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The addition of heteroatoms to pristine carbon quantum dots (CQDs) change their structure and optical properties. In this study, fluorine (F)- and chlorine (Cl)-doped CQDs are prepared by the one-step green hydrothermal route from sodium fluoride, sodium chloride, urea, and citric acid as the starting precursors. Microscopy analysis reveals that the average size of these quantum dots is 5 ± 2 nm, whereas the chemical study shows the existence of C–F and C–Cl bonds. The produced F- and Cl-doped CQDs have fluorescence quantum yields of 0.151 and 0.284, respectively, at an excitation wavelength of 450 nm. Charge transfer resistance of F- and Cl-doped CQDs films is 2 orders of magnitude higher than in the pristine CQD films. Transport band gap of the doped CQDs is 2 eV bigger than that of pristine CQDs. Radical scavenging activity shows very good antioxidant activity of doped CQDs. Antibacterial testing reveals poor antibacterial activity against Staphylococcus aureus and Escherichia coli. The F- and Cl-doped CQDs are successfully used as fluorescent probes for cell imaging as shown by confocal microscopy.

Hypoxia is a phenomenon often arising in solid tumours, linked to aggressive malignancy, bad prognosis and resistance to therapy. Hypoxia-inducible factor-1 has been identified as a key mediator of cell and tissue adaptation to hypoxic conditions through transcriptional activation of many genes involved in glucose metabolism and other cancer-related processes, such as angiogenesis, cell survival and cell invasion. Cyclic adenosine 3′5′-monophosphate is one of the most ancient and evolutionarily conserved signalling molecules and the cAMP/PKA signalling pathway plays an important role in cellular adaptation to hypoxia. We have investigated possible new mechanisms behind hypoxic activation of the cAMP/PKA pathway. For the first time, we have shown that hypoxia induces transcriptional up-regulation of the system of adenylyl cyclases, enzymes responsible for cAMP production, in a panel of carcinoma cell lines of various origin. Our data prove functional relevance of the hypoxic increase of adenylyl cyclases VI and VII at least partially mediated by HIF-1 transcription factor. We have identified adenylyl cyclase VI and VII isoforms as mediators of cellular response to hypoxia, which led to the elevation of cAMP levels and enhanced PKA activity, with an impact on cell migration and pH regulation.

Lymphocytic choriomeningitis virus (LCMV) isLymphocytic choriomeningitis virus (LCMV) is a prototypic arenavirus with bisegmented single-stranded RNA genome. Both segments (small [S] and large [L]) contain two open reading frames in mutually opposite orientations and utilize an ambisense coding strategy (19). The S RNA encodes a major viral protein nucleoprotein (NP) and a glycoprotein precursor (GP-C), which is cotranslationally cleaved into peripheral glycoprotein 1 (GP1) and transmembrane glycoprotein 2 (GP2) (30). The L RNA segment encodes an RNA-dependent RNA polymerase (L) and a regulatory ring finger Z protein (ZP) (2, 26). Virus replication starts with the L polymerase-driven transcription of the 3Ј RNA genome arms of negative polarity and produces mRNAs that are subsequently translated to NP and L polymerase. These viral proteins assist in the transcription of the RNA genome to virus cRNA, serving as a template for the synthesis of the new genomic RNA molecules as well as for the subgenomic mRNAs translated to GP-C and ZP (2, 19). This unusual two-stage replication strategy facilitates establishment of virus persistence, which can be sustained by the virus ribonucleoprotein composed of NP, the RNA genome, and L polymerase in the absence of mature virion production caused by absent or limited expression of glycoproteins (2, 33). LCMV can easily set up persistent infection in a wide variety of cell types derived from various species, where it does not perturb vital cell functions but modulates nonessential phenotypic features (20,22,33).In vivo, LCMV readily causes persistent infection of common house mouse (Mus musculus), its natural host and reservoir. Humans are generally infected through the respiratory tract after direct or indirect contact with infected rodents or pets. In immunocompetent individuals, LCMV causes illnesses varying from mild flu-like symptoms to rare severe encephalitis (15,22). Infection with this virus during pregnancy has been linked to spontaneous abortions and malformations (16). More strikingly, fatal cases of LCMV infections transmitted via transplanted organs from infected donors to immunosuppressed recipients were recently reported and call for more attention to this seemingly innocent virus (1, 7).The MX strain of LCMV was originally identified in the human MaTu cell line, which was presumably derived from a mammary tumor as described earlier (21,25). Analysis of the MX LCMV coding regions has revealed sequence differences supporting the view that MX is a separate LCMV strain (9,25,32). MX LCMV does not cause any cellular damage, and its host range is restricted to only few cell lines. It is transmissible by direct cell-to-cell contact or cell extract but not by filtered medium of infected cells (25). Furthermore, cells infected with MX LCMV accumulate high cytoplasmic levels of NP and ZP and contain deleted RNAs (9,25,32).Despite NP being the most abundant viral protein expressed in persistently infected cells and a major component of the minimal infection unit of the vir...

BACKGROUND: Carbonic anhydrase IX (CA IX) is a hypoxia-induced enzyme regulating tumour pH and facilitating cell migration/ invasion. It is primarily expressed as a transmembrane cell-surface protein, but its ectodomain can be shed by ADAM17 to extracellular space. This study aims to elucidate the impact of CA IX shedding on cancer cells. METHODS: We generated a non-shed CA IX mutant by deletion of amino acids 393-402 from the stalk region and studied its phenotypic effects compared to full-length, shedding-competent CA IX using a range of assays based on immunodetection, confocal microscopy, in vitro real-time cell monitoring and in vivo tumour cell inoculation using xenografted NMRI and C57BL/6J female mice. RESULTS: We demonstrated that the impairment of shedding does not alter the ability of CA IX to bind ADAM17, internalise, form oligomers and regulate pH, but induces cancer-promoting changes in extracellular proteome. Moreover, it affects intrinsic properties of cells expressing the non-shed variant, in terms of their increased ability to migrate, generate primary tumours and form metastatic lesions in lungs. CONCLUSIONS: Our results show that the ectodomain shedding controls pro-tumorigenic and pro-metastatic roles of the cellassociated CA IX and suggest that this phenomenon should be considered when developing CA IX-targeted therapeutic strategies.

The physiological context of virus-infected cells can markedly affect multiplication and spread of the virus progeny. During persistent infection, the virus exploits the host cell without disturbing its vital functions. However, microenvironmental hypoxia can uncouple this intimate relationship and escalate virus pathogenesis. Accumulating evidence suggests that hypoxia-inducible factor (HIF) modulates gene expression of the viruses that pass through a DNA stage, contain hypoxia-responsive promoter elements, and replicate in the nucleus. Here we show that hypoxia can influence the gene expression and transmission of the cytoplasmic RNA virus lymphocytic choriomeningitis virus (LCMV), which is a neglected human pathogen and teratogen. The MX strain of LCMV, which we used as a model, replicates in a persistent mode in human HeLa cells, fails to produce mature envelope glycoproteins, and spreads through cell-cell contacts in the absence of extracellular infectious virions. Both exposure of MX-infected HeLa cells to chronic hypoxia and gene transfer approaches led to increased virus RNA transcription and higher levels of the viral proteins via a HIFdependent mechanism. Moreover, hypoxia enhanced the formation of infectious virions capable of transmitting LCMV by cell-free medium. This LCMV "reactivation" might have health-compromising consequences in hypoxia-associated situations, such as fetal development and ischemia-related pathologies.The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) provides an important model for investigations of the mechanisms of viral persistence and pathogenesis. Studies using this model led to major advances in virology and immunology that apply universally to other viral and microbial infections of humans (5,7,43,45). Even though LCMV infections are mostly asymptomatic and often remain unnoticed, compelling evidence indicates that LCMV is a neglected human pathogen of clinical significance, especially in cases of congenital infections leading to an increased risk of spontaneous abortion or central nervous system (CNS) disorders and chorioretinitis (3,4,17,44). Moreover, LCMV poses a special threat to immunocompromised individuals, as tragically illustrated by recent cases of transplant-associated infections by LCMV with fatal outcomes in the United States (13) and Australia (25). LCMV has a bisegmented single-stranded RNA genome and a life cycle confined to the cell cytoplasm. The genome consists of a small segment (S) (3.4 kb) and a large segment (L) (7.2 kb). Each genomic segment uses an ambisense coding strategy to direct the synthesis of two polypeptides from two opposite open reading frames separated by an intergenic region. The S segment encodes a major viral protein nucleoprotein (NP) and a glycoprotein precursor (GPC), which is posttranslationally cleaved into peripheral glycoprotein 1 (GP1) and transmembrane glycoprotein 2 (GP2). The L segment encodes a viral RNA-dependent RNA polymerase (L) and a small regulatory RING domain-containing Z protein (Z) (6, 42). S...

BackgroundCarbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors. Expression of CA IX is driven predominantly by the hypoxia-inducible factor (HIF) pathway and helps to maintain intracellular pH homeostasis under hypoxic conditions, resulting in acidification of the tumor microenvironment. Carnosine (β-alanyl-L-histidine) is an anti-tumorigenic agent that inhibits the proliferation of cancer cells. In this study, we investigated the role of CA IX in carnosine-mediated antitumor activity and whether the underlying mechanism involves transcriptional and translational modulation of HIF-1α and CA IX and/or altered CA IX function.MethodsThe effect of carnosine was studied using two-dimensional cell monolayers of several cell lines with endogenous CA IX expression as well as Madin Darby canine kidney transfectants, three-dimensional HeLa spheroids, and an in vivo model of HeLa xenografts in nude mice. mRNA and protein expression and protein localization were analyzed by real-time PCR, western blot analysis, and immunofluorescence staining, respectively. Cell viability was measured by a flow cytometric assay. Expression of HIF-1α and CA IX in tumors was assessed by immunohistochemical staining. Real-time measurement of pH was performed using a sensor dish reader. Binding of CA IX to specific antibodies and metabolon partners was investigated by competitive ELISA and proximity ligation assays, respectively.ResultsCarnosine increased the expression levels of HIF-1α and HIF targets and increased the extracellular pH, suggesting an inhibitory effect on CA IX-mediated acidosis. Moreover, carnosine significantly inhibited the growth of three-dimensional spheroids and tumor xenografts compared with untreated controls. Competitive ELISA showed that carnosine disrupted binding between CA IX and antibodies specific for its catalytic domain. This finding was supported by reduced formation of the functional metabolon of CA IX and anion exchanger 2 in the presence of carnosine.ConclusionsOur results indicate that interaction of carnosine with CA IX leads to conformational changes of CA IX and impaired formation of its metabolon, which in turn disrupts CA IX function. These findings suggest that carnosine could be a promising anticancer drug through its ability to attenuate the activity of CA IX.

Inositol 1,4,5-trisphosphate (IP(3)) receptors are emerging as key sites for regulation by pro- and anti-apoptotic factors. Induction of apoptosis for 3 h increased mRNA and protein levels of type 1 IP(3) receptors in non-differentiated (ND), but not in differentiated (D) PC12 cells. Inhibitors of the IP(3) R's calcium release-2-aminoethoxydiphenyl borate (2-APB) and xestospongin-completely prevented Bax and caspase-3 mRNA increase after treatment with the apoptosis inducer set (AIK), and this reinforces the importance of IP(3) R1 in the apoptosis of ND PC12 cells. Apoptosis induction not only increases the IP(3) R1 protein, but it also causes formation of IP(3) R1 clusters in the nucleus which most likely result from fusion of the nucleoplasmic reticulum and/or IP(3) R1 translocation to the nucleus. This is quite similar to the observations noted after overexpression of IP(3) R1 in PC12 cells. The amount of IP(3) induced calcium release was higher in control than in AIK-treated cells. From our results we propose that after the apoptosis induction the amount of intranuclear calcium decreased dramatically due to the increase of calcium permeability of the nuclear calcium store vesicles. Therefore, increase of the calcium permeability may result from IP(3) receptors translocation to nuclei that can boost the calcium transport through IP(3) receptors.