Comprehensive biothreat cluster identification by PCR/electrospray-ionization mass spectrometry

Technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short. The ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. A technological solution to this problem can be achieved by coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS). The biothreat assay described here detects ten bacterial and four viral biothreat clusters on the NIAID priority pathogen and HHS/USDA select agent lists. Detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. Analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. Further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. The biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. Coupling biothreat cluster-specific PCR to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry.     

The role of oxidised regenerated cellulose/collagen in chronic wound repair and its potential mechanism of action

Normal wound healing is a carefully controlled balance of destructive processes necessary to remove damaged tissue and repair processes which lead to new tissue formation. Proteases and growth factors play a pivotal role in regulating this balance, and if disrupted in favour of degradation then delayed healing ensues; a trait of chronic wounds. Whilst there are many types of chronic wounds, biochemically they are thought to be similar in that they are characterised by a prolonged inflammatory phase, which results in elevated levels of proteases and diminished growth factor activity. This increase in proteolytic activity and subsequent degradation of growth factors is thought to contribute to the net tissue loss associated with these chronic wounds.

In this study, we describe a new wound treatment, comprising oxidised regenerated cellulose and collagen (ORC/collagen), which can redress this imbalance and modify the chronic wound environment. We demonstrate that ORC/collagen can inactivate potentially harmful factors such as proteases, oxygen free radicals and excess metal ions present in chronic wound fluid, whilst simultaneously protecting positive factors such as growth factors and delivering them back to the wound.

These characteristics suggest a beneficial role for this material in helping to re-balance the chronic wound environment and therefore promote healing.     

Evolution of aquatic insect behaviours across a gradient of disturbance predictability

Natural disturbance regimes--cycles of fire, flood, drought or other events--range from highly predictable (disturbances occur regularly in time or in concert with a proximate cue) to highly unpredictable. While theory predicts how populations should evolve under different degrees of disturbance predictability, there is little empirical evidence of how this occurs in nature. Here, we demonstrate local adaptation in populations of an aquatic insect occupying sites along a natural gradient of disturbance predictability, where predictability was defined as the ability of a proximate cue (rainfall) to signal a disturbance (flash flood). In controlled behavioural experiments, populations from predictable environments responded to rainfall events by quickly exiting the water and moving sufficiently far from the stream to escape flash floods. By contrast, populations from less predictable environments had longer response times and lower response rates, reflecting the uncertainty inherent to these environments. Analysis with signal detection theory showed that for 13 out of 15 populations, observed response times were an optimal compromise between the competing risks of abandoning versus remaining in the stream, mediated by the rainfall-flood correlation of the local environment. Our study provides the first demonstration that populations can evolve in response to differences in disturbance predictability, and provides evidence that populations can adapt to among-stream differences in flow regime.     

Insulin-Responsive Compartments Containing GLUT4 in 3T3-L1 and CHO Cells: Regulation by Amino Acid Concentrations

In fat and muscle, insulin stimulates glucose uptake by rapidly mobilizing the GLUT4 glucose transporter from a specialized intracellular compartment to the plasma membrane. We describe a method to quantify the relative proportion of GLUT4 at the plasma membrane, using flow cytometry to measure a ratio of fluorescence intensities corresponding to the cell surface and total amounts of a tagged GLUT4 reporter in individual living cells. Using this assay, we demonstrate that both 3T3-L1 and CHO cells contain intracellular compartments from which GLUT4 is rapidly mobilized by insulin and that the initial magnitude and kinetics of redistribution to the plasma membrane are similar in these two cell types when they are cultured identically. Targeting of GLUT4 to a highly insulin-responsive compartment in CHO cells is modulated by culture conditions. In particular, we find that amino acids regulate distribution of GLUT4 to this kinetically defined compartment through a rapamycin-sensitive pathway. Amino acids also modulate the magnitude of insulin-stimulated translocation in 3T3-L1 adipocytes. Our results indicate a novel link between glucose and amino acid metabolism.     

The glucose transporter 4-regulating protein TUG is essential for highly insulin-responsive glucose uptake in 3T3-L1 adipocytes

Insulin stimulates glucose uptake in fat and muscle by redistributing GLUT4 glucose transporters from intracellular membranes to the cell surface. We previously proposed that, in 3T3-L1 adipocytes, TUG retains GLUT4 within unstimulated cells and insulin mobilizes this retained GLUT4 by stimulating its dissociation from TUG. Yet the relative importance of this action in the overall control of glucose uptake remains uncertain. Here we report that transient, small interfering RNA-mediated depletion of TUG causes GLUT4 translocation and enhances glucose uptake in unstimulated 3T3-L1 adipocytes, similar to insulin. Stable TUG depletion or expression of a dominant negative fragment likewise stimulates GLUT4 redistribution and glucose uptake, and insulin causes a 2-fold further increase. Microscopy shows that TUG governs the accumulation of GLUT4 in perinuclear membranes distinct from endosomes and indicates that it is this pool of GLUT4 that is mobilized by TUG disruption. Interestingly, in addition to translocating GLUT4 and enhancing glucose uptake, TUG disruption appears to accelerate the degradation of GLUT4 in lysosomes. Finally, we find that TUG binds directly and specifically to a large intracellular loop in GLUT4. Together, these findings demonstrate that TUG is required to retain GLUT4 intracellularly in 3T3-L1 adipocytes in the absence of insulin and further implicate the insulin-stimulated dissociation of TUG and GLUT4 as an important action by which insulin stimulates glucose uptake.     

Evolution at the tip and base of the X chromosome in an African population of Drosophila melanogaster

Hitchhiking effects of advantageous mutations have been invoked to explain reduced polymorphism in regions of low crossing-over in Drosophila. Besides reducing DNA heterozygosity, hitchhiking effects should produce strong linkage disequilibrium and a frequency spectrum skewed toward an excess of rare polymorphisms (compared to the neutral expectation). We measured DNA polymorphism in a Zimbabwe population of D. melanogaster at three loci, yellow, achaete, and suppressor of forked, located in regions of reduced crossing-over. Similar to previously published surveys of these genomic regions in other populations, we observed low levels of nucleotide variability. However, the frequency spectrum was compatible with a neutral model, and there was abundant evidence for recombination in the history of the yellow and ac genes. Thus, some aspects of the data cannot be accounted for by a simple hitchhiking model. An alternative hypothesis, background selection, might be compatible with the observed patterns of linkage disequilibrium and the frequency spectrum. However, this model cannot account for the observed reduction in nucleotide heterozygosity. Thus, there is currently no satisfactory theoretical model for the data from the tip and base of the X chromosome in D. melanogaster.