Monitoring blood loss with near infrared spectroscopy

Experimental research has shown correlation between near infrared spectroscopy (NIRS) and blood loss, but these findings have not been validated in man. Ten blood donors were monitored before, during and for 10 min after blood collection (470 ml) with NIRS. A Somanetics INVOS 4100 oximeter monitored regional haemoglobin saturation in the cerebral cortex (cSO(2)-left frontal area) and from the left calf (pSO(2)). A Critikon 2001 Cerebral Redox Model monitored total (tHb), oxygenated (O(2)Hb) and deoxygenated (HHb) haemoglobin from the right calf. The oxygenation index [HbD]=[O(2)Hb]-[HHb] was derived from the data. cSO(2) (P<0.001), pSO(2) (P<0.001) and HbD (P=0.001) decreased during blood collection. Maximum changes occurred 10 minutes after collection for cSO(2), with a mean fall (95% C.I.) of 2.5 (-0.06-4.86)%, at the end of blood collection for pSO(2), with a mean fall (95% C.I.) of 3 (0.74-5.26)% and after 8% of blood volume loss for HbD, with a mean fall (95% C.I.) of 7.2 (2.25-12.16). Cerebral and peripheral oxygenation did not recover after blood collection. There was good correlation between NIRS parameters and blood loss. NIRS is a potentially useful technique for monitoring blood loss in humans. Further research is needed to define its role in clinical practice.     

Virus hazards from food, water and other contaminated environments

Numerous viruses of human or animal origin can spread in the environment and infect people via water and food, mostly through ingestion and occasionally through skin contact. These viruses are released into the environment by various routes including water run-offs and aerosols. Furthermore, zoonotic viruses may infect humans exposed to contaminated surface waters. Foodstuffs of animal origin can be contaminated, and their consumption may cause human infection if the viruses are not inactivated during food processing. Molecular epidemiology and surveillance of environmental samples are necessary to elucidate the public health hazards associated with exposure to environmental viruses. Whereas monitoring of viral nucleic acids by PCR methods is relatively straightforward and well documented, detection of infectious virus particles is technically more demanding and not always possible (e.g. human norovirus or hepatitis E virus). The human pathogenic viruses that are most relevant in this context are nonenveloped and belong to the families of the Caliciviridae, Adenoviridae, Hepeviridae, Picornaviridae and Reoviridae. Sampling methods and strategies, first-choice detection methods and evaluation criteria are reviewed.     

Evidence for an adenosine A2/Ra receptor on human basophils

5'-N-ethylcarboxamideadenosine (NECA) greater than 2-chloroadenosine greater than adenosine greater than (-)-N6-(R-phenyl-isopropyl)-adenosine greater than (+)-N6-(S-phenylisopropyl)-adenosine, in that order of potency, inhibited in vitro antigen-induced histamine release from human basophils in a dose-dependent fashion. Inhibition occurred only during the first stage of antigen-induced histamine release and the nucleosides failed to inhibit the release caused by the Ca2+ ionophore, A23187. 6-nitrobenzylthioinosine and dipyridamole, which inhibit adenosine uptake, and erythro-9-(2-hydroxy-3-nonyl)adenine, which blocks adenosine metabolism, did not impair the inhibition caused by NECA and adenosine. 8-phenyltheophylline and theophylline, two competitive antagonists of adenosine receptors, blocked the inhibition caused by NECA and adenosine. These data suggest that NECA and other adenosine analogs activate a specific cell surface adenosine receptor which possesses properties similar to those of an adenosine A2/Ra receptor.     

Conformational changes of calpain from human erythrocytes in the presence of Ca2+

Small angle x-ray scattering has been used to monitor calpain structural transitions during the activation process triggered by Ca(2+) binding. The scattering pattern of the unliganded enzyme in solution does not display any significant difference with that calculated from the crystal structure. The addition of Ca(2+) promotes the formation of large aggregates, indicating the exposure of hydrophobic patches on the surface of the protease. In contrast, Ca(2+) addition in the presence of the thiol proteinase inhibitor E64 or of the inhibitor leupeptin causes a small conformational change with no dissociation of the heterodimer. The resulting conformation appears to be slightly more extended than the unliganded form. From the comparison between ab initio models derived from our data with the crystal structure, the major observable conformational change appears to be localized at level of the L-subunit and in particular seems to confirm the mutual movement already observed by the crystallographic analysis of the dII (dIIb) and the dI (dIIa) domains creating a functional active site. This work not only provides another piece of supporting evidence for the calpain conformational change in the presence of Ca(2+), but actually constitutes the first experimental observation of this change for intact heterodimeric calpain in solution.     

Adult cardiac stem cells are multipotent and support myocardial regeneration

The notion of the adult heart as terminally differentiated organ without self-renewal potential has been undermined by the existence of a subpopulation of replicating myocytes in normal and pathological states. The origin and significance of these cells has remained obscure for lack of a proper biological context. We report the existence of Lin(-) c-kit(POS) cells with the properties of cardiac stem cells. They are self-renewing, clonogenic, and multipotent, giving rise to myocytes, smooth muscle, and endothelial cells. When injected into an ischemic heart, these cells or their clonal progeny reconstitute well-differentiated myocardium, formed by blood-carrying new vessels and myocytes with the characteristics of young cells, encompassing approximately 70% of the ventricle. Thus, the adult heart, like the brain, is mainly composed of terminally differentiated cells, but is not a terminally differentiated organ because it contains stem cells supporting its regeneration. The existence of these cells opens new opportunities for myocardial repair.     

Functional proteomic screens reveal an essential extracellular role for hsp90 alpha in cancer cell invasiveness

Tumour cell invasiveness is crucial for cancer metastasis and is not yet understood. Here we describe two functional screens for proteins required for the invasion of fibrosarcoma cells that identified the molecular chaperone heat shock protein 90 (hsp90). The hsp90 alpha isoform, but not hsp90 beta, is expressed extracellularly where it interacts with the matrix metalloproteinase 2 (MMP2). Inhibition of extracellular hsp90 alpha decreases both MMP2 activity and invasiveness. This role for extracellular hsp90 alpha in MMP2 activation indicates that cell-impermeant anti-hsp90 drugs might decrease invasiveness without the concerns inherent in inhibiting intracellular hsp90.     

Long-Lived Intracellular Single-Molecule Fluorescence Using Electroporated Molecules

Studies of biomolecules in vivo are crucial to understand their function in a natural, biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins to study their expression, localization, and action; however, the scope of such studies would be increased considerably by using organic fluorophores, which are smaller and more photostable than their fluorescent protein counterparts. Here, we describe a straightforward, versatile, and high-throughput method to internalize DNA fragments and proteins labeled with organic fluorophores into live Escherichia coli by employing electroporation. We studied the copy numbers, diffusion profiles, and structure of internalized molecules at the single-molecule level in vivo, and were able to extend single-molecule observation times by two orders of magnitude compared to green fluorescent protein, allowing continuous monitoring of molecular processes occurring from seconds to minutes. We also exploited the desirable properties of organic fluorophores to perform single-molecule Förster resonance energy transfer measurements in the cytoplasm of live bacteria, both for DNA and proteins. Finally, we demonstrate internalization of labeled proteins and DNA into yeast Saccharomyces cerevisiae, a model eukaryotic system. Our method should broaden the range of biological questions addressable in microbes by single-molecule fluorescence.