Population genomics of Sinorhizobium medicae based on low-coverage sequencing of sympatric isolates

We investigated the genomic diversity of a local population of the symbiotic bacterium Sinorhizobium medicae, isolated from the roots of wild Medicago lupulina plants, in order to assess genomic diversity, to identify genomic regions influenced by duplication, deletion or strong selection, and to explore the composition of the pan-genome. Partial genome sequences of 12 isolates were obtained by Roche 454 shotgun sequencing (average 5.3 Mb per isolate) and compared with the published sequence of S. medicae WSM 419. Homologous recombination appears to have less impact on the polymorphism patterns of the chromosome than on the chromid pSMED01 and megaplasmid pSMED02. Moreover, pSMED02 is a hot spot of insertions and deletions. The whole chromosome is characterized by low sequence polymorphism, consistent with the high density of housekeeping genes. Similarly, the level of polymorphism of symbiosis genes (low) and of genes involved in polysaccharide synthesis (high) may reflect different selection. Finally, some isolates carry genes that may confer adaptations that S. medicae WSM 419 lacks, including homologues of genes encoding rhizobitoxine synthesis, iron uptake, response to autoinducer-2, and synthesis of distinct polysaccharides. The presence or absence of these genes was confirmed by PCR in each of these 12 isolates and a further 27 isolates from the same population. All isolates had rhizobitoxine genes, while the other genes were co-distributed, suggesting that they may be on the same mobile element. These results are discussed in relation to the ecology of Medicago symbionts and in the perspective of population genomics studies.     

ProADD: A database on Protein Aggregation Diseases

ProADD, a database for protein aggregation diseases, is developed to organize the data under a single platform to facilitate easy access for researchers. Diseases caused due to protein aggregation and the proteins involved in each of these diseases are integrated. The database helps in classification of proteins involved in the protein aggregation diseases based on sequence and structural analysis. Analysis of proteins can be done to mine patterns prevailing among the aggregating proteins.

Availability

http://bicmku.in/ProADD     

Transition of healthy to diseased synovial tissue in rheumatoid arthritis is associated with gain of mesenchymal/fibrotic characteristics

The healthy synovial lining layer consists of a single cell layer that regulates the transport between the joint cavity and the surrounding tissue. It has been suggested that abnormalities such as somatic mutations in the p53 tumor-suppressor gene contribute to synovial hyperplasia and invasion in rheumatoid arthritis (RA). In this study, expression of epithelial markers on healthy and diseased synovial lining tissue was examined. In addition, we investigated whether a regulated process, resembling epithelial to mesenchymal transition (EMT)/fibrosis, could be responsible for the altered phenotype of the synovial lining layer in RA. Synovial tissue from healthy subjects and RA patients was obtained during arthroscopy. To detect signs of EMT, expression of E-cadherin (epithelial marker), collagen type IV (indicator of the presence of a basement membrane) and alpha-smooth muscle actin (alpha-sma; a myofibroblast marker) was investigated on frozen tissue sections using immunohistochemistry. Fibroblast-like synoviocytes (FLSs) from healthy subjects were isolated and subjected to stimulation with synovial fluid (SF) from two RA patients and to transforming growth factor (TGF)-beta. To detect whether EMT/fibrotic markers were increased, expression of collagen type I, alpha-sma and telopeptide lysylhydroxylase (TLH) was measured by real time PCR. Expression of E-cadherin and collagen type IV was found in healthy and arthritic synovial tissue. Expression of alpha-sma was only found in the synovial lining layer of RA patients. Stimulation of healthy FLSs with SF resulted in an upregulation of alpha-sma and TLH mRNA. Collagen type I and TLH mRNA were upregulated after stimulation with TGF-beta. Addition of bone morphogenetic protein (BMP)-7 to healthy FLS stimulated with SF inhibited the expression of alpha-sma mRNA. The finding that E-cadherin and collagen type IV are expressed in the lining layer of healthy and arthritic synovium indicates that these lining cells display an epithelial-like phenotype. In addition, the presence of alpha-sma in the synovial lining layer of RA patients and induction of fibrotic markers in healthy FLSs by SF from RA patients indicate that a regulated process comparable to EMT might cause the alteration in phenotype of RA FLSs. Therefore, BMP-7 may represent a promising agent to counteract the transition imposed on synoviocytes in the RA joint.     

A new model for nuclear envelope breakdown

Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane.     

Antioxidant activity of different dihydropyridines

Lacidipine, a dihydropyridine-based calcium antagonist (DHP), has already been demonstrated to possess antioxidant activity and to reduce the intracellular production of reactive oxygen species (ROS). To verify if this effect is a peculiarity of this molecule, or belongs to other DHPs, the activity of lacidipine was compared with those of amlodipine, lercanidipine, nimodipine, and nifedipine. The DHPs were incorporated in bovine aortic endothelial cells (BAECs). Cu(2+)-oxidized LDL (ox-LDL, 5 microM) was incubated with BAECs for 5 min. 2',7'-Dichlorofluorescein (DCF) as expression of intracellular ROS production was measured by flow cytometry. Ox-LDL induced a strong increase in intracellular ROS formation (p<0.001) that was significantly reduced only with lacidipine and lercanidipine (p from <0.05 to <0.01); the effect of lacidipine, however, resulted in being much more evident than lercanidipine (p<0.01); amlodipine, nimodopine, and nifedipine had no effect on ROS formation. The lowest IC50s, i.e. the concentrations determining the 50% reduction of ROS, were obtained with lacidipine (p<0.01). The inhibitory effect of lacidipine on ox-LDL-induced ROS production in endothelial cells is a peculiarity of this molecule through its antioxidant activity.     

Multiphoton excited fabrication of collagen matrixes cross-linked by a modified benzophenone dimer: bioactivity and enzymatic degradation

Multiphoton excited (MPE) photochemistry is used to fabricate model tissue engineering scaffolds directly from types I, II, and IV collagen. A modified benzophenone dimer (BPD) provides the photoactivation and becomes incorporated into the resulting collagen matrixes. Unlike xanthene photochemistries, the benzophenone dimer can be used in acidic environments, where most forms of collagen have the greatest solubility. The minimum feature sizes are investigated by using two- and three-photon excitation, where the latter provides for superior "resolution" and suggests that collagen structures can be fabricated on the size scales of focal contacts. The resulting structures display excellent retention of bioactivity as evidenced by highly specific cell adhesion as well as immunofluorescence labeling. Structural and chemical aspects of the collagen matrixes are probed through measuring the enzymatic degradation through specific and nonspecific proteases, as the resulting relative rates are consistent with the activity of these enzymes. The degradation rates can also be controlled through varying the cross-link density in the matrixes, which is achieved through tuning the exposure dose during the fabrication process. The degradation rates are also found to be consistent with swelling/shrinking measurements and thus the average mesh size of the matrixes. In all cases the enzymatic degradations are well-fit single exponentials, suggesting that the matrixes can be fabricated with a priori knowledge of their structural properties. These results coupled with the resulting bioactivity suggest that the multiphoton fabrication process may be a powerful tool for the creation of cell-sized tissue engineering scaffolds.     

CD134 as target for specific drug delivery to auto-aggressive CD4<Superscript>+</Superscript>T cells in adjuvant arthritis

T cells have an important role during the development of autoimmune diseases. In adjuvant arthritis, a model for rheumatoid arthritis, we found that the percentage of CD4⁺ T cells expressing the activation marker CD134 (OX40 antigen) was elevated before disease onset. Moreover, these CD134⁺ T cells showed a specific proliferative response to the disease-associated epitope of mycobacterial heat shock protein 60, indicating that this subset contains auto-aggressive T cells. We studied the usefulness of CD134 as a molecular target for immune intervention in arthritis by using liposomes coated with a CD134-directed monoclonal antibody as a drug targeting system. Injection of anti-CD134 liposomes subcutaneously in the hind paws of pre-arthritic rats resulted in targeting of the majority of CD4⁺CD134⁺ T cells in the popliteal lymph nodes. Furthermore, we showed that anti-CD134 liposomes bound to activated T cells were not internalized. However, drug delivery by these liposomes could be established by loading anti-CD134 liposomes with the dipalmitate-derivatized cytostatic agent 5'-fluorodeoxyuridine. These liposomes specifically inhibited the proliferation of activated CD134⁺ T cells in vitro, and treatment with anti-CD134 liposomes containing 5'-fluorodeoxyuridine resulted in the amelioration of adjuvant arthritis. Thus, CD134 can be used as a marker for auto-aggressive CD4⁺ T cells early in arthritis, and specific liposomal targeting of drugs to these cells via CD134 can be employed to downregulate disease development.