Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source.

BACKGROUND: The filamentous fungus Trichoderma reesei has been used as a host organism for the production of lignocellulosic biomass-degrading enzymes. Although this microorganism has high potential for protein production, it has not yet been widely used for heterologous recombinant protein production. Transcriptional induction of the cellulase genes is essential for high-level protein production in T. reesei; however, glucose represses this transcriptional induction. Therefore, cellulose is commonly used as a carbon source for providing its degraded sugars such as cellobiose, which act as inducers to activate the strong promoters of the major cellulase (cellobiohydrolase 1 and 2 (cbh1 and cbh2) genes. However, replacement of cbh1 and/or cbh2 with a gene encoding the protein of interest (POI) for high productivity and occupancy of recombinant proteins remarkably impairs the ability to release soluble inducers from cellulose, consequently reducing the production of POI. To overcome this challenge, we first used an inducer-free biomass-degrading enzyme expression system, previously developed to produce cellulases and hemicellulases using glucose as the sole carbon source, for recombinant protein production using T. reesei. RESULTS: We chose endogenous secretory enzymes and heterologous camelid small antibodies (nanobody) as model proteins. By using the inducer-free strain as a parent, replacement of cbh1 with genes encoding two intrinsic enzymes (aspartic protease and glucoamylase) and three different nanobodies (1ZVH, caplacizumab, and ozoralizumab) resulted in their high secretory productions using glucose medium without inducers such as cellulose. Based on signal sequences (carrier polypeptides) and protease inhibitors, additional replacement of cbh2 with the nanobody gene increased the percentage of POI to about 20% of total secreted proteins in T. reesei. This allowed the production of caplacizumab, a bivalent nanobody, to be increased to 9.49-fold (508 mg/L) compared to the initial inducer-free strain. CONCLUSIONS: In general, whereas the replacement of major cellulase genes leads to extreme decrease in the degradation capacity of cellulose, our inducer-free system enabled it and achieved high secretory production of POI with increased occupancy in glucose medium. This system would be a novel platform for heterologous recombinant protein production in T. reesei.

Use of FDG-PET to detect a chronic odontogenic infection as a possible source of the brain abscess.

This study describes the use of (18)F-fluoro-2-deoxyglucose positron emission tomography (FDG-PET) to detect a chronic odontogenic infection as the possible origin of a brain abscess (BA). A 74-year-old man with esophageal carcinoma was referred to our department to determine the origin of a BA in his oral cavity. He had no acute odontogenic infections. The BA was drained, and bacteria of the Staphylococcus milleri group were detected. Whole body FDG-PET revealed that the only sites of definite uptake of FDG were the esophageal carcinoma and the left upper maxillary region (SUVmax: 4.5). These findings suggested that the BA may have originated from a chronic periodontal infection. Six teeth with progressive chronic periodontal disease were extracted to remove the possible source of BA. These findings excluded the possibility of direct spread of bacteria from the odontogenic infectious lesion to the intracranial cavity. After extraction, there was no relapse of BA.

Anti-BP180-type mucous membrane pemphigoid: report of two cases.

We describe two patients with anti-BP180-type mucous membrane pemphigoid (MMP), who were correctly diagnosed and treated in early stages through the cooperation of dentists and dermatologists. Patient 1 was a 74-year-old woman who visited our dental department due to blisters over the oral mucosa and eruptions on the skin. She had also experienced bleeding of the gingiva and palate mucosa. Biopsy specimens from the oral mucosa revealed detachment of epithelial basement membrane and subepithelial lamina propria with slight chronic inflammation. Direct immunofluorescence (DIF) revealed linear IgG and IgA deposits along the basement membrane zone (BMZ). Indirect immunofluorescence (IIF) using 1 M-NaCl split normal human skin showed binding of IgG and IgA on the epidermal side. On immunoblot analysis, IgG and IgA autoantibodies reacted with the C-terminal protein of BP180. These findings indicated a diagnosis of anti-BP180-type MMP. Patient 2 was a 59-year-old woman who was referred to our dental department with a history of blisters and large erosions on the gingiva. Biopsy specimens from the oral mucosa revealed partial junctional separation at the level of the basement membrane. DIF showed linear depositions of IgG and C3 along the BMZ. IIF, using 1 M-NaCl split normal human skin, revealed circulating anti-BMZ-IgG antibodies bound to the epidermal side. These findings indicated a diagnosis of anti-BP180-type MMP. Both patients were treated successfully with systemic or topical steroids and oral health care. In conclusion, appropriate clinical examination and cooperation among medical specialists are important for the early diagnosis and treatment of patients with recurrent and chronic stomatitis and for their good prognosis.

Differences in sequential posttreatment salivary IL-6 levels between patients with and patients without locoregional recurrences of oral squamous cell carcinoma: Part III of a cohort study.

OBJECTIVE: Sequential postoperative salivary interleukin-6 (IL-6) concentrations were examined in patients with oral squamous cell carcinoma (OSCC) who had early or late locoregional recurrences or those who did not. STUDY DESIGN: Twenty-seven consecutive patients with OSCC were originally included in the study. All patients underwent radical surgery. Four saliva samples were collected before (periods I and II) and after (periods III and IV) surgery, and IL-6 concentrations were measured. RESULTS: Although postoperative (period III: at the time of discharge) salivary IL-6 level was significantly higher in patients with early locoregional recurrence (P = .02) than in those without, no such relationships were observed for preoperative IL-6 concentrations (periods I and II). Postoperative (period IV: 24 months after surgery) IL-6 level was significantly higher in patients with late locoregional recurrence (P = .03) than in those without, but no such relationships were observed for IL-6 concentrations in periods I, II, and III. CONCLUSIONS: Sequential postoperative salivary IL-6 concentration may be a useful marker for diagnosis of early and late locoregional recurrence in OSCC.