Novel Biological Substrates of Human Kallikrein 7 Identified through Degradomics

Kallikrein-related peptidases (KLKs) are a group of serine proteases widely expressed in various tissues and involved in a wide range of physiological and pathological processes. Although our understanding of the pathophysiological roles of most KLKs has blossomed in recent years, identification of the direct endogenous substrates of human KLKs remains an unmet objective. In this study we employed a degradomics approach to systemically investigate the endogenous substrates of KLK7 in an effort to understand the molecular pathways underlying KLK7 action in skin. We identified several previously known as well as novel protein substrates. Our most promising candidates were further validated with the use of targeted quantitative proteomics (selected reaction monitoring methods) and in vitro recombinant protein digestion assays. Our study revealed midkine, CYR61, and tenascin-C as endogenous substrates for KLK7. Interestingly, some of these substrates (e.g. midkine) were prone to proteolytic cleavage only by KLK7 (and not by other skin-associated KLKs), whereas others (e.g. CYR61 and tenascin-C) could be digested by several KLKs. Furthermore, using melanoma cell line, we show that KLK7-mediated cleavage of midkine results in an overall reduction in the pro-proliferative and pro-migratory effect of midkine. An inverse relation between KLK7 and midkine is also observed in human melanoma tissues. In summary, our degradomics approach revealed three novel endogenous substrates for KLK7, which may shed more light on the pathobiological roles of KLK7 in human skin. Similar substrate screening approaches could be applied for the discovery of biological substrates of other protease.     

Monocyte attachment to laminin in diabetes mellitus: The role of ATP

Monocyte-extracellular matrix interactions have been implicated in atherosclerosis pathophysiology. Laminin, the main basement membrane protein contains cell binding domains that can be cryptic, presented only after protein modification. In the present study we evaluated monocyte attachment to laminin-1 in the presence of ATP. Monocytes were derived from either healthy volunteers or patients with diabetes mellitus type II. For the estimation of monocyte attachment to laminin the myeloperoxidase assay was used. Monocytes derived from diabetic patients, showed an increased ability to attach to laminin (p = 0.0055). The presence of ATP increased the attachment of control monocytes to laminin (p = 0.0022). On the contrary, the presence of ATP did not affect the attachment of monocytes derived from diabetic patients to laminin. Our results indicate a modified interaction between monocytes and laminin-1 in diabetes mellitusKey words: monocytes, ATP, laminin-1, diabetes mellitus, attachment     

Fatty acid retention under temporally heterogeneous dietary intake in a cladoceran

Omega‐3 (ω3) and ‐6 (ω6) polyunsaturated fatty acids (PUFA) are essential for all aquatic animals, but their dietary availability can be highly heterogeneous in space and time. The way consumers retain PUFA across such heterogeneous feeding conditions remains poorly understood. In a series of feeding experiments, we investigated how retention efficiencies (i.e. amount in consumer biomass/amount ingested) of PUFA and bulk carbon responded to heterogeneous PUFA intake in Daphnia magna. Heterogeneous PUFA intake was achieved by exposing D. magna to algal diets of different PUFA content and composition for specific time periods. The retention efficiency of carbon did not change among dietary treatments. At shorter exposure to PUFA‐rich diet, retention efficiencies of most PUFA were 2–3 times higher than that of bulk carbon, clearly indicating PUFA bioaccumulation in D. magna. Increasing exposure to PUFA‐rich diet caused exponential decrease of retention efficiencies for most PUFA. However, D. magna receiving more PUFA were richer in these compounds despite lower retention efficiency. Eicosapentaenoic (20:5ω3) and arachidonic acid (20:4ω6) and their precursors were always supplied in the same proportions (3.6:1), but the 20:5ω3/20:4ω6 ratio in D. magna (an important measure of nutritional quality for consumers) increased with exposure time to these PUFA from 2.2:1 to 3.8:1, thus eventually matching the diet. Our results suggest that D. magna is an efficient gatherer, accumulator, and repository of PUFA under low/fragmented dietary availability. However, at higher availabilities, PUFA are not always bioaccumulated in D. magna. Hence, the efficiency of PUFA transfer by daphnids in food webs may depend on temporal PUFA availability and its range of variation. Finally, we show that heterogeneity in PUFA intake may also affect higher trophic levels by influencing nutritionally critical PUFA ratios of zooplankton.     

Genetic Identification and Phylogeny of Three Species of the Genus <Emphasis Type="Italic">Trachurus</Emphasis> Based on Mitochondrial DNA Analysis

The genetic identification and the phylogenetic relationships of 3 European species of the genus Trachurus (T. trachurus, T. mediterraneus, and T. picturatus) across their geographical distribution, have been investigated by mitochondrial DNA analysis. Both cytochrome b and 16S ribosomal DNA sequence analysis revealed the existence of several species-specific positions that distinguish the 3 studied species. Genetic distances between the species indicated that T. mediterraneus and T. picturatus are more closely related than T. trachurus. Similar topologies have been produced by neighbor-joining, maximum-likelihood, and maximum-parsimony trees, and they were in accordance with previous taxonomic classification. Internucleotide and intranucleotide diversity of T. picturatus was 2 times higher than that of T. mediterraneus and T. trachurus, possibly owing to the low levels of fishing pressure for T. picturatus. This is the first report of the phylogenetic relationships of the 3 Trachurus species and provides a possible scenario of the time of divergence related to the closure of the Gibraltar Straits. In addition, the present results can be used for genetic identification of the 3 species, even from the early stage of eggs, and for detection of commercial fraud.     

Comparative study of flow in right-sided and left-sided aortas: numerical simulations in patient-based models

A right-sided aorta is a rare malformation which may be associated with other various types of congenital heart disease. We utilised haemodynamic, echocardiographic measurements, computerised tomography and image reconstruction software packages that were integrated in a computational fluid dynamics model to determine blood flow patterns in patient-based aortas. In the left-sided aorta, a systolic clockwise rotational component was present, while helical flow was depicted in the aortic arch that was converted in the descending aorta as counter-rotating vortices with accompanying retrograde flow. The right-sided configuration has not altered the orientation of the three-dimensional vortex, but intensification of polymorphic flow patterns, alterations in wall shear stress distribution and development of a lateral pressure gradient at the area of an aneurysmal anomaly was observed. Moreover, increments of Reynolds, Womersley and Dean numbers were evident. These phenomena along with the formation of the aneurysm might influence cardiovascular risk in patients with right-sided aortas.     

Reduced Intestinal Tumorigenesis in APCmin Mice Lacking Melanin-Concentrating Hormone

Background

Melanin-concentrating hormone (MCH) is an evolutionary conserved hypothalamic neuropeptide that in mammals primarily regulates appetite and energy balance. We have recently identified a novel role for MCH in intestinal inflammation by demonstrating attenuated experimental colitis in MCH deficient mice or wild type mice treated with an anti-MCH antibody. Therefore, targeting MCH has been proposed for the treatment of inflammatory bowel disease. Given the link between chronic intestinal inflammation and colorectal cancer, in the present study we sought to investigate whether blocking MCH might have effects on intestinal tumorigenesis that are independent of inflammation.

Methodology

Tumor development was evaluated in MCH-deficient mice crossed to the APCmin mice which develop spontaneously intestinal adenomas. A different cohort of MCH−/− and MCH+/+ mice in the APCmin background was treated with dextran sodium sulphate (DSS) to induce inflammation-dependent colorectal tumors. In Caco2 human colorectal adenocarcinoma cells, the role of MCH on cell survival, proliferation and apoptosis was investigated.

Results

APCmin mice lacking MCH developed fewer, smaller and less dysplastic tumors in the intestine and colon which at the molecular level are characterized by attenuated activation of the wnt/beta-catenin signaling pathway and increased apoptotic indices. Form a mechanistic point of view, MCH increased the survival of colonic adenocarcinoma Caco2 cells via inhibiting apoptosis, consistent with the mouse studies.

Conclusion

In addition to modulating inflammation, MCH was found to promote intestinal tumorigenesis at least in part by inhibiting epithelial cell apoptosis. Thereby, blocking MCH as a therapeutic approach is expected to decrease the risk for colorectal cancer.     

Tumour necrosis factor-alpha (TNFalpha) and interleukin-10 are crucial mediators in post-operative systemic inflammatory response and determine the occurrence of complications after major abdominal surgery

BACKGROUND: The course of serum cytokine levels in patients with postoperative systemic inflammatory response syndrome (SIRS) after major abdominal surgery remains currently unclear. METHODS: Blood was sampled pre- and post-operatively and on days 1 and 2 in 40 patients undergoing major abdominal surgery. Concentrations of tumour necrosis factor-alpha (TNFalpha), interleukin (IL) -6, IL-8, and IL-10 were measured by the LINCOplex assay; those of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) by an enzyme immunoassay. RESULTS: Compared to their pre-operative values, sTREM-1 was elevated on day 2; TNFalpha on day 1; IL-6 and IL-10 post-operatively and on days 1 and 2; and IL-8 post-operatively and on day 1. The duration of operation correlated with TNFalpha and IL-10 at all sampling times, and with IL-6 post-operatively. There were no differences in cytokine concentrations between patients who exhibited post-operative complications and those who did not. IL-10/TNFalpha below 30 was found in all patients with complications (100%) and in 20 patients without complications (64.5%, p: 0.043). CONCLUSIONS: SIRS following major surgery is characterised by complex alterations in cytokine concentrations. The balance between TNFalpha and IL-10 seems to determine the occurrence of post-operative complications.     

Laminin,gamma 2 (LAMC2): A Promising New Putative Pancreatic Cancer Biomarker Identified by Proteomic Analysis of Pancreatic Adenocarcinoma Tissues

In pancreatic cancer, the incidence and mortality curves coincide. One major reason for this high mortality rate in pancreatic ductal adenocarcinoma (PDAC) patients is the dearth of effective diagnostic, prognostic, and disease-monitoring biomarkers. Unfortunately, existing tumor markers, as well as current imaging modalities, are not sufficiently sensitive and/or specific for early-stage diagnosis. There is, therefore, an urgent need for improved serum markers of the disease. Herein, we performed Orbitrap® mass spectrometry proteomic analysis of four PDAC tissues and their adjacent benign tissues and identified a total of 2190 nonredundant proteins. Sixteen promising candidates were selected for further scrutiny using a systematic scoring algorithm. Our preliminary serum verification of the top four candidates (DSP, LAMC2, GP73, and DSG2) in 20 patients diagnosed with pancreatic cancer and 20 with benign pancreatic cysts, showed a significant (p < 0.05) elevation of LAMC2 in pancreatic cancer serum. Extensive validation of LAMC2 in healthy, benign, and PDAC sera from geographically diverse cohorts (n = 425) (Japan, Europe, and USA) demonstrated a significant increase in levels in early-stage PDAC compared with benign diseases. The sensitivity of LAMC2 was comparable to CA19.9 in all data sets, with an AUC value greater than 0.85 in discriminating healthy patients from early-stage PDAC patients. LAMC2 exhibited diagnostic complementarity with CA19.9 by showing significant (p < 0.001 in two out of three cohorts) elevation in PDAC patients with clinically low CA19.9 levels.Pancreatic ductal adenocarcinoma (PDAC)¹ is one of the most devastating cancers and the fourth leading cause of cancer-related deaths in North America (1). Ninety-five percent of patients will not survive beyond five years; this high mortality rate is primarily attributed to the lack of effective diagnostic techniques and treatment regimens. The hallmark features of pancreatic cancer (PC) are late presentation and aggressive metastatic progression (2, 3). The National Cancer Institute statistics estimate that approximately $1.9 billion is being spent in the United States alone each year on PC diagnosis and treatment. PDAC is classified into resectable (∼10–20%), locally advanced unresectable (∼30–40%), and metastatic (∼50%) (3). PDAC diagnosed at resectable stage can possibly be cured with complete surgical removal. This could improve the survival rates and considerably lower treatment costs. It is projected that 20–40% of patients with resectable PDAC survive more than five years after complete surgical removal, highlighting the importance of early-stage diagnosis. Unfortunately, carbohydrate antigen 19–9 (CA19.9), the current standard serum tumor marker for PDAC, has certain limitations as an early detection biomarker (its sensitivity for small tumors {<3 cm} is ∼50% and it is significantly elevated in many benign conditions (e.g. biliary obstruction, hepatic cirrhosis, chronic pancreatitis)) (4, 5). In light of the scarcity of other, more reliable markers, CA19.9 is currently used in the clinic as a prognostic and surveillance marker. Undoubtedly, the need for a more reliable consistent biomarker (or biomarker panel) for early PDAC diagnosis remains unmet. In pursuit of novel PDAC biomarker candidates, we have previously delineated the proteomes of malignant pancreatic ascitic fluids, pools of pancreatic juice, and pancreatic cancer cell lines (BxPC3, CAPAN, CFPAC1, MIA-Paca2, PANC1, and SU.86.86). We identified a panel of five potential candidate biomarkers, which, in combination, slightly outperformed CA19.9 in a pilot verification study (40 individuals; 20 healthy, and 20 PDAC) (6).From a different perspective, in the current study, we deployed a comparative quantitative tissue proteomic methodology to compare the proteome of malignant pancreatic tissues with that of their adjacent normal counterparts. A total of 2190 nonredundant proteins were identified, which were further scrutinized using a systematic scoring algorithm based on their quantified cancer-versus-normal ratios, on their identification in malignant pancreatic ascites fluid, on their cancer-specific nature, and on their tissue-expression profiles. Our analysis resulted in sixteen promising candidate biomarkers, which fulfilled our criteria and selected for further validation studies. In a multistep validation approach, the selected candidates were first verified in serum samples obtained from 20 patients with benign pancreatic diseases and 20 patients with pancreatic cancer, using commercially available ELISA kits. The best candidate (LAMC2) was further tested in three geographically diverse cohorts from Germany, Japan, and the US composed of 435 serum samples from healthy, benign, and early and late stage cancer patients. Our approach brought to light a previously unknown, promising PDAC candidate biomarker, LAMC2.     

Granulation mechanism of a UASB reactor supplemented with iron

The aim of this paper was to propose a granulation mechanism in order to interpret all the experimental observations that arose during experiments on two UASB reactors, where one was supplemented with ferrous iron at a dose of 0.01 g Fe(2+) per g COD feed. This supplementation with ferrous iron allowed COD removal of more than 98% at a loading rate of 9 g COD/L per day, which was 24% higher than for the reactor not receiving the ferrous iron. Moreover, in the Fe(2+)-dosed reactor, a higher increase of the granule diameter was observed. Indeed, the granule diameter in the Fe(2+)-dosed reactor at the end of the experiments was 56% greater than that of the control reactor. This mechanism describes the course of anaerobic granule growth. The formation of the inorganic precipitate of ferrous sulphide constitutes the inert nuclei around which the biomass is attached. This initiates the formation of new granules.