SOUND PRODUCTION IN THE LARVAE OF GEOTRUPES SPINIGER (MARSHAM) (COLEOPTERA: GEOTRUPIDAE)

ABSTRACT

This paper focuses on sound production in the larvae of Geotrupes spiniger (Marsham) (Coleoptera: Geotrupidae). A single stridulatory apparatus was identified and then analysed from a structural point of view. Sound was studied using spectography and results included a quantitative analysis with characterizing physical parameters. Results highlight the need to study stridulation in Geotrupidae more in detail, to improve our understanding of its evolutionary and biological meaning.     

Comparison of dynamics of wildtype and V94M human UDP-galactose 4-epimerase—A computational perspective on severe epimerase-deficiency galactosemia

UDP-galactose 4′-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr157) and the enzyme-bound NAD⁺ cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (k_cat), with less substantial effects on K_m. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia.     

Assessing the effect of sound file compression and background noise on measures of acoustic signal structure

The study of animal acoustic signals is a central tool for many fields in ecology and evolution, but the diversity of analytical methods and sources of animal sound recordings poses important challenges for carrying out robust acoustic analyses. Sound file compression and background noise may both affect acoustic analysis, although little attention has been paid to their respective effects. We evaluated the effect of these factors by assessing the systematic deviation (i.e. bias) and measurement error (i.e. precision) that they generate on spectrographic parameters and two (dis)similarity methods (dynamic time warping on frequency contours and cross-correlation), which represent the most common methods currently used for quantitative characterization of acoustic signals. Measurements were taken across a wide range of signals from a diverse group of bird species, and compared between uncompressed files and decompressed files obtained from mp3-encoded files generated using the two most common mp3 encoders (Fraunhofer and LAME). Measurements were also compared across a range of synthetically-generated background noise levels. Compression did not significantly bias any of the acoustic or similarity measurements. However, the precision of acoustic parameters representing single extreme values (e.g. peak frequency) as well as dynamic time warping distances, was strongly affected by compression. High background noise biased most energy distribution-related parameters (e.g. spectral entropy) and affected the precision of most acoustic parameters and dynamic time warping. Overall, compression and background noise did have considerable effects on acoustic analyses. We provide recommendations to avoid potential pitfalls and maximize the information that can be reliably obtained.     

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.     

Sugar transport together with environmental conditions controls time lags between xylem and stem diameter changes

In the present study the seasonal patterns of time lags between diurnal xylem and whole stem diameter variations at the top and at the base of two Scots pine trees (Pinus sylvestris L.) were compared. The diameter variations were measured during the summers of 2001 and 2002. Time lags were determined using the cross‐correlation method. The lags were found to vary in time according to the different stages of growth. At the top the xylem lagged behind the whole stem between the beginning of stem growth and the end of shoot growth in both years. In 2001 the time lags at the base showed a similar behaviour during stem growth. That kind of seasonal pattern of the time lags would result from the changes in the sink strength due to changing growth rate at different parts of the tree and the differences in the annual rhythm of growth and water availability in the soil (based on precipitation measurements) between the years 2001 and 2002 were reflected in the patterns. The time lags of shrinking and swelling periods during high and low photosynthetic activity (measured using a shoot chamber) were also compared. It was found, for example, that in 2001 in the middle of the growing season at the top of the tree the whole stem lagged on average 15 min more behind the xylem on the days of high photosynthetic activity than on the days of low or moderate. These results show for the first time that the transportation of carbohydrates and variable sink activity could be detected during the growing season in field conditions using stem and xylem diameter variation measurements. Furthermore, these results provide evidence of the pressure gradient‐driven flow also in the phloem of gymnosperms.     

CorTectorTM SX100：一款桌面式荧光相关光谱仪的原理和应用

荧光相关光谱检测技术具有超灵敏(单分子)、快速(数秒至数分钟)和多功能(检测分子浓度、大小和相互作用)等技术优点,且无需反应物分离,因此有潜力成为一种新型均相、高敏荧光免疫检测技术,适用于在溶液中或单个活细胞内检测生物分子特性．本文首先介绍荧光相关光谱检测技术的原理和研究进展,然后结合项目团队自主研发的目前全球唯一一款可靠、易使用的桌面式荧光相关光谱仪,进一步探讨荧光相关光谱检测技术的具体实现和潜在应用．     

Applications of dual-color fluorescence cross-correlation spectroscopy in antibody binding studies

Two-photon dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) was applied to study the binding interactions of monoclonal antibodies (mAbs) and protein antigens. We measured the binding constant of the interaction of a 32-amino acid brain natriuretic peptide (BNP) with a mAbs and demonstrated the utility of DC-FCCS in studies of antibody sandwiches, trimolecular formations, where two different antibodies bind the same antigen simultaneously. We also show the use of DC-FCCS for monitoring competitive displacement of the labeled antibody in antibody-antigen complexes and subsequent determination of the pertinent dissociation rate (off-rate). The off-rate measurements were performed for two mAbs toward tissue inhibitor 1 of metalloproteinases (TIMP-1). From a methodological perspective, selection of the best labeling protocols and careful optimization of the FCCS instrumentation are essential to achieve the highest cross-correlation signal. When working in vitro, it is practical to generate a complete binding curve using the normalized cross-correlation signal and then fit the experimental points to a binding model. DC-FCCS offers the sensitivity and all other advantages of a solution phase fluorescence-based technique. For systems containing proteins of a similar size that interact without substantial changes in the fluorescence intensity, DC-FCCS serves as a preferred means of measuring solution phase binding constants.     

Interplay between oncogenic K-Ras and wild-type H-Ras in Caco2 cell transformation

Mutation of RAS genes is one of the most common oncogenic alterations in cancer and acquisition of activating RAS mutations has been demonstrated to cause progression of colorectal adenoma to cancer. The aim of this study was to identify changes in the proteome of the intermediate-stage colorectal cancer cell line Caco2, induced by ectopic expression of two distinct RAS proteins, KRAS(V12) and HRAS(V12), in their mutated, constitutively active form. Using 2D-gel electrophoresis, followed by LC-MS/MS we identified almost 200 differentially expressed proteins in pair-wise comparisons of Caco2 vs Caco2-KRAS(V12) and Caco2 vs Caco2-HRAS(V12). Although many of the affected proteins were unique for each pair, there were also substantial similarities. Interestingly, transformation by the mutant KRAS(V12) gene resulted in elevated expression levels and activity of endogenous H-ras protein. Silencing the latter with a specific RNAi reversed several proteomic changes observed in KRAS(V12)-transformed cells, suggesting that oncogenic K-ras partly exerts its effects through endogenous H-ras activation. Alterations in the expression of cytoskeletal and cell adhesion proteins, caused by HRAS siRNA treatment, correlated with a reduction in the invasive properties of Caco2-KRAS(V12) cells. Our data suggest a novel interplay between K-ras and H-ras, with possible implications for colorectal carcinogenesis.