The elimination of two restriction enzyme genes allows for electroporation-based transformation and CRISPR-Cas9-based base editing in the non-competent Gram-negative bacterium Acinetobacter sp. Tol 5.

Environmental isolates are promising candidates for new chassis of synthetic biology because of their inherent capabilities, which include efficiently converting a wide range of substrates into valuable products and resilience to environmental stresses; however, many remain genetically intractable and unamenable to established genetic tools tailored for model bacteria. Acinetobacter sp. Tol 5, an environmentally isolated Gram-negative bacterium, possesses intriguing properties for use in synthetic biology applications. Despite the previous development of genetic tools for the engineering of strain Tol 5, its genetic manipulation has been hindered by low transformation efficiency via electroporation, rendering the process laborious and time-consuming. This study demonstrated the genetic refinement of the Tol 5 strain, achieving efficient transformation via electroporation. We deleted two genes encoding type I and type III restriction enzymes. The resulting mutant strain not only exhibited marked efficiency of electrotransformation but also proved receptive to both in vitro and in vivo DNA assembly technologies, thereby facilitating the construction of recombinant DNA without reliance on intermediate Escherichia coli constructs. In addition, we successfully adapted a CRISPR-Cas9-based base-editing platform developed for other Acinetobacter species. Our findings provide genetic modification strategies that allow for the domestication of environmentally isolated bacteria, streamlining their utilization in synthetic biology applications.IMPORTANCERecent synthetic biology has sought diverse bacterial chassis from environmental sources to circumvent the limitations of laboratory Escherichia coli strains for industrial and environmental applications. One of the critical barriers in cell engineering of bacterial chassis is their inherent resistance to recombinant DNA, propagated either in vitro or within E. coli cells. Environmental bacteria have evolved defense mechanisms against foreign DNA as a response to the constant threat of phage infection. The ubiquity of phages in natural settings accounts for the genetic intractability of environmental isolates. The significance of our research is in demonstrating genetic modification strategies for the cell engineering of such genetically intractable bacteria. This research marks a pivotal step in the domestication of environmentally isolated bacteria, promising candidates for emerging synthetic biology chassis. Our work thus significantly contributes to advancing their applications across industrial, environmental, and biomedical fields.

Accurate Selection of Sublobar Resection for Small Non-small Cell Lung Cancer.

BACKGROUND: Although sublobar resection (wedge resection [Wed] or segmentectomy [Seg]) has become a standard operative procedure for clinical stages IA1 and IA2 non-small cell lung cancer (NSCLC) in Japan, the impact of this procedure on the prognosis and postoperative complications in real-world clinical practice is unknown. METHODS: This study retrospectively analyzed risk factors for a poor prognosis and postoperative complications of 470 patients with clinical stage ≤ IA2 NSCLC who underwent surgery from 2012 to 2021. RESULTS: Among the patients with a consolidation-to-tumor ratio (CTR) higher than 0.5, the 5-year relapse-free survival (RFS) rate was significantly lower in the Wed group (72.1%) than in the Seg (85.8%) and Lob (86.8%) groups (p < 0.01), but the difference between the Seg and Lob groups was not significant. Among patients with a CTR of 0.5 or lower, the 5-year RFS rate did not differ significantly among the three groups. Multivariable analysis of RFS showed that the prognosis was significantly worse in the Wed group than in the Lob group (hazard ratio, 2.83; p < 0.01), but the difference between the Wed and Seg groups or the between Seg and Lob groups was not significant. Multivariable analysis of postoperative complications showed a significantly lower risk in the Wed group than in the Seg group (odds ratio, 0.31; p < 0.01). CONCLUSIONS: Seg could become the standard operative procedure for clinical stages IA1 and IA2 NSCLC patients. Wed is suggested to be an option for patients with a CTR of 0.5 or lower and has the advantage of avoiding postoperative complications.

Predictive Value of Recurrence of Solid and Micropapillary Subtypes in Lung Adenocarcinoma.

INTRODUCTION: Although histological subtype in lung adenocarcinoma has been reported as a poor prognostic factor in several studies, its utility has not yet been revealed as an adaptation criterion of postoperative adjuvant chemotherapy. METHODS: Four hundred ninety-four lung adenocarcinoma patients were enrolled in this retrospective study. A subanalysis was performed in 420 lung adenocarcinoma patients with pathological stage 0-I disease for risk factors of postoperative recurrence. RESULTS: Maximum standardized uptake value (SUVmax) (p < 0.01), pathological stage ≥II (p < 0.04), and adjuvant chemotherapy (p < 0.01) were risk factors for recurrence in the multivariate analysis, whereas histological subtype was not a significant factor for recurrence at all stages. In the subanalysis, univariate analysis showed that carcinoembryonic antigen expression (p < 0.01), prognostic nutrition index (p = 0.03), SUVmax (p < 0.01), lymphatic invasion (p < 0.01), vascular invasion (p < 0.01), grade 3-4 differentiation (p < 0.01), pathological stage ≥IA3 (p < 0.01), and histological subtype (p = 0.03) were significant risk factors of recurrence. SUVmax (p < 0.01) was the only risk factor for recurrence in the multivariate analysis, whereas histological subtype was not (p = 0.07). Relapse-free survival (RFS) was significantly worse in the micropapillary- and solid-predominant subtype groups than in the other subtypes (p = 0.01). On the other hand, RFS with or without uracil-tegafur as adjuvant chemotherapy in lung micropapillary- or solid-predominant adenocarcinoma patients with pathological stage IA-IB disease was not significantly different. CONCLUSION: This study suggested that histological subtypes, such as micropapillary- or solid-predominant pattern, are risk factors for recurrence in pathological stage 0-I lung adenocarcinoma and may be necessary adjuvant chemotherapy instead of uracil-tegafur.

Prognostic Factors among Patients with Resected Non-Adenocarcinoma of the Lung.

INTRODUCTION: Few studies have investigated the prognostic factors for non-adenocarcinoma of the lung. We retrospectively evaluated the prognostic factors on the basis of histological type of non-adenocarcinoma of the lung treated by pulmonary resection. METHODS: We enrolled 266 patients with non-adenocarcinoma of the lung in this retrospective study: 196 with squamous cell carcinoma (SCC) and 70 with non-SCC. RESULTS: Relapse-free survival (RFS) did not differ significantly between SCC and non-SCC patients (p = 0.33). For SCC patients, RFS differed significantly between patients who underwent wedge resection and non-wedge resection (p < 0.01) and between patients with Clavien-Dindo grade ≥3a and 0-2 postoperative complications (p < 0.01). For non-SCC patients, RFS rates were significantly different in the groups divided at neutrophil-to-lymphocyte ratio = 2.40 (p = 0.02), maximum standardized uptake value (SUVmax) = 8.39 (p < 0.01), between patients with pathological stage (pStage) 0-I and with pStage more than II (p < 0.01). For SCC patients, male sex (p = 0.04), wedge resection (p = 0.01), and Clavien-Dindo grade ≥3a (p = 0.02) were significant factors for RFS in multivariate analysis. For non-SCC patients, neutrophil-to-lymphocyte ratio >2.40 (p < 0.01), SUVmax >8.39 (p = 0.01), and pStage ≥II (p = 0.03) were significant factors for RFS in multivariate analysis. CONCLUSION: RFS did not differ significantly differently between SCC and non-SCC patients. It is necessary to perform more than segmentectomy and to avoid severe postoperative complications for SCC patients. SUVmax might be an adaptation criterion of adjuvant chemotherapy for patients with non-adenocarcinoma and non-SCC of the lung.

Analysis of risk factors of postoperative complication for non-small cell lung cancer.

BACKGROUND: The relationship between risk factors of common postoperative complications after pulmonary resection, such as air leakage, atelectasis, and arrhythmia, and patient characteristics, including nutritional status or perioperative factors, has not been sufficiently elucidated. METHODS: One thousand one hundred thirty-nine non-small cell lung cancer patients who underwent pulmonary resection were retrospectively analyzed for risk factors of common postoperative complications. RESULTS: In a multivariate analysis, male sex (P = 0.01), age ≥ 65 years (P < 0.01), coexistence of chronic obstructive pulmonary disease (COPD) (P < 0.01), upper lobe (P < 0.01), surgery time ≥ 155 min (P < 0.01), and presence of lymphatic invasion (P = 0.01) were significant factors for postoperative complication. Male sex (P < 0.01), age ≥ 65 years (P = 0.02), body mass index (BMI) < 21.68 (P < 0.01), coexistence of COPD (P = 0.02), and surgery time ≥ 155 min (P = 0.01) were significant factors for severe postoperative complication. Male sex (P = 0.01), BMI < 21.68 (P < 0.01), thoracoscopic surgery (P < 0.01), and surgery time ≥ 155 min (P < 0.01) were significant risk factors for postoperative air leakage. Coexistence of COPD (P = 0.01) and coexistence of asthma (P < 0.01) were significant risk factors for postoperative atelectasis. Prognostic nutrition index (PNI) < 45.52 (P < 0.01), lobectomy or extended resection more than lobectomy (P = 0.01), and surgery time ≥ 155 min (P < 0.01) were significant risk factors for postoperative arrhythmia. CONCLUSION: Low BMI, thoracoscopic surgery, and longer surgery time were significant risk factors for postoperative air leakage. Coexistence of COPD and coexistence of asthma were significant risk factors for postoperative atelectasis. PNI, surgery time, and surgical procedure were revealed as risk factors of postoperative arrhythmia. Patients with these factors should be monitored for postoperative complication. TRIAL REGISTRATION: The Institutional Review Board of Kanazawa Medical University approved the protocol of this retrospective study (approval number: I392), and written informed consent was obtained from all patients.

Relative efficacies of EGFR-TKIs and immune checkpoint inhibitors for treatment of recurrent non-small cell lung cancer after surgery.

INTRODUCTION: The relative efficacies of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and immune checkpoint inhibitors (ICIs) for the treatment of recurrent non-small cell lung cancer (NSCLC) after surgery remain unclear. METHODS: Among 801 patients with NSCLC who underwent pulmonary resection at Kanazawa Medical University between 2017 and 2021, 64 patients had recurrence. We retrospectively compared the efficacies of EGFR-TKIs and ICIs in these patients with recurrent NSCLC who underwent pulmonary resection. RESULTS: The 3-year overall survival rates after recurrence were 79.3% in patients who received EGFR-TKIs, 69.5% in patients who received ICIs, and 43.7% in patients who received cytotoxic agents. There was no significant difference in overall survival between patients treated with EGFR-TKIs and ICIs (p=0.14) or between patients treated with ICIs and cytotoxic agents (p=0.23), but overall survival was significantly higher in patients treated with EGFR-TKIs compared with cytotoxic agents (p<0.01) The probabilities of a 2-year response were 88.5%, 61.6%, and 25.9% in patients treated with EGFR-TKIs, ICIs, and cytotoxic agents, respectively. There was no significant difference in response periods between patients treated with EGFR-TKIs and ICIs (p=0.18), but the response period was significantly better in patients treated with EGFR-TKIs (p<0.01) or ICIs (p=0.03) compared with cytotoxic agents. Percent-predicted vital capacity (p=0.03) and epidermal growth factor receptor gene mutation (p<0.01) were significant factors affecting the overall response to chemotherapy in multivariate analysis. CONCLUSION: EGFR-TKIs and ICIs are effective for treating recurrent NSCLC after surgery. Although adjuvant chemotherapy for completely resected pathological stage II to IIIA NSCLC, atezolizumab or Osimertinib, has also been recently approved as adjuvant chemotherapy, there is a risk that patients who relapse after adjuvant chemotherapy will have less choice.

Association between lower lobe location and early recurrence for non-small cell lung cancer.

OBJECTIVES: It is unclear whether a lower lobe origin is a risk factor for early recurrence of non-small cell lung cancer (NSCLC) in patients who underwent pulmonary resection. MATERIALS AND METHODS: The risk factors for early recurrence, defined as recurrence occurring within 1 year after surgery, were analyzed in 476 patients with NSCLC who underwent pulmonary resection without wedge resection. RESULTS: The proportion of men, Brinkman's index, carcinoembryonic antigen levels, and the maximum standardized uptake value (SUVmax) were significantly higher in patients with early recurrence than in those without early recurrence. Furthermore, the rates of lower lobe origin, extended resection beyond lobectomy, lymphatic invasion, vascular invasion, and advanced-stage disease were significantly higher in patients with early recurrence. Age (odds ratio [OR] = 4.46, p < 0.01), SUVmax (OR = 5.78, p = 0.02), a lower lobe origin (OR = 3.06, p = 0.01), and pathological stage (OR = 3.34, p = 0.01) were risk factors for early recurrence in multivariate analysis. Furthermore, only early recurrence (OR = 3.34, p = 0.01) was a risk factor for overall survival in multivariate analysis, and overall survival outcomes and prognoses significantly differed between patients with and without early recurrence (p < 0.01). CONCLUSIONS: Age, SUVmax, a lower lobe origin, and pathological stage are risk factors for early recurrence. These results suggest that for patients with NSCLC who underwent pulmonary resection, SUVmax and a lower lobe origin are important for deciding the indication for adjuvant chemotherapy in addition to pathological stage.

The Utility of SUVmax as an Adaptation Criterion for Limited Resection in Stage IA Non-Small Cell Lung Cancer.

INTRODUCTION: Although the consolidation diameter of a tumor on computed tomography (CT) is an adaptation criterion for limited resection in early-stage non-small cell lung cancer (NSCLC), whether the maximum standardized uptake value (SUVmax) is also an adaptation criterion for limited resection has not been evaluated. METHODS: In total, 478 NSCLC patients with clinical stage IA disease were analyzed, among whom 383 were used to perform a sub-analysis. RESULTS: Multivariate analysis showed that consolidation diameter (odds ratio [OR]: 3.05, p = 0.01), SUVmax (OR: 10.74, p = 0.02), and lymphatic invasion (OR: 10.34, p < 0.01) were risk factors for lymph node metastasis in clinical stage IA NSCLC patients. Furthermore, age (OR: 2.98, p = 0.03), SUVmax (OR: 13.07, p = 0.02), and lymphatic invasion (OR: 5.88, p = 0.02) were risk factors for lymph node metastasis in clinical stage IA lung adenocarcinoma patients according to multivariate analysis. CONCLUSION: Consolidation diameter of a tumor on CT, SUVmax, and lymphatic invasion are risk factors for lymph node metastasis. However, SUVmax was a risk factor for lymph node metastasis rather than consolidation diameter on CT in lung adenocarcinoma patients. These results suggest that for early-stage lung adenocarcinoma patients, SUVmax is more important for deciding the indication of limited resection than consolidation diameter of the tumor on CT.

Improvements in perioperative outcomes for non-small cell lung cancer: a decade-long analysis.

BACKGROUND: Video-assisted thoracic surgery (VATS) procedures for non-small cell lung cancer (NSCLC) have steadily increased and have become the gold standard, but their prognostic advantage compared with thoracotomy has not been elucidated. This study retrospectively evaluated perioperative characteristics of VATS for NSCLC over time. METHODS: We collected the clinical data of 760 patients with NSCLC who underwent pulmonary resection over the past decade, classifying patients into early (2011-2015) and late (2016-2020) periods. Changes in NSCLC patient characteristics, surgical approaches, perioperative factors, postoperative morbidities, and prognoses were analyzed. RESULTS: Patients in the late period were older (p = 0.01), had more comorbidities (p = 0.01), and had earlier-stage cancer (p < 0.01) than those in the early period. The late period had significantly fewer surgical procedures for lobectomy or extended resection beyond lobectomy (p < 0.01), open thoracotomies (p < 0.01), postoperative (p = 0.02) and severe morbidities (p < 0.01), and a significantly shorter postoperative hospital stay than the early period. Surgical procedures of lobectomy or extended resection beyond lobectomy (p < 0.01) were significant risk factors for postoperative morbidity, and being in the early period (p < 0.01) and surgical procedures of lobectomy or extended resection beyond lobectomy (p < 0.01) were significant risk factors for severe postoperative morbidities. The overall survival prognosis significantly differed between the groups (p = 0.02) but progression-free survival did not (p = 0.89). CONCLUSIONS: The incidence of postoperative morbidities decreased over time in older patients and patients with more comorbidities. The prognosis of patients with NSCLC did not change with increasing VATS or sublobar resection.

Prognostic Impact of Cancer Inflammation Prognostic Index for Non-small Cell Lung Cancer.

PURPOSE: Cancer-inflammation prognostic index (CIPI) is calculated by multiplying the concentration of carcinoembryonic antigen by neutrophil-to-lymphocyte ratio. CIPI has been reported as a prognostic factor for colorectal cancer. Although carcinoembryonic antigen and neutrophil-to-lymphocyte ratio have been reported as prognostic factors for non-small cell lung cancer (NSCLC), it has not been investigated whether CIPI is a useful marker. METHODS: We analyzed the prognostic factors, including CIPI, in 700 NSCLC patients treated by pulmonary resection. We also analyzed a subgroup of 482 patients with pathological stage I NSCLC. RESULT: CIPI > 14.59 (P < 0.01), maximum standardized uptake value (SUVmax) > 5.35 (P < 0.01), lymphatic invasion (P = 0.01), and pathological stage (P < 0.01) were significant factors for relapse-free survival (RFS) in multivariate analysis. SUVmax > 5.35 (P < 0.01) and pathological stage (P < 0.01) were revealed as significant factors for overall survival in the multivariate analysis. In the subanalysis, CIPI > 14.88 (P = 0.01) and SUVmax > 5.07 (P < 0.01) were significant factors for RFS of pathological stage I NSCLC in multivariate analysis. CONCLUSION: CIPI was a significant factor for RFS in NSCLC patients treated surgically, even in those with pathological stage I disease. SUVmax was also a significant factor for RFS and overall survival in NSCLC patients treated surgically, and for RFS in patients with pathological stage I NSCLC. TRIAL REGISTRATION: The Institutional Review Board of Kanazawa Medical University approved the protocol of this retrospective study (Approval Number: I392), and written informed consent was obtained from all patients.

PD-L1 Expression is not a Predictive Factor for Recurrence in Resected Non-small Cell Lung Cancer.

PURPOSE: Although targeting programmed death-1 (PD-1) and its ligand, programmed death-ligand 1 (PD-L1), is an established treatment modality for non-small cell lung cancer (NSCLC), the prognostic relevance of PD-L1 expression in NSCLC patients who undergo pulmonary resection is controversial. METHODS: Two hundred thirty-seven NSCLC patients who underwent pulmonary resection were enrolled and the relationship between PD-L1 and various clinicopathological factors, as well as the prognostic relevance of PD-L1, was evaluated. RESULTS: PD-L1 expression was significantly higher in male patients (p < 0.01), lymphatic invasion (p < 0.01), vascular invasion (p < 0.01), grade 3-4 differentiation (p < 0.01), squamous cell carcinoma (p < 0.01), and pathological stage > II (p < 0.01), but significantly lower in those who were epithelial growth factor receptor (EGFR) mutation negative (p < 0.01). Relapse-free survival was significantly worse in patients with PD-L1 expression (p = 0.04). Univariate analysis showed that male sex (p = 0.04), carcinoembryonic antigen expression (CEA) (p < 0.01), maximum standardized uptake value (p < 0.01), lymphatic invasion (p < 0.01), vascular invasion (p < 0.01), grade 3-4 differentiation (p < 0.01), lower lobe disease (p = 0.04), PD-L1 expression (p = 0.03), and pathological stage (p < 0.01) were significant risk factors of recurrence. In multivariate analysis, CEA expression (p = 0.01), lymphatic invasion (p = 0.04), and pathological stage (p < 0.01) were risk factors for recurrence, whereas PD-L1 expression was not a significant factor of recurrence (p = 0.62). CONCLUSION: PD-L1 expression was not a risk factor of recurrence but tumor progression tended to increase PD-L1 expression. TRIAL REGISTRATION: The Institutional Review Board of Kanazawa Medical University approved the protocol of this retrospective study (approval number: I392), and written informed consent was obtained from all patients.

[Pulmonary Nodule Localization by Pleural Marking Using Virtual Thoracoscopic Imaging].

Percutaneous or transbronchial markings are performed to localize pulmonary nodules preoperatively. We present a novel intraoperative procedure that utilizes virtual thoracoscopic imaging-assisted pleural marking. In this procedure, a virtual thoracoscopic image is created preoperatively, and the coordinates of the pleural point above the tumor are determined. The pleural marker is intraoperatively placed on the coordinates, and dye is transferred to the visceral pleura with two lung ventilations. We present the specific procedures and countermeasures for cases when nodules are not palpable. Additionally, we present a comparison between the various methods of preoperative marking and this method.

Analysis of risk factors for postoperative complications in non-small cell lung cancer: comparison with the Japanese National Clinical Database risk calculator.

BACKGROUND: Although the risk calculator of the National Clinical Database (RC-NCD) has been widely used to predict the occurrence of mortality and major morbidity in Japan, it has not been demonstrated whether a correlation between the calculated RC-NCD risk score and the actual occurrence of mortality and severe morbidity exists. METHODS: The clinical data of 585 patients who underwent pulmonary resection for non-small cell lung cancer were collected, and the risk factors for postoperative morbidity were analyzed to verify the validity of the RC-NCD. RESULTS: The coexistence of asthma (p = 0.02), nutrition lymphocyte ratio (p = 0.04), and pulmonary lobe (p < 0.01) were significant risk factors for postoperative morbidity in the present study, and the percent-predicted vital capacity (p < 0.01), pulmonary lobe (p = 0.03), and type of operative procedure (p = 0.01) were significant risk factors for severe postoperative morbidity. Furthermore, in patients received lobectomy, coexistence of asthma (p = 0.01) and pulmonary lobe (p < 0.01) were identified as significant risk factors for postoperative morbidity. Meanwhile, male sex (p = 0.01), high BMI (p < 0.01), low vital capacity (p = 0.04), and pulmonary lobe (p = 0.03) were identified as significant risk factors for severe postoperative morbidity. CONCLUSIONS: Given that the pulmonary lobe was a significant risk factor for postoperative morbidity in patients received pulmonary resection and for severe postoperative morbidity in patients received lobectomy, the RC-NCD for postoperative morbidity needs to be modified according to high-risk lobes. TRIAL REGISTRATION: The Institutional Review Board of Kanazawa Medical University approved the protocol of this retrospective study (approval number: I392), and written informed consent was obtained from all patients.

Individualization of risk factors for postoperative complication after lung cancer surgery: a retrospective study.

BACKGROUND: The risk factors for postoperative complications after pulmonary resection in patients with non-small cell lung cancer (NSCLC) have not been elucidated. METHODS: Clinical data of 956 patients with NSCLC were analyzed. Patient factors such as sex, age, comorbidities, smoking history, respiratory function, and the lobe involved in lung cancer and operative factors such as operative approach and operative procedures were collected and analyzed. RESULTS: Male sex (odds ratio [OR]: 1.73, 95% confidence interval [CI]: 1.09-2.75, p = 0.01), coexistence of asthma (OR 2.68, 95% CI 1.19-6.02, p = 0.01), low percentage of forced expiratory volume in 1 s (FEV1) (OR 1.41, 95% CI 1.02-1.95, p = 0.03), and lobectomy or greater resection (OR 2.47, 95% CI 1.66-3.68, p < 0.01) were identified as significant risk factors for postoperative complications. Male sex (OR 1.98; 95% CI 1.03-3.81, p = 0.03) and complete video-assisted thoracic surgery and robot-assisted thoracic surgery (OR 1.64; 95% CI 1.09-2.45; p = 0.01) were identified as significant risk factors for postoperative air leakage. Coexistence of asthma (OR 9.97; 95% CI 3.66-27.38; p < 0.01) was identified as a significant risk factor for postoperative atelectasis or pneumonia. Lobectomy or greater resection (OR 19.71; 95% CI 2.70-143.57; p < 0.01) was identified as a significant risk factor for postoperative arrhythmia. CONCLUSION: Male sex, coexistence of asthma, low percentage of FEV1, and operative procedure were significant risk factors for postoperative complications. Furthermore, risk factors varied according to postoperative complications.

Congenital partial pericardial defect discovered incidentally during surgery for lung cancer: a case report and literature review.

BACKGROUND: Congenital pericardial defects are rare congenital anomalies, often asymptomatic and incidentally detected during thoracic surgery. CASE PRESENTATION: A 74-year-old man with primary lung cancer (cT1cN0M0, Stage IA3) underwent thoracoscopic radical lobectomy. At the time of thoracotomy, a pericardial defect was found on the ventral side of the hilar region, and the left atrial appendage was exposed. Due to concern that contact between the bronchial stump and the left atrial appendage may lead to postoperative bleeding and heart hernia, the pericardial defect was closed with an expanded polytetrafluoroethylene GoreTex® membrane. Preoperative computed tomography was reanalyzed with a 1 mm slice, congenital pericardial defect was detected as the pericardium had penetrated under the left atrial appendage. CONCLUSIONS: In congenital partial pericardial defect, contact between the left atrial appendage and bronchial stump, due to movement of the lung or heart, increases the risk of bleeding after lung resection. Therefore, closure of the defect should be considered. Although it is difficult to diagnose congenital partial pericardial defect preoperatively, computed tomography taken with a slice thickness of 1 mm is useful for diagnosis.

Bottom-up Creation of an Artificial Cell Covered with the Adhesive Bacterionanofiber Protein AtaA.

The bacterial cell surface structure has important roles for various cellular functions. However, research on reconstituting bacterial cell surface structures is limited. This study aimed to bottom-up create a cell-sized liposome covered with AtaA, the adhesive bacterionanofiber protein localized on the cell surface of Acinetobacter sp. Tol 5, without the use of the protein secretion and assembly machineries. Liposomes containing a benzylguanine derivative-modified phospholipid were decorated with a truncated AtaA protein fused to a SNAP-tag expressed in a soluble fraction in Escherichia coli. The obtained liposome showed a similar surface structure and function to that of native Tol 5 cells and adhered to both hydrophobic and hydrophilic solid surfaces. Furthermore, this artificial cell was able to drive an enzymatic reaction in the adhesive state. The developed artificial cellular system will allow for analysis of not only AtaA, but also other cell surface proteins under a cell-mimicking environment. In addition, AtaA-decorated artificial cells may inspire the development of biotechnological applications that require immobilization of cells onto a variety of solid surfaces, in particular, in environments where the use of genetically modified organisms is prohibited.

The endogenous redox rhythm is controlled by a central circadian oscillator in cyanobacterium Synechococcus elongatus PCC7942.

The intracellular redox and the circadian clock in photosynthetic organisms are two major regulators globally affecting various biological functions. Both of the global control systems have evolved as systems to adapt to regularly or irregularly changing light environments. Here, we report that the two global regulators mutually interact in cyanobacterium Synechococcus elongatus PCC7942, a model photosynthetic organism whose clock molecular mechanism is well known. Electrochemical assay using a transmembrane electron mediator revealed that intracellular redox of S. elongatus PCC7942 cell exhibited circadian rhythms under constant light conditions. The redox rhythm disappeared when transcription/translation of clock genes is defunctionalized, indicating that the transcription/translation controlled by a core KaiABC oscillator generates the circadian redox rhythm. Importantly, the amplitude of the redox rhythm at a constant light condition was large enough to affect the KaiABC oscillator. The findings indicated that the intracellular redox state is actively controlled to change in a 24-h cycle under constant light conditions by the circadian clock system.

Cell-Membrane Permeable Redox Phospholipid Polymers Induce Apoptosis in MDA-MB-231 Human Breast Cancer Cells.

Induction of oxidative stress is an effective approach to causing apoptotic death of cancer cells. Since oxidative stress is generally caused by an intracellular redox imbalance, altering the intracellular redox is a promising strategy toward the growth suppression of cancer cells. Here, we attempted to induce apoptosis in MDA-MB-231 human breast cancer cells by adding a cell-membrane permeable redox phospholipid polymer that can alter the intracellular redox. We found that apoptosis and the deactivation of oxidative phosphorylation were induced in the MDA-MB-231 cells in the presence of the oxidized form of the redox polymer. Remarkably, such phenomena were not observed in the presence of the reduced form of the redox polymer that cannot intercept metabolic electrons. These results indicate that the redox polymer that mediates extracellular electron transfer (EET) generates oxidative stress, leading to the apoptosis of the cancer cells.

Efficient Counterselection for Methylococcus capsulatus (Bath) by Using a Mutated pheS Gene.

Methylococcus capsulatus (Bath) is a representative gammaproteobacterial methanotroph that has been studied extensively in diverse research fields. The sacB gene, which encodes levansucrase, causing cell death in the presence of sucrose, is widely used as a counterselectable marker for disruption of a target gene in Gram-negative bacteria. However, sacB is not applicable to all Gram-negative bacteria, and its efficiency for the counterselection of M. capsulatus (Bath) is low. Here, we report the construction of an alternative counterselectable marker, pheS*, by introduction of two point mutations (A306G and T252A) into the pheS gene from M. capsulatus (Bath), which encodes the α-subunit of phenylalanyl-tRNA synthetase. The transformant harboring pheS* on an expression plasmid showed sensitivity to 10 mM p-chloro-phenylalanine, whereas the transformant harboring an empty plasmid showed no sensitivity, indicating the availability of pheS* as a counterselectable marker in M. capsulatus (Bath). To validate the utility of the pheS* marker in counterselection, we attempted to obtain an unmarked mutant of xoxF, a gene encoding the major subunit of Xox methanol dehydrogenase, which we failed to obtain by counterselection using the sacB marker. PCR, immunodetection using an anti-XoxF antiserum, and a cell growth assay in the absence of calcium demonstrated successful disruption of the xoxF gene in M. capsulatus (Bath). The difference in counterselection efficiencies of the markers indicated that pheS* is more suitable than sacB for counterselection in M. capsulatus (Bath). This study provides a new genetic tool enabling efficient counterselection in M. capsulatus (Bath).IMPORTANCE Methanotrophs have long been considered promising strains for biologically reducing methane from the environment and converting it into valuable products, because they can oxidize methane at ambient temperatures and pressures. Although several methodologies and tools for the genetic manipulation of methanotrophs have been developed, their mutagenic efficiency remains lower than that of tractable strains such as Escherichia coli Therefore, further improvements are still desired. The significance of our study is that we increased the efficiency of counterselection in M. capsulatus (Bath) by employing pheS*, which was newly constructed as a counterselectable marker. This will allow for the efficient production of gene-disrupted and gene-integrated mutants of M. capsulatus (Bath). We anticipate that this counterselection system will be utilized widely by the methanotroph research community, leading to improved productivity of methane-based bioproduction and new insights into methanotrophy.