Outcome of surgery for chronic suppurative otitis media with resistant Pseudomonas aeruginosa.

OBJECTIVE: To assess the effectiveness of surgical treatment of active chronic suppurative otitis media (CSOM) with ciprofloxacin-resistant Pseudomonas aeruginosa (P. aeruginosa) that failed comprehensive local and systemic treatment. STUDY DESIGN: Retrospective case review. SETTING: Tertiary referral center. PATIENTS: Eleven patients with ciprofloxacin-resistant P. aeruginosa CSOM that remained active despite comprehensive local and systemic treatment. All patients were operated by a single surgeon between February 2016 and July 2019 INTERVENTION(S): Tympanoplasty alone was performed in seven cases and accompanied by mastoidectomy in the other four cases. MAIN OUTCOME MEASURE(S): Resolution of infection and tympanic graft take on otoscopy. The secondary outcome measure is hearing. RESULTS: Tympanic graft take was successful and the infectious process was resolved in 8 out of the 11 cases, yielding a success rate of 73%. The average follow-up was 20 months. No surgical complications occurred. CONCLUSIONS: Tympanoplasty, with or without mastoidectomy, is safe and yields acceptable anatomical and functional success rates when intensive local and systemic treatment fails to stop the purulent discharge.

[Dentinal hypersensitivity].

Dentinal hypersensitivity is defined as short and transient painful response of exposed dentin, usually cervical, to different stimuli, such as thermal, mechanical osmotic or chemical. The etiology of dentinal hypersensitivity is open tubules (because of enamel loss or gingival recession), allowing painful stimulus to reach the pulp. The hydrodynamic theory explains the mechanism through which pain is aroused. When treating dentinal hypersensitivity, dentists always have to rule out other pathologies, such as carries, leakage, postoperative sensitivity, cracked tooth etc., and only then assess pain intensity and treat the tooth. Treatment always starts with prevention of both stimulus and exposure of dentin, and reducing predisposing factors. The treatment options include OTC products, such as fluoride and/or potassium enriched mouth washes and dentifrices, or in-office treatments, such as high content fluoride varnishes and gels, potassium oxalate chelating agents, Glutaraldehyde containing tissue fixating agents, bonding materials, low viscosity glass ionomers and even non-conservative treatments such as root canal therapy or mucogingival surgical interventions.

A retrospective analysis of temporomandibular findings among Israeli-born patients based on the RDC/TMD.

The purpose of this study was to evaluate temporomandibular disorders (TMD) Axis I and II among Israeli-Jewish patients using the Hebrew version of the Research Diagnostic Criteria (RDC) for TMD and to compare the results with Swedish, United States, Asian and Israeli-Arab populations. The study consisted of 298 Israeli-born, Jewish patients (male/female ratio 3.5:1), arriving at an Orofacial Pain Clinic during the year 2001-2004. A complete clinical examination was carried out according to the RDC/TMD protocol. Axis I diagnoses: 65% of the Israeli-Jewish patients exhibited myofacial pain (Group I disorder), 38% disc displacement (Group II disorder) and 18% arthralgia, osteoarthritis or osteoarthrosis (Group III disorder). Axis II diagnoses: 20% of the patients scored severe depression and 35% scored somatization. Pain was reported in 82% of the patients (mean pain duration 35.7-33.8 months for women, 44.1 for men). Patients had an average disability score of 30.0 +/- 30.2. Chronic pain grade IV was present in 4% of the patients. Israeli-Jewish temporomandibular disorder patients showed results similar to those reported for other countries, further supporting the use of the RDC/TMD internationally as a reliable epidemiological tool. Globally, Axis I scores were similar, while Axis II scores were more susceptible to geographic/ethnic differences. Gender can influence Axis I and Axis II as well as possible gender specific association with socio-economic status. In future comparisons, men and women should be considered separately.

Expression, purification and applications of staphylococcal protein A fused to cellulose-binding domain.

Because staphylococcal Protein A (ProtA) binds specifically to IgG, it has been used for many immunological manipulations, most notably antibody purification and diagnostics. Immobilization is required for most of these applications. Here we describe a genetic-engineering approach to immobilizing ProtA on cellulose, by fusing it to cellulose-binding domain (CBD) derived from the cellulose-binding Protein A of Clostridium cellulovorans. The bifunctional fusion protein was expressed in Escherichia coli, recovered on a cellulose column and purified by elution at alkaline pH. ProtA-CBD was used to purify IgG from rabbit serum and its ability to bind IgG from different sources was determined. The bifunctional chimaeric protein can bind up to 23.4 mg/ml human IgG at a ratio of 1 mol of ProtA-CBD/2 mol of human IgG, and can purify up to 11.6 mg/ml rabbit IgG from a serum. The ability to bind functionally active CBD-affinity reagents to cellulosic microtitre plates was demonstrated. Our results indicate that a combination of CBD-affinity reagents and cellulosic microtitre plates is an attractive diagnostics matrix for the following reasons: (i) cellulose exhibits very low non-specific binding; and (ii) CBD-fusion proteins bind directly to cellulose at high density. A unique signal-amplification method was developed based on the ability of ProtA-CBD to link stained cellulose particles to primary antibody in a Western blot.

Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger beta-glucosidase.

The beta-glucosidase from Aspergillus niger (CMI CC 324262) was purified, and an N-terminal sequence and two internal sequences were determined. BglI genomic gene and the cDNA were cloned from a genomic library and by reverse transcriptase-polymerase chain reaction, respectively. The cDNA was successfully expressed in Saccharomyces cerevisiae and Pichia pastoris. Sequence analysis revealed that the gene encodes a 92-kDa enzyme that is a member of glycosidase family 3. (1)H-NMR analysis of the reaction catalyzed by this enzyme confirmed that, in common with other family 3 glycosidases, this enzyme hydrolyzes with net retention of anomeric configuration. Accordingly, the enzyme was inactivated by 2-deoxy-2-fluoro beta-glucosyl fluoride, with kinetic parameters of k(i) = 4.5 min(-1), K(I) = 35.4 mM, through the trapping of a covalent glycosyl enzyme intermediate. The catalytic competence of this intermediate was demonstrated by the fact that incubation with linamarin resulted in reactivation, presumably via a transglycosylation mechanism. Peptic digestion of the 2-deoxy-2-fluoroglucosyl enzyme and subsequent analysis of high pressure liquid chromatography eluates by electrospray ionization triple quadrupole mass spectrometry in the neutral loss mode allowed the localization of a 2-deoxy-2-fluoroglucosyl-peptide. Sequence determination of this labeled peptide by tandem mass spectrometry in the daughter ion scan mode permitted the identification of Asp-261 as the catalytic nucleophile within the sequence VMSDW. Asp-261 is fully conserved within this family, consistent with its key role, and aligns with the aspartic acid residue previously identified in the Aspergillus wentii enzyme by labeling with conduritol B epoxide (Bause, E., and Legler, G. (1974) Hoppe-Seyler's Z. Physiol. Chem. 355, 438-442).

Immobilization of recombinant heparinase I fused to cellulose-binding domain.

Immobilization of biologically active proteins is of great importance to research and industry. Cellulose is an attractive matrix and cellulose-binding domain (CBD) an excellent affinity tag protein for the purification and immobilization of many of these proteins. We constructed two vectors to enable the cloning and expression of proteins fused to the N- or C-terminus of CBD. Their usefulness was demonstrated by fusing the heparin-degrading protein heparinase I to CBD (CBD-HepI and HepI-CBD). The fusion proteins were over-expressed in Escherichia coli under the control of a T7 promoter and found to accumulate in inclusion bodies. The inclusion bodies were recovered by centrifugation, the proteins were refolded and recovered on a cellulose column. The bifunctional fusion protein retained its abilities to bind to cellulose and degrade heparin. C-terminal fusion of heparinase I to CBD was somewhat superior to N-terminal fusion: Although specific activities in solution were comparable, the latter exhibited impaired binding capacity to cellulose. CBD-HepI-cellulose bioreactor was operated continuously and degraded heparin for over 40 h without any significant loss of activity. By varying the flow rate, the mean molecular weight of the heparin oligosaccharide produced could be controlled. The molecular weight distribution profiles, obtained from heparin depolymerization by free heparinase I, free CBD-HepI, and cellulose-immobilized CBD-HepI, were compared. The profiles obtained by free heparinase I and CBD-HepI were indistinguishable, however, immobilized CBD-HepI produced much lower molecular weight fragments at the same percentage of depolymerization. Thus, CBD can be used for the efficient production of bioreactors, combining purification and immobilization into essentially a single step.

A-protein from achromogenic atypical Aeromonas salmonicida: molecular cloning, expression, purification, and characterization.

Achromogenic atypical Aeromonas salmonicida is the causative agent of goldfish ulcer disease. Virulence of this bacterium is associated with the production of a paracrystalline outer membrane A-layer protein. The species-specific structural gene for the monomeric form of A-protein was cloned into a pET-3d plasmid in order to express and produce a recombinant form of the protein in Escherichia coli BL21(DE3). The induced protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by ion exchange chromatography on Q-Sepharose to over 95% pure monomeric protein. Recombinant A-protein was compared by biochemical, immunological, and molecular methods with the A-protein isolated from atypical A. salmonicida bacterial cells by the glycine and the membrane extraction methods. The recombinant form was found to be undistinguishable from the wild type when examined by SDS-PAGE and gel filtration chromatography. The immunological similarity of the protein samples was demonstrated by employing polyclonal and monoclonal antibodies in ELISA and Western blot techniques. All forms of A-protein were found to activate the secretion of tumor necrosis factor alpha from murine macrophage. To date, this represents the first large-scale production of biologically active recombinant A-protein.

A cellulose-binding domain-fused recombinant human T cell connective tissue-activating peptide-III manifests heparanase activity.

The chemokine connective tissue-activating peptide (CTAP)-III, which belongs to the leukocyte-derived growth factor family of mediators, was previously shown to be mitogenic for fibroblasts. However, it has recently been shown that CTAP-III, released from platelets, can act like a heparanase enzyme and degrade heparan sulfate. This suggests that CTAP-III may also function as a proinflammatory mediator. We have successfully cloned CTAP-III from a lambdagt11 cDNA library of PHA-activated human CD4(+) T cells and produced recombinant CTAP-III as a fusion protein with a cellulose-binding domain moiety. This recombinant CTAP-III exhibited heparanase activity and released degradation products from metabolically labeled, naturally produced extracellular matrix. We have also developed polyclonal and monoclonal antibodies, and these antibodies against the recombinant CTAP-III detected the CTAP-III molecule in human T cells, polymorphonuclear leukocytes, and placental extracts. Thus, our study provides tools to examine further immune cell behavior in inflamed sites rich with extracellular moieties and proinflammatory mediators.

Cloning and characterization of elongation specific endo-1,4-beta-glucanase (cel1) from Arabidopsis thaliana.

The isolation of an elongation-specific endo-1,4-beta-glucanase-cel1 from Arabidopsis thaliana was made possible by the fact that considerable homology exists between different endo-1,4-beta-glucanase (EGase) genes from different plants. Degenerate primers were synthesized based on two conserved regions from the avocado and tomato cellulase amino acid sequences. The A.thaliana cel1 cDNA gene was found to encode a 54kDa protein; sequence comparison with the avocado EGase revealed 56% identity. Northern blot analysis of cel1 suggested its developmental regulation. RNA transcripts were undetectable in fully expanded leaves as well as at the basal internode of flowering stems. However, a strong transcript signal was detected in the elongating zone of flowering stems of normal plants. The RNA transcript level of cel1 in the elongating zone of dwarf flowering stems was significantly lower than in the corresponding zone in normal plants. This suggests cel1's involvement in cell elongation in A. thaliana. Transgenic tobacco plants transformed with the putative cel1 promoter region fused to the gus reporter gene, showed a significant GUS staining both in shoot and root elongating zones. These results further substantiate the link between cel1 expression and plant cell elongation.