Study on the method to quantify viable bacterial cellson the surface of endotracheal suction catheters

気管内吸引カテーテルに付着した一般細菌の生菌数測定方法について,超音波法およびチューブミキサーによ攪拌法を用いて検討した｡まず,人工的に緑膿菌を付着させた気管内吸引カテーテルを超音波処理することにより生菌数を測定した｡その結果,処理時間が1分を経過すると生菌数は減少をはじめ経時的に減少傾向を示した｡一方,攪拌法では0.5分の処理をピーク値としてその後の減少傾向は認められなかった｡次に,在宅療養患者に使用したカテーテルをチューブミキサーで0.5分攪拌後,生菌数の測定を行った｡また,同じカテーテルを用いて走査型電子顕微鏡による観察を行った結果,画面上の細菌数の印象と生菌数の測定結果に矛盾はなかった｡これらのことから気管内吸引カテーテルに付着した一般細菌の生菌数測定方法として,生理食塩水に入れたカテーテルをチューブミキサーで攪拌する方法が有用であると考えられた｡As a method for the detection of viable bacteria attached to endotracheal suction catheters, we evaluated sonication and dissociation using a tube mixer. The catheter fragments with Pseudomonas aeruginosa PAO1 were treated by each of the two methods, and viable cells in the elutions were counted. The highest number of viable cells was observed at 0.5 min by either method. The viable cell count decreased when the sonication time exceeded 1 min, while only a slight decrease of viable cells was observed by using a tube mixer. The catheters used for patients receiving care at home were fragmented and treated by a tube mixer to detach bacteria, and viable cells were counted. Electron microscopy observation showed an association between the viable cell count and morphology of surfaces of the catheters. These results suggest that adequate removal of bacteria attached to endotracheal suction catheters is possible by agitating catheter fragments for 0.5 min in physiological saline using a tube mixer

IMiDs uniquely synergize with TKIs to upregulate apoptosis of Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressing a dominant-negative IKZF1 isoform

Abstract The long-term prognosis of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL) is still unsatisfactory even after the emergence of tyrosine kinase inhibitors (TKIs) against chimeric BCR-ABL, and this is associated with the high incidence of genetic alterations of Ikaros family zinc finger 1 (IKZF1), most frequently the hemi-allelic loss of exons 4–7 expressing a dominant-negative isoform Ik6. We found that lenalidomide (LEN), a representative of immunomodulatory drugs (IMiDs), which have been long used for the treatment of multiple myeloma, specifically induced accumulation of Ik6 with the disappearance of functional isoforms within 24 h (i.e., abrupt and complete shut-down of the IKZF1 activity) in Ik6-positive Ph+ALL cells in a neddylation-dependent manner. The functional IKZF3 isoforms expression was also abruptly and markedly downregulated. The LEN treatment specifically suppressed proliferation of Ik6-positive-Ph+ALL cells by inducing cell cycle arrest via downregulation of cyclins D3 and E and CDK2, and of importance, markedly upregulated their apoptosis in synergy with the TKI imatinib (IM). Apoptosis of IM-resistant Ph+ALL cells with T315I mutation of BCR-ABL was also upregulated by LEN in the presence of the newly developed TKI ponatinib. Analyses of flow cytometry, western blot, and oligonucleotide array revealed that apoptosis was caspase-/p53-dependent and associated with upregulation of pro-apoptotic Bax/Bim, enhanced dephosphorylation of BCR-ABL/Akt, and downregulation of oncogenic helicase genes HILLS, CDC6, and MCMs4 and 8. Further, the synergism of LEN with IM was clearly documented as a significant prolongation of survival in the xenograft mice model. Because this synergism was further potentiated in vitro by dexamethasone, a key drug for ALL treatment, the strategy of repositioning IMiDs for the treatment of Ik6-positive Ph+ALL patients certainly shed new light on an outpatient-based treatment option for achieving their long-term durable remission and higher QOL, particularly for those who are not tolerable to intensified therapeutic approaches

Involvement of BCR::ABL1 in laminin adhesion of Philadelphia chromosome‐positive acute lymphoblastic leukemia through upregulation of integrin α6

Abstract Background Adhesion of cancer cells to extracellular matrix laminin through the integrin superfamily reportedly induces drug resistance. Heterodimers of integrin α6 (CD49f) with integrin β1 (CD29) or β4 (CD104) are major functional receptors for laminin. Higher CD49f expression is reportedly associated with a poorer response to induction therapy in childhood B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL). Moreover, a xenograft mouse model transplanted with primary BCP‐ALL cells revealed that neutralized antibody against CD49f improved survival after chemotherapy. Aims Considering the poor outcomes in Philadelphia chromosome (Ph)‐positive ALL treated with conventional chemotherapy without tyrosine kinase inhibitors, we sought to investigate an involvement of the laminin adhesion. Methods and results Ph‐positive ALL cell lines expressed the highest levels of CD49f among the BCP‐ALL cell lines with representative translocations, while CD29 and CD104 were ubiquitously expressed in BCP‐ALL cell lines. The association of Ph‐positive ALL with high levels of CD49f gene expression was also confirmed in two databases of childhood ALL cohorts. Ph‐positive ALL cell lines attached to laminin and their laminin‐binding properties were disrupted by blocking antibodies against CD49f and CD29 but not CD104. The cell surface expression of CD49f, but not CD29 and CD104, was downregulated by imatinib treatment in Ph‐positive ALL cell lines, but not in their T315I‐acquired sublines. Consistently, the laminin‐binding properties were disrupted by the imatinib pre‐treatment in the Ph‐positive ALL cell line, but not in its T315I‐acquired subline. Conclusion BCR::ABL1 plays an essential role in the laminin adhesion of Ph‐positive ALL cells through upregulation of CD49f

A Stand Structure Dominated by Betula maximowicziana in the Chichibu Mountains, Central Japan

1．秩父山地の標高1,300m地点にあるウダイカンバ優占林分内に0.96haの調査区（以下，秩父区）を設置し，毎木調査から林分構造を，樹齢測定から林齢を明らかにした。2．秩父区内に出現した樹種数は39種，立木本数は1,177本/ha，BA合計は42.0m2/ha，材積は330.0m3/haであった。そのうちウダイカンバは本数が83本/ha（7.1％），BA合計が15.8m2/ha（37.5％），材積が154.7m3/ha（46.9％）であった。3．秩父区では22m以上の階層でウダイカンバが圧倒的に優占する第一層，14～22mの階層はミズメ，ミズナラ，イヌシデなどの多い第二層，および4～14mの階層はカエデ類，シデ類，アラゲアオダモ，リョウブの多い第三層の3つの階層が確認された。4．秩父区内とその周辺にある枯死木20本の樹齢測定の結果から，調査木の年輪数は83～120年の範囲にあり，そのうち近年枯死した5個体の年輪数から秩父区は約120年前に更新したものと推定された。5．秩父区と，山火事後に更新したウダイカンバ優占二次林である東京大学北海道演習林固定標準地の北演5133（3回間伐，林齢91年），北演5236（無間伐，林齢86年）を比較したところ，秩父区は本数密度と年直径成長量で北演5133に近い値を示し，平均胸高直径と材積で北演5133と北演5236より大きかった。しかし，秩父区のウダイカンバは用材として評価した場合，北演5133のそれより劣っており，秩父演習林に多い林齢100年未満のウダイカンバ二次林で用材生産をする場合には，優良木を選木してその直径成長を促すための間伐を実施する必要があると考えられた。6．秩父区ではミズナラがウダイカンバに比べて寿命が長く，耐陰性も高いことから，今後の長い遷移過程において徐々にウダイカンバが枯死し，それに代わって現在第一層，第二層にあるミズナラが優占していくものと推察された。In order to investigate the floristic composition and structure of a stand dominated by Betula maximowizciana, a research plot with an area of 0.96 ha was established at an altitude of 1,300m a.s.l. in the Chichibu Mountains, central Japan. Diameter at breast height (dbh) and the height of all the trees with dbh larger than 5.0cm were measured in the plot. Thirty-nine tree species were recorded in the plot. The number of trees, basal area and stand volume were 1,177/ha, 42.0m2/ha and 330.0m3/ha, respectively. For B. maximowicziana, these values were 83/ha (7.1%), 15.8m2/ha (37.5%) and 154.7m3/ha (46.9%), respectively. The stand was stratified into three layers, the first-layer (height of layer: hl>22m) in which the predominant species was B. maximowicziana, the second-layer (14<hl≦22m) in which B. grossa, Quercus crispula and Carpinus tschonoskii were abundant and the third-layer (4<hl≦14m) in which Acer spp. Carpinus spp., Fraxinus languinosa and Clethra barvinervis were abundant. From the age analysis of twenty dead trees sampled within the plot and its surroundings, tree ages were in the range of 83 to 120 years. From the age analysis of five sample trees, which seemed to die recently, it was estimated that this stand was regenerated about 120 years ago. Numerical values of the stand in Chichibu plot were compared to those of the stands dominated by B. maximowicziana in two plots (plot no. 5133 and 5236) in the Tokyo Univ. Forest in Hokkaido, northern Japan. They were regenerated after forest fire and the stand age of plot no. 5133 and 5236 were 91 and 86 years, respectively. Qualitative thinning has been conducted three times in the former plot but this was not the case in the latter. Tree density and mean annual diameter growth rate of the Chichibu plot were similar to those of plot no. 5133. On the other hand, mean dbh and stand volume at the Chichibu plot was larger than those of the two plots in Hokkaido. However, from the viewpoint of the commercial value of wood utilization, the morphological characteristics of the B. maximowizciana trees in the Chichibu plot are inferior to those of the trees in plot no. 5133 where qualitative thinning has been conducted. Hence, for the production of high-quality large diameter trees, qualitative thinning should also be conducted especially to B. maximowicziana stands with an age less than 100 years. These are abundant in the Tokyo Univ. Forest in Chichibu. It is thought that the B. maximowicziana trees composing the canopy layer in the Chichibu plot at present are gradually being replaced by Quercus crispula in the long course of succession, since the latter, characterized by its longer life span and higher shade tolerance, has an advantage over the former

Anti-leukemic activity of bortezomib and carfilzomib on B-cell precursor ALL cell lines

<div><p>Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated <i>in vitro</i> BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+) ALL, <i>IKZF1</i> deletion, and biallelic loss of <i>CDKN2A</i> were associated with favorable response. Even in Ph-negative ALL cell lines, <i>IKZF1</i> deletion and bilallelic loss of <i>CDKN2A</i> were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin) in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ), a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17;19) ALL cell lines, and <i>IKZF1</i> deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17;19) ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17;19) ALL cell line to CFZ. These observations suggested that <i>IKZF1</i> deletion could be a useful biomarker to predict good sensitivity to CFZ and BTZ, and that CFZ combination chemotherapy may be a new therapeutic option with higher anti-leukemic activity for refractory ALL that contain P-glycoprotein-negative leukemia cells.</p></div

Long-term phenology data on woody plants at The University of Tokyo Hokkaido Forest from 1930 to 2010

地球温暖化自体注目され始めたのが1980 年代に入ってからであるため，気候変動の研究に資する長期データは世界的にも限られている。東京大学北海道演習林では1930 年から樹木のフェノロジーを調査してきた。しかし，元データが公表された最初の8 年間を除くと，解析した結果が公表されたのみで，外部の研究者が貴重な長期データを利用できる状況になかった。一方で，元データが部分的に紛失した期間があり，調査対象個体の継続性についても未整理の状態で，内部の研究者でも解析できる状況になかった。そこで，本報告では，しばしば変更された調査項目や調査個体と個体数，観測者を整理して，時系列データの接続性を検討した。そして，できる限り調査個体すべての元データを数値データとして公表した。The number of long-term datasets available for research into climate change is limited because global warming has only been a major research subject since the 1980s. The University of Tokyo Hokkaido Forest (UTHF) has accumulated phenology data of woody plants since 1930. However, researchers outside the UTHF have not been able to utilize these valuable long-term data because only analyzed data have been published, with the exception of the first 8 years when original data were published. Also, it is even difficult for researchers at the UTHF to use these data because the continuity of the time-series is not clear and because the original data for the 13 years from 1938 to 1950 was lost. In this paper, the continuity of the long-term time-series data was clarified by organizing observed events, observed species and individuals, the number of individual trees for each species, and observers, which have been frequently changed. We have tried to publish the original numerical data (=dates) for all the individual trees that have been observed

Effect of overexpression and knockout of P-glycoprotein on carfilzomib sensitivity.

<p>(A) Cell surface expression of P-glycoprotein in parental 697 cells and 697R cells. Dotted and solid lines indicate fluorescence intensities of isotype control and anti-P-glycoprotein (P-gp) antibodies in parental cells, respectively. Shade indicates fluorescence intensity of anti-P-glycoprotein antibody in 697R cells. (B) Effects of P-glycoprotein (P-gp) inhibitors on carfilzomib (upper panels) and bortezomib (lower panels) sensitivities in parental 697 cells (left panels) and 697R cells (right panels). The vertical axes indicate % viability determined by alamarBlue cell viability assay and the horizontal axes indicate the concentration of carfilzomib or bortezomib. Blue, red, and green lines indicate dose response curves of carfilzomib or bortezomib alone, carfilzomib or bortezomib in combination with 0.8 μM of nilotinib, and carfilzomib or bortezomib in combination with 5 μM of verapamil, respectively. Mean values in triplicated analyses are indicated, and asterisks are indicated when p value in t-test is <0.01. (C) Induction of apoptotic cell death by carfilzomib in combination with P-glycoprotein (P-gp) inhibitors. Parental 697 cells (left panels) and 697R cells (right panels) were cultured with 20 nM of carfilzomib in the presence or absence of 0.8 μM of nilotinib or 5 μM of verapamil for 18 hours, and analyzed with Annexin V-binding (horizontal axis) and 7AAD-staining (vertical axis) using flow cytometry. The percentages of living cells (Annexin V-negative/7AAD-negative) and early (Annexin V-positive/ 7AAD-negative) and late (Annexin V-positive/ 7AAD-positive) apoptotic cells are indicated. (D) Effect of P-glycoprotein knockout on bortezomib and carfilzomib sensitivities in HALO1 cells. P-glycoprotein-positive HALO1 cells were electroporated with an <i>ABCB1</i>-specific CRISPR/Cas 9 vector that contains cDNA of Cas9 fused to human CD4 cDNA via a 2A peptide sequence. 48 hours after electroporation, CD4-positive cells were harvested using anti-CD4 antibody. Upper middle panel indicates flow cytometric analysis of CD4 expression 3 days after electroporation. Upper left and upper right panels indicate flow cytometric analyses of P-glycoprotein (P-gp) expression in parental and CD4-positive population of HALO1 cells, respectively. Bottom panels indicate two color analysis of P-glycoprotein (P-gp) expression (vertical axis) and Annexin V-binding (horizontal axis) by flow cytometry in the CD4-positive populations of HALO1 cells treated with 20nM of bortezomib and 20nM of carfilzomib for 12 hours. Cell viabilities in P-glycoprotein (P-gp)-positive and negative populations are indicated at the left side of each panel.</p