Clinical risk factors of colorectal cancer in patients with serrated polyposis syndrome: a multicentre cohort analysis.

OBJECTIVE: Serrated polyposis syndrome (SPS) is accompanied by an increased risk of colorectal cancer (CRC). Patients fulfilling the clinical criteria, as defined by the WHO, have a wide variation in CRC risk. We aimed to assess risk factors for CRC in a large cohort of patients with SPS and to evaluate the risk of CRC during surveillance. DESIGN: In this retrospective cohort analysis, all patients with SPS from seven centres in the Netherlands and two in the UK were enrolled. WHO criteria were used to diagnose SPS. Patients who only fulfilled WHO criterion-2, with IBD and/or a known hereditary CRC syndrome were excluded. RESULTS: In total, 434 patients with SPS were included for analysis; 127 (29.3%) were diagnosed with CRC. In a per-patient analysis ≥1 serrated polyp (SP) with dysplasia (OR 2.07; 95% CI 1.28 to 3.33), ≥1 advanced adenoma (OR 2.30; 95% CI 1.47 to 3.67) and the fulfilment of both WHO criteria 1 and 3 (OR 1.60; 95% CI 1.04 to 2.51) were associated with CRC, while a history of smoking was inversely associated with CRC (OR 0.36; 95% CI 0.23 to 0.56). Overall, 260 patients underwent surveillance after clearing of all relevant lesions, during which two patients were diagnosed with CRC, corresponding to 1.9 events/1000 person-years surveillance (95% CI 0.3 to 6.4). CONCLUSION: The presence of SPs containing dysplasia, advanced adenomas and/or combined WHO criteria 1 and 3 phenotype is associated with CRC in patients with SPS. Patients with a history of smoking show a lower risk of CRC, possibly due to a different pathogenesis of disease. The risk of developing CRC during surveillance is lower than previously reported in literature, which may reflect a more mature multicentre cohort with less selection bias.

The effects of sarcolipin over-expression in mouse skeletal muscle on metabolic activity.

Studies in sarcolipin knockout mice have led to the suggestion that skeletal muscle sarcolipin plays a role in thermogenesis. The mechanism proposed is uncoupling of the sarcoplasmic reticulum calcium pump. However, in other work sarcolipin was not detected in mouse skeletal tissue. We have therefore measured sarcolipin levels in mouse skeletal muscle using semi-quantitative western blotting and synthetic mouse sarcolipin. Sarcolipin levels were so low that it is unlikely that knocking out sarcolipin would have a measurable effect on thermogenesis by SERCA. In addition, overexpression of neither wild type nor FLAG-tagged variants of mouse sarcolipin in transgenic mice had any major significant effects on body mass, energy expenditure, even when mice were fed on a high fat diet.

Impact of the consideration of serrated polyps to the interval of colonoscopic surveillance in the NHS Bowel Cancer Screening Programme.

AIM: Most international post polypectomy surveillance guidelines do not recommend surveillance for serrated polyps. In the present study the additional impact of serrated polyps on surveillance intervals from international adenoma surveillance guidelines was investigated. METHOD: Endoscopic and pathology records were audited of participants in the NHS Bowel Cancer Screening Programme (guaiac faecal occult blood test, gFOBT) in 2011. Surveillance intervals were calculated for current guidelines and also for serrated polyps based on previously described aggressive and conservative strategies. RESULTS: In total, 389 patients were included of whom 141 (36.2%) were high risk (advanced adenoma: adenoma ≥ 10 mm, villous elements, high grade dysplasia, or adenoma ≥ 3 in number) needing surveillance at ≤ 3 years. Thirty-three (8.5%) had significant serrated polyps, of whom 18 (4.6% of the total) had significant serrated lesions and simultaneous advanced adenoma or ≥ 3 adenomas. Adopting an aggressive surveillance strategy, the mean overall absolute additional proportion of all such patients in the surveillance group at 3 years or less was 4.0% (3.9% - 4.1%; 4.2% women; 3.8% men). These proportions varied according to endoscopist from 2.3% to 4.7%. For more conservative strategies the increase was only 1%. CONCLUSION: The impact of including serrated polyps in current guidelines would result in a small increase in surveillance intervals for FOBT based bowel cancer screening. About half of those who might need surveillance for serrated polyps would already receive surveillance for being in a high risk adenoma group.

Ratios derived from an array of standard haematological indices predict the oncological outcome in colon cancer.

AIM: The interaction between inflammation and cancer is well established. Surrogate markers of systemic inflammation, such as the neutrophil/lymphocyte ratio (NLR), may be associated with the long-term oncological outcome. The present study aimed to characterize the relationship between several ratios derived from haematological indices using a classification and regression tree analysis. METHOD: Haematological white-cell ratios were established for all patients undergoing colonic cancer resection with curative intent (n = 436) in a regional cancer centre. The optimal ratios associated with overall survival (OS) were established in a training set (n = 386) using a classification and regression tree (CRT) technique. The association between ratios and OS was assessed in a separate test set (n = 50). Within the test set, two groups were generated based on each ratio (one group above and one group below the cut-off value identified in the training set). The association between ratios and OS was assessed using a stepwise Cox proportional-hazards regression model. RESULTS: The following ratios, identified by the CRT, were associated with adverse OS in the test set: an NLR of ≥ 3.4 [hazard ratio (HR) = 3.4, P < 0.001]; and a white-cell-count/lymphocyte ratio (WLR) of ≥ 5.28 (HR = 4.1, P = 0.03). CONCLUSION: This is the first study to apply recursive partitioning in determining the relationship between haematological ratios and OS in colon cancer. Haematological ratios were predictive of oncological outcome. What does this paper add to the literature? This study suggests an association between systemic inflammation and oncological outcome.

Effects of lipid structure on the state of aggregation of potassium channel KcsA.

The state of aggregation of potassium channel KcsA was determined as a function of lipid:protein molar ratio in bilayer membranes of the zwitterionic lipid phosphatidylcholine (PC) and of the anionic lipid phosphatidylglycerol (PG). EPR (electron paramagnetic resonance) with spin-labeled phospholipids was used to determine the number of motionally restricted lipids per KcsA tetramer. Unexpectedly, this number decreased with a decreasing lipid:KcsA tetramer molar ratio in the range of 88:1 to 30:1, consistent with sharing of annular lipid shells and KcsA-KcsA contact at high mole fractions of protein. Fluorescence quenching experiments with brominated phospholipids showed a decrease in fluorescence quenching at low lipid:KcsA tetramer mole ratios, also consistent with KcsA-KcsA contact at high mole fractions of protein. The effects of low mole ratios of lipid seen in EPR and fluorescence quenching experiments were more marked in bilayers of PC than in bilayers of PG, suggesting stronger association of PG than PC with KcsA. This was confirmed by direct measurement of lipid association constants using spin-labeled phospholipids, showing higher association constants for all anionic lipids than for PC. The results show that the probability of contacts between KcsA tetramers will be very low at lipid:protein molar ratios that are typical of native biological membranes.

Characterizing the fatty acid binding site in the cavity of potassium channel KcsA.

We show that interactions of fatty acids with the central cavity of potassium channel KcsA can be characterized using the fluorescence probe 11-dansylaminoundecanoic acid (Dauda). The fluorescence emission spectrum of Dauda bound to KcsA in bilayers of dioleoylphosphatidylcholine contains three components, which can be attributed to KcsA-bound and lipid-bound Dauda together with unbound Dauda. The binding of Dauda to KcsA was characterized by a dissociation constant of 0.47 ± 0.10 µM with 0.94 ± 0.06 binding site per KcsA tetramer. Displacement of KcsA-bound Dauda by the tetrabutylammonium (TBA) ion confirmed that the Dauda binding site was in the central cavity of KcsA. Dissociation constants for a range of fatty acids were determined by displacement of Dauda: binding of fatty acids increased in strength with an increasing chain length from C14 to C20 but then decreased in strength from C20 to C22. Increasing the number of double bonds in the chain from one to four had little effect on binding, dissociation constants for oleic acid and arachidonic acid, for example, being 2.9 ± 0.2 and 3.0 ± 0.4 µM, respectively. Binding of TBA to KcsA was very slow, whereas binding of Dauda was fast, suggesting that TBA can enter the cavity only through an open channel whereas Dauda can bind to the closed channel, presumably entering the cavity via the lipid bilayer.

Multiple binding sites for fatty acids on the potassium channel KcsA.

Interactions of fatty acids with the potassium channel KcsA were studied using Trp fluorescence quenching and electron paramagnetic resonance (EPR) techniques. The brominated analogue of oleic acid was shown to bind to annular sites on KcsA and to the nonannular sites at each protein-protein interface in the homotetrameric structure with binding constants relative to dioleoylphosphatidylcholine of 0.67 ± 0.04 and 0.87 ± 0.08, respectively. Mutation of the two Arg residues close to the nonannular binding sites had no effect on fatty acid binding. EPR studies with a spin-labeled analogue of stearic acid detected a high-affinity binding site for the fatty acid with strong immobilization. Fluorescence quenching studies with the spin-labeled analogue showed that the binding site detected in the EPR experiments could not be one of the annular or nonannular binding sites. Instead, it is proposed that the EPR studies detect binding to the central hydrophobic cavity of the channel, with a binding constant in the range of ~0.1-1 µM.

A randomized, controlled trial of narrow-band imaging vs high-definition white light for adenoma detection in patients at high risk of adenomas.

AIM: The study aimed to investigate whether narrow-band imaging (NBI) can enhance adenoma detection in patients at high risk for adenomas compared with high-definition white-light endoscopy (WLE). High risk was defined as three or more adenomas at last colonoscopy, history of colorectal cancer and positive faecal occult blood test. METHOD: Two hundred and fourteen patients were randomized 1:1 to examination with NBI or WLE. The primary outcome measure was the proportion of patients with at least one adenoma detected. Secondary outcomes included total adenomas and polyps, flat adenomas, nonadenomatous polyps, advanced adenomas and patients with three or five or more adenomas. A post hoc analysis to examine the effect of endoscopist and bowel preparation was performed. RESULTS: There was no significant difference in the proportion of patients with at least one adenoma: NBI 73%vs WLE 66%, odds ratio 1.40 (95% CI 0.78-2.52), P = 0.26. There was no significant difference for any secondary outcome measure except for the number of flat adenomas which was significantly greater with NBI [comparison ratio 2.66 (95% CI 1.52-4.63), P = 0.001]. Post hoc analysis indicated that one of three endoscopists performed significantly better for adenoma detection with NBI than WLE [comparison ratio 1.92 (95% CI 1.07-3.44), P = 0.03]. Good bowel preparation was associated with significantly improved adenoma detection with NBI [comparison ratio 1.55 (95% CI 1.01-2.22), P = 0.04] but not with fair preparation. CONCLUSION: Overall NBI did not improve detection compared with WLE in a group of patients at high risk for colorectal adenomas, but specific subgroups might benefit.

Achieving high quality colonoscopy: using graphical representation to measure performance and reset standards.

AIM: Completeness and thoroughness of colonoscopy are measured by the caecal intubation rate (CIR) and the adenoma detection rate (ADR). National standards are ≥ 90% and ≥ 10% respectively. Variability in CIR and ADR have been demonstrated but comparison between individuals and units is difficult. We aimed to assess the performance of colonoscopy in endoscopy units in the northeast of England. METHOD: Data on colonoscopy performance and sedation use were collected over 3 months from 12 units. Colonoscopies performed by screening colonoscopists were included for the CIR only. Funnel plots with upper and lower 95% confidence limits for CIR and ADR were created. RESULTS: CIR was 92.5% (n = 5720) and ADR 15.9% (n = 4748). All units and 128 (99.2%) colonoscopists were above the lower limit for CIR. All units achieved the ADR standard with 10 above the upper limit. Ninety-nine (76.7%) colonoscopists were above 10%, 16 (12.4%) above the upper limit and 7 (5.4%) below the lower limit. Median medication doses were 2.2 mg midazolam, 29.4 mg pethidine and 83.3 µg fentanyl. In all, 15.1% of colonoscopies were unsedated. Complications were bleeding (0.10%) and perforation (0.02%). There was one death possibly related to bowel preparation. CONCLUSION: Results indicate that colonoscopies are performed safely and to a high standard. Funnel plots can highlight variability and areas for improvement. Analyses of ADR presented graphically around the global mean suggest that the national standard should be reset at 15%.

The localization of the ER retrieval sequence for the calcium pump SERCA1.

A number of studies using chimeric constructs made by fusing endoplasmic/sarcoplasmic reticulum calcium pump (SERCA) sequences with those of the plasma membrane located calcium pump (PMCA) have suggested that the retention/retrieval signal responsible for maintaining SERCA in the endoplasmic reticulum (ER) is located within the N-terminus of these pumps. Because of the difficulties in identifying the presence of constructs at the plasma membrane we have used a trans-Golgi network (TGN) marker to evaluate whether chimeric proteins are retained by the ER or have lost their retention/retrieval sequences and are able to enter the wider endomembrane system and reach the TGN. In this study, attempts to locate this retention/retrieval sequence demonstrate that the retention sequences are located not in the N-terminus, as previously suggested, but in the largely transmembranous C-terminal domain of SERCA. Further attempts to identify the precise retention/retrieval motif using SERCA1/PMCA3 chimeras were unsuccessful. This may be due to the fact that introducing SERCA1 sequences into the C-terminal PMCA3 sequence and vice versa disrupts the organization of the closely packed transmembrane helices leading to retention of such constructs by the quality control mechanisms of the ER. An alternative explanation is that SERCAs have targeting motifs that are non-linear, being made up of several segments of sequence to form a patch that interacts with the retrieval machinery.

Retrieval from the ER-golgi intermediate compartment is key to the targeting of c-terminally anchored ER-resident proteins.

Endoplasmic reticulum (ER) resident proteins may be maintained in the ER by retention, where the leak into post-ER compartments is absent or slow, or retrieval, where a significant leak is countered by retrieval from post-ER compartments. Here the targeting of the C-terminally anchored protein ER-resident protein, cytochrome b5a (cytb5a), considered to be maintained in the ER mainly by the process of retention, is compared with that of sarcolipin (SLN) and phospholamban (PLB); also C-terminally anchored ER-residents. Laser confocal microscopy, and cell fractionation of green fluorescent protein-tagged constructs expressed in COS 7 cells indicate that while calnexin appears to be retained in the ER with no evidence of leak into the ER-Golgi intermediate compartment (ERGIC), significant amounts of cytb5a, SLN, and PLB are detectable in the ERGIC, indicating that there is considerable leak from the ER. This is supported by an in vitro budding assay that shows that while small amounts of calnexin appear in the transport vesicles budding off from the ER, significant amounts of cytb5a and SLN are found in such vesicles. These data support the hypothesis that retrieval plays a major role in ensuring that C-terminally anchored proteins are maintained in the ER.

Recent advances in Membrane Biochemistry.

This Biochemical Society Annual Symposium on Recent Advances in Membrane Biochemistry was organized to bring together experts from across the spectrum of biomembrane disciplines from the biological to the biophysical/structural, with the intention of promoting interactions and collaborations across the field. We were keen that the potential for improving human health that stems from a deeper understanding of membrane structure/function should be acknowledged, especially in the light of the increasing numbers of membrane protein structures that continue to be made available to the biomembrane community. This foreword provides an idea of what was communicated in the various sessions and, we hope, gives an impression of the excitement generated by the speakers and delegates at this over-subscribed Symposium.

A diversity of SERCA Ca2+ pump inhibitors.

The SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) is probably the most extensively studied membrane protein transporter. There is a vast array of diverse inhibitors for the Ca2+ pump, and many have proved significant in helping to elucidate both the mechanism of transport and gaining conformational structures. Some SERCA inhibitors such as thapsigargin have been used extensively as pharmacological tools to probe the roles of Ca2+ stores in Ca2+ signalling processes. Furthermore, some inhibitors have been implicated in the cause of diseases associated with endocrine disruption by environmental pollutants, whereas others are being developed as potential anticancer agents. The present review therefore aims to highlight some of the wide range of chemically diverse inhibitors that are known, their mechanisms of action and their binding location on the Ca2+ ATPase. Additionally, some ideas for the future development of more useful isoform-specific inhibitors and anticancer drugs are presented.

What is the most reliable imaging modality for small colonic polyp characterization? Study of white-light, autofluorescence, and narrow-band imaging.

BACKGROUND AND STUDY AIMS: In vivo optical diagnosis of small colorectal polyps has potential clinical and cost advantages, but requires accuracy and high interobserver agreement for clinically acceptability. We aimed to assess interobserver variability and diagnostic performance of endoscopic imaging modalities in characterizing small colonic polyps. METHODS: High quality still images of 80 polyps < 1 cm were recorded using white-light endoscopy (WLE), autofluorescence imaging (AFI) and narrow-band imaging with and without magnification (NBI and NBImag). All images were assessed for quality, prediction of polyp histology, and vascular pattern intensity (with NBI) by nine experienced colonoscopists (four experts in advanced imaging) from five UK centers. Interobserver agreement (kappa statistic), sensitivity, specificity, and accuracy were calculated compared with histopathological findings. RESULTS: Interobserver agreement for predicting polyp histology using NBImag was significantly better for experts (κ = 0.63, substantial) compared with nonexperts (κ = 0.30, fair; P < 0.001), and was moderate for all colonoscopists with WLE, AFI and NBI. Interobserver agreement for vascular pattern intensity using NBI was 0.69 (substantial) for experts and 0.57 (good) for nonexperts. NBImag had higher sensitivity than WLE (experts, 0.93 vs. 0.68, P < 0.001; nonexperts, 0.90 vs. 0.52, P < 0.001) and higher overall accuracy (experts, 0.76 vs. 0.64, P = 0.003; nonexperts 0.61 vs. 0.40, P < 0.001). AFI had worse accuracy than WLE for both expert colonoscopists (0.53 vs. 0.64, P = 0.02) and nonexperts (0.32 vs. 0.40, P = 0.04). CONCLUSIONS: Of the imaging modalities tested, NBImag appeared to have the best overall accuracy and interobserver agreement, although not adequate for in vivo diagnosis. NBI and AFI did not have better sensitivity, specificity, or accuracy compared with WLE.

Faecal immunochemical testing (FIT): sources of result variation based on three years of routine testing of symptomatic patients in English primary care.

Introduction: We aimed to determine the analytical capabilities of a commonly used faecal immunochemical test (FIT) to detect faecal haemoglobin (Hb) in symptomatic people attending primary care in the context of the English NICE DG30 guidance.Materials and Methods: Data obtained from independent verification studies and clinical testing of the HM-JACKarc FIT method in routine primary care practice were analysed to derive performance characteristics.Results: Detection capabilities for the FIT method were 0.5 µg/g (limit of blank), 1.3 µg/g (limit of detection) and 3.0 µg/g (limit of quantitation). Of 33 non-homogenized specimens, 31 (93.9%) analysed in triplicate were consistently categorized relative to 10 µg/g, compared to all 33 (100%) homogenized specimens. Imprecision was higher (median 27.8%, (range 20.5% to 48.6%)) in non-homogenized specimens than in homogenized specimens (10.2%, (7.0 to 13.5%)). Considerable variation was observed in sequential clinical specimens from individual patients but no positive or negative trend in specimen degradation was observed over time (p = 0.26).Discussion: The FIT immunoassay evaluated is capable of detecting faecal Hb at concentrations well below the DG30 threshold of 10 µg/g and is suitable for application in this context. The greatest practical challenge to FIT performance is reproducible sampling, the pre-analytical step associated with most variability. Further research should focus on reducing sampling variability, particularly as post-COVID-19 guidance recommends greater FIT utilization.

Thymus-dependent areas in the lymphoid organs of neonatally thymectomized mice.

Specific areas of lymphocyte depletion, termed thymus-dependent areas, have been delineated in neonatally thymectomized C3H/Bi and F(1) (C57BL x C3H/Bi) mice. They occur within the lymphoid follicles of the spleen immediately surrounding the central arterioles, and constitute the mid and deep cortical zones of the lymph nodes. These depleted areas appear in healthy thymectomized mice as early as 3 wk after operation but, in mice which survive for more than 6 to 7 wk, the thymus-dependent areas are repopulated by rapidly dividing pyroninophilic cells, the majority of which are immature plasma cells. Syngeneic thymus cells, labeled in vitro with tritiated adenosine localize preferentially in the thymus-dependent areas after intravenous injection. Similarly labeled spleen cells also accumulate in these areas but, in addition, are distributed at the periphery of splenic follicles and in the outer cortical zone of the lymph nodes. Many more spleen than thymus cells enter the lymphoid tissues and the spleen appears to be the primary target. The apparent paradox that syngeneic thymus cells are less efficient than spleen cells in restoring neonatally thymectomized mice to normality is discussed in the light of these results and possible routes by which the migrating cells could enter the lymphoid tissues are considered. The origin of the plasma cells which repopulate the lymphocyte depleted areas is also discussed. It is concluded that the normal thymus produces cells which contribute directly to the migratory or circulatory lymphocyte population but that there also exists another source of supply for the plasma cell series. These two systems may function synergistically so that the thymus may control, directly or indirectly, the balance of cell populations within the body.

Retrospective study of technical aspects and complications of endoscopic submucosal dissection for laterally spreading tumors of the colorectum.

BACKGROUND AND STUDY AIMS: Laterally spreading tumors - non granular type (LST-NG) are more often considered candidates for endoscopic submucosal dissection (ESD) than laterally spreading tumors - granular type (LST-G), because of their higher potential for submucosal invasion. However, ESD for LST-NG can be technically difficult. The aim of our study was to compare our ESD results for LST-NG and for LST-G. PATIENTS AND METHODS: Ninety-nine LST-NG and 169 LST-G measuring 20 mm in size or more were removed by ESD. We retrospectively evaluated the clinicopathological features of the tumors and treatment results (en bloc resection rate, procedure time and speed, rate of use of ancillary devices, and complication and recurrence rates). RESULTS: Histopathology revealed that there were more submucosally invasive lesions in the LST-NG than in the LST-G group (28 % vs. 9 %; P < 0.0001). The en bloc resection rate, en bloc R0 resection rate, and en bloc curative resection rate of LST-NG were similar to those of LST-G (LST-NG: 99 %, 98 %, and 88 %; LST-G: 99 %, 98 %, and 91 %). In LST-NG, the median procedure time tended to be longer (LST-NG: 69 min; LST-G: 60 min) and the median procedure speed was slower (LST-NG: 0.15 cm (2)/min; LST-G: 0.25 cm (2)/min; P < 0.0001). Use of ancillary devices was higher for LST-NG (38 % vs. 15 % for LST-G; P < 0.0001), as was the perforation rate (5.1 % vs. 0.6 % for LST-G; P = 0.027). No recurrence was seen in either group. CONCLUSIONS: ESD was an effective treatment method for both LST-NG and LST-G. However, the degree of technical difficulty appears higher for LST-NG than for LST-G lesions, as shown by the lower dissection speed and higher perforation rate. ESD for LST-NG should probably be performed by those with significant experience of colorectal ESD.

Artemisinins target the SERCA of Plasmodium falciparum.

Artemisinins are extracted from sweet wormwood (Artemisia annua) and are the most potent antimalarials available, rapidly killing all asexual stages of Plasmodium falciparum. Artemisinins are sesquiterpene lactones widely used to treat multidrug-resistant malaria, a disease that annually claims 1 million lives. Despite extensive clinical and laboratory experience their molecular target is not yet identified. Activated artemisinins form adducts with a variety of biological macromolecules, including haem, translationally controlled tumour protein (TCTP) and other higher-molecular-weight proteins. Here we show that artemisinins, but not quinine or chloroquine, inhibit the SERCA orthologue (PfATP6) of Plasmodium falciparum in Xenopus oocytes with similar potency to thapsigargin (another sesquiterpene lactone and highly specific SERCA inhibitor). As predicted, thapsigargin also antagonizes the parasiticidal activity of artemisinin. Desoxyartemisinin lacks an endoperoxide bridge and is ineffective both as an inhibitor of PfATP6 and as an antimalarial. Chelation of iron by desferrioxamine abrogates the antiparasitic activity of artemisinins and correspondingly attenuates inhibition of PfATP6. Imaging of parasites with BODIPY-thapsigargin labels the cytosolic compartment and is competed by artemisinin. Fluorescent artemisinin labels parasites similarly and irreversibly in an Fe2+-dependent manner. These data provide compelling evidence that artemisinins act by inhibiting PfATP6 outside the food vacuole after activation by iron.