Managing and mining protein crystallization data

The crystallization of macromolecules remains a major bottleneck in structural biology. The routine screening of more than one thousand crystallization conditions and subsequent optimization by fine screening presents a challenge to conventional laboratory notebook keeping. In addition, the development of high-throughput robotic crystallization and imaging systems presents a pressing need for low-cost laboratory information management system (LIMS). Here we describe CLIMS2, a crystallization LIMS that features a simple, user-friendly graphical interface, allowing the storage, management, retrieval and mining of crystallization data. The CLIMS2 executable and documentation is freely available at http://clims.med.monash.edu.au.     

Biodiversity management of fens and fen meadows by grazing,cutting and burning

Question: Can the biodiversity of fens in Europe and North America be maintained through the use of grazing (especially cattle grazing), fire, and/or cutting? Location: European and North American fens. Methods: This paper is a review of the literature on the effects of grazing, fire and cutting on fens, to explore the relationship between management and biodiversity in fens. Results: A reduction of cattle grazing, mowing and burning in fens has led to a reduction in biodiversity in fens. The vegetation of abandoned fens shifts to trees and shrubs after 10–15 years, which shade the smaller and rarer species of these wetlands. While careful use of fire is used to manage fens in North America, it is not widely used in European fens, perhaps because the peat of drained fens may catch fire. Cattle grazing cannot be considered a natural disturbance in North America, since cattle did not evolve on that continent. In Europe, cattle do not generally graze in unaltered fens, but they do use slightly drained fen meadows. Conclusions: Three approaches have been used to control the dominance of tall woody and herbaceous species in abandoned fens, including the re‐introduction of cattle, mowing, and burning. Overgrazing results in a permanent reduction in biodiversity, therefore cattle re‐introduction must be approached cautiously. In Europe, but not in North America, mowing has been an important management tool, and mowing has been successful in maintaining species richness, particularly in fens that have been mowed annually for centuries. Fire has been the most common and successful management tool in North America although it is not effective in removing shrubs that have become large. Because the problems and solutions are similar, the literature of both European and North American fen management can be analyzed to better assess the management of fens on both continents. Many management questions require further study and these are listed in the paper.     

Detection of nucleotide binding to Na,K-ATPase in an aqueous membrane suspension by 13C cross-polarization magic-angle spinning NMR spectroscopy

Binding of uniformly (13)C labelled ATP to Na,K-ATPase was studied by (13)C cross-polarization magic-angle spinning (CP-MAS) NMR. In the presence of 30 mM Na(+) , and with sample- and time-averaging, NMR spectra obtained at 4 degrees C exhibited several resonances for the bound nucleotide. Chemical shifts suggested that site-specific changes in the micro-environment or conformation of the nucleotide occurred in the high affinity binding site. These experiments permit further studies of nucleotide dynamics, structure and binding under physiologically relevant conditions.     

Viral gastroenteritis associated with genogroup II norovirus among U.S. military personnel in Turkey, 2009

The present study demonstrates that multiple NoV genotypes belonging to genogroup II contributed to an acute gastroenteritis outbreak at a US military facility in Turkey that was associated with significant negative operational impact. Norovirus (NoV) is an important pathogen associated with acute gastroenteritis among military populations. We describe the genotypes of NoV outbreak occurred at a United States military facility in Turkey. Stool samples were collected from 37 out of 97 patients presenting to the clinic on base with acute gastroenteritis and evaluated for bacterial and viral pathogens. NoV genogroup II (GII) was identified by RT-PCR in 43% (16/37) stool samples. Phylogenetic analysis of a 260 base pair fragment of the NoV capsid gene from ten stool samples indicated the circulation of multiple and rare genotypes of GII NoV during the outbreak. We detected four GII.8 isolates, three GII.15, two GII.9 and a sole GII.10 NoV. Viral sequences could be grouped into four clusters, three of which have not been previously reported in Turkey. The fact that current NoV outbreak was caused by rare genotypes highlights the importance of norovirus strain typing. While NoV genogroup II is recognized as causative agent of outbreak, circulation of current genotypes has been rarely observed in large number of outbreaks.     

The effects of chemokine, adhesion and extracellular matrix molecules on binding of mesenchymal stromal cells to poly(l-lactic acid)

Abstract Background aims. Mesenchymal stromal cells (MSC) are pluripotent adult stem cells capable of osteogenesis and chondrogenesis to form bone and cartilage. This characteristic gives them the potential for bone and cartilage regeneration. Synthetic polymers have been studied to examine whether they could be used as a scaffold for tissue engineering. In the current study a two-dimensional (2-D) poly(l-lactic acid) (PLLA) scaffold was treated with chemokine, adhesion and extracellular matrix molecules with the aim of using biologic molecules to improve the attachment of human MSC. Methods. MSC were isolated from human bone marrow and applied to a 2-D PLLA scaffold. Chemokines ligand (CXCL12 and CXCL13), adhesion molecules [P-selectin, vascular cell adhesion molecule (VCAM)-1 and heparin] and extracellular matrix molecules (fibronectin and type IV collagen) were coated on the scaffold and their effects on the number of MSC that adhered were recorded. Results. When used alone CXCL12 and CXCL13 enhanced MSC adhesion, as did VCAM-1, P-selectin, fibronectin and collagen, but not heparin. The effects of VCAM-1, P-selectin and heparin were enhanced by the addition of CXCL12. Incubation of MSC with antibodies to integrins α4 and α5β1 inhibited their adhesion to VCAM-1 and fibronectin-treated PLLA respectively, suggesting that these integrins were involved in the MSC interactions. Conclusions. The use of certain chemokines and adhesion and extracellular matrix molecules, alone or in combination, is beneficial for the attachment of MSC to PLLA, and may be helpful as natural molecules in scaffolds for regenerative medicine.     

Cedar Virus: A Novel Henipavirus Isolated from Australian Bats

The genus Henipavirus in the family Paramyxoviridae contains two viruses, Hendra virus (HeV) and Nipah virus (NiV) for which pteropid bats act as the main natural reservoir. Each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. Here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, Cedar virus (CedPV), which shares significant features with the known henipaviruses. The genome size (18,162 nt) and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin- B2) for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. In this context, it is interesting to note that the major genetic difference between CedPV and HeV or NiV lies within the coding strategy of the P gene, which is known to play an important role in evading the host innate immune system. Unlike HeV, NiV, and almost all known paramyxoviruses, the CedPV P gene lacks both RNA editing and also the coding capacity for the highly conserved V protein. Preliminary study indicated that CedPV infection of human cells induces a more robust IFN-β response than HeV.     

Phenotypic effects of the "mini-muscle" allele in a large HR x C57BL/6J mouse backcross

From outbred Hsd:ICR mice, we selectively bred 4 replicate lines for high running (High-Runner [HR] lines) on wheels while maintaining 4 nonselected lines as controls (C lines). An apparent Mendelian recessive, the "mini-muscle" (MM) allele, whose main phenotypic effect is to reduce hindlimb muscle mass by 50%, was discovered in 2 HR lines and 1 C line. This gene of major effect has gone to fixation in one selected line, remains polymorphic in another, and is now undetectable in the one C line. Homozygotes exhibit various pleiotropic effects, including a doubling of mass-specific muscle aerobic capacity, and larger hearts, livers, and spleens. To create a population suitable for mapping the genomic location of the MM allele and to better characterize its pleiotropic effects, we crossed females fixed for the MM allele with male C57BL/6J. F(1) males were then backcrossed to the MM parent females. Backcross (BC) mice (N = 404) were dissected, and a 50:50 ratio of normal to MM phenotype was observed with no overlap in relative muscle mass. In the BC, analysis of covariance revealed that MM individuals ran significantly more on days 5 and 6 of a 6-day exposure to running wheels (as in the routine selective-breeding protocol), were smaller in body mass, and had larger ventricles and spleens.     

Transaldolase deficiency influences the pentose phosphate pathway, mitochondrial homoeostasis and apoptosis signal processing

TAL (transaldolase) was originally described in the yeast as an enzyme of the PPP (pentose phosphate pathway). However, certain organisms and mammalian tissues lack TAL, and the overall reason for its existence is unclear. Recently, deletion of Ser(171) (TALDeltaS171) was found in five patients causing inactivation, proteasome-mediated degradation and complete deficiency of TAL. In the present study, microarray and follow-up Western-blot, enzyme-activity and metabolic studies of TALDeltaS171 TD (TAL-deficient) lymphoblasts revealed co-ordinated changes in the expression of genes involved in the PPP, mitochondrial biogenesis, oxidative stress, and Ca(2+) fluxing. Sedoheptulose 7-phosphate was accumulated, whereas G6P (glucose 6-phosphate) was depleted, indicating a failure to recycle G6P for the oxidative branch of the PPP. Nucleotide analysis showed depletion of NADPH and NAD(+) and accumulation of ADP-ribose. TD cells have diminished Deltapsi(m) (mitochondrial transmembrane potential) and increased mitochondrial mass associated with increased production of nitric oxide and ATP. TAL deficiency resulted in enhanced spontaneous and H(2)O(2)-induced apoptosis. TD lymphoblasts showed increased expression of CD38, which hydrolyses NAD(+) into ADP-ribose, a trigger of Ca(2+) release from the endoplasmic reticulum that, in turn, facilitated CD20-induced apoptosis. By contrast, TD cells were resistant to CD95/Fas-induced apoptosis, owing to a dependence of caspase activity on redox-sensitive cysteine residues. Normalization of TAL activity by adeno-associated-virus-mediated gene transfer reversed the elevated CD38 expression, ATP and Ca(2+) levels, suppressed H(2)O(2)- and CD20-induced apoptosis and enhanced Fas-induced cell death. The present study identified the TAL deficiency as a modulator of mitochondrial homoeostasis, Ca(2+) fluxing and apoptosis.     

The use of modelling and simulation approach in reconstructing past landscapes from fossil pollen data: a review and results from the POLLANDCAL network

Information on past land cover in terms of absolute areas of different landscape units (forest, open land, pasture land, cultivated land, etc.) at local to regional scales is needed to test hypotheses and answer questions related to climate change (e.g. feedbacks effects of land-cover change), archaeological research, and nature conservancy (e.g. management strategy). The palaeoecological technique best suited to achieve quantitative reconstruction of past vegetation is pollen analysis. A simulation approach developed by Sugita (the computer model POLLSCAPE) which uses models based on the theory of pollen analysis is presented together with examples of application. POLLSCAPE has been adopted as the central tool for POLLANDCAL (POLlen/LANdscape CALibration), an international research network focusing on this topic. The theory behind models of the pollen–vegetation relationship and POLLSCAPE is reviewed. The two model outputs which receive greatest attention in this paper are the relevant source area of pollen (RSAP) and pollen loading in mires and lakes. Six examples of application of POLLSCAPE are presented, each of which explores a possible use of the POLLANDCAL tools and a means of validating or evaluating the models with empirical data. The landscape and vegetation factors influencing the size of the RSAP, the importance of pollen productivity estimates (PPEs) for the model outputs, the detection of small and rare patches of plant taxa in pollen records, and quantitative reconstructions of past vegetation and landscapes are discussed on the basis of these examples. The simulation approach is seen to be useful both for exploring different vegetation/landscape scenarios and for refuting hypotheses.