Challenges for the effective molecular imprinting of proteins.

Molecular imprinting is a technique that is used to create artificial receptors by the formation of a polymer network around a template molecule. This technique has proven to be particularly effective for molecules with low molecular weight (<1500 Da), and during the past five years the number of research articles on the imprinting of larger (bio)templates is increasing considerably. However, expanding the methodology toward imprinted materials for selective recognition of proteins, DNA, viruses and bacteria appears to be extremely challenging. This paper presents a critical analysis of data presented by several authors and our own experiments, showing that the molecular imprinting of proteins still faces some fundamental challenges. The main topics of concern are proper monomer selection, washing method/template removal, quantification of the rebinding and reproducibility. Use of charged monomers can lead to strong electrostatic interactions between monomers and template but also to undesired high aspecific binding. Up till now, it has not been convincingly shown that electrostatic interactions lead to better imprinting results. The combination of a detergent (SDS) and AcOH, commonly used for template removal, can lead to experimental artifacts, and should ideally be avoided. In many cases template rebinding is unreliably quantified, results are not evaluated critically and lack statistical analysis. Therefore, it can be argued that presently, in numerous publications the scientific evidence of molecular imprinting of proteins is not convincing.

Comparative multiplexed mass spectrometric analyses of endogenously expressed yeast nuclear and cytoplasmic exosomes.

Here we combined tandem affinity purification with several mass-spectrometry-based approaches to gain more insight into the composition and structure of the yeast nuclear-cytoplasmic exosome protein complex. The yeast exosome fulfills several different functions in RNA metabolism and can be localized in both the cytoplasm and the nucleus. These two exosome complexes differ in protein composition, although they share several constituents. We focused on these differences in composition by selecting a nuclear-specific exosome protein (Rrp6) and a cytoplasmic-specific protein (Ski7) as the tandem-affinity-purification-tagged affinity bait protein. First, we investigated both these purified exosome assemblies by macromolecular mass spectrometry (MS) to determine the stability and mass of the intact protein complexes and to obtain information on composition and core constituents. We used tandem MS on these intact protein complexes to further probe the composition and to obtain insight into the peripheral nature of some of the constituents. Finally, we combine stable isotope labeling with MS to quantitate differences in exosome composition and posttranslational modifications. We identified a few phosphorylation sites that are differentially regulated between the cytoplasmic exosome and the nuclear exosome. From all of these data, we conclude that the yeast nuclear exosome and the cytoplasmic exosome share a common stable core complex, but are decorated with quite a few differing peripheral proteins. We show that the nuclear exosome selectively copurifies with the alpha/beta importin heterodimer, which is known to be involved in the transport of proteins across the nuclear membrane.

Assessment of local changes of cerebral perfusion and blood concentration by near infrared spectroscopy and ultrasound contrast densitometry.

The objective of this study is to correlate regional cerebral blood concentration measurements made with near infrared spectroscopy to simultaneous local measurements of ultrasound contrast agent (CA) densitometry. Experiments were performed with piglets (7 kg) under general anesthesia. The cerebral blood flow (CBF) and volume (CBV) were changed by inducing various degrees of hypercapnia. NIRS measurements were performed with a quasi-continuous wave system, using an optode distance of 3-6 cm. The concentration changes in oxygenated and deoxygenated hemoglobin and their sum and difference (cO2Hb, cHHb, ctHb, cHbD) were continuously calculated. Ultrasound contrast agent (SF6) was administered as a short intra-venous bolus. Ultrasound equipment was used in pulse inversion second harmonic gray scale imaging mode at low transmit power setting. Three regions-of-interest (0.25 cm2) were analyzed in each image. Wash-in curves were constructed as spatial mean gray level vs. time. The variables collected with both methods changed according to the induced changes in the physiological condition. Changes in the PaCO2, pH and carotid flow induced highly correlated changes in cO2Hb, cHHb, ctHb and cHbD, and in the variables derived from CA analyses. NIRS and CA methods measure regional, respectively, local changes in CBV and CBF. Moreover, NIRS can yield complementary information about the cerebral oxygenation.