Differential expression and localization of brain-type and mitochondrial creatine kinase isoenzymes during development of the chicken retina: Mi-CK as a marker for differentiation of photoreceptor cells

The expression and the cellular- as well as subcellular-distribution of brain-type B-CK and mitochondrial Mi-CK during development of the chicken retina was studied by immunoblotting, immunofluorescence and immunogold methods. B-CK expression and accumulation in retina was high from early stages of embryonic development on, decreased slightly around hatching and remained high again during adulthood. At early stages of development (days 2-5), B-CK was more or less evenly distributed over the entire retina with the exception of ganglion cells, which were stained more strongly for B-CK than other retinal precursor cells. Then, at around day 10, the beginning of stratified immunostaining by anti-B-CK antibody was noted concomitant with progressing differentiation. Finally, a dramatic increase in staining of the differentiating photoreceptor cells was seen before hatching (day 18) with weaker staining of other cell types. At hatching, as in the adult state, most of the B-CK was localized within rods and cones. Thus, during retinal development marked changes in the immunostaining pattern for B-CK were evident. By contrast, Mi-CK expression was low during development in ovo and rose just before hatching with a predominant accumulation of this isoenzyme within the ellipsoid portion of the inner photoreceptor cell segments. Mi-CK accumulation in the retina coincided with functional maturation of photoreceptors and therefore represents a good marker for terminal differentiation of these cells. B-CK, present from early stages of retina development, seems to be relevant for the energetics of retinal cell proliferation, migration and differentiation, whereas the simultaneous expression of both B- and Mi-CK around the time of hatching indicates a coordinated function of the two CK isoforms as constituents of a PCr-circuit involved in the energetics of vision, which, in autophagous birds, has to be operational at this point in time.     

Experimental and computational characterization of mass transfer in high turndown bioreactors

Single-use bioreactors (SUBs, or disposable bioreactors) are extensively used for the clinical and commercial production of biologics. Despite widespread application, minimal results have been reported utilizing the turndown ratio; an operation mode where the working range of the bioreactor can be expanded to include low fluid volumes. In this work, a systematic investigation into free surface mass transfer and cell growth in high turndown single-use bioreactors is presented. This approach, which combines experimental mass transfer measurements with numerical simulation, deconvolutes the combined effects of headspace mixing and the free surface convective mass transfer on cell growth. Under optimized conditions, mass transfer across the interface alone may be sufficient to satisfy oxygen demands of the cell culture. Within the context of high turndown bioreactors, this finding provides a counterpoint to traditional sparge-based bioreactor operational philosophy. Multiple monoclonal antibody-producing cell lines grown using this high turndown approach showed similar viable cell densities to those cells expanded using a traditional cell bag rocker. Furthermore, cells taken directly from the turndown expansion and placed into production showed identical growth characteristics to traditionally expanded cultures. Taken together, these results suggest that the Xcellerex SUB can be run at a 5:1 working volume as a seed to itself, with no need for system modifications, potentially simplifying preculture operations.     

Influence of sarmesin on the cardiac and vascular actions of angiotensin II

Summary The angiotensin II (ANG II) receptor blocker properties of sarmesin and its influence on the homotropic cooperativity of ANG II receptors were studied in two experimental models: isolated rabbit aorta and isolated rabbit atria. The results show that: (i) sarmesin is a specific competitive antagonist of ANG II receptors, with high affinity (pA₂=8.93 in the isolated aorta and 8.66 in the isolated atria); and (ii) the slope of the concentration-response curves to ANG II and the Hill coefficient increased in the presence of sarmesin, the latter suggesting an enhancement of the positive homotropic cooperativity of ANG II receptors. These results may be explained overall by the reciprocal negative modulation of receptor affinity between sarmesin and ANG II, due, possibly, to opposite effects on the binding of G-proteins to ANG II receptors.     

Genetic variability in the European minnow,Phoxinus phoxinus (L.)

An electrophoretic study of genetic variation at 11 loci was performedfor a population of European minnows, Phoxinus phoxinus (L.). Ten loci, EST-1 ^*, EST-2 ^* EST-3 ^*,GPD-1 ^*,GPD-2 ^*,GPI-1 ^*,GPI-2 ^*,MPI ^*,6PGD ^* and PGM ^* were polymorphic. IDH ^*wasmonomorphic. The mean number of heterozygotic loci over all 176 fish was 3.05 ± 0.104(SE). Observed mean heterozygosity was 0.28±0.058(SE) and expected mean heterozygosity was 0.27±0.054(SE). EST-2 ^*, EST-3 ^* andPGM ^* were not in Hardy-Weinberg equilibrium. Length,condition, parasite numbers or male breeding characters, i.e. red colorationand tubercles, were not influenced by single enzyme loci.     

Myeloid-derived growth factor regulates neutrophil motility in interstitial tissue damage

Neutrophil recruitment to tissue damage is essential for host defense but can also impede tissue repair. The cues that differentially regulate neutrophil responses to tissue damage and infection remain unclear. Here, we report that the paracrine factor myeloid-derived growth factor (MYDGF) is induced by tissue damage and regulates neutrophil motility to damaged, but not infected, tissues in zebrafish larvae. Depletion of MYDGF impairs wound healing, and this phenotype is rescued by depleting neutrophils. Live imaging and photoconversion reveal impaired neutrophil reverse migration and inflammation resolution in mydgf mutants. We found that persistent neutrophil inflammation in tissues of mydgf mutants was dependent on the HIF-1α pathway. Taken together, our data suggest that MYDGF is a damage signal that regulates neutrophil interstitial motility and inflammation through a HIF-1α pathway in response to tissue damage.     

Metabolic signature associated with parameters of the complete blood count in apparently healthy individuals

Metabolomics studies now approach large sample sizes and the health characterization of the study population often include complete blood count (CBC) results. Upon careful interpretation the CBC aids diagnosis and provides insight into the health status of the patient within a clinical setting. Uncovering metabolic signatures associated with parameters of the CBC in apparently healthy individuals may facilitate interpretation of metabolomics studies in general and related to diseases. For this purpose 879 subjects from the population‐based Study of Health in Pomerania (SHIP)‐TREND were included. Using metabolomics data resulting from mass‐spectrometry based measurements in plasma samples associations of specific CBC parameters with metabolites were determined by linear regression models. In total, 118 metabolites significantly associated with at least one of the CBC parameters. Strongest associations were observed with metabolites of heme degradation and energy production/consumption. Inverse association seen with mean corpuscular volume and mean corpuscular haemoglobin comprised metabolites potentially related to kidney function. The presently identified metabolic signatures are likely derived from the general function and formation/elimination of blood cells. The wealth of associated metabolites strongly argues to consider CBC in the interpretation of metabolomics studies, in particular if mutual effects on those parameters by the disease of interest are known.     

Independent component analysis of Raman spectra: Application on paraffin-embedded skin biopsies

Raman spectra provide wealthy but complex information about the chemical constituents of biological samples. Digital processing techniques are usually needed to extract the spectra of chemical constituents and their associated concentration profiles. However, spectral signatures may admit transformations from those recorded on pure constituents and these techniques require a priori knowledge of spectra to be estimated. We propose in this study to analyse paraffin-embedded skin biopsies of malignant and benign tumors dedicated to oncology researches by Raman spectroscopy and advanced signal processing methods. We show that the commonly used principal component analysis (PCA) does not give physically interpretable estimators of spectra and associated concentration profiles. Based on a linear model and taking into account the statistical properties of spectra, independent component analysis (ICA) is used to better estimate the spectra of chemical constituents. The estimators of associated concentration profiles are no longer orthogonal and have only positive values, contrary to PCA. ICA allows to model the paraffin by three Raman spectra and provides good estimators of underlying spectra of the human skin, which is of great interest in oncology since the retrieval of spectral features of different types of skin tumors is sufficient for their discrimination.     

Mechanisms of cytotoxicity induced by horseradish peroxidase/indole-3-acetic acid gene therapy

We have previously proposed the horseradish peroxidase (HRP) and the non-toxic plant hormone indole-3-acetic acid (IAA) as a novel system for gene-directed enzyme/prodrug therapy (GDEPT). The cytotoxic potential of HRP/IAA GDEPT and the induction of a bystander effect were demonstrated in vitro under normoxic as well as hypoxic tumour conditions. To date, the chemical agents and the cellular targets involved in HRP/IAA-mediated toxicity have not been identified. In the present work, some of the molecular and morphological features of the cells treated with HRP/IAA gene therapy were analysed. Human T24 bladder carcinoma cells transiently transfected with the HRP cDNA and exposed to the prodrug IAA showed chromatin condensation, formation of apoptotic bodies, DNA fragmentation, and Annexin V binding. Similar effects were observed when the cells were incubated with the apoptotic agent cisplatin. Caspases appeared to be involved as effectors in HRP/IAA-mediated apoptosis, since treatment with a general caspase inhibitor decreased the fraction of cells with micronuclei (MN) by 30%, with fragmented DNA by 50%, and with condensed chromatin by 60%. However, very little degradation of one of the downstream targets of caspase-3, PARP, could be detected, and apoptosis alone did not appear to account for the killing levels measured with a clonogenic assay. The effect of HRP/IAA treatment on cell cycle progression was also investigated, and a rapid cytostatic effect, equally affecting all phases of the division cycle, was observed.     

Tumour class prediction and discovery by microarray-based DNA methylation analysis

Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation occurs in a tumour type-specific manner. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput assays for methylation detection. We have developed the first microarray-based technique which allows genome-wide assessment of selected CpG dinucleotides as well as quantification of methylation at each site. Several hundred CpG sites were screened in 76 samples from four different human tumour types and corresponding healthy controls. Discriminative CpG dinucleotides were identified for different tissue type distinctions and used to predict the tumour class of as yet unknown samples with high accuracy using machine learning techniques. Some CpG dinucleotides correlate with progression to malignancy, whereas others are methylated in a tissue-specific manner independent of malignancy. Our results demonstrate that genome-wide analysis of methylation patterns combined with supervised and unsupervised machine learning techniques constitute a powerful novel tool to classify human cancers.