Colon tumour secretopeptidome: Insights into endogenous proteolytic cleavage events in the colon tumour microenvironment

The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-M_r (< 3 kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤ 1% q-value, ≤ 5% PEP) — a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell–cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To confirm reproducibility of peptide fragment abundance in this study, we report the identification of a specific cleaved peptide fragment in the secretopeptidome from the colon-specific GPA33 antigen in 4/14 human CRC models. This improved secretopeptidome isolation and characterisation strategy has extended our understanding of endogenous peptides generated through proteolysis of classical/non-classical secreted proteins, extracellular proteolytic processing of cell surface membrane proteins, and peptides generated through RIP. The novel peptide cleavage site information in this study provides a useful first step in detailing proteolytic cleavage associated with tumourigenesis and the extracellular environment. This article is part of a Special Issue entitled: An Updated Secretome.     

连续性血液净化在肾衰危重病人抢救中的应用及效果评价

目的:探讨连续性血液净化在危重病人抢救中的应用及效果评价.方法:选取2010年1月～2012年11月我院收治的危重病人共26例,均采用连续性静脉-静脉血液滤过方法治疗,比较治疗前后的心脏血流动力学参数、肾功能与电解质的变化,观察全部患者治疗前后的临床症状改善情况及临床转归.结果:治疗前后的心率、中心静脉压、平均动脉压、心指数及心排出量差异均存在统计学意义(P＜0.05);治疗后患者的血尿素氮、肌酐及钾水平均明显低于治疗前(P＜0.05);治疗后患者的低血压、低血氧、昏迷改善率分别为66.67％、71.43％及62.50％;26例患者中死亡14例(53.85％),存活12例(46.15％).结论:连续性血液净化可以积极有效地治疗危重病患者,选择治疗的时候需要针对患者的情况进行综合判断和全面评估.     

The surface structure of a complex substrate revealed by enzyme kinetics and Freundlich constants for α-amylase interaction with the surface of starch

Background

Starch is a main source of carbohydrate in human diets, but differences are observed in postprandial glycaemia following ingestion of different foods containing identical starch contents. Such differences reflect variations in rates at which different starches are digested in the intestine. In seeking explanations for these differences, we have studied the interaction of α-amylase with starch granules. Understanding this key step in digestion should help with a molecular understanding for observed differences in starch digestion rates.

Methods

For enzymes acting upon solid substrates, a Freundlich equation relates reaction rate to enzyme adsorption at the surface. The Freundlich exponent (n) equals 2/3 for a liquid-smooth surface interface, 1/3 for adsorption to exposed edges of ordered structures and 1.0 for solution–solution interfaces. The topography of a number of different starch granules, revealed by Freundlich exponents, was compared with structural data obtained by differential scanning calorimetry and Fourier transform infrared spectroscopy with attenuated total internal reflectance (FTIR-ATR).

Results

Enzyme binding rate and FTIR-ATR peak ratio were directly proportional to n and Δ_gelH was inversely related to n. Amylase binds fastest to solubilised starch and to granules possessing smooth surfaces at the solid–liquid interface and slowest to granules possessing ordered crystalline surfaces.

Conclusions

Freundlich exponents provide information about surface blocklet structures of starch that supplements knowledge obtained from physical methods.

General Significance

Nanoscale structures at the surface of starch granules influence hydrolysis by α-amylase. This can be important in understanding how dietary starch is digested with relevance to diabetes, cardiovascular health and cancer.     

Thermoregulatory behavior and oxygen consumption of Octopus mimus paralarvae: The effect of age

Octopus mimus is an important cephalopod species in the coastal zone of Peru and Chile that is exposed to temperature variations from time to time due to El Niño/Southern Oscillation (ENSO) episodes when surface temperatures can reach 24 °C, 6 °C above typical temperatures in their habitat. The relationships between temperature and food availability are important factors that determine the recruitment of juveniles into the O. mimus population. The present study was to evaluate the relationship between thermoregulatory behavior and the age of paralarvae (summer population) to determine whether changes in this behavior occur during internal yolk consumption, making larvae more vulnerable to environmental temperature change. Oxygen consumption of paralarvae when 1–4 d old was determined to establish if respiration could be used to monitor the physiological changes that occur during yolk consumption. Horizontal thermal selection (17–30 °C), critical thermal maxima (CTMax), minima (CTMin), and oxygen consumption experiments were conducted with fasting paralarvae 1–4 d old at 20 °C. Preferred temperatures were dependent on the age of O. mimus paralarvae. One day old paralarvae selected a temperature 1.1 °C (23·4 °C) higher than 2 – 4 d old paralarvae (22·3 °C). The CTMax of paralarvae increased with age with values of 31·9±1.1 °C in 1-d-olds and 33·4±0.3 to 4-d-olds. CTMin also changed with age with low values in 2-d-old paralarvae (9.1±1·3 °C) and 11·9±0·9 °C in 4-d-old animals. The temperature tolerance range of paralarvae was age-dependent (TTD=difference between CTMax and CTMin) with higher values in 2 and 3 d old paralarvae (25–26 °C) as compared to 1 d old (23·1 °C) and 4 d old animals (22.7 °C). Oxygen consumption was not affected by the age of paralarvae, suggesting that mechanisms exist that compensate their metabloism until at least 4 d of age. The temperature tolerance range of a planktonic paralarvae of octopus species is presented for the first time. This range was dependent on the age of paralarvae, and so rendered the paralarvae more vunerable to a combination of high temperature and food deprivation during first days of life. Results in the present study provide evidence that O. mimus could be under ecological pressure if a climate change causes increased or decreased temperatures into their distribution range.     

Impact of flow velocity on the dynamic behaviour of biofilm bacteria

Abstract

The impact of flow velocity (FV) on the growth dynamics of biofilms and bulk water heterotrophic plate count (HPC) bacteria in drinking water distribution systems was quantified and modeled by combining a logistic growth model with mass balance equations. The dynamic variations in the specific growth and release rates of biofilm bacteria were also quantified. The experimental results showed that the maximum biofilm biomass did not change when flow velocity was increased from 20 to 40 cm s^?1, but was significantly affected when flow velocity was further increased to 60 cm s^?1. Although the concentration of biofilm bacteria was substantially reduced by the higher shear stress, the concentration of bacteria in the bulk fluid was slightly increased. From this it is estimated that the specific growth rate and specific release rate of biofilm bacteria had doubled. The specific release (detachment) rate was dependent on the specific growth rate of the biofilm bacteria.     

Molecular dynamics simulations of valveless pumping in a closed microfluidic tube-system

In this paper, we study the flow which is generated by a valveless pumping mechanism in a closed micro-fluidic tube-system consisting of two tubes with different radii. This system has been investigated by Ottesen [J. T. Ottesen, J. Math. Biol. 46, 309, (2003)] at a macroscopic level. We find that the results from the microscopic simulations qualitatively agree with the macroscopic results. Especially, it is shown that the direction of the flow changes with changing pumping frequency and the location of the pumping device. We study the local flow generated in the system and show that the stream velocities away from the pumping area can be modeled by a superposition of a plug flow and a zero-mean oscillatory flow.     

Role of G‐proteins in the effects of leptin on pedunculopontine nucleus neurons

The pedunculopontine nucleus (PPN), the cholinergic arm of the reticular activating system, regulates waking and rapid eye movement sleep. Here, we demonstrate immunohistochemical labeling of the leptin receptor signaling isoform in PPN neurons, and investigated the effects of G‐protein modulation and the leptin triple antagonist (TA) on the action of leptin in the PPN. Whole‐cell patch clamp recordings were performed in rat brainstem slices from 9 to 17 day old pups. Previous results showed that leptin caused a partial blockade of sodium (I_Na) and h‐current (I_H) in PPN neurons. TA (100 nM) reduced the blockade of I_Na (~ 50% reduction) and I_H (~ 93% reduction) caused by leptin. Intracellular guanosine 5′‐[β‐thio]diphosphate trilithium salt (a G‐protein inhibitor) significantly reduced the effect of leptin on I_Na(~ 60% reduction) but not on I_H (~ 25% reduction). Intracellular GTPγS (a G‐protein activator) reduced the effect of leptin on both I_Na (~ 80% reduction) and I_H (~ 90% reduction). These results suggest that the effects of leptin on the intrinsic properties of PPN neurons are leptin receptor‐ and G‐protein dependent. We also found that leptin enhanced NMDA receptor‐mediated responses in single neurons and in the PPN population as a whole, an effect blocked by TA. These experiments further strengthen the association between leptin dysregulation and sleep disturbances.

     

Periodicity of extinction and recolonization of the West Indian topshell Cittarium pica in the Quaternary of Bermuda (Gastropoda: Trochoidea)

Hermit‐crab transported shells of the West Indian top shell Cittarium pica occur in numerous terrestrial fossil deposits on Bermuda, which is the most remote outpost of this Caribbean species. Cittarium is so far known only from deposits of interglacial ages corresponding to marine isotope stages (MIS) 11, 9, 5e, and 1 (Holocene). In at least the cases of MIS 11 and 5e, Cittarium appears at the very beginning of the interglacial. The species is definitely absent from well‐stratified cave deposits of the last glacial (MIS 4–2). It is hypothesized that colder sea‐surface temperatures (SSTs) during glacial periods caused Cittarium to be extirpated on Bermuda, and that it has made at least four independent colonizations of the island in the past 400 000 years. Because of the limited larval life of Cittarium, the window for colonization may be a very narrow period at the onset of interglacials when the SST has risen sufficiently to make Bermuda habitable but the velocity of the Gulf Stream is still sufficiently rapid to transport viable larvae from the Bahamas. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 235–243.     

Cardiomyocyte dysfunction during the chronic phase of Chagas disease

Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of heart failure. We investigated modifications in the cellular electrophysiological and calcium-handling characteristics of an infected mouse heart during the chronic phase of the disease. The patch-clamp technique was used to record action potentials (APs) and L-type Ca²⁺ and transient outward K⁺ currents. [Ca²⁺]_i changes were determined using confocal microscopy. Infected ventricular cells showed prolonged APs, reduced transient outward K⁺ and L-type Ca²⁺ currents and reduced Ca²⁺ release from the sarcoplasmic reticulum. Thus, the chronic phase of Chagas disease is characterised by cardiomyocyte dysfunction, which could lead to heart failure.