Functional evidence for a dual route to amygdala

The amygdala plays a central role in evaluating the behavioral importance of sensory information. Anatomical subcortical pathways provide direct input to the amygdala from early sensory systems and may support an adaptively valuable rapid appraisal of salient information. However, the functional significance of these subcortical inputs remains controversial. We recorded magnetoencephalographic activity evoked by tones in the context of emotionally valent faces and tested two competing biologically motivated dynamic causal models against these data: the dual and cortical models. The dual model comprised two parallel (cortical and subcortical) routes to the amygdala, whereas the cortical model excluded the subcortical path. We found that neuronal responses elicited by salient information were better explained when a subcortical pathway was included. In keeping with its putative functional role of rapid stimulus appraisal, the subcortical pathway was most important early in stimulus processing. However, as often assumed, its action was not limited to the context of fear, pointing to a more widespread information processing role. Thus, our data supports the idea that an expedited evaluation of sensory input is best explained by an architecture that involves a subcortical path to the amygdala.     

Generation and characterization of anti-MUC4 monoclonal antibodies reactive with normal and cancer cells in humans.

We have previously cloned the full-length cDNA (approximately 28 Kb) and established the complete genomic organization (25 exons/introns over 100 kb) of the human MUC4 mucin. This large molecule is predicted to protrude over 2 microm above the cell surface, in which MUC4alpha is an extracellular mucin-type glycoprotein subunit and MUC4beta is the transmembrane subunit. Over two thirds of the encoded protein sequence consists of 16-amino-acid tandem repeats (TR), which are flanked by unique sequences. In this study we generated and characterized monoclonal antibodies (MAbs) directed against the TR region of MUC4. Mice were immunized with a KLH-conjugated MUC4 TR peptide, STGDTTPLPVTDTSSV. Several clones were purified by three rounds of limited dilutions and stable clones presenting a sustained antibody production were selected for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the MUC4 peptide and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the MAbs (8G7) was strongly reactive against the MUC4 peptide and with native MUC4 from human tissues or pancreatic cancer cells in Western blotting, immunohistochemistry, and confocal analysis. Anti-MUC4 MAb may represent a powerful tool for the study of MUC4 function under normal and pathological conditions and for diagnosis of solid tumors including those in the breast, pancreas, lungs, and ovaries.     

A Structured Model of Video Reproduces Primary Visual Cortical Organisation

The visual system must learn to infer the presence of objects and features in the world from the images it encounters, and as such it must, either implicitly or explicitly, model the way these elements interact to create the image. Do the response properties of cells in the mammalian visual system reflect this constraint? To address this question, we constructed a probabilistic model in which the identity and attributes of simple visual elements were represented explicitly and learnt the parameters of this model from unparsed, natural video sequences. After learning, the behaviour and grouping of variables in the probabilistic model corresponded closely to functional and anatomical properties of simple and complex cells in the primary visual cortex (V1). In particular, feature identity variables were activated in a way that resembled the activity of complex cells, while feature attribute variables responded much like simple cells. Furthermore, the grouping of the attributes within the model closely parallelled the reported anatomical grouping of simple cells in cat V1. Thus, this generative model makes explicit an interpretation of complex and simple cells as elements in the segmentation of a visual scene into basic independent features, along with a parametrisation of their moment-by-moment appearances. We speculate that such a segmentation may form the initial stage of a hierarchical system that progressively separates the identity and appearance of more articulated visual elements, culminating in view-invariant object recognition.     

Morphological development,cell number,and allocation of cells to trophectoderm and inner cell mass of in vitro fertilized and parthenogenetically developed buffalo embryos: The effect of IGF-I

The morphology and number of cells in the trophectoderm (TE) and inner cell mass (ICM) of buffalo blastocysts derived from in vitro fertilization and cultured in the presence or absence of insulin-like growth factor-I (IGF-I) were analyzed by differential fluorochrome staining technique. The total cell number (TCN), TE number, and ICM cell number were significantly higher in blastocysts developed in vitro in the presence of IGF-I as compared to blastocysts developed without IGF-I (P < 0.01). It was observed that the buffalo blastocyst took 5–9 days postfertilization to develop in vitro. In order to correlate the time required for blastocyst development and the allocation of cells to TE and ICM, blastocysts were designated as fast (developing on or before day 7) or slow (developing after day 7). The TCN, TE, and ICM cells of fast-developing blastocysts cultured in the presence of IGF-I were significantly higher than slow-developing blastocysts (P < 0.01). The blastocysts developed on day 6 had a mean total cell number 118.6 ± 21.4, which significantly decreased to 85.6 ± 17.4, 62.0 ± 14.5, and 17.0 ± 4.0 on days 7, 8, and 9, respectively (P < 0.05). Normal development of buffalo embryo showed that, on average, embryos reached compact morula stage at the earliest between days 4.5–5.5. Blastocysts developed, at the earliest, between days 5.0–6.0, and it took them, on average, 6.5 days to hatch from the zona pellucida. TCN, TE, and ICM increased three times from morula to blastocyst; however, the proportion of ICM to TCN remained the same, in both embryonic stages. TE approximately doubled in hatched blastocysts, as compared to unhatched blastocysts (P < 0.05). However, ICM cells were decreased. The time required for development of parthenogenetic blastocysts was observed to be greater as compared to in vitro fertilized (IVF) blastocysts. The total cell number of parthenogenetic blastocysts was 100.8 ± 11.3, including 59.2 ± 8.4 cells of TE and 42.1 ± 6.9 cells of ICM. © 1996 Wiley-Liss, Inc.     

Transvenous pacing in complex post-operative congenital heart disease guided by angiography: A case report

Transvenous pacing in patients with postoperative complex congenital heart disease (CHD) can be challenging and pose technical challenges to lead placement because of the complex anatomy, distortions produced by the surgical procedures, and the altered relationship of cardiac chambers. We describe the utility of angiography for transvenous dual chamber pacemaker implantation in a post-operative complex congenital heart disease.     

Reactive oxygen species mediate microRNA-302 regulation of AT-rich interacting domain 4a and C-C motif ligand 5 expression during transitions between quiescence and proliferation

Normal cell growth consists of two distinct phases, quiescence and proliferation. Quiescence, or G(0), is a reversible growth arrest in which cells retain the ability to reenter the proliferative cycle (G(1), S, G(2), and M). Although not actively dividing, quiescent cells are metabolically active and quiescence is actively maintained. Our results from microRNA PCR arrays and Taqman PCR assays showed a significant decrease (4-fold) in miR-302 levels during quiescence compared to proliferating normal human fibroblasts, suggesting that miR-302 could regulate cellular proliferation. Results from a Q-RT-PCR and dual-luciferase-3'-UTR reporter assays identified ARID4a (AT-rich interacting domain 4a, also known as RBP1) and CCL5 (C-C motif ligand 5) as targets for miR-302. Ionizing radiation decreased miR-302 levels, which was associated with an increase in its target mRNA levels, ARID4a and CCL5. Such an inverse correlation was also observed in cells treated with hydrogen peroxide as well as SOD2-overexpressing cells. Overexpression of miR-302 suppresses ARID4a and CCL5 mRNA levels, and increased the percentage of S-phase cells. These results identified miR-302 as an ROS-sensitive regulator of ARID4a and CCL5 mRNAs as well as demonstrate a regulatory role of miR-302 during quiescence and proliferation.     

Behavioral observations of adolescent Holstein heifers cloned from adult somatic cells

Cloning using somatic cells offers many potential applications in biomedicine and basic research. The objective of this study was to test whether clones from the same genotype can be used as models to study the genetic influences of behavior. Specifically, several aspects of the behavior of four prepubertal heifers cloned from somatic cells of a 13-year-old Holstein cow along with age-matched control heifers were compared to determine whether juvenile clones from an aged adult behave similarly to their age-matched controls, and whether clones with identical genetic makeup exhibit any behavioral trends. Behavioral observations or behavior challenge tests were conducted to compare the following traits: vocalization, play behavior, movement frequencies, grooming, curiosity, and companion preference, as well as dominance and aggressiveness. From play behavior, movements and vocalization, we observed that these four juvenile clones of an aged genetic donor did not show behavioral indications of aging and were similar to their counterparts of comparable chronological age except that they tended to play less than controls. Behavioral trends were also observed in the clones that indicated that they exhibited higher levels of curiosity, more grooming activities and were more aggressive and dominant than controls. Furthermore, these four clones preferred each other or the donor as companions, which may indicate genetic kin recognition.     

Harnessing asymmetrical substrate recognition by thermostable EndoV to achieve balanced linear amplification in multiplexed SNP typing.

Multiplexed amplification of specific DNA sequences, by PCR or by strand-displacement amplification, is an intrinsically biased process. The relative abundance of amplified DNA can be altered significantly from the original representation and, in extreme cases, allele dropout can occur. In this paper, we present a method of linear amplification of DNA that relies on the cooperative, sequence-dependent functioning of the DNA mismatch-repair enzyme endonuclease V (EndoV) from Thermotoga maritima (Tma) and Bacillus stearothermophilus (Bst) DNA polymerase. Tma EndoV can nick one strand of unmodified duplex DNA, allowing extension by Bst polymerase. By controlling the bases surrounding a mismatch and the mismatch itself, the efficiency of nicking by EndoV and extension by Bst polymerase can be controlled. The method currently allows 100-fold multiplexed amplification of target molecules to be performed isothermally, with an average change of <1.3-fold in their original representation. Because only a single primer is necessary, primer artefacts and nonspecific amplification products are minimized.     

High sensitivity EndoV mutation scanning through real-time ligase proofreading

The ability to associate mutations in cancer genes with the disease and its subtypes is critical for understanding oncogenesis and identifying biomarkers for clinical diagnosis. A two-step mutation scanning method that sequentially used endonuclease V (EndoV) to nick at mismatches and DNA ligase to reseal incorrectly or nonspecifically nicked sites was previously developed in our laboratory. Herein we report an optimized single-step assay that enables ligase to proofread EndoV cleavage in real-time under a compromise between buffer conditions. Real-time proofreading results in a dramatic reduction of background cleavage. A universal PCR strategy that employs both unlabeled gene-specific primers and labeled universal primers, allows for multiplexed gene amplification and precludes amplification of primer dimers. Internally labeled PCR primers eliminate EndoV cleavage at the 5′ terminus, enabling high-throughput capillary electrophoresis readout. Furthermore, signal intensity is increased and artifacts are reduced by generating heteroduplexes containing only one of the two possible mismatches (e.g. either A/C or G/T). The single-step assay improves sensitivity to 1:50 and 1:100 (mutant:wild type) for unknown mutations in the p53 and K-ras genes, respectively, opening prospects as an early detection tool.