The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF.

Vascular endothelial growth factor (VEGF)mRNA undergoes alternative splicing events that generate four different homodimeric isoforms, VEGF121, VEGF165, VEGF189, or VEGF206. VEGF121 is a nonheparin-binding acidic protein, which is freely diffusible. The longer forms, VEGF189 or VEGF206, are highly basic proteins tightly bound to extracellular heparin-containing proteoglycans. VEGF165 has intermediate properties. To determine the localization of VEGF isoforms, transfected human embryonic kidney CEN4 cells expressing VEGF165, VEGF189, or VEGF206 were stained by immunofluorescence with a specific monoclonal antibody. The staining was found in patches and streaks suggestive of extracellular matrix (ECM). VEGF165 was observed largely in Golgi apparatus-like structures. Immunogold labeling of cells expressing VEGF189 or VEGF206 revealed that the staining was localized to the subepithelial ECM. VEGF associated with the ECM was bioactive, because endothelial cells cultured on ECM derived from cells expressing VEGF189 or VEGF206 were markedly stimulated to proliferate. In addition, ECM-bound VEGF can be released into a soluble and bioactive form by heparin or plasmin. ECM-bound VEGF189 and VEGF206 have molecular masses consistent with the intact polypeptides. The ECM may represent an important source of VEGF and angiogenic potential.     

Progressive search in tandem mass spectrometry

A cell exhibits a variety of responses to internal and external cues. These responses are possible, in part, due to the presence of an elaborate gene regulatory network (GRN) in every single cell. In the past 20 years, many groups worked on reconstructing the topological structure of GRNs from large-scale gene expression data using a variety of inference algorithms. Insights gained about participating players in GRNs may ultimately lead to therapeutic benefits. Mutual information (MI) is a widely used metric within this inference/reconstruction pipeline as it can detect any correlation (linear and non-linear) between any number of variables (n-dimensions). However, the use of MI with continuous data (for example, normalized fluorescence intensity measurement of gene expression levels) is sensitive to data size, correlation strength and underlying distributions, and often requires laborious and, at times, ad hoc optimization. In this work, we first show that estimating MI of a bi- and tri-variate Gaussian distribution using k-nearest neighbor (kNN) MI estimation results in significant error reduction as compared to commonly used methods based on fixed binning. Second, we demonstrate that implementing the MI-based kNN Kraskov–Stoögbauer–Grassberger (KSG) algorithm leads to a significant improvement in GRN reconstruction for popular inference algorithms, such as Context Likelihood of Relatedness (CLR). Finally, through extensive in-silico benchmarking we show that a new inference algorithm CMIA (Conditional Mutual Information Augmentation), inspired by CLR, in combination with the KSG-MI estimator, outperforms commonly used methods. Using three canonical datasets containing 15 synthetic networks, the newly developed method for GRN reconstruction—which combines CMIA, and the KSG-MI estimator—achieves an improvement of 20–35% in precision-recall measures over the current gold standard in the field. This new method will enable researchers to discover new gene interactions or better choose gene candidates for experimental validations.     

Distribution of antarctic zooplankton around Elephant Island during the austral summers of 1988, 1989, and 1990

Year to year variation and vertical distributions of epipelagic Zooplankton around Elephant Island and King George Island were examined with samples collected with bongo nets and a 1 m² MOCNESS during the austral summers (Jan–Feb.) of 1988, 1989 and 1990. Copepods were the major components of epipelagic Zooplankton (in numbers) with dominance of Metridia gerlachei (1988 and 1989) and small calanoids and cyclopoids (1990). Euphausiids and salps were the next most abundant groups. The percent composition of euphausiids decreased from 1988 to 1990 while that of salps increased. The abundance of salps exceeded euphausiids and major taxa of copepods in 1990. Local patches of polychaetes and amphipods were also found. Statistically significant annual variations with increased numbers in 1990 were found by analyses of variance in total abundance, abundances of copepods, salps, chaetognaths and amphipods, but abundances of euphausiids, polychaetes and fishes showed no significant annual variations. When the study area was divided geographically, horizontal variability in abundance within each year showed no significance in total abundance, abundances of copepods, euphausiids, amphipods and fishes, but significance in salps, polychaetes and chaetognaths. Results of site clustering based on covariances of abundances of eleven major taxa were well matched, though not perfect, with the distribution of surface water temperatures which could be used as a tracer of source water masses suggesting that spatial variation was related to hydrodynamic conditions. Factor analyses showed that annual and spatial variations in abundance were mainly caused by only two taxa, Metridia gerlachei and salps (mostly Salpa thompsoni). These two taxa were responsible for about 60% and 30% of total variance, respectively, and were useful indicators of the interannual variation. That is, 1988 and 1989 were the years of M. gerlachei, and 1990 was the year of salps. From vertically stratified MOCNESS samples, it was shown that the major taxa in this study area were active vertical migrators. While most samples obtained by relatively shallow tows (uppermost 100 m depth) were composed of exclusively one or two taxa, those from relatively deep tows (down to 200 m) showed various patterns of vertical stratification suggesting that the patterns of vertical migration were species specific. Species specific ontogenetic vertical migration associated with elevated habitat temperatures also seemed to be responsible for the annual variation in zooplankton distribution in the upper water column.     

Integrating sample similarities into latent class analysis: a tree-structured shrinkage approach

This paper is concerned with using multivariate binary observations to estimate the probabilities of unobserved classes with scientific meanings. We focus on the setting where additional information about sample similarities is available and represented by a rooted weighted tree. Every leaf in the given tree contains multiple samples. Shorter distances over the tree between the leaves indicate a priori higher similarity in class probability vectors. We propose a novel data integrative extension to classical latent class models with tree-structured shrinkage. The proposed approach enables (1) borrowing of information across leaves, (2) estimating data-driven leaf groups with distinct vectors of class probabilities, and (3) individual-level probabilistic class assignment given the observed multivariate binary measurements. We derive and implement a scalable posterior inference algorithm in a variational Bayes framework. Extensive simulations show more accurate estimation of class probabilities than alternatives that suboptimally use the additional sample similarity information. A zoonotic infectious disease application is used to illustrate the proposed approach. The paper concludes by a brief discussion on model limitations and extensions.     

Phosphorylation states greatly regulate the activity and gating properties of Cav3.1 T-type Ca2+ channels

Ca_v3.1 T-type Ca²⁺ channels play pivotal roles in neuronal low-threshold spikes, visceral pain, and pacemaker activity. Phosphorylation has been reported to potently regulate the activity and gating properties of Ca_v3.1 channels. However, systematic identification of phosphorylation sites (phosphosites) in Ca_v3.1 channel has been poorly investigated. In this work, we analyzed rat Ca_v3.1 protein expressed in HEK-293 cells by mass spectrometry, identified 30 phosphosites located at the cytoplasmic regions, and illustrated them as a Ca_v3.1 phosphorylation map which includes the reported mouse Ca_v3.1 phosphosites. Site-directed mutagenesis of the phosphosites to Ala residues and functional analysis of the phospho-silent Ca_v3.1 mutants expressed in Xenopus oocytes showed that the phospho-silent mutation of the N-terminal Ser18 reduced its current amplitude with accelerated current kinetics and negatively shifted channel availability. Remarkably, the phospho-silent mutations of the C-terminal Ser residues (Ser1924, Ser2001, Ser2163, Ser2166, or Ser2189) greatly reduced their current amplitude without altering the voltage-dependent gating properties. In contrast, the phosphomimetic Asp mutations of Ca_v3.1 on the N- and C-terminal Ser residues reversed the effects of the phospho-silent mutations. Collectively, these findings demonstrate that the multiple phosphosites of Ca_v3.1 at the N- and C-terminal regions play crucial roles in the regulation of the channel activity and voltage-dependent gating properties.     

CHAPS solubilization of a G-protein sensitive 5-HT1A receptor from bovine hippocampus

The binding of [3H] 8-OH-DPAT to membrane-bound 5-HT1A receptors from bovine hippocampus was saturable and corresponded to a single high-affinity state. Solubilization of the bovine hippocampal membranes with 10 mM CHAPS containing 200 mM NaCl, renders a preparation which binds [3H] 8-OH-DPAT with high-affinity (Kd = 1.9 nM) and is guanine nucleotide sensitive and ketanserin insensitive. 50% of [3H] 8-OH-DPAT binding activity is solubilized. The presence of GMP-P(NH)P promotes a low-affinity (Kd = 9.6 nM) state which is characteristic of receptors coupled to G-proteins. GMP-P(NH)P markedly accelerates the dissociation [3H] 8-OH-DPAT from solubilized membranes while having negligible effects on association. Thus, the agonist can activate the terniary complex rather than to promote its formation. 8-OH DPAT, WB 4101 and 5-carboxamidotryptamine dose responsively inhibit soluble [3H] 8-OH-DPAT binding with IC(50) values of 16.1, 15.6 and 1.3 nM, respectively. The CHAPS solubilized membrane preparation retains many of the [3H] 8-OH-DPAT binding characteristics of the membrane bound form.     

Object-oriented graphical modeling of FMSs

Presented in the article is a method for constructing a graphical model of an FMS by using a new modeling tool called JR-net (Job Resource relation-net). JR-net is an object-oriented graphical tool for modeling automated manufacturing systems (AMSs), such as FMSs, FASs, and AS/RSs. As with the object-oriented modeling paradigm of Rumbaugh et al. (1991), the JR-net modeling framework supports the three stages of models: static layout model (object model); job flow model (functional model); and supervisory control model (dynamic model). In this article, the existing JR-net structure (Park 1992, Han et al., 1995) is extended further to make it a graphical tool for FMS modeling. Using the extended JR-net, a step-by-step procedure for constructing a graphical model of FMSs is presented. Also addressed are issues of classifying FMSs in terms of their generic functions and of utilizing the JR-net model of FMSs.     

Optimization of the separation and detection of the enantiomers of isoproterenol in microdialysis samples by cyclodextrin-modified capillary electrophoresis using electrochemical detection

Isoproterenol is a chiral catecholamine with a half-life of elimination of less than 10 min. In order to study the pharmacokinetics of this compound using microdialysis sampling, an analytical method was needed which could resolve the individual enantiomers of isoproterenol and required less than 1 μl of sample. A capillary electrophoretic method using a run buffer containing methyl-O-β-cyclodextrin as a chiral recognition agent was developed which could resolve the enantiomers of isoproterenol. The detection limits using UV absorbance detection were found to be too high to determine the concentration of isoproterenol in plasma for a sufficient time following administration to establish the pharmacokinetics. The detection limits were decreased three orders of magnitude to 3 ng/ml by using an amperometric detector. The detection limits were decreased to 0.6 ng/ml using an on-column concentration technique in which peak stacking was accomplished by following the sample injection with a plug of acid.