A quantification method of somatic mutations in normal tissues and their accumulation in pediatric patients with chemotherapy.

Somatic mutations are accumulated in normal human tissues with aging and exposure to carcinogens. If we can accurately count any passenger mutations in any single DNA molecule, since their quantity is much larger than driver mutations, we can sensitively detect mutation accumulation in polyclonal normal tissues. Duplex sequencing, which tags both DNA strands in one DNA molecule, enables accurate count of such mutations, but requires a very large number of sequencing reads for each single sample of human-genome size. Here, we reduced the genome size to 1/90 using the BamHI restriction enzyme and established a cost-effective pipeline. The enzymatically cleaved and optimal sequencing (EcoSeq) method was able to count somatic mutations in a single DNA molecule with a sensitivity of as low as 3 × 10-8 per base pair (bp), as assessed by measuring artificially prepared mutations. Taking advantages of EcoSeq, we analyzed normal peripheral blood cells of pediatric sarcoma patients who received chemotherapy (n = 10) and those who did not (n = 10). The former had a mutation frequency of 31.2 ± 13.4 × 10-8 per base pair while the latter had 9.0 ± 4.5 × 10-8 per base pair (P < 0.001). The increase in mutation frequency was confirmed by analysis of the same patients before and after chemotherapy, and increased mutation frequencies persisted 46 to 64 mo after chemotherapy, indicating that the mutation accumulation constitutes a risk of secondary leukemia. EcoSeq has the potential to reveal accumulation of somatic mutations and exposure to environmental factors in any DNA samples and will contribute to cancer risk estimation.

Contributions of the N-terminal flanking residues of an antigenic peptide from the Japanese cedar pollen allergen Cry j 1 to the T-cell activation by HLA-DP5.

Cry j 1 is a major allergen present in Japanese cedar (Cryptomeria japonica) pollens. Peptides with the core sequence of KVTVAFNQF from Cry j 1 ('pCj1') bind to HLA-DP5 and activate Th2 cells. In this study, we noticed that Ser and Lys at positions -2 and -3, respectively, in the N-terminal flanking (NF) region to pCj1 are conserved well in HLA-DP5-binding allergen peptides. A competitive binding assay showed that the double mutation of Ser(-2) and Lys(-3) to Glu [S(P-2)E/K(P-3)E] in a 13-residue Cry j 1 peptide (NF-pCj1) decreased its affinity for HLA-DP5 by about 2-fold. Similarly, this double mutation reduced, by about 2-fold, the amount of NF-pCj1 presented on the surface of mouse antigen-presenting dendritic cell line 1 (mDC1) cells stably expressing HLA-DP5. We established NF-pCj1-specific and HLA-DP5-restricted CD4+ T-cell clones from HLA-DP5 positive cedar pollinosis (CP) patients, and analyzed their IL-2 production due to the activation of mouse TG40 cells expressing the cloned T-cell receptor by the NF-pCj1-presenting mDC1 cells. The T-cell activation was actually decreased by the S(P-2)E/K(P-3)E mutation, corresponding to the reduction in the peptide presentation by this mutation. In contrast, the affinity of NF-pCj1·HLA-DP5 for the T-cell receptor was not affected by the S(P-2)E/K(P-3)E mutation, as analyzed by surface plasmon resonance. Considering the positional and side-chain differences of these NF residues from previously reported T-cell activating sequences, the mechanisms of enhanced T-cell activation by Ser(-2) and Lys(-3) of NF-pCj1 may be novel.

Response to pembrolizumab in a patient with primary lung adenocarcinoma originated from indium lung.

BACKGROUND: Indium is a metal used as a compound called indium-tin oxide for liquid crystal display. Its inhalation causes lung toxicity, resulting in a new occupational lung disease called indium lung. Although the carcinogenicity of indium has been reported in an animal model, its carcinogenicity in humans is unknown. CASE PRESENTATION: This is the first reported case of a primary lung cancer originating from indium lung. In this report, we describe a 46-year-old man with interstitial pneumonia-type indium lung diagnosed 16 years ago. The initial symptom was left chest pain, and computed tomography showed a mass adjacent to the aorta with left pleural effusion. Specimens collected using video-assisted thoracoscopy revealed an adenocarcinoma with a high expression of programmed cell death-ligand 1 (cT4N0M1a stage IVA). Although the lesions showed a remarkable aggressive nature, the patient benefited from pembrolizumab, a monoclonal antibody against programmed cell death 1, which was used as second-line therapy for 2 years. CONCLUSIONS: It is important for clinicians to be aware of lung cancer development in indium-exposed workers or in patients with indium lung, as this could have an aggressive behavior. Treatment with immune checkpoint inhibitors is an option even in patients with interstitial pneumonia-type indium lung.

Nitrosoallene-Mediated endo-Cyclizations for the Synthesis of (Hetero)cyclic α-Substituted exo-Unsaturated Oximes.

Nitrosoallene-mediated endo-dig cyclization reactions producing (hetero)cyclic exo-unsaturated oximes (enoximes) are described. The intramolecular 1,4-type addition to in situ generated nitrosoallenes afforded α-substituted cyclic enoximes with exo-methylene units, which are the favored conformation for further cyclizations. The strong electron-withdrawing ability of the nitroso group facilitated the construction of five-to-seven-membered ring systems via C-O, C-N, C-S, and C-C bond formations, including a quaternary carbon center, at low temperatures.

[Displaced B1+2 Found at Video Assisted Thoracic Surgery for Lung Cancer of Left Upper Lobe].

We report the case of an 83-year-old woman who presented with an abnormal pulmonary nodule suspected to be lung cancer in the left S3 segment. Bronchoscopy showed that the left main bronchus branched off into the B1+2, B3 plus lingular bronchus, and lower bronchus. Video-assisted thoracic surgery was performed, and the nodule was pathologically diagnosed as a primary lung cancer. Subsequently, left upper lobectomy was performed, and an abnormal bronchus was observed behind the main pulmonary artery. Intraoperative bronchoscopy indicated that the bronchus was the displaced B1+2. The B3 plus lingular bronchus existed at the common place of the upper bronchus. The displaced B1+2 and the other upper bronchus were transected separately. No other abnormalities were observed in the pulmonary arteries, veins, or bronchi. Preoperative examination is the best way to detect this bronchial abnormality;identification with intraoperative bronchoscopy can play a crucial role in determining the perioperative strategy.

A case of enormous retroperitoneal liposarcoma with prolapse from the left inguinal canal following hernia repair.

BACKGROUND: Liposarcomas represent ~9.8-16% of soft tissue sarcomas, with the extremities and retroperitoneum being the primary sites of occurrence. While liposarcoma in the inguinal region is uncommon, few reported cases originate from the retroperitoneum and protrude into the scrotum through the inguinal canal. Here, we present a case of a retroperitoneal liposarcoma with prolapse from the left inguinal canal into the scrotum following hernia repair with a mesh plug. CASE PRESENTATION: A 55-year-old male patient underwent a CT scan for a suspected recurrent inguinal hernia, which revealed a sizeable adipose-dense tumor by the left kidney extruded through the left inguinal canal surrounding the scrotum. The patient had undergone mesh plug repair for a left inguinal hernia at another hospital one year ago and noticed ipsilateral inguinal swelling after the hernia repair. The patient was referred to our hospital. The tumor resection was completed with combined resection of potentially involved organs: left side colon, left kidney, and left adrenal gland. Also, complete excision of the tumor was accomplished through surgical resection of the posterior wall of the inguinal canal, the mesh plug, and the tumor extending into the scrotum. Given the nearly complete absence of the inguinal canal's posterior wall and the anterior wall's torn state, sutures were employed to close the external obturator tenosynovitis. Additionally, the inguinal ligament was closed using a tension-free incision technique. Only a mesh was subsequently placed. The resected tumor measured 47 × 30 × 15 cm and 7.5 kg in weight. After surgical resection, a retroperitoneal liposarcoma diagnosis was established. After 2 years and 6 months following the surgical resection, no recurrence has been observed for either liposarcoma or inguinal hernia. CONCLUSION: The previous inguinal hernia in this case must be a prolapse of retroperitoneal liposarcoma. Thus, it is recommended to conduct a preoperative examination, which should include a CT scan, since the presence of a fatty mass within the hernia may indicate the presence of a retroperitoneal liposarcoma. Even if a preoperative diagnosis cannot be made, a long-term prognosis can be expected if the retroperitoneal liposarcoma can be completely resected at reoperation.

A case of laparoscopic distal gastrectomy and median arcuate ligament release for a gastric cancer patient with median arcuate ligament syndrome.

BACKGROUND: The median arcuate ligament syndrome (MALS) is a disease in which the celiac artery is compressed by the arcuate ligament and causes stenosis. If abdominal pain or an aneurysm is observed in the head of the pancreas, it is necessary to release the arcuate ligament, and recently laparoscopic surgery has been reported. However, the indication for treatment in asymptomatic cases is unknown. The treatment for asymptomatic MALS in patients with gastric cancer who are indicated for surgery is also novel. CASE PRESENTATION: A 70-year-old female was found with early gastric cancer in the middle body of the stomach. An enhanced CT scan showed no metastasis, but a gallstone and stenosis of the celiac artery due to the MALS were found. The patient underwent releasing median arcuate ligament after lymph node dissection. A median arcuate ligament was located on the ventral side of the left gastric artery stump, and the celiac artery was exposed when cutting it off. The operation time was 4 h and 59 min, and the bleeding was 6 ml. It took about 5 min to dissect the medial arcuate ligament. The postoperative course was satisfactory, and the patient was discharged 7 days after the operation. CT scan and 3-D CT angiography were performed about 2 months after the operation, and the findings revealed that the celiac artery's stenosis resolved. CONCLUSION: The patient underwent laparoscopic gastrectomy and simultaneously the median arcuate ligament release under an excellent visual field. Therefore, median arcuate ligament release may be considered if MALS is found in a gastrectomy case.

Influence of degree of DNA degradation in formalin-fixed and paraffin-embedded tissue samples on accuracy of genome-wide DNA methylation analysis.

Aim: Depending upon the degree of DNA degradation of formalin-fixed and paraffin-embedded tissue samples, accuracy of measurement by Infinium MethylationEPIC BeadChip assay (Illumina, CA, USA) was assessed. Materials & methods: DNA quality of six formalin-fixed and paraffin-embedded lung tissue samples with different formalin fixation periods was assessed by Illumina quality control, DNA copy number and DNA integrity number value. Infinium data from restored bisulfite treated DNA were compared with datum from a fresh-frozen sample. Results: The correlation coefficient decreased from 0.993 to 0.970 depending upon DNA degradation, even if the Illumina quality control was met. Exclusion of specific probes improved the correlation regardless of tissue. Conclusion: Poor DNA quality can be assessed as an amplifiable DNA copy number and DNA integrity number value. Probe filtering has the potential to improve assay accuracy.

Trajectory and profile of quality of life in patients undergoing lung resection for lung cancer during hospitalization according to the EQ-5D.

OBJECTIVE: To clarify the trajectory and profile of quality of life (QOL) in lung cancer patients undergoing surgery during hospitalization. METHODS: Among prospectively enrolled 279 patients between 2015 and 2018, we used the EQ-5D-5 levels (EQ-5D-5L), which consist of a descriptive system of five dimensions and a visual analog scale (VAS) (from 0 to 100). The five dimensions are mobility, self-care, usual activities, pain/discomfort, and anxiety/depression, which are rated according to a 5-grade evaluation. QOL assessments were performed preoperatively (Pre) and on postoperative day 1/3/5/7 (D1/D3/D5/D7). To observe the trajectory of QOL, the EQ-5D VAS was used. To define the profile of QOL, we applied univariate linear regression analysis to predict EQ-5D VAS based on the five dimensions of EQ-5D as explanatory variables and to determine the rank of absolute values of the standardized coefficient of each dimension that represented strength of the effect on the EQ-5D VAS. RESULTS: The means of VAS scores were as follows: Pre/D1/D3/D5/D7 = 79/45/58/64/71. Younger age, fewer comorbidities, and surgical indications decreased the VAS at D1. More comorbidities, advanced cancer stage, postoperative adverse events, and chest tube placements hampered restoration of the VAS. Regarding QOL profiles, anxiety/depression was the highest rank preoperatively while usual activity, but not pain/discomfort, was the highest postoperatively. CONCLUSIONS: This is the first study to visualize the trajectory of QOL in surgical patients with lung cancer during hospitalization. This information may help improve perioperative patient care.

Preoperative predictors of restoration in quality of life after surgery for lung cancer.

BACKGROUND: The preoperative predictors of quality of life (QOL) in patients who undergo lung resection for lung cancer are poorly known. Here, we investigated these predictors in such patients using two QOL measures. METHODS: In this single-institutional prospective cohort study, we administered the EQ-5D-5 levels (EQ-5D-5L) from January 2015, and the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire with 30 items from April 2015 to April 2018 preoperatively (Pre) and at one month postoperatively (M1), and one year postoperatively (Y1). General health status was measured by the EQ-5D visual analogue scale (VAS) and EORTC global health status/QOL (GHS) scores. Multivariable linear regression analyses were used to explore the preoperative predictors of QOL at Y1. RESULTS: A total of 223 patients were included in the study. The EQ-5D VAS and EORTC GHS scores, at Pre, M1, and Y1, were 80 ± 15, 77 ± 15, and 84 ± 11; and 74 ± 19, 65 ± 20, and 78 ± 17, respectively. In the multivariable analyses, the albumin level, preoperative VAS score, and preoperative pain/discomfort and anxiety/depression were identified as predictors by the EQ-5D VAS score. The preoperative EORTC GHS score, absence of diabetes mellitus, preoperative cognitive function score, and preoperative symptom score of pain were identified as predictors by the EORTC GHS score. CONCLUSIONS: The EQ-5D VAS and EORTC GHS scores traced similar trajectories of QOL. In both QOL measures, preoperative pain was found as a common predictor. These predictors may help improve patient/survivor care in the future.

Successful use of extracorporeal membrane oxygenation for airway-obstructing lung adenocarcinoma.

Endobronchial-invasive lung cancers are generally diagnosed at advanced stages and may require emergency treatment for airway obstruction. Stent implantation is a common intervention for such obstructed airways but certain subsets of patients cannot receive adequate treatment without respiratory support. Veno-venous extracorporeal membrane oxygenation (ECMO) is a salvage therapy for respiratory failure but its usefulness in managing patients with advanced lung cancer remains unclear given the poor prognosis. In recent years, molecular targeted agents for patients with driver mutations offer rapid responses and may be administered even while under critical care. In this report, we describe the case of 39-year-old female who presented to our emergency department with severe respiratory distress. A computed tomography scan revealed a large mediastinal tumor invading the tracheal carina causing severe stenosis of the left main bronchus and right main pulmonary artery. ECMO support was required as the respiratory condition remained unstable despite high pressure ventilation. Under ECMO support, the patient underwent bronchial stent implantation and was successfully weaned off ECMO. The tumor was histologically diagnosed as pulmonary adenocarcinoma with anaplastic lymphoma kinase gene rearrangement. Treatment with a tyrosine kinase inhibitor, alectinib, induced a marked tumor reduction within a short period. The patient recovered well and is now in remission one year later. This case indicates that intensive respiratory support with ECMO may become a bridge through the critical period for selected patients with respiratory failure secondary to advanced lung cancer. KEY POINTS: SIGNIFICANT FINDINGS OF THIS STUDY: ECMO was important to maintain oxygenation during airway intervention for acute respiratory failure due to critical lung adenocarcinoma with ALK gene rearrangement. WHAT THIS STUDY ADDS: With the development of targeted therapies and the improvement in therapeutic bronchoscopy, intensive respiratory support with ECMO may be helpful especially in selected lung cancer patients with oncogenic driver mutations.

Thymectomy during Myasthenic Crisis under Artificial Respiration.

A 34-year-old man was diagnosed with thymoma, which was evaluated preoperatively as stage II or III, with myasthenia gravis (MG). The size of the tumor was 70 × 44 × 80 mm. No invasion to neighboring organs was observed. Prednisolone was prescribed for stabilization of MG. However, a myasthenic crisis (MC) occurred, and intensive care, including emergent endobronchial intubation followed by artificial ventilation, pulse steroid therapy, high-dose intravenous immunoglobulin, and tacrolimus hydrate, was initiated. A chest computed tomography on day 6 revealed tumor reduction to 50 × 30 × 60 mm. An extended total thymectomy by median sternotomy was performed, and artificial ventilation was continued after that. Scheduled artificial ventilation and steroid therapy together can, therefore, enable complete resection of thymoma in patients undergoing treatment for MC. While ventilation helps avert a respiratory failure, the steroid therapy temporarily reduces the tumor size, making resection easier.

Long-term efficacy of infliximab treatment and the predictors of treatment outcomes in patients with refractory uveitis associated with Behçet's disease.

OBJECTIVE: To assess the long-term efficacy and safety of infliximab (IFX) treatment for refractory uveitis associated with Behçet's disease (BD) and to identify predictors of long-term IFX therapy outcomes. METHODS: We retrospectively studied 44 consecutive BD patients with uveitis who were started on IFX therapy and analyzed the efficacy and safety of IFX and the treatment continuation rate. To determine predictors of IFX responsiveness, we analyzed the clinical characteristics of the patients who received regular maintenance therapy and those who required treatment intensification. The serum cytokine levels prior to IFX were measured through the Bio-Plex human cytokine assays. RESULTS: IFX significantly reduced the frequency of ocular attacks and improved the visual acuity of patients with BD-related uveitis. However, approximately half of the patients required dose escalations, necessitating a shortening of the intervals between IFX infusions due to loss of efficacy during the 5-year treatment. The frequency of ocular attacks was significantly higher in patients with complete BD than in patients with incomplete BD. A multiplex cytokine analysis revealed that patients with BD-related uveitis exhibited increased serum IL-2, IL-6, IL-8, and MCP-1 levels. Moreover, among BD patients, the serum IL-2 and IL-6 levels were particularly high in those who maintained remission and received regular IFX treatments. CONCLUSION: We confirmed the long-term efficacy and tolerability of IFX in patients with BD-related uveitis. Our results indicate that complete BD may be less responsive to IFX and that the pretreatment serum cytokine profiles may be useful for predicting the long-term IFX therapy outcomes.

In situ detection of the activation of Rac1 and RalA small GTPases in mouse adipocytes by immunofluorescent microscopy following in vivo and ex vivo insulin stimulation.

Rac1 has been implicated in insulin-dependent glucose uptake by mechanisms involving plasma membrane translocation of the glucose transporter GLUT4 in skeletal muscle. Although the uptake of glucose is also stimulated by insulin in adipose tissue, the role for Rac1 in adipocyte insulin signaling remains controversial. As a step to reveal the role for Rac1 in adipocytes, we aimed to establish immunofluorescent microscopy to detect the intracellular distribution of activated Rac1. The epitope-tagged Rac1-binding domain of a Rac1-specific target was utilized as a probe that specifically recognizes the activated form of Rac1. Rac1 activation in response to ex vivo and in vivo insulin stimulations in primary adipocyte culture and mouse white adipose tissue, respectively, was successfully observed by immunofluorescent microscopy. These Rac1 activations were mediated by phosphoinositide 3-kinase. Another small GTPase RalA has also been implicated in insulin-stimulated glucose uptake in skeletal muscle and adipose tissue. Similarly to Rac1, immunofluorescent microscopy using an activated RalA-specific polypeptide probe allowed us to detect intracellular distribution of insulin-activated RalA in adipocytes. These novel approaches to visualize the activation status of small GTPases in adipocytes will largely contribute to the understanding of signal transduction mechanisms particularly for insulin action.

Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with aerolysin-type architecture.

Cry46Ab is a Cry toxin derived from Bacillus thuringiensis TK-E6. Cry46Ab is not significantly homologous to other mosquitocidal Cry or Cyt toxins and is classified as an aerolysin-type pore-forming toxin based on structural similarity. In this study, the potency of Cry46Ab was assessed for its potential application to mosquito control. A synthetic Cry46Ab gene, cry46Ab-S1, was designed to produce recombinant Cry46Ab as a glutathione-S-transferase fusion in Escherichia coli. Recombinant Cry46Ab showed apparent toxicity to Culex pipiens larvae, with a 50% lethal dose of 1.02 µg/ml. In an artificial lipid bilayer, Cry46Ab activated by trypsin caused typical current transitions between open and closed states, suggesting it functions as a pore-forming toxin similar to other Cry and Cyt toxins. The single-channel conductance was 103.3 ± 4.1 pS in 150 mM KCl. Co-administration of recombinant Cry46Ab with other mosquitocidal Cry toxins, especially the combination of Cry4Aa and Cry46Ab, resulted in significant synergistic toxicity against C. pipiens larvae. Co-administration of multiple toxins exhibiting different modes of action is believed to prevent the onset of resistance in insects. Our data, taken in consideration with the differences in its structure, suggest that Cry46Ab could be useful in not only reducing resistance levels but also improving the insecticidal activity of Bt-based bio-insecticides.

Primary and Secondary Spontaneous Pneumothorax: Prevalence, Clinical Features, and In-Hospital Mortality.

Background. Optimal treatment practices and factors associated with in-hospital mortality in spontaneous pneumothorax (SP) are not fully understood. We evaluated prevalence, clinical characteristics, and in-hospital mortality among Japanese patients with primary or secondary SP (PSP/SSP). Methods. We retrospectively reviewed and stratified 938 instances of pneumothorax in 751 consecutive patients diagnosed with SP into the PSP and SSP groups. Factors associated with in-hospital mortality in SSP were identified by multiple logistic regression analysis. Results. In the SSP group (n = 327; 34.9%), patient age, requirement for emergency transport, and length of stay were greater (all, p < 0.001), while the prevalence of smoking (p = 0.023) and number of surgical interventions (p < 0.001) were lower compared to those in the PSP group (n = 611; 65.1%). Among the 16 in-hospital deceased patients, 12 (75.0%) received emergency transportation and 10 (62.5%) exhibited performance status (PS) of 3-4. In the SSP group, emergency transportation was an independent factor for in-hospital mortality (odds ratio 16.37; 95% confidence interval, 4.85-55.20; p < 0.001). Conclusions. The prevalence and clinical characteristics of PSP and SSP differ considerably. Patients with SSP receiving emergency transportation should receive careful attention.

Dimensional dependence of electronic structure of fullerene polymers.

Fullerene polymers made of C(60) are systematically investigated by means of a first-principles pseudopotential approach within the local density approximation of the density functional theory. We assume 10 different structures of fullerene polymers. The first three are C(60) polymer networks cross-linked by [2+2] cycloadditional four-membered rings, and the other seven are composed of peanut-shaped fused C(60) polymer chains cross-linked by either seven-membered rings or eight-membered rings. Owing to the overlap of wave functions as well as the hybrid networks of sp(2)-like (3-fold coordinated) and sp(3)-like (4-fold coordinated) carbon atoms, the electronic structure is considerably different from each other. We find that the resulting electronic structure is either semiconductor or semimetal depending on the spatial dimensionality of materials.

Invasive Thymoma with Pure Red Cell Aplasia and Amegakaryocytic Thrombocytopenia.

We here describe a case involving a 67-yearold female patient who was referred to our hospital due to severe anemia (hemoglobin, 5.0 g/dL), thrombocytopenia (platelet count, 0.6 × 104/µL), and a mediastinal shadow with calcification noted on X-ray. On admission, an anterior mediastinal tumor was detected, and bone marrow biopsy revealed few megakaryocytes and severely reduced numbers of erythroid cells. The diagnosis was thymoma with pure red cell aplasia (PRCA) and acquired amegakaryocytic thrombocytopenia (AAMT). On Day 8 of admission, the patient received immunosuppressive therapy together with cyclosporine for the 2 severe hematologic diseases, which were stabilized within 2 months. Subsequently, total thymectomy was performed. The diagnosis of the tumor invading the left lung was invasive thymoma, Masaokakoga stage III. The histological diagnosis was World Health Organization type AB. Thymoma accompanied with PRCA and AAMT is very rare, and, based on our case, immunotherapeutic therapy for the hematologic disorders should precede surgical intervention.

A comprehensive software suite for the analysis of cDNAs.

We have developed a comprehensive software suite for bioinformatics research of cDNAs; it is aimed at rapid characterization of the features of genes and the proteins they code. Methods implemented include the detection of translation initiation and termination signals, statistical analysis of codon usage, comparative study of amino acid composition, comparative modeling of the structures of product proteins, prediction of alternative splice forms, and metabolic pathway reconstruction.