein, gelsolin, which have established roles in cell motility, are overexpressed in metastatic pancreatic cancer; and to describe their pattern of expression in pancreatic cancer tissue and their effect on cell motility in pancreatic cancer cell lines. METHODS: CapG was identified by mass spectrometry and immunoblotting. CapG and gelsolin expression was assessed by immunohistochemical analysis on a pancreatic cancer tissue microarray and correlated with clinical and pathological parameters. CapG and gelsolin levels were reduced using RNA interface in Suit-2, Panc-1 and MiaPaCa-2 cells. Cell motility was assessed using modified Boyden chamber or wound-healing assays. RESULTS: Multiple isoforms of CapG were detected in pancreatic cancer tissue and cell lines. Immunohistochemical analysis of benign (n = 44 patients) and malignant (n = 69) pancreatic ductal cells showed significantly higher CapG staining intensity in nuclear (p<0.001) and cytoplasmic (p<0.001) compartments of malignant cells. Similarly, gelsolin immunostaining of benign (n = 24 patients) and malignant (n = 68 patients) pancreatic ductal cells showed higher expression in both compartments (both p<0.001). High nuclear CapG was associated with increased tumour size (p = 0.001). High nuclear gelsolin was associated with reduced survival (p = 0.01). Reduction of CapG or gelsolin expression in cell lines by RNAi was accompanied by significantly impaired motility. CONCLUSIONS: Up regulation of these actin-capping proteins in pancreatic cancer and their ability to modulate cell motility in vitro suggest their potentially important role in pancreatic cancer cell motility and consequently dissemination.

Evaluating an optimal gastric closure method for transgastric surgery.

BACKGROUND: The transgastric approach is currently being studied as a potentially less invasive alternative to conventional laparoscopy for intra-abdominal surgery. A major obstacle to overcome is the closure of the transgastric incision in a rapid, reproducible, and safe manner. The effectiveness of various techniques for gastrotomy closure were compared by assessing leak pressures in an ex vivo porcine stomach model. METHODS: Whole stomachs from adult white pigs were suspended in a Plexiglas box to facilitate endoscopic technique. Standard gastrotomies were made by needle knife incision and dilation with a controlled radial expansion (CRE) balloon. The first arm used standard QuickClips; the second, a prototype device developed by LSI Solutions; the third, hand-sewn by a senior surgeon; the final, a control with open gastrotomy. Five stomachs were tested per study arm. After closure, each stomach was inflated by an automated pressure gauge. The pressures to achieve air leakage and liquid leakage were recorded. RESULTS: The unclosed controls demonstrated air leakage at a median pressure of 15 mmHg, representing baseline system resistance. The QuickClip closures leaked air at a median pressure of 33 mmHg. The prototype gastrotomy device yielded the highest median air leak pressure of 85 mmHg while dramatically diminishing time for incision and gastrotomy closure to approximately 5 min. The hand-sewn closures leaked air at a median pressure of 47 mmHg. Using Kruskal-Wallis statistical analysis, the comparisons were significant (p = 0.0019). Post hoc paired comparisons using MULTTEST procedure with both Bonferroni and bootstrap adjustments revealed that the difference between prototype and clips was significant; prototype versus hand-sewn was not. Liquid-leak pressures produced similar results. CONCLUSIONS: The prototype device decreases procedure time and yields leak-resistant gastrotomy closures that are superior to clips and rival hand-sewn interrupted stitches.

Peroral endoscopic reduction of dilated gastrojejunal anastomosis after Roux-en-Y gastric bypass: a possible new option for patients with weight regain.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) is an effective treatment for severe obesity. However, many patients regain weight over time. The mechanisms for this are unclear, and several factors may contribute, including dilation of the gastrojejunal anastomosis. This study aimed to assess the feasibility of endoscopic gastrojejunal anastomotic tightening and to determine the effect of tightening on body weight. METHODS: Eight patients with significant weight regain and dilated gastrojejunal anastomosis after RYGB were included in this pilot study. Sutures were placed endoscopically at the rim of the anastomosis. When tightened, the sutures formed tissue placations, reducing the size of the anastomotic aperture. RESULTS: The average preprocedure body mass index (BMI) was 40.5, and the patients had regained a mean of 24 kg from their post-RYGB nadir. The average pouch length was 5.7 cm, and the average anastomotic diameter was 25 mm. The average postreduction diameter was 10.0 mm (68% reduction). Six of the eight patients showed weight loss (mean, 10 kg) at 4 months. Repeat procedures were performed for three patients who had lost 4, 5, and 9 kg, respectively with the initial procedure. After the second anastomotic reduction, the final diameters were, respectively, 14, 5, and 5 mm. The first patient did not have further weight loss. The remaining two patients showed a total weight loss of 19 and 20 kg, respectively, at 5 months. All 11 reductions were accomplished without significant complication. The average postreduction BMI was 37.7, and the percentage of excess weight loss was 23.4%. CONCLUSION: Peroral endoscopic suturing to tighten dilated gastrojejunal anastomoses appears technically feasible and safe. This procedure is associated with variable but significant weight loss, and preliminary results suggest that it may offer a new treatment option for postbypass weight regain in selected patients.

Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis.

We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.

Photobacterium rosenbergii sp. nov. and Enterovibrio coralii sp. nov., vibrios associated with coral bleaching.

Six new Vibrio-like isolates originating from different species of bleached and healthy corals around Magnetic Island (Australia) were investigated using a polyphasic approach. Phylogenetic analyses based on 16S rRNA, recA and rpoA gene sequences split the isolates in two new groups. Strains LMG 22223(T), LMG 22224, LMG 22225, LMG 22226 and LMG 22227 were phylogenetic neighbours of Photobacterium leiognathi LMG 4228(T) (95.6 % 16S rRNA gene sequence similarity), whereas strain LMG 22228(T) was related to Enterovibrio norvegicus LMG 19839(T) (95.5 % 16S rRNA gene sequence similarity). The two new groups can be distinguished from closely related species on the basis of several phenotypic features, including fermentation of d-mannitol, melibiose and sucrose, and utilization of different compounds as carbon sources, arginine dihydrolase activity, nitrate reduction, resistance to the vibriostatic agent O/129 and the presence of fatty acids 15 : 0 iso and 17 : 0 iso. The names Photobacterium rosenbergii sp. nov. (type strain LMG 22223(T)=CBMAI 622(T)=CC1(T)) and Enterovibrio coralii sp. nov. (type strain LMG 22228(T)=CBMAI 623(T)=CC17(T)) are proposed to accommodate these new isolates. The G+C contents of the DNA of the two type strains are respectively 47.6 and 48.2 mol%.

Use of recA as an alternative phylogenetic marker in the family Vibrionaceae.

This study analysed the usefulness of recA gene sequences as an alternative phylogenetic and/or identification marker for vibrios. The recA sequences suggest that the genus Vibrio is polyphyletic. The high heterogeneity observed within vibrios was congruent with former polyphasic taxonomic studies on this group. Photobacterium species clustered together and apparently nested within vibrios, while Grimontia hollisae was apart from other vibrios. Within the vibrios, Vibrio cholerae and Vibrio mimicus clustered apart from the other genus members. Vibrio harveyi- and Vibrio splendidus-related species formed compact separated groups. On the other hand, species related to Vibrio tubiashii appeared scattered in the phylogenetic tree. The pairs Vibrio coralliilyticus and Vibrio neptunius, Vibrio nereis and Vibrio xuii and V. tubiashii and Vibrio brasiliensis clustered completely apart from each other. There was a correlation of 0.58 between recA and 16S rDNA pairwise similarities. Strains of the same species have at least 94 % recA sequence similarity. recA gene sequences are much more discriminatory than 16S rDNA. For 16S rDNA similarity values above 98 % there was a wide range of recA similarities, from 83 to 99 %.

Vibrio kanaloae sp. nov., Vibrio pomeroyi sp. nov. and Vibrio chagasii sp. nov., from sea water and marine animals.

The taxonomic position of the fluorescent amplified fragment length polymorphism fingerprinting groups A46 (five isolates), A51 (six isolates), A52 (five isolates) and A53 (seven isolates) obtained in a previous study were further analysed through a polyphasic approach. The 23 isolates were phylogenetically related to Vibrio splendidus, but DNA-DNA hybridization experiments proved that they belong to three novel species. Chemotaxonomic and phenotypic analyses further disclosed several features that differentiate between the 23 isolates and known Vibrio species. The names Vibrio kanaloae sp. nov. (type strain LMG 20539(T) = CAIM 485(T); EMBL accession no. AJ316193; G + C content 44.7 mol%), Vibrio pomeroyi sp. nov. (type strain LMG 20537(T) = CAIM 578(T); EMBL accession no. AJ491290; G +C content 44.1 mol%) and Vibrio chagasii sp. nov. (type strain LMG 21353(T) = CAIM 431(T); EMBL accession no. AJ316199; G + C content 44.6 mol%) are respectively proposed to encompass the five isolates of A46, the six isolates of A51 and the 12 isolates of A52/A53. The three novel species can be distinguished from known Vibrio species by several phenotypic features, including utilization and fermentation of various carbon sources, beta-galactosidase activity and fatty acid content (particularly of 12 : 0, 14: 0, 14 : 0 iso and 16 : 0 iso).

Vibrio tasmaniensis sp. nov., isolated from Atlantic salmon (Salmo salar L.).

We describe the polyphasic characterization of four Vibrio isolates which formed a tight AFLP group in a former study. The group was closely related to V. cyclitrophicus, V. lentus and V. splendidus (98.2-98.9% similarity) on the basis of the 16S rDNA sequence analysis, but by DNA-DNA hybridisation experiments it had at maximum 61% DNA similarity towards V. splendidus. Thus, we propose that the isolates represent a new Vibrio species i.e. V. tasmaniensis (LMG 20012T; EMBL under the accession numbers AJ316192; mol% G+C of DNA of the type strain is 44.7). Useful phenotypical features for discrimination of V. tasmaniensis from other Vibrio species include gelatinase and beta-galactosidase activity, fatty acid composition (particularly 14:0), utilisation and fermentation of different compounds (e.g. sucrose, melibiose and D-galactose) as sole carbon source.

Enterovibrio norvegicus gen. nov., sp. nov., isolated from the gut of turbot (Scophthalmus maximus) larvae: a new member of the family Vibrionaceae.

Twenty-two isolates originating from the gut of healthy cultured turbot larvae in Norway were investigated using a polyphasic approach. Amplified fragment length polymorphism fingerprinting analysis showed that the isolates have typical patterns and form two main groups. Phylogenetic analysis revealed that the isolates belong to the gamma-Proteobacteria, with Vibrio hollisae as their closest neighbour. DNA-DNA hybridization, chemotaxonomic and phenotypic analyses further proved that these isolates represent a tight novel taxon that differs from currently described species in the family Vibrionaceae. It is proposed that these novel isolates be accommodated in a new genus, Enterovibrio gen. nov., with Enterovibrio norvegicus sp. nov. as the type species. Isolates were motile by a polar flagellum, positive for oxidase, catalase, arginine dihydrolase and beta-galactosidase, but negative for the Voges-Proskauer reaction. They produced indole, did not reduce nitrate and were resistant to the vibriostatic agent O/129. The DNA G+C content of E. norvegicus was 47.1-47.9 mol%. The type strain is E. norvegicus LMG 19839(T) (= CAIM 430(T)).

Late G1 accumulation after 2 Gy of gamma-irradiation is related to endogenous Raf-1 protein expression and intrinsic radiosensitivity in human cells.

We have previously reported a correlation between high endogenous expression of the protein product of the RAF-1 proto-oncogene, intrinsic cellular radiosensitivity and rapid exit from a G2/M delay induced by 2 Gy of gamma-irradiation. Raf1 is a positive serine/threonine kinase signal transduction factor that relays signals from the cell membrane to the MAP kinase system further downstream and is believed to be involved in an ionizing radiation signal transduction pathway modulating the G1/S checkpoint. We therefore extended our flow cytometric studies to investigate relationships between radiosensitivity, endogenous expression of the Raf1 protein and perturbation of cell cycle checkpoints, leading to alterations in the G1, S and G2/M populations after 2 Gy of gamma-irradiation. Differences in intrinsic radiosensitivity after modulation of the G1/S checkpoint have generally been understood to involve p53 function up to the present time. A role for dominant oncogenes in control of G1/S transit in radiation-treated cells has not been identified previously. Here, we show in 12 human in vitro cancer cell lines that late G1 accumulation after 2 Gy of radiation is related to both Raf1 expression (r = 0.91, P = 0.0001) and the radiosensitivity parameter SF2 (r = -0.71, P = 0.009).