Osteopontin Levels Are Persistently Elevated for 4 weeks Following Minimally Invasive Colorectal Cancer Resection.

INTRODUCTION: Osteopontin (OPN) is an integrin binding phosphorylated glycoprotein secreted by macrophages and leukocytes that is found in extracellular fluids and sites of inflammation; various forms of CD44 serve as receptors. Osteopontin, expressed by numerous cancers, enhances tumor progression and angiogenesis via the PI3K/AKT and ERK mediated pathways in concert with Vascular Endothelial Growth Factor (VEGF); OPN also plays a role in wound healing. The impact of minimally invasive colorectal resection (MICR) for colorectal cancer (CRC) on plasma OPN levels is unknown. This study's goal was to assess blood levels during the first month after MICR. METHOD: Patients undergoing MICR for CRC who were enrolled in an IRB approved tissue/prospective data bank for whom preoperative, postop Day (POD) 1, POD 3, and at least 1 late postop plasma sample (POD 7-34) were available were studied. Osteopontin levels were determined in duplicate via enzyme linked immunosorbent assay (ELISA) (results reported as mean ± SD). The Wilcoxon signed rank test was used for analysis (significance P < .05). RESULTS: A total of 101 CRC patients (63% colon and 37% rectal) met study criteria. The mean preop OPN level was 89.2 ± 36.8 (ng/ml) for the entire group. Significantly elevated (P < .001) mean plasma levels were detected, vs preop, on POD1 (198.0 ± 67.4; n = 101), POD 3 (186.0 ± 72.6, n = 101), POD 7-13 (154.1 ± 70.2, n = 70), POD14-20 (146.7 ± 53.4, n=32), and POD 21-27 (123.0 ± 56.9, n = 25). No difference was noted at the POD 27-34 timepoint (P > .05). CONCLUSION: Plasma OPN levels are significantly elevated over baseline for a month after MICR for CRC. The early rise in OPN levels may be related to the postop acute inflammatory response. The persistent elevation noted in weeks 2-4, however, may be a manifestation of wound healing in which OPN plays a role. Similar persistent plasma elevations of VEGF, angiopoietin 2 (ANG 2), and 11 other proangiogenic proteins have been noted and, collectively, may promote angiogenesis in residual tumors.

Plasma MMP-2 and MMP-7 levels are elevated first month after surgery and may promote growth of residual metastases.

BACKGROUND: MMP-2 also known as gelatinase A and MMP-7 (matrilysin) are members of the zinc-dependent family of MMPs (Matrix metalloproteinase). MMP-2 and MMP-7 are remodeling enzymes that digest extracellular matrix; MMP-2 is extensively expressed during development and is upregulated at sites of tissue damage, inflammation, and in stromal cells of metastatic tumors. MMP-7 is expressed in the epithelial cells and in a variety of cancers including colon tumors. Plasma MMP-2 and MMP-7 levels were assessed before and after minimally invasive colorectal resection for cancer pathology. AIM: To determine plasma MMP-2 and MMP-7 levels before and after minimally invasive colorectal resection for cancer pathology. METHODS: Patients enrolled in a plasma bank for whom plasma was available were eligible. Plasma obtained from preoperative (Preop) and postoperative blood samples was used. Only colorectal cancer (CRC) patients who underwent elective minimally invasive cancer resection with preop, post-operative day (POD) 1, 3 and at least 1 late postop sample (POD 7-34) were included. Late samples were bundled into 7 d blocks (POD 7-13, 14-20, etc.) and treated as single time points. Plasma MMP-2 and MMP-7 levels were determined via enzyme-linked immunosorbent assay in duplicate. RESULTS: Total 88 minimally invasive CRC resection CRC patients were studied (right colectomy, 37%; sigmoid, 24%; and LAR/AR 18%). Cancer stages were: 1, 31%; 2, 30%; 3, 34%; and 4, 5%. Mean Preop MMP-2 plasma level (ng/mL) was 179.3 ± 40.9 (n = 88). Elevated mean levels were noted on POD1 (214.3 ± 51.2, n = 87, P < 0.001), POD3 (258.0 ± 63.9, n = 80, P < 0.001), POD7-13 (229.9 ± 62.3, n = 65, P < 0.001), POD 14-20 (234.9 ± 47.5, n = 25, P < 0.001), POD 21-27 (237.0 ± 63.5, n = 17, P < 0.001,) and POD 28-34 (255.4 ± 59.7, n = 15, P < 0.001). Mean Preop MMP-7 level was 3.9 ± 1.9 (n = 88). No significant differences were noted on POD 1 or 3, however, significantly elevated levels were noted on POD 7-13 (5.7 ± 2.5, n = 65, P < 0.001), POD 14-20 (5.9 ± 2.5, n = 25, P < 0.001), POD 21-27 (6.1 ± 3.6, n = 17, P = 0.002) and on POD 28-34 (6.8 ± 3.3, n = 15 P < 0.001,) vs preop levels. CONCLUSION: MMP-2 levels are elevated for 5 wk and MMP-7 levels elevated for weeks 2-6. The etiology of these changes in unclear, trauma and wound healing likely play a role. These changes may promote residual tumor growth and metastasis.

Are the curli proteins CsgE and CsgF intrinsically disordered?

Curli are a type of proteinaceous cell surface filament produced by enteric bacteria such as Escherichia and Salmonella that facilitate cell adhesion and invasion, bio-film formation, and environmental persistence. Curli assembly involves 6 proteins encoded by the curli specific genes A, B, C, E, F, and G. Although CsgA is the major structural component of curli, CsgE, and CsgF, are thought to play important chaperone like functions in the assembly of CsgA into curli. Given that some proteins with chaperone like function have been observed to contain disordered regions, sequence analysis and circular dichroism spectroscopy was used to investigate the possibility that structures of CsgE and CsgF were also disordered. Sequence analysis based on charge and hydrophobicity, as well as using the disorder prediction software PONDR, indicates that both proteins have significant regions of disorder. The secondary structure and unfolding, of CsgE and CsgF, analyzed using circular dichroism spectroscopy suggests that both proteins lack a well defined and stable structure. These observations support the hypothesis that the curli assembly proteins CsgE and CsgF are disordered proteins containing intrinsically disordered regions.

Membrane Curvature-sensing and Curvature-inducing Activity of Islet Amyloid Polypeptide and Its Implications for Membrane Disruption.

Islet amyloid polypeptide (IAPP) is a 37-amino acid amyloid protein intimately associated with pancreatic islet ß-cell dysfunction and death in type II diabetes. In this study, we combine spectroscopic methods and microscopy to investigate α-helical IAPP-membrane interactions. Using light scattering and fluorescence microscopy, we observe that larger vesicles become smaller upon treatment with human or rat IAPP. Electron microscopy shows the formation of various highly curved structures such as tubules or smaller vesicles in a membrane-remodeling process, and spectrofluorometric detection of vesicle leakage shows disruption of membrane integrity. This effect is stronger for human IAPP than for the less toxic rat IAPP. From CD spectra in the presence of different-sized vesicles, we also uncover the membrane curvature-sensing ability of IAPP and find that it transitions from inducing to sensing membrane curvature when lipid negative charge is decreased. Our in vivo EM images of immunogold-labeled rat IAPP and human IAPP show both forms to localize to mitochondrial cristae, which contain not only locally curved membranes but also phosphatidylethanolamine and cardiolipin, lipids with high spontaneous negative curvature. Disruption of membrane integrity by induction of membrane curvature could apply more broadly to other amyloid proteins and be responsible for membrane damage observed in other amyloid diseases as well.

Plasma levels of angiopoietin-like protein 4 (ANGPTL4) are significantly lower preoperatively in colorectal cancer patients than in cancer-free patients and are further decreased during the first month after minimally invasive colorectal resection.

BACKGROUND: Surgery has been associated with proangiogenic plasma protein changes that may promote tumor growth. Angiopoietin-like protein 4 (ANGPTL4) is expressed by endothelial cells and other tissues in response to hypoxia. Both intact ANGPTL4 and its partly degraded C-terminal fragment may promote tumor angiogenesis. This study had two purposes: to measure and compare preoperative plasma ANGPTL4 levels in patients with colorectal cancer (CRC) and benign colorectal disease (BCD) and to determine plasma levels after minimally invasive colorectal resection (MICR) for CRC. METHODS: Plasma was obtained from an IRB-approved plasma/data bank. Preoperative plasma ANGPTL4 levels were measured for CRC and BCD patients, but postoperative levels were determined only for CRC patients for whom a preoperative, a postoperative day (POD) 3, and at least one late postoperative sample (POD 7-55) were available. Late samples were bundled into four time blocks and considered as single time points. ANGPTL4 levels (mean ± SD) were measured via ELISA and compared (significance, p < 0.01 after Bonferroni correction). RESULTS: Eighty CRC (71 % colon, 29 % rectal) and 60 BCD (62 % diverticulitis, 38 % adenoma) patients were studied. The mean preoperative plasma ANGPTL4 level in CRC patients (247.2 ± 230.7 ng/ml) was lower than the BCD group result (330.8 ± 239.0 ng/ml, p = 0.01). There was an inverse relationship between plasma levels and advanced CRC as judged by three criteria. In regard to the postoperative CRC analysis, the "n" for each time point varied: lower plasma levels (p < 0.001) were noted on POD 3 (161.4 ± 140.4 ng/ml, n = 80), POD 7-13 (144.6 ± 134.5 ng/ml, n = 46), POD 14-20 (139.0 ± 117.8 ng/ml, n = 27), POD 21-27 (138.9 ± 202.4, n = 20), and POD 28-55 (160.1 ± 179.0, n = 42) when compared to preoperative results. CONCLUSION: CRC is associated with lower preoperative plasma ANGPTL4 levels compared with BCD, and the levels may vary inversely with disease severity. After MICR for CRC, levels are significantly lower for over a month compared with the preoperative level; the cause for this persistent decrease is unclear. The implications of both the lower preoperative level and the persistently decreased postoperative levels are unclear. Further studies are needed.

Structure of alpha-helical membrane-bound human islet amyloid polypeptide and its implications for membrane-mediated misfolding.

Human islet amyloid polypeptide (hIAPP) misfolding is thought to play an important role in the pathogenesis of type II diabetes mellitus. It has recently been shown that membranes can catalyze the misfolding of hIAPP via an alpha-helical intermediate of unknown structure. To better understand the mechanism of membrane-mediated misfolding, we used site-directed spin labeling and EPR spectroscopy to generate a three-dimensional structural model of this membrane-bound form. We find that hIAPP forms a single alpha-helix encompassing residues 9-22. The helix is flanked by N- and C-terminal regions that do not take up a clearly detectable secondary structure and are less ordered. Residues 21 and 22 are located in a transitional region between the alpha-helical structure and C terminus and exhibit significant mobility. The alpha-helical structure presented here has important implications for membrane-mediated aggregation. Anchoring hIAPP to the membrane not only increases the local concentration but also reduces the encounter between peptides to essentially a two-dimensional process. It is significant to note that the alpha-helical membrane-bound form leaves much of an important amyloidogenic region of hIAPP (residues 20-29) exposed for misfolding. Misfolding of this and other regions is likely further aided by the low dielectric environment near the membrane that is known to promote secondary structure formation. Based upon these considerations, a structural model for membrane-mediated aggregation is discussed.

CT scan findings in oesophagogastric perforation after out of hospital cardiopulmonary resuscitation.

Rupture of the oesophagus or stomach at the time of cardiopulmonary resuscitation can occur with accidental oesophageal intubation. The common site of rupture is the lesser curvature of the stomach, but can also occur at the oesophagogastric junction. The patient presented with a massive pneumoperitoneum after an out of hospital ventricular fibrillation arrest. CT scanning was helpful in making the diagnosis. In out of hospital resuscitation, current JRCALC (Joint Royal Colleges Ambulance Liaison Committee) recommendations may not avoid this complication.

Membrane interaction of islet amyloid polypeptide.

Increasing evidence suggests that the misfolding and deposition of IAPP plays an important role in the pathogenesis of type II, or non-insulin-dependent diabetes mellitus (T2DM). Membranes have been implicated in IAPP-dependent toxicity in several ways: Lipid membranes have been shown to promote the misfolding and aggregation of IAPP. Thus, potentially toxic forms of IAPP can be generated when IAPP interacts with cellular membranes. In addition, membranes have been implicated as the target of IAPP toxicity. IAPP has been shown to disrupt membrane integrity and to permeabilize membranes. Since disruption of cellular membranes is highly toxic, such a mechanism has been suggested to explain the observed IAPP toxicity. Here, we review IAPP-membrane interaction in the context of (1) catalyzing IAPP misfolding and (2) being a potential origin of IAPP toxicity.

Lipid membranes modulate the structure of islet amyloid polypeptide.

The 37-residue islet amyloid polypeptide (IAPP) is thought to play an important role in the pathogenesis of type II diabetes. Despite a growing body of evidence implicating membrane interaction in IAPP toxicity, the membrane-bound form has not yet been well characterized. Here we used circular dichroism (CD) and fluorescence spectroscopy to investigate the molecular details of the interaction of IAPP with lipid membranes of varying composition. In the presence of membranes containing negatively charged phosphatidylserine (PS), we observed significant acceleration in the formation of IAPP aggregates. This acceleration is strongly modulated by the PS concentration and ionic strength, and is also observed at physiologically relevant PS concentrations. CD spectra of IAPP obtained immediately after the addition of membranes containing PS revealed features characteristic of an alpha-helical conformation approximately approximately 15-19 residues in length. After a longer incubation with membranes, IAPP gave rise to CD spectra characteristic of a beta-sheet conformation. Taken together, our CD and fluorescence data indicate that conditions that promote weakly stable alpha-helical conformations may promote IAPP aggregation. The potential roles of IAPP-membrane interaction and the novel membrane-bound alpha-helical conformation in IAPP aggregation are discussed.

Identifying structural features of fibrillar islet amyloid polypeptide using site-directed spin labeling.

Pancreatic amyloid deposits, composed primarily of the 37-residue islet amyloid polypeptide (IAPP), are a characteristic feature found in more than 90% of patients with type II diabetes. Although IAPP amyloid deposits are associated with areas of pancreatic islet beta-cell dysfunction and depletion and are thought to play a role in disease, their structure is unknown. We used electron paramagnetic resonance spectroscopy to analyze eight spin-labeled derivatives of IAPP in an effort to determine structural features of the peptide. In solution, all eight derivatives gave rise to electron paramagnetic resonance spectra with sharp lines indicative of rapid motion on the sub-nanosecond time scale. These spectra are consistent with a rapidly tumbling and highly dynamic peptide. In contrast, spectra for the fibrillar form exhibit reduced mobility and the presence of strong intermolecular spin-spin interactions. The latter implies that the peptide subunits are ordered and that the same residues from neighboring peptides are in close proximity to one another. Our data are consistent with a parallel arrangement of IAPP peptides within the amyloid fibril. Analysis of spin label mobility indicates a high degree of order throughout the peptide, although the N-terminal region is slightly less ordered. Possible similarities with respect to the domain organization and parallelism of Alzheimer's amyloid beta peptide fibrils are discussed.