Management of suspected anastomotic leak after bariatric laparoscopic Roux-en-y gastric bypass.

BACKGROUND: Anastomotic leak is one of the most serious complications following bariatric laparoscopic Roux-en-Y gastric bypass (LRYGB), and associated with high morbidity rates and prolonged hospital stay. Timely management is of utmost importance for the clinical outcome. This study evaluated the approach to suspected leakage in a high-volume bariatric surgery unit. METHODS: All consecutive patients who underwent LRYGB performed by the same team of surgeons were registered prospectively in a clinical database from September 2005 to June 2012. Suspected leaks were identified based on either clinical suspicion and/or associated laboratory values, or by a complication severity grade of at least II using the Clavien-Dindo score. RESULTS: A total of 6030 patients underwent LRYGB during the study period. The leakage rate was 1·1 per cent (64 patients). Forty-five leaks (70 per cent) were treated surgically and 19 (30 per cent) conservatively. Eight (13 per cent) of 64 patients needed intensive care and the mortality rate was 3 per cent (2 of 64). Early leaks (developing in 5 days or fewer after LRYGB) were treated by suture of the defect in 20 of 22 patients and/or operative drainage in 13. Late leaks (after 5 days) were managed with operative drainage in 19 of 23 patients and insertion of a gastrostomy tube in 15. Patients who underwent surgical treatment early after the symptoms of leakage developed had a shorter hospital stay than those who had symptoms for more than 24 h before reoperation (12·5 versus 24·4 days respectively; P < 0·001). CONCLUSION: Clinical suspicion of an anastomotic leak should prompt an aggressive surgical approach without undue delay. Early operative treatment was associated with shorter hospital stay. Delays in treatment, including patient delay, after symptom development were associated with adverse outcomes.

High-volume bariatric surgery in a single center: safety, quality, cost-efficacy and teaching aspects in 2,000 consecutive cases.

BACKGROUND: Obesity surgery is the most effective treatment for morbid obesity and the fastest growing area in surgery. Laparoscopic Roux-en-Y gastric bypass (LRYGB) is the gold standard procedure in many countries. Optimization of the treatment process is important in order to keep the morbidity rate down and cost of treatment as low as possible. METHODS: In September 2005, we established a bariatric surgery program. Until December 2010, 2,000 patients underwent LRYGB. Clinical pathways were established, with focus on safety, fast-track methodology and training of surgeons. Time recordings from all parts of the treatment, as well as clinical outcome, were prospectively registered. RESULTS: Time consumption for the total procedure in the operating theater was reduced from 102 to 54 min (P < 0.001). With only 11 min turnover between patients, the total time for one patient has been reduced to 65 min, enabling us to perform six operations in a single operating theater during ordinary daytime. Early complication rate was 2.8%, and mean hospital stay was 2.3 days. We were able to double the patients treated in 2010 compared to 2007 with only 10% increase in staff. Three surgeons were trained during the period into fully qualified senior bariatric surgeons. CONCLUSIONS: Multimodal evidence-based care within the fast-track methodology and routine time recordings was successful in order to increase the production volumes and reduce costs, without compromising the safety or quality for the patients. This kind of approach may be transferred to other types of standardized surgery.

Disseminated tumour cells as a prognostic biomarker in colorectal cancer.

BACKGROUND: The study was performed to determine detection rate and prognostic relevance of disseminated tumour cells (DTC) in patients receiving curatively intended surgery for colorectal cancer (CRC). METHODS: The study population consisted of 235 patients with CRC prospectively recruited from five hospitals in the Oslo region. Bone marrow (BM) aspirates were collected at the time of surgery and the presence of DTC was determined by two immunological methods; immunomagnetic selection (using an anti-EpCAM antibody) and immunocytochemistry (using a pan-cytokeratin antibody). Associations between the presence of DTC and metastasis-free, disease-specific and overall survival were analysed using univariate and multivariate methods. RESULTS: Disseminated tumour cells were detected in 41 (17%) and 28 (12%) of the 235 examined BM samples by immunomagnetic selection and immunocytochemistry, respectively, with only five samples being positive with both methods. The presence of DTC was associated with adverse outcome (metastasis-free, disease-specific and overall survival) in univariate and multivariate analyses. CONCLUSION: The presence of DTC was associated with adverse prognosis in this cohort of patients curatively resected for CRC, suggesting that DTC detection still holds promise as a biomarker in CRC.

Transgenic peas (Pisum sativum) expressing polygalacturonase inhibiting protein from raspberry (Rubus idaeus) and stilbene synthase from grape (Vitis vinifera).

The pea (Pisum sativum L.) varieties Baroness (United Kingdome) and Baccara (France) were transformed via Agrobacterium tumefaciens-mediated gene transfer with pGPTV binary vectors containing the bar gene in combination with two different antifungal genes coding for polygalacturonase-inhibiting protein (PGIP) from raspberry (Rubus idaeus L.) driven by a double 35S promoter, or the stilbene synthase (Vst1) from grape (Vitis vinifera L.) driven by its own elicitor-inducible promoter. Transgenic lines were established and transgenes combined via conventional crossing. Resveratrol, produced by Vst1 transgenic plants, was detected using HPLC and the PGIP expression was determined in functional inhibition assays against fungal polygalacturonases. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated.

Use of a herbicide or lysine plus threonine for non-antibiotic selection of transgenic chickpea.

A desensitized aspartate kinase (AK) gene has been developed as a non-antibiotic selection marker for use in the production of transgenic chickpea ( Cicer arietinum L.). Transgenic shoots regenerated from embryo explants bombarded with the desensitized AK gene were selected on media containing two amino acids, lysine and threonine (LT). Approximately 15% of the putative transgenic shoots of vars. P-362 and P-1042 survived after 4 weeks of growth on MSB5 medium (MS mineral salts and B5 vitamins) containing 2 microM thidiazuron (TDZ) and 2 mM lysine and 2 m M threonine. These shoots were subsequently grown on MSB5 medium supplemented with 2 micro M TDZ and 5 mM lysine and 5 mM threonine, and nearly 1% continued to grow after 16 weeks of selection. A phosphinothricin (PPT) selection system for Agrobacterium-mediated chickpea transformation was also developed. Three varieties of chickpea, P-362, P-1042 and P-1043, were successfully used for Agrobacterium transformation. Following Agrobacterium infection, 3-8% of the regenerated shoots remained green and continued to grow on MSB5 medium supplemented with 2.5 mg l(-1 )PPT. Increasing the concentrations of PPT to 15 mg l(-1) reduced transgenic shoot production in P-362, P-1042 and P-1043 to 0.7%, 1.2% and 1.1%, respectively. Selected putatively transformed shoots of all three varieties were rooted and grown to maturity. Southern hybridization analysis revealed single as well as multiple integration of genes in selected transgenic lines. The level of AK activity detected in LT-selected plants was higher than that detected in the non-transformed control.

Transformation of apple ( Malus domestica Borkh.) with the stilbene synthase gene from grapevine ( Vitis vinifera L.) and a PGIP gene from kiwi ( Actinidia deliciosa).

The objective of the present research was to introduce genes with antifungal potential into the commercially important apple cvs. Elstar and Holsteiner Cox in order to establish resistance against fungal diseases. The gene encoding the stilbene synthase (Vst1) from Vitis vinifera L., responsible for the synthesis of the phytoalexin resveratrol in grapevine, and the gene for a polygalacturonase-inhibiting protein (PGIP) from kiwi ( Actinidia deliciosa) were transferred into Holsteiner Cox and Elstar via Agrobacterium tumefaciens-mediated transformation. A total of nine transgenic Holsteiner Cox clones and one transgenic E clone carrying the stilbene-synthase gene as well as three transgenic Holsteiner Cox lines harbouring the polygalacturonase-inhibiting protein from Kiwi were identified via polymerase chain reaction and Southern blot analysis. High performance liquid chromatography analysis revealed the accumulation of a resveratrol-derivate, a glycoside, in transgenic Vst1 plants.

Identification of AFLP and STS markers closely linked to the def locus in pea.

The recessive mutation of the def gene of pea (Pisum sativum L.) leads to the loss of the hilum, the abscission zone between the seed and the pod. Thereby, it reduces the free dispersal of the seeds through pod shattering. As a prerequisite for a gene isolation via a map-based cloning approach, bulked segregant analysis followed by single plant analyses of over 200 homozygous individuals of a population of 476 F2 plants derived from a cross between 'DGV' (def wild-type) and 'PF' (def mutant), were used to detect markers closely linked to the def locus. The AFLP technique in combination with silver staining was used to maximize numbers of reproducible marker loci. Fifteen AFLP loci showed a genetic distance less than 5 and two of them less than 1 centiMorgans (cM) to the gene of interest. AFLPs were converted into sequence tagged sites (STSs) and into a newly refined AFLP-based single locus marker named the 'sequence specified AFLP' (ssAFLP).

Cloning of a gene for an acyl-CoA dehydrogenase from Pisum sativum L. and purification and characterization of its product as an isovaleryl-CoA dehydrogenase.

Isovaleryl-CoA dehydrogenase (IVD, EC ) catalyzes the third step in the catabolism of leucine in mammals. Deficiency of this enzyme leads to the clinical disorder isovaleric acidemia. IVD has been purified and characterized from human and rat liver, and the x-ray crystallographic structure of purified recombinant human IVD has been reported. Nothing is known about IVD activity in plants, although cDNA clones from Arabidopsis thaliana and partial sequences from Gossypium hirsutum and Oryza sativa have been identified as putative IVDs based on sequence homology and immuno cross-reactivity. In this report we describe the identification and characterization of an IVD from pea, purification of the enzyme using a novel and rapid auxin affinity chromatography matrix, and cloning of the corresponding gene. At the amino acid level, pea IVD is 60% similar to human and rat IVD. The specific activity and abundance of plant IVD was found to be significantly lower than for its human counterpart and exhibits developmental regulation. Substrate specificity of the plant enzyme is similar to the human IVD, and it cross-reacts to anti-human IVD antibodies. Molecular modeling of the pea enzyme based on the structure of human IVD indicates a high degree of structural similarity among these enzymes. Glu-244, shown to function as the catalytic base in human IVD along with most of the amino acids that make up the acyl CoA binding pocket, is conserved in pea IVD. The genomic structure of the plant IVD gene consists of 13 exons and 12 introns, spanning approximately 4 kilobases, and the predicted RNA splicing sites exhibit the extended consensus sequence described for other plant genes.

Assignment of the auxin binding abilities of ABP44 in gel.

Several approaches were successfully performed to directly assign and characterize auxin binding of ABP44 in gel. The 44 kDa high affinity auxin binding protein ABP44 from pea was tested for its ability to bind 5-azido-[7-3H]-IAA in photoaffinity labeling experiments. Competition experiments with several auxin analogues confirm data published previously (Reinard and Jacobsen 1995). Critical reflections of the limitations of the method are also discussed. Immunostaining using the antibody D16 (Napier and Venis 1992), which is directed against the putative binding site of ABP1, revealed that ABP44's auxin binding site is at least partially related to the corresponding site of ABP1. Nevertheless, both proteins do not share any further immunological relationships. Our results with D16 recommend a careful reconsideration of data published by other authors. Furthermore, a 80 kDa, dimeric glutathione dependent formaldehyde dehydrogenase (FDH) from mung bean, described recently, was found to be different from ABP44. In contrast to the described FDH, ABP44 exhibited no FDH activity.

Transfer and mapping of the shoot-differentiation locus Shd1 in barley chromosome 2.

The locus Shd1, which we previously mapped to the long arm of chromosome 2 of Hordeum vulgare L., controls the differentiation of shoots from immature barley embryo callus. The locus has major effects and its action explains more than 65% of the total genetic variance in the shoot-differentiation rate. The allele of cultivar Kanto Nakate Gold designated Shd1K has a significant positive effect on the shoot-differentiation rate, whereas Shd1A of cultivar Azumamugi does not promote shoot differentiation. To identify gene products and characterize the function of Shd1, a set of near-isogenic lines is essential. In this study we produced BC5F1 plants by repeated backcrossing of 'Azumamugi' to F1 plants ('Azumamugi' x 'Kanto Nakate Gold'). The BC5F1 plants were examined for their RFLP genotype and for the shoot-differentiation ability of immature embryo-derived callus. The results indicated that the Shd1 locus was located in a chromosomal region between MWG2081 and MWG503 that flanks the MWG801, cMWG699, v (ear type), and MWG865 loci. Shd1K from 'Kanto Nakate Gold' functions effectively in the genetic background of 'Azumamugi', an indication that backcross breeding is possible for production of near-isogenic lines that would be very suitable for tissue culture.

Thidiazuron-induced high frequency of shoot induction and plant regeneration in protoplast derived pea callus.

Protoplasts isolated from lateral shoot buds of cotyledon-free pea embryo axes were regenerated to callus. Protoplast derived calluses with a diameter of about 1cm were transferred to shoot induction media, containing different concentrations (1-50µM) of thidiazuron. Shoot formation was observed after 16 weeks up to 12% efficiency. Thidiazuron (10µM) was the most effective concentration in all experiments. Shoot buds elongated in medium supplemented with N-isopentenyl adenine and indole-3-butyric acid. Since rooting was almost impossible in these thidiazuron-induced shoots, shoots were grafted onto young pea seedlings and regenerated to fertile plants.

Interspecific hybridization between common and tepary beans: increased hybrid embryo growth, fertility, and efficiency of hybridization through recurrent and congruity backcrossing.

Cultivated common bean (Phaseolus vulgaris L.) and tepary bean (Phaseolus acutifolius A. Gray) genotypes possessing desirable agronomic traits were hybridized. The F1 hybrids were backcrossed twice with the common bean (i.e., recurrent backcrossing). Also, alternate backcrosses with common and tepary beans (i.e., congruity backcrossing) were carried out. Embryo culture was necessary for all initial interspecific crosses, and its requirement was proportionally lower when the common bean was used as the recurrent parent and as the last parent of congruity backcrosses. Modification of the embryo culture technique was necessary to produce congruity hybrids. Effects of both tepary and common bean genotypes on the success rate of hybridization were observed. Tepary accession G 40001 and common bean cultivar ICA Pijao facilitated interspecies hybridization. Growth of hybrid embryos before rescue, recovery of mature hybrid plants, and the vigor and fertility of F1 hybrids all increased with increased recurrent and congruity backcrosses and intermatings between male-sterile F1 and selected fertile F2 plants of the third and fifth congruity backcrosses. Introgression of tepary genes was verified by means of seed protein electrophoretic analysis and morphological markers. The results suggest that congruity backcrossing can help to gradually reduce or overcome P. vulgaris x P. acutifolius hybridization barriers such as genotype incompatibility, early embryo abortion, hybrid sterility, and lower frequencies of hybridization.

Physiological and biochemical characterization of glyoxalase I, a general marker for cell proliferation, from a soybean cell suspension.

Using a strictly auxin-dependent soybean (Glycine max (L.) Merr.) cell suspension, we studied the correlation of auxin-dependent cell proliferation and the activity of glyoxalase I (S-lactoylglutathione-lyase EC 4.4.1.5), and enzyme generally associated with cell proliferation in animal, microbial and, as reported recently, also plant systems. We found the activity of glyoxalase I to be modulated during the proliferation cycle, with a maximal activity between day 2 and day 4 of culture growth. After starving the culture of auxins for three subsequent periods, both the enzyme activity and cell-growth could be re-initiated with auxin. Enzyme activity reached its maximum 1 d before cell number was at a maximum. The enzyme was purified to homogeneity and characterized.

Agrobacterium tumefaciens-mediated transformation of Pisum sativum L. using binary and cointegrate vectors.

Epicotyl segments and nodus expiants from etiolated seedlings of Pisum sativum were transformed using Agrobacterium tumefaciens strains GV 2260 (p35S GUS INT) and GV 3850 HPT carrying either a neomycin- or hygromycinphosphotransferase-gene as selectable markers. The transgenic character of hygromycin- or kananamycin-resistant tissue was confirmed by detection of nopaline or neomycinphosphotransferase-II- and ß-glucuronidase activity in crude extracts of resistant tissues. Up to 5 % of developing shoots from shoot proliferating nodi were regenerated via organogenesis to kanamycin-resistant plantlets. Transformation frequency in vitro was found to be influenced by expiant source, A. tumefaciens strain, pea genotype and duration of cocultivation. Acetosyringone did not increase the transformation rate.

An inexpensive small volume equilibrium dialysis system for protein-ligand binding assays.

A simple and inexpensive system for equilibrium dialysis-based binding assays has been developed employing 1.5-ml microtubes. The main advantages are small volume of test buffers, no time-consuming assembly of the test vials, and inexpensive test cells. A comparison of the results derived from this equilibrium dialysis system with those of polyethylenimine-filtration assay and those of a commercially available equilibrium dialysis test system (Diachema from Dianorm) shows good agreement.

Plant regeneration from pea protoplasts via somatic embyogenesis.

Plant regeneration via somatic embryogenesis was obtained from pea protoplasts. Strong auxins (picloram or 2.4-D) and increased osmolarity of the medium were necessary for embryo induction. Relatively high amounts of embryogenic calli could be obtained in 2 genotypes. After a period on hormone-free medium, a second induction of somatic embryos was possible. Further development of somatic embryos was accomplished on GA3 - containing medium.

Modulation of soluble auxin-binding proteins in soybean cell suspensions.

Rapidly growing cell suspensions of soybean were analyzed for the presence of cytoplasmic high-affinity binding sites for auxin. Cytosol preparations were studied in lag, log and early stationary phase of the growth cycle. Two binding sites were detected, which show some similarities with binding sites previously reported from etiolated pea epicotyls. While the number of both sites declined in the cytoplasm during the growth cycle, the number of one of the two sites increased at the onset of rapid cell divisions. In parallel, both sites exhibited an increase in binding affinity during the growth cycle. The data will be discussed in relation to other reports on soluble auxin binding as well as to possible physiological functions.

Marker proteins for embryogenic differentiation patterns in pea callus.

Polypeptide pattern alterations during somatic embryogenesis were investigated using callus cultures of two Pisum sativum genotypes. Both genotypes show the formation of two different callus lines from the same explant after six to eight weeks in culture: a nodular yellowish callus line, which forms somatic embryoids in suspension cultures (e(+)) and a white compact callus line with no regenerative capacity (e(-)). The cytosol proteins of the two different callus lines were separated in a semi-preparative two-dimensional system and the polypeptide patterns were compared. Two protein bands were found (P1: Mr=45000 D, pI=7.0-7.1; P2: Mr=7000 D, pI=<4.5), which were characteristic of the putatively embryogenic (e(+)) callus line in all tissues investigated (two genotypes × two explant sources). These proteins found in nodular (e(+)) pea cultures are very similar to two proteins found in Daucus carota suspension cultures preceding the formation of somatic embryos.

Plant regeneration via somatic embryogenesis in pea (Pisum sativum L.).

Whole plant regeneration via somatic embryogenesis was obtained in pea (Pisum sativum L.) using explants from immature embryos or shoot apex segments. The induction of somatic embryos required picloram or 2,4-D. Germination of fully-developed embryos was accomplished by subculture on medium with only cytokinin and then on medium supplemented with cytokinins in combination with a reduced auxin concentration. Plantlets obtained from both zygotic embryos and shoot apices were transferred to soil and were grown to maturity. Nine plants were examined cytologically, revealing three tetraploids (2n=4x=28) and six diploids (2n=2x=14).