PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-5 (5)
 

Clipboard (0)
None
Journals
Authors
more »
Year of Publication
Document Types
1.  Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER  
Local structural similarity restraints (LSSR) provide a novel method for exploiting NCS or structural similarity to an external target structure. Two examples are given where BUSTER re-refinement of PDB entries with LSSR produces marked improvements, enabling further structural features to be modelled.
Maximum-likelihood X-ray macromolecular structure refinement in BUSTER has been extended with restraints facilitating the exploitation of structural similarity. The similarity can be between two or more chains within the structure being refined, thus favouring NCS, or to a distinct ‘target’ structure that remains fixed during refinement. The local structural similarity restraints (LSSR) approach considers all distances less than 5.5 Å between pairs of atoms in the chain to be restrained. For each, the difference from the distance between the corresponding atoms in the related chain is found. LSSR applies a restraint penalty on each difference. A functional form that reaches a plateau for large differences is used to avoid the restraints distorting parts of the structure that are not similar. Because LSSR are local, there is no need to separate out domains. Some restraint pruning is still necessary, but this has been automated. LSSR have been available to academic users of BUSTER since 2009 with the easy-to-use -autoncs and -­target target.pdb options. The use of LSSR is illustrated in the re-refinement of PDB entries 5rnt, where -target enables the correct ligand-binding structure to be found, and 1osg, where -autoncs contributes to the location of an additional copy of the cyclic peptide ligand.
doi:10.1107/S0907444911056058
PMCID: PMC3322596  PMID: 22505257
BUSTER; NCS restraints; target-structure restraints; local structural similarity restraints
2.  Relief from Zmp1-Mediated Arrest of Phagosome Maturation Is Associated with Facilitated Presentation and Enhanced Immunogenicity of Mycobacterial Antigens▿ 
Pathogenic mycobacteria escape host innate immune responses by blocking phagosome-lysosome fusion. Avoiding lysosomal delivery may also be involved in the capacity of mycobacteria to evade major histocompatibility complex (MHC) class I- or II-dependent T-cell responses. In this study, we used a genetic mutant of Mycobacterium bovis BCG that is unable to escape lysosomal transfer and show that presentation of mycobacterial antigens is affected by the site of intracellular residence. Compared to infection with wild-type BCG, infection of murine bone marrow-derived dendritic cells with a mycobacterial mutant deficient in zinc metalloprotease 1 (Zmp1) resulted in increased presentation of MHC class II-restricted antigens, as assessed by activation of mycobacterial Ag85A-specific T-cell hybridomas. The zmp1 deletion mutant was more immunogenic in vivo, as measured by delayed-type hypersensitivity (DTH), antigen-specific lymphocyte proliferation, and the frequency of antigen-specific gamma interferon (IFN-γ)-producing lymphocytes of both CD4 and CD8 subsets. In conclusion, our results suggest that phagosome maturation and lysosomal delivery of BCG facilitate mycobacterial antigen presentation and enhance immunogenicity.
doi:10.1128/CVI.00015-11
PMCID: PMC3122614  PMID: 21471301
3.  Platelet Recruitment during Multiple Donor Platelet Apheresis Differs between Cell Separators 
Summary
Background
Recruitment of platelets (PLT) during donor PLT apheresis may facilitate the harvest of multiple units within a single donation.
Methods
We compared two PLT apheresis procedures (Amicus and Trima Accel) in a prospective, randomized, paired cross-over study in 60 donors. The 120 donations were compared for depletion of circulating PLT in the donors, PLT yields and PLT recruitment. A recruitment was defined as ratio of total PLT yield and donor PLT depletion > 1.
Results
Despite comparable differences of pre- and post-apheresis PLT counts (87 × 109/l in Trima Accel vs. 92 × 109/l in Amicus, p = 0.383), PLT yields were higher with Trima Accel (7.48 × 1011 vs. 6.06 × 1011, p < 0.001), corresponding to a higher PLT recruitment (1.90 vs. 1.42, p < 0.001). We observed a different increase of WBC counts after aphereses, which was more pronounced with Trima Accel than with Amicus (1.30 × 109/l vs. 0.46 × 109/l, p < 0.001).
Conclusion
Both procedures induced PLT recruitment. This was higher in Trima Accel, contributing to a higher yield in spite of a comparable depletion of circulating PLT in the donors. This recruitment facilitates the harvest of multiple units within a single donation and seems to be influenced by the procedure utilized. The different increases of circulating donor white blood cells after donation need further investigation.
doi:10.1159/000328634
PMCID: PMC3128152  PMID: 21760764
Platelet apheresis; Platelet recruitment; Cell separator
4.  Data processing and analysis with the autoPROC toolbox 
Typical topics and problems encountered during data processing of diffraction experiments are discussed and the tools provided in the autoPROC software are described.
A typical diffraction experiment will generate many images and data sets from different crystals in a very short time. This creates a challenge for the high-throughput operation of modern synchrotron beamlines as well as for the subsequent data processing. Novice users in particular may feel overwhelmed by the tables, plots and numbers that the different data-processing programs and software packages present to them. Here, some of the more common problems that a user has to deal with when processing a set of images that will finally make up a processed data set are shown, concentrating on difficulties that may often show up during the first steps along the path of turning the experiment (i.e. data collection) into a model (i.e. interpreted electron density). Difficulties such as unexpected crystal forms, issues in crystal handling and suboptimal choices of data-collection strategies can often be dealt with, or at least diagnosed, by analysing specific data characteristics during processing. In the end, one wants to distinguish problems over which one has no immediate control once the experiment is finished from problems that can be remedied a posteriori. A new software package, autoPROC, is also presented that combines third-party processing programs with new tools and an automated workflow script that is intended to provide users with both guidance and insight into the offline processing of data affected by the difficulties mentioned above, with particular emphasis on the automated treatment of multi-sweep data sets collected on multi-axis goniostats.
doi:10.1107/S0907444911007773
PMCID: PMC3069744  PMID: 21460447
autoPROC; data processing
5.  Fes-Cre Targets Phosphatidylinositol Glycan Class a (Piga) Inactivation to Hematopoietic Stem Cells in the Bone Marrow 
A somatic mutation in the X-linked phosphatidylinositol glycan class A (PIGA) gene causes the loss of glycosyl phosphatidylinositol (GPI)-linked proteins on blood cells from patients with paroxysmal nocturnal hemoglobinuria. Because all blood cell lineages may be affected it is thought that the mutation occurs in a hematopoietic stem cell. In transgenic mice, germline transmission of an inactive Piga gene is embryonic lethal. To inactivate the murine Piga gene in early hematopoiesis we therefore chose conditional gene inactivation using the Cre/loxP system. We expressed Cre recombinase under the transcription regulatory sequences of the human c-fes gene. FES-Cre inactivated PIGA in hematopoietic cells of mice carrying a floxed Piga allele (LF mice). PIGA− cells were found in all hematopoietic lineages of definitive but not primitive hematopoiesis. Their proportions were low in newborn mice but subsequently increased continuously to produce for the first time mice that have almost exclusively PIGA− blood cells. The loss of GPI-linked proteins occurred mainly in c-kit+CD34+Lin− progenitor cells before the CFU-GEMM stage. Using bone marrow reconstitution experiments with purified PIGA− cells we demonstrate that LF mice have long-term bone marrow repopulating cells that lack GPI-linked proteins, indicating that recombination of the floxed Piga allele occurs in the hematopoietic stem cell.
PMCID: PMC2195941  PMID: 11535627
hematopoiesis; stem cell; c-fes; paroxysmal nocturnal hemoglobinuria; conditional gene inactivation

Results 1-5 (5)