Activity Of Target Cells Research Articles

Activity-Dependent Global Downscaling of Evoked Neurotransmitter Release across Glutamatergic Inputs in Drosophila.

Within mammalian brain circuits, activity-dependent synaptic adaptations, such as synaptic scaling, stabilize neuronal activity in the face of perturbations. Stability afforded through synaptic scaling involves uniform scaling of quantal amplitudes across all synaptic inputs formed on neurons, as well as on the postsynaptic side. It remains unclear whether activity-dependent uniform scaling also operates within peripheral circuits. We tested for such scaling in a Drosophila larval neuromuscular circuit, where the muscle receives synaptic inputs from different motoneurons. We used motoneuron-specific genetic manipulations to increase the activity of only one motoneuron and recordings of postsynaptic currents from inputs formed by the different motoneurons. We discovered an adaptation which caused uniform downscaling of evoked neurotransmitter release across all inputs through decreases in release probabilities. This "presynaptic downscaling" maintained the relative differences in neurotransmitter release across all inputs around a homeostatic set point, caused a compensatory decrease in synaptic drive to the muscle affording robust and stable muscle activity, and was induced within hours. Presynaptic downscaling was associated with an activity-dependent increase in Drosophila vesicular glutamate transporter expression. Activity-dependent uniform scaling can therefore manifest also on the presynaptic side to produce robust and stable circuit outputs. Within brain circuits, uniform downscaling on the postsynaptic side is implicated in sleep- and memory-related processes. Our results suggest that evaluation of such processes might be broadened to include uniform downscaling on the presynaptic side.SIGNIFICANCE STATEMENT To date, compensatory adaptations which stabilise target cell activity through activity-dependent global scaling have been observed only within central circuits, and on the postsynaptic side. Considering that maintenance of stable activity is imperative for the robust function of the nervous system as a whole, we tested whether activity-dependent global scaling could also manifest within peripheral circuits. We uncovered a compensatory adaptation which causes global scaling within a peripheral circuit and on the presynaptic side through uniform downscaling of evoked neurotransmitter release. Unlike in central circuits, uniform scaling maintains functionality over a wide, rather than a narrow, operational range, affording robust and stable activity. Activity-dependent global scaling therefore operates on both the presynaptic and postsynaptic sides to maintain target cell activity.

Molecular Biology of Escherichia Coli Shiga Toxins' Effects on Mammalian Cells.

Shiga toxins (Stxs), syn. Vero(cyto)toxins, are potent bacterial exotoxins and the principal virulence factor of enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin-producing E. coli (STEC). EHEC strains, e.g., strains of serovars O157:H7 and O104:H4, may cause individual cases as well as large outbreaks of life-threatening diseases in humans. Stxs primarily exert a ribotoxic activity in the eukaryotic target cells of the mammalian host resulting in rapid protein synthesis inhibition and cell death. Damage of endothelial cells in the kidneys and the central nervous system by Stxs is central in the pathogenesis of hemolytic uremic syndrome (HUS) in humans and edema disease in pigs. Probably even more important, the toxins also are capable of modulating a plethora of essential cellular functions, which eventually disturb intercellular communication. The review aims at providing a comprehensive overview of the current knowledge of the time course and the consecutive steps of Stx/cell interactions at the molecular level. Intervention measures deduced from an in-depth understanding of this molecular interplay may foster our basic understanding of cellular biology and microbial pathogenesis and pave the way to the creation of host-directed active compounds to mitigate the pathological conditions of STEC infections in the mammalian body.

Anti-tumor efficacy of folate modified PLGA-based nanoparticles for the co-delivery of drugs in ovarian cancer.

Background: Ovarian cancer is a leading cause of death in gynecologic malignancies. The high mortality is mainly caused by advanced stage at presentation in most patients. Even after the combination of cytoreductive surgery and systemic platinum and taxane treatment, most patients relapse and eventually succumb to the disease. Therefore, there is an urgent need for new treatments. Purpose: A novel folate (FA)-targeted co-delivery of docetaxel (DTX) and gemcitabine (GEM) nanoparticles (NPs) was developed to overcome ovarian cancer. Materials and methods: Physicochemical characteristics of NPs such as size, morphology, and release profiles were explored. In vitro and in vivo studies were carried out to assess the efficacy of their antitumor activity in target cells. Results: FA modified DTX and GEM co-loaded NPs were prepared using the solvent evaporation method. The NPs with a particle size of ~120nm were stable in the observation period. The hemolysis results indicated that FA-PEG2000-PLGA was potentially feasible for targeted antitumor drug delivery through blood circulation. In vitro release study suggested that in comparison with the free drug, PLGA-DTX/GEM NPs and FA-PEG2000-PLGA-DTX/GEM NPs had sustained-release properties. However, there was no obvious difference between the two NPs with the same drug in the release profile. Ovarian cancer cells in vitro efficiently took up the non-targeted and FA-targeted NPs; improved cytotoxicity was observed in the FA-targeted NPs, showing a 3.59- fold drop in the IC50 in SKOV-3 cells as compared to DTX/GEM alone. Cellular uptake showed that through surface modification, more drugs entered the cell successfully. Pharmacodynamics results showed a statistically significant effect on the rate of reduction of tumor volume for FA-PEG2000-PLGA-DTX/GEM NPs than other groups and no toxicity of organs.Conclusion: The present study indicates that the FA-PEG2000-PLGA-DTX/GEM NPs provides a promising platform for the treatment of ovarian cancer.

Structural mechanisms of selectivity and gating in anion channelrhodopsins.

Both designed and natural anion-conducting channelrhodopsins (dACRs and nACRs, respectively) have been widely applied in optogenetics (enabling selective inhibition of target-cell activity during animal behaviour studies), but each class exhibits performance limitations, underscoring trade-offs in channel structure-function relationships. Therefore, molecular and structural insights into dACRs and nACRs will be critical not only for understanding the fundamental mechanisms of these light-gated anionchannels, but also to create next-generation optogenetic tools. Here we report crystal structures of the dACR iC++, along with spectroscopic, electrophysiological and computational analyses that provide unexpected insights into pH dependence, substrate recognition, channel gating and ion selectivity of both dACRs and nACRs. These results enabled us to create an anion-conducting channelrhodopsin integrating the key features of large photocurrent and fast kinetics alongside exclusive anion selectivity.

Single-Fluorescent Protein Reporters Allow Parallel Quantification of Natural Killer Cell-Mediated Granzyme and Caspase Activities in Single Target Cells.

Natural killer (NK) cells eliminate infected and tumorigenic cells through delivery of granzymes via perforin pores or by activation of caspases via death receptors. In order to understand how NK cells combine different cell death mechanisms, it is important to quantify target cell responses on a single cell level. However, currently existing reporters do not allow the measurement of several protease activities inside the same cell. Here, we present a strategy for the comparison of two different proteases at a time inside individual target cells upon engagement by NK cells. We developed single-fluorescent protein reporters containing the RIEAD or the VGPD cleavage site for the measurement of granzyme B activity. We show that these two granzyme B reporters can be applied in combination with caspase-8 or caspase-3 reporters. While we did not find that caspase-8 was activated by granzyme B, our method revealed that caspase-3 activity follows granzyme B activity with a delay of about 6 min. Finally, we illustrate the comparison of several different reporters for granzyme A, M, K, and H. The approach presented here is a valuable means for the investigation of the temporal evolution of cell death mediated by cytotoxic lymphocytes.

Dexamethasone Modulation of the Interaction between Two Types of Phagocytes of the Holothurian Eupentacta fraudatrix (Djakonov et Baranova, 1958) (Holothuroidea: Dendrochirotida)

This study examines the interaction between two types of phagocytes (P1 and P2) of the holothurian Eupentacta fraudatrix and its in vitro modulation by dexamethasone. Our results indicate that inhibition of apoptosis in P1 phagocytes by P2 phagocytes was accompanied by increased activities of antioxidant enzymes and reduced synthesis of interleukin-1α-like substances. We hypothesize that P1-phagocyte-related effects occurred in response to a high level of hydrogen peroxide produced by P2 phagocytes. The reduced anti-apoptotic effect of P2-phagocyte supernatant during prolonged incubation (24 h) was accompanied by a decline in defense reactions in P1 phagocytes due to depletion of antioxidant enzymes (catalase, glutathione reductase, and glutathione transferase). Inhibition of apoptotis in P1 phagocytes associated with upregulation of antioxidant enzyme defense in response to P2 phagocytes preincubated with dexamethasone (100 µM) indicates that P2 phagocytes affect P1 phagocytes via a ROS-associated mechanism. Thus, our data provide evidence that P1 and P2 phagocytes exhibit their maximum activity at different stages of the immune response, thus causing inhibition of activity in target cells during prolonged exposure. Dexamethasone enhances these effects.

Insulin Modulates Hippo Signaling by Impairing YAP Function

Yes‐associated protein (YAP) is the main transcriptional coactivator of the Hippo pathway, which regulates organ size, tissue homeostasis, and regeneration. YAP integrates multiple inputs from different signaling cascades. Recent evidence implicates YAP also in the control of nutrient and energy status of the cell, but the mechanisms underlying this function are unclear. In this study, we show that insulin modulates YAP activity in classic insulin target cells. We found by subcellular fractionation and Western blotting that insulin increases YAP phosphorylation, with a significant decrease in the abundance of YAP in the nuclei of HepG2 liver cells. Further, this regulation is likely mediated through the insulin receptor (IR), as overexpression of IR increased YAP phosphorylation. Proximity ligation assay analysis revealed a marked reduction in the interaction of YAP with TEA domain (TEAD) transcription factors in the nuclei of insulin‐treated cells. Consistent with these findings, insulin significantly impaired YAP/TEAD‐mediated transcription as measured by a dual luciferase assay in both HepG2 and C2C12 muscle cells. Further, quantitative PCR of HepG2 cells treated with insulin revealed reduced transcription of YAP regulated genes. Insulin alters transcription of numerous genes relevant to energy homeostasis, and we observed that knockdown of YAP by siRNA alters expression of genes in gluconeogenesis. Insulin stimulates activation of the phosphoinositide 3‐kinase (PI3K) pathway. The inhibitory effect of insulin on YAP transcription was abrogated by small‐molecule inhibitors of PI3K. Collectively, our results identify insulin as a previously undescribed suppressor of YAP activity and provide insight into an important link between the insulin and Hippo pathways.Support or Funding InformationThis work was supported by the National Institutes of Health Intramural Research Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Photodynamic Therapy Mediated by Aloe-Emodin Inhibited Angiogenesis and Cell Metastasis Through Activating MAPK Signaling Pathway on HUVECs.

Photodynamic therapy is a clinically used, minimally invasive therapeutic procedure that involves the application of photosensitizers which can locate in target cells and so be irradiated at a corresponding wavelength. Laser light irradiation activation of photosensitizers generates free reactive oxygen species, which induces selective cytotoxic activity in target cells. Within recent years, aloe-emodin as a photosensitizer has been successfully applied in photodynamic therapy applications. Angiogenesis plays an important role in tumor growth and metastasis; thus, the development of a novel target treatment for angiogenesis is essential in order to improve treatment therapeutics for cancer treatment. An essential step in angiogenesis involves the formation of tube-like structures during matrix degradation, rearrangement, and apoptosis of endothelial cells. In the present study, we investigated the mechanisms of photocytotoxicity induced by aloe-emodin in human umbilical vein endothelial cells. Analysis of cell proliferation results noted a significant decrease in cultured cells which received various concentrations of aloe-emodin and photodynamic therapy–induced light doses. Additionally, mitochondrial mechanisms of apoptotic cell death were observed in aloe-emodin photodynamic therapy–treated cells, as tube formation assays noted angiogenesis suppression after treatment. The capacity of migration and invasion of human umbilical vein endothelial cells was measured using the transwell assay and demonstrated that aloe-emodin photodynamic therapy significantly inhibited the migration and invasion of human umbilical vein endothelial cells. The expression of p38, extracellular signal-regulated kinase, the c-Jun N-terminal kinases, and vascular endothelial growth factor suggested that the cellular metastasis was related to mitogen-activated protein kinase signal pathway. Furthermore, disorganization of F action cytoskeleton components was observed after aloe-emodin photodynamic therapy. Overall, the findings from this study suggest that aloe-emodin photodynamic therapy inhibited angiogenesis and cellular metastasis in human umbilical vein endothelial cells by activating the mitogen-activated protein kinase apoptotic signaling cell death pathway.

Quantifying the relative contributions of different solute carriers to aggregate substrate transport

Determining the contributions of different transporter species to overall cellular transport is fundamental for understanding the physiological regulation of solutes. We calculated the relative activities of Solute Carrier (SLC) transporters using the Michaelis-Menten equation and global fitting to estimate the normalized maximum transport rate for each transporter (Vmax). Data input were the normalized measured uptake of the essential neutral amino acid (AA) L-leucine (Leu) from concentration-dependence assays performed using Xenopus laevis oocytes. Our methodology was verified by calculating Leu and L-phenylalanine (Phe) data in the presence of competitive substrates and/or inhibitors. Among 9 potentially expressed endogenous X. laevis oocyte Leu transporter species, activities of only the uniporters SLC43A2/LAT4 (and/or SLC43A1/LAT3) and the sodium symporter SLC6A19/B0AT1 were required to account for total uptake. Furthermore, Leu and Phe uptake by heterologously expressed human SLC6A14/ATB0,+ and SLC43A2/LAT4 was accurately calculated. This versatile systems biology approach is useful for analyses where the kinetics of each active protein species can be represented by the Hill equation. Furthermore, its applicable even in the absence of protein expression data. It could potentially be applied, for example, to quantify drug transporter activities in target cells to improve specificity.

Assessing Anti-miR Pharmacology with miRNA Polysome Shift Assay.

Target engagement measurements are critical for evaluating developmental drug candidates and their pharmacological activity. microRNA (miRNA) Polysome Shift Assay enables measurement of anti-miR drug target engagement (i.e. extent of miRNA inhibition) without the need to pre-identify or pre-validate downstream miRNA-regulated genes. This makes it useful for assessing anti-miR activity in target tissues or cells where biology of the inhibited miRNAs may not be well understood. In addition, miRNA Polysome Shift Assay can be multiplexed to assess inhibition of multiple miRNAs by a single anti-miR, thus guiding drug optimization for enhancing or avoiding these activities as desired. This chapter outlines the miRNA Polysome Shift Assay technique, describes sample preparation and quality control, and how to calculate and interpret results.

Real-time intravital imaging of pH variation associated with osteoclast activity.

Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.

A single-loop recombinant pseudotyped-virus-based assay to detect HIV-1 phenotypic resistance.

HIV/AIDS is a leading public health concern throughout the world. Currently, treatment of HIV/AIDS still depends on highly active antiretroviral therapy (HAART); however, there is increasing evidence showing the emergence of resistance to antiretroviral drugs in HIV-1 strains, making ART less effective over time. Intensive monitoring of HIV-1 drug resistance is therefore of great importance to evaluate the current sensitivity of antiretroviral agents and is urgently needed. The aim of this study was to develop a single-loop recombinant pseudotyped-virus-based assay to detect phenotypic resistance in clinical HIV-1 strains. HIV-1 RNA was extracted from HIV-1-infected human plasma samples, and an approximately 3-kb fragment containing p7/p1/p6 cleavage sites and full-length protease (PR), reverse transcriptase (RT), thermonuclease (TNase), and integrase (1-280 aa) genes was amplified by nested RT-PCR. A retroviral vector was constructed using the HIV-1 infectious molecular clone pLWJ to test antiretroviral drug susceptibility. pLWJ-SV40-Luc contained a luciferase expression cassette inserted within a deleted region of the envelope (env) gene as an indicator gene. Resistance test vectors (RTVs) were constructed by incorporating amplified target genes into pLWJ-SV40-Luc by using ApaI or AgeI and AarI restriction sites and conventional cloning methods. The virus stocks used for drug susceptibility test were produced by co-transfecting 293T cells with RTVs and a plasmid that provided vesicular stomatitis virus glycoprotein (VSV-G). Viral replication was monitored by measuring luciferase activity in infected target cells at approximately 48 h postinfection. A total of 35 clinical plasma samples from HIV-1-infected humans were tested, and target fragments were successfully amplified from 34 samples (97.1 %) and 33 RTVs were successfully constructed by directional cloning, with an overall success rate of 94.3 %. A clear-cut dose-dependent relationship was detected between virus production and luciferase activity in the constructed phenotypic resistance testing system. The highest coefficient of determination (R(2)) was found between luciferase activity and drug concentration and viral inhibition at 293T cell concentrations of 5 × 10(4) cells per well. The phenotypic profiles of the viruses from 29 clinical samples almost completely matched the observed genotypes. The results demonstrate that a single-loop recombinant pseudotyped-virus-based assay was successfully developed, and this testing system has high stability and appears to be applicable for testing phenotypic resistance of clinical HIV-1 strains to commonly used antiretroviral agents.

Role of ITPR1/HIF-2α axis in protecting renal cancer cells against natural killer cells.

417 Background: Clear cell renal cell carcinomas frequently display inactivation of VHL gene leading to increased level of hypoxia inducible factors (HIFs). The specific role of VHL mutations and selective activation of HIF-2 alpha in modulating RCC susceptibility to cytotoxic immune response remains largely unknown. Methods: In this study, we used 786-0 RCC VHL-mutated cell line selectively induces HIF-2 alpha stabilization.Chromium release cytotoxicity assay was regularly used to evaluate tumor target cells sensitivity to NK-mediated lysis. Confocal microscopy was aso used to analyse immunologic synapse formation and for autophagy studies. Immunochemistry staining using RCC tissue microarrays was performed to evaluate HIF-2 alpha and ITPR1 expression in RCC patients. Results: We demonstrated that the RCC cell line 786-0 with mutated VHL was resistant to NK-mediated lysis as compared to the VHL-corrected cell line (WT7). This resistance was found to require HIF-2α stabilization. Based on global gene expression profiling and ChIP assay, we found ITPR1 (inositol 1,4,5-trisphosphate receptor, type 1) as a direct novel target of HIF-2α and targeting ITPR1 significantly increased susceptibility of 786-0 cells to NK-mediated lysis. Interestingly, using a large group (235) of RCC patients,we demonstrated the existence of a significant correlation between HIF-2α and ITPR1 expression Mechanistically, HIF-2α in 786-0 cells lead to overexpression of ITPR1, which subsequently regulated the NK mediated killing through the activation of autophagy in target cells by NK derived signal. Both ITPR1 and Beclin-1 silencing in 786-0 cells inhibited NK-induced autophagy and subsequently increased Granzyme B activity in target cells. Finally, in vivo ITPR1 targeting significantly enhanced the NK-mediated tumor regression. Conclusions: Our data provide insights into the link between HIF-2α, the ITPR1-related pathway and natural immunity and strongly suggest a role for the HIF-2α /ITPR1 axis in regulating RCC cell survival. Futur NK cell-based immunotherapy should integrate HIF-2/ITPR1 axis as intrinsic feature of resistance tumor cells to improve NK cell response in RCC patients.

Mouse lymphomyeloid cells can function with significantly decreased expression levels of cytochrome C.

Cytochrome c is an indispensable electron carrier in the mitochondrial respiratory chain and also an important mediator of the internal pathway triggering apoptosis. Mice with a complete deficiency of the Cycs gene encoding the somatic cytochrome c die during the embryogenesis. Using the technology of LoxP-cre-dependent tissue-specific recombination, we obtained some mouse strains with significantly reduced expression of cytochrome c in certain cell types ("conditional genetic knockdown"). This knockdown was achieved by abrogation of the normal splicing of the Cycs locus pre-mRNA due to an additional acceptor site inside the stop-cassette neo(r). Previously, we observed embryonic lethality in homozygous mice with the same knockdown of cytochrome c in all cells of the organism. In the present work we studied two novel mouse strains with conditional knockdown of the Cycs gene in T lymphocytes and macrophages. Somewhat surprisingly, the mice of these two strains under normal conditions were not phenotypically different from the wild-type mice, either on the whole organism level or on the level of activity of individual target cells. Thus, the amount of cytochrome c in lymphomyeloid cells does not affect their development and normal functioning.

ITPR1 protects renal cancer cells against natural killer cells by inducing autophagy.

Clear cell renal cell carcinomas (RCC) frequently display inactivation of von Hippel-Lindau (VHL) gene leading to increased level of hypoxia-inducible factors (HIF). In this study, we investigated the potential role of HIF2α in regulating RCC susceptibility to natural killer (NK) cell-mediated killing. We demonstrated that the RCC cell line 786-0 with mutated VHL was resistant to NK-mediated lysis as compared with the VHL-corrected cell line (WT7). This resistance was found to require HIF2α stabilization. On the basis of global gene expression profiling and chromatin immunoprecipitation assay, we found ITPR1 (inositol 1,4,5-trisphosphate receptor, type 1) as a direct novel target of HIF2α and that targeting ITPR1 significantly increased susceptibility of 786-0 cells to NK-mediated lysis. Mechanistically, HIF2α in 786-0 cells lead to overexpression of ITPR1, which subsequently regulated the NK-mediated killing through the activation of autophagy in target cells by NK-derived signal. Interestingly, both ITPR1 and Beclin-1 silencing in 786-0 cells inhibited NK-induced autophagy and subsequently increased granzyme B activity in target cells. Finally, in vivo ITPR1 targeting significantly enhanced the NK-mediated tumor regression. Our data provide insight into the link between HIF2α, the ITPR1-related pathway, and natural immunity and strongly suggest a role for the HIF2α/ITPR1 axis in regulating RCC cell survival.

Real-time detection of CTL function reveals distinct patterns of caspase activation mediated by Fas versus granzyme B.

Activation of caspase-mediated apoptosis is reported to be a hallmark of both granzyme B- and Fas-mediated pathways of killing by CTLs; however, the kinetics of caspase activation remain undefined owing to an inability to monitor target cell-specific apoptosis in real time. We have overcome this limitation by developing a novel biosensor assay that detects continuous, protease-specific activity in target cells. Biosensors were engineered from a circularly permuted luciferase, linked internally by either caspase 3/7 or granzyme B/caspase 8 cleavage sites, thus allowing activation upon proteolytic cleavage by the respective proteases. Coincubation of murine CTLs with target cells expressing either type of biosensor led to a robust luminescent signal within minutes of cell contact. The signal was modulated by the strength of TCR signaling, the ratio of CTL/target cells, and the type of biosensor used. Additionally, the luciferase signal at 30 min correlated with target cell death, as measured by a (51)Cr-release assay. The rate of caspase 3/7 biosensor activation was unexpectedly rapid following granzyme B- compared with Fas-mediated signal induction in murine CTLs; the latter appeared gradually after a 90-min delay in perforin- or granzyme B-deficient CTLs. Remarkably, the Fas-dependent, caspase 3/7 biosensor signal induced by perforin-deficient human CTLs was also detectable after a 90-min delay when measured by redirected killing. Thus, we have used a novel, real-time assay to demonstrate the distinct pattern of caspase activation induced by granzyme B versus Fas in human and murine CTLs.

How to Train Your Dragon: Targeted Delivery of MicroRNA to Cancer Cells In Vivo

The fundamental role of microRNAs (miRNAs) in both initiation and progression of cancer has made them attractive therapeutic targets.1 miRNAs can simultaneously alter expression of multiple target genes and often disrupt entire signaling networks, resulting in efficient changes in the activity and phenotype of target cells. At the same time, their broad functionality can be a liability that results in difficult-to-control off-target effects and toxicities. A report by Esposito et al. in this issue of Molecular Therapy provides a possible solution to the problem of cell-specific delivery of therapeutic miRNAs and represents an important step in the development of targeted miRNA mimics.2 The authors present a novel strategy for the delivery of tumor suppressor let-7g miRNA to specific cancer cells using unformulated oligonucleotides in the form of an aptamer specific for the oncogenic tyrosine kinase receptor, Axl (Figure 1).2

14-3-3 and β-catenin are secreted on extracellular vesicles to activate the oncogenic Wnt pathway

Aberrant activation of the canonical Wnt signal transduction pathway is involved in a large number of human diseases. β-catenin, the key effector protein of the canonical Wnt pathway, functions in the nucleus with T-cell factor/lymphoid enhancer factor (TCF/LEF) to activate expression of Wnt target genes. Here we show that members of the 14-3-3 protein family bind disheveled-2 (Dvl-2) and glycogen synthase-3β (GSK-3β) to attenuate the interaction between GSK-3β and β-catenin. Importantly, 14-3-3 and β-catenin form “bleb-like” structures and are secreted via extracellular vesicles to induce Wnt signaling activity in target cells. Our data suggest a novel way of transducing the oncogenic Wnt signal in which β-catenin is regulated by 14-3-3ζ through the formation of “oncosomes” that contain both the 14-3-3 and β-catenin proteins.

Potency Of Bacteriocin For Animal Health And Food Safety

The emergence of antibiotic resistance in many bacteria related to animal and public health stresses the importance of decreasing the use of antibiotics in animal production. The reduction of antibiotic application in livestock can only be achieved if alternative antimicrobial strategies are available. A number of strategies have been explored to control microbial pathogens and to improve growth and feed efficiency in livestock without the use of antibiotics. Bacteriocins have been more extensively studied and proposed as potential alternatives to conventional antibiotics in animal husbandry. Bacteriocins are antimicrobial peptides ribosomally synthesized by many species of Bacteria and some strains of Archaea. In general, bacteriocins just exhibited bactericidal or bacteriostatic activity against other bacteria that are closely related to the producing strain. The main mechanisms of bacteriocin activity vary from pore formation in cytoplasmic membranes to the inhibition of cell wall biosynthesis and enzyme activities (RNAse or DNAse) in target cells. The use of bacteriocins in probiotic applications, as preservatives, and most excitingly as alternatives to conventional antibiotics is being broadly explored and studied. This review will describe the bacteriocins potency for animal health and food safety, as well as the results of bacteriocin study that had been conducted in Indonesia.Key words: Bacteriocin, animal health, food safety

Early Growth Response-1 (EGR-1) and Nuclear Factor of Activated T Cells (NFAT) Cooperate to Mediate CD40L Expression in Megakaryocytes and Platelets

Increasing evidence implicates circulating platelets as mediators of chronic inflammatory and autoimmune diseases via the expression and release of CD40L, an important modulator of inflammation and adaptive immune responses traditionally associated with activated T cells. Emerging evidence suggests that platelet CD40L is dynamically regulated in several chronic inflammatory and autoimmune diseases and may mediate progression and secondary pathology associated with those disease states. The present study identifies NFATc2 as a key transcriptional modulator of CD40L expression in megakaryocytes and inflammatory activity of platelets. Furthermore, the current data show that EGR-1, a member of the early growth response family of zinc finger transcription factors, modulates NFATc2-dependent regulation of CD40L expression in megakaryocytes. Our novel demonstration that in vivo biochemical or genetic inhibition of NFATc2 activity in megakaryocyte diminishes platelet CD40L implicates the NFATc2/EGR-1 axis as a key regulatory pathway of inflammatory and immunomodulatory activity in platelets and represents a target for the development of therapeutics for the potential treatment of chronic inflammatory and autoimmune diseases.