Vascular access surgery training in the United Kingdom is currently perceived (by trainees) to be inadequate.

INTRODUCTION: Vascular access surgery (VAS) involves the creation and maintenance of arteriovenous access to facilitate haemodialysis. The prevalence of haemodialysis is rising despite increases in kidney transplants on a yearly basis. There is currently only one access surgery fellowship accredited by the Royal College of Surgeons of England. We aimed to establish the experience and perceived competence in access surgery of senior vascular surgery trainees. METHODS: A short questionnaire (SurveyMonkey) was used to survey all senior (ST6-ST8) vascular surgery trainees in Health Education England (HEE) vascular surgery training programmes. The short survey asked trainees to report their: (1) training grade; (2) training deanery; (3) experience of access surgery; and (4) whether senior trainees thought they would be able to independently undertake primary access surgery post-completion of training (post Certificate of Completion of Training). The survey was circulated via HEE deaneries and the vascular surgery trainees' society: the Rouleaux Club. RESULTS: Twenty-eight senior (ST6-ST8) vascular surgery trainees responded to the survey: 29.6% were ST6 level, 33.3% were ST7 and 37.1% were ST8. Deanery respondence was evenly spread, although London was overrepresented (37.1%). In total, 28.6% had been involved in fewer than 10 cases, 35.7% in 10-25 cases, and 35.7% in more than 25 cases. Almost 54% of senior vascular surgery trainees believed they would not be able to undertake independent access surgery once they had completed training. CONCLUSIONS: Competence in access surgery is an increasing requirement of a consultant vascular surgeon. More formalised training is required to adequately train the next generation of vascular surgeons.

Vascular access surgery training in the United Kingdom is currently perceived (by trainees) to be inadequate.

INTRODUCTION: Vascular access surgery (VAS) involves the creation and maintenance of arteriovenous access to facilitate haemodialysis. The prevalence of haemodialysis is rising despite increases in kidney transplants on a yearly basis. There is currently only one access surgery fellowship accredited by the Royal College of Surgeons of England. We aimed to establish the experience and perceived competence in access surgery of senior vascular surgery trainees. METHODS: A short questionnaire (SurveyMonkey) was used to survey all senior (ST6-ST8) vascular surgery trainees in Health Education England (HEE) vascular surgery training programmes. The short survey asked trainees to report their: (1) training grade; (2) training deanery; (3) experience of access surgery; and (4) whether senior trainees thought they would be able to independently undertake primary access surgery post-completion of training (post Certificate of Completion of Training). The survey was circulated via HEE deaneries and the vascular surgery trainees' society: the Rouleaux Club. RESULTS: Twenty-eight senior (ST6-ST8) vascular surgery trainees responded to the survey: 29.6% were ST6 level, 33.3% were ST7 and 37.1% were ST8. Deanery respondence was evenly spread, although London was overrepresented (37.1%). In total, 28.6% had been involved in fewer than 10 cases, 35.7% in 10-25 cases, and 35.7% in more than 25 cases. Almost 54% of senior vascular surgery trainees believed they would not be able to undertake independent access surgery once they had completed training. CONCLUSIONS: Competence in access surgery is an increasing requirement of a consultant vascular surgeon. More formalised training is required to adequately train the next generation of vascular surgeons.

Traumatic arterial spasm causing transient limb ischaemia: a genuine clinical entity.

Traumatic arterial spasm is a phenomenon that has long been questioned by clinicians. Indeed, some would argue that surgical exploration is mandatory whenever there are signs of distal ischaemia following limb trauma. We present a case of angiographically demonstrated tibial artery spasm following gunshot injury. Exploration was unnecessary and distal perfusion was reestablished spontaneously. This case demonstrates the existence of traumatic arterial spasm as a genuine clinical entity and suggests that immediate surgical exploration may not be necessary in all cases.

Hybrid treatment of complex aortic arch disease with supra-aortic debranching and endovascular stent graft repair.

BACKGROUND: Aortic arch disease has conventionally been the domain of open surgical repair. Hybrid open and endovascular repair has evolved as an alternative, less invasive, treatment option with promising results. A systematic literature review and analysis of the reported outcomes was undertaken. METHODS: An Internet-based literature search using MEDLINE was performed to identify all studies reporting on hybrid aortic arch repair with supra-aortic branch revascularisation and subsequent stent graft deployment. Debranching should involve at least one carotid artery, so that patients merely requiring a carotid-subclavian bypass were not included. Only reports of five patients or more were included in the analysis. Outcome measures were technical success, perioperative, 30-day and late morbidity and mortality. RESULTS: Eighteen studies fulfilled our search criteria, and data from 195 patients were entered for the analysis. No comparative studies of hybrid aortic arch repair with other conventional or innovative treatment modalities were identified. Complete arch repair was performed in 122 patients (63%). The overall technical success rate was 86% (167/195). The most common reason for technical failure was endoleak (9%, 17/195). Overall perioperative morbidity and mortality rates were 21% (41/195) and 9% (18/195), respectively. The most common perioperative complication was stroke (7%, 14/195). Four aneurysm-related deaths were reported during follow-up (2%). No long-term data on hybrid aortic arch repair were identified. CONCLUSIONS: Hybrid repair of complex aortic arch disease is an alternative treatment option with acceptable short-term results. Stroke remains a frequent complication and mortality rates are significant. Further research with large comparative studies and longer follow-up is required.

Fatal cerebellar stroke following emergency endovascular stent grafting of a leaking thoracic aortic aneurysm: A case report.

Stent-grafting of thoracic aortic diseases has developed as an alternative therapeutic modality in thoracic aneurysm management. Postprocedural complications include mortality, endoleaks, paraplegia and stroke. Other complications that may arise in cases of overstenting the origin of the left subclavian arther include left upper limb ischemia, subclavian steal syndrome and stroke. Posterior circulation strokes due to vertebral artery insufficiency have been reported in the past. In the present case, a fatal stroke caused by a cerebellar infarct culminating in the death of a patient with a leaking thoracic aortic aneurysm is reported. Medical personnel as well as patients should be aware of this possible complication. Vigilance in assessing the contralateral cerebral circulation before the procedure is a prerequisite in less acute circumstances.

An integrated care pathway to save the critically ischaemic diabetic foot.

This prospective study describes and evaluates the efficacy of an integrated care pathway for the management of the critically ischaemic diabetic foot patients by a multidisciplinary team. A weekly joint diabetes/vascular/podiatry ward round and outpatient clinic was established where patients were assessed within 7 days of referral by clinical examination, ankle-brachial-index-pressures, duplex angiogram and transcutaneous oxygen pressures. An angiogram +/- angioplasty or alternatively a magnetic resonance angiography prior to surgical revascularisation was performed in patients deemed not suitable for angioplasty based on the above vascular assessment. Between January 2002 and June 2003(18 months), 128 diabetic patients with lower limb ischaemia were seen. Thirty-four (26.6%) patients received medical treatment alone, and 18 (14.1%) were deemed 'palliative' due to their significant co-morbidities. The remaining 76 (59.4%) patients underwent either angioplasty (n = 56), surgical reconstruction (n = 18), primary major amputation (n = 2) or secondary amputation after surgical revascularisation (n = 1). Minor toe amputations were required in 35 patients. The mortality in the intervention group was 14% (11/76). This integrated multidisciplinary approach offers a consistent and equitable service to diabetic patients with critically ischaemic feet and appears to have a beneficial major/minor amputation ratio.

Association of malignant disease with critical leg ischaemia.

BACKGROUND: The aim of the study was to determine the prevalence of malignant disease in patients with critical leg ischaemia (CLI). METHODS: : Data for all patients with CLI presenting to a tertiary vascular unit over an 18-month interval were collected prospectively. Patients with clinical, laboratory or radiological features suggestive of malignancy were evaluated further. RESULTS: Of 192 patients admitted with CLI, 22 (11.5 per cent) were found to have an associated malignancy; ten had lung cancer. Fifteen were anaemic on presentation. The prevalence of occult malignancy in patients with acute leg ischaemia was 16 per cent (ten of 62) compared with 9.2 per cent (12 of 130) in those with chronic CLI. Eleven of 22 of patients with CLI and malignancy died within 6 months, compared with 35 (20.6 per cent) of 170 patients with no evidence of malignancy. CONCLUSION: A high prevalence of occult cancer was found in patients presenting with CLI; this was associated with a significantly increased mortality rate at 6 months.

Molecular breeding of transgenic rice expressing a xylanase domain of the xynA gene from Clostridium thermocellum.

The gene encoding the catalytic domain of thermostable xylanase from Clostridium thermocellum F1 was expressed in rice plants under the control of a constitutive promoter. The gene encoding Xylanase A was modified to encode the catalytic domain of family 11 xylanase without the signal sequence (xynA1), and was introduced into rice plants and expressed under the control of a modified cauliflower mosaic virus 35S promoter. Zymogram analysis indicated that the recombinant xylanase was produced in rice plants. The xynA1 gene was stably expressed in rice straw and seed grains. No phenotypic effect of xylanase expression was noted. The enzyme was detected in the desiccated grain. High levels of enzyme activity were maintained in the cell-free extract during incubation at 60 degrees C for 24 h. The results indicated that high levels of xylanase can be produced in rice plants.

A novel beta-N-acetylglucosaminidase of Clostridium paraputrificum M-21 with high activity on chitobiose.

A beta- N-acetylglucosaminidase gene ( nag3A) from Clostridium paraputrificum M-21 was cloned in Escherichia coli. The nag3A gene consists of an open reading frame of 1,239-bp, encoding 413 amino acids with a deduced molecular weight of 45,531 Da. Nag3A is a single domain enzyme containing a family 3 glycoside hydrolase catalytic domain. Nag3A was purified from recombinant E. coli and characterized. The enzyme hydrolyzed chitooligomers such as di- N-acetylchitobiose, tri- N-acetylchitotriose, tetra- N-acetylchitotetraose, penta- N-acetylchitopentaose, hexa- N-acetylchitohexaose, ball-milled chitin, and synthetic substrates such as 4-methylumbelliferyl N-acetyl beta- D-glucosaminide [4-MU-(GlcNAc)], but had no activity at all against p-nitrophenyl-beta- D-glucoside, p-nitrophenyl-beta- D-xyloside, or p-nitrophenyl-beta- D-galactosamine. The enzyme was optimally active at 50 degrees C and pH 7.0, and the apparent K(m) and V(max) values for 4-MU-(GlcNAc) were 7.9 micro M and 21.8 micro mol min(-1) mg protein(-1), respectively. SDS-PAGE, zymogram, and immunological analyses suggested that this enzyme is induced by ball-milled chitin.

Clostridium thermocellum cellulase CelT, a family 9 endoglucanase without an Ig-like domain or family 3c carbohydrate-binding module.

The celT gene of Clostridium thermocellum strain F1 was found downstream of the mannanase gene man26B [Kurokawa J et al. (2001) Biosci Biotechnol Biochem 65:548-554] in pKS305. The open reading frame of celT consists of 1,833 nucleotides encoding a protein of 611 amino acids with a predicted molecular weight of 68,510. The mature form of CelT consists of a family 9 cellulase domain and a dockerin domain responsible for cellulosome assembly, but lacks a family 3c carbohydrate-binding module (CBM) and an immunoglobulin (Ig)-like domain, which are often found with family 9 catalytic domains. CelT devoid of the dockerin domain (CelTDeltadoc) was constructed and purified from a recombinant Escherichia coli, and its enzyme properties were examined. CelTDeltadoc showed strong activity toward carboxymethylcellulose (CMC) and barley beta-glucan, and low activity toward xylan. The V(max) and K(m) values were 137 micro mol min(-1) mg(-1) and 16.7 mg/ml, respectively, for CMC. Immunological analysis indicated that CelT is a catalytic component of the C. thermocellum F1 cellulosome. This is the first report describing the characterization of a family 9 cellulase without an Ig-like domain or family 3c CBM.

Sequence of the Clostridium thermocellum mannanase gene man26B and characterization of the translated product.

The man26B gene of Clostridium thermocellum strain F1 was found in pKS305, which had been selected as a recombinant plasmid conferring endoglucanase activity on Escherichia coli. The open reading frame of man26B consists of 1,773 nucleotides encoding a protein of 591 amino acids with a predicted molecular weight of 67,047. Man26B is a modular enzyme composed of an N-terminal signal peptide and three domains in the following order: a mannan-binding domain, a family 26 mannanase domain, and a dockerin domain responsible for cellulosome assembly. We found that this gene was a homologue of the man26A gene of C. thermocellum strain YS but that there were insertion or deletion mutations that caused a frame-shift mutation affecting a stretch of 26 amino acids in the catalytic domain. Man26B devoid of the dockerin domain was constructed and purified from a recombinant E. coli, and its enzyme properties were examined. Immunological analysis indicated that Man26B was a catalytic component of the C. thermocellum F1 cellulosome.

The multidomain xylanase Xyn10B as a cellulose-binding protein in Clostridium stercorarium.

The cells of Clostridium stercorarium F-9 grown on cellobiose bound to insoluble cellulose allomorphs such as phosphoric acid-swollen cellulose (ASC). Treatment of the cells with 3 M guanidine hydrochloride extracted surface-layer proteins from the cells and abolished the affinity of the cells for ASC. SDS-polyacrylamide gel electrophoresis, zymogram, and immunological analyses indicated that one of the major surface layer proteins was Xyn10B, which is a modular xylanase comprising two family 22 carbohydrate-binding modules (CBMs), a family 10 catalytic domain of glycosyl hydrolases, a family 9 CBM, and two S-layer homologous (SLH) domains. The C. stercorarium F-9 cells treated with guanidine hydrochloride coprecipitated with ASC upon the addition of a derivative of Xyn10B containing both a CBM and SLH domain in addition to a catalytic domain, but not a derivative without Xyn10B-SLH domains, suggesting that Xyn10B functioned as a cellulose-binding protein in C. stercorarium F-9.

[Alleviation of type I restriction in Escherichia coli K12 in the presence of the arsR gene from pKW301 of Acidiphilium multivorum AIU 301]. / Oslablenie restriktsii tipa I u Escherichia coli K12 v prisutstvii gena arsR pKW301 Acidiphilium multivorum AIU 301.

A study was made of the antirestriction activity of Acidiphilium multivorum AIU 301 ArsR, a repressor of the ars operon which confers resistance to arsenite and arsenate and is on pKW301. In Escherichia coli, arsR cloned under the control of Plac in a multi-copy vector alleviated restriction of nonmodified lambda DNA by a factor of 120, six times more efficiently than its analogs of conjugal plasmids R64 (incI1) and R773 (incFI). Amino acid sequence analysis showed that the three ArsR proteins have a homologous region of 38 residues, including the antirestriction motif, in their N domains, whereas the motif is in the C domain in the Ard proteins. The other regions are nonhomologous, and pKW301 ArsR is 33 residues shorter than R64 and R773 ArsRs. The total charge is -4 in pKW301 ArsR and +2 in R64 and R733 ArsRs. A total negative charge was assumed to contribute to the antirestriction activity.

Importance of the carbohydrate-binding module of Clostridium stercorarium Xyn10B to xylan hydrolysis.

The Clostridium stercorarium xylanase Xyn10B is a modular enzyme comprising two thermostabilizing domains, a family 10 catalytic domain of glycosyl hydrolases, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains [Biosci. Biotechnol. Biochem., 63, 1596-1604 (1999)]. To investigate the role of this CBM, we constructed two derivatives of Xyn10B and compared their hydrolytic activity toward xylan and some preparations of plant cell walls; Xyn10BdeltaCBM consists of a catalytic domain only, and Xyn10B-CBM comprises a catalytic domain and a CBM. Xyn10B-CBM bound to various insoluble polysaccharides including Avicel, acid-swollen cellulose, ball-milled chitin, Sephadex G-25, and amylose-resin. A cellulose binding assay in the presence of soluble saccharides suggested that the CBM of Xyn10B had an affinity for even monosaccharides such as glucose, galactose, xylose, mannose and ribose. Removal of the CBM from the enzyme negated its cellulose- and xylan-binding abilities and severely reduced its enzyme activity toward insoluble xylan and plant cell walls but not soluble xylan. These findings clearly indicated that the CBM of Xyn10B is important in the hydrolysis of insoluble xylan. This is the first report of a family 9 CBM with an affinity for insoluble xylan in addition to crystalline cellulose and the ability to increase hydrolytic activity toward insoluble xylan.

Conversion of chitinous wastes to hydrogen gas by Clostridium paraputrificum M-21.

The chitinolytic bacterium Clostridium paraputrificum strain M-21 produced 2.2 and 1.5 mol hydrogen gas from 1 mol N-acetyl-D-glucosamine (GlcNAc) and ball-milled chitin equivalent to 1 mol of GlcNAc, respectively, at pH 6.0. In addition, strain M-21 efficiently degraded and fermented ball-milled raw shrimp and lobster shells to produce hydrogen gas: 11.4 mmol H2 from 2.6 g of the former and 7.8 mmol H2 from 1.5 g of the latter. Hydrogen evolution from these shell wastes were enhanced two fold by employing acid and alkali pretreatment. Waste from the starch industry was also converted to hydrogen. When C. paraputrificum M-21 was cultivated on ball-milled chitin and ball-milled shrimp shells for 14 and 12 h, respectively, chitinases ChiA and/or ChiB were detected as the major chitinase species in the supernatant of the cultures, suggesting that the play a critical role in the degradation of chitinous materials.

Characterization of Clostridium paraputrificum chitinase A from a recombinant Escherichia coli.

Clostridium paraputrificum chitinase A (ChiA) was purified from a recombinant Escherichia coli. ChiA was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)2]. ChiA showed maximum activity at pH 6.0 and its optimum temperature was 45 degrees C. ChiA was stable between pH 6.0 and 9.0 and at temperatures up to 40 degrees C. The K(m) and V(max) values of ChiA for 4-MU-(GlcNAc)2 were estimated to be 6.9 microM and 43 micromol/min/mg, respectively. Thin-layer chromatography indicated that ChiA hydrolyzes chitooligosaccharides to mainly chitobiose. ChiA was found to adsorb not only chitinous polymers but also cellulosic polymers.

Sequence of celQ and properties of celQ, a component of the Clostridium thermocellum cellulosome.

The nucleotide sequence of the Clostridium thermocellum F1 celQ gene, which codes for the endoglucanase CelQ, consists of 2,130 bp encoding 710 amino acids. The precursor form of CelQ has a molecular weight of 79,809 and is composed of a signal peptide, a family 9 cellulase domain, a family IIIc carbohydrate-binding module (CBM), and a dockerin domain. Truncated derivatives of CelQ were constructed: CelQdeltadoc consisted of the catalytic domain and the CBM; CelQcat consisted of the catalytic domain only. CelQdeltadoc showed strong activity toward carboxymethylcellulose (CMC) and barley beta-glucan and low activity toward Avicel, acid-swollen cellulose, lichenan, and xylan. The Vmax and Km values were 235 micromol/min/mg and 3.3 mg/ml, respectively, for CMC. By contrast, CelQcat, which was devoid of the CBM, showed negligible activity toward CMC, i.e., about 1/1,000 of the activity of CelQdeltadoc, supporting the previously proposed idea that family IIIc CBMs participate in the catalytic function of the enzyme. Immunological analysis using an antiserum raised against CelQdeltadoc confirmed that CelQ is a component of the C. thermocellum cellulosome.

Purification, characterization, and molecular cloning of acidophilic xylanase from penicillium sp.40.

Penicillum sp. 40, which can grow in an extremely acidic medium at pH 2.0 was screened from an acidic soil. This fungus produces xylanases when grown in a medium containing xylan as a sole carbon source. A major xylanase was purified from the culture supernatant of Penicillium sp. 40 and designated XynA. The molecular mass of XynA was estimated to be 25,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. XynA has an optimum pH at 2.0 and is stable in pH 2.0-5.0. Western blot analysis using anit-XynA antibody showed that XynA was induced by xylan and repressed by glucose. Also, its production was increased by an acidic medium. The gene encoding XynA (xynA) was isolated from the genomic library of Penicillium sp. 40. The structural part of xynA was found to be 721 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynA was interrupted by a single intron which was 58 bp in size and encoded 221 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynA had a signal peptide composed of 31 amino acids. The molecular mass caliculated from the deduced amino acid sequence of XynA is 20,713. This is lower than that estimated by gel electrophoresis, suggesting that XynA is a glycoprotein. The predicted amino acid sequence of XynA has strong similarity to other family xylanases from fungi.

Characterization of the cellulolytic complex (cellulosome) from Ruminococcus albus.

The cellulolytic complex was isolated from the culture supernatant of Ruminococcus albus strain F-40 grown on cellulose by a Sephacryl S-300HR column chromatography. The molecular mass of the cellulolytic complex was found to be larger than 1.5 x 10(6) Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the cellulolytic complex contained at least 15 proteins with molecular weights from 40kDa to 250 kDa. Among them, 11 proteins showed endoglucanase and/or xylanase activities on the zymograms. Immunological analysis using an antiserum raised against the dockerin domain of endoglucanase VII of R. albus (DocVII) suggested that at least 7 proteins in the cellulolytic complex contained a dockerin domain immunoreactive with the anti-Doc-VII antiserum. Furthermore, DocVII was shown to specifically interact with a 40-kDa protein of the cellulolytic complex by Far-Western blot analysis. These results strongly suggest that the cellulolytic complex produced by R. albus resembles the cellulosome specified for the cellulolytic complex of several clostridia such as Clostridium thermocellum and respective components are assembled into the cellulosome by the mechanism common in all of the cellulolytic clostridia, i.e., the cellulosome is formed by the interaction between a dockerin domain of catalytic components and a cohesin domain of a scaffolding protein.

Sequence of egV and properties of EgV, a Ruminococcus albus endoglucanase containing a dockerin domain.

The Ruminococcus albus F-40 egV gene, encoding endoglucanase V (EGV), consists of an open reading frame of 1,833 nucleotides and encodes 611 amino acids with a deduced molecular weight of 67,103. The deduced EGV is a modular enzyme composed of a catalytic domain of family 5 of glycosyl hydrolases, a domain of unknown function, and a dockerin domain responsible for cellulosome assembly, suggesting that R. albus F-40 produces a cellulosome, and EGV is a component of the cellulosome. A truncated form of EGV with an apparent molecular weight of 42,000 was purified from a recombinant Escherichia coli and characterized since EGV suffered from partial proteolysis by E. coli protease(s). The truncated EGV was active toward carboxylmethyl cellulose, xylan, lichenan, and acid-swollen cellulose. The pH and temperature optima of the enzyme were 7.0 and 40 degrees C, respectively. By Western blot analysis using the antiserum raised against the truncated enzyme, EGV was detected in the whole cells but not in the culture supernatant of R. alubus F-40, suggesting that EGV was located on the cell surface.