Novel, highly expressed late nodulin gene (LjNOD16) from Lotus japonicus.

We have isolated a Lotus japonicus cDNA corresponding to a highly abundant, late nodule-specific RNA species that encodes a polypeptide with a predicted molecular mass of 15.6 kD. The protein and its corresponding gene were designated Nlj16 and LjNOD16, respectively. LjNOD16 was found to be expressed only in the infected cells of L. japonicus nodules. Related DNA sequences could be identified in the genomes of both Glycine max and Medicago sativa. In the latter, a homologous mRNA species was detected in the nodules. Unlike LjNOD16, its alfalfa homologs appear to represent low-abundance mRNA species. However, the proteins corresponding to the LjNOD16 and its alfalfa homolog could be detected at similar levels in nodules but not in roots of both legume species. The predicted amino acid sequence analysis of nodulin Nlj16 revealed the presence of a long alpha-helical region and a positively charged C terminus. The former domain has a very high propensity to form a coiled-coil type structure, indicating that nodulin Nlj16 may interact with an as-yet-unidentified protein target(s) in the nodule-infected cells. Homology searches revealed no significant similarities to any known sequences in the databases, with the exception of two related, anonymous Arabidopsis expressed sequence tags.     

Extensive Management Promotes Plant and Microbial Nitrogen Retention in Temperate Grassland

Leaching losses of nitrogen (N) from soil and atmospheric N deposition have led to widespread changes in plant community and microbial community composition, but our knowledge of the factors that determine ecosystem N retention is limited. A common feature of extensively managed, species-rich grasslands is that they have fungal-dominated microbial communities, which might reduce soil N losses and increase ecosystem N retention, which is pivotal for pollution mitigation and sustainable food production. However, the mechanisms that underpin improved N retention in extensively managed, species-rich grasslands are unclear. We combined a landscape-scale field study and glasshouse experiment to test how grassland management affects plant and soil N retention. Specifically, we hypothesised that extensively managed, species-rich grasslands of high conservation value would have lower N loss and greater N retention than intensively managed, species-poor grasslands, and that this would be due to a greater immobilisation of N by a more fungal-dominated microbial community. In the field study, we found that extensively managed, species-rich grasslands had lower N leaching losses. Soil inorganic N availability decreased with increasing abundance of fungi relative to bacteria, although the best predictor of soil N leaching was the C/N ratio of aboveground plant biomass. In the associated glasshouse experiment we found that retention of added ¹⁵N was greater in extensively than in intensively managed grasslands, which was attributed to a combination of greater root uptake and microbial immobilisation of ¹⁵N in the former, and that microbial immobilisation increased with increasing biomass and abundance of fungi. These findings show that grassland management affects mechanisms of N retention in soil through changes in root and microbial uptake of N. Moreover, they support the notion that microbial communities might be the key to improved N retention through tightening linkages between plants and microbes and reducing N availability.     

Binding Interactions of Keratin-Based Hair Fiber Extract to Gold,Keratin, and BMP-2

Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines) have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2) has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (K_D) of 1.8 × 10⁻⁴ M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals) were at work. The association of BMP-2 to kerateine was found to be greater (K_D = 1.1 × 10⁻⁷ M), within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS) shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (K_D = 3.2 × 10⁻⁵ M). BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks), suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5), below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the development of better, more clinically relevant BMP-2-conjugated systems for bone repair and regeneration.     

Enthoprotin: a novel clathrin-associated protein identified through subcellular proteomics

Despite numerous advances in the identification of the molecular machinery for clathrin-mediated budding at the plasma membrane, the mechanistic details of this process remain incomplete. Moreover, relatively little is known regarding the regulation of clathrin-mediated budding at other membrane systems. To address these issues, we have utilized the powerful new approach of subcellular proteomics to identify novel proteins present on highly enriched clathrin-coated vesicles (CCVs). Among the ten novel proteins identified is the rat homologue of a predicted gene product from human, mouse, and Drosophila genomics projects, which we named enthoprotin. Enthoprotin is highly enriched on CCVs isolated from rat brain and liver extracts. In cells, enthoprotin demonstrates a punctate staining pattern that is concentrated in a perinuclear compartment where it colocalizes with clathrin and the clathrin adaptor protein (AP)1. Enthoprotin interacts with the clathrin adaptors AP1 and with Golgi-localized, gamma-ear-containing, Arf-binding protein 2. Through its COOH-terminal domain, enthoprotin binds to the terminal domain of the clathrin heavy chain and stimulates clathrin assembly. These data suggest a role for enthoprotin in clathrin-mediated budding on internal membranes. Our study reveals the utility of proteomics in the identification of novel vesicle trafficking proteins.     

EPR, ENDOR, and TRIPLE resonance spectroscopy on the neutral flavin radical in Escherichia coli DNA photolyase

Ultraviolet radiation promotes the formation of a cyclobutane ring between adjacent pyrimidine residues on the same DNA strand to form a pyrimidine dimer. Such dimers may be restored to their monomeric forms through the action of a light-absorbing enzyme named DNA photolyase. The redox-active cofactor involved in the light-induced electron transfer reactions of DNA repair and enzyme photoactivation is a noncovalently bound FAD. In this paper, the FAD cofactor of Escherichia coli DNA photolyase was characterized as the neutral flavin semiquinone by EPR spectroscopy at 9.68 and 94.5 GHz. From the high-frequency/high-field EPR spectrum, the principal values of the axially symmetric g-matrix of FADH(*) were extracted. Both EPR spectra show an emerging hyperfine splitting of 0.85 mT that could be assigned to the isotropic hyperfine coupling constant (hfc) of the proton at N(5). To obtain more information about the electron spin density distribution ENDOR and TRIPLE resonance spectroscopies were applied. All major proton hfc's could be measured and unambiguously assigned to molecular positions at the isoalloxazin moiety of FAD. The isotropic hfc's of the protons at C(8alpha) and C(6) are among the smallest values reported for protein-bound neutral flavin semiquinones so far, suggesting a highly restricted delocalization of the unpaired electron spin on the isoalloxazin moiety. Two further hfc's have been detected and assigned to the inequivalent protons at C(1'). Some conclusions about the geometrical arrangement of the ribityl side chain with respect to the isoalloxazin ring could be drawn: Assuming tetrahedral angles at C(1') the dihedral angle between the C(1')-C(2') bond and the 2p(z)() orbital at N(10) has been estimated to be 170.4 degrees +/- 1 degrees.     

Characterization of a novel Obg-like ATPase in the protozoan Trypanosoma cruzi

We characterized a gene encoding an YchF-related protein, TcYchF, potentially associated with the protein translation machinery of Trypanosoma cruzi. YchF belongs to the translation factor-related (TRAFAC) class of P-loop NTPases. The coding region of the gene is 1185 bp long and encodes a 44.3 kDa protein. BlastX searches showed TcYchF to be very similar (45-86%) to putative GTP-binding proteins from eukaryotes, including some species of trypanosomatids (Leishmania major and Trypanosoma brucei). A lower but significant level of similarity (38-43%) was also found between the predicted sequences of TcYchF and bacterial YyaF/YchF GTPases of the Spo0B-associated GTP-binding protein (Obg) family. Some of the most important features of the G domain of this family of GTPases are conserved in TcYchF. However, we found that TcYchF preferentially hydrolyzed ATP rather than GTP. The function of YyaF/YchF is unknown, but other members of the Obg family are known to be associated with ribosomal subunits. Immunoblots of the polysome fraction from sucrose gradients showed that TcYchF was associated with ribosomal subunits and polysomes. Immunoprecipitation assays showed that TcYchF was also associated with the proteasome of T. cruzi. Furthermore, inactivation of the T. brucei homolog of TcYchF by RNA interference inhibited the growth of procyclic forms of the parasite. These data suggest that this protein plays an important role in the translation machinery of trypanosomes.     

Automated system for gene annotation and metabolic pathway reconstruction using general sequence databases

Despite the growing number of genomes published or currently being sequenced, there is a relative paucity of software for functional classification of newly discovered genes and their assignment to metabolic pathways. Available software for such analyses has a very steep learning curve and requires the installation, configuration, and maintenance of large amounts of complex infrastructure, including complementary software and databases. Many such tools are restricted to one or a few data sources and classification schemes. In this work, we report an automated system for gene annotation and metabolic pathway reconstruction (ASGARD), which was designed to be powerful and generalizable, yet simple for the biologist to install and run on centralized, commonly available computers. It avoids the requirement for complex resources such as relational databases and web servers, as well as the need for administrator access to the operating system. Our methodology contributes to a more rapid investigation of the potential biochemical capabilities of genes and genomes by the biological researcher, and is useful in biochemical as well as comparative and evolutionary studies of pathways and networks.     

A clinical return-to-work rule for patients with back pain

Background

Tools for early identification of workers with back pain who are at high risk of adverse occupational outcome would help concentrate clinical attention on the patients who need it most, while helping reduce unnecessary interventions (and costs) among the others. This study was conducted to develop and validate clinical rules to predict the 2-year work disability status of people consulting for nonspecific back pain in primary care settings.

Methods

This was a 2-year prospective cohort study conducted in 7 primary care settings in the Quebec City area. The study enrolled 1007 workers (participation, 68.4% of potential participants expected to be eligible) aged 18–64 years who consulted for nonspecific back pain associated with at least 1 day''s absence from work. The majority (86%) completed 5 telephone interviews documenting a large array of variables. Clinical information was abstracted from the medical files. The outcome measure was “return to work in good health” at 2 years, a variable that combined patients'' occupational status, functional limitations and recurrences of work absence. Predictive models of 2-year outcome were developed with a recursive partitioning approach on a 40% random sample of our study subjects, then validated on the rest.

Results

The best predictive model included 7 baseline variables (patient''s recovery expectations, radiating pain, previous back surgery, pain intensity, frequent change of position because of back pain, irritability and bad temper, and difficulty sleeping) and was particularly efficient at identifying patients with no adverse occupational outcome (negative predictive value 78%– 94%).

Interpretation

A clinical prediction rule accurately identified a large proportion of workers with back pain consulting in a primary care setting who were at a low risk of an adverse occupational outcome.Since the 1950s, back pain has taken on the proportions of a veritable epidemic, counting now among the 5 most frequent reasons for visits to physicians'' offices in North America^1,2,3 and ranking sixth among health problems generating the highest direct medical costs.⁴ Because of its high incidence and associated expense, effective intervention for back pain has great potential for improving population health and for freeing up extensive societal resources.So-called red flags to identify pain that is specific (i.e., pain in the back originating from tumours, fractures, infections, cauda equina syndrome, visceral pain and systemic disease)⁵ account for about 3% of all cases of back pain.⁶ The overwhelming majority of back-pain problems are thus nonspecific. One important feature of nonspecific back pain among workers is that a small proportion of cases (< 10%) accounts for most of the costs (> 70%).^{7,8,9,10,11,12,13,14} This fact has led investigators to focus on the early identification of patients who are at higher risk of disability, so that specialized interventions can be provided earlier, whereas other patients can be expected to recover with conservative care.^{9,15,16,17,18,19,20,21,22,23,24,25} Although this goal has become much sought-after in back-pain research, most available studies in this area have 3 methodological problems:

• Potential predictors are often limited to administrative or clinical data, whereas it is clear that back pain is a multidimensional health problem.
• The outcome variable is most often a 1-point dichotomous measure of return to work, time off work or duration of compensation, although some authors have warned against the use of first return to work as a measure of recovery. Baldwin and colleagues,²⁶ for instance, point out that first return to work is frequently followed by recurrences of work absence.
• Most published prediction rules developed for back pain have not been successfully validated on any additional samples of patients.

Our study aimed to build a simple predictive tool that could be used by primary care physicians to identify workers with nonspecific back pain who are at higher risk of long-term adverse occupational outcomes, and then to validate this tool on a fresh sample of subjects.     

The role of landscape change and paleoclimatic events in shaping the evolutionary history of the Polioptila gnatcatchers (Passeriformes,Polioptilidae) with emphasis on species associated with open habitats

We conducted a large‐scale phylogenetic and biogeographical inference of the Poliptila gnatcatchers and investigated the evolutionary history of two closely related neotropical bird species linked to open habitats, Polioptila dumicola and Polioptila plumbea. A Bayesian inference was employed based on the NADH subunit 2 gene to reconstruct the phylogenetic relationship of the gnatcatchers, and ancestral area reconstructions were estimated using BioGeoBEARS. For the phylogeographic analysis, we analyzed two mitochondrial genes, cytochrome b and ND2, of 102 individuals from P. dumicola and P. plumbea distributed throughout the complete range of both species. To reconstruct the dates related to the splitting events, we included a subset of sequences from the nuclear gene beta‐fibrinogen intron‐7. A striking result was the recovery of the sister relationship between the lineages of P. dumicola /plumbea and the paraphyly among the subspecies of P. plumbea: the first group was formed by P. dumicola, P. p. plumbea, P. p. parvirostris, P. p. atricapilla and P. lactea, occurring mainly on the Brazilian shield; while the second group consisted of lineages from north of the Amazon, west of the Andes, and Central America, and included P. maior, P. p. cinericia, P. p. bilineata and P. p. innotata. Significant phylogeographic structure was evident within lineages attributed to P. plumbea, with high levels of differentiation in the well‐defined clades according to all phylogenetic analyses. Our biogeographic analyses support distinct evolutionary histories related to founder events and vicariance, occurring during the late Pliocene and early Pleistocene. Several dispersal episodes between North/Central America and South America led to the establishment of populations which became differentiated due to landscape changes, such as the establishment of riverine barriers, the uplift of the Andes and the formation of the Panama Isthmus.