A novel strategy to over-express and purify homologous proteins from Streptococcus pneumoniae

Functional studies of Streptococcus pneumoniae virulence factors are facilitated by the development of complementation/mutagenesis systems. These methods usually result in poor expression yields; therefore, biochemical and structural/functional characterizations are mostly performed with proteins expressed and purified from heterologous systems (e.g. Escherichia coli). However, heterologous expression does not guarantee correct protein structure and function. In this work, we developed a method to over-express and purify homologous proteins from S. pneumoniae. The system relies on the combined use of the shuttle plasmid pMU1328 and a natural constitutive pneumococcal promoter, P₉₆. Efficient over-expression of secreted, membrane or surface anchored proteins, either wild type or mutant, was achieved. As proof of principle the S. pneumoniae pilus-1 backbone RrgB was successfully purified as a His-tag secreted protein (RrgB-His_SP) from pneumococcal culture supernatants. N-terminal sequencing and mass spectrometry analysis of RrgB-His_SP allowed the determination of the leader sequence cleavage site in pneumococcus, while proteolysis studies confirmed the stability of RrgB-His_SP to trypsin digestion. The data presented here support the use of this novel homologous expression method for all S. pneumoniae proteins for which extensive characterization studies are planned. Moreover, given the promiscuity of the pMU1328 replicon, this system could be used in diverse bacterial species.     

Low sclerostin levels: a predictive marker of persistent inflammation in ankylosing spondylitis during anti-tumor necrosis factor therapy?

Introduction

Sclerostin levels have been reported to be low in ankylosing spondylitis (AS), but there is no data regarding the possible role of this Wnt inhibitor during anti-tumor necrosis factor (TNF) therapy. The present study longitudinally evaluated sclerostin levels, inflammatory markers and bone mineral density (BMD) in AS patients under anti-TNF therapy.

Methods

Thirty active AS patients were assessed at baseline, 6 and 12 months after anti-TNF therapy regarding clinical parameters, inflammatory markers, BMD and baseline radiographic damage (mSASSS). Thirty age- and sex-matched healthy individuals comprised the control group. Patients'' sclerostin levels, sclerostin binding low-density lipoprotein receptor-related protein 6 (LRP6) and BMD were evaluated at the same time points and compared to controls.

Results

At baseline, AS patients had lower sclerostin levels (60.5 ± 32.7 vs. 96.7 ± 52.9 pmol/L, P = 0.002) and comparable sclerostin binding to LRP6 (P = 0.387) than controls. Improvement of Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Metrology Index (BASMI), Ankylosing Spondylitis quality of life (ASQoL) was observed at baseline vs. 6 vs. 12 months (P < 0.01). Concomitantly, a gradual increase in spine BMD (P < 0.001) and a positive correlation between baseline mSASSS and spine BMD was found (r = 0.468, P < 0.01). Inflammatory parameters reduction was observed comparing baseline vs. 6 vs. 12 months (P <0.01). Sclerostin levels progressively increased [baseline (60.5 ± 32.7) vs. 6 months (67.1 ± 31.9) vs. 12 months (72.7 ± 32.3) pmol/L, P <0.001]. At 12 months, the sclerostin levels remained significantly lower in patients compared to controls (72.7 ± 32.3 vs. 96.70 ± 52.85 pmol/L, P = 0.038). Moreover, sclerostin serum levels at 12 months were lower in the 10 patients with high C reactive protein (CRP) (≥ 5 mg/l) compared to the other 20 patients with normal CRP (P = 0.004). Of note, these 10 patients with persistent inflammation also had lower sclerostin serum levels at baseline compared to the other patients (P = 0.023). Univariate logistic regression analysis demonstrated that AS patients with lower sclerostin serum levels had an increased risk to have high CRP at 12 months (odds ratio = 7.43, 95% CI 1.23 to 45.01, P = 0.020) than those with higher sclerostin values.

Conclusions

Persistent low sclerostin levels may underlie continuous inflammation in AS patients under anti-TNF therapy.     

Naturally Occurring Carboxypeptidase A6 Mutations: EFFECT ON ENZYME FUNCTION AND ASSOCIATION WITH EPILEPSY*

Carboxypeptidase A6 (CPA6) is a member of the A/B subfamily of M14 metallocarboxypeptidases that is expressed in brain and many other tissues during development. Recently, two mutations in human CPA6 were associated with febrile seizures and/or temporal lobe epilepsy. In this study we screened for additional CPA6 mutations in patients with febrile seizures and focal epilepsy, which encompasses the temporal lobe epilepsy subtype. Mutations found from this analysis as well as CPA6 mutations reported in databases of single nucleotide polymorphisms were further screened by analysis of the modeled proCPA6 protein structure and the functional role of the mutated amino acid. The point mutations predicted to affect activity and/or protein folding were tested by expression of the mutant in HEK293 cells and analysis of the resulting CPA6 protein. Common polymorphisms in CPA6 were also included in this analysis. Several mutations resulted in reduced enzyme activity or CPA6 protein levels in the extracellular matrix. The mutants with reduced extracellular CPA6 protein levels showed normal levels of 50-kDa proCPA6 in the cell, and this could be converted into 37-kDa CPA6 by trypsin, suggesting that protein folding was not greatly affected by the mutations. Interestingly, three of the mutations that reduced extracellular CPA6 protein levels were found in patients with epilepsy. Taken together, these results provide further evidence for the involvement of CPA6 mutations in human epilepsy and reveal additional rare mutations that inactivate CPA6 and could, therefore, also be associated with epileptic phenotypes.     

Robotics-based synthesis of human motion

The synthesis of human motion is a complex procedure that involves accurate reconstruction of movement sequences, modeling of musculoskeletal kinematics, dynamics and actuation, and characterization of reliable performance criteria. Many of these processes have much in common with the problems found in robotics research. Task-based methods used in robotics may be leveraged to provide novel musculoskeletal modeling methods and physiologically accurate performance predictions. In this paper, we present (i) a new method for the real-time reconstruction of human motion trajectories using direct marker tracking, (ii) a task-driven muscular effort minimization criterion and (iii) new human performance metrics for dynamic characterization of athletic skills. Dynamic motion reconstruction is achieved through the control of a simulated human model to follow the captured marker trajectories in real-time. The operational space control and real-time simulation provide human dynamics at any configuration of the performance. A new criteria of muscular effort minimization has been introduced to analyze human static postures. Extensive motion capture experiments were conducted to validate the new minimization criterion. Finally, new human performance metrics were introduced to study in details an athletic skill. These metrics include the effort expenditure and the feasible set of operational space accelerations during the performance of the skill. The dynamic characterization takes into account skeletal kinematics as well as muscle routing kinematics and force generating capacities. The developments draw upon an advanced musculoskeletal modeling platform and a task-oriented framework for the effective integration of biomechanics and robotics methods.     

Analysis of non-TIR NBS-LRR resistance gene analogs in <Emphasis Type="Italic">Musa acuminata</Emphasis> Colla: Isolation,RFLP marker development,and physical mapping

Background  

Many commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes). The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence.     

Candidate SNP of CACNA2D1 Gene Associated with Clinical Mastitis and Production Traits in Sahiwal (Bos taurus indicus) and Karan Fries (Bos taurus taurus × Bos taurus indicus)

The present study was conducted to identify polymorphisms in CACNA2D1 gene and their association with clinical mastitis and production traits. Exon 18 and its flanking regions were screened for the presence of SNPs. Statistical analysis was performed to identify association of period of birth, breed, and genotype with mastitis incidence on randomly selected 103 Sahiwal and 102 Karan Fries cattle. PCR-RFLP analysis revealed that g.38819398G?>?A mutation in exon 18 (269?bp amplicon) of CACNA2D1 gene resolved into AA, AG, and GG genotypes in Sahiwal and Karan Fries cattle. Wald chi-square analysis revealed that the period of birth, breed, and genotype were significantly associated with mastitis incidence. GG genotyped cattle were found to be less susceptible to mastitis. Least square analysis revealed that GG and AG genotype animals of G38819398A SNP of CACNA2D1 gene in Sahiwal as well as in Karan Fries cattle were associated with higher average milk yields during 1st, 2nd, and 3rd lactations (P?<?0.01). These observations and their differential association with the incidence of mastitis and production traits can be utilized as an aid to selection for simultaneous improvement of both antagonistic traits; however, validation of results on large number of animals is warranted.     

dabPNA: design, synthesis, and DNA binding studies

In continuing our research efforts for developing new oligodeoxynucleotide (ODN)-like drugs and diagnostics, we designed diaminobutyric peptide nucleic acids (dabPNAs), nucleopeptides characterized by a diaminobutyric-based building block that is an isomer of the aminoethylglycyl PNA (aegPNA) unit and the acyclic modification of the aminoprolyl PNA (ampPNA) monomer. In this work we present the solid phase synthesis of a dabPNA oligomer and of two aegPNAs containing a single dabPNA unit. A study relative to their binding ability towards DNA is also reported even in comparison with the well known aegPNAs.