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Sample records for single-cell stat5 signal

  1. Stat5 signaling specifies basal versus stress erythropoietic responses through distinct binary and graded dynamic modalities.

    PubMed

    Porpiglia, Ermelinda; Hidalgo, Daniel; Koulnis, Miroslav; Tzafriri, Abraham R; Socolovsky, Merav

    2012-08-01

    Erythropoietin (Epo)-induced Stat5 phosphorylation (p-Stat5) is essential for both basal erythropoiesis and for its acceleration during hypoxic stress. A key challenge lies in understanding how Stat5 signaling elicits distinct functions during basal and stress erythropoiesis. Here we asked whether these distinct functions might be specified by the dynamic behavior of the Stat5 signal. We used flow cytometry to analyze Stat5 phosphorylation dynamics in primary erythropoietic tissue in vivo and in vitro, identifying two signaling modalities. In later (basophilic) erythroblasts, Epo stimulation triggers a low intensity but decisive, binary (digital) p-Stat5 signal. In early erythroblasts the binary signal is superseded by a high-intensity graded (analog) p-Stat5 response. We elucidated the biological functions of binary and graded Stat5 signaling using the EpoR-HM mice, which express a "knocked-in" EpoR mutant lacking cytoplasmic phosphotyrosines. Strikingly, EpoR-HM mice are restricted to the binary signaling mode, which rescues these mice from fatal perinatal anemia by promoting binary survival decisions in erythroblasts. However, the absence of the graded p-Stat5 response in the EpoR-HM mice prevents them from accelerating red cell production in response to stress, including a failure to upregulate the transferrin receptor, which we show is a novel stress target. We found that Stat5 protein levels decline with erythroblast differentiation, governing the transition from high-intensity graded signaling in early erythroblasts to low-intensity binary signaling in later erythroblasts. Thus, using exogenous Stat5, we converted later erythroblasts into high-intensity graded signal transducers capable of eliciting a downstream stress response. Unlike the Stat5 protein, EpoR expression in erythroblasts does not limit the Stat5 signaling response, a non-Michaelian paradigm with therapeutic implications in myeloproliferative disease. Our findings show how the binary and

  2. Dasatinib (BMS-354825) inhibits Stat5 signaling associated with apoptosis in chronic myelogenous leukemia cells.

    PubMed

    Nam, Sangkil; Williams, Ann; Vultur, Adina; List, Alan; Bhalla, Kapil; Smith, David; Lee, Francis Y; Jove, Richard

    2007-04-01

    Dasatinib (BMS-354825) is a novel, oral, potent, multi-targeted kinase inhibitor of Bcr-Abl and Src family kinases (SFK) and is a promising cancer therapeutic agent. Preclinical data indicate that dasatinib is 325-fold more potent than imatinib against cells expressing wild-type Bcr-Abl, and that dasatinib is active against 18 of 19 Bcr-Abl mutations known to cause imatinib resistance. Phase I clinical data show that dasatinib is well tolerated and highly effective for the treatment of imatinib-resistant/imatinib-intolerant chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia. However, the molecular mechanism of action of dasatinib is not fully understood. In this study, we confirm that dasatinib inhibits tyrosine phosphorylation of SFKs, including Src, Hck, and Lyn, in K562 human CML cells. Significantly, downstream signal transducer and activator of transcription 5 (Stat5) signaling is also blocked by dasatinib as shown by decreases in levels of phosphorylated Stat5 and Stat5 DNA-binding activities. In addition, dasatinib down-regulates expression of Stat5 target genes, including Bcl-x, Mcl-1, and cyclin D1. Consistent with these results, blockade of Stat5 signaling by dasatinib is accompanied by inhibition of cell proliferation and induction of apoptosis. Surprisingly, Stat5 DNA-binding activities are enhanced with increasing cell density, which is associated with resistance to apoptosis by dasatinib. Our findings indicate that inhibition of Stat5 signaling downstream of Bcr-Abl/SFKs contributes to the action of dasatinib, and, conversely, that increasing cell density up-regulates Stat5 activation and confers resistance to dasatinib. Moreover, the level of phosphorylated Stat5 in CML cells represents a mechanistically relevant biomarker for monitoring inhibition of Bcr-Abl signaling by dasatinib in CML patients using convenient immunocytochemical assays.

  3. GH/STAT5 signaling during the growth period in livers of mice overexpressing GH.

    PubMed

    Martinez, Carolina S; Piazza, Verónica G; Díaz, María E; Boparai, Ravneet K; Arum, Oge; Ramírez, María C; González, Lorena; Becú-Villalobos, Damasia; Bartke, Andrzej; Turyn, Daniel; Miquet, Johanna G; Sotelo, Ana I

    2015-04-01

    GH/STAT5 signaling is desensitized in the liver in adult transgenic mice overexpressing GH; however, these animals present greater body size. To assess whether the STAT5 pathway is active during the growth period in the liver in these animals, and how signaling modulators participate in this process, growing transgenic mice and normal siblings were evaluated. STAT5 does not respond to an acute GH-stimulus, but displays higher basal phosphorylation in the livers of growing GH-overexpressing mice. GH receptor and the positive modulators glucocorticoid receptor and HNF1 display greater abundance in transgenic animals, supporting the activity of STAT5. The negative modulators cytokine-induced suppressor and PTP1B are increased in GH-overexpressing mice. The suppressors SOCS2 and SOCS3 exhibit higher mRNA levels in transgenic mice but lower protein content, indicating that they are being actively degraded. Therefore, STAT5 signaling is increased in the liver in GH-transgenic mice during the growth period, with a balance between positive and negative effectors resulting in accelerated but controlled growth.

  4. Indirubin derivatives induce apoptosis of chronic myelogenous leukemia cells involving inhibition of Stat5 signaling.

    PubMed

    Nam, Sangkil; Scuto, Anna; Yang, Fan; Chen, Wenyong; Park, Sungman; Yoo, Hwa-Seung; Konig, Heiko; Bhatia, Ravi; Cheng, Xinlai; Merz, Karl-Heinz; Eisenbrand, Gerhard; Jove, Richard

    2012-06-01

    Indirubin is the major active anti-tumor component of a traditional Chinese herbal medicine used for treatment of chronic myelogenous leukemia (CML). While previous studies indicate that indirubin is a promising therapeutic agent for CML, the molecular mechanism of action of indirubin is not fully understood. We report here that indirubin derivatives (IRDs) potently inhibit Signal Transducer and Activator of Transcription 5 (Stat5) protein in CML cells. Compound E804, which is the most potent in this series of IRDs, blocked Stat5 signaling in human K562 CML cells, imatinib-resistant human KCL-22 CML cells expressing the T315I mutant Bcr-Abl (KCL-22M), and CD34-positive primary CML cells from patients. Autophosphorylation of Src family kinases (SFKs) was strongly inhibited in K562 and KCL-22M cells at 5 μM E804, and in primary CML cells at 10 μM E804, although higher concentrations partially inhibited autophosphorylation of Bcr-Abl. Previous studies indicate that SFKs cooperate with Bcr-Abl to activate downstream Stat5 signaling. Activation of Stat5 was strongly blocked by E804 in CML cells. E804 down-regulated expression of Stat5 target proteins Bcl-x(L) and Mcl-1, associated with induction of apoptosis. In sum, our findings identify IRDs as potent inhibitors of the SFK/Stat5 signaling pathway downstream of Bcr-Abl, leading to apoptosis of K562, KCL-22M and primary CML cells. IRDs represent a promising structural class for development of new therapeutics for wild type or T315I mutant Bcr-Abl-positive CML patients.

  5. Growth hormone STAT5-mediated signaling and its modulation in mice liver during the growth period.

    PubMed

    Martinez, Carolina S; Piazza, Verónica G; Ratner, Laura D; Matos, Marina N; González, Lorena; Rulli, Susana B; Miquet, Johanna G; Sotelo, Ana I

    2013-01-01

    Postnatal growth exhibits two instances of rapid growth in mice: the first is perinatal and independent of growth hormone (GH), the second is peripuberal and GH-dependent. Signal transducer and activator of transcription 5b (STAT5b) is the main GH-signaling mediator and it is related to IGF1 synthesis and somatic growth. The aim of this work was to assess differential STAT5 sensitivity to GH during the growth period in mouse liver of both sexes. Three representative ages were selected: 1-week-old animals, in the GH-independent phase of growth; 2.5-week-old mice, at the onset of the GH-dependent phase of growth; and 9-week-old young adults. GH-signaling mediators were assessed by immunoblotting, quantitative RT-PCR and immunohistochemistry. GH-induced STAT5 phosphorylation is low at one-week and maximal at 2.5-weeks of age when compared to young adults, accompanied by higher protein content at the onset of growth. Suppressor CIS and phosphatase PTP1B exhibit high levels in one-week animals, which gradually decline, while SOCS2 and SOCS3 display higher levels at adulthood. Nuclear phosphorylated STAT5 is low in one-week animals while in 2.5-week animals it is similar to 9-week control; expression of SOCS3, an early response GH-target gene, mimics this pattern. STAT5 coactivators glucocorticoid receptor (GR) and hepatic nuclear factor 1 (HNF1) abundance is higher in adulthood. Therefore, GH-induced STAT5 signaling presents age-dependent activity in liver, with its maximum coinciding with the onset of GH-dependent phase of growth, accompanied by an age-dependent variation of modulating factors. This work contributes to elucidate the molecular mechanisms implicated in GH responsiveness during growth.

  6. How Intrinsic Molecular Dynamics Control Intramolecular Communication in Signal Transducers and Activators of Transcription Factor STAT5

    PubMed Central

    Langenfeld, Florent; Guarracino, Yann; Arock, Michel; Trouvé, Alain; Tchertanov, Luba

    2015-01-01

    Signal Transducer and Activator of Transcription STAT5 is a key mediator of cell proliferation, differentiation and survival. While STAT5 activity is tightly regulated in normal cells, its constitutive activation directly contributes to oncogenesis and is associated with a broad range of hematological and solid tumor cancers. Therefore the development of compounds able to modulate pathogenic activation of this protein is a very challenging endeavor. A crucial step of drug design is the understanding of the protein conformational features and the definition of putative binding site(s) for such modulators. Currently, there is no structural data available for human STAT5 and our study is the first footprint towards the description of structure and dynamics of this protein. We investigated structural and dynamical features of the two STAT5 isoforms, STAT5a and STAT5b, taken into account their phosphorylation status. The study was based on the exploration of molecular dynamics simulations by different analytical methods. Despite the overall folding similarity of STAT5 proteins, the MD conformations display specific structural and dynamical features for each protein, indicating first, sequence-encoded structural properties and second, phosphorylation-induced effects which contribute to local and long-distance structural rearrangements interpreted as allosteric event. Further examination of the dynamical coupling between distant sites provides evidence for alternative profiles of the communication pathways inside and between the STAT5 domains. These results add a new insight to the understanding of the crucial role of intrinsic molecular dynamics in mediating intramolecular signaling in STAT5. Two pockets, localized in close proximity to the phosphotyrosine-binding site and adjacent to the channel for communication pathways across STAT5, may constitute valid targets to develop inhibitors able to modulate the function-related communication properties of this signaling

  7. FYN expression potentiates FLT3-ITD induced STAT5 signaling in acute myeloid leukemia

    PubMed Central

    Chougule, Rohit A.; Kazi, Julhash U.; Rönnstrand, Lars

    2016-01-01

    FYN is a non-receptor tyrosine kinase belonging to the SRC family of kinases, which are frequently over-expressed in human cancers, and play key roles in cancer biology. SRC has long been recognized as an important oncogene, but little attention has been given to its other family members. In this report, we have studied the role of FYN in FLT3 signaling in respect to acute myeloid leukemia (AML). We observed that FYN displays a strong association with wild-type FLT3 as well as oncogenic FLT3-ITD and is dependent on the kinase activity of FLT3 and the SH2 domain of FYN. We identified multiple FYN binding sites in FLT3, which partially overlapped with SRC binding sites. To understand the role of FYN in FLT3 signaling, we generated FYN overexpressing cells. We observed that expression of FYN resulted in slightly enhanced phosphorylation of AKT, ERK1/2 and p38 in response to ligand stimulation. Furthermore, FYN expression led to a slight increase in FLT3-ITD-dependent cell proliferation, but potent enhancement of STAT5 phosphorylation as well as colony formation. We also observed that FYN expression is deregulated in AML patient samples and that higher expression of FYN, in combination with FLT3-ITD mutation, resulted in enrichment of the STAT5 signaling pathway and correlated with poor prognosis in AML. Taken together our data suggest that FYN cooperates with oncogenic FLT3-ITD in cellular transformation by selective activation of the STAT5 pathway. Therefore, inhibition of FYN, in combination with FLT3 inhibition, will most likely be beneficial for this group of AML patients. PMID:26848862

  8. Neuronal STAT5 signaling is required for maintaining lactation but not for postpartum maternal behaviors in mice.

    PubMed

    Buonfiglio, Daniella C; Ramos-Lobo, Angela M; Silveira, Marina A; Furigo, Isadora C; Hennighausen, Lothar; Frazão, Renata; Donato, Jose

    2015-05-01

    Prolactin and placental lactogens control mammary development and lactation as well as play an important role in maternal behaviors. However, the molecular mechanisms in the brain responsible for this regulation remain largely unknown. Therefore, the present study investigated whether Signal Transducer and Activator of Transcription 5 (STAT5) signaling in the brain, the key transcriptional factor recruited by prolactin receptor and other hormones, is required for postpartum maternal behavior, maintenance of lactation and offspring growth. Neuronal ablation of STAT5 impaired the control of prolactin secretion and reduced the hypothalamic expression of suppressors of cytokine signaling (i.e., SOCS3 and CISH). In addition, neuronal STAT5 deletion attenuated the hyperphagia commonly observed during lactation by decreasing the hypothalamic expression of orexigenic neurotransmitters such as the neuropeptide Y and agouti-related protein. The lower food intake of lactating neuron-specific STAT5 knockout females resulted in reduced milk production and offspring growth. Unexpectedly, postpartum maternal behavior expression was not impaired in neuron-specific STAT5 knockout females. On the contrary, the latency to retrieve and group the pups into the nest was reduced in mutant dams. Finally, we demonstrated that approximately 30% of recorded neurons in the medial preoptic area were acutely depolarized by prolactin suggesting that fast STAT5-independent signaling pathways may be involved in the regulation of maternal behaviors. Overall, our results revealed important information about the molecular mechanisms recruited by hormones to orchestrate the activation of neural circuitries engaged in the induction of maternal care.

  9. A multi-level model accounting for the effects of JAK2-STAT5 signal modulation in erythropoiesis.

    PubMed

    Lai, Xin; Nikolov, Svetoslav; Wolkenhauer, Olaf; Vera, Julio

    2009-08-01

    We develop a multi-level model, using ordinary differential equations, based on quantitative experimental data, accounting for murine erythropoiesis. At the sub-cellular level, the model includes a description of the regulation of red blood cell differentiation through Epo-stimulated JAK2-STAT5 signalling activation, while at the cell population level the model describes the dynamics of (STAT5-mediated) red blood cell differentiation from their progenitors. Furthermore, the model includes equations depicting the hypoxia-mediated regulation of hormone erythropoietin blood levels. Take all together, the model constitutes a multi-level, feedback loop-regulated biological system, involving processes in different organs and at different organisational levels. We use our model to investigate the effect of deregulation in the proteins involved in the JAK2-STAT5 signalling pathway in red blood cells. Our analysis results suggest that down-regulation in any of the three signalling system components affects the hematocrit level in an individual considerably. In addition, our analysis predicts that exogenous Epo injection (an already existing treatment for several blood diseases) may compensate the effects of single down-regulation of Epo hormone level, STAT5 or EpoR/JAK2 expression level, and that it may be insufficient to counterpart a combined down-regulation of all the elements in the JAK2-STAT5 signalling cascade. PMID:19660986

  10. Suppressor of Cytokine Signalling-6 Promotes Neurite Outgrowth via JAK2/STAT5-Mediated Signalling Pathway, Involving Negative Feedback Inhibition

    PubMed Central

    Gupta, Sakshi; Mishra, Kanchan; Surolia, Avadhesha; Banerjee, Kakoli

    2011-01-01

    Background Suppressors of cytokine signalling (SOCS) protein family are key regulators of cellular responses to cytokines and play an important role in the nervous system. The SOCS6 protein, a less extensively studied SOCS family member, has been shown to induce insulin resistance in the retina and promote survival of the retinal neurons. But no reports are available about the role of SOCS6 in neuritogenesis. In this study, we examined the role of SOCS6 in neurite outgrowth and neuronal cell signalling. Methodology/Principal Findings The effect of SOCS6 in neural stem cells differentiation was studied in neural stem cells and PC12 cell line. Highly elevated levels of SOCS6 were found upon neural cell differentiation both at the mRNA and protein level. Furthermore, SOCS6 over-expression lead to increase in neurite outgrowth and degree of branching, whereas SOCS6 knockdown with specific siRNAs, lead to a significant decrease in neurite initiation and extension. Insulin-like growth factor-1 (IGF-1) stimulation which enhanced neurite outgrowth of neural cells resulted in further enhancement of SOCS6 expression. Jak/Stat (Janus Kinase/Signal Transducer And Activator Of Transcription) pathway was found to be involved in the SOCS6 mediated neurite outgrowth. Bioinformatics study revealed presence of putative Stat binding sites in the SOCS6 promoter region. Transcription factors Stat5a and Stat5b were involved in SOCS6 gene upregulation leading to neuronal differentiation. Following differentiation, SOCS6 was found to form a ternary complex with IGFR (Insulin Like Growth Factor-1 Receptor) and JAK2 which acted in a negative feedback loop to inhibit pStat5 activation. Conclusion/Significance The current paradigm for the first time states that SOCS6, a SOCS family member, plays an important role in the process of neuronal differentiation. These findings define a novel molecular mechanism for Jak2/Stat5 mediated SOCS6 signalling. PMID:22125600

  11. Division of labor by dual feedback regulators controls JAK2/STAT5 signaling over broad ligand range

    PubMed Central

    Bachmann, Julie; Raue, Andreas; Schilling, Marcel; Böhm, Martin E; Kreutz, Clemens; Kaschek, Daniel; Busch, Hauke; Gretz, Norbert; Lehmann, Wolf D; Timmer, Jens; Klingmüller, Ursula

    2011-01-01

    Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. The specific contributions of individual feedback regulators, however, remain unclear. Based on extensive time-resolved data sets in primary erythroid progenitor cells, we established a dynamic pathway model to dissect the roles of the two transcriptional negative feedback regulators of the suppressor of cytokine signaling (SOCS) family, CIS and SOCS3, in JAK2/STAT5 signaling. Facilitated by the model, we calculated the STAT5 response for experimentally unobservable Epo concentrations and provide a quantitative link between cell survival and the integrated response of STAT5 in the nucleus. Model predictions show that the two feedbacks CIS and SOCS3 are most effective at different ligand concentration ranges due to their distinct inhibitory mechanisms. This divided function of dual feedback regulation enables control of STAT5 responses for Epo concentrations that can vary 1000-fold in vivo. Our modeling approach reveals dose-dependent feedback control as key property to regulate STAT5-mediated survival decisions over a broad range of ligand concentrations. PMID:21772264

  12. Erbb4 Signaling in the Mammary Gland Is Required for Lobuloalveolar Development and Stat5 Activation during Lactation

    PubMed Central

    Jones, Frank E.; Welte, Thomas; Fu, Xin-Yuan; Stern, David F.

    1999-01-01

    Signaling by members of the epidermal growth factor receptor family plays an important role in breast development and breast cancer. Earlier work suggested that one of these receptors, ErbB4, is coupled to unique responses in this tissue. To determine the function of ErbB4 signaling in the normal mouse mammary gland, we inactivated ErbB4 signaling by expressing a COOH terminally deleted dominant-negative allele of ErbB4 (ErbB4ΔIC) as a transgene in the mammary gland. Despite the expression of ErbB4ΔIC from puberty through later stages of mammary development, an ErbB4ΔIC-specific phenotype was not observed until mid-lactation. At 12-d postpartum, lobuloalveoli expressing ErbB4ΔIC protein were condensed and lacked normal lumenal lactation products. In these lobuloalveoli, β-casein mRNA, detected by in situ hybridization, was normal. However, whey acidic protein mRNA was reduced, and α-lactalbumin mRNA was undetectable. Stat5 expression was detected by immunohistochemistry in ErbB4ΔIC-expressing tissue. However, Stat5 was not phosphorylated at Y694 and was, therefore, probably inactive. When expressed transiently in 293T cells, ErbB4 induced phosphorylation of Stat5. This phosphorylation required an intact Stat5 SH2 domain. In summary, our results demonstrate that ErbB4 signaling is necessary for mammary terminal differentiation and Stat5 activation at mid-lactation. PMID:10508857

  13. MSM Enhances GH Signaling via the Jak2/STAT5b Pathway in Osteoblast-Like Cells and Osteoblast Differentiation through the Activation of STAT5b in MSCs

    PubMed Central

    Joung, Youn Hee; Lim, Eun Joung; Darvin, Pramod; Chung, So Chung; Jang, Ju Woong; Do Park, Kyung; Lee, Hak Kyo; Kim, Heui Soo; Park, Taekyu; Yang, Young Mok

    2012-01-01

    Methylsulfonylmethane (MSM) is a naturally occurring sulfur compound with well-known anti-oxidant properties and anti-inflammatory activities. But, its effects on bone are unknown. Growth hormone (GH) is regulator of bone growth and bone metabolism. GH activates several signaling pathways such as the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) pathway, thereby regulating expression of genes including insulin-like growth factor (IGF)-1. GH exerts effects both directly and via IGF-1, which signals by activating the IGF-1 receptor (IGF-1R). In this study, we investigated the effects of MSM on the GH signaling via the Jak/STAT pathway in osteoblasts and the differentiation of primary bone marrow mesenchymal stem cells (MSCs). MSM was not toxic to osteoblastic cells and MSCs. MSM increased the expression of GH-related proteins including IGF-1R, p-IGF-1R, STAT5b, p-STAT5b, and Jak2 in osteoblastic cells and MSCs. MSM increased IGF-1R and GHR mRNA expression in osteoblastic cells. The expression of MSM-induced IGF-1R and GHR was inhibited by AG490, a Jak2 kinase inhibitor. MSM induced binding of STAT5 to the IGF-1R and increased IGF-1 and IGF-1R promoter activities. Analysis of cell extracts by immunoprecipitation and Western blot showed that MSM enhanced GH-induced activation of Jak2/STAT5b. We found that MSM and GH, separately or in combination, activated GH signaling via the Jak2/STAT5b pathway in UMR-106 cells. Using siRNA analysis, we found that STAT5b plays an essential role in GH signaling activation in C3H10T1/2 cells. Osteogenic marker genes (ALP, ON, OCN, BSP, OSX, and Runx2) were activated by MSM, and siRNA-mediated STAT5b knockdown inhibited MSM-induced expression of osteogenic markers. Furthermore, MSM increased ALP activity and the mineralization of MSCs. Taken together, these results indicated that MSM can promote osteogenic differentiation of MSCs through activation of STAT5b. PMID:23071812

  14. SnoN regulates mammary gland alveologenesis and onset of lactation by promoting prolactin/Stat5 signaling.

    PubMed

    Jahchan, Nadine S; Wang, Douglas; Bissell, Mina J; Luo, Kunxin

    2012-09-01

    Mammary epithelial cells undergo structural and functional differentiation at late pregnancy and parturition to produce and secrete milk. Both TGF-β and prolactin pathways are crucial regulators of this process. However, how the activities of these two antagonistic pathways are orchestrated to initiate lactation has not been well defined. Here, we show that SnoN, a negative regulator of TGF-β signaling, coordinates TGF-β and prolactin signaling to control alveologenesis and lactogenesis. SnoN expression is induced at late pregnancy by the coordinated actions of TGF-β and prolactin. The elevated SnoN promotes Stat5 signaling by enhancing its stability, thereby sharply increasing the activity of prolactin signaling at the onset of lactation. SnoN-/- mice display severe defects in alveologenesis and lactogenesis, and mammary epithelial cells from these mice fail to undergo proper morphogenesis. These defects can be rescued by an active Stat5. Thus, our study has identified a new player in the regulation of milk production and revealed a novel function of SnoN in mammary alveologenesis and lactogenesis in vivo through promotion of Stat5 signaling.

  15. Deletion of IGF-I Receptor (IGF-IR) in Primary Osteoblasts Reduces GH-Induced STAT5 Signaling

    PubMed Central

    Gan, Yujun; Zhang, Yue; DiGirolamo, Douglas J.; Jiang, Jing; Wang, Xiangdong; Cao, Xuemei; Zinn, Kurt R.; Carbone, David P.; Clemens, Thomas L.; Frank, Stuart J.

    2010-01-01

    GH promotes longitudinal growth and regulates multiple cellular functions in humans and animals. GH signals by binding to GH receptor (GHR) to activate the tyrosine kinase, Janus kinase 2 (JAK2), and downstream pathways including signal transducer and activator of transcription 5 (STAT5), thereby regulating expression of genes including IGF-I. GH exerts effects both directly and via IGF-I, which signals by activating the IGF-I receptor (IGF-IR). IGF-IR is a cell surface receptor that contains intrinsic tyrosine kinase activity within its intracellular domain. In this study, we examined the potential role of IGF-IR in facilitating GH-induced signal transduction, using mouse primary calvarial osteoblasts with Lox-P sites flanking both IGF-IR alleles. These cells respond to both GH and IGF-I and in vitro infection with an adenovirus that drives expression of Cre recombinase (Ad-Cre) dramatically reduces IGF-IR abundance without affecting the abundance of GHR, JAK2, STAT5, or ERK. Notably, infection with Ad-Cre, but not a control adenovirus, markedly inhibited acute GH-induced STAT5 activity (more than doubling the ED50 and reducing the maximum activity by nearly 50%), while sparing GH-induced ERK activity, and markedly inhibited GH-induced transactivation of a STAT5-dependent luciferase reporter. The effect of Ad-Cre on GH signaling was specific, as platelet-derived growth factor-induced signaling was unaffected by Ad-Cre-mediated reduction of IGF-IR. Ad-Cre-mediated inhibition of GH signaling was reversed by adenoviral reexpression of IGF-IR, but not by infection with an adenovirus that drives expression of a hemagglutination-tagged somatostatin receptor, which drives expression of the unrelated somatostatin receptor, and Ad-Cre infection of nonfloxed osteoblasts did not affect GH signaling. Notably, infection with an adenovirus encoding a C-terminally truncated IGF-IR that lacks the tyrosine kinase domain partially rescued both acute GH-induced STAT5 activity and GH

  16. STAT5-regulated microRNA-193b controls haematopoietic stem and progenitor cell expansion by modulating cytokine receptor signalling

    PubMed Central

    Haetscher, Nadine; Feuermann, Yonatan; Wingert, Susanne; Rehage, Maike; Thalheimer, Frederic B.; Weiser, Christian; Bohnenberger, Hanibal; Jung, Klaus; Schroeder, Timm; Serve, Hubert; Oellerich, Thomas; Hennighausen, Lothar; Rieger, Michael A.

    2015-01-01

    Haematopoietic stem cells (HSCs) require the right composition of microRNAs (miR) for proper life-long balanced blood regeneration. Here we show a regulatory circuit that prevents excessive HSC self-renewal by upregulation of miR-193b upon self-renewal promoting thrombopoietin (TPO)-MPL-STAT5 signalling. In turn, miR-193b restricts cytokine signalling, by targeting the receptor tyrosine kinase c-KIT. We generated a miR-193b knockout mouse model to unravel the physiological function of miR-193b in haematopoiesis. MiR-193b−/− mice show a selective gradual enrichment of functional HSCs, which are fully competent in multilineage blood reconstitution upon transplantation. The absence of miR-193b causes an accelerated expansion of HSCs, without altering cell cycle or survival, but by decelerating differentiation. Conversely, ectopic miR-193b expression restricts long-term repopulating HSC expansion and blood reconstitution. MiR-193b-deficient haematopoietic stem and progenitor cells exhibit increased basal and cytokine-induced STAT5 and AKT signalling. This STAT5-induced microRNA provides a negative feedback for excessive signalling to restrict uncontrolled HSC expansion. PMID:26603207

  17. Mitochondrial translocation of signal transducer and activator of transcription 5 (STAT5) in leukemic T cells and cytokine-stimulated cells

    SciTech Connect

    Chueh, Fu-Yu; Leong, King-Fu; Yu, Chao-Lan

    2010-11-26

    Research highlights: {yields} STAT5 interacts with a mitochondrial protein PDC-E2 in a leukemic T cell line LSTRA. {yields} Tyrosine-phosphorylated STAT5, but not STAT3, is present in LSTRA mitochondria. {yields} Cytokines induce mitochondrial translocation of STAT5, but not STAT1 or STAT3. {yields} Cytokine-induced mitochondrial translocation of tyrosine-phosphorylated STAT5 is transient. {yields} Mitochondrial STAT5 binds to a putative STAT5 site in the mitochondrial DNA in vitro. -- Abstract: Signal transducers and activators of transcription (STATs) were first identified as key signaling molecules in response to cytokines. Constitutive STAT activation also has been widely implicated in oncogenesis. We analyzed STAT5-associated proteins in a leukemic T cell line LSTRA, which exhibits constitutive tyrosine phosphorylation and activation of STAT5. A cellular protein was found to specifically interact with STAT5 in LSTRA cells by co-immunoprecipitation. Sequencing analysis and subsequent immunoblotting confirmed the identity of this STAT5-associated protein as the E2 component of mitochondrial pyruvate dehydrogenase complex (PDC-E2). Consistent with this interaction, both subcellular fractionation and immunofluorescence microscopy revealed mitochondrial localization of STAT5 in LSTRA cells. Mitochondrial localization of tyrosine-phosphorylated STAT5 also occurred in cytokine-stimulated cells. A time course experiment further demonstrated the transient kinetics of STAT5 mitochondrial translocation after cytokine stimulation. In contrast, cytokine-induced STAT1 and STAT3 activation did not result in their translocation into mitochondria. Furthermore, we showed that mitochondrial STAT5 bound to the D-loop regulatory region of mitochondrial DNA in vitro. It suggests a potential role of STAT5 in regulating the mitochondrial genome. Proliferative metabolism toward aerobic glycolysis is well known in cancer cells as the Warburg effect and is also observed in cytokine

  18. Co-operating STAT5 and AKT signaling pathways in chronic myeloid leukemia and mastocytosis: possible new targets of therapy

    PubMed Central

    Bibi, Siham; Arslanhan, Melis Dilara; Langenfeld, Florent; Jeanningros, Sylvie; Cerny-Reiterer, Sabine; Hadzijusufovic, Emir; Tchertanov, Luba; Moriggl, Richard; Valent, Peter; Arock, Michel

    2014-01-01

    Chronic myeloid leukemia and systemic mastocytosis are myeloid neoplasms sharing a number of pathogenetic and clinical features. In both conditions, an aberrantly activated oncoprotein with tyrosine kinase activity, namely BCR-ABL1 in chronic myeloid leukemia, and mutant KIT, mostly KIT D816V, in systemic mastocytosis, is key to disease evolution. The appreciation of the role of such tyrosine kinases in these diseases has led to the development of improved therapies with tyrosine kinase-targeted inhibitors. However, most drugs, including new KIT D816V-blocking agents, have failed to achieve long-lasting remissions in advanced systemic mastocytosis, and there is a similar problem in chronic myeloid leukemia, where imatinib-resistant patients sometimes fail to achieve remission, even with second- or third-line BCR-ABL1 specific tyrosine kinase inhibitors. During disease progression, additional signaling pathways become activated in neoplastic cells, but most converge into major downstream networks. Among these, the AKT and STAT5 pathways appear most critical and may result in drug-resistant chronic myeloid leukemia and systemic mastocytosis. Inhibition of phosphorylation of these targets has proven their crucial role in disease-evolution in both malignancies. Together, these observations suggest that STAT5 and AKT are key drivers of oncogenesis in drug-resistant forms of the diseases, and that targeting STAT5 and AKT might be an interesting approach in these malignancies. The present article provides an overview of our current knowledge about the critical role of AKT and STAT5 in the pathophysiology of chronic myeloid leukemia and systemic mastocytosis and on their potential value as therapeutic targets in these neoplasms. PMID:24598853

  19. Increased STAT5 signaling in the ring dove brain in response to prolactin administration and spontaneous elevations in prolactin during the breeding cycle.

    PubMed

    Buntin, John D; Buntin, Linda

    2014-05-01

    Prolactin acts on target cells in the central nervous system (CNS) to stimulate behavioral changes associated with parental care in birds, but the signaling mechanisms that mediate these actions have not been characterized. In mammals, the Janus Kinase 2-Signal Transducer and Activator of Transcription 5 (JAK2-STAT5) signaling pathway mediates many of the actions of prolactin. To assess the importance of this pathway in prolactin-sensitive target cells in the avian brain, we measured changes in activated (phosphorylated) STAT5 (pSTAT5) in the forebrain of female ring doves sampled as plasma prolactin levels change during the breeding cycle and in prolactin-treated, non-breeding females. The anatomical distribution of cells exhibiting pSTAT5 immunoreactivity in dove brain closely paralleled the distribution of prolactin receptors in this species. The density of pSTAT5 immunoreactive (pSTAT5-ir) cells was highest in the preoptic area, the suprachiasmatic, paraventricular, and ventromedial hypothalamic nuclei, the lateral and tuberal hypothalamic regions, the lateral bed nucleus of the stria terminalis, and the lateral septum. Mean pSTAT5-ir cell densities in these eight brain areas were several fold higher in breeding females during late incubation/early post-hatching when plasma prolactin levels have been observed to peak than in non-breeding females or breeding females sampled at earlier stages when prolactin titers have been reported to be lower. Similar differences were observed between prolactin-treated and vehicle-treated females in all three of the forebrain regions that were compared. We conclude that JAK2-STAT5 signaling is strongly activated in response to prolactin stimulation in the ring dove brain and could potentially mediate some of the centrally-mediated behavioral effects of this hormone.

  20. Sleeping Beauty transposon screen identifies signaling modules that cooperate with STAT5 activation to induce B-cell acute lymphoblastic leukemia.

    PubMed

    Heltemes-Harris, L M; Larson, J D; Starr, T K; Hubbard, G K; Sarver, A L; Largaespada, D A; Farrar, M A

    2016-06-30

    Signal transducer and activator of transcription 5 (STAT5) activation occurs frequently in human progenitor B-cell acute lymphoblastic leukemia (B-ALL). To identify gene alterations that cooperate with STAT5 activation to initiate leukemia, we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) with mice in which a mutagenic Sleeping Beauty transposon (T2/Onc) was mobilized only in B cells. Stat5b-CA mice typically do not develop B-ALL (<2% penetrance); in contrast, 89% of Stat5b-CA mice in which the T2/Onc transposon had been mobilized died of B-ALL by 3 months of age. High-throughput sequencing approaches were used to identify genes frequently targeted by the T2/Onc transposon; these included Sos1 (74%), Kdm2a (35%), Jak1 (26%), Bmi1 (19%), Prdm14 or Ncoa2 (13%), Cdkn2a (10%), Ikzf1 (8%), Caap1 (6%) and Klf3 (6%). Collectively, these mutations target three major cellular processes: (i) the Janus kinase/STAT5 pathway (ii) progenitor B-cell differentiation and (iii) the CDKN2A tumor-suppressor pathway. Transposon insertions typically resulted in altered expression of these genes, as well as downstream pathways including STAT5, extracellular signal-regulated kinase (Erk) and p38. Importantly, expression of Sos1 and Kdm2a, and activation of p38, correlated with survival, further underscoring the role these genes and associated pathways have in B-ALL.

  1. Sleeping Beauty transposon screen identifies signaling modules that cooperate with STAT5 activation to induce B-cell acute lymphoblastic leukemia.

    PubMed

    Heltemes-Harris, L M; Larson, J D; Starr, T K; Hubbard, G K; Sarver, A L; Largaespada, D A; Farrar, M A

    2016-06-30

    Signal transducer and activator of transcription 5 (STAT5) activation occurs frequently in human progenitor B-cell acute lymphoblastic leukemia (B-ALL). To identify gene alterations that cooperate with STAT5 activation to initiate leukemia, we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) with mice in which a mutagenic Sleeping Beauty transposon (T2/Onc) was mobilized only in B cells. Stat5b-CA mice typically do not develop B-ALL (<2% penetrance); in contrast, 89% of Stat5b-CA mice in which the T2/Onc transposon had been mobilized died of B-ALL by 3 months of age. High-throughput sequencing approaches were used to identify genes frequently targeted by the T2/Onc transposon; these included Sos1 (74%), Kdm2a (35%), Jak1 (26%), Bmi1 (19%), Prdm14 or Ncoa2 (13%), Cdkn2a (10%), Ikzf1 (8%), Caap1 (6%) and Klf3 (6%). Collectively, these mutations target three major cellular processes: (i) the Janus kinase/STAT5 pathway (ii) progenitor B-cell differentiation and (iii) the CDKN2A tumor-suppressor pathway. Transposon insertions typically resulted in altered expression of these genes, as well as downstream pathways including STAT5, extracellular signal-regulated kinase (Erk) and p38. Importantly, expression of Sos1 and Kdm2a, and activation of p38, correlated with survival, further underscoring the role these genes and associated pathways have in B-ALL. PMID:26500062

  2. BM microenvironmental protection of CML cells from imatinib through Stat5/NF-κB signaling and reversal by Wogonin

    PubMed Central

    Xu, Xuefen; Zhang, Xiaobo; Liu, Yicheng; Yang, Lin; Huang, Shaoliang; Lu, Lu; Wang, Shuhao; Guo, Qinglong; Zhao, Li

    2016-01-01

    Constitutive Stat5 activation enhanced cell survival and resistance to imatinib (IM) in chronic myelogenous leukemia (CML) cells. However, the mechanism of Stat5 activation in mediating resistance to IM in bone marrow (BM) microenvironment has not been evaluated precisely. In this study, we reported HS-5-derived conditioned medium (CM) significantly enhanced IM resistance in K562 and KU812. Interestingly, upregulation of the proportion of CD34+ subpopulation was found in CML cells. Subsequently, the BCR/ABL-independent activation of Stat5 increased P-glycoprotein (P-gp) activity in CM-mediated protection of CML stem cells (LSCs) from IM. Further research revealed Stat5 activation increased the DNA binding activity of NF-κB though binding of p-Stat5 and p-RelA in nucleus. Moreover, highly acetylated RelA was required for Stat5-mediated RelA nuclear binding. The study further confirmed that Wogonin potentiated the inhibitory effects of IM on leukemia development by suppressing Stat5 pathway both in CM model and the K562 xenograft model. In summary, results clearly demonstrated BCR/ABL-independent Stat5 survival pathway could contribute to resistance of CML LSCs to IM in BM microenvironment and suggested that natural durgs effectively inhibiting Stat5 may be an attractive approach to overcome resistance to BCR/ABL kinase inhibitors. PMID:27027438

  3. Identification of STAT5A and STAT5B target genes in human T cells.

    PubMed

    Kanai, Takahiro; Seki, Scott; Jenks, Jennifer A; Kohli, Arunima; Kawli, Trupti; Martin, Dorrelyn Patacsil; Snyder, Michael; Bacchetta, Rosa; Nadeau, Kari C

    2014-01-01

    Signal transducer and activator of transcription (STAT) comprises a family of universal transcription factors that help cells sense and respond to environmental signals. STAT5 refers to two highly related proteins, STAT5A and STAT5B, with critical function: their complete deficiency is lethal in mice; in humans, STAT5B deficiency alone leads to endocrine and immunological problems, while STAT5A deficiency has not been reported. STAT5A and STAT5B show peptide sequence similarities greater than 90%, but subtle structural differences suggest possible non-redundant roles in gene regulation. However, these roles remain unclear in humans. We applied chromatin immunoprecipitation followed by DNA sequencing using human CD4(+) T cells to detect candidate genes regulated by STAT5A and/or STAT5B, and quantitative-PCR in STAT5A or STAT5B knock-down (KD) human CD4(+) T cells to validate the findings. Our data show STAT5A and STAT5B play redundant roles in cell proliferation and apoptosis via SGK1 interaction. Interestingly, we found a novel, unique role for STAT5A in binding to genes involved in neural development and function (NDRG1, DNAJC6, and SSH2), while STAT5B appears to play a distinct role in T cell development and function via DOCK8, SNX9, FOXP3 and IL2RA binding. Our results also suggest that one or more co-activators for STAT5A and/or STAT5B may play important roles in establishing different binding abilities and gene regulation behaviors. The new identification of these genes regulated by STAT5A and/or STAT5B has major implications for understanding the pathophysiology of cancer progression, neural disorders, and immune abnormalities. PMID:24497979

  4. Sleeping Beauty transposon screen identifies signaling modules that cooperate with STAT5 activation to induce B cell acute lymphoblastic leukemia

    PubMed Central

    Heltemes-Harris, Lynn M.; Larson, Jon D.; Starr, Timothy K.; Hubbard, Gregory K.; Sarver, Aaron L.; Largaespada, David A.; Farrar, Michael A.

    2015-01-01

    STAT5 activation occurs frequently in human progenitor B cell acute lymphoblastic leukemia (B-ALL). To identify gene alterations that cooperate with STAT5 activation to initiate leukemia we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) to mice in which a mutagenic Sleeping Beauty transposon (T2/Onc) was mobilized only in B cells. Stat5b-CA mice typically do not develop B-ALL (<2% penetrance); in contrast, 89% of Stat5b–CA mice in which the T2/Onc transposon had been mobilized died of B-ALL by 3 months of age. High-throughput sequencing approaches were used to identify genes frequently targeted by the T2/Onc transposon; these included Sos1 (74%), Kdm2a (35%), Jak1 (26%), Bmi1 (19%), Prdm14 or Ncoa2 (13%), Cdkn2a (10%), Ikzf1 (8%), Caap1 (6%) and Klf3 (6%). Collectively, these mutations target three major cellular processes: (i) the JAK/STAT5 pathway (ii) progenitor B cell differentiation and (iii) the CDKN2A tumor suppressor pathway. Transposon insertions typically resulted in altered expression of these genes, as well as downstream pathways including STAT5, ERK and p38. Importantly, expression of Sos1 and Kdm2a, and activation of p38, correlated with survival, further underscoring the role these genes and associated pathways play in B-ALL. PMID:26500062

  5. JAK2V617F/STAT5 signaling pathway promotes cell proliferation through activation of Pituitary Tumor Transforming Gene 1 expression

    SciTech Connect

    Shen, Xu-Liang; Wei, Wu; Xu, Hong-Liang; Zhang, Mei-Xiang; Qin, Xiao-Qi; Shi, Wen-Zhi; Jiang, Zhi-Ping; Chen, Yi-Jian; Chen, Fang-Ping

    2010-08-06

    Research highlights: {yields} AG490, a member of tyrosine kinase inhibitors, could inhibit the JAK2V617F/STAT5 signaling pathway in HEL cell which harbor JAK2V617F mutation. {yields} Inhibition of the JAK2V617F/STAT5 signaling pathway inhibited the growth of HEL cells. {yields} JAK2V617F mutation promotes cell proliferation through activation of PTTG1 expression. {yields} JAK2V617F/STAT5 signaling pathway regulate PTTG1 expression at transcriptional level. -- Abstract: Gain-of-function mutations of JAK2 play crucial roles in the development of myeloproliferative neoplasms; however, the underlying downstream events of this activated signaling pathway are not fully understood. Our experiment was designed and performed to address one aspect of this issue. Here we report that AG490, a potent JAK2V617F kinase inhibitor, effectively inhibits the proliferation of HEL cells. Interestingly, AG490 also decreases the expression of PTTG1, a possible target gene of the aberrant signaling pathway, in a dose- and time-dependent manner. Furthermore, the promoter activity analyses reveal that the inhibition of the PTTG1 expression is affected at the transcriptional level. Thus, our results suggest that the JAK2V617F/STAT5 signaling pathway promotes cell proliferation through the transcriptional activation of PTTG1.

  6. Signal transducer and activator of transcription 5 (STAT5) paralog dose governs T cell effector and regulatory functions

    PubMed Central

    Villarino, Alejandro; Laurence, Arian; Robinson, Gertraud W; Bonelli, Michael; Dema, Barbara; Afzali, Behdad; Shih, Han-Yu; Sun, Hong-Wei; Brooks, Stephen R; Hennighausen, Lothar; Kanno, Yuka; O'Shea, John J

    2016-01-01

    The transcription factor STAT5 is fundamental to the mammalian immune system. However, the relationship between its two paralogs, STAT5A and STAT5B, and the extent to which they are functionally distinct, remain uncertain. Using mouse models of paralog deficiency, we demonstrate that they are not equivalent for CD4+ 'helper' T cells, the principal orchestrators of adaptive immunity. Instead, we find that STAT5B is dominant for both effector and regulatory (Treg) responses and, therefore, uniquely necessary for immunological tolerance. Comparative analysis of genomic distribution and transcriptomic output confirm that STAT5B has fargreater impact but, surprisingly, the data point towards asymmetric expression (i.e. paralog dose), rather than distinct functional properties, as the key distinguishing feature. Thus, we propose a quantitative model of STAT5 paralog activity whereby relative abundance imposes functional specificity (or dominance) in the face of widespread structural homology. DOI: http://dx.doi.org/10.7554/eLife.08384.001 PMID:26999798

  7. The dipeptide Pro-Asp promotes IGF-1 secretion and expression in hepatocytes by enhancing JAK2/STAT5 signaling pathway.

    PubMed

    Wang, Songbo; Wang, Guoqing; Zhang, Mengyuan; Zhuang, Lu; Wan, Xiaojuan; Xu, Jingren; Wang, Lina; Zhu, Xiaotong; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Shu, Gang; Jiang, Qingyan

    2016-11-15

    It has been implicated that IGF-1 secretion can be regulated by dietary protein. However, whether the dipeptides, one of digested products of dietary protein, have influence on IGF-1 secretion remain largely unknown. Our study aimed to investigate the effects of the dipeptide Pro-Asp on IGF-1 secretion and expression in hepatocytes and to explore the possible underlying mechanisms. Our findings demonstrated that Pro-Asp promoted the secretion and gene expression of IGF-1 in HepG2 cells and primary porcine hepatocytes. Meanwhile, Pro-Asp activated the ERK and Akt signaling pathways, downstream of IGF-1. In addition, Pro-Asp enhanced GH-mediated JAK2/STAT5 signaling pathway, while inhibition of JAK2/STAT5 blocked the promotive effect of Pro-Asp on IGF-1 secretion and expression. Moreover, acute injection of Pro-Asp stimulated IGF-1 expression and activated JAK2/STAT5 signaling pathway in mice liver. Together, these results suggested that the dipeptide Pro-Asp promoted IGF-1 secretion and expression in hepatocytes by enhancing GH-mediated JAK2/STAT5 signaling pathway.

  8. Caveolin-1-deficient Mice Show Accelerated Mammary Gland Development During Pregnancy, Premature Lactation, and Hyperactivation of the Jak-2/STAT5a Signaling Cascade

    PubMed Central

    Park, David S.; Lee, Hyangkyu; Frank, Philippe G.; Razani, Babak; Nguyen, Andrew V.; Parlow, Albert F.; Russell, Robert G.; Hulit, James; Pestell, Richard G.; Lisanti, Michael P.

    2002-01-01

    It is well established that mammary gland development and lactation are tightly controlled by prolactin signaling. Binding of prolactin to its cognate receptor (Prl-R) leads to activation of the Jak-2 tyrosine kinase and the recruitment/tyrosine phosphorylation of STAT5a. However, the mechanisms for attenuating the Prl-R/Jak-2/STAT5a signaling cascade are just now being elucidated. Here, we present evidence that caveolin-1 functions as a novel suppressor of cytokine signaling in the mammary gland, akin to the SOCS family of proteins. Specifically, we show that caveolin-1 expression blocks prolactin-induced activation of a STAT5a-responsive luciferase reporter in mammary epithelial cells. Furthermore, caveolin-1 expression inhibited prolactin-induced STAT5a tyrosine phosphorylation and DNA binding activity, suggesting that caveolin-1 may negatively regulate the Jak-2 tyrosine kinase. Because the caveolin-scaffolding domain bears a striking resemblance to the SOCS pseudosubstrate domain, we examined whether Jak-2 associates with caveolin-1. In accordance with this homology, we demonstrate that Jak-2 cofractionates and coimmunoprecipitates with caveolin-1. We next tested the in vivo relevance of these findings using female Cav-1 (−/−) null mice. If caveolin-1 normally functions as a suppressor of cytokine signaling in the mammary gland, then Cav-1 null mice should show premature development of the lobuloalveolar compartment because of hyperactivation of the prolactin signaling cascade via disinhibition of Jak-2. In accordance with this prediction, Cav-1 null mice show accelerated development of the lobuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a (pY694) at its Jak-2 phosphorylation site. In addition, the Ras-p42/44 MAPK cascade is hyper-activated. Because a similar premature lactation phenotype is observed in SOCS1 (−/−) null mice, we conclude that caveolin-1 is a novel suppressor of cytokine signaling. PMID:12388746

  9. Antiapoptotic effects of erythropoietin in differentiated neuroblastoma SH-SY5Y cells require activation of both the STAT5 and AKT signaling pathways.

    PubMed

    Um, Moonkyoung; Lodish, Harvey F

    2006-03-01

    The hematopoietic cytokine erythropoietin (Epo) prevents neuronal death during ischemic events in the brain and in neurodegenerative diseases, presumably through its antiapoptotic effects. To explore the role of different signaling pathways in Epo-mediated antiapoptotic effects in differentiated human neuroblastoma SH-SY5Y cells, we employed a prolactin receptor (PrlR)/erythropoietin receptor (EpoR) chimera system, in which binding of prolactin (Prl) to the extracellular domain activates EpoR signaling in the cytosol. On induction of apoptosis by staurosporine, Prl supports survival of the SH-SY5Y cells expressing the wild-type PrlR/EpoR chimera. In these cells Prl treatment strongly activates the STAT5, AKT, and MAPK signaling pathways and induces weak activation of the p65 NF-kappaB factor. Selective mutation of the eight tyrosine residues of the EpoR cytoplasmic domain results in impaired or absent activation of either STAT5 (mutation of Tyr(343)) or AKT (mutation of Tyr(479)) or both (mutation of all eight tyrosine residues). Most interestingly, Prl treatment does not prevent apoptosis in cells expressing mutant PrlR/EpoR chimeras in which either the STAT5 or the AKT signaling pathways are not activated. In contrast, ERK 1/2 is fully activated by all mutant PrlR/EpoR chimeras, comparable with the level seen with the wild-type PrlR/EpoR chimera, implying that activation of the MAPK signaling pathway per se is not sufficient for antiapoptotic activity. Therefore, the antiapoptotic effects of Epo in neuronal cells require the combinatorial activation of multiple signaling pathways, including STAT5, AKT, and potentially MAPK as well, in a manner similar to that observed in hematopoietic cells.

  10. Prolactin-induced prostate tumorigenesis links sustained Stat5 signaling with the amplification of basal/stem cells and emergence of putative luminal progenitors.

    PubMed

    Sackmann-Sala, Lucila; Chiche, Aurélie; Mosquera-Garrote, Nerea; Boutillon, Florence; Cordier, Corinne; Pourmir, Ivan; Pascual-Mathey, Luz; Kessal, Karima; Pigat, Natascha; Camparo, Philippe; Goffin, Vincent

    2014-11-01

    Current androgen ablation therapies for prostate cancer are initially successful, but the frequent development of castration resistance urges the generation of alternative therapies and represents an important health concern. Prolactin/signal transducer and activator of transcription 5 (STAT5) signaling is emerging as a putative target for alternative treatment for prostate cancer. However, mechanistic data for its role in development or progression of prostate tumors are scarce. In vivo mouse studies found that local prolactin induced the amplification of prostate epithelial basal/stem cells. Because these cells are proposed cells of origin for prostate cancer and disease recurrence, we looked further into this amplification. Our results indicated that sustained Stat5 activation was associated with the occurrence of abnormal basal/stem cell clusters in prostate epithelium of prostate-specific prolactin-transgenic mice. Analysis of epithelial areas containing these clusters found high proliferation, Stat5 activation, and expression of stem cell antigen 1. Furthermore, enhanced prolactin signaling also led to amplification of a luminal cell population that was positive for stem cell antigen 1. These cells may originate from amplified basal/stem cells and might represent important progenitors for tumor development in prostate epithelium. These data provide a deeper understanding of the initial stages of prostate tumorigenesis induced by prolactin to help determine whether this hormone or its downstream messengers could be useful targets for prostate cancer treatment in the future.

  11. ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling

    PubMed Central

    Wang, Jun; Rouse, Clay; Jasper, Jeff S.; Pendergast, Ann Marie

    2016-01-01

    Bone metastases occur in up to 70% of advanced breast cancer. For most patients with breast cancer, bone metastases are predominantly osteolytic. Interactions between tumor cells and stromal cells in the bone microenvironment drive osteolytic bone metastasis, a process that requires the activation of osteoclasts, cells that break down bone. Here, we report that ABL kinases promoted metastasis of breast cancer cells to bone by regulating the crosstalk between tumor and the bone microenvironment. ABL kinases protected tumor cells from apoptosis induced by TRAIL (TNF-related apoptosis-inducing ligand), activated the transcription factor STAT5, and promoted osteolysis through the STAT5-dependent expression of genes encoding the osteoclast activating factors interleukin 6 (IL6) and matrix metalloproteinase-1 (MMP1). Furthermore, ABL kinases increased the abundance of the Hippo pathway mediator TAZ and the expression of TAZ-dependent target genes that promote bone metastasis. Knockdown of ABL kinases or treatment with ABL-specific allosteric inhibitor impaired osteolytic metastasis of breast cancer cells in mice. These findings revealed a role for ABL kinases in regulating tumor-bone interactions and provide a rationale for targeting both tumor and the bone microenvironment with ABL-specific inhibitors. PMID:26838548

  12. ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling.

    PubMed

    Wang, Jun; Rouse, Clay; Jasper, Jeff S; Pendergast, Ann Marie

    2016-02-01

    Bone metastases occur in up to 70% of advanced breast cancer. For most patients with breast cancer, bone metastases are predominantly osteolytic. Interactions between tumor cells and stromal cells in the bone microenvironment drive osteolytic bone metastasis, a process that requires the activation of osteoclasts, cells that break down bone. We report that ABL kinases promoted metastasis of breast cancer cells to bone by regulating the crosstalk between tumor cells and the bone microenvironment. ABL kinases protected tumor cells from apoptosis induced by TRAIL (TNF-related apoptosis-inducing ligand), activated the transcription factor STAT5, and promoted osteolysis through the STAT5-dependent expression of genes encoding the osteoclast-activating factors interleukin-6 (IL-6) and matrix metalloproteinase 1 (MMP1). Furthermore, in breast cancer cells, ABL kinases increased the abundance of the Hippo pathway mediator TAZ and the expression of TAZ-dependent target genes that promote bone metastasis. Knockdown of ABL kinases or treatment with ABL-specific allosteric inhibitor impaired osteolytic metastasis of breast cancer cells in mice. These findings revealed a role for ABL kinases in regulating tumor-bone interactions and provide a rationale for using ABL-specific inhibitors to limit breast cancer metastasis to bone. PMID:26838548

  13. Environmentally relevant concentrations of arsenite and monomethylarsonous acid inhibit IL-7/STAT5 cytokine signaling pathways in mouse CD3+CD4-CD8- double negative thymus cells.

    PubMed

    Xu, Huan; Lauer, Fredine T; Liu, Ke Jian; Hudson, Laurie G; Burchiel, Scott W

    2016-04-15

    Environmental arsenic exposure is a public health issue. Immunotoxicity induced by arsenic has been reported in humans and animal models. The purpose of this study was to evaluate mechanisms of As(+3) and MMA(+3) toxicity in mouse thymus cells. Because we know that MMA(+3) inhibits IL-7 signaling in mouse bone marrow pre-B cells, we studied the influence of As(+3) and MMA(+3) on T cell development in the thymus at the earliest stage of T cell development (CD4-CD8-, double negative, DN) which requires IL-7 dependent signaling. We found in a DN thymus cell line (D1) that a low concentration of MMA(+3) (50 nM) suppressed IL-7 dependent JAK1, 3 and STAT5 signaling. As(+3) suppressed STAT5 and JAK3 at higher concentrations (500 nM). Cell surface expression of the IL-7 receptor (CD127) was also suppressed by 50 nM MMA(+)3, but was increased by 500 NM As(+3), indicating possible differences in the mechanisms of action of these agents. A decrease in cyclin D1 protein expression was observed in D1 cells exposed to As(+3) at 500 nM and MMA(+3) starting at 50 nM, suggesting that arsenic at these environmentally-relevant doses suppresses early T cell development through the inhibition of IL-7 signaling pathway. PMID:26921788

  14. Environmentally relevant concentrations of arsenite and monomethylarsonous acid inhibit IL-7/STAT5 cytokine signaling pathways in mouse CD3+CD4-CD8- double negative thymus cells.

    PubMed

    Xu, Huan; Lauer, Fredine T; Liu, Ke Jian; Hudson, Laurie G; Burchiel, Scott W

    2016-04-15

    Environmental arsenic exposure is a public health issue. Immunotoxicity induced by arsenic has been reported in humans and animal models. The purpose of this study was to evaluate mechanisms of As(+3) and MMA(+3) toxicity in mouse thymus cells. Because we know that MMA(+3) inhibits IL-7 signaling in mouse bone marrow pre-B cells, we studied the influence of As(+3) and MMA(+3) on T cell development in the thymus at the earliest stage of T cell development (CD4-CD8-, double negative, DN) which requires IL-7 dependent signaling. We found in a DN thymus cell line (D1) that a low concentration of MMA(+3) (50 nM) suppressed IL-7 dependent JAK1, 3 and STAT5 signaling. As(+3) suppressed STAT5 and JAK3 at higher concentrations (500 nM). Cell surface expression of the IL-7 receptor (CD127) was also suppressed by 50 nM MMA(+)3, but was increased by 500 NM As(+3), indicating possible differences in the mechanisms of action of these agents. A decrease in cyclin D1 protein expression was observed in D1 cells exposed to As(+3) at 500 nM and MMA(+3) starting at 50 nM, suggesting that arsenic at these environmentally-relevant doses suppresses early T cell development through the inhibition of IL-7 signaling pathway.

  15. An invertebrate signal transducer and activator of transcription 5 (STAT5) ortholog from the disk abalone, Haliotis discus discus: Genomic structure, early developmental expression, and immune responses to bacterial and viral stresses.

    PubMed

    Bathige, S D N K; Umasuthan, Navaneethaiyer; Park, Hae-Chul; Lee, Jehee

    2016-03-01

    Signal transducer and activator of transcription (STAT) family members are key signaling molecules that transduce cellular responses from the cell membrane to the nucleus upon Janus kinase (JAK) activation. Although seven STAT members have been reported in mammals, very limited information on STAT genes in molluscans is available. In this study, we identified and characterized a STAT paralog that is homologous to STAT5 from the disk abalone, Haliotis discus discus, and designated as AbSTAT5. Comparison of the deduced amino acid sequence for AbSTAT5 (790 amino acids) with other counterparts revealed conserved residues important for functions and typical domain regions, including the N-terminal domain, coiled-coil domain, DNA-binding domain, linker domain, and Src homology 2 (SH2) domains as mammalian counterparts. Analysis of STAT phylogeny revealed that AbSTAT5 was clustered with the molluscan subgroup in STAT5 clade with distinct evolution. According to the genomic structure of AbSTAT5, the coding sequence was distributed into 20 exons with 19 introns. Immunologically essential transcription factor-binding sites, such as GATA-1, HNF, SP1, C/EBP, Oct-1, AP1, c-Jun, and Sox-2, were predicted at the 5'-proximal region of AbSTAT5. Expression of AbSTAT5 mRNA was detected in different stages of embryonic development and observed at considerably higher levels in the morula and late veliger stages. Tissue-specific expressional studies revealed that the highest level of AbSTAT5 transcripts was detected in hemocytes, followed by gill tissues. Temporal expressions of AbSTAT5 were analyzed upon live bacterial (Vibrio parahemolyticus and Listeria monocytogenes), viral (viral hemorrhagic septicemia virus), and pathogen-associated molecular pattern (lipopolysaccharides and Poly I:C) stimulations, and significant elevations indicated immune modulation. These results suggest that AbSTAT5 may be involved in maintaining innate immune responses from developmental to adult stages in

  16. An invertebrate signal transducer and activator of transcription 5 (STAT5) ortholog from the disk abalone, Haliotis discus discus: Genomic structure, early developmental expression, and immune responses to bacterial and viral stresses.

    PubMed

    Bathige, S D N K; Umasuthan, Navaneethaiyer; Park, Hae-Chul; Lee, Jehee

    2016-03-01

    Signal transducer and activator of transcription (STAT) family members are key signaling molecules that transduce cellular responses from the cell membrane to the nucleus upon Janus kinase (JAK) activation. Although seven STAT members have been reported in mammals, very limited information on STAT genes in molluscans is available. In this study, we identified and characterized a STAT paralog that is homologous to STAT5 from the disk abalone, Haliotis discus discus, and designated as AbSTAT5. Comparison of the deduced amino acid sequence for AbSTAT5 (790 amino acids) with other counterparts revealed conserved residues important for functions and typical domain regions, including the N-terminal domain, coiled-coil domain, DNA-binding domain, linker domain, and Src homology 2 (SH2) domains as mammalian counterparts. Analysis of STAT phylogeny revealed that AbSTAT5 was clustered with the molluscan subgroup in STAT5 clade with distinct evolution. According to the genomic structure of AbSTAT5, the coding sequence was distributed into 20 exons with 19 introns. Immunologically essential transcription factor-binding sites, such as GATA-1, HNF, SP1, C/EBP, Oct-1, AP1, c-Jun, and Sox-2, were predicted at the 5'-proximal region of AbSTAT5. Expression of AbSTAT5 mRNA was detected in different stages of embryonic development and observed at considerably higher levels in the morula and late veliger stages. Tissue-specific expressional studies revealed that the highest level of AbSTAT5 transcripts was detected in hemocytes, followed by gill tissues. Temporal expressions of AbSTAT5 were analyzed upon live bacterial (Vibrio parahemolyticus and Listeria monocytogenes), viral (viral hemorrhagic septicemia virus), and pathogen-associated molecular pattern (lipopolysaccharides and Poly I:C) stimulations, and significant elevations indicated immune modulation. These results suggest that AbSTAT5 may be involved in maintaining innate immune responses from developmental to adult stages in

  17. Redefining Signaling Pathways with an Expanding Single-Cell Toolbox.

    PubMed

    Gaudet, Suzanne; Miller-Jensen, Kathryn

    2016-06-01

    Genetically identical cells respond heterogeneously to uniform environmental stimuli. Consequently, investigating the signaling networks that control these cell responses using 'average' bulk cell measurements can obscure underlying mechanisms and misses information emerging from cell-to-cell variability. Here we review recent technological advances including live-cell fluorescence imaging-based approaches and microfluidic devices that enable measurements of signaling networks, dynamics, and responses in single cells. We discuss how these single-cell tools have uncovered novel mechanistic insights for canonical signaling pathways that control cell proliferation (ERK), DNA-damage responses (p53), and innate immune and stress responses (NF-κB). Future improvements in throughput and multiplexing, analytical pipelines, and in vivo applicability will all significantly expand the biological information gained from single-cell measurements of signaling pathways. PMID:26968612

  18. Redefining Signaling Pathways with an Expanding Single-Cell Toolbox.

    PubMed

    Gaudet, Suzanne; Miller-Jensen, Kathryn

    2016-06-01

    Genetically identical cells respond heterogeneously to uniform environmental stimuli. Consequently, investigating the signaling networks that control these cell responses using 'average' bulk cell measurements can obscure underlying mechanisms and misses information emerging from cell-to-cell variability. Here we review recent technological advances including live-cell fluorescence imaging-based approaches and microfluidic devices that enable measurements of signaling networks, dynamics, and responses in single cells. We discuss how these single-cell tools have uncovered novel mechanistic insights for canonical signaling pathways that control cell proliferation (ERK), DNA-damage responses (p53), and innate immune and stress responses (NF-κB). Future improvements in throughput and multiplexing, analytical pipelines, and in vivo applicability will all significantly expand the biological information gained from single-cell measurements of signaling pathways.

  19. JAK2/STAT5/Bcl-xL signalling is essential for erythropoietin-mediated protection against apoptosis induced in PC12 cells by the amyloid β−peptide Aβ25–35

    PubMed Central

    Ma, Rong; Hu, Jing; Huang, Chengfang; Wang, Min; Xiang, Jizhou; Li, Gang

    2014-01-01

    BACKGROUND AND PURPOSE Erythropoietin (EPO) exerts neuroprotective actions in the CNS, including protection against apoptosis induced by the amyloid β−peptide Aβ25–35. However, it remains unclear which signalling pathway activated by EPO is involved in this neuroprotection. Here, we have investigated whether JAK2/STAT5/Bcl-xL and ERK1/2 signalling pathways are essential for EPO-mediated protection against apoptosis induced by Aβ25–35. EXPERIMENTAL APPROACH EPO was added to cultures of PC12 cells, 1 h before Aβ25–35. For kinase inhibitor studies, AG490 and PD98059 were added to PC12 cells, 0.5 h before the addition of EPO. Transfection with siRNA was used to knockdown STAT5. Activation of JAK2/STAT5/Bcl-xL and ERK1/2 signalling pathways were investigated by Western blotting. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide assay and apoptosis was detected by TUNEL and acridine orange–ethidium bromide double staining. KEY RESULTS EPO increased phosphorylation of JAK2 and STAT5 in PC12 cells treated with Aβ25–35. Furthermore, EPO modulated the nuclear translocation of phospho-STAT5, which increased expression of Bcl-xL and decreased levels of caspase-3. These beneficial effects were blocked by the JAK2 inhibitor, AG490 or STAT5 knockdown. However, the ERK1/2 pathway did not play a crucial role in our model. CONCLUSIONS AND IMPLICATIONS EPO protected PC12 cells against Aβ25–35-induced neurotoxicity. Activation of JAK2/STAT5/Bcl-xL pathway was important in EPO-mediated neuroprotection. EPO may serve as a novel protective agent against Aβ25–35-induced cytotoxicity in, for instance, Alzheimer's disease. PMID:24597613

  20. The STAT5b Pathway Defect and Autoimmunity

    PubMed Central

    Kanai, Takahiro; Jenks, Jennifer; Nadeau, Kari Christine

    2012-01-01

    The signal transducer and activator of transcription (STAT) 5b is a universal transcription factor that plays key biological roles in allergic diseases, immunodeficiencies, autoimmunities, cancers, hematological diseases, growth disorders, and lung diseases. The identification of distinct pathological manifestations of STAT5b deficiency in humans has highlighted the critical role of the STAT5b pathway. Proper gene transcription at IL-2R α, FOXP3, Bcl-2, and growth hormone (GH) associated loci are thought to be associated with normal STAT5b transcriptional activity. These genes are thought to play important roles in allergy/autoimmunity, immunodeficiency, cancer/anemia, and growth, respectively. The STAT5A and STAT5B genes are collocated on 17q11. Although these two monomeric proteins exhibit peptide sequence similarities of >90%, it is known through observations of STAT5b deficient subjects that STAT5a and STAT5b are not fully redundant in humans. Patients with STAT5b deficiency have decreased numbers of regulatory CD4+CD25high T cell (Treg) despite their STAT5a levels being normal. Prior studies on STAT5b deficient subjects have revealed immunological aberrations associated with the following disease phenotype: modest lymphopenia and decreased populations of Treg, γ−δ T cells, and natural killer (NK) cells. Most subjects with STAT5b deficiency show severe eczema, and autoimmune disease (juvenile idiopathic arthritis, autoimmune thyroiditis, idiopathic thrombocytic purpura) which are thought to be associated with Treg dysfunction. We will review the likely pathophysiological mechanisms associated with STAT5b deficiency. PMID:22912632

  1. An autoregulatory enhancer controls mammary-specific STAT5 functions

    PubMed Central

    Metser, Gil; Shin, Ha Youn; Wang, Chaochen; Yoo, Kyung Hyun; Oh, Sumin; Villarino, Alejandro V.; O'Shea, John J.; Kang, Keunsoo; Hennighausen, Lothar

    2016-01-01

    Signal Transducers and Activators of Transcription (STATs) are principal transcription factors downstream of cytokine receptors. Although STAT5A is expressed in most tissues it remains to be understood why its premier, non-redundant functions are restricted to prolactin-induced mammary gland development and function. We report that the ubiquitously expressed Stat5a/b locus is subject to additional lineage-specific transcriptional control in mammary epithelium. Genome-wide surveys of epigenetic status and transcription factor occupancy uncovered a putative mammary-specific enhancer within the intergenic sequences separating the two Stat5 genes. This region exhibited several hallmarks of genomic enhancers, including DNaseI hypersensitivity, H3K27 acetylation and binding by GR, NFIB, ELF5 and MED1. Mammary-specific STAT5 binding was obtained at two canonical STAT5 binding motifs. CRISPR/Cas9-mediated genome editing was used to delete these sites in mice and determine their biological function. Mutant animals exhibited an 80% reduction of Stat5 levels in mammary epithelium and a concomitant reduction of STAT5-dependent gene expression. Transcriptome analysis identified a class of mammary-restricted genes that was particularly dependent on high STAT5 levels as a result of the intergenic enhancer. Taken together, the mammary-specific enhancer enables a positive feedback circuit that contributes to the remarkable abundance of STAT5 and, in turn, to the efficacy of STAT5-dependent mammary physiology. PMID:26446995

  2. An autoregulatory enhancer controls mammary-specific STAT5 functions.

    PubMed

    Metser, Gil; Shin, Ha Youn; Wang, Chaochen; Yoo, Kyung Hyun; Oh, Sumin; Villarino, Alejandro V; O'Shea, John J; Kang, Keunsoo; Hennighausen, Lothar

    2016-02-18

    Signal Transducers and Activators of Transcription (STATs) are principal transcription factors downstream of cytokine receptors. Although STAT5A is expressed in most tissues it remains to be understood why its premier, non-redundant functions are restricted to prolactin-induced mammary gland development and function. We report that the ubiquitously expressed Stat5a/b locus is subject to additional lineage-specific transcriptional control in mammary epithelium. Genome-wide surveys of epigenetic status and transcription factor occupancy uncovered a putative mammary-specific enhancer within the intergenic sequences separating the two Stat5 genes. This region exhibited several hallmarks of genomic enhancers, including DNaseI hypersensitivity, H3K27 acetylation and binding by GR, NFIB, ELF5 and MED1. Mammary-specific STAT5 binding was obtained at two canonical STAT5 binding motifs. CRISPR/Cas9-mediated genome editing was used to delete these sites in mice and determine their biological function. Mutant animals exhibited an 80% reduction of Stat5 levels in mammary epithelium and a concomitant reduction of STAT5-dependent gene expression. Transcriptome analysis identified a class of mammary-restricted genes that was particularly dependent on high STAT5 levels as a result of the intergenic enhancer. Taken together, the mammary-specific enhancer enables a positive feedback circuit that contributes to the remarkable abundance of STAT5 and, in turn, to the efficacy of STAT5-dependent mammary physiology.

  3. Papillomavirus-specific CD4+ T cells exhibit reduced STAT-5 signaling and altered cytokine profiles in patients with recurrent respiratory papillomatosis.

    PubMed

    James, Eddie A; DeVoti, James A; Rosenthal, David W; Hatam, Lynda J; Steinberg, Bettie M; Abramson, Allan L; Kwok, William W; Bonagura, Vincent R

    2011-06-01

    Recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6) or HPV-11. Specific HLA-DR haplotypes DRB1*01:02 and DRB1*03:01 are associated with the development of RRP, disease severity, and Th2-like responses to HPV early proteins. Th1-like responses to HPV proteins have been shown to be protective in animal models. Therefore, we investigated the hypothesis that RRP patients have dysfunctional Th1-like, HPV-specific T cell responses. Using MHC class II tetramers, we identified immunogenic peptides within HPV-11 early proteins. Two distinct peptides (E6(113-132) and E2(1-20)) contained DRB1*01:02- or DRB1*03:01-restricted epitopes, respectively. An additional peptide (E2(281-300)) contained an epitope presented by both alleles. Peptide binding, tetramer, and proliferation assays identified minimal epitopes within these peptides. These epitopes elicited E2/E6-specific CD4(+) T cell responses in RRP patients and healthy control subjects, allowing the isolation of HPV-specific T cell lines using tetramers. The cytokine profiles and STAT signaling of these tetramer-positive T cells were measured to compare the polarization and responsiveness of HPV-specific T cells from patients with RRP and healthy subjects. HPV-specific IFN-γ secretion was substantially lower in T cells from RRP patients. HPV-specific IL-13 secretion was seen at modest levels in T cells from RRP patients and was absent in T cells from healthy control subjects. HPV-specific T cells from RRP patients exhibited reduced STAT-5 phosphorylation and reduced IL-2 secretion, suggesting anergy. Levels of STAT-5 phosphorylation and IFN-γ secretion could be improved through addition of IL-2 to HPV-specific T cell lines from RRP patients. Therapeutic vaccination or interventions aimed at restoring Th1-like cytokine responses to HPV proteins and reversing anergy could improve clinical outcomes for RRP patients.

  4. Pathologic complete response after preoperative anti-HER2 therapy correlates with alterations in PTEN, FOXO, phosphorylated Stat5, and autophagy protein signaling

    PubMed Central

    2013-01-01

    Background To define protein molecular characteristics of tumor cells prior to, and immediately following, preoperative human epidermal growth factor receptor 2 (HER2)-targeted therapy that correlate with pathologic complete response (pCR) or non response (no pCR) to preoperative HER2-directed therapy and chemotherapy. Methods This open-label, phase II study randomized patients with HER2-positive stage II or III invasive breast cancer to trastuzumab, lapatinib, or both, 2 weeks prior to and during chemotherapy with FEC75 for 4 courses; then paclitaxel 80 mg/m2 weekly for 12 courses, then surgery. Core needle biopsies were collected at baseline and after 2 weeks of anti-HER2 therapy prior to chemotherapy. Data were correlated with pCR, defined as absence of invasive tumor in breast and lymph nodes. Results Of 100 enrolled patients, the analysis population included those who had surgery and received ≥75% chemotherapy (78% [n = 78]). pCRs by arm are: trastuzumab (n = 26), 54% [n = 14]; lapatinib (n = 29), 45% [n = 13]; trastuzumab plus lapatinib (n = 23), 74% [n = 17]). Paired biopsy specimens were available for 49 patients (63%). Tumor cells of patients with pCR in the trastuzumab or lapatinib treatment arms showed nonphosphorylated FOXO, phosphorylated Stat5, and sparse signal-transduction protein network crosstalk representing different patterns of connections with PI3K and autophagy proteins compared with no pCR. Conclusion In this exploratory study, pCR with preoperative anti-HER2 therapy and chemotherapy correlated with the levels and phosphorylation status of specific baseline signal pathway proteins in tumor cells. These data may provide candidate biomarkers to stratify initial treatment and potential combination therapies for future study. Tissue preservation technology introduced here makes this procedure widely feasible. Trial registration ClinicalTrials.gov: NCT00524303 PMID:24304724

  5. Suppression of beta-casein gene expression by inhibition of protein synthesis in mouse mammary epithelial cells is associated with stimulation of NF-kappaB activity and blockage of prolactin-Stat5 signaling.

    PubMed

    Beaton, Angela; Broadhurst, Marita K; Wilkins, Richard J; Wheeler, Thomas T

    2003-02-01

    The protein synthesis inhibitor cycloheximide (Chx) suppresses prolactin-induced beta-casein gene expression in the mammary epithelial cell line COMMA-D. As the mechanism underlying this effect is unclear, the effects of protein synthesis inhibitors on interactions of transcription factors with the beta-casein promoter were examined. Suppression of prolactin-induced beta-casein gene expression occurred in both COMMA-D cells and primary mammary cell cultures with as little as 2 h protein synthesis inhibition. This was associated with changes in transcription factors interacting at a response element in the proximal region of the rat beta-casein promoter. Inhibition of protein synthesis was associated with NF-kappaB binding at a site immediately 3' to the Stat5-binding site at position 97-89 of the beta-casein promoter, suppression of Stat5 DNA-binding activity, and inhibition of Stat5 tyrosine phosphorylation. Treatment with the NF-kappaB inhibitor parthenolide failed to restore prolactin responsiveness. These results show that protein synthesis inhibition is associated with both blockage of prolactin-Stat5 signaling and NF-kappaB binding to the beta-casein promoter, but that the latter is not necessary for the suppression of beta-casein expression.

  6. Surviving apoptosis: life-death signaling in single cells

    PubMed Central

    Flusberg, Deborah A.; Sorger, Peter K.

    2015-01-01

    Tissue development and homeostasis are regulated by opposing pro-survival and pro-death signals. An interesting feature of the Tumor Necrosis Factor (TNF) family of ligands is that they simultaneously activate opposing signals within a single cell via the same ligand-receptor complex. The magnitude of pro-death events such as caspase activation and pro-survival events such as NF-κB activation vary not only from one cell type to the next but also among individual cells of the same type due to intrinsic and extrinsic noise. The molecules involved in these pro-survival/pro-death pathways, and the different phenotypes that result from their activities, have been recently reviewed. Here we focus on the impact of cell-to-cell variability in the strength of these opposing signals on shaping cell fate decisions. PMID:25920803

  7. Age-Related Differences in Percentages of Regulatory and Effector T Lymphocytes and Their Subsets in Healthy Individuals and Characteristic STAT1/STAT5 Signalling Response in Helper T Lymphocytes

    PubMed Central

    Holcar, Marija; Goropevšek, Aleš; Ihan, Alojz; Avčin, Tadej

    2015-01-01

    The dynamic process of the development of the immune system can in itself result in age-related immune malfunctions. In this study, we analysed lymphocyte subsets in the peripheral blood of 60 healthy donors, divided into groups of children, adolescents, and adults, focusing on effector (Teff) and regulatory (Treg) T lymphocytes and STAT1/STAT5 signalling response in helper T lymphocytes (Th) in adults, using flow cytometry. Our results demonstrate a decrease in the percentage of total Tregs and an increase in the percentage of total Teffs with age and a consequential immense increase in the Teff/Treg ratio. The increase of Teffs was most apparent in Th1, Th1Th17, and Th17CD161− subsets. Significant Th lymphocyte STAT1 expression differences were observed between children and adolescents, which were associated with the decrease in activated Tregs. Higher expression of STAT1 was found in FoxP3hi than in FoxP3low Th lymphocytes, while significant IL-2 induced STAT5 phosphorylation differences were found among the subsets of Th lymphocytes in adults. Our study demonstrates age-related changes in circulating Teff and Treg, as well as significant differences in STAT5/STAT1 signalling among FoxP3+ Th lymphocytes, providing new advances in the understanding of immunosenescence. PMID:26525134

  8. Age-Related Differences in Percentages of Regulatory and Effector T Lymphocytes and Their Subsets in Healthy Individuals and Characteristic STAT1/STAT5 Signalling Response in Helper T Lymphocytes.

    PubMed

    Holcar, Marija; Goropevšek, Aleš; Ihan, Alojz; Avčin, Tadej

    2015-01-01

    The dynamic process of the development of the immune system can in itself result in age-related immune malfunctions. In this study, we analysed lymphocyte subsets in the peripheral blood of 60 healthy donors, divided into groups of children, adolescents, and adults, focusing on effector (Teff) and regulatory (Treg) T lymphocytes and STAT1/STAT5 signalling response in helper T lymphocytes (Th) in adults, using flow cytometry. Our results demonstrate a decrease in the percentage of total Tregs and an increase in the percentage of total Teffs with age and a consequential immense increase in the Teff/Treg ratio. The increase of Teffs was most apparent in Th1, Th1Th17, and Th17CD161- subsets. Significant Th lymphocyte STAT1 expression differences were observed between children and adolescents, which were associated with the decrease in activated Tregs. Higher expression of STAT1 was found in FoxP3hi than in FoxP3low Th lymphocytes, while significant IL-2 induced STAT5 phosphorylation differences were found among the subsets of Th lymphocytes in adults. Our study demonstrates age-related changes in circulating Teff and Treg, as well as significant differences in STAT5/STAT1 signalling among FoxP3+ Th lymphocytes, providing new advances in the understanding of immunosenescence. PMID:26525134

  9. Gravity perception and signal transduction in single cells

    NASA Astrophysics Data System (ADS)

    Block, I.; Wolke, A.; Briegleb, W.; Ivanova, K.

    Cellular signal processing in multi-, as well as in unicellular organisms, has to rely on fundamentally similar mechanisms. Free-living single cells often use the gravity vector for their spatial orientation (gravitaxis) and show distinct gravisensitivities. In this investigation the gravisensitive giant ameboid cell Physarum polycephalum (Myxomycetes, acellular slime molds) is used. Its gravitaxis and the modulation of its intrinsic rhythmic contraction activity by gravity was demonstrated in 180 °turn experiments and in simulated, as well as in actual, near-weightlessness studies (fast-rotating clinostat; Spacelab D1, IML-1). The stimulus perception was addressed in an IML-2 experiment, which provided information on the gravireceptor itself by the determination of the cell's acceleration-sensitivity threshold. Ground-based experiments designed to elucidate the subsequent steps in signal transduction leading to a motor response, suggest that an acceleration stimulus induces changes in the level of second messenger, adenosine 3',5'-cyclic monophosphate (cAMP), indicating also that the acceleration-stimulus signal transduction chain of Physarum uses an ubiquitous second messenger pathway.

  10. Persistent STAT5 activation in myeloid neoplasms recruits p53 into gene regulation.

    PubMed

    Girardot, M; Pecquet, C; Chachoua, I; Van Hees, J; Guibert, S; Ferrant, A; Knoops, L; Baxter, E J; Beer, P A; Giraudier, S; Moriggl, R; Vainchenker, W; Green, A R; Constantinescu, S N

    2015-03-01

    STAT (Signal Transducer and Activator of Transcription) transcription factors are constitutively activated in most hematopoietic cancers. We previously identified a target gene, LPP/miR-28 (LIM domain containing preferred translocation partner in lipoma), induced by constitutive activation of STAT5, but not by transient cytokine-activated STAT5. miR-28 exerts negative effects on thrombopoietin receptor signaling and platelet formation. Here, we demonstrate that, in transformed hematopoietic cells, STAT5 and p53 must be synergistically bound to chromatin for induction of LPP/miR-28 transcription. Genome-wide association studies show that both STAT5 and p53 are co-localized on the chromatin at 463 genomic positions in proximal promoters. Chromatin binding of p53 is dependent on persistent STAT5 activation at these proximal promoters. The transcriptional activity of selected promoters bound by STAT5 and p53 was significantly changed upon STAT5 or p53 inhibition. Abnormal expression of several STAT5-p53 target genes (LEP, ATP5J, GTF2A2, VEGFC, NPY1R and NPY5R) is frequently detected in platelets of myeloproliferative neoplasm (MPN) patients, but not in platelets from healthy controls. In conclusion, persistently active STAT5 can recruit normal p53, like in the case of MPN cells, but also p53 mutants, such as p53 M133K in human erythroleukemia cells, leading to pathologic gene expression that differs from canonical STAT5 or p53 transcriptional programs.

  11. STAT5 regulation of BCL10 parallels constitutive NFκB activation in lymphoid tumor cells

    PubMed Central

    Nagy, Zsuzsanna S; LeBaron, Matthew J; Ross, Jeremy A; Mitra, Abhisek; Rui, Hallgeir; Kirken, Robert A

    2009-01-01

    Background Signal Transducer and Activator of Transcription 5 A and B (STAT5) are key survival factors in cells of the lymphoid lineage. Identification of novel, tissue-specific STAT5 regulated genes would advance the ability to combat diseases due to aberrant STAT5 signaling. In the present work a library of human STAT5 bound genomic elements was created and validated. Results Of several STAT5 responsive genomic regulatory elements identified, one was located within the first intron of the human BCL10 gene. Chromatin immuno-precipitation reactions confirmed constitutive in vivo STAT5 binding to this intronic fragment in various human lymphoid tumor cell lines. Interestingly, non-phosphorylated STAT5 was found in the nuclei of Kit225 and YT cells in the absence of cytokine stimulation that paralleled constitutive NFκB activation. Inhibition of the hyperactive JAK3/STAT5 pathway in MT-2 cells via the Mannich-base, NC1153, diminished the constitutive in vivo occupancy of BCL10-SBR by STAT5, reduced NFκB activity and BCL10 protein expression in a dose dependent manner. Moreover, depletion of STAT5 via selective antisense oligonucleotide treatment similarly resulted in decreased BCL10 mRNA and protein expression, cellular viability and impaired NFκB activity independent of IL-2. Conclusion These results suggest that the NFκB regulator BCL10 is an IL-2-independent STAT5 target gene. These findings proffer a model in which un-activated STAT5 can regulate pathways critical for lymphoid cell survival and inhibitors that disrupt STAT5 function independent of tyrosine phosphorylation may be therapeutically effective in treating certain leukemias/lymphomas. PMID:19709433

  12. Subcellular optogenetics – controlling signaling and single-cell behavior

    PubMed Central

    Karunarathne, W. K. Ajith; O'Neill, Patrick R.; Gautam, Narasimhan

    2015-01-01

    ABSTRACT Variation in signaling activity across a cell plays a crucial role in processes such as cell migration. Signaling activity specific to organelles within a cell also likely plays a key role in regulating cellular functions. To understand how such spatially confined signaling within a cell regulates cell behavior, tools that exert experimental control over subcellular signaling activity are required. Here, we discuss the advantages of using optogenetic approaches to achieve this control. We focus on a set of optical triggers that allow subcellular control over signaling through the activation of G-protein-coupled receptors (GPCRs), receptor tyrosine kinases and downstream signaling proteins, as well as those that inhibit endogenous signaling proteins. We also discuss the specific insights with regard to signaling and cell behavior that these subcellular optogenetic approaches can provide. PMID:25433038

  13. Chemical and Hormonal Effects on STAT5b-Dependent Sexual Dimorphism of the Liver Transcriptome.

    EPA Science Inventory

    The growth hormone (GH)-activated transcription factor signal transducer and activator of transcription 5b (STAT5b) is a key regulator of sexually dimorphic gene expression in the liver. Suppression of hepatic STAT5b signaling is associated with lipid metabolic dysfunction leadi...

  14. Signal Transduction at the Single-Cell Level: Approaches to Study the Dynamic Nature of Signaling Networks.

    PubMed

    Handly, L Naomi; Yao, Jason; Wollman, Roy

    2016-09-25

    Signal transduction, or how cells interpret and react to external events, is a fundamental aspect of cellular function. Traditional study of signal transduction pathways involves mapping cellular signaling pathways at the population level. However, population-averaged readouts do not adequately illuminate the complex dynamics and heterogeneous responses found at the single-cell level. Recent technological advances that observe cellular response, computationally model signaling pathways, and experimentally manipulate cells now enable studying signal transduction at the single-cell level. These studies will enable deeper insights into the dynamic nature of signaling networks.

  15. Coregulation of genetic programs by the transcription factors NFIB and STAT5.

    PubMed

    Robinson, Gertraud W; Kang, Keunsoo; Yoo, Kyung Hyun; Tang, Yong; Zhu, Bing-Mei; Yamaji, Daisuke; Colditz, Vera; Jang, Seung Jian; Gronostajski, Richard M; Hennighausen, Lothar

    2014-05-01

    Mammary-specific genetic programs are activated during pregnancy by the common transcription factor signal transducer and activator of transcription (STAT) 5. More than one third of these genes carry nuclear factor I/B (NFIB) binding motifs that coincide with STAT5 in vivo binding, suggesting functional synergy between these two transcription factors. The role of NFIB in this governance was investigated in mice from which Nfib had been inactivated in mammary stem cells or in differentiating alveolar epithelium. Although NFIB was not required for alveolar expansion, the combined absence of NFIB and STAT5 prevented the formation of functional alveoli. NFIB controlled the expression of mammary-specific and STAT5-regulated genes and chromatin immunoprecipitation-sequencing established STAT5 and NFIB binding at composite regulatory elements containing histone H3 lysine dimethylation enhancer marks and progesterone receptor binding. By integrating previously published chromatin immunoprecipitation-sequencing data sets, the presence of NFIB-STAT5 modules in other cell types was investigated. Notably, genomic sites bound by NFIB in hair follicle stem cells were also occupied by STAT5 in mammary epithelium and coincided with enhancer marks. Many of these genes were under NFIB control in both hair follicle stem cells and mammary alveolar epithelium. We propose that NFIB-STAT5 modules, possibly in conjunction with other transcription factors, control cell-specific genetic programs.

  16. Regulation of the interferon regulatory factor-8 (IRF-8) tumor suppressor gene by the signal transducer and activator of transcription 5 (STAT5) transcription factor in chronic myeloid leukemia.

    PubMed

    Waight, Jeremy D; Banik, Debarati; Griffiths, Elizabeth A; Nemeth, Michael J; Abrams, Scott I

    2014-05-30

    Tyrosine kinase inhibitors such as imatinib can effectively target the BCR-ABL oncoprotein in a majority of patients with chronic myeloid leukemia (CML). Unfortunately, some patients are resistant primarily to imatinib and others develop drug resistance, prompting interest in the discovery of new drug targets. Although much of this resistance can be explained by the presence of mutations within the tyrosine kinase domain of BCR-ABL, such mutations are not universally identified. Interferon regulatory factor-8 (IRF-8) is a transcription factor that is essential for myelopoiesis. Depressed IRF-8 levels are observed in a majority of CML patients and Irf-8(-/-) mice exhibit a CML-like disease. The underlying mechanisms of IRF-8 loss in CML are unknown. We hypothesized that BCR-ABL suppresses transcription of IRF-8 through STAT5, a proximal BCR-ABL target. Treatment of primary cells from newly diagnosed CML patients in chronic phase as well as BCR-ABL(+) cell lines with imatinib increased IRF-8 transcription. Furthermore, IRF-8 expression in cell line models was necessary for imatinib-induced antitumor responses. We have demonstrated that IRF-8 is a direct target of STAT5 and that silencing of STAT5 induced IRF-8 expression. Conversely, activating STAT5 suppressed IRF-8 transcription. Finally, we showed that STAT5 blockade using a recently discovered antagonist increased IRF-8 expression in patient samples. These data reveal a previously unrecognized BCR-ABL-STAT5-IRF-8 network, which widens the repertoire of potentially new anti-CML targets.

  17. Dual-Specificity Phosphatase 4 Regulates STAT5 Protein Stability and Helper T Cell Polarization*

    PubMed Central

    Liao, Fang-Hsuean; Chan, Yi-Chiao; Huang, Ching-Yu

    2015-01-01

    Immune responses are critically regulated by the functions of CD4 helper T cells. Based on their secreted cytokines, helper T cells are further categorized into different subsets like Treg or Th17 cells, which suppress or promote inflammatory responses, respectively. Signals from IL-2 activate the transcription factor STAT5 to promote Treg but suppress Th17 cell differentiation. Our previous results found that the deficiency of a dual-specificity phosphatase, DUSP4, induced STAT5 hyper-activation, enhanced IL-2 signaling, and increased T cell proliferation. In this report, we examined the effects of DUSP4 deficiency on helper T cell differentiation and STAT5 regulation. Our in vivo data showed that DUSP4 mice were more resistant to the induction of autoimmune encephalitis, while in vitro differentiations revealed enhanced iTreg and reduced Th17 polarization in DUSP4-deficient T cells. To study the cause of this altered helper T cell polarization, we performed luciferase reporter assays and confirmed that, as predicted by our previous report, DUSP4 over-expression suppressed the transcription factor activity of STAT5. Surprisingly, we also found that DUSP4-deficient T but not B cells exhibited elevated STAT5 protein levels, and over-expressed DUSP4 destabilized STAT5 in vitro; moreover, this destabilization required the phosphatase activity of DUSP4, and was insensitive to MG132 treatment. Finally, domain-mapping results showed that both the substrate-interacting and the phosphatase domains of DUSP4 were required for its optimal interaction with STAT5, while the coiled-coil domain of STAT5 appeared to hinder this interaction. Our data thus provide the first genetic evidence that DUSP4 is important for helper T cell development. In addition, they also help uncover the novel, DUSP4-mediated regulation of STAT5 protein stability. PMID:26710253

  18. STAT5 is a key transcription factor for IL-3-mediated inhibition of RANKL-induced osteoclastogenesis

    PubMed Central

    Lee, Jongwon; Seong, Semun; Kim, Jung Ha; Kim, Kabsun; Kim, Inyoung; Jeong, Byung-chul; Nam, Kwang-Il; Kim, Kyung Keun; Hennighausen, Lothar; Kim, Nacksung

    2016-01-01

    Among the diverse cytokines involved in osteoclast differentiation, interleukin (IL)-3 inhibits RANKL-induced osteoclastogenesis. However, the mechanism underlying IL-3-mediated inhibition of osteoclast differentiation is not fully understood. Here we demonstrate that the activation of signal transducers and activators of transcription 5 (STAT5) by IL-3 inhibits RANKL-induced osteoclastogenesis through the induction of the expression of Id genes. We found that STAT5 overexpression inhibited RANKL-induced osteoclastogenesis. However, RANKL did not regulate the expression or activation of STAT5 during osteoclast differentiation. STAT5 deficiency prevented IL-3-mediated inhibition of osteoclastogenesis, suggesting a key role of STAT5 in IL-3-mediated inhibition of osteoclast differentiation. In addition, IL-3-induced STAT5 activation upregulated the expression of Id1 and Id2, which are negative regulators of osteoclastogenesis. Overexpression of ID1 or ID2 in STAT5-deficient cells reversed osteoclast development recovered from IL-3-mediated inhibition. Importantly, microcomputed tomography and histomorphometric analysis revealed that STAT5 conditional knockout mice showed reduced bone mass, with an increased number of osteoclasts. Furthermore, IL-3 inhibited RANKL-induced osteoclast differentiation less effectively in the STAT5 conditional knockout mice than in the wild-type mice after RANKL injection. Taken together, our findings indicate that STAT5 contributes to the remarkable IL-3-mediated inhibition of RANKL-induced osteoclastogenesis by activating Id genes and their associated pathways. PMID:27485735

  19. STAT5 is a key transcription factor for IL-3-mediated inhibition of RANKL-induced osteoclastogenesis.

    PubMed

    Lee, Jongwon; Seong, Semun; Kim, Jung Ha; Kim, Kabsun; Kim, Inyoung; Jeong, Byung-Chul; Nam, Kwang-Il; Kim, Kyung Keun; Hennighausen, Lothar; Kim, Nacksung

    2016-01-01

    Among the diverse cytokines involved in osteoclast differentiation, interleukin (IL)-3 inhibits RANKL-induced osteoclastogenesis. However, the mechanism underlying IL-3-mediated inhibition of osteoclast differentiation is not fully understood. Here we demonstrate that the activation of signal transducers and activators of transcription 5 (STAT5) by IL-3 inhibits RANKL-induced osteoclastogenesis through the induction of the expression of Id genes. We found that STAT5 overexpression inhibited RANKL-induced osteoclastogenesis. However, RANKL did not regulate the expression or activation of STAT5 during osteoclast differentiation. STAT5 deficiency prevented IL-3-mediated inhibition of osteoclastogenesis, suggesting a key role of STAT5 in IL-3-mediated inhibition of osteoclast differentiation. In addition, IL-3-induced STAT5 activation upregulated the expression of Id1 and Id2, which are negative regulators of osteoclastogenesis. Overexpression of ID1 or ID2 in STAT5-deficient cells reversed osteoclast development recovered from IL-3-mediated inhibition. Importantly, microcomputed tomography and histomorphometric analysis revealed that STAT5 conditional knockout mice showed reduced bone mass, with an increased number of osteoclasts. Furthermore, IL-3 inhibited RANKL-induced osteoclast differentiation less effectively in the STAT5 conditional knockout mice than in the wild-type mice after RANKL injection. Taken together, our findings indicate that STAT5 contributes to the remarkable IL-3-mediated inhibition of RANKL-induced osteoclastogenesis by activating Id genes and their associated pathways. PMID:27485735

  20. Conditional Density-based Analysis of T cell Signaling in Single Cell Data

    PubMed Central

    Krishnaswamy, Smita; Spitzer, Matthew H.; Mingueneau, Michael; Bendall, Sean C; Litvin, Oren; Stone, Erica; Pe’er, Dana; Nolan, Garry P

    2015-01-01

    Cellular circuits sense the environment, process signals, and compute decisions using networks of interacting proteins. To model such a system, the abundance of each activated protein species can be described as a stochastic function of the abundance of other proteins. High-dimensional single-cell technologies, like mass cytometry, offer an opportunity to characterize signaling circuit-wide. However, the challenge of developing and applying computational approaches to interpret such complex data remains. Here, we developed computational methods, based on established statistical concepts, to characterize signaling network relationships by quantifying the strengths of network edges and deriving signaling response functions. In comparing signaling between naïve and antigen-exposed CD4+ T-lymphocytes, we find that although these two cell subtypes had similarly-wired networks, naïve cells transmitted more information along a key signaling cascade than did antigen-exposed cells. We validated our characterization on mice lacking the extracellular-regulated MAP kinase (ERK2), which showed stronger influence of pERK on pS6 (phosphorylated-ribosomal protein S6), in naïve cells compared to antigen-exposed cells, as predicted. We demonstrate that by using cell-to-cell variation inherent in single cell data, we can algorithmically derive response functions underlying molecular circuits and drive the understanding of how cells process signals. PMID:25342659

  1. Nongenomic STAT5-dependent effects on Golgi apparatus and endoplasmic reticulum structure and function.

    PubMed

    Lee, Jason E; Yang, Yang-Ming; Liang, Feng-Xia; Gough, Daniel J; Levy, David E; Sehgal, Pravin B

    2012-03-01

    We report unexpected nongenomic functions of signal transducer and activator of transcription (STAT) 5 species in the cytoplasm aimed at preserving the structure and function of the Golgi apparatus and rough endoplasmic reticulum (ER) in vascular cells. Immunoimaging and green fluorescent protein-tagged-STAT5a protein localization studies showed the constitutive association of nonphosphorylated STAT5a, and to a lesser extent STAT5b, with the Golgi apparatus and of STAT5a with centrosomes in human pulmonary arterial endothelial and smooth muscle cells. Acute knockdown of STAT5a/b species using small interfering RNAs (siRNAs), including in the presence of an mRNA synthesis inhibitor (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole), produced a dramatic phenotype within 1 day, consisting of dilatation and fragmentation of Golgi cisternae, a marked tubule-to-cyst change in the ER, increased accumulation of reticulon-4 (RTN4)/Nogo-B and atlastin-3 (ATL3) at cyst-zone boundaries, cystic separation of the outer and inner nuclear membranes, accompanied by scalloped/lunate distortion of the nucleus, with accumulation of RTN4 on convex sides of distorted nuclei. These cells showed inhibition of vesicular stomatitis virus G protein glycoprotein trafficking, mitochondrial fragmentation, and reduced mitochondrial function. STAT5a/b(-/-) mouse embryo fibroblasts also showed altered ER/Golgi dynamics. RTN4 knockdown using siRNA did not affect development of the cystic phenotype; ATL3 siRNA led to effacement of cyst-zone boundaries. In magnetic-bead cross-immunopanning assays, ATL3 bound both STAT5a and STAT5b. Remarkably, this novel cystic ER/lunate nucleus phenotype was characteristic of vascular cells in arterial lesions of idiopathic pulmonary hypertension, an unrelentingly fatal human disease. These data provide evidence of a STAT-family protein regulating the structure of a cytoplasmic organelle and implicate this mechanism in the pathogenesis of a human disease.

  2. Single cell kinase signaling assay using pinched flow coupled droplet microfluidics.

    PubMed

    Ramji, Ramesh; Wang, Ming; Bhagat, Ali Asgar S; Tan Shao Weng, Daniel; Thakor, Nitish V; Teck Lim, Chwee; Chen, Chia-Hung

    2014-05-01

    Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib. PMID:24926389

  3. Single cell kinase signaling assay using pinched flow coupled droplet microfluidics

    PubMed Central

    Ramji, Ramesh; Wang, Ming; Bhagat, Ali Asgar S.; Tan Shao Weng, Daniel; Thakor, Nitish V.; Teck Lim, Chwee; Chen, Chia-Hung

    2014-01-01

    Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib. PMID:24926389

  4. Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function.

    PubMed

    Pinz, Sophia; Unser, Samy; Buob, Dominik; Fischer, Philipp; Jobst, Belinda; Rascle, Anne

    2015-04-20

    Signal transducer and activator of transcription STAT5 is essential for the regulation of proliferation and survival genes. Its activity is tightly regulated through cytokine signaling and is often upregulated in cancer. We showed previously that the deacetylase inhibitor trichostatin A (TSA) inhibits STAT5-mediated transcription by preventing recruitment of the transcriptional machinery at a step following STAT5 binding to DNA. The mechanism and factors involved in this inhibition remain unknown. We now show that deacetylase inhibitors do not target STAT5 acetylation, as we initially hypothesized. Instead, they induce a rapid increase in global histone acetylation apparently resulting in the delocalization of the bromodomain and extra-terminal (BET) protein Brd2 and of the Brd2-associated factor TBP to hyperacetylated chromatin. Treatment with the BET inhibitor (+)-JQ1 inhibited expression of STAT5 target genes, supporting a role of BET proteins in the regulation of STAT5 activity. Accordingly, chromatin immunoprecipitation demonstrated that Brd2 is associated with the transcriptionally active STAT5 target gene Cis and is displaced upon TSA treatment. Our data therefore indicate that Brd2 is required for the proper recruitment of the transcriptional machinery at STAT5 target genes and that deacetylase inhibitors suppress STAT5-mediated transcription by interfering with Brd2 function.

  5. A microchip platform for interrogating tumor-macrophage paracrine signaling at the single-cell level.

    PubMed

    Elitas, Meltem; Brower, Kara; Lu, Yao; Chen, Jonathan J; Fan, Rong

    2014-09-21

    It is increasingly recognized that infiltrating immune cells contribute to the pathogenesis of a wide range of solid tumors. The paracrine signaling between the tumor and the immune cells alters the functional state of individual tumor cells and, correspondingly, the anticipated response to radiation or chemotherapies, which is of great importance to clinical oncology. Here we present a high-density microchip platform capable of measuring a panel of paracrine signals associated with heterotypic tumor-immune cell interactions in the single-cell, pair-wise manner. The device features a high-content cell capture array of 5000+ sub-nanoliter microchambers for the isolation of single and multi-cell combinations and a multi-plex antibody "barcode" array for multiplexed protein secretion analysis from each microchamber. In this work, we measured a panel of 16 proteins produced from individual glioma cells, individual macrophage cells and varying heterotypic multi-cell combinations of both on the same device. The results show changes of tumor cell functional phenotypes that cannot be explained by an additive effect from isolated single cells and, presumably, can be attributed to the paracrine signaling between macrophage and glioma cells. The protein correlation analysis reveals the key signaling nodes altered by tumor-macrophage communication. This platform enables the novel pair-wise interrogation of heterotypic cell-cell paracrine signaling at the individual cell level with an in-depth analysis of the changing functional phenotypes for different co-culture cell combinations.

  6. Disruption of STAT5b-Regulated Sexual Dimorphism of the Liver Transcriptome by Diverse Factors Is a Common Event

    PubMed Central

    Oshida, Keiyu; Vasani, Naresh; Waxman, David J.; Corton, J. Christopher

    2016-01-01

    Signal transducer and activator of transcription 5b (STAT5b) is a growth hormone (GH)-activated transcription factor and a master regulator of sexually dimorphic gene expression in the liver. Disruption of the GH hypothalamo-pituitary-liver axis controlling STAT5b activation can lead to metabolic dysregulation, steatosis, and liver cancer. Computational approaches were developed to identify factors that disrupt STAT5b function in a mouse liver gene expression compendium. A biomarker comprised of 144 STAT5b-dependent genes was derived using comparisons between wild-type male and wild-type female mice and between STAT5b-null and wild-type mice. Correlations between the STAT5b biomarker gene set and a test set comprised of expression datasets (biosets) with known effects on STAT5b function were evaluated using a rank-based test (the Running Fisher algorithm). Using a similarity p-value ≤ 10−4, the test achieved a balanced accuracy of 99% and 97% for detection of STAT5b activation or STAT5b suppression, respectively. The STAT5b biomarker gene set was then used to identify factors that activate (masculinize) or suppress (feminize) STAT5b function in an annotated mouse liver and primary hepatocyte gene expression compendium of ~1,850 datasets. Disruption of GH-regulated STAT5b is a common phenomenon in liver in vivo, with 5% and 29% of the male datasets, and 11% and 13% of the female datasets, associated with masculinization or feminization, respectively. As expected, liver STAT5b activation/masculinization occurred at puberty and suppression/feminization occurred during aging and in mutant mice with defects in GH signaling. A total of 70 genes were identified that have effects on STAT5b activation in genetic models in which the gene was inactivated or overexpressed. Other factors that affected liver STAT5b function were shown to include fasting, caloric restriction and infections. Together, these findings identify diverse factors that perturb the hypothalamo

  7. Intramolecular hydrophobic interactions are critical mediators of STAT5 dimerization

    PubMed Central

    Fahrenkamp, Dirk; Li, Jinyu; Ernst, Sabrina; Schmitz-Van de Leur, Hildegard; Chatain, Nicolas; Küster, Andrea; Koschmieder, Steffen; Lüscher, Bernhard; Rossetti, Giulia; Müller-Newen, Gerhard

    2016-01-01

    STAT5 is an essential transcription factor in hematopoiesis, which is activated through tyrosine phosphorylation in response to cytokine stimulation. Constitutive activation of STAT5 is a hallmark of myeloid and lymphoblastic leukemia. Using homology modeling and molecular dynamics simulations, a model of the STAT5 phosphotyrosine-SH2 domain interface was generated providing first structural information on the activated STAT5 dimer including a sequence, for which no structural information is available for any of the STAT proteins. We identified a novel intramolecular interaction mediated through F706, adjacent to the phosphotyrosine motif, and a unique hydrophobic interface on the surface of the SH2 domain. Analysis of corresponding STAT5 mutants revealed that this interaction is dispensable for Epo receptor-mediated phosphorylation of STAT5 but essential for dimer formation and subsequent nuclear accumulation. Moreover, the herein presented model clarifies molecular mechanisms of recently discovered leukemic STAT5 mutants and will help to guide future drug development. PMID:27752093

  8. Unidirectional signal propagation in primary neurons micropatterned at a single-cell resolution

    NASA Astrophysics Data System (ADS)

    Yamamoto, H.; Matsumura, R.; Takaoki, H.; Katsurabayashi, S.; Hirano-Iwata, A.; Niwano, M.

    2016-07-01

    The structure and connectivity of cultured neuronal networks can be controlled by using micropatterned surfaces. Here, we demonstrate that the direction of signal propagation can be precisely controlled at a single-cell resolution by growing primary neurons on micropatterns. To achieve this, we first examined the process by which axons develop and how synapses form in micropatterned primary neurons using immunocytochemistry. By aligning asymmetric micropatterns with a marginal gap, it was possible to pattern primary neurons with a directed polarization axis at the single-cell level. We then examined how synapses develop on micropatterned hippocampal neurons. Three types of micropatterns with different numbers of short paths for dendrite growth were compared. A normal development in synapse density was observed when micropatterns with three or more short paths were used. Finally, we performed double patch clamp recordings on micropatterned neurons to confirm that these synapses are indeed functional, and that the neuronal signal is transmitted unidirectionally in the intended orientation. This work provides a practical guideline for patterning single neurons to design functional neuronal networks in vitro with the direction of signal propagation being controlled.

  9. Stat5 is indispensable for the maintenance of bcr/abl-positive leukaemia

    PubMed Central

    Hoelbl, Andrea; Schuster, Christian; Kovacic, Boris; Zhu, Bingmei; Wickre, Mark; Hoelzl, Maria A; Fajmann, Sabine; Grebien, Florian; Warsch, Wolfgang; Stengl, Gabriele; Hennighausen, Lothar; Poli, Valeria; Beug, Hartmut; Moriggl, Richard; Sexl, Veronika

    2010-01-01

    Tumourigenesis caused by the Bcr/Abl oncoprotein is a multi-step process proceeding from initial to tumour-maintaining events and finally results in a complex tumour-supporting network. A key to successful cancer therapy is the identification of critical functional nodes in an oncogenic network required for disease maintenance. So far, the transcription factors Stat3 and Stat5a/b have been implicated in bcr/abl-induced initial transformation. However, to qualify as a potential drug target, a signalling pathway must be required for the maintenance of the leukaemic state. Data on the roles of Stat3 or Stat5a/b in leukaemia maintenance are elusive. Here, we show that both, Stat3 and Stat5 are necessary for initial transformation. However, Stat5- but not Stat3-deletion induces G0/G1 cell cycle arrest and apoptosis of imatinib-sensitive and imatinib-resistant stable leukaemic cells in vitro. Accordingly, Stat5-abrogation led to effective elimination of myeloid and lymphoid leukaemia maintenance in vivo. Hence, we identified Stat5 as a vulnerable point in the oncogenic network downstream of Bcr/Abl representing a case of non-oncogene addiction (NOA). PMID:20201032

  10. Study of Signal Detection, Integration, and Propagation in Quorum Sensing at the Single Cell Level

    NASA Astrophysics Data System (ADS)

    Long, Tao; Bassler, Bonnie; Wingreen, Ned

    2007-03-01

    Bacteria respond to their environment and to each other and accordingly adjust their gene-expression levels. Accurate signal detection, appropriate signal integration, and faithful signal propagation are essential for a cell to make correct adjustments in response to various extracellular cues. To better understand this information processing by living cells, we studied a model system -- the quorum-sensing circuit in Vibrio harveyi. Quorum sensing is a process in which bacteria communicate with each other by diffusible chemical molecules, termed ``autoinducers'', to commit to coordinated developmental decisions. Three types of autoinducers are detected coincidently by three parallel receptors. The signals are then integrated into the same signaling pathway and propagated by phosphorylation or dephosphorylation of the pathway components. To quantitatively measure the intracellular response, we applied a fluorescent protein reporter, whose production is regulated by a phosphorylated protein in the pathway. By single-cell microscopy, we can explore features of this information-processing circuit such as coincidence detection, signal integration, noise reduction or filtering, and especially the fidelity in signal processing achieved in the presence of inevitable fluctuations.

  11. Chemical and Hormonal Effects on STAT5b-Dependent Sexual Dimorphism of the Liver Transcriptome

    PubMed Central

    Oshida, Keiyu; Waxman, David J.; Corton, J. Christopher

    2016-01-01

    The growth hormone (GH)-activated transcription factor signal transducer and activator of transcription 5b (STAT5b) is a key regulator of sexually dimorphic gene expression in the liver. Suppression of hepatic STAT5b signaling is associated with lipid metabolic dysfunction leading to steatosis and liver cancer. In the companion publication, a STAT5b biomarker gene set was identified and used in a rank-based test to predict both increases and decreases in liver STAT5b activation status/function with high (≥ 97%) accuracy. Here, this computational approach was used to identify chemicals and hormones that activate (masculinize) or suppress (feminize) STAT5b function in a large, annotated mouse liver and primary hepatocyte gene expression compendium. Exposure to dihydrotestosterone and thyroid hormone caused liver masculinization, whereas glucocorticoids, fibroblast growth factor 15, and angiotensin II caused liver feminization. In mouse models of diabetes and obesity, liver feminization was consistently observed and was at least partially reversed by leptin or resveratrol exposure. Chemical-induced feminization of male mouse liver gene expression profiles was a relatively frequent phenomenon: of 156 gene expression biosets from chemically-treated male mice, 29% showed feminization of liver STAT5b function, while <1% showed masculinization. Most (93%) of the biosets that exhibited feminization of male liver were also associated with activation of one or more xenobiotic-responsive receptors, most commonly constitutive activated receptor (CAR) or peroxisome proliferator-activated receptor alpha (PPARα). Feminization was consistently associated with increased expression of peroxisome proliferator-activated receptor gamma (Pparg) but not other lipogenic transcription factors linked to steatosis. GH-activated STAT5b signaling in mouse liver is thus commonly altered by diverse chemicals, and provides a linkage between chemical exposure and dysregulated gene expression

  12. Protein signaling networks from single cell fluctuations and information theory profiling.

    PubMed

    Shin, Young Shik; Remacle, F; Fan, Rong; Hwang, Kiwook; Wei, Wei; Ahmad, Habib; Levine, R D; Heath, James R

    2011-05-18

    Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network. PMID:21575571

  13. Targeting STAT5 in Hematological Malignancies through Inhibition of the Bromodomain and Extra-Terminal (BET) Bromodomain Protein BRD2

    PubMed Central

    Liu, Suhu; Walker, Sarah R.; Nelson, Erik A.; Cerulli, Robert; Xiang, Michael; Toniolo, Patricia A.; Qi, Jun; Stone, Richard M.; Wadleigh, Martha; Bradner, James E.; Frank, David A.

    2014-01-01

    The transcription factor signal transducer and activator of transcription 5 (STAT5) is constitutively activated in a wide range of leukemias and lymphomas, and drives the expression of genes necessary for proliferation, survival, and self-renewal. Thus, targeting STAT5 is an appealing therapeutic strategy for hematological malignancies. Given the importance of bromodomain-containing proteins in transcriptional regulation, we considered the hypothesis that a pharmacological bromodomain inhibitor could inhibit STAT5-dependent gene expression. We found that the small molecule bromodomain and extra-terminal (BET) bromodomain inhibitor JQ1 decreases STAT5-dependent (but not STAT3-dependent) transcription of both heterologous reporter genes and endogenous STAT5 target genes. JQ1 reduces STAT5 function in leukemia and lymphoma cells with constitutive STAT5 activation, or inducibly activated by cytokine stimulation. Among the BET bromodomain sub-family of proteins, it appears that BRD2 is the critical mediator for STAT5 activity. In experimental models of acute T cell lymphoblastic leukemias, where activated STAT5 contributes to leukemia cell survival, Brd2 knock-down or JQ1 treatment shows strong synergy with tyrosine kinase inhibitors in inducing leukemia cells apoptosis. By contrast, mononuclear cells isolated form umbilical cord blood, which is enriched in normal hematopoietic precursor cells, were unaffected by these combinations. These findings indicate a unique functional association between BRD2 and STAT5, and suggest that combinations of JQ1 and tyrosine kinase inhibitors may be an important rational strategy for treating leukemias and lymphomas driven by constitutive STAT5 activation. PMID:24435449

  14. Inherent growth hormone resistance in the skeletal muscle of the fine flounder is modulated by nutritional status and is characterized by high contents of truncated GHR, impairment in the JAK2/STAT5 signaling pathway, and low IGF-I expression.

    PubMed

    Fuentes, Eduardo N; Einarsdottir, Ingibjörg Eir; Valdes, Juan Antonio; Alvarez, Marco; Molina, Alfredo; Björnsson, Björn Thrandur

    2012-01-01

    A detailed understanding of how the GH and IGF-I regulate muscle growth, especially in early vertebrates, is still lacking. The fine flounder is a flatfish species exhibiting remarkably slow growth, representing an intriguing model for elucidating growth regulatory mechanisms. Key components of the GH system were examined in groups of fish during periods of feeding, fasting, and refeeding. Under feeding conditions, there is an inherent systemic and local (muscle) GH resistance, characterized by higher levels of plasma GH than of IGF-I, skeletal muscle with a greater content of the truncated GH receptor (GHRt) than of full-length GHR (GHRfl), an impaired activation of the Janus kinase 2 (JAK2)-signal transducers and activators of transcription 5 (STAT5) signaling pathway, and low IGF-I expression. Fasting leads to further elevation of plasma GH levels concomitant with suppressed IGF-I levels. The ratio of GHRfl to GHRt in muscle decreases during fasting, causing an inactivation of the JAK2/STAT5 signaling pathway and suppressed IGF-I expression, further impairing growth. When fish are returned to nutritionally favorable conditions, plasma GH levels decrease, and the ratio of GHRfl to GHRt in muscle increases, triggering JAK2/STAT5 reactivation and local IGF-I expression, concomitant with increased growth. The study suggests that systemic IGF-I is supporting basal slow growth in this species, without ruling out that local IGF-I is participating in muscle growth. These results reveal for the first time a unique model of inherent GH resistance in the skeletal muscle of a nonmammalian species and contribute to novel insights of the endocrine and molecular basis of growth regulation in earlier vertebrates.

  15. Stat5 activation enables erythropoiesis in the absence of EpoR and Jak2.

    PubMed

    Grebien, Florian; Kerenyi, Marc A; Kovacic, Boris; Kolbe, Thomas; Becker, Verena; Dolznig, Helmut; Pfeffer, Klaus; Klingmüller, Ursula; Müller, Mathias; Beug, Hartmut; Müllner, Ernst W; Moriggl, Richard

    2008-05-01

    Erythropoiesis requires erythropoietin (Epo) and stem cell factor (SCF) signaling via their receptors EpoR and c-Kit. EpoR, like many other receptors involved in hematopoiesis, acts via the kinase Jak2. Deletion of EpoR or Janus kinase 2 (Jak2) causes embryonic lethality as a result of defective erythropoiesis. The contribution of distinct EpoR/Jak2-induced signaling pathways (mitogen-activated protein kinase, phosphatidylinositol 3-kinase, signal transducer and activator of transcription 5 [Stat5]) to functional erythropoiesis is incompletely understood. Here we demonstrate that expression of a constitutively activated Stat5a mutant (cS5) was sufficient to relieve the proliferation defect of Jak2(-/-) and EpoR(-/-) cells in an Epo-independent manner. In addition, tamoxifen-induced DNA binding of a Stat5a-estrogen receptor (ER)* fusion construct enabled erythropoiesis in the absence of Epo. Furthermore, c-Kit was able to enhance signaling through the Jak2-Stat5 axis, particularly in lymphoid and myeloid progenitors. Although abundance of hematopoietic stem cells was 2.5-fold reduced in Jak2(-/-) fetal livers, transplantation of Jak2(-/-)-cS5 fetal liver cells into irradiated mice gave rise to mature erythroid and myeloid cells of donor origin up to 6 months after transplantation. Cytokine- and c-Kit pathways do not function independently of each other in hematopoiesis but cooperate to attain full Jak2/Stat5 activation. In conclusion, activated Stat5 is a critical downstream effector of Jak2 in erythropoiesis/myelopoiesis, and Jak2 functionally links cytokine- with c-Kit-receptor tyrosine kinase signaling.

  16. The Status of STAT3 and STAT5 in Human Breast Atypical Ductal Hyperplasia.

    PubMed

    Shi, Aiping; Dong, Jie; Hilsenbeck, Susan; Bi, Lirong; Zhang, Hong; Li, Yi

    2015-01-01

    Signal Transducer and Activation of Transcription factors (STAT3 and STAT5) play important roles in breast epithelial cell differentiation, proliferation, and apoptosis. They have been investigated extensively in established breast cancer, but their activation status in precancerous lesions has not been reported. Formalin-fixed, paraffin-embedded archival tissues from 59 cases of atypical ductal hyperplasia (ADH) and 31 cases of normal human breast tissue as well as 21 cases of usual ductal hyperplasias (UDH) were obtained from the First Hospital of Jilin University, China, and stained for pSTAT3 and pSTAT5 by immunohistochemistry. The median percentage of pSTAT5+ cells in ADH was 12%, not significantly deviant from that in normal breast. The median percentage of pSTAT3+ cells in ADH was 30%, significantly higher than that of normal breast. pSTAT3 and pSTAT5 were exclusive of each other--they were detected in different ADHs or in different cells within the same ADHs. In addition, both pSTAT3 and pSTAT5 were produced in similar percentages of cells in ADHs from cancer-free patients vs. ADHs that were adjacent to an invasive cancer. Our finding of a complementary expression pattern of pSTAT3 and pSTAT5 in ADH suggests that these two transcription factors may have feedback inhibitory effects on each other during early stages of breast cancer evolution, and that disruption of this inverse relationship may be important in the progression from early lesions to cancer, which exhibits positive association between pSTAT3 and pSTAT5. PMID:26146825

  17. The Status of STAT3 and STAT5 in Human Breast Atypical Ductal Hyperplasia

    PubMed Central

    Shi, Aiping; Dong, Jie; Hilsenbeck, Susan; Bi, Lirong; Zhang, Hong; Li, Yi

    2015-01-01

    Signal Transducer and Activation of Transcription factors (STAT3 and STAT5) play important roles in breast epithelial cell differentiation, proliferation, and apoptosis. They have been investigated extensively in established breast cancer, but their activation status in precancerous lesions has not been reported. Formalin-fixed, paraffin-embedded archival tissues from 59 cases of atypical ductal hyperplasia (ADH) and 31 cases of normal human breast tissue as well as 21 cases of usual ductal hyperplasias (UDH) were obtained from the First Hospital of Jilin University, China, and stained for pSTAT3 and pSTAT5 by immunohistochemistry. The median percentage of pSTAT5+ cells in ADH was 12%, not significantly deviant from that in normal breast. The median percentage of pSTAT3+ cells in ADH was 30%, significantly higher than that of normal breast. pSTAT3 and pSTAT5 were exclusive of each other—they were detected in different ADHs or in different cells within the same ADHs. In addition, both pSTAT3 and pSTAT5 were produced in similar percentages of cells in ADHs from cancer-free patients vs. ADHs that were adjacent to an invasive cancer. Our finding of a complementary expression pattern of pSTAT3 and pSTAT5 in ADH suggests that these two transcription factors may have feedback inhibitory effects on each other during early stages of breast cancer evolution, and that disruption of this inverse relationship may be important in the progression from early lesions to cancer, which exhibits positive association between pSTAT3 and pSTAT5. PMID:26146825

  18. Evidence for Extracellular ATP as a Stress Signal in a Single-Celled Organism

    PubMed Central

    Sivaramakrishnan, Venketesh

    2015-01-01

    ATP is omnipresent in biology and acts as an extracellular signaling molecule in mammals. Information regarding the signaling function of extracellular ATP in single-celled eukaryotes is lacking. Here, we explore the role of extracellular ATP in cell volume recovery during osmotic swelling in the amoeba Dictyostelium. Release of micromolar ATP could be detected during cell swelling and regulatory cell volume decrease (RVD) phases during hypotonic challenge. Scavenging ATP with apyrase caused profound cell swelling and loss of RVD. Apyrase-induced swelling could be rescued by 100 μM βγ-imidoATP. N-Ethylmalemide (NEM), an inhibitor of vesicular exocytosis, caused heightened cell swelling, loss of RVD, and inhibition of ATP release. Amoebas with impaired contractile vacuole (CV) fusion (drainin knockout [KO] cells) displayed increased swelling but intact ATP release. One hundred micromolar Gd3+ caused cell swelling while blocking any recovery by βγ-imidoATP. ATP release was 4-fold higher in the presence of Gd3+. Cell swelling was associated with an increase in intracellular nitric oxide (NO), with NO-scavenging agents causing cell swelling. Swelling-induced NO production was inhibited by both apyrase and Gd3+, while NO donors rescued apyrase- and Gd3+-induced swelling. These data suggest extracellular ATP released during cell swelling is an important signal that elicits RVD. Though the cell surface receptor for ATP in Dictyostelium remains elusive, we suggest ATP operates through a Gd3+-sensitive receptor that is coupled with intracellular NO production. PMID:26048010

  19. Inhibition of Stat5a/b enhances proteasomal degradation of androgen receptor liganded by antiandrogens in prostate cancer

    PubMed Central

    Hoang, David T.; Gu, Lei; Liao, Zhiyong; Talati, Pooja G.; Shen, Feng; Koptyra, Mateusz; Tan, Shyh-Han; Ellsworth, Elyse; Gupta, Shilpa; Montie, Heather; Dagvadorj, Ayush; Savolainen, Saija; Leiby, Benjamin; Mirtti, Tuomas; Merry, Diane E.; Nevalainen, Marja T.

    2015-01-01

    Although poorly understood, androgen receptor (AR) signaling is sustained despite treatment of prostate cancer with antiandrogens and potentially underlies development of incurable castrate-resistant prostate cancer. However, therapies targeting the AR signaling axis eventually fail when prostate cancer progresses to the castrate-resistant stage. Stat5a/b, a candidate therapeutic target protein in prostate cancer, synergizes with AR to reciprocally enhance signaling of both proteins. In this work, we demonstrate that Stat5a/b sequesters antiandrogen-liganded (MDV3100, Bicalutamide, Flutamide) AR in prostate cancer cells and protects it against proteasomal degradation in prostate cancer. Active Stat5a/b increased nuclear levels of both unliganded and antiandrogen-liganded AR, as demonstrated in prostate cancer cell lines, xenograft tumors and clinical patient-derived prostate cancer samples. Physical interaction between Stat5a/b and AR in prostate cancer cells was mediated by the DNA-binding domain of Stat5a/b and the N-terminal domain of AR. Moreover, active Stat5a/b increased AR occupancy of the Prostate Specific Antigen promoter and AR-regulated gene expression in prostate cancer cells. Mechanistically, both Stat5a/b genetic knockdown and antiandrogen treatment induced proteasomal degradation of AR in prostate cancer cells, with combined inhibition of Stat5a/b and AR leading to maximal loss of AR protein and prostate cancer cell viability. Our results indicate that therapeutic targeting of AR in prostate cancer using antiandrogens may be substantially improved by targeting of Stat5a/b. PMID:25552366

  20. Signaling profiling at the single-cell level identifies a distinct signaling signature in murine hematopoietic stem cells

    PubMed Central

    Du, Juan; Wang, Jinyong; Kong, Guangyao; Jiang, Jing; Zhang, Jingfang; Liu, Yangang; Tong, Wei; Zhang, Jing

    2012-01-01

    Hematopoietic stem cell (HSC) function is tightly regulated by cytokine signaling. Although phospho-flow cytometry allows us to study signaling in defined populations of cells, there has been tremendous hurdle to carry out this study in rare HSCs due to unrecoverable critical HSC markers, low HSC number, and poor cell recovery rate. Here, we overcame these difficulties and developed a “HSC phospho-flow” method to analyze cytokine signaling in murine HSCs at the single-cell level and compare HSC signaling profile to that of multipotent progenitors (MPPs), a cell type immediately downstream of HSCs, and commonly used Lin− cKit+ cells (LK cells, enriched for myeloid progenitors). We chose to study signaling evoked from three representative cytokines, stem cell factor (SCF) and thrombopoietin (TPO) that are essential for HSC function, and granulocyte macrophage-colony stimulating factor (GM-CSF) that is dispensable for HSCs. HSCs display a distinct TPO and GM-CSF signaling signature from MPPs and LK cells, which highly correlates with receptor surface expression. In contrast, although majority of LK cells express lower levels of cKit than HSCs and MPPs, SCF-evoked ERK1/2 activation in LK cells shows a significantly increased magnitude for a prolonged period. These results suggest that specific cellular context plays a more important role than receptor surface expression in SCF signaling. Our study of HSC signaling at the homeostasis stage paves the way to investigate signaling changes in HSCs under conditions of stress, aging, and hematopoietic diseases. PMID:22628264

  1. Activated Stat5 trafficking Via Endothelial Cell-derived Extracellular Vesicles Controls IL-3 Pro-angiogenic Paracrine Action.

    PubMed

    Lombardo, Giusy; Dentelli, Patrizia; Togliatto, Gabriele; Rosso, Arturo; Gili, Maddalena; Gallo, Sara; Deregibus, Maria Chiara; Camussi, Giovanni; Brizzi, Maria Felice

    2016-05-09

    Soluble factors and cell-derived extracellular vesicles (EVs) control vascular cell fate during inflammation. The present study investigates the impact of Interleukin 3 (IL-3) on EV release by endothelial cells (ECs), the mechanisms involved in EV release and paracrine actions. We found that IL-3 increases EV release, which is prevented by IL-3Ralpha blockade. EVs released upon IL-3 stimulation were able to induce pro-angiogenic signals as shown by chromatin immunoprecipitation (ChIP) assay performed on the promoter region of cyclin D1 and tridimensional tube-like structure formation. We herein demonstrate that these effects rely on the transfer of miR-126-3p, pre-miR-126 and, more importantly, of activated signal transduction and activator of transcription 5 (pSTAT5) from IL-3-EV cargo into recipient ECs. We show, using the dominant negative form (ΔN)STAT5 and an activated STAT5 (1*6STAT5) constructs, that STAT5 drives IL-3-mediated EV release, miR-126-3p and pSTAT5 content. Finally, using EVs recovered from ΔNSTAT5 expressing ECs, we provide evidence that miR-126-3p and pSTAT5 trafficking is relevant for IL-3-mediated paracrine pro-angiogenic signals. These results indicate that IL-3 regulates EC-EV release, cargo and IL-3 angiogenic paracrine action via STAT5. Moreover, these results provide evidence that EC-derived IL-3-EVs can serve as pro-angiogenic clinical delivery wound healing devices.

  2. Activated Stat5 trafficking Via Endothelial Cell-derived Extracellular Vesicles Controls IL-3 Pro-angiogenic Paracrine Action

    PubMed Central

    Lombardo, Giusy; Dentelli, Patrizia; Togliatto, Gabriele; Rosso, Arturo; Gili, Maddalena; Gallo, Sara; Deregibus, Maria Chiara; Camussi, Giovanni; Brizzi, Maria Felice

    2016-01-01

    Soluble factors and cell-derived extracellular vesicles (EVs) control vascular cell fate during inflammation. The present study investigates the impact of Interleukin 3 (IL-3) on EV release by endothelial cells (ECs), the mechanisms involved in EV release and paracrine actions. We found that IL-3 increases EV release, which is prevented by IL-3Ralpha blockade. EVs released upon IL-3 stimulation were able to induce pro-angiogenic signals as shown by chromatin immunoprecipitation (ChIP) assay performed on the promoter region of cyclin D1 and tridimensional tube-like structure formation. We herein demonstrate that these effects rely on the transfer of miR-126-3p, pre-miR-126 and, more importantly, of activated signal transduction and activator of transcription 5 (pSTAT5) from IL-3-EV cargo into recipient ECs. We show, using the dominant negative form (ΔN)STAT5 and an activated STAT5 (1*6STAT5) constructs, that STAT5 drives IL-3-mediated EV release, miR-126-3p and pSTAT5 content. Finally, using EVs recovered from ΔNSTAT5 expressing ECs, we provide evidence that miR-126-3p and pSTAT5 trafficking is relevant for IL-3-mediated paracrine pro-angiogenic signals. These results indicate that IL-3 regulates EC-EV release, cargo and IL-3 angiogenic paracrine action via STAT5. Moreover, these results provide evidence that EC-derived IL-3-EVs can serve as pro-angiogenic clinical delivery wound healing devices. PMID:27157262

  3. Regulation of Stat5 by FAK and PAK1 in Oncogenic FLT3- and KIT-Driven Leukemogenesis.

    PubMed

    Chatterjee, Anindya; Ghosh, Joydeep; Ramdas, Baskar; Mali, Raghuveer Singh; Martin, Holly; Kobayashi, Michihiro; Vemula, Sasidhar; Canela, Victor H; Waskow, Emily R; Visconte, Valeria; Tiu, Ramon V; Smith, Catherine C; Shah, Neil; Bunting, Kevin D; Boswell, H Scott; Liu, Yan; Chan, Rebecca J; Kapur, Reuben

    2014-11-20

    Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs), and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription, is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK) whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis.

  4. Validation of a multicolor staining to monitor phosphoSTAT5 levels in regulatory T-cell subsets

    PubMed Central

    Ehx, Grégory; Hannon, Muriel; Beguin, Yves; Humblet-Baron, Stéphanie; Baron, Frédéric

    2015-01-01

    BACKGROUND Regulatory T cells (Tregs) are key players in immune tolerance. They express the transcription factor FOXP3 and are dependent of the STAT5 signaling for their homeostasis. So far, the study of phosphorylated epitopes by flow cytometry required treating the cells with methanol, which is harmful for several epitopes. METHODS Here we assessed whether the PerFix EXPOSE reagent kit (PFE)(Beckman Coulter) allowed monitoring the phosphorylation level of STAT5 in Treg subpopulations together with complex immunophenotyping. Results observed with the PFE kit were compared to those observed without cell permeabilization for surface markers, with paraformaldehyde permeabilization for non-phosphorylated intracellular epitopes, and with methanol-based permeabilization for phosphoSTAT5 staining. RESULTS In human PBMCs, the PFE kit allowed the detection of surface antigens, FOXP3, KI67 and phosphoSTAT5 in similar proportions to what was observed without permeabilization (for surface antigens), or with PFA or methanol permeabilizations for FOXP3/KI67 and phosphoSTAT5, respectively. Comparable observations were made with murine splenocytes. Further, the PFE kit allowed determining the response of different human and murine Treg subsets to IL-2. It also allowed demonstrating that human Treg subsets with the highest levels of phosphoSTAT5 had also the highest suppressive activity in vitro, and that anti-thymocyte glogulin (ATG) induced Treg independently of the STAT5 pathway, both in vitro and in vivo. CONCLUSIONS We have validated a multicolor staining method that allows monitoring phosphoSTAT5 levels in Treg subsets. This staining could be useful to monitor responses of various Treg subsets to IL-2 therapy. PMID:26657728

  5. Miz1 Deficiency in the Mammary Gland Causes a Lactation Defect by Attenuated Stat5 Expression and Phosphorylation

    PubMed Central

    Sanz-Moreno, Adrián; Fuhrmann, David; Wolf, Elmar; von Eyss, Björn; Eilers, Martin; Elsässer, Hans-Peter

    2014-01-01

    Miz1 is a zinc finger transcription factor with an N-terminal POZ domain. Complexes with Myc, Bcl-6 or Gfi-1 repress expression of genes like Cdkn2b (p15Ink4) or Cdkn1a (p21Cip1). The role of Miz1 in normal mammary gland development has not been addressed so far. Conditional knockout of the Miz1 POZ domain in luminal cells during pregnancy caused a lactation defect with a transient reduction of glandular tissue, reduced proliferation and attenuated differentiation. This was recapitulated in vitro using mouse mammary gland derived HC11 cells. Further analysis revealed decreased Stat5 activity in Miz1ΔPOZ mammary glands and an attenuated expression of Stat5 targets. Gene expression of the Prolactin receptor (PrlR) and ErbB4, both critical for Stat5 phosphorylation (pStat5) or pStat5 nuclear translocation, was decreased in Miz1ΔPOZ females. Microarray, ChIP-Seq and gene set enrichment analysis revealed a down-regulation of Miz1 target genes being involved in vesicular transport processes. Our data suggest that deranged intracellular transport and localization of PrlR and ErbB4 disrupt the Stat5 signalling pathway in mutant glands and cause the observed lactation phenotype. PMID:24586582

  6. Constitutive STAT5 Activation Correlates With Better Survival in Cervical Cancer Patients Treated With Radiation Therapy

    SciTech Connect

    Chen, Helen H.W.; Chou, Cheng-Yang; Wu, Yuan-Hua; Hsueh, Wei-Ting; Hsu, Chiung-Hui; Guo, How-Ran; Lee, Wen-Ying; Su, Wu-Chou

    2012-02-01

    Purpose: Constitutively activated signal transducers and activators of transcription (STAT) factors, in particular STAT1, STAT3, and STAT5, have been detected in a wide variety of human primary tumors and have been demonstrated to directly contribute to oncogenesis. However, the expression pattern of these STATs in cervical carcinoma is still unknown, as is whether or not they have prognostic significance. This study investigated the expression patterns of STAT1, STAT3, and STAT5 in cervical cancer and their associations with clinical outcomes in patients treated with radical radiation therapy. Methods and Materials: A total of 165 consecutive patients with International Federation of Gynecology and Obstetrics (FIGO) Stages IB to IVA cervical cancer underwent radical radiation therapy, including external beam and/or high-dose-rate brachytherapy between 1989 and 2002. Immunohistochemical studies of their formalin-fixed, paraffin-embedded tissues were performed. Univariate and multivariate analyses were performed to identify and to evaluate the effects of these factors affecting patient survival. Results: Constitutive activations of STAT1, STAT3, and STAT5 were observed in 11%, 22%, and 61% of the participants, respectively. While STAT5 activation was associated with significantly better metastasis-free survival (p < 0.01) and overall survival (p = 0.04), STAT1 and STAT3 activation were not. Multivariate analyses showed that STAT5 activation, bulky tumor ({>=}4 cm), advanced stage (FIGO Stages III and IV), and brachytherapy (yes vs. no) were independent prognostic factors for cause-specific overall survival. None of the STATs was associated with local relapse. STAT5 activation (odds ratio = 0.29, 95% confidence interval = 0.13-0.63) and advanced stage (odds ratio = 2.54; 95% confidence interval = 1.03-6.26) were independent predictors of distant metastasis. Conclusions: This is the first report to provide the overall expression patterns and prognostic significance of

  7. Single cell analysis of Vibrio harveyi uncovers functional heterogeneity in response to quorum sensing signals

    PubMed Central

    2012-01-01

    Background Vibrio harveyi and closely related species are important pathogens in aquaculture. A complex quorum sensing cascade involving three autoinducers controls bioluminescence and several genes encoding virulence factors. Single cell analysis of a V. harveyi population has already indicated intercellular heterogeneity in the production of bioluminescence. This study was undertaken to analyze the expression of various autoinducer-dependent genes in individual cells. Results Here we used reporter strains bearing promoter::gfp fusions to monitor the induction/repression of three autoinducer-regulated genes in wild type conjugates at the single cell level. Two genes involved in pathogenesis - vhp and vscP, which code for an exoprotease and a component of the type III secretion system, respectively, and luxC (the first gene in the lux operon) were chosen for analysis. The lux operon and the exoprotease gene are induced, while vscP is repressed at high cell density. As controls luxS and recA, whose expression is not dependent on autoinducers, were examined. The responses of the promoter::gfp fusions in individual cells from the same culture ranged from no to high induction. Importantly, simultaneous analysis of two autoinducer induced phenotypes, bioluminescence (light detection) and exoproteolytic activity (fluorescence of a promoter::gfp fusion), in single cells provided evidence for functional heterogeneity within a V. harveyi population. Conclusions Autoinducers are not only an indicator for cell density, but play a pivotal role in the coordination of physiological activities within the population. PMID:22985329

  8. Visual detection of STAT5B gene expression in living cell using the hairpin DNA modified gold nanoparticle beacon.

    PubMed

    Xue, Jianpeng; Shan, Lingling; Chen, Haiyan; Li, Yang; Zhu, Hongyan; Deng, Dawei; Qian, Zhiyu; Achilefu, Samuel; Gu, Yueqing

    2013-03-15

    Signal transducer and activator of transcription 5B (STAT5B) is an important protein in JAK-STAT signaling pathway that is responsible for the metastasis and proliferation of tumor cells. Determination of the STAT5B messenger Ribonucleic Acid (mRNA) relating to the STAT5B expression provides insight into the mechanism of tumor progression. In this study, we designed and used a special hairpin deoxyribonucleic acid (DNA) for human STAT5B mRNA to functionalize gold nanoparticles, which served as a beacon for detecting human STAT5B expression. Up to 90% quenching efficiency was achieved. Upon hybridizing with the target mRNA, the hairpin DNA modified gold nanoparticle beacons (hDAuNP beacons) release the fluorophores attached at 5' end of the oligonucleotide sequence. The fluorescence properties of the beacon before and after the hybridization with the complementary DNA were confirmed in vitro. The stability of hDAuNP beacons against degradation by DNase I and GSH indicated that the prepared beacon is stable inside cells. The detected fluorescence in MCF-7 cancer cells correlates with the specific STAT5B mRNA expression, which is consistent with the result from PCR measurement. Fluorescence microscopy showed that the hDAuNP beacons internalized in cells without using transfection agents, with intracellular distribution in the cytoplasm rather than the nucleus. The results demonstrated that this beacon could directly provide quantitative measurement of the intracellular STAT5B mRNA in living cells. Compared to the previous approaches, this beacon has advantages of higher target to background ratio of detection and an increased resistance to nuclease degradation. The strategy reported in this study is a promising approach for the intracellular measurement of RNA or protein expression in living cells, and has great potential in the study of drug screening and discovery.

  9. Single-Cell Measurements of IgE-Mediated FcεRI Signaling Using an Integrated Microfluidic Platform

    SciTech Connect

    Liu, Yanli; Barua, Dipak; Liu, Peng; Wilson, Bridget S.; Oliver, Janet M.; Hlavacek, William S.; Singh, Anup K.

    2013-03-27

    Heterogeneity in responses of cells to a stimulus, such as a pathogen or allergen, can potentially play an important role in deciding the fate of the responding cell population and the overall systemic response. Measuring heterogeneous responses requires tools capable of interrogating individual cells. Cell signaling studies commonly do not have single-cell resolution because of the limitations of techniques used such as Westerns, ELISAs, mass spectrometry, and DNA microarrays. Microfluidics devices are increasingly being used to overcome these limitations. In this paper, we report on a microfluidic platform for cell signaling analysis that combines two orthogonal single-cell measurement technologies: on-chip flow cytometry and optical imaging. The device seamlessly integrates cell culture, stimulation, and preparation with downstream measurements permitting hands-free, automated analysis to minimize experimental variability. The platform was used to interrogate IgE receptor (FcεRI) signaling, which is responsible for triggering allergic reactions, in RBL-2H3 cells. Following on-chip crosslinking of IgE-FcεRI complexes by multivalent antigen, we monitored signaling events including protein phosphorylation, calcium mobilization and the release of inflammatory mediators. The results demonstrate the ability of our platform to produce quantitative measurements on a cell-by-cell basis from just a few hundred cells. Finally, model-based analysis of the Syk phosphorylation data suggests that heterogeneity in Syk phosphorylation can be attributed to protein copy number variations, with the level of Syk phosphorylation being particularly sensitive to the copy number of Lyn.

  10. Single-Cell Measurements of IgE-Mediated FcεRI Signaling Using an Integrated Microfluidic Platform

    PubMed Central

    Liu, Yanli; Barua, Dipak; Liu, Peng; Wilson, Bridget S.; Oliver, Janet M.; Hlavacek, William S.; Singh, Anup K.

    2013-01-01

    Heterogeneity in responses of cells to a stimulus, such as a pathogen or allergen, can potentially play an important role in deciding the fate of the responding cell population and the overall systemic response. Measuring heterogeneous responses requires tools capable of interrogating individual cells. Cell signaling studies commonly do not have single-cell resolution because of the limitations of techniques used such as Westerns, ELISAs, mass spectrometry, and DNA microarrays. Microfluidics devices are increasingly being used to overcome these limitations. Here, we report on a microfluidic platform for cell signaling analysis that combines two orthogonal single-cell measurement technologies: on-chip flow cytometry and optical imaging. The device seamlessly integrates cell culture, stimulation, and preparation with downstream measurements permitting hands-free, automated analysis to minimize experimental variability. The platform was used to interrogate IgE receptor (FcεRI) signaling, which is responsible for triggering allergic reactions, in RBL-2H3 cells. Following on-chip crosslinking of IgE-FcεRI complexes by multivalent antigen, we monitored signaling events including protein phosphorylation, calcium mobilization and the release of inflammatory mediators. The results demonstrate the ability of our platform to produce quantitative measurements on a cell-by-cell basis from just a few hundred cells. Model-based analysis of the Syk phosphorylation data suggests that heterogeneity in Syk phosphorylation can be attributed to protein copy number variations, with the level of Syk phosphorylation being particularly sensitive to the copy number of Lyn. PMID:23544131

  11. Resolution of Conflicting Signals at the Single-Cell Level in the Regulation of Cyanobacterial Photosynthesis and Nitrogen Fixation

    PubMed Central

    Mohr, Wiebke; Vagner, Tomas; Kuypers, Marcel M. M.; Ackermann, Martin; LaRoche, Julie

    2013-01-01

    Unicellular, diazotrophic cyanobacteria temporally separate dinitrogen (N2) fixation and photosynthesis to prevent inactivation of the nitrogenase by oxygen. This temporal segregation is regulated by a circadian clock with oscillating activities of N2 fixation in the dark and photosynthesis in the light. On the population level, this separation is not always complete, since the two processes can overlap during transitions from dark to light. How do single cells avoid inactivation of nitrogenase during these periods? One possibility is that phenotypic heterogeneity in populations leads to segregation of the two processes. Here, we measured N2 fixation and photosynthesis of individual cells using nanometer-scale secondary ion mass spectrometry (nanoSIMS) to assess both processes in a culture of the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii during a dark-light and a continuous light phase. We compared single-cell rates with bulk rates and gene expression profiles. During the regular dark and light phases, C. watsonii exhibited the temporal segregation of N2 fixation and photosynthesis commonly observed. However, N2 fixation and photosynthesis were concurrently measurable at the population level during the subjective dark phase in which cells were kept in the light rather than returned to the expected dark phase. At the single-cell level, though, cells discriminated against either one of the two processes. Cells that showed high levels of photosynthesis had low nitrogen fixing activities, and vice versa. These results suggest that, under ambiguous environmental signals, single cells discriminate against either photosynthesis or nitrogen fixation, and thereby might reduce costs associated with running incompatible processes in the same cell. PMID:23805199

  12. STAT5B mutations in heterozygous state have negative impact on height: another clue in human stature heritability

    PubMed Central

    Scalco, Renata C; Hwa, Vivian; Domené, Horacio M.; Jasper, Héctor G.; Belgorosky, Alicia; Marino, Roxana; Pereira, Alberto M.; Tonelli, Carlos A.; Wit, Jan M.; Rosenfeld, Ron G.; Jorge, Alexander A.L.

    2016-01-01

    Context and objective Growth hormone insensitivity with immune dysfunction caused by signal transducer and activator of transcription 5B (STAT5B) mutations is an autosomal recessive condition. Heterozygous mutations in other genes involved in growth regulation were previously associated with a mild height reduction. Our objective was to assess for the first time the phenotype of heterozygous STAT5B mutations. Methods We genotyped and performed clinical and laboratorial evaluations in 52 relatives of 2 previously described Brazilian brothers with homozygous STAT5B c.424_427del mutation (21 heterozygous). Additionally, we obtained height data and genotype from 1,104 adult control individuals from the same region in Brazil and identified 5 additional families harboring the same mutation (18 individuals, 11 heterozygous). Furthermore, we gathered the available height data from first-degree relatives of patients with homozygous STAT5B mutations (17 individuals from 7 families). Data from heterozygous individuals and non-carriers were compared. Results Individuals carrying heterozygous STAT5B c.424_427del mutation were 0.6 SDS shorter than their non-carrier relatives (p= 0.009). Heterozygous subjects also had significantly lower SDS for serum concentrations of IGF-1 (p=0.028) and IGFBP-3 (p=0.02) than their non-carrier relatives. The 17 heterozygous first-degree relatives of patients carrying homozygous STAT5B mutations had an average height SDS of −1.4 ± 0.8 when compared with population-matched controls (p < 0.001). Conclusions STAT5B mutations in heterozygous state have a significant negative impact on height (approximately 3.9 cm). This effect is milder than the effect seen in the homozygous state, with height usually within the normal range. Our results support the hypothesis that heterozygosity of rare pathogenic variants contributes to normal height heritability. PMID:26034074

  13. Prolactin/Stat5 and androgen R1881 coactivate carboxypeptidase-D gene in breast cancer cells.

    PubMed

    Koirala, Samir; Thomas, Lynn N; Too, Catherine K L

    2014-03-01

    Plasma membrane-bound carboxypeptidase-D (CPD) cleaves C-terminal arginine from extracellular substrates. In the cell, arginine is converted to nitric oxide (NO). We have reported that up-regulation of CPD mRNA/protein levels by 17β-estradiol and prolactin (PRL) in breast cancer cells, and by testosterone in prostate cancer cells, increased NO production and cell survival. The CPD promoter contains a consensus γ-interferon-activated sequence (GAS) and 3 putative androgen response elements (ARE.1, ARE.2, ARE.3) that could potentially bind PRL-activated transcription factor Stat5 (signal transducer and activator of transcription 5) and the liganded androgen receptor (AR), respectively. This study showed that synthetic androgen R1881 and PRL elevated CPD mRNA/protein levels in human MCF-7 and T47D breast cancer cells in a time-/dose-dependent manner. PRL/R1881-elevated CPD expression was blocked by actinomycin-D, and a CPD promoter construct containing these GAS and AREs was stimulated by PRL or R1881, indicating transcriptional regulation by both hormones. Luciferase reporter assays showed that GAS and the adjacent ARE.1 only were active. Mutation of GAS in the ΔGAS-CPD construct (ARE.1 intact) abolished CPD promoter activity in response to PRL and, surprisingly, to R1881 as well. ΔGAS-CPD promoter activity was restored by PRL+R1881 in combination, and enhanced by ectopic Stat5, but abolished by Stat5 gene knockdown. Chromatin immunoprecipitation analysis confirmed binding of activated Stat5 and liganded AR to GAS and ARE.1, respectively. Activated Stat5 also induced binding of unliganded AR to ARE.1, and liganded AR induced binding of unactivated Stat5 to GAS. In summary, PRL and R1881, acting through Stat5 and AR, act cooperatively to stimulate CPD gene transcription in breast cancer cells. PMID:24433040

  14. Prolactin/Stat5 and androgen R1881 coactivate carboxypeptidase-D gene in breast cancer cells.

    PubMed

    Koirala, Samir; Thomas, Lynn N; Too, Catherine K L

    2014-03-01

    Plasma membrane-bound carboxypeptidase-D (CPD) cleaves C-terminal arginine from extracellular substrates. In the cell, arginine is converted to nitric oxide (NO). We have reported that up-regulation of CPD mRNA/protein levels by 17β-estradiol and prolactin (PRL) in breast cancer cells, and by testosterone in prostate cancer cells, increased NO production and cell survival. The CPD promoter contains a consensus γ-interferon-activated sequence (GAS) and 3 putative androgen response elements (ARE.1, ARE.2, ARE.3) that could potentially bind PRL-activated transcription factor Stat5 (signal transducer and activator of transcription 5) and the liganded androgen receptor (AR), respectively. This study showed that synthetic androgen R1881 and PRL elevated CPD mRNA/protein levels in human MCF-7 and T47D breast cancer cells in a time-/dose-dependent manner. PRL/R1881-elevated CPD expression was blocked by actinomycin-D, and a CPD promoter construct containing these GAS and AREs was stimulated by PRL or R1881, indicating transcriptional regulation by both hormones. Luciferase reporter assays showed that GAS and the adjacent ARE.1 only were active. Mutation of GAS in the ΔGAS-CPD construct (ARE.1 intact) abolished CPD promoter activity in response to PRL and, surprisingly, to R1881 as well. ΔGAS-CPD promoter activity was restored by PRL+R1881 in combination, and enhanced by ectopic Stat5, but abolished by Stat5 gene knockdown. Chromatin immunoprecipitation analysis confirmed binding of activated Stat5 and liganded AR to GAS and ARE.1, respectively. Activated Stat5 also induced binding of unliganded AR to ARE.1, and liganded AR induced binding of unactivated Stat5 to GAS. In summary, PRL and R1881, acting through Stat5 and AR, act cooperatively to stimulate CPD gene transcription in breast cancer cells.

  15. A microfluidic platform for systems pathology: multiparameter single-cell signaling measurements of clinical brain tumor specimens.

    PubMed

    Sun, Jing; Masterman-Smith, Michael D; Graham, Nicholas A; Jiao, Jing; Mottahedeh, Jack; Laks, Dan R; Ohashi, Minori; DeJesus, Jason; Kamei, Ken-ichiro; Lee, Ki-Bum; Wang, Hao; Yu, Zeta T F; Lu, Yi-Tsung; Hou, Shuang; Li, Keyu; Liu, Max; Zhang, Nangang; Wang, Shutao; Angenieux, Brigitte; Panosyan, Eduard; Samuels, Eric R; Park, Jun; Williams, Dirk; Konkankit, Vera; Nathanson, David; van Dam, R Michael; Phelps, Michael E; Wu, Hong; Liau, Linda M; Mischel, Paul S; Lazareff, Jorge A; Kornblum, Harley I; Yong, William H; Graeber, Thomas G; Tseng, Hsian-Rong

    2010-08-01

    The clinical practice of oncology is being transformed by molecular diagnostics that will enable predictive and personalized medicine. Current technologies for quantitation of the cancer proteome are either qualitative (e.g., immunohistochemistry) or require large sample sizes (e.g., flow cytometry). Here, we report a microfluidic platform-microfluidic image cytometry (MIC)-capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 1,000 to 2,800 cells. Using cultured cell lines, we show simultaneous measurement of four critical signaling proteins (EGFR, PTEN, phospho-Akt, and phospho-S6) within the oncogenic phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. To show the clinical application of the MIC platform to solid tumors, we analyzed a panel of 19 human brain tumor biopsies, including glioblastomas. Our MIC measurements were validated by clinical immunohistochemistry and confirmed the striking intertumoral and intratumoral heterogeneity characteristic of glioblastoma. To interpret the multiparameter, single-cell MIC measurements, we adapted bioinformatic methods including self-organizing maps that stratify patients into clusters that predict tumor progression and patient survival. Together with bioinformatic analysis, the MIC platform represents a robust, enabling in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine. PMID:20631065

  16. A microfluidic platform for systems pathology: multiparameter single-cell signaling measurements of clinical brain tumor specimens

    PubMed Central

    Sun, Jing; Masterman-Smith, Michael; Graham, Nicholas A.; Jiao, Jing; Mottahedeh, Jack; Laks, Dan R.; Ohashi, Minori; DeJesus, Jason; Kamei, Ken-ichiro; Lee, Ki-Bum; Wang, Hao; Yu, Zeta T.F.; Lu, Yi-Tsung; Hou, Shuang; Li, Keyu; Liu, Max; Zhang, Nangang; Wang, Shutao; Angenieux, Brigitte; Panosyan, Eduard; Samuels, Eric R.; Park, Jun; Williams, Dirk; Konkankit, Vera; Nathanson, David; van Dam, R. Michael; Phelps, Michael E.; Wu, Hong; Liau, Linda M.; Mischel, Paul S.; Lazareff, Jorge A.; Kornblum, Harley I.; Yong, William H.; Graeber, Thomas G.; Tseng, Hsian-Rong

    2011-01-01

    The clinical practice of oncology is being transformed by molecular diagnostics that will enable predictive and personalized medicine. Current technologies for quantitation of the cancer proteome are either qualitative (e.g., immunohistochemistry) or require large sample sizes (e.g., flow cytometry). Here, we report a microfluidic platform, Microfluidic Image Cytometry (MIC), capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 1,000-2,800 cells. Using cultured cell lines, we demonstrate simultaneous measurement of four critical signaling proteins (EGFR, PTEN, phospho-Akt and phospho-S6) within the oncogenic PI3K/Akt/mTOR signaling pathway. To demonstrate the clinical application of the MIC platform to solid tumors, we analyzed a panel of 19 human brain tumor biopsies, including glioblastomas. Our MIC measurements were validated by clinical immunohistochemistry and confirmed the striking inter- and intra-tumoral heterogeneity characteristic of glioblastoma. To interpret the multiparameter, single-cell MIC measurements, we adapted bioinformatic methods including self-organizing maps that stratify patients into clusters which predict tumor progression and patient survival. Together with bioinformatic analysis, the MIC platform represents a robust, enabling in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine. PMID:20631065

  17. A microfluidic platform for systems pathology: multiparameter single-cell signaling measurements of clinical brain tumor specimens.

    PubMed

    Sun, Jing; Masterman-Smith, Michael D; Graham, Nicholas A; Jiao, Jing; Mottahedeh, Jack; Laks, Dan R; Ohashi, Minori; DeJesus, Jason; Kamei, Ken-ichiro; Lee, Ki-Bum; Wang, Hao; Yu, Zeta T F; Lu, Yi-Tsung; Hou, Shuang; Li, Keyu; Liu, Max; Zhang, Nangang; Wang, Shutao; Angenieux, Brigitte; Panosyan, Eduard; Samuels, Eric R; Park, Jun; Williams, Dirk; Konkankit, Vera; Nathanson, David; van Dam, R Michael; Phelps, Michael E; Wu, Hong; Liau, Linda M; Mischel, Paul S; Lazareff, Jorge A; Kornblum, Harley I; Yong, William H; Graeber, Thomas G; Tseng, Hsian-Rong

    2010-08-01

    The clinical practice of oncology is being transformed by molecular diagnostics that will enable predictive and personalized medicine. Current technologies for quantitation of the cancer proteome are either qualitative (e.g., immunohistochemistry) or require large sample sizes (e.g., flow cytometry). Here, we report a microfluidic platform-microfluidic image cytometry (MIC)-capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 1,000 to 2,800 cells. Using cultured cell lines, we show simultaneous measurement of four critical signaling proteins (EGFR, PTEN, phospho-Akt, and phospho-S6) within the oncogenic phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. To show the clinical application of the MIC platform to solid tumors, we analyzed a panel of 19 human brain tumor biopsies, including glioblastomas. Our MIC measurements were validated by clinical immunohistochemistry and confirmed the striking intertumoral and intratumoral heterogeneity characteristic of glioblastoma. To interpret the multiparameter, single-cell MIC measurements, we adapted bioinformatic methods including self-organizing maps that stratify patients into clusters that predict tumor progression and patient survival. Together with bioinformatic analysis, the MIC platform represents a robust, enabling in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine.

  18. Clarifying the role of Stat5 in lymphoid development and Abelson-induced transformation

    PubMed Central

    Hoelbl, Andrea; Kovacic, Boris; Kerenyi, Marc A.; Simma, Olivia; Warsch, Wolfgang; Cui, Yongzhi; Beug, Hartmut; Hennighausen, Lothar; Moriggl, Richard; Sexl, Veronika

    2010-01-01

    The Stat5 transcription factors Stat5a and Stat5b have been implicated in lymphoid development and transformation. Most studies have employed Stat5a/b-deficient mice where gene targeting disrupted the first protein-coding exon, resulting in the expression of N-terminally truncated forms of Stat5a/b (Stat5a/bΔN/ΔN mice). We have now reanalyzed lymphoid development in Stat5a/bnull/null mice having a complete deletion of the Stat5a/b gene locus. The few surviving Stat5a/bnull/null mice lacked CD8+ T lymphocytes. A massive reduction of CD8+ T cells was also found in Stat5a/bfl/fl lck-cre transgenic animals. While γδ T-cell receptor–positive (γδTCR+) cells were expressed at normal levels in Stat5a/bΔN/ΔN mice, they were completely absent in Stat5a/bnull/null animals. Moreover, B-cell maturation was abrogated at the pre–pro-B-cell stage in Stat5a/bnull/null mice, whereas Stat5a/bΔN/ΔN B-lymphoid cells developed to the early pro-B-cell stage. In vitro assays using fetal liver-cell cultures confirmed this observation. Most strikingly, Stat5a/bnull/null cells were resistant to transformation and leukemia development induced by Abelson oncogenes, whereas Stat5a/bΔN/ΔN-derived cells readily transformed. These findings show distinct lymphoid defects for Stat5a/bΔN/ΔN and Stat5a/bnull/null mice and define a novel functional role for the N-termini of Stat5a/b in B-lymphoid transformation. PMID:16493008

  19. Stat5 is critical for the development and maintenance of myeloproliferative neoplasm initiated by Nf1 deficiency

    PubMed Central

    Sachs, Zohar; Been, Raha A.; DeCoursin, Krista J.; Nguyen, Hanh T.; Mohd Hassan, Nurul A.; Noble-Orcutt, Klara E.; Eckfeldt, Craig E.; Pomeroy, Emily J.; Diaz-Flores, Ernesto; Geurts, Jennifer L.; Diers, Miechaleen D.; Hasz, Diane E.; Morgan, Kelly J.; MacMillan, Margaret L.; Shannon, Kevin M.; Largaespada, David A.; Wiesner, Stephen M.

    2016-01-01

    Juvenile myelomonocytic leukemia is a rare myeloproliferative neoplasm characterized by hyperactive RAS signaling. Neurofibromin1 (encoded by the NF1 gene) is a negative regulator of RAS activation. Patients with neurofibromatosis type 1 harbor loss-of-function mutations in NF1 and have a 200- to 500-fold increased risk of juvenile myelomonocytic leukemia. Leukemia cells from patients with juvenile myelomonocytic leukemia display hypersensitivity to certain cytokines, such as granulocyte-macrophage colony-stimulating factor. The granulocyte-macrophage colony-stimulating factor receptor utilizes pre-associated JAK2 to initiate signals after ligand binding. JAK2 subsequently activates STAT5, among other downstream effectors. Although STAT5 is gaining recognition as an important mediator of growth factor signaling in myeloid leukemias, the contribution of STAT5 to the development of hyperactive RAS-initiated myeloproliferative disease has not been well described. In this study, we investigated the consequence of STAT5 attenuation via genetic and pharmacological approaches in Nf1-deficient murine models of juvenile myelomonocytic leukemia. We found that homozygous Stat5 deficiency extended the lifespan of Nf1-deficient mice and eliminated the development of myeloproliferative neoplasm associated with Nf1 gene loss. Likewise, we found that JAK inhibition with ruxolitinib attenuated myeloproliferative neoplasm in Nf1-deficient mice. Finally, we found that primary cells from a patient with KRAS-mutant juvenile myelomonocytic leukemia displayed reduced colony formation in response to JAK2 inhibition. Our findings establish a central role for STAT5 activation in the pathogenesis of juvenile myelomonocytic leukemia and suggest that targeting this pathway may be of clinical utility in these patients. PMID:27418650

  20. Single-cell variation leads to population invariance in NF-κB signaling dynamics

    PubMed Central

    Hughey, Jacob J.; Gutschow, Miriam V.; Bajar, Bryce T.; Covert, Markus W.

    2015-01-01

    The activation dynamics of nuclear factor (NF)-κB have been shown to affect downstream gene expression. On activation, NF-κB shuttles back and forth across the nuclear envelope. Many dynamic features of this shuttling have been characterized, and most features vary significantly with respect to ligand type and concentration. Here, we report an invariant feature with regard to NF-κB dynamics in cellular populations: the distribution—the average, as well as the variance—of the time between two nuclear entries (the period). We find that this period is conserved, regardless of concentration and across several different ligands. Intriguingly, the distributions observed at the population level are not observed in individual cells over 20-h time courses. Instead, the average period of NF-κB nuclear translocation varies considerably among single cells, and the variance is much smaller within a cell than that of the population. Finally, analysis of daughter-cell pairs and isogenic populations indicates that the dynamics of the NF-κB response is heritable but diverges over multiple divisions, on the time scale of weeks to months. These observations are contrary to the existing theory of NF-κB dynamics and suggest an additional level of control that regulates the overall distribution of translocation timing at the population level. PMID:25473117

  1. Single-Cell Measurements of IgE-Mediated FcεRI Signaling Using an Integrated Microfluidic Platform

    DOE PAGES

    Liu, Yanli; Barua, Dipak; Liu, Peng; Wilson, Bridget S.; Oliver, Janet M.; Hlavacek, William S.; Singh, Anup K.

    2013-03-27

    Heterogeneity in responses of cells to a stimulus, such as a pathogen or allergen, can potentially play an important role in deciding the fate of the responding cell population and the overall systemic response. Measuring heterogeneous responses requires tools capable of interrogating individual cells. Cell signaling studies commonly do not have single-cell resolution because of the limitations of techniques used such as Westerns, ELISAs, mass spectrometry, and DNA microarrays. Microfluidics devices are increasingly being used to overcome these limitations. In this paper, we report on a microfluidic platform for cell signaling analysis that combines two orthogonal single-cell measurement technologies: on-chipmore » flow cytometry and optical imaging. The device seamlessly integrates cell culture, stimulation, and preparation with downstream measurements permitting hands-free, automated analysis to minimize experimental variability. The platform was used to interrogate IgE receptor (FcεRI) signaling, which is responsible for triggering allergic reactions, in RBL-2H3 cells. Following on-chip crosslinking of IgE-FcεRI complexes by multivalent antigen, we monitored signaling events including protein phosphorylation, calcium mobilization and the release of inflammatory mediators. The results demonstrate the ability of our platform to produce quantitative measurements on a cell-by-cell basis from just a few hundred cells. Finally, model-based analysis of the Syk phosphorylation data suggests that heterogeneity in Syk phosphorylation can be attributed to protein copy number variations, with the level of Syk phosphorylation being particularly sensitive to the copy number of Lyn.« less

  2. IL-15 prolongs CD154 expression on human CD4 T cells via STAT5 binding to the CD154 transcriptional promoter

    PubMed Central

    Lowe, RM; Genin, A; Orgun, N; Cron, RQ

    2014-01-01

    Activation induced CD154 expression on CD4 T cells is prolonged in systemic lupus erythematosus but the mechanism(s) for its dysregulation are unknown. The studies reported herein demonstrate that IL-15 is capable of prolonging CD154 expression on PHA activated CD4 T cells. Since IL-15 signals through STAT5, predicted STAT5 binding sites in the human CD154 transcriptional promoter were identified, and STAT5 binding to the proximal CD154 promoter in vitro and in vivo following primary CD4 T cell activation was demonstrated. Moreover, overexpression of wild-type(WT) STAT5 in primary human CD4 T cells augmented CD154 transcription, whereas overexpression of a dominant negative (DN) STAT5 protein inhibited CD154 transcription. Mutation of the most proximal STAT5 binding site in the CD154 promoter resulted in diminished DNA binding and reduced CD154 transcriptional activity. Interestingly, STAT5-specific siRNA inhibited CD154 surface expression at 48 but not 24 hours after T cell activation. Thus, these findings provide some of the first evidence to support a possible mechanistic link to explain how the overexpression of IL-15 observed in lupus patients may be involved in the prolonged expression of CD154 that has also been observed on lupus CD4 T cells. PMID:24500400

  3. Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma.

    PubMed

    Ramos, Grasieli de Oliveira; Bernardi, Lisiane; Lauxen, Isabel; Sant'Ana Filho, Manoel; Horwitz, Alan Rick; Lamers, Marcelo Lazzaron

    2016-01-01

    Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization. PMID:26978651

  4. Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma

    PubMed Central

    Ramos, Grasieli de Oliveira; Bernardi, Lisiane; Lauxen, Isabel; Sant’Ana Filho, Manoel; Horwitz, Alan Rick; Lamers, Marcelo Lazzaron

    2016-01-01

    Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization. PMID:26978651

  5. Label-Free Imaging of Dynamic and Transient Calcium Signaling in Single Cells.

    PubMed

    Lu, Jin; Li, Jinghong

    2015-11-01

    Cell signaling consists of diverse events that occur at various temporal and spatial scales, ranging from milliseconds to hours and from single biomolecules to cell populations. The pathway complexities require the development of new techniques that detect the overall signaling activities and are not limited to quantifying a single event. A plasmonic-based electrochemical impedance microscope (P-EIM) that can provide such data with excellent temporal and spatial resolution and does not require the addition of any labels for detection has now been developed. The highly dynamic and transient calcium signaling activities at the early stage of G-protein-coupled receptor (GPCR) stimulation were thus studied. It could be shown that a subpopulation of cells is more responsive towards agonist stimulation, and the heterogeneity of the local distributions and the transient activities of the ion channels during agonist-activated calcium flux in single HeLa cells were investigated.

  6. Single-Cell E. coli Response to an Instantaneously Applied Chemotactic Signal

    PubMed Central

    Sagawa, Takashi; Kikuchi, Yu; Inoue, Yuichi; Takahashi, Hiroto; Muraoka, Takahiro; Kinbara, Kazushi; Ishijima, Akihiko; Fukuoka, Hajime

    2014-01-01

    In response to an attractant or repellant, an Escherichia coli cell controls the rotational direction of its flagellar motor by a chemotaxis system. When an E. coli cell senses an attractant, a reduction in the intracellular concentration of a chemotaxis protein, phosphorylated CheY (CheY-P), induces counterclockwise (CCW) rotation of the flagellar motor, and this cellular response is thought to occur in several hundred milliseconds. Here, to measure the signaling process occurring inside a single E. coli cell, including the recognition of an attractant by a receptor cluster, the inactivation of histidine kinase CheA, and the diffusion of CheY and CheY-P molecules, we applied a serine stimulus by instantaneous photorelease from a caged compound and examined the cellular response at a temporal resolution of several hundred microseconds. We quantified the clockwise (CW) and CCW durations immediately after the photorelease of serine as the response time and the duration of the response, respectively. The results showed that the response time depended on the distance between the receptor and motor, indicating that the decreased CheY-P concentration induced by serine propagates through the cytoplasm from the receptor-kinase cluster toward the motor with a timing that is explained by the diffusion of CheY and CheY-P molecules. The response time included 240 ms for enzymatic reactions in addition to the time required for diffusion of the signaling molecule. The measured response time and duration of the response also revealed that the E. coli cell senses a similar serine concentration regardless of whether the serine concentration is increasing or decreasing. These detailed quantitative findings increase our understanding of the signal transduction process that occurs inside cells during bacterial chemotaxis. PMID:25099812

  7. Truncated pStat5B is associated with the Idd4 locus in NOD mice

    SciTech Connect

    Davoodi-Semiromi, Abdoreza . E-mail: semiromi@pathology.ufl.edu; McDuffie, Marcia; Litherland, Sally; Clare-Salzler, Michael

    2007-05-11

    We investigate JAK-STAT5 activation and its relationship to full-length Stat5B (FL-Stat5) and constitutive phosphorylated carboxy-truncated Stat5B (ct-pStat5) in four different strains of mouse. Our electrophoresis mobility shift assays data indicate constitutive phosphorylation of full-length-Stat5 (p < 0.001) and DNA binding in NOD but not in B6 mice. Our data suggest that the relative ratio of FL-Stat5: ct-Stat5 in NOD is 5- to 8-fold lower (p < 0.0001) when compared with normal B6 mice. Additionally, EMSAs data from B6.NOD/c11 suggest contribution of Idd4 susceptibility locus on chromosome 11 in constitutive phosphorylation of Stat5 in NOD mice. The presence of ct-pStat5 in regulatory T cells of NOD mice suggests this form of Stat5 is associated with impaired function of Tregs in NOD mouse. In agreement with our previous report the JAK-Stat5B defective pathway in NOD mice along with other defective factors is associated with the pathogenesis of autoimmune type 1 diabetes in NOD mice.

  8. Reduced Levels of Hspa9 Attenuates Stat5 Activation in Mouse B-cells

    PubMed Central

    Krysiak, Kilannin; Tibbitts, Justin F.; Shao, Jin; Liu, Tuoen; Ndonwi, Matthew; Walter, Matthew J.

    2014-01-01

    HSPA9 is located on chromosome 5q31.2 in humans, a region that is commonly deleted in patients with myeloid malignancies [del(5q)], including myelodysplastic syndromes (MDS). HSPA9 expression is reduced by 50% in patients with del(5q)-associated MDS, consistent with haploinsufficient levels. Zebrafish mutants and knockdown studies in human and mouse cells have implicated a role for HSPA9 in hematopoiesis. To comprehensively evaluate the effects of Hspa9 haploinsufficiency on hematopoiesis, we generated an Hspa9 knockout mouse model. While homozygous knockout of Hspa9 is embryonic lethal, mice with heterozygous deletion of Hspa9 (Hspa9+/−) are viable and have a 50% reduction in Hspa9 expression. Hspa9+/− mice have normal basal hematopoiesis and do not develop MDS. However, Hspa9+/− mice have a cell- intrinsic reduction in bone marrow CFU-PreB colony formation without alterations in the number of B-cell progenitors in vivo, consistent with a functional defect in Hspa9+/− B-cell progenitors. We further reduced Hspa9 expression (<50%) using RNAi and observe reduced B-cell progenitors in vivo, indicating that appropriate levels (≥50%) of Hspa9 are required for normal B- lymphopoiesis in vivo. Knockdown of Hspa9 in an IL-7 dependent mouse B-cell line reduced Stat5 phosphorylation following IL-7 receptor stimulation, supporting a role for Hspa9 in Stat5 signaling in B-cells. Collectively, these data implicate a role for Hspa9 in B-lymphopoiesis and Stat5 activation downstream of IL-7 signaling. PMID:25550197

  9. Mechanics governs single-cell signaling and multi-cell robustness in biofilm infections

    NASA Astrophysics Data System (ADS)

    Gordon, Vernita

    In biofilms, bacteria and other microbes are embedded in extracellular polymers (EPS). Multiple types of EPS can be produced by a single bacterial strain - the reasons for this redundancy are not well-understood. Our work suggests that different polymers may confer distinct mechanical benefits. Our model organism is Pseudomonas aeruginosa, an opportunistic human pathogen that forms chronic biofilm infections associated with increased antibiotic resistance and evasion of the immune defense. Biofilms initiate when bacteria attach to a surface, sense the surface, and change their gene expression. Changes in gene expression are regulated by a chemical signal, cyclic-di-GMP. We find that one EPS material, called ``PEL,'' enhances surface sensing by increasing mechanical coupling of single bacteria to the surface. Measurements of bacterial motility suggest that PEL may increase frictional interactions between the surface and the bacteria. Consistent with this, we show that bacteria increase cyclic-di-GMP signaling in response to mechanical shear stress. Mechanosensing has long been known to be important to the function of cells in higher eukaryotes, but this is one of only a handful of studies showing that bacteria can sense and respond to mechanical forces. For the mature biofilm, the embedding polymer matrix can protect bacteria both chemically and mechanically. P. aeruginosa infections in the cystic fibrosis (CF) lung often last for decades, ample time for the infecting strain(s) to evolve. Production of another EPS material, alginate, is well-known to tend to increase over time in CF infections. Alginate chemically protects biofilms, but also makes them softer and weaker. Recently, it is being increasingly recognized that bacteria in chronic CF infections also evolve to increase PSL production. We use oscillatory bulk rheology to determine the unique contributions of EPS materials to biofilm mechanics. Unlike alginate, increased PSL stiffens biofilms. Increasing both

  10. Mechanical signals in plant development: a new method for single cell studies

    NASA Technical Reports Server (NTRS)

    Lynch, T. M.; Lintilhac, P. M.

    1997-01-01

    Cell division, which is critical to plant development and morphology, requires the orchestration of hundreds of intracellular processes. In the end, however, cells must make critical decisions, based on a discrete set of mechanical signals such as stress, strain, and shear, to divide in such a way that they will survive the mechanical loads generated by turgor pressure and cell enlargement within the growing tissues. Here we report on a method whereby tobacco protoplasts swirled into a 1.5% agarose entrapment medium will survive and divide. The application of a controlled mechanical load to agarose blocks containing protoplasts orients the primary division plane of the embedded cells. Photoelastic analysis of the agarose entrapment medium can identify the lines of principal stress within the agarose, confirming the hypothesis that cells divide either parallel or perpendicular to the principal stress tensors. The coincidence between the orientation of the new division wall and the orientation of the principal stress tensors suggests that the perception of mechanical stress is a characteristic of individual plant cells. The ability of a cell to determine a shear-free orientation for a new partition wall may be related to the applied load through the deformation of the matrix material. In an isotropic matrix a uniaxial load will produce a rotationally symmetric strain field, which will define a shear-free plane. Where high stress intensities combine with the loading geometry to produce multiaxial loads there will be no axis of rotational symmetry and hence no shear free plane. This suggests that two mechanisms may be orienting the division plane, one a mechanism that works in rotationally symmetrical fields, yielding divisions perpendicular to the compressive tensor, parallel to the long axis of the cell, and one in asymmetric fields, yielding divisions parallel to the short axis of the cell and the compressive tensor.

  11. HMGN2 inducibly binds a novel transactivation domain in nuclear PRLr to coordinate Stat5a-mediated transcription.

    PubMed

    Fiorillo, Alyson A; Medler, Terry R; Feeney, Yvonne B; Liu, Yi; Tommerdahl, Kalie L; Clevenger, Charles V

    2011-09-01

    The direct actions of transmembrane receptors within the nucleus remain enigmatic. In this report, we demonstrate that the prolactin receptor (PRLr) localizes to the nucleus where it functions as a coactivator through its interactions with the latent transcription factor signal transducer and activator of transcription 5a (Stat5a) and the high-mobility group N2 protein (HMGN2). We identify a novel transactivation domain within the PRLr that is activated by ligand-induced phosphorylation, an event coupled to HMGN2 binding. The association of the PRLr with HMGN2 enables Stat5a-responsive promoter binding, thus facilitating transcriptional activation and promoting anchorage-independent growth. We propose that HMGN2 serves as a critical regulatory factor in Stat5a-driven gene expression by facilitating the assembly of PRLr/Stat5a onto chromatin and that these events may serve to promote biological events that contribute to a tumorigenic phenotype. Our data imply that phosphorylation may be the molecular switch that activates a cell surface receptor transactivation domain, enabling it to tether chromatin-modifying factors, such as HMGN2, to target promoter regions in a sequence-specific manner.

  12. Stat5a serine 725 and 779 phosphorylation is a prerequisite for hematopoietic transformation

    PubMed Central

    Friedbichler, Katrin; Kerenyi, Marc A.; Kovacic, Boris; Li, Geqiang; Hoelbl, Andrea; Yahiaoui, Saliha; Sexl, Veronika; Müllner, Ernst W.; Fajmann, Sabine; Cerny-Reiterer, Sabine; Valent, Peter; Beug, Hartmut; Gouilleux, Fabrice; Bunting, Kevin D.

    2010-01-01

    Stat5 transcription factors are essential gene regulators promoting proliferation, survival, and differentiation of all hematopoietic cell types. Mutations or fusions of oncogenic tyrosine kinases often result in constitutive Stat5 activation. We have modeled persistent Stat5 activity by using an oncogenic Stat5a variant (cS5). To analyze the hitherto unrecognized role of Stat5 serine phosphorylation in this context, we have generated cS5 constructs with mutated C-terminal serines 725 and 779, either alone or in combination. Genetic complementation assays in primary Stat5null/null mast cells and Stat5ΔN T cells demonstrated reconstitution of proliferation with these mutants. Similarly, an in vivo reconstitution experiment of transduced Stat5null/null fetal liver cells transplanted into irradiated wild-type recipients revealed that these mutants exhibit biologic activity in lineage differentiation. By contrast, the leukemogenic potential of cS5 in bone marrow transplants decreased dramatically in cS5 single-serine mutants or was completely absent upon loss of both serine phosphorylation sites. Our data suggest that Stat5a serine phosphorylation is a prerequisite for cS5-mediated leukemogenesis. Hence, interference with Stat5a serine phosphorylation might provide a new therapeutic option for leukemia and myeloid dysplasias without affecting major functions of Stat5 in normal hematopoiesis. PMID:20508164

  13. STAT5A/B Gene Locus Undergoes Amplification during Human Prostate Cancer Progression

    PubMed Central

    Haddad, Bassem R.; Gu, Lei; Mirtti, Tuomas; Dagvadorj, Ayush; Vogiatzi, Paraskevi; Hoang, David T.; Bajaj, Renu; Leiby, Benjamin; Ellsworth, Elyse; Blackmon, Shauna; Ruiz, Christian; Curtis, Mark; Fortina, Paolo; Ertel, Adam; Liu, Chengbao; Rui, Hallgeir; Visakorpi, Tapio; Bubendorf, Lukas; Lallas, Costas D.; Trabulsi, Edouard J.; McCue, Peter; Gomella, Leonard; Nevalainen, Marja T.

    2014-01-01

    The molecular mechanisms underlying progression of prostate cancer (PCa) to castrate-resistant (CR) and metastatic disease are poorly understood. Our previous mechanistic work shows that inhibition of transcription factor Stat5 by multiple alternative methods induces extensive rapid apoptotic death of Stat5-positive PCa cells in vitro and inhibits PCa xenograft tumor growth in nude mice. Furthermore, STAT5A/B induces invasive behavior of PCa cells in vitro and in vivo, suggesting involvement of STAT5A/B in PCa progression. Nuclear STAT5A/B protein levels are increased in high-grade PCas, CR PCas, and distant metastases, and high nuclear STAT5A/B expression predicts early disease recurrence and PCa-specific death in clinical PCas. Based on these findings, STAT5A/B represents a therapeutic target protein for advanced PCa. The mechanisms underlying increased Stat5 protein levels in PCa are unclear. Herein, we demonstrate amplification at the STAT5A/B gene locus in a significant fraction of clinical PCa specimens. STAT5A/B gene amplification was more frequently found in PCas of high histologic grades and in CR distant metastases. Quantitative in situ analysis revealed that STAT5A/B gene amplification was associated with increased STAT5A/B protein expression in PCa. Functional studies showed that increased STAT5A/B copy numbers conferred growth advantage in PCa cells in vitro and as xenograft tumors in vivo. The work presented herein provides the first evidence of somatic STAT5A/B gene amplification in clinical PCas. PMID:23660011

  14. STAT5A/B gene locus undergoes amplification during human prostate cancer progression.

    PubMed

    Haddad, Bassem R; Gu, Lei; Mirtti, Tuomas; Dagvadorj, Ayush; Vogiatzi, Paraskevi; Hoang, David T; Bajaj, Renu; Leiby, Benjamin; Ellsworth, Elyse; Blackmon, Shauna; Ruiz, Christian; Curtis, Mark; Fortina, Paolo; Ertel, Adam; Liu, Chengbao; Rui, Hallgeir; Visakorpi, Tapio; Bubendorf, Lukas; Lallas, Costas D; Trabulsi, Edouard J; McCue, Peter; Gomella, Leonard; Nevalainen, Marja T

    2013-06-01

    The molecular mechanisms underlying progression of prostate cancer (PCa) to castrate-resistant (CR) and metastatic disease are poorly understood. Our previous mechanistic work shows that inhibition of transcription factor Stat5 by multiple alternative methods induces extensive rapid apoptotic death of Stat5-positive PCa cells in vitro and inhibits PCa xenograft tumor growth in nude mice. Furthermore, STAT5A/B induces invasive behavior of PCa cells in vitro and in vivo, suggesting involvement of STAT5A/B in PCa progression. Nuclear STAT5A/B protein levels are increased in high-grade PCas, CR PCas, and distant metastases, and high nuclear STAT5A/B expression predicts early disease recurrence and PCa-specific death in clinical PCas. Based on these findings, STAT5A/B represents a therapeutic target protein for advanced PCa. The mechanisms underlying increased Stat5 protein levels in PCa are unclear. Herein, we demonstrate amplification at the STAT5A/B gene locus in a significant fraction of clinical PCa specimens. STAT5A/B gene amplification was more frequently found in PCas of high histologic grades and in CR distant metastases. Quantitative in situ analysis revealed that STAT5A/B gene amplification was associated with increased STAT5A/B protein expression in PCa. Functional studies showed that increased STAT5A/B copy numbers conferred growth advantage in PCa cells in vitro and as xenograft tumors in vivo. The work presented herein provides the first evidence of somatic STAT5A/B gene amplification in clinical PCas.

  15. Hypothesis: Neuroendocrine Mechanisms (Hypothalamus–Growth Hormone–STAT5 Axis) Contribute to Sex Bias in Pulmonary Hypertension

    PubMed Central

    Sehgal, Pravin B; Yang, Yang-Ming; Miller, Edmund J

    2015-01-01

    Pulmonary hypertension (PH) is a disease with high morbidity and mortality. The prevalence of idiopathic pulmonary arterial hypertension (IPAH) and hereditary pulmonary arterial hypertension (HPAH) is approximately two- to four-fold higher in women than in men. Paradoxically, there is an opposite male bias in typical rodent models of PH (chronic hypoxia or monocrotaline); in these models, administration of estrogenic compounds (for example, estradiol-17β [E2]) is protective. Further complexities are observed in humans ingesting anorexigens (female bias) and in rodent models, such as after hypoxia plus SU5416/Sugen (little sex bias) or involving serotonin transporter overexpression or dexfenfluramine administration (female bias). These complexities in sex bias in PH remain incompletely understood. We recently discovered that conditional deletion of signal transducer and activator of transcription 5a/b (STAT5a/b) in vascular smooth muscle cells abrogated the male bias in PH in hypoxic mice and that late-stage obliterative lesions in patients of both sexes with IPAH and HPAH showed reduced STAT5a/b, reduced Tyr-P-STAT5 and reduced B-cell lymphoma 6 protein (BCL6). In trying to understand the significance of these observations, we realized that there existed a well-characterized E2-sensitive central neuroendocrine mechanism of sex bias, studied over the last 40 years, that, at its peripheral end, culminated in species-specific male (“pulsatile”) versus female (“more continuous”) temporal patterns of circulating growth hormone (GH) levels leading to male versus female patterned activation of STAT5a/b in peripheral tissues and thus sex-biased expression of hundreds of genes. In this report, we consider the contribution of this neuroendocrine mechanism (hypothalamus-GH-STAT5) in the generation of sex bias in different PH situations. PMID:26252185

  16. Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes.

    PubMed

    Preston, Gavin C; Sinclair, Linda V; Kaskar, Aneesa; Hukelmann, Jens L; Navarro, Maria N; Ferrero, Isabel; MacDonald, H Robson; Cowling, Victoria H; Cantrell, Doreen A

    2015-08-01

    Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses. PMID:26136212

  17. Pyrvinium selectively induces apoptosis of lymphoma cells through impairing mitochondrial functions and JAK2/STAT5.

    PubMed

    Xiao, Meifang; Zhang, Liming; Zhou, Yizheng; Rajoria, Pasupati; Wang, Changfu

    2016-01-15

    Targeting mitochondrial respiration has emerged as an attractive therapeutic strategy in blood cancer due to their unique metabolic dependencies. In this study, we show that pyrvinium, a FDA-approved anthelmintic drug, selectively targets lymphoma T-cells though inhibition of mitochondrial functions and JAK2/STAT5. Pyrvinium induces apoptosis of malignant T-cell line Jurkat and primary T-cells from lymphoma patients while sparing T-cells from healthy donors. Increased level of active caspase-3 and decreased levels of Bcl-2 and Mcl-1 were also observed in Jurkat and lymphoma T-cells but not normal T-cells treated with pyrvinium. In addition, pyrvinium impairs mitochondrial functions by inhibit mitochondrial respiration, suppressing mitochondrial respiratory complex I activity, increasing ROS and decreasing ATP levels. However, the effects of pyrvinium were abolished in mitochondrial respiration-deficient Jurkat ρ(0) cells, confirming that pyrvinium acts on lymphoma T-cells via targeting mitochondrial respiration. We further show that lymphoma T-cells derived from patients depend more on mitochondrial respiration than normal T-cells, and this explains the selective toxicity of pyrvinium in lymphoma versus normal T-cells. Finally, we demonstrate that pyrvinium also suppresses JAK2/STAT5 signaling pathway in Jurkat cells. Our study suggests that pyrvinium is a useful addition to T-cell lymphoma treatment, and emphasizes the potential therapeutic value of the differences in the mitochondrial characteristics between malignant and normal T-cells in blood cancer.

  18. Effects of insulin and IGF-I on growth hormone- induced STAT5 activation in 3T3-F442A adipocytes

    PubMed Central

    2013-01-01

    Background Growth hormone (GH) and insulin signaling pathways are known important regulators of adipose homeostasis. The cross-talk between GH and insulin signaling pathways in mature adipocytes is poorly understood. Methods In the present study, the impact of insulin on GH-mediated signaling in differentiated 3T3-F442A adipocytes and primary mice adipocytes was examined. Results Insulin alone did not induce STAT5 tyrosine phosphorylation, but enhanced GH-induced STAT5 activation. This effect was more pronounced when insulin was added 20 min prior to GH treatment. The above results were further confirmed by in vivo study, showing that insulin pretreatment potentiated GH- induced STAT5 tyrosine phosphorylation in visceral adipose tissues of C57/BL6 mice. In addition, our in vitro results showed that IGF-I had similar potentiating effect as insulin on GH-induced STAT5 activation. In vitro, insulin and IGF-I had an additive effect on GH- induced MAPK activation. Conclusion These results indicate that both insulin and IGF-I specifically potentiated GH mediated STAT5 activation in mature adipose cells. These findings suggest that insulin and GH, usually with antagonistic functions, might act synergistically to regulate some specific functions in mature adipocytes. PMID:23631823

  19. Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function

    SciTech Connect

    Xu, Ren; Nelson, Celeste M.; Muschler, John L.; Veiseh, Mandana; Vonderhaar, Barbara K.; Bissell, Mina J.

    2009-06-03

    Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We show that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and {beta}-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression.

  20. Comparison of the transactivation domains of Stat5 and Stat6 in lymphoid cells and mammary epithelial cells.

    PubMed Central

    Moriggl, R; Berchtold, S; Friedrich, K; Standke, G J; Kammer, W; Heim, M; Wissler, M; Stöcklin, E; Gouilleux, F; Groner, B

    1997-01-01

    Stat (signal transducers and activators of transcription) and Jak (Janus kinases) proteins are central components in the signal transduction events in hematopoietic and epithelial cells. They are rapidly activated by various cytokines, hormones, and growth factors. Upon ligand binding and cytokine receptor dimerization, Stat proteins are phosphorylated on tyrosine residues by Jak kinases. Activated Stat proteins form homo- or heterodimers, translocate to the nucleus, and induce transcription from responsive genes. Stat5 and Stat6 are transcription factors active in mammary epithelial cells and immune cells. Prolactin activates Stat5, and interleukin-4 (IL-4) activates Stat6. Both cytokines are able to stimulate cell proliferation, differentiation, and survival. We investigated the transactivation potential of Stat6 and found that it is not restricted to lymphocytes. IL-4-dependent activation of Stat6 was also observed in HC11 mammary epithelial cells. In these cells, Stat6 activation led to the induction of the beta-casein gene promoter. The induction of this promoter was confirmed in COS7 cells. The glucocorticoid receptor was able to further enhance IL-4-induced gene transcription through the action of Stat6. Deletion analysis of the carboxyl-terminal region of Stat6 and recombination of this region with a heterologous DNA binding domain allowed the delimitation and characterization of the transactivation domain of Stat6. The potencies of the transactivation domains of Stat5, Stat6, and viral protein VP16 were compared. Stat6 had a transactivation domain which was about 10-fold stronger than that of Stat5. In pre-B cells (Ba/F3), the transactivation domain of Stat6 was IL-4 regulated, independently from its DNA binding function. PMID:9199300

  1. The STAT5 inhibitor pimozide decreases survival of chronic myelogenous leukemia cells resistant to kinase inhibitors

    PubMed Central

    Nelson, Erik A.; Walker, Sarah R.; Weisberg, Ellen; Bar-Natan, Michal; Barrett, Rosemary; Gashin, Laurie B.; Terrell, Shariya; Klitgaard, Josephine L.; Santo, Loredana; Addorio, Martha R.; Ebert, Benjamin L.; Griffin, James D.

    2011-01-01

    The transcription factor STAT5 is an essential mediator of the pathogenesis of chronic myelogenous leukemia (CML). In CML, the BCR/ABL fusion kinase causes the constitutive activation of STAT5, thereby driving the expression of genes promoting survival. BCR/ABL kinase inhibitors have become the mainstay of therapy for CML, although CML cells can develop resistance through mutations in BCR/ABL. To overcome this problem, we used a cell-based screen to identify drugs that inhibit STAT-dependent gene expression. Using this approach, we identified the psychotropic drug pimozide as a STAT5 inhibitor. Pimozide decreases STAT5 tyrosine phosphorylation, although it does not inhibit BCR/ABL or other tyrosine kinases. Furthermore, pimozide decreases the expression of STAT5 target genes and induces cell cycle arrest and apoptosis in CML cell lines. Pimozide also selectively inhibits colony formation of CD34+ bone marrow cells from CML patients. Importantly, pimozide induces similar effects in the presence of the T315I BCR/ABL mutation that renders the kinase resistant to presently available inhibitors. Simultaneously inhibiting STAT5 with pimozide and the kinase inhibitors imatinib or nilotinib shows enhanced effects in inhibiting STAT5 phosphorylation and in inducing apoptosis. Thus, targeting STAT5 may be an effective strategy for the treatment of CML and other myeloproliferative diseases. PMID:21233313

  2. Bottom-up assembly of RNA nanoparticles containing phi29 motor pRNA to silence the asthma STAT5b gene.

    PubMed

    Qiu, C; Peng, W K; Shi, F; Zhang, T

    2012-09-13

    Activation of the transcription factor signal transducer and activator of transcription 5b (STAT5b) is a key event in the development of asthma. The potent ability of small interfering RNA (siRNA) to inhibit the expression of STAT5b mRNA has provided a new class of therapeutics for asthma. However, efficient delivery of siRNAs remains a key obstacle to their successful application. A targeted intracellular delivery approach for siRNA to specific cell types would be highly desirable. We used packaging RNA (pRNA), a component of the bacteriophage phi29-packaging motor, to deliver STAT5b siRNA to asthmatic spleen lymphocytes. This pRNA was able to spontaneously carry siRNA/STAT5b and aptamer/CD4, which is a ligand to CD4 molecule. Based on RT-PCR data, the pRNA dimer effectively inhibited STAT5b gene mRNA expression of asthmatic spleen lymphocytes, without the need for additional transfections. We conclude that the pRNA dimer carrying both siRNA and aptamer can deliver functional siRNA to cells; possibly, the aptamer acts as a ligand to interact with specific receptors. The pRNAs were evaluated with a CCK-8 kit and were found to have little cytotoxicity. We conclude that pRNA as a novel nanovehicle for RNA worth further study.

  3. GRAM domain-containing protein 1A (GRAMD1A) promotes the expansion of hepatocellular carcinoma stem cell and hepatocellular carcinoma growth through STAT5

    PubMed Central

    Fu, Binsheng; Meng, Wei; Zhao, Hui; Zhang, Bing; Tang, Hui; Zou, Ying; Yao, Jia; Li, Heping; Zhang, Tong

    2016-01-01

    Hepatocellular carcinoma (HCC) is the leading cause for cancer death worldwide, new prognostic factors and targets are critical for HCC treatment. Here, we found GRAMD1A was upregulated in HCC tissues, patients with high GRAMD1A levels had poor outcome, statistical analyses found GRAMD1A expression was positively correlated with pathologic differentiation and survival or mortality. It was an unfavorable prognostic factor for HCC patients. Functional analyses revealed GRAMD1A contributed to the self-renewal of HCC stem cells, resistance to chemotherapy and tumor growth of HCC determined by hepatosphere formation assay, side population (SP) analysis, TUNEL assay, soft agar growth ability assay and tumor growth model in vivo. Mechanism analyses found signal transducer and activator of transcription 5 (STAT5) was the target of GRAMD1A, GRAMD1A regulated the target genes of STAT5 and the transcriptional activity of STAT5. Inhibition of STAT5 in indicated HCC cells overexpressing GRAMD1A suppressed the effects of GRAMD1A on the self-renewal of HCC stem cell, resistance to chemotherapy and tumor growth, suggesting GRAMD1A promoted the self-renewal of HCC stem cells and the development of HCC by increasing STAT5 level. GRAMD1A might be a useful biomarker and target for HCC. PMID:27585821

  4. GRAM domain-containing protein 1A (GRAMD1A) promotes the expansion of hepatocellular carcinoma stem cell and hepatocellular carcinoma growth through STAT5.

    PubMed

    Fu, Binsheng; Meng, Wei; Zhao, Hui; Zhang, Bing; Tang, Hui; Zou, Ying; Yao, Jia; Li, Heping; Zhang, Tong

    2016-01-01

    Hepatocellular carcinoma (HCC) is the leading cause for cancer death worldwide, new prognostic factors and targets are critical for HCC treatment. Here, we found GRAMD1A was upregulated in HCC tissues, patients with high GRAMD1A levels had poor outcome, statistical analyses found GRAMD1A expression was positively correlated with pathologic differentiation and survival or mortality. It was an unfavorable prognostic factor for HCC patients. Functional analyses revealed GRAMD1A contributed to the self-renewal of HCC stem cells, resistance to chemotherapy and tumor growth of HCC determined by hepatosphere formation assay, side population (SP) analysis, TUNEL assay, soft agar growth ability assay and tumor growth model in vivo. Mechanism analyses found signal transducer and activator of transcription 5 (STAT5) was the target of GRAMD1A, GRAMD1A regulated the target genes of STAT5 and the transcriptional activity of STAT5. Inhibition of STAT5 in indicated HCC cells overexpressing GRAMD1A suppressed the effects of GRAMD1A on the self-renewal of HCC stem cell, resistance to chemotherapy and tumor growth, suggesting GRAMD1A promoted the self-renewal of HCC stem cells and the development of HCC by increasing STAT5 level. GRAMD1A might be a useful biomarker and target for HCC. PMID:27585821

  5. STAT5 transcriptional activity is impaired by LIF in a mammary epithelial cell line.

    PubMed

    Granillo, Agustina Rodriguez; Boffi, Juan Carlos; Barañao, Lino; Kordon, Edith; Pecci, Adali; Guberman, Alejandra

    2007-05-11

    Gene regulation mediated by STAT factors has been implicated in cellular functions with relevance to a variety of processes. Particularly, STAT5 and STAT3 play a crucial role in mammary epithelium displaying reciprocal activation kinetics during pregnancy, lactation and involution. Here, we show that LIF treatment of mammary epithelial HC11 cells reduces the phosphorylation levels and transcriptional activity of p-STAT5 in correlation with STAT3 phosphorylation. We have also found that STAT5 activity is negatively modulated by this cytokine, both on a gene whose expression is induced, as well as on a promoter repressed by STAT5. Besides, our results show that lactogenic hormones increase LIF effect on gene induction without modifying STAT3 phosphorylation state. Our findings strongly suggest that there is crosstalk between STAT5 and STAT3 pathways that could modulate their ability to regulate gene expression.

  6. STAT5 Outcompetes STAT3 To Regulate the Expression of the Oncogenic Transcriptional Modulator BCL6

    PubMed Central

    Walker, Sarah R.; Nelson, Erik A.; Yeh, Jennifer E.; Pinello, Luca; Yuan, Guo-Cheng

    2013-01-01

    Inappropriate activation of the transcription factors STAT3 and STAT5 has been shown to drive cancer pathogenesis through dysregulation of genes involved in cell survival, growth, and differentiation. Although STAT3 and STAT5 are structurally related, they can have opposite effects on key genes, including BCL6. BCL6, a transcriptional repressor, has been shown to be oncogenic in diffuse large B cell lymphoma. BCL6 also plays an important role in breast cancer pathogenesis, a disease in which STAT3 and STAT5 can be activated individually or concomitantly. To determine the mechanism by which these oncogenic transcription factors regulate BCL6 transcription, we analyzed their effects at the levels of chromatin and gene expression. We found that STAT3 increases expression of BCL6 and enhances recruitment of RNA polymerase II phosphorylated at a site associated with transcriptional initiation. STAT5, in contrast, represses BCL6 expression below basal levels and decreases the association of RNA polymerase II at the gene. Furthermore, the repression mediated by STAT5 is dominant over STAT3-mediated induction. STAT5 exerts this effect by displacing STAT3 from one of the two regulatory regions to which it binds. These findings may underlie the divergent biology of breast cancers containing activated STAT3 alone or in conjunction with activated STAT5. PMID:23716595

  7. STAT-5 is activated constitutively in T cells, B cells and monocytes from patients with primary Sjögren's syndrome

    PubMed Central

    Pertovaara, M; Silvennoinen, O; Isomäki, P

    2015-01-01

    The expression and phosphorylation of signal transducer and activator of transcription-1 (STAT-1) have been shown to be markedly increased in the salivary gland epithelial cells of patients with primary Sjögren's syndrome (pSS). The present aim was to investigate the activation status of different STAT proteins in peripheral blood (PB) lymphocytes and monocytes, and their correlations with clinical parameters in patients with pSS. To this end, PB samples were drawn from 16 patients with active pSS and 16 healthy blood donors, and the phosphorylation of STAT-1, -3, -4, -5 and -6 proteins was studied in T cells, B cells and monocytes using multi-colour flow cytometry. In addition, mRNA expression of STAT molecules in PB mononuclear cells (PBMC) was studied with quantitative reverse transcriptase–polymerase chain reaction (RT–PCR). Basal phosphorylation of STAT-5 was found to be significantly higher in PB T cells, B cells and monocytes in patients with pSS than in healthy controls. The expression of STAT-5 mRNA was not increased in PBMC. pSTAT-5 levels in B cells and monocytes showed a significant correlation with serum immunoglobulin (Ig)G levels and anti-SSB antibody titres. Constitutive STAT-5 activation in monocytes and CD4+ T cells was associated with purpura. There were no major differences in the activation of other STATs between pSS patients and healthy controls. In conclusion, STAT-5 is activated constitutively in PB leucocytes in patients with pSS, and basal STAT-5 phosphorylation seems to associate with hypergammaglobulinaemia, anti-SSB antibody production and purpura. PMID:25736842

  8. STAT5a/b contribute to sex bias in vascular disease: A neuroendocrine perspective

    PubMed Central

    Sehgal, Pravin B; Yang, Yang-Ming; Yuan, Huijuan; Miller, Edmund J

    2015-01-01

    abstract Previous studies have elucidated a neuroendocrine mechanism consisting of the hypothalamus (growth hormone releasing hormone, GHRH) – pituitary (growth hormone, GH) – STAT5a/b axis that underlies sex-biased gene expression in the liver. It is now established that male vs female patterned secretion of GHRH, and thus of circulating GH levels (“pulsatile” vs “more continuous” respectively), leading to differently patterned activation of PY-STAT5a/b in hepatocytes results in sex-biased gene expression of cohorts of hundreds of downstream genes. This review outlines new data in support of a STAT5a/b-based mechanism of sex bias in the vascular disease pulmonary hypertension (PH). Puzzling observations in PH include its 2-4-fold higher prevalence in women but a male-dominance in many rodent models, and, paradoxically, inhibition of PH development by estrogens in such models. We observed that conditional deletion of STAT5a/b in vascular smooth muscle cells (SMC) in mice converted the male-dominant model of chronic hypoxia-induced PH into a female-dominant phenotype. In human idiopathic PH, there was reduced STAT5a/b and PY-STAT5 in cells in late-stage obliterative pulmonary arterial lesions in both men and women. A juxtaposition of the prior liver data with the newer PH-related data drew attention to the hypothalamus-GH-STAT5 axis, which is the major target of estrogens at the level of the hypothalamus. This hypothesis explains many of the puzzling aspects of sex bias in PH in humans and rodent models. The extension of STAT5-anchored mechanisms of sex bias to vascular disease emphasizes the contribution of central neuroendocrine processes in generating sexual dimorphism in different tissues and cell types. PMID:27141328

  9. Gap junctions mediate STAT5-independent β-casein expression in CID-9 mammary epithelial cells.

    PubMed

    Talhouk, Rabih S; Khalil, Antoine A; Bajjani, Rachid; Rahme, Gilbert J; El-Sabban, Marwan E

    2011-10-01

    Crosstalk between gap junction intracellular communication (GJIC), STAT5 and OCT-1 in gap junction (GJ)-dependent β-casein expression was investigated. CID-9 mammary cells plated with prolactin on non-adherent substratum (poly-HEMA) expressed β-casein independent of STAT5 only in the presence of the GJIC inducer, cAMP. Nuclear STAT5 levels were not detectable. By contrast, cells on EHS-drip expressed β-casein in a STAT5-dependent manner and nuclear STAT5 levels were up-regulated. A 75 kDa OCT-1 isoform was detected in conditions that induced β-casein expression regardless of substratum. Interestingly, 40 and 28 kDa OCT-1 isoforms were induced in cells on polyHEMA with cAMP. Electrophoretic mobility shift assays (EMSA) for OCT-1 revealed two band shifts in cells on polyHEMA with cAMP and on EHS-drip, which were repressed by the GJIC inhibitor, 18α-GA. These studies demonstrated that mammary cells on polyHEMA expressed β-casein in response to prolactin in a pathway that involves GJIC and OCT-1 and is independent of STAT5 nuclear translocation.

  10. Rapamycin reduces fibroblast proliferation without causing quiescence and induces STAT5A/B-mediated cytokine production.

    PubMed

    Gillespie, Zoe E; MacKay, Kimberly; Sander, Michelle; Trost, Brett; Dawicki, Wojciech; Wickramarathna, Aruna; Gordon, John; Eramian, Mark; Kill, Ian R; Bridger, Joanna M; Kusalik, Anthony; Mitchell, Jennifer A; Eskiw, Christopher H

    2015-01-01

    Rapamycin is a well-known inhibitor of the Target of Rapamycin (TOR) signaling cascade; however, the impact of this drug on global genome function and organization in normal primary cells is poorly understood. To explore this impact, we treated primary human foreskin fibroblasts with rapamycin and observed a decrease in cell proliferation without causing cell death. Upon rapamycin treatment chromosomes 18 and 10 were repositioned to a location similar to that of fibroblasts induced into quiescence by serum reduction. Although similar changes in positioning occurred, comparative transcriptome analyses demonstrated significant divergence in gene expression patterns between rapamycin-treated and quiescence-induced fibroblasts. Rapamycin treatment induced the upregulation of cytokine genes, including those from the Interleukin (IL)-6 signaling network, such as IL-8 and the Leukemia Inhibitory Factor (LIF), while quiescent fibroblasts demonstrated up-regulation of genes involved in the complement and coagulation cascade. In addition, genes significantly up-regulated by rapamycin treatment demonstrated increased promoter occupancy of the transcription factor Signal Transducer and Activator of Transcription 5A/B (STAT5A/B). In summary, we demonstrated that the treatment of fibroblasts with rapamycin decreased proliferation, caused chromosome territory repositioning and induced STAT5A/B-mediated changes in gene expression enriched for cytokines. PMID:26652669

  11. Single-cell network profiling of peripheral blood mononuclear cells from healthy donors reveals age- and race-associated differences in immune signaling pathway activation.

    PubMed

    Longo, Diane M; Louie, Brent; Putta, Santosh; Evensen, Erik; Ptacek, Jason; Cordeiro, James; Wang, Ena; Pos, Zoltan; Hawtin, Rachael E; Marincola, Francesco M; Cesano, Alessandra

    2012-02-15

    A greater understanding of the function of the human immune system at the single-cell level in healthy individuals is critical for discerning aberrant cellular behavior that occurs in settings such as autoimmunity, immunosenescence, and cancer. To achieve this goal, a systems-level approach capable of capturing the response of the interdependent immune cell types to external stimuli is required. In this study, an extensive characterization of signaling responses in multiple immune cell subpopulations within PBMCs from a cohort of 60 healthy donors was performed using single-cell network profiling (SCNP). SCNP is a multiparametric flow cytometry-based approach that enables the simultaneous measurement of basal and evoked signaling in multiple cell subsets within heterogeneous populations. In addition to establishing the interindividual degree of variation within a broad panel of immune signaling responses, the possible association of any observed variation with demographic variables including age and race was investigated. Using half of the donors as a training set, multiple age- and race-associated variations in signaling responses in discrete cell subsets were identified, and several were subsequently confirmed in the remaining samples (test set). Such associations may provide insight into age-related immune alterations associated with high infection rates and diminished protection following vaccination and into the basis for ethnic differences in autoimmune disease incidence and treatment response. SCNP allowed for the generation of a functional map of healthy immune cell signaling responses that can provide clinically relevant information regarding both the mechanisms underlying immune pathological conditions and the selection and effect of therapeutics.

  12. Prolactin signalling in the mouse hypothalamus is primarily mediated by signal transducer and activator of transcription factor 5b but not 5a.

    PubMed

    Yip, S H; Eguchi, R; Grattan, D R; Bunn, S J

    2012-12-01

    Prolactin acts at multiple targets throughout the body, including the mammary gland, heart, liver, muscle and brain. Upon binding to its receptors, prolactin signals through the phosphorylation and thus activation of signal transducer and activator of transcription 5 (STAT5). There are two very similar STAT5 isoforms, termed STAT5a and STAT5b, which are selectively activated by prolactin in specific tissues. Various brain regions, including the hypothalamus, are prolactin responsive, although the STAT5 isoform involved in these actions is unknown. Immunohistochemical and western blot analysis were used to determine the expression and activation of STAT5a and STAT5b throughout the hypothalamus in adult wild-type and STAT5b-deficient mice. Both groups were pretreated with bromocriptine to suppress endogenous prolactin levels followed by the administration of ovine prolactin (10 mg/kg) for 45 min. STAT5a and STAT5b were expressed throughout the hypothalamus of wild-type mice. As expected, only STAT5a was detected in STAT5b-deficient mice, although, unexpectedly, there was a marked reduction in its expression compared to wild-type mice. When stimulated with prolactin, phosphorylated STAT5 was observed in the hypothalamus of wild-type but not STAT5b-deficient mice. By contrast, phosphorylated STAT5 was detected in mammary gland epithelial cells and adipocytes of STAT5b-deficient animals. Thus, although STAT5a was still expressed in the STAT5b-deficient mice, it was not phosphorylated in the hypothalamus in response to prolactin. These observations indicate that STAT5b but not STAT5a is the primary mediator of the action of prolactin in the hypothalamus. Despite the similarity between the two STAT5 isoforms, STAT5a was unable to compensate for the absence of STAT5b, suggesting that each isoform exhibits a unique biological activity.

  13. Study on the STAT5A/AvaI polymorphism in Jersey cows and association with milk production traits.

    PubMed

    Dario, Cataldo; Selvaggi, Maria

    2011-11-01

    In mammals, the STAT proteins (signal transducers and activators of transcription) are a group of cytoplasmic transcription factors that mediate the actions of many peptide hormones and cytokines within target cells. In this study, the STAT5A/AvaI polymorphism was investigated with the PCR-RFLP technique in a sample of 191 Jersey cows reared in southern Italy. This polymorphism is localized in the coding region of the bovine STAT5A gene. It is a substitution C → T at position 6853 within the exon 7. All the possible genotypes for the C/T polymorphism were identified. The overall frequencies of alleles C and T were 0.75 and 0.25 respectively. In order to study the relationship between this SNP and milk production traits, data for 305-day milk production were used. Significant differences between CC and CT genotypes were found in milk yield (MY), fat yield (FY), solids not fat (SNF) yield, total solids (TS) yield (P < 0.01) and protein yield (PY) (P < 0.05). In particular the CC cows produced more milk than CT ones (6196.07 vs. 5744.66 kg) without differences in protein and fat content. As a consequence, CC cows yielded more fat, protein, solids not fat and total solids than CT. As far as concerns lactose and ash contents and the fat/protein ratio, no significant difference was found between the two genotypes. Even if further studies should be carry out to assess the role of this SNP on production traits, it is possible to conclude that STAT5A/AvaI polymorphism seems to be a promising indirect marker to improve milk production traits in cattle.

  14. Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity.

    PubMed

    Beuvink, I; Hess, D; Flotow, H; Hofsteenge, J; Groner, B; Hynes, N E

    2000-04-01

    The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779)-specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration. PMID:10744710

  15. Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity.

    PubMed

    Beuvink, I; Hess, D; Flotow, H; Hofsteenge, J; Groner, B; Hynes, N E

    2000-04-01

    The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779)-specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration.

  16. Single cell analysis of low-power laser irradiation-induced activation of signaling pathway in cell proliferation

    NASA Astrophysics Data System (ADS)

    Xing, Da; Gao, Xuejuan

    2007-02-01

    Low-power laser irradiation (LPLI) has been shown to promote cell proliferation in various cell types, yet the mechanism of which has not been fully clarified. Investigating the signaling pathways involved in the laser irradiation is important for understanding these processes. The small G protein Ras works as a binary switch in many important intracellular signaling pathways and, therefore, has been one of the focal targets of signal-transduction investigations and drug development. The Ras/Raf/MEK/ERK (extracellular-signal-regulated kinase) signaling pathway is a network that governs proliferation, differentiation and cell survival. Recent studies suggest that Ras/Raf signaling pathway is involved in the LPLI-induced cell proliferation. On the other hand, Protein kinase Cs (PKCs), the Ca 2+ activated, phospholipid-dependent serine/threonine protein kinases, have been recently presumed to be involved in the regulation of cell proliferation induced by LPLI. In this report, to monitor the direct activations of Ras and PKCs after LPLI treatment in living cells in real time, Raichu-Ras reporter and C kinase activity reporter (CKAR) were utilized, both of which were constructed based on fluorescence resonance energy transfer (FRET) technique. The direct activation of Ras is predominantly initiated from the different microdomains of the plasma membrane. The results are monitored during cell proliferation induced by LPLI (0.8 J/cm2) in serum-starved COS-7 cells expressing Raichu-Ras reporter using FRET imaging on laser scanning confocal microscope. Furthermore, the increasing activation of PKCs is also monitored during cell proliferation induced by LPLI (0.8 J/cm2) in serum-starved human lung adenocarcinoma cells (ASTC-a-1) expressing CKAR reporter using the similar way. Taken together, the dynamic increases of H-Ras and PKCs activities are observed during the processes of cell proliferation induced by LPLI.

  17. Identification of STAT3 and STAT5 proteins in the rat suprachiasmatic nucleus and the Day/Night difference in astrocytic STAT3 phosphorylation in response to lipopolysaccharide.

    PubMed

    Moravcová, Simona; Červená, Kateřina; Pačesová, Dominika; Bendová, Zdeňka

    2016-01-01

    Signal transducers and activators of transcription (STAT) proteins regulate many aspects of cellular physiology from growth and differentiations to immune responses. Using immunohistochemistry, we show the daily rhythm of STAT3 protein in the rat suprachiasmatic nucleus (SCN), with low but significant amplitude peaking in the morning. We also reveal the strong expression of STAT5A in astrocytes of the SCN and the STAT5B signal in nonastrocytic cells. Administration of lipopolysaccharide (LPS) acutely induced phosphorylation of STAT3 on Tyr705 during both the day and the night and induced phosphorylation on Ser727 but only after the daytime application. The LPS-induced phospho-STAT3 (Tyr705) remained elevated for 24 hr after the daytime application but declined within 8 hr when LPS was applied at night.

  18. Automation of a phospho-STAT5 staining procedure for flow cytometry for application in drug discovery.

    PubMed

    Malergue, Fabrice; van Agthoven, Andreas; Scifo, Caroline; Egan, Dave; Strous, Ger J

    2015-03-01

    Drug discovery often requires the screening of compound libraries on tissue cultured cells. Some major targets in drug discovery belong to signal transduction pathways, and PerFix EXPOSE* allows easy flow cytometry phospho assays. We thus investigated the possibility to further simplify and automate this assay, to allow the direct screening of drugs targeting signaling pathways. We show here the sensitivity of this fully automated assay on human growth hormone (hGH)-driven JAK/STAT5-activated IM-9 cells, and we discuss the throughput of this system, which is compatible with medium-throughput drug screening. Because the kit works directly on whole blood samples, ex-vivo assays are also possible with this approach, which could allow for the screening of drugs under more physiological conditions.

  19. SERS assay of telomerase activity at single-cell level and colon cancer tissues via quadratic signal amplification.

    PubMed

    Shi, Muling; Zheng, Jing; Liu, Changhui; Tan, Guixiang; Qing, Zhihe; Yang, Sheng; Yang, Jinfeng; Tan, Yongjun; Yang, Ronghua

    2016-03-15

    As an important biomarker and therapeutic target, telomerase has attracted extensive attention concerning its detection and monitoring. Recently, enzyme-assisted amplification approaches have provided useful platforms for the telomerase activity detection, however, further improvement in sensitivity is still hindered by the single-step signal amplification. Herein, we develop a quadratic signal amplification strategy for ultrasensitive surface-enhanced Raman scattering (SERS) detection of telomerase activity. The central idea of our design is using telomerase-induced silver nanoparticles (AgNPs) assembly and silver ions (Ag(+))-mediated cascade amplification. In our approach, each telomerase-aided DNA sequence extension could trigger the formation of a long double-stranded DNA (dsDNA), making numerous AgNPs assembling along with this long strand through specific Ag-S bond, to form a primary amplification element. For secondary amplification, each conjugated AgNP was dissolved into Ag(+), which can effectively induce the 4-aminobenzenethiol (4-ABT) modified gold nanoparticles (AuNPs@4-ABT) to undergo aggregation to form numerous "hot-spots". Through quadratic amplifications, a limit of detection down to single HeLa cell was achieved. More importantly, this method demonstrated good performance when applied to tissues from colon cancer patients, which exhibits great potential in the practical application of telomerase-based cancer diagnosis in early stages. To demonstrate the potential in screening the telomerase inhibitors and telomerase-targeted drugs, the proposed design is successfully employed to measure the inhibition of telomerase activity by 3'-azido-3'-deoxythymidine.

  20. Single-Cell Analysis Reveals that Insulation Maintains Signaling Specificity between Two Yeast MAPK Pathways with Common Components

    PubMed Central

    Patterson, Jesse C.; Klimenko, Evguenia S.; Thorner, Jeremy

    2014-01-01

    Eukaryotic cells use multiple mitogen-activated protein kinase (MAPK) cascades to evoke appropriate responses to external stimuli. In Saccharomyces cerevisiae, the MAPK Fus3 is activated by pheromone-binding G protein-coupled receptors to promote mating, whereas the MAPK Hog1 is activated by hyperosmotic stress to elicit the high osmolarity glycerol (HOG) response. Although these MAPK pathways share several upstream components, exposure to either pheromone or osmolyte alone triggers only the appropriate response. We used fluorescent localization- and transcription-specific reporters to assess activation of these pathways in individual cells on the minute and hour timescale, respectively. Dual activation of these two MAPK pathways occurred over a broad range of stimulant concentrations and temporal regimes in wild-type cells subjected to co-stimulation. Thus, signaling specificity is achieved through an “insulation” mechanism, not a “cross-inhibition” mechanism. Furthermore, we showed that there was a critical period during which Hog1 activity had to occur for proper insulation of the HOG pathway. PMID:20959523

  1. Over-expression of Stat5b confers protection against diabetes in the non-obese diabetic (NOD) mice via up-regulation of CD4{sup +}CD25{sup +} regulatory T cells

    SciTech Connect

    Jin, Yulan; Purohit, Sharad; Chen, Xueqin; Yi, Bing; She, Jin-Xiong

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer This is the first study to provide direct evidence of the role of Stat5b in NOD mice. Black-Right-Pointing-Pointer Over-expression of wild type Stat5b transgene protects NOD mice against diabetes. Black-Right-Pointing-Pointer This protection may be mediated by the up-regulation of CD4{sup +}CD25{sup +} Tregs. -- Abstract: The signal transducers and activators of transcription (STAT) family of proteins play a critical role in cytokine signaling required for fine tuning of immune regulation. Previous reports showed that a mutation (L327M) in the Stat5b protein leads to aberrant cytokine signaling in the NOD mice. To further elaborate the role of Stat5b in diabetes, we established a NOD transgenic mouse that over-expresses the wild type Stat5b gene. The incidences of spontaneous diabetes as well as cyclophosphamide-induced diabetes were significantly reduced and delayed in the Stat5b transgenic NOD mice compared to their littermate controls. The total cell numbers of CD4{sup +} T cells and especially CD8{sup +} T cells in the spleen and pancreatic lymph node were increased in the Stat5b transgenic NOD mice. Consistent with these findings, CD4{sup +} and CD8{sup +} T cells from the Stat5b transgenic NOD mice showed a higher proliferation capacity and up-regulation of multiple cytokines including IL-2, IFN-{gamma}, TNF-{alpha} and IL-10 as well as anti-apoptotic gene Bcl-xl. Furthermore, the number and proportion of CD4{sup +}CD25{sup +} regulatory T cells were significantly increased in transgenic mice although in vitro suppression ability of the regulatory T-cells was not affected by the transgene. Our results suggest that Stat5b confers protection against diabetes in the NOD mice by regulating the numbers and function of multiple immune cell types, especially by up-regulating CD4{sup +}CD25{sup +} regulatory T cells.

  2. Regulation by the extracellular matrix (ECM) of prolactin-induced alpha s1-casein gene expression in rabbit primary mammary cells: role of STAT5, C/EBP, and chromatin structure.

    PubMed

    Jolivet, Geneviève; Pantano, Thaïs; Houdebine, Louis Marie

    2005-05-15

    The aim of the present study was to understand how the extracellular matrix (ECM) regulates at the gene level the prolactin (Prl)-induced signal transducer and activator of transcription 5 (STAT5)-dependent expression of the alpha s1-casein gene in mammary epithelial cells. CCAAT enhancer binding proteins (C/EBPs) are assumed regulators of beta-casein gene expression. Rabbit primary mammary cells express alpha s1-casein gene when cultured on collagen and not on plastic. Similar C/EBPbeta, C/EBPdelta, STAT5, and Prl-activated STAT5 were found under all culture conditions. Thus the ECM does not act through C/EBPs or STAT5. This was confirmed by transfections of rabbit primary mammary cells by a construct sensitive to ovine prolactin (oPrl) and ECM (6i TK luc) encompassing STAT5 and C/EBP binding sites. The mutation of C/EBPs binding sites showed that these sites were not mandatory for Prl-induced expression of the construct. Interestingly, chromatin immunoprecipitation by the anti-acetylhistone H4 antibody (ChIP) showed that the ECM (and not Prl) maintained a high amount of histone H4 acetylation upstream of the alpha s1-casein gene especially at the level of a distal Prl- and ECM-sensitive enhancer. Alpha6 integrin (a membrane receptor of laminin, the principal active component of the mammary ECM) was found at the surface of cells cultured on collagen but not on plastic. In cells cultured on collagen in the presence of anti-alpha6 integrin antibody, Prl-induced transcription of the endogenous alpha s1-casein gene was significantly reduced, without modifying C/EBPs and STAT5. Besides, histone H4 acetylation was reduced. Thus, we propose that the ECM regulates rabbit alpha s1-casein protein expression by local modification of chromatin structure, independently of STAT5 and C/EBPs.

  3. Intrinsic HER4/4ICD transcriptional activation domains are required for STAT5A activated gene expression.

    PubMed

    Han, Wen; Sfondouris, Mary E; Semmes, Eleanor C; Meyer, Alicia M; Jones, Frank E

    2016-10-30

    The epidermal growth factor receptor family member HER4 undergoes proteolytic processing at the cell surface to release the HER4 intracellular domain (4ICD) nuclear protein. Interestingly, 4ICD directly interacts with STAT5 and functions as an obligate STAT5 nuclear chaperone. Once in the nucleus 4ICD binds with STAT5 at STAT5 target genes, dramatically potentiating STAT5 transcriptional activation. These observations raise the possibility that 4ICD directly coactivates STAT5 gene expression. Using both yeast and mammalian transactivation reporter assays, we performed truncations of 4ICD fused to a GAL4 DNA binding domain and identified two independent 4ICD transactivation domains located between residues 1022 and 1090 (TAD1) and 1192 and 1225 (TAD2). The ability of the 4ICD DNA binding domain fusions to transactivate reporter gene expression required deletion of the intrinsic tyrosine kinase domain. In addition, we identified the 4ICD carboxyl terminal TVV residues, a PDZ domain binding motif (PDZ-DBM), as a potent transcriptional repressor. The transactivation activity of the HER4 carboxyl terminal domain lacking the tyrosine kinase (CTD) was significantly lower than similar EGFR or HER2 CTD. However, deletion of the HER4 CTD PDZ-DBM enhanced HER4 CTD transactivation to levels equivalent to the EGFR and HER2 CTDs. To determine if 4ICD TAD1 and TAD2 have a physiologically relevant role in STAT5 transactivation, we coexpressed 4ICD or 4ICD lacking TAD2 or both TAD1 and TAD2 with STAT5 in a luciferase reporter assay. Our results demonstrate that each 4ICD TAD contributes additively to STAT5A transactivation and the ability of STAT5A to transactivate the β-casein promoter requires the 4ICD TADs. Taken together, published data and our current results demonstrate that both 4ICD nuclear chaperone and intrinsic coactivation activities are essential for STAT5 regulated gene expression. PMID:27502417

  4. Distinct roles of STAT3 and STAT5 in the pathogenesis and targeted therapy of breast cancer

    PubMed Central

    Walker, Sarah R.; Xiang, Michael; Frank, David A.

    2013-01-01

    The transcription factors STAT3 and STAT5 play important roles in the regulation of mammary gland function during pregnancy, lactation, and involution. Given that STAT3 and STAT5 regulate genes involved in proliferation and survival, it is not surprising that inappropriate activation of STAT3 and STAT5 occurs commonly in breast cancer. Although these proteins are structurally similar, they have divergent and opposing effects on gene expression and cellular phenotype. Notably, when STAT5 and STAT3 are activated simultaneously, STAT5 has a dominant effect, and leads to decreased proliferation and increased sensitivity to cell death. Similarly, in breast cancer, activation of both STAT5 and STAT3 is associated with longer patient survival than activation of STAT3 alone. Pharmacological inhibitors of STAT3 and STAT5 are being developed for cancer therapy, though understanding the activation state and functional interaction of STAT3 and STAT5 in a patient's tumor may be critical for the optimal use of this strategy. PMID:23531638

  5. Differential expression of STAT5 and Bcl-xL, and high expression of Neu and STAT3 in non-small-cell lung carcinoma.

    PubMed

    Sánchez-Ceja, S G; Reyes-Maldonado, E; Vázquez-Manríquez, M E; López-Luna, J J; Belmont, A; Gutiérrez-Castellanos, S

    2006-11-01

    Experimental evidence suggests that in lung cancer, development, progression and an increased proliferation rate can be linked to apoptosis-related factors. The objective of this study is to evaluate the status of Neu, signal transducer and activator of transcription (STAT)-3, STAT5 and Bcl-xL expression in non-small-cell lung cancer. We investigated the immunohistochemical expression of these proteins in 92 non-small-cell lung cancer specimens to establish their role in lung cancer pathogenesis. Neu was overexpressed in 65% of cases, and although STAT3 was overexpressed in 52.1% in cytoplasm, it was expressed in nucleus (activated) in 60.8%. Meanwhile, STAT5 was found overexpressed in 41.3% in cytoplasm and 32.6% in nucleus. Thus, Bcl-xL was overexpressed in cytoplasm in 81.5%. Interestingly, we found nuclear expression of Bcl-xL in 30.4% of cases. Finally, we found correlation among histological types of lung cancer and nuclear expression of both STAT5 (P=0.005) and nuclear Bcl-xL (P=0.003). Besides, nuclear expression of Bcl-xL was correlated with TNM stage IV (distant metastasis) (P=0.02). These results suggest for the first time, a relevant role for STAT5 and Bcl-xL as apoptosis-regulatory proteins in the pathogenesis of lung cancer, and overexpression of both Neu and activated STAT3, could be related with the proliferation rate in lung carcinoma cells.

  6. NF-κB/Rel, not STAT5, regulates nitric oxide synthase transcription in Apostichopus japonicus.

    PubMed

    Shao, Yina; Wang, Zhenhui; Lv, Zhimeng; Li, Chenghua; Zhang, Weiwei; Li, Ye; Duan, Xuemei

    2016-08-01

    Nitric oxide (NO) is an important signaling molecular in the immune system of all vertebrates and invertebrates for pathologic and physiologic process, and it is largely produced by inducible nitric oxide synthase (iNOS). To uncover key mechanisms regulating NOS expression in sea cucumber Apostichopus japonicus, we amplified a fragment of the NOS promoter by genome walking approach and characterized putative transcription factor binding motifs using luciferase assay. Transient transfection of EPC cells using 5'-deletion constructs linked to luciferase reporter revealed that the region -614/+39 contributed importantly to expression of the AjNOS gene, and the -614 bp of the 5'-flanking region of the AjNOS gene responded well to LPS. Analysis of the functional promoter region revealed the presence of two potential NF-κB (-375 bp to -366 bp, -76 bp to -67 bp) and three STAT binding sites (-284 bp to -276 bp, -95 bp to 87 bp, -81 bp to -73 bp). When luciferase reporter vector and expression vector co-transfected revealed that NF-κB/Rel, but not STAT5, activate the AjNOS promoter fragment. Furthermore, two truncated reporter vectors co-transfected with vector expressing NF-κB/Rel revealed that the first NF-κB binding site (-375 bp to -366 bp) was essential for the ability of this promoter to induce AjNOS transcription. In addition, blocking the AjRel by SN50 (NF-κB inhibitory peptide) depressed the AjNOS expression and NO production both in vivo and in vitro, respectively, revealing that AjRel might directly modulate AjNOS. All our findings confirmed that NF-κB dependent mechanisms regulating expression of AjNOS and suggested a means of linking NO production to the immune response. PMID:27005898

  7. The STAT5-GATA2 Pathway Is Critical in Basophil and Mast Cell Differentiation and Maintenance

    PubMed Central

    Li, Yapeng; Qi, Xiaopeng; Liu, Bing; Huang, Hua

    2015-01-01

    Transcription factor GATA2 plays critical roles in hematopoietic stem cell survival and proliferation, GMP differentiation, and basophil and mast cell differentiation. However, precise roles of GATA2 in basophil and mast cell differentiation and maintenance have not been delineated. We have identified GATA2 as an essential transcription factor in differentiation of newly identified common basophil and mast cell progenitors into basophils and mast cells. We observed Gata2 haploinsufficiency for mast cell differentiation but not for basophil differentiation. We examined the precise role of GATA2 in maintaining the expression of a wide range of genes that are important for performing basophil or mast cell functions. The effects of GATA2 on gene expression were broadly based. We demonstrated that GATA2 was required for maintaining Fcer1a mRNA and FcεRIα protein expression on both basophils and mast cells as well as for maintaining Kit mRNA and c-Kit protein expression on mast cells. GATA2 was required for histamine synthesis and was also critical for Il4 mRNA expression in basophils and Il13 mRNA expression in mast cells. We demonstrate a STAT5-GATA2 connection, showing that the STAT5 transcription factor directly bound to the promoter and an intronic region of the Gata2 gene. Overexpression of the Gata2 gene was sufficient to direct basophil and mast cell differentiation in the absence of the Stat5 gene. Our study reveals that the STAT5-GATA2 pathway is critical for basophil and mast cell differentiation and maintenance. PMID:25801432

  8. Evaluation of STAT5A Gene Expression in Aflatoxin B1 Treated Bovine Mammary Epithelial Cells

    PubMed Central

    Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

    2013-01-01

    Purpose: Aflatoxin B1 (AFB1) is a potent mycotoxin which has been produced by fungi such as Aspergillus flavus and Aspergillus parasiticus as secondary metabolites due to their growth on food stuffs and induces hepatocellular carcinoma in many animal species, including humans. In the present study, the effect of AFB1 on STAT5A gene expression was investigated in bovine mammary epithelial cells using real time RT-PCR. Methods: Bovine mammary epithelial cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, cells were treated with AFB1 and incubated for 8 h. For real time PCR reaction, total RNA from the cultured and treated cells was extracted and used for complementary DNA synthesis. Results: The expression of STAT5A gene was significantly down regulated by AFB1 in dose- dependent manner and led to the reduction of proliferation and differentiation of epithelial cells, which has direct effect in milk protein quantity and quality. Conclusion: According to the results, it seems that down regulation of STAT5A gene in AFB1-treated cells maybe due to DNA damage induced by AFB1 in bovine mammary epithelial cells. PMID:24312879

  9. Hierarchy within the mammary STAT5-driven Wap super-enhancer.

    PubMed

    Shin, Ha Youn; Willi, Michaela; Yoo, Kyung Hyun; Zeng, Xianke; Wang, Chaochen; Metser, Gil; Hennighausen, Lothar

    2016-08-01

    Super-enhancers comprise dense transcription factor platforms highly enriched for active chromatin marks. A paucity of functional data led us to investigate the role of super-enhancers in the mammary gland, an organ characterized by exceptional gene regulatory dynamics during pregnancy. ChIP-seq analysis for the master regulator STAT5A, the glucocorticoid receptor, H3K27ac and MED1 identified 440 mammary-specific super-enhancers, half of which were associated with genes activated during pregnancy. We interrogated the Wap super-enhancer, generating mice carrying mutations in STAT5-binding sites within its constituent enhancers. Individually, the most distal site displayed the greatest enhancer activity. However, combinatorial mutation analysis showed that the 1,000-fold induction in gene expression during pregnancy relied on all enhancers. Disabling the binding sites of STAT5, NFIB and ELF5 in the proximal enhancer incapacitated the entire super-enhancer. Altogether, these data suggest a temporal and functional enhancer hierarchy. The identification of mammary-specific super-enhancers and the mechanistic exploration of the Wap locus provide insights into the regulation of cell-type-specific expression of hormone-sensing genes. PMID:27376239

  10. Loss of EZH2 results in precocious mammary gland development and activation of STAT5-dependent genes

    PubMed Central

    Yoo, Kyung Hyun; Oh, Sumin; Kang, Keunsoo; Hensel, Tim; Robinson, Gertraud W.; Hennighausen, Lothar

    2015-01-01

    Establishment and differentiation of mammary alveoli during pregnancy are controlled by prolactin through the transcription factors STAT5A and STAT5B (STAT5), which also regulate temporal activation of mammary signature genes. This study addressed the question whether the methyltransferase and transcriptional co-activator EZH2 controls the differentiation clock of mammary epithelium. Ablation of Ezh2 from mammary stem cells resulted in precocious differentiation of alveolar epithelium during pregnancy and the activation of mammary-specific STAT5 target genes. This coincided with enhanced occupancy of these loci by STAT5, EZH1 and RNA Pol II. Limited activation of differentiation-specific genes was observed in mammary epithelium lacking both EZH2 and STAT5, suggesting a modulating but not mandatory role for STAT5. Loss of EZH2 did not result in overt changes in genome-wide and gene-specific H3K27me3 profiles, suggesting compensation through enhanced EZH1 recruitment. Differentiated mammary epithelia did not form in the combined absence of EZH1 and EZH2. Transplantation experiments failed to demonstrate a role for EZH2 in the activity of mammary stem and progenitor cells. In summary, while EZH1 and EZH2 serve redundant functions in the establishment of H3K27me3 marks and the formation of mammary alveoli, the presence of EZH2 is required to control progressive differentiation of milk secreting epithelium during pregnancy. PMID:26250110

  11. SINGLE CELL GENOME SEQUENCING

    PubMed Central

    Yilmaz, Suzan; Singh, Anup K.

    2011-01-01

    Whole genome amplification and next-generation sequencing of single cells has become a powerful approach for studying uncultivated microorganisms that represent 90–99 % of all environmental microbes. Single cell sequencing enables not only the identification of microbes but also linking of functions to species, a feat not achievable by metagenomic techniques. Moreover, it allows the analysis of low abundance species that may be missed in community-based analyses. It has also proved very useful in complementing metagenomics in the assembly and binning of single genomes. With the advent of drastically cheaper and higher throughput sequencing technologies, it is expected that single cell sequencing will become a standard tool in studying the genome and transcriptome of microbial communities. PMID:22154471

  12. Diphlorethohydroxycarmalol Inhibits Interleukin-6 Production by Regulating NF-κB, STAT5 and SOCS1 in Lipopolysaccharide-Stimulated RAW264.7 Cells

    PubMed Central

    Kang, Na-Jin; Han, Sang-Chul; Kang, Gyeoung-Jin; Koo, Dong-Hwan; Koh, Young-Sang; Hyun, Jin-Won; Lee, Nam-Ho; Ko, Mi-Hee; Kang, Hee-Kyoung; Yoo, Eun-Sook

    2015-01-01

    Diphlorethohydroxycarmalol (DPHC) is a phlorotannin compound isolated from Ishige okamuarae, a brown alga. This study was conducted to investigate the anti-inflammatory effect and action mechanism of DPHC in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We found that DPHC strongly reduces the production of interleukin 6 (IL-6), but not that of tumor necrosis factor-alpha (TNF-α) induced by LPS. DPHC (12.5 and 100 μM) suppressed the phosphorylation and the nuclear translocation of NF-kappaB (NF-κB), a central signaling molecule in the inflammation process induced by LPS. The suppressor of cytokine signaling 1 (SOCS1) is a negative feedback regulator of Janus kinase (Jak)-signal transducer and activator of transcription (STAT) signaling. In this study, DPHC inhibited STAT5 expression and upregulated that of SOCS1 at a concentration of 100 μM. Furthermore, N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (a specific NF-κB inhibitor) and JI (a specific Jak2 inhibitor) reduced the production of IL-6, but not that of tumor necrosis factor-alpha (TNF-α) in LPS-stimulated RAW 264.7 macrophages. These findings demonstrate that DPHC inhibits IL-6 production via the downregulation of NF-κB and Jak2-STAT5 pathway and upregulation of SOCS1. PMID:25871292

  13. Single Cell Oncogenesis

    NASA Astrophysics Data System (ADS)

    Lu, Xin

    It is believed that cancer originates from a single cell that has gone through generations of evolution of genetic and epigenetic changes that associate with the hallmarks of cancer. In some cancers such as various types of leukemia, cancer is clonal. Yet in other cancers like glioblastoma (GBM), there is tremendous tumor heterogeneity that is likely to be caused by simultaneous evolution of multiple subclones within the same tissue. It is obvious that understanding how a single cell develops into a clonal tumor upon genetic alterations, at molecular and cellular levels, holds the key to the real appreciation of tumor etiology and ultimate solution for therapeutics. Surprisingly very little is known about the process of spontaneous tumorigenesis from single cells in human or vertebrate animal models. The main reason is the lack of technology to track the natural process of single cell changes from a homeostatic state to a progressively cancerous state. Recently, we developed a patented compound, photoactivatable (''caged'') tamoxifen analogue 4-OHC and associated technique called optochemogenetic switch (OCG switch), which we believe opens the opportunity to address this urgent biological as well as clinical question about cancer. We propose to combine OCG switch with genetically engineered mouse models of head and neck squamous cell carcinoma and high grade astrocytoma (including GBM) to study how single cells, when transformed through acute loss of tumor suppressor genes PTEN and TP53 and gain of oncogenic KRAS, can develop into tumor colonies with cellular and molecular heterogeneity in these tissues. The abstract is for my invited talk in session ``Beyond Darwin: Evolution in Single Cells'' 3/18/2016 11:15 AM.

  14. MiR-21 is under control of STAT5 but is dispensable for mammary development and lactation.

    PubMed

    Feuermann, Yonatan; Kang, Keunsoo; Shamay, Avi; Robinson, Gertraud W; Hennighausen, Lothar

    2014-01-01

    Development of mammary alveolar epithelium during pregnancy is controlled by prolactin, through the transcription factors STAT5A/B that activate specific sets of target genes. Here we asked whether some of STAT5's functions are mediated by microRNAs. The miR-21 promoter sequence contains a bona-fide STAT5 binding site and miR-21 levels increased in HC11 mammary cells upon prolactin treatment. In vivo miR-21 was abundantly expressed in mammary epithelium at day 6 of pregnancy. Analysis of mice lacking miR-21 revealed that their mammary tissue developed normally during pregnancy and dams were able to nurse their pups. Our study demonstrated that although expression of miR-21 is under prolactin control through the transcription factors STAT5A/B its presence is dispensable for mammary development and lactation. PMID:24497923

  15. MiR-21 Is under Control of STAT5 but Is Dispensable for Mammary Development and Lactation

    PubMed Central

    Feuermann, Yonatan; Kang, Keunsoo; Shamay, Avi; Robinson, Gertraud W.; Hennighausen, Lothar

    2014-01-01

    Development of mammary alveolar epithelium during pregnancy is controlled by prolactin, through the transcription factors STAT5A/B that activate specific sets of target genes. Here we asked whether some of STAT5's functions are mediated by microRNAs. The miR-21 promoter sequence contains a bona-fide STAT5 binding site and miR-21 levels increased in HC11 mammary cells upon prolactin treatment. In vivo miR-21 was abundantly expressed in mammary epithelium at day 6 of pregnancy. Analysis of mice lacking miR-21 revealed that their mammary tissue developed normally during pregnancy and dams were able to nurse their pups. Our study demonstrated that although expression of miR-21 is under prolactin control through the transcription factors STAT5A/B its presence is dispensable for mammary development and lactation. PMID:24497923

  16. Spatiotemporally controlled single cell sonoporation

    PubMed Central

    Fan, Zhenzhen; Liu, Haiyan; Mayer, Michael; Deng, Cheri X.

    2012-01-01

    This paper presents unique approaches to enable control and quantification of ultrasound-mediated cell membrane disruption, or sonoporation, at the single-cell level. Ultrasound excitation of microbubbles that were targeted to the plasma membrane of HEK-293 cells generated spatially and temporally controlled membrane disruption with high repeatability. Using whole-cell patch clamp recording combined with fluorescence microscopy, we obtained time-resolved measurements of single-cell sonoporation and quantified the size and resealing rate of pores. We measured the intracellular diffusion coefficient of cytoplasmic RNA/DNA from sonoporation-induced transport of an intercalating fluorescent dye into and within single cells. We achieved spatiotemporally controlled delivery with subcellular precision and calcium signaling in targeted cells by selective excitation of microbubbles. Finally, we utilized sonoporation to deliver calcein, a membrane-impermeant substrate of multidrug resistance protein-1 (MRP1), into HEK-MRP1 cells, which overexpress MRP1, and monitored the calcein efflux by MRP1. This approach made it possible to measure the efflux rate in individual cells and to compare it directly to the efflux rate in parental control cells that do not express MRP1. PMID:23012425

  17. The STAT5 Inhibitor Pimozide Displays Efficacy in Models of Acute Myelogenous Leukemia Driven by FLT3 Mutations

    PubMed Central

    Nelson, Erik A.; Walker, Sarah R.; Xiang, Michael; Weisberg, Ellen; Bar-Natan, Michal; Barrett, Rosemary; Liu, Suiyang; Kharbanda, Surender; Christie, Amanda L.; Nicolais, Maria; Griffin, James D.; Stone, Richard M.; Kung, Andrew L.

    2012-01-01

    Activation of the transcription factor STAT5 is essential for the pathogenesis of acute myelogenous leukemia (AML) containing the FLT3 internal tandem duplication (ITD) mutation. FLT3 ITD is a constitutively active tyrosine kinase that drives the activation of STAT5, leading to the growth and survival of AML cells. Although there has been some success in identifying tyrosine kinase inhibitors that block the function of FLT3 ITD, there remains a continued need for effective treatment of this disease. We have identified the psychotropic drug pimozide as an effective inhibitor of STAT5 function. Pimozide inhibits the tyrosine phosphorylation of STAT5, leading to the death of AML cells through the induction of apoptosis. Pimozide shows a combinatorial effect with the tyrosine kinase inhibitors midostaurin (PKC412) and sunitinib in the inhibition of STAT5 tyrosine phosphorylation and the induction of apoptosis. Significantly, pimozide reduces the tumor burden in a mouse model of FLT3-driven AML. Therefore, identifying STAT5 inhibitors may provide a new avenue for the treatment of AML, and these may be effective alone or in combination with tyrosine kinase inhibitors. PMID:23264850

  18. Mutations of the tumor suppressor gene SOCS-1 in classical Hodgkin lymphoma are frequent and associated with nuclear phospho-STAT5 accumulation.

    PubMed

    Weniger, M A; Melzner, I; Menz, C K; Wegener, S; Bucur, A J; Dorsch, K; Mattfeldt, T; Barth, T F E; Möller, P

    2006-04-27

    The suppressors of cytokine signaling (SOCS) are critically involved in the regulation of cellular proliferation, survival, and apoptosis via cytokine-induced JAK/STAT signaling. SOCS-1 silencing by aberrant DNA methylation contributes to oncogenesis in various B-cell neoplasias and carcinomas. Recently, we showed an alternative loss of SOCS-1 function due to deleterious SOCS-1 mutations in a major subset of primary mediastinal B-cell lymphoma (PMBL) and in the PMBL line MedB-1, and a biallelic SOCS-1 deletion in PMBL line Karpas1106P. For both cell lines our previous data demonstrated retarded JAK2 degradation and sustained phospho-JAK2 action leading to enhanced DNA binding of phospho-STAT5. Here, we analysed SOCS-1 in laser-microdissected Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). We detected SOCS-1 mutations in HRS cells of eight of 19 cHL samples and in three of five Hodgkin lymphoma (HL)-derived cell lines by sequencing analysis. Moreover, we found a significant association between mutated SOCS-1 of isolated HRS cells and nuclear phospho-STAT5 accumulation in HRS cells of cHL tumor tissue (P < 0.01). Collectively, these findings support the concept that PMBL and cHL share many overlapping features, and that defective tumor suppressor gene SOCS-1 triggers an oncogenic pathway operative in both lymphomas. PMID:16532038

  19. Diverse phosphorylation patterns of B cell receptor-associated signaling in naïve and memory human B cells revealed by phosphoflow, a powerful technique to study signaling at the single cell level

    PubMed Central

    Toapanta, Franklin R.; Bernal, Paula J.; Sztein, Marcelo B.

    2012-01-01

    Following interaction with cognate antigens, B cells undergo cell activation, proliferation, and differentiation. Ligation of the B cell receptor (BCR) leads to the phosphorylation of BCR-associated signaling proteins within minutes of antigen binding, a process with profound consequences for the fate of the cells and development of effector immunity. Phosphoflow allows a rapid evaluation of various signaling pathways in complex heterogenous cell subsets. This novel technique was used in combination with multi-chromatic flow cytometry (FC) and fluorescent-cell barcoding (FCB) to study phosphorylation of BCR-associated signaling pathways in naïve and memory human B cell subsets. Proteins of the initiation (Syk), propagation (Btk, Akt), and integration (p38MAPK and Erk1/2) signaling units were studied. Switched memory (Sm) CD27+ and Sm CD27− phosphorylation patterns were similar when stimulated with anti-IgA or -IgG. In contrast, naïve and unswitched memory (Um) cells showed significant differences following IgM stimulation. Enhanced phosphorylation of Syk was observed in Um cells, suggesting a lower activation threshold. This is likely the result of higher amounts of IgM on the cell surface, higher pan-Syk levels, and enhanced susceptibility to phosphatase inhibition. All other signaling proteins evaluated also showed some degree of enhanced phosphorylation in Um cells. Furthermore, both the phospholipase C-γ2 (PLC-γ2) and phosphatidylinositol 3-kinase (PI3K) pathways were activated in Um cells, while only the PI3K pathway was activated on naïve cells. Um cells were the only ones that activated signaling pathways when stimulated with fluorescently labeled S. Typhi and S. pneumoniae. Finally, simultaneous evaluation of signaling proteins at the single cell level (multiphosphorylated cells) revealed that interaction with gram positive and negative bacteria resulted in complex and diverse signaling patterns. Phosphoflow holds great potential to accelerate

  20. Models at the Single Cell Level

    PubMed Central

    Cheong, Raymond; Paliwal, Saurabh

    2014-01-01

    Many cellular behaviors cannot be completely captured or appropriately described at the cell population level. Noise induced by stochastic chemical reactions, spatially polarized signaling networks and heterogeneous cell-cell communication are among the many phenomena that require fine-grained analysis. Accordingly, the mathematical models used to describe such systems must be capable of single cell or subcellular resolution. Here, we review techniques for modeling single cells, including models of stochastic chemical kinetics, spatially heterogeneous intracellular signaling, and spatial stochastic systems. We also briefly discuss applications of each type of model. PMID:20836009

  1. Single cell heterogeneity

    PubMed Central

    Abdallah, Batoul Y; Horne, Steven D; Stevens, Joshua B; Liu, Guo; Ying, Andrew Y; Vanderhyden, Barbara; Krawetz, Stephen A; Gorelick, Root; Heng, Henry HQ

    2013-01-01

    Multi-level heterogeneity is a fundamental but underappreciated feature of cancer. Most technical and analytical methods either completely ignore heterogeneity or do not fully account for it, as heterogeneity has been considered noise that needs to be eliminated. We have used single-cell and population-based assays to describe an instability-mediated mechanism where genome heterogeneity drastically affects cell growth and cannot be accurately measured using conventional averages. First, we show that most unstable cancer cell populations exhibit high levels of karyotype heterogeneity, where it is difficult, if not impossible, to karyotypically clone cells. Second, by comparing stable and unstable cell populations, we show that instability-mediated karyotype heterogeneity leads to growth heterogeneity, where outliers dominantly contribute to population growth and exhibit shorter cell cycles. Predictability of population growth is more difficult for heterogeneous cell populations than for homogenous cell populations. Since “outliers” play an important role in cancer evolution, where genome instability is the key feature, averaging methods used to characterize cell populations are misleading. Variances quantify heterogeneity; means (averages) smooth heterogeneity, invariably hiding it. Cell populations of pathological conditions with high genome instability, like cancer, behave differently than karyotypically homogeneous cell populations. Single-cell analysis is thus needed when cells are not genomically identical. Despite increased attention given to single-cell variation mediated heterogeneity of cancer cells, continued use of average-based methods is not only inaccurate but deceptive, as the “average” cancer cell clearly does not exist. Genome-level heterogeneity also may explain population heterogeneity, drug resistance, and cancer evolution. PMID:24091732

  2. Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen.

    PubMed

    Deng, Dawei; Li, Yang; Xue, Jianpeng; Wang, Jie; Ai, Guanhua; Li, Xin; Gu, Yueqing

    2015-01-01

    Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem-loop-stem oligonucleotide sequences attached by Au-S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.

  3. Single Cell Physiology

    NASA Astrophysics Data System (ADS)

    Neveu, Pierre; Sinha, Deepak Kumar; Kettunen, Petronella; Vriz, Sophie; Jullien, Ludovic; Bensimon, David

    The possibility to control at specific times and specific places the activity of biomolecules (enzymes, transcription factors, RNA, hormones, etc.) is opening up new opportunities in the study of physiological processes at the single cell level in a live organism. Most existing gene expression systems allow for tissue specific induction upon feeding the organism with exogenous inducers (e.g., tetracycline). Local genetic control has earlier been achieved by micro-injection of the relevant inducer/repressor molecule, but this is an invasive and possibly traumatic technique. In this chapter, we present the requirements for a noninvasive optical control of the activity of biomolecules and review the recent advances in this new field of research.

  4. Single cell optical transfection.

    PubMed

    Stevenson, David J; Gunn-Moore, Frank J; Campbell, Paul; Dholakia, Kishan

    2010-06-01

    The plasma membrane of a eukaryotic cell is impermeable to most hydrophilic substances, yet the insertion of these materials into cells is an extremely important and universal requirement for the cell biologist. To address this need, many transfection techniques have been developed including viral, lipoplex, polyplex, capillary microinjection, gene gun and electroporation. The current discussion explores a procedure called optical injection, where a laser field transiently increases the membrane permeability to allow species to be internalized. If the internalized substance is a nucleic acid, such as DNA, RNA or small interfering RNA (siRNA), then the process is called optical transfection. This contactless, aseptic, single cell transfection method provides a key nanosurgical tool to the microscopist-the intracellular delivery of reagents and single nanoscopic objects. The experimental possibilities enabled by this technology are only beginning to be realized. A review of optical transfection is presented, along with a forecast of future applications of this rapidly developing and exciting technology. PMID:20064901

  5. Structure-based screen identifies a potent small-molecule inhibitor of Stat5a/b with therapeutic potential for prostate cancer and chronic myeloid leukemia

    PubMed Central

    Liao, Zhiyong; Gu, Lei; Vergalli, Jenny; Mariani, Samanta A.; De Dominici, Marco; Lokareddy, Ravi K.; Dagvadorj, Ayush; Purushottamachar, Puranik; McCue, Peter A.; Trabulsi, Edouard; Lallas, Costas D.; Gupta, Shilpa; Ellsworth, Elyse; Blackmon, Shauna; Ertel, Adam; Fortina, Paolo; Leiby, Benjamin; Xia, Guanjun; Rui, Hallgeir; Hoang, David T.; Gomella, Leonard G.; Cingolani, Gino; Njar, Vincent; Pattabiraman, Nagarajan; Calabretta, Bruno; Nevalainen, Marja T.

    2015-01-01

    Bypassing tyrosine kinases responsible for Stat5a/b phosphorylation would be advantageous for therapy development for Stat5a/b-regulated cancers. Here, we sought to identify small-molecule inhibitors of Stat5a/b for lead optimization and therapy development for prostate cancer (PC) and Bcr-Abl-driven leukemias. In silico screening of chemical structure databases combined with medicinal chemistry was used for identification of a panel of small-molecule inhibitors to block SH2-domain-mediated docking of Stat5a/b to the receptor-kinase complex and subsequent phosphorylation and dimerization. We tested the efficacy of the lead-compound, IST5-002, in experimental models and patient samples of two known Stat5a/b-driven cancers, prostate cancer (PC) and chronic myeloid leukemia (CML). The lead compound Inhibitor of Stat5-002 (IST5-002) prevented both Jak2 and Bcr-Abl-mediated phosphorylation and dimerization of Stat5a/b, and selectively inhibited transcriptional activity of Stat5a (IC50 1.5 μM) and Stat5b (IC50 3.5 μM). IST5-002 suppressed nuclear translocation of Stat5a/b, binding to DNA and Stat5a/b target gene expression. IST5-002 induced extensive apoptosis of PC cells, impaired growth of PC xenograft tumors and induced cell death in patient-derived PCs when tested ex vivo in explant organ cultures. Importantly, IST5-002 induced robust apoptotic death not only of imatinib-sensitive but also imatinib-resistant chronic myeloid leukemia (CML) cell lines and primary CML cells from patients. IST5-002 provides a lead structure for further chemical modifications for clinical development for Stat5a/b-driven solid tumors and hematological malignancies. PMID:26026053

  6. Coffee Polyphenols Change the Expression of STAT5B and ATF-2 Modifying Cyclin D1 Levels in Cancer Cells

    PubMed Central

    Oleaga, Carlota; Ciudad, Carlos J.; Noé, Véronique; Izquierdo-Pulido, Maria

    2012-01-01

    Background. Epidemiological studies suggest that coffee consumption reduces the risk of cancer, but the molecular mechanisms of its chemopreventive effects remain unknown. Objective. To identify differentially expressed genes upon incubation of HT29 colon cancer cells with instant caffeinated coffee (ICC) or caffeic acid (CA) using whole-genome microarrays. Results. ICC incubation of HT29 cells caused the overexpression of 57 genes and the underexpression of 161, while CA incubation induced the overexpression of 12 genes and the underexpression of 32. Using Venn-Diagrams, we built a list of five overexpressed genes and twelve underexpressed genes in common between the two experimental conditions. This list was used to generate a biological association network in which STAT5B and ATF-2 appeared as highly interconnected nodes. STAT5B overexpression was confirmed at the mRNA and protein levels. For ATF-2, the changes in mRNA levels were confirmed for both ICC and CA, whereas the decrease in protein levels was only observed in CA-treated cells. The levels of cyclin D1, a target gene for both STAT5B and ATF-2, were downregulated by CA in colon cancer cells and by ICC and CA in breast cancer cells. Conclusions. Coffee polyphenols are able to affect cyclin D1 expression in cancer cells through the modulation of STAT5B and ATF-2. PMID:22919439

  7. Depletion of STAT5 blocks TEL-SYK-induced APMF-type leukemia with myelofibrosis and myelodysplasia in mice.

    PubMed

    Sprissler, C; Belenki, D; Maurer, H; Aumann, K; Pfeifer, D; Klein, C; Müller, T A; Kissel, S; Hülsdünker, J; Alexandrovski, J; Brummer, T; Jumaa, H; Duyster, J; Dierks, C

    2014-08-22

    The spleen tyrosine kinase (SYK) was identified as an oncogenic driver in a broad spectrum of hematologic malignancies. The in vivo comparison of three SYK containing oncogenes, SYK(wt), TEL-SYK and IL-2-inducible T-cell kinase (ITK)-SYK revealed a general myeloexpansion and the establishment of three different hematologic (pre)diseases. SYK(wt) enhanced the myeloid and T-cell compartment, without leukemia/lymphoma development. ITK-SYK caused lethal T-cell lymphomas and the cytoplasmic TEL-SYK fusion induced an acute panmyelosis with myelofibrosis-type acute myeloid leukemia (AML) with up to 50% immature megakaryoblasts infiltrating bone marrow, spleen and liver, additional MPN features (myelofibrosis and granulocyte expansion) and MDS stigmata with megakaryocytic and erythroid dysplasia. LKS cells were reduced and all subsets (LT/ST/MPP) showed reduced proliferation rates. SYK inhibitor treatment (R788) of diseased TEL-SYK mice reduced leukocytosis, spleen and liver infiltration, enhanced the hematocrit and prolonged survival time, but could not significantly reduce myelofibrosis. Stat5 was identified as a major downstream mediator of TEL-SYK in vitro as well as in vivo. Consequently, targeted deletion of Stat5 in vivo completely abrogated TEL-SYK-induced AML and myelofibrosis development, proving Stat5 as a major driver of SYK-induced transformation. Our experiments highlight the important role of SYK in AML and myelofibrosis and prove SYK and STAT5 inhibitors as potent treatment options for those diseases.

  8. Identification of CD25 as STAT5-Dependent Growth-Regulator of Leukemic Stem Cells in Ph+ CML

    PubMed Central

    Sadovnik, Irina; Hoelbl-Kovacic, Andrea; Herrmann, Harald; Eisenwort, Gregor; Warsch, Wolfgang; Hoermann, Gregor; Greiner, Georg; Blatt, Katharina; Peter, Barbara; Stefanzl, Gabriele; Berger, Daniela; Bilban, Martin; Herndlhofer, Susanne; Sill, Heinz; Sperr, Wolfgang R.; Streubel, Berthold; Mannhalter, Christine; Holyoake, Tessa L.; Sexl, Veronika; Valent, Peter

    2015-01-01

    Purpose In chronic myeloid leukemia (CML), leukemic stem cells (LSCs) represent a critical target of therapy. However, little is known about markers and targets expressed by LSCs. The aim of this project was to identify novel interesting markers of CML LSCs. Experimental Design CML LSCs were examined by flow cytometry, qPCR, and various bioassays. In addition, we examined the multipotent CD25+ CML cell line KU812. Results In contrast to normal hematopoietic stem cells, CD34+/CD38− CML LSCs expressed the interleukin-2 receptor alpha chain, IL-2RA (CD25). STAT5 was found to induce expression of CD25 in Lin−/Sca-1+/Kit+ stem cells in C57Bl/6 mice. Correspondingly, shRNA-induced STAT5-depletion resulted in decreased CD25 expression in KU812 cells. Moreover, the BCR/ABL1 inhibitors nilotinib and ponatinib were found to decrease STAT5 activity and CD25 expression in KU812 cells and primary CML LSCs. A CD25-targeting shRNA was found to augment proliferation of KU812 cells in vitro and their engraftment in vivo in NOD/SCID-IL-2Rγ−/− mice. In drug-screening experiments, the PI3-Kinase/mTOR blocker BEZ235 promoted the expression of STAT5 and CD25 in CML cells. Finally, we found that BEZ235 produces synergistic anti-neoplastic effects on CML cells when applied in combination with nilotinib or ponatinib. Conclusion CD25 is a novel STAT5-dependent marker of CML LSCs and may be useful for LSC detection and LSC isolation in clinical practice and basic science. Moreover, CD25 serves as a growth-regulator of CML LSCs, which may have biological and clinical implications and may pave the way for the development of new more effective LSC-eradicating treatment strategies in CML. PMID:26607600

  9. Deletion of STAT5a/b in Vascular Smooth Muscle Abrogates the Male Bias in Hypoxic Pulmonary Hypertension in Mice: Implications in the Human Disease

    PubMed Central

    Yang, Yang-Ming; Yuan, Huijuan; Edwards, John G; Skayian, Yester; Ochani, Kanta; Miller, Edmund J; Sehgal, Pravin B

    2014-01-01

    Chronic hypoxia typically elicits pulmonary hypertension (PH) in mice with a male-dominant phenotype. There is an opposite-sex bias in human PH, with a higher prevalence in women, but greater survival (the “estrogen paradox”). We investigated the involvement of the STAT5a/b species, previously established to mediate sexual dimorphism in other contexts, in the sex bias in PH. Mice with heterozygous or homozygous deletions of the STAT5a/b locus in vascular smooth muscle cells (SMCs) were generated in crosses between STAT5a/bfl/fl and transgelin (SM22α)-Cre+/+ parents. Wild-type (wt ) males subjected to chronic hypoxia showed significant PH and pulmonary arterial remodeling, with wt females showing minimal changes (a male-dominant phenotype). However, in conditional STAT5+/− or STAT5−/− mice, hypoxic females showed the severest manifestations of PH (a female-dominant phenotype). Immunofluorescence studies on human lung sections showed that obliterative pulmonary arterial lesions in patients with idiopathic pulmonary arterial hypertension (IPAH) or hereditary pulmonary arterial hypertension (HPAH), both male and female, overall had reduced STAT5a/b, reduced PY-STAT5 and reduced endoplasmic reticulum (ER) GTPase atlastin-3 (ATL3). Studies of SMCs and endothelial cell (EC) lines derived from vessels isolated from lungs of male and female IPAH patients and controls revealed instances of coordinate reductions in STAT5a, STAT5b and ATL3 in IPAH-derived cells, including SMCs and ECs from the same patient. Taken together, these data provide the first definitive evidence for a contribution of STAT5a/b to the sex bias in PH in the hypoxic mouse and implicate reduced STAT5 in the pathogenesis of the human disease. PMID:25470773

  10. Digital microfluidic immunocytochemistry in single cells

    PubMed Central

    Ng, Alphonsus H. C.; Dean Chamberlain, M.; Situ, Haozhong; Lee, Victor; Wheeler, Aaron R.

    2015-01-01

    We report a new technique called Digital microfluidic Immunocytochemistry in Single Cells (DISC). DISC automates protocols for cell culture, stimulation and immunocytochemistry, enabling the interrogation of protein phosphorylation on pulsing with stimulus for as little as 3 s. DISC was used to probe the phosphorylation states of platelet-derived growth factor receptor (PDGFR) and the downstream signalling protein, Akt, to evaluate concentration- and time-dependent effects of stimulation. The high time resolution of the technique allowed for surprising new observations—for example, a 10 s pulse stimulus of a low concentration of PDGF is sufficient to cause >30% of adherent fibroblasts to commit to Akt activation. With the ability to quantitatively probe signalling events with high time resolution at the single-cell level, we propose that DISC may be an important new technique for a wide range of applications, especially for screening signalling responses of a heterogeneous cell population. PMID:26104298

  11. Chemical Analysis of Single Cells

    NASA Astrophysics Data System (ADS)

    Borland, Laura M.; Kottegoda, Sumith; Phillips, K. Scott; Allbritton, Nancy L.

    2008-07-01

    Chemical analysis of single cells requires methods for quickly and quantitatively detecting a diverse array of analytes from extremely small volumes (femtoliters to nanoliters) with very high sensitivity and selectivity. Microelectrophoretic separations, using both traditional capillary electrophoresis and emerging microfluidic methods, are well suited for handling the unique size of single cells and limited numbers of intracellular molecules. Numerous analytes, ranging from small molecules such as amino acids and neurotransmitters to large proteins and subcellular organelles, have been quantified in single cells using microelectrophoretic separation techniques. Microseparation techniques, coupled to varying detection schemes including absorbance and fluorescence detection, electrochemical detection, and mass spectrometry, have allowed researchers to examine a number of processes inside single cells. This review also touches on a promising direction in single cell cytometry: the development of microfluidics for integrated cellular manipulation, chemical processing, and separation of cellular contents.

  12. Single cell microfluidics for systems oncology

    NASA Astrophysics Data System (ADS)

    Fan, Rong

    2012-02-01

    The singular term ``cancer'' is never one kind of disease, but deceivingly encompasses a large number of heterogeneous disease states, which makes it impossible to completely treat cancer using a generic approach. Rather systems approaches are urgently required to assess cancer heterogeneity, stratify patients and enable the most effective, individualized treatment. The heterogeneity of tumors at the single cell level is reflected by the hierarchical complexity of the tumor microenvironment. To identify all the cellular components, including both tumor and infiltrating immune cells, and to delineate the associated cell-to-cell signaling network that dictates tumor initiation, progression and metastasis, we developed a single cell microfluidics chip that can analyze a panel of proteins that are potentially associated inter-cellular signaling network in tumor microenvironment from hundreds of single cells in parallel. This platform integrates two advanced technologies -- microfluidic single cell handling and ultra-high density protein array. This device was first tested for highly multiplexed profiling of secreted proteins including tumor-immune signaling molecules from monocytic leukemia cells. We observed profound cellular heterogeneity with all functional phenotypes quantitatively identified. Correlation analysis further indicated the existence of an intercellular cytokine network in which TNFα-induced secondary signaling cascades further increased functional cellular diversity. It was also exploited to evaluate polyfunctionality of tumor antigen-specific T cells from melanoma patients being treated with adoptive T cell transfer immunotherapy. This platform could be further extended to analyze both solid tumor cells (e.g. human lung carcinoma cells) and infiltrating immune cells (e.g. macrophages) so as to enable systems analysis of the complex tumor microenvironment from small amounts of clinical specimens, e.g. skinny needle biopsies. Thus, it could potentially

  13. Bovine prolactin elevates hTF expression directed by a tissue-specific goat β-casein promoter through prolactin receptor-mediated STAT5a activation.

    PubMed

    Jiang, Shizhong; Ren, Zhaorui; Xie, Fei; Yan, Jingbin; Huang, Shuzhen; Zeng, Yitao

    2012-11-01

    Prolactin promotes the expression of exogenous human transferrin gene in the milk of transgenic mice. To elucidate this, a recombinant plasmid of bovine prolactin plus human transferrin vector was co-transfected into cultured murine mammary gland epithelial cells. Prolactin-receptor antagonist and shRNA corresponding to prolactin-receptor mRNA were added into the cell culture mixture to investigate the relations between prolactin-receptor and human transferrin expression after bovine prolactin inducement. Levels of human transferrin in the supernatants were increased under the presentation of bovine prolactin (from 1,076 ± 115 to 1,886 ± 114 pg/ml). With the treatment of prolactin-receptor antagonist or shRNA, human transferrin in cells was declined (1,886 ± 113 vs. 1,233 ± 85 pg/ml or 1,114 ± 75 pg/ml, respectively). An inverse correlation was found between the dosage of prolactin-receptor antagonist and expression level of human transferrin. Real-time qRT-PCR analysis showed that the relative level of signal transducer and activator of transcription 5a (STAT5a) transcript in transfected cells correlated with expression levels of human transferrin in the supernatant of the same cells. Bovine prolactin thus improved the expression of human transferrin through such a possible mechanism that bovine prolactin activated STAT5a transcription expression via combined with prolactin-receptor and suggest a potential utility of the bovine prolactin for efficient expression of valuable pharmaceutical proteins in mammary glands of transgenic animals.

  14. Ultrasensitive proteomic quantitation of cellular signaling by digitized nanoparticle-protein counting

    PubMed Central

    Jacob, Thomas; Agarwal, Anupriya; Ramunno-Johnson, Damien; O’Hare, Thomas; Gönen, Mehmet; Tyner, Jeffrey W.; Druker, Brian J.; Vu, Tania Q.

    2016-01-01

    Many important signaling and regulatory proteins are expressed at low abundance and are difficult to measure in single cells. We report a molecular imaging approach to quantitate protein levels by digitized, discrete counting of nanoparticle-tagged proteins. Digitized protein counting provides ultrasensitive molecular detection of proteins in single cells that surpasses conventional methods of quantitating total diffuse fluorescence, and offers a substantial improvement in protein quantitation. We implement this digitized proteomic approach in an integrated imaging platform, the single cell-quantum dot platform (SC-QDP), to execute sensitive single cell phosphoquantitation in response to multiple drug treatment conditions and using limited primary patient material. The SC-QDP: 1) identified pAKT and pERK phospho-heterogeneity and insensitivity in individual leukemia cells treated with a multi-drug panel of FDA-approved kinase inhibitors, and 2) revealed subpopulations of drug-insensitive CD34+ stem cells with high pCRKL and pSTAT5 signaling in chronic myeloid leukemia patient blood samples. This ultrasensitive digitized protein detection approach is valuable for uncovering subtle but important differences in signaling, drug insensitivity, and other key cellular processes amongst single cells. PMID:27320899

  15. Single-cell Transcriptome Study as Big Data

    PubMed Central

    Yu, Pingjian; Lin, Wei

    2016-01-01

    The rapid growth of single-cell RNA-seq studies (scRNA-seq) demands efficient data storage, processing, and analysis. Big-data technology provides a framework that facilitates the comprehensive discovery of biological signals from inter-institutional scRNA-seq datasets. The strategies to solve the stochastic and heterogeneous single-cell transcriptome signal are discussed in this article. After extensively reviewing the available big-data applications of next-generation sequencing (NGS)-based studies, we propose a workflow that accounts for the unique characteristics of scRNA-seq data and primary objectives of single-cell studies. PMID:26876720

  16. Single-cell Transcriptome Study as Big Data.

    PubMed

    Yu, Pingjian; Lin, Wei

    2016-02-01

    The rapid growth of single-cell RNA-seq studies (scRNA-seq) demands efficient data storage, processing, and analysis. Big-data technology provides a framework that facilitates the comprehensive discovery of biological signals from inter-institutional scRNA-seq datasets. The strategies to solve the stochastic and heterogeneous single-cell transcriptome signal are discussed in this article. After extensively reviewing the available big-data applications of next-generation sequencing (NGS)-based studies, we propose a workflow that accounts for the unique characteristics of scRNA-seq data and primary objectives of single-cell studies. PMID:26876720

  17. Specificity of g protein-coupled receptor kinase 6-mediated phosphorylation and regulation of single-cell m3 muscarinic acetylcholine receptor signaling.

    PubMed

    Willets, Jonathon M; Mistry, Rajendra; Nahorski, Stefan R; Challiss, R A John

    2003-11-01

    Previously we have shown that G protein-coupled receptor kinase (GRK) 6 plays a major role in the regulation of the human M3 muscarinic acetylcholine receptor (M3 mAChR) in the human neuroblastoma SH-SY5Y. However, 30-fold overexpression of the catalytically inactive, dominant-negative K215RGRK6 produced only a 50% suppression of M3 mAChR phosphorylation and desensitization. Here, we have attempted to determine whether other endogenous kinases play a role in the regulation of M3 mAChR signaling. In contrast to the clear attenuating effect of K215RGRK6 expression on M3 mAChR regulation, dominant-negative forms of GRKs (K220RGRK2, K220RGRK3, K215RGRK5) and casein kinase 1alpha (K46RCK1alpha) were without effect. In addition, inhibition of a variety of second-messenger-regulated kinases and the tyrosine kinase Src also had no effect upon agonist-stimulated M3 mAChR regulation. To investigate further the desensitization process we have followed changes in inositol 1,4,5-trisphosphate in single SHSY5Y cells using the pleckstrin homology domain of PLCdelta1 tagged with green fluorescent protein (eGFP-PHPLCdelta1). Stimulation of cells with approximate EC50 concentrations of agonist before and after a desensitizing period of agonist exposure resulted in a marked attenuation of the latter response. Altered GRK6 activity, through overexpression of wild-type GRK6 or K215RGRK6, enhanced or reduced the degree of M3 mAChR desensitization, respectively. Taken together, our data indicate that M3 mAChR desensitization is mediated by GRK6 in human SH-SY5Y cells, and we show that receptor desensitization of phospholipase C signaling can be monitored in 'real-time' in single, living cells. PMID:14573754

  18. Single Cell Electrical Characterization Techniques.

    PubMed

    Mansor, Muhammad Asraf; Ahmad, Mohd Ridzuan

    2015-01-01

    Electrical properties of living cells have been proven to play significant roles in understanding of various biological activities including disease progression both at the cellular and molecular levels. Since two decades ago, many researchers have developed tools to analyze the cell's electrical states especially in single cell analysis (SCA). In depth analysis and more fully described activities of cell differentiation and cancer can only be accomplished with single cell analysis. This growing interest was supported by the emergence of various microfluidic techniques to fulfill high precisions screening, reduced equipment cost and low analysis time for characterization of the single cell's electrical properties, as compared to classical bulky technique. This paper presents a historical review of single cell electrical properties analysis development from classical techniques to recent advances in microfluidic techniques. Technical details of the different microfluidic techniques are highlighted, and the advantages and limitations of various microfluidic devices are discussed. PMID:26053399

  19. Quantification of Circadian Rhythms in Single Cells

    PubMed Central

    Westermark, Pål O.; Welsh, David K.; Okamura, Hitoshi; Herzel, Hanspeter

    2009-01-01

    Bioluminescence techniques allow accurate monitoring of the circadian clock in single cells. We have analyzed bioluminescence data of Per gene expression in mouse SCN neurons and fibroblasts. From these data, we extracted parameters such as damping rate and noise intensity using two simple mathematical models, one describing a damped oscillator driven by noise, and one describing a self-sustained noisy oscillator. Both models describe the data well and enabled us to quantitatively characterize both wild-type cells and several mutants. It has been suggested that the circadian clock is self-sustained at the single cell level, but we conclude that present data are not sufficient to determine whether the circadian clock of single SCN neurons and fibroblasts is a damped or a self-sustained oscillator. We show how to settle this question, however, by testing the models' predictions of different phases and amplitudes in response to a periodic entrainment signal (zeitgeber). PMID:19956762

  20. Plant single-cell and single-cell-type metabolomics.

    PubMed

    Misra, Biswapriya B; Assmann, Sarah M; Chen, Sixue

    2014-10-01

    In conjunction with genomics, transcriptomics, and proteomics, plant metabolomics is providing large data sets that are paving the way towards a comprehensive and holistic understanding of plant growth, development, defense, and productivity. However, dilution effects from organ- and tissue-based sampling of metabolomes have limited our understanding of the intricate regulation of metabolic pathways and networks at the cellular level. Recent advances in metabolomics methodologies, along with the post-genomic expansion of bioinformatics knowledge and functional genomics tools, have allowed the gathering of enriched information on individual cells and single cell types. Here we review progress, current status, opportunities, and challenges presented by single cell-based metabolomics research in plants.

  1. Single-cell western blotting

    PubMed Central

    Hughes, Alex J.; Spelke, Dawn P.; Xu, Zhuchen; Kang, Chi-Chih; Schaffer, David V.; Herr, Amy E.

    2014-01-01

    To measure cell-to-cell variation in protein-mediated functions — a hallmark of biological processes — we developed an approach to conduct ~103 concurrent single-cell western blots (scWesterns) in ~4 hours. A microscope slide supporting a 30 µm-thick photoactive polyacrylamide gel enables western blotting comprised of: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins, and antibody probing. We apply this scWestern to monitor single rat neural stem cell differentiation and responses to mitogen stimulation. The scWestern quantifies target proteins even with off-target antibody binding, multiplexes to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supports analyses of low starting cell numbers (~200) when integrated with fluorescence activated cell sorting. The scWestern thus overcomes limitations in single-cell protein analysis (i.e., antibody fidelity, sensitivity, and starting cell number) and constitutes a versatile tool for the study of complex cell populations at single-cell resolution. PMID:24880876

  2. Signal transducer and activator of transcription 5 as a key signaling pathway in normal mammary gland developmental biology and breast cancer.

    PubMed

    Furth, Priscilla A; Nakles, Rebecca E; Millman, Sarah; Diaz-Cruz, Edgar S; Cabrera, M Carla

    2011-10-12

    STAT5 consists of two proteins, STAT5A/B, that impact mammary cell differentiation, proliferation, and survival. In normal development, STAT5 expression and activity are regulated by prolactin signaling with JAK2/ELF5, EGF signaling networks that include c-Src, and growth hormone, insulin growth factor, estrogen, and progesterone signaling pathways. In cancer, erythropoietin signaling can also regulate STAT5. Activation levels are influenced by AKT, caveolin, PIKE-A, Pak1, c-Myb, Brk, beta-integrin, dystroglycan, other STATs, and STAT pathway molecules JAK1, Shp2, and SOCS. TGF-β and PTPN9 can downregulate prolactin- and EGF-mediated STAT5 activation, respectively. IGF, AKT, RANKL, cyclin D1, BCL6, and HSP90A lie downstream of STAT5.

  3. Single Cell Electrical Characterization Techniques

    PubMed Central

    Mansor, Muhammad Asraf; Ahmad, Mohd Ridzuan

    2015-01-01

    Electrical properties of living cells have been proven to play significant roles in understanding of various biological activities including disease progression both at the cellular and molecular levels. Since two decades ago, many researchers have developed tools to analyze the cell’s electrical states especially in single cell analysis (SCA). In depth analysis and more fully described activities of cell differentiation and cancer can only be accomplished with single cell analysis. This growing interest was supported by the emergence of various microfluidic techniques to fulfill high precisions screening, reduced equipment cost and low analysis time for characterization of the single cell’s electrical properties, as compared to classical bulky technique. This paper presents a historical review of single cell electrical properties analysis development from classical techniques to recent advances in microfluidic techniques. Technical details of the different microfluidic techniques are highlighted, and the advantages and limitations of various microfluidic devices are discussed. PMID:26053399

  4. Cutaneous T-cell lymphoma (CTCL) expresses immunosuppressive CD80 (B7-1) cell-surface protein in the STAT5-dependent manner

    PubMed Central

    Zhang, Qian; Wang, Hong Yi; Wei, Fang; Liu, Xiaobin; Paterson, Jennifer C.; Roy, Darshan; Mihova, Daniela; Woetmann, Anders; Ptasznik, Andrzej; Odum, Niels; Schuster, Stephen J.; Marafioti, Teresa; Riley, James; Wasik, Mariusz A.

    2014-01-01

    Here we report that cutaneous T-cell lymphoma (CTCL) cells and tissues ubiquitously express the immunosuppressive cell-surface protein CD80 (B7-1). CD80 expression in CTCL cells is strictly dependent on the expression of both members of STAT5 family: STAT5a and STAT5b and their joint ability to transcriptionally activate the CD80 gene. In the IL-2-dependent CTCL cells, CD80 expression is induced by the cytokine in the Jak1/3 and STAT5a/b-dependent manner, while in the CTCL cells with the constitutive STAT5 activation, CD80 expression is also STAT5a/b-dependent but independent of Jak activity. While depletion of CD80 expression does not affect the proliferative rate and viability of the CTCL cells, induced expression of the cell-inhibitory receptor of CD80; CD152 (CTLA-4), impairs growth of the cells. Co-culture of CTCL cells with normal T lymphocytes comprised of either both CD4+ and CD8+ populations, or the CD4+ subset alone, transfected with CD152 mRNA, inhibits proliferation of the normal T-cells in the CD152- and CD80-dependent manner. These data identify a new mechanism of immune evasion in CTCL and suggest that the CD80-CD152 axis may become a therapeutic target in this type of lymphoma. PMID:24523507

  5. Lyn- and PLC-beta3-dependent regulation of SHP-1 phosphorylation controls Stat5 activity and myelomonocytic leukemia-like disease.

    PubMed

    Xiao, Wenbin; Ando, Tomoaki; Wang, Huan-You; Kawakami, Yuko; Kawakami, Toshiaki

    2010-12-23

    Hyperactivation of the transcription factor Stat5 leads to various leukemias. Stat5 activity is regulated by the protein phosphatase SHP-1 in a phospholipase C (PLC)-β3-dependent manner. Thus, PLC-β3-deficient mice develop myeloproliferative neoplasm, like Lyn (Src family kinase)- deficient mice. Here we show that Lyn/PLC-β3 doubly deficient lyn(-/-);PLC-β3(-/-) mice develop a Stat5-dependent, fatal myelodysplastic/myeloproliferative neoplasm, similar to human chronic myelomonocytic leukemia (CMML). In hematopoietic stem cells of lyn(-/-);PLC-β3(-/-) mice that cause the CMML-like disease, phosphorylation of SHP-1 at Tyr(536) and Tyr(564) is abrogated, resulting in reduced phosphatase activity and constitutive activation of Stat5. Furthermore, SHP-1 phosphorylation at Tyr(564) by Lyn is indispensable for maximal phosphatase activity and for suppression of the CMML-like disease in these mice. On the other hand, Tyr(536) in SHP-1 can be phosphorylated by Lyn and another kinase(s) and is necessary for efficient interaction with Stat5. Therefore, we identify a novel Lyn/PLC-β3-mediated regulatory mechanism of SHP-1 and Stat5 activities.

  6. Single-cell photonic nanocavity probes

    PubMed Central

    Shambat, Gary; Kothapalli, Sri-Rajasekhar; Provine, J; Sarmiento, Tomas; Harris, James; Gambhir, Sanjiv Sam; Vučković, Jelena

    2013-01-01

    Single-cell interrogation at the nanoscale can take on many forms—namely, pillars1, tubes2–5, and wires6–8 as probes for gentle insertion into a cell’s interior, allowing for studies on single-cell cargo delivery4, electrochemistry3 or electrophysiology1, and optical signaling9–10. However, optical probes such as nanowires9 or tapered fibers10 are passive in nature and have only served as conduits to guide light into and out of a cell. Here we demonstrate a nanobeam photonic crystal (PC) cavity as an advanced cellular nanoprobe, active in nature, and configurable to provide a multitude of actions for both intracellular sensing and control. Our semiconductor nanocavity probes emit photoluminescence (PL) from embedded quantum dots (QD) and sustain high quality resonant photonic modes inside cells. The beams can be loaded in cells and tracked for days at a time, with cells undergoing regular division with the beams. Furthermore, we present in vitro label-free protein sensing with our probes as a path towards real-time biomarker detection in single cells. PMID:23387382

  7. Nanokit for single-cell electrochemical analyses

    PubMed Central

    Pan, Rongrong; Xu, Mingchen; Jiang, Dechen; Burgess, Jame D.; Chen, Hong-Yuan

    2016-01-01

    The development of more intricate devices for the analysis of small molecules and protein activity in single cells would advance our knowledge of cellular heterogeneity and signaling cascades. Therefore, in this study, a nanokit was produced by filling a nanometer-sized capillary with a ring electrode at the tip with components from traditional kits, which could be egressed outside the capillary by electrochemical pumping. At the tip, femtoliter amounts of the kit components were reacted with the analyte to generate hydrogen peroxide for the electrochemical measurement by the ring electrode. Taking advantage of the nanotip and small volume injection, the nanokit was easily inserted into a single cell to determine the intracellular glucose levels and sphingomyelinase (SMase) activity, which had rarely been achieved. High cellular heterogeneities of these two molecules were observed, showing the significance of the nanokit. Compared with the current methods that use a complicated structural design or surface functionalization for the recognition of the analytes, the nanokit has adapted features of the well-established kits and integrated the kit components and detector in one nanometer-sized capillary, which provides a specific device to characterize the reactivity and concentrations of cellular compounds in single cells. PMID:27671654

  8. Single cell-resolution western blotting.

    PubMed

    Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E

    2016-08-01

    This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4-6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine. PMID:27466711

  9. Potentials of single-cell biology in identification and validation of disease biomarkers.

    PubMed

    Niu, Furong; Wang, Diane C; Lu, Jiapei; Wu, Wei; Wang, Xiangdong

    2016-09-01

    Single-cell biology is considered a new approach to identify and validate disease-specific biomarkers. However, the concern raised by clinicians is how to apply single-cell measurements for clinical practice, translate the message of single-cell systems biology into clinical phenotype or explain alterations of single-cell gene sequencing and function in patient response to therapies. This study is to address the importance and necessity of single-cell gene sequencing in the identification and development of disease-specific biomarkers, the definition and significance of single-cell biology and single-cell systems biology in the understanding of single-cell full picture, the development and establishment of whole-cell models in the validation of targeted biological function and the figure and meaning of single-molecule imaging in single cell to trace intra-single-cell molecule expression, signal, interaction and location. We headline the important role of single-cell biology in the discovery and development of disease-specific biomarkers with a special emphasis on understanding single-cell biological functions, e.g. mechanical phenotypes, single-cell biology, heterogeneity and organization of genome function. We have reason to believe that such multi-dimensional, multi-layer, multi-crossing and stereoscopic single-cell biology definitely benefits the discovery and development of disease-specific biomarkers.

  10. Ursolic acid-mediated apoptosis of K562 cells involves Stat5/Akt pathway inhibition through the induction of Gfi-1.

    PubMed

    Lin, Ze; Jiang, Jikai; Liu, Xiao-Shan

    2016-01-01

    Ursolic acid (UA) is a promising natural compound for cancer prevention and therapy. We previously reported that UA induced apoptosis in CML-derived K562 cells. Here we show that the apoptotic process is accompanied by down-regulation of Bcl-xL and Mcl-1 expression and dephosphorylation of Bad. These events are associated with Stat5 inhibition, which is partially mediated through elevated expression of transcriptional repressor Gfi-1. Gfi-1 knockdown using siRNA abrogates the ability of UA to decrease Stat5b expression and attenuates apoptosis induction by UA. We also demonstrate that UA suppresses the Akt kinase activity by inhibiting Akt1/2 expression, which correlates with Stat5 inhibition. Stat5 activity inhibited by a chemical inhibitor or siRNA, Akt1/2 mRNA expression is suppressed. Moreover, we show that UA exerts growth-inhibition in Imatinib-resistant K562/G01. UA has synergistic effects when used in combination with Imatinib in both K562 and K562/G01. Altogether, the data provide evidence that UA's pro-apoptotic effect in K562 cells is associated with the Gfi-1/Stat5/Akt pathway. The findings indicate that UA could potentially be a useful agent in the treatment of CML. PMID:27634378

  11. Ursolic acid-mediated apoptosis of K562 cells involves Stat5/Akt pathway inhibition through the induction of Gfi-1.

    PubMed

    Lin, Ze; Jiang, Jikai; Liu, Xiao-Shan

    2016-01-01

    Ursolic acid (UA) is a promising natural compound for cancer prevention and therapy. We previously reported that UA induced apoptosis in CML-derived K562 cells. Here we show that the apoptotic process is accompanied by down-regulation of Bcl-xL and Mcl-1 expression and dephosphorylation of Bad. These events are associated with Stat5 inhibition, which is partially mediated through elevated expression of transcriptional repressor Gfi-1. Gfi-1 knockdown using siRNA abrogates the ability of UA to decrease Stat5b expression and attenuates apoptosis induction by UA. We also demonstrate that UA suppresses the Akt kinase activity by inhibiting Akt1/2 expression, which correlates with Stat5 inhibition. Stat5 activity inhibited by a chemical inhibitor or siRNA, Akt1/2 mRNA expression is suppressed. Moreover, we show that UA exerts growth-inhibition in Imatinib-resistant K562/G01. UA has synergistic effects when used in combination with Imatinib in both K562 and K562/G01. Altogether, the data provide evidence that UA's pro-apoptotic effect in K562 cells is associated with the Gfi-1/Stat5/Akt pathway. The findings indicate that UA could potentially be a useful agent in the treatment of CML.

  12. Ursolic acid-mediated apoptosis of K562 cells involves Stat5/Akt pathway inhibition through the induction of Gfi-1

    PubMed Central

    Lin, Ze; Jiang, Jikai; Liu, Xiao-Shan

    2016-01-01

    Ursolic acid (UA) is a promising natural compound for cancer prevention and therapy. We previously reported that UA induced apoptosis in CML-derived K562 cells. Here we show that the apoptotic process is accompanied by down-regulation of Bcl-xL and Mcl-1 expression and dephosphorylation of Bad. These events are associated with Stat5 inhibition, which is partially mediated through elevated expression of transcriptional repressor Gfi-1. Gfi-1 knockdown using siRNA abrogates the ability of UA to decrease Stat5b expression and attenuates apoptosis induction by UA. We also demonstrate that UA suppresses the Akt kinase activity by inhibiting Akt1/2 expression, which correlates with Stat5 inhibition. Stat5 activity inhibited by a chemical inhibitor or siRNA, Akt1/2 mRNA expression is suppressed. Moreover, we show that UA exerts growth-inhibition in Imatinib-resistant K562/G01. UA has synergistic effects when used in combination with Imatinib in both K562 and K562/G01. Altogether, the data provide evidence that UA’s pro-apoptotic effect in K562 cells is associated with the Gfi-1/Stat5/Akt pathway. The findings indicate that UA could potentially be a useful agent in the treatment of CML. PMID:27634378

  13. Pseudotime estimation: deconfounding single cell time series

    PubMed Central

    Reid, John E.; Wernisch, Lorenz

    2016-01-01

    Motivation: Repeated cross-sectional time series single cell data confound several sources of variation, with contributions from measurement noise, stochastic cell-to-cell variation and cell progression at different rates. Time series from single cell assays are particularly susceptible to confounding as the measurements are not averaged over populations of cells. When several genes are assayed in parallel these effects can be estimated and corrected for under certain smoothness assumptions on cell progression. Results: We present a principled probabilistic model with a Bayesian inference scheme to analyse such data. We demonstrate our method’s utility on public microarray, nCounter and RNA-seq datasets from three organisms. Our method almost perfectly recovers withheld capture times in an Arabidopsis dataset, it accurately estimates cell cycle peak times in a human prostate cancer cell line and it correctly identifies two precocious cells in a study of paracrine signalling in mouse dendritic cells. Furthermore, our method compares favourably with Monocle, a state-of-the-art technique. We also show using held-out data that uncertainty in the temporal dimension is a common confounder and should be accounted for in analyses of repeated cross-sectional time series. Availability and Implementation: Our method is available on CRAN in the DeLorean package. Contact: john.reid@mrc-bsu.cam.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27318198

  14. Magnetic levitation of single cells.

    PubMed

    Durmus, Naside Gozde; Tekin, H Cumhur; Guven, Sinan; Sridhar, Kaushik; Arslan Yildiz, Ahu; Calibasi, Gizem; Ghiran, Ionita; Davis, Ronald W; Steinmetz, Lars M; Demirci, Utkan

    2015-07-14

    Several cellular events cause permanent or transient changes in inherent magnetic and density properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, we use magnetic levitation principles for biological characterization and monitoring of cells and cellular events. We demonstrate that each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which we distinguish at an unprecedented resolution of 1 × 10(-4) g ⋅ mL(-1). We have identified unique differences in levitation and density blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogenous cell populations. Furthermore, we demonstrate that changes in cellular density and levitation profiles can be monitored in real time at single-cell resolution, allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish density as a powerful biomarker for investigating living systems and their responses. Thereby, our method enables rapid, density-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biological changes under various physiological conditions, including antibiotic or cancer treatment in personalized medicine. PMID:26124131

  15. Magnetic levitation of single cells

    PubMed Central

    Durmus, Naside Gozde; Tekin, H. Cumhur; Guven, Sinan; Sridhar, Kaushik; Arslan Yildiz, Ahu; Calibasi, Gizem; Davis, Ronald W.; Steinmetz, Lars M.; Demirci, Utkan

    2015-01-01

    Several cellular events cause permanent or transient changes in inherent magnetic and density properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, we use magnetic levitation principles for biological characterization and monitoring of cells and cellular events. We demonstrate that each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which we distinguish at an unprecedented resolution of 1 × 10−4 g⋅mL−1. We have identified unique differences in levitation and density blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogenous cell populations. Furthermore, we demonstrate that changes in cellular density and levitation profiles can be monitored in real time at single-cell resolution, allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish density as a powerful biomarker for investigating living systems and their responses. Thereby, our method enables rapid, density-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biological changes under various physiological conditions, including antibiotic or cancer treatment in personalized medicine. PMID:26124131

  16. Transcriptional (ChIP-Chip) Analysis of ELF1, ETS2, RUNX1 and STAT5 in Human Abdominal Aortic Aneurysm

    PubMed Central

    Pahl, Matthew C.; Erdman, Robert; Kuivaniemi, Helena; Lillvis, John H.; Elmore, James R.; Tromp, Gerard

    2015-01-01

    We investigated transcriptional control of gene expression in human abdominal aortic aneurysm (AAA). We previously identified 3274 differentially expressed genes in human AAA tissue compared to non-aneurysmal controls. Four expressed transcription factors (ELF1, ETS2, STAT5 and RUNX1) were selected for genome-wide chromatin immunoprecipitation. Transcription factor binding was enriched in 4760 distinct genes (FDR < 0.05), of which 713 were differentially expressed in AAA. Functional classification using Gene Ontology (GO), KEGG, and Network Analysis revealed enrichment in several biological processes including “leukocyte migration” (FDR = 3.09 × 10−05) and “intracellular protein kinase cascade” (FDR = 6.48 × 10−05). In the control aorta, the most significant GO categories differed from those in the AAA samples and included “cytoskeleton organization” (FDR = 1.24 × 10−06) and “small GTPase mediated signal transduction” (FDR = 1.24 × 10−06). Genes up-regulated in AAA tissue showed a highly significant enrichment for GO categories “leukocyte migration” (FDR = 1.62 × 10−11), “activation of immune response” (FDR = 8.44 × 10−11), “T cell activation” (FDR = 4.14 × 10−10) and “regulation of lymphocyte activation” (FDR = 2.45 × 10−09), whereas the down-regulated genes were enriched in GO categories “cytoskeleton organization” (FDR = 7.84 × 10−05), “muscle cell development” (FDR = 1.00 × 10−04), and “organ morphogenesis” (FDR = 3.00 × 10−04). Quantitative PCR assays confirmed a sub-set of the transcription factor binding sites including those in MTMR11, DUSP10, ITGAM, MARCH1, HDAC8, MMP14, MAGI1, THBD and SPOCK1. PMID:25993293

  17. Molecular circuits for associative learning in single-celled organisms.

    PubMed

    Fernando, Chrisantha T; Liekens, Anthony M L; Bingle, Lewis E H; Beck, Christian; Lenser, Thorsten; Stekel, Dov J; Rowe, Jonathan E

    2009-05-01

    We demonstrate how a single-celled organism could undertake associative learning. Although to date only one previous study has found experimental evidence for such learning, there is no reason in principle why it should not occur. We propose a gene regulatory network that is capable of associative learning between any pre-specified set of chemical signals, in a Hebbian manner, within a single cell. A mathematical model is developed, and simulations show a clear learned response. A preliminary design for implementing this model using plasmids within Escherichia coli is presented, along with an alternative approach, based on double-phosphorylated protein kinases.

  18. Molecular circuits for associative learning in single-celled organisms

    PubMed Central

    Fernando, Chrisantha T.; Liekens, Anthony M.L.; Bingle, Lewis E.H.; Beck, Christian; Lenser, Thorsten; Stekel, Dov J.; Rowe, Jonathan E.

    2008-01-01

    We demonstrate how a single-celled organism could undertake associative learning. Although to date only one previous study has found experimental evidence for such learning, there is no reason in principle why it should not occur. We propose a gene regulatory network that is capable of associative learning between any pre-specified set of chemical signals, in a Hebbian manner, within a single cell. A mathematical model is developed, and simulations show a clear learned response. A preliminary design for implementing this model using plasmids within Escherichia coli is presented, along with an alternative approach, based on double-phosphorylated protein kinases. PMID:18835803

  19. A New Toolbox for Assessing Single Cells

    PubMed Central

    Tsioris, Konstantinos; Torres, Alexis J.; Douce, Thomas B.; Love, J. Christopher

    2015-01-01

    Unprecedented access to the biology of single cells is now feasible, enabled by recent technological advancements that allow us to manipulate and measure sparse samples and achieve a new level of resolution in space and time. This review focuses on advances in tools to study single cells for specific areas of biology. We examine both mature and nascent techniques to study single cells at the genomics, transcriptomics, and proteomics level. In addition, we provide an overview of tools that are well suited for following biological responses to defined perturbations with single-cell resolution. Techniques to analyze and manipulate single cells through soluble and chemical ligands, the microenvironment, and cell-cell interactions are provided. For each of these topics, we highlight the biological motivation, applications, methods, recent advances, and opportunities for improvement. The toolbox presented in this review can function as a starting point for the design of single-cell experiments. PMID:24910919

  20. A new toolbox for assessing single cells.

    PubMed

    Tsioris, Konstantinos; Torres, Alexis J; Douce, Thomas B; Love, J Christopher

    2014-01-01

    Unprecedented access to the biology of single cells is now feasible, enabled by recent technological advancements that allow us to manipulate and measure sparse samples and achieve a new level of resolution in space and time. This review focuses on advances in tools to study single cells for specific areas of biology. We examine both mature and nascent techniques to study single cells at the genomics, transcriptomics, and proteomics level. In addition, we provide an overview of tools that are well suited for following biological responses to defined perturbations with single-cell resolution. Techniques to analyze and manipulate single cells through soluble and chemical ligands, the microenvironment, and cell-cell interactions are provided. For each of these topics, we highlight the biological motivation, applications, methods, recent advances, and opportunities for improvement. The toolbox presented in this review can function as a starting point for the design of single-cell experiments.

  1. A new toolbox for assessing single cells.

    PubMed

    Tsioris, Konstantinos; Torres, Alexis J; Douce, Thomas B; Love, J Christopher

    2014-01-01

    Unprecedented access to the biology of single cells is now feasible, enabled by recent technological advancements that allow us to manipulate and measure sparse samples and achieve a new level of resolution in space and time. This review focuses on advances in tools to study single cells for specific areas of biology. We examine both mature and nascent techniques to study single cells at the genomics, transcriptomics, and proteomics level. In addition, we provide an overview of tools that are well suited for following biological responses to defined perturbations with single-cell resolution. Techniques to analyze and manipulate single cells through soluble and chemical ligands, the microenvironment, and cell-cell interactions are provided. For each of these topics, we highlight the biological motivation, applications, methods, recent advances, and opportunities for improvement. The toolbox presented in this review can function as a starting point for the design of single-cell experiments. PMID:24910919

  2. Single-Cell Genomics for Virology

    PubMed Central

    Ciuffi, Angela; Rato, Sylvie; Telenti, Amalio

    2016-01-01

    Single-cell sequencing technologies, i.e., single cell analysis followed by deep sequencing investigate cellular heterogeneity in many biological settings. It was only in the past year that single-cell sequencing analyses has been applied in the field of virology, providing new ways to explore viral diversity and cell response to viral infection, which are summarized in the present review. PMID:27153082

  3. Single-Cell Genomics for Virology.

    PubMed

    Ciuffi, Angela; Rato, Sylvie; Telenti, Amalio

    2016-01-01

    Single-cell sequencing technologies, i.e., single cell analysis followed by deep sequencing investigate cellular heterogeneity in many biological settings. It was only in the past year that single-cell sequencing analyses has been applied in the field of virology, providing new ways to explore viral diversity and cell response to viral infection, which are summarized in the present review. PMID:27153082

  4. Association study of single nucleotide polymorphisms in JAK2 and STAT5B genes and their differential mRNA expression with mastitis susceptibility in Chinese Holstein cattle.

    PubMed

    Usman, T; Wang, Y; Liu, C; Wang, X; Zhang, Y; Yu, Y

    2015-08-01

    The JAK-STAT pathway plays a key role in mediating immune responses. The genetic effects of single nucleotide polymorphisms (SNPs) in JAK2 and STAT5B were investigated for serum cytokines, mastitis indicators and productions traits in a population of 468 Chinese Holstein cattle. Pooled DNA sequencing revealed one SNP (BTA8:g.39645396A>G) in JAK2 and two SNPs (BTA19:g.43673888A>G and BTA19:g.43660093T>C) in STAT5B. A fixed effect model considering the effects of SNPs, parity, herd, season and year of calving was used by way of the general linear model procedure of sas. Genotype frequencies of these SNPs in the population were in Hardy-Weinberg equilibrium (P > 0.05). A novel SNP (g.39645396A>G) in JAK2 was predicted to change the amino acid from lysine to asparagine and was significantly associated with the somatic cell count (SCC) and somatic cell score (SCS), whereas g.43673888A>G in STAT5B was significantly associated with SCC, SCS and interleukin-4 (IL-4) (P < 0.05). The dominant effect of g.39645396A>G in JAK2 was significant for SCS, and its additive effect was significant for SCC, whereas the dominant effect of g.43673888A>G in STAT5B was significant for SCS and IL-4 (P < 0.05). The combination of g.39645396A>G in JAK2 and g.43673888A>G in STAT5B showed a significant effect on SCC, SCS, IL-4 and TNF-α (P < 0.05). As for mRNA expression analysis, the AA genotype g.39645396A>G and GG genotype g.43673888A>G indicated higher mRNA expression level and were significantly different from other genotypes (P < 0.05). The results imply that JAK2 and STAT5B genes could be useful candidate genes, and the identified polymorphisms might potentially be strong genetic markers for selection of dairy cattle against mastitis development.

  5. The role of nanotechnology in single-cell detection: a review.

    PubMed

    Wang, Changling; Zhang, Yuxiang; Xia, Mingdian; Zhu, Xingxi; Qi, Shitao; Shen, Huaqiang; Liu, Tiebing; Tang, Liming

    2014-10-01

    Biological processes in single cells, such as signal transduction, DNA duplication, and protein synthesis and trafficking, occur in subcellular compartments at nanoscale level. Achieving high spatial-temporal resolution, high sensitivity, and high specificity in single-cell detection poses a great challenge. Nanotechnology, which has been widely applied in the fields of medicine, electronics, biomaterials, and energy production, has the potential to provide solutions for single-cell detection. Here we present a review of the use of nanotechnology in single-cell detection over the past two decades. First, we review the main areas of scientific interest, including morphology, ion concentration, DNA, RNA, protein, intracellular temperature, elements, and mechanical properties. Second, four categories of application of nanotechnology to single-cell detection are described: nanomanipulation, nanodevices, nanomaterials as labels, and nano Secondary ion mass spectrometry. Finally, the prospects and future trends in single-cell detection and analysis are discussed. PMID:25992411

  6. The role of nanotechnology in single-cell detection: a review.

    PubMed

    Wang, Changling; Zhang, Yuxiang; Xia, Mingdian; Zhu, Xingxi; Qi, Shitao; Shen, Huaqiang; Liu, Tiebing; Tang, Liming

    2014-10-01

    Biological processes in single cells, such as signal transduction, DNA duplication, and protein synthesis and trafficking, occur in subcellular compartments at nanoscale level. Achieving high spatial-temporal resolution, high sensitivity, and high specificity in single-cell detection poses a great challenge. Nanotechnology, which has been widely applied in the fields of medicine, electronics, biomaterials, and energy production, has the potential to provide solutions for single-cell detection. Here we present a review of the use of nanotechnology in single-cell detection over the past two decades. First, we review the main areas of scientific interest, including morphology, ion concentration, DNA, RNA, protein, intracellular temperature, elements, and mechanical properties. Second, four categories of application of nanotechnology to single-cell detection are described: nanomanipulation, nanodevices, nanomaterials as labels, and nano Secondary ion mass spectrometry. Finally, the prospects and future trends in single-cell detection and analysis are discussed.

  7. Activation of Stat 5b in erythroid progenitors correlates with the ability of ErbB to induce sustained cell proliferation.

    PubMed Central

    Mellitzer, G; Wessely, O; Decker, T; Meinke, A; Hayman, M J; Beug, H

    1996-01-01

    Self renewal of normal erythroid progenitors is induced by the receptor tyrosine kinase c-ErbB, whereas other receptors (c-Kit/Epo-R) regulate erythroid differentiation. To address possible mechanisms that could explain this selective activity of c-ErbB, we analyzed the ability of these receptors to activate the different members of the Stat transcription factor family. Ligand activation of c-ErbB induced the tyrosine phosphorylation, DNA-binding, and reporter gene transcription of Stat 5b in erythroblasts. In contrast, ligand activation of c-Kit was unable to induce any of these effects in the same cells. Activation of the erythropoietin receptor caused specific DNA-binding of Stat 5b, but failed to induce reporter gene transcription. These biochemical findings correlate perfectly with the selective ability of c-ErbB to cause sustained self renewal in erythroid progenitors. Images Fig. 1 Fig. 3 Fig. 4 PMID:8790376

  8. Integrated Electrowetting Nanoinjector for Single Cell Transfection

    PubMed Central

    Shekaramiz, Elaheh; Varadarajalu, Ganeshkumar; Day, Philip J.; Wickramasinghe, H. Kumar

    2016-01-01

    Single cell transfection techniques are essential to understand the heterogeneity between cells. We have developed an integrated electrowetting nanoinjector (INENI) to transfect single cells. The high transfection efficiency, controlled dosage delivery and ease of INENI fabrication promote the widespread application of the INENI in cell transfection assays. PMID:27374766

  9. Electrochemical synthesis on single cells as templates.

    PubMed

    Tam, Jasper; Salgado, Shehan; Miltenburg, Mark; Maheshwari, Vivek

    2013-10-01

    The cell surface is made electrochemically active by interfacing with graphene sheets. The electrical and thermal properties of graphene allow the control of cell surface potential for electrochemical synthesis. Using this approach radially projecting ZnO nanorods are templated on the surface of single cells. This reported single cell photosensor has superior performance than similar devices made on planar surfaces.

  10. High-Dimensional Single-Cell Cancer Biology

    PubMed Central

    Doxie, Deon B.

    2014-01-01

    Cancer cells are distinguished from each other and from healthy cells by features that drive clonal evolution and therapy resistance. New advances in high-dimensional flow cytometry make it possible to systematically measure mechanisms of tumor initiation, progression, and therapy resistance on millions of cells from human tumors. Here we describe flow cytometry techniques that enable a ‘single-cell systems biology’ view of cancer. High-dimensional techniques like mass cytometry enable multiplexed single-cell analysis of cell identity, clinical biomarkers, signaling network phospho-proteins, transcription factors, and functional readouts of proliferation, cell cycle status, and apoptosis. This capability pairs well with a signaling profiles approach that dissects mechanism by systematically perturbing and measuring many nodes in a signaling network. Single-cell approaches enable study of cellular heterogeneity of primary tissues and turn cell subsets into experimental controls or opportunities for new discovery. Rare populations of stem cells or therapy resistant cancer cells can be identified and compared to other types of cells within the same sample. In the long term, these techniques will enable tracking of minimal residual disease (MRD) and disease progression. By better understanding biological systems that control development and cell-cell interactions in healthy and diseased contexts, we can learn to program cells to become therapeutic agents or target malignant signaling events to specifically kill cancer cells. Single-cell approaches that provide deep insight into cell signaling and fate decisions will be critical to optimizing the next generation of cancer treatments combining targeted approaches and immunotherapy. PMID:24671264

  11. Microfluidic-based single cell trapping using a combination of stagnation point flow and physical barrier

    NASA Astrophysics Data System (ADS)

    Yu, Miao; Chen, Zongzheng; Xiang, Cheng; Liu, Bo; Xie, Handi; Qin, Kairong

    2016-06-01

    Single cell trapping in vitro by microfluidic device is an emerging approach for the study of the relationship between single cells and their dynamic biochemical microenvironments. In this paper, a hydrodynamic-based microfluidic device for single cell trapping is designed using a combination of stagnation point flow and physical barrier. The microfluidic device overcomes the weakness of the traditional ones, which have been only based upon either stagnation point flows or physical barriers, and can conveniently load dynamic biochemical signals to the trapped cell. In addition, it can connect with a programmable syringe pump and a microscope to constitute an integrated experimental system. It is experimentally verified that the microfluidic system can trap single cells in vitro even under flow disturbance and conveniently load biochemical signals to the trapped cell. The designed micro-device would provide a simple yet effective experimental platform for further study of the interactions between single cells and their microenvironments.

  12. Studying bacterial quorum-sensing at the single cell level

    NASA Astrophysics Data System (ADS)

    Delfino Perez, Pablo; Pelakh, Leslie; Young, Jonathan; Johnson, Elaine; Hagen, Stephen

    2010-03-01

    Like many bacterial species, Vibrio fischeri can detect its own population density through a quorum sensing (QS) mechanism. The bacterium releases a signal molecule (AI, autoinducer), which accumulates at high population density and triggers a genetic switch. In V.fischeri this leads to bioluminescence. Little is known about how stochastic gene expression affects QS at the level of single cells. We are imaging the luminescence of individual V.fischeri cells in a flow chamber and directly measuring the intercell variability in AI activation of the QS circuit. Our single-cell luminescence experiments allow us to track cells over time and characterize variations in their response to AI levels. We find heterogeneous response to the external signal: at a given AI concentration some cells may be strongly luminescent while others are virtually dark. The analysis of noise in the individual cell response can eventually lead to a better understanding of how cells use QS to gather information about their environment.

  13. Single-cell chromatin accessibility reveals principles of regulatory variation

    PubMed Central

    Buenrostro, Jason D.; Wu, Beijing; Litzenburger, Ulrike M.; Ruff, Dave; Gonzales, Michael L.; Snyder, Michael P.; Chang, Howard Y.; Greenleaf, William J.

    2015-01-01

    Cell-to-cell variation is a universal feature of life that impacts a wide range of biological phenomena, from developmental plasticity1,2 to tumor heterogeneity3. While recent advances have improved our ability to document cellular phenotypic variation4–8 the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of cellular DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells via assay for transposase-accessible chromatin using sequencing (ATAC-seq). Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single-cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provides insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type specific accessibility variance across 8 cell types. Targeted perturbations of cell cycle or transcription factor signaling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome topological domains9 de novo, linking single-cell accessibility variation to three-dimensional genome organization. All together, single-cell analysis of DNA accessibility provides new insight into cellular variation of the “regulome.” PMID:26083756

  14. Single-cell chromatin accessibility reveals principles of regulatory variation.

    PubMed

    Buenrostro, Jason D; Wu, Beijing; Litzenburger, Ulrike M; Ruff, Dave; Gonzales, Michael L; Snyder, Michael P; Chang, Howard Y; Greenleaf, William J

    2015-07-23

    Cell-to-cell variation is a universal feature of life that affects a wide range of biological phenomena, from developmental plasticity to tumour heterogeneity. Although recent advances have improved our ability to document cellular phenotypic variation, the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of mammalian DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells by assay for transposase-accessible chromatin using sequencing (ATAC-seq) integrated into a programmable microfluidics platform. Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provide insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types. Targeted perturbations of cell cycle or transcription factor signalling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome compartments de novo, linking single-cell accessibility variation to three-dimensional genome organization. Single-cell analysis of DNA accessibility provides new insight into cellular variation of the 'regulome'.

  15. Automated Single Cell Data Decontamination Pipeline

    SciTech Connect

    Tennessen, Kristin; Pati, Amrita

    2014-03-21

    Recent technological advancements in single-cell genomics have encouraged the classification and functional assessment of microorganisms from a wide span of the biospheres phylogeny.1,2 Environmental processes of interest to the DOE, such as bioremediation and carbon cycling, can be elucidated through the genomic lens of these unculturable microbes. However, contamination can occur at various stages of the single-cell sequencing process. Contaminated data can lead to wasted time and effort on meaningless analyses, inaccurate or erroneous conclusions, and pollution of public databases. A fully automated decontamination tool is necessary to prevent these instances and increase the throughput of the single-cell sequencing process

  16. Single Cell Transcriptome Amplification with MALBAC

    PubMed Central

    Tan, Longzhi; Tang, Fuchou; Xie, X. Sunney

    2015-01-01

    Recently, Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) has been developed for whole genome amplification of an individual cell, relying on quasilinear instead of exponential amplification to achieve high coverage. Here we adapt MALBAC for single-cell transcriptome amplification, which gives consistently high detection efficiency, accuracy and reproducibility. With this newly developed technique, we successfully amplified and sequenced single cells from 3 germ layers from mouse embryos in the early gastrulation stage, and examined the epithelial-mesenchymal transition (EMT) program among cells in the mesoderm layer on a single-cell level. PMID:25822772

  17. Single-cell approaches for molecular classification of endocrine tumors

    PubMed Central

    Koh, James; Allbritton, Nancy L.; Sosa, Julie A.

    2015-01-01

    Purpose of review In this review, we summarize recent developments in single-cell technologies that can be employed for the functional and molecular classification of endocrine cells in normal and neoplastic tissue. Recent findings The emergence of new platforms for the isolation, analysis, and dynamic assessment of individual cell identity and reactive behavior enables experimental deconstruction of intratumoral heterogeneity and other contexts, where variability in cell signaling and biochemical responsiveness inform biological function and clinical presentation. These tools are particularly appropriate for examining and classifying endocrine neoplasias, as the clinical sequelae of these tumors are often driven by disrupted hormonal responsiveness secondary to compromised cell signaling. Single-cell methods allow for multidimensional experimental designs incorporating both spatial and temporal parameters with the capacity to probe dynamic cell signaling behaviors and kinetic response patterns dependent upon sequential agonist challenge. Summary Intratumoral heterogeneity in the provenance, composition, and biological activity of different forms of endocrine neoplasia presents a significant challenge for prognostic assessment. Single-cell technologies provide an array of powerful new approaches uniquely well suited for dissecting complex endocrine tumors. Studies examining the relationship between clinical behavior and tumor compositional variations in cellular activity are now possible, providing new opportunities to deconstruct the underlying mechanisms of endocrine neoplasia. PMID:26632769

  18. Single-Cell Analysis in Cancer Genomics.

    PubMed

    Saadatpour, Assieh; Lai, Shujing; Guo, Guoji; Yuan, Guo-Cheng

    2015-10-01

    Genetic changes and environmental differences result in cellular heterogeneity among cancer cells within the same tumor, thereby complicating treatment outcomes. Recent advances in single-cell technologies have opened new avenues to characterize the intra-tumor cellular heterogeneity, identify rare cell types, measure mutation rates, and, ultimately, guide diagnosis and treatment. In this paper we review the recent single-cell technological and computational advances at the genomic, transcriptomic, and proteomic levels, and discuss their applications in cancer research. PMID:26450340

  19. Efficient Synergistic Single-Cell Genome Assembly.

    PubMed

    Movahedi, Narjes S; Embree, Mallory; Nagarajan, Harish; Zengler, Karsten; Chitsaz, Hamidreza

    2016-01-01

    As the vast majority of all microbes are unculturable, single-cell sequencing has become a significant method to gain insight into microbial physiology. Single-cell sequencing methods, currently powered by multiple displacement genome amplification (MDA), have passed important milestones such as finishing and closing the genome of a prokaryote. However, the quality and reliability of genome assemblies from single cells are still unsatisfactory due to uneven coverage depth and the absence of scattered chunks of the genome in the final collection of reads caused by MDA bias. In this work, our new algorithm Hybrid De novo Assembler (HyDA) demonstrates the power of coassembly of multiple single-cell genomic data sets through significant improvement of the assembly quality in terms of predicted functional elements and length statistics. Coassemblies contain significantly more base pairs and protein coding genes, cover more subsystems, and consist of longer contigs compared to individual assemblies by the same algorithm as well as state-of-the-art single-cell assemblers SPAdes and IDBA-UD. Hybrid De novo Assembler (HyDA) is also able to avoid chimeric assemblies by detecting and separating shared and exclusive pieces of sequence for input data sets. By replacing one deep single-cell sequencing experiment with a few single-cell sequencing experiments of lower depth, the coassembly method can hedge against the risk of failure and loss of the sample, without significantly increasing sequencing cost. Application of the single-cell coassembler HyDA to the study of three uncultured members of an alkane-degrading methanogenic community validated the usefulness of the coassembly concept. HyDA is open source and publicly available at http://chitsazlab.org/software.html, and the raw reads are available at http://chitsazlab.org/research.html. PMID:27243002

  20. Efficient Synergistic Single-Cell Genome Assembly

    PubMed Central

    Movahedi, Narjes S.; Embree, Mallory; Nagarajan, Harish; Zengler, Karsten; Chitsaz, Hamidreza

    2016-01-01

    As the vast majority of all microbes are unculturable, single-cell sequencing has become a significant method to gain insight into microbial physiology. Single-cell sequencing methods, currently powered by multiple displacement genome amplification (MDA), have passed important milestones such as finishing and closing the genome of a prokaryote. However, the quality and reliability of genome assemblies from single cells are still unsatisfactory due to uneven coverage depth and the absence of scattered chunks of the genome in the final collection of reads caused by MDA bias. In this work, our new algorithm Hybrid De novo Assembler (HyDA) demonstrates the power of coassembly of multiple single-cell genomic data sets through significant improvement of the assembly quality in terms of predicted functional elements and length statistics. Coassemblies contain significantly more base pairs and protein coding genes, cover more subsystems, and consist of longer contigs compared to individual assemblies by the same algorithm as well as state-of-the-art single-cell assemblers SPAdes and IDBA-UD. Hybrid De novo Assembler (HyDA) is also able to avoid chimeric assemblies by detecting and separating shared and exclusive pieces of sequence for input data sets. By replacing one deep single-cell sequencing experiment with a few single-cell sequencing experiments of lower depth, the coassembly method can hedge against the risk of failure and loss of the sample, without significantly increasing sequencing cost. Application of the single-cell coassembler HyDA to the study of three uncultured members of an alkane-degrading methanogenic community validated the usefulness of the coassembly concept. HyDA is open source and publicly available at http://chitsazlab.org/software.html, and the raw reads are available at http://chitsazlab.org/research.html. PMID:27243002

  1. Single-cell transcriptomics for microbial eukaryotes.

    PubMed

    Kolisko, Martin; Boscaro, Vittorio; Burki, Fabien; Lynn, Denis H; Keeling, Patrick J

    2014-11-17

    One of the greatest hindrances to a comprehensive understanding of microbial genomics, cell biology, ecology, and evolution is that most microbial life is not in culture. Solutions to this problem have mainly focused on whole-community surveys like metagenomics, but these analyses inevitably loose information and present particular challenges for eukaryotes, which are relatively rare and possess large, gene-sparse genomes. Single-cell analyses present an alternative solution that allows for specific species to be targeted, while retaining information on cellular identity, morphology, and partitioning of activities within microbial communities. Single-cell transcriptomics, pioneered in medical research, offers particular potential advantages for uncultivated eukaryotes, but the efficiency and biases have not been tested. Here we describe a simple and reproducible method for single-cell transcriptomics using manually isolated cells from five model ciliate species; we examine impacts of amplification bias and contamination, and compare the efficacy of gene discovery to traditional culture-based transcriptomics. Gene discovery using single-cell transcriptomes was found to be comparable to mass-culture methods, suggesting single-cell transcriptomics is an efficient entry point into genomic data from the vast majority of eukaryotic biodiversity.

  2. Technologies for Single-Cell Isolation

    PubMed Central

    Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

    2015-01-01

    The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field. PMID:26213926

  3. Technologies for Single-Cell Isolation.

    PubMed

    Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

    2015-01-01

    The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field. PMID:26213926

  4. Technologies for Single-Cell Isolation.

    PubMed

    Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

    2015-01-01

    The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

  5. Thermomicrocapillaries as temperature biosensors in single cells

    NASA Astrophysics Data System (ADS)

    Herth, Simone; Giesguth, Miriam; Wedel, Waldemar; Reiss, Günther; Dietz, Karl-Josef

    2013-03-01

    Temperature is an important physical parameter in biology and its deviation from optimum can cause damage in biosystems. Thermocouples based on the Seebeck effect can be structured on glass microcapillaries to obtain thermomicrocapillaries (TMCs) usable in a micromanipulation setup. The suitability of the setup was proven by monitoring the temperature increase upon illumination of leaves and single cells following insertion of the TMC. The increase was 1.5 K in green tissue and 0.75 K in white leaf sections due to lower absorption. In single cells of trichomes, the increase was 0.5 K due to heat dissipation to the surrounding air.

  6. Limiting Energy Dissipation Induces Glassy Kinetics in Single-Cell High-Precision Responses

    NASA Astrophysics Data System (ADS)

    Das, Jayajit

    2016-03-01

    Single cells often generate precise responses by involving dissipative out-of-thermodynamic equilibrium processes in signaling networks. The available free energy to fuel these processes could become limited depending on the metabolic state of an individual cell. How does limiting dissipation affect the kinetics of high precision responses in single cells? I address this question in the context of a kinetic proofreading scheme used in a simple model of early time T cell signaling. I show using exact analytical calculations and numerical simulations that limiting dissipation qualitatively changes the kinetics in single cells marked by emergence of slow kinetics, large cell-to-cell variations of copy numbers, temporally correlated stochastic events (dynamic facilitation), and, ergodicity breaking. Thus, constraints in energy dissipation, in addition to negatively affecting ligand discrimination in T cells, can create a fundamental difficulty in interpreting single cell kinetics from cell population level results.

  7. Microchip platforms for multiplex single-cell functional proteomics with applications to immunology and cancer research

    PubMed Central

    2013-01-01

    Single-cell functional proteomics assays can connect genomic information to biological function through quantitative and multiplex protein measurements. Tools for single-cell proteomics have developed rapidly over the past 5 years and are providing approaches for directly elucidating phosphoprotein signaling networks in cancer cells or for capturing high-resolution snapshots of immune system function in patients with various disease conditions. We discuss advances in single-cell proteomics platforms, with an emphasis on microchip methods. These methods can provide a direct correlation of morphological, functional and molecular signatures at the single-cell level. We also provide examples of how those platforms are being applied to both fundamental biology and clinical studies, focusing on immune-system monitoring and phosphoprotein signaling networks in cancer. PMID:23998271

  8. Hydrogel Pore-Size Modulation for Enhanced Single-Cell Western Blotting.

    PubMed

    Duncombe, Todd A; Kang, Chi-Chih; Maity, Santanu; Ward, Toby M; Pegram, Mark D; Murthy, Niren; Herr, Amy E

    2016-01-13

    Pore-gradient microgel arrays enable thousands of parallel high-resolution single-cell protein electrophoresis separations for targets accross a wide molecular mass (25-289 kDa), yet within 1 mm separation distances. Dual crosslinked hydrogels facilitate gel-pore expansion after electrophoresis for efficient and uniform immunoprobing. The photopatterned, light-activated, and acid-expandable hydrogel underpins single-cell protein analysis, here for oncoprotein-related signaling in human breast biopsy.

  9. Exploring symbioses by single-cell genomics.

    PubMed

    Kamke, Janine; Bayer, Kristina; Woyke, Tanja; Hentschel, Ute

    2012-08-01

    Single-cell genomics has advanced the field of microbiology from the analysis of microbial metagenomes where information is "drowning in a sea of sequences," to recognizing each microbial cell as a separate and unique entity. Single-cell genomics employs Phi29 polymerase-mediated whole-genome amplification to yield microgram-range genomic DNA from single microbial cells. This method has now been applied to a handful of symbiotic systems, including bacterial symbionts of marine sponges, insects (grasshoppers, termites), and vertebrates (mouse, human). In each case, novel insights were obtained into the functional genomic repertoire of the bacterial partner, which, in turn, led to an improved understanding of the corresponding host. Single-cell genomics is particularly valuable when dealing with uncultivated microorganisms, as is still the case for many bacterial symbionts. In this review, we explore the power of single-cell genomics for symbiosis research and highlight recent insights into the symbiotic systems that were obtained by this approach. PMID:22983031

  10. Capillary Electrophoretic Technologies for Single Cell Metabolomics

    ERIC Educational Resources Information Center

    Lapainis, Theodore E.

    2009-01-01

    Understanding the functioning of the brain is hindered by a lack of knowledge of the full complement of neurotransmitters and neuromodulatory compounds. Single cell measurements aid in the discovery of neurotransmitters used by small subsets of neurons that would be diluted below detection limits or masked by ubiquitous compounds when working with…

  11. Microfluidic devices for measuring gene network dynamics in single cells

    PubMed Central

    Bennett, Matthew R.; Hasty, Jeff

    2010-01-01

    The dynamics governing gene regulation have an important role in determining the phenotype of a cell or organism. From processing extracellular signals to generating internal rhythms, gene networks are central to many time-dependent cellular processes. Recent technological advances now make it possible to track the dynamics of gene networks in single cells under various environmental conditions using microfluidic ‘lab-on-a-chip’ devices, and researchers are using these new techniques to analyse cellular dynamics and discover regulatory mechanisms. These technologies are expected to yield novel insights and allow the construction of mathematical models that more accurately describe the complex dynamics of gene regulation. PMID:19668248

  12. Condensing Raman spectrum for single-cell phenotype analysis

    PubMed Central

    2015-01-01

    Background In recent years, high throughput and non-invasive Raman spectrometry technique has matured as an effective approach to identification of individual cells by species, even in complex, mixed populations. Raman profiling is an appealing optical microscopic method to achieve this. To fully utilize Raman proling for single-cell analysis, an extensive understanding of Raman spectra is necessary to answer questions such as which filtering methodologies are effective for pre-processing of Raman spectra, what strains can be distinguished by Raman spectra, and what features serve best as Raman-based biomarkers for single-cells, etc. Results In this work, we have proposed an approach called rDisc to discretize the original Raman spectrum into only a few (usually less than 20) representative peaks (Raman shifts). The approach has advantages in removing noises, and condensing the original spectrum. In particular, effective signal processing procedures were designed to eliminate noise, utilising wavelet transform denoising, baseline correction, and signal normalization. In the discretizing process, representative peaks were selected to signicantly decrease the Raman data size. More importantly, the selected peaks are chosen as suitable to serve as key biological markers to differentiate species and other cellular features. Additionally, the classication performance of discretized spectra was found to be comparable to full spectrum having more than 1000 Raman shifts. Overall, the discretized spectrum needs about 5storage space of a full spectrum and the processing speed is considerably faster. This makes rDisc clearly superior to other methods for single-cell classication. PMID:26681607

  13. Breakdown of the FLT3-ITD/STAT5 axis and synergistic apoptosis induction by the histone deacetylase inhibitor panobinostat and FLT3-specific inhibitors.

    PubMed

    Pietschmann, Kristin; Bolck, Hella Anna; Buchwald, Marc; Spielberg, Steffi; Polzer, Harald; Spiekermann, Karsten; Bug, Gesine; Heinzel, Thorsten; Böhmer, Frank-Dietmar; Krämer, Oliver H

    2012-11-01

    Activating mutations of the class III receptor tyrosine kinase FLT3 are the most frequent molecular aberration in acute myeloid leukemia (AML). Mutant FLT3 accelerates proliferation, suppresses apoptosis, and correlates with poor prognosis. Therefore, it is a promising therapeutic target. Here, we show that RNA interference against FLT3 with an internal tandem duplication (FLT3-ITD) potentiates the efficacy of the histone deacetylase inhibitor (HDACi) panobinostat (LBH589) against AML cells expressing FLT3-ITD. Similar to RNA interference, tyrosine kinase inhibitors (TKI; AC220/cpd.102/PKC412) in combination with LBH589 exhibit superior activity against AML cells. Median dose-effect analyses of drug-induced apoptosis rates of AML cells (MV4-11 and MOLM-13) revealed combination index (CI) values indicating strong synergism. AC220, the most potent and FLT3-specific TKI, shows highest synergism with LBH589 in the low nanomolar range. A 4-hour exposure to LBH589 + AC220 already generates more than 50% apoptosis after 24 hours. Different cell lines lacking FLT3-ITD as well as normal peripheral blood mononuclear cells are not significantly affected by LBH589 + TKI, showing the specificity of this treatment regimen. Immunoblot analyses show that LBH589 + TKI induce apoptosis via degradation of FLT3-ITD and its prosurvival target STAT5. Previously, we showed the LBH589-induced proteasomal degradation of FLT3-ITD. Here, we show that activated caspase-3 also contributes to the degradation of FLT3-ITD and that STAT5 is a direct target of this protease. Our data strongly emphasize HDACi/TKI drug combinations as promising modality for the treatment of FLT3-ITD-positive AMLs. PMID:22942377

  14. Automated micropipette aspiration of single cells.

    PubMed

    Shojaei-Baghini, Ehsan; Zheng, Yi; Sun, Yu

    2013-06-01

    This paper presents a system for mechanically characterizing single cells using automated micropipette aspiration. Using vision-based control and position control, the system controls a micromanipulator, a motorized translation stage, and a custom-built pressure system to position a micropipette (4 μm opening) to approach a cell, form a seal, and aspirate the cell into the micropipette for quantifying the cell's elastic and viscoelastic parameters as well as viscosity. Image processing algorithms were developed to provide controllers with real-time visual feedback and to accurately measure cell deformation behavior on line. Experiments on both solid-like and liquid-like cells demonstrated that the system is capable of efficiently performing single-cell micropipette aspiration and has low operator skill requirements.

  15. Relating Single Cell Heterogeneity To Genotype During Cancer Progression

    NASA Astrophysics Data System (ADS)

    Rajaram, Satwik

    2013-03-01

    Progression of normal cells towards cancer is driven by a series of genetic changes. Traditional population-averaged measurements have found that cell signalling activities are increasingly altered during this progression. Despite the fact that cancer cells are known to be highly heterogeneous, the response of individual pathways to specific genetic changes remains poorly characterized at a single cell level. Do signalling alterations in a pathway reflect a shift of the whole population, or changes to specific subpopulations? Are alterations to pathways independent, or are cells with alterations in one pathway more likely to be abnormal in another due to crosstalk? We are building a computational framework that analyzes immunofluorescence microscopy images of cells to identify alterations in individual pathways at a single-cell level. A primary novelty of our approach is a ``change of basis'' that allows us to understand signalling in cancer cells in terms of the much better understood patterns of signalling in normal cells. This allows us to model heterogeneous populations of cancer cells as a mixture of distinct subpopulations, each with a specific combination of signalling pathways altered beyond the normal baseline. We used this framework to analyze human bronchial epithelial cell lines containing a series of genetic modifications commonly seen in lung cancer. We confirmed expected trends (such as a population-wide epithelial mesenchymal transition following the last of our series of modifications) and are presently studying the relation between the mutational profiles of cancer cells and pathway crosstalk. Our framework will help establish a more natural basis for future investigations into the phenotype-genotype relationship in heterogeneous populations.

  16. Progesterone receptor repression of prolactin/signal transducer and activator of transcription 5-mediated transcription of the beta-casein gene in mammary epithelial cells.

    PubMed

    Buser, Adam C; Gass-Handel, Elizabeth K; Wyszomierski, Shannon L; Doppler, Wolfgang; Leonhardt, Susan A; Schaack, Jerome; Rosen, Jeffrey M; Watkin, Harriet; Anderson, Steven M; Edwards, Dean P

    2007-01-01

    Prolactin (PRL) and glucocorticoids act synergistically to stimulate transcription of the beta-casein milk protein gene. Signal transducer and activator of transcription 5 (Stat5) mediates PRL-dependent trans-activation, and glucocorticoid potentiation occurs through cross talk between glucocorticoid receptor (GR) and Stat5 at the beta-casein promoter. In the mouse, progesterone withdrawal leads to terminal differentiation and secretory activation of the mammary gland at parturition, indicating progesterone's role in repressing milk protein gene expression during pregnancy. To investigate the mechanism of the inhibitory action of progesterone, experiments were performed with cell culture systems reconstituted to express progesterone receptor (PR), the PRL receptor/Stat5 signaling pathway, and GR, enabling evaluation of PR, GR, and Stat5 interactions at the beta-casein promoter. With COS-1, normal murine mammary gland, HC-11, and primary mammary epithelial cells, progestin-PR directly repressed the PRL receptor/Stat5a signaling pathway's mediation of PRL-induced beta-casein transcription. Progestin-PR also inhibited glucocorticoid-GR enhancement of PRL induced trans-activation of beta-casein. Inhibition depended on a functional PR DNA binding domain and specific PR-DNA interactions at the beta-casein promoter. Chromatin immunoprecipitation assays in HC-11 cells revealed recruitment of PR and Stat5a to the beta-casein promoter by progestin or PRL, respectively. Recruitment was disrupted by cotreatment with progestin and PRL, suggesting a mutual interference between activated PR and Stat5a. Without PRL, progestin-PR also recruited Stat5a to the beta-casein promoter, suggesting that recruitment of an unactivated form of Stat5a may contribute to inhibition of beta-casein by progesterone. These results define a negative cross talk between PR and Stat5a/GR that may contribute to the physiological role of progesterone to repress lactogenic hormone induction of the beta

  17. Single-cell SNP analyses and interpretations based on RNA-Seq data for colon cancer research

    PubMed Central

    Chen, Jiahuan; Zhou, Qian; Wang, Yangfan; Ning, Kang

    2016-01-01

    Single-cell sequencing is useful for illustrating the cellular heterogeneities inherent in many intricate biological systems, particularly in human cancer. However, owing to the difficulties in acquiring, amplifying and analyzing single-cell genetic material, obstacles remain for single-cell diversity assessments such as single nucleotide polymorphism (SNP) analyses, rendering biological interpretations of single-cell omics data elusive. We used RNA-Seq data from single-cell and bulk colon cancer samples to analyze the SNP profiles for both structural and functional comparisons. Colon cancer-related pathways with single-cell level SNP enrichment, including the TGF-β and p53 signaling pathways, were also investigated based on both their SNP enrichment patterns and gene expression. We also detected a certain number of fusion transcripts, which may promote tumorigenesis, at the single-cell level. Based on these results, single-cell analyses not only recapitulated the SNP analysis results from the bulk samples but also detected cell-to-cell and cell-to-bulk variations, thereby aiding in early diagnosis and in identifying the precise mechanisms underlying cancers at the single-cell level. PMID:27677461

  18. Get to Understand More from Single-Cells: Current Studies of Microfluidic-Based Techniques for Single-Cell Analysis

    PubMed Central

    Lo, Shih-Jie; Yao, Da-Jeng

    2015-01-01

    This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell. PMID:26213918

  19. Parallel single-cell analysis microfluidic platform.

    PubMed

    van den Brink, Floris T G; Gool, Elmar; Frimat, Jean-Philippe; Bomer, Johan; van den Berg, Albert; Le Gac, Séverine

    2011-11-01

    We report a PDMS microfluidic platform for parallel single-cell analysis (PaSCAl) as a powerful tool to decipher the heterogeneity found in cell populations. Cells are trapped individually in dedicated pockets, and thereafter, a number of invasive or non-invasive analysis schemes are performed. First, we report single-cell trapping in a fast (2-5  min) and reproducible manner with a single-cell capture yield of 85% using two cell lines (P3x63Ag8 and MCF-7), employing a protocol which is scalable and easily amenable to automation. Following this, a mixed population of P3x63Ag8 and MCF-7 cells is stained in situ using the nucleic acid probe (Hoechst) and a phycoerythrin-labeled monoclonal antibody directed at EpCAM present on the surface of the breast cancer cells MCF-7 and absent on the myeloma cells P3x63Ag8 to illustrate the potential of the device to analyze cell population heterogeneity. Next, cells are porated in situ using chemicals in a reversible (digitonin) or irreversible way (lithium dodecyl sulfate). This is visualized by the transportation of fluorescent dyes through the membrane (propidium iodide and calcein). Finally, an electrical protocol is developed for combined cell permeabilization and electroosmotic flow (EOF)-based extraction of the cell content. It is validated here using calcein-loaded cells and visualized through the progressive recovery of calcein in the side channels, indicating successful retrieval of individual cell content. PMID:22025223

  20. Single Cell Transcriptomics: Methods and Applications

    PubMed Central

    Kanter, Itamar; Kalisky, Tomer

    2015-01-01

    Traditionally, gene expression measurements were performed on “bulk” samples containing populations of thousands of cells. Recent advances in genomic technology have made it possible to measure gene expression in hundreds of individual cells at a time. As a result, cellular properties that were previously masked in “bulk” measurements can now be observed directly. In this review, we will survey emerging technologies for single cell transcriptomics, and describe how they are used to study complex disease such as cancer, as well as other biological phenomena such as tissue regeneration, embryonic development, and immune response. PMID:25806353

  1. Thermoresponsive Micropatterned Substrates for Single Cell Studies

    PubMed Central

    Mandal, Kalpana; Balland, Martial; Bureau, Lionel

    2012-01-01

    We describe the design of micropatterned surfaces for single cell studies, based on thermoresponsive polymer brushes. We show that brushes made of poly(N-isopropylacrylamide) grafted at high surface density display excellent protein and cell anti-adhesive properties. Such brushes are readily patterned at the micron scale via deep UV photolithography. A proper choice of the adhesive pattern shapes, combined with the temperature-dependent swelling properties of PNIPAM, allow us to use the polymer brush as a microactuator which induces cell detachment when the temperature is reduced below C. PMID:22701519

  2. Tracing haematopoietic stem cell formation at single-cell resolution.

    PubMed

    Zhou, Fan; Li, Xianlong; Wang, Weili; Zhu, Ping; Zhou, Jie; He, Wenyan; Ding, Meng; Xiong, Fuyin; Zheng, Xiaona; Li, Zhuan; Ni, Yanli; Mu, Xiaohuan; Wen, Lu; Cheng, Tao; Lan, Yu; Yuan, Weiping; Tang, Fuchou; Liu, Bing

    2016-05-18

    Haematopoietic stem cells (HSCs) are derived early from embryonic precursors, such as haemogenic endothelial cells and pre-haematopoietic stem cells (pre-HSCs), the molecular identity of which still remains elusive. Here we use potent surface markers to capture the nascent pre-HSCs at high purity, as rigorously validated by single-cell-initiated serial transplantation. Then we apply single-cell RNA sequencing to analyse endothelial cells, CD45(-) and CD45(+) pre-HSCs in the aorta-gonad-mesonephros region, and HSCs in fetal liver. Pre-HSCs show unique features in transcriptional machinery, arterial signature, metabolism state, signalling pathway, and transcription factor network. Functionally, activation of mechanistic targets of rapamycin (mTOR) is shown to be indispensable for the emergence of HSCs but not haematopoietic progenitors. Transcriptome data-based functional analysis reveals remarkable heterogeneity in cell-cycle status of pre-HSCs. Finally, the core molecular signature of pre-HSCs is identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating stepwise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical applications.

  3. Tracing haematopoietic stem cell formation at single-cell resolution.

    PubMed

    Zhou, Fan; Li, Xianlong; Wang, Weili; Zhu, Ping; Zhou, Jie; He, Wenyan; Ding, Meng; Xiong, Fuyin; Zheng, Xiaona; Li, Zhuan; Ni, Yanli; Mu, Xiaohuan; Wen, Lu; Cheng, Tao; Lan, Yu; Yuan, Weiping; Tang, Fuchou; Liu, Bing

    2016-05-26

    Haematopoietic stem cells (HSCs) are derived early from embryonic precursors, such as haemogenic endothelial cells and pre-haematopoietic stem cells (pre-HSCs), the molecular identity of which still remains elusive. Here we use potent surface markers to capture the nascent pre-HSCs at high purity, as rigorously validated by single-cell-initiated serial transplantation. Then we apply single-cell RNA sequencing to analyse endothelial cells, CD45(-) and CD45(+) pre-HSCs in the aorta-gonad-mesonephros region, and HSCs in fetal liver. Pre-HSCs show unique features in transcriptional machinery, arterial signature, metabolism state, signalling pathway, and transcription factor network. Functionally, activation of mechanistic targets of rapamycin (mTOR) is shown to be indispensable for the emergence of HSCs but not haematopoietic progenitors. Transcriptome data-based functional analysis reveals remarkable heterogeneity in cell-cycle status of pre-HSCs. Finally, the core molecular signature of pre-HSCs is identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating stepwise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical applications. PMID:27225119

  4. Optical manipulation and microfluidics for studies of single cell dynamics

    NASA Astrophysics Data System (ADS)

    Eriksson, E.; Scrimgeour, J.; Granéli, A.; Ramser, K.; Wellander, R.; Enger, J.; Hanstorp, D.; Goksör, M.

    2007-08-01

    Most research on optical manipulation aims towards investigation and development of the system itself. In this paper we show how optical manipulation, imaging and microfluidics can be combined for investigations of single cells. Microfluidic systems have been fabricated and are used, in combination with optical tweezers, to enable environmental changes for single cells. The environment within the microfluidic system has been modelled to ensure control of the process. Three biological model systems have been studied with different combinations of optical manipulation, imaging techniques and microfluidics. In Saccharomyces cerevisiae, environmentally induced size modulations and spatial localization of proteins have been studied to elucidate various signalling pathways. In a similar manner the oxygenation cycle of single red blood cells was triggered and mapped using Raman spectroscopy. In the third experiment the forces between the endoplasmic reticulum and chloroplasts were studied in Pisum sativum and Arabidopsis thaliana. By combining different techniques we make advanced biological research possible, revealing information on a cellular level that is impossible to obtain with traditional techniques.

  5. Degradation of the transcription factors NF-κB, STAT3, and STAT5 is involved in Entamoeba histolytica-induced cell death in Caco-2 colonic epithelial cells.

    PubMed

    Kim, Kyeong Ah; Min, Arim; Lee, Young Ah; Shin, Myeong Heon

    2014-10-01

    Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-κB (p65) in Caco-2 cells. However, IκB, an inhibitor of NF-κB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-κB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-κB and STATs in colonic epithelial cells, which ultimately accelerates cell death.

  6. Single Molecule and Single Cell Epigenomics

    PubMed Central

    Hyun, Byung-Ryool; McElwee, John L.; Soloway, Paul D.

    2014-01-01

    Dynamically regulated changes in chromatin states are vital for normal development and can produce disease when they go awry. Accordingly, much effort has been devoted to characterizing these states under normal and pathological conditions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most widely used method to characterize where in the genome transcription factors, modified histones, modified nucleotides and chromatin binding proteins are found; bisulfite sequencing (BS-seq) and its variants are commonly used to characterize the locations of DNA modifications. Though very powerful, these methods are not without limitations. Notably, they are best at characterizing one chromatin feature at a time, yet chromatin features arise and function in combination. Investigators commonly superimpose separate ChIP-seq or BS-seq datasets, and then infer where chromatin features are found together. While these inferences might be correct, they can be misleading when the chromatin source has distinct cell types, or when a given cell type exhibits any cell to cell variation in chromatin state. These ambiguities can be eliminated by robust methods that directly characterize the existence and genomic locations of combinations of chromatin features in very small inputs of cells or ideally, single cells. Here we review single molecule epigenomic methods under development to overcome these limitations, the technical challenges associated with single molecule methods and their potential application to single cells. PMID:25204781

  7. Single molecule and single cell epigenomics.

    PubMed

    Hyun, Byung-Ryool; McElwee, John L; Soloway, Paul D

    2015-01-15

    Dynamically regulated changes in chromatin states are vital for normal development and can produce disease when they go awry. Accordingly, much effort has been devoted to characterizing these states under normal and pathological conditions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most widely used method to characterize where in the genome transcription factors, modified histones, modified nucleotides and chromatin binding proteins are found; bisulfite sequencing (BS-seq) and its variants are commonly used to characterize the locations of DNA modifications. Though very powerful, these methods are not without limitations. Notably, they are best at characterizing one chromatin feature at a time, yet chromatin features arise and function in combination. Investigators commonly superimpose separate ChIP-seq or BS-seq datasets, and then infer where chromatin features are found together. While these inferences might be correct, they can be misleading when the chromatin source has distinct cell types, or when a given cell type exhibits any cell to cell variation in chromatin state. These ambiguities can be eliminated by robust methods that directly characterize the existence and genomic locations of combinations of chromatin features in very small inputs of cells or ideally, single cells. Here we review single molecule epigenomic methods under development to overcome these limitations, the technical challenges associated with single molecule methods and their potential application to single cells.

  8. Single Cell Chromatography, LDRD Feasibility Study

    SciTech Connect

    Knize, M G; Bailey, C G

    2007-02-22

    A limitation in the mass spectrometry of biological materials is the reduced ion formation caused by sample complexity. We proposed to develop an enabling technology, single cell planar chromatography, which will greatly increase the amount of chemical information that can be obtained from single biological cells when using imaging mass spectrometry or other surface analysis methods. The sample preparation methods were developed for the time-of-flight secondary mass spectrometer (ToF-SIMS) at LLNL. This instrument has a measured zeptomole (10{sup -21} mole, 600 atoms) limit-of-detection for a molecule with a mass to charge ratio of 225[1]. Our goal was to use planar chromatographic separation to approach similar low limits of detection even with the chemically complex contents of a single cell. The process was proposed to reduce ion suppression and at the same time expose more of the cell contents to the ion beam. The method of work was to deposit biological cells on a silicon chip with suitable chromatographic and electrical properties, dissolve the cell with a droplet of solvent, allow the solvent to evaporate, and then allow the movement of cell contents laterally by immersing an edge of the chip in to a chromatographic solvent, that then moves through the chromatographic matrix allowing the components to interact with, and be separated by, the chromatographic substrate. This process is a miniaturized version of thin layer chromatography with detection by surface mass spectrometry.

  9. Self-Folding Single Cell Grippers

    PubMed Central

    2015-01-01

    Given the heterogeneous nature of cultures, tumors, and tissues, the ability to capture, contain, and analyze single cells is important for genomics, proteomics, diagnostics, therapeutics, and surgery. Moreover, for surgical applications in small conduits in the body such as in the cardiovascular system, there is a need for tiny tools that approach the size of the single red blood cells that traverse the blood vessels and capillaries. We describe the fabrication of arrayed or untethered single cell grippers composed of biocompatible and bioresorbable silicon monoxide and silicon dioxide. The energy required to actuate these grippers is derived from the release of residual stress in 3–27 nm thick films, did not require any wires, tethers, or batteries, and resulted in folding angles over 100° with folding radii as small as 765 nm. We developed and applied a finite element model to predict these folding angles. Finally, we demonstrated the capture of live mouse fibroblast cells in an array of grippers and individual red blood cells in untethered grippers which could be released from the substrate to illustrate the potential utility for in vivo operations. PMID:24937214

  10. Reassessment of H&E stained clot specimens and immunohistochemistry of phosphorylated Stat5 for histological diagnosis of MDS/MPN.

    PubMed

    Tsuruyama, Tatsuaki; Aini, Wulamujiang; Hiratsuka, Takuya

    2015-12-01

    Few studies have comprehensively analysed histopathological findings of bone marrow clots for diagnosis of haematopoietic cell dysplasia. In particular, a limited number of studies have assessed the use of haematoxylin and eosin (H&E) staining, which is generally considered less informative than May-Giemsa staining. In the current study, the utility of bone marrow clot specimens for diagnosis was examined using H&E staining and immunohistochemistry. Patients with myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasm (MDS/MPN), including chronic myelomonocytic leukaemia (CMML), atypical chronic myeloid leukaemia (aCML) lacking Philadelphia chromosome, and juvenile myelomonocytic leukaemia (JMML), were selected for histological evaluation. H&E stained specimens were advantageous for observation of atypical basophilic staining of the cytoplasm and nucleus related to dysplasia. This finding was significantly supported for both MDS and MDS/MPN (p < 0.05 versus May-Giemsa staining); therefore, we concluded that H&E staining could be used for identification of dysplastic cells. In addition, despite the loss of tissue structure, phosphorylated Stat5 immunostaining was sufficiently useful for the observation of myelodysplastic blasts. Thus, clot specimens are useful for diagnosis of haematopoietic dysplasia by pathologists.

  11. Quantum dot imaging platform for single-cell molecular profiling

    NASA Astrophysics Data System (ADS)

    Zrazhevskiy, Pavel; Gao, Xiaohu

    2013-03-01

    Study of normal cell physiology and disease pathogenesis heavily relies on untangling the complexity of intracellular molecular mechanisms and pathways. To achieve this goal, comprehensive molecular profiling of individual cells within the context of microenvironment is required. Here we report the development of a multicolour multicycle in situ imaging technology capable of creating detailed quantitative molecular profiles for individual cells at the resolution of optical imaging. A library of stoichiometric fluorescent probes is prepared by linking target-specific antibodies to a universal quantum dot-based platform via protein A in a quick and simple procedure. Surprisingly, despite the potential for multivalent binding between protein A and antibody and the intermediate affinity of this non-covalent bond, fully assembled probes do not aggregate or exchange antibodies, facilitating highly multiplexed parallel staining. This single-cell molecular profiling technology is expected to open new opportunities in systems biology, gene expression studies, signalling pathway analysis and molecular diagnostics.

  12. Limits of sensing temporal concentration changes by single cells.

    PubMed

    Mora, Thierry; Wingreen, Ned S

    2010-06-18

    Berg and Purcell [Biophys. J. 20, 193 (1977)] calculated how the accuracy of concentration sensing by single-celled organisms is limited by noise from the small number of counted molecules. Here we generalize their results to the sensing of concentration ramps, which is often the biologically relevant situation (e.g., during bacterial chemotaxis). We calculate lower bounds on the uncertainty of ramp sensing by three measurement devices: a single receptor, an absorbing sphere, and a monitoring sphere. We contrast two strategies, simple linear regression of the input signal versus maximum likelihood estimation, and show that the latter can be twice as accurate as the former. Finally, we consider biological implementations of these two strategies, and identify possible signatures that maximum likelihood estimation is implemented by real biological systems.

  13. Limits of Sensing Temporal Concentration Changes by Single Cells

    NASA Astrophysics Data System (ADS)

    Mora, Thierry; Wingreen, Ned S.

    2010-06-01

    Berg and Purcell [Biophys. J. 20, 193 (1977)BIOJAU0006-349510.1016/S0006-3495(77)85544-6] calculated how the accuracy of concentration sensing by single-celled organisms is limited by noise from the small number of counted molecules. Here we generalize their results to the sensing of concentration ramps, which is often the biologically relevant situation (e.g., during bacterial chemotaxis). We calculate lower bounds on the uncertainty of ramp sensing by three measurement devices: a single receptor, an absorbing sphere, and a monitoring sphere. We contrast two strategies, simple linear regression of the input signal versus maximum likelihood estimation, and show that the latter can be twice as accurate as the former. Finally, we consider biological implementations of these two strategies, and identify possible signatures that maximum likelihood estimation is implemented by real biological systems.

  14. Digital Microfluidics for Manipulation and Analysis of a Single Cell

    PubMed Central

    He, Jie-Long; Chen, An-Te; Lee, Jyong-Huei; Fan, Shih-Kang

    2015-01-01

    The basic structural and functional unit of a living organism is a single cell. To understand the variability and to improve the biomedical requirement of a single cell, its analysis has become a key technique in biological and biomedical research. With a physical boundary of microchannels and microstructures, single cells are efficiently captured and analyzed, whereas electric forces sort and position single cells. Various microfluidic techniques have been exploited to manipulate single cells through hydrodynamic and electric forces. Digital microfluidics (DMF), the manipulation of individual droplets holding minute reagents and cells of interest by electric forces, has received more attention recently. Because of ease of fabrication, compactness and prospective automation, DMF has become a powerful approach for biological application. We review recent developments of various microfluidic chips for analysis of a single cell and for efficient genetic screening. In addition, perspectives to develop analysis of single cells based on DMF and emerging functionality with high throughput are discussed. PMID:26389890

  15. Digital Microfluidics for Manipulation and Analysis of a Single Cell.

    PubMed

    He, Jie-Long; Chen, An-Te; Lee, Jyong-Huei; Fan, Shih-Kang

    2015-09-15

    The basic structural and functional unit of a living organism is a single cell. To understand the variability and to improve the biomedical requirement of a single cell, its analysis has become a key technique in biological and biomedical research. With a physical boundary of microchannels and microstructures, single cells are efficiently captured and analyzed, whereas electric forces sort and position single cells. Various microfluidic techniques have been exploited to manipulate single cells through hydrodynamic and electric forces. Digital microfluidics (DMF), the manipulation of individual droplets holding minute reagents and cells of interest by electric forces, has received more attention recently. Because of ease of fabrication, compactness and prospective automation, DMF has become a powerful approach for biological application. We review recent developments of various microfluidic chips for analysis of a single cell and for efficient genetic screening. In addition, perspectives to develop analysis of single cells based on DMF and emerging functionality with high throughput are discussed.

  16. Single cell protein as an occupational hazard.

    PubMed Central

    Ekenvall, L; Dölling, B; Göthe, C J; Ebbinghaus, L; von Stedingk, L V; Wasserman, J

    1983-01-01

    Single cell protein (SCP) intended for animal feed purposes was produced in a pilot plant. The SCP consisted of Methylomonas methanolica, a pseudomonas species which is an obligate methanol user. The SCP was cultured in fermenters and later dewatered and dried in a spray-drier. Seven of eight research workers had febrile reactions 6-12 hours after exposure to SCP dust. All workers had high titres of IgG and IgM antibodies against the pseudomonas species as measured with indirect ELISA and passive haemagglutination techniques. The mechanism behind the febrile reaction is judged to be a non-immunological reaction caused by endotoxins. By increasing the particle size of the SCP through using different drying procedures, a product which generated less dust was obtained. PMID:6830720

  17. Single-cell protein from waste cellulose

    NASA Technical Reports Server (NTRS)

    Dunlap, C. E.; Callihan, C. D.

    1973-01-01

    The recycle, reuse, or reclamation of single cell protein from liquid and solid agricultural waste fibers by a fermentation process is reported. It is shown that cellulose comprises the bulk of the fibers at 50% to 55% of the dry weight of the refuse and that its biodegradability is of prime importance in the choice of a substrate. The application of sodium hydroxide followed by heat and pressure serves to de-polymerize and disrupt lignin structure while swelling the cellulose to increase water uptake and pore volume. Some of the lignin, hemi-celluloses, ash, and cellulose of the material is hydrolized and solubilized. Introduction of microorganisms to the substrate fibers mixed with nutrients produces continuous fermentation of cellulose for further protein extraction and purification.

  18. JGI Genomic Single-cell Assembly Workflow

    SciTech Connect

    Trong, S.

    2011-09-16

    JIGSAW is a software package disigned to quality control and assemble genomic DNA sequences from single-cell bacterial and archaeal genomes. Amplification of singel-cell genomes using multiple displacement amplification technology presents challenges that magnify the amount of contaminants in the sample and produce non uniform depth of sequence coverage. these factors pose problems whan assembling the genomic data using currently availible short read assembles. The software addresses these problems by removing contaminants and normalizing the sequence read coverage prior to assemble. A hybrid assembly approach using two different open source genome assembly tools is then applied to piece together the DNA fragments. Additional reporting of QC metrics for the input sample and the genome assembly is provided for further analysis.

  19. Kinetics of virus production from single cells.

    PubMed

    Timm, Andrea; Yin, John

    2012-03-01

    The production of virus by infected cells is an essential process for the spread and persistence of viral diseases, the effectiveness of live-viral vaccines, and the manufacture of viruses for diverse applications. Yet despite its importance, methods to precisely measure virus production from cells are lacking. Most methods test infected-cell populations, masking how individual cells behave. Here we measured the kinetics of virus production from single cells. We combined simple steps of liquid-phase infection, serial dilution, centrifugation, and harvesting, without specialized equipment, to track the production of virus particles from BHK cells infected with vesicular stomatitis virus. Remarkably, cell-to-cell differences in latent times to virus release were within a factor of two, while production rates and virus yields spanned over 300-fold, highlighting an extreme diversity in virus production for cells from the same population. These findings have fundamental and technological implications for health and disease.

  20. JGI Genomic Single-cell Assembly Workflow

    2011-09-16

    JIGSAW is a software package disigned to quality control and assemble genomic DNA sequences from single-cell bacterial and archaeal genomes. Amplification of singel-cell genomes using multiple displacement amplification technology presents challenges that magnify the amount of contaminants in the sample and produce non uniform depth of sequence coverage. these factors pose problems whan assembling the genomic data using currently availible short read assembles. The software addresses these problems by removing contaminants and normalizing the sequencemore » read coverage prior to assemble. A hybrid assembly approach using two different open source genome assembly tools is then applied to piece together the DNA fragments. Additional reporting of QC metrics for the input sample and the genome assembly is provided for further analysis.« less

  1. Linked T Cell Receptor and Cytokine Signaling Govern the Development of the Regulatory T cell Repertoire

    PubMed Central

    Burchill, Matthew A.; Yang, Jianying; Vang, Kieng B.; Moon, James J.; Chu, H. Hamlet; Lio, Chan-Wang J.; Vegoe, Amanda L.; Hsieh, Chyi-Song; Jenkins, Marc K.; Farrar, Michael A.

    2008-01-01

    Summary Appropriate development of regulatory T cells (Tregs) is necessary to prevent autoimmunity. Neonatal mice, unlike adults, lack factors required for Treg development. It is unclear what these missing factors are. However, signals emanating from the TCR, CD28 and γc-dependent cytokine receptors are required for Treg development. Herein we demonstrate that expression of a constitutively-active STAT5b transgene (STAT5b-CA) allows for Treg development in neonatal mice and restores Treg numbers in CD28−/− mice. Sequence analysis of TCR genes in STAT5b-CA Tregs indicates that ectopic STAT5 activation results in a TCR repertoire that more closely resembles that of naïve T cells. Using MHCII tetramers to identify antigen-specific T cells, we demonstrate that STAT5 signals divert thymocytes normally destined to become naïve T cells into the Treg lineage. Our data support a two-step model of Treg differentiation in which TCR/CD28 signals induce cytokine responsiveness; STAT5-inducing cytokines then complete the program of Treg differentiation. PMID:18199418

  2. Chronic uremia attenuates growth hormone-induced signal transduction in skeletal muscle.

    PubMed

    Sun, Di Fei; Zheng, Zhilan; Tummala, Padmaja; Oh, Jun; Schaefer, Franz; Rabkin, Ralph

    2004-10-01

    Malnutrition and muscle wasting are common in chronic renal failure (CRF) and adversely affect morbidity and mortality. Contributing to the muscle wasting is resistance to growth hormone (GH). For testing whether impaired GH signaling is a cause of the skeletal muscle GH resistance and for elucidating its mechanisms, muscle GH signaling and action were studied in GH-deficient rats with surgically induced CRF and sham-operated pairfed control rats. GH treatment increased gastrocnemius muscle IGF-1 mRNA levels significantly in control but not in CRF rats. GH-activated Janus-associated kinase 2 (JAK2)-signal transducers and activators of transcription 5 (STAT5) signaling was impaired in CRF rats, despite normal GH receptor (GHR), JAK2, and STAT5 protein levels. Phosphorylation of the GHR, JAK2, and STAT5 in response to GH was depressed by nearly half in CRF (P < 0.05), and nuclear phospho-STAT5 levels were depressed by approximately one third (P < 0.01). GH-stimulated suppressors of cytokine signaling 2 mRNA levels were significantly higher in CRF. This may be related to inflammatory cytokine activity because C-reactive protein levels were elevated. Muscle protein-tyrosine phosphatase activity was also increased significantly by twofold. In conclusion, rats with CRF acquire skeletal muscle resistance to GH that is caused at least in part by impaired JAK2-GHR-STAT5 phosphorylation and nuclear STAT5 translocation. Furthermore, it seems that the attenuated JAK2-STAT5 phosphorylation may be caused by at least two different processes. One involves depressed phosphorylation of the signaling proteins because of increased suppressors of cytokine signaling 2 expression that may be linked to low-grade inflammation. The other may involve increased signaling protein dephosphorylation because of heightened protein-tyrosine phosphatase activity.

  3. Interleukin-6 inhibits hepatic growth hormone signaling via upregulation of Cis and Socs-3.

    PubMed

    Denson, Lee A; Held, Matthew A; Menon, Ram K; Frank, Stuart J; Parlow, Albert F; Arnold, Dodie L

    2003-04-01

    Cytokines may cause an acquired growth hormone (GH) resistance in patients with inflammatory diseases. Anabolic effects of GH are mediated through activation of STAT5 transcription factors. We have reported that TNF-alpha suppresses hepatic GH receptor (GHR) gene expression, whereas the cytokine-inducible SH2-containing protein 1 (Cis)/suppressors of cytokine signaling (Socs) genes are upregulated by TNF-alpha and IL-6 and inhibit GH activation of STAT5. However, the relative importance of these mechanisms in inflammatory GH resistance was not known. We hypothesized that IL-6 would prevent GH activation of STAT5 and that this would involve Cis/Socs protein upregulation. GH +/- LPS was administered to TNF receptor 1 (TNFR1) or IL-6 null mice and wild-type (WT) controls. STAT5, STAT3, GHR, Socs 1-3, and Cis phosphorylation and abundance were assessed by using immunoblots, EMSA, and/or real time RT-PCR. TNF-alpha and IL-6 abundance were assessed by using ELISA. GH activated STAT5 in WT and TNFR1 or IL-6 null mice. LPS pretreatment prevented STAT5 activation in WT and TNFR1 null mice; however, STAT5 activation was preserved in IL-6 null mice. GHR abundance did not change with LPS administration. Inhibition of STAT5 activation by LPS was temporally associated with phosphorylation of STAT3 and upregulation of Cis and Socs-3 protein in WT and TNFR1 null mice; STAT3, Cis, and Socs-3 were not induced in IL-6 null mice. IL-6 inhibits hepatic GH signaling by upregulating Cis and Socs-3, which may involve activation of STAT3. Therapies that block IL-6 may enhance GH signaling in inflammatory diseases.

  4. Self-Digitization Microfluidic Chip for Absolute Quantification of mRNA in Single Cells

    PubMed Central

    2015-01-01

    Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques. PMID:25390242

  5. Self-digitization microfluidic chip for absolute quantification of mRNA in single cells.

    PubMed

    Thompson, Alison M; Gansen, Alexander; Paguirigan, Amy L; Kreutz, Jason E; Radich, Jerald P; Chiu, Daniel T

    2014-12-16

    Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques.

  6. Fast and high resolution single-cell BRET imaging.

    PubMed

    Goyet, Elise; Bouquier, Nathalie; Ollendorff, Vincent; Perroy, Julie

    2016-01-01

    Resonance Energy Transfer (RET)-based technologies are used to report protein-protein interactions in living cells. Among them, Bioluminescence-initiated RET (BRET) provides excellent sensitivity but the low light intensity intrinsic to the bioluminescent process hampers its use for the localization of protein complexes at the sub-cellular level. Herein we have characterized the methodological conditions required to reliably perform single-cell BRET imaging using an extremely bright luciferase, Nanoluciferase (Nluc). With this, we achieved an unprecedented performance in the field of protein-protein interaction imaging in terms of temporal and spatial resolution, duration of signal stability, signal sensitivity and dynamic range. As proof-of-principle, an Nluc-containing BRET-based sensor of ERK activity enabled the detection of subtle, transient and localized variations in ERK activity in neuronal dendritic spines, induced by the activation of endogenous synaptic NMDA receptors. This development will improve our comprehension of both the spatio-temporal dynamics of protein-protein interactions and the activation patterns of specific signaling pathways. PMID:27302735

  7. Fast and high resolution single-cell BRET imaging

    PubMed Central

    Goyet, Elise; Bouquier, Nathalie; Ollendorff, Vincent; Perroy, Julie

    2016-01-01

    Resonance Energy Transfer (RET)-based technologies are used to report protein-protein interactions in living cells. Among them, Bioluminescence-initiated RET (BRET) provides excellent sensitivity but the low light intensity intrinsic to the bioluminescent process hampers its use for the localization of protein complexes at the sub-cellular level. Herein we have characterized the methodological conditions required to reliably perform single-cell BRET imaging using an extremely bright luciferase, Nanoluciferase (Nluc). With this, we achieved an unprecedented performance in the field of protein-protein interaction imaging in terms of temporal and spatial resolution, duration of signal stability, signal sensitivity and dynamic range. As proof-of-principle, an Nluc-containing BRET-based sensor of ERK activity enabled the detection of subtle, transient and localized variations in ERK activity in neuronal dendritic spines, induced by the activation of endogenous synaptic NMDA receptors. This development will improve our comprehension of both the spatio-temporal dynamics of protein-protein interactions and the activation patterns of specific signaling pathways. PMID:27302735

  8. Signal transducer and activator of transcription 5 is implicated in disease activity in adult and juvenile onset systemic lupus erythematosus.

    PubMed

    Meshaal, Safa; El Refai, Rasha; El Saie, Ahmed; El Hawary, Rabab

    2016-06-01

    The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is one of a handful of pleiotropic cascades used to transduce a multitude of signals for development and homeostasis in humans. It is the principal signaling mechanism for a wide array of cytokines and growth factors. Dysregulated cytokine action on immune cells plays an important role in the initiation and progress of systemic lupus erythematosus (SLE). In this study, we tried to assess the role of STAT5 in systemic lupus erythematosus and correlate its phosphorylation level with the disease activity. The activation of the STAT5 was assessed by measuring the level of expression of phosphorylated STAT5 (pSTAT5) using flow cytometry on the peripheral blood T and B cells in 58 SLE patients (40 adult and 18 juvenile onset) and on 23 healthy age- and sex-matched controls for both groups. Serum prolactin level was also assessed in the patients and control by ELISA. The study revealed that the level of pSTAT5 was higher in adult SLE patients than in healthy control (p = 0.001) and in juvenile-onset SLE patients versus age-matched control (p = 0.031). A positive correlation existed between the pSTAT5 levels and Systemic Lupus Activity Measure (SLAM) score and also with multiple clinical manifestations indicating a potential role of STAT5 signaling in pathogenesis SLE. The pSTAT5 signaling is implicated in the disease activity of SLE and may be a useful target of therapy by correcting the dysregulation of cytokines involved in the disease pathogenesis.

  9. Signal transducer and activator of transcription 5 is implicated in disease activity in adult and juvenile onset systemic lupus erythematosus.

    PubMed

    Meshaal, Safa; El Refai, Rasha; El Saie, Ahmed; El Hawary, Rabab

    2016-06-01

    The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is one of a handful of pleiotropic cascades used to transduce a multitude of signals for development and homeostasis in humans. It is the principal signaling mechanism for a wide array of cytokines and growth factors. Dysregulated cytokine action on immune cells plays an important role in the initiation and progress of systemic lupus erythematosus (SLE). In this study, we tried to assess the role of STAT5 in systemic lupus erythematosus and correlate its phosphorylation level with the disease activity. The activation of the STAT5 was assessed by measuring the level of expression of phosphorylated STAT5 (pSTAT5) using flow cytometry on the peripheral blood T and B cells in 58 SLE patients (40 adult and 18 juvenile onset) and on 23 healthy age- and sex-matched controls for both groups. Serum prolactin level was also assessed in the patients and control by ELISA. The study revealed that the level of pSTAT5 was higher in adult SLE patients than in healthy control (p = 0.001) and in juvenile-onset SLE patients versus age-matched control (p = 0.031). A positive correlation existed between the pSTAT5 levels and Systemic Lupus Activity Measure (SLAM) score and also with multiple clinical manifestations indicating a potential role of STAT5 signaling in pathogenesis SLE. The pSTAT5 signaling is implicated in the disease activity of SLE and may be a useful target of therapy by correcting the dysregulation of cytokines involved in the disease pathogenesis. PMID:27041383

  10. Front-Rear Polarization by Mechanical Cues: From Single Cells to Tissues.

    PubMed

    Ladoux, Benoit; Mège, René-Marc; Trepat, Xavier

    2016-06-01

    Directed cell migration is a complex process that involves front-rear polarization, characterized by cell adhesion and cytoskeleton-based protrusion, retraction, and contraction of either a single cell or a cell collective. Single cell polarization depends on a variety of mechanochemical signals including external adhesive cues, substrate stiffness, and confinement. In cell ensembles, coordinated polarization of migrating tissues results not only from the application of traction forces on the extracellular matrix but also from the transmission of mechanical stress through intercellular junctions. We focus here on the impact of mechanical cues on the establishment and maintenance of front-rear polarization from single cell to collective cell behaviors through local or large-scale mechanisms. PMID:26920934

  11. Kinetics of small molecule interactions with membrane proteins in single cells measured with mechanical amplification

    PubMed Central

    Guan, Yan; Shan, Xiaonan; Zhang, Fenni; Wang, Shaopeng; Chen, Hong-Yuan; Tao, Nongjian

    2015-01-01

    Measuring small molecule interactions with membrane proteins in single cells is critical for understanding many cellular processes and for screening drugs. However, developing such a capability has been a difficult challenge. We show that molecular interactions with membrane proteins induce a mechanical deformation in the cellular membrane, and real-time monitoring of the deformation with subnanometer resolution allows quantitative analysis of small molecule–membrane protein interaction kinetics in single cells. This new strategy provides mechanical amplification of small binding signals, making it possible to detect small molecule interactions with membrane proteins. This capability, together with spatial resolution, also allows the study of the heterogeneous nature of cells by analyzing the interaction kinetics variability between different cells and between different regions of a single cell. PMID:26601298

  12. Plasma membrane and cytoskeleton dynamics during single-cell wound healing.

    PubMed

    Boucher, Eric; Mandato, Craig A

    2015-10-01

    Wounding leads not only to plasma membrane disruption, but also to compromised cytoskeleton structures. This results not only in unwarranted exchanges between the cytosol and extracellular milieu, but also in loss of tensegrity, which may further endanger the cell. Tensegrity can be described as the interplay between the tensile forces generated by the apparent membrane tension, actomyosin contraction, and the cytoskeletal structures resisting those changes (e.g., microtubules). It is responsible for the structural integrity of the cell and for its ability to sense mechanical signals. Recent reviews dealing with single-cell healing mostly focused on the molecular machineries controlling the traffic and fusion of specific vesicles, or their role in different pathologies. In this review, we aim to take a broader view of the different modes of single cell repair, while focussing on the different ways the changes in plasmalemma surface area and composition, plasmalemma tension, and cytoskeletal dynamics may influence and affect single-cell repair.

  13. Front-Rear Polarization by Mechanical Cues: From Single Cells to Tissues.

    PubMed

    Ladoux, Benoit; Mège, René-Marc; Trepat, Xavier

    2016-06-01

    Directed cell migration is a complex process that involves front-rear polarization, characterized by cell adhesion and cytoskeleton-based protrusion, retraction, and contraction of either a single cell or a cell collective. Single cell polarization depends on a variety of mechanochemical signals including external adhesive cues, substrate stiffness, and confinement. In cell ensembles, coordinated polarization of migrating tissues results not only from the application of traction forces on the extracellular matrix but also from the transmission of mechanical stress through intercellular junctions. We focus here on the impact of mechanical cues on the establishment and maintenance of front-rear polarization from single cell to collective cell behaviors through local or large-scale mechanisms.

  14. Plasma membrane and cytoskeleton dynamics during single-cell wound healing.

    PubMed

    Boucher, Eric; Mandato, Craig A

    2015-10-01

    Wounding leads not only to plasma membrane disruption, but also to compromised cytoskeleton structures. This results not only in unwarranted exchanges between the cytosol and extracellular milieu, but also in loss of tensegrity, which may further endanger the cell. Tensegrity can be described as the interplay between the tensile forces generated by the apparent membrane tension, actomyosin contraction, and the cytoskeletal structures resisting those changes (e.g., microtubules). It is responsible for the structural integrity of the cell and for its ability to sense mechanical signals. Recent reviews dealing with single-cell healing mostly focused on the molecular machineries controlling the traffic and fusion of specific vesicles, or their role in different pathologies. In this review, we aim to take a broader view of the different modes of single cell repair, while focussing on the different ways the changes in plasmalemma surface area and composition, plasmalemma tension, and cytoskeletal dynamics may influence and affect single-cell repair. PMID:26209916

  15. Noninvasive measurement of hydrogen and potassium ion flux from single cells and epithelial structures.

    PubMed

    Smith, P J; Trimarchi, J

    2001-01-01

    This review introduces new developments in a technique for measuring the movement of ions across the plasma membrane. With the use of a self-referencing ion-selective (Seris) probe, transport mechanisms can be studied on a variety of preparations ranging from tissues to single cells. In this paper we illustrate this versatility with examples from the vas deferens and inner ear epithelium to large and small single cells represented by mouse single-cell embryos and rat microglia. Potassium and hydrogen ion fluxes are studied and pharmacological manipulation of the signals are reported. The strengths of the self-referencing technique are reviewed with regard to biological applications, and the expansion of self-referencing probes to include electrochemical and enzyme-based sensors is discussed.

  16. Characterizing Phenotypes and Signaling Networks of Single Human Cells by Mass Cytometry.

    PubMed

    Leelatian, Nalin; Diggins, Kirsten E; Irish, Jonathan M

    2015-01-01

    Single cell mass cytometry is revolutionizing our ability to quantitatively characterize cellular biomarkers and signaling networks. Mass cytometry experiments routinely measure 25-35 features of each cell in primary human tissue samples. The relative ease with which a novice user can generate a large amount of high quality data and the novelty of the approach have created a need for example protocols, analysis strategies, and datasets. In this chapter, we present detailed protocols for two mass cytometry experiments designed as training tools. The first protocol describes detection of 26 features on the surface of human peripheral blood mononuclear cells. In the second protocol, a mass cytometry signaling network profile measures 25 node states comprised of five key signaling effectors (AKT, ERK1/2, STAT1, STAT5, and p38) quantified under five conditions (Basal, FLT3L, SCF, IL-3, and IFNγ). This chapter compares manual and unsupervised data analysis approaches, including bivariate plots, heatmaps, histogram overlays, SPADE, and viSNE. Data files in this chapter have been shared online using Cytobank ( http://www.cytobank.org/irishlab/ ). PMID:26542718

  17. Characterizing Phenotypes and Signaling Networks of Single Human Cells by Mass Cytometry.

    PubMed

    Leelatian, Nalin; Diggins, Kirsten E; Irish, Jonathan M

    2015-01-01

    Single cell mass cytometry is revolutionizing our ability to quantitatively characterize cellular biomarkers and signaling networks. Mass cytometry experiments routinely measure 25-35 features of each cell in primary human tissue samples. The relative ease with which a novice user can generate a large amount of high quality data and the novelty of the approach have created a need for example protocols, analysis strategies, and datasets. In this chapter, we present detailed protocols for two mass cytometry experiments designed as training tools. The first protocol describes detection of 26 features on the surface of human peripheral blood mononuclear cells. In the second protocol, a mass cytometry signaling network profile measures 25 node states comprised of five key signaling effectors (AKT, ERK1/2, STAT1, STAT5, and p38) quantified under five conditions (Basal, FLT3L, SCF, IL-3, and IFNγ). This chapter compares manual and unsupervised data analysis approaches, including bivariate plots, heatmaps, histogram overlays, SPADE, and viSNE. Data files in this chapter have been shared online using Cytobank ( http://www.cytobank.org/irishlab/ ).

  18. PHASE I SINGLE CELL ELECTROLYZER TEST RESULTS

    SciTech Connect

    Steimke, J; Timothy Steeper, T

    2008-08-05

    This document reports the results of Phase I Single Cell testing of an SO{sub 2}-Depolarized Water Electrolyzer. Testing was performed primarily during the first quarter of FY 2008 at the Savannah River National Laboratory (SRNL) using an electrolyzer cell designed and built at SRNL. Other facility hardware were also designed and built at SRNL. This test further advances this technology for which work began at SRNL in 2005. This research is valuable in achieving the ultimate goal of an economical hydrogen production process based on the Hybrid Sulfur (HyS) Cycle. The focus of this work was to conduct single cell electrolyzer tests to further develop the technology of SO{sub 2}-depolarized electrolysis as part of the HyS Cycle. The HyS Cycle is a hybrid thermochemical cycle that may be used in conjunction with advanced nuclear reactors or centralized solar receivers to produce hydrogen by water-splitting. Like all other sulfur-based cycles, HyS utilizes the high temperature thermal decomposition of sulfuric acid to produce oxygen and regenerate sulfur dioxide. The unique aspect of HyS is the generation of hydrogen in a water electrolyzer that is operated under conditions where dissolved sulfur dioxide depolarizes the anodic reaction, resulting in substantial voltage reduction. Low cell voltage is essential for both thermodynamic efficiency and hydrogen cost. Sulfur dioxide is oxidized at the anode, producing sulfuric acid that is sent to the high temperature acid decomposition portion of the cycle. The electrolyzer cell uses the membrane electrode assembly (MEA) concept. The anode and cathode are formed by spraying platinum containing catalyst on both sides of a Proton Exchange Membrane (PEM). In most testing the material of the PEM was NafionR. The electrolyzer cell active area can be as large as 54.8 cm{sup 2}. Feed to the anode of the electrolyzer is a sulfuric acid solution containing sulfur dioxide. The partial pressure of sulfur dioxide could be varied in the

  19. The potential of single-cell profiling in plants.

    PubMed

    Efroni, Idan; Birnbaum, Kenneth D

    2016-01-01

    Single-cell transcriptomics has been employed in a growing number of animal studies, but the technique has yet to be widely used in plants. Nonetheless, early studies indicate that single-cell RNA-seq protocols developed for animal cells produce informative datasets in plants. We argue that single-cell transcriptomics has the potential to provide a new perspective on plant problems, such as the nature of the stem cells or initials, the plasticity of plant cells, and the extent of localized cellular responses to environmental inputs. Single-cell experimental outputs require different analytical approaches compared with pooled cell profiles and new tools tailored to single-cell assays are being developed. Here, we highlight promising new single-cell profiling approaches, their limitations as applied to plants, and their potential to address fundamental questions in plant biology. PMID:27048384

  20. Live single-cell laser tag

    PubMed Central

    Binan, Loïc; Mazzaferri, Javier; Choquet, Karine; Lorenzo, Louis-Etienne; Wang, Yu Chang; Affar, El Bachir; De Koninck, Yves; Ragoussis, Jiannis; Kleinman, Claudia L.; Costantino, Santiago

    2016-01-01

    The ability to conduct image-based, non-invasive cell tagging, independent of genetic engineering, is key to cell biology applications. Here we introduce cell labelling via photobleaching (CLaP), a method that enables instant, specific tagging of individual cells based on a wide array of criteria such as shape, behaviour or positional information. CLaP uses laser illumination to crosslink biotin onto the plasma membrane, coupled with streptavidin conjugates to label individual cells for genomic, cell-tracking, flow cytometry or ultra-microscopy applications. We show that the incorporated mark is stable, non-toxic, retained for several days, and transferred by cell division but not to adjacent cells in culture. To demonstrate the potential of CLaP for genomic applications, we combine CLaP with microfluidics-based single-cell capture followed by transcriptome-wide next-generation sequencing. Finally, we show that CLaP can also be exploited for inducing transient cell adhesion to substrates for microengineering cultures with spatially patterned cell types. PMID:27198043

  1. Silicon dioxide thin film mediated single cell nucleic acid isolation.

    PubMed

    Bogdanov, Evgeny; Dominova, Irina; Shusharina, Natalia; Botman, Stepan; Kasymov, Vitaliy; Patrushev, Maksim

    2013-01-01

    A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube.

  2. Silicon Dioxide Thin Film Mediated Single Cell Nucleic Acid Isolation

    PubMed Central

    Bogdanov, Evgeny; Dominova, Irina; Shusharina, Natalia; Botman, Stepan; Kasymov, Vitaliy; Patrushev, Maksim

    2013-01-01

    A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube. PMID:23874571

  3. Single cell analysis: the new frontier in 'Omics'

    SciTech Connect

    Wang, Daojing; Bodovitz, Steven

    2010-01-14

    Cellular heterogeneity arising from stochastic expression of genes, proteins, and metabolites is a fundamental principle of cell biology, but single cell analysis has been beyond the capabilities of 'Omics' technologies. This is rapidly changing with the recent examples of single cell genomics, transcriptomics, proteomics, and metabolomics. The rate of change is expected to accelerate owing to emerging technologies that range from micro/nanofluidics to microfabricated interfaces for mass spectrometry to third- and fourth-generation automated DNA sequencers. As described in this review, single cell analysis is the new frontier in Omics, and single cell Omics has the potential to transform systems biology through new discoveries derived from cellular heterogeneity.

  4. Laser tweezers Raman spectroscopy of single cells

    NASA Astrophysics Data System (ADS)

    Chen, De

    Raman scattering is an inelastic collision between the vibrating molecules inside the sample and the incident photons. During this process, energy exchange takes place between the photon and the scattering molecule. By measuring the energy change of the photon, the molecular vibration mode can be probed. The vibrational spectrum contains valuable information about the disposition of atomic nuclei and chemical bonds within a molecule, the chemical compositions and the interactions between the molecule and its surroundings. In this dissertation, laser tweezers Raman spectroscopy (LTRS) technique is applied for the analysis of biological cells and human cells at single cell level. In LTRS, an individual cell is trapped in aqueous medium with laser tweezers, and Raman scattering spectra from the trapped cell are recorded in real-time. The Raman spectra of these cells can be used to reveal the dynamical processes of cell growth, cell response to environment changes, and can be used as the finger print for the identification of a bacterial cell species. Several biophysical experiments were carried out using LTRS: (1) the dynamic germination process of individual spores of Bacillus thuringiensis was detected via Ca-DPA, a spore-specific biomarker molecule; (2) inactivation and killing of Bacillus subtilis spores by microwave irradiation and wet heat were studied at single cell level; (3) the heat shock activation process of single B. subtilis spores were analyzed, in which the reversible transition from glass-like state at low temperature to liquid-like state at high temperature in spore was revealed at the molecular level; (4) the kinetic processes of bacterial cell lysis of E. coli by lysozyme and by temperature induction of lambda phage were detected real-time; (5) the fixation and rehydration of human platelets were quantitatively evaluated and characterized with Raman spectroscopy method, which provided a rapid way to quantify the quality of freeze-dried therapeutic

  5. Single cell mechanics of keratinocyte cells.

    PubMed

    Lulevich, Valentin; Yang, Hsin-ya; Isseroff, R Rivkah; Liu, Gang-yu

    2010-11-01

    Keratinocytes represent the major cell type of the uppermost layer of human skin, the epidermis. Using AFM-based single cell compression, the ability of individual keratinocytes to resist external pressure and global rupturing forces is investigated and compared with various cell types. Keratinocytes are found to be 6-70 times stiffer than other cell types, such as white blood, breast epithelial, fibroblast, or neuronal cells, and in contrast to other cell types they retain high mechanic strength even after the cell's death. The absence of membrane rupturing peaks in the force-deformation profiles of keratinocytes and their high stiffness during a second load cycle suggests that their unique mechanical resistance is dictated by the cytoskeleton. A simple analytical model enables the quantification of Young's modulus of keratinocyte cytoskeleton, as high as 120-340 Pa. Selective disruption of the two major cytoskeletal networks, actin filaments and microtubules, does not significantly affect keratinocyte mechanics. F-actin is found to impact cell deformation under pressure. During keratinocyte compression, the plasma membrane stretches to form peripheral blebs. Instead of blebbing, cells with depolymerized F-actin respond to pressure by detaching the plasma membrane from the cytoskeleton underneath. On the other hand, the compression force of keratinocytes expressing a mutated keratin (cell line, KEB-7) is 1.6-2.2 times less than that for the control cell line that has normal keratin networks. Therefore, we infer that the keratin intermediate filament network is responsible for the extremely high keratinocyte stiffness and resilience. This could manifest into the rugged protective nature of the human epidermis. PMID:20728993

  6. Single cell genomics of subsurface microorganisms

    NASA Astrophysics Data System (ADS)

    Stepanauskas, R.; Onstott, T. C.; Lau, C.; Kieft, T. L.; Woyke, T.; Rinke, C.; Sczyrba, A.; van Heerden, E.

    2012-12-01

    Recent studies have revealed unexpected abundance and diversity of microorganisms in terrestrial and marine subsurface, providing new perspectives over their biogeochemical significance, evolution, and the limits of life. The now commonly used research tools, such as metagenomics and PCR-based gene surveys enabled cultivation-unbiased analysis of genes encoded by natural microbial communities. However, these methods seldom provide direct evidence for how the discovered genes are organized inside genomes and from which organisms do they come from. Here we evaluated the feasibility of an alternative, single cell genomics approach, in the analysis of subsurface microbial community composition, metabolic potential and microevolution at the Sanford Underground Research Facility (SURF), South Dakota, and the Witwaterstrand Basin, South Africa. We successfully recovered genomic DNA from individual microbial cells from multiple locations, including ultra-deep (down to 3,500 m) and low-biomass (down to 10^3 cells mL^-1) fracture water. The obtained single amplified genomes (SAGs) from SURF contained multiple representatives of the candidate divisions OP3, OP11, OD1 and uncharacterized archaea. By sequencing eight of these SAGs, we obtained the first genome content information for these phylum-level lineages that do not contain a single cultured representative. The Witwaterstrand samples were collected from deep fractures, biogeochemical dating of which suggests isolation from tens of thousands to tens of millions of years. Thus, these fractures may be viewed as "underground Galapagos", a natural, long-term experiment of microbial evolution within well-defined temporal and spatial boundaries. We are analyzing multiple SAGs from these environments, which will provide detailed information about adaptations to life in deep subsurface, mutation rates, selective pressures and gene flux within and across microbial populations.

  7. Chronic Inhibition of STAT3/STAT5 in Treatment-Resistant Human Breast Cancer Cell Subtypes: Convergence on the ROS/SUMO Pathway and Its Effects on xCT Expression and System xc- Activity.

    PubMed

    Linher-Melville, Katja; Nashed, Mina G; Ungard, Robert G; Haftchenary, Sina; Rosa, David A; Gunning, Patrick T; Singh, Gurmit

    2016-01-01

    Pharmacologically targeting activated STAT3 and/or STAT5 has been an active area of cancer research. The cystine/glutamate antiporter, system xc-, contributes to redox balance and export of intracellularly produced glutamate in response to up-regulated glutaminolysis in cancer cells. We have previously shown that blocking STAT3/5 using the small molecule inhibitor, SH-4-54, which targets the SH2 domains of both proteins, increases xCT expression, thereby increasing system xc- activity in human breast cancer cells. The current investigation demonstrates that chronic SH-4-54 administration, followed by clonal selection of treatment-resistant MDA-MB-231 and T47D breast cancer cells, elicits distinct subtype-dependent effects. xCT mRNA and protein levels, glutamate release, and cystine uptake are decreased relative to untreated passage-matched controls in triple-negative MDA-MB-231 cells, with the inverse occurring in estrogen-responsive T47D cells. This "ying-yang" effect is linked with a shifted balance between the phosphorylation status of STAT3 and STAT5, intracellular ROS levels, and STAT5 SUMOylation/de-SUMOylation. STAT5 emerged as a definitive negative regulator of xCT at the transcriptional level, while STAT3 activation is coupled with increased system xc- activity. We propose that careful classification of a patient's breast cancer subtype is central to effectively targeting STAT3/5 as a therapeutic means of treating breast cancer, particularly given that xCT is emerging as an important biomarker of aggressive cancers.

  8. Chronic Inhibition of STAT3/STAT5 in Treatment-Resistant Human Breast Cancer Cell Subtypes: Convergence on the ROS/SUMO Pathway and Its Effects on xCT Expression and System xc- Activity

    PubMed Central

    Linher-Melville, Katja; Nashed, Mina G.; Ungard, Robert G.; Haftchenary, Sina; Rosa, David A.; Gunning, Patrick T.; Singh, Gurmit

    2016-01-01

    Pharmacologically targeting activated STAT3 and/or STAT5 has been an active area of cancer research. The cystine/glutamate antiporter, system xc-, contributes to redox balance and export of intracellularly produced glutamate in response to up-regulated glutaminolysis in cancer cells. We have previously shown that blocking STAT3/5 using the small molecule inhibitor, SH-4-54, which targets the SH2 domains of both proteins, increases xCT expression, thereby increasing system xc- activity in human breast cancer cells. The current investigation demonstrates that chronic SH-4-54 administration, followed by clonal selection of treatment-resistant MDA-MB-231 and T47D breast cancer cells, elicits distinct subtype-dependent effects. xCT mRNA and protein levels, glutamate release, and cystine uptake are decreased relative to untreated passage-matched controls in triple-negative MDA-MB-231 cells, with the inverse occurring in estrogen-responsive T47D cells. This “ying-yang” effect is linked with a shifted balance between the phosphorylation status of STAT3 and STAT5, intracellular ROS levels, and STAT5 SUMOylation/de-SUMOylation. STAT5 emerged as a definitive negative regulator of xCT at the transcriptional level, while STAT3 activation is coupled with increased system xc- activity. We propose that careful classification of a patient’s breast cancer subtype is central to effectively targeting STAT3/5 as a therapeutic means of treating breast cancer, particularly given that xCT is emerging as an important biomarker of aggressive cancers. PMID:27513743

  9. Chronic Inhibition of STAT3/STAT5 in Treatment-Resistant Human Breast Cancer Cell Subtypes: Convergence on the ROS/SUMO Pathway and Its Effects on xCT Expression and System xc- Activity.

    PubMed

    Linher-Melville, Katja; Nashed, Mina G; Ungard, Robert G; Haftchenary, Sina; Rosa, David A; Gunning, Patrick T; Singh, Gurmit

    2016-01-01

    Pharmacologically targeting activated STAT3 and/or STAT5 has been an active area of cancer research. The cystine/glutamate antiporter, system xc-, contributes to redox balance and export of intracellularly produced glutamate in response to up-regulated glutaminolysis in cancer cells. We have previously shown that blocking STAT3/5 using the small molecule inhibitor, SH-4-54, which targets the SH2 domains of both proteins, increases xCT expression, thereby increasing system xc- activity in human breast cancer cells. The current investigation demonstrates that chronic SH-4-54 administration, followed by clonal selection of treatment-resistant MDA-MB-231 and T47D breast cancer cells, elicits distinct subtype-dependent effects. xCT mRNA and protein levels, glutamate release, and cystine uptake are decreased relative to untreated passage-matched controls in triple-negative MDA-MB-231 cells, with the inverse occurring in estrogen-responsive T47D cells. This "ying-yang" effect is linked with a shifted balance between the phosphorylation status of STAT3 and STAT5, intracellular ROS levels, and STAT5 SUMOylation/de-SUMOylation. STAT5 emerged as a definitive negative regulator of xCT at the transcriptional level, while STAT3 activation is coupled with increased system xc- activity. We propose that careful classification of a patient's breast cancer subtype is central to effectively targeting STAT3/5 as a therapeutic means of treating breast cancer, particularly given that xCT is emerging as an important biomarker of aggressive cancers. PMID:27513743

  10. Hepatic growth hormone and glucocorticoid receptor signaling in body growth, steatosis and metabolic liver cancer development.

    PubMed

    Mueller, Kristina M; Themanns, Madeleine; Friedbichler, Katrin; Kornfeld, Jan-Wilhelm; Esterbauer, Harald; Tuckermann, Jan P; Moriggl, Richard

    2012-09-25

    Growth hormone (GH) and glucocorticoids (GCs) are involved in the control of processes that are essential for the maintenance of vital body functions including energy supply and growth control. GH and GCs have been well characterized to regulate systemic energy homeostasis, particular during certain conditions of physical stress. However, dysfunctional signaling in both pathways is linked to various metabolic disorders associated with aberrant carbohydrate and lipid metabolism. In liver, GH-dependent activation of the transcription factor signal transducer and activator of transcription (STAT) 5 controls a variety of physiologic functions within hepatocytes. Similarly, GCs, through activation of the glucocorticoid receptor (GR), influence many important liver functions such as gluconeogenesis. Studies in hepatic Stat5 or GR knockout mice have revealed that they similarly control liver function on their target gene level and indeed, the GR functions often as a cofactor of STAT5 for GH-induced genes. Gene sets, which require physical STAT5-GR interaction, include those controlling body growth and maturation. More recently, it has become evident that impairment of GH-STAT5 signaling in different experimental models correlates with metabolic liver disease, ranging from hepatic steatosis to hepatocellular carcinoma (HCC). While GH-activated STAT5 has a protective role in chronic liver disease, experimental disruption of GC-GR signaling rather seems to ameliorate metabolic disorders under metabolic challenge. In this review, we focus on the current knowledge about hepatic GH-STAT5 and GC-GR signaling in body growth, metabolism, and protection from fatty liver disease and HCC development.

  11. Single-Cell Approaches: Pandora's Box of Developmental Mechanisms.

    PubMed

    Hadjantonakis, Anna-Katerina; Arias, Alfonso Martinez

    2016-09-26

    Single-cell approaches are providing a new lexicon of developmental cell biology by revealing heterogeneities in seemingly uniform cellular populations. By bridging scales, single-cell approaches should, in principle, galvanize our understanding of how individual cells adopt distinct fates as they build complex tissues. PMID:27676428

  12. UV Decontamination of MDA Reagents for Single Cell Genomics

    SciTech Connect

    Lee, Janey; Tighe, Damon; Sczyrba, Alexander; Malmatrom, Rex; Clingenpeel, Scott; Malfatti, Stephanie; Rinke, Christian; Wang, Zhong; Stepanauskas, Ramunas; Cheng, Jan-Fang; Woyke, Tanja

    2011-03-18

    Single cell genomics, the amplification and sequencing of genomes from single cells, can provide a glimpse into the genetic make-up and thus life style of the vast majority of uncultured microbial cells, making it an immensely powerful and increasingly popular tool. This is accomplished by use of multiple displacement amplification (MDA), which can generate billions of copies of a single bacterial genome producing microgram-range DNA required for shotgun sequencing. Here, we address a key challenge inherent to this approach and propose a solution for the improved recovery of single cell genomes. While DNA-free reagents for the amplification of a single cell genome are a prerequisite for successful single cell sequencing and analysis, DNA contamination has been detected in various reagents, which poses a considerable challenge. Our study demonstrates the effect of UV irradiation in efficient elimination of exogenous contaminant DNA found in MDA reagents, while maintaining Phi29 activity. Consequently, we also find that increased UV exposure to Phi29 does not adversely affect genome coverage of MDA amplified single cells. While additional challenges in single cell genomics remain to be resolved, the proposed methodology is relatively quick and simple and we believe that its application will be of high value for future single cell sequencing projects.

  13. Single Nanowire Probe for Single Cell Endoscopy and Sensing

    NASA Astrophysics Data System (ADS)

    Yan, Ruoxue

    The ability to manipulate light in subwavelength photonic and plasmonic structures has shown great potentials in revolutionizing how information is generated, transformed and processed. Chemically synthesized nanowires, in particular, offers a unique toolbox not only for highly compact and integrated photonic modules and devices, including coherent and incoherent light sources, waveguides, photodetectors and photovoltaics, but also for new types of nanoscopic bio-probes for spot cargo delivery and in-situ single cell endoscopy and sensing. Such nanowire probes would enable us to carry out intracellular imaging and probing with high spatial resolution, monitor in-vivo biological processes within single living cells and greatly improve our fundamental understanding of cell functions, intracellular physiological processes, and cellular signal pathways. My work is aimed at developing a material and instrumental platform for such single nanowire probe. Successful optical integration of Ag nanowire plasmonic waveguides, which offers deep subwavelength mode confinement, and conventional photonic waveguides was demonstrated on a single nanowire level. The highest plasmonic-photonic coupling efficiency coupling was found at small coupling angles and low input frequencies. The frequency dependent propagation loss was observed in Ag nanowire and was confirmed by quantitative measurement and in agreement with theoretical expectations. Rational integration of dielectric and Ag nanowire waveguide components into hybrid optical-plasmonic routing devices has been demonstrated. This capability is essential for incorporating sub-100nm Ag nanowire waveguides into optical fiber based nanoprobes for single cell endoscopy. The nanoprobe system based on single nanowire waveguides was demonstrated by optically coupling semiconductor or metal nanowire with an optical fiber with tapered tip. This nanoprobe design requires minimal instrumentation which makes it cost efficient and readily

  14. Single Cell Mass Cytometry for Analysis of Immune System Functional States

    PubMed Central

    Bjornson, Zach B.; Nolan, Garry P.; Fantl, Wendy J.

    2013-01-01

    Single cell mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with antibody panels (upwards of 40) in which each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the Lanthanide series of the periodic table. Isotope labelled antibodies recognize surface markers to delineate cell types and intracellular signaling molecules to provide a measure of the network state—and thereby demarcating multiple cell state functions such as apoptosis, DNA damage and cell cycle. By measuring all these parameters simultaneously, the signaling state of an individual cell can be measured at its network state. This review will cover the basics of mass cytometry as well as outline steps already taken to allow it to stand aside traditional fluorescence based cytometry in the immunologist’s analytical arsenal in their study of immune states during infection. PMID:23999316

  15. Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis.

    PubMed

    Shin, Jaehoon; Berg, Daniel A; Zhu, Yunhua; Shin, Joseph Y; Song, Juan; Bonaguidi, Michael A; Enikolopov, Grigori; Nauen, David W; Christian, Kimberly M; Ming, Guo-li; Song, Hongjun

    2015-09-01

    Somatic stem cells contribute to tissue ontogenesis, homeostasis, and regeneration through sequential processes. Systematic molecular analysis of stem cell behavior is challenging because classic approaches cannot resolve cellular heterogeneity or capture developmental dynamics. Here we provide a comprehensive resource of single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny. We further developed Waterfall, a bioinformatic pipeline, to statistically quantify singe-cell gene expression along a de novo reconstructed continuous developmental trajectory. Our study reveals molecular signatures of adult qNSCs, characterized by active niche signaling integration and low protein translation capacity. Our analyses further delineate molecular cascades underlying qNSC activation and neurogenesis initiation, exemplified by decreased extrinsic signaling capacity, primed translational machinery, and regulatory switches in transcription factors, metabolism, and energy sources. Our study reveals the molecular continuum underlying adult neurogenesis and illustrates how Waterfall can be used for single-cell omics analyses of various continuous biological processes.

  16. Single cell deposition and patterning with a robotic system.

    PubMed

    Lu, Zhe; Moraes, Christopher; Ye, George; Simmons, Craig A; Sun, Yu

    2010-01-01

    Integrating single-cell manipulation techniques in traditional and emerging biological culture systems is challenging. Microfabricated devices for single cell studies in particular often require cells to be spatially positioned at specific culture sites on the device surface. This paper presents a robotic micromanipulation system for pick-and-place positioning of single cells. By integrating computer vision and motion control algorithms, the system visually tracks a cell in real time and controls multiple positioning devices simultaneously to accurately pick up a single cell, transfer it to a desired substrate, and deposit it at a specified location. A traditional glass micropipette is used, and whole- and partial-cell aspiration techniques are investigated to manipulate single cells. Partially aspirating cells resulted in an operation speed of 15 seconds per cell and a 95% success rate. In contrast, the whole-cell aspiration method required 30 seconds per cell and achieved a success rate of 80%. The broad applicability of this robotic manipulation technique is demonstrated using multiple cell types on traditional substrates and on open-top microfabricated devices, without requiring modifications to device designs. Furthermore, we used this serial deposition process in conjunction with an established parallel cell manipulation technique to improve the efficiency of single cell capture from ∼80% to 100%. Using a robotic micromanipulation system to position single cells on a substrate is demonstrated as an effective stand-alone or bolstering technology for single-cell studies, eliminating some of the drawbacks associated with standard single-cell handling and manipulation techniques.

  17. CellStress - open source image analysis program for single-cell analysis

    NASA Astrophysics Data System (ADS)

    Smedh, Maria; Beck, Caroline; Sott, Kristin; Goksör, Mattias

    2010-08-01

    This work describes our image-analysis software, CellStress, which has been developed in Matlab and is issued under a GPL license. CellStress was developed in order to analyze migration of fluorescent proteins inside single cells during changing environmental conditions. CellStress can also be used to score information regarding protein aggregation in single cells over time, which is especially useful when monitoring cell signaling pathways involved in e.g. Alzheimer's or Huntington's disease. Parallel single-cell analysis of large numbers of cells is an important part of the research conducted in systems biology and quantitative biology in order to mathematically describe cellular processes. To quantify properties for single cells, large amounts of data acquired during extended time periods are needed. Manual analyses of such data involve huge efforts and could also include a bias, which complicates the use and comparison of data for further simulations or modeling. Therefore, it is necessary to have an automated and unbiased image analysis procedure, which is the aim of CellStress. CellStress utilizes cell contours detected by CellStat (developed at Fraunhofer-Chalmers Centre), which identifies cell boundaries using bright field images, and thus reduces the fluorescent labeling needed.

  18. Single cell measurements of vacuolar rupture caused by intracellular pathogens.

    PubMed

    Keller, Charlotte; Mellouk, Nora; Danckaert, Anne; Simeone, Roxane; Brosch, Roland; Enninga, Jost; Bobard, Alexandre

    2013-06-12

    Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to β-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, β-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

  19. Supramolecular Probes for Assessing Glutamine Uptake Enable Semi-Quantitative Metabolic Models in Single Cells

    PubMed Central

    Xue, Min; Wei, Wei; Su, Yapeng; Johnson, Dazy; Heath, James R.

    2016-01-01

    We describe a supramolecular surface competition assay for quantifying glutamine uptake from single cells. Cy3-labeled cyclodextrins were immobilized on a glass surface as a supramolecular host/FRET donor, and adamantane-BHQ2 conjugates were employed as the guest/quencher. An adamantane-labeled glutamine analog was selected through screening a library of compounds and validated by cell uptake experiments. When integrated onto a single cell barcode chip (SCBC) with a multiplex panel of 15 other metabolites, associated metabolic enzymes, and phosphoproteins, the resultant data provided input for a steady state model that describes energy potential in single cells, and correlates that potential with receptor tyrosine kinase signaling. We utilize this integrated assay to interrogate a dose-dependent response of model brain cancer cells to EGFR inhibition. We find that low dose (1 μM erlotinib) drugging actually increases cellular energy potential even as glucose uptake and phosphoprotein signaling is repressed. We also identify new interactions between phosphoprotein signaling and cellular energy processes that may help explain the facile resistance exhibited by certain cancer patients to EGFR inhibitors. PMID:26916347

  20. Visualizing Wnt Palmitoylation in Single Cells.

    PubMed

    Gao, Xinxin; Hannoush, Rami N

    2016-01-01

    Wnt palmitoylation regulates its secretion and signaling activity in cells. Methods to monitor cellular Wnt palmitoylation are instrumental in investigating Wnt activity, secretion, and its interaction with cellular membrane compartments. This protocol describes a method we have recently developed to detect cellular Wnt palmitoylation. The method, combining click chemistry, bio-orthogonal fatty acid probes, and proximity ligation assay (PLA), provides high sensitivity and subcellular resolution for detection of Wnt palmitoylation. It is also compatible with multiple imaging platforms, and is applicable to detecting palmitoylated forms of other fatty acylated proteins. PMID:27590146

  1. Cloning of Plasmodium falciparum by single-cell sorting.

    PubMed

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-10-01

    Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples.

  2. Advances in measuring single-cell pharmacology in vivo.

    PubMed

    Vinegoni, Claudio; Dubach, J Matthew; Thurber, Greg M; Miller, Miles A; Mazitschek, Ralph; Weissleder, Ralph

    2015-09-01

    Measuring key pharmacokinetic and pharmacodynamic parameters in vivo at the single cell level is likely to enhance drug discovery and development. In this review, we summarize recent advances in this field and highlight current and future capabilities. PMID:26024776

  3. Droplet microfluidics--a tool for single-cell analysis.

    PubMed

    Joensson, Haakan N; Andersson Svahn, Helene

    2012-12-01

    Droplet microfluidics allows the isolation of single cells and reagents in monodisperse picoliter liquid capsules and manipulations at a throughput of thousands of droplets per second. These qualities allow many of the challenges in single-cell analysis to be overcome. Monodispersity enables quantitative control of solute concentrations, while encapsulation in droplets provides an isolated compartment for the single cell and its immediate environment. The high throughput allows the processing and analysis of the tens of thousands to millions of cells that must be analyzed to accurately describe a heterogeneous cell population so as to find rare cell types or access sufficient biological space to find hits in a directed evolution experiment. The low volumes of the droplets make very large screens economically viable. This Review gives an overview of the current state of single-cell analysis involving droplet microfluidics and offers examples where droplet microfluidics can further biological understanding.

  4. Computational and analytical challenges in single-cell transcriptomics.

    PubMed

    Stegle, Oliver; Teichmann, Sarah A; Marioni, John C

    2015-03-01

    The development of high-throughput RNA sequencing (RNA-seq) at the single-cell level has already led to profound new discoveries in biology, ranging from the identification of novel cell types to the study of global patterns of stochastic gene expression. Alongside the technological breakthroughs that have facilitated the large-scale generation of single-cell transcriptomic data, it is important to consider the specific computational and analytical challenges that still have to be overcome. Although some tools for analysing RNA-seq data from bulk cell populations can be readily applied to single-cell RNA-seq data, many new computational strategies are required to fully exploit this data type and to enable a comprehensive yet detailed study of gene expression at the single-cell level.

  5. Single-cell bioelectrical impedance platform for monitoring cellular response to drug treatment.

    PubMed

    Asphahani, Fareid; Wang, Kui; Thein, Myo; Veiseh, Omid; Yung, Sandy; Xu, Jian; Zhang, Miqin

    2011-02-01

    The response of cells to a chemical or biological agent in terms of their impedance changes in real-time is a useful mechanism that can be utilized for a wide variety of biomedical and environmental applications. The use of a single-cell-based analytical platform could be an effective approach to acquiring more sensitive cell impedance measurements, particularly in applications where only diminutive changes in impedance are expected. Here, we report the development of an on-chip cell impedance biosensor with two types of electrodes that host individual cells and cell populations, respectively, to study its efficacy in detecting cellular response. Human glioblastoma (U87MG) cells were patterned on single- and multi-cell electrodes through ligand-mediated natural cell adhesion. We comparatively investigated how these cancer cells on both types of electrodes respond to an ion channel inhibitor, chlorotoxin (CTX), in terms of their shape alternations and impedance changes to exploit the fine detectability of the single-cell-based system. The detecting electrodes hosting single cells exhibited a significant reduction in the real impedance signal, while electrodes hosting confluent monolayer of cells showed little to no impedance change. When single-cell electrodes were treated with CTX of different doses, a dose-dependent impedance change was observed. This enables us to identify the effective dose needed for this particular treatment. Our study demonstrated that this single-cell impedance system may potentially serve as a useful analytical tool for biomedical applications such as environmental toxin detection and drug evaluation.

  6. Single-cell bioelectrical impedance platform for monitoring cellular response to drug treatment

    NASA Astrophysics Data System (ADS)

    Asphahani, Fareid; Wang, Kui; Thein, Myo; Veiseh, Omid; Yung, Sandy; Xu, Jian; Zhang, Miqin

    2011-02-01

    The response of cells to a chemical or biological agent in terms of their impedance changes in real-time is a useful mechanism that can be utilized for a wide variety of biomedical and environmental applications. The use of a single-cell-based analytical platform could be an effective approach to acquiring more sensitive cell impedance measurements, particularly in applications where only diminutive changes in impedance are expected. Here, we report the development of an on-chip cell impedance biosensor with two types of electrodes that host individual cells and cell populations, respectively, to study its efficacy in detecting cellular response. Human glioblastoma (U87MG) cells were patterned on single- and multi-cell electrodes through ligand-mediated natural cell adhesion. We comparatively investigated how these cancer cells on both types of electrodes respond to an ion channel inhibitor, chlorotoxin (CTX), in terms of their shape alternations and impedance changes to exploit the fine detectability of the single-cell-based system. The detecting electrodes hosting single cells exhibited a significant reduction in the real impedance signal, while electrodes hosting confluent monolayer of cells showed little to no impedance change. When single-cell electrodes were treated with CTX of different doses, a dose-dependent impedance change was observed. This enables us to identify the effective dose needed for this particular treatment. Our study demonstrated that this single-cell impedance system may potentially serve as a useful analytical tool for biomedical applications such as environmental toxin detection and drug evaluation.

  7. Inside Single Cells: Quantitative Analysis with Advanced Optics and Nanomaterials

    PubMed Central

    Cui, Yi; Irudayaraj, Joseph

    2014-01-01

    Single cell explorations offer a unique window to inspect molecules and events relevant to mechanisms and heterogeneity constituting the central dogma of biology. A large number of nucleic acids, proteins, metabolites and small molecules are involved in determining and fine-tuning the state and function of a single cell at a given time point. Advanced optical platforms and nanotools provide tremendous opportunities to probe intracellular components with single-molecule accuracy, as well as promising tools to adjust single cell activity. In order to obtain quantitative information (e.g. molecular quantity, kinetics and stoichiometry) within an intact cell, achieving the observation with comparable spatiotemporal resolution is a challenge. For single cell studies both the method of detection and the biocompatibility are critical factors as they determine the feasibility, especially when considering live cell analysis. Although a considerable proportion of single cell methodologies depend on specialized expertise and expensive instruments, it is our expectation that the information content and implication will outweigh the costs given the impact on life science enabled by single cell analysis. PMID:25430077

  8. Biased Allelic Expression in Human Primary Fibroblast Single Cells

    PubMed Central

    Borel, Christelle; Ferreira, Pedro G.; Santoni, Federico; Delaneau, Olivier; Fort, Alexandre; Popadin, Konstantin Y.; Garieri, Marco; Falconnet, Emilie; Ribaux, Pascale; Guipponi, Michel; Padioleau, Ismael; Carninci, Piero; Dermitzakis, Emmanouil T.; Antonarakis, Stylianos E.

    2015-01-01

    The study of gene expression in mammalian single cells via genomic technologies now provides the possibility to investigate the patterns of allelic gene expression. We used single-cell RNA sequencing to detect the allele-specific mRNA level in 203 single human primary fibroblasts over 133,633 unique heterozygous single-nucleotide variants (hetSNVs). We observed that at the snapshot of analyses, each cell contained mostly transcripts from one allele from the majority of genes; indeed, 76.4% of the hetSNVs displayed stochastic monoallelic expression in single cells. Remarkably, adjacent hetSNVs exhibited a haplotype-consistent allelic ratio; in contrast, distant sites located in two different genes were independent of the haplotype structure. Moreover, the allele-specific expression in single cells correlated with the abundance of the cellular transcript. We observed that genes expressing both alleles in the majority of the single cells at a given time point were rare and enriched with highly expressed genes. The relative abundance of each allele in a cell was controlled by some regulatory mechanisms given that we observed related single-cell allelic profiles according to genes. Overall, these results have direct implications in cellular phenotypic variability. PMID:25557783

  9. Microfluidic single-cell analysis of intracellular compounds

    PubMed Central

    Chao, Tzu-Chiao; Ros, Alexandra

    2008-01-01

    Biological analyses traditionally probe cell ensembles in the range of 103–106 cells, thereby completely averaging over relevant individual cell responses, such as differences in cell proliferation, responses to external stimuli or disease onset. In past years, this fact has been realized and increasing interest has evolved for single-cell analytical methods, which could give exciting new insights into genomics, proteomics, transcriptomics and systems biology. Microfluidic or lab-on-a-chip devices are the method of choice for single-cell analytical tools as they allow the integration of a variety of necessary process steps involved in single-cell analysis, such as selection, navigation, positioning or lysis of single cells as well as separation and detection of cellular analytes. Along with this advantageous integration, microfluidic devices confine single cells in compartments near their intrinsic volume, thus minimizing dilution effects and increasing detection sensitivity. This review overviews the developments and achievements of microfluidic single-cell analysis of intracellular compounds in the past few years, from proof-of-principle devices to applications demonstrating a high biological relevance. PMID:18682362

  10. Genetic programs constructed from layered logic gates in single cells

    PubMed Central

    Moon, Tae Seok; Lou, Chunbo; Tamsir, Alvin; Stanton, Brynne C.; Voigt, Christopher A.

    2014-01-01

    Genetic programs function to integrate environmental sensors, implement signal processing algorithms and control expression dynamics1. These programs consist of integrated genetic circuits that individually implement operations ranging from digital logic to dynamic circuits2–6, and they have been used in various cellular engineering applications, including the implementation of process control in metabolic networks and the coordination of spatial differentiation in artificial tissues. A key limitation is that the circuits are based on biochemical interactions occurring in the confined volume of the cell, so the size of programs has been limited to a few circuits1,7. Here we apply part mining and directed evolution to build a set of transcriptional AND gates in Escherichia coli. Each AND gate integrates two promoter inputs and controls one promoter output. This allows the gates to be layered by having the output promoter of an upstream circuit serve as the input promoter for a downstream circuit. Each gate consists of a transcription factor that requires a second chaperone protein to activate the output promoter. Multiple activator–chaperone pairs are identified from type III secretion pathways in different strains of bacteria. Directed evolution is applied to increase the dynamic range and orthogonality of the circuits. These gates are connected in different permutations to form programs, the largest of which is a 4-input AND gate that consists of 3 circuits that integrate 4 inducible systems, thus requiring 11 regulatory proteins. Measuring the performance of individual gates is sufficient to capture the behaviour of the complete program. Errors in the output due to delays (faults), a common problem for layered circuits, are not observed. This work demonstrates the successful layering of orthogonal logic gates, a design strategy that could enable the construction of large, integrated circuits in single cells. PMID:23041931

  11. Information Processing in Single Cells and Small Networks: Insights from Compartmental Models

    NASA Astrophysics Data System (ADS)

    Poirazi, Panayiota

    2009-03-01

    The goal of this paper is to present a set of predictions generated by detailed compartmental models regarding the ways in which information may be processed, encoded and propagated by single cells and neural assemblies. Towards this goal, I will review a number of modelling studies from our lab that investigate how single pyramidal neurons and small neural networks in different brain regions process incoming signals that are associated with learning and memory. I will first discuss the computational capabilities of individual pyramidal neurons in the hippocampus [1-3] and how these properties may allow a single cell to discriminate between different memories [4]. I will then present biophysical models of prefrontal layer V neurons and small networks that exhibit sustained activity under realistic synaptic stimulation and discuss their potential role in working memory [5].

  12. Investigating IL-1β Secretion Using Real-Time Single-Cell Imaging.

    PubMed

    Diamond, Catherine; Bagnall, James; Spiller, David G; White, Michael R; Mortellaro, Alessandra; Paszek, Pawel; Brough, David

    2016-01-01

    The pro-inflammatory cytokine interleukin (IL)-1β is an important mediator of the inflammatory response. In order to perform its role in the inflammatory cascade, IL-1β must be secreted from the cell, yet it lacks a signal peptide that is required for conventional secretion, and the exact mechanism of release remains undefined. Conventional biochemical methods have limited the investigation into the processes involved in IL-1β secretion to population dynamics, yet heterogeneity between cells has been observed at a single-cell level. Here, greater sensitivity is achieved with the use of a newly developed vector that codes for a fluorescently labelled version of IL-1β. Combining this with real-time single-cell confocal microscopy using the methods described here, we have developed an effective protocol for investigating the mechanisms of IL-1β secretion and the testing of the hypothesis that IL-1β secretion requires membrane permeabilisation. PMID:27221482

  13. A microfluidic approach to parallelized transcriptional profiling of single cells

    PubMed Central

    Sun, Hao; Olsen, Timothy; Zhu, Jing; Tao, Jianguo; Ponnaiya, Brian; Amundson, Sally A.; Brenner, David J.; Lin, Qiao

    2016-01-01

    The ability to correlate single-cell genetic information with cellular phenotypes is of great importance to biology and medicine, as it holds the potential to gain insight into disease pathways that is unavailable from ensemble measurements. We present a microfluidic approach to parallelized, rapid, quantitative analysis of messenger RNA from single cells via RT-qPCR. The approach leverages an array of single-cell RT-qPCR analysis units formed by a set of parallel microchannels concurrently controlled by elastomeric pneumatic valves, thereby enabling parallelized handling and processing of single cells in a drastically simplified operation procedure using a relatively small number of microvalves. All steps for single-cell RT-qPCR, including cell isolation and immobilization, cell lysis, mRNA purification, reverse transcription and qPCR, are integrated on a single chip, eliminating the need for off-chip manual cell and reagent transfer and qPCR amplification as commonly used in existing approaches. Additionally, the approach incorporates optically transparent microfluidic components to allow monitoring of single-cell trapping without the need for molecular labeling that can potentially alter the targeted gene expression and utilizes a polycarbonate film as a barrier against evaporation to minimize the loss of reagents at elevated temperatures during the analysis. We demonstrate the utility of the approach by the transcriptional profiling for the induction of the cyclin-dependent kinase inhibitor 1a and the glyceraldehyde 3-phosphate dehydrogenase in single cells from the MCF-7 breast cancer cell line. Furthermore, the methyl methanesulfonate is employed to allow measurement of the expression of the genes in individual cells responding to a genotoxic stress. PMID:27194954

  14. Microfluidic single-cell whole-transcriptome sequencing

    PubMed Central

    Streets, Aaron M.; Zhang, Xiannian; Cao, Chen; Pang, Yuhong; Wu, Xinglong; Xiong, Liang; Yang, Lu; Fu, Yusi; Zhao, Liang; Tang, Fuchou; Huang, Yanyi

    2014-01-01

    Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis. PMID:24782542

  15. Single-cell analysis delineates a trajectory toward the human early otic lineage.

    PubMed

    Ealy, Megan; Ellwanger, Daniel C; Kosaric, Nina; Stapper, Andres P; Heller, Stefan

    2016-07-26

    Efficient pluripotent stem cell guidance protocols for the production of human posterior cranial placodes such as the otic placode that gives rise to the inner ear do not exist. Here we use a systematic approach including defined monolayer culture, signaling modulation, and single-cell gene expression analysis to delineate a developmental trajectory for human otic lineage specification in vitro. We found that modulation of bone morphogenetic protein (BMP) and WNT signaling combined with FGF and retinoic acid treatments over the course of 18 days generates cell populations that develop chronological expression of marker genes of non-neural ectoderm, preplacodal ectoderm, and early otic lineage. Gene expression along this differentiation path is distinct from other lineages such as endoderm, mesendoderm, and neural ectoderm. Single-cell analysis exposed the heterogeneity of differentiating cells and allowed discrimination of non-neural ectoderm and otic lineage cells from off-target populations. Pseudotemporal ordering of human embryonic stem cell and induced pluripotent stem cell-derived single-cell gene expression profiles revealed an initially synchronous guidance toward non-neural ectoderm, followed by comparatively asynchronous occurrences of preplacodal and otic marker genes. Positive correlation of marker gene expression between both cell lines and resemblance to mouse embryonic day 10.5 otocyst cells implied reasonable robustness of the guidance protocol. Single-cell trajectory analysis further revealed that otic progenitor cell types are induced in monolayer cultures, but further development appears impeded, likely because of lack of a lineage-stabilizing microenvironment. Our results provide a framework for future exploration of stabilizing microenvironments for efficient differentiation of stem cell-generated human otic cell types. PMID:27402757

  16. Automated single cell isolation from suspension with computer vision

    PubMed Central

    Ungai-Salánki, Rita; Gerecsei, Tamás; Fürjes, Péter; Orgovan, Norbert; Sándor, Noémi; Holczer, Eszter; Horvath, Robert; Szabó, Bálint

    2016-01-01

    Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1–2 nanoliters from a thin (~100 μm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1 μm positioning precision, adaptive cell targeting and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved. PMID:26856740

  17. Single-cell measurement of red blood cell oxygen affinity

    PubMed Central

    Di Caprio, Giuseppe; Stokes, Chris; Higgins, John M.; Schonbrun, Ethan

    2015-01-01

    Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen–Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2–3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability. PMID:26216973

  18. Automated single cell isolation from suspension with computer vision.

    PubMed

    Ungai-Salánki, Rita; Gerecsei, Tamás; Fürjes, Péter; Orgovan, Norbert; Sándor, Noémi; Holczer, Eszter; Horvath, Robert; Szabó, Bálint

    2016-02-09

    Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1-2 nanoliters from a thin (~100 μm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1 μm positioning precision, adaptive cell targeting and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved.

  19. Electrical impedance tomographic imaging of a single cell electroporation.

    PubMed

    Meir, Arie; Rubinsky, Boris

    2014-06-01

    A living cell placed in a high strength electric field, can undergo a process known as electroporation. It is believed that during electroporation nano-scale defects (pores) occur in the membrane of the cell, causing dramatic changes to the permeability of its membrane. Electroporation is an important technique in biotechnology and medicine and numerous methods are being developed to improve the understanding and use of the technology. We propose to extend the toolbox available for studying electroporation by generating impedance distribution images of the cell as it undergoes electroporation using Electrical Impedance Tomography (EIT). To investigate the feasibility of this concept, we develop a mathematical model of the process of electroporation in a single cell and of EIT of the process and show simulation results of a computer-based finite element model (FEM). Our work is an attempt to develop a new imaging tool for visualizing electroporation in a single cell, offering a different temporal and spatial resolution compared to the state of the art, which includes bulk measurements of electrical properties during single cell electroporation, patch clamp and voltage clamp measurement in single cells and optical imaging with colorimetric dyes during single cell electroporation. This paper is a preliminary theoretic feasibility study.

  20. Overview of single-cell elastic light scattering techniques.

    PubMed

    Kinnunen, Matti; Karmenyan, Artashes

    2015-05-01

    We present and discuss several modern optical methods based on elastic light scattering (ELS), along with their technical features and applications in biomedicine and life sciences. In particular, we review some ELS experiments at the single-cell level and explore new directions of applications. Due to recent developments in experimental systems (as shown in the literature), ELS lends itself to useful applications in the life sciences. Of the developed methods, we cover elastic scattering spectroscopy, optical tweezer-assisted measurement, goniometers, Fourier transform light scattering (FTLS), and microscopic methods. FTLS significantly extends the potential analysis of single cells by allowing monitoring of dynamical changes at the single-cell level. The main aim of our review is to demonstrate developments in the experimental investigation of ELS in single cells including issues related to theoretical “representations” and modeling of biological systems (cells, cellular systems, tissues, and so on). Goniometric measurements of ELS from optically trapped single cells are shown and the importance of the experimental verification of theoretical models of ELS in the context of biomedical applications is discussed.

  1. Single-cell printer: automated, on demand, and label free.

    PubMed

    Gross, Andre; Schöndube, Jonas; Niekrawitz, Sonja; Streule, Wolfgang; Riegger, Lutz; Zengerle, Roland; Koltay, Peter

    2013-12-01

    Within the past years, single-cell analysis has developed into a key topic in cell biology to study cellular functions that are not accessible by investigation of larger cell populations. Engineering approaches aiming to access single cells to extract information about their physiology, phenotype, and genotype at the single-cell level are going manifold ways, meanwhile allowing separation, sorting, culturing, and analysis of individual cells. Based on our earlier research toward inkjet-like printing of single cells, this article presents further characterization results obtained with a fully automated prototype instrument for printing of single living cells in a noncontact inkjet-like manner. The presented technology is based on a transparent microfluidic drop-on-demand dispenser chip coupled with a camera-assisted automatic detection system. Cells inside the chip are detected and classified with this detection system before they are expelled from the nozzle confined in microdroplets, thus enabling a "one cell per droplet" printing mode. To demonstrate the prototype instrument's suitability for biological and biomedical applications, basic experiments such as printing of single-bead and cell arrays as well as deposition and culture of single cells in microwell plates are presented. Printing efficiencies greater than 80% and viability rates about 90% were achieved.

  2. Single-cell technologies to study the immune system.

    PubMed

    Proserpio, Valentina; Mahata, Bidesh

    2016-02-01

    The immune system is composed of a variety of cells that act in a coordinated fashion to protect the organism against a multitude of different pathogens. The great variability of existing pathogens corresponds to a similar high heterogeneity of the immune cells. The study of individual immune cells, the fundamental unit of immunity, has recently transformed from a qualitative microscopic imaging to a nearly complete quantitative transcriptomic analysis. This shift has been driven by the rapid development of multiple single-cell technologies. These new advances are expected to boost the detection of less frequent cell types and transient or intermediate cell states. They will highlight the individuality of each single cell and greatly expand the resolution of current available classifications and differentiation trajectories. In this review we discuss the recent advancement and application of single-cell technologies, their limitations and future applications to study the immune system.

  3. Single-cell RNA-seq: advances and future challenges

    PubMed Central

    Saliba, Antoine-Emmanuel; Westermann, Alexander J.; Gorski, Stanislaw A.; Vogel, Jörg

    2014-01-01

    Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Single-cell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNA-seq protocols have been introduced and are reviewed here—each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field. PMID:25053837

  4. Disentangling neural cell diversity using single-cell transcriptomics.

    PubMed

    Poulin, Jean-Francois; Tasic, Bosiljka; Hjerling-Leffler, Jens; Trimarchi, Jeffrey M; Awatramani, Rajeshwar

    2016-08-26

    Cellular specialization is particularly prominent in mammalian nervous systems, which are composed of millions to billions of neurons that appear in thousands of different 'flavors' and contribute to a variety of functions. Even in a single brain region, individual neurons differ greatly in their morphology, connectivity and electrophysiological properties. Systematic classification of all mammalian neurons is a key goal towards deconstructing the nervous system into its basic components. With the recent advances in single-cell gene expression profiling technologies, it is now possible to undertake the enormous task of disentangling neuronal heterogeneity. High-throughput single-cell RNA sequencing and multiplexed quantitative RT-PCR have become more accessible, and these technologies enable systematic categorization of individual neurons into groups with similar molecular properties. Here we provide a conceptual and practical guide to classification of neural cell types using single-cell gene expression profiling technologies. PMID:27571192

  5. Spatial reconstruction of single-cell gene expression

    PubMed Central

    Satija, Rahul; Farrell, Jeffrey A.; Gennert, David; Schier, Alexander F.; Regev, Aviv

    2015-01-01

    Spatial localization is a key determinant of cellular fate and behavior, but spatial RNA assays traditionally rely on staining for a limited number of RNA species. In contrast, single-cell RNA-seq allows for deep profiling of cellular gene expression, but established methods separate cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos, inferring a transcriptome-wide map of spatial patterning. We confirmed Seurat’s accuracy using several experimental approaches, and used it to identify a set of archetypal expression patterns and spatial markers. Additionally, Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems. PMID:25867923

  6. Disentangling neural cell diversity using single-cell transcriptomics.

    PubMed

    Poulin, Jean-Francois; Tasic, Bosiljka; Hjerling-Leffler, Jens; Trimarchi, Jeffrey M; Awatramani, Rajeshwar

    2016-08-26

    Cellular specialization is particularly prominent in mammalian nervous systems, which are composed of millions to billions of neurons that appear in thousands of different 'flavors' and contribute to a variety of functions. Even in a single brain region, individual neurons differ greatly in their morphology, connectivity and electrophysiological properties. Systematic classification of all mammalian neurons is a key goal towards deconstructing the nervous system into its basic components. With the recent advances in single-cell gene expression profiling technologies, it is now possible to undertake the enormous task of disentangling neuronal heterogeneity. High-throughput single-cell RNA sequencing and multiplexed quantitative RT-PCR have become more accessible, and these technologies enable systematic categorization of individual neurons into groups with similar molecular properties. Here we provide a conceptual and practical guide to classification of neural cell types using single-cell gene expression profiling technologies.

  7. Defining heterogeneity within bacterial populations via single cell approaches.

    PubMed

    Davis, Kimberly M; Isberg, Ralph R

    2016-08-01

    Bacterial populations are heterogeneous, which in many cases can provide a selective advantage during changes in environmental conditions. In some instances, heterogeneity exists at the genetic level, in which significant allelic variation occurs within a population seeded by a single cell. In other cases, heterogeneity exists due to phenotypic differences within a clonal, genetically identical population. A variety of mechanisms can drive this latter strategy. Stochastic fluctuations can drive differential gene expression, but heterogeneity in gene expression can also be driven by environmental changes sensed by individual cells residing in distinct locales. Utilizing multiple single cell approaches, workers have started to uncover the extent of heterogeneity within bacterial populations. This review will first describe several examples of phenotypic and genetic heterogeneity, and then discuss many single cell approaches that have recently been applied to define heterogeneity within bacterial populations. PMID:27273675

  8. Single-cell sequencing in stem cell biology.

    PubMed

    Wen, Lu; Tang, Fuchou

    2016-04-15

    Cell-to-cell variation and heterogeneity are fundamental and intrinsic characteristics of stem cell populations, but these differences are masked when bulk cells are used for omic analysis. Single-cell sequencing technologies serve as powerful tools to dissect cellular heterogeneity comprehensively and to identify distinct phenotypic cell types, even within a 'homogeneous' stem cell population. These technologies, including single-cell genome, epigenome, and transcriptome sequencing technologies, have been developing rapidly in recent years. The application of these methods to different types of stem cells, including pluripotent stem cells and tissue-specific stem cells, has led to exciting new findings in the stem cell field. In this review, we discuss the recent progress as well as future perspectives in the methodologies and applications of single-cell omic sequencing technologies.

  9. Review of methods to probe single cell metabolism and bioenergetics

    PubMed Central

    Vasdekis, Andreas E.; Stephanopoulos, Gregory

    2015-01-01

    Single cell investigations have enabled unexpected discoveries, such as the existence of biological noise and phenotypic switching in infection, metabolism and treatment. Herein, we review methods that enable such single cell investigations specific to metabolism and bioenergetics. Firstly, we discuss how to isolate and immobilize individuals from a cell suspension, including both permanent and reversible approaches. We also highlight specific advances in microbiology for its implications in metabolic engineering. Methods for probing single cell physiology and metabolism are subsequently reviewed. The primary focus therein is on dynamic and high-content profiling strategies based on label-free and fluorescence microspectroscopy and microscopy. Non-dynamic approaches, such as mass spectrometry and nuclear magnetic resonance, are also briefly discussed. PMID:25448400

  10. Single-cell technologies for monitoring immune systems

    PubMed Central

    Chattopadhyay, Pratip K; Gierahn, Todd M; Roederer, Mario; Love, J Christopher

    2014-01-01

    The complex heterogeneity of cells, and their interconnectedness with each other, are major challenges to identifying clinically relevant measurements that reflect the state and capability of the immune system. Highly multiplexed, single-cell technologies may be critical for identifying correlates of disease or immunological interventions as well as for elucidating the underlying mechanisms of immunity. Here we review limitations of bulk measurements and explore advances in single-cell technologies that overcome these problems by expanding the depth and breadth of functional and phenotypic analysis in space and time. The geometric increases in complexity of data make formidable hurdles for exploring, analyzing and presenting results. We summarize recent approaches to making such computations tractable and discuss challenges for integrating heterogeneous data obtained using these single-cell technologies. PMID:24448570

  11. Automated single cell sorting and deposition in submicroliter drops

    NASA Astrophysics Data System (ADS)

    Salánki, Rita; Gerecsei, Tamás; Orgovan, Norbert; Sándor, Noémi; Péter, Beatrix; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

    2014-08-01

    Automated manipulation and sorting of single cells are challenging, when intact cells are needed for further investigations, e.g., RNA or DNA sequencing. We applied a computer controlled micropipette on a microscope admitting 80 PCR (Polymerase Chain Reaction) tubes to be filled with single cells in a cycle. Due to the Laplace pressure, fluid starts to flow out from the micropipette only above a critical pressure preventing the precise control of drop volume in the submicroliter range. We found an anomalous pressure additive to the Laplace pressure that we attribute to the evaporation of the drop. We have overcome the problem of the critical dropping pressure with sequentially operated fast fluidic valves timed with a millisecond precision. Minimum drop volume was 0.4-0.7 μl with a sorting speed of 15-20 s per cell. After picking NE-4C neuroectodermal mouse stem cells and human primary monocytes from a standard plastic Petri dish we could gently deposit single cells inside tiny drops. 94 ± 3% and 54 ± 7% of the deposited drops contained single cells for NE-4C and monocytes, respectively. 7.5 ± 4% of the drops contained multiple cells in case of monocytes. Remaining drops were empty. Number of cells deposited in a drop could be documented by imaging the Petri dish before and after sorting. We tuned the adhesion force of cells to make the manipulation successful without the application of microstructures for trapping cells on the surface. We propose that our straightforward and flexible setup opens an avenue for single cell isolation, critically needed for the rapidly growing field of single cell biology.

  12. CalQuo: automated, simultaneous single-cell and population-level quantification of global intracellular Ca2+ responses

    NASA Astrophysics Data System (ADS)

    Fritzsche, Marco; Fernandes, Ricardo A.; Colin-York, Huw; Santos, Ana M.; Lee, Steven F.; Lagerholm, B. Christoffer; Davis, Simon J.; Eggeling, Christian

    2015-11-01

    Detecting intracellular calcium signaling with fluorescent calcium indicator dyes is often coupled with microscopy techniques to follow the activation state of non-excitable cells, including lymphocytes. However, the analysis of global intracellular calcium responses both at the single-cell level and in large ensembles simultaneously has yet to be automated. Here, we present a new software package, CalQuo (Calcium Quantification), which allows the automated analysis and simultaneous monitoring of global fluorescent calcium reporter-based signaling responses in up to 1000 single cells per experiment, at temporal resolutions of sub-seconds to seconds. CalQuo quantifies the number and fraction of responding cells, the temporal dependence of calcium signaling and provides global and individual calcium-reporter fluorescence intensity profiles. We demonstrate the utility of the new method by comparing the calcium-based signaling responses of genetically manipulated human lymphocytic cell lines.

  13. Dichotomy of cellular inhibition by small-molecule inhibitors revealed by single-cell analysis

    PubMed Central

    Vogel, Robert M.; Erez, Amir; Altan-Bonnet, Grégoire

    2016-01-01

    Despite progress in drug development, a quantitative and physiological understanding of how small-molecule inhibitors act on cells is lacking. Here, we measure the signalling and proliferative response of individual primary T-lymphocytes to a combination of antigen, cytokine and drug. We uncover two distinct modes of signalling inhibition: digital inhibition (the activated fraction of cells diminishes upon drug treatment, but active cells appear unperturbed), versus analogue inhibition (the activated fraction is unperturbed whereas activation response is diminished). We introduce a computational model of the signalling cascade that accounts for such inhibition dichotomy, and test the model predictions for the phenotypic variability of cellular responses. Finally, we demonstrate that the digital/analogue dichotomy of cellular response as revealed on short (signal transduction) timescales, translates into similar dichotomy on longer (proliferation) timescales. Our single-cell analysis of drug action illustrates the strength of quantitative approaches to translate in vitro pharmacology into functionally relevant cellular settings. PMID:27687249

  14. A single cell penetration system by ultrasonic driving

    NASA Astrophysics Data System (ADS)

    Zhou, Zhaoying; Xiao, Mingfei; Yang, Xing; Wu, Ting

    2008-12-01

    The researches of single cell's control and operation are the hotspots in whole world. Among the various technologies, the transmission of ectogenic genetic materials between cell membrane is very significant. Imitating the Chinese traditional acupuncture therapy, a new ultrasonic resonance driving method, is imported to drive a cell's penetration probe. A set of the single cell penetration system was established to perform this function. This system includes four subsystems: driving part, micromanipulation part, observation and measurement part, and actuation part. Some fish egg experiments indicate that this system is workable and effective.

  15. Single-cell epigenomics: techniques and emerging applications.

    PubMed

    Schwartzman, Omer; Tanay, Amos

    2015-12-01

    Epigenomics is the study of the physical modifications, associations and conformations of genomic DNA sequences, with the aim of linking these with epigenetic memory, cellular identity and tissue-specific functions. While current techniques in the field are characterizing the average epigenomic features across large cell ensembles, the increasing interest in the epigenetics within complex and heterogeneous tissues is driving the development of single-cell epigenomics. We review emerging single-cell methods for capturing DNA methylation, chromatin accessibility, histone modifications, chromosome conformation and replication dynamics. Together, these techniques are rapidly becoming a powerful tool in studies of cellular plasticity and diversity, as seen in stem cells and cancer.

  16. The technology and biology of single-cell RNA sequencing.

    PubMed

    Kolodziejczyk, Aleksandra A; Kim, Jong Kyoung; Svensson, Valentine; Marioni, John C; Teichmann, Sarah A

    2015-05-21

    The differences between individual cells can have profound functional consequences, in both unicellular and multicellular organisms. Recently developed single-cell mRNA-sequencing methods enable unbiased, high-throughput, and high-resolution transcriptomic analysis of individual cells. This provides an additional dimension to transcriptomic information relative to traditional methods that profile bulk populations of cells. Already, single-cell RNA-sequencing methods have revealed new biology in terms of the composition of tissues, the dynamics of transcription, and the regulatory relationships between genes. Rapid technological developments at the level of cell capture, phenotyping, molecular biology, and bioinformatics promise an exciting future with numerous biological and medical applications. PMID:26000846

  17. Functionalized nanopipettes: toward label-free, single cell biosensors

    PubMed Central

    Actis, Paolo; Mak, Andy C.

    2010-01-01

    Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study single cell dynamics. This review is focused on creative applications of nanopipette technology for biosensing. We highlight the potential of this technology with a particular attention to integration of this biosensor with single cell manipulation platforms. PMID:20730113

  18. Single-Cell Isolation and Gene Analysis: Pitfalls and Possibilities

    PubMed Central

    Hodne, Kjetil; Weltzien, Finn-Arne

    2015-01-01

    During the last two decades single-cell analysis (SCA) has revealed extensive phenotypic differences within homogenous cell populations. These phenotypic differences are reflected in the stochastic nature of gene regulation, which is often masked by qualitatively and quantitatively averaging in whole tissue analyses. The ability to isolate transcripts and investigate how genes are regulated at the single cell level requires highly sensitive and refined methods. This paper reviews different strategies currently used for SCA, including harvesting, reverse transcription, and amplification of the RNA, followed by methods for transcript quantification. The review provides the historical background to SCA, discusses limitations, and current and future possibilities in this exciting field of research. PMID:26569222

  19. Single cell pattern formation and transient cytoskeletal arrays

    PubMed Central

    Bement, William M.; von Dassow, George

    2015-01-01

    A major goal of developmental biology is to explain the emergence of pattern in cell layers, tissues and organs. Developmental biologists now accept that reaction diffusion-based mechanisms are broadly employed in developing organisms to direct pattern formation. Here we briefly consider these mechanisms and then apply some of the concepts derived from them to several processes that occur in single cells: wound repair, yeast budding, and cytokinesis. Two conclusions emerge from this analysis: first, there is considerable overlap at the level of general mechanisms between developmental and single cell pattern formation; second, dynamic structures based on the actin cytoskeleton may be far more ordered than is generally recognized. PMID:24529246

  20. Microfluidic extraction and stretching of chromosomal DNA from single cell nuclei for DNA fluorescence in situ hybridization

    PubMed Central

    Wang, Xiaozhu; Takebayashi, Shin-ichiro; Bernardin, Evans; Gilbert, David M.; Chella, Ravindran

    2012-01-01

    We have developed a novel method for genetic characterization of single cells by integrating microfluidic stretching of chromosomal DNA and fiber fluorescence in situ hybridization (FISH). In this method, individually isolated cell nuclei were immobilized in a microchannel. Chromosomal DNA was released from the nuclei and stretched by a pressure-driven flow. We analyzed and optimized flow conditions to generate a millimeter-long band of stretched DNA from each nucleus. Telomere fiber FISH was successfully performed on the stretched chromosomal DNA. Individual telomere fiber FISH signals from single cells could be resolved and their lengths measured, demonstrating the ability of the method to quantify genetic features at the level of single cells. PMID:22231286

  1. Single-Cell Gene Expression Analysis of Cholinergic Neurons in the Arcuate Nucleus of the Hypothalamus

    PubMed Central

    Chua, Streamson; Jo, Young-Hwan

    2016-01-01

    The cholinoceptive system in the hypothalamus, in particular in the arcuate nucleus (ARC), plays a role in regulating food intake. Neurons in the ARC contain multiple neuropeptides, amines, and neurotransmitters. To study molecular and neurochemical heterogeneity of ARC neurons, we combine single-cell qRT-PCR and single-cell whole transcriptome amplification methods to analyze expression patterns of our hand-picked 60 genes in individual neurons in the ARC. Immunohistochemical and single-cell qRT-PCR analyses show choline acetyltransferase (ChAT)-expressing neurons in the ARC. Gene expression patterns are remarkably distinct in each individual cholinergic neuron. Two-thirds of cholinergic neurons express tyrosine hydroxylase (Th) mRNA. A large subset of these Th-positive cholinergic neurons is GABAergic as they express the GABA synthesizing enzyme glutamate decarboxylase and vesicular GABA transporter transcripts. Some cholinergic neurons also express the vesicular glutamate transporter transcript gene. POMC and POMC-processing enzyme transcripts are found in a subpopulation of cholinergic neurons. Despite this heterogeneity, gene expression patterns in individual cholinergic cells appear to be highly regulated in a cell-specific manner. In fact, membrane receptor transcripts are clustered with their respective intracellular signaling and downstream targets. This novel population of cholinergic neurons may be part of the neural circuitries that detect homeostatic need for food and control the drive to eat. PMID:27611685

  2. Single-Cell Gene Expression Analysis of Cholinergic Neurons in the Arcuate Nucleus of the Hypothalamus.

    PubMed

    Jeong, Jae Hoon; Woo, Young Jae; Chua, Streamson; Jo, Young-Hwan

    2016-01-01

    The cholinoceptive system in the hypothalamus, in particular in the arcuate nucleus (ARC), plays a role in regulating food intake. Neurons in the ARC contain multiple neuropeptides, amines, and neurotransmitters. To study molecular and neurochemical heterogeneity of ARC neurons, we combine single-cell qRT-PCR and single-cell whole transcriptome amplification methods to analyze expression patterns of our hand-picked 60 genes in individual neurons in the ARC. Immunohistochemical and single-cell qRT-PCR analyses show choline acetyltransferase (ChAT)-expressing neurons in the ARC. Gene expression patterns are remarkably distinct in each individual cholinergic neuron. Two-thirds of cholinergic neurons express tyrosine hydroxylase (Th) mRNA. A large subset of these Th-positive cholinergic neurons is GABAergic as they express the GABA synthesizing enzyme glutamate decarboxylase and vesicular GABA transporter transcripts. Some cholinergic neurons also express the vesicular glutamate transporter transcript gene. POMC and POMC-processing enzyme transcripts are found in a subpopulation of cholinergic neurons. Despite this heterogeneity, gene expression patterns in individual cholinergic cells appear to be highly regulated in a cell-specific manner. In fact, membrane receptor transcripts are clustered with their respective intracellular signaling and downstream targets. This novel population of cholinergic neurons may be part of the neural circuitries that detect homeostatic need for food and control the drive to eat. PMID:27611685

  3. Single Cell Response to Time-dependent Stimuli using a Microfluidic Bioreactor

    NASA Astrophysics Data System (ADS)

    Johnson-Chavarria, Eric M.; Agrawal, Utsav; Tanyeri, Melikhan; Kuhlman, Thomas E.; Schroeder, Charles M.

    2014-03-01

    Cellular adaptation is critical for survival under uncertain or dynamic environmental conditions. Recent studies have reported the ability of biological systems to implement low-pass filters to distinguish high frequency noise in environmental stimuli from lower frequency input signals, yet we still lack a complete understanding of this phenomenon. In this work, we report a microfluidic-based platform for single cell analysis that provides dynamic control over periodic, time-dependent culture media. Single cells are confined in free solution by the sole action of gentle fluid flow, thereby enabling non-perturbative trapping of cells for long time scales. In this way, our microfluidic-based technique provides the ability to control external stimuli with precise methods while observing non-adherent cells over long timescales. Using this approach, we observed intranucleoid diffusion of genetic repressor proteins released from a chromosomal binding array. Overall, this microfluidic approach provides a direct method for sustaining periodic environmental conditions, measuring growth rates, and detecting gene expression of single cells in free solution. Funded by NIH Pathway to Independence (PI) Award, 4R00HG004183-03. This work was supported by the National Science Foundation through a Graduate Research Fellowship to Eric M. Johnson-Chavarria.

  4. Systematic Reconstruction of Molecular Cascades Regulating GP Development Using Single-Cell RNA-Seq.

    PubMed

    Li, Junxiang; Luo, Haofei; Wang, Rui; Lang, Jidong; Zhu, Siyu; Zhang, Zhenming; Fang, Jianhuo; Qu, Keke; Lin, Yuting; Long, Haizhou; Yao, Yi; Tian, Geng; Wu, Qiong

    2016-05-17

    The growth plate (GP) comprising sequentially differentiated cell layers is a critical structure for bone elongation and regeneration. Although several key regulators in GP development have been identified using genetic perturbation, systematic understanding is still limited. Here, we used single-cell RNA-sequencing (RNA-seq) to determine the gene expression profiles of 217 single cells from GPs and developed a bioinformatics pipeline named Sinova to de novo reconstruct physiological GP development in both temporal and spatial high resolution. Our unsupervised model not only confirmed prior knowledge, but also enabled the systematic discovery of genes, potential signal pathways, and surface markers CD9/CD200 to precisely depict development. Sinova further identified the effective combination of transcriptional factors (TFs) that regulates GP maturation, and the result was validated using an in vitro EGFP-Col10a screening system. Our case systematically reconstructed molecular cascades in GP development through single-cell profiling, and the bioinformatics pipeline is applicable to other developmental processes. VIDEO ABSTRACT. PMID:27160914

  5. Analysis of Intracellular Glucose at Single Cells Using Electrochemiluminescence Imaging.

    PubMed

    Xu, Jingjing; Huang, Peiyuan; Qin, Yu; Jiang, Dechen; Chen, Hong-Yuan

    2016-05-01

    Here, luminol electrochemiluminescence was first applied to analyze intracellular molecules, such as glucose, at single cells. The individual cells were retained in cell-sized microwells on a gold coated indium tin oxide (ITO) slide, which were treated with luminol, triton X-100, and glucose oxidase simultaneously. The broken cellular membrane in the presence of triton X-100 released intracellular glucose into the microwell and reacted with glucose oxidase to generate hydrogen peroxide, which induced luminol luminescence under positive potential. To achieve fast analysis, the luminescences from 64 individual cells on one ITO slide were imaged in 60 s using a charge-coupled device (CCD). More luminescence was observed at all the microwells after the introduction of triton X-100 and glucose oxidase suggested that intracellular glucose was detected at single cells. The starvation of cells to decrease intracellular glucose produced less luminescence, which confirmed that our luminescence intensity was correlated with the concentration of intracellular glucose. Large deviations in glucose concentration at observed single cells revealed high cellular heterogeneity in intracellular glucose for the first time. This developed electrochemiluminescence assay will be potentially applied for fast analysis of more intracellular molecules in single cells to elucidate cellular heterogeneity. PMID:27094779

  6. Selective single cell isolation for genomics using microraft arrays

    PubMed Central

    Welch, Joshua D.; Williams, Lindsay A.; DiSalvo, Matthew; Brandt, Alicia T.; Marayati, Raoud; Sims, Christopher E.; Allbritton, Nancy L.; Prins, Jan F.; Yeh, Jen Jen; Jones, Corbin D.

    2016-01-01

    Genomic methods are used increasingly to interrogate the individual cells that compose specific tissues. However, current methods for single cell isolation struggle to phenotypically differentiate specific cells in a heterogeneous population and rely primarily on the use of fluorescent markers. Many cellular phenotypes of interest are too complex to be measured by this approach, making it difficult to connect genotype and phenotype at the level of individual cells. Here we demonstrate that microraft arrays, which are arrays containing thousands of individual cell culture sites, can be used to select single cells based on a variety of phenotypes, such as cell surface markers, cell proliferation and drug response. We then show that a common genomic procedure, RNA-seq, can be readily adapted to the single cells isolated from these rafts. We show that data generated using microrafts and our modified RNA-seq protocol compared favorably with the Fluidigm C1. We then used microraft arrays to select pancreatic cancer cells that proliferate in spite of cytotoxic drug treatment. Our single cell RNA-seq data identified several expected and novel gene expression changes associated with early drug resistance. PMID:27530426

  7. Tumor Heterogeneity, Single-Cell Sequencing, and Drug Resistance

    PubMed Central

    Schmidt, Felix; Efferth, Thomas

    2016-01-01

    Tumor heterogeneity has been compared with Darwinian evolution and survival of the fittest. The evolutionary ecosystem of tumors consisting of heterogeneous tumor cell populations represents a considerable challenge to tumor therapy, since all genetically and phenotypically different subpopulations have to be efficiently killed by therapy. Otherwise, even small surviving subpopulations may cause repopulation and refractory tumors. Single-cell sequencing allows for a better understanding of the genomic principles of tumor heterogeneity and represents the basis for more successful tumor treatments. The isolation and sequencing of single tumor cells still represents a considerable technical challenge and consists of three major steps: (1) single cell isolation (e.g., by laser-capture microdissection), fluorescence-activated cell sorting, micromanipulation, whole genome amplification (e.g., with the help of Phi29 DNA polymerase), and transcriptome-wide next generation sequencing technologies (e.g., 454 pyrosequencing, Illumina sequencing, and other systems). Data demonstrating the feasibility of single-cell sequencing for monitoring the emergence of drug-resistant cell clones in patient samples are discussed herein. It is envisioned that single-cell sequencing will be a valuable asset to assist the design of regimens for personalized tumor therapies based on tumor subpopulation-specific genetic alterations in individual patients. PMID:27322289

  8. Single cell metastatic phenotyping using pulsed nanomechanical indentations

    NASA Astrophysics Data System (ADS)

    Babahosseini, Hesam; Strobl, Jeannine S.; Agah, Masoud

    2015-09-01

    The existing approach to characterize cell biomechanical properties typically utilizes switch-like models of mechanotransduction in which cell responses are analyzed in response to a single nanomechanical indentation or a transient pulsed stress. Although this approach provides effective descriptors at population-level, at a single-cell-level, there are significant overlaps in the biomechanical descriptors of non-metastatic and metastatic cells which precludes the use of biomechanical markers for single cell metastatic phenotyping. This study presents a new promising marker for biosensing metastatic and non-metastatic cells at a single-cell-level using the effects of a dynamic microenvironment on the biomechanical properties of cells. Two non-metastatic and two metastatic epithelial breast cell lines are subjected to a pulsed stresses regimen exerted by atomic force microscopy. The force-time data obtained for the cells revealed that the non-metastatic cells increase their resistance against deformation and become more stiffened when subjected to a series of nanomechanical indentations. On the other hand, metastatic cells become slightly softened when their mechanical microenvironment is subjected to a similar dynamical changes. This distinct behavior of the non-metastatic and metastatic cells to the pulsed stresses paradigm provided a signature for single-cell-level metastatic phenotyping with a high confidence level of ∼95%.

  9. Single-cell electroporation using a multifunctional pipette†

    PubMed Central

    Ainla, Alar; Xu, Shijun; Sanchez, Nicolas; Jeffries, Gavin D. M.

    2013-01-01

    We present here a novel platform combination, using a multifunctional pipette to individually electroporate single-cells and to locally deliver an analyte, while in their culture environment. We demonstrate a method to fabricate low-resistance metallic electrodes into a PDMS pipette, followed by characterization of its effectiveness, benefits and limits in comparison with an external carbon microelectrode. PMID:22810424

  10. Multi-Omics of Single Cells: Strategies and Applications.

    PubMed

    Bock, Christoph; Farlik, Matthias; Sheffield, Nathan C

    2016-08-01

    Most genome-wide assays provide averages across large numbers of cells, but recent technological advances promise to overcome this limitation. Pioneering single-cell assays are now available for genome, epigenome, transcriptome, proteome, and metabolome profiling. Here, we describe how these different dimensions can be combined into multi-omics assays that provide comprehensive profiles of the same cell. PMID:27212022

  11. Single-cell genomics for dissection of complex malaria infections

    PubMed Central

    Nair, Shalini; Nkhoma, Standwell C.; Serre, David; Zimmerman, Peter A.; Gorena, Karla; Daniel, Benjamin J.; Nosten, François; Anderson, Timothy J.C.; Cheeseman, Ian H.

    2014-01-01

    Most malaria infections contain complex mixtures of distinct parasite lineages. These multiple-genotype infections (MGIs) impact virulence evolution, drug resistance, intra-host dynamics, and recombination, but are poorly understood. To address this we have developed a single-cell genomics approach to dissect MGIs. By combining cell sorting and whole-genome amplification (WGA), we are able to generate high-quality material from parasite-infected red blood cells (RBCs) for genotyping and next-generation sequencing. We optimized our approach through analysis of >260 single-cell assays. To quantify accuracy, we decomposed mixtures of known parasite genotypes and obtained highly accurate (>99%) single-cell genotypes. We applied this validated approach directly to infections of two major malaria species, Plasmodium falciparum, for which long term culture is possible, and Plasmodium vivax, for which no long-term culture is feasible. We demonstrate that our single-cell genomics approach can be used to generate parasite genome sequences directly from patient blood in order to unravel the complexity of P. vivax and P. falciparum infections. These methods open the door for large-scale analysis of within-host variation of malaria infections, and reveal information on relatedness and drug resistance haplotypes that is inaccessible through conventional sequencing of infections. PMID:24812326

  12. Modeling genome coverage in single-cell sequencing

    PubMed Central

    Daley, Timothy; Smith, Andrew D.

    2014-01-01

    Motivation: Single-cell DNA sequencing is necessary for examining genetic variation at the cellular level, which remains hidden in bulk sequencing experiments. But because they begin with such small amounts of starting material, the amount of information that is obtained from single-cell sequencing experiment is highly sensitive to the choice of protocol employed and variability in library preparation. In particular, the fraction of the genome represented in single-cell sequencing libraries exhibits extreme variability due to quantitative biases in amplification and loss of genetic material. Results: We propose a method to predict the genome coverage of a deep sequencing experiment using information from an initial shallow sequencing experiment mapped to a reference genome. The observed coverage statistics are used in a non-parametric empirical Bayes Poisson model to estimate the gain in coverage from deeper sequencing. This approach allows researchers to know statistical features of deep sequencing experiments without actually sequencing deeply, providing a basis for optimizing and comparing single-cell sequencing protocols or screening libraries. Availability and implementation: The method is available as part of the preseq software package. Source code is available at http://smithlabresearch.org/preseq. Contact: andrewds@usc.edu Supplementary information: Supplementary material is available at Bioinformatics online. PMID:25107873

  13. Parameter screening in microfluidics based hydrodynamic single-cell trapping.

    PubMed

    Deng, B; Li, X F; Chen, D Y; You, L D; Wang, J B; Chen, J

    2014-01-01

    Microfluidic cell-based arraying technology is widely used in the field of single-cell analysis. However, among developed devices, there is a compromise between cellular loading efficiencies and trapped cell densities, which deserves further analysis and optimization. To address this issue, the cell trapping efficiency of a microfluidic device with two parallel micro channels interconnected with cellular trapping sites was studied in this paper. By regulating channel inlet and outlet status, the microfluidic trapping structure can mimic key functioning units of previously reported devices. Numerical simulations were used to model this cellular trapping structure, quantifying the effects of channel on/off status and trapping structure geometries on the cellular trapping efficiency. Furthermore, the microfluidic device was fabricated based on conventional microfabrication and the cellular trapping efficiency was quantified in experiments. Experimental results showed that, besides geometry parameters, cellular travelling velocities and sizes also affected the single-cell trapping efficiency. By fine tuning parameters, more than 95% of trapping sites were taken by individual cells. This study may lay foundation in further studies of single-cell positioning in microfluidics and push forward the study of single-cell analysis.

  14. Selective single cell isolation for genomics using microraft arrays.

    PubMed

    Welch, Joshua D; Williams, Lindsay A; DiSalvo, Matthew; Brandt, Alicia T; Marayati, Raoud; Sims, Christopher E; Allbritton, Nancy L; Prins, Jan F; Yeh, Jen Jen; Jones, Corbin D

    2016-09-30

    Genomic methods are used increasingly to interrogate the individual cells that compose specific tissues. However, current methods for single cell isolation struggle to phenotypically differentiate specific cells in a heterogeneous population and rely primarily on the use of fluorescent markers. Many cellular phenotypes of interest are too complex to be measured by this approach, making it difficult to connect genotype and phenotype at the level of individual cells. Here we demonstrate that microraft arrays, which are arrays containing thousands of individual cell culture sites, can be used to select single cells based on a variety of phenotypes, such as cell surface markers, cell proliferation and drug response. We then show that a common genomic procedure, RNA-seq, can be readily adapted to the single cells isolated from these rafts. We show that data generated using microrafts and our modified RNA-seq protocol compared favorably with the Fluidigm C1. We then used microraft arrays to select pancreatic cancer cells that proliferate in spite of cytotoxic drug treatment. Our single cell RNA-seq data identified several expected and novel gene expression changes associated with early drug resistance.

  15. Genomic Analysis at the Single-Cell Level

    PubMed Central

    Kalisky, Tomer; Blainey, Paul; Quake, Stephen R.

    2013-01-01

    Studying complex biological systems such as a developing embryo, a tumor, or a microbial ecosystem often involves understanding the behavior and heterogeneity of the individual cells that constitute the system and their interactions. In this review, we discuss a variety of approaches to single-cell genomic analysis. PMID:21942365

  16. Single-Cell Analysis of Mast Cell Degranulation Induced by Airway Smooth Muscle-Secreted Chemokines

    PubMed Central

    Manning, Benjamin M.; Meyer, Audrey F.; Gruba, Sarah M.; Haynes, Christy L.

    2015-01-01

    Background Asthma is a chronic inflammatory disease characterized by narrowed airways, bronchial hyper-responsiveness, mucus hyper-secretion, and airway remodeling. Mast cell (MC) infiltration into airway smooth muscle (ASM) is a defining feature of asthma, and ASM regulates the inflammatory response by secreting chemokines, including CXCL10 and CCL5. Single cell analysis offers a unique approach to study specific cellular signaling interactions within large and complex signaling networks such as the inflammatory microenvironment in asthma. Methods Carbon fiber microelectrode amperometry was used to study the effects of ASM–secreted chemokines on mouse peritoneal MC degranulation. Results MC degranulation in response to CXCL10 and CCL5 was monitored at the single cell level. Relative to IgE-mediated degranulation, CXCL10- and CCL5-stimulated MCs released a decreased amount of serotonin per granule with fewer release events per cell. Decreased serotonin released per granule was correlated with increased spike half-width and rise-time values. Conclusions MCs are directly activated with ASM-associated chemokines. CXCL10 and CCL5 induce less robust MC degranulation compared to IgE- and A23187-stimulation. The kinetics of MC degranulation are signaling pathway-dependent, suggesting a biophysical mechanism of regulated degranulation that incorporates control over granule trafficking, transport, and docking machinery. General Significance The biophysical mechanisms, including variations in number of exocytotic release events, serotonin released per granule, and the membrane kinetics of exocytosis that underlie MC degranulation in response to CXCL10 and CCL5 were characterized at the single cell level. These findings clarify the function of ASM-derived chemokines as instigators of MC degranulation relative to classical mechanisms of MC stimulation. PMID:25986989

  17. Single-cell transcriptome analysis of endometrial tissue

    PubMed Central

    Krjutškov, K.; Katayama, S.; Saare, M.; Vera-Rodriguez, M.; Lubenets, D.; Samuel, K.; Laisk-Podar, T.; Teder, H.; Einarsdottir, E.; Salumets, A.; Kere, J.

    2016-01-01

    STUDY QUESTION How can we study the full transcriptome of endometrial stromal and epithelial cells at the single-cell level? SUMMARY ANSWER By compiling and developing novel analytical tools for biopsy, tissue cryopreservation and disaggregation, single-cell sorting, library preparation, RNA sequencing (RNA-seq) and statistical data analysis. WHAT IS KNOWN ALREADY Although single-cell transcriptome analyses from various biopsied tissues have been published recently, corresponding protocols for human endometrium have not been described. STUDY DESIGN, SIZE, DURATION The frozen-thawed endometrial biopsies were fluorescence-activated cell sorted (FACS) to distinguish CD13-positive stromal and CD9-positive epithelial cells and single-cell transcriptome analysis performed from biopsied tissues without culturing the cells. We studied gene transcription, applying a modern and efficient RNA-seq protocol. In parallel, endometrial stromal cells were cultured and global expression profiles were compared with uncultured cells. PARTICIPANTS/MATERIALS, SETTING, METHODS For method validation, we used two endometrial biopsies, one from mid-secretory phase (Day 21, LH+8) and another from late-secretory phase (Day 25). The samples underwent single-cell FACS sorting, single-cell RNA-seq library preparation and Illumina sequencing. MAIN RESULTS AND THE ROLE OF CHANCE Here we present a complete pipeline for single-cell gene-expression studies, from clinical sampling to statistical data analysis. Tissue manipulation, starting from disaggregation and cell-type-specific labelling and ending with single-cell automated sorting, is managed within 90 min at low temperature to minimize changes in the gene expression profile. The single living stromal and epithelial cells were sorted using CD13- and CD9-specific antibodies, respectively. Of the 8622 detected genes, 2661 were more active in cultured stromal cells than in biopsy cells. In the comparison of biopsy versus cultured cells, 5603

  18. Single-cell intracellular nano-pH probes†

    PubMed Central

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2016-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution. PMID:27708772

  19. Single-cell intracellular nano-pH probes†

    PubMed Central

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2016-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

  20. Single-cell imaging detection of nanobarcoded nanoparticle biodistributions in tissues for nanomedicine

    NASA Astrophysics Data System (ADS)

    Eustaquio, Trisha; Cooper, Christy L.; Leary, James F.

    2011-03-01

    In nanomedicine, biodistribution studies are critical to evaluate the safety and efficacy of nanoparticles. Currently, extensive biodistribution studies are hampered by the limitations of bulk tissue and single-cell imaging techniques. To ameliorate these limitations, we have developed a novel method for single nanoparticle detection that incorporates a conjugated oligonucleotide as a "nanobarcode" for detection via in situ PCR. This strategy magnifies the detection signal from single nanoparticles, facilitating rapid evaluation of nanoparticle uptake by cell type over larger areas. The nanobarcoding method can enable precise analysis of nanoparticle biodistributions and expedite translation of these nanoparticles to the clinic.

  1. Optical Clearing Delivers Ultrasensitive Hyperspectral Dark-Field Imaging for Single-Cell Evaluation.

    PubMed

    Cui, Yi; Wang, Xiaolei; Ren, Wen; Liu, Jing; Irudayaraj, Joseph

    2016-03-22

    A single-cell optical clearing methodology is developed and demonstrated in hyperspectral dark-field microscopy (HSDFM) and imaging of plasmonic nanoprobes. Our strategy relies on a combination of delipidation and refractive index (RI) matching with highly biocompatible and affordable agents. Before applying the RI-matching solution, the delipidation step by using a mild solvent effectively eliminates those high-density, lipid-enriched granular structures which emit strong scattering. Upon treatment, the background scattering from cellular organelles could be repressed to a negligible level while the scattering signals from plasmonic nanomaterials increase, leading to a significant improvement of the signal-to-noise ratio (SNR). With this method established, the versatility and applicability of HSDFM are greatly enhanced. In our demonstration, quantitative mapping of the dimerization-activated receptor kinase HER2 is achieved in a single cancer cell by a nonfluorescent approach. High-resolution imaging for oncogenic mRNAs, namely ER, PR, and HER2, is performed with single labeling. More importantly, in situ multiplex detection of mRNA and protein is made possible by HSDFM since it overcomes the difficulties of complex staining and signal imbalance suffered by the conventional optical imaging. Last, we show that with optical clearing, characterization of intracellularly grown gold particulates is accomplished at an unprecedented spatiotemporal resolution. Taken together, the uniqueness of optical clearing and HSDFM is expected to open ample avenues for single-cell studies and biomedical engineering. PMID:26895095

  2. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation

    NASA Astrophysics Data System (ADS)

    Shalek, Alex K.; Satija, Rahul; Shuga, Joe; Trombetta, John J.; Gennert, Dave; Lu, Diana; Chen, Peilin; Gertner, Rona S.; Gaublomme, Jellert T.; Yosef, Nir; Schwartz, Schraga; Fowler, Brian; Weaver, Suzanne; Wang, Jing; Wang, Xiaohui; Ding, Ruihua; Raychowdhury, Raktima; Friedman, Nir; Hacohen, Nir; Park, Hongkun; May, Andrew P.; Regev, Aviv

    2014-06-01

    High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript's level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a `core' module of antiviral genes is expressed very early by a few `precocious' cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced `peaked' inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses.

  3. Molecular Models of STAT5A Tetramers Complexed to DNA Predict Relative Genome-Wide Frequencies of the Spacing between the Two Dimer Binding Motifs of the Tetramer Binding Sites

    PubMed Central

    Sathyanarayana, Bangalore K.; Li, Peng; Lin, Jian-Xin; Leonard, Warren J.

    2016-01-01

    STAT proteins bind DNA as dimers and tetramers to control cellular development, differentiation, survival, and expansion. The tetramer binding sites are comprised of two dimer-binding sites repeated in tandem. The genome-wide distribution of the spacings between the dimer binding sites shows a distinctive, non-random pattern. Here, we report on estimating the feasibility of building possible molecular models of STAT5A tetramers bound to a DNA double helix with all possible spacings between the dimer binding sites. We found that the calculated feasibility estimates correlated well with the experimentally measured frequency of tetramer-binding sites. This suggests that the feasibility of forming the tetramer complex was a major factor in the evolution of this DNA sequence variation. PMID:27537504

  4. Molecular Models of STAT5A Tetramers Complexed to DNA Predict Relative Genome-Wide Frequencies of the Spacing between the Two Dimer Binding Motifs of the Tetramer Binding Sites.

    PubMed

    Sathyanarayana, Bangalore K; Li, Peng; Lin, Jian-Xin; Leonard, Warren J; Lee, Byungkook

    2016-01-01

    STAT proteins bind DNA as dimers and tetramers to control cellular development, differentiation, survival, and expansion. The tetramer binding sites are comprised of two dimer-binding sites repeated in tandem. The genome-wide distribution of the spacings between the dimer binding sites shows a distinctive, non-random pattern. Here, we report on estimating the feasibility of building possible molecular models of STAT5A tetramers bound to a DNA double helix with all possible spacings between the dimer binding sites. We found that the calculated feasibility estimates correlated well with the experimentally measured frequency of tetramer-binding sites. This suggests that the feasibility of forming the tetramer complex was a major factor in the evolution of this DNA sequence variation. PMID:27537504

  5. Electrochemical Visualization of Intracellular Hydrogen Peroxide at Single Cells.

    PubMed

    He, Ruiqin; Tang, Huifen; Jiang, Dechen; Chen, Hong-yuan

    2016-02-16

    In this Letter, the electrochemical visualization of hydrogen peroxide inside one cell was achieved first using a comprehensive Au-luminol-microelectrode and electrochemiluminescence. The capillary with a tip opening of 1-2 μm was filled with the mixture of chitosan and luminol, which was coated with the thin layers of polyvinyl chloride/nitrophenyloctyl ether (PVC/NPOE) and gold as the microelectrode. Upon contact with the aqueous hydrogen peroxide, hydrogen peroxide and luminol in contact with the gold layer were oxidized under the positive potential resulting in luminescence for the imaging. Due to the small diameter of the electrode, the microelectrode tip was inserted into one cell and the bright luminescence observed at the tip confirmed the visualization of intracellular hydrogen peroxide. The further coupling of oxidase on the electrode surface could open the field in the electrochemical imaging of intracellular biomolecules at single cells, which benefited the single cell electrochemical detection. PMID:26879364

  6. Understanding hematopoiesis from a single-cell standpoint.

    PubMed

    Kokkaliaris, Konstantinos D; Lucas, Daniel; Beerman, Isabel; Kent, David G; Perié, Leïla

    2016-06-01

    The cellular diversity of the hematopoietic system has been extensively studied, and a plethora of cell surface markers have been used to discriminate and prospectively purify different blood cell types. However, even within phenotypically identical fractions of hematopoietic stem and progenitor cells or lineage-restricted progenitors, significant functional heterogeneity is observed when single cells are analyzed. To address these challenges, researchers are now using techniques to follow single cells and their progeny to improve our understanding of the underlying functional heterogeneity. On November 19, 2015, Dr. David Kent and Dr. Leïla Perié, two emerging young group leaders, presented their recent efforts to dissect the functional properties of individual cells with a webinar series organized by the International Society for Experimental Hematology. Here, we provide a summary of the presented methods for cell labeling and clonal tracking and discuss how these different techniques have been employed to study hematopoiesis.

  7. Adult Mouse Cortical Cell Taxonomy by Single Cell Transcriptomics

    PubMed Central

    Tasic, Bosiljka; Menon, Vilas; Nguyen, Thuc Nghi; Kim, Tae Kyung; Jarsky, Tim; Yao, Zizhen; Levi, Boaz; Gray, Lucas T.; Sorensen, Staci A.; Dolbeare, Tim; Bertagnolli, Darren; Goldy, Jeff; Shapovalova, Nadiya; Parry, Sheana; Lee, Changkyu; Smith, Kimberly; Bernard, Amy; Madisen, Linda; Sunkin, Susan M.; Hawrylycz, Michael; Koch, Christof; Zeng, Hongkui

    2016-01-01

    Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. Here, we construct a cellular taxonomy of one cortical region, primary visual cortex, in adult mice based on single cell RNA-sequencing. We identify 49 transcriptomic cell types including 23 GABAergic, 19 glutamatergic and seven non-neuronal types. We also analyze cell-type specific mRNA processing and characterize genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we show that some of our transcriptomic cell types display specific and differential electrophysiological and axon projection properties, thereby confirming that the single cell transcriptomic signatures can be associated with specific cellular properties. PMID:26727548

  8. Parameter estimation for the distribution of single cell lag times.

    PubMed

    Baranyi, József; George, Susan M; Kutalik, Zoltán

    2009-07-01

    In Quantitative Microbial Risk Assessment, it is vital to understand how lag times of individual cells are distributed over a bacterial population. Such identified distributions can be used to predict the time by which, in a growth-supporting environment, a few pathogenic cells can multiply to a poisoning concentration level. We model the lag time of a single cell, inoculated into a new environment, by the delay of the growth function characterizing the generated subpopulation. We introduce an easy-to-implement procedure, based on the method of moments, to estimate the parameters of the distribution of single cell lag times. The advantage of the method is especially apparent for cases where the initial number of cells is small and random, and the culture is detectable only in the exponential growth phase.

  9. Adult mouse cortical cell taxonomy revealed by single cell transcriptomics.

    PubMed

    Tasic, Bosiljka; Menon, Vilas; Nguyen, Thuc Nghi; Kim, Tae Kyung; Jarsky, Tim; Yao, Zizhen; Levi, Boaz; Gray, Lucas T; Sorensen, Staci A; Dolbeare, Tim; Bertagnolli, Darren; Goldy, Jeff; Shapovalova, Nadiya; Parry, Sheana; Lee, Changkyu; Smith, Kimberly; Bernard, Amy; Madisen, Linda; Sunkin, Susan M; Hawrylycz, Michael; Koch, Christof; Zeng, Hongkui

    2016-02-01

    Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. We constructed a cellular taxonomy of one cortical region, primary visual cortex, in adult mice on the basis of single-cell RNA sequencing. We identified 49 transcriptomic cell types, including 23 GABAergic, 19 glutamatergic and 7 non-neuronal types. We also analyzed cell type-specific mRNA processing and characterized genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we found that some of our transcriptomic cell types displayed specific and differential electrophysiological and axon projection properties, thereby confirming that the single-cell transcriptomic signatures can be associated with specific cellular properties.

  10. Cellular identity at the single-cell level.

    PubMed

    Coskun, Ahmet F; Eser, Umut; Islam, Saiful

    2016-10-20

    A single cell creates surprising heterogeneity in a multicellular organism. While every organismal cell shares almost an identical genome, molecular interactions in cells alter the use of DNA sequences to modulate the gene of interest for specialization of cellular functions. Each cell gains a unique identity through molecular coding across the DNA, RNA, and protein conversions. On the other hand, loss of cellular identity leads to critical diseases such as cancer. Most cell identity dissection studies are based on bulk molecular assays that mask differences in individual cells. To probe cell-to-cell variability in a population, we discuss single cell approaches to decode the genetic, epigenetic, transcriptional, and translational mechanisms for cell identity formation. In combination with molecular instructions, the physical principles behind cell identity determination are examined. Deciphering and reprogramming cellular types impact biology and medicine.

  11. Electrochemical Visualization of Intracellular Hydrogen Peroxide at Single Cells.

    PubMed

    He, Ruiqin; Tang, Huifen; Jiang, Dechen; Chen, Hong-yuan

    2016-02-16

    In this Letter, the electrochemical visualization of hydrogen peroxide inside one cell was achieved first using a comprehensive Au-luminol-microelectrode and electrochemiluminescence. The capillary with a tip opening of 1-2 μm was filled with the mixture of chitosan and luminol, which was coated with the thin layers of polyvinyl chloride/nitrophenyloctyl ether (PVC/NPOE) and gold as the microelectrode. Upon contact with the aqueous hydrogen peroxide, hydrogen peroxide and luminol in contact with the gold layer were oxidized under the positive potential resulting in luminescence for the imaging. Due to the small diameter of the electrode, the microelectrode tip was inserted into one cell and the bright luminescence observed at the tip confirmed the visualization of intracellular hydrogen peroxide. The further coupling of oxidase on the electrode surface could open the field in the electrochemical imaging of intracellular biomolecules at single cells, which benefited the single cell electrochemical detection.

  12. Advances and Applications of Single Cell Sequencing Technologies

    PubMed Central

    Wang, Yong; Navin, Nicholas E.

    2015-01-01

    Single cell sequencing (SCS) has emerged as a powerful new set of technologies for studying rare cells and delineating complex populations. Over the past 5 years, SCS methods for DNA and RNA have had a broad impact on many diverse fields of biology, including microbiology, neurobiology, development, tissue mosaicism, immunology and cancer research. In this review, we will discuss SCS technologies and applications, as well as translational applications in the clinic. PMID:26000845

  13. Bifurcation Analysis of Fractional Order Single Cell with Delay

    NASA Astrophysics Data System (ADS)

    Çelik, Vedat

    This paper presents the bifurcation analysis of fractional order model of delayed single cell which is proposed for delayed cellular neural networks with respect to the time delay τ. The bifurcation points, time delay τc, are determined by modified Mikhailov stability criterion for a range of fractional delayed cell order 0.3 ≤ q < 1. Numerical results obtained from Adams-Bashforth-Moulton method demonstrate that the supercritical Hopf bifurcation occurs in the system.

  14. Microwave-induced thermogenetic activation of single cells

    SciTech Connect

    Safronov, N. A.; Fedotov, I. V.; Ermakova, Yu. G.; Matlashov, M. E.; Belousov, V. V.; Sidorov-Biryukov, D. A.; Fedotov, A. B.; Zheltikov, A. M.

    2015-04-20

    Exposure to a microwave field is shown to enable thermogenetic activation of individual cells in a culture of cell expressing thermosensitive ion channels. Integration of a microwave transmission line with an optical fiber and a diamond quantum thermometer has been shown to allow thermogenetic single-cell activation to be combined with accurate local online temperature measurements based on an optical detection of electron spin resonance in nitrogen–vacancy centers in diamond.

  15. Mie scatter corrections in single cell infrared microspectroscopy.

    PubMed

    Konevskikh, Tatiana; Lukacs, Rozalia; Blümel, Reinhold; Ponossov, Arkadi; Kohler, Achim

    2016-06-23

    Strong Mie scattering signatures hamper the chemical interpretation and multivariate analysis of the infrared microscopy spectra of single cells and tissues. During recent years, several numerical Mie scatter correction algorithms for the infrared spectroscopy of single cells have been published. In the paper at hand, we critically reviewed existing algorithms for the correction of Mie scattering and suggest improvements. We developed an iterative algorithm based on Extended Multiplicative Scatter Correction (EMSC), for the retrieval of pure absorbance spectra from highly distorted infrared spectra of single cells. The new algorithm uses the van de Hulst approximation formula for the extinction efficiency employing a complex refractive index. The iterative algorithm involves the establishment of an EMSC meta-model. While existing iterative algorithms for the correction of resonant Mie scattering employ three independent parameters for establishing a meta-model, we could decrease the number of parameters from three to two independent parameters, which reduced the calculation time for the Mie scattering curves for the iterative EMSC meta-model by a factor of 10. Moreover, by employing the Hilbert transform for evaluating the Kramers-Kronig relations based on a FFT algorithm in Matlab, we further improved the speed of the algorithm by a factor of 100. For testing the algorithm we simulate distorted apparent absorbance spectra by utilizing the exact theory for the scattering of infrared light at absorbing spheres, taking into account the high numerical aperture of infrared microscopes employed for the analysis of single cells and tissues. In addition, the algorithm was applied to measured absorbance spectra of single lung cancer cells. PMID:27034998

  16. Single cell magnetic imaging using a quantum diamond microscope

    PubMed Central

    Park, H.; Weissleder, R.; Yacoby, A.; Lukin, M. D.; Lee, H.; Walsworth, R. L.; Connolly, C. B.

    2015-01-01

    We apply a quantum diamond microscope to detection and imaging of immunomagnetically labeled cells. This instrument uses nitrogen-vacancy (NV) centers in diamond for correlated magnetic and fluorescence imaging. Our device provides single-cell resolution and two orders of magnitude larger field of view (~1 mm2) than previous NV imaging technologies, enabling practical applications. To illustrate, we quantify cancer biomarkers expressed by rare tumor cells in a large population of healthy cells. PMID:26098019

  17. Mie scatter corrections in single cell infrared microspectroscopy.

    PubMed

    Konevskikh, Tatiana; Lukacs, Rozalia; Blümel, Reinhold; Ponossov, Arkadi; Kohler, Achim

    2016-06-23

    Strong Mie scattering signatures hamper the chemical interpretation and multivariate analysis of the infrared microscopy spectra of single cells and tissues. During recent years, several numerical Mie scatter correction algorithms for the infrared spectroscopy of single cells have been published. In the paper at hand, we critically reviewed existing algorithms for the correction of Mie scattering and suggest improvements. We developed an iterative algorithm based on Extended Multiplicative Scatter Correction (EMSC), for the retrieval of pure absorbance spectra from highly distorted infrared spectra of single cells. The new algorithm uses the van de Hulst approximation formula for the extinction efficiency employing a complex refractive index. The iterative algorithm involves the establishment of an EMSC meta-model. While existing iterative algorithms for the correction of resonant Mie scattering employ three independent parameters for establishing a meta-model, we could decrease the number of parameters from three to two independent parameters, which reduced the calculation time for the Mie scattering curves for the iterative EMSC meta-model by a factor of 10. Moreover, by employing the Hilbert transform for evaluating the Kramers-Kronig relations based on a FFT algorithm in Matlab, we further improved the speed of the algorithm by a factor of 100. For testing the algorithm we simulate distorted apparent absorbance spectra by utilizing the exact theory for the scattering of infrared light at absorbing spheres, taking into account the high numerical aperture of infrared microscopes employed for the analysis of single cells and tissues. In addition, the algorithm was applied to measured absorbance spectra of single lung cancer cells.

  18. Single-cell magnetic imaging using a quantum diamond microscope.

    PubMed

    Glenn, David R; Lee, Kyungheon; Park, Hongkun; Weissleder, Ralph; Yacoby, Amir; Lukin, Mikhail D; Lee, Hakho; Walsworth, Ronald L; Connolly, Colin B

    2015-08-01

    We apply a quantum diamond microscope for detection and imaging of immunomagnetically labeled cells. This instrument uses nitrogen-vacancy (NV) centers in diamond for correlated magnetic and fluorescence imaging. Our device provides single-cell resolution and a field of view (∼1 mm(2)) two orders of magnitude larger than that of previous NV imaging technologies, enabling practical applications. To illustrate, we quantified cancer biomarkers expressed by rare tumor cells in a large population of healthy cells.

  19. Single cell transcriptomics of neighboring hyphae of Aspergillus niger

    PubMed Central

    2011-01-01

    Single cell profiling was performed to assess differences in RNA accumulation in neighboring hyphae of the fungus Aspergillus niger. A protocol was developed to isolate and amplify RNA from single hyphae or parts thereof. Microarray analysis resulted in a present call for 4 to 7% of the A. niger genes, of which 12% showed heterogeneous RNA levels. These genes belonged to a wide range of gene categories. PMID:21816052

  20. Single Cell Analysis of a Bacterial Sender-Receiver System

    PubMed Central

    Mückl, Andrea; Kapsner, Korbinian; Gerland, Ulrich; Simmel, Friedrich C.

    2016-01-01

    Monitoring gene expression dynamics on the single cell level provides important information on cellular heterogeneity and stochasticity, and potentially allows for more accurate quantitation of gene expression processes. We here study bacterial senders and receivers genetically engineered with components of the quorum sensing system derived from Aliivibrio fischeri on the single cell level using microfluidics-based bacterial chemostats and fluorescence video microscopy. We track large numbers of bacteria over extended periods of time, which allows us to determine bacterial lineages and filter out subpopulations within a heterogeneous population. We quantitatively determine the dynamic gene expression response of receiver bacteria to varying amounts of the quorum sensing inducer N-3-oxo-C6-homoserine lactone (AHL). From this we construct AHL response curves and characterize gene expression dynamics of whole bacterial populations by investigating the statistical distribution of gene expression activity over time. The bacteria are found to display heterogeneous induction behavior within the population. We therefore also characterize gene expression in a homogeneous bacterial subpopulation by focusing on single cell trajectories derived only from bacteria with similar induction behavior. The response at the single cell level is found to be more cooperative than that obtained for the heterogeneous total population. For the analysis of systems containing both AHL senders and receiver cells, we utilize the receiver cells as ‘bacterial sensors’ for AHL. Based on a simple gene expression model and the response curves obtained in receiver-only experiments, the effective AHL concentration established by the senders and their ‘sending power’ is determined. PMID:26808777

  1. Recent advances in microbial single cell genomics technology and applications

    NASA Astrophysics Data System (ADS)

    Stepanauskas, R.

    2015-12-01

    Single cell genomics is increasingly utilized as a powerful tool to decipher the metabolic potential, evolutionary histories and in situ interactions of environmental microorganisms. I will present several new developments of this exciting technology, which improve genomic data recovery from individual cells and allow its integration with cell's phenotypic properties. I will also demonstrate how these new technical capabilities help understanding the biology of the "microbial dark matter" inhabiting marine and terrestrial subsurface environments.

  2. [Technological advances in single-cell genomic analyses].

    PubMed

    Pan, Xing-Hua; Zhu, Hai-Ying; Marjani, Sadie L

    2011-01-01

    The technological progress of the genomics has transformed life science research. The main objectives of genomics are sequencing of new genomes and genome-wide identification of the function and the interaction of genes and their products. The recently developed second generation or next generation sequencing platforms and DNA microarray technology are immensely important and powerful tools for functional genomic analyses. However, their application is limited by the requirement of sufficient amounts of high quality nucleic acid samples. Therefore, when only a single cell or a very small number of cells are available or are preferred, the whole genomic sequencing or functional genomic objectives cannot be achieved conventionally and require a robust amplification method. This review highlights DNA amplification technologies and summarizes the strategies currently utilized for whole genome sequencing of a single cell, with specific focus on studies investigating microorganisms; An outline for targeted re-sequencing enabling the analysis of larger genomes is also provided. Furthermore, the review presents the emerging functional genomic applications using next-generation sequencing or microarray analysis to examine genome-wide transcriptional profile, chromatin modification and other types of protein-DNA binding profile, and CpG methylation mapping in a single cell or a very low quantity of cells. The nature of these technologies and their prospects are also addressed.

  3. Microarray analysis of copy number variation in single cells.

    PubMed

    Konings, Peter; Vanneste, Evelyne; Jackmaert, Sigrun; Ampe, Michèle; Verbeke, Geert; Moreau, Yves; Vermeesch, Joris Robert; Voet, Thierry

    2012-02-01

    We present a protocol for reliably detecting DNA copy number aberrations in a single human cell. Multiple displacement-amplified DNAs of a cell are hybridized to a 3,000-bacterial artificial chromosome (BAC) array and to an Affymetrix 250,000 (250K)-SNP array. Subsequent copy number calling is based on the integration of BAC probe-specific copy number probabilities that are estimated by comparing probe intensities with a single-cell whole-genome amplification (WGA) reference model for diploid chromosomes, as well as SNP copy number and loss-of-heterozygosity states estimated by hidden Markov models (HMM). All methods for detecting DNA copy number aberrations in single human cells have difficulty in confidently discriminating WGA artifacts from true genetic variants. Furthermore, some methods lack thorough validation for segmental DNA imbalance detection. Our protocol minimizes false-positive variant calling and enables uniparental isodisomy detection in single cells. Additionally, it provides quality assessment, allowing the exclusion of uninterpretable single-cell WGA samples. The protocol takes 5-7 d. PMID:22262009

  4. Single cell dissection of early kidney development: multilineage priming.

    PubMed

    Brunskill, Eric W; Park, Joo-Seop; Chung, Eunah; Chen, Feng; Magella, Bliss; Potter, S Steven

    2014-08-01

    We used a single cell RNA-seq strategy to create an atlas of gene expression patterns in the developing kidney. At several stages of kidney development, histologically uniform populations of cells give rise to multiple distinct lineages. We performed single cell RNA-seq analysis of total mouse kidneys at E11.5 and E12.5, as well as the renal vesicles at P4. We define an early stage of progenitor cell induction driven primarily by gene repression. Surprising stochastic expression of marker genes associated with differentiated cell types was observed in E11.5 progenitors. We provide a global view of the polarized gene expression already present in the renal vesicle, the first epithelial precursor of the nephron. We show that Hox gene read-through transcripts can be spliced to produce intergenic homeobox swaps. We also identify a surprising number of genes with partially degraded noncoding RNA. Perhaps most interesting, at early developmental times single cells often expressed genes related to several developmental pathways. This provides powerful evidence that initial organogenesis involves a process of multilineage priming. This is followed by a combination of gene repression, which turns off the genes associated with most possible lineages, and the activation of increasing numbers of genes driving the chosen developmental direction. PMID:25053437

  5. Single cell dissection of early kidney development: multilineage priming

    PubMed Central

    Brunskill, Eric W.; Park, Joo-Seop; Chung, Eunah; Chen, Feng; Magella, Bliss; Potter, S. Steven

    2014-01-01

    We used a single cell RNA-seq strategy to create an atlas of gene expression patterns in the developing kidney. At several stages of kidney development, histologically uniform populations of cells give rise to multiple distinct lineages. We performed single cell RNA-seq analysis of total mouse kidneys at E11.5 and E12.5, as well as the renal vesicles at P4. We define an early stage of progenitor cell induction driven primarily by gene repression. Surprising stochastic expression of marker genes associated with differentiated cell types was observed in E11.5 progenitors. We provide a global view of the polarized gene expression already present in the renal vesicle, the first epithelial precursor of the nephron. We show that Hox gene read-through transcripts can be spliced to produce intergenic homeobox swaps. We also identify a surprising number of genes with partially degraded noncoding RNA. Perhaps most interesting, at early developmental times single cells often expressed genes related to several developmental pathways. This provides powerful evidence that initial organogenesis involves a process of multilineage priming. This is followed by a combination of gene repression, which turns off the genes associated with most possible lineages, and the activation of increasing numbers of genes driving the chosen developmental direction. PMID:25053437

  6. Review of methods to probe single cell metabolism and bioenergetics

    DOE PAGES

    Vasdekis, Andreas E.; Stephanopoulos, Gregory

    2014-10-31

    The sampling and manipulation of cells down to the individual has been of substantial interest since the very beginning of Life Sciences. Herein, our objective is to highlight the most recent developments in single cell manipulation, as well as pioneering ones. First, flow-through methods will be discussed, namely methods in which the single cells flow continuously in an ordered manner during their analysis. This section will be followed by confinement techniques that enable cell isolation and confinement in one, two- or three-dimensions. Flow cytometry and droplet microfluidics are the two most common methods of flow-through analysis. While both are high-throughputmore » techniques, their difference lays in the fact that the droplet encapsulated cells experience a restricted and personal microenvironment, while in flow cytometry cells experience similar nutrient and stimuli initial concentrations. These methods are rather well established; however, they recently enabled immense strides in single cell phenotypic analysis, namely the identification and analysis of metabolically distinct individuals from an isogenic population using both droplet microfluidics and flow cytometry.« less

  7. Review of methods to probe single cell metabolism and bioenergetics

    SciTech Connect

    Vasdekis, Andreas E.; Stephanopoulos, Gregory

    2014-10-31

    The sampling and manipulation of cells down to the individual has been of substantial interest since the very beginning of Life Sciences. Herein, our objective is to highlight the most recent developments in single cell manipulation, as well as pioneering ones. First, flow-through methods will be discussed, namely methods in which the single cells flow continuously in an ordered manner during their analysis. This section will be followed by confinement techniques that enable cell isolation and confinement in one, two- or three-dimensions. Flow cytometry and droplet microfluidics are the two most common methods of flow-through analysis. While both are high-throughput techniques, their difference lays in the fact that the droplet encapsulated cells experience a restricted and personal microenvironment, while in flow cytometry cells experience similar nutrient and stimuli initial concentrations. These methods are rather well established; however, they recently enabled immense strides in single cell phenotypic analysis, namely the identification and analysis of metabolically distinct individuals from an isogenic population using both droplet microfluidics and flow cytometry.

  8. Single Cell Genomics and Transcriptomics for Unicellular Eukaryotes

    SciTech Connect

    Ciobanu, Doina; Clum, Alicia; Singh, Vasanth; Salamov, Asaf; Han, James; Copeland, Alex; Grigoriev, Igor; James, Timothy; Singer, Steven; Woyke, Tanja; Malmstrom, Rex; Cheng, Jan-Fang

    2014-03-14

    Despite their small size, unicellular eukaryotes have complex genomes with a high degree of plasticity that allow them to adapt quickly to environmental changes. Unicellular eukaryotes live with prokaryotes and higher eukaryotes, frequently in symbiotic or parasitic niches. To this day their contribution to the dynamics of the environmental communities remains to be understood. Unfortunately, the vast majority of eukaryotic microorganisms are either uncultured or unculturable, making genome sequencing impossible using traditional approaches. We have developed an approach to isolate unicellular eukaryotes of interest from environmental samples, and to sequence and analyze their genomes and transcriptomes. We have tested our methods with six species: an uncharacterized protist from cellulose-enriched compost identified as Platyophrya, a close relative of P. vorax; the fungus Metschnikowia bicuspidate, a parasite of water flea Daphnia; the mycoparasitic fungi Piptocephalis cylindrospora, a parasite of Cokeromyces and Mucor; Caulochytrium protosteloides, a parasite of Sordaria; Rozella allomycis, a parasite of the water mold Allomyces; and the microalgae Chlamydomonas reinhardtii. Here, we present the four components of our approach: pre-sequencing methods, sequence analysis for single cell genome assembly, sequence analysis of single cell transcriptomes, and genome annotation. This technology has the potential to uncover the complexity of single cell eukaryotes and their role in the environmental samples.

  9. Isolating single cells in a neurosphere assay using inertial microfluidics

    PubMed Central

    Nathamgari, S. Shiva P.; Dong, Biqin; Zhou, Fan; Kang, Wonmo; Giraldo-Vela, Juan P.; McGuire, Tammy; McNaughton, Rebecca L.; Sun, Cheng; Kessler, John A.; Espinosa, Horacio D.

    2015-01-01

    Sphere forming assays are routinely used for in vitro propagation and differentiation of stem cells. Because the stem cell clusters can become heterogeneous and polyclonal, they must first be dissociated into a single cell suspension for further clonal analysis or differentiation studies. The dissociated population is marred by the presence of doublets, triplets and semi-cleaved/intact clusters which makes identification and further analysis of differentiation pathways difficult. In this work, we use inertial microfluidics to separate the single cells and clusters in a population of chemically dissociated neurospheres. In contrast to previous microfluidic sorting technologies which operated at high flow rates, we implement the spiral microfluidic channel in a novel focusing regime that occurs at lower flow rates. In this regime, the curvature-induced Dean’s force focuses the smaller, single cells towards the inner wall and the larger clusters towards the center. We further demonstrate that sorting in this low flow rate (and hence low shear stress) regime yields a high percentage (> 90%) of viable cells and preserves multipotency by differentiating the sorted neural stem cell population into neurons and astrocytes. The modularity of the device allows easy integration with other lab-on-a-chip devices for upstream mechanical dissociation and downstream high-throughput clonal analysis, localized electroporation and sampling. Although demonstrated in the case of the neurosphere assay, the method is equally applicable to other sphere forming assays. PMID:26511875

  10. Single-Cell Force Spectroscopy of Probiotic Bacteria

    PubMed Central

    Beaussart, Audrey; El-Kirat-Chatel, Sofiane; Herman, Philippe; Alsteens, David; Mahillon, Jacques; Hols, Pascal; Dufrêne, Yves F.

    2013-01-01

    Single-cell force spectroscopy is a powerful atomic force microscopy modality in which a single living cell is attached to the atomic force microscopy cantilever to quantify the forces that drive cell-cell and cell-substrate interactions. Although various single-cell force spectroscopy protocols are well established for animal cells, application of the method to individual bacterial cells remains challenging, mainly owing to the lack of appropriate methods for the controlled attachment of single live cells on cantilevers. We present a nondestructive protocol for single-bacterial cell force spectroscopy, which combines the use of colloidal probe cantilevers and of a bioinspired polydopamine wet adhesive. Living cells from the probiotic species Lactobacillus plantarum are picked up with a polydopamine-coated colloidal probe, enabling us to quantify the adhesion forces between single bacteria and biotic (lectin monolayer) or abiotic (hydrophobic monolayer) surfaces. These minimally invasive single-cell experiments provide novel, to our knowledge, insight into the specific and nonspecific forces driving the adhesion of L. plantarum, and represent a generic platform for studying the molecular mechanisms of cell adhesion in probiotic and pathogenic bacteria. PMID:23663831

  11. Defining cell types and states with single-cell genomics

    PubMed Central

    Trapnell, Cole

    2015-01-01

    A revolution in cellular measurement technology is under way: For the first time, we have the ability to monitor global gene regulation in thousands of individual cells in a single experiment. Such experiments will allow us to discover new cell types and states and trace their developmental origins. They overcome fundamental limitations inherent in measurements of bulk cell population that have frustrated efforts to resolve cellular states. Single-cell genomics and proteomics enable not only precise characterization of cell state, but also provide a stunningly high-resolution view of transitions between states. These measurements may finally make explicit the metaphor that C.H. Waddington posed nearly 60 years ago to explain cellular plasticity: Cells are residents of a vast “landscape” of possible states, over which they travel during development and in disease. Single-cell technology helps not only locate cells on this landscape, but illuminates the molecular mechanisms that shape the landscape itself. However, single-cell genomics is a field in its infancy, with many experimental and computational advances needed to fully realize its full potential. PMID:26430159

  12. Single-Cell-Precision Microplasma-Induced Cancer Cell Apoptosis

    PubMed Central

    Lu, Xinpei; He, Guangyuan; Ostrikov, Kostya

    2014-01-01

    The issue of single-cell control has recently attracted enormous interest. However, in spite of the presently achievable intracellular-level physiological probing through bio-photonics, nano-probe-based, and some other techniques, the issue of inducing selective, single-cell-precision apoptosis, without affecting neighbouring cells remains essentially open. Here we resolve this issue and report on the effective single-cell-precision cancer cell treatment using the reactive chemistry of the localized corona-type plasma discharge around a needle-like electrode with the spot size ∼1 µm. When the electrode is positioned with the micrometer precision against a selected cell, a focused and highly-localized micro-plasma discharge induces apoptosis in the selected individual HepG2 and HeLa cancer cells only, without affecting any surrounding cells, even in small cell clusters. This is confirmed by the real-time monitoring of the morphological and structural changes at the cellular and cell nucleus levels after the plasma exposure. PMID:24971517

  13. A stochastic transcriptional switch model for single cell imaging data

    PubMed Central

    Hey, Kirsty L.; Momiji, Hiroshi; Featherstone, Karen; Davis, Julian R.E.; White, Michael R.H.; Rand, David A.; Finkenstädt, Bärbel

    2015-01-01

    Gene expression is made up of inherently stochastic processes within single cells and can be modeled through stochastic reaction networks (SRNs). In particular, SRNs capture the features of intrinsic variability arising from intracellular biochemical processes. We extend current models for gene expression to allow the transcriptional process within an SRN to follow a random step or switch function which may be estimated using reversible jump Markov chain Monte Carlo (MCMC). This stochastic switch model provides a generic framework to capture many different dynamic features observed in single cell gene expression. Inference for such SRNs is challenging due to the intractability of the transition densities. We derive a model-specific birth–death approximation and study its use for inference in comparison with the linear noise approximation where both approximations are considered within the unifying framework of state-space models. The methodology is applied to synthetic as well as experimental single cell imaging data measuring expression of the human prolactin gene in pituitary cells. PMID:25819987

  14. Microfluidics-based single-cell functional proteomics for fundamental and applied biomedical applications.

    PubMed

    Yu, Jing; Zhou, Jing; Sutherland, Alex; Wei, Wei; Shin, Young Shik; Xue, Min; Heath, James R

    2014-01-01

    We review an emerging microfluidics-based toolkit for single-cell functional proteomics. Functional proteins include, but are not limited to, the secreted signaling proteins that can reflect the biological behaviors of immune cells or the intracellular phosphoproteins associated with growth factor-stimulated signaling networks. Advantages of the microfluidics platforms are multiple. First, 20 or more functional proteins may be assayed simultaneously from statistical numbers of single cells. Second, cell behaviors (e.g., motility) may be correlated with protein assays. Third, extensions to quantized cell populations can permit measurements of cell-cell interactions. Fourth, rare cells can be functionally identified and then separated for further analysis or culturing. Finally, certain assay types can provide a conduit between biology and the physicochemical laws. We discuss the history and challenges of the field then review design concepts and uses of the microchip platforms that have been reported, with an eye toward biomedical applications. We then look to the future of the field.

  15. Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis.

    PubMed

    Shin, Jaehoon; Berg, Daniel A; Zhu, Yunhua; Shin, Joseph Y; Song, Juan; Bonaguidi, Michael A; Enikolopov, Grigori; Nauen, David W; Christian, Kimberly M; Ming, Guo-li; Song, Hongjun

    2015-09-01

    Somatic stem cells contribute to tissue ontogenesis, homeostasis, and regeneration through sequential processes. Systematic molecular analysis of stem cell behavior is challenging because classic approaches cannot resolve cellular heterogeneity or capture developmental dynamics. Here we provide a comprehensive resource of single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny. We further developed Waterfall, a bioinformatic pipeline, to statistically quantify singe-cell gene expression along a de novo reconstructed continuous developmental trajectory. Our study reveals molecular signatures of adult qNSCs, characterized by active niche signaling integration and low protein translation capacity. Our analyses further delineate molecular cascades underlying qNSC activation and neurogenesis initiation, exemplified by decreased extrinsic signaling capacity, primed translational machinery, and regulatory switches in transcription factors, metabolism, and energy sources. Our study reveals the molecular continuum underlying adult neurogenesis and illustrates how Waterfall can be used for single-cell omics analyses of various continuous biological processes. PMID:26299571

  16. Resonant waveguide grating imagers for single cell analysis and high throughput screening

    NASA Astrophysics Data System (ADS)

    Fang, Ye

    2015-08-01

    Resonant waveguide grating (RWG) systems illuminate an array of diffractive nanograting waveguide structures in microtiter plate to establish evanescent wave for measuring tiny changes in local refractive index arising from the dynamic mass redistribution of living cells upon stimulation. Whole-plate RWG imager enables high-throughput profiling and screening of drugs. Microfluidics RWG imager not only manifests distinct receptor signaling waves, but also differentiates long-acting agonism and antagonism. Spatially resolved RWG imager allows for single cell analysis including receptor signaling heterogeneity and the invasion of cancer cells in a spheroidal structure through 3-dimensional extracellular matrix. High frequency RWG imager permits real-time detection of drug-induced cardiotoxicity. The wide coverage in target, pathway, assay, and cell phenotype has made RWG systems powerful tool in both basic research and early drug discovery process.

  17. Rapid multiplex analysis of lipid raft components with single-cell resolution.

    PubMed

    Schatzlmaier, Philipp; Supper, Verena; Göschl, Lisa; Zwirzitz, Alexander; Eckerstorfer, Paul; Ellmeier, Wilfried; Huppa, Johannes B; Stockinger, Hannes

    2015-09-22

    Lipid rafts, a distinct class of highly dynamic cell membrane microdomains, are integral to cell homeostasis, differentiation, and signaling. However, their quantitative examination is challenging when working with rare cells, developmentally heterogeneous cell populations, or molecules that only associate weakly with lipid rafts. We present a fast biochemical method, which is based on lipid raft components associating with the nucleus upon partial lysis during centrifugation through nonionic detergent. Requiring little starting material or effort, our protocol enabled the multidimensional flow cytometric quantitation of raft-resident proteins with single-cell resolution, thereby assessing the membrane components from a few cells in complex cell populations, as well as their dynamics resulting from cell signaling, differentiation, or genetic mutation.

  18. Bidirectional Promoter Engineering for Single Cell MicroRNA Sensors in Embryonic Stem Cells

    PubMed Central

    Sladitschek, Hanna L.

    2016-01-01

    MicroRNAs have emerged as important markers and regulators of cell identity. Precise measurements of cellular miRNA levels rely traditionally on RNA extraction and thus do not allow to follow miRNA expression dynamics at the level of single cells. Non-invasive miRNA sensors present an ideal solution but they critically depend on the performance of suitable ubiquitous promoters that reliably drive expression both in pluripotent and differentiated cell types. Here we describe the engineering of bidirectional promoters that drive the expression of precise ratiometric fluorescent miRNA sensors in single mouse embryonic stem cells (mESCs) and their differentiated derivatives. These promoters are based on combinations of the widely used CAG, EF1α and PGK promoters as well as the CMV and PGK enhancers. miR-142-3p, which is known to be bimodally expressed in mESCs, served as a model miRNA to gauge the precision of the sensors. The performance of the resulting miRNA sensors was assessed by flow cytometry in single stable transgenic mESCs undergoing self-renewal or differentiation. EF1α promoters arranged back-to-back failed to drive the robustly correlated expression of two transgenes. Back-to-back PGK promoters were shut down during mESC differentiation. However, we found that a back-to-back arrangement of CAG promoters with four CMV enhancers provided both robust expression in mESCs undergoing differentiation and the best signal-to-noise for measurement of miRNA activity in single cells among all the sensors we tested. Such a bidirectional promoter is therefore particularly well suited to study the dynamics of miRNA expression during cell fate transitions at the single cell level. PMID:27152616

  19. Bidirectional Promoter Engineering for Single Cell MicroRNA Sensors in Embryonic Stem Cells.

    PubMed

    Sladitschek, Hanna L; Neveu, Pierre A

    2016-01-01

    MicroRNAs have emerged as important markers and regulators of cell identity. Precise measurements of cellular miRNA levels rely traditionally on RNA extraction and thus do not allow to follow miRNA expression dynamics at the level of single cells. Non-invasive miRNA sensors present an ideal solution but they critically depend on the performance of suitable ubiquitous promoters that reliably drive expression both in pluripotent and differentiated cell types. Here we describe the engineering of bidirectional promoters that drive the expression of precise ratiometric fluorescent miRNA sensors in single mouse embryonic stem cells (mESCs) and their differentiated derivatives. These promoters are based on combinations of the widely used CAG, EF1α and PGK promoters as well as the CMV and PGK enhancers. miR-142-3p, which is known to be bimodally expressed in mESCs, served as a model miRNA to gauge the precision of the sensors. The performance of the resulting miRNA sensors was assessed by flow cytometry in single stable transgenic mESCs undergoing self-renewal or differentiation. EF1α promoters arranged back-to-back failed to drive the robustly correlated expression of two transgenes. Back-to-back PGK promoters were shut down during mESC differentiation. However, we found that a back-to-back arrangement of CAG promoters with four CMV enhancers provided both robust expression in mESCs undergoing differentiation and the best signal-to-noise for measurement of miRNA activity in single cells among all the sensors we tested. Such a bidirectional promoter is therefore particularly well suited to study the dynamics of miRNA expression during cell fate transitions at the single cell level.

  20. Bidirectional Promoter Engineering for Single Cell MicroRNA Sensors in Embryonic Stem Cells.

    PubMed

    Sladitschek, Hanna L; Neveu, Pierre A

    2016-01-01

    MicroRNAs have emerged as important markers and regulators of cell identity. Precise measurements of cellular miRNA levels rely traditionally on RNA extraction and thus do not allow to follow miRNA expression dynamics at the level of single cells. Non-invasive miRNA sensors present an ideal solution but they critically depend on the performance of suitable ubiquitous promoters that reliably drive expression both in pluripotent and differentiated cell types. Here we describe the engineering of bidirectional promoters that drive the expression of precise ratiometric fluorescent miRNA sensors in single mouse embryonic stem cells (mESCs) and their differentiated derivatives. These promoters are based on combinations of the widely used CAG, EF1α and PGK promoters as well as the CMV and PGK enhancers. miR-142-3p, which is known to be bimodally expressed in mESCs, served as a model miRNA to gauge the precision of the sensors. The performance of the resulting miRNA sensors was assessed by flow cytometry in single stable transgenic mESCs undergoing self-renewal or differentiation. EF1α promoters arranged back-to-back failed to drive the robustly correlated expression of two transgenes. Back-to-back PGK promoters were shut down during mESC differentiation. However, we found that a back-to-back arrangement of CAG promoters with four CMV enhancers provided both robust expression in mESCs undergoing differentiation and the best signal-to-noise for measurement of miRNA activity in single cells among all the sensors we tested. Such a bidirectional promoter is therefore particularly well suited to study the dynamics of miRNA expression during cell fate transitions at the single cell level. PMID:27152616

  1. Single-cell codetection of metabolic activity, intracellular functional proteins, and genetic mutations from rare circulating tumor cells.

    PubMed

    Zhang, Yu; Tang, Yin; Sun, Shuai; Wang, Zhihua; Wu, Wenjun; Zhao, Xiaodong; Czajkowsky, Daniel M; Li, Yan; Tian, Jianhui; Xu, Ling; Wei, Wei; Deng, Yuliang; Shi, Qihui

    2015-10-01

    The high glucose uptake and activation of oncogenic signaling pathways in cancer cells has long made these features, together with the mutational spectrum, prime diagnostic targets of circulating tumor cells (CTCs). Further, an ability to characterize these properties at a single cell resolution is widely believed to be essential, as the known extensive heterogeneity in CTCs can obscure important correlations in data obtained from cell population-based methods. However, to date, it has not been possible to quantitatively measure metabolic, proteomic, and genetic data from a single CTC. Here we report a microchip-based approach that allows for the codetection of glucose uptake, intracellular functional proteins, and genetic mutations at the single-cell level from rare tumor cells. The microchip contains thousands of nanoliter grooves (nanowells) that isolate individual CTCs and allow for the assessment of their glucose uptake via imaging of a fluorescent glucose analog, quantification of a panel of intracellular signaling proteins using a miniaturized antibody barcode microarray, and retrieval of the individual cell nuclei for subsequent off-chip genome amplification and sequencing. This approach integrates molecular-scale information on the metabolic, proteomic, and genetic status of single cells and permits the inference of associations between genetic signatures, energy consumption, and phosphoproteins oncogenic signaling activities in CTCs isolated from blood samples of patients. Importantly, this microchip chip-based approach achieves this multidimensional molecular analysis with minimal cell loss (<20%), which is the bottleneck of the rare cell analysis. PMID:26378744

  2. Single Cell Adhesion Assay Using Computer Controlled Micropipette

    PubMed Central

    Salánki, Rita; Hős, Csaba; Orgovan, Norbert; Péter, Beatrix; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

    2014-01-01

    Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today’s techniques typically have an extremely low throughput (5–10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub

  3. Single cell adhesion assay using computer controlled micropipette.

    PubMed

    Salánki, Rita; Hős, Csaba; Orgovan, Norbert; Péter, Beatrix; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

    2014-01-01

    Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today's techniques typically have an extremely low throughput (5-10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub-population of

  4. Single-cell Genomics using Droplet-based Microfluidics

    NASA Astrophysics Data System (ADS)

    Basu, Anindita; Macosko, Evan; Shalek, Alex; McCarroll, Steven; Regev, Aviv; Weitz, Dave

    2014-03-01

    We develop a system to profile the transcriptome of mammalian cells in isolation using reverse emulsion droplet-based microfluidic techniques. This is accomplished by (a) encapsulating and lysing one cell per emulsion droplet, and (b) uniquely barcoding the RNA contents from each cell using unique DNA-barcoded microgel beads. This enables us to study the transcriptional behavior of a large number of cells at single-cell resolution. We then use these techniques to study transcriptional responses of isolated immune cells to precisely controlled chemical and pathological stimuli provided in the emulsion droplet.

  5. Modeling single cell antibody excretion on a biosensor.

    PubMed

    Stojanović, Ivan; Baumgartner, Wolfgang; van der Velden, Thomas J G; Terstappen, Leon W M M; Schasfoort, Richard B M

    2016-07-01

    We simulated, using Comsol Multiphysics, the excretion of antibodies by single hybridoma cells and their subsequent binding on a surface plasmon resonance imaging (SPRi) sensor. The purpose was to confirm that SPRi is suitable to accurately quantify antibody (anti-EpCAM) excretion. The model showed that antibody loss by diffusion away from the sensor was less than 1%. Unexpectedly, more than 99% of the excreted antibodies were captured on the sensor. These data prove the remarkable phenomenon that the SPRi output of cellular antibody excretion and its subsequent binding, performed under the conditions described here, is directly usable for quantification of single cell antibody production rates. PMID:27040182

  6. TOPAZ-2 single-cell TFE electric insulation properties study

    SciTech Connect

    Vasilchenko, A.V.; Izhvanov, O.L.

    1996-03-01

    TOPAZ-II single cell thermoinic fuel element (TFE) electric insulation parameters under testing with electric heating were measured. TFE electric design schematic, experimental procedure and measurements results are described. Collector resistance was measured in helium at 420{endash}890 K. Metal ceramic ceals insulation properties were measured in vacuum P=10{sup {minus}4} Pa and in cesium vapor P=10{sup {minus}1}{minus}260 Pa, at 420{endash}730 K. Results of separate TFE are compared with the data; that were measured during nuclear power system (NPS) Ya-21U test. Based upon this data NPS power losses were estimated. {copyright} {ital 1996 American Institute of Physics.}

  7. Single-cell analyses of circulating tumor cells

    PubMed Central

    Chen, Xi-Xi; Bai, Fan

    2015-01-01

    Circulating tumor cells (CTCs) are a population of tumor cells mediating metastasis, which results in most of the cancer related deaths. The number of CTCs in the peripheral blood of patients is rare, and many platforms have been launched for detection and enrichment of CTCs. Enumeration of CTCs has already been used as a prognosis marker predicting the survival rate of cancer patients. Yet CTCs should be more potential. Studies on CTCs at single cell level may help revealing the underlying mechanism of tumorigenesis and metastasis. Though far from developed, this area of study holds much promise in providing new clinical application and deep understanding towards metastasis and cancer development. PMID:26487963

  8. Single-cell analyses of circulating tumor cells.

    PubMed

    Chen, Xi-Xi; Bai, Fan

    2015-09-01

    Circulating tumor cells (CTCs) are a population of tumor cells mediating metastasis, which results in most of the cancer related deaths. The number of CTCs in the peripheral blood of patients is rare, and many platforms have been launched for detection and enrichment of CTCs. Enumeration of CTCs has already been used as a prognosis marker predicting the survival rate of cancer patients. Yet CTCs should be more potential. Studies on CTCs at single cell level may help revealing the underlying mechanism of tumorigenesis and metastasis. Though far from developed, this area of study holds much promise in providing new clinical application and deep understanding towards metastasis and cancer development.

  9. Virtual microfluidics for digital quantification and single-cell sequencing.

    PubMed

    Xu, Liyi; Brito, Ilana L; Alm, Eric J; Blainey, Paul C

    2016-09-01

    We have developed hydrogel-based virtual microfluidics as a simple and robust alternative to complex engineered microfluidic systems for the compartmentalization of nucleic acid amplification reactions. We applied in-gel digital multiple displacement amplification (dMDA) to purified DNA templates, cultured bacterial cells and human microbiome samples in the virtual microfluidics system, and demonstrated whole-genome sequencing of single-cell MDA products with excellent coverage uniformity and markedly reduced chimerism compared with products of liquid MDA reactions. PMID:27479330

  10. Modeling single cell antibody excretion on a biosensor.

    PubMed

    Stojanović, Ivan; Baumgartner, Wolfgang; van der Velden, Thomas J G; Terstappen, Leon W M M; Schasfoort, Richard B M

    2016-07-01

    We simulated, using Comsol Multiphysics, the excretion of antibodies by single hybridoma cells and their subsequent binding on a surface plasmon resonance imaging (SPRi) sensor. The purpose was to confirm that SPRi is suitable to accurately quantify antibody (anti-EpCAM) excretion. The model showed that antibody loss by diffusion away from the sensor was less than 1%. Unexpectedly, more than 99% of the excreted antibodies were captured on the sensor. These data prove the remarkable phenomenon that the SPRi output of cellular antibody excretion and its subsequent binding, performed under the conditions described here, is directly usable for quantification of single cell antibody production rates.

  11. Single cell analytic tools for drug discovery and development

    PubMed Central

    Heath, James R.; Ribas, Antoni; Mischel, Paul S.

    2016-01-01

    The genetic, functional, or compositional heterogeneity of healthy and diseased tissues presents major challenges in drug discovery and development.1-3 In cancers, heterogeneity may be essential for tumor stability,4 but its precise role in tumor biology is poorly resolved. This challenges the design of accurate disease models for use in drug development, and can confound the interpretation of biomarker levels, and of patient responses to specific therapies. The complex nature of heterogeneous tissues has motivated the development of tools for single cell genomic, transcriptomic, and multiplex proteomic analysis. We review these tools, assess their advantages and limitations, and explore their potential applications in drug discovery and development. PMID:26669673

  12. The mechanisms shaping the single-cell transcriptional landscape.

    PubMed

    Martinez-Jimenez, Celia Pilar; Odom, Duncan T

    2016-04-01

    Recent technological and computational advances in understanding the transcriptional and chromatin features of single cells have begun answering longstanding questions in the extent and impact of biological heterogeneity. Here, we outline the intrinsic and extrinsic mechanisms that underlie the transcriptional and functional diversity within superficially homogeneous populations, and we discuss how fascinating new studies have afforded novel insight into each mechanism. The studies are chosen in part to include initial reports of novel functional genomics tools where the eventual applications will clearly have profound impact on our understanding the dynamics of cell-to-cell transcriptional variation-from individual cells to whole organisms.

  13. Deregulation in STAT signaling is important for cutaneous T-cell lymphoma (CTCL) pathogenesis and cancer progression.

    PubMed

    Netchiporouk, Elena; Litvinov, Ivan V; Moreau, Linda; Gilbert, Martin; Sasseville, Denis; Duvic, Madeleine

    2014-01-01

    Deregulation of STAT signaling has been implicated in the pathogenesis for a variety of cancers, including CTCL. Constitutive activation of STAT5 and STAT3 was observed in early and late stages of CTCL, respectively. In early stages, IL-2, IL-7 and IL-15 signaling via JAK1 and JAK3 kinases is believed to be responsible for activating STAT5, while in advanced stages development of IL-21 autocrine signaling is thought to be important for STAT3 activation. Recent molecular evidence further suggests that upregulation of STAT5 in early disease stages results in increased expression of oncogenic miR-155 microRNA that subsequently targets STAT4 expression on mRNA level. STAT4 signaling is known to be critical for T helper (Th) 1 phenotype differentiation and its loss results in a switch from Th1 to Th2 phenotype in malignant T cells. During this switch the expression of STAT6 is often upregulated in CTCL. In advanced stages, activation of STAT3 and STAT5 may become completely cytokine-independent and be driven only via constitutively active JAK1 and JAK3 kinases. Further research into the molecular pathogenesis of JAK/STAT signaling in this cancer may enable us to develop effective therapies for our patients. PMID:25485578

  14. Deciphering the Receptor Repertoire Encoding Specific Odorants by Time-Lapse Single-Cell Array Cytometry

    PubMed Central

    Suzuki, Masato; Yoshimoto, Nobuo; Shimono, Ken; Kuroda, Shun’ichi

    2016-01-01

    Mammals can recognize a vast number of odorants by using olfactory receptors (ORs) known as G protein-coupled receptors. The OR gene family is one of the most diverse gene families in mammalian genomes. Because of the vast combinations of ORs and odorants, few ORs have thus far been linked to specific odorants. Here, we established a functional screening method for OR genes by using a microchamber array containing >5,400 single olfactory epithelium-derived cells from mice applied to time-lapse single-cell array cytometry. This method facilitated the prompt isolation of single olfactory sensory neurons (OSNs) responding to the odorant of interest. Subsequent single-cell RT-PCR allowed us to isolate the genes encoding respective ORs. By using volatile molecules recognized as biomarkers for lung cancers, this method could deorphanize ORs and thereby reconstitute the OR-mediated signaling cascade in HEK293T cells. Thus, our system could be applied to identify any receptor by using specific ligands in the fields of physiopathology and pharmacology. PMID:26832639

  15. Single-cell transcriptome analysis reveals dynamic changes in lncRNA expression during reprogramming.

    PubMed

    Kim, Daniel H; Marinov, Georgi K; Pepke, Shirley; Singer, Zakary S; He, Peng; Williams, Brian; Schroth, Gary P; Elowitz, Michael B; Wold, Barbara J

    2015-01-01

    Cellular reprogramming highlights the epigenetic plasticity of the somatic cell state. Long noncoding RNAs (lncRNAs) have emerging roles in epigenetic regulation, but their potential functions in reprogramming cell fate have been largely unexplored. We used single-cell RNA sequencing to characterize the expression patterns of over 16,000 genes, including 437 lncRNAs, during defined stages of reprogramming to pluripotency. Self-organizing maps (SOMs) were used as an intuitive way to structure and interrogate transcriptome data at the single-cell level. Early molecular events during reprogramming involved the activation of Ras signaling pathways, along with hundreds of lncRNAs. Loss-of-function studies showed that activated lncRNAs can repress lineage-specific genes, while lncRNAs activated in multiple reprogramming cell types can regulate metabolic gene expression. Our findings demonstrate that reprogramming cells activate defined sets of functionally relevant lncRNAs and provide a resource to further investigate how dynamic changes in the transcriptome reprogram cell state.

  16. Metabolic Differentiation of Neuronal Phenotypes by Single Cell CE-ESI-MS

    PubMed Central

    Nemes, Peter; Knolhoff, Ann M.; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

    2011-01-01

    Single cell mass spectrometry (MS) is a rapidly emerging field in metabolic investigations. The inherent chemical complexity of most biological samples poses analytical challenges when using MS platforms to measure sample content without prior chemical separation. Here, a single-cell capillary electrophoresis (CE) system was coupled with electrospray ionization (ESI) MS to enable the simultaneous measurement of a vast array of endogenous compounds in over 50 identified and isolated large neurons from the Aplysia californica central nervous system. More than 300 distinct ion signals (m/z values) were detected from a single neuron in positive ion mode, 140 of which were selected for chemometric data analysis. Metabolic features were evaluated among six different neuron types (B1, B2, left pleural 1 (LPl1), metacerebral cell (MCC), R2, and R15), chosen for their various physiological functions. The results indicated chemical similarities among some neuron types (B1 to B2 and LPl1 to R2) and distinctive features for others (MCC and R15 cells). The quantitative nature of the MS platform allowed the comparison of metabolite levels for specific neurons. The CE-ESI-MS approach for examination of individual nanoliter-volume cells as described herein is readily adaptable to other volume-limited samples. PMID:21809850

  17. Time-Resolved Study of Nanoparticle Induced Apoptosis Using Microfabricated Single Cell Arrays

    PubMed Central

    Röttgermann, Peter J. F.; Dawson, Kenneth A.; Rädler, Joachim O.

    2016-01-01

    Cell fate decisions like apoptosis are heterogeneously implemented within a cell population and, consequently, the population response is recognized as sum of many individual dynamic events. Here, we report on the use of micro-patterned single-cell arrays for real-time tracking of nanoparticle-induced (NP) cell death in sets of thousands of cells in parallel. Annexin (pSIVA) and propidium iodide (PI), two fluorescent indicators of apoptosis, are simultaneously monitored after exposure to functionalized polystyrene (PS−NH2) nanobeads as a model system. We find that the distribution of Annexin onset times shifts to later times and broadens as a function of decreasing NP dose. We discuss the mean time-to-death as a function of dose, and show how the EC50 value depends both on dose and time of measurement. In addition, the correlations between the early and late apoptotic markers indicate a systematic shift from apoptotic towards necrotic cell death during the course of the experiment. Thus, our work demonstrates the potential of array-based single cell cytometry for kinetic analysis of signaling cascades in a high-throughput format. PMID:27600074

  18. Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method.

    PubMed

    Lin, Jia-Ren; Fallahi-Sichani, Mohammad; Sorger, Peter K

    2015-01-01

    Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, particularly in the case of highly multiplexed methods such as mass cytometry (CyTOF) able to correlate the levels of multiple signalling, differentiation and cell fate markers. Immunofluorescence (IF) microscopy adds information on cell morphology and the microenvironment that are not obtained using flow-based techniques, but the multiplicity of conventional IF is limited. This has motivated development of imaging methods that require specialized instrumentation, exotic reagents or proprietary protocols that are difficult to reproduce in most laboratories. Here we report a public-domain method for achieving high multiplicity single-cell IF using cyclic immunofluorescence (CycIF), a simple and versatile procedure in which four-colour staining alternates with chemical inactivation of fluorophores to progressively build a multichannel image. Because CycIF uses standard reagents and instrumentation and is no more expensive than conventional IF, it is suitable for high-throughput assays and screening applications. PMID:26399630

  19. Emerging methods to study bacteriophage infection at the single-cell level.

    PubMed

    Dang, Vinh T; Sullivan, Matthew B

    2014-01-01

    Bacteria and their viruses (phages) are abundant across diverse ecosystems and their interactions influence global biogeochemical cycles and incidence of disease. Problematically, both classical and metagenomic methods insufficiently assess the host specificity of phages and phage-host infection dynamics in nature. Here we review emerging methods to study phage-host interaction and infection dynamics with a focus on those that offer resolution at the single-cell level. These methods leverage ever-increasing sequence data to identify virus signals from single-cell amplified genome datasets or to produce primers/probes to target particular phage-bacteria pairs (digital PCR and phageFISH), even in complex communities. All three methods enable study of phage infection of uncultured bacteria from environmental samples, while the latter also discriminates between phage-host interaction outcomes (e.g., lytic, chronic, lysogenic) in model systems. Together these techniques enable quantitative, spatiotemporal studies of phage-bacteria interactions from environmental samples of any ecosystem, which will help elucidate and predict the ecological and evolutionary impacts of specific phage-host pairings in nature. PMID:25566233

  20. Emerging methods to study bacteriophage infection at the single-cell level.

    PubMed

    Dang, Vinh T; Sullivan, Matthew B

    2014-01-01

    Bacteria and their viruses (phages) are abundant across diverse ecosystems and their interactions influence global biogeochemical cycles and incidence of disease. Problematically, both classical and metagenomic methods insufficiently assess the host specificity of phages and phage-host infection dynamics in nature. Here we review emerging methods to study phage-host interaction and infection dynamics with a focus on those that offer resolution at the single-cell level. These methods leverage ever-increasing sequence data to identify virus signals from single-cell amplified genome datasets or to produce primers/probes to target particular phage-bacteria pairs (digital PCR and phageFISH), even in complex communities. All three methods enable study of phage infection of uncultured bacteria from environmental samples, while the latter also discriminates between phage-host interaction outcomes (e.g., lytic, chronic, lysogenic) in model systems. Together these techniques enable quantitative, spatiotemporal studies of phage-bacteria interactions from environmental samples of any ecosystem, which will help elucidate and predict the ecological and evolutionary impacts of specific phage-host pairings in nature.

  1. 2D light scattering static cytometry for label-free single cell analysis with submicron resolution.

    PubMed

    Xie, Linyan; Yang, Yan; Sun, Xuming; Qiao, Xu; Liu, Qiao; Song, Kun; Kong, Beihua; Su, Xuantao

    2015-11-01

    Conventional optical cytometric techniques usually measure fluorescence or scattering signals at fixed angles from flowing cells in a liquid stream. Here we develop a novel cytometer that employs a scanning optical fiber to illuminate single static cells on a glass slide, which requires neither microfluidic fabrication nor flow control. This static cytometric technique measures two dimensional (2D) light scattering patterns via a small numerical aperture (0.25) microscope objective for label-free single cell analysis. Good agreement is obtained between the yeast cell experimental and Mie theory simulated patterns. It is demonstrated that the static cytometer with a microscope objective of a low resolution around 1.30 μm has the potential to perform high resolution analysis on yeast cells with distributed sizes. The capability of the static cytometer for size determination with submicron resolution is validated via measurements on standard microspheres with mean diameters of 3.87 and 4.19 μm. Our 2D light scattering static cytometric technique may provide an easy-to-use, label-free, and flow-free method for single cell diagnostics.

  2. Single-cell analysis and sorting using droplet-based microfluidics

    PubMed Central

    Mazutis, Linas; Gilbert, John; Ung, W Lloyd; Weitz, David A; Griffiths, Andrew D; Heyman, John A

    2014-01-01

    We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. as an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. the beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ~200 Hz as well as cell enrichment. the microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ~1 million cells, the microfluidic operations require 2–6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5–7 d. PMID:23558786

  3. Time-Resolved Study of Nanoparticle Induced Apoptosis Using Microfabricated Single Cell Arrays.

    PubMed

    Röttgermann, Peter J F; Dawson, Kenneth A; Rädler, Joachim O

    2016-01-01

    Cell fate decisions like apoptosis are heterogeneously implemented within a cell population and, consequently, the population response is recognized as sum of many individual dynamic events. Here, we report on the use of micro-patterned single-cell arrays for real-time tracking of nanoparticle-induced (NP) cell death in sets of thousands of cells in parallel. Annexin (pSIVA) and propidium iodide (PI), two fluorescent indicators of apoptosis, are simultaneously monitored after exposure to functionalized polystyrene (PS - NH 2) nanobeads as a model system. We find that the distribution of Annexin onset times shifts to later times and broadens as a function of decreasing NP dose. We discuss the mean time-to-death as a function of dose, and show how the EC 50 value depends both on dose and time of measurement. In addition, the correlations between the early and late apoptotic markers indicate a systematic shift from apoptotic towards necrotic cell death during the course of the experiment. Thus, our work demonstrates the potential of array-based single cell cytometry for kinetic analysis of signaling cascades in a high-throughput format. PMID:27600074

  4. Single-cell analysis and sorting using droplet-based microfluidics.

    PubMed

    Mazutis, Linas; Gilbert, John; Ung, W Lloyd; Weitz, David A; Griffiths, Andrew D; Heyman, John A

    2013-05-01

    We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2-6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5-7 d.

  5. Deciphering the Receptor Repertoire Encoding Specific Odorants by Time-Lapse Single-Cell Array Cytometry.

    PubMed

    Suzuki, Masato; Yoshimoto, Nobuo; Shimono, Ken; Kuroda, Shun'ichi

    2016-01-01

    Mammals can recognize a vast number of odorants by using olfactory receptors (ORs) known as G protein-coupled receptors. The OR gene family is one of the most diverse gene families in mammalian genomes. Because of the vast combinations of ORs and odorants, few ORs have thus far been linked to specific odorants. Here, we established a functional screening method for OR genes by using a microchamber array containing >5,400 single olfactory epithelium-derived cells from mice applied to time-lapse single-cell array cytometry. This method facilitated the prompt isolation of single olfactory sensory neurons (OSNs) responding to the odorant of interest. Subsequent single-cell RT-PCR allowed us to isolate the genes encoding respective ORs. By using volatile molecules recognized as biomarkers for lung cancers, this method could deorphanize ORs and thereby reconstitute the OR-mediated signaling cascade in HEK293T cells. Thus, our system could be applied to identify any receptor by using specific ligands in the fields of physiopathology and pharmacology. PMID:26832639

  6. Myeloid neoplasm demonstrating a STAT5B-RARA rearrangement and genetic alterations associated with all-trans retinoic acid resistance identified by a custom next-generation sequencing assay.

    PubMed

    Kluk, Michael J; Abo, Ryan P; Brown, Ronald D; Kuo, Frank C; Dal Cin, Paola; Pozdnyakova, Olga; Morgan, Elizabeth A; Lindeman, Neal I; DeAngelo, Daniel J; Aster, Jon C

    2015-10-01

    We describe the case of a patient presenting with several weeks of symptoms related to pancytopenia associated with a maturation arrest at the late promyelocyte/early myelocyte stage of granulocyte differentiation. A diagnosis of acute promyelocytic leukemia was considered, but the morphologic features were atypical for this entity and conventional tests for the presence of a PML-RARA fusion gene were negative. Additional analysis using a custom next-generation sequencing assay revealed a rearrangement producing a STAT5B-RARA fusion gene, which was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and supplementary cytogenetic studies, allowing the diagnosis of a morphologically atypical form of acute promyelocytic leukemia to be made. Analysis of the sequencing data permitted characterization of both chromosomal breakpoints and revealed two additional alterations, a small deletion in RARA exon 9 and a RARA R276W substitution, that have been linked to resistance to all-trans retinoic acid. This case highlights how next-generation sequencing can augment currently standard testing to establish diagnoses in difficult cases, and in doing so help guide selection of therapy. PMID:27148563

  7. 3,3'-Diindolylmethane Inhibits Flt3L/GM-CSF-induced-bone Marrow-derived CD103+ Dendritic Cell Differentiation Regulating Phosphorylation of STAT3 and STAT5

    PubMed Central

    Choi, Ah-Jeong; Kim, Soo-Ji; Jeong, So-Yeon

    2015-01-01

    The intestinal immune system maintains oral tolerance to harmless antigens or nutrients. One mechanism of oral tolerance is mediated by regulatory T cell (Treg)s, of which differentiation is regulated by a subset of dendritic cell (DC)s, primarily CD103+ DCs. The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, plays an important role in regulating immunity. The intestines are exposed to various AhR ligands, including endogenous metabolites and phytochemicals. It was previously reported that AhR activation induced tolerogenic DCs in mice or in cultures of bone marrow-derived DCs. However, given the variety of tolerogenic DCs, which type of tolerogenic DCs is regulated by AhR remains unknown. In this study, we found that AhR ligand 3,3'-diindolylmethane (DIM) inhibited the development of CD103+ DCs from mouse bone marrow cells stimulated with Flt3L and GM-CSF. DIM interfered with phosphorylation of STAT3 and STAT5 inhibiting the expression of genes, including Id2, E2-2, IDO-1, and Aldh1a2, which are associated with DC differentiation and functions. Finally, DIM suppressed the ability of CD103+ DCs to induce Foxp3+ Tregs. PMID:26770182

  8. Current Developments in Prokaryotic Single Cell Whole Genome Amplification

    SciTech Connect

    Goudeau, Danielle; Nath, Nandita; Ciobanu, Doina; Cheng, Jan-Fang; Malmstrom, Rex

    2014-03-14

    Our approach to prokaryotic single-cell Whole Genome Amplification at the JGI continues to evolve. To increase both the quality and number of single-cell genomes produced, we explore all aspects of the process from cell sorting to sequencing. For example, we now utilize specialized reagents, acoustic liquid handling, and reduced reaction volumes eliminate non-target DNA contamination in WGA reactions. More specifically, we use a cleaner commercial WGA kit from Qiagen that employs a UV decontamination procedure initially developed at the JGI, and we use the Labcyte Echo for tip-less liquid transfer to set up 2uL reactions. Acoustic liquid handling also dramatically reduces reagent costs. In addition, we are exploring new cell lysis methods including treatment with Proteinase K, lysozyme, and other detergents, in order to complement standard alkaline lysis and allow for more efficient disruption of a wider range of cells. Incomplete lysis represents a major hurdle for WGA on some environmental samples, especially rhizosphere, peatland, and other soils. Finding effective lysis strategies that are also compatible with WGA is challenging, and we are currently assessing the impact of various strategies on genome recovery.

  9. Predicting stochastic gene expression dynamics in single cells.

    PubMed

    Mettetal, Jerome T; Muzzey, Dale; Pedraza, Juan M; Ozbudak, Ertugrul M; van Oudenaarden, Alexander

    2006-05-01

    Fluctuations in protein numbers (noise) due to inherent stochastic effects in single cells can have large effects on the dynamic behavior of gene regulatory networks. Although deterministic models can predict the average network behavior, they fail to incorporate the stochasticity characteristic of gene expression, thereby limiting their relevance when single cell behaviors deviate from the population average. Recently, stochastic models have been used to predict distributions of steady-state protein levels within a population but not to predict the dynamic, presteady-state distributions. In the present work, we experimentally examine a system whose dynamics are heavily influenced by stochastic effects. We measure population distributions of protein numbers as a function of time in the Escherichia coli lactose uptake network (lac operon). We then introduce a dynamic stochastic model and show that prediction of dynamic distributions requires only a few noise parameters in addition to the rates that characterize a deterministic model. Whereas the deterministic model cannot fully capture the observed behavior, our stochastic model correctly predicts the experimental dynamics without any fit parameters. Our results provide a proof of principle for the possibility of faithfully predicting dynamic population distributions from deterministic models supplemented by a stochastic component that captures the major noise sources. PMID:16648266

  10. Block-Cell-Printing for live single-cell printing

    PubMed Central

    Zhang, Kai; Chou, Chao-Kai; Xia, Xiaofeng; Hung, Mien-Chie; Qin, Lidong

    2014-01-01

    A unique live-cell printing technique, termed “Block-Cell-Printing” (BloC-Printing), allows for convenient, precise, multiplexed, and high-throughput printing of functional single-cell arrays. Adapted from woodblock printing techniques, the approach employs microfluidic arrays of hook-shaped traps to hold cells at designated positions and directly transfer the anchored cells onto various substrates. BloC-Printing has a minimum turnaround time of 0.5 h, a maximum resolution of 5 µm, close to 100% cell viability, the ability to handle multiple cell types, and efficiently construct protrusion-connected single-cell arrays. The approach enables the large-scale formation of heterotypic cell pairs with controlled morphology and allows for material transport through gap junction intercellular communication. When six types of breast cancer cells are allowed to extend membrane protrusions in the BloC-Printing device for 3 h, multiple biophysical characteristics of cells—including the protrusion percentage, extension rate, and cell length—are easily quantified and found to correlate well with their migration levels. In light of this discovery, BloC-Printing may serve as a rapid and high-throughput cell protrusion characterization tool to measure the invasion and migration capability of cancer cells. Furthermore, primary neurons are also compatible with BloC-Printing. PMID:24516129

  11. Microdevices for biomolecular detection and single cell analysis

    NASA Astrophysics Data System (ADS)

    Manalis, Scott

    2008-03-01

    Recent advances towards developing biomolecular and single cell applications for a mass-based biosensor known as the suspended microchannel resonator (SMR) will be presented. In SMR detection, target molecules or cells flow through a vibrating suspended microchannel and are captured by receptor molecules attached to the interior channel walls. What separates the SMR from the existing resonant mass sensors is that the receptors, targets, and their aqueous environment are confined inside the resonator, while the resonator itself can oscillate at high Q in an external vacuum environment, thus yielding extraordinarily high sensitivity. This approach solves the problem of viscous damping that degrades the sensitivity of cantilever resonators in solution. We have achieved a resolution of approximately 1 femtogram (1 Hz bandwidth) which is represents an improvement of six order of magnitude improvement over a high-end commercial quartz crystal microbalance. This gives access to intriguing applications such as mass based flow cytometry, real-time monitoring of single cell growth, and the direct detection of protein biomarkers.

  12. Limitations of fitting angular scattering from single cells (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Fan, Xing; Cannaday, Ashley E.; Berger, Andrew J.

    2016-04-01

    The literature contains several reports of Mie-like fits to angular-domain elastic scattering measurements from multiple cells or isolated mitochondria. In these studies, the sampling volume typically contains hundreds or thousands of mitochondria, allowing for the size distribution of mitochondria to be modeled as a smooth function, (e.g. Gaussian or log-normal) with a small number of free parameters. In the case of a single-cell volume containing significantly fewer mitochondria, the true size distribution will no longer be as smooth. Increasing the number of free parameters can lead to unstable fits, however, as the forward-directed angular scattering pattern from such a population illuminated with 785 nm light is a monotonically decaying radial function with few distinct features. Using simulations, we have investigated the limitations of modeling single-cell mitochondrial scattering using smooth population distributions of Mie scatterers. In different instances, the fidelity of the estimated size information can be limited by the number of organelles, the angular detection range, or the non-ideality of the data (both speckle and shot noise). We will describe the conditions under which each of these effects dominates. We will also discuss whether mean and standard deviation are the best sizes to report from such Mie modeling, or if there are other size parameters that have greater fidelity to the true, non-smooth size distributions.

  13. Cell tracing dyes significantly change single cell mechanics

    PubMed Central

    Lulevich, Valentin; Shih, Yi-Ping; Lo, Su Hao; Liu, Gang-yu

    2009-01-01

    Cell tracing dyes are very frequently utilized in cellular biology research because they provide highly sensitive fluorescent tags that do not compromise cellular functions such as growth and proliferation. In many investigations concerning cellular adhesion and mechanics, fluorescent dyes have been employed with the assumption of little impact on the results. Using the single-cell compression technique developed by our team, the single-cell mechanics of MDA-MB-468 and MLC-SV40 cells were investigated as a function of dye uptake. Cell tracing dyes increase living cell stiffness 3-6 times and cell-to-probe adhesion up to 7 times. These results suggest a more significant effect than toxins, such as Thrombin. A simple analytical model was derived to enable the extraction of the Young’s moduli of the cell membrane and cytoskeleton from the force-deformation profiles measured for individual cells. The increase in Young’s modulus of the membrane is 3-7 times, which is more significant than that of the cytoskeleton (1.1-3.4 times). We propose that changes in cell mechanics upon the addition of fluorescent tracing dye are primarily due to incorporation of amphiphilic dye molecules into the cellular plasma membrane, which increases the lateral interaction among phospholipid chains and thus enhances their rigidity and adhesion. PMID:19366241

  14. Ciliary heterogeneity within a single cell: the Paramecium model.

    PubMed

    Aubusson-Fleury, Anne; Cohen, Jean; Lemullois, Michel

    2015-01-01

    Paramecium is a single cell able to divide in its morphologically differentiated stage that has many cilia anchored at its cell surface. Many thousands of cilia are thus assembled in a short period of time during division to duplicate the cell pattern while the cell continues swimming. Most, but not all, of these sensory cilia are motile and involved in two main functions: prey capture and cell locomotion. These cilia display heterogeneity, both in their length and their biochemical properties. Thanks to these properties, as well as to the availability of many postgenomic tools and the possibility to follow the regrowth of cilia after deciliation, Paramecium offers a nice opportunity to study the assembly of the cilia, as well as the genesis of their diversity within a single cell. In this paper, after a brief survey of Paramecium morphology and cilia properties, we describe the tools and the protocols currently used for immunofluorescence, transmission electron microscopy, and ultrastructural immunocytochemistry to analyze cilia, with special recommendations to overcome the problem raised by cilium diversity. PMID:25837404

  15. In vivo lipidomics using single-cell Raman spectroscopy

    PubMed Central

    Wu, Huawen; Volponi, Joanne V.; Oliver, Ann E.; Parikh, Atul N.; Simmons, Blake A.; Singh, Seema

    2011-01-01

    We describe a method for direct, quantitative, in vivo lipid profiling of oil-producing microalgae using single-cell laser-trapping Raman spectroscopy. This approach is demonstrated in the quantitative determination of the degree of unsaturation and transition temperatures of constituent lipids within microalgae. These properties are important markers for determining engine compatibility and performance metrics of algal biodiesel. We show that these factors can be directly measured from a single living microalgal cell held in place with an optical trap while simultaneously collecting Raman data. Cellular response to different growth conditions is monitored in real time. Our approach circumvents the need for lipid extraction and analysis that is both slow and invasive. Furthermore, this technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of impermeability, toxicity, and specificity of the fluorescent probes common in currently used protocols. Although the single-cell Raman spectroscopy demonstrated here is focused on the study of the microalgal lipids with biofuel applications, the analytical capability and quantitation algorithms demonstrated are applicable to many different organisms and should prove useful for a diverse range of applications in lipidomics. PMID:21310969

  16. Ciliary heterogeneity within a single cell: the Paramecium model.

    PubMed

    Aubusson-Fleury, Anne; Cohen, Jean; Lemullois, Michel

    2015-01-01

    Paramecium is a single cell able to divide in its morphologically differentiated stage that has many cilia anchored at its cell surface. Many thousands of cilia are thus assembled in a short period of time during division to duplicate the cell pattern while the cell continues swimming. Most, but not all, of these sensory cilia are motile and involved in two main functions: prey capture and cell locomotion. These cilia display heterogeneity, both in their length and their biochemical properties. Thanks to these properties, as well as to the availability of many postgenomic tools and the possibility to follow the regrowth of cilia after deciliation, Paramecium offers a nice opportunity to study the assembly of the cilia, as well as the genesis of their diversity within a single cell. In this paper, after a brief survey of Paramecium morphology and cilia properties, we describe the tools and the protocols currently used for immunofluorescence, transmission electron microscopy, and ultrastructural immunocytochemistry to analyze cilia, with special recommendations to overcome the problem raised by cilium diversity.

  17. Single-cell force spectroscopy of pili-mediated adhesion

    NASA Astrophysics Data System (ADS)

    Sullan, Ruby May A.; Beaussart, Audrey; Tripathi, Prachi; Derclaye, Sylvie; El-Kirat-Chatel, Sofiane; Li, James K.; Schneider, Yves-Jacques; Vanderleyden, Jos; Lebeer, Sarah; Dufrêne, Yves F.

    2013-12-01

    Although bacterial pili are known to mediate cell adhesion to a variety of substrates, the molecular interactions behind this process are poorly understood. We report the direct measurement of the forces guiding pili-mediated adhesion, focusing on the medically important probiotic bacterium Lactobacillus rhamnosus GG (LGG). Using non-invasive single-cell force spectroscopy (SCFS), we quantify the adhesion forces between individual bacteria and biotic (mucin, intestinal cells) or abiotic (hydrophobic monolayers) surfaces. On hydrophobic surfaces, bacterial pili strengthen adhesion through remarkable nanospring properties, which - presumably - enable the bacteria to resist high shear forces under physiological conditions. On mucin, nanosprings are more frequent and adhesion forces larger, reflecting the influence of specific pili-mucin bonds. Interestingly, these mechanical responses are no longer observed on human intestinal Caco-2 cells. Rather, force curves exhibit constant force plateaus with extended ruptures reflecting the extraction of membrane nanotethers. These single-cell analyses provide novel insights into the molecular mechanisms by which piliated bacteria colonize surfaces (nanosprings, nanotethers), and offer exciting avenues in nanomedicine for understanding and controlling the adhesion of microbial cells (probiotics, pathogens).

  18. Detecting Bacterial Surface Organelles on Single Cells Using Optical Tweezers.

    PubMed

    Zakrisson, Johan; Singh, Bhupender; Svenmarker, Pontus; Wiklund, Krister; Zhang, Hanqing; Hakobyan, Shoghik; Ramstedt, Madeleine; Andersson, Magnus

    2016-05-10

    Bacterial cells display a diverse array of surface organelles that are important for a range of processes such as intercellular communication, motility and adhesion leading to biofilm formation, infections, and bacterial spread. More specifically, attachment to host cells by Gram-negative bacteria are mediated by adhesion pili, which are nanometers wide and micrometers long fibrous organelles. Since these pili are significantly thinner than the wavelength of visible light, they cannot be detected using standard light microscopy techniques. At present, there is no fast and simple method available to investigate if a single cell expresses pili while keeping the cell alive for further studies. In this study, we present a method to determine the presence of pili on a single bacterium. The protocol involves imaging the bacterium to measure its size, followed by predicting the fluid drag based on its size using an analytical model, and thereafter oscillating the sample while a single bacterium is trapped by an optical tweezer to measure its effective fluid drag. Comparison between the predicted and the measured fluid drag thereby indicate the presence of pili. Herein, we verify the method using polymer coated silica microspheres and Escherichia coli bacteria expressing adhesion pili. Our protocol can in real time and within seconds assist single cell studies by distinguishing between piliated and nonpiliated bacteria.

  19. Microtrap electrode devices for single cell trapping and impedance measurement.

    PubMed

    Mondal, D; Roychaudhuri, C; Das, L; Chatterjee, J

    2012-10-01

    This paper reports the design and fabrication of electrode microtraps for single cell trapping and impedance measurement. In this work, the microtrap electrodes of parallel and elliptical geometry have been fabricated by electroplating of gold electrodes of optimum thickness. This has enabled the formation of electrode traps without requiring any precision alignment between separate insulating traps like PDMS and the bottom gold electrodes. Further the improved uniformity of the electric field between the trapping electrodes as observed from COVENTORWARE simulation significantly reduces the effect of cell position inside the microwell on the electrical measurement unlike previous reports. This makes it possible to directly extract the equivalent cell parameters from the electrical measurement without introducing any correction factor corresponding to cell position. We have performed impedance spectroscopy with both the microwell electrode structures with single HeLa cell at two different positions of trapping. It has been observed that there is almost no change in the extracted values of cell resistance and capacitance for different positions within parallel electrodes and there is only 0.7 % and 0.85 % change in cell resistance and capacitance for the two positions within elliptical electrodes. Thus these microwell electrode structures can be used as an improved and a more convenient platform for single cell electrical characterization. PMID:22767244

  20. Chip Based Single Cell Analysis for Nanotoxicity Assessment

    PubMed Central

    Shah, Pratikkumar; Kaushik, Ajeet; Zhu, Xuena; Zhang, Chengxiao; Li, Chen-Zhong

    2014-01-01

    Nanomaterials, because of their tunable properties and performances, have been utilized extensively in everyday life related consumable products and technology. On exposure, beyond physiological range, nanomaterials cause health risks via affecting the function of organisms, genomic systems, and even central nervous system. Thus, new analytical approaches for nanotoxicity assessment to verify the feasibility of nanomaterials for future use are in demand. The conventional analytical techniques, such as spectrophotometric assay-based techniques usually require a lengthy and time-consuming process and many times produces false positives, and many times cannot be implemented at a single cell level measurement for studying cell behavior without interference of their surrounding environment. Hence, there is demand of a precise, accurate, sensitive assessment for toxicity using single cell. Recently, due to advantages of automation of fluids and minimization of human error, the integration of cell-on-a-chip (CoC) with microfluidic system is in practice for nanotoxicity assessments. This review explains nanotoxicity and assessment approaches with advantages/limitations and new approaches to overcome the confines of traditional techniques. Recent advances in nanotoxicity assessment using CoC integrated with microfluidic system are also discussed in this review, which may be of use for nanotoxicity assessment and diagnostics. PMID:24567949

  1. RF Breakdown in Normal Conducting Single-Cell Structures

    SciTech Connect

    Dolgashev, V.A.; Nantista, C.D.; Tantawi, S.G.; Higashi, Y.; Higo, T.; /KEK, Tsukuba

    2006-02-22

    Operating accelerating gradient in normal conducting accelerating structures is often limited by rf breakdown. The limit depends on multiple parameters, including input rf power, rf circuit, cavity shape and material. Experimental and theoretical study of the effects of these parameters on the breakdown limit in full scale structures is difficult and costly. We use 11.4 GHz single-cell traveling wave and standing wave accelerating structures for experiments and modeling of rf breakdown behavior. These test structures are designed so that the electromagnetic fields in one cell mimic the fields in prototype multicell structures for the X-band linear collider. Fields elsewhere in the test structures are significantly lower than that of the single cell. The setup uses matched mode converters that launch the circular TM{sub 01} mode into short test structures. The test structures are connected to the mode launchers with vacuum rf flanges. This setup allows economic testing of different cell geometries, cell materials and preparation techniques with short turn-around time. Simple 2D geometry of the test structures simplifies modeling of the breakdown currents and their thermal effects.

  2. Single-cell analysis of endothelial morphogenesis in vivo

    PubMed Central

    Yu, Jianxin A.; Castranova, Daniel; Pham, Van N.; Weinstein, Brant M.

    2015-01-01

    Vessel formation has been extensively studied at the tissue level, but the difficulty in imaging the endothelium with cellular resolution has hampered study of the morphogenesis and behavior of endothelial cells (ECs) in vivo. We are using endothelial-specific transgenes and high-resolution imaging to examine single ECs in zebrafish. By generating mosaics with transgenes that simultaneously mark endothelial nuclei and membranes we are able to definitively identify and study the morphology and behavior of individual ECs during vessel sprouting and lumen formation. Using these methods, we show that developing trunk vessels are composed of ECs of varying morphology, and that single-cell analysis can be used to quantitate alterations in morphology and dynamics in ECs that are defective in proper guidance and patterning. Finally, we use single-cell analysis of intersegmental vessels undergoing lumen formation to demonstrate the coexistence of seamless transcellular lumens and single or multicellular enclosed lumens with autocellular or intercellular junctions, suggesting that heterogeneous mechanisms contribute to vascular lumen formation in vivo. The tools that we have developed for single EC analysis should facilitate further rigorous qualitative and quantitative analysis of EC morphology and behavior in vivo. PMID:26253401

  3. Exploring Arabidopsis thaliana Root Endophytes via Single-Cell Genomics

    SciTech Connect

    Lundberg, Derek; Woyke, Tanja; Tringe, Susannah; Dangl, Jeff

    2014-03-19

    Land plants grow in association with microbial communities both on their surfaces and inside the plant (endophytes). The relationships between microbes and their host can vary from pathogenic to mutualistic. Colonization of the endophyte compartment occurs in the presence of a sophisticated plant immune system, implying finely tuned discrimination of pathogens from mutualists and commensals. Despite the importance of the microbiome to the plant, relatively little is known about the specific interactions between plants and microbes, especially in the case of endophytes. The vast majority of microbes have not been grown in the lab, and thus one of the few ways of studying them is by examining their DNA. Although metagenomics is a powerful tool for examining microbial communities, its application to endophyte samples is technically difficult due to the presence of large amounts of host plant DNA in the sample. One method to address these difficulties is single-cell genomics where a single microbial cell is isolated from a sample, lysed, and its genome amplified by multiple displacement amplification (MDA) to produce enough DNA for genome sequencing. This produces a single-cell amplified genome (SAG). We have applied this technology to study the endophytic microbes in Arabidopsis thaliana roots. Extensive 16S gene profiling of the microbial communities in the roots of multiple inbred A. thaliana strains has identified 164 OTUs as being significantly enriched in all the root endophyte samples compared to their presence in bulk soil.

  4. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters.

    PubMed

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  5. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters

    PubMed Central

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  6. The dynamics of p53 in single cells: physiologically based ODE and reaction-diffusion PDE models

    NASA Astrophysics Data System (ADS)

    Eliaš, Ján; Dimitrio, Luna; Clairambault, Jean; Natalini, Roberto

    2014-08-01

    The intracellular signalling network of the p53 protein plays important roles in genome protection and the control of cell cycle phase transitions. Recently observed oscillatory behaviour in single cells under stress conditions has inspired several research groups to simulate and study the dynamics of the protein with the aim of gaining a proper understanding of the physiological meanings of the oscillations. We propose compartmental ODE and PDE models of p53 activation and regulation in single cells following DNA damage and we show that the p53 oscillations can be retrieved by plainly involving p53-Mdm2 and ATM-p53-Wip1 negative feedbacks, which are sufficient for oscillations experimentally, with no further need to introduce any delays into the protein responses and without considering additional positive feedback.

  7. Preparation of Single Cell Suspensions from Mouse Aorta

    PubMed Central

    Hu, Desheng; Yin, Changjun; Mohanta, Sarajo K.; Weber, Christian; Habenicht, Andreas J. R.

    2016-01-01

    Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by lipid deposition, plaque formation, and immune cell infiltration. Innate and adaptive immune cells infiltrate the artery during development of the disease. Moreover, advanced disease leads to formation of artery tertiary lymphoid organs in the adventitia (Grabner et al., 2009; Hu et al., 2015). Various and diverse types of immune cells have been identified in the aorta adventitia vs atherosclerotic plaques (Elewa et al., 2016; Galkina et al., 2006; Lotzer et al., 2010; Mohanta et al., 2016; Mohanta et al., 2014; Moos et al., 2005; Srikakulapu et al., 2016; Zhao et al., 2004). There are conflicting reports on the number and subtypes of immune cells in the aorta depending on the age of the animals, the protocol that is used to obtain single cell suspensions, and the dietary conditions of the mice (Campbell et al., 2012; Clement et al., 2015; Galkina et al., 2006; Kyaw et al., 2012). The number of immune cells in the aorta differs as much as tenfold using different protocols (Butcher et al., 2012; Galkina et al., 2006; Gjurich et al., 2015; Grabner et al., 2009; Hu et al., 2015). These discrepant results call for a protocol that robustly documents bona fide aorta cells rather than those in the surrounding tissues or blood. Critical methodological hurdles include the removal of adjacent adipose tissue and small paraaortic lymph nodes lining the entire aortic tree that are not visible by the naked eye. A dissection microscope is therefore recommended. Moreover protocols of aorta preparations should ascertain that lymphocyte aggregates referred to as fat associated lymphoid clusters (FALCs) (Benezech et al., 2015; Elewa et al., 2015) that are often present at the border between the adipose tissue and the adventitia are removed before enzyme digestion. We propose - besides other approaches (Hu et al., 2015; Mohanta et al., 2014) - a combination of immunohistochemical staining and

  8. Nitrogen assimilation by single cells in hot springs

    NASA Astrophysics Data System (ADS)

    Poret-peterson, A. T.; Romaniello, S. J.; Bose, M.; Williams, P.; Elser, J. J.; Shock, E.; Anbar, A. D.; Hartnett, H. E.

    2012-12-01

    Microorganisms drive biogeochemical cycles and require nutrients, such as ammonium and nitrate, to function. As a result, following nutrient flows provides opportunities to study how microbial activity influences ecosystem-level processes. Most past measurements of microbial nutrient uptake rely on bulk measurements, which are informative but provide little information about heterogeneity among community members involved in elemental transformations, nor about possible effects of physiological state or taxonomic identity. Since microbial communities tend to be phylogenetically and physiologically diverse, it is reasonable to expect that community members will respond differently to nutrient addition. Here, we examine nitrogen assimilation (via addition of 15N-labeled ammonium or nitrate) in Yellowstone hot spring microbial communities. Using the NanoSIMS, we imaged cells at a very high spatial resolution (nanometer scale) necessary to determine 15N enrichments in single micron-sized cells. We compare the N isotopic enrichments observed in single cells to that determined in bulk sediments by standard isotope ratio mass spectrometry. NanoSIMS imaging of 56 individual cells from sediments of an acidic hot spring (pH 4.7, T=67oC) incubated with 15N-ammonium shows that about two-thirds of the cells (38) exhibited 15N-enrichment. Most cells had 15N enrichments from 0.39 to 0.91 atom %, while some cells were much more significantly enriched. Bulk analyses of sediments show that ammonium assimilation and nitrate assimilation readily occurred at this spring. These findings show that microbes in this hot spring may differentially take up ammonium, which may arise from a number of factors including differences in cellular N requirements, growth rates, and the ability to transport ammonium. This work represents some of the first single-cell isotopic measurements from an extreme environment. Efforts are underway to image sediment samples from other hot springs and to pair Nano

  9. Simultaneous photoacoustic and optical attenuation imaging of single cells using photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Moore, Michael J.; Strohm, Eric M.; Kolios, Michael C.

    2016-03-01

    A new technique for simultaneously acquiring photoacoustic images as well as images based on the optical attenuation of single cells in a human blood smear was developed. An ultra-high frequency photoacoustic microscope equipped with a 1 GHz transducer and a pulsed 532 nm laser was used to generate the images. The transducer and 20X optical objective used for laser focusing were aligned coaxially on opposing sides of the sample. Absorption of laser photons by the sample yielded conventional photoacoustic (PA) signals, while incident photons which were not attenuated by the sample were absorbed by the transducer, resulting in the formation of a photoacoustic signal (tPA) within the transducer itself. Both PA and tPA signals, which are separated in time, were recorded by the system in a single RF-line. Areas of strong signal in the PA images corresponded to dark regions in the tPA images. Additional details, including the clear delineation of the cell cytoplasm and features in red blood cells, were visible in the tPA image but not the corresponding PA image. This imaging method has applications in probing the optical absorption and attenuation characteristics of biological cells with sub-cellular resolution.

  10. A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

    PubMed Central

    Raghu, Deepa; Christodoulides, Joseph A.; Delehanty, James B.; Byers, Jeff M.; Raphael, Marc P.

    2015-01-01

    Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required. In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging. A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells. PMID:26650542

  11. Ablation and analysis of small cell populations and single cells by consecutive laser pulses

    NASA Astrophysics Data System (ADS)

    Shrestha, Bindesh; Nemes, Peter; Vertes, Akos

    2010-10-01

    Laser ablation of single cells through a sharpened optical fiber is used for the detection of metabolites by laser ablation electrospray ionization (LAESI) mass spectrometry (MS). Ablation of the same Allium cepa epidermal cell by consecutive pulses indicates the rupture of the cell wall by the second shot. Intracellular sucrose heterogeneity is detected by subsequent laser pulses pointing to rupturing the vacuolar membrane by the third exposure. Ion production by bursts of laser pulses shows that the drying of ruptured A. cepa cells occurs in ˜50 s at low pulse rates (10 pulses/s bursts) and significantly faster at high pulse rates (100 pulses/s bursts). These results point to the competing role of cytoplasm ejection and evaporative drying in diminishing the LAESI-MS signal in ˜50 s or 100 laser pulses, whichever occurs first.

  12. JAK/STAT/SOCS-signaling pathway and colon and rectal cancer

    PubMed Central

    Slattery, Martha L.; Lundgreen, Abbie; Kadlubar, Susan A.; Bondurant, Kristina L.; Wolff, Roger K.

    2012-01-01

    The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway is involved in immune function and cell growth. We evaluated the association between genetic variation in JAK1 (10 SNPs), JAK2 (9 SNPs), TYK2 (5 SNPs), SOCS1 (2 SNPs), SOCS2 (2 SNPs), STAT1 (16 SNPs), STAT2 (2 SNPs), STAT3 (6 SNPs), STAT4 (21 SNPs), STAT5A (2 SNPs), STAT5B (3 SNPs), STAT6 (4 SNPs) with risk of colorectal cancer. We used data from population-based case-control studies (colon cancer n=1555 cases, 1956 controls; rectal cancer n=754 cases, 959 controls). JAK2, SOCS2, STAT1, STAT3, STAT5A, STAT5B, and STAT6 were associated with colon cancer; STAT3, STAT4, STAT6, and TYK2 were associated with rectal cancer. Given the biological role of the JAK/STAT-signaling pathway and cytokines, we evaluated interaction with IFNG, TNF, and IL6; numerous statistically significant associations after adjustment for multiple comparisons were observed. The following statistically significant interactions were observed: TYK2 with aspirin/NSAID use; STAT1, STAT4, and TYK2 with estrogen status; and JAK2, STAT2, STAT4, STAT5A, STAT5B, and STAT6 with smoking status and colon cancer risk; JAK2, STAT6, and TYK2 with aspirin/NSAID use; JAK1 with estrogen status; STAT2 with cigarette smoking and rectal cancer. JAK2, SOCS1, STAT3, STAT5, and TYK2 were associated with colon cancer survival (HRR of 3.3 95% CI 2.01, 5.42 for high mutational load). JAK2, SOCS1, STAT1, STAT4, and TYK2 were associated with rectal cancer survival (HRR 2.80 95 %CI 1.63, 4.80). These data support the importance of the JAK/STAT-signaling pathway in colorectal cancer and suggest targets for intervention. PMID:22121102

  13. Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

    PubMed Central

    Gerdes, Michael J.; Sevinsky, Christopher J.; Sood, Anup; Adak, Sudeshna; Bello, Musodiq O.; Bordwell, Alexander; Can, Ali; Corwin, Alex; Dinn, Sean; Filkins, Robert J.; Hollman, Denise; Kamath, Vidya; Kaanumalle, Sireesha; Kenny, Kevin; Larsen, Melinda; Lazare, Michael; Lowes, Christina; McCulloch, Colin C.; McDonough, Elizabeth; Pang, Zhengyu; Rittscher, Jens; Santamaria-Pang, Alberto; Sarachan, Brion D.; Seel, Maximilian L.; Seppo, Antti; Shaikh, Kashan; Sui, Yunxia; Zhang, Jingyu; Ginty, Fiona

    2013-01-01

    Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics. PMID:23818604

  14. Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis

    NASA Astrophysics Data System (ADS)

    Weng, Shinuo; Shao, Yue; Chen, Weiqiang; Fu, Jianping

    2016-09-01

    Mechanical homeostasis--a fundamental process by which cells maintain stable states under environmental perturbations--is regulated by two subcellular mechanotransducers: cytoskeleton tension and integrin-mediated focal adhesions (FAs). Here, we show that single-cell mechanical homeostasis is collectively driven by the distinct, graduated dynamics (rheostasis) of subcellular cytoskeleton tension and FAs. Such rheostasis involves a mechanosensitive pattern wherein ground states of cytoskeleton tension and FA determine their distinct reactive paths through either relaxation or reinforcement. Pharmacological perturbations of the cytoskeleton and molecularly modulated integrin catch-slip bonds biased the rheostasis and induced non-homeostasis of FAs, but not of cytoskeleton tension, suggesting a unique sensitivity of FAs in regulating homeostasis. Theoretical modelling revealed myosin-mediated cytoskeleton contractility and catch-slip-bond-like behaviours in FAs and the cytoskeleton as sufficient and necessary mechanisms for quantitatively recapitulating mechanosensitive rheostasis. Our findings highlight the previously underappreciated physical nature of the mechanical homeostasis of cells.

  15. Laser-induced microbubble poration of localized single cells.

    PubMed

    Fan, Qihui; Hu, Wenqi; Ohta, Aaron T

    2014-05-01

    Laser-induced microbubbles were used to porate the cell membranes of localized single NIH/3T3 fibroblasts. Microsecond laser pulses were focused on an optically absorbent substrate, creating a vapour microbubble that oscillated in size at the laser focal point in a fluidic chamber. The shear stress accompanying the bubble size oscillation was able to porate nearby cells. Cell poration was demonstrated with the delivery of FITC-dextran dye with various molecular weights. Under optimal poration conditions, the cell poration efficiency was up to 95.2 ± 4.8%, while maintaining 97.6 ± 2.4% cell viability. The poration system is able to target a single cell without disturbing surrounding cells. PMID:24632785

  16. Rotational manipulation of single cells and organisms using acoustic waves.

    PubMed

    Ahmed, Daniel; Ozcelik, Adem; Bojanala, Nagagireesh; Nama, Nitesh; Upadhyay, Awani; Chen, Yuchao; Hanna-Rose, Wendy; Huang, Tony Jun

    2016-01-01

    The precise rotational manipulation of single cells or organisms is invaluable to many applications in biology, chemistry, physics and medicine. In this article, we describe an acoustic-based, on-chip manipulation method that can rotate single microparticles, cells and organisms. To achieve this, we trapped microbubbles within predefined sidewall microcavities inside a microchannel. In an acoustic field, trapped microbubbles were driven into oscillatory motion generating steady microvortices which were utilized to precisely rotate colloids, cells and entire organisms (that is, C. elegans). We have tested the capabilities of our method by analysing reproductive system pathologies and nervous system morphology in C. elegans. Using our device, we revealed the underlying abnormal cell fusion causing defective vulval morphology in mutant worms. Our acoustofluidic rotational manipulation (ARM) technique is an easy-to-use, compact, and biocompatible method, permitting rotation regardless of optical, magnetic or electrical properties of the sample under investigation. PMID:27004764

  17. Sample Targeting During Single-Particle Single-Cell Irradiation

    NASA Astrophysics Data System (ADS)

    Bigelow, A. W.; Randers-Pehrson, G.; Michel, K. A.; Brenner, D. J.; Dymnikov, A. D.

    2003-08-01

    An apertured microbeam is used for single-particle single-cell irradiation to study radiobiological effects at the Radiological Research Accelerator Facility (RARAF), Center for Radiological Research, Columbia University. The present sample targeting system involves imaging techniques and a stepping motor stage to sequentially position a cell nucleus above a vertical ion beam. An interest expressed by the biology research community in targeting subnuclear components has spurred the development of microbeam II, a next-generation facility to include a focused ion beam and a more precise sample manipulator, a voice coil stage. Sample positioning precision will rely on a feedback circuit incorporating linear variable differential transformer (LVDT) position measurements. In addition, post-lens electrostatic deflection is a contender for a point-and-shoot system that could speed up the cell irradiation process for cells within an image frame. Crucial to this development is that ion beam blow up must be minimal during deflection.

  18. Sample Targeting During Single-Particle Single-Cell Irradiation

    SciTech Connect

    Bigelow, A.W.; Randers-Pehrson, G.; Michel, K.A.; Brenner, D.J.; Dymnikov, A.D.

    2003-08-26

    An apertured microbeam is used for single-particle single-cell irradiation to study radiobiological effects at the Radiological Research Accelerator Facility (RARAF), Center for Radiological Research, Columbia University. The present sample targeting system involves imaging techniques and a stepping motor stage to sequentially position a cell nucleus above a vertical ion beam. An interest expressed by the biology research community in targeting subnuclear components has spurred the development of microbeam II, a next-generation facility to include a focused ion beam and a more precise sample manipulator, a voice coil stage. Sample positioning precision will rely on a feedback circuit incorporating linear variable differential transformer (LVDT) position measurements. In addition, post-lens electrostatic deflection is a contender for a point-and-shoot system that could speed up the cell irradiation process for cells within an image frame. Crucial to this development is that ion beam blow up must be minimal during deflection.

  19. Rotational manipulation of single cells and organisms using acoustic waves.

    PubMed

    Ahmed, Daniel; Ozcelik, Adem; Bojanala, Nagagireesh; Nama, Nitesh; Upadhyay, Awani; Chen, Yuchao; Hanna-Rose, Wendy; Huang, Tony Jun

    2016-01-01

    The precise rotational manipulation of single cells or organisms is invaluable to many applications in biology, chemistry, physics and medicine. In this article, we describe an acoustic-based, on-chip manipulation method that can rotate single microparticles, cells and organisms. To achieve this, we trapped microbubbles within predefined sidewall microcavities inside a microchannel. In an acoustic field, trapped microbubbles were driven into oscillatory motion generating steady microvortices which were utilized to precisely rotate colloids, cells and entire organisms (that is, C. elegans). We have tested the capabilities of our method by analysing reproductive system pathologies and nervous system morphology in C. elegans. Using our device, we revealed the underlying abnormal cell fusion causing defective vulval morphology in mutant worms. Our acoustofluidic rotational manipulation (ARM) technique is an easy-to-use, compact, and biocompatible method, permitting rotation regardless of optical, magnetic or electrical properties of the sample under investigation.

  20. Genome wide copy number analysis of single cells

    PubMed Central

    Baslan, Timour; Kendall, Jude; Rodgers, Linda; Cox, Hilary; Riggs, Mike; Stepansky, Asya; Troge, Jennifer; Ravi, Kandasamy; Esposito, Diane; Lakshmi, B.; Wigler, Michael; Navin, Nicholas; Hicks, James

    2016-01-01

    Summary Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells, where information regarding genetic heterogeneity is lost. Here, we present a protocol that allows for the genome wide copy number analysis of single nuclei isolated from mixed populations of cells. Single nucleus sequencing (SNS), combines flow sorting of single nuclei based on DNA content, whole genome amplification (WGA), followed by next generation sequencing to quantize genomic intervals in a genome wide manner. Multiplexing of single cells is discussed. Additionally, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes ~3 days from flow cytometry to sequence-ready DNA libraries. PMID:22555242

  1. Emergent Collective Chemotaxis without Single-Cell Gradient Sensing

    NASA Astrophysics Data System (ADS)

    Camley, Brian A.; Zimmermann, Juliane; Levine, Herbert; Rappel, Wouter-Jan

    2016-03-01

    Many eukaryotic cells chemotax, sensing and following chemical gradients. However, experiments show that even under conditions when single cells cannot chemotax, small clusters may still follow a gradient. This behavior is observed in neural crest cells, in lymphocytes, and during border cell migration in Drosophila, but its origin remains puzzling. Here, we propose a new mechanism underlying this "collective guidance," and study a model based on this mechanism both analytically and computationally. Our approach posits that contact inhibition of locomotion, where cells polarize away from cell-cell contact, is regulated by the chemoattractant. Individual cells must measure the mean attractant value, but need not measure its gradient, to give rise to directional motility for a cell cluster. We present analytic formulas for how the cluster velocity and chemotactic index depend on the number and organization of cells in the cluster. The presence of strong orientation effects provides a simple test for our theory of collective guidance.

  2. Single Cell Magnetic Measurements with a Superconducting Quantum Interference Device

    NASA Astrophysics Data System (ADS)

    Palmstrom, Johanna C.; Arps, Jennifer; Dwyer, Bo; Kalisky, Beena; Kirtley, John R.; Moler, Kathryn A.; Qian, Lisa C.; Rosenberg, Aaron J.; Rutt, Brian; Tee, Sui Seng; Theis, Eric; Urbach, Elana; Wang, Yihua

    2014-03-01

    Magnetic nanoparticles play an important role in numerous biomedical applications such as magnetic resonance imaging and targeted drug delivery. There is a need for tools to characterize individual magnetic nanoparticles and the magnetic properties of individual cells. We use a scanning supercond