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Calreticulin (CRT) is a high-capacity Ca2+ protein whose expression is up-regulated during cellular transformation and is associated with disease progression in multiple types of malignancies. At the same time, CRT has been characterized as an important stress-response protein capable of inducing immunogenic cell death (ICD) when translocated to the cell surface. It remains unclear why CRT expression is preserved by malignant cells during the course of transformation despite its immunogenic properties. In this study, we identify a novel, critical function of CRT as a cell survival factor in multiple types of human solid-tissue malignancies. CRT knockdown activates p53, which mediates cell-death response independent of executioner caspase activity and accompanied full-length poly ADP ribose polymerase (PARP) cleavage. Mechanistically, we show that down-regulation of CRT results in mitochondrial Ca2+ overload and induction of mitochondria permeability transition pore (mPTP)-dependent cell death, which can be significantly rescued by the mPTP inhibitor, Cyclosporin A (CsA). The clinical importance of CRT expression was revealed in the analysis of the large cohort of cancer patients (N = 2,058) to demonstrate that high levels of CRT inversely correlates with patient survival. Our study identifies intracellular CRT as an important therapeutic target for tumors whose survival relies on its expression. [ABSTRACT FROM AUTHOR]

Ligand binding to death receptors activates apoptosis in cancer cells. Stimulation of death receptors results in the formation of intracellular multiprotein platforms that either activate the apoptotic initiator Caspase-8 to trigger cell death, or signal through kinases to initiate inflammatory and cell survival signalling. Two of these platforms, the Death-Inducing Signalling Complex (DISC) and the RIPoptosome, also initiate necroptosis by building filamentous scaffolds that lead to the activation of mixed lineage kinase domain-like pseudokinase. To explain cell decision making downstream of death receptor activation, we developed a semi-stochastic model of DISC/RIPoptosome formation. The model is a hybrid of a direct Gillespie stochastic simulation algorithm for slow assembly of the RIPoptosome and a deterministic model of downstream caspase activation. The model explains how alterations in the level of death receptor-ligand complexes, their clustering properties and intrinsic molecular fluctuations in RIPoptosome assembly drive heterogeneous dynamics of Caspase-8 activation. The model highlights how kinetic proofreading leads to heterogeneous cell responses and results in fractional cell killing at low levels of receptor stimulation. It reveals that the noise in Caspase-8 activation—exclusively caused by the stochastic molecular assembly of the DISC/RIPoptosome platform—has a key function in extrinsic apoptotic stimuli recognition. [ABSTRACT FROM AUTHOR]

Malignant melanoma is an aggressive type of cancer and the deadliest form of skin cancer. Even though enormous efforts have been undertaken, in particular the treatment options against the metastasizing form are challenging and the prognosis is generally poor. A novel therapeutical approach is the application of secondary plant constituents occurring in food and food products. Herein, the effect of the dietary chalcone cardamonin, inter alia found in Alpinia species, was tested using human malignant melanoma cells. These data were compared to cardamonin treated normal melanocytes and dermal fibroblasts representing healthy cells. To investigate the impact of cardamonin on tumor and normal cells, it was added to monolayer cell cultures and cytotoxicity, proliferation, tumor invasion, and apoptosis were studied with appropriate cell biological and biochemical methods. Cardamonin treatment resulted in an apoptosis-mediated increase in cytotoxicity towards tumor cells, a decrease in their proliferation rate, and a lowered invasive capacity, whereas the viability of melanocytes and fibroblasts was hardly affected at such concentrations. A selective cytotoxic effect of cardamonin on melanoma cells compared to normal (healthy) cells was shown in vitro. This study along with others highlights that dietary chalcones may be a valuable tool in anticancer therapies which has to be proven in the future in vivo. [ABSTRACT FROM AUTHOR]

Podoplanin, a transmembrane glycoprotein, is overexpressed in certain types of tumors and induces platelet aggregation by binding to C-type lectin-like receptor 2 (CLEC-2) on the platelet membrane. Activated platelets release granule components, which in turn, trigger epithelial-mesenchymal transition and confer invasive capacity to the tumor cells. Therefore, blocking the podoplanin-CLEC-2 interaction by a small-molecule compound is a potential therapeutic strategy to prevent cancer metastasis and invasion. To effectively identify such inhibitory compounds, we have developed a pull-down-based inhibitory compound screening system. An immunoglobulin Fc domain-CLEC-2 fusion protein was used as a bait to capture podoplanin derived from podoplanin-overexpressing HeLa cells in the presence and absence of the test compound. The protein complex was then pulled down using protein A beads. To shorten the turnaround time, increase throughput, and decrease the workload for the operators, centrifugal filter units were employed to separate free and bound podoplanin, instead of using customary aspiration-centrifugation washing cycles. Slot blotting was also utilized in lieu of gel electrophoresis and electrical transfer. Thus, the use of our pull down screening system could facilitate the effective selection of potential inhibitor compounds of the podoplanin-CLEC-2 interaction for cancer therapy. Importantly, our methodology is also applicable to targeting other protein-protein interactions. [ABSTRACT FROM AUTHOR]

To escape CD8+ T-cell immunity, human cytomegalovirus (HCMV) US11 redirects MHC-I for rapid ER-associated proteolytic degradation (ERAD). In humans, classical MHC-I molecules are encoded by the highly polymorphic HLA-A, -B and -C gene loci. While HLA-C resists US11 degradation, the specificity for HLA-A and HLA-B products has not been systematically studied. In this study we analyzed the MHC-I peptide ligands in HCMV-infected cells. A US11-dependent loss of HLA-A ligands was observed, but not of HLA-B. We revealed a general ability of HLA-B to assemble with β2m and exit from the ER in the presence of US11. Surprisingly, a low-complexity region between the signal peptide sequence and the Ig-like domain of US11, was necessary to form a stable interaction with assembled MHC-I and, moreover, this region was also responsible for changing the pool of HLA-B ligands. Our data suggest a two-pronged strategy by US11 to escape CD8+ T-cell immunity, firstly, by degrading HLA-A molecules, and secondly, by manipulating the HLA-B ligandome. [ABSTRACT FROM AUTHOR]

Practical laboratory classes teaching molecular pharmacology approaches employed in the development of therapeutic strategies are of great interest for students of courses in Biotechnology, Applied Biology, Pharmaceutic and Technology Chemistry, Translational Oncology. Unfortunately, in most cases the technology to be transferred to learning students is complex and requires multi-step approaches. In this respect, simple and straightforward experimental protocols might be of great interest. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. microRNA therapeutics, and (b) on the employment of biomolecules of great interest in applied biology and pharmacology, i.e. peptide nucleic acids (PNAs). The aims of the practical laboratory were to determine: (a) the possible PNA-mediated arrest in RT-qPCR, to be eventually used to demonstrate PNA targeting of selected miRNAs; (b) the possible lack of activity on mutated PNA sequences; (c) the effects (if any) on the amplification of other unrelated miRNA sequences. The results which can be obtained support the following conclusions: PNA-mediated arrest in RT-qPCR can be analyzed in a easy way; mutated PNA sequences are completely inactive; the effects of the employed PNAs are specific and no inhibitory effect occurs on other unrelated miRNA sequences. This activity is simple (cell culture, RNA extraction, RT-qPCR are all well-established technologies), fast (starting from isolated and characterized RNA, few hours are just necessary), highly reproducible (therefore easily employed by even untrained students). On the other hand, these laboratory lessons require some facilities, the most critical being the availability of instruments for PCR. While this might be a problem in the case these instruments are not available, we would like to underline that determination of the presence or of a lack of amplified product can be also obtained using standard analytical approaches based on agarose gel electrophoresis. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic E. coli (EHEC) is a human intestinal pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. No vaccines or specific therapies are currently available to prevent or treat these infections. EHEC tightly attaches to the intestinal epithelium by injecting the intimin receptor Tir into the host cell via a type III secretion system (T3SS). In this project, we identified a camelid single domain antibody (nanobody), named TD4, that recognizes a conserved Tir epitope overlapping the binding site of its natural ligand intimin with high affinity and stability. We show that TD4 inhibits attachment of EHEC to cultured human HeLa cells by preventing Tir clustering by intimin, activation of downstream actin polymerization and pedestal formation. Furthermore, we demonstrate that TD4 significantly reduces EHEC adherence to human colonic mucosa in in vitro organ cultures. Altogether, these results suggest that nanobody-based therapies hold potential in the development of much needed treatment and prevention strategies against EHEC infection. [ABSTRACT FROM AUTHOR]

Most intracellular pathogens that reside in a vacuole prevent transit of their compartment to lysosomal organelles. Effector mechanisms induced by the pro-inflammatory cytokine Interferon-gamma (IFNγ) can promote the delivery of pathogen-occupied vacuoles to lysosomes for proteolytic degradation and are therefore important for host defense against intracellular pathogens. The bacterial pathogen Coxiella burnetii is unique in that, transport to the lysosome is essential for replication. The bacterium modulates membrane traffic to create a specialized autophagolysosomal compartment called the Coxiella-containing vacuole (CCV). Importantly, IFNγ signaling inhibits intracellular replication of C. burnetii, raising the question of which IFNγ-activated mechanisms restrict replication of a lysosome-adapted pathogen. To address this question, siRNA was used to silence a panel of IFNγ-induced genes in HeLa cells to identify genes required for restriction of C. burnetii intracellular replication. This screen demonstrated that Indoleamine 2,3-dioxygenase 1 (IDO1) contributes to IFNγ-mediated restriction of C. burnetii. IDO1 is an enzyme that catabolizes cellular tryptophan to kynurenine metabolites thereby reducing tryptophan availability in cells. Cells deficient in IDO1 function were more permissive for C. burnetii replication when treated with IFNγ, and supplementing IFNγ-treated cells with tryptophan enhanced intracellular replication. Additionally, ectopic expression of IDO1 in host cells was sufficient to restrict replication of C. burnetii in the absence of IFNγ signaling. Using differentiated THP1 macrophage-like cells it was determined that IFNγ-activation resulted in IDO1 production, and that supplementation of IFNγ-activated THP1 cells with tryptophan enhanced C. burnetii replication. Thus, this study identifies IDO1 production as a key cell-autonomous defense mechanism that limits infection by C. burnetii, which suggests that peptides derived from hydrolysis of proteins in the CCV do not provide an adequate supply of tryptophan for bacterial replication. [ABSTRACT FROM AUTHOR]

The transport of macromolecules into the cell nucleus occurs through nuclear pore complexes (NPCs) and is mediated by cellular receptors. Recently, a novel mechanism of nuclear entry, in which actin polymerization provides a propulsive force driving the transport through the NPC, has been proposed. This mechanism is used by the nucleocapsid from baculovirus, one of the largest viruses to replicate in the nucleus of their host cells, which crosses the NPC and enters the nucleus independently of cellular receptors. The baculovirus nucleocapsid contains a protein that hijacks the cellular actin polymerization machinery to assemble actin filaments that propel the nucleocapsid through the host cell cytoplasm. In this study, we functionalized carbon nanotubes by covalently attaching a protein domain responsible for inducing actin polymerization and investigated their nuclear entry. We found that the functionalized carbon nanotubes were able to enter the cell nucleus under permissive conditions for actin polymerization, but not when this process was inhibited. We conclude that the mechanical force generated by actin polymerization can drive cargo entry into the cell nucleus. Our results support a novel force-driven mechanism for molecular entry into the cell nucleus. [ABSTRACT FROM AUTHOR]

The tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis. Vibrio vulnificus, an opportunistic pathogen, harbors three distinct tad loci. Among them, only tad1 locus was highly upregulated in in vivo growing bacteria compared to in vitro culture condition. To understand the pathogenic roles of the three tad loci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only the Δtad123 triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on the Δtad123 mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. The Δtad123 mutant cells showed attenuated host cell adhesion, decreased biofilm formation, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. The Δtad123 mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Complement depletion by treating with anti-C5 antibody rescued the viable count of Δtad123 in infected mouse bloodstream to the level comparable to wild type strain. Taken together, all three tad loci cooperate to confer successful invasion of V. vulnificus into deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia. [ABSTRACT FROM AUTHOR]

Enhancer of zeste homolog 2 (EZH2) tri-methylates histone 3 at position lysine 27 (H3K27me3). Overexpression and gain-of-function mutations in EZH2 are regarded as oncogenic drivers in lymphoma and other malignancies due to the silencing of tumor suppressors and differentiation genes. EZH2 inhibition is sought to represent a good strategy for tumor therapy. In this study, we treated Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) cell lines with 3-deazaneplanocin—A (DZNep), an indirect EZH2 inhibitor which possesses anticancer properties both in-vitro and in-vivo. We aimed to address the impact of the lymphoma type, EZH2 mutation status, as well as MYC, BCL2 and BCL6 translocations on the sensitivity of the lymphoma cell lines to DZNep-mediated apoptosis. We show that DZNep inhibits proliferation and induces apoptosis of these cell lines independent of the type of lymphoma, the EZH2 mutation status and the MYC, BCL2 and BCL6 rearrangement status. Furthermore, DZNep induced a much stronger apoptosis in majority of these cell lines at a lower concentration, and within a shorter period when compared with EPZ-6438, a direct EZH2 inhibitor currently in phase II clinical trials. Apoptosis induction by DZNep was both concentration-dependent and time-dependent, and was associated with the inhibition of EZH2 and subsequent downregulation of H3K27me3 in DZNep-sensitive cell lines. Although EZH2, MYC, BCL2 and BCL6 are important prognostic biomarkers for lymphomas, our study shows that they poorly influence the sensitivity of lymphoma cell lines to DZNep-mediated apoptosis. [ABSTRACT FROM AUTHOR]

Cystic Fibrosis is caused by mutations in the CFTR anion channel, many of which cause its misfolding and degradation. CFTR folding depends on the Hsc70 and Hsp70 chaperones and their co-chaperone DNAJA1, but Hsc70/Hsp70 is also involved in CFTR degradation. Here, we address how these opposing functions are balanced. DNAJA2 and DNAJA1 were both important for CFTR folding, however overexpressing DNAJA2 but not DNAJA1 enhanced CFTR degradation at the endoplasmic reticulum by Hsc70/Hsp70 and the E3 ubiquitin ligase CHIP. Excess Hsp70 also promoted CFTR degradation, but this occurred through the lysosomal pathway and required CHIP but not complex formation with HOP and Hsp90. Notably, the Hsp70 inhibitor MKT077 enhanced levels of mature CFTR and the most common disease variant ΔF508-CFTR, by slowing turnover and allowing delayed maturation, respectively. MKT077 also boosted the channel activity of ΔF508-CFTR when combined with the corrector compound VX809. Thus, the Hsp70 system is the major determinant of CFTR degradation, and its modulation can partially relieve the misfolding phenotype. [ABSTRACT FROM AUTHOR]

Telomerase is a ribonucleoprotein ribonucleic enzyme that is essential for cellular immortalization via elongation of telomere repeat sequences at the end of chromosomes. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase holoenzyme, is a key regulator of telomerase activity. Telomerase activity, which has been detected in the majority of cancer cells, is accompanied by hTERT expression, suggesting that this enzyme activity contributes to an unlimited replication potential of cancer cells via regulation of telomere length. Thus, hTERT is an attractive target for cancer-specific treatments. We previously reported that pared-like homeodomain 1 (PITX1) is a negative regulator of hTERT through direct binding to the hTERT promoter. However, the mechanism by which the function of PITX1 contributes to transcriptional silencing of the hTERT gene remains to be clarified. Here, we show that PITX1 and zinc finger CCHC-type containing 10 (ZCCHC10) proteins cooperate to facilitate the transcriptional regulation of the hTERT gene by functional studies via FLAG pull-down assay. Co-expression of PITX1 and ZCCHC10 resulted in inhibition of hTERT transcription, in melanoma cell lines, whereas mutate-deletion of homeodomain in PITX1 that interact with ZCCHC10 did not induce similar phenotypes. In addition, ZCCHC10 expression levels showed marked decrease in the majority of melanoma cell lines and tissues. Taken together, these results suggest that ZCCHC10-PITX1 complex is the functional unit that suppresses hTERT transcription, and may play a crucial role as a novel tumor suppressor complex. [ABSTRACT FROM AUTHOR]

Adoptive transfer of ex vivo expanded tumor infiltrating lymphocytes (TILs) has led to clinical benefit in some patients with melanoma but has not demonstrated convincing efficacy in other solid cancers. Whilst the presence of TILs in many types of cancer is often associated with better clinical prognosis, their function has not been systematically evaluated across cancer types. Responses to immunological checkpoint inhibitors in a wide range of cancers, including those for which adoptive transfer of expanded TILs has not shown clinical benefit, has clearly delineated a number of tumor type associated with tumor-reactive lymphocytes capable of effecting tumor remissions. Neuroblastoma is an aggressive childhood solid cancer in which immunotherapy with GD2-directed antibodies confers a proven survival advantage through incompletely understood mechanisms. We therefore evaluated the feasibility of ex vivo expansion of TILs from freshly resected neuroblastoma tumors and the potential therapeutic utility of TIL expansions. TILs were successfully expanded from both tumor biopsies or resections. Significant numbers of NKT and γδT cells were identified alongside the mixed population of cytotoxic (CD8+) and helper (CD4+) T cells of both effector and central memory phenotypes. Isolated TILs were broadly non-reactive against autologous tumor and neuroblastoma cell lines, so enhancement of neuroblastoma killing was attained by transducing TILs with a second-generation chimeric antigen receptor (CAR) targeting GD2. CAR-TILs demonstrated antigen-specific cytotoxicity against tumor cell lines. This study is the first to show reproducible expansion of TILs from pediatric neuroblastoma, the high proportion of innate-like lymphocytes, and the feasibility to use CAR-TILs therapeutically. [ABSTRACT FROM AUTHOR]

Mutations in ATP13A2 cause Kufor-Rakeb syndrome (KRS), a juvenile form of Parkinson’s disease (PD) with dementia. However, the mechanisms by which mutations in ATP13A2 cause KRS is not understood. The mutations lead to misfolding of the translated Atp13a2 protein and its premature degradation in the endoplasmic reticulum, never reaching the lysosome where the protein is thought to function. Atp13a2 is a P-type ATPase, a class of proteins that function in ion transport. Indeed, studies of human, mouse, and yeast Atp13a2 proteins suggest a possible involvement in regulation of heavy metal toxicity. Here we report on the cytoprotective function of Atp13a2 on HeLa cells and dopamine neurons of Caenorhabditis elegans (C. elegans). HeLa cells stably overexpressing V5- tagged Atp13a2Isoform-1 protein were more resistant to elevated manganese exposure and to starvation-induced cell death compared to cells not overexpressing the protein. Because PD is characterized by loss of dopamine neurons, we generated transgenic C. elegans expressing GFP-tagged human Atp13a2 protein in dopamine neurons. The transgenic animals exhibited higher resistance to dopamine neuron degeneration after acute exposure to manganese compared to nematodes that expressed GFP alone. The results suggest Atp13a2 Isoform-1 protein confers cytoprotection against toxic insults, including those that cause PD syndromes. [ABSTRACT FROM AUTHOR]

The enteric bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), utilizes two type III secretion systems (T3SSs) to invade host cells, survive and replicate intracellularly. T3SS1 and its dedicated effector proteins are required for bacterial entry into non-phagocytic cells and establishment and trafficking of the nascent Salmonella-containing vacuole (SCV). Here we identify the first T3SS1 effector required to maintain the integrity of the nascent SCV as SopF. SopF associates with host cell membranes, either when translocated by bacteria or ectopically expressed. Recombinant SopF binds to multiple phosphoinositides in protein-lipid overlays, suggesting that it targets eukaryotic cell membranes via phospholipid interactions. In yeast, the subcellular localization of SopF is dependent on the activity of Mss4, a phosphatidylinositol 4-phosphate 5-kinase that generates PI(4,5)P2 from PI(4)P, indicating that membrane recruitment of SopF requires specific phospholipids. Ectopically expressed SopF partially colocalizes with specific phosphoinositide pools present on the plasma membrane in mammalian cells and with cytoskeletal-associated markers at the leading edge of cells. Translocated SopF concentrates on plasma membrane ruffles and around intracellular bacteria, presumably on the SCV. SopF is not required for bacterial invasion of non-phagocytic cells but is required for maintenance of the internalization vacuole membrane as infection with a S. Typhimurium ΔsopF mutant led to increased lysis of the SCV compared to wild type bacteria. Our structure-function analysis shows that the carboxy-terminal seven amino acids of SopF are essential for its membrane association in host cells and to promote SCV membrane stability. We also describe that SopF and another T3SS1 effector, SopB, act antagonistically to modulate nascent SCV membrane dynamics. In summary, our study highlights that a delicate balance of type III effector activities regulates the stability of the Salmonella internalization vacuole. [ABSTRACT FROM AUTHOR]

Transformation in chromatin organization is one of the most universal markers of carcinogenesis. Microscale chromatin alterations have been a staple of histopathological diagnosis of neoplasia, and nanoscale alterations have emerged as a promising marker for cancer prognostication and the detection of predysplastic changes. While numerous methods have been developed to detect these alterations, most methods for sample preparation remain largely validated via conventional microscopy and have not been examined with nanoscale sensitive imaging techniques. For these nanoscale sensitive techniques to become standard of care screening tools, new histological protocols must be developed that preserve nanoscale information. Partial Wave Spectroscopic (PWS) microscopy has recently emerged as a novel imaging technique sensitive to length scales ranging between 20 and 200 nanometers. As a label-free, high-throughput, and non-invasive imaging technique, PWS microscopy is an ideal tool to quantify structural information during sample preparation. Therefore, in this work we applied PWS microscopy to systematically evaluate the effects of cytological preparation on the nanoscales changes of chromatin using two live cell models: a drug-based model of Hela cells differentially treated with daunorubicin and a cell line comparison model of two cells lines with inherently distinct chromatin organizations. Notably, we show that existing cytological preparation can be modified in order to maintain clinically relevant nanoscopic differences, paving the way for the emerging field of nanopathology. [ABSTRACT FROM AUTHOR]

Human papillomaviruses (HPVs) are causative agents of various diseases associated with cellular hyperproliferation, including cervical cancer, one of the most prevalent tumors in women. E7 is one of the two HPV-encoded oncoproteins and directs recruitment and subsequent degradation of tumor-suppressive proteins such as retinoblastoma protein (pRb) via its LxCxE motif. E7 also triggers tumorigenesis in a pRb-independent pathway through its C-terminal domain, which has yet been largely undetermined, with a lack of structural information in a complex form with a host protein. Herein, we present the crystal structure of the E7 C-terminal domain of HPV18 belonging to the high-risk HPV genotypes bound to the catalytic domain of human nonreceptor-type protein tyrosine phosphatase 14 (PTPN14). They interact directly and potently with each other, with a dissociation constant of 18.2 nM. Ensuing structural analysis revealed the molecular basis of the PTPN14-binding specificity of E7 over other protein tyrosine phosphatases and also led to the identification of PTPN21 as a direct interacting partner of E7. Disruption of HPV18 E7 binding to PTPN14 by structure-based mutagenesis impaired E7’s ability to promote keratinocyte proliferation and migration. Likewise, E7 binding-defective PTPN14 was resistant for degradation via proteasome, and it was much more effective than wild-type PTPN14 in attenuating the activity of downstream effectors of Hippo signaling and negatively regulating cell proliferation, migration, and invasion when examined in HPV18-positive HeLa cells. These results therefore demonstrated the significance and therapeutic potential of the intermolecular interaction between HPV E7 and host PTPN14 in HPV-mediated cell transformation and tumorigenesis. [ABSTRACT FROM AUTHOR]

It is important that bacterium can coordinately deliver several effectors into host cells to disturb the cellular progress during infection, however, the precise role of effectors in host cell cytosol remains to be resolved. In this study, we identified a new bacterial virulence effector from pathogenic Edwardsiella piscicida, which presents conserved crystal structure to thioredoxin family members and is defined as a thioredoxin-like protein (Trxlp). Unlike the classical bacterial thioredoxins, Trxlp can be translocated into host cells, mimicking endogenous thioredoxin to abrogate ASK1 homophilic interaction and phosphorylation, then suppressing the phosphorylation of downstream Erk1/2- and p38-MAPK signaling cascades. Moreover, Trxlp-mediated inhibition of ASK1-Erk/p38-MAPK axis promotes the pathogenesis of E. piscicida in zebrafish larvae infection model. Taken together, these data provide insights into the mechanism underlying the bacterial thioredoxin as a virulence effector in downmodulating the innate immune responses during E. piscicida infection. [ABSTRACT FROM AUTHOR]

Glioblastoma is a high-grade glioma with poor prognosis even after surgery and standard therapy. Here, we asked whether carnosine (β-alanyl-L-histidine), a naturally occurring dipeptide, exert its anti-neoplastic effect on glioblastoma cells via PI3K/Akt/mTOR signaling. Therefore, glioblastoma cells from the lines U87 and T98G were exposed to carnosine, to the mTOR inhibitor rapamycin and to the PI3K inhibitor Ly-294,002. Pyruvate dehydrogenase kinase (PDK4) expression, known to be a target of PI3K/Akt/mTOR, and which is also affected by carnosine, was analyzed by RT-qPCR, and reporter gene assays with the human PDK4 promoter were performed. Cell viability was assessed by cell-based assays and mTOR and Akt phosphorylation by Western blotting. Rapamycin and Ly-294,002 increased PDK4 mRNA expression in both cell lines but significance was only reached in U87. Carnosine significantly increased expression in both lines. A significant combinatorial effect of carnosine was only detected in U87 when the dipeptide was combined with Ly-294,002. Reporter gene assays revealed no specific effect of carnosine on the human PDK4 promoter, whereas both inhibitors increased reporter gene expression. Rapamycin reduced phosphorylation of mTOR, and Ly-294,002 that of Akt. A significant reduction of Akt phosphorylation was observed in the presence of carnosine in U87 but not in T98G, and carnosine had no effect on mTOR phosphorylation. Cell viability as determined by ATP in cell lysates was reduced only in the presence of carnosine. We conclude that carnosine’s anti-neoplastic effect is independent from PI3K/Akt/mTOR signaling. As the dipeptide reduced viability in tumor cells that do not respond to PI3K or mTOR inhibitors, it appears to be worth to further investigate the mechanisms by which carnosine exerts its anti-tumor effect and to consider it for therapy, especially as it is a naturally occurring compound that has already been used for the treatment of other diseases without indication of side-effects. [ABSTRACT FROM AUTHOR]

Adult T-cell Leukemia (ATL) is a lymphoproliferative disease of CD4+ T-cells infected with Human T-cell Leukemia Virus type I (HTLV-1). With the exception of allogeneic hematopoietic stem cell transplantation, there are no effective treatments to cure ATL, and ATL cells often acquire resistance to conventional chemotherapeutic agents. Accumulating evidence shows that development and maintenance of ATL requires key contributions from the viral protein, HTLV-1 basic leucine zipper factor (HBZ). In this study we found that HBZ activates expression of Heme Oxygenase 1 (HMOX-1), a component of the oxidative stress response that functions to detoxify free heme. Transcription of HMOX1 and other antioxidant genes is regulated by the small Mafs. These cellular basic leucine zipper (bZIP) factors control transcription by forming homo- or heterodimers among themselves or with other cellular bZIP factors that then bind Maf responsive elements (MAREs) in promoters or enhancers of antioxidant genes. Our data support a model in which HBZ activates HMOX1 transcription by forming heterodimers with the small Mafs that bind MAREs located in an upstream enhancer region. Consistent with this model, we found that HMOX-1 is upregulated in HTLV-1-transformed T-cell lines and confers these cells with resistance to heme-induced cytotoxicity. In this context, HBZ-mediated activation of HMOX-1 expression may contribute to resistance of ATL cells to certain chemotherapeutic agents. We also provide evidence that HBZ counteracts oxidative stress caused by two other HTLV-1-encoded proteins, Tax and p13. Tax induces oxidative stress as a byproduct of driving mitotic expansion of infected cells, and p13 is believed to induce oxidative stress to eliminate infected cells that have become transformed. Therefore, in this context, HBZ-mediated activation of HMOX-1 expression may facilitate transformation. Overall, this study characterizes a novel function of HBZ that may support the development and maintenance of ATL. [ABSTRACT FROM AUTHOR]

Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts. This activity, which subverts numerous signaling and membrane trafficking pathways in the infected cells, is thought to contribute to pathogen virulence. The molecular and cellular mechanisms underlying these events are not well understood. We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis, recycling and transcytosis in epithelial host cells. To this end, we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells. We show that type-III secreted components prompt the recruitment of clathrin (clathrin and AP2), early (Rab5a and EEA1) and recycling (Rab4a, Rab11a, Rab11b, FIP2, Myo5b) endocytic machineries to peripheral plasma membrane infection sites. Protein cargoes, e.g. transferrin receptors, β1 integrins and aquaporins, which exploit the endocytic pathways mediated by these machineries, were also found to be recruited to these sites. Moreover, the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover (i.e. endocytosis and recycling) and transcytosis to the infected plasma membrane. The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells, and mostly on EspF in polarized epithelial cells. These data suggest a model whereby EPEC effectors hijack endosomal recycling machineries to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane. We hypothesize that these activities contribute to bacterial colonization and virulence. [ABSTRACT FROM AUTHOR]

Persistent human papillomavirus (HPV) infection is the leading cause of cervical cancer. Although the fundamental link between HPV infection and oncogenesis is established, the specific mechanisms of virus-mediated transformation are not fully understood. We previously demonstrated that the HPV encoded E6 protein increases the activity of the proto-oncogenic transcription factor STAT3 in primary human keratinocytes; however, the molecular basis for STAT3 activation in cervical cancer remains unclear. Here, we show that STAT3 phosphorylation in HPV positive cervical cancer cells is mediated primarily via autocrine activation by the pro-inflammatory cytokine Interleukin 6 (IL-6). Antibody-mediated blockade of IL-6 signalling in HPV positive cells inhibits STAT3 phosphorylation, whereas both recombinant IL-6 and conditioned media from HPV positive cells leads to increased STAT3 phosphorylation within HPV negative cervical cancer cells. Interestingly, we demonstrate that activation of the transcription factor NFκB, involving the small GTPase Rac1, is required for IL-6 production and subsequent STAT3 activation. Our data provides new insights into the molecular re-wiring of cancer cells by HPV E6. We reveal that activation of an IL-6 signalling axis drives the autocrine and paracrine phosphorylation of STAT3 within HPV positive cervical cancers cells and that activation of this pathway is essential for cervical cancer cell proliferation and survival. Greater understanding of this pathway provides a potential opportunity for the use of existing clinically approved drugs for the treatment of HPV-mediated cervical cancer. [ABSTRACT FROM AUTHOR]

Infection of mammalian cells with vesicular stomatitis virus (VSV) results in the inhibition of cellular translation while viral translation proceeds efficiently. VSV RNA synthesis occurs entirely within the cytoplasm, where during transcription the viral polymerase produces 5 mRNAs that are structurally indistinct to cellular mRNAs with respect to their 5′ cap-structure and 3′-polyadenylate tail. Using the global approach of massively parallel sequencing of total cytoplasmic, monosome- and polysome-associated mRNA, we interrogate the impact of VSV infection of HeLa cells on translation. Analysis of sequence reads in the different fractions shows >60% of total cytoplasmic and polysome-associated reads map to the 5 viral genes by 6 hours post-infection, a time point at which robust host cell translational shut-off is observed. Consistent with an overwhelming abundance of viral mRNA in the polysome fraction, the reads mapping to cellular genes were reduced. The cellular mRNAs that remain most polysome-associated following infection had longer half-lives, were typically larger, and were more AU rich, features that are shared with the viral mRNAs. Several of those mRNAs encode proteins known to positively affect viral replication, and using chemical inhibition and siRNA depletion we confirm that the host chaperone heat shock protein 90 (hsp90) and eukaryotic translation initiation factor 3A (eIF3A)—encoded by 2 such mRNAs—support viral replication. Correspondingly, regulated in development and DNA damage 1 (Redd1) encoded by a host mRNA with reduced polysome association inhibits viral infection. These data underscore the importance of viral mRNA abundance in the shut-off of host translation in VSV infected cells and link the differential translatability of some cellular mRNAs with pro- or antiviral function. [ABSTRACT FROM AUTHOR]

Due to the poor prognosis of advanced metastatic melanoma, it is crucial to find early biomarkers that help identify which melanomas will metastasize. By comparing the gene expression data from primary and cutaneous melanoma samples from The Cancer Genome Atlas (TCGA), we identified GPC6 among a set of genes whose expression levels can distinguish between primary melanoma and regional cutaneous/subcutaneous metastases. Glypicans are thought to play a role in tumor growth by regulating the signaling pathways of Wnt, Hedgehogs, fibroblast growth factors (FGFs), and bone morphogenetic proteins (BMPs). We showed that GPC6 expression was up-regulated in a melanoma cell line compared to normal melanocytes and in metastatic melanoma compared to primary melanoma. Furthermore, GPC6 expression was positively correlated with genes largely involved in cell adhesion and migration in both melanoma samples and in RNA-seq samples from other TCGA tumors. Our results suggest that GPC6 may play a role in tumor metastatic progression. In TCGA melanoma samples, we also showed that GPC6 expression was negatively correlated with miR-509-3p, which has previously been shown to function as a tumor suppressor in various cancer cell lines. We overexpressed miR-509-3p in A375 melanoma cells and showed that GPC6 expression was significantly suppressed. This result suggested that GPC6 was a putative target of miR-509-3p in melanoma. Together, our findings identified GPC6 as an early biomarker for melanoma metastatic progression, one that can be regulated by miR-509-3p. [ABSTRACT FROM AUTHOR]

Targeted therapies have become the focus of much of the cancer therapy research conducted in the United States. While these therapies have made vast improvements in the treatment of cancer, their results have been somewhat disappointing due to acquired resistances, high cost, and limited populations of susceptible patients. As a result, targeted therapeutics are often combined with other targeted therapeutics or chemotherapies. Compounds which target more than one cancer related pathway are rare, but have the potential to synergize multiple components of therapeutic cocktails. Natural products, as opposed to targeted therapies, typically interact with multiple cellular targets simultaneously, making them a potential source of synergistic cancer treatments. In this study, a rare natural product, deacetylnemorone, was shown to inhibit cell growth in a broad spectrum of cancer cell lines, selectively induce cell death in melanoma cells, and inhibit angiogenesis and invasion. Combined, these results demonstrate that deacetylnemorone affects multiple cancer-related targets associated with tumor growth, drug resistance, and metastasis. Thus, the multi-targeting natural product, deacetylnemorone, has the potential to enhance the efficacy of current cancer treatments as well as reduce commonly acquired treatment resistance. [ABSTRACT FROM AUTHOR]

Phenotypic plasticity is associated with non-genetic drug tolerance in several cancers. Such plasticity can arise from chromatin remodeling, transcriptomic reprogramming, and/or protein signaling rewiring, and is characterized as a cell state transition in response to molecular or physical perturbations. This, in turn, can confound interpretations of drug responses and resistance development. Using BRAF-mutant melanoma cell lines as the prototype, we report on a joint theoretical and experimental investigation of the cell-state transition dynamics associated with BRAF inhibitor drug tolerance. Thermodynamically motivated surprisal analysis of transcriptome data was used to treat the cell population as an entropy maximizing system under the influence of time-dependent constraints. This permits the extraction of an epigenetic potential landscape for drug-induced phenotypic evolution. Single-cell flow cytometry data of the same system were modeled with a modified Fokker-Planck-type kinetic model. The two approaches yield a consistent picture that accounts for the phenotypic heterogeneity observed over the course of drug tolerance development. The results reveal that, in certain plastic cancers, the population heterogeneity and evolution of cell phenotypes may be understood by accounting for the competing interactions of the epigenetic potential landscape and state-dependent cell proliferation. Accounting for such competition permits accurate, experimentally verifiable predictions that can potentially guide the design of effective treatment strategies. [ABSTRACT FROM AUTHOR]

Graphene Oxide (GO) has recently attracted substantial attention in biomedical field as an effective platform for biological sensing, tissue scaffolds and in vitro fluorescence imaging. However, the targeting modality and the capability of its in vivo detection have not been explored. To enhance the functionality of GO, we combine it with superparamagnetic iron oxide nanoparticles (Fe3O4 NPs) serving as a biocompatible magnetic drug delivery addends and magnetic resonance contrast agent for MRI. Synthesized GO-Fe3O4 conjugates have an average size of 260 nm and show low cytotoxicity comparable to that of GO. Fe3O4 nanoparticles provide superparamagnetic properties for magnetic targeted drug delivery allowing simple manipulation by the magnetic field and magnetic resonance imaging with high r2/r1 relaxivity ratios of ~10.7. GO-Fe3O4 retains pH-sensing capabilities of GO used in this work to detect cancer versus healthy environments in vitro and exhibits fluorescence in the visible for bioimaging. As a drug delivery platform GO-Fe3O4 shows successful fluorescence-tracked transport of hydrophobic doxorubicin non-covalently conjugated to GO with substantial loading and 2.5-fold improved efficacy. As a result, we propose GO-Fe3O4 nanoparticles as a novel multifunctional magnetic targeted platform for high efficacy drug delivery traced in vitro by GO fluorescence and in vivo via MRI capable of optical cancer detection. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis infections are the leading cause of sexually transmitted infections of bacterial origin. Lower genital tract infections are often asymptomatic, and therefore left untreated, leading to ascending infections that have long-term consequences on female reproductive health. Human pathology can be recapitulated in mice with the mouse adapted strain C. muridarum. Eight years into the post-genetic era, significant advances to expand the Chlamydia genetic toolbox have been made to facilitate the study of this important human pathogen. However, the need for additional tools remains, especially for C. muridarum. Here, we describe a new set of spectinomycin resistant E. coli-Chlamydia shuttle vectors, for C. trachomatis and C. muridarum. These versatile vectors allow for expression and localization studies of Chlamydia effectors, such as Inc proteins, and will be instrumental for mutant complementation studies. In addition, we have exploited the differential expression of specific Chlamydia genes during the developmental cycle to engineer an omcA: :gfp fluorescent transcriptional reporter. This novel tool allows for monitoring RB to EB conversion at the bacterial level. Spatiotemporal tracking of GFP expression within individual inclusions revealed that RB to EB conversion initiates in bacteria located at the edge of the inclusion and correlates with the time post initiation of bacterial replication and inclusion size. Comparison between primary and secondary inclusions potentially suggests that the environment in which the inclusions develop influences the timing of conversion. Altogether, the Chlamydia genetic tools described here will benefit the field, as we continue to investigate the molecular mechanisms underlying Chlamydia-host interaction and pathogenesis. [ABSTRACT FROM AUTHOR]

Demethoxycurcumin (DMC) is a safe and natural food-coloring additive, as well as an agent with several therapeutic properties. However, extensive glucuronidation in vivo has resulted in its poor bioavailability. In this study, we aimed to investigate the formation of DMC-O-glucuronides by uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1) and its transport by breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs) in HeLa cells stably transfected with UGT1A1 (named HeLa1A1 cells). The chemical inhibitors Ko143 (a selective BCRP inhibitor) and MK571 (a pan-MRP inhibitor) both induced an obvious decrease in the excretion rate of DMC-O-glucuronides and a significant increase in intracellular DMC-O-glucuronide concentrations. Furthermore, BCRP knock-down resulted in a marked reduction in the level of excreted DMC-O-glucuronides (maximal 55.6%), whereas MRP1 and MRP4 silencing significantly decreased the levels of excreted DMC-O-glucuronides (a maximum of 42.9% for MRP1 and a maximum of 29.9% for MRP3), respectively. In contrast, neither the levels of excreted DMC-O-glucuronides nor the accumulation of DMC-O-glucuronides were significantly altered in the MRP4 knock-down HeLa cells. The BCRP, MRP1 and MRP3 transporters were identified as the most important contributors to the excretion of DMC-O-glucuronides. These results may significantly contribute to improving our understanding of mechanisms underlying the cellular disposition of DMC via UGT-mediated metabolism. [ABSTRACT FROM AUTHOR]

Organelle selective imaging can reveal structural and functional characters of cells undergoing external stimuli, and is considered critical in revealing biological fundamentals, designing targeted delivery system, and screening potential drugs and therapeutics. This paper describes the nucleoli targeting ability of nanoscale carbon dots (including nanodiamond) that are hydrothermally made with controlled surface charges. The surface charges of carbon dots are controlled in the range of -17.9 to -2.84 mV by changing the molar ratio of two precursors, citric acid (CA) and ethylenediamine (EDA). All carbon dots samples show strong fluorescence under wide excitation wavelength, and samples with both negative and positve charges show strong fluorescent contrast from stained nucleoli. The nucleoli selective imaging of live cell has been confirmed with Hoechst staining and nucleoli specific staining (SYTO RNA-select green), and is explained as surface charge heterogeneity on carbon dots. Carbon dots with both negative and positive charges have better ability to penetrate cell and nucleus membranes, and the charge heterogeneity helps carbon dots to bind preferentially to nucleoli, where the electrostatic environment is favored. [ABSTRACT FROM AUTHOR]

The function of AarF domain-containing kinase 1 (ADCK1) has not been thoroughly revealed. Here we identified that ADCK1 utilizes YME1-like 1 ATPase (YME1L1) to control optic atrophy 1 (OPA1) and inner membrane mitochondrial protein (IMMT) in regulating mitochondrial dynamics and cristae structure. We firstly observed that a serious developmental impairment occurred in Drosophila ADCK1 (dADCK1) deletion mutant, resulting in premature death before adulthood. By using temperature sensitive ubiquitously expression driver tub-Gal80ts/tub-Gal4 or muscle-specific expression driver mhc-Gal4, we observed severely defective locomotive activities and structural abnormality in the muscle along with increased mitochondrial fusion in the dADCK1 knockdown flies. Moreover, decreased mitochondrial membrane potential, ATP production and survival rate along with increased ROS and apoptosis in the flies further demonstrated that the structural abnormalities of mitochondria induced by dADCK1 knockdown led to their functional abnormalities. Consistent with the ADCK1 loss-of-function data in Drosophila, ADCK1 over-expression induced mitochondrial fission and clustering in addition to destruction of the cristae structure in Drosophila and mammalian cells. Interestingly, knockdown of YME1L1 rescued the phenotypes of ADCK1 over-expression. Furthermore, genetic epistasis from fly genetics and mammalian cell biology experiments led us to discover the interactions among IMMT, OPA1 and ADCK1. Collectively, these results established a mitochondrial signaling pathway composed of ADCK1, YME1L1, OPA1 and IMMT, which has essential roles in maintaining mitochondrial morphologies and functions in the muscle. [ABSTRACT FROM AUTHOR]

Many receptor tyrosine kinases (RTKs, such as EGFR, MET) are negatively regulated by ubiquitination and degradation mediated by Cbl proteins, a family of RING finger (RF) ubiquitin ligases (E3s). Loss of Cbl protein function is associated with malignant transformation driven by increased RTK activity. RF E3s, such as the Cbl proteins, interact with a ubiquitin-conjugating enzyme (E2) to confer specificity to the ubiquitination process and direct the transfer of ubiquitin from the E2 to one or more lysines on the target proteins. Using in vitro E3 assays and yeast two-hybrid screens, we found that Ube2d, Ube2e families, Ube2n/2v1, and Ube2w catalyze autoubiquitination of the Cbl protein and Ube2d2, Ube2e1, and Ube 2n/2v1 catalyze Cbl-mediated substrate ubiquitination of the EGFR and SYK. Phosphorylation of the Cbl protein by by Src resulted in increased E3 activity compared to unphosphorylated cbl or Cbl containing a phosphomimetic Y371E mutation. Ubiquitin chain formation depended on the E2 tested with Cbl with Ube2d2 forming both K48 and K63 linked chains, Ube2n/2v1 forming only K63 linked chains, and Ube2w inducing monoubiquitination. In cells, the Ube2d family, Ube2e family, and Ube2n/2v1 contributed to EGFR ubiquitination. Our data suggest that multiple E2s can interact with Cbl and modulate its E3 activity in vitro and in cells. [ABSTRACT FROM AUTHOR]

Background: Syphilis affects approximately 11 million people each year globally, and is the third most prevalent sexually transmitted bacterial infection in the United States. Inability to independently culture and genetically manipulate Treponema pallidum subsp. pallidum, the causative agent of this disease, has hindered our understanding of the molecular mechanisms of syphilis pathogenesis. Here, we used the non-infectious and poorly adherent B314 strain of the Lyme disease-causing spirochete, Borrelia burgdorferi, to express two variants of a known fibronectin-binding adhesin, Tp0136, from T. pallidum SS14 and Nichols strains. Using this surrogate system, we investigated the ability of Tp0136 in facilitating differential binding to mammalian cell lines offering insight into the possible role of this virulence factor in colonization of specific tissues by T. pallidum during infection. Principal findings: Expression of Tp0136 could be detected on the surface of B. burgdorferi by indirect immunofluorescence assay using sera from a secondary syphilis patient that does not react with intact B314 spirochetes transformed with the empty vector. Increase in Tp0136-mediated adherence of B314 strain to human epithelial HEK293 cells was observed with comparable levels of binding exhibited by both Tp0136 alleles. Adherence of Tp0136-expressing B314 was highest to epithelial HEK293 and C6 glioma cells. Gain in binding of B314 strain expressing Tp0136 to purified fibronectin and poor binding of these spirochetes to the fibronectin-deficient cell line (HEp-2) indicated that Tp0136 interaction with this host receptor plays an important role in spirochetal attachment to mammalian cells. Furthermore, preincubation of these cell lines with fibronectin-binding peptide from Staphylococcus aureus FnbA-2 protein significantly inhibited binding of B314 expressing Tp0136. Conclusions: Our results show that Tp0136 facilitates differential level of binding to cell lines representing various host tissues, which highlights the importance of this protein in colonization of human organs by T. pallidum and resulting syphilis pathogenesis. [ABSTRACT FROM AUTHOR]

Cell culture is a ubiquitous and flexible research method. However, it heavily relies on plastic consumables generating millions of tonnes of plastic waste yearly. Plastic waste is a major and growing global concern. Here we describe a new cell culture dish that offers a culture area equivalent to three petri dishes but that is on average 61% lighter and occupies 67% less volume. Our dish is composed of a lid and three thin containers surrounded by a light outer shell. Cell culture can be performed in each of the containers sequentially. The outer shell provides the appropriate structure for the manipulation of the dish as a whole. The prototype was tested by sequentially growing cells in each of its containers. As a control, sequential cultures in groups of 3 petri dishes were performed. No statistical differences were found between the prototype and the control in terms of cell number, cell viability or cell distribution. [ABSTRACT FROM AUTHOR]

In their comment, Chen and Chuang [] pointed out several weak points of our recent paper []. Here we respond in detail to clarify the dataset we used in our work. We also discuss the three confounding factors they listed in their comment. [ABSTRACT FROM AUTHOR]

Survival rates of osteosarcoma patients could not be significantly improved by conventional chemotherapeutic treatment regimens since the introduction of high-dose chemotherapy 35 years ago. Therefore, there is a strong clinical need for new therapeutic targets and personalized treatment strategies, requiring reliable in vivo model systems for the identification and testing of potential new treatment approaches. Conventional in vivo rodent experiments face ethical issues, are time consuming and costly, being of particular relevance in orphan diseases like osteosarcoma. An attractive alternative to such animal experiments is the chicken chorioallantoic membrane (CAM) assay. The CAM is a highly vascularized, non-innervated extra-embryonic membrane that is perfectly suited for the engraftment of tumor cells. However, only few reports are available for osteosarcoma and reported data are inconsistent. Therefore, the aim of this study was the adaptation and optimization of the CAM assay for its application in osteosarcoma research. Tumor take rates and volumes of osteosarcoma that developed on the CAM were analyzed after modification of several experimental parameters, including egg windowing, CAM pretreatment, inoculation technique and many more. Eight osteosarcoma cell lines were investigated. Our optimized OS-CAM-assay was finally validated against a rat animal xenograft model. Using the cell line MNNG HOS as reference we could improve the tumor take rates from 51% to 94%, the viability of the embryos from initially 40% to >80% and achieved a threefold increase of the tumor volumes. We were able to generate solid tumors from all eight osteosarcoma cell lines used in this study and could reproduce results that were obtained using an osteosarcoma rat animal model. The CAM assay can bridge the gap between in vitro cell culture and in vivo animal experiments. As reliable in vivo model for osteosarcoma research the optimized CAM assay may speed up preclinical data collection and simplifies research on potential new agents towards personalized treatment strategies. Further, in accordance with Russell’s and Burch’s “Principles of Humane Experimental Technique” the reasonable use of this model provides a refinement by minimizing pain and suffering of animals and supports a considerable reduction and/or replacement of animal experiments. [ABSTRACT FROM AUTHOR]

While investigating the role played by de novo lipid (DNL) biosynthesis in cancer cells, we sought a medium condition that would support cell proliferation without providing any serum lipids. Here we report that a defined serum free cell culture medium condition containing insulin, transferrin and selenium (ITS) supports controlled study of transcriptional regulation of de novo fatty acid (DNFA) production and de novo cholesterol synthesis (DNCS) in melanoma cell lines. This lipid-free ITS medium is able to support continuous proliferation of several melanoma cell lines that utilize DNL to support their lipid requirements. We show that the ITS medium stimulates gene transcription in support of both DNFA and DNCS, specifically mediated by SREBP1/2 in melanoma cells. We further found that the ITS medium promoted SREBP1 nuclear localization and occupancy on DNFA gene promoters. Our data show clear utility of this serum and lipid-free medium for melanoma cancer cell culture and lipid-related areas of investigation. [ABSTRACT FROM AUTHOR]

p21, an inhibitor of cyclin-dependent kinase, functions as an oncogene or tumor suppressor depending on the context of a variety of extracellular and intracellular signals. The expression of p21 could be regulated at the transcriptional and/or post-translational levels. The p21 gene is well-known to be regulated in both p53-dependent and -independent manners. However, the detailed regulatory mechanisms of p21 messenger RNA and protein expression via statins remain unknown, and the possible application of statins as anticancer reagents remains to be controversial. Our data showed that the statins—fluvastatin and lovastatin—induced p21 expression as general histone deacetylase inhibitors in a p53-independent manner, which is mediated through various pathways, such as apoptosis, autophagy, cell cycle progression, and DNA damage, to be involved in the function of p21 in HeLa cells. The curative effect repositioning of digoxin, a cardiovascular medication, was combined with fluvastatin and lovastatin, and the results further implied that p21 induction is involved in a p53-dependent and p53-independent manner. Digoxin modified the effects of statins on ATF3, p21, p53, and cyclin D1 expression, while fluvastatin boosted its DNA damage effect and lovastatin impeded its DNA damage effect. Fluvastatin and lovastatin combined with digoxin further support the localization specificity of their interactivity with our subcellular localization data. This study will not only clarify the regulatory mechanisms of p21 induction by statins but will also shed light on the repurposing of widely cardiovascular medications for the treatment of cervical cancer. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection, responsible for millions of infections each year. Despite this high prevalence, the elucidation of the molecular mechanisms of Chlamydia pathogenesis has been difficult due to limitations in genetic tools and its intracellular developmental cycle. Within a host epithelial cell, chlamydiae replicate within a vacuole called the inclusion. Many Chlamydia–host interactions are thought to be mediated by the Inc family of type III secreted proteins that are anchored in the inclusion membrane, but their array of host targets are largely unknown. To investigate how the inclusion membrane proteome changes over the course of an infected cell, we have adapted the APEX2 system of proximity-dependent biotinylation. APEX2 is capable of specifically labeling proteins within a 20 nm radius in living cells. We transformed C. trachomatis to express the enzyme APEX2 fused to known inclusion membrane proteins, allowing biotinylation and purification of inclusion-associated proteins. Using quantitative mass spectrometry against APEX2 labeled samples, we identified over 400 proteins associated with the inclusion membrane at early, middle, and late stages of epithelial cell infection. This system was sensitive enough to detect inclusion interacting proteins early in the developmental cycle, at 8 hours post infection, a previously intractable time point. Mass spectrometry analysis revealed a novel, early association between C. trachomatis inclusions and endoplasmic reticulum exit sites (ERES), functional regions of the ER where COPII-coated vesicles originate. Pharmacological and genetic disruption of ERES function severely restricted early chlamydial growth and the development of infectious progeny. APEX2 is therefore a powerful in situ approach for identifying critical protein interactions on the membranes of pathogen-containing vacuoles. Furthermore, the data derived from proteomic mapping of Chlamydia inclusions has illuminated an important functional role for ERES in promoting chlamydial developmental growth. [ABSTRACT FROM AUTHOR]

Cultured cell populations are composed of heterogeneous cells, and previous single-cell lineage tracking analysis of individual HeLa cells provided empirical evidence for significant heterogeneity of the rate of cell proliferation and induction of cell death. Nevertheless, such cell lines have been used for investigations of cellular responses to various substances, resulting in incomplete characterizations. This problem caused by heterogeneity within cell lines could be overcome by investigating the spatiotemporal responses of individual cells to a substance. However, no approach to investigate the responses by analyzing spatiotemporal data is currently available. Thus, this study aimed to analyze the spatiotemporal responses of individual HeLa cells to cytotoxic, sub-cytotoxic, and non-cytotoxic doses of the well-characterized carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although cytotoxic doses of MNNG are known to induce cell death, the single-cell tracking approach revealed that cell death occurred following at least four different cellular events, suggesting that cell death is induced via multiple processes. We also found that HeLa cells exposed to a sub-cytotoxic dose of MNNG were in a state of equilibrium between cell proliferation and cell death, with cell death again induced through different processes. However, exposure of cells to a non-cytotoxic dose of MNNG promoted growth by reducing the cell doubling time, thus promoting the growth of a sub-population of cells. A single-cell lineage tracking approach could dissect processes leading to cell death in a spatiotemporal manner and the results suggest that spatiotemporal data obtained by tracking individual cells can be used as a new type of bioinformatics data resource that enables the examination of cellular responses to various external substances. [ABSTRACT FROM AUTHOR]

The family of oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs) mediate sterol and phospholipid transfer and signaling at membrane contact sites (MCS). The activity of OSBP at MCS is regulated by phosphorylation, but whether this applies to ORPs is unknown. Here we report the functional characterization of a unique proline/serine-rich phosphorylation motif (S762SPSSPSS769) in the lipid binding OSBP-related domain of full-length ORP4L and a truncated variant ORP4S. Phosphorylation was confirmed by mass spectrometry and [32P]PO4 incorporation, and in silico and in vitro assays using purified ORP4L identified putative proline-directed kinases that phosphorylate the site. The functional significance of the phospho-site was assessed by mutating serine 762, S763, S766 and S768 to aspartate or alanine to produce phosphomimetic (S4D) and phosphorylation-deficient (S4A) mutants, respectively. Solution binding of 25-hydroxycholesterol and cholesterol by recombinant ORP4L-S4D and -S4A was similar to wild-type but ORP4L-S4D more effectively extracted cholesterol from liposomes. ORP4L homo-dimerization was unaffected by phosphorylation but gel filtration of ORP4L-S4D indicated that the native conformation was affected. Confocal microscopy revealed that ORP4L-S4D also strongly associated with bundled vimentin filaments, a feature shared with ORP4S which lacks the PH and dimerization domains. We conclude that phosphorylation of a unique serine/proline motif in the ORD induces a conformation change in ORP4L that enhances interaction with vimentin and cholesterol extraction from membranes. [ABSTRACT FROM AUTHOR]

The Cytolethal Distending Toxin (CDT) is produced by many pathogenic bacteria. CDT is known to induce genomic DNA damage to host eukaryotic cells through its catalytic subunit, CdtB. CdtB is structurally homologous to DNase I and has a nuclease activity, dependent on several key residues. Yet some differences between various CdtB subunit activities, and discrepancies between biochemical and cellular data, have been observed. To better characterise the role of CdtB in the induction of DNA damage, we affinity-purified wild-type and mutants of CdtB, issued from E. coli and H. ducreyi, under native and denaturing conditions. We then compared their nuclease activity by a classic in vitro assay using plasmid DNA, and two different eukaryotic assays–the first assay where host cells were transfected with a plasmid encoding CdtB, the second assay where host cells were directly transfected with purified CdtB. We show here that in vitro nuclease activities are difficult to quantify, whereas CdtB activities in host cells can be easily interpreted and confirmed the loss of function of the catalytic mutant. Our results highlight the importance of performing multiple assays while studying the effects of bacterial genotoxins, and indicate that the classic in vitro assay should be complemented with cellular assays. [ABSTRACT FROM AUTHOR]

Tumour cell immune evasion is a principal hallmark of successful metastasis. Tumour cells in the vasculature adopt a platelet cloak that efficiently suppresses the innate immune system by directly inhibiting Natural Killer (NK) cells, which normally function to neutralise spreading cancers. Here we describe two novel mechanisms of tumour cell evasion of NK cell anti-tumour functions. The first, an ‘immune decoy’ mechanism in which platelets induce the release of soluble NKG2D ligands from the tumour cell to mask detection and actively suppress NK cell degranulation and inflammatory cytokine (IFNγ) production, concomitantly. This represents a double-hit to immune clearance of malignant cells during metastasis. The second mechanism, a platelet-derived TGFβ-mediated suppression of the CD226/CD96-CD112/CD155 axis, is a novel pathway with poorly understood anti-cancer functions. We have demonstrated that platelets robustly suppress surface expression of CD226 and CD96 on the NK cell surface and their associated ligands on the tumour cell to further enhance NK cell suppression. These highly evolved mechanisms promote successful tumour immune evasion during metastasis and provide a unique opportunity for studying the complexity of cellular interactions in the metastatic cascade and thus novel targets for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

The enzymes GALNTs add GalNAc sugar to Ser and Thr residues, forming the Tn glycan. GALNTs are activated by trafficking from Golgi to ER, a process driven by the Src kinase and negatively regulated by ERK8. This GALNTs activation (aka GALA) pathway induces high Tn levels and is a key driver of liver tumor growth. Recently, Tabak and colleagues have contested our previous data that EGF stimulation can induce GALNTs relocation. Here, we show that relocation induced by EGF is actually detectable in the very images acquired by Tabak et al. Furthermore, we show that over-expression of EGFR strongly enhances EGF-induced relocation and that EGFR appears required to drive relocation induced by ERK8 depletion. Direct co-localisation of GALNT with the ER marker Calnexin is observed after EGF stimulation. We furthermore propose that quantification of O-glycosylation of the ER resident protein PDIA4 provides a mean to quantify GALA independently of imaging. In sum, we demonstrate that the claimed non-reproducibility was due to experimental imaging conditions, that EGFR is indeed a driver of GALA and propose additional markers to facilitate the study of this pathway. [ABSTRACT FROM AUTHOR]

Diarrheic diseases account for the annual death of approximately 1.9 million children under the age of 5 years, and it is a major cause of work absenteeism in developed countries. As diarrheagenic bacteria, enteropathogenic Escherichia coli (EPEC) attach to cells in the small intestine, causing local disappearance of microvilli and inducing the formation of actin-rich pedestals that disrupt the intestinal barrier and help EPEC adhere to and infect intestinal cells. Antibiotics and other bioactive compounds can often be found by analyzing traditional medicines. Here a crude aqueous extract of Hibiscus sabdariffa, which typically grows in subtropical and tropical areas and is a popular medicinal tisane in many countries, was analyzed for antibacterial activity against EPEC. In standard microdilution assays, the extract showed a minimum inhibitory concentration of 6.5 mg/ml against EPEC growth. Time-kill kinetics assays demonstrated significant 24 h bactericidal activity at 25 mg/ml. The extract is able to impede pedestal induction. Not only did the extract inhibit preformed pedestals but it prevented pedestal induction as well. Remarkably, it also promoted the formation of EPEC filaments, as observed with other antibiotics. Our results in vitro support the potential of Hibiscus sabdariffa as an antimicrobial agent against EPEC. [ABSTRACT FROM AUTHOR]

Leprosy is a chronic infection where the skin and peripheral nervous system is invaded by Mycobacterium leprae. The infection mechanism remains unknown in part because culture methods have not been established yet for M. leprae. Mce1A protein (442 aa) is coded by mce1A (1326 bp) of M. leprae. The Mce1A homolog in Mycobacterium tuberculosis is known to be associated with M. tuberculosis epithelial cell entry, and survival and multiplication within macrophages. Studies using recombinant proteins have indicated that Mce1A of M. leprae is also associated with epithelial cell entry. This study is aimed at identifying particular sequences within Mce1A associated with M. leprae epithelial cell entry. Recombinant proteins having N-terminus and C-terminus truncations of the Mce1A region of M. leprae were created in Escherichia coli. Entry activity of latex beads, coated with these truncated proteins (r-lep37 kDa and r-lep27 kDa), into HeLa cells was observed by electron microscopy. The entry activity was preserved even when 315 bp (105 aa) and 922 bp (308 aa) was truncated from the N-terminus and C-terminus, respectively. This 316–921 bp region was divided into three sub-regions: 316–531 bp (InvX), 532–753 bp (InvY), and 754–921 bp (InvZ). Each sub-region was cloned into an AIDA vector and expressed on the surface of E. coli. Entry of these E. coli into monolayer-cultured HeLa and RPMI2650 cells was observed by electron microscopy. Only E. coli harboring the InvX sub-region exhibited cell entry. InvX was further divided into 4 domains, InvXa—InvXd, containing sequences 1–24 aa, 25–46 aa, 47–57 aa, and 58–72 aa, respectively. Recombinant E. coli, expressing each of InvXa—InvXd on the surface, were treated with antibodies against these domains, then added to monolayer cultured RPMI cells. The effectiveness of these antibodies in preventing cell entry was studied by colony counting. Entry activity was suppressed by antibodies against InvXa, InvXb, and InvXd. This suggests that these three InvX domains of Mce1A are important for M. leprae invasion into nasal epithelial cells. [ABSTRACT FROM AUTHOR]

Adolescent Idiopathic Scoliosis (AIS) is a spinal deformity that affects approximately 3 percent of human adolescents. Although the etiology and molecular basis of AIS is unclear, several genes such as POC5 have been identified as possible causes of the condition. In order to understand the role of POC5 in the pathogenesis of AIS, we investigated the subcellular localization of POC5 in cilia of cells over-expressing either the wild type (wt) or an AIS-related POC5 variant POC5A429V. Mutation of POC5 was found to alter its subcellular localization and to induce ciliary retraction. Furthermore, we observed an impaired cell-cycle progression with the accumulation of cells in the S-phase in cells expressing POC5A429V. Using immunoprecipitation coupled to mass spectrometry, we identified specific protein interaction partners of POC5, most of which were components of cilia and cytoskeleton. Several of these interactions were altered upon mutation of POC5. Altogether, our results demonstrate major cellular alterations, disturbances in centrosome protein interactions and cilia retraction in cells expressing an AIS-related POC5 mutation. Our study suggests that defects in centrosomes and cilia may underlie AIS pathogenesis. [ABSTRACT FROM AUTHOR]

Glioblastoma is the leading malignant glioma with a poor prognosis. This study aimed to investigate the antitumor effects of natural killer cells in combination with temozolomide as the standard chemotherapeutic agent for glioblastoma. Using a simple, feeder-less, and chemically defined culture method, we expanded human peripheral blood mononuclear cells and assessed the receptor expression, natural killer cell activity, and regulatory T cell frequency in expanded cells. Next, using the standard human glioblastoma cell lines (temozolomide-sensitive U87MG, temozolomide-resistant T98G, and LN-18), we assessed the ligand expressions of receptors on natural killer cells. Furthermore, the antitumor effects of the combination of the expanded natural killer cells and temozolomide were assessed using growth inhibition assays, apoptosis detection assays, and senescence-associated β-galactosidase activity assays in the glioblastoma cell lines. Novel culture systems were sufficient to attain highly purified (>98%), expanded (>440-fold) CD3−/CD56+ peripheral blood-derived natural killer cells. We designated the expanded population as genuine induced natural killer cells. Genuine induced natural killer cells exhibited a high natural killer activity and low regulatory T cell frequency compared with lymphokine-activated killer cells. Growth inhibition assays revealed that genuine induced natural killer cells inhibited the glioblastoma cell line growth but enhanced temozolomide-induced inhibition effects in U87MG. Apoptosis detection assays revealed that genuine induced natural killer cells induced apoptosis in the glioblastoma cell lines. Furthermore, senescence-associated β-galactosidase activity assays revealed that temozolomide induced senescence in U87MG. Genuine induced natural killer cells induce apoptosis in temozolomide-sensitive and temozolomide-resistant glioblastoma cells and enhances temozolomide-induced antitumor effects in different mechanisms. Hence, the combination of genuine induced natural killer cells and temozolomide may prove to be a promising immunochemotherapeutic approach in patients with glioblastoma if the antitumor effects in vivo can be demonstrated. [ABSTRACT FROM AUTHOR]

This study describes the comparative expression and purification of hepatitis B surface antigen (HBsAg) particles produced upon infection of human primary hepatocytes and human hepatoma cell lines (HuH-7 and HepG2) with recombinant vaccinia viruses. The highest levels of HBsAg expression were found in HuH-7 hepatoma cells following infection with recombinant vaccinia viruses, which contain the S gene under control of a 7.5 k-promoter. Four different methods for purification of the HBsAg particles were examined: isopycnic ultracentrifugation, sucrose cushion sedimentation, isocratic column gel filtration, and binding to anti-HBs-coated microparticles. The highest degree of purity of HBsAg particles was reached by the method based on anti-HBs-coated microparticles. The resulting product was >98% pure. Biochemical analysis and characterization of purified HBsAg particles were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and electron microscopy. The HBsAg, purified from human hepatoma cell lines and from human primary hepatocytes, consisted of both the non-glycosylated (p25) and the glycosylated (gp27) form and assembled into typical 22-nm particles, and thus may be of great interest and importance for research, diagnostics, and medical treatments. [ABSTRACT FROM AUTHOR]