The diagnostic and prognostic values of inflammatory markers in intraductal papillary mucinous neoplasm.

BACKGROUND: Intraductal papillary mucinous neoplasm (IPMN) is an broad-spectrum disease from benign to malignant. Inflammatory markers are known as prognostic predictors in various diseases. The purpose of this study was to determine the predictive value of inflammatory markers for prognosis in IPMN. METHODS: From April 1995 to December 2016, patients who underwent pancreatectomy with pathologically confirmed IPMN at four tertiary centers were enrolled. Patients with a history of pancreatitis or cholangitis, and other malignancies were excluded. Neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and advanced lung cancer inflammation index (ALI) were calculated. RESULTS: Of all, ninety-eight patients (26.8%) were diagnosed as invasive IPMN. The NLR and PLR were significantly elevated in invasive IPMN than in non-invasive disease (2.0 vs 1.8, p = 0.004; 117.1 vs 107.4, p = 0.009, respectively). ALI was significantly higher in non-invasive IPMN than in invasive disease (58.1 vs 45.9, p < 0.001). In multivariate analysis, only NLR showed significant association among the inflammatory markers studied (p = 0.044). In invasive IPMN, the five-year recurrence-free survival rate for NLR less than 3.5 was superior to the rest (59.1 vs 42.2, p = 0.023). CONCLUSION: NLR may help to rightly select IPMN patients who will require surgery and may serve as a useful prognostic factor.

The effects of sarcopenia and sarcopenic obesity after pancreaticoduodenectomy in patients with pancreatic head cancer.

BACKGROUND: Recently, several studies have reported that sarcopenia and sarcopenic obesity (SO) could worsen postoperative complications after PD. This study aims to evaluate the effects of preoperative sarcopenia and SO following PD in pancreatic head cancer (PHD). METHODS: Preoperative sarcopenia and SO were assessed in 548 patients undergoing PD for PHC at Samsung Medical Centre between 2007 and 2016. The visceral adipose tissue-to-skeletal muscle ratio was calculated from cross-sectional visceral fat and muscle areas on preoperative CT images. The overall survival (OS) and rate of clinically relevant postoperative pancreatic fistula (CR-POPF) among postoperative complications were extracted from prospectively maintained databases. RESULTS: Preoperative sarcopenia was present in 252 patients (45.9%). The 5-year survival rates of patients with non-sarcopenia and sarcopenia were 28.4% and 23.4% (p = 0.046). Preoperative SO was present in 202 patients (36.9%). After multivariable analysis, the presence of SO was the only independent risk factor for CR-POPF (p = 0.018). CONCLUSION: Sarcopenia can be a risk factor affecting decreased OS after PD in patients with PHC. SO is the only predictive factor for CR-POPF after PD in patients with PHC. More observational studies are needed to evaluate the effects of sarcopenia and SO on survival after PD.

Nomogram for predicting postoperative pancreatic fistula.

BACKGROUND: Previous studies analyzed risk factors for postoperative pancreatic fistula (POPF) and developed risk prediction tool using scoring system. However, no study has built a nomogram based on individual risk factors. This study aimed to evaluate individual risks of POPF and propose a nomogram for predicting POPF. METHODS: From 2007 to 2016, medical records of 1771 patients undergoing pancreaticoduodenctomy were reviewed retrospectively. Variables with p < 0.05 in multivariate logistic regression analysis were included in the nomogram. Internal performance validation was executed using a repeated cross validation method. RESULTS: Of 1771 patients, 222 (12.5%) experienced POPF. In multivariable analysis, sex (p = 0.004), body mass index (BMI) (p < 0.001), ASA score (p = 0.039), preoperative albumin (p = 0.035), pancreatic duct diameter (p = 0.002), and location of tumor (p < 0.001) were identified as independent predictors for POPF. Based on these six variables, a POPF nomogram was developed. The area under the curve (AUC) estimated from the receiver operating characteristic (ROC) graph was 0.709 in the train set and 0.652 in the test set. CONCLUSIONS: A POPF nomogram was developed. This nomogram may be useful for selecting patients who need more intensified therapy and establishing customized treatment strategy.

Neoadjuvant therapy versus upfront surgery for borderline-resectable pancreatic cancer.

BACKGROUND: Neoadjuvant therapy is recommended for patients with borderline-resectable pancreatic cancer (BRPC). In this study, we compare survival outcomes of neoadjuvant therapy with upfront surgery. METHODS: From January 2011 to June 2016, 1415 patients underwent treatments for pancreatic cancer in Samsung Medical Center. Among them, 112 (7.9%) patients were categorized as BRPC by the NCCN 2016 guideline. They were classified by type of initial treatments into neoadjuvant group (NA, N.=26) and upfront surgery group (US, N.=86). RESULTS: The median survival duration of all patients was 18.3 months. Patients in the NA group had more T4 disease than those in the US group (38.5% in NA versus 15.1% in the US group; P=0.010). Arterial involvement was more frequent in the NA group (42.3% versus 15.1%; P=0.003). In the NA group, ten (38.5%) patients underwent surgery, and seven of them had complete R0 resection. In the US group, 83 (96.5%) patients received radical surgery, and 42 (48.8%) had R0 resection. In survival analysis according to intent to treat, the overall two-year survival rate was 51.1% in the US group and 36.7% in the NA group (P=0.001). However, among patients who underwent surgery (N.=96), the two-year overall survival rate was not significantly different between the two groups (P=0.089). According to involved vessels, the survival rate was not different between patients with arterial or both arterial and venous involvement and in patients with only venous involvement (P=0.649). CONCLUSIONS: It is necessary to demonstrate the efficacy of neoadjuvant therapy and to standardize the regimens through large-scale, multicenter, randomized controlled studies.

NMR, mass spectrometry and chemical evidence reveal a different chemical structure for methanobactin that contains oxazolone rings.

Methanobactin (mb) is a small copper-binding peptide produced by methanotrophic bacteria and is intimately involved in both their copper metabolism and their role in the global carbon cycle. The structure for methanobactin comprises seven amino acids plus two chromophoric residues that appear unique to methanobactin. In a previously published structure, both chromophoric residues contain a thiocarbonyl attached to a hydroxyimidazolate ring. In addition, one is attached to a pyrrolidine ring, while the other is attached to an isopropyl ester. A published X-ray determined structure for methanobactin shows these two chromophoric groups forming an N2S2 binding site for a single Cu(I) ion with a distorted tetrahedral geometry. In this report we show that NMR, mass spectrometry, and chemical data reveal a chemical structure that is significantly different than the previously published one. Specifically, the 1H and 13C NMR assignments are inconsistent with an N-terminal isopropyl ester and point instead to a 3-methylbutanoyl group. Our data also indicate that oxazolone rings instead of hydroxyimidazolate rings form the core of the two chromophoric residues. Because these rings are directly involved in the binding of Cu(I) and other metals by methanobactin and are likely involved in the many chemical activities displayed by methanobactin, their correct identity is central to developing an accurate and detailed understanding of methanobactin's many chemical and biological roles. For example, the oxazolone rings make methanobactin structurally more similar to other bacterially produced bactins and siderophores and suggest pathways for its biosynthesis.

Mössbauer studies of the membrane-associated methane monooxygenase from Methylococcus capsulatus bath: evidence for a Diiron center.

Two methane monooxygenase (MMO) systems have been identified in methanotrophic bacteria, namely, a soluble or cytoplasmic MMO and a membrane-associated or particulate MMO. The active site of the well-characterized soluble MMO contains a bis-mu-hydroxo-bridged diiron cluster. X-ray crystallographic studies of the particulate enzyme, pMMO, have identified two copper centers on the alpha subunit (pmoB) of the alphabetagamma trimer and a site at the interface of the betagamma subunits filled by a Zn, apparently from the crystallization buffer. In our hands, pMMO preparations containing 1-2 iron atoms per alphabetagamma show the highest catalytic activity. We have employed Mössbauer spectroscopy to characterize the iron in our preparations. Interestingly, we find in pMMO a component with the same spectral properties as the antiferromagnetically coupled diiron(III) cluster in the soluble enzyme. In whole cells, we find nearly 1 diiron center per alphabetagamma of pMMO; in purified enzyme preparations, only 10% of the sites appear to be occupied. These occupancies correlate well with the measured specific activities of purified pMMO and pMMO in whole cells. We suggest that it is the "Zn site" that accommodates the diiron center in active pMMO.

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b.

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV-visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.

Localization and surgical treatment of the pancreatic insulinomas.

OBJECTIVES: Insulinomas are rare tumours that originate from the islet cells of the pancreas. The aims of this study were to gain an understanding of the clinical features of insulinomas and to establish the diagnostic and therapeutic strategies. METHODS: A review was carried out in 20 patients with insulinoma surgically treated in our institution over the last 10 years. Presenting symptoms, biochemical studies, preoperative and intraoperative localization studies, operative management and complications were analysed. RESULTS: The male-to-female ratio was 8:12, with a mean age of 46.4 years. Each patient suffered from significant neuroglycopenic symptoms, usually manifested by dizziness, sweating, headache and confusion. The preoperative median serum levels of glucose, insulin and C-peptide at the termination of the fast were 37.5 mg/dL, 23.5 microU/mL, 5.6 ng/mL, respectively. Preoperative tumour localization was achieved by means of ultrasonography (US), computed tomography, selective angiography or intra-arterial calcium injection with hepatic venous sampling, and sensitivities of these examinations were 81.8, 73.7, 94.1 and 100%, respectively. Intraoperative localization was carried out by a combination of manual palpation and intraoperative US with retrospective sensitivities of 80 and 100%, respectively. Enucleation was carried out in 16 patients and distal pancreatectomy in 4. The mortality and morbidity rates were 0 and 10%, respectively. One patient developed late diabetes mellitus type 1 after distal pancreatectomy. CONCLUSIONS: We conclude that the diagnosis of insulinoma can be made on the basis of the results of a supervised fast, careful palpation with intraoperative US is essential for intraoperative detection of insulinomas and surgical resection is the best choice for treatment of benign insulinomas.

Long-term outcome of portomesenteric vein invasion and prognostic factors in pancreas head adenocarcinoma.

BACKGROUND: The purpose of this study was to clarify the post-operative prognosis of pancreatic head cancer with pathologic portal vein (PV) or superior mesenteric vein (SMV) invasion. METHODS: From May 1995 to December 2009, preoperative, intra-operative and post-operative data from 276 patients who underwent pancreaticoduodenectomy for pancreatic ductal adenocarcinoma were reviewed retrospectively. The long-term prognosis was compared between patients with a pathologic PV-SMV invasion and those without invasion. RESULTS: Fourty-six patients (16.7%) underwent PV-SMV resection during pancreaticoduodenectomy. Pathologic PV-SMV invasion was observed in 30 (65.2%). Post-operative severe morbidity (grade 3 or 4) was similar for patients with and without PV-SMV resection (8.7% with versus 7.0% without P = 0.754). The mortality rate was 2.2% with PV-SMV resection and 0.9% without PV-SMV resection (P = 0.423). Survival of PV-SMV resection and no resection group had no significant difference (median survival, 16 versus 12 months; P = 0.086). No significant difference in overall survival was seen between patients with and without pathologic PV-SMV invasion (median survival, 13 versus 16 months; P = 0.663). Tumour differentiation, R status, tumour size and type of operation were revealed as independent prognostic factors. CONCLUSIONS: 34.8% of patients who underwent PV-SMV resection had no pathologic invasion. And PV-SMV resection did not increase the rate of severe complications and mortality. Furthermore, the prognosis for patients with pathologic PV-SMV invasion may be nearly the same as patients with no invasion. So, PV-SMV resection with reconstruction should be considered in pancreatic head cancer patients with suspected PV-SMV invasion.

Spectral and copper binding properties of methanobactin from the facultative methanotroph Methylocystis strain SB2.

Methanobactin (mb) is the first characterized example of a chalkophore, a class of copper-binding chromopeptides similar to iron-binding siderophores. Structural, redox, themodynamic, and spectral studies on chalkophores have focused almost exclusively on the mb from Methylosinus trichosporium OB3b (mb-OB3b). The structural characterization of a second mb from Methylocystis strain SB2 (mb-SB2) provides a means to examine the core structural features and metal binding properties of this group of chromopeptides. With the exception of the 5-membered rings (either oxazolone or imidazolone), enethiol groups, and the N-terminus oxo group, the structure of mb-SB2 differs markedly from mb-OB3b. In particular the amino acids commonly associated with metal coordination and redox activity found in mb-OB3b, Cys, Met, and Try, are replaced by Ala or are missing in mb-SB2. In this report the spectral and thermodynamic properties of mb-SB2 are presented and compared to mb-OB3b. The results demonstrate that the spectral and basic copper binding properties of both methanobactins are similar and the unique copper binding capacity of both methanobactins lies primarily in the pair of five-membered rings and associated enethiol groups. The remaining portions of the methanobactin appear to provide the scaffolding that brings together of the two ring systems to produce the tetrahedral binding site for copper binding.

Isolation of methanobactin from the spent media of methane-oxidizing bacteria.

Chalkophores are low molecular mass modified peptides involved in copper acquisition in methane-oxidizing bacteria (MOB). A screening method for the detection of this copper-binding molecule is presented in Chapter 16. Here we describe methods to (1) maximize expression and secretion of chalkophores, (2) concentrate chalkophores from the spent media of MOB, and (3) purify chalkophores.

Spectral and thermodynamic properties of methanobactin from γ-proteobacterial methane oxidizing bacteria: a case for copper competition on a molecular level.

Methanobactin (mb) is a low molecular mass copper-binding molecule analogous to iron-binding siderophores. The molecule is produced by many methanotrophic or methane oxidizing bacteria (MOB), but has only been characterized to date in one MOB, Methylosinus trichosporium OB3b. To explore the potential molecular diversity in this novel class of metal binding compound, the spectral (UV-visible, fluorescent, and electron paramagnetic resonance) and thermodynamic properties of mb from two γ-proteobacterial MOB, Methylococcus capsulatus Bath and Methylomicrobium album BG8, were determined and compared to the mb from the α-proteobacterial MOB, M. trichosporium OB3b. The mb from both γ-proteobacterial MOB differed from the mb from M. trichosporium OB3b in molecular mass and spectral properties. Compared to mb from M. trichosporium OB3b, the extracellular concentrations were low, as were copper-binding constants of mb from both γ-proteobacterial MOB. In addition, the mb from M. trichosporium OB3b removed Cu(I) from the mb of both γ-proteobacterial MOB. Taken together the results suggest mb may be a factor in regulating methanotrophic community structure in copper-limited environments.

Oxidase, superoxide dismutase, and hydrogen peroxide reductase activities of methanobactin from types I and II methanotrophs.

Methanobactin (mb) is a copper-binding chromopeptide that appears to be involved in oxidation of methane by the membrane-associated or particulate methane monooxygenase (pMMO). To examine this potential physiological role, the redox and catalytic properties of mb from three different methanotrophs were examined in the absence and presence of O(2). Metal free mb from the type II methanotroph Methylosinus trichosporium OB3b, but not from the type I methanotrophs Methylococcus capsulatus Bath or Methylomicrobium album BG8, were reduced by a variety of reductants, including NADH and duroquinol, and catalyzed the reduction of O(2) to O(2)(-). Copper-containing mb (Cu-mb) from all three methanotrophs showed several interesting properties, including reductase dependent oxidase activity, dismutation of O(2)(-) to H(2)O(2), and the reductant dependent reduction of H(2)O(2) to H(2)O. The superoxide dismutase-like and hydrogen peroxide reductase activities of Cu-mb were 4 and 1 order(s) of magnitude higher, respectively, than the observed oxidase activity. The results demonstrate that Cu-mb from all three methanotrophs are redox-active molecules and oxygen radical scavengers, with the capacity to detoxify both superoxide and hydrogen peroxide without the formation of the hydroxyl radicals associated with Fenton reactions. As previously observed with Cu-mb from Ms. trichosporium OB3b, Cu-mb from both type I methanotrophs stimulated pMMO activity. However, in contrast to previous studies using mb from Ms. trichosporium OB3b, pMMO activity was not inhibited by mb from the two type I methanotrophs at low copper to mb ratios.

Spectral, kinetic, and thermodynamic properties of Cu(I) and Cu(II) binding by methanobactin from Methylosinus trichosporium OB3b.

To examine the potential role of methanobactin (mb) as the extracellular component of a copper acquisition system in Methylosinus trichosporium OB3b, the metal binding properties of mb were examined. Spectral (UV-visible, fluorescence, and circular dichroism), kinetic, and thermodynamic data suggested copper coordination changes at different Cu(II):mb ratios. Mb appeared to initially bind Cu(II) as a homodimer with a comparatively high copper affinity at Cu(II):mb ratios below 0.2, with a binding constant (K) greater than that of EDTA (log K = 18.8) and an approximate DeltaG degrees of -47 kcal/mol. At Cu(II):mb ratios between 0.2 and 0.45, the K dropped to (2.6 +/- 0.46) x 10(8) with a DeltaG degrees of -11.46 kcal/mol followed by another K of (1.40 +/- 0.21) x 10(6) and a DeltaG degrees of -8.38 kcal/mol at Cu(II):mb ratios of 0.45-0.85. The kinetic and spectral changes also suggested Cu(II) was initially coordinated to the 4-thiocarbonyl-5-hydroxy imidazolate (THI) and possibly Tyr, followed by reduction to Cu(I), and then coordination of Cu(I) to 4-hydroxy-5-thiocarbonyl imidazolate (HTI) resulting in the final coordination of Cu(I) by THI and HTI. The rate constant (k(obsI)) of binding of Cu(II) to THI exceeded that of the stopped flow apparatus that was used, i.e., >640 s(-)(1), whereas the coordination of copper to HTI showed a 6-8 ms lag time followed by a k(obsII) of 121 +/- 9 s(-)(1). Mb also solubilized and bound Cu(I) with a k(obsI) to THI of >640 s(-)(1), but with a slower rate constant to HTI (k(obsII) = 8.27 +/- 0.16 s(-)(1)), and appeared to initially bind Cu(I) as a monomer.

Effect of methanobactin on the activity and electron paramagnetic resonance spectra of the membrane-associated methane monooxygenase in Methylococcus capsulatus Bath.

Improvements in the purification of methanobactin (mb) from either Methylosinus trichosporium OB3b(T) or Methylococcus capsulatus Bath resulted in preparations that stimulated methane-oxidation activity in both whole-cell and cell-free fractions of Methylococcus capsulatus Bath expressing the membrane-associated methane monooxygenase (pMMO). By using washed membrane factions with pMMO activities in the 290 nmol propylene oxidized min(-1) (mg protein)(-1) range, activities approaching 400 nmol propylene oxidized min(-1) (mg protein)(-1) were commonly observed following addition of copper-containing mb (Cu-mb), which represented 50-75 % of the total whole-cell activity. The stimulation of methane-oxidation activity by Cu-mb was similar to or greater than that observed with equimolar concentrations of Cu(II), without the inhibitory effects observed with high copper concentrations. Stimulation of pMMO activity was not observed with copper-free mb, nor was it observed when the copper-to-mb ratio was <0.5 Cu atoms per mb. The electron paramagnetic resonance (EPR) spectra of mb differed depending on the copper-to-mb ratio. At copper-to-mb ratios of <0.4 Cu(II) per mb, Cu(II) addition to mb showed an initial coordination by both sulfur and nitrogen, followed by reduction to Cu(I) in <2 min. At Cu(II)-to-mb ratios between 0.4 and 0.9 Cu(II) per mb, the intensity of the Cu(II) signal in EPR spectra was more representative of the Cu(II) added and indicated more nitrogen coordination. The EPR spectral properties of mb and pMMO were also examined in the washed membrane fraction following the addition of Cu(II), mb and Cu-mb in the presence or absence of reductants (NADH or duroquinol) and substrates (CH4 and/or O2). The results indicated that Cu-mb increased electron flow to the pMMO, increased the free radical formed following the addition of O2 and decreased the residual free radical following the addition of O2 plus CH4. The increase in pMMO activity and EPR spectral changes to the pMMO following Cu-mb addition represent the first positive evidence of interactions between the pMMO and Cu-mb.

The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH:quinone oxidoreductase complex from Methylococcus capsulatus Bath.

Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol.min(-1).mg of protein(-1). Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 microM) to 95 microM. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.