Synthesis, characterization and DNA binding properties of oligopyridine-ruthenium(II)-amino acid conjugates

The DNA-binding properties of a number of ruthenium oligopyridine complexes with conjugated amino acids having the general formulae [Ru(terpy)(4-COY-4'-Mebpy)(X)](n)(+), X=NO (n=3), X=Cl (n=1) and NO(2) (n=1) and Y=AlaCONH(2) and TrpCONH(2) are reported. The new complexes were spectroscopically characterized and their DNA-binding properties were studied by means of circular dichroism (CD), (23)Na and (31)P NMR spectroscopy. The results show that the chlorido complexes interact by coordination to the DNA bases with the conjugated amino acid able to provide an additional interaction with the DNA helix. In addition, electrostatic interactions between all studied complexes and the DNA polyanion were observed. The nitro complexes were found to be insignificant, affecting only the (31)P NMR signal, probably due to changes in the hydration sphere of the DNA close to the phosphates.     

Haplotype analysis of vernalization loci in European barley germplasm reveals novel <Emphasis Type="Italic">VRN-H1</Emphasis> alleles and a predominant winter <Emphasis Type="Italic">VRN-H1/VRN-H2</Emphasis> multi-locus haplotype

In barley, variation in the requirement for vernalization (an extended period of low temperature before flowering can occur) is determined by the VRN-H1, -H2 and -H3 loci. In European cultivated germplasm, most variation in vernalization requirement is accounted for by alleles at VRN-H1 and VRN-H2 only, but the range of allelic variation is largely unexplored. Here we characterise VRN-H1 and VRN-H2 haplotypes in 429 varieties representing a large portion of the acreage sown to barley in Western Europe over the last 60 years. Analysis of genotype, intron I sequencing data and growth habit tests identified three novel VRN-H1 alleles and determined the most frequent VRN-H1 intron I rearrangements. Combined analysis of VRN-H1 and VRN-H2 alleles resulted in the classification of seventeen VRN-H1/VRN-H2 multi-locus haplotypes, three of which account for 79% of varieties. The molecular markers employed here represent powerful diagnostic tools for prediction of growth habit and assessment of varietal purity. These markers will also allow development of germplasm to test the behaviour of individual alleles with the aim of understanding the relationship between allelic variation and adaptation to specific agri-environments.     

A THEMIS:SHP1 complex promotes T‐cell survival

THEMIS is critical for conventional T‐cell development, but its precise molecular function remains elusive. Here, we show that THEMIS constitutively associates with the phosphatases SHP1 and SHP2. This complex requires the adapter GRB2, which bridges SHP to THEMIS in a Tyr‐phosphorylation‐independent fashion. Rather, SHP1 and THEMIS engage with the N‐SH3 and C‐SH3 domains of GRB2, respectively, a configuration that allows GRB2‐SH2 to recruit the complex onto LAT. Consistent with THEMIS‐mediated recruitment of SHP to the TCR signalosome, THEMIS knock‐down increased TCR‐induced CD3‐ζ phosphorylation, Erk activation and CD69 expression, but not LCK phosphorylation. This generalized TCR signalling increase led to augmented apoptosis, a phenotype mirrored by SHP1 knock‐down. Remarkably, a KI mutation of LCK Ser59, previously suggested to be key in ERK‐mediated resistance towards SHP1 negative feedback, did not affect TCR signalling nor ligand discrimination in vivo. Thus, the THEMIS:SHP complex dampens early TCR signalling by a previously unknown molecular mechanism that favours T‐cell survival. We discuss possible implications of this mechanism in modulating TCR output signals towards conventional T‐cell development and differentiation.     

Conjugation of a peptide to mannan and its confirmation by tricine sodium dodecyl sulfate–polyacrylamide gel electrophoresis

The conjugation of polysaccharides to peptides is essential for antigen delivery and vaccine development. Herein, we show that tricine SDS-PAGE in combination with Coomassie Blue staining was adequate to determine the conjugation efficacy of a peptide (epitope 35–55 of myelin oligodendrocyte glycoprotein) to mannan. In addition, tricine SDS-PAGE and periodic acid–Schiff stains were able to monitor the redox state of mannan. Using the described protocol, more than 99.9% of a peptide containing five lysines at its N-terminus was confirmed conjugated to mannan.     

Computational investigation of blood cell transport in retinal microaneurysms

Microaneurysms (MAs) are one of the earliest clinically visible signs of diabetic retinopathy (DR). MA leakage or rupture may precipitate local pathology in the surrounding neural retina that impacts visual function. Thrombosis in MAs may affect their turnover time, an indicator associated with visual and anatomic outcomes in the diabetic eyes. In this work, we perform computational modeling of blood flow in microchannels containing various MAs to investigate the pathologies of MAs in DR. The particle-based model employed in this study can explicitly represent red blood cells (RBCs) and platelets as well as their interaction in the blood flow, a process that is very difficult to observe in vivo. Our simulations illustrate that while the main blood flow from the parent vessels can perfuse the entire lumen of MAs with small body-to-neck ratio (BNR), it can only perfuse part of the lumen in MAs with large BNR, particularly at a low hematocrit level, leading to possible hypoxic conditions inside MAs. We also quantify the impacts of the size of MAs, blood flow velocity, hematocrit and RBC stiffness and adhesion on the likelihood of platelets entering MAs as well as their residence time inside, two factors that are thought to be associated with thrombus formation in MAs. Our results show that enlarged MA size, increased blood velocity and hematocrit in the parent vessel of MAs as well as the RBC-RBC adhesion promote the migration of platelets into MAs and also prolong their residence time, thereby increasing the propensity of thrombosis within MAs. Overall, our work suggests that computational simulations using particle-based models can help to understand the microvascular pathology pertaining to MAs in DR and provide insights to stimulate and steer new experimental and computational studies in this area.     

SALL4 controls cell fate in response to DNA base composition

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Leishmania proteins derived from recombinant DNA: current status and next steps

The parasite Leishmania is a major cause of disease worldwide. In the past 15 years, many groups have analysed DNA-derived proteins from Leishmania. Large amounts of data obtained by these groups can be collated to direct future research into Leishmania and to find novel immunological mechanisms and information about its metabolism. International coordination will increase both the basic knowledge about Leishmania and the capacity to apply this knowledge to combat leishmaniases.     

Active remodeling in idiopathic interstitial pneumonias: evaluation of collagen types XII and XIV.

Fibril-associated collagens with interrupted triple helices (FACITs) XII and XIV act as fibril organizers and assist in the maintenance of uniform fibril size. We investigated the spatial expression patterns of collagens XII and XIV in cryptogenic organizing pneumonia (COP)/organizing pneumonia (OP) and in idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia (UIP) and compared them to normal human lung. Study subjects included 10 patients with COP/OP, 10 patients with IPF/UIP, and 8 control subjects. Immunostaining for collagens XII and XIV was carried out in paraffin-embedded human lung tissue sections. Picrosirius red histochemical staining for collagen I expression and electron microcopy to evaluate fibril diameter were also performed. In normal lung, collagens XII and XIV were expressed in perivascular and subpleural connective tissue. In COP/OP, both collagens showed intense staining in perivascular connective tissue, thickened alveolar septae, and subpleural areas. In IPF/UIP, XII and XIV were expressed in perivascular connective tissue, in areas of established fibrosis, and in areas of subpleural thickening. Only collagen XII was expressed in granulation tissue plugs in COP/OP and in fibroblastic foci in IPF/UIP. Collagen type I was overexpressed in fibrotic areas. Electron micrographs revealed obvious fibril diameter alteration and fusion in the same areas. FACITs XII and XIV are expressed in normal and fibrotic lung. Unlike collagen XIV, collagen XII was expressed in granulation tissue plugs in COP/OP and in fibroblast foci in IPF/UIP. This may suggest a possible distinct role for both collagens in the modulation of the extracellular matrix during the onset of fibrotic process.