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Transplantation of pancreatic islets has been shown to be effective, in some patients, for the long-term treatment of type 1 diabetes. However, transplantation of islets into either the portal vein or the subcutaneous space can be limited by insufficient oxygen transfer, leading to islet loss. Furthermore, oxygen diffusion limitations can be magnified when islet numbers are increased dramatically, as in translating from rodent studies to human-scale treatments. To address these limitations, an islet transplantation approach using an acellular vascular graft as a vascular scaffold has been developed, termed the B io V ascular P ancreas (BVP). To create the BVP, islets are seeded as an outer coating on the surface of an acellular vascular graft, using fibrin as a hydrogel carrier. The BVP can then be anastomosed as an arterial (or arteriovenous) graft, which allows fully oxygenated arterial blood with a pO2 of roughly 100 mmHg to flow through the graft lumen and thereby supply oxygen to the islets. In silico simulations and in vitro bioreactor experiments show that the BVP design provides adequate survivability for islets and helps avoid islet hypoxia. When implanted as end-to-end abdominal aorta grafts in nude rats, BVPs were able to restore near-normoglycemia durably for 90 days and developed robust microvascular infiltration from the host. Furthermore, pilot implantations in pigs were performed, which demonstrated the scalability of the technology. Given the potential benefits provided by the BVP, this tissue design may eventually serve as a solution for transplantation of pancreatic islets to treat or cure type 1 diabetes. [ABSTRACT FROM AUTHOR]

Transplantation of pancreatic islets has been shown to be effective, in some patients, for the long-term treatment of type 1 diabetes. However, transplantation of islets into either the portal vein or the subcutaneous space can be limited by insufficient oxygen transfer, leading to islet loss. Furthermore, oxygen diffusion limitations can be magnified when islet numbers are increased dramatically, as in translating from rodent studies to human-scale treatments. To address these limitations, an islet transplantation approach using an acellular vascular graft as a vascular scaffold has been developed, termed the B io V ascular P ancreas (BVP). To create the BVP, islets are seeded as an outer coating on the surface of an acellular vascular graft, using fibrin as a hydrogel carrier. The BVP can then be anastomosed as an arterial (or arteriovenous) graft, which allows fully oxygenated arterial blood with a pO2 of roughly 100 mmHg to flow through the graft lumen and thereby supply oxygen to the islets. In silico simulations and in vitro bioreactor experiments show that the BVP design provides adequate survivability for islets and helps avoid islet hypoxia. When implanted as end-to-end abdominal aorta grafts in nude rats, BVPs were able to restore near-normoglycemia durably for 90 days and developed robust microvascular infiltration from the host. Furthermore, pilot implantations in pigs were performed, which demonstrated the scalability of the technology. Given the potential benefits provided by the BVP, this tissue design may eventually serve as a solution for transplantation of pancreatic islets to treat or cure type 1 diabetes. [ABSTRACT FROM AUTHOR]

Network activity is strongly tied to animal movement; however, hippocampal circuits selectively engaged during locomotion or immobility remain poorly characterized. Here we examined whether distinct locomotor states are encoded differentially in genetically defined classes of hippocampal interneurons. To characterize the relationship between interneuron activity and movement, we used in vivo, two-photon calcium imaging in CA1 of male and female mice, as animals performed a virtual-reality (VR) track running task. We found that activity in most somatostatin-expressing and parvalbumin-expressing interneurons positively correlated with locomotion. Surprisingly, nearly one in five somatostatin or one in seven parvalbumin interneurons were inhibited during locomotion and activated during periods of immobility. Anatomically, the somata of somatostatin immobility-activated neurons were smaller than those of movementactivated neurons. Furthermore, immobility-activated interneurons were distributed across cell layers, with somatostatin-expressing cells predominantly in stratum oriens and parvalbumin-expressing cells mostly in stratum pyramidale. Importantly, each cell's correlation between activity and movement was stable both over time and across VR environments. Our findings suggest that hippocampal interneuronal microcircuits are preferentially active during either movement or immobility periods. These inhibitory networks may regulate information flow in "labeled lines" within the hippocampus to process information during distinct behavioral states. [ABSTRACT FROM AUTHOR]

A psychophysical paradigm for investigating unconscious perception was used to test the hypothesis of dissociation between detection and identification of phobic stimuli. Spider-phobic and non-phobic participants were presented with masked images of spiders and flowers and an equal number of control stimuli in a random sequence. After each masked stimulus was flashed, participants first reported whether or not an object was presented. Then they identified each stimulus as either a spider or a flower, regardless of their prior detection response. Phobic participants identified both detected and undetected spiders better than chance, as assessed by two measures of response bias. They did not exhibit dissociation between detection and identification for flowers. Non-phobic participants did not exhibit detection–identification dissociation for either spiders or flowers. These results are consistent with the interpretation that phobic individuals unconsciously perceive their feared stimulus, and constitute the first direct demonstration of such for emotional stimuli. [ABSTRACT FROM AUTHOR]

Synapses onto distal dendritic tufts are believed to function by modulating time-locked proximal inputs; however, the role of these synapses when proximal inputs are asynchronous or silent is unknown. Surprisingly, we found that activation of apical tuft synapses alone resulted in heterosynaptic potentiation of proximal synapses. In mouse adult hippocampal CA 1 pyramidal neurons, we show that activation of distal inputs from the entorhinal cortex (EC) specifically strengthens proximal synapses projecting from CA3. This slow AMPA receptor-mediated potentiation is accompanied by increased synaptic GluN2B-containing NMDA receptors, which are normally restricted to juvenile animals. These two synaptic modifications interact to generate striking bidirectional metaplastic changes. Heterosynaptically potentiated synapses become resistant to subsequent long-term potentiation (LTP) as the two forms of AMPA receptor-mediated potentiation occlude. However, this is only true when the LTP induction protocol is relatively weak. When it is strong and repeated, the magnitude of LTP after heterosynaptic plasticity is greatly increased, specifically through the activation of GluN2B-containing NMDA receptors. Thus, CA1 neurons expressing heterosynaptic potentiation induced by external sensory input from the EC become more strongly driven by internally generated environmental representations from CA3. Furthermore, subsequent SC LTP in this ensemble is shifted to potentiate only strongly activated CA3 inputs, while endowing these synapses with enhanced potentiation. These results show that one set of inputs can exert long-lasting heterosynaptic control over another, allowing the coupling of two functionally and spatially distinct pathways, thereby greatly expanding the repertoire of cellular and network plasticity. [ABSTRACT FROM AUTHOR]

In response to decreased activity, neurons make global compensatory increases in excitatory synaptic strength. However, how neuronal maturity affects this process is unclear. We silenced cultured hippocampal neurons with TTX at 7 days in vitro, during rapid synaptogenesis, and at 14 days, when major synaptogenesis is complete. For each age, we have explored the effects of short (1 day) and longer (2 days) periods of silencing. We have confirmed that the changes in synaptic strength depend on 2 main mechanisms, one presynaptic and the other postsynaptic. The presynaptic mechanism involves an increase in the probability of neurotransmitter release, mostly arising through an increase in the number of synaptic vesicles available for release. The postsynaptic mechanism operates through an increase in the number of postsynaptic receptors for the excitatory neurotransmitter glutamate. When neurons are silenced for 1 day, young neurons employ the postsynaptic mechanism, whereas more mature neurons increase their strength through the presynaptic mechanism. The postsynaptic strengthening in young neurons does not depend on gene transcription, whereas the presynaptic mechanism does. If neurons are silenced for 2 days, younger and older neurons employ both pre and postsynaptic mechanisms for synaptic strengthening. We also found evidence for 2 additional mechanisms that increased the effective synaptic coupling between neurons after 2 days of silencing: an increase in the number of synapses, and an increase in the electrotonic length of dendrites. These results expand our basic understanding of neuronal homeostasis, and reveal the developmental regulation of its expression mechanisms. [ABSTRACT FROM AUTHOR]

Examines the cell cycle control of anchorage-independent growth in nontransformed fibroblasts. Regulation of the anchorage-independent growth; Regulation by adhesion; Requirement for the proliferation of cells.

Despite considerable advances in prostate cancer research, there is a major need for a systemic delivery platform that efficiently targets anti-cancer drugs to sites of disseminated prostate cancer while minimizing host toxicity. In this proof-of-principle study, human mesenchymal stem cells (MSCs) were loaded with poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) that encapsulate the macromolecule G114, a thapsigargin-based prostate specific antigen (PSA)-activated prodrug. G114-particles (∼950 nm in size) were internalized by MSCs, followed by the release of G114 as an intact prodrug from loaded cells. Moreover, G114 released from G114 MP-loaded MSCs selectively induced death of the PSA-secreting PCa cell line, LNCaP. Finally, G114 MP-loaded MSCs inhibited tumor growth when used in proof-of-concept co-inoculation studies with CWR22 PCa xenografts, suggesting that cell-based delivery of G114 did not compromise the potency of this pro-drug in-vitro or in-vivo . This study demonstrates a potentially promising approach to assemble a cell-based drug delivery platform, which inhibits cancer growth in-vivo without the need of genetic engineering. We envision that upon achieving efficient homing of systemically infused MSCs to cancer sites, this MSC-based platform may be developed into an effective, systemic ‘Trojan Horse’ therapy for targeted delivery of therapeutic agents to sites of metastatic PCa. [ABSTRACT FROM AUTHOR]

Abstract: This experiment compared the effects of exposure to masked phobic stimuli at a very brief stimulus-onset asynchrony on spider-phobic and non-phobic individuals. Participants were identified through a widely used questionnaire and a Behavioral Avoidance Test (BAT) with a live, caged tarantula to establish baseline levels of avoidance. One week later, they were individually administered one of two continuous series of masked images: spiders or flowers. Preliminary masking experiments showed that independent samples of participants from the same populations failed to recognize these stimuli. Participants in the main experiment reported ratings of subjective distress immediately before and after the exposure manipulation. Then they engaged in the BAT once again. Very brief exposure to images of spiders reduced phobic participants’ avoidance of the tarantula. No effects were evidenced on subjective distress, or on non-phobic participants. Theoretical implications for the non-conscious basis of fear are discussed. [Copyright &y& Elsevier]

Mutations in doublecortin (DCX) are associated with intractable epilepsy in humans, due to a severe disorganization of the neocortex and hippocampus known as classical lissencephaly. However, the basis of the epilepsy in lissencephaly remains unclear. To address potential functional redundancy with murin Dcx, we targeted one of the closest homologues, doublecortin-like kinase 2 (DcIk2). Here, we report that DcxDclk2-null mice display frequent spontaneous seizures that originate in the hippocampus, with most animals dying in the first few months of life. Elevated hippocampal expression of c-fos and loss of somatostatin-positive interneurons were identified, both known to correlate with epilepsy. Dcx and Dclk2 are coexpresseci in developing hippocampus, and, in their absence, there is dosagedependent disrupted hippocampal lamination associated with a cellautonomous simplification of pyramidal dendritic arborizations leading to reduced inhibitory synaptic tone. These data suggest that hippocampal dysmaturation and insufficient receptive field for inhibitory input may underlie the epilepsy in lissencephaly, and suggest potential therapeutic strategies for controlling epilepsy in these patients. [ABSTRACT FROM AUTHOR]

Akt is a serine/threonine kinase that transduces survival signals from survival/growth factors. Deregulation and signal imbalance in cancer cells make them prone to apoptosis. Upregulation or activation of Akt to aid the survival of cancer cells is a common theme in human malignancies. We have developed small-molecule Akt inhibitors that are potent and specific. These Akt inhibitors can inhibit Akt activity and block phosphorylation by Akt on multiple downstream targets in cells. Synergy in apoptosis induction was observed when Akt inhibitors were combined with doxorubicin or camptothecin. Akt inhibitor-induced enhancement of topoisomerase inhibitor cytotoxicity was also evident in long-term cell survival assay. Synergy with paclitaxel in apoptosis induction was evident in cells pretreated with paclitaxel, and enhancement of tumor delay by paclitaxel was demonstrated through cotreatment with Akt inhibitor Compound A (A-443654). Combination with other classes of chemotherapeutic agents did not yield any enhancement of cytotoxicity. These findings provide important guidance in selecting appropriate classes of chemotherapeutic agents for combination with Akt inhibitors in cancer treatment. [ABSTRACT FROM AUTHOR]

The serine/threonine kinases, Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ, play a critical role in preventing cancer cells from undergoing apoptosis. However, the function of individual Akt isoforms in the tumorigenicity of cancer cells is still not well defined. In the current study, we used an Akt1 antisense oligonucleotide (AS) to specifically downregulate Akt1 protein in both cancer and normal cells. Our data indicate that Akt1 AS treatment inhibits the ability of MiaPaCa-2, H460, HCT-15, and HT1080 cells to grow in soft agar. The treatment also induces apoptosis in these cancer cells as demonstrated by FACS analysis and a caspase activity assay. Conversely, Akt1 AS treatment has little effect on the cell growth and survival of normal human cells including normal human fibroblast (NHF), fibroblast from muscle (FBM), and mammary gland epithelial 184B5 cells. In addition, Akt1 AS specifically sensitizes cancer cells to typical chemotherapeutic agents. Thus, Akt1 is indispensable for maintaining the tumorigenicity of cancer cells. Inhibition of Akt1 may provide a powerful sensitization agent for chemotherapy specifically in cancer cells. Neoplasia (2001) 3, 278–286. [ABSTRACT FROM AUTHOR]

A transplanted stem cell's engagement with a pathologic niche is the first step in its restoring homeostasis to that site. Inflammatory chemokines are constitutively produced in such a niche; their binding to receptors on the stem cell helps direct that cell's "pathotropism." Neural stem cells (NSCs), which express CXCR4, migrate to sites of CNS injury or degeneration in part because astrocytes and vasculature produce the inflammatory chemokine CXCL12. Binding of CXCL12 to CXCR4 (a G protein-coupled receptor, GPCR) triggers repair processes within the NSC. Although a tool directing NSCs to where needed has been long-sought, one would not inject this chemokine in vivo because undesirable inflammation also follows CXCL12-CXCR4 coupling. Alternatively, we chemically "mutated" CXCL12, creating a CXCR4 agonist that contained a strong pure binding motif linked to a signaling motif devoid of sequences responsible for synthetic functions. This synthetic dual-moity CXCR4 agonist not only elicited more extensive and persistent human NSC migration and distribution than did native CXCL 12, but induced no host inflammation (or other adverse effects); rather, there was predominantly reparative gene expression. When coadministered with transplanted human induced pluripotent stem cell-derived hNSCs in a mouse model of a prototypical neurodegenerative disease, the agonist enhanced migration, dissemination, and integration of donor-derived cells into the diseased cerebral cortex (including as electrophysiologically-active cortical neurons) where their secreted cross-corrective enzyme mediated a therapeutic impact unachieved by cells alone. Such a "designer" cytokine receptor-agonist peptide illustrates that treatments can be controlled and optimized by exploiting fundamental stem cell properties (e.g., "inflammo-attraction"). [ABSTRACT FROM AUTHOR]

Dysregulated translation of mRNA plays a major role in tumorigenesis. Mitogen-activated protein kinase interacting kinases (MNK)1/2 are key regulators of mRNA translation integrating signals from oncogenic and immune signaling pathways through phosphorylation of eIF4E and other mRNA binding proteins. Modulation of these key effector proteins regulates mRNA, which controls tumor/stromal cell signaling. Compound 23 (eFT508), an exquisitely selective, potent dual MNK1/2 inhibitor, was designed to assess the potential for control of oncogene signaling at the level of mRNA translation. The crystal structure-guided design leverages stereoelectronic interactions unique to MNK culminating in a novel pyridone–aminal structure described for the first time in the kinase literature. Compound 23 has potent in vivo antitumor activity in models of diffuse large cell B-cell lymphoma and solid tumors, suggesting that controlling dysregulated translation has real therapeutic potential. Compound 23 is currently being evaluated in Phase 2 clinical trials in solid tumors and lymphoma. Compound 23 is the first highly selective dual MNK inhibitor targeting dysregulated translation being assessed clinically. [ABSTRACT FROM AUTHOR]

Abstract: We have investigated the SAR of a series of pyrimidinone-containing Cdc7 kinase inhibitors. A wide range of amine substitutions give potent compounds with activities (K i) less than 1nM. Kinase selectivity is reasonable and cytotoxicity corresponds to inhibition of MCM2 phosphorylation. [Copyright &y& Elsevier]

Abstract: A series of heteroaryl-pyridine containing inhibitors of Akt are reported. The synthesis and structure–activity relationships are discussed, leading to the discovery of a indazole-pyridine analogue (K i =0.16nM). These compounds bind in the ATP binding site, are potent, ATP competitive, and reversible inhibitors of Akt activity. No selectivity amongst the Akt isoforms is observed for this analogue, but there is good selectivity against an panel of other kinases. It is least selective for other members of the AGC family of kinases but is nonetheless 40-fold selective for Akt over PKA. The compound shows cellular activity and significantly slows tumor growth in vivo. [Copyright &y& Elsevier]

Checkpoint kinase 1 (Chk1) is a checkpoint gene that is activated after DNA damage. It phosphorylates and inactivates the Cdc2 activating phosphatase Cdc25C. This in turn inactivates Cdc2, which leads to G2/M arrest. We report that blocking Chk1 expression by antisense or ribozymes in mammalian cells induces apoptosis and interferes with the G2/M arrest induced by adriamycin. The Chk1 inhibitor UCN-01 also blocks the G2 arrest after DNA damage and renders cells more susceptible to adriamycin. These results indicate that Chk1 is an essential gene for the checkpoint mechanism during normal cell proliferation as well as in the DNA damage response. Neoplasia (2001) 3, 411–419. [ABSTRACT FROM AUTHOR]

To clarify the roles of specific isoforms of PKC in regulating growth and cell cycle progression of the HC11 mammary epithelial cell line, we investigated the effects of activating endogenous PKC isoforms with the phorbol ester tumor promoter TPA, and also the effects of TPA on genetically engineered cells containing increased levels of individual PKC isoforms. We found that TPA treatment of HC11 cells induced a transient cell cycle arrest in G0/G1. Western blot analyses of the TPA treated cells provided evidence that the endogenous PKCα present in these cells mediated these effects. Indeed, derivatives of the HC11 cell line that inducibly overexpress an exogenous PKCα or ectopic PKCβ1 exhibited more marked growth inhibition by TPA than control cells. Immunohistochemical staining of cells following treatment with TPA revealed selective translocation of PKCα into the nucleus, whereas PKCβ1 remained in the cytoplasm. The transient arrest of HC11 cells following treatment with TPA was associated with marked induction of both p21cip1 mRNA and protein. This induction was exaggerated in the derivatives that overexpressed either PKCα or PKCβ1. Therefore, in mouse mammary epithelial cells activation of the endogenous PKCα can transiently arrest cells in G0/G1 which may be due, at least in part, to induction of the transcription of p21cip1. [ABSTRACT FROM AUTHOR]

The perivascular niche (PVN) plays an essential role in brain tumor stem‐like cell (BTSC) fate control, tumor invasion, and therapeutic resistance. Here, a microvasculature‐on‐a‐chip system as a PVN model is used to evaluate the ex vivo dynamics of BTSCs from ten glioblastoma patients. BTSCs are found to preferentially localize in the perivascular zone, where they exhibit either the lowest motility, as in quiescent cells, or the highest motility, as in the invasive phenotype, with migration over long distance. These results indicate that PVN is a niche for BTSCs, while the microvascular tracks may serve as a path for tumor cell migration. The degree of colocalization between tumor cells and microvessels varies significantly across patients. To validate these results, single‐cell transcriptome sequencing (10 patients and 21 750 single cells in total) is performed to identify tumor cell subtypes. The colocalization coefficient is found to positively correlate with proneural (stem‐like) or mesenchymal (invasive) but not classical (proliferative) tumor cells. Furthermore, a gene signature profile including PDGFRA correlates strongly with the "homing" of tumor cells to the PVN. These findings demonstrate that the model can recapitulate in vivo tumor cell dynamics and heterogeneity, representing a new route to study patient‐specific tumor cell functions. [ABSTRACT FROM AUTHOR]