High sequence coverage by in-capillary proteolysis of native proteins and simultaneous analysis of the resulting peptides by nanoelectrospray ionization-mass spectrometry and tandem mass spectrometry

Losses of proteolytic peptides during extraction and/or purification procedures succeeding in-gel or in-solution digests of proteins frequently occur in the course of protein identification investigations. In order to overcome this disadvantage, the method of in-capillary digest was developed: native proteins were incubated in the presence of endoproteases in the electrospray capillary and the resulting peptides were analyzed by nanoelectrospray-mass spectrometry during the ongoing proteolysis. In-capillary digest of apomyglobin by use of trypsin in a molar ratio of 25:1 yielded complete degradation already after 15 min. The sequence coverage based on formation of molecular ions was 100% and peptide ions could be fragmented by collision-induced dissociation and sequenced. When myoglobin was incubated in the electrospray capillary with trypsin in a molar ratio of 500:1, a clear shift from molecular ions and miscleaved peptide ions to the expected final tryptic peptide ions was observed over a 2 h period. The peptide spectra obtained from tryptic in-capillary proteolysis of bovine serum albumin and apotransferrin, respectively, gave rise to sequence coverages of more than 40% for both proteins. The data obtained from the peptide maps as well as from collision-induced dissociation (CID) of selected peptides were more than sufficient for protein identification by database searches. An elephant milk protein preparation was used to demonstrate the application of in-capillary proteolysis on protein mixtures. Tryptic digest, simultaneous analysis of the proteolytic peptides by use of CID, and subsequent sequencing allowed the identification of lactoferrin, alphas1-casein, beta-casein, delta-casein, and kappa-casein by homology search.     

A pharmacogenomic method for individualized prediction of drug sensitivity

Identifying the best drug for each cancer patient requires an efficient individualized strategy. We present MATCH (M erging genomic and pharmacologic A nalyses for T herapy CH oice), an approach using public genomic resources and drug testing of fresh tumor samples to link drugs to patients. Valproic acid (VPA) is highlighted as a proof‐of‐principle. In order to predict specific tumor types with high probability of drug sensitivity, we create drug response signatures using publically available gene expression data and assess sensitivity in a data set of >40 cancer types. Next, we evaluate drug sensitivity in matched tumor and normal tissue and exclude cancer types that are no more sensitive than normal tissue. From these analyses, breast tumors are predicted to be sensitive to VPA. A meta‐analysis across breast cancer data sets shows that aggressive subtypes are most likely to be sensitive to VPA, but all subtypes have sensitive tumors. MATCH predictions correlate significantly with growth inhibition in cancer cell lines and three‐dimensional cultures of fresh tumor samples. MATCH accurately predicts reduction in tumor growth rate following VPA treatment in patient tumor xenografts. MATCH uses genomic analysis with in vitro testing of patient tumors to select optimal drug regimens before clinical trial initiation.     

Notch-signalling is required for head regeneration and tentacle patterning in Hydra

Local self-activation and long ranging inhibition provide a mechanism for setting up organising regions as signalling centres for the development of structures in the surrounding tissue. The adult hydra hypostome functions as head organiser. After hydra head removal it is newly formed and complete heads can be regenerated. The molecular components of this organising region involve Wnt-signalling and β-catenin. However, it is not known how correct patterning of hypostome and tentacles are achieved in the hydra head and whether other signals in addition to HyWnt3 are needed for re-establishing the new organiser after head removal. Here we show that Notch-signalling is required for re-establishing the organiser during regeneration and that this is due to its role in restricting tentacle activation. Blocking Notch-signalling leads to the formation of irregular head structures characterised by excess tentacle tissue and aberrant expression of genes that mark the tentacle boundaries. This indicates a role for Notch-signalling in defining the tentacle pattern in the hydra head. Moreover, lateral inhibition by HvNotch and its target HyHes are required for head regeneration and without this the formation of the β-catenin/Wnt dependent head organiser is impaired. Work on prebilaterian model organisms has shown that the Wnt-pathway is important for setting up signalling centres for axial patterning in early multicellular animals. Our data suggest that the integration of Wnt-signalling with Notch-Delta activity was also involved in the evolution of defined body plans in animals.     

The lantern of Aristotle: organization of its coelom and origin of its muscles (Echinodermata,Echinoida)

Summary Comparative ultrastructural analyses of the muscles that work the lantern of Aristotle support the opinion that the muscles in question are myoepithelially organized or derivatives of myoepithelia. They are part of the epithelium of the peripharyngeal cavity (=lantern coelom). The coelom epithelium may become multiplelayered in certain regions and is composed of (1) a layer of muscle cells that vary in number and size, (2) nerve cells and their processes that are interspersed between the muscle layer and (3) monociliated adluminal cells that build a continuous cell lining and completely cover the muscle layer. According to their complexity, the lantern muscles exhibit consecutive stages of myoepithelial variations and may finally simulate subepithelial musculature. The results of this study support the hypothesis of a histological development of subepithelial musculature from simple myoepithelia, although both epithelial and mesenchymal musculature may occur in the Echinodermata. Detailed knowledge of the organization of the lantern's coelom space was a prerequisite for the present study. In contrast to previous examinations the lantern coelom is not a continuous space, but is subdivided into several cavities that are partially completely separated from each other. On the one hand, this subdivision is probably caused by the sophisticated arrangement of the lantern's ossicles and on the other by the septa that give rise to muscles that fulfill different functions. lanter's ossicles and on the other by the septa that give     

Combinatorial G-CSF/AMD3100 Treatment in Cardiac Repair after Myocardial Infarction

Aims

Several studies suggest that circulating bone marrow derived stem cells promote the regeneration of ischemic tissues. For hematopoietic stem cell transplantation combinatorial granulocyte-colony stimulating factor (G-CSF)/Plerixafor (AMD3100) administration was shown to enhance mobilization of bone marrow derived stem cells compared to G-CSF monotherapy. Here we tested the hypothesis whether combinatorial G-CSF/AMD3100 therapy has beneficial effects in cardiac recovery in a mouse model of myocardial infarction.

Methods

We analyzed the effect of single G-CSF (250 µg/kg/day) and combinatorial G-CSF/AMD3100 (100 µg/kg/day) treatment on cardiac morphology, vascularization, and hemodynamics 28 days after permanent ligation of the left anterior descending artery (LAD). G-CSF treatment started directly after induction of myocardial infarction (MI) for 3 consecutive days followed by a single AMD3100 application on day three after MI in the G-CSF/AMD3100 group. Cell mobilization was assessed by flow cytometry of blood samples drawn from tail vein on day 0, 7, and 14.

Results

Peripheral blood analysis 7 days after MI showed enhanced mobilization of white blood cells (WBC) and endothelial progenitor cells (EPC) upon G-CSF and combinatorial G-CSF/AMD3100 treatment. However, single or combinatorial treatment showed no improvement in survival, left ventricular function, and infarction size compared to the saline treated control group 28 days after MI. Furthermore, no differences in histology and vascularization of infarcted hearts could be observed.

Conclusion

Although the implemented treatment regimen caused no adverse effects, our data show that combinatorial G-CSF/AMD therapy does not promote myocardial regeneration after permanent LAD occlusion.     

Image interpolation allows accurate quantitative bone morphometry in registered micro-computed tomography scans

Time-lapsed in vivo micro-computed tomography is a powerful tool to analyse longitudinal changes in the bone micro-architecture. Registration can overcome problems associated with spatial misalignment between scans; however, it requires image interpolation which might affect the outcome of a subsequent bone morphometric analysis. The impact of the interpolation error itself, though, has not been quantified to date. Therefore, the purpose of this ex vivo study was to elaborate the effect of different interpolator schemes [nearest neighbour, tri-linear and B-spline (BSP)] on bone morphometric indices. None of the interpolator schemes led to significant differences between interpolated and non-interpolated images, with the lowest interpolation error found for BSPs (1.4%). Furthermore, depending on the interpolator, the processing order of registration, Gaussian filtration and binarisation played a role. Independent from the interpolator, the present findings suggest that the evaluation of bone morphometry should be done with images registered using greyscale information.     

Distinguishing Neurocognitive Processes Reflected by P600 Effects: Evidence from ERPs and Neural Oscillations

Research on language comprehension using event-related potentials (ERPs) reported distinct ERP components reliably related to the processing of semantic (N400) and syntactic information (P600). Recent ERP studies have challenged this well-defined distinction by showing P600 effects for semantic and pragmatic anomalies. So far, it is still unresolved whether the P600 reflects specific or rather common processes. The present study addresses this question by investigating ERPs in response to a syntactic and pragmatic (irony) manipulation, as well as a combined syntactic and pragmatic manipulation. For the syntactic condition, a morphosyntactic violation was applied, whereas for the pragmatic condition, such as “That is rich”, either an ironic or literal interpretation was achieved, depending on the prior context. The ERPs at the critical word showed a LAN-P600 pattern for syntactically incorrect sentences relative to correct ones. For ironic compared to literal sentences, ERPs showed a P200 effect followed by a P600 component. In comparison of the syntax-related P600 to the irony-related P600, distributional differences were found. Moreover, for the P600 time window (i.e., 500–900 ms), different changes in theta power between the syntax and pragmatics effects were found, suggesting that different patterns of neural activity contributed to each respective effect. Thus, both late positivities seem to be differently sensitive to these two types of linguistic information, and might reflect distinct neurocognitive processes, such as reanalysis of the sentence structure versus pragmatic reanalysis.     

The mutational dynamics of the SARS-CoV-2 virus in serial passages in vitro

Since its outbreak in 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) keeps surprising the medical community by evolving diverse immune escape mutations in a rapid and effective manner. To gain deeper insight into mutation frequency and dynamics, we isolated ten ancestral strains of SARS-CoV-2 and performed consecutive serial incubation in ten replications in a suitable and common cell line and subsequently analysed them using RT-qPCR and whole genome sequencing. Along those lines we hoped to gain fundamental insights into the evolutionary capacity of SARS-CoV-2 in vitro. Our results identified a series of adaptive genetic changes, ranging from unique convergent substitutional mutations and hitherto undescribed insertions. The region coding for spike proved to be a mutational hotspot, evolving a number of mutational changes including the already known substitutions at positions S:484 and S:501. We discussed the evolution of all specific adaptations as well as possible reasons for the seemingly inhomogeneous potential of SARS-CoV-2 in the adaptation to cell culture. The combination of serial passage in vitro with whole genome sequencing uncovers the immense mutational potential of some SARS-CoV-2 strains. The observed genetic changes of SARS-CoV-2 in vitro could not be explained solely by selectively neutral mutations but possibly resulted from the action of directional selection accumulating favourable genetic changes in the evolving variants, along the path of increasing potency of the strain. Competition among a high number of quasi-species in the SARS-CoV-2 in vitro population gene pool may reinforce directional selection and boost the speed of evolutionary change.