電顕でtime-lapse

Science 2010; 328:187 - 193

Four-Dimensional Electron Microscopy

Ahmed H. Zewail

The discovery of the electron over a century ago and the realization of its dual character have given birth to one of the two most powerful imaging instruments: the electron microscope. The electron microscope’s ability to resolve three-dimensional (3D) structures on the atomic scale is continuing to affect different fields, including materials science and biology. In this Review, we highlight recent developments and inventions made by introducing the fourth dimension of time in electron microscopy. Today, ultrafast electron microscopy (4D UEM) enables a resolution that is 10 orders of magnitude better than that of conventional microscopes, which are limited by the video-camera rate of recording. After presenting the central concept involved, that of single-electron stroboscopic imaging, we discuss prototypical applications, which include the visualization of complex structures when unfolding on different length and time scales. The developed UEM variant techniques are several, and here we illucidate convergent-beam and near-field imaging, as well as tomography and scanning-pulse microscopy. We conclude with current explorations in imaging of nanomaterials and biostructures and an outlook on possible future directions in space-time, 4D electron microscopy.

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生体材料で本当に電顕－計時観察が実現すると、かなりたくさんのことがわかりそうな気がする。

Proc Natl Acad Sci U S A. 2010;107(8):3829-33

Thalamic deactivation at sleep onset precedes that of the cerebral cortex in humans.

Magnin M, Rey M, Bastuji H, Guillemant P, Mauguiere F, Garcia-Larrea L.

Thalamic and cortical activities are assumed to be time-locked throughout all vigilance states. Using simultaneous intracortical and intrathalamic recordings, 

we demonstrate here that the thalamic deactivation occurring at sleep onset most often precedes that of the cortex by several minutes, whereas reactivation of both structures during awakening is synchronized. Delays between thalamus and cortex deactivations can vary from one subject to another when a similar cortical region is considered. In addition, heterogeneity in activity levels throughout the cortical mantle is larger than previously thought during the descent into sleep. Thus, asynchronous thalamo-cortical deactivation while falling asleep probably explains the production of hypnagogic hallucinations by a still-activated cortex and the common self-overestimation of the time needed to fall asleep.

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●入眠時にThalamusのほうがcortexより活動が早く（数分～数十分）ことを、ヒト検体を用いて検証。覚醒時はほぼ同時。

●the dimention of activation (DA)という、波形解析のアルゴリズムを用いて脳波を解析→大きな同期性徐波が出る睡眠時は数値が低下、ランダムで細かな波がでる覚醒時は数値が増加

fMRIによる、ヒト脳の機能的マップ

Nature Methods 7, 253 (2010) 

"A new study, pooling brain-imaging data from 35 centers across the world, shows the power of data sharing and demonstrates a universal architecture of functional connections in the human brain."

該当サイトはこちら。

Dev Biol. 331:210-21, 2009

Active cell movements coupled to positional induction are involved in lineage segregation in the mouse blastocyst.

In the mouse blastocyst, some cells of the inner cell mass (ICM) develop into primitive endoderm (PE) at the surface, while deeper cells form the epiblast. It remained unclear whether the position of cells determines their fate, such that gene expression is adjusted to cell position, or if cells are pre-specified at random positions and then sort. We have tracked and characterised dynamics of all ICM cells from the early to late blastocyst stage. Time-lapse microscopy in H2B-EGFP embryos shows that a large proportion of ICM cells change position between the surface and deeper compartments. Most of this cell movement depends on actin and is associated with cell protrusions. We also find that while most cells are precursors for only one lineage, some give rise to both, indicating that lineage segregation is not complete in the early ICM. Finally, changing the expression levels of the PE marker Gata6 reveals that it is required in surface cells but not sufficient for the re-positioning of deeper cells. We provide evidence that Wnt9A, known to be expressed in the surface ICM, facilitates re-positioning of Gata6-expressing cells. Combining these experimental results with computer modelling suggests that PE formation involves both cell sorting movements and position-dependent induction.

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●Blastcyst内の細胞の動きをtime-lapseでモニターし、epi-PEのsegrigationを説明する3つのモデル（Positional, Salt&Pepper, Cell sorting+positonal induction）のうち、最後のモデルに妥当性があることをコンピューターシミュレーションを用いて示す。
●Gata6+細胞はICMの表層側に来るが、この移動にはWnt9aが必要

●4/7数理ゼミで議論された論文

Dev Cell. 16:398-410, 2009

The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass.

Outside cells of the preimplantation mouse embryo form the trophectoderm (TE), a process requiring the transcription factor Tead4. Here, we show that transcriptionally active Tead4 can induce Cdx2 and other trophoblast genes in parallel in embryonic stem cells. In embryos, the Tead4 coactivator protein Yap localizes to nuclei of outside cells, and modulation of Tead4 or Yap activity leads to changes in Cdx2 expression. In inside cells, Yap is phosphorylated and cytoplasmic, and this involves the Hippo signaling pathway component Lats. We propose that active Tead4 promotes TE development in outside cells, whereas Tead4 activity is suppressed in inside cells by cell contact- and Lats-mediated inhibition of nuclear Yap localization. Thus, differential signaling between inside and outside cell populations leads to changes in cell fate specification during TE formation.

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CDBより。
●Tead4-Cdx2 pathwayによるTE分化制御について
●Tead4の発現はpan-embryonicなのに、なぜTEでのみ機能するか？

●Cell-cell contacts→Hippo→Lats1/2---|Yorkie/Yap1→Tead4経路活性化

●活性化型Tead4として、Tead4VP16を使用（いいのか？）
●Tead4haCdx2を含むTE細胞分化にかかわる因子群を制御（Fig1～2）
●核内YapとCdx2の発現がcorrelate、かつYapでCdx2の発現が制御される（Fig.3，4)
●Wwtr1（Yap-related)がcompensativeに機能
●Yap/Wwtr1のkinaseであるLats1/2がYapのリン酸化を介して核外排出を促進
●E-cadherin阻害剤のECCD1を用いると、Yapの核外排出とリン酸化が阻害されることから、ICMのcell-cell interactionが（Hippoを介して）Yapのリン酸化+核外排出に重要（Fig6)
●胚をばらしてreaggregateしたもの/immunosurgeryでICMだけ出してcultureしたもので、cell positionによってYapの核局在が規定されていることを証明（Fig7）
●モデル（Fig7)

Immunosurgeryについての文献は。
http://www.pnas.org/content/72/12/5099.full.pdf
http://media.wiley.com/product_data/excerpt/68/04700335/0470033568.pdf

Molecular Psychiatry 15:154-165, 2010

Conditional corticotropin-releasing hormone overexpression in the mouse forebrain enhances rapid eye movement sleep

Impaired sleep and enhanced stress hormone secretion are the hallmarks of stress-related disorders, including major depression. The central neuropeptide, corticotropin-releasing hormone (CRH), is a key hormone that regulates humoral and behavioral adaptation to stress. Its prolonged hypersecretion is believed to play a key role in the development and course of depressive symptoms, and is associated with sleep impairment. To investigate the specific effects of central CRH overexpression on sleep, we used conditional mouse mutants that overexpress CRH in the entire central nervous system (CRH-COE-Nes) or only in the forebrain, including limbic structures (CRH-COE-Cam). Compared with wild-type or control mice during baseline, both homozygous CRH-COE-Nes and -Cam mice showed constantly increased rapid eye movement (REM) sleep, whereas slightly suppressed non-REM sleep was detected only in CRH-COE-Nes mice during the light period. In response to 6-h sleep deprivation, elevated levels of REM sleep also became evident in heterozygous CRH-COE-Nes and -Cam mice during recovery, which was reversed by treatment with a CRH receptor type 1 (CRHR1) antagonist in heterozygous and homozygous CRH-COE-Nes mice. The peripheral stress hormone levels were not elevated at baseline, and even after sleep deprivation they were indistinguishable across genotypes. As the stress axis was not altered, sleep changes, in particular enhanced REM sleep, occurring in these models are most likely induced by the forebrain CRH through the activation of CRHR1. CRH hypersecretion in the forebrain seems to drive REM sleep, supporting the notion that enhanced REM sleep may serve as biomarker for clinical conditions associated with enhanced CRH secretion.

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●Camk2a-CRE x R26-CRHの系で、BFにCRHを発現
●ストレス→corticosterone系について。しかしデータは微妙。上がるのがREMってのも・・・

Trends Cell Biol. 2010 Mar 29. [Epub ahead of print]

The Beclin 1-VPS34 complex - at the crossroads of autophagy and beyond.

An increasing body of research on autophagy provides overwhelming evidence for its connection to diverse biological functions and human diseases. Beclin 1, the first mammalian autophagy protein to be described, appears to act as a nexus point between autophagy, endosomal, and perhaps also cell death pathways. Beclin 1 performs these roles as part of a core complex that contains vacuolar sorting protein 34 (VPS34), a class III phosphatidylinositol-3 kinase. The precise mechanism of Beclin 1-mediated regulation of these cellular functions is unclear, but substantial progress has recently been made in identifying new players and their functions in Beclin 1-VSP34 complexes. Here we review emerging studies that are beginning to unveil the physiological functions of Beclin 1-VPS34 in the central control of autophagic activity and other trafficking events through the formation of distinct Beclin 1-VPS34 protein complexes.

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Beclin1-Vps34複合体とその周辺についてよくまとまったレビュー
UVRAGとRubiconについてがメイン。機能的役割分担がよくわかる図が有り。

noise&signal

ICT Resultsより

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When noise becomes the signal

European researchers have developed a new class of electronics that uses noise ? normally a problem ? as part of the signal. It means better, faster electronics.

　 ：

These are simple computing, logic gates. Their actions resemble the firing of signals as they are observed between neurons. The SUBTLE team believes that its devices can be thus used in the future to mimic neuron action in artificial networks and to serve as sensors for signals usually hidden under the noise.

   ：

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ノイズを利用してシグナルを増幅する試み？
神経回路との類似性について議論されている。

Cell Stem Cell, 6:323-335,2010

MicroRNA-9 Coordinates Proliferation and Migration of Human Embryonic Stem Cell-Derived Neural Progenitors

Human pluripotent stem cells offer promise for use in cell-based therapies for brain injury and diseases. However, their cellular behavior is poorly understood. Here we show that the expression of the brain-specific microRNA-9 (miR-9) is turned on in human neural progenitor cells (hNPCs) derived from human embryonic stem cells. Loss of miR-9 suppressed proliferation but promoted migration of hNPCs cultured in vitro. hNPCs without miR-9 activity also showed enhanced migration when transplanted into mouse embryonic brains or adult brains of a mouse model of stroke. These effects were not due to precocious differentiation of hNPCs. One of the key targets directly regulated by miR-9 encodes stathmin, which increases microtubule instability and whose expression in hNPCs correlates inversely with that of miR-9. Partial inhibition of stathmin activity suppressed the effects of miR-9 loss on proliferation and migration of human or embryonic rat neural progenitors. These results identify miR-9 as a novel regulator that coordinates the proliferation and migration of hNPCs.

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ヒトESだが、neural progenitorの発生で機能するmicro RNAの論文。
loss of miR-9でmigrationが過剰になる？

PLoS ONE 4(12):e8530, 2009

SNAI1 and SNAI2 Are Asymmetrically Expressed at the 2-Cell Stage and Become Segregated to the TE in the Mouse Blastocyst

SNAI1 and SNAI2 are transcription factors that initiate Epithelial-to-Mesenchymal cell transitions throughout development and in cancer metastasis. Here we show novel expression of SNAI1 and SNAI2 throughout mouse preimplantation development revealing asymmetrical localization of both SNAI1 and SNAI2 in individual blastomeres beginning at the 2-cell stage through to the 8-cell stage where SNAI1 and SNAI2 are then only detected in outer cells and not inner cells of the blastocyst. This study implicates SNAI1 and SNAI2 in the lineage segregation of the trophectoderm and inner cell mass, and provides new insight into these oncogenes.

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Trophectoderm(TE)の分化に重要？な転写因子であるSNAI1/2の発現パターンを見た論文。

予備知識として、
SNAI1/2→E-cadherin/Na-K ATPase→TE

SNAI1KO→neural crestの発育以上
SNAI2KO→survive at birth
compensatoryなメカニズムか？

PLoS ONE 4(12): e8295, 2010

A Conceptual Mathematical Model of the Dynamic Self-Organisation of Distinct Cellular Organelles

Formation, degradation and renewal of cellular organelles is a dynamic process based on permanent budding, fusion and inter-organelle traffic of vesicles. These processes include many regulatory proteins such as SNAREs, Rabs and coats. Given this complex machinery, a controversially debated issue is the definition of a minimal set of generic mechanisms necessary to enable the self-organization of organelles differing in number, size and chemical composition. We present a conceptual mathematical model of dynamic organelle formation based on interacting vesicles which carry different types of fusogenic proteins (FP) playing the role of characteristic marker proteins. Our simulations (ODEs) show that a de novo formation of non-identical organelles, each accumulating a different type of FP, requires a certain degree of disproportionation of FPs during budding. More importantly however, the fusion kinetics must indispensably exhibit positive cooperativity among these FPs, particularly for the formation of larger organelles. We compared different types of cooperativity: sequential alignment of corresponding FPs on opposite vesicle/organelles during fusion and pre-formation of FP-aggregates (equivalent, e.g., to SNARE clusters) prior to fusion described by Hill kinetics. This showed that the average organelle size in the system is much more sensitive to the disproportionation strength of FPs during budding if the vesicular transport system gets along with a fusion mechanism based on sequential alignments of FPs. Therefore, pre-formation of FP aggregates within the membranes prior to fusion introduce robustness with respect to organelle size. Our findings provide a plausible explanation for the evolution of a relatively large number of molecules to confer specificity on the fusion machinery compared to the relatively small number involved in the budding process. Moreover, we could speculate that a specific cooperativity which may be described by Hill kinetics (aggregates or Rab/SNARE complex formation) is suitable if maturation/identity switching of organelles play a role (bistability).

 ===

Menbrane fusionとbuddingのモデリングの話。

Nature Cell Biology 10:429 - 436, 2008

Tensile forces govern germ-layer organization in zebrafish

Understanding the factors that direct tissue organization during development is one of the most fundamental goals in developmental biology. Various hypotheses explain cell sorting and tissue organization on the basis of the adhesive and mechanical properties of the constituent cells1. However, validating these hypotheses has been difficult due to the lack of appropriate tools to measure these parameters. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of individual ectoderm, mesoderm and endoderm progenitor cells from gastrulating zebrafish embryos. Combining these data with tissue self-assembly in vitro and the sorting behaviour of progenitors in vivo, we have shown that differential actomyosin-dependent cell-cortex tension, regulated by Nodal/TGFbeta-signalling (transforming growth factor beta), constitutes a key factor that directs progenitor-cell sorting. These results demonstrate a previously unrecognized role for Nodal-controlled cell-cortex tension in germ-layer organization during gastrulation.

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細胞表面のcortical tensionが細胞の種類によって異なっていることをAFMを用いて証明。
それがprogenitor-cell sortingの主要なfactorであることを示す。

なかなか面白い仮説。

MCB 26:7539-7549, 2006

The Grb2/Mek Pathway Represses Nanog in Murine Embryonic Stem Cells

The homeobox gene Nanog is a key intrinsic determinant of self renewal in embryonic stem (ES) cells, and its repression leads ES cells to selectively differentiate into primitive endoderm. Although Nanog repression occurs at the outermost layer of ES cell aggregates independent of the leukemia inhibitory factor (LIF)/STAT3 pathway, it is largely undetermined what external cues and intracellular signals cause the event. Of interest, addition of the tyrosine phosphatase inhibitor, sodium vanadate, selectively repressed Nanog transcription without any detectable changes in upstream transcriptional regulators Oct3/4 and Sox2. Furthermore, sodium vanadate induced primitive endoderm differentiation, even in the inner cells of ES cell aggregates. Expression of Gata6 and Zfp42, two putative downstream Nanog effectors, was also increased and decreased by the addition of sodium vanadate, respectively, but these changes were eliminated by exogenous Nanog expression. The effects of sodium vanadate were abrogated by Grb2 deficiency or by the addition of the Mek inhibitor, PD98059. Indeed, PD98059 prevented Nanog repression induced by ES cell aggregation as well. Furthermore, transfection of a constitutive active Mek mutant into ES cells induced Nanog repression and primitive endoderm differentiation. These data indicate that the Grb2/Mek pathway primarily mediates Nanog gene repression upon ES cell differentiation into primitive endoderm.

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タイトル通りの内容。

●SU5402→FGFR inhibitor
●Fig8にまとめた図あり

IntroにFGF-PI3K-AKT経路の寄与について言及あり（Ref.）

Development 137:715-24, 2010

FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst.

Primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Recent studies showed that EPI and PE progenitors expressing the lineage-specific transcriptional factors Nanog and Gata6, respectively, arise progressively as the ICM develops. Subsequent sorting of the two progenitors during blastocyst maturation results in the ormation of morphologically distinct EPI and PE layers at E4.5. It is, however, unknown how the initial differences between the two populations become established in the E3.5 blastocyst. Because the ICM cells are derived from two distinct rounds of polarized cell divisions during cleavage, a possible role for cell lineage history in promoting EPI versus PE fate has been proposed. We followed cell lineage from the eight-cell stage by live cell tracing and could find no clear linkage between developmental history of individual ICM cells and later cell fate. However, modulating FGF signaling levels by inhibition of the receptor/MAP kinase pathway or by addition of exogenous FGF shifted the fate of ICM cells to become either EPI or PE, respectively. Nanog- or Gata6-expressing progenitors could still be shifted towards the alternative fate by modulating FGF signaling during blastocyst maturation, suggesting that the ICM progenitors are not fully committed to their final fate at the time that initial segregation of gene expression occurs. In conclusion, we propose a model in which stochastic and progressive specification of EPI and PE lineages occurs during maturation of the blastocyst in an FGF/MAP kinase signal-dependent manner.

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初期胚の細胞分化の様子やシグナルの関与を細かくみた秀作。
IntroやDisの情報もよくreviewされてて有用。

●8cell stageの細胞の一つにH2B-RFPとmembrane-RFPを入れて、その後の分裂の様子をライブでフォロー（Fig1, Table1)。primary inside cellとsecondary inside cellとの寄与度は差がないことを証明。

● FGF/MAPKシグナルのinhibitorをE2.5～4.5の間のさまざまな時間でtreatして、どこからが不可逆なポイントの検討、PE/EPIへの分化の影響などを検討。（Fig2-4）→E4.5で最終的にplasciticyが失われるが、それまではNanog/Gata6のどちらかをふらふらしている。
Gata4の発現がcritical?（Discussion）

●High-doseなFGF4（+heparin）がEPI formationのブロックに十分であることを証明（Fig4, 5）

●PE(Gata6+)/EPI(Nanog+)の細胞が"pepper-salt pattern"でICM内に散在していることから、stochasticなメカニズムでヘテロな細胞集団が生じた後、どちらかのlineageにcommitすると考えられる（Discussion）←ここの決定機構は？？

●inner cell-outer cellのcontactについて、cell表面のcortical tensionの寄与？（Fig1D）(Ref.)

●FGFR/MEK inhibitorがtrophoectodermの細胞運命に影響を与えていない。
ICM内のFGF4の発現はOct4/Sox2による（Ref.1&2）

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過去の報告のまとめ

●ICM内の細胞でのtranscriptomeの揺らぎ、position independent？

●NanogとGata6の発現は、mutually exclusive

●FGF4、FGFR2、Grb2、MAPKなどのシグナルの寄与、過去文献（Hamazaki et al. MCB 2006など、次の記事でまとめ）→Ffg4, Fgfr2, Grb2KOマウスはPEの形成が完全に失われてEPIになる、DN-FGFRをESに入れるとPE形成能が失われる、一方でexogenous FGFはPE形成をenhanceしない（今回の論文と結論が異なるので条件が違う？）、PTPase inhibitor(vanadate)やCA-MAPKはPE形成を促進する、など

●8細胞期のpolarizationと8-16細胞期のsymmetric-asymmetric division (Ref.)

●平均のouter:inner cellの割合は10.8:5.2

Autophagy論文いくつか

Proc Natl Acad Sci U S A. 2010 Mar 15. [Epub ahead of print]
Mechanisms of acute axonal degeneration in the optic nerve in vivo.

Axonal degeneration is an initial key step in traumatic and neurodegenerative CNS disorders. We established a unique in vivo epifluorescence imaging paradigm to characterize very early events in axonal degeneration in the rat optic nerve. Single retinal ganglion cell axons were visualized by AAV-mediated expression of dsRed and this allowed the quantification of postlesional acute axonal degeneration (AAD). EM analysis revealed severe structural alterations of the cytoskeleton, cytoplasmatic vacuolization, and the appearance of autophagosomes within the first hours after lesion. Inhibition of autophagy resulted in an attenuation of acute axonal degeneration. Furthermore, a rapid increase of intraaxonal calcium levels following crush lesion could be visualized using a calcium-sensitive dye. Application of calcium channel inhibitors prevented crush-induced calcium increase and markedly attenuated axonal degeneration, whereas application of a calcium ionophore aggravated the degenerative phenotype. We finally demonstrate that increased postlesional autophagy is calcium dependent and thus mechanistically link autophagy and intraaxonal calcium levels. Both processes are proposed to be major targets for the manipulation of axonal degeneration in future therapeutic settings.

 ====

Eur J Cancer. 2010 Mar 13. [Epub ahead of print]
Inhibition of autophagy augments 5-fluorouracil chemotherapy in human colon cancer in vitro and in vivo model.

Although 5-fluorouracil (5-FU)-based adjuvant chemotherapy is widely used in the treatment of colorectal cancer, novel therapeutic strategies need to be explored. It has been reported that autophagy is extensively implicated in cancer. However, the function of autophagy is not fully understood. In the present study, apoptosis induced by 5-FU in 3 human colon cancer cell lines (HCT116, DLD-1, and DLD-1/5-FU (a specific 5-FU-resistant sub-line)) was measured using MTT assay, DNA fragmentation assay, Hoechst 33342 staining, and caspase-3 immunoblotting. The autophagy activation induced by 5-FU treatment was revealed by microtubule-associated protein 1 light chain 3 (LC3) immunofluorescence and immunoblotting and p62 immunoblotting. Inhibition of autophagy by 3-methyladenine (3-MA) or small interference RNA targeting Atg7 (Atg7 siRNA) significantly augmented 5-FU-induced apoptosis. This synergistic effect of 5-FU and 3-MA was further confirmed in the DLD-1 xenograft tumour model. Tumour growth was suppressed more significantly with combination treatment than 5-FU treatment alone. In conclusion, autophagy was activated as a protective mechanism against 5-FU-induced apoptosis and its inhibition could be a promising strategy for adjuvant chemotherapy in colon cancer.

 ====

BMC Cancer. 2010 Mar 16;10(1):98. [Epub ahead of print]
Genetic and epigenetic silencing of the beclin 1 gene in sporadic breast tumors.

ABSTRACT: BACKGROUND: : Beclin 1, an important autophagy-related protein in human cells, is involved in cell death and cell survival. Beclin 1 mapped to human chromosome 17q21. It is widely expressed in normal mammary epithelial cells. Although down-regulated expression with mono-allelic deletions of beclin 1 gene was frequently observed in breast tumors, whether there was other regulatory mechanism of beclin 1 was to be investigated. We studied the expression of beclin 1 and explored the possible regulatory mechanisms on its expression in breast tumors. METHOD: S: 20 pairs of tumors and adjacent normal tissues from patients with sporadic breast invasive ductal cancer (IDCs) were collected. The mRNA expression of beclin 1 was detected by real-time quantitative RT-PCR. Loss of heterozygosity (LOH) was determined by real-time quantitative PCR and microsatellite methods. The protein expression of beclin 1, p53, BRCA1 and BRCA2 was assessed by immunohistochemistry. CpG islands in 5' genomic region of beclin 1 gene were identified using MethylPrimer Program. Sodium bisulfite sequencing was used in examining the methylation status of each CpG island. RESULTS: : Decreased beclin 1 mRNA expression was detected in 70% of the breast tumors, and the protein levels were co-related to the mRNA levels. Expression of beclin 1 mRNA was demonstrated to be much higher in the BRCA1 positive tumors than that in the BRCA1 negative ones. Loss of heterozygosity was detected in more than 45% of the breast tumors, and a dense cluster of CpG islands was found from the 5' end to the intron 2 of the beclin 1 gene. Methylation analysis showed that the promoter and the intron 2 of beclin 1 were aberrantly methylated in the tumors with decreased expression. CONCLUSIONS: : These data indicated that LOH and aberrant DNA methylation might be the possible reasons of the decreased expression of beclin 1 in the breast tumors. The findings here shed some new light on the regulatory mechanisms of beclin 1 in breast cancer.

Trends in Neurosciences 30:350-356, 2007

Thalamic synchrony and dynamic regulation of global forebrain oscillations

The circuitry within the thalamus creates an intrinsic oscillatory unit whose function depends critically on reciprocal synaptic connectivity between excitatory thalamocortical relay neurons and inhibitory thalamic reticular neurons along with a robust post-inhibitory rebound mechanism in relay neurons. Feedforward and feedback connections between cortex and thalamus reinforce the thalamic oscillatory activity into larger thalamocortical networks to generate sleep spindles and spike-wave discharge of generalized absence epilepsy. The degree of synchrony within the thalamic network seems to be crucial in determining whether normal (spindle) or pathological (spike-wave) oscillations occur, and recent studies show that regulation of excitability in the reticular nucleus leads to dynamical modulation of the state of the thalamic circuit and provide a basis for explaining how a variety of unrelated genetic alterations might lead to the spike-wave phenotype. In addition, given the central role of the reticular nucleus in generating spike-wave discharge, these studies have suggested specific interventions that would prevent seizures while still allowing normal spindle generation to occur. This review is part of the INMED/TINS special issue Physiogenic and pathogenic oscillations: the beauty and the beast, based on presentations at the annual INMED/TINS symposium (http://inmednet.com).

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Neuro-circuitをどのように考えるか、という一例として。

 　→　→　→　→
↑ Core network ↓
 　←　←　←　←
     ↑
●Structure-excitatory/inhibitory/connection?
●Main molecule/neural subtype?
●Modificator?
     ↑
inhibitionで、ネットワークのoutput（synchronization）が影響するか？

PNAS 103:17949-17954, 2006

 Lipocalin-type prostaglandin D synthase produces prostaglandin D2 involved in regulation of physiological sleep

Prostaglandin (PG) D2 has been proposed to be essential for the initiation and maintenance of the physiological sleep of rats because intracerebroventricular administration of selenium tetrachloride (SeCl4), a selective inhibitor of PGD synthase (PGDS), was shown to reduce promptly and effectively the amounts of sleep during the period of infusion. However, gene knockout (KO) mice of PGDS and prostaglandin D receptor (DP1R) showed essentially the same circadian profiles and daily amounts of sleep as wild-type (WT) mice, raising questions about the involvement of PGD2 in regulating physiological sleep. Here we examined the effect of SeCl4 on the sleep of WT and KO mice for PGDS and DP1R and that of a DP1R antagonist, ONO-4127Na, on the sleep of rats. The i.p. injection of SeCl4 into WT mice decreased the PGD2 content in the brain without affecting the amounts of PGE2 and PGF2α. It inhibited sleep dose-dependently and immediately after the administration during the light period when mice normally sleep, increasing the wake time; and the treatment with this compound resulted in a distinct sleep rebound during the following dark period. The SeCl4-induced insomnia was observed in hematopoietic PGDS KO mice but not at all in lipocalin-type PGDS KO, hematopoietic and lipocalin-type PGDS double KO or DP1R KO mice. Furthermore, the DP1R antagonist ONO-4127Na reduced sleep of rats by 30% during infusion into the subarachnoid space under the rostral basal forebrain at 200 pmol/min. These results clearly show that the lipocalin-type PGDS/PGD2/DP1R system plays pivotal roles in the regulation of physiological sleep.

 　＝＝＝

●SeCl4（PGD synthaseの阻害剤）を入れると、sleep↓

●DP1R antagonistのONO-4127Naを入れると、sleep↓

●しかし、DP1R-KOマウスでは、sleepの量もcircadianも差がない

●L-PGDS-KOマウスでも、sleepの量もcircadianも差がない

  (Ref.)

↓

●PGD2以外のalternative pathwayによるcompensationの問題/阻害剤のspecificityの問題

●DP1R-KOマウスでSeCl4を入れるとどうなる？

↓

●H-PGDS、L-PGDS、DP1R-KOマウスでSeCl4を入れたら、、L-PGDS、DP1R-KOマウスではsleep↓の効果が見えなくなった。ということは、acuteの効果はやはり見える。

●sleep↓の後のリバウンドは、L&H-PGDS-DKOにすると消える。

●いずれにしても、circadianはがっつり残る→睡眠ホメオスタシスと日内変動のメカニズムがどう絡み合うのか？？

 

細胞内ナノマシン作製の試み

記事引用

＝＝＝

Future bio-nanotechnology will use computer chips inside living cells

(Nanowerk Spotlight) Continuing miniaturization has moved the semiconductor industry well into the nano realm with leading chip manufacturers on their way to CMOS using 22nm process technology. With transistors the size of tens of nanometers, researchers have begun to explore the interface of biology and electronics by integrating nanoelectronic components and living cells. While researchers have already experimented with integrating living cells into semiconductor materials (see "Scientists integrate living brain cells into organic semiconductors") other research is exploring the opposite way, i.e. integrating nanoelectronics into living cells.

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applicationについて、折を見て考える。

Current Opinion in Pharmacology 7:33-38, 2007 (Review)

Prostaglandins and adenosine in the regulation of sleep and wakefulness

Prostaglandin (PG) D2 and adenosine are potent humoral sleep-inducing factors that accumulate in the brain during prolonged wakefulness. PGD2 is produced in the brain by lipocalin-type PGD synthase, which is localized mainly in the leptomeninges, choroid plexus and oligodendrocytes, and circulates in the cerebrospinal fluid as a sleep hormone. It stimulates DP1 receptors on leptomeningeal cells of the basal forebrain to release adenosine as a paracrine signaling molecule to promote sleep. Adenosine activates adenosine A2A receptor-expressing sleep-active neurons in the basal forebrain and the ventrolateral preoptic area. Sleep-promoting neurons in the ventrolateral preoptic area send inhibitory signals to suppress the histaminergic neurons in the tuberomammillary nucleus, which contribute to arousal through histamine H1 receptors. Increased knowledge of the molecular mechanisms by which PGD2 induces sleep through activation of adenosine A2A receptors and inhibition of the histaminergic arousal system will be useful both for a better understanding of sleep/wake regulation and for the development of novel types of sleeping pills or anti-doze drugs.

＝＝＝

●sleep deprivationでは、PGD2もadenosineも量が上がる。

●DP1R-KOでもcircadianに差がないそう・・・
adenosineの上流であることは分かった。

●A1R→BFのCholinergic neuron？
あまり重要な役回りではなさそう

●A2AR→caffeineのターゲット

●＜重要＞A1R-KOも、A2AR-KOも、normalなcircadian profilesを示し、NREM/REMの量もWTと変わらない。reboundについてはA2AR-KOでは差が出る（A1R-KOは差なし)。

●PGD2/A2ARアゴニスト投与後のc-Fos染色で、sleep-active neurons（VLPO）が同定された。
ref.: 32, 33

●NA, Ach, A1R signal---| VLPO

（serotonineとadenosineへの反応性の違いで、Type1/2に分けられている）
●5-HT ---| VLPO Type1 neuron
   ↓
VLPO Type2 neuron
●CGS21680　→　VLPO Type2 neuron

●Figure 1がよくまとまっている。

●全体的なインプレ：
・KOマウスで差が出ない～通常のS-Wサイクルで働いているというより、S-Wサイクルのコントロールをしているニューロンの活性を著しく変動させている、という感じ？Adenosineは日内変動がある不思議。

・Thresholdの形成要因は？細胞の外部/内部因子の関わり？

●VLPOのGABA neuron　→　TMNのヒスタミン、となっているが、それだけだろうか？

その研究を始める前に

vikingさんのブログより、一部引用。

＝＝＝

だからその論文はリジェクトされる

  (途中略）

では、最後のまとめとして「リジェクトされない論文を書くには」・・・

   1. まずそもそも研究に着手する前に、総説が1本書けるぐらい
　　　先行研究をサーベイする
   2. まずそもそも実験をする前に、実験計画に過不足がないよう
　　　じっくりプランを練る
   3. まずそもそも解析をする前に、解析手法の原理をきちんと勉強する
   4. まずそもそも論文を書く前に、有意であるべき統計値が有意になる

　　　ようにロジックを考える

　（後略）

＝＝＝

ついつい準備不足で手を突っ込んでしまう傾向があるので、自戒を込めて。

Li et al. PNAS 107:1402-1407, 2010

 Stk40 links the pluripotency factor Oct4 to the Erk/MAPK pathway and controls extraembryonic endoderm differentiation

Self-renewal and differentiation of embryonic stem cells (ESCs) are controlled by intracellular transcriptional factors and extracellular factor-activated signaling pathways. Transcription factor Oct4 is a key player maintaining ESCs in an undifferentiated state, whereas the Erk/MAPK pathway is known to be important for ESC differentiation. However, the manner in which intracellular pluripotency factors modulate extracellular factor-activated signaling pathways in ESCs is not well understood. Here, we report identification of a target gene of Oct4, serine/threonine kinase 40 (Stk40), which is able to activate the Erk/MAPK pathway and induce extraembryonic--endoderm (ExEn) differentiation in mouse ESCs. Interestingly, cells overexpressing Stk40 exclusively contribute to the ExEn layer of chimeric embryos when injected into host blastocysts. In contrast, deletion of Stk40 in ESCs markedly reduces ExEn differentiation in vitro. Mechanistically, Stk40 interacts with Rcn2, which also activates Erk1/2 to induce ExEn specification in mouse ESCs. Moreover, Rcn2 proteins are specifically located in the cytoplasm of the ExEn layer of early mouse embryos. Importantly, knockdown of Rcn2 blocks Stk40-activated Erk1/2 and ESC differentiation. Therefore, our study establishes a link between the pluripotency factor Oct4 and the Erk/MAPK signaling pathway, and it uncovers cooperating signals in the Erk/MAPK activation that control ExEn differentiation.

＝＝＝

 　Oct4 --| Skt40 (kinase)-Rcn2　→　Ras/MAPK　→　ExEn
                                                          （機序不明）

一応Skt40はkinaseのようだが。

科学とアートの境界線

science book cafe 2009
先端バイオロジーが加速する、科学とアートのゆくえ

より、一部引用

いま、遺伝子工学、再生医療、マイクロマシンといった先端科学技術は、「人間が人工的に細胞（生命の基本単位）をつくりだす」ところまできている。岩崎秀雄氏は、そこで使われる技術やバイオ素材を使い、独自の切り絵の世界と組み合わせた、かつてない芸術表現を追究するアーティストだ。岩崎氏はアーティストであると同時に、科学者でもある。いや、科学が本業といってもいい。早稲田大学で生物リズムを研究し、日本の若手研究者による学会「細胞を創る研究会」を率先する。科学にとってアートは、アートにとって科学は、どのように存在し、どこへ向かうのか？科学とアートの境界面に立つ岩崎氏に、デザインジャーナリストの藤崎圭一郎氏が聞いた。
2009年8月29日開催の青山ブックセンターでのトークイベント「先端バイオロジーが加速する、科学とアートのゆくえ」をもとに構成。

（中略）

藤崎 ── どこまで科学技術がやれば、人間が生命をつくった、と言い切れるのですか？

岩崎 ── 私は「細胞を創る研究会」の発起人の1人で、その問いには非常に興味をもっています。研究者にとっても難しい問いです。藤崎さんはどう思いますか？

藤崎 ── 増殖して構成成分を見ただけでは考えられないような性質が備わったら、細胞、つまり生命ができた、というイメージです。

岩崎 ── 私もいつもバクテリアを見ている微生物屋なので、「増殖＝細胞＝生命」とイメージします。けれども、研究会の発起人の中でも，哺乳類の脳の生物時計の研究者（理化学研究所　上田泰己博士）がいて、彼は細胞の増殖よりも、細胞同士がネットワークをつくる能力を再構成したいといいます。また、マイクロマシンをつくる研究者（東京大学　竹内昌治准教授）は、とにかく動くものが好きで、動いていないと生き物という感じがしないといいます。

つまり、「私がつくりたい細胞（＝生命の基本単位）」のイメージは、研究者ごとにすごく違うのです。状況はロボット研究と似ています。どこまでいったらヒューマノイドをつくったといえるのか、という問題のように。面白いところではありますが。

 （中略）

藤崎 ── 岩崎さんの場合は、細胞をつくる、なんていうものすごいアーティスティックな科学のプロジェクトをやりながら、一方で切り絵をやっていらっしゃる。その使い分けはどうなっているのですか？　科学の仕事とアートの仕事は、ぜんぜん違うのですよね。

岩崎 ── 私は科学とアートの境界面に興味があります。境界面は明確にあるのでなく、たゆたっています。その境界面の上に立って、両サイドをのぞきこみたい。そもそも、科学とアートを対比すること自体がおかしな概念だと思います。アートの表現は、世界全部をのみこんでしまうほど巨大にもなるのですが、サイエンスの研究は、かなり限られた世界です。

科学は「これをやっちゃいけない」という項目が多い。シャープな解をえぐり出すために、彫刻で石から像を切り出すように、余分な部分をどんどん削ぎ落としていく行為です。そのための色々な手続きが制度化されています。だからこそ非常に強いし、多くのことがわかるシステムで、素晴らしい。でも逆にいえば、削ぎ落とされるものが必ずある。アートは科学が削ぎ落とすものを拾い上げることができます。私はサイエンスを相対化するためのエネルギーとして、アートが重要であると思っています。

＝＝＝

ある意味、自分の考える理想的な姿だなあ。科学もアートも、どちらも超のつく一流の人ならではのスタンス。

Autophagy & Cancer

Autophagy. 2010 Apr 26;6(3)
Targeting the prodeath and prosurvival functions of autophagy as novel therapeutic strategies in cancer.

Autophagy is an evolutionarily conserved lysosomal pathway for degrading cytoplasmic proteins, macromolecules, and organelles. While autophagy has become one of the most attractive topics in cancer research, the current autophagy literature is often viewed as confusing, because of its association with apparently contradictory roles, such as survival and cell death. Autophagy can serve as a tumor suppressor, as a partial reduction in autophagic capacity or defective autophagy (e.g., heterozygous knockdown BECN1 (+/-) in mice) provides an oncogenic stimulus, causing malignant transformation and spontaneous tumors. In addition, autophagy seems to function as a protective cell survival mechanism against environmental and cellular stress (e.g., nutrient deprivation, hypoxia and therapeutic stress) and causes resistance to antineoplastic therapies. Recent studies have demonstrated that the inhibition of autophagy in cancer cells may be therapeutically beneficial in some circumstances, as it can sensitize cancer cells to different therapies, including DNA-damaging agents, antihormone therapies (e.g., tamoxifen), and radiation therapy. This supports the hypothesis that inhibiting autophagy can negatively influence cancer cell survival and increase cell death when combined with anticancer agents, providing a therapeutic advantage against cancer. On the other hand, the induction of autophagy by the inhibition of anti-autophagic proteins, such as Bcl-2, PKCdelta, and tissue transglutaminase 2 (TG2), may lead to autophagic cell death in some apoptosis-resistant cancers (i.e., breast and pancreatic cancers), indicating that the induction of autophagy alone may also be used as a potential therapy. Overall, the data suggest that, depending on the cellular features, either the induction or the inhibition of autophagy can provide therapeutic benefits to patients and that the design and synthesis of the first-generation modulators of autophagy may provide the tools for proof of concept experiments and the impetus for translational studies that may ultimately lead to new therapeutic strategies in cancer.

＝＝＝

癌細胞によって、survivalに働いたりcell deathに働いたりするので、個々の細胞ごとにeffectを確認しないといけない。

Nat Neuro 13:369-378, 2010

Stimulus onset quenches neural variability: a widespread cortical phenomenon

Neural responses are typically characterized by computing the mean firing rate, but response variability can exist across trials. Many studies have examined the effect of a stimulus on the mean response, but few have examined the effect on response variability. We measured neural variability in 13 extracellularly recorded datasets and one intracellularly recorded dataset from seven areas spanning the four cortical lobes in monkeys and cats. In every case, stimulus onset caused a decline in neural variability. This occurred even when the stimulus produced little change in mean firing rate. The variability decline was observed in membrane potential recordings, in the spiking of individual neurons and in correlated spiking variability measured with implanted 96-electrode arrays. The variability decline was observed for all stimuli tested, regardless of whether the animal was awake, behaving or anaesthetized. This widespread variability decline suggests a rather general property of cortex, that its state is stabilized by an input.

＝＝＝

慣れない分野でタフな論文。理解が結構怪しいが。

●刺激後にneuronの活動（membrane potencial/firing rates）のvariabilityが低下することを、いくつかの指標を定義して証明。その指標を変化させるほかのfactorが影響しているわけではないことをなにやらごちゃごちゃと検討している・・・らしい・・・

●平均化することで失われてしまう情報に意味があるのではないかという問題提起

とすれば・・・

・個々のneuronのvariabilityがノイズであると考えれば、network全体として機能することでbufferingしている？

・むしろ個々のneuronのvariabilityはそれぞれの機能（処理している情報）の違いを反映している？

●Discussionより

"Mechanistically, the variance decline inplies that cortical curcuits become more stable when driven."

→こういうタイプのネットワークはいくつか考えられて、recurrent networkもその一つ。

視床下部神経核とホルモンの機能（まとめサイト）

滋賀医大のサイトでいいまとめがあったのでメモ。

サイトはこちら

Sleep. 33(1):19-28, 2010.

Genetic evidence for a role for protein kinase A in the maintenance of sleep and thalamocortical oscillations.

STUDY OBJECTIVES: Genetic manipulation of cAMP-dependent protein kinase A (PKA) in Drosophila has implicated an important role for PKA in sleeplwake state regulation. Here, we characterize the role of this signaling pathway in the regulation of sleep using electroencephalographic (EEG) and electromyographic (EMG) recordings in R(AB) transgenic mice that express a dominant negative form of the regulatory subunit of PKA in neurons within cortex and hippocampus. Previous studies have revealed that these mutant mice have reduced PKA activity that results in the impairment of hippocampus-dependent long-term memory and long-lasting forms of hippocampal synaptic plasticity. DESIGN: PKA assays, in situ hybridization, immunoblots, and sleep studies were performed in R(AB) transgenic mice and wild-type control mice. MEASUREMENTS AND RESULTS: We have found that R(AB) transgenic mice have reduced PKA activity within cortex and reduced Ser845 phosphorylation of the glutamate receptor subunit GluR1. R(AB) transgenic mice exhibit non-rapid eye movement (NREM) sleep fragmentation and increased amounts of rapid eye movement (REM) sleep relative to wild-type mice. Further, R(AB) transgenic mice have more delta power but less sigma power during NREM sleep relative to wild-type mice. After sleep deprivation, the amounts of NREM and REM sleep were comparable between wild-type and R(AB) transgenic mice. However, the homeostatic rebound of sigma power in R(AB) transgenic mice was reduced. CONCLUSIONS: Alterations in cortical synaptic receptors, impairments in sleep continuity, and alterations in sleep oscillations in R(AB) mice imply that PKA is involved not only in synaptic plasticity and memory storage but also in the regulation of sleep/wake states.

＝＝＝

PKA-GluRの経路について、Tgマウスによる考察。

＝＝＝

Neurosci Lett.469(1):1-5, 2010

Sleep fragmentation reduces hippocampal CA1 pyramidal cell excitability and response to adenosine.

Sleep fragmentation (SF) impairs the restorative/cognitive benefits of sleep via as yet unidentified alterations in neural physiology. Previously, we found that hippocampal synaptic plasticity and spatial learning are impaired in a rat model of SF which utilizes a treadmill to awaken the animals every 2 min, mimicking the frequency of awakenings observed in human sleep apnea patients. Here, we investigated the cellular mechanisms responsible for these effects, using whole-cell patch-clamp recordings. 24h of SF decreased the excitability of hippocampal CA1 pyramidal neurons via decreased input resistance, without alterations in other intrinsic membrane or action potential properties (when compared to cage controls, or to exercise controls that experienced the same total amount of treadmill movement as SF rats). Contrary to our initial prediction, the hyperpolarizing response to bath applied adenosine (30 microM) was reduced in the CA1 neurons of SF treated rats. Our initial prediction was based on the evidence that sleep loss upregulates cortical adenosine A1 receptors; however, the present findings are consistent with a very recent report that hippocampal A1 receptors are not elevated by sleep loss. Thus, increased adenosinergic inhibition is unlikely to be responsible for reduced hippocampal long-term potentiation in SF rats. Instead, the reduced excitability of CA1 pyramidal neurons observed here may contribute to the loss of hippocampal long-term potentiation and hippocampus-dependent cognitive impairments associated with sleep disruption. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

＝＝＝

電気生理のFigが2個だけ。

Oct4によって制御される遺伝子群

Molecular Systems Biology 6 Article number: 354  doi:10.1038/msb.2010.9
Published online: 9 March 2010

Zebrafish Pou5f1-dependent transcriptional networks in temporal control of early development

Pou5f1/Oct4の下流遺伝子を、ゼブラフィッシュ胚を用いて解析。

●マウスなど他種のターゲットとかぶっていて、マウスOct4はゼブラPou5f1mutantをレスキューできる。

●Pou5f1/Oct4の転写制御に関する数学的モデルを提唱。

GLYCOLIPO

in vivo delivery用のliposome試薬を業者さんが売り込みに来ていたのでチェック。

片山化学工業
標的指向性リポソームを利用した生体分子イメージングと薬物送達　「GLYCOLIPO(TM)」

GEからも類似品？
GLYCOLIPO　分子イメージング試薬（標的指向性リポソーム）

もうひとつ類似品。
Carestream Molecular Imaging: imaging of cancer biology and relevant pathways in vivo
Nature Methods 6 December 2009

Greased Lightbox

→←+-↻

読み込み中

クリックでキャンセルします

画像が存在しません

大規模解析の方法と視覚化

Nature Methods, March 2010
Technology featureより

なんでもomeをつければいいってもんじゃないとも思うけど、epigenome解析においてDNAメチル化を網羅的に解析する意義は大きいので。
DNAメチル化を解析する方法の開発に、$1.2 millionのグラントが出たとの記載もある。


しかし最終的には、「何を」「どのように」制御しているか、というところまで話が落ちないと、何もわからないだろうと推測。

ついでに、mass dataのvisualization toolsをいくつか。
Nature Methods 7, S5 - S15 (2010)
Visualizing genomes: techniques and challenges

Nature Methods 7, S56 - S68 (2010)
Visualization of omics data for systems biology

New optogenetic tools

Nature Methods March, 2010のHighlightより、
新しいoptogenetic toolを見つける試みと、既存のツールをmutagenesisで改良する試みを紹介。

refs.
Chow et al. Nature 463:98-102,2010
Gunaydin et al. Nat. Neurosci. 13:387-392,2010

そのうち読む。

Autophagy関連論文いくつか

AMPK-mediated autophagy inhibits apoptosis in cisplatin-treated tumour cells
Journal of Cellular and Molecular Medicine, 13:3644 - 3654, 2010

The role of autophagy in cisplatin anticancer action was investigated using human U251 glioma, rat C6 glioma and mouse L929 fibrosarcoma cell lines. A dose- and time-dependent induction of autophagy was observed in tumour cells following cisplatin treatment, as demonstrated by up-regulation of autophagy-inducing protein beclin-1 and subsequent appearance of acridine orange-stained acidic autophagic vesicles. The presence of autophagosomes in cisplatin-treated cells was also confirmed by electron microscopy. Inhibition of autophagy with lysosomal inhibitors bafilomycin A1 and chloroquine, or a PI3 kinase inhibitor wortmannin, markedly augmented cisplatin-triggered oxidative stress and caspase activation, leading to an increase in DNA fragmentation and apoptotic cell death. The mechanisms underlying the protective effect of autophagy apparently involved the interference with cisplatin-induced modulation of Bcl-2 family proteins, as inhibition of autophagy potentiated cisplatin-mediated up-regulation of proapoptotic Bax and down-regulation of anti-apoptotic Bcl-2. Autophagy induction in cisplatin-treated cells was preceded by activation of adenosine monophosphate-activated protein kinase (AMPK) and concomitant down-regulation of mammalian target of rapamycin (mTOR)-mediated phosphorylation of p70S6 kinase. The ability of cisplatin to trigger autophagy was reduced by small interfering RNA (siRNA)-mediated AMPK silencing, while transfection with mTOR siRNA was sufficient to trigger autophagy in tumour cells. Finally, siRNA-mediated AMPK down-regulation and AMPK inhibitor compound C increased cisplatin-induced tumour cell death, while mTOR siRNA and AMPK activator metformin protected tumour cells from cisplatin. Taken together, these data suggest that cisplatin-triggered activation of AMPK and subsequent suppression of mTOR activity can induce an autophagic response that protects tumour cells from cisplatin-mediated apoptotic death.

AMPKとautophagyのリンク。備忘的に。

＝＝＝

Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice

JCI 2010 online in March

Injury and loss of podocytes are leading factors of glomerular disease and renal failure. The postmitotic podocyte is the primary glomerular target for toxic, immune, metabolic, and oxidant stress, but little is known about how this cell type copes with stress. Recently, autophagy has been identified as a major pathway that delivers damaged proteins and organelles to lysosomes in order to maintain cellular homeostasis. Here we report that podocytes exhibit an unusually high level of constitutive autophagy. Podocyte-specific deletion of autophagy-related 5 (Atg5) led to a glomerulopathy in aging mice that was accompanied by an accumulation of oxidized and ubiquitinated proteins, ER stress, and proteinuria. These changes resulted ultimately in podocyte loss and late-onset glomerulosclerosis. Analysis of pathophysiological conditions indicated that autophagy was substantially increased in glomeruli from mice with induced proteinuria and in glomeruli from patients with acquired proteinuric diseases. Further, mice lacking Atg5 in podocytes exhibited strongly increased susceptibility to models of glomerular disease. These findings highlight the importance of induced autophagy as a key homeostatic mechanism to maintain podocyte integrity. We postulate that constitutive and induced autophagy is a major protective mechanism against podocyte aging and glomerular injury, representing a putative target to ameliorate human glomerular disease and aging-related loss of renal function.

●podocyteでbasal autophagyが高い（Fig1)→podocyte cell lineを作製！。

●ubiquitin-proteasome systemとfunctional cross-talk（UPSによるcompensation）があることを示す（Fig3)。が、age-dependentにlate-onset  glomeruosclerosisを生じる（Fig4)。

●ER stress (Calnexin)、oxidized and ubiquitinated protein aggregate（Ub, p62など）の蓄積を伴う（Fig5)。逆に、proteinuric diseaseでautophagyがupregulateしていることを示す（Fig6、BSAのoverloadで人工的にproteinuriaにしたサンプルと、ヒト疾患のbiopsy標本)。

●ATG5ノックアウトを用いて、stress adaptationにautophagyが必須であることを示す（Fig7、puromycin aminonucleoside (PAN)、adriamycinで負荷）。

＝＝＝

Induction of autophagy-dependent necroptosis is required for childhood acute lymphoblastic leukemia cells to overcome glucocorticoid resistance

JCI 2010 online in March

In vivo resistance to first-line chemotherapy, including to glucocorticoids, is a strong predictor of poor outcome in children with acute lymphoblastic leukemia (ALL). Modulation of cell death regulators represents an attractive strategy for subverting such drug resistance. Here we report complete resensitization of multidrug-resistant childhood ALL cells to glucocorticoids and other cytotoxic agents with subcytotoxic concentrations of obatoclax, a putative antagonist of BCL-2 family members. The reversal of glucocorticoid resistance occurred through rapid activation of autophagy-dependent necroptosis, which bypassed the block in mitochondrial apoptosis. This effect was associated with dissociation of the autophagy inducer beclin-1 from the antiapoptotic BCL-2 family member myeloid cell leukemia sequence 1 (MCL-1) and with a marked decrease in mammalian target of rapamycin (mTOR) activity. Consistent with a protective role for mTOR in glucocorticoid resistance in childhood ALL, combination of rapamycin with the glucocorticoid dexamethasone triggered autophagy-dependent cell death, with characteristic features of necroptosis. Execution of cell death, but not induction of autophagy, was strictly dependent on expression of receptor-interacting protein (RIP-1) kinase and cylindromatosis (turban tumor syndrome) (CYLD), two key regulators of necroptosis. Accordingly, both inhibition of RIP-1 and interference with CYLD restored glucocorticoid resistance completely. Together with evidence for a chemosensitizing activity of obatoclax in vivo, our data provide a compelling rationale for clinical translation of this pharmacological approach into treatments for patients with refractory ALL.

薬剤耐性（特にglucocorticoid耐性）ALLにBCL2-familyのputative antagonistであるobatoclaxを用いると、autophagy dependent necroptosisを起こして耐性が解除される、という内容。

●DEXとobatoclaxの組み合わせで、ALL細胞のviabilityが低下、これはBCL-2及びBCL-XLの特異的阻害剤であるABT-737では同様の効果が得られないことから、BCL-2ファミリーのMCL-1によるものと（Fig1、refでMCL-1がglucocorticoid耐性の主要因子であることが示されている)。

●この耐性解除はapoptosis independent、autophagy dependent（Fig2-4）。

●DEX＋obatoclaxでmTOR活性が低下（Fig5、機序は？）。

●ObatoclaxがBeclin-1とMCL-1との結合を抑制することを証明（Fig6)。ここで見られる細胞死がnecroticであることを示す（Fig7A,B、plasma membraneのintegrityが消失することを電顕で示す）。さらに、necrotic deathに関係するRIP-1 kinaseと、その制御因子であるdeUb enzymeのCYLDがこの経路のpositive regulatorであることを示す（Fig7C-G）。

●ヒト検体でこの現象の臨床的重要性を証明（Fig8）。

●Beclin1はhaploinsufficientなoncosupressor→autophagyの癌抑制的な機能を示唆。上流のあの分子と同じだ。。。

Early Embryogenesisの障害を引き起こす薬剤

Early embryonic losses in mice induced by diethylstilbestrol

Congenital Anomalies　49:269-273, 2010

Estrogens cause embryonic lethality and the disturbance of early placental development in mice. Diethylstilbestrol (DES) at 1, 10, or 100 ?g/kg was orally administered to Institute of Cancer Research mice on gestational days (GD) 4 through 8, and the uterus and placenta were examined histopathologically on GD 9. Decidua of DES-treated mice showed insufficient development, and the uterine lumen at the implantation site did not effectively minimize. The trophoblast giant cell layer was not separated from the uterine lumen by the decidua capsularis, and hemorrhage from the denuded trophoblast giant cell layer into the uterine lumen was noted at the peripheral part of the decidua basalis. The results of the present study suggest that decidual hypoplasia and subsequent placental hemorrhage causes fetal death due to the administration of DES during the early stage of pregnancy.

ゴールから振り返る

何か大きなことを成し遂げるときに、最初にやること 

より、一部引用

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「何かを成し遂げようと思うときは、今自分がいる位置からどう積み上げるか、と考えてはダメだ。

出来上がった本なり、目指してるものを自分が今手にしていることを想像して、今の自分がいる位置を振り返ってみるのが大切だ。」

ゴールにたどり着くには、様々な道があり、どの道を通っていけばいいのか、全く分からない。

でも、ゴールにたどり着いた自分から見れば、道は自分が通ってきた一本だけだ。

ゴールにいる自分をよく想像する。

そこから今の自分を振り返ると、一本の通るべき道が見える。

その道をよく見ると、今の自分には大きく欠けている部分（Missing part）がいくつか見えるだろう。

そのMissing Partsこそが、最初に取り組むべきことだ。

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「引き寄せの法則」とかも同じことを言っていて、「自分の望みを実現した状態を強くイメージすることが大事」と。

「集中力」が増す3つの仕かけ

プレジデント　2010年2月12日(金) 

より、一部引用

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●自己報酬神経群とは、その名の通り「自分へのごほうび」をモチベーションに働く部位であり、この部位が活発に働かないと脳は活性化しない。
重要なのは、活性化はごほうびが得られたという「結果」によって起こるのではなく、ごほうびが得られそうだという「期待」によって起こる点だ。ごほうびが得られた、つまり結果を手にしたと思うと、むしろ脳の機能は低下してしまうのである。
集中力を持続するには、この脳の仕組みを利用すればいい。ゴールの仕方に集中する。あるいは、目標よりも遠くにゴールを定めるのだ。そうすれば、実際のゴールの手前で脳のパフォーマンスが落ちることはなくなる。


●脳には自己保存本能がある。（中略）自己保存本能は人間にとって大切なものだが、「失敗するかもしれない」という否定語は、この自己保存本能に過剰反応を起こさせて、脳の働きにブレーキをかけてしまう。それゆえ、コツコツやるという人は、自分が現在持っている以上の力を発揮することが難しいのである。
反対に、とても到達できそうにない目的に向かって一気にかけ上がろうと考えると、脳は信じられないほど高いパフォーマンスを示してくれる。つまり、実際は長距離走の場合でも、短距離走のつもりで全力疾走を繰り返すことで、あるところから人間の能力はぐーっと伸びてくる。
そして一気、一気でダッシュを繰り返して、ふと気付くと、到底超えられそうもなかった壁を突破しているものなのだ。そんな人のことを世間は、異様な集中力を持った人と呼ぶ。


●「（競泳の北島選手に対して）ハンセンをライバルだと思っちゃいけない。自分を高めるためのツールだと思いなさい。そして、最後の10メートルをKゾーン（北島ゾーン）と名づけて、水と仲間になり、ぶっちぎりの、感動的な泳ぎを見せる舞台だと思いなさい」
つまり、ハンセンとも水とも「仲間になれ」とアドバイスしたのだ。（中略）
結果を求めるあまり能力を発揮できない愚を避けるには、目標達成の「仕方」にこだわるのがいい。（中略）結果を求めず、達成の仕方に全力投球するとき、人間は信じられない集中力を発揮する。ポイントは、「損得勘定抜きに」だ。損得勘定とは、実は、結果を求める気持ちにほかならないからである。


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脳科学的なこじつけが正しいかどうか疑問のところはあるが、役に立つならまあ良し。
集中力の問題はここ暫くの課題。「昴」のボレロ編で主人公が見せたような集中力が再現よく出せるようになるのが理想。

ARACNE

ベイジアンネットワークを改良したネットワーク解析アルゴリズム。遺伝子間の相互作用の強さを評価して、間接的な相互作用とみなせる関係を排除し、直接的な相互作用を高い精度で推定することを目指した。

ARACNE (Algorithm for the Reconstruction of Accurate Cellular Networks), a novel algorithm, using microarray expression profiles, specifically designed to scale up to the complexity of regulatory networks in mammalian cells, yet general enough to address a wider range of network deconvolution problems. This method uses an information theoretic approach to eliminate the vast majority of indirect interactions typically inferred by pairwise analysis.

On synthetic datasets ARACNE achieves extremely low error rates and significantly outperforms established methods, such as Relevance Networks and Bayesian Networks. Application to the deconvolution of genetic networks in human B cells demonstrates ARACNE’s ability to infer validated transcriptional targets of the c-MYC proto-oncogene.

Ex）ヒトB細胞の遺伝子間相互作用推定

Reverse engineering of regulatory networks in human B cells. Nature Genetics. 2005 Apr;37(4):382-90. (Download PDF)

PANTHER Classification system

遺伝子、タンパク、パスウェイ等の公開データベース。

The PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System is a unique resource that classifies genes by their functions, using published scientific experimental evidence and evolutionary relationships to predict function even in the absence of direct experimental evidence. Proteins are classified by expert biologists into families and subfamilies of shared function, which are then categorized by molecular function and biological processontology terms. For an increasing number of proteins, detailed biochemical interactions in canonical pathways are captured and can be viewed interactively.To get started, try either a text search, browsing by function, or take a look at interactive pie charts that summarize the functions of whole genomes for Human, Mouse, Rat and Drosophila melanogaster.
PANTHER is supported by a research grant from the National Institute of General Medical Sciences [grant GM081084] and maintained by theEvolutionary Systems Biology Group at SRI.

Our database of genes, transcripts and proteins in the following species: Human, Mouse, Rat, and Drosophila melanogaster. Search by keyword Search by location Browse by function More...

A library (PANTHER/LIB) of protein families and subfamilies and associated data such as phylogenetic trees, multiple sequence alignments and HMMs. Search PANTHER Families Score a sequence against PANTHER HMMs More...

A database of 165 expert-curated signaling and metabolic pathways mapped to protein sequences in PANTHER Library, viewable in standard CellDesigner notation, a simplified activity flow notation, and Systems Biology Graphical Notation (SBGN) using our interactive pathway applet. Browse Pathways Search Pathways More...

A collection of terms (PANTHER/X) describing protein molecular functions and biological processes. Browse ontologies PANTHER to Gene Ontology™ mapping Gene Ontology™ to PANTHER mapping

マイクロアレイのパスウェイ解析も可能。