In vivo measurements of diffuse reflectance and time‐resolved autofluorescence emission spectra of basal cell carcinomas

We present a clinical investigation of diffuse reflectance and time‐resolved autofluorescence spectra of skin cancer with an emphasis on basal cell carcinoma. A total of 25 patients were measured using a compact steady‐state diffuse reflectance/fluorescence spectrometer and a fibre‐optic‐coupled multispectral time‐resolved spectrofluorometer. Measurements were performed in vivo prior to surgical excision of the investigated region. Singular value decomposition was used to reduce the dimensionality of steady state diffuse reflectance and fluorescence spectra. Linear discriminant analysis was then applied to the measurements of basal cell carcinomas (BCCs) and used to predict the tissue disease state with a leave‐one‐out methodology. This approach was able to correctly diagnose 87% of the BCCs. With 445 nm excitation a decrease in the spectrally averaged fluorescence lifetime was observed between normal tissue and BCC lesions with a mean value of 886 ps. Furthermore, the fluorescence lifetime for BCCs was lower than that of the surrounding healthy tissue in all cases and statistical analysis of the data revealed that this decrease was significant (p = 0.002). (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Persistence of antibiotic resistant bacteria

Bacterial adaptation to antibiotics has been very successful and over the past decade the increase in antibiotic resistance has generated considerable medical problems. Even though many drug resistances confer a fitness cost, suggesting that they might disappear by reducing the volume of antibiotic use, increasing evidence obtained from laboratory and epidemiological studies indicate that several processes will act to cause long-term persistence of resistant bacteria. Compensatory evolution that ameliorates the costs of resistance, the occurrence of cost-free resistances and genetic linkage between non-selected and selected resistances will confer a stabilization of the resistant bacteria. Thus, it is of importance that we forcefully implement strategies to reduce the rate of appearance and spread of resistant bacteria to allow new drug discovery to catch up with bacterial resistance development.     

A cyanobacterial gene in nonphotosynthetic protists--an early chloroplast acquisition in eukaryotes?

Since the incorporation of mitochondria and chloroplasts (plastids) into the eukaryotic cell by endosymbiosis, genes have been transferred from the organellar genomes to the nucleus of the host, via an ongoing process known as endosymbiotic gene transfer. Accordingly, in photosynthetic eukaryotes, nuclear genes with cyanobacterial affinity are believed to have originated from endosymbiotic gene transfer from chloroplasts. Analysis of the Arabidopsis thaliana genome has shown that a significant fraction (2%-9%) of the nuclear genes have such an endosymbiotic origin. Recently, it was argued that 6-phosphogluconate dehydrogenase (gnd)-the second enzyme in the oxidative pentose phosphate pathway-was one such example. Here we show that gnd genes with cyanobacterial affinity also are present in several nonphotosynthetic protistan lineages, such as Heterolobosea, Apicomplexa, and parasitic Heterokonta. Current data cannot definitively resolve whether these groups acquired the gnd gene by primary and/or secondary endosymbiosis or via an independent lateral gene transfer event. Nevertheless, our data suggest that chloroplasts were introduced into eukaryotes much earlier than previously thought and that several major groups of heterotrophic eukaryotes have secondarily lost photosynthetic plastids.     

Oxidative metabolism of dopamine: a colour reaction from human midbrain analysed by mass spectrometry

In order to identify the protein responsible for a dopamine peroxidizing activity, previously described in human normal and parkinsonian substantia nigra by our group, we developed non-denaturing polyacrylamide gel electrophoresis conditions, mimicking the characteristic colour in vitro reaction, resulting from cyclic oxidation of dopamine (DA). After separating protein mixtures from human normal midbrain homogenates on two sets of identical native gels, one gel set was subjected to specific activity staining by using DA and hydrogen peroxide. An activity red/orange band appeared in midbrain tissue lanes, similarly to the lane where commercial horseradish peroxidase (HRP) was present as control of peroxidative activity. The second set of gels, stained with Coomassie Blue, showed other, not enzymatically active protein bands. Mass spectrometry analysis of the bands containing the activity and the corresponding Coomassie Blue bands revealed the presence of proteins that may play a role in neurodegenerative disease, highlighting a possible functional link among dopamine/dopaminochrome redox cycle and protein metabolism.     

Isolation of potato proteins using simulated moving bed technology

The simulated moving bed (SMB) concept of chromatography was applied to treat potato juice from production of starch. The aim was to harvest proteins. SMB offers possibilities to operate with different process strategies and in this study it was shown possible to harvest up to 80% of the protein in a process utilizing very little extra water besides that already present in the juice. After depleting protein from the juice in the adsorption step, the flow through was used to recondition the column after elution. The present study illustrates a new concept of applying chromatography as a capturing step of bulk products.     

Estimation of the size of the alloreactive NK cell repertoire: studies in individuals homozygous for the group A KIR haplotype

Stem cell transplantation across HLA barriers may trigger NK cell-mediated graft-vs-leukemia effects leading to improved survival for patients with hematological malignancies. However, the genetic algorithm based on killer cell Ig-like receptor (KIR) and HLA genes used to predict NK cell alloreactivity have yielded discrepant results. Accordingly, it has been difficult to define transplantation settings that favor NK cell alloreactivity. In this study, we have used multiparameter flow cytometry to simultaneously analyze the cell surface expression of all four major inhibitory KIR and CD94/NKG2A to determine the size of the alloreactive NK cell repertoires in 31 individuals homozygous for the group A KIR haplotype. We observed a vast variability in the frequencies of cells with an alloreactive potential, ranging from 0 to 62% of the total NK cell population depending on which, and how many, KIR ligands were missing in theoretical recipients. This analysis required a functional examination of KIR3DL2-single positive NK cells, showing that this subset was hyporesponsive in individuals harboring the cognate ligands HLA-A3/A11. The results provide new insights into the variability of the functional alloreactive NK cell repertoire and have implications for donor selection in hematopoietic stem cell transplantation and adoptive NK cell-based immunotherapy.     

Ambient temperature effects on photo induced gonadal cycles and hormonal secretion patterns in Great Tits from three different breeding latitudes

The present study determines how populations of Great Tits (Parus major) breeding in southern, mid and northern European latitudes have adjusted their reproductive endocrinology to differences in the ambient temperature during the gonadal cycle. A study based on long-term breeding data, using the Colwell predictability model, showed that the start of the breeding season has a high predictability (≈ 0.8-0.9) at all latitudes, and that the environmental information factor (I_e) progressively decreased from mid Italy (I_e > 4) to northern Finland (I_e < 1). The results indicate that integration of supplementary information, such as ambient temperature, with photoperiodic initial predictive information (day length), becomes progressively more important in maintaining the predictability of the breeding season with decreasing latitude. This hypothesis was verified by exposing photosensitive Great Tits from northern Norway, southern Sweden and northern Italy to sub-maximal photo-stimulatory day lengths (13L:11D) under two different ambient temperature regimes (+ 4 °C and + 20 °C). Changes in testicular size, plasma levels of LH and testosterone were measured. The main results were: (1) Initial testicular growth rate, as well as LH secretion, was affected by temperature in the Italian, but not in birds from the two Scandinavian populations. (2) Maximum testicular size, maximum LH and testosterone levels were maintained for a progressively shorter period of time with increasing latitude, regardless of whether the birds were kept on a low or a high ambient temperature. (3) In birds from all latitudes, the development of photorefractoriness, as indicated by testicular regression and a decrease in plasma levels of LH and testosterone, started much earlier (with the exception for LH Great Tits from northern Scandinavia) when kept on + 20 °C than when kept on + 4 °C. The prolonging effects of a low temperature was more pronounced in Mediterranean birds, than in birds from Scandinavia, and more pronounced in Great Tits from southern Scandinavia than in Great Tits from northern Scandinavia. Ecological implications of the results are discussed, as well as possible impact of global warming on the breeding success of European Great Tits from different breeding latitudes.     

The crystal structure of peroxymyoglobin generated through cryoradiolytic reduction of myoglobin compound III during data collection

Myoglobin has the ability to react with hydrogen peroxide, generating high-valent complexes similar to peroxidases (compounds I and II), and in the presence of excess hydrogen peroxide a third intermediate, compound III, with an oxymyoglobin-type structure is generated from compound II. The compound III is, however, easily one-electron reduced to peroxymyoglobin by synchrotron radiation during crystallographic data collection. We have generated and solved the 1.30 A (1 A=0.1 nm) resolution crystal structure of the peroxymyoglobin intermediate, which is isoelectric to compound 0 and has a Fe-O distance of 1.8 A and O-O bond of 1.3 A in accordance with a Fe(II)-O-O- (or Fe(III)-O-O2-) structure. The generation of the peroxy intermediate through reduction of compound III by X-rays shows the importance of using single-crystal microspectrophotometry when doing crystallography on metalloproteins. After having collected crystallographic data on a peroxy-generated myoglobin crystal, we were able (by a short annealing) to break the O-O bond leading to formation of compound II. These results indicate that the cryoradiolytic-generated peroxymyoglobin is biologically relevant through its conversion into compound II upon heating. Additionally, we have observed that the Xe1 site is occupied by a water molecule, which might be the leaving group in the compound II to compound III reaction.     

Adaptation of human embryonic stem cells to feeder-free and matrix-free culture conditions directly on plastic surfaces

Previous studies have shown that cultivation of undifferentiated human embryonic stem (hES) cells requires human fibroblasts (hF) or mouse embryonic fibroblast (mEF) feeders or a coating matrix such as laminin, fibronectin or Matrigel in combination with mEF or hF conditioned medium. We here demonstrate a successful feeder-free and matrix-free culture system in which undifferentiated hES cells can be cultured directly on plastic surfaces without any supportive coating, in a hF conditioned medium. The hES cells cultured directly on plastic surfaces grow as colonies with morphology very similar to cells cultured on Matrigel(TM). Two hES cell lines SA167 and AS034.1 were adapted to matrix-free growth (MFG) and have so far been cultured up to 43 passages and cryopreserved successfully. The lines maintained a normal karyotype and expressed the expected marker profile of undifferentiated hES cells for Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and SSEA-1. The hES cells formed teratomas in SCID mice and differentiated in vitro into derivates of all three germ layers. Thus, the MFG-adapted hES cells appear to retain pluripotency and to remain undifferentiated. The present culture system has a clear potential to be scaleable up to a manufacturing level and become the preferred culture system for various applications such as cell therapy and toxicity testing.