Predictors of recurrence following local excision for early-stage anal squamous cell carcinoma.

INTRODUCTION: There is increasing use of local excision (LE) for definitive treatment of early-stage anal squamous cell carcinoma (ASCC) to avoid the morbidity associated with chemoradiotherapy (CRT). However, the importance of different histological variables on risk of recurrence is poorly understood. METHODS: A detailed analysis of patient characteristics, histology results, recurrence patterns and salvage treatment was conducted in consecutive T1/T2N0 ASCC patients treated by LE 2010-2021 across a UK regional cancer network multi-disciplinary team (MDT). Associations between potential predictors of disease recurrence were explored using chi-squared and Kruskal-Wallis tests for categorical and continuous variables respectively. RESULTS: Of 621 ASCC patients discussed in the network MDT, 164 had early-stage disease (T1/T2 N0). Of these, 36 (22%) were deemed suitable for LE (median age 61 years, female to male ratio 2:1). Twenty-two LE tumours were T1; 14 were T2. There were 12 well-differentiated tumours, 21 moderate and 3 poorly-differentiated. Seven out of 36 LE patients (19.4%) developed recurrence, all of whom went on to have salvage treatment with CRT (n = 4), re-excision (n = 2) or radiotherapy (n = 1). Predictors of disease recurrence following LE were: tumour differentiation (p = 0.024), tumour depth (p = 0.033) and R1 resection margin (p = 0.034). Tumour stage and site (margin/canal) were non-significant. CONCLUSION: LE for T1/T2 N0 ASCC of the margin or canal is a viable treatment strategy to avoid the morbidity associated with CRT and salvage treatments are still available for patients that develop recurrence. Tumour differentiation, depth and margin status are all important factors to consider when discussing management of early-stage ASCC.

360° virtual reality video for the acquisition of knot tying skills: A randomised controlled trial.

BACKGROUND: 360° virtual reality (VR) video is an exciting and evolving field. Current technology promotes a totally immersive, 3-dimensional (3D), 360° experience anywhere in the world using simply a smart phone and virtual reality headset. The potential for its application in the field of surgical education is enormous. The aim of this study was to determine knot tying skills taught with a 360-degree VR video compared to conventional 2D video teaching. MATERIAL AND METHODS: This trial was a prospective, randomised controlled study. 40 foundation year doctors (first year postgraduate) were randomised to either the 360-degree VR video (n = 20) or 2D video teaching (n = 20). Participants were given 15 min to watch their allocated video. Ability to tie a single handed reef knot was then assessed against a marking criteria developed for the Royal College of Surgeons, England, (RCSeng) Basic Surgical Skills (BSS) course, by a blinded assessor competent in knot tying. Each candidate then underwent further teaching using Peyton's four step model. Knot tying technique was then re-assessed. RESULTS: Knot tying scores were significantly better in the VR video teaching arm when compared with conventional (median knot score 5.0 vs 4.0 p = 0.04). When used in combination with face to face skills teaching this difference persisted (median knot score 9.5 vs 9.0 p = 0.01). More people in the VR arm constructed a complete reef knot than in the 2D arm following face to face teaching (17/20 vs 12/20). No difference between the groups existed in the time taken to construct a reef knot following video and teaching (median time 31.0s vs 30.5s p = 0.89). CONCLUSION: This study shows there is significant merit in the application of 360-degree VR video technology in surgical training, both as an independent teaching aid and when used as an adjunct to traditional face to face teaching.

Emergency General Surgery: evolution of a subspecialty by stealth.

BACKGROUND: Emergency surgical patients account for around half of all NHS surgical workload and 80 % of surgical deaths. Few trainees opt to CCT in General Surgery, and there is no recognised subspecialty training program in Emergency General Surgery (EGS). Despite this lack of training and relevant assessment by examination, there appears to be an increasing number of EGS posts advertised. This study aims to provide information about potential future employment opportunities for surgical trainees. METHODS: All consultant surgeon posts, advertised in the British Medical Journal between January 2009 and December 2014 were included. Data collected included specialty, region and institute of advertised post. For the purposes of statistical analysis, data was divided into two separate year bands: 2009-2011 and 2012-2014. Statistical analysis was by Chi-squared test; p <0.01 was considered statistically significant. An online tool was also used to determine experience and attitudes towards EGS amongst Consultant members of the ASGBI and all UK trainees in national training number (NTN) posts. RESULTS: Over the six-year study period, there were 1240 consultant job adverts in a general surgical specialty. Nine hundred and 75 were substantive posts; the region with the most jobs was London and the South East (n = 278). There were 55 jobs advertised in EGS, either with (20) or without (35) another subspecialty. The number of EGS adverts increased significantly in 2012-14 compared to 2009-11 (p = 0.008). 229 (28 %) Consultants and 309 (22 %) trainees responded to the survey. 16 % of consultants work in NHS institutions with Emergency General Surgeons. Only 21 % of trainees believe EGS will be delivered by EGS consultants in the future whilst 8.2 % of trainees stated EGS as their career plan. Less than half of all UK consultant surgeons see EGS as a subspecialty. CONCLUSIONS: This data demonstrates increasing societal need for EGS consultants over the last six years and the emergence of Emergency Surgery as a new subspecialty. In order to meet the EGS needs of the NHS, general surgical training and the examination system need to be revised.

Digital histology quantification of intra-hepatic fat in patients undergoing liver resection.

BACKGROUND: High intra-hepatic fat (IHF) content is associated with insulin resistance, visceral adiposity, and increased morbidity and mortality following liver resection. However, in clinical practice, IHF is assessed indirectly by pre-operative imaging [for example, chemical-shift magnetic resonance (CS-MR)]. We used the opportunity in patients undergoing liver resection to quantify IHF by digital histology (D-IHF) and relate this to CT-derived anthropometrics, insulin-related serum biomarkers, and IHF estimated by CS-MR. METHODS: A reproducible method for quantification of D-IHF using 7 histology slides (inter- and intra-rater concordance: 0.97 and 0.98) was developed. In 35 patients undergoing resection for colorectal cancer metastases, we measured: CT-derived subcutaneous and visceral adipose tissue volumes, Homeostasis Model Assessment of Insulin Resistance (HOMA-IR), fasting serum adiponectin, leptin and fetuin-A. We estimated relative IHF using CS-MR and developed prediction models for IHF using a factor-clustered approach. RESULTS: The multivariate linear regression models showed that D-IHF was best predicted by HOMA-IR (Beta coefficient(per doubling): 2.410, 95% CI: 1.093, 5.313) and adiponectin (ß(per doubling): 0.197, 95% CI: 0.058, 0.667), but not by anthropometrics. MR-derived IHF correlated with D-IHF (rho: 0.626; p = 0.0001), but levels of agreement deviated in upper range values (CS-MR over-estimated IHF: regression versus zero, p = 0.009); this could be adjusted for by a correction factor (CF: 0.7816). CONCLUSIONS: Our findings show IHF is associated with measures of insulin resistance, but not measures of visceral adiposity. CS-MR over-estimated IHF in the upper range. Larger studies are indicated to test whether a correction of imaging-derived IHF estimates is valid.

Equivalent survival in patients with and without steatosis undergoing resection for colorectal liver metastases following pre-operative chemotherapy.

BACKGROUND: We previously reported that the presence of steatosis did not adversely influence survival in patients undergoing resection for colorectal liver metastases (CLM) without pre-operative chemotherapy. Here, this hypothesis is tested in patients undergoing resection for CLM following pre-operative chemotherapy. METHODS: We assessed the effects of background liver pathology, categorized as 'normal', 'steatosis' and 'other', on perioperative mortality, overall survival (OS) and cancer-specific survival (CSS) in LiverMetSurvey patients. Survival analyses included log-rank tests and multivariate Cox models, incorporating well-established prognosticators. In secondary analyses, re-populating the model with non-chemotherapy patients, the effect modification of chemotherapy on the impact of steatosis on survival was tested. RESULTS: Of 4329 patients undergoing first-time liver resection following pre-operative chemotherapy, histologies were normal in 1913 (44%), steatosis in 1675 (39%), and other abnormal pathologies in 741 (17%). For normal, steatosis and other, 90-day mortalities were 2.1%, 2.3%, and 3.5% (P = 0.103). For the three histo-pathological groups, 5-year OS rates were 39%, 42%, and 36% (Plogrank = 0.363); 5-year CSS rates were 43%, 45% and 41% (Plogrank = 0.496), respectively. The associations of steatosis with OS and CSS were materially unchanged in the multivariate models. Chemotherapy did not interact with the effect of steatosis on survival. CONCLUSION: The findings of equivalent survivals challenge the common perception that steatosis in CLM patients after pre-operative chemotherapy is associated with increased peri-operative mortality and poorer long-term survival.

Differential effects of glycosphingolipids on the detergent-insolubility of the glycosylphosphatidylinositol-anchored membrane dipeptidase.

The insolubility of glycosylphosphatidylinositol (GPI)-anchored proteins in certain detergents appears to be an intrinsic property of their association with sphingolipids and cholesterol in lipid rafts. We show that the GPI-anchored protein membrane dipeptidase is localized in detergent-insoluble lipid rafts isolated from porcine kidney microvillar membranes, and that these rafts, which lack caveolin, are enriched not only in sphingomyelin and cholesterol, but also in the glycosphingolipid lactosylceramide (LacCer). Dipeptidase purified from porcine kidney was reconstituted into artificial liposomes in order to investigate the relationship between glycosphingolipids and GPI-anchored protein detergent-insolubility. Dipeptidase was insoluble in liposomes containing extremely low concentrations of LacCer. In contrast, identical concentrations of glucosylceramide or galactosylceramide failed to promote significant detergent-insolubility. Cholesterol was shown to enhance the detergent-insoluble effect of LacCer. GC-MS analysis revealed dramatic differences between the fatty acyl compositions of LacCer and those of the other glycosphingolipids. However, despite these differences, we show that the unusually marked effect of LacCer to promote the detergent-insolubility of dipeptidase cannot be singularly attributed to the fatty acyl composition of this glycosphingolipid molecule. Instead, we suggest that the ability of LacCer to confer detergent-insolubility on this GPI-anchored protein is dependent on the structure of the lipid molecule in its entirety, and that this glycosphingolipid may have an important role to play in the stabilization of lipid rafts, particularly the caveolin-free glycosphingolipid signalling domains.

A point mutation in the juxtamembrane stalk of human angiotensin I-converting enzyme invokes the action of a distinct secretase.

Angiotensin I-converting enzyme (ACE) is one of a number of integral membrane proteins that is proteolytically shed from the cell surface by a zinc metallosecretase. Mutagenesis of Asn(631) to Gln in the juxtamembrane stalk region of ACE resulted in more efficient secretion of the mutant protein (ACE(NQ)) as determined by pulse-chase analysis. In contrast to the wild-type ACE, the cleavage of ACE(NQ) was not blocked by the metallosecretase inhibitor batimastat but by the serine protease inhibitor, 1,3-dichloroisocoumarin. Incubation of the cells at 15 degrees C revealed that ACE(NQ) was cleaved in the endoplasmic reticulum, and mass spectrometric analysis of the secreted form of the protein indicated that it had been cleaved at the Asn(635)-Ser(636) bond, three residues N-terminal to the normal secretase cleavage site at Arg(638)-Ser(639). These data clearly show that a point mutation in the juxtamembrane region of an integral membrane protein can invoke the action of a mechanistically and spatially distinct secretase. In light of this observation, previous data on the effect of mutations in the juxtamembrane stalk of shed proteins being accommodated by a single secretase having a relaxed specificity need to be re-evaluated.

The role of proteolysis in Alzheimer's disease.

Alzheimer's disease is characterised by the progressive deposition of the 4 kDa beta-amyloid peptide (A beta) in extracellular senile plaques in the brain. A beta is derived by proteolytic cleavage of the amyloid precursor protein (APP) by various proteinases termed secretases. alpha-Secretase is inhibited by hydroxamate-based zinc metalloproteinase inhibitors such as batimastat with I50 values in the low micromolar range, and displays many properties in common with the secretase that releases angiotensin converting enzyme. A cell impermeant biotinylated derivative of one such inhibitor completely blocked the release of APP from the surface of neuronal cells, indicating that alpha-secretase cleaves APP at the cell-surface. A range of hydroxamate-based compounds have been used to distinguish between alpha-secretase and tumour necrosis factor-alpha convertase, a member of the ADAMs (a disintegrin and metalloproteinase-like) family of zinc metalloproteinases. Recent data suggests that the presenilins may be aspartyl proteinases with the specificity of gamma-secretase. Although APP and the presenilins are present in detergent-insoluble, cholesterol- and glycosphingolipid-rich lipid rafts, they do not behave as typical lipid raft proteins, and thus it is unclear whether these membrane domains are the sites for proteolytic processing of APP.

Distribution of Presenilins and Amyloid Precursor Protein (APP) in Detergent-Insoluble Membrane Domains.

Until recently, the detergent insolubility of certain membrane-associated proteins was singularly attributed to an association with the cytoskeleton. However, in 1988 we observed that a number of glycosyl-phosphatidylinositol (GPI)-anchored proteins were resistant to solubilization by nonionic detergents such as Triton X-100 (1). This detergent insolubility is acquired as the proteins pass through the endoplasmic reticulum and on to the Golgi apparatus (2), and arises not from a direct interaction of the GPI-anchored proteins with cytoskeletal elements but as a result of the specific lipid composition of the membrane domains with which these proteins associate (3,4). Mammalian cell membranes contain hundreds of individual lipid species which can be grouped under several major headings (e.g., glycerophospholipids, sphingomyelins, ceramides, glycosphingolipids, and cholesterol) (2,5,6). Glycerophospholipids, such as phosphatidylcholine and phosphatidylethanolamine, predominate in the membrane milieu. Consequently, the bulk of the cell membrane is fluid and in a continual state of flux. However, the membrane domains with which GPI-anchored proteins associate are enriched with sphingolipids and cholesterol, making them less fluid than the membrane milieu (2,4). Such membrane domains have been referred to as "lipid rafts" (7) and there has been some controversy as to whether they exist in vivo or whether they form as an artefact of the procedures employed in their isolation (8). However, recent studies in both artificial lipid bilayers and living cell membranes using such techniques.

Amyloid precursor protein, although partially detergent-insoluble in mouse cerebral cortex, behaves as an atypical lipid raft protein.

Lipid rafts are regions of the plasma membrane that are enriched in cholesterol, glycosphingolipids and acylated proteins, and which have been proposed as sites for the proteolytic processing of the Alzheimer's amyloid precursor protein (APP). Lipid rafts can be isolated on the basis of their insolubility in Triton X-100 at 4 degrees C, with the resulting low-density, detergent-insoluble glycolipid-enriched fraction (DIG) being isolated by flotation through a sucrose density gradient. The detergent-insolubility of APP in mouse cerebral cortex relative to a variety of DIG marker proteins (alkaline phosphatase, flotillin, F3 protein and prion protein) and non-DIG proteins (alkaline phosphodiesterase I, aminopeptidase A and clathrin) has been examined. Alkaline phosphatase, flotillin, F3 protein and the prion protein were present exclusively in the DIG region of the sucrose gradient over a range of protein/detergent ratios used to solubilize the membranes and displayed a characteristic enrichment in the low-density fraction as the protein/detergent ratio was decreased. In contrast, most of the APP, alkaline phosphodiesterase I, aminopeptidase A and clathrin was effectively solubilized at all of the protein/detergent ratios examined. However, a minor proportion of these latter proteins was detected in DIGs at levels which remained constant irrespective of the protein/detergent ratio. When DIGs were isolated from the sucrose gradients and treated with excess Triton X-100, both the DIG marker proteins and APP, alkaline phosphodiesterase I and clathrin were predominantly resistant to detergent extraction at 37 degrees C. These results show that, although a minor proportion of APP is present in DIGs, where it is detergent-insoluble even at 37 degrees C, it behaves as an atypical lipid raft protein and raises questions as to whether lipid rafts are a site for its proteolytic processing.

Control of motor seizures by brotizolam with maintenance of stable refractory periods for self-stimulation.

In recent years, we have been pursuing our mapping investigations of the substrate for brain-stimulation reward in regions of the anterior hypothalamic and lateral preoptic areas. However, one problem is that stimulation of these sites often generates overt seizures so that their suppression via a pharmacological means would be very useful. The sedative-hypnotic benzodiazepine, brotizolam, is reportedly a long-lasting anticonvulsant. Hence, its effects on motor seizures elicited from stimulation of the lateral preoptic area were evaluated in the first experiment. Both tested doses (5.0 and 7.5 mg/kg) of the drug were shown to significantly decrease the number, and marginally, the severity of stimulation-induced seizures; furthermore, this effect was relatively long lasting, up to about 3 h. The higher dose of brotizolam did not alter the single-pulse thresholds for self-stimulation, a requirement for evaluations of poststimulation excitability, the purpose of the second experiment. Here, our interest was in documenting whether the membrane properties of the stimulated neurons, as assessed by refractory periods, were altered by brotizolam. No differences in the time course of recovery were observed; refractoriness began between 0.4 and 0.8 ms, and reached 50% recovery by 2.0 ms, which is consistent with the pattern of poststimulation excitability typically measured at these sites. Thus, in addition to its long-lasting suppression of motor seizures in rats, brotizolam does not alter the time course of recovery from refractoriness of the neurons that mediate brain-stimulation reward in the lateral preoptic area.

Characterization of detergent-insoluble complexes containing the familial Alzheimer's disease-associated presenilins.

Many cases of early-onset familial Alzheimer's disease have been linked to mutations within two genes encoding the proteins presenilin-1 and presenilin-2. The presenilins are 48-56-kDa proteins that can be proteolytically cleaved to generate an N-terminal fragment (approximately 25-35 kDa) and a C-terminal fragment (approximately 17-20 kDa). The N- and C-terminal fragments of presenilin-1, but not full-length presenilin-1, were readily detected in both human and mouse cerebral cortex and in neuronal and glioma cell lines. In contrast, presenilin-2 was detected almost exclusively in cerebral cortex as the full-length molecule with a molecular mass of 56 kDa. The association of the presenilins with detergent-insoluble, low-density membrane microdomains, following the isolation of these structures from cerebral cortex by solubilization in Triton X-100 and subsequent sucrose density gradient centrifugation, was also examined. A minor fraction (10%) of both the N- and C-terminal fragments of presenilin-1 was associated with the detergent-insoluble, low-density membrane microdomains, whereas a considerably larger proportion of full-length presenilin-2 was present in the same membrane microdomains. In addition, a significant proportion of full-length presenilin-2 was present in a high-density, detergent-insoluble cytoskeletal pellet enriched in beta-actin. The presence of the presenilins in detergent-insoluble, low-density membrane microdomains indicates a possible role for these specialized regions of the membrane in the lateral separation of Alzheimer's disease-associated proteins within the lipid bilayer and/or in the distinct functions of these proteins.

Modulation of phosphatidylcholine biosynthesis in celery by exogenous fatty acids.

The effects of C16 and C18 fatty acids on the synthesis of phosphatidylcholine were studied in Apium graveolens cell suspension cultures and postmitochondrial supernatants. When cells were exposed to exogenous oleic acid, the rate of phosphatidylcholine biosynthesis increased 1.4-fold within 5 min of the addition of the fatty acid to the culture medium. The sensitivity of microsomal CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) to saturated and unsaturated fatty acids was monitored through the addition of unesterified fatty acids to postmitochondrial supernatants. The saturated fatty acids, palmitic and stearic, appeared to have little effect on CTP:cholinephosphate cytidylyltransferase activity, whereas exposure to oleic, linoleic and cis-vaccenic acids resulted in significant increases in enzyme activity. Optimal microsomal CTP:cholinephosphate cytidylyltransferase activities were achieved by the incubation of postmitochondrial supernatants with 500 microM oleate. The exogenous fatty acids were found to be incorporated into microsomal membranes in their unesterified form. Removal of unesterified fatty acids by incubation of microsomal membranes with defatted bovine serum albumin resulted in the reduction of microsomal CTP:cholinephosphate cytidylyltransferase activity; demonstrating that the enzyme requires unesterified unsaturated fatty acids.

The amyloid precursor protein is not enriched in caveolae-like, detergent-insoluble membrane microdomains.

The amyloid precursor protein may be processed by several different pathways, one of which produces the amyloid beta-peptide betaA4 present in the amyloid plaques characteristic of Alzheimer's disease. A recent report suggested that axonal-amyloid precursor protein is present in a membrane fraction "with caveolae-like properties." In the present study we have isolated detergent-insoluble, caveolae-like membranes from both mouse cerebellum and the human neuroblastoma cell line SH-SY5Y. Detergent-insoluble membranes from mouse cerebellum retained nearly all of the glycosylphosphatidylinositol-anchored proteins--alkaline phosphatase, 5'-nucleotidase, and the F3 protein--while excluding the majority of the plasmalemmal marker protein alkaline phosphodiesterase I. Although the inositol trisphosphate receptor was highly enriched in this detergent-insoluble fraction, neither amyloid precursor protein nor clathrin immunoreactivity could be detected. Similar results were obtained with SH-SY5Y cells, where 5'-nucleotidase activity was enriched at least 30-fold in the detergent-insoluble membranes, but no amyloid precursor protein or clathrin immunoreactivity could be detected. Caveolin could not be detected in microsomal membranes from either mouse cerebellum or SH-SY5Y cells. These observations suggest that amyloid precursor protein is not normally present in detergent-insoluble, caveolae-like membrane microdomains.

Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes.

The Triton-insoluble complex from porcine lung membranes has been separated into two distinct subfractions visible as discrete light-scattering bands following buoyant density-gradient centrifugation in sucrose. Both of these detergent-insoluble complexes were enriched in the glycosyl-phosphatidylinositol (GPI)-anchored ectoenzymes alkaline phosphatase, aminopeptidase P and 5'-nucleotidase, and both complexes excluded the polypeptide-anchored ectoenzymes angiotensin-converting enzyme, dipeptidyl peptidase IV and aminopeptidases A and N. The GPI-anchored proteins in both complexes were susceptible to release by phosphatidylinositol-specific phospholipase C. Both complexes were also enriched in cholesterol and glycosphingolipids, and in caveolin/VIP21, although only the higher-density fraction was enriched in the plasmalemmal caveolar marker proteins Ca(2+)-ATPase and the inositol 1,4,5-trisphosphate receptor. Among the annexin family of proteins, annexins I and IV were absent from the two detergent-insoluble complexes, annexin V was present in both, and annexins II and VI were only enriched in the higher-density fraction. When the mental chelator EGTA was present in the isolation buffers, annexins II and VI dissociated from the higher-density detergent-insoluble complex and only a single light-scattering band was observed on the sucrose gradient, at the same position as for the lower-density complex. In contrast, in the presence of excess calcium only a single detergent-insoluble complex was isolated from the sucrose gradients, at an intermediate density. Thus the detergent-insoluble membrane complex can be subfractionated on the basis of what appears to be calcium-dependent, annexin-mediated, vesicle aggregation into two distinct populations, only one of which is enriched in plasmalemmal caveolar marker proteins.