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Rho-ROCK and Rac-PAK Signaling Pathways Have Opposing Effects on the Cell-to-Cell Spread of Marek’s Disease Virus

Rho-ROCK and Rac-PAK Signaling Pathways Have Opposing Effects on the Cell-to-Cell Spread of Marek’s Disease Virus

Cell-to-cell spread efficiency is the result of the virus cell-to-cell spread itself and of the virus cycle from the entry to the assembly of infectious virions, and probably not as a simple sum of virus release and entry [5]. Since several years, growing evidences show the role of actin and various RhoGTPases during early and/or late steps of herpesviruses cycle as thoroughly reviewed [65,66,67,68,69]. Therefore for MDV, as cell-to-cell spread cannot be separated from replication, entry and egress need also to be taken into account in the interpretation of the cell-to-cell spread results. We are now discussing if one of these steps could explain the favorable effect of Rho-ROCK pathway and the negative one of Rac-PAK that we observed in MDV spread. With respect to entry, for other herpesviruses the involvement of RhoGTPases was always evaluated a short time post-infection with cell-free virions at moderate moi, experimental conditions which are significantly different from ours (infection at low moi with cell- associated virus, long duration of infection with multiple virus cycles, on a dense cell layer). Among theses studies, a few depicted a positive effect of RhoA or ROCKs on the early stages of infection [70,71,72,73]. For HSV-1, the entry in primary human corneal fibroblasts and nectin-1-CHO cells was described to involve RhoA activation and to be mediated by a phagocytosis-like uptake RhoA-dependent [70]. This process requires, before RhoA activation, the formation of membrane ruffles which depends on Rac1 and PAK1 [70,74], a pathway that is not favorable for MDV cell-to-cell spread. This led us to conclude that a phagocytosis-like uptake in CESCs cannot explain by itself the effect of Rho/Rac signaling on MDV spread. Another example is EHV-1 [71], which enters via an endocytic mechanism or by direct fusion at the cell surface according to the cells, like HSV-1. In both situations, EHV-1 requires ROCK activity for a productive EHV-1 infection through a non-elucidated mechanism, as demonstrated in presence of Y-27632 or after overexpression of Gem, a negative ROCK1 regulator. A similar mechanism is therefore possible for MDV. The last example concerns KSHV, which was found to activate RhoA early after infection in human fibroblast cells, with RhoA having a role in the nuclear delivery of viral DNA [72]. Here the role of RhoA was associated to Dia2, another of its downstream effector [73] and the MTs stabilization, a mechanism
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A bacterial regulatory RNA attenuates virulence, spread and human host cell phagocytosis.

A bacterial regulatory RNA attenuates virulence, spread and human host cell phagocytosis.

MATERIALS AND METHODS Strains, culture conditions, genetic manipulations and oxida- tive stress experiments All the bacterial strains and plasmids used are listed in Sup- plementary Table S1. S. aureus strains were grown at 37 ◦ C in brain heart infusion broth (BHI, Oxoid). When neces- sary, chloramphenicol and erythromycin were added at a 10 ␮g/ml concentration. For the oxidative stress, overnight cultures were diluted (1 /100), incubated with 5 mM hy- drogen peroxide (Aldrich Chemical, St Louis, MO) and optical density was measured using the spectrophotome- ter Biotek Synergy 2. A chromosomal gene disruption mutant was constructed by deletion of the targeted gene and insertion of an erythromycin-resistance gene by us- ing the temperature-sensitive vector pBT2 ( 19 ) according to ( 15 ). Double-crossover events corresponding to the de- sired gene disruptions were confirmed by polymerase chain reaction (PCR) and sequenced. In all the sRNA overex- pression genetic constructions, the RNAs are expressed from their endogenous promoters. In pCN35 sprC and pCN38 sprC, the nucleotide sequence of sprC containing 300 upstream nucleotides was amplified from Newman ge- nomic DNA as a 460-bp fragment possessing EcoRI and PstI restriction sites at each end. The PCR product was cloned in pCN35erm and pCN38cat. E. coli DH5 ␣ were transformed with pCN35 sprC and pCN38sprC and se- lected for ampicillin resistance. The plasmids pCN35-sprC and pCN38-sprC were transferred into RN4220 and se- lected for erythromycin and chloramphenicol resistance re- spectively, then finally transferred to Newman WT and Newman sprC.
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Correlation of cell membrane dynamics and cell motility

Correlation of cell membrane dynamics and cell motility

proteins [5]. A series of studies using quantitative fluores- cent speckle microscopy have revealed the power of com- puter assisted high throughput analysis of time-lapse microscopy images: an analysis of the number of speckles suggested distinct regulation of actin polymerization- depolymerization dynamics in different intracellular regions [6,7]. The ratio of protrusive to inactive cell peri- meter has been used as the measure of cell edge activity [8]. Difference of the cell membrane boundary was reported in the study of cell spread dynamics [9] and its role in actin transport for protruding lamellipodia [10], formation of filopodia downstream of SCAR (Suppressor of cAMP receptor) [11], and the role of cofilin as a pro- moter of actin polymerization leading to protrusion [12]. Alternatively protrusion rates are measured at multiple locations of the cell boundary. The morphological changes have been studied by placing markers in the cell boundary at regular intervals and tracking their displace- ment in orthogonal directions to the cell boundary [13]. Instead of direct displacement of tracking, cell bound- aries can be analyzed with kymographs [14]. This techni- que involves high resolution time-lapse microscopy to capture subcellular motion which is widely used for rela- tively small sample sizes due to highly magnified imaging and for relatively short periods of time. However, these approaches are not suitable for high throughput applica- tions due to computational complexity compounded by elaborate cell shapes and its ever changing dynamics.
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Cell invasion in the spheroid sprouting assay: a spatial organisation analysis adaptable to cell behaviour.

Cell invasion in the spheroid sprouting assay: a spatial organisation analysis adaptable to cell behaviour.

2D-projected Images of the Spheroid Assay When embedded in a collagen gel, cells can adopt different modes of invasion as depicted in Figure 1 a. During invasion, cells can remain linked to the spheroid, leading to the generation of a rough spheroid, spread out from the spheroid or detach from it to migrate as single cells or aligned in tube-like structures. This cell invasion process can lead to empty regions in the core spheroid. Image processing prior to image measurement classically consists of filtering the images to eliminate noise and increase the contrast followed by image binarisation, in which objects of interest, in this case cells, are assigned a pixel value of 1, with the background assigned a value of 0. To achieve this, we proceeded in several steps (Figure 1). At t = 0, the spheroid image was usually very well contrasted (Figure 1 b), and the choice of an appropriate threshold for binarisation was straightforward (Figure 1 c). At later time points (t.0), the spheroid consisted of an expanded spheroid core and migrating cells (Figure 1 d). These features were discriminated using the Frangi multiscale filter [23] (Figure 1 e), which allows for the identification of a wide range of object sizes, elongations and orientations in images presenting non-homogeneous intensities. In some cases, morphological filters [24] were used to eliminate any remaining noise in the images.
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Correlation of cell membrane dynamics and cell motility

Correlation of cell membrane dynamics and cell motility

proteins [5]. A series of studies using quantitative fluores- cent speckle microscopy have revealed the power of com- puter assisted high throughput analysis of time-lapse microscopy images: an analysis of the number of speckles suggested distinct regulation of actin polymerization- depolymerization dynamics in different intracellular regions [6,7]. The ratio of protrusive to inactive cell peri- meter has been used as the measure of cell edge activity [8]. Difference of the cell membrane boundary was reported in the study of cell spread dynamics [9] and its role in actin transport for protruding lamellipodia [10], formation of filopodia downstream of SCAR (Suppressor of cAMP receptor) [11], and the role of cofilin as a pro- moter of actin polymerization leading to protrusion [12]. Alternatively protrusion rates are measured at multiple locations of the cell boundary. The morphological changes have been studied by placing markers in the cell boundary at regular intervals and tracking their displace- ment in orthogonal directions to the cell boundary [13]. Instead of direct displacement of tracking, cell bound- aries can be analyzed with kymographs [14]. This techni- que involves high resolution time-lapse microscopy to capture subcellular motion which is widely used for rela- tively small sample sizes due to highly magnified imaging and for relatively short periods of time. However, these approaches are not suitable for high throughput applica- tions due to computational complexity compounded by elaborate cell shapes and its ever changing dynamics.
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Cell-Intrinsic and cell-extrinsic resistance to classical chemotherapies

Cell-Intrinsic and cell-extrinsic resistance to classical chemotherapies

played an integral role in keeping me sane, if not healthy. I am forever indebted to my two best friends from college, Rachel Amouyal née Ellman, and Leah Mosenkis née Robsman. There is far far too much to write here, but I certainly would not have reached this day without the two of them. Without Cosco I likely would not have done this research, perhaps wouldn’t have even gone to grad school. The lessons and experiences from that time continue to reverberate in my life in ever sweeter ways ( ב ה ). I am so thankful we have managed to keep in touch as our ׳׳ friendship maintains me in many ways. I only wish we didn’t live spread over 9 hours of time zones. I am also grateful for the friendship of Ranit Patel, who I have known nearly since birth. Defenestrating ourselves and re-wiring her house as children were fun, but her presence during my 5 th year of graduate school which began horribly and ended
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Cell-to-Cell Spreading of HIV-1 in Myeloid Target Cells Escapes SAMHD1 Restriction

Cell-to-Cell Spreading of HIV-1 in Myeloid Target Cells Escapes SAMHD1 Restriction

While HIV-1 infection with cell-free viruses has been largely documented, cell-to-cell transmission probably represents the dominant mode of infection (23, 24). This prop- agation route is very efficient and involves the different cell types targeted by HIV-1, including cells of the myeloid lineage (i.e., macrophages, DCs, and OCs) (22, 24–26), which are not easily infected by cell-free viruses, mainly because of the high expression of cellular restriction factors, including SAMHD1, an enzyme that cleaves deoxynucleo- side triphosphates (dNTPs) and depletes the pool of intracellular nucleotides necessary for efficient HIV-1 replication in these noncycling myeloid cells (16, 27–31). This cell-to-cell mode of infection likely plays important roles at the level of genital and rectal mucosa and then for virus spread in numerous host tissues. However, there is still a paucity of knowledge of the mechanisms that control virus dissemination by cell-to- cell transfer in myeloid cell targets. Only two recent reports have been published regarding virus cell-to-cell dissemination to macrophages from infected T cells. While it was reported previously by the group of Quentin Sattentau that macrophages could be infected via selective capture or phagocytosis of HIV-1-infected T cells (26), we have reported that HIV-1 is mainly transferred to macrophages from infected T cells by a two-step cell fusion process (25).
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Cell interactions and cell signaling during hematopoietic development

Cell interactions and cell signaling during hematopoietic development

2 CNRS, UMR 7622, Inserm U 1156, IBPS, Laboratoire de Biologie du Développement, 75005 Paris 3 Corresponding author. E-mail : thierry.jaffredo@upmc.fr Abstract Hematopoiesis is a key process that leads to the formation of all blood cell lineages from a specialized, multipotent cell, named the Hematopoietic Stem Cell (HSC). During development, the embryo produces several waves of hematopoiesis that produce specialized subsets of hematopoietic cells. Tissue interactions and cell signaling play an essential role in developmental hematopoiesis by allowing the formation of hematopoietic and endothelial cells (EC) from the mesoderm in particular in the yolk sac and by instructing the different generations of hematopoietic cells (HC). The embryonic aorta is another site wherein tissue interaction is essential for the production of the first HSCs that is achieved from a specialized subset of hemogenic endothelial cells. This production is tightly time- and space-controlled with the transcription factor Runx1 and the Notch signaling pathway playing a key role in this process and the surrounding tissues controlling the aortic shape and fate. Here we shall briefly review how hemogenic EC differentiate from the mesoderm, how the different aortic components assemble coordinately to establish the dorso-ventral polarity resulting in the initiation of Runx1 expression in hemogenic EC and the initiation of the hematopoietic program through modulation of the Notch- Runx1 axis. These data should help elucidate the first steps in HSC commitment and bring further insights into the manipulation of adult HSCs.
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Bispecific, T-Cell-Recruiting Antibodies in B-Cell Malignancies.

Bispecific, T-Cell-Recruiting Antibodies in B-Cell Malignancies.

Non-Hodgkin Lymphomas Non-Hodgkin lymphomas are B-cell malignancies that are primarily located in lymph nodes. The disease progression is driven by precursor lymphocytes, where 85% of the cases emerge from B-cell precursors ( 69 ). The 5-year survival rates vary highly, from 30% to 86%, among the subtypes of NHL ( 70 ). These subtypes are mainly categorized into two groups. Aggressive lymphomas are rapidly evolving entities with a high tumor cell proliferation rates, but potentially curable when responding to high-dose chemotherapy. In contrast, indolent subtypes represent low grade lymphomas and are incurable ( 71 ). Specific translocations enhance the expression of oncogenic proteins and disrupt DNA damage control mechanisms and will finally result in the development of various NHL subtypes ( 69 ). To target these cells, cell surface Ags CD19, CD20 and CD30 are widely used targets ( 72 ).The diagnosis is established by tissue biopsy, followed by immunohistochemistry and genetic studies ( 71 ). Further evaluation of the disease progression can be obtained by staging systems, such as international prognostic index (IPI) and combined Positron Emission Tomography – Computed Tomography (PET-CT) ( 73 ).
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Cell Wall

Cell Wall

Three types of CWP can be distinguished on the basis of their interactions with cell wall components (11). CWP can have little or no interactions with cell wall polysaccharides or other CWP and thus move in the extracellular space. Such proteins can be found in liquid culture media of cell suspensions and seedlings or can be extracted with low ionic strength buffers. We call this fraction “labile proteins”. Most of them have acidic pI ranging from 2 to 6. Alternatively, CWP can be weakly bound to the matrix by Van der Waals interactions, hydrogen bonds, hydrophobic or ionic interactions. Such proteins can be extracted by salts. Most of them have basic pI ranging from 8 to 11, so that they are positively charged at the acidic pH of cell walls. Even though most of the cell wall polysaccharides are neutral, pectins contain polygalacturonic acid residues that provide negative charges for interactions with basic proteins. Such interactions would be modulated by pH, degree of pectin esterification, Ca 2+ concentration, as well as mobility and diffusion coefficients of these macromolecules (8). Two protein domains involved in interactions with pectins have been described. A four Arg residues-domain of a peroxidase was shown to have a high affinity for Ca 2+ -pectate in vitro (12). A domain of two polygalacturonase-inhibiting proteins comprising four residues of Arg and Lys interacts in vitro with pectin (13). Finally, CWP can be strongly bound to cell wall components so that they are resistant to salt-extraction. As examples, extensins are cross- linked by covalent links (14).
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Biodiversity as a barrier to glioma cell invasion.

Biodiversity as a barrier to glioma cell invasion.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignemen[r]

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Defining the contributors to mammalian cell mass

Defining the contributors to mammalian cell mass

mTORCI senses amino acid sufficiency; when amino acids are abundant, this kinase activates downstream biosynthetic pathways (such as protein synthesis) to generate new c[r]

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Bacterial cell proliferation: from molecules to cells

Bacterial cell proliferation: from molecules to cells

ORIGINAL UNEDITED MANUSCRIPT Figure 2. Phases of early inhibition and late activation drive the coordination between cell division and DNA segregation. The choreography of the chromosome along the cell cycle drives the temporal switch between inhibition and by spatially re-organizing key regulatory factors. On panels A., B. and C. are represented the positive (green) or negative (red) effects of key factors on the coordination between DNA segregation and cell division. Chromosomes are represented in white with their origin of replication, ori (white circle) and dif site (black and white square). The names of the major positive (green) and negative (red) regulators described in the text for each model organism is listed below each schematic. The black color for ParB indicates a dual role. A. In Enterobacteriaceae, the early inhibition of divisome assembly is mediated by the oscillatory Min system and the nucleoid occlusion factor SlmA. At later stages, SlmA has cleared away from mid-cell and multiple activators drive the localization of the ter macrodomain, centered around dif, at the division site and coordinate late segregation events with cytokinesis. B. In B. subtilis and C. crescentus, ParB, Noc and Min (ParB and MipZ in C. crescentus) organized at the pole(s) and around ori cooperate to inhibit Z ring formation at mid-cell. At later stages, the relocation of the two ori copies to both poles lifts the early inhibition. The crosstalk between late segregation steps and the division machinery via FtsK homologs (SpoIIIE and SftA in B. subtilis) remains to be established. C. In S. pneumoniae, the Min system is absent and poles do not serve as organizing centers. Instead, the segregation of the two copies of ori lifts the inhibition of the division machinery activity at mid cell and drives the assembly new division machineries at ¼ - ¾ positions in coordination with MapZ. The role the DNA translocase FtsK has not been
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Cell cycle: The bacterial approach to coordination

Cell cycle: The bacterial approach to coordination

Control of replication initiation in C. crescentus C. crescentus has been a fertile system for analysing how the cell cycle and differentiation are coordinated. Recent work has characterized a transcription factor that coordinates the initiation of DNA replication with several other cellular processes [16,17]. Growing cultures of C. crescentus contain two distinct cell types: a sessile, stalked cell that is compe- tent to initiate DNA replication, and a motile, swarmer cell. The swarmer cell is unable to initiate the cell cycle until it differentiates into a stalked cell, losing its flagellum and developing a prosthetic ‘stalk’ at the same pole. The newly formed stalked cell can then initiate DNA replication, eventually dividing asymmetrically to produce a swarmer cell and a stalked cell.
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Regulatory T Cell-Enhancing Therapies to Treat Atherosclerosis

Regulatory T Cell-Enhancing Therapies to Treat Atherosclerosis

To be efficient, the T cell immune response must be tightly regulated, in order to effectively respond to pathogens, prevent responses to self-antigens, and avoid mounting harmful responses to innocuous antigens. Cell-intrinsic mechanisms of tolerance in the thy- mus and periphery ensure the physical elimination of self-reactive T cells by clonal deletion or their functional inactivation by clonal anergy. In addition, cell-extrinsic mechanisms, mediated in part by specialized CD4 + T cells with immunosuppressive activity, Tregs, help regulate Th cell activity [ 43 ]. Naturally-occurring Tregs are generated in the thymus during T cell development. They comprise 5–10% of all peripheral CD4 + T cells. Treg lineage commitment essentially depends on TCR engagement by high avidity for self-peptide– MHC and costimulatory signals mediated by CD28, resulting in the upregulation of CD25. IL-2 or IL-15 are required for fully committed Tregs expressing the forkhead/winged helix transcription factor FoxP3, which is crucial for their development and function. Deficiency of FoxP3 in both humans and mice results in a lack of Tregs, and the development of severe systemic inflammatory diseases manifested by autoimmunity, colitis, and allergies [ 44 ]. The second route for the generation of FoxP3 + Tregs, termed induced Tregs (iTregs), is the differentiation of naïve CD4 + T cells in the periphery following TCR stimulation in the presence of IL-2 and TGF-β. Although iTregs compose only a small percentage of Tregs as a whole, this cell subpopulation is particularly enriched in certain tissues, such as the gut and maternal placenta, and crucially participates in the establishment of tolerance against commensal bacteria, foods, allergens, and the fetus in a pregnant mother [ 45 ]. In addition to the phenotypic and functional heterogeneity of Tregs, it has been suggested that Tregs can become unstable under certain inflammatory conditions and acquire a phenotype more characteristic of effector T cells, which promotes rather than suppresses inflammation [ 43 ]. 4.2. Treg Suppressive Activity
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Contribution to the development of IDEAL-Cell, a new concept of intermediate temperature fuel cell

Contribution to the development of IDEAL-Cell, a new concept of intermediate temperature fuel cell

Proposing a new concept of fuel cell to the scientific community wouldn’t make sense if it couldn’t bring advantages with respect to the already existing concepts. What was written in the two previous sections allows us to conclude the advantages expected from IDEAL-Cell. The innovation of this concept lies on the central membrane, which is the core of the device [13]. The idea of a mixed dedicated proton and oxygen ion conducting membrane that can allow water formation and evacuation simplifies the role the electrodes have to play. Compared to the already existing systems, the constraints in terms of microstructures are decreased. Thus, materials and microstructures of the anode and the cathode can now be optimized to their main purpose: fuel and combustive diffusion and their electrochemical reactions. It is no longer necessary to take into account the gas counter-flow to evacuate water out of the electrodes microstructures. Since water is not present in the electrodes, the electrochemical sites activity is no longer decreased. In fact, oxygen and hydrogen chambers are almost closed (single gas flow, no exhaust pipe); the stack architecture design is therefore made simpler than that of SOFC and PCFC, and offers the possibility of applying a pressure independently at both anode and cathode sides. This will allow the optimization of operating conditions and will drastically limit the polarization losses. In the anode, fuel is not diluted and the cell’s potential is not decreased. In the cathode, a dry oxidizing atmosphere is less demanding both for the cathode and the interconnects materials. Since some oxygen ion electrolyte materials have been reported to have good performances at 600 ◦
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Cell-cell and cell-medium interactions in the growth of mouse embryonic stem cells

Cell-cell and cell-medium interactions in the growth of mouse embryonic stem cells

3.3.3 Self­renewal potential and its variation with the number of cells in a cluster  We next decided to investigate a more comprehensive measure of self‐renewal. To do this we first need  to  define  a  quantitative  measure  of  self‐renewal.  Since  the  process  of  self‐renewal  consists  of  cell  division while maintaining the pluripotency of cells, we wished to determine the growth rate as well as  the level of pluripotency of cells (defined as the percentage of pluripotent cells in a colony) in the colony  resulting from a cluster of cells. Pluripotency can be assessed via a number of assays. The least stringent  functional  assay  for  the  developmental  potential  of  a  cultured  cell  is  in  vitro  differentiation  followed,  with increasing stringency, by the generation of teratomas (germ cell tumors), chimera formation, and  germ line contribution 90 . The most rigorous test for developmental potency is the injection of cells into  4n  host  blastocysts 91, 92 ,  which  results  in  animals  composed  only  of  the  injected  donor  cells  (“all  ES”  embryos  or  animals)  rather  than  a  chimeric  composite  of  injected  and  host‐derived  cells.  However,  these  approaches  are  challenging  to  perform  with  single  cells,  and  when  one  is  interested  in  assaying  large  numbers  of  cells.  A  commonly  used  alternative  is  to  measure  the  expression  level  of  certain  proteins  that  are  referred  to  as  pluripotency  markers 157, 158 .  These  proteins  are  highly  expressed  in  ES  cells,  but  not  in  differentiated  cell  types.  Three  proteins  that  are  widely  accepted  as  pluripotency  markers  for  mESCs  are  the  transcription  factors  Oct‐4,  Sox‐2,  and  Nanog 23 .  Ideally  one  would  like  to  separately  measure  the  growth  rate  and  marker  expression  of  cells.  However,  as  mentioned  before,  measuring  the  growth  rate  for  ESCs  is  challenging  in  a  system  such  as  ours  where  the  readout  is  microscopy  based.  Therefore  we  decided  to  instead  measure  the  total  Oct‐4  expression  of  the  colony  formed from a cluster of cells, i.e., the product of the Oct‐4 expression level per cell and the number of  cells in the colony. We call this quantity the self‐renewal potential (SRP) of a cluster of cells. Since the  SRP  depends  on  the  Oct‐4  expression  level  as  well  as  the  number  of  cells  in  the  resulting  colony,  it  encompasses both features of self‐renewal. Our choice of Oct‐4 over Sox‐2 or Nanog was determined by  the availability of a cell‐line expressing green fluorescent protein (GFP) under the control of the Oct‐4  promoter (ABJ1 cell‐line 84 ), which allowed for an easy, microscopy‐based determination of the SRP. 
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Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency

Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency

. Specifically, which is the precise correlation of these different pluripotency states with the in vivo equivalents is still a question of debate. Known molecular markers of such plasticity are mainly transcription factors operating within a pluripotency gene regulatory network 9 . More recently, metabolites are emerging as key regulators of stem cell plasticity, acting as epigenetic modifiers 10,11 ; however, much less is known on the role of microenvironment. Indeed, elucidation of the extrinsic mechanisms that control stem cell plasticity is crucial for understanding both early embryo development and controlling the differentiation potential of pluripotent stem cells 12 . In the attempt to shed lights on this issue, we focused on the glycosylphosphatidylinositol (GPI)-anchored extracellular protein Cripto. Cripto is a key developmental factor and a multifunctional signalling molecule 13 . In the mouse embryo, Cripto is essential for primitive streak formation and patterning of the anterior–posterior axis during gastrulation 14 and it negatively regulates ESC neural differentiation while permitting cardiac differentiation 15 . Although largely considered as a stem cell surface marker 16 , no studies so far have directly investigated its functional role in pluripotency. In this study, we report the consequences of genetic and pharmacological modulation of Cripto signalling on the generation and/or maintenance of mEpiSCs and hESCs.
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Analysis of Single-Cell RNA-Seq Identifies Cell-Cell Communication Associated with Tumor Characteristics

Analysis of Single-Cell RNA-Seq Identifies Cell-Cell Communication Associated with Tumor Characteristics

Classification of Cell Types Based on scRNA-Seq Data of Syngeneic Mouse Tumor Models To begin identifying cell type specific cell-cell communication, we first identified the cell type of each single cell. Because of lim- itations in scRNA-seq technology, such as mRNA capture efficiency, the collected data contained undetected genes ( Ko- lodziejczyk et al., 2015 ). This phenomenon is collectively called ‘‘zero dropout’’ and makes identification of cell types based on individual marker genes infeasible for all cells in the dataset. We therefore refined a previously published supervised classifi- cation approach for assigning cell types ( Schelker et al., 2017 ). We first manually defined a list of cell types for which to search in the dataset and then specified marker genes that define each cell type ( Table S3 ; Determining Gene Markers for Synge- neic Tumor Models ). To assign individual cells as positive or negative for each marker gene, we fit Gaussian mixture models to the expression values of each marker gene and then assigned each cell in the dataset to one of the mixture components. We tested Gaussian mixture models containing one through five components to allow for the possibility of multi-modal gene expression ( Fitting Gaussian Mixture Models to Determine Marker Expression ). However, in all cases except one (Rpl29), mixture models containing two components best fit the gene expression profile using the Bayesian information criteria (BIC) as a metric for model selection ( Figure S2 C).
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Cytolytic T-cell response to the PASD1 cancer testis antigenin patients with diffuse large B-cell lymphoma

Cytolytic T-cell response to the PASD1 cancer testis antigenin patients with diffuse large B-cell lymphoma

Results c-IFN release assay The results of the c-IFN response ELISPOT assay are summarized in Table II. A significant c-IFN response was detected in 21/29 (72%) HLA-A*0201-positive DLBCL patients after short-term culture with PASD1 peptides com- pared to those results obtained from the control cultures (cells stimulated with the irrelevant HIV peptide or medium only, P < 0Æ05). Of these, 18 patients had de novo DLBCL of which two patients had relapsed DLBCL, two patients developed DLBCL via transformation of their FL and one patient had T-cell rich B-cell lymphoma. Thirteen patients responded to two or more peptides and, of these, two patients responded to all five PASD1 peptides, one patient to four peptides and six patients to three peptides. The frequencies of PASD1-respond- ing T cells varied between patients, ranging from 1:600 (0Æ17%) PBMCs in Patient 1 to 1:2000 (0Æ05%) in Patient 2. No significant c-IFN responses were obtained from the HLA- A*0201-negative patients with either de novo (n = 12), trans- formed (n = 8) DLBCL or T-cell rich B-cell lymphoma (n = 1). Results are shown in Table SI. Furthermore, none of the PBMCs obtained from the four HLA-A*0201-positive and 2 HLA-A*0201-negative healthy subjects recognized the PASD1 peptides. Although only relatively small numbers of patients were studied, it is notable that of those patients who were able to recognize the PASD1 peptides, 12 achieved complete remission, six are currently in partial remission while three patients have died. This is in contrast to the situation with the eight HLA-A*0201-positive patients who were unable to recognize PASD1 peptides where only one achieved complete remission, three are in partial remission and four have died during the course of this study.
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