Characterization of the 31-kDa antigen gene of Haemophilus somnus and its product

Consistent immunoreactivities of the 31-, 40- and 78-kDa proteins of Haemophilus somnus were observed in immunoblots with bovine antisera to H. somnus. A genomic library of H. somnus 8025 DNA was constructed in plasmid pUC19 and 45 recombinants expressed immunoreactive proteins in Western blots using bovine antiserum. Ten of the recombinants expressing a 31-kDa protein caused lysis of bovine erythrocytes. Restriction endonuclease mapping indicated that the hemolytic recombinants shared an approximately 1.7-kb BglII fragment. Southern blot analysis using the BglII fragment as a probe revealed homology among the recombinants and the presence of an identically sized BglII fragment in the chromosome of all H. somnus isolates tested. Sequence analysis indicated the presence of an 822-bp open reading frame within the 1.7-kb BglII fragment. Deletion of this open reading frame resulted in the loss of hemolytic activity and protein expression in recombinant Escherichia coli, suggesting the possible role of the 31-kDa protein as a hemolysin. An amino acid sequence deduced from the DNA sequence shared homology with outer membrane protein A of E. coli, Salmonella typhimurium and Shigella dysenteriae, with P6 of Haemophilus influenzae and with PIII of Neisseria gonorrhoeae;To determine the protective ability of the recombinant expressing the 31-kDa protein of H. somnus, mice were vaccinated 2-3 times with the heat-killed recombinant, followed by a challenge with H. somnus. Vaccination with the recombinant expressing the 31-kDa antigen of H. somnus provided 5-fold greater protection compared to E. coli with the vector only. Potential diagnostic use of monoclonal and polyclonal antibodies raised against the recombinant was evaluated. Monoclonal mouse antibodies against a crude membrane protein preparation or formalin-killed recombinant were evaluated on ELISA and Western blot against H. somnus antigens. Polyclonal Guinea pig sera against different preparations of the recombinant showed different immunoreactivities on various immunological tests. Polyclonal Guinea pig sera against the heat-killed recombinant and formalin-killed H. somnus strongly detected H. somnus in immunohistochemical staining of formalin-fixed tissue sections of bovine lungs

A Prospective Randomized Clinical Trial Comparing Bone Union Rate Following Anterior Cervical Discectomy and Fusion Using a Polyetheretherketone Cage: Hydroxyapatite/B-Tricalcium Phosphate Mixture versus Hydroxyapatite/Demineralized Bone Matrix Mixture

Study DesignProspective randomized noninferiority trial.PurposeTo evaluate whether the union rate of anterior cervical discectomy and fusion (ACDF) using a polyetheretherketone (PEEK) cage filled with a mixture of hydroxyapatite (HA) and demineralized bone matrix (DBM) is inferior to that of a mixture of β-tricalcium phosphate (β-TCP) and HA.Overview of LiteratureThere have been no clinical trials investigating the outcomes of a mixture of HA and DBM in a PEEK cage in ACDF.MethodsEighty-five eligible patients were randomly assigned to group B (n=43), in which a PEEK cage with a mixture of HA and DBM was used, or group C (n=42), in which a PEEK cage with a mixture of HA and β-TCP was used. The primary study endpoint was the fusion rate, which was assessed with dynamic radiographs and computed tomography (CT) scans. Secondary endpoints included pain intensity using a visual analogue scale, functional outcome using a neck disability index score, laboratory tests of inflammatory profiles, and the infection rate.ResultsSeventy-seven patients (38 in group B and 39 in group C) were included in the final analysis. One year postoperatively, bone fusion was achieved in 87% of group B patients and 87% of group C patients on dynamic radiographs, and 87% of group B patients and 72% of group C patients on CT scans (p=1.00 and 0.16, respectively). There were also no between-groups differences with respect to the secondary endpoints.ConclusionsA HA/DBM mixture inside a PEEK cage can provide noninferior outcomes compared to a HA/TCP mixture in ACDF

Colon cancer-derived oncogenic EGFR G724S mutant identified by whole genome sequence analysis is dependent on asymmetric dimerization and sensitive to cetuximab

Background: Inhibition of the activated epidermal growth factor receptor (EGFR) with either enzymatic kinase inhibitors or anti-EGFR antibodies such as cetuximab, is an effective modality of treatment for multiple human cancers. Enzymatic EGFR inhibitors are effective for lung adenocarcinomas with somatic kinase domain EGFR mutations while, paradoxically, anti-EGFR antibodies are more effective in colon and head and neck cancers where EGFR mutations occur less frequently. In colorectal cancer, anti-EGFR antibodies are routinely used as second-line therapy of KRAS wild-type tumors. However, detailed mechanisms and genomic predictors for pharmacological response to these antibodies in colon cancer remain unclear. Findings: We describe a case of colorectal adenocarcinoma, which was found to harbor a kinase domain mutation, G724S, in EGFR through whole genome sequencing. We show that G724S mutant EGFR is oncogenic and that it differs from classic lung cancer derived EGFR mutants in that it is cetuximab responsive in vitro, yet relatively insensitive to small molecule kinase inhibitors. Through biochemical and cellular pharmacologic studies, we have determined that cells harboring the colon cancer-derived G719S and G724S mutants are responsive to cetuximab therapy in vitro and found that the requirement for asymmetric dimerization of these mutant EGFR to promote cellular transformation may explain their greater inhibition by cetuximab than small-molecule kinase inhibitors. Conclusion: The colon-cancer derived G719S and G724S mutants are oncogenic and sensitive in vitro to cetuximab. These data suggest that patients with these mutations may benefit from the use of anti-EGFR antibodies as part of the first-line therapy

Characterization of the 31-kDa antigen gene of Haemophilus somnus and its product

Consistent immunoreactivities of the 31-, 40- and 78-kDa proteins of Haemophilus somnus were observed in immunoblots with bovine antisera to H. somnus. A genomic library of H. somnus 8025 DNA was constructed in plasmid pUC19 and 45 recombinants expressed immunoreactive proteins in Western blots using bovine antiserum. Ten of the recombinants expressing a 31-kDa protein caused lysis of bovine erythrocytes. Restriction endonuclease mapping indicated that the hemolytic recombinants shared an approximately 1.7-kb BglII fragment. Southern blot analysis using the BglII fragment as a probe revealed homology among the recombinants and the presence of an identically sized BglII fragment in the chromosome of all H. somnus isolates tested. Sequence analysis indicated the presence of an 822-bp open reading frame within the 1.7-kb BglII fragment. Deletion of this open reading frame resulted in the loss of hemolytic activity and protein expression in recombinant Escherichia coli, suggesting the possible role of the 31-kDa protein as a hemolysin. An amino acid sequence deduced from the DNA sequence shared homology with outer membrane protein A of E. coli, Salmonella typhimurium and Shigella dysenteriae, with P6 of Haemophilus influenzae and with PIII of Neisseria gonorrhoeae;To determine the protective ability of the recombinant expressing the 31-kDa protein of H. somnus, mice were vaccinated 2-3 times with the heat-killed recombinant, followed by a challenge with H. somnus. Vaccination with the recombinant expressing the 31-kDa antigen of H. somnus provided 5-fold greater protection compared to E. coli with the vector only. Potential diagnostic use of monoclonal and polyclonal antibodies raised against the recombinant was evaluated. Monoclonal mouse antibodies against a crude membrane protein preparation or formalin-killed recombinant were evaluated on ELISA and Western blot against H. somnus antigens. Polyclonal Guinea pig sera against different preparations of the recombinant showed different immunoreactivities on various immunological tests. Polyclonal Guinea pig sera against the heat-killed recombinant and formalin-killed H. somnus strongly detected H. somnus in immunohistochemical staining of formalin-fixed tissue sections of bovine lungs.</p

A SIMPLE METHOD TO CALCULATE THE DISPLACEMENT DAMAGE CROSS SECTION OF SILICON CARBIDE

We developed a simple method to prepare the displacement damage cross section of SiC using NJOY and SRIM/TRIM. The number of displacements per atom (DPA) dependent on primary knock-on atom (PKA) energy was computed using SRIM/TRIM and it is directly used by NJOY/HEATR to compute the neutron energy dependent DPA cross sections which are required to estimate the accumulated DPA of nuclear material. SiC DPA cross section is published as a table in DeCART 47 energy group structure. Proposed methodology can be easily extended to other materials

Epigenetic Modification of CFTR in Head and Neck Cancer

Cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP (cAMP)-regulated chloride channel, is critical for secretion and absorption across diverse epithelia. Mutations or absence of CFTR result in pathogeneses, including cancer. While CFTR has been proposed as a tumor suppressing gene in tumors of the intestine, lung, and breast cancers, its effects in head and neck cancer (HNC) have yet to be investigated. This study aimed to define expression patterns and epigenetic modifications of CFTR in HNC. CFTR was expressed in normal but not in HNC cells and tissues. Treatment with 5-aza-2&prime;-deoxycytidine (5-Aza-CdR) was associated with rescued expression of CFTR, whose function was confirmed by patch clamp technique. Further experiments demonstrated that CFTR CpG islands were hypermethylated in cancer cells and tissues and hypomethylated in normal cells and tissue. Our results suggest that CFTR epigenetic modifications are critical in both down-regulation and up-regulation of CFTR expression in HNC and normal cells respectively. We then investigated the impact of CFTR on expressions and functions of cancer-related genes. CFTR silencing was closely associated with changes to other cancer-related genes, suppressing apoptosis while enhancing proliferation, cell motility, and invasion in HNC. Our findings demonstrate that hypermethylation of CFTR CpG islands and CFTR deficiency is closely related to HNC