Increased levels of free circulating DNA in patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is difficult to diagnose because of numerous interstitial lung diseases with similar symptoms. As serum DNA has proven useful for early lung cancer detection, we aimed to define the relevance of this marker in discriminating IPF from other fibrotic and nonfibrotic/nonmalignant lung diseases. DNA was quantified in 191 subjects: 64 healthy individuals, 58 patients with IPF, 17 patients with nonspecific pulmonary fibrosis (13 idiopathic nonspecific interstitial pneumonia, 4 chronic hypersensitivity pneumonitis), and 52 patients with other diffuse/nonmalignant lung diseases. The median value of free DNA in IPF patients was 61.1 ng/mL (range 7.1-405), which was significantly higher than that of healthy donors (median 6.8, range 2.2-184) (p<0.001) and that of patients with other diffuse/nonmalignant lung diseases (median 28.0, range 4.2-281) (p=0.004). The area under the ROC curve was 0.926 (95% CI 0.879-0.973) when IPF patients were compared with healthy donors, and 0.702 (95% CI 0.609-0.796) when a comparison was made with non-IPF pulmonary diseases. In conclusion, we observed significantly higher levels of free circulating DNA in patients with IPF than in those with other fibrotic or diffuse/nonmalignant lung diseases.     

Synthesis of structurally simplified analogues of aplidinone A,a pro-apoptotic marine thiazinoquinone

The synthesis of analogues of aplidinone A (7), a prenylated quinone isolated from the Mediterranean ascidian Aplidium conicum, has been performed. This work not only allowed confirming the structural assignment of aplidinone A, previously made with the support of GIAO shielding calculations, but, above all, made a series of structurally related quinone derivatives (compounds 8–13 and the natural metabolite) available for a screening in vitro for cytotoxic and pro-apoptotic activity and for SAR studies. The study evidenced one of the synthetic analogues (11) as a potent cytotoxic and pro-apoptotic agent against several tumor cell lines which also inhibits the TNFα-induced NF-κB activation in a human leukemia T cell line. This exemplifies the potential of a natural product to qualify as lead structure for medicinal chemistry campaigns, affording simplified analogues with better bioactivity and easier to synthesize.     

Effect of cell seeding and mechanical loading on vascularization and tissue formation inside a scaffold: A mechano-biological model using a lattice approach to simulate cell activity

Achieving successful vascularization remains one of the main problems in bone tissue engineering. After scaffold implantation, the growth of capillaries into the porous construct may be too slow to provide adequate nutrients to the cells in the scaffold interior and this inhibits tissue formation in the scaffold core. Often, prior to implantation, a controlled cell culture environment is used to stimulate cell proliferation and, once in place, the mechanical environment acting on the tissue construct is determined by the loading conditions at the implantation site. To what extent do cell seeding conditions and the construct loading environment have an effect on scaffold vascularization and tissue growth? In this study, a mechano-biological model for tissue differentiation and blood vessel growth was used to determine the influence of cell seeding on vascular network development and tissue growth inside a regular-structured bone scaffold under different loading conditions. It is predicted that increasing the number of cells seeded homogeneously reduces the rate of vascularization and the maximum penetration of the vascular network, which in turn reduces bone tissue formation. The seeding of cells in the periphery of the scaffold was predicted to be beneficial for vascularization and therefore for bone growth; however, tissue formation occurred more slowly during the first weeks after implantation compared to homogeneous seeding. Low levels of mechanical loading stimulated bone formation while high levels of loading inhibited bone formation and capillary growth. This study demonstrates the feasibility of computational design approaches for bone tissue engineering.     

Kinematic and kinetic features of normal level walking in patellofemoral pain syndrome: More than a sagittal plane alteration

Patients with patellofemoral pain syndrome (PFPS) often report discomfort and pain during walking. To date, most of the studies conducted to determine gait alterations in PFPS patients have focused on sagittal plane alterations. Physiological and biomechanical factors, however, suggest that frontal and transverse plane alterations may be involved in PFPS. We therefore decided to conduct a kinematic and kinetic evaluation on all three planes in 9 PFPS subjects and 9 healthy sex- and age-matched controls. General gait characteristics were similar in patients and controls, with the exception of swing velocity, which was lower in PFPS patients. Patients also displayed an increased knee abductor and external rotator moments in loading response, and reduced knee extensor moment both in loading response and in terminal stance. We speculate that these findings may be linked both to a pain-avoiding gait pattern and to alterations in the timing of activation of different components of the quadriceps muscle, which is typical of PFPS. The relevance for clinicians is this gait pattern may represent a biomechanical risk factor for future knee osteoarthritis. We therefore recommend that treatments aimed at PFPS should also attempt to restore a correct walking pattern.     

Structural Characterization of Protein-Protein Complexes by Integrating Computational Docking with Small-angle Scattering Data

X-ray crystallography and NMR can provide detailed structural information of protein-protein complexes, but technical problems make their application challenging in the high-throughput regime. Other methods such as small-angle X-ray scattering (SAXS) are more promising for large-scale application, but at the cost of lower resolution, which is a problem that can be solved by complementing SAXS data with theoretical simulations. Here, we propose a novel strategy that combines SAXS data and accurate protein-protein docking simulations. The approach has been benchmarked on a large pool of known structures with synthetic SAXS data, and on three experimental examples. The combined approach (pyDockSAXS) provided a significantly better success rate (43% for the top 10 predictions) than either of the two methods alone. Further analysis of the influence of different docking parameters made it possible to increase the success rates for specific cases, and to define guidelines for improving the data-driven protein-protein docking protocols.     

BacA Is Essential for Bacteroid Development in Nodules of Galegoid,but not Phaseoloid,Legumes

BacA is an integral membrane protein, the mutation of which leads to increased resistance to the antimicrobial peptides bleomycin and Bac7_1-35 and a greater sensitivity to SDS and vancomycin in Rhizobium leguminosarum bv. viciae, R. leguminosarum bv. phaseoli, and Rhizobium etli. The growth of Rhizobium strains on dicarboxylates as a sole carbon source was impaired in bacA mutants but was overcome by elevating the calcium level. While bacA mutants elicited indeterminate nodule formation on peas, which belong to the galegoid tribe of legumes, bacteria lysed after release from infection threads and mature bacteroids were not formed. Microarray analysis revealed almost no change in a bacA mutant of R. leguminosarum bv. viciae in free-living culture. In contrast, 45 genes were more-than 3-fold upregulated in a bacA mutant isolated from pea nodules. Almost half of these genes code for cell membrane components, suggesting that BacA is crucial to alterations that occur in the cell envelope during bacteroid development. In stark contrast, bacA mutants of R. leguminosarum bv. phaseoli and R. etli elicited the formation of normal determinate nodules on their bean host, which belongs to the phaseoloid tribe of legumes. Bacteroids from these nodules were indistinguishable from the wild type in morphology and nitrogen fixation. Thus, while bacA mutants of bacteria that infect galegoid or phaseoloid legumes have similar phenotypes in free-living culture, BacA is essential only for bacteroid development in indeterminate galegoid nodules.Bacteria of the family Rhizobiaceae are alphaproteobacteria, which form a species-specific symbiotic relationship with leguminous plants. Plants release flavonoids that typically induce the synthesis of lipochitooligosaccharides by rhizobia, which in turn initiate a signaling cascade in the plant, leading to nodule formation (34). Rhizobia become trapped by curling root hairs, which they enter via infection threads that grow and ramify into the root cortex, where newly induced meristematic cells form the nodule (34). Bacteria are released from infection threads and engulfed by a plant-derived symbiosome membrane. In galegoid legumes (a clade in the subfamily Papilionoideae, such as Medicago, Pisum, or Vicia), which form indeterminate nodules that have a persistent meristem, bacteria undergo the endoreduplication of their chromosome, resulting in dramatic increases in size, shape, and DNA content to become terminally differentiated bacteroids (32). However, in phaseoloid legumes (e.g., lotus, bean, and soybean), which form determinate nodules with a transient meristem, bacteria do not undergo endoreduplication and therefore do not enlarge substantially. These bacteroids retain a normal DNA content and can regrow after isolation from nodules (32). The endoreduplication of bacteroids is controlled by the plant, and it is believed that nodule-specific cysteine-rich (NCR) peptides, which are made in indeterminate, but not in determinate, nodules, may be responsible for inducing and maintaining bacteroid development (31, 32). Finally, mature bacteroids receive dicarboxylic acids from the plant, which they use as a carbon, reductant, and energy source for the reduction of N₂ to ammonia (38). The ammonia is secreted to the plant, where it is assimilated into amino acids or ureides, depending on the legume, for export to the shoot.Sinorhizobium meliloti BacA protein was the first bacterial factor identified to be essential for bacteroid development (15). More recently, it also has been shown to be essential for the Mesorhizobium-Astragalus symbiosis (42). S. meliloti elicits the formation of indeterminate nodules on alfalfa, and while S. meliloti bacA null mutants induce nodule formation, bacteria lyse soon after endocytosis but prior to bacteroid differentiation (15, 20). BacA is a cytoplasmic membrane protein that shares 64% identity with SbmA from Escherichia coli (15, 25). SbmA/BacA proteins belong to the ATP binding cassette (ABC) superfamily and share sequence similarity with a family of eukaryotic peroxisomal membrane proteins, including the human adrenoleukodystrophy protein, which is required for the efficient transport of very-long-chain fatty acids (VLCFAs) out of the cytoplasm (9). Consistent with this, S. meliloti BacA is required for the complete modification of lipid A with VLCFAs (9). However, since S. meliloti mutants, which are directly involved in the biosynthesis of VLCFA-modified lipid A, show bacteroid abnormalities but still can form a successful alfalfa symbiosis, the effect of BacA on lipid A VLCFA modification does not fully account for its essential role in bacteroid development (10, 11, 16). Strains mutated in bacA also have an increased resistance to the glycopeptide bleomycin, a low-level resistance to aminoglycoside antibiotics, and an increased sensitivity to ethanol, sodium dodecyl sulfate (SDS), and deoxycholate relative to the sensitivities of the parent strain (12, 18, 25). More recently it has been shown that an S. meliloti bacA null mutant has an increased resistance to a truncated form of a eukaryotic proline-rich peptide, Bac7_1-16, and was unable to accumulate a fluorescently labeled form of this peptide (28). This finding, combined with the increased resistance of an S. meliloti bacA null mutant to bleomycin, led to the hypothesis that BacA is itself a putative peptide transporter (BacA mediated) or able to alter the activity of such a transporter (BacA influenced) (11, 15, 18, 28).As the increased resistance of the S. meliloti bacA null mutant to bleomycin and Bac7_1-16 appears to be independent of the VLCFA modification of lipid A (11, 28), this suggested that either BacA-mediated or BacA-influenced peptide uptake into S. meliloti plays a role in bacteroid development. Since indeterminate galegoid nodules contain hundreds of NCR peptides, whereas determinate phaseoloid nodules lack these host peptides (31), we considered it important to assess the role of BacA in bacteroid development during the formation of both nodule types.Here, we show that bacA mutants of Rhizobium leguminosarum bv. viciae strains 3841 and A34 failed to develop bacteroids and did not fix nitrogen in indeterminate pea (Pisum sativum) nodules. However, bacA mutants of both R. leguminosarum bv. phaseoli 4292 and Rhizobium etli CE3 formed normal bacteroids and fixed nitrogen at wild-type rates in determinate bean (Phaseolus vulgaris) nodules. This is consistent with BacA being a key component of bacteroid development in indeterminate galegoid nodules that is not required for functional bacteroid formation in determinate phaseoloid nodules.     

The reaction of [Os6(CO)18] with [(SPPh2)2NH]: Facile rearrangement of the metal framework

The reaction of [Os₆(CO)₁₈] 1 with [(SPPh₂)₂NH] in the presence of Me₃NO produces a purple compound characterized spectroscopically and by X-ray crystallography, as [HOs₆(CO)₁₇(SPPh₂)₂N] 2. The structure shows the hexanuclear fragment to have suffered a geometrical rearrangement to give a metal framework that can be described as an edge-bridged tetrahedron with an additional terminal osmium atom bonded to one of the bridged metal atoms. The ligand acts as a bimetallic tetraconnective unit through both sulphur atoms between two non-bonded osmium atoms.