MiR-223 Suppresses Cell Proliferation by Targeting IGF-1R

To study the roles of microRNA-223 (miR-223) in regulation of cell growth, we established a miR-223 over-expression model in HeLa cells infected with miR-223 by Lentivirus pLL3.7 system. We observed in this model that miR-223 significantly suppressed the proliferation, growth rate, colony formation of HeLa cells in vitro, and in vivo tumorigenicity or tumor formation in nude mice. To investigate the mechanisms involved, we scanned and examined the potential and putative target molecules of miR-223 by informatics, quantitative PCR and Western blot, and found that insulin-like growth factor-1 receptor (IGF-1R) was the functional target of miR-223 inhibition of cell proliferation. Targeting IGF-1R by miR-223 was not only seen in HeLa cells, but also in leukemia and hepatoma cells. The downstream pathway, Akt/mTOR/p70S6K, to which the signal was mediated by IGF-1R, was inhibited as well. The relative luciferase activity of the reporter containing wild-type 3′UTR(3′untranslated region) of IGF-1R was significantly suppressed, but the mutant not. Silence of IGF-1R expression by vector-based short hairpin RNA resulted in the similar inhibition with miR-223. Contrarily, rescued IGF-1R expression in the cells that over-expressed miR-223, reversed the inhibition caused by miR-223 via introducing IGF-1R cDNA that didn't contain the 3′UTR. Meanwhile, we also noted that miR-223 targeted Rasa1, but the downstream molecules mediated by Rasa1 was neither targeted nor regulated. Therefore we believed that IGF-1R was the functional target for miR-223 suppression of cell proliferation and its downstream PI3K/Akt/mTOR/p70S6K pathway suppressed by miR-223 was by targeting IGF-1R

Identification and Behavioral Evaluation of Sex Pheromone Components of the Chinese Pine Caterpillar Moth, Dendrolimus tabulaeformis

Background: The Chinese pine caterpillar moth, Dendrolimus tabulaeformis Tsai and Liu (Lepidoptera: Lasiocampidae) is the most important defoliator of coniferous trees in northern China. Outbreaks occur over enormous areas and often lead to the death of forests during 2–3 successive years of defoliation. The sex pheromone of D. tabulaeformis was investigated to define its chemistry and behavioral activity. Methodology/Principal Findings: Sex pheromone was collected from calling female D. tabulaeformis by headspace solid phase microextraction (SPME) and by solvent extraction of pheromone glands. Extracts were analyzed by coupled gas chromatography/mass spectrometry (GC-MS) and coupled GC-electroantennographic detection (GC-EAD), using antennae from male moths. Five components from the extracts elicited antennal responses. These compounds were identified by a combination of retention indices, electron impact mass spectral matches, and derivatization as (Z)-5-dodecenyl acetate (Z5-12:OAc), (Z)-5-dodecenyl alcohol (Z5-12:OH), (5Z,7E)-5,7-dodecadien-1-yl acetate (Z5,E7-12:OAc), (5Z,7E)-5,7-dodecadien-1-yl propionate (Z5,E7-12:OPr), and (5Z,7E)-5,7-dodecadien-1-ol (Z5,E7-12:OH). Behavioral assays showed that male D. tabulaeformis strongly discriminated against incomplete and aberrant blend ratios. The correct ratio of Z5,E7-12:OAc, Z5,E7-12:OH, and Z5,E7-12:OPr was essential for optimal upwind flight and source contact. The two monoenes, Z5-12:OAc and Z5-12:OH, alone or binary mixtures, had no effect on behavioral responses when added to the optimal threecomponen

From In Vivo to In Vitro: Dynamic Analysis of Plasmodium falciparum var Gene Expression Patterns of Patient Isolates during Adaptation to Culture

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var gene family, plays a crucial role in disease virulence through its involvement in binding to various host cellular receptors during infection. Growing evidence suggests that differential expression of the various var subgroups may be involved in parasite virulence. To further explore this issue, we have collected isolates from symptomatic patients in south China-Myanmar border, and characterized their sequence diversity and transcription profiles over time of var gene family, and cytoadherence properties from the time of their initial collection and extending through a two month period of adaptation to culture. Initially, we established a highly diverse, DBLα (4 cysteines) subtype-enriched, but unique local repertoire of var-DBL1α sequences by cDNA cloning and sequencing. Next we observed a rapid transcriptional decline of upsA- and upsB-subtype var genes at ring stage through qRT-PCR assays, and a switching event from initial ICAM-I binding to the CD36-binding activity during the first week of adaptive cultivation in vitro. Moreover, predominant transcription of upsA var genes was observed to be correlated with those isolates that showed a higher parasitemia at the time of collection and the ICAM-1-binding phenotype in culture. Taken together, these data indicate that the initial stage of adaptive process in vitro significantly influences the transcription of virulence-related var subtypes and expression of PfEMP1 variants. Further, the specific upregulation of the upsA var genes is likely linked to the rapid propagation of the parasite during natural infection due to the A-type PfEMP1 variant-mediated growth advantages

Expression of CD82 in Human Trophoblast and Its Role in Trophoblast Invasion

BACKGROUND: Well-controlled trophoblast invasion at maternal-fetal interface is a critical event for the normal development of placenta. CD82 is a member of transmembrane 4 superfamily, which showed important role in inhibiting tumor cell invasion and migration. We surmised that CD82 are participates in trophoblast differentiation during placenta development. METHODOLOGY/PRINCIPAL FINDINGS: CD82 was found to be strongly expressed in human first trimester placental villous and extravillous trophoblast cells as well as in trophoblast cell lines. To investigate whether CD82 plays a role in trophoblast invasion and migration, we further utilized human villous explants culture model on matrigel and invasion/migration assay of trophoblast cell line HTR8/SVneo. CD82 siRNA significantly promoted outgrowth of villous explants in vitro (P<0.01), as well as invasion and migration of HTR8/SVneo cells (P<0.05), whereas the trophoblast proliferation was not affected. The enhanced effect of CD82 siRNA on invasion and migration of trophoblast cells was found associated with increased gelatinolytic activities of matrix metalloproteinase MMP9 while over-expression of CD82 markedly decreased trphoblast cell invasion and migration as well as MMP9 activities. CONCLUSIONS/SIGNIFICANCE: These findings suggest that CD82 is an important negative regulator at maternal-fetal interface during early pregnancy, inhibiting human trophoblast invasion and migration

Efflux Pump, the Masked Side of ß-Lactam Resistance in Klebsiella pneumoniae Clinical Isolates

International audienceBACKGROUND: Beta-lactamase production and porin decrease are the well-recognized mechanisms of acquired beta-lactam resistance in Klebsiella pneumoniae isolates. However, such mechanisms proved to be absent in K. pneumoniae isolates that are non susceptible to cefoxitin (FOX) and susceptible to amoxicillin+clavulanic acid in our hospital. Assessing the role of efflux pumps in this beta-lactam phenotype was the aim of this study. METHODOLOGY/FINDINGS: MICs of 9 beta-lactams, including cloxacillin (CLX), and other antibiotic families were tested alone and with an efflux pump inhibitor (EPI), then with both CLX (subinhibitory concentrations) and EPI against 11 unique bacteremia K. pneumoniae isolates displaying the unusual phenotype, and 2 ATCC strains. CLX and EPI-dose dependent effects were studied on 4 representatives strains. CLX MICs significantly decreased when tested with EPI. A similar phenomenon was observed with piperacillin+tazobactam whereas MICs of the other beta-lactams significantly decreased only in the presence of both EPI and CLX. Thus, FOX MICs decreased 128 fold in the K. pneumoniae isolates but also 16 fold in ATCC strain. Restoration of FOX activity was CLX dose-dependent suggesting a competitive relationship between CLX and the other beta-lactams with regard to their efflux. For chloramphenicol, erythromycin and nalidixic acid whose resistance was also due to efflux, adding CLX to EPI did not increase their activity suggesting differences between the efflux process of these molecules and that of beta-lactams. CONCLUSION: This is the first study demonstrating that efflux mechanism plays a key role in the beta-lactam susceptibility of clinical isolates of K. pneumoniae. Such data clearly evidence that the involvement of efflux pumps in beta-lactam resistance is specially underestimated in clinical isolates

Several domains from VAR2CSA can induce Plasmodium falciparum adhesion-blocking antibodies

<p>Abstract</p> <p>Background</p> <p>Malaria caused by <it>Plasmodium falciparum </it>can result in several different syndromes with severe clinical consequences for the about 200 million individuals infected each year. During pregnancy, women living in endemic areas become susceptible to malaria due to lack of antibodies against a unique <it>P. falciparum </it>membrane protein, named VAR2CSA. This antigen is not expressed in childhood infections, since it binds chondroitin sulphate A (CSA) expressed on the intervillous space in the placenta. A vaccine appears possible because women acquire protective antibodies hindering sequestration in the placenta as a function of parity. A challenge for vaccine development is to design small constructs of this large antigen, which can induce broadly protective antibodies. It has previously been shown that one domain of VAR2CSA, DBL4-FCR3, induces parasite adhesion-blocking antibodies. In this study, it is demonstrated that other domains of VAR2CSA also can induce antibodies with inhibitory activity.</p> <p>Methods</p> <p>All VAR2CSA domains from the 3D7 and HB3 parasites were produced in <it>Baculovirus</it>-transfected insect cells. Groups of three rats per protein were immunized and anti-sera were tested for surface reactivity against infected erythrocytes expressing FCR3 VAR2CSA and for the ability to inhibit FCR3CSA parasite adhesion to CSA. The fine specificity of the immune sera was analysed by VAR2CSA peptide arrays.</p> <p>Results</p> <p>Inhibitory antibodies were induced by immunization with DBL3-HB3 T1 and DBL1-3D7. However, unlike the previously characterised DBL4-FCR3 response the inhibitory response against DBL1-3D7 and DBL3-HB3 T1 was poorly reproduced in the second rounds of immunizations.</p> <p>Conclusion</p> <p>It is possible to induce parasite adhesion-blocking antibodies when immunizing with a number of different VAR2CSA domains. This indicates that the CSA binding site in VAR2CSA is comprised of epitopes from different domains.</p

PfRH5: A Novel Reticulocyte-Binding Family Homolog of Plasmodium falciparum that Binds to the Erythrocyte, and an Investigation of Its Receptor

Multiple interactions between parasite ligands and their receptors on the human erythrocyte are a condition of successful Plasmodium falciparum invasion. The identification and characterization of these receptors presents a major challenge in the effort to understand the mechanism of invasion and to develop the means to prevent it. We describe here a novel member of the reticulocyte-binding family homolog (RH) of P. falciparum, PfRH5, and show that it binds to a previously unrecognized receptor on the RBC. PfRH5 is expressed as a 63 kDa protein and localized at the apical end of the invasive merozoite. We have expressed a fragment of PfRH5 which contains the RBC-binding domain and exhibits the same pattern of interactions with the RBC as the parent protein. Attachment is inhibited if the target cells are exposed to high concentrations of trypsin, but not to lower concentrations or to chymotrypsin or neuraminidase. We have determined the affinity, copy number and apparent molecular mass of the receptor protein. Thus, we have shown that PfRH5 is a novel erythrocyte-binding ligand and the identification and partial characterization of the new RBC receptor may indicate the existence of an unrecognized P. falciparum invasion pathwa

In Situ Synthesis of Reduced Graphene Oxide and Gold Nanocomposites for Nanoelectronics and Biosensing

In this study, an in situ chemical synthesis approach has been developed to prepare graphene–Au nanocomposites from chemically reduced graphene oxide (rGO) in aqueous media. UV–Vis absorption, atomic force microscopy, scanning electron microscopy, transmission electron microscopy, and Raman spectroscopy were used to demonstrate the successful attachment of Au nanoparticles to graphene sheets. Configured as field-effect transistors (FETs), the as-synthesized single-layered rGO-Au nanocomposites exhibit higher hole mobility and conductance when compared to the rGO sheets, promising its applications in nanoelectronics. Furthermore, we demonstrate that the rGO-Au FETs are able to label-freely detect DNA hybridization with high sensitivity, indicating its potentials in nanoelectronic biosensing

Children with cerebral malaria or severe malarial anaemia lack immunity to distinct variant surface antigen subsets

Abstract Variant surface antigens (VSAs) play a critical role in severe malaria pathogenesis. Defining gaps, or “lacunae”, in immunity to these Plasmodium falciparum antigens in children with severe malaria would improve our understanding of vulnerability to severe malaria and how protective immunity develops. Using a protein microarray with 179 antigen variants from three VSA families as well as more than 300 variants of three other blood stage P. falciparum antigens, reactivity was measured in sera from Malian children with cerebral malaria or severe malarial anaemia and age-matched controls. Sera from children with severe malaria recognized fewer extracellular PfEMP1 fragments and were less reactive to specific fragments compared to controls. Following recovery from severe malaria, convalescent sera had increased reactivity to certain non-CD36 binding PfEMP1s, but not other malaria antigens. Sera from children with severe malarial anaemia reacted to fewer VSAs than did sera from children with cerebral malaria, and both of these groups had lacunae in their seroreactivity profiles in common with children who had both cerebral malaria and severe malarial anaemia. This microarray-based approach may identify a subset of VSAs that could inform the development of a vaccine to prevent severe disease or a diagnostic test to predict at-risk children

Dynamic Activation and Repression of the Plasmodium falciparum rif Gene Family and Their Relation to Chromatin Modification

The regulation of variant gene expression in Plasmodium falciparum is still only partially understood. Regulation of var genes, the most studied gene family involved in antigenic variation, is orchestrated by a dynamic pattern of inherited chromatin states. Although recent evidence pointed to epigenetic regulation of transcribed and repressed rif loci, little is known about specific on/off associated histone modifications of individual rif genes. To investigate the chromatin marks for transcribed and repressed rif loci, we cultivated parasites and evaluated the transcriptional status of chosen rif targets by qRT-PCR and performed ChIP assays using H3K9ac and H3K9me3 antibodies. We then monitored changes in the epigenetic patterns in parasites after several reinvasions and also evaluated the “poised” mark in trophozoites and schizonts of the same erythrocytic cycle by ChIP using H3K4me2 specific antibodies. Our results show that H3K9 is acetylated in transcribed rif loci and trimethylated or even unmodified in repressed rif loci. These transcriptional and epigenetic states are inherited after several reinvasions. The poised modification H3K4me2 showed a tendency to be more present in loci in trophozoites that upon progression to schizonts strongly transcribe the respective locus. However, this effect was not consistently observed for all monitored loci. While our data show important similarities to var transcription-associated chromatin modifications, the observed swiftly occurring modifications at rif loci and the absence of H3K9 modification point to a different dynamic of recruitment of chromatin modifying enzymes