Bloom syndrome DNA helicase mitigates mismatch repair-dependent apoptosis.

Generation of O6-methylguanine (O6-meG) by DNA-alkylating agents such as N-methyl N-nitrosourea (MNU) activates the multiprotein mismatch repair (MMR) complex and the checkpoint response involving ATR/CHK1 and ATM/CHK2 kinases, which may in turn trigger cell cycle arrest and apoptosis. The Bloom syndrome DNA helicase BLM interacts with the MMR complex, suggesting functional relevance to repair and checkpoint responses. We observed a strong interaction of BLM with MMR proteins in HeLa cells upon treatment with MNU as evidenced by co-immunoprecipitation as well as colocalization in the nucleus as revealed by dual immunofluorescence staining. Knockout of BLM sensitized HeLa MR cells to MNU-induced cell cycle disruption and enhanced expression of the apoptosis markers cleaved caspase-9 and PARP1. MNU-treated BLM-deficient cells also exhibited a greater number of 53BP1 foci and greater phosphorylation levels of H2AX at S139 and RPA32 at S8, indicating the accumulation of DNA double-strand breaks. These findings suggest that BLM prevents double-strand DNA breaks during the MMR-dependent DNA damage response and mitigates O6-meG-induced apoptosis.

FANCD2 counteracts O6-methylguanine-induced mismatch repair-dependent apoptosis.

BACKGROUND: Sn1-type alkylating agents methylate the oxygen atom on guanine bases thereby producing O6-methylguanine. This modified base could pair with thymine and cytosine, resulting in the formation of O6-methylguanine/thymine mismatch during DNA replication, recognized by the mismatch repair (MMR) complex, which then initiates the DNA damage response and subsequent apoptotic processes. In our investigation of the molecular mechanisms underlying MMR-dependent apoptosis, we observed FANCD2 modification upon the activity of alkylating agent N-methyl-N-nitrosourea (MNU). This observation led us to hypothesize a relevant role for FANCD2 in the apoptosis induction process. METHODS AND RESULTS: We generated FANCD2 knockout cells using the CRISPR/Cas9 method in the human cervical cancer cell line HeLa MR. FANCD2-deficient cells exhibited MNU hypersensitivity. Upon MNU exposure, FANCD2 colocalized with the MMR complex. MNU-treated FANCD2 knockout cells displayed severe S phase delay followed by increased G2/M arrest and MMR-dependent apoptotic cell death. Moreover, FANCD2 knockout cells exhibited impaired CtIP and RAD51 recruitment to the damaged chromatin and DNA double-strand break accumulation, indicated by simultaneously observed increased γH2AX signal and 53BP1 foci. CONCLUSIONS: Our data suggest that FANCD2 is crucial for recruiting homologous recombination factors to the sites of the MMR-dependent replication stress to resolve the arrested replication fork and counteract O6-methylguanine-triggered MMR-dependent apoptosis.

Severe subglottic stenosis after resection of anterior mediastinal tumor using a double­lumen tube: a case report.

Background: Double­lumen tubes (DLTs) are commonly used for differential pulmonary ventilation during thoracic surgery. Few reports exist on subglottic stenosis among patients who underwent surgery involving DLTs; we lack immediate postoperative period documentation leading up to the onset and subsequent recovery of subglottic stenosis. Herein, we present a case of a 75-year-old woman successfully treated for subglottic stenosis after DLT. Case Description: A 75-year-old woman presented to our hospital with an abnormal chest shadow, which was identified during a medical examination. Chest computed tomography revealed an anterior mediastinal mass with a poor contrast effect measuring 6.0 cm × 3.1 cm × 1.9 cm, which grew from 2.2 to 6.0 cm over 21 months. Low and high signals were detected on T1- and T2-weighted thoracic magnetic resonance imaging, respectively. Concordantly, a thymic cyst was suspected. The patient underwent robotic-assisted thoracoscopic resection via the right lateral approach. A 35-Fr left-sided DLT was used for intubation and differential lung ventilation. Hoarseness and stridor were observed on postoperative day (POD) 1. Laryngoscopy showed submucosal hemorrhage around the vocal cords and mild subglottic stenosis; however, there was no arytenoid dislocation or findings necessitating emergency treatment. On POD 4, her stridor became more severe and laryngoscopy was re-performed and revealed subglottic stenosis progression prompting emergency tracheotomy. The stenosis further progressed, and almost complete airway obstruction was observed on POD 7. By POD 9, partially improving the subglottic stenosis, thereafter the subglottic stenosis was almost completely alleviated by POD 12. The tracheal cannula was removed on POD 22. Trachea-cutaneous fistula closure was performed on POD 35, and she was discharged on POD 42, remaining well. The pathological examination of the anterior mediastinal tumor confirmed the diagnosis of thymic cyst. Conclusions: Airway obstruction owing to subglottic stenosis may occur several days post-surgery with a DLT. Prompt tracheostomy is recommended to prevent complete airway obstruction in patients with progressive subglottic stenosis.

Effect of Particle Sizes and Contents of Surface Pre-Reacted Glass Ionomer Filler on Mechanical Properties of Auto-Polymerizing Resin.

Herein, the mechanical properties of an auto-polymerizing resin incorporated with a surface pre-reacted glass ionomer (S-PRG) filler were evaluated. For this, S-PRG fillers with particle sizes of 1 µm (S-PRG-1) and 3 µm (S-PRG-3) were mixed at 10, 20, 30, and 40 wt% to prepare experimental resin powders. The powders and a liquid (powder/liquid ratio = 1.0 g/0.5 mL) were kneaded and filled into a silicone mold to obtain rectangular specimens. The flexural strength and modulus (n = 12) were recorded via a three-point bending test. The flexural strengths of S-PRG-1 at 10 wt% (62.14 MPa) and S-PRG-3 at 10 and 20 wt% (68.68 and 62.70 MPa, respectively) were adequate (>60 MPa). The flexural modulus of the S-PRG-3-containing specimen was significantly higher than that of the S-PRG-1-containing specimen. Scanning electron microscopy observations of the specimen fracture surfaces after bending revealed that the S-PRG fillers were tightly embedded and scattered in the resin matrix. The Vickers hardness increased with an increasing filler content and size. The Vickers hardness of S-PRG-3 (14.86-15.48 HV) was higher than that of S-PRG-1 (13.48-14.97 HV). Thus, the particle size and content of the S-PRG filler affect the mechanical properties of the experimental auto-polymerizing resin.

Contact Angle and Cell Adhesion of Micro/Nano-Structured Poly(lactic-co-glycolic acid) Membranes for Dental Regenerative Therapy.

Biodegradable membranes are used in regenerative dentistry for guided tissue regeneration (GTR) and guided bone regeneration (GBR). In this study, patterned poly(lactic-co-glycolic acid) (PLGA) membranes with groove, pillar, and hole structures were successfully fabricated by thermal nanoimprinting. Their surfaces were evaluated for topography by scanning electron microscopy and laser microscopy, for hydrophobicity/hydrophilicity by contact angle analysis, and for MC3T3-E1 cell adhesion. The sizes of the patterns on the surfaces of the membranes were 0.5, 1.0, and 2.0 µm, respectively, with the height/depth being 1.0 µm. The pillared and holed PLGA membranes were significantly more hydrophobic than the non-patterned PLGA membranes (p < 0.05). However, the 0.5 µm- and 1.0 µm-grooved PLGA membranes were significantly more hydrophilic than the non-patterned PLGA membranes (p < 0.05). The 0.5 µm-grooved, pillared, and holed membranes exhibited significantly superior adhesion to the MC3T3-E1 cells than the non-patterned PLGA (p < 0.05). These results suggest that patterned PLGA membranes can be clinically used for GTR and GBR in the dental regeneration field.

Substrate recognition mechanism and substrate-dependent conformational changes of an ROK family glucokinase from Streptomyces griseus.

Carbon catabolite repression (CCR) is a widespread phenomenon in many bacteria that is defined as the repression of catabolic enzyme activities for an unfavorable carbon source by the presence of a preferable carbon source. In Streptomyces, secondary metabolite production often is negatively affected by the carbon source, indicating the involvement of CCR in secondary metabolism. Although the CCR mechanism in Streptomyces still is unclear, glucokinase is presumably a central player in CCR. SgGlkA, a glucokinase from S. griseus, belongs to the ROK family glucokinases, which have two consensus sequence motifs (1 and 2). Here, we report the crystal structures of apo-SgGlkA, SgGlkA in complex with glucose, and SgGlkA in complex with glucose and adenylyl imidodiphosphate (AMPPNP), which are the first structures of an ROK family glucokinase. SgGlkA is divided into a small α/ß domain and a large α+ß domain, and it forms a dimer-of-dimer tetrameric configuration. SgGlkA binds a ß-anomer of glucose between the two domains, and His157 in consensus sequence 1 plays an important role in the glucose-binding mechanism and anomer specificity of SgGlkA. In the structures of SgGlkA, His157 forms an HC3-type zinc finger motif with three cysteine residues in consensus sequence 2 to bind a zinc ion, and it forms two hydrogen bonds with the C1 and C2 hydroxyls of glucose. When the three structures are compared, the structure of SgGlkA is found to be modified by the binding of substrates. The substrate-dependent conformational changes of SgGlkA may be related to the CCR mechanism in Streptomyces.

A case of extranodal NK/T-cell lymphoma, nasal type mimicking typical manifestations of adult-onset Still's disease (AOSD) with hemophagocytic syndrome: diagnostic consideration between malignant lymphoma without lymphadenopathy and AOSD.

A 25-year-old Japanese man was suffering from high fever, sore throat, arthralgia, and macular salmon-pink eruption. The superficial lymph node was not palpable, and computed tomographic scans from the neck to pelvis demonstrated hepatosplenomegaly without apparent lymphadenopathy. Therefore, the possibility of malignant lymphoma was considered to be extremely low. Serology for Epstein Barr virus (EBV) and cytomegalovirus showed a postinfectious state, and blood culture was negative. Serum rheumatoid factor and antinuclear antibody were negative. Leukocytopenia (2.4 x 10(3)/mul) was observed, and thus a diagnosis of adult-onset Still's disease (AOSD) with hemophagocytic syndrome (HPS) was made. Fifty-five milligrams of prednisolone daily improved his symptoms and leukocytopenia promptly, but high fever with severe and progressive thrombocytopenia occurred 12 days later. Bone marrow aspiration revealed the presence of lymphoma cells and hemophagocytosis, and the CD45 gating analysis showed expanding population of CD2(+), CD3(-), and CD56(+) cells. Further, mucosal ulceration in the nasal cavity was detected. Therefore, a diagnosis of extranodal natural killer (NK)/T-cell lymphoma, nasal type, concomitant with HPS was made, and treatment with dexamethasone, etoposide, ifosfamide, carboplatin (DeVIC) regimen ameliorated his symptoms and platelet transfusion dependency. Later, a high titer of serum EBV-DNA was detected, which supported the diagnosis. Diagnosing AOSD, extranodal presentation of malignant lymphoma such as extranodal NK/T-cell lymphoma, nasal type, should be carefully considered.

[Eight cases of neuroendcrine carcinoma of the head and neck].

Small cell neuroendocrine carcinoma of the head and neck is rare, and diagnosis may be difficult. Malignancy is high, as is the incidence of distant metastasis. We reported eight cases of stage IV small cell neuroendocrine carcinoma of the head and neck, all in men with a mean onset age of 62 years (range: 45 to 80 years). Three cases arose from the maxillary sinus, two from the ethmoid sinus, one from the parotid gland, one from the tonsil, and one from the larynx. Histological analysis by hematoxylin-eosin staining tentatively revealed malignant lymphoma and undifferentiated carcinoma in two cases each, while immunohistological and/or electron microscopy analysis confirmed histological diagnosis. All were treated by chemotherapy (VP-16, CDDP) and seven cases with radiotherapy based on the schedule of small cell carcinoma of the lung and two cases with lesional resection. Chemotherapy and radiotherapy were effective locally. Five patients died of distant metastasis to the brain, bone, lung, liver, or skin within 12 months. One is alive with liver metastasis. Two have no evidence of disease--one for eight years and the other for one year. Long-term survival thus requires the effective treatment of distant metastasis.