Perioperative humoral stress response to laparoscopic versus open aortobifemoral bypass surgery.

Minimally invasive surgery seems to reduce hormonal stress response to surgery, but has not previously been examined in major abdominal vascular surgery. Aortic cross-clamping time and operation time is known to be longer in the totally laparoscopic aortobifemoral bypass (LABF) as compared to open aortobifemoral bypass (OABF). The main objective of our study was to measure the hormonal stress response during surgery and aortic cross-clamping in patients undergoing a totally laparoscopic versus an open aortobifemoral bypass. This was a sub-study of a larger randomized controlled multicentre trial. Thirty consecutive patients with severe aortoiliac occlusive disease were randomized to either a laparoscopic (LABF) or an open (OABF) procedure. The surgical stress response was measured by perioperative monitoring of adrenocorticotropic hormone (ACTH), aldosterone, metanephrine and cortisol at eight different time-points. During surgery. there was an increase in all humoral stress markers in both groups. The analysis of covariance showed increased levels of cortisol and ACTH in open group at 24 h time-point as compared to the baseline and this difference was statistically significant between the two groups, which indicate an earlier return to baseline levels in the laparoscopic group. Results from the General Estimated Equations (GEE) model indicate that LABF generates a lower level of metanephrine and higher level of aldosterone as compared to OABF. In conclusion, although they have higher levels of ACTH, aldosterone and cortisol during surgery, the patients operated with a laparoscopic aortobifemoral bypass achieve earlier hormonal homeostasis after surgery compared to open aortobifemoral bypass.

Functional characterization of key enzymes involved in L-glutamate synthesis and degradation in the thermotolerant and methylotrophic bacterium Bacillus methanolicus.

Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter(-1) of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low Km value for ammonium (10 mM) and a high Km value for l-glutamate (250 mM), and the Vmax value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar Km values (1 to 1.4 mM) and Vmax values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism.

Genome sequence of thermotolerant Bacillus methanolicus: features and regulation related to methylotrophy and production of L-lysine and L-glutamate from methanol.

Bacillus methanolicus can utilize methanol as its sole carbon and energy source, and the scientific interest in this thermotolerant bacterium has focused largely on exploring its potential as a biocatalyst for the conversion of methanol into L-lysine and L-glutamate. We present here the genome sequences of the important B. methanolicus model strain MGA3 (ATCC 53907) and the alternative wild-type strain PB1 (NCIMB13113). The physiological diversity of these two strains was demonstrated by a comparative fed-batch methanol cultivation displaying highly different methanol consumption and respiration profiles, as well as major differences in their L-glutamate production levels (406 mmol liter(-1) and 11 mmol liter(-1), respectively). Both genomes are small (ca 3.4 Mbp) compared to those of other related bacilli, and MGA3 has two plasmids (pBM19 and pBM69), while PB1 has only one (pBM20). In particular, we focus here on genes representing biochemical pathways for methanol oxidation and concomitant formaldehyde assimilation and dissimilation, the important phosphoenol pyruvate/pyruvate anaplerotic node, the tricarboxylic acid cycle including the glyoxylate pathway, and the biosynthetic pathways for L-lysine and L-glutamate. Several unique findings were made, including the discovery of three different methanol dehydrogenase genes in each of the two B. methanolicus strains, and the genomic analyses were accompanied by gene expression studies. Our results provide new insight into a number of peculiar physiological and metabolic traits of B. methanolicus and open up possibilities for system-level metabolic engineering of this bacterium for the production of amino acids and other useful compounds from methanol.

Quality of Life (QoL) Assessment in the Patients Operated with Either Laparoscopic or an Open Aortobifemoral Bypass for Aortoiliac Occlusive Disease (AIOD): 2 Years Results of a Randomized Controlled Trial.

BACKGROUND: QoL assessment within surgical treatment is seldom investigated and sparsely reported in the medical literature. This study aimed to compare QoL in a randomized fashion in the patients treated with either a laparoscopic aortobifemoral bypass (LABFB) or an open aortobifemoral bypass (OABFB) for the treatment of AIOD. PATIENTS AND METHODS: Seventy-one consecutive patients with AIOD, Trans-Atlantic Inter-Society Consensus II Type D lesions (TASC II, Type D) were randomized to LABFB or OABFB. Thirty-five patients in the LABFB and thirty-six in the OABFB groups were compared for the changes in the QoL, with the short-form health survey (SF-36), EuroQol 5 dimensions (EQ-5D), and EQ-5D visual analog scale (VAS) preoperatively, and postoperatively at 1, 3, 6, 12 and 24 months. Mann-Whitney U-Test and Wilcoxon sign-rank test were used for group comparison. Mixed model analysis was performed to examine the effect of different variables on the QoL. RESULTS: In the patients treated with LABFB, physical component score (PCS) and mental component score (MCS) in SF-36 were significantly higher than OABFB, at 1 and 3 months postoperatively. PCS was also significantly higher in the LABFB group than OABFB at 24 months postoperatively. The preoperative QoL scores for both the laparoscopy and the open group were significantly lower than the age-matched general Norwegian population. EQ-5D median scores were significantly higher in the LABFB at all postoperative follow-up time points up to 12 months. The patients in the LABFB group also had a statistically significant increase in EQ-5D VAS compared to OABFB, at 1 and 12 months postoperatively (p = 0.005, and p = 0.037, respectively). CONCLUSION: QoL seems better in patients treated with LABFB than OABFB, particularly during the early months after surgery.

Bacillus methanolicus pyruvate carboxylase and homoserine dehydrogenase I and II and their roles for L-lysine production from methanol at 50 degrees C.

We here present the pyc gene encoding pyruvate carboxylase (PC), and the hom-1 and hom-2 genes encoding two active homoserine dehydrogenase (HD) proteins, in methylotrophic Bacillus methanolicus MGA3. In general, both PC and HD are regarded as key targets for improving bacterial L-lysine production; PC plays a role in precursor oxaloacetate (OAA) supply while HD controls an important branch point in the L-lysine biosynthetic pathway. The hom-1 and hom-2 genes were strongly repressed by L-threonine and L-methionine, respectively. Wild-type MGA3 cells secreted 0.4 g/l L-lysine and 59 g/l L-glutamate under optimised fed batch methanol fermentation. The hom-1 mutant M168-20 constructed herein secreted 11 g/l L-lysine and 69 g/l of L-glutamate, while a sixfold higher L-lysine overproduction (65 g/l) of the previously constructed classical B. methanolicus mutant NOA2#13A52-8A66 was accompanied with reduced L-glutamate production (28 g/l) and threefold elevated pyc transcription level. Overproduction of PC and its mutant enzyme P455S in M168-20 had no positive effect on the volumetric L-lysine yield and the L-lysine yield on methanol, and caused significantly reduced volumetric L-glutamate yield and L: -glutamate yield on methanol. Our results demonstrated that hom-1 represents one key target for achieving L-lysine overproduction, PC activity plays an important role in controlling L-glutamate production from methanol, and that OAA precursor supply is not a major bottleneck for L-lysine overproduction by B. methanolicus.

Totally laparoscopic aortobifemoral bypass surgery in the treatment of aortoiliac occlusive disease or abdominal aortic aneurysms - a systematic review and critical appraisal of literature.

PURPOSE: This systematic review aims to evaluate the published literature regarding totally laparoscopic aortobifemoral bypass (LABF) surgery in the treatment of aortoiliac occlusive disease (AIOD) or abdominal aortic aneurysms (AAA), compared with open aortobifemoral bypass surgery. MATERIALS AND METHODS: A systematic review of the medical literature between 1990 and 2016 was performed, searching the medical databases Cochrane Library, OVID Medline, Embase and PubMed. Studies concerning totally LABF with or without control group and containing more than 10 patients were included in the analysis. Operative and aortic cross-clamping times, blood loss, rate of conversion to open surgery, mortality and morbidity within the first 30 postoperative days, hospital stay and primary and secondary patency of the graft were extracted and compared with open surgery when possible. RESULTS: Sixty-six studies were deemed eligible for inclusion in this review, 16 of them matched the inclusion criteria for quantitative synthesis. The patient material consisted of 588 patients undergoing totally LABF, 22 due to AAA, and the remaining 566 for AIOD. Five comparative studies regarding AIOD compared 211 totally LABF procedures with 246 open procedures. Only one study concerning AAA was eligible for inclusion, and this study did not provide a comparison against an open group. The operating and aortic cross-clamping times were shorter in the open group. Conversion rates ranged from 0% to 27%. There was no statistically significant difference in mortality between the two groups (p=0.64). Hospital stays ranged from 4.0 to 12.1 and 5.0 to 12.8 days in the laparoscopic group and open group, respectively. Most of the studies provided low levels of evidence, mainly due to lack of blinding, randomization and correction of bias. CONCLUSION: Totally laparoscopic aortoiliac surgery seems to be a feasible technique with unaffected mortality and trend toward benefits in hospital stay and possibly also in complication rates. The literature published this far is sparse and with inconsistent results. More randomized controlled trials are required before this method can be widely implemented.

Patient-perceived health-related quality of life before and after laparoscopic aortobifemoral bypass.

BACKGROUND: In patients operated with laparoscopic aortobifemoral bypass (LABFB) for atherosclerotic obstruction in aortoiliac segment, the main focus of the reports published during the last two decades has been morbidity and mortality. The primary objective of this study was to examine the health-related quality of life (HRQL) in these patients before and after LABFB. PATIENTS AND METHODS: Fifty consecutive patients (27 females) with Trans-Atlantic Inter-Society Consensus II, type D lesions were prospectively included. Short-Form 36 (SF-36) questionnaire was used to get information about the HRQL before LABFB and at 1, 3 and 6 months after the operation. Main indication for LABFB was intermittent claudication. Linear mixed-effect models were used to assess changes in HRQL over time. Age, gender, smoking, blood loss, operation time, concomitant operation, the American Society of Anesthesiologists category, length of hospital stay, previous vascular procedures and aorta cross-clamping were used as fixed factors and their impact on the physical components of the SF-36, as well as the summary scores were determined with univariate analysis. Variables with P<0.2 were included in the multivariate regression analysis. P<0.05 was considered statistically significant. RESULTS: Statistically significant improvement was found in all SF-36 domains as well as in the summary scores after LABFB compared to the preoperative scores. The improvement in scores was substantial already at 1 month and the effect was maintained at 3 and 6 months survey time points. Concomitant operations had a statistically significant negative impact on the physical components of SF-36. Data completeness of item questionnaires was 93% in the whole material. Reliability scale and homogeneity estimates for the eight domains had high internal consistency. CONCLUSION: Patients operated with LABFB for Trans-Atlantic Inter-Society Consensus II, type D lesions have reduced HRQL. LABFB leads to substantial and statistically significant improvement in the patients' HRQL, when examined with SF-36. These results need to be replicated by a randomized clinical trial.

Cost-utility analysis comparing laparoscopic vs open aortobifemoral bypass surgery.

OBJECTIVES: Laparoscopic aortobifemoral bypass has become an established treatment option for symptomatic aortoiliac obstructive disease at dedicated centers. Minimally invasive surgical techniques like laparoscopic surgery have often been shown to reduce expenses and increase patients' health-related quality of life. The main objective of our study was to measure quality-adjusted life years (QALYs) and costs after totally laparoscopic and open aortobifemoral bypass. PATIENTS AND METHODS: This was a within trial analysis in a larger ongoing randomized controlled prospective multicenter trial, Norwegian Laparoscopic Aortic Surgery Trial. Fifty consecutive patients suffering from symptomatic aortoiliac occlusive disease suitable for aortobifemoral bypass surgery were randomized to either totally laparoscopic (n=25) or open surgical procedure (n=25). One patient dropped out of the study before surgery. We measured health-related quality of life using the EuroQol (EQ-5D-5L) questionnaire at 4 different time points, before surgery and for 6 months during follow-up. We calculated the QALYs gained by using the area under the curve for both groups. Costs were calculated based on prices for surgical equipment, vascular prosthesis and hospital stay. RESULTS: We found a significantly higher increase in QALYs after laparoscopic vs open aortobifemoral bypass surgery, with a difference of 0.07 QALYs, (p=0.001) in favor of laparoscopic aortobifemoral bypass. The total cost of surgery, equipment and hospital stay after laparoscopic surgery (9,953 €) was less than open surgery (17,260 €), (p=0.001). CONCLUSION: Laparoscopic aortobifemoral bypass seems to be cost-effective compared with open surgery, due to an increase in QALYs and lower procedure-related costs.

l-lysine production by Bacillus methanolicus: Genome-based mutational analysis and l-lysine secretion engineering.

Bacillus methanolicus is a methylotrophic bacterium with an increasing interest in academic research and for biotechnological applications. This bacterium was previously applied for methanol-based production of l-glutamate, l-lysine and the five-carbon diamine cadaverine by wild type, classical mutant and recombinant strains. The genomes of two different l-lysine secreting B. methanolicus classical mutant strains, NOA2#13A52-8A66 and M168-20, were sequenced. We focused on mutational mapping in genes present in l-lysine and other relevant amino acid biosynthetic pathways, as well as in the primary cell metabolism important for precursor supply. In addition to mutations in the aspartate pathway genes dapG, lysA and hom-1, new mutational target genes like alr, proA, proB1, leuC, odhA and pdhD were identified. Surprisingly, no mutations were found in the putative l-lysine transporter gene lysEMGA3. Inspection of the wild type B. methanolicus strain PB1 genome sequence identified two homologous putative l-lysine transporter genes, lysEPB1 and lysE2PB1. The biological role of these putative l-lysine transporter genes, together with the heterologous l-lysine exporter gene lysECg from Corynebacterium glutamicum, were therefore investigated. Our results demonstrated that the titer of secreted l-lysine in B. methanolicus was significantly increased by overexpression of lysECg while overexpression of lysEMGA3, lysEPB1 and lysE2PB1 had no measurable effect.

Streamlined Preparation of Immobilized Candida antarctica Lipase B.

Candida antarctica lipase B (CalB) was efficiently expressed (6.2 g L-1) in Escherichia coli by utilizing an N-terminal tag cassette and the XylS/Pm expression system in a fed-batch bioreactor; subsequent direct binding to EziG from crude extracts resulted in an immobilized catalyst with superior activity to Novozym 435.

Whole blood gene expression in infants with respiratory syncytial virus bronchiolitis.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of viral bronchiolitis in infants worldwide, and environmental, viral and host factors are all of importance for disease susceptibility and severity. To study the systemic host response to this disease we used the microarray technology to measure mRNA gene expression levels in whole blood of five male infants hospitalised with acute RSV, subtype B, bronchiolitis versus five one year old male controls exposed to RSV during infancy without bronchiolitis. The gene expression levels were further evaluated in a new experiment using quantitative real-time polymerase chain reaction (QRT-PCR) both in the five infants selected for microarray and in 13 other infants hospitalised with the same disease. RESULTS: Among the 30 genes most differentially expressed by microarray nearly 50% were involved in immunological processes. We found the highly upregulated interferon, alpha-inducible protein 27 (IFI27) and the highly downregulated gene Charcot-Leyden crystal protein (CLC) to be the two most differentially expressed genes in the microarray study. When performing QRT-PCR on these genes IFI27 was upregulated in all but one infant, and CLC was downregulated in all 18 infants, and similar to that given by microarray. CONCLUSION: The gene IFI27 is upregulated and the gene CLC is downregulated in whole blood of infants hospitalised with RSV, subtype B, bronchiolitis and is not reported before. More studies are needed to elucidate the specificity of these gene expressions in association with host response to this virus in bronchiolitis of moderate severity.

Comparison of the acute-phase response after laparoscopic versus open aortobifemoral bypass surgery: a substudy of a randomized controlled trial.

PURPOSE: Minimally invasive surgical techniques have been shown to reduce the inflammatory response related to a surgical procedure. The main objective of our study was to measure the inflammatory response in patients undergoing a totally laparoscopic versus open aortobifemoral bypass surgery. This is the first randomized trial on subjects in this population. PATIENTS AND METHODS: This is a substudy of a larger randomized controlled multicenter trial (Norwegian Laparoscopic Aortic Surgery Trial). Thirty consecutive patients with severe aortoiliac occlusive disease eligible for aortobifemoral bypass surgery were randomized to either a totally laparoscopic (n=14) or an open surgical procedure (n=16). The inflammatory response was measured by perioperative monitoring of serum interleukin-6 (IL-6), IL-8, and C-reactive protein (CRP) at six different time points. RESULTS: The inflammatory reaction caused by the laparoscopic procedure was reduced compared with open surgery. IL-6 was significantly lower after the laparoscopic procedure, measured by comparing area under the curve (AUC), and after adjusting for the confounding effect of coronary heart disease (P=0.010). The differences in serum levels of IL-8 and CRP did not reach statistical significance. CONCLUSION: In this substudy of a randomized controlled trial comparing laparoscopic and open aortobifemoral bypass surgeries, we found a decreased perioperative inflammatory response after the laparoscopic procedure measured by comparing AUC for serum IL-6.

A comparative cohort study of totally laparoscopic and open aortobifemoral bypass for the treatment of advanced atherosclerosis.

BACKGROUND: Totally laparoscopic aortobifemoral bypass (LABF) procedure has been shown to be feasible for the treatment of advanced aortoiliac occlusive disease (AIOD). This study compares the LABF with the open aortobifemoral bypass (OABF) operation. METHODS: In this prospective comparative cohort study, 50 consecutive patients with type D atherosclerotic lesions in the aortoiliac segment were treated with an LABF operation. The group was compared with 30 patients who were operated on with the OABF procedure for the same disease and time period. We had an explanatory strategy, and our research hypothesis was to compare the two surgical procedures based on a composite event (all-cause mortality, graft occlusion, and systemic morbidity). Stratification analysis was performed by using the Mantel-Haenszel method with the patient-time model. Cox multivariate regression method was used to adjust for confounding effect after considering the proportional hazard assumption. Cox proportional cause-specific hazard regression model was used for competing risk endpoint. RESULTS: There was a higher frequency of comorbidity in the OABF group. A significant reduction of composite event, 82% (hazard ratio 0.18; 95% CI 0.08-0.42, P=0.0001) was found in the LABF group when compared with OABF group, during a median follow-up time period of 4.12 years (range from 1 day to 9.32 years). In addition, less operative bleeding and shorter length of hospital stay were observed in the LABF group when compared with the OABF group. All components of the composite event showed the same positive effect in favor of LABF procedure. CONCLUSION: LABF for the treatment of AIOD, Trans-Atlantic Inter-Society Consensus II type D lesions, seems to result in a less composite event when compared with the OABF procedure. To conclude, our results need to be replicated by a randomized clinical trial.

Methylotrophic Bacillus methanolicus encodes two chromosomal and one plasmid born NAD+ dependent methanol dehydrogenase paralogs with different catalytic and biochemical properties.

Bacillus methanolicus can utilize methanol as the sole carbon source for growth and it encodes an NAD(+)-dependent methanol dehydrogenase (Mdh), catalyzing the oxidation of methanol to formaldehyde. Recently, the genomes of the B. methanolicus strains MGA3 (ATCC53907) and PB1 (NCIMB13113) were sequenced and found to harbor three different putative Mdh encoding genes, each belonging to the type III Fe-NAD(+)-dependent alcohol dehydrogenases. In each strain, two of these genes are encoded on the chromosome and one on a plasmid; only one chromosomal act gene encoding the previously described activator protein ACT was found. The six Mdhs and the ACT proteins were produced recombinantly in Escherichia coli, purified, and characterized. All Mdhs required NAD(+) as cosubstrate, were catalytically stimulated by ACT, exhibited a broad and different substrate specificity range and displayed both dehydrogenase and reductase activities. All Mdhs catalyzed the oxidation of methanol; however the catalytic activity for methanol was considerably lower than for most other alcohols tested, suggesting that these enzymes represent a novel class of alcohol dehydrogenases. The kinetic constants for the Mdhs were comparable when acting as pure enzymes, but together with ACT the differences were more pronounced. Quantitative PCR experiments revealed major differences with respect to transcriptional regulation of the paralogous genes. Taken together our data indicate that the repertoire of methanol oxidizing enzymes in thermotolerant bacilli is larger than expected with complex mechanisms involved in their regulation.