Distinct cortical and collicular mechanisms of inhibition of return revealed with S cone stimuli

Visual orienting of attention and gaze are widely considered to be mediated by shared neural pathways, with automatic phenomena such as inhibition of return (IOR)--the bias against returning to recently visited locations--being generated via the direct pathway from retina to superior colliculus (SC). Here, we show that IOR occurs without direct access to the SC, by using a technique that employs stimuli visible only to short-wave-sensitive (S) cones. We found that these stimuli, to which the SC is blind , were quite capable of eliciting IOR, measured by traditional manual responses. Critically, however, we found that S cone stimuli did not cause IOR when saccadic eye movement responses were required. This demonstrates that saccadic IOR is not the same as traditional IOR, providing support for two separate cortical and collicular mechanisms of IOR. These findings represent a clear dissociation between visual orienting of attention and gaze.     

Enhanced growth,sporulation and toxin production by Bacillus thuringiensis subsp. kurstaki in oil seed meal extract media containing cystine

A mineral salts medium supplemented with peptone and 40mg% cystine supported enhanced sporulation (10¹¹spores/ ml) and high yields of insecticidal crystal protein (17.2mg/ml) in Bacillus thuringiensis subsp. kurstaki (3a3b) in 2.0l baffled aerated cylinders. These high yields could also be achieved with defatted soybean and groundnut seed meal extracts when supplemented with cystine.     

The 2003 anatomy ceremony: a service of gratitude

In keeping with a long-standing tradition, Yale Medical and Physician Associate students gather at a ceremony each year after the completion of the anatomy course. The ceremony is a chance to reflect and to give thanks. It gives students the opportunity to articulate their gratitude to the selfless individuals who donated their bodies for the benefit of education. Many family members of the donors attend the ceremony. By reading poetry, performing musical pieces, and presenting works of art, the students and their teachers express some of the emotions and thoughts that the anatomy course has evoked. The following are some of the contributions presented at this year's ceremony.     

Potential applications of DNA microarrays in biodefense-related diagnostics

Recent years have witnessed a logarithmic growth in the number of applications involving DNA microarrays. Extrapolation of their use for infectious diagnostics and biodefense-related diagnostics seems obvious. Nevertheless, the application of DNA microarrays to biodefense-related diagnostics will depend on solving a set of substantial, yet approachable, technical and logistical problems that encompass diverse topics from amplification efficiency to bioinformatics.     

Cost effectiveness analysis of screening for sight threatening diabetic eye disease

ObjectiveTo measure the cost effectiveness of systematic photographic screening for sight threatening diabetic eye disease compared with existing practice.DesignCost effectiveness analysisSettingLiverpool.SubjectsA target population of 5000 diabetic patients invited for screening.ResultsBaseline prevalence of sight threatening eye disease was 14.1%. The cost effectiveness of the systematic programme was £209 (sensitivity 89%, specificity 86%, compliance 80%, annual cost £104 996) and of the opportunistic programme was £289 (combined sensitivity 63%, specificity 92%, compliance 78%, annual cost £99 981). The incremental cost effectiveness of completely replacing the opportunistic programme was £32. Absolute values of cost effectiveness were highly sensitive to varying prevalence, sensitivity and specificity, compliance, and programme size.ConclusionReplacing existing programmes with systematic screening for diabetic eye disease is justified.     

Detection and identification of bacteria using antibiotic susceptibility and a multi-array electrochemical sensor with pattern recognition

This work proposes the use of amperometric signals generated by a 96-well multi-array dissolved oxygen multi-electrode sensor (DOX) coupled with principal component analysis for continuous monitoring, identification and differentiation of bacteria. Two types of differentiation mechanisms were tested: (1) direct monitoring of respiratory activity via oxygen consumption and (2) quantification of the effect of three broad-spectrum antibiotics on bacteria growth and respiration over time. Five species of bacteria were examined including: Escherichia coli, Escherichia adecarboxylata, Comamonas acidovorans, Corynebacterium glutamicum and Staphylococcus epidermidis. The addition of small concentrations of antibiotics to the growth medium alters the oxygen consumption of the cells and a unique fingerprint is created for a specific cell. This fingerprint is shown to evolve over a specific concentration range that is dependant of instrumental constraints of the DOX system. The application of principal component analysis (PCA) to classify the data was also examined. It was shown that bacteria could be classified simply by their oxygen consumption rates over a varying concentration range. Discrimination between species can also be increased by the effects of the antibiotics on the oxygen consumption of varying concentrations of cells. The proposed DOX-PCA system illustrates a generic template that can be tailored to meet specific research goals by the selection of specific cell/antibiotic combinations and concentrations.     

Membrane insertion of the FYVE domain is modulated by pH

The FYVE domain associates with phosphatidylinositol 3‐phosphate [PtdIns(3)P] in membranes of early endosomes and penetrates bilayers. Here, we detail principles of membrane anchoring and show that the FYVE domain insertion into PtdIns(3)P‐enriched membranes and membrane‐mimetics is substantially increased in acidic conditions. The EEA1 FYVE domain binds to POPC/POPE/PtdIns(3)P vesicles with a Kd of 49 nM at pH 6.0, however associates ～24 fold weaker at pH 8.0. The decrease in the affinity is primarily due to much faster dissociation of the protein from the bilayers in basic media. Lowering the pH enhances the interaction of the Hrs, RUFY1, Vps27p and WDFY1 FYVE domains with PtdIns(3)P‐containing membranes in vitro and in vivo, indicating that pH‐dependency is a general function of the FYVE finger family. The PtdIns(3)P binding and membrane insertion of the FYVE domain is modulated by the two adjacent His residues of the R(R/K)HHCRXCG signature motif. Mutation of either His residue abolishes the pH‐sensitivity. Both protonation of the His residues and nonspecific electrostatic contacts stabilize the FYVE domain in the lipid‐bound form, promoting its penetration and increasing the membrane residence time. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Locked nucleic acid flow cytometry-fluorescence in situ hybridization (LNA flow-FISH): a method for bacterial small RNA detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination, cellular identification and gene expression, monitoring viral multiplication in infected cells, and bacterial community analysis and enumeration. Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (T(m)) of these analogs for natural nucleic acids. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes and have been successfully used to detect eukaryotic mRNA and viral RNA in mammalian cells. Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).     

Isoenzymes of phosphofructokinase in the rat. Demonstration of the three non-identical subunits by biochemical, immunochemical and kinetic studies.

In man and the rabbit, 6-phosphofructokinase (PFK, EC 2.7.1.11) exists in tetrameric isoenzymic forms composed of muscle (M or A), liver (L or B) and platelet or brain (P or C) subunits, which are under separate genetic control. In contrast, the genetic control of the rat PFK has not yet been conclusively established; it is unclear whether the P-type or C-type subunit exists in this species. To resolve this question, we investigated the enzyme from the skeletal muscle, liver and brain of rats of Wag/Rij strain. Our studies demonstrate that the rat PFK is also under the control of three structural loci and that the homotetramers M4, P4 and L4 exhibit unique chromatographic, immunological and kinetic-regulatory properties. Skeletal-muscle and brain PFKs consist of isolated M4 and P4 homotetramers respectively. Although liver PFK consists predominantly of L4 homotetramer, it also contains small amounts of PL3 and P2L2 species. All three PFKs exhibit allosteric properties: co-operativity with fructose 6-phosphate and inhibition by ATP decrease in the order P4 greater than L4 greater than M4. P4 and M4 tetramers are the most sensitive to citrate inhibition, whereas L4 tetramer is the least sensitive. More importantly, P4 and L4 isoenzymes are the most sensitive to activation by fructose 2,6-bisphosphate, whereas M4 isoenzyme is the least sensitive. These results indicate that the brain PFK in this strain of rat is a unique tetramer, P4, which also exhibits allosteric kinetics, as do the well-studied M4 and L4 isoenzymes. The reported differences in the number and nature of isoenzymes present in the rat brain and liver most probably reflect the differences in the strains studied by previous investigators. Since the nature of the rat PFK isoenzymes and nomenclatures reported by previous investigators have been now reconciled, it is proposed that, for the sake of uniformity, only well-established nomenclatures used for the rabbit or human PFK isoenzymes be used for the rat isoenzymes.     

Building the bacterial orisome: high‐affinity DnaA recognition plays a role in setting the conformation of oriC DNA

During assembly of the E. coli pre‐replicative complex (pre‐RC), initiator DnaA oligomers are nucleated from three widely separated high‐affinity DnaA recognition sites in oriC. Oligomer assembly is then guided by low‐affinity DnaA recognition sites, but is also regulated by a switch‐like conformational change in oriC mediated by sequential binding of two DNA bending proteins, Fis and IHF, serving as inhibitor and activator respectively. Although their recognition sites are separated by up to 90 bp, Fis represses IHF binding and weak DnaA interactions until accumulating DnaA displaces Fis from oriC. It remains unclear whether high‐affinity DnaA binding plays any role in Fis repression at a distance and it is also not known whether all high‐affinity DnaA recognition sites play an equivalent role in oligomer formation. To examine these issues, we developed origin‐selective recombineering methods to mutate E. coli chromosomal oriC. We found that, although oligomers were assembled in the absence of any individual high‐affinity DnaA binding site, loss of DnaA binding at peripheral sites eliminated Fis repression, and made binding of both Fis and IHF essential. We propose a model in which interaction of DnaA molecules at high‐affinity sites regulates oriC DNA conformation.