Structure and Dynamics of the Native HIV-1 Env Trimer

HIV-1/AIDS remains one of the worst pandemics in human history. Despite tremendous efforts, no effective vaccine has been found. Recent reports give new insights into the structure and dynamics of the HIV-1 Env trimer and renew hopes that a better understanding of Env will translate into new vaccine candidates and more-effective antiretroviral therapies.     

Between-session reliability of four hop tests and the agility T-test

The purposes of this study were firstly to investigate whether learning affects were present in the administration of 4 hop tests and the Agility T-test and secondly to assess the between-session reliability of these tests. Twenty-two recreational athletes (11 women: age 22.3 ± 3.7 years, height 167.7 ± 6.2 cm, weight 59.2 ± 6.9 kg and 11 men: age 22.8 ± 3.1 years, height 179.8 ± 4 cm, weight 79.6 ± 10 kg) took part in the study. The subjects performed 6 repetitions of each hop test and 4 repetitions of the Agility T-test once a week over a period of 3 weeks. Distances were normalized to leg length and presented as a percentage value for the single, triple and crossover hop. Results showed that there were significant differences in scores between genders and that learning affects were present in all tests. Intraclass correlation coefficients ranged from 0.76 to 0.92 for the hop tests and 0.82 to 0.96 for the Agility T-test. The results indicated that the hop and Agility T-tests are reliable tests for use with subjects in a clinical or team sport environment. The error measurement statistics presented could be of help to practitioners to determine whether changes in individuals' scores in the hop and Agility T-tests are because of a true change in performance or measurement error. Of most importance was the fact that all subjects achieved at least 90% limb symmetry index on all 4 hop tests. Therefore, we recommend that a minimum limb symmetry value of 90%, rather than previously recommended 85%, should be adopted during rehabilitation and conditioning.     

Transplantation of organic surface horizons of boreal soils into warmer regions alters microbiology but not the temperature sensitivity of decomposition

Changes in soil carbon, the largest terrestrial carbon pool, are critical for the global carbon cycle, atmospheric CO₂ levels and climate. Climate warming is predicted to be most pronounced in the northern regions and therefore the large soil carbon pool residing in boreal forests will be subject to larger global warming impact than soil carbon pools in the temperate or the tropical forest. A major uncertainty in current estimates of the terrestrial carbon balance is related to decomposition of soil organic matter (SOM). We hypothesized that when soils are exposed to warmer climate the structure of the ground vegetation will change much more rapidly than the dominant tree species. This change will alter the quality and amount of litter input to the soil and induce changes in microbial communities, thus possibly altering the temperature sensitivity of SOM decomposition. We transferred organic surface soil sections from the northern borders of the boreal forest zone to corresponding forest sites in the southern borders of the boreal forest zone and studied the effects of warmer climate after an adaptation period of 2 years. The results showed that initially ground vegetation and soil microbial community structure and community functions were different in northern and southern forest sites and that 2 years of exposure to warmer climate was long enough to cause changes in these ecological indicators. The rate of SOM decomposition was approximately equally sensitive to temperature irrespective of changes in vegetation or microbial communities in the studied forest sites. However, as temperature sensitivity of the decomposition increases with decreasing temperature regime, the proportional increase in the decomposition rate in northern latitudes could lead to significant carbon losses from the soils.     

On the evolution of diet and landscape during the Upper Paleolithic through Mesolithic at Franchthi Cave (Peloponnese, Greece)

Franchthi Cave in southern Greece preserves one of the most remarkable records of socioeconomic change of the Late Pleistocene through early Holocene. Located on the southern end of the Argolid Peninsula, the area around the site was greatly affected by climate variation and marine transgression. This study examines the complex interplay of site formation processes (material deposition rates), climate-driven landscape change, and human hunting systems during the Upper Paleolithic through Mesolithic at Franchthi Cave based on the H1B faunal series. Building on earlier work, we establish the full spectrum of the meat diet using taphonomic evidence, and we analyze these data for trends in socioeconomic reorganization. Foraging patterns during the Aurignacian and “Gravettoid” occupations at Franchthi were terrestrial and already rather diversified in comparison to Middle Paleolithic diets in southern Greece. Hunting shifted abruptly to a mixed marine-terrestrial pattern during the Final Paleolithic, and fishing activities intensified though the Mesolithic. The zooarchaeological data indicate two consecutive trends of increasing dietary breadth, the first within an exclusively terrestrial context, and the second as marine habitats came into use through the end of the Mesolithic. The intensity of the human occupations at this site increased in tandem with intensified use of animal and plants. Comparison to the inland site of Klissoura Cave 1 indicates that the trend toward broader diets was regional as well as local.     

Analysis of expressed sequence tags (ESTs) and gene expression changes under different growth conditions for the ciliate Anophryoides haemophila, the causative agent of bumper car disease in the American lobster (Homarus americanus)

The scuticociliate Anophryoides haemophila, causes bumper car disease in American lobster (Homarus americanus) in commercial holding facilities in Atlantic Canada. While the parasite has been recognized since the 1970s and much has been learned about its biology, minimal molecular characterization exists. With genome consortiums turning to model organisms like the ciliates Tetrahymena and Paramecium, the amount of relevant sequence data available has made sequence surveys more attractive for gene discovery in related ciliates. We sequenced 9984 expressed sequence tags (ESTs) from a non-normalized A. haemophila cDNA library to characterize gene expression patterns, functional gene distribution and to discover novel genes related to the parasitic life history. The A. haemophila ESTs were grouped into 843 clusters and singletons with 658 EST clusters having identifiable homologs, while 159 ESTs were unique and had no similarity to any sequences in the public databases. Not unexpectedly, about 67% of the A. haemophila ESTs have similarity to annotated and hypothetical genes from the related oligohymenophorean ciliate, Tetrahymena. Numerous cysteine proteases, hypothetical proteins and novel sequences possess putative secretory signal peptides suggesting that they may contribute to the pathogenesis of bumper car disease in lobster. Real time RT-qPCR analysis of cathepsin L and two homologs of cathepsin B did not show any changes in gene expression under varying in vitro growth conditions or during a modified-in vivo infection which may be suggestive of the opportunistic life history strategy of this ciliate.     

Flavocytochrome P450 BM3 mutant W1046A is a NADH-dependent fatty acid hydroxylase: Implications for the mechanism of electron transfer in the P450 BM3 dimer

Bacillus megaterium P450 BM3 (BM3) is a P450/P450 reductase fusion enzyme, where the dimer is considered the active form in NADPH-dependent fatty acid hydroxylation. The BM3 W1046A mutant was generated, removing an aromatic “shield” from its FAD isoalloxazine ring. W1046A BM3 is a catalytically active NADH-dependent lauric acid hydroxylase, with product formation slightly superior to the NADPH-driven enzyme. The W1046A BM3 K_m for NADH is 20-fold lower than wild-type BM3, and catalytic efficiency of W1046A BM3 with NADH and NADPH are similar in lauric acid oxidation. Wild-type BM3 also catalyzes NADH-dependent lauric acid hydroxylation, but less efficiently than W1046A BM3. A hypothesis that W1046A BM3 is inactive [15] helped underpin a model of electron transfer from FAD in one BM3 monomer to FMN in the other in order to drive fatty acid hydroxylation in native BM3. Our data showing W1046A BM3 is a functional fatty acid hydroxylase are consistent instead with a BM3 catalytic model involving electron transfer within a reductase monomer, and from FMN of one monomer to heme of the other [12]. W1046A BM3 is an efficient NADH-utilizing fatty acid hydroxylase with potential biotechnological applications.     

Multiphoton Multispectral Fluorescence Lifetime Tomography for the Evaluation of Basal Cell Carcinomas

We present the first detailed study using multispectral multiphoton fluorescence lifetime imaging to differentiate basal cell carcinoma cells (BCCs) from normal keratinocytes. Images were acquired from 19 freshly excised BCCs and 27 samples of normal skin (in & ex vivo). Features from fluorescence lifetime images were used to discriminate BCCs with a sensitivity/specificity of 79%/93% respectively. A mosaic of BCC fluorescence lifetime images covering >1 mm² is also presented, demonstrating the potential for tumour margin delineation. Using 10,462 manually segmented cells from the image data, we quantify the cellular morphology and spectroscopic differences between BCCs and normal skin for the first time. Statistically significant increases were found in the fluorescence lifetimes of cells from BCCs in all spectral channels, ranging from 19.9% (425–515 nm spectral emission) to 39.8% (620–655 nm emission). A discriminant analysis based diagnostic algorithm allowed the fraction of cells classified as malignant to be calculated for each patient. This yielded a receiver operator characteristic area under the curve for the detection of BCC of 0.83. We have used both morphological and spectroscopic parameters to discriminate BCC from normal skin, and provide a comprehensive base for how this technique could be used for BCC assessment in clinical practice.     

Proteomic and physiological experiments to test Thermotoga neapolitana constraint-based model hypotheses of carbon source utilization

Constraint-based models of biochemical reaction networks require experimental validation to test model-derived hypotheses and iteratively improve the model. Physiological and proteomic analysis of Thermotoga neapolitana growth on cellotetraose was conducted to identify gene products related to growth on cellotetraose to improve a constraint-based model of T. neapolitana central carbon metabolism with incomplete cellotetraose pathways. In physiological experiments comparing cellotetraose to cellobiose and glucose as growth substrates, product formation yields on cellotetraose, cellobiose, and glucose were similar; however cell yields per mol carbon consumed were higher on cellotetraose than on cellobiose or glucose. Proteomic analysis showed increased expression of several proteins from cells grown on cellotetraose compared with glucose cell cultures, including cellobiose phosphorylase (CTN_0783), endo-1,4-β-glucosidase (CTN_1106), and an ATP-binding protein (CTN_1296). The CTN_1296 gene product should be evaluated further for participation in cellotetraose metabolism and is included as one of two hypothetical gene-protein-reaction associations in the T. neapolitana constraint-based model to reinstate cellotetraose metabolism in model simulations.     

Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis

Background

Membrane-bound organelles are a defining feature of eukaryotic cells, and play a central role in most of their fundamental processes. The Rab G proteins are the single largest family of proteins that participate in the traffic between organelles, with 66 Rabs encoded in the human genome. Rabs direct the organelle-specific recruitment of vesicle tethering factors, motor proteins, and regulators of membrane traffic. Each organelle or vesicle class is typically associated with one or more Rab, with the Rabs present in a particular cell reflecting that cell's complement of organelles and trafficking routes.

Results

Through iterative use of hidden Markov models and tree building, we classified Rabs across the eukaryotic kingdom to provide the most comprehensive view of Rab evolution obtained to date. A strikingly large repertoire of at least 20 Rabs appears to have been present in the last eukaryotic common ancestor (LECA), consistent with the 'complexity early' view of eukaryotic evolution. We were able to place these Rabs into six supergroups, giving a deep view into eukaryotic prehistory.

Conclusions

Tracing the fate of the LECA Rabs revealed extensive losses with many extant eukaryotes having fewer Rabs, and none having the full complement. We found that other Rabs have expanded and diversified, including a large expansion at the dawn of metazoans, which could be followed to provide an account of the evolutionary history of all human Rabs. Some Rab changes could be correlated with differences in cellular organization, and the relative lack of variation in other families of membrane-traffic proteins suggests that it is the changes in Rabs that primarily underlies the variation in organelles between species and cell types.     

Special issue: Cytochrome P450 structure and function: introduction

The 17th International Conference on Cytochrome P450 Biochemistry, Biophysics and Structure was held in Manchester, UK from 26-30 June 2011. This issue of FEBS J. contains review and primary research articles reflecting the breadth of science covered at this conference, and reflecting the impact of P450-related research in fields as diverse as steroid metabolism, plant biochemistry, structural biology and biotechnology.