Modification of a neuronal network direction using stepwise photo-thermal etching of an agarose architecture

Control over spatial distribution of individual neurons and the pattern of neural network provides an important tool for studying information processing pathways during neural network formation. Moreover, the knowledge of the direction of synaptic connections between cells in each neural network can provide detailed information on the relationship between the forward and feedback signaling. We have developed a method for topographical control of the direction of synaptic connections within a living neuronal network using a new type of individual-cell-based on-chip cell-cultivation system with an agarose microchamber array (AMCA). The advantages of this system include the possibility to control positions and number of cultured cells as well as flexible control of the direction of elongation of axons through stepwise melting of narrow grooves. Such micrometer-order microchannels are obtained by photo-thermal etching of agarose where a portion of the gel is melted with a 1064-nm infrared laser beam. Using this system, we created neural network from individual Rat hippocampal cells. We were able to control elongation of individual axons during cultivation (from cells contained within the AMCA) by non-destructive stepwise photo-thermal etching. We have demonstrated the potential of our on-chip AMCA cell cultivation system for the controlled development of individual cell-based neural networks

Significance of Smoking as a Postoperative Prognostic Factor in Patients with Non-small Cell Lung Cancer

IntroductionIn this study, we investigated the influence of smoking on the postoperative prognosis in patients with non-small cell lung cancer.MethodsThe subjects consisted of 770 patients who underwent a resection of lung cancer in our department between 1994 and 2005. We compared the clinico-pathological findings between the smoking and never-smoking groups. The pack-year index (PYI) was used as a smoking index.ResultsThe smoking group consisted of 569 patients (74%), and the never-smoking group consisted of 201 patients (26%). The smokers were composed of 492 men and 77 women. Among the adenocarcinoma patients, there were 293 (61%) smokers and 185 (39%) never-smokers. The patients with squamous cell carcinoma included 204 (95%) smokers and 10 (5%) never-smokers. The proportion of patients with stage IA disease was significantly higher in the never-smokers than that of the smokers. The 5-year survival rate after surgery was 66% in the never-smoking group; however, the rates were 56% in patients with a PYI more than or equal to 20, and 55% in those with PYI more than 20. Seventy-nine (13.9%) patients in the smoking group and seven (3.5%) patients in the never-smoking group died of other diseases, with a significant difference (p < 0.01). Of these patients, 44 (56%) and 13 (16%) in the smoking group died of respiratory and cardiovascular disorders, respectively. In our series, excluding those who died of other diseases, there were no significant differences in the postoperative prognosis.ConclusionsIn the smoking group, the prognosis was poorer than that in the never-smoking group. The higher proportion of early stage disease (stage IA) and female gender were major causes of the better prognosis of the never-smokers. Nevertheless, the high pulmonary/cardiovascular complication-related mortality was another cause of the poor prognosis of the smokers with lung cancer

Improvement of a Plate Culture Method for Thiobacillus thiooxidans and Isolation of a New Strain

Thiobacillus thiooxidansは固体平板培地上でのコロニー形成が悪く,遺伝子操作を行うために欠かせない技術であるクローンの選抜が困難であったため,遺伝学的研究は従属細菌のそれに比して著しく遅れていた.本論文では,T.thiooxidansの個体平板培養法の改良を目的として,エネルギー源,ゲル化剤について検討し,得られた新しい固体培養法を用いて固体平板培地上で生育の速い菌株を分離した.新しい培地はSilvermanとLundgrenの9K培地の無機塩にエネルギー源として0.3% の四チオン酸,ゲル化剤として0.6% のジェランガムを用いた.分離株には,従属栄養生育能は認められなかったが,元素硫黄および四チオン酸を含む9K培地での良好な生育は確認できた.さらに,キノンがユビキノン8であり,菌体脂肪酸には3-ヒドロキシミリスチン酸が含まれること,GC含量が52% であること等の菌学的諸性質から分離株はT.thiooxidansに属する菌株であることが確認できた.この菌株と新しい平板培地を用いると,従属栄養細菌に匹敵する大型コロニーが得られ,細胞選抜を容易に行うことが出来る

Activity-dependent oligodendrocyte calcium dynamics and their changes in Alzheimer’s disease

Oligodendrocytes (OCs) form myelin around axons, which is dependent on neuronal activity. This activity-dependent myelination plays a crucial role in training and learning. Previous studies have suggested that neuronal activity regulates proliferation and differentiation of oligodendrocyte precursor cells (OPCs) and myelination. In addition, deficient activity-dependent myelination results in impaired motor learning. However, the functional response of OC responsible for neuronal activity and their pathological changes is not fully elucidated. In this research, we aimed to understand the activity-dependent OC responses and their different properties by observing OCs using in vivo two-photon microscopy. We clarified that the Ca2+ activity in OCs is neuronal activity dependent and differentially regulated by neurotransmitters such as glutamate or adenosine triphosphate (ATP). Furthermore, in 5-month-old mice models of Alzheimer’s disease, a period before the appearance of behavioral abnormalities, the elevated Ca2+ responses in OCs are ATP dependent, suggesting that OCs receive ATP from damaged tissue. We anticipate that our research will help in determining the correct therapeutic strategy for neurodegenerative diseases beyond the synapse

Expanding the Repertoire of Optogenetically Targeted Cells with an Enhanced Gene Expression System

Optogenetics has been enthusiastically pursued in recent neuroscience research, and the causal relationship between neural activity and behavior is becoming ever more accessible. Here, we established knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction (KENGE-tet) and succeeded in generating transgenic mice expressing a highly light-sensitive channelrhodopsin-2 mutant at levels sufficient to drive the activities of multiple cell types. This method requires two lines of mice: one that controls the pattern of expression and another that determines the protein to be produced. The generation of new lines of either type readily expands the repertoire to choose from. In addition to neurons, we were able to manipulate the activity of nonexcitable glial cells in vivo. This shows that our system is applicable not only to neuroscience but also to any biomedical study that requires understanding of how the activity of a selected population of cells propagates through the intricate organic systems

A randomized phase II study of S-1 monotherapy versus cisplatin with vinorelbine for completely resected stage II/IIIA non-small cell lung cancer: rationale and study protocol design for the LOGIK1702 study

Background: The current standard postoperative treatment for stage II-IIIA non-small cell lung cancer (NSCLC) is a regimen of platinum doublet adjuvant chemotherapy. These regimens, which are the same as for solid NSCLC tumors, often cause severe adverse reactions in the treated patients. Therefore, an effective treatment regimen with fewer side effects is needed.Methods/design: The purpose of this study is to evaluate the effectiveness and safety of S-1 monotherapy (80 mg/m2 orally administrated twice daily, at day 1–14, 16 cycles) and cisplatin with vinorelbine combination therapy (cisplatin 80 mg/m2 at day 1,vinorelbine 25 mg/m2 at day 1, 8, 4 cycles) in patients with II/IIIA stage non-small-cell lung cancer who underwent a total resection. In addition, we will also evaluate the level of treatment side effects by assessing quality of life (QOL), work productivity and activity performance. The primary endpoint is a 2-year relapse free survival (RFS) and the second primary endpoints are 2-year overall survival (OS), rate of treatment completion, safety, work productivity and activity, and quality of adjusted life years (QALY). At the same time, we aim to obtain precise information required to perform future phase 3 randomized controlled trials. The study is designed to estimate the primary endpoint with accuracy determined as the width of its 95% confidence interval to be less than 20%. Recruitment started in May 2017 and is ongoing.Discussion: This study has been conceived to establish a superior regimen for completely resected NSCLC based on efficacy, safety and QOL.Trial registration: Registry number: UMIN000027435. Registered May 22, 2017

Randomized phase II study of pemetrexed or pemetrexed plus bevacizumab for elderly patients with previously untreated non-squamous non-small cell lung cancer: Results of the Lung Oncology Group in Kyushu (LOGIK1201)

Objectives: To evaluate the efficacy and safety, we conducted a randomized phase II study of pemetrexed (Pem) versus Pem + bevacizumab (Bev) for elderly patients with non-squamous non-small cell lung cancer (NSqNSCLC). Patients and methods: The eligibility criteria were as follows: NSqNSCLC, no prior therapy,stage IIIB/IV disease or postoperative recurrence, age: ?75 years, performance status (PS): 0?1, and adequate bone marrow function. The patients were randomly assigned (1:1 ratio)to receive Pem or Pem + Bev. The primary endpoint was progression-free survival (PFS).The secondary endpoints were the response rate, OS, toxicities, and cost-effectiveness. Results: Forty-one patients were enrolled and 40 (20 from each group) were assessable. Their characteristics were as follows: male/female = 23/17; median age (range) = 78 (75?83); stage IIIB/IV/postoperative recurrence = 1/30/9; PS 0/1 = 11/29. All cases involved adenocarcinoma.There was no significant intergroup difference in PFS and the median PFS (95% confidence interval) values of the Pem and Pem + Bev groups were 5.4 (3.0?7.4) and 5.5 (3.6?9.9) months, respectively (p = 0.66). The response rate was significantly higher in the Pem + Bev group(15% vs. 55%, p = 0.0146), and there was no significant difference in OS (median: 16.0 vs. 16.4 months, p = 0.58). Grade 3 and 4 leukopenia, neutropenia,and thrombocytopenia were seen in 10 and 30, 20 and 55, and 5 and 5 cases, respectively. Drug costs were higher in the Pem + Bev group (median: 1,522,008 vs. 3,368,428 JPY, p = 0.01). No treatment-related deaths occurred. Conclusions: Adding Bev to Pem did not result in improved survival in the elderly NSqNSCLC patients. Compared with Pem + Bev, Pem monotherapy had similar effects on survival, a more favorable toxicity profile, and was more cost-effective in elderly NSqNSCLC patients. Pem monotherapy might be one of the optional regimen for NSqNSCLC patients aged ?75 years

Integrated genetic and clinical prognostic factors for aggressive adult T-cell leukemia/lymphoma

成人T細胞白血病リンパ腫（ATL）におけるゲノム情報と臨床情報を統合したリスクモデルを確立 --ATLの個別化医療を推進--. 京都大学プレスリリース. 2023-04-10.The prognosis of aggressive adult T-cell leukemia/lymphoma (ATL) is poor, and allogeneic hematopoietic stem-cell transplantation (allo-HSCT) is a curative treatment. To identify favorable prognostic patients after intensive chemotherapy, and who therefore might not require upfront allo-HSCT, we aimed to improve risk stratification of aggressive ATL patients aged <70 years. The clinical risk factors and genetic mutations were incorporated into risk modeling for overall survival (OS). We generated the m7-ATLPI, a clinicogenetic risk model for OS, that included the ATL prognostic index (PI) (ATL-PI) risk category, and non-silent mutations in seven genes, namely TP53, IRF4, RHOA, PRKCB, CARD11, CCR7, and GATA3. In the training cohort of 99 patients, the m7-ATLPI identified a low-, intermediate-, and high-risk group with 2-year OS of 100%, 43%, and 19%, respectively (hazard ratio [HR] 5.46, p < 0.0001). The m7-ATLPI achieved superior risk stratification compared to the current ATL-PI (C-index 0.92 vs. 0.85, respectively). In the validation cohort of 84 patients, the m7-ATLPI defined low-, intermediate-, and high-risk groups with a 2-year OS of 81%, 30%, and 0%, respectively (HR 2.33, p = 0.0094), and the model again outperformed the ATL-PI (C-index 0.72 vs. 0.70, respectively). The simplified m7-ATLPI, which is easier to use in clinical practice, achieved superior risk stratification compared to the ATL-PI, as did the original m7-ATLPI; the simplified version was calculated by summing the following: high-risk ATL-PI category (+10), low-risk ATL-PI category (−4), and non-silent mutations in TP53 (+4), IRF4 (+3), RHOA (+1), PRKCB (+1), CARD11 (+0.5), CCR7 (−2), and GATA3 (−3)

Characterization of two fungal lipoxygenases expressed in Aspergillus oryzae.

International audienceTwo fungal lipoxygenase genes were cloned from a rice pathogen, Magnaporthe salvinii, and the take-all fungus, Gaeumannomyces graminis var. tritici, and successfully expressed in Aspergillus oryzae in secreted form. The lipoxygenases expressed, termed MLOX and GLOX, were purified and characterized to evaluate suitability for industrial applications. Both enzymes were active broadly at pH 4-11 and had optimum temperatures around 60 °C, but they were largely different in substrate specificity. Where MLOX was active broadly on arachidonic acid, EPA and DHA, and even on derivatives of fatty acids, such as methyl linoleate or linoleoyl alcohol, GLOX was more specific to linoleic acid and linolenic acid. The most remarkable difference between the two fungal LOXs was the positional and stereo-specificity of oxygenation reactions on polyunsaturated fatty acids. When using linoleic acid as the substrate, the product of MLOX was 9S-hydroperoxy-(E,Z)-octadecadienoic acid (9S(E,Z)-HPODE), on the other hand, the product of GLOX was 13R(E,Z)-HPODE. The enzymes were evaluated for a couple of potential applications and found to be effective on bleaching colored compounds such as carotenoids