Perayot Pamonsinlapatham

Neuropilin-1 regulates a new VEGF-induced gene, Phactr-1, which controls tubulogenesis and modulates lamellipodial dynamics in human endothelial cells

Recently, we identified a new Vascular Endothelial Growth Factor (VEGF)-A(165)-induced gene Phactr-1, (Phosphatase Actin Regulator-1). We reported that Phactr-1 gene silencing inhibited tube formation in human umbilical endothelial cells (HUVECs) indicating a key role for Phactr-1 in tubulogenesis in vitro. In this study, we investigated the role of Phactr-1 in several cellular processes related to angiogenesis. We found that neuropilin-1 (NRP-1) and VEGF-R1 depletion inhibited Phactr-1 mRNA expression while NRP-2 and VEGF-R2 depletion had no effect. We described a new interaction site of VEGF-A(165) to VEGF-R1 in peptides encoded by exons 7 and 8 of VEGF-A(165). The specific inhibition of VEGF-A(165) binding on NRP-1 and VEGF-R1 by ERTCRC and CDKPRR peptides decreased the Phactr-1 mRNA levels in HUVECs indicating that VEGF-A(165)-dependent regulation of Phactr-1 expression required both NRP-1 and VEGF-R1 receptors. In addition, upon VEGFA(165)-stimulation Phactr-1 promotes formation and maintenance of cellular tubes through NRP-1 and VEGFR1. Phactr-1 was previously identified as protein phosphatase 1 (PP1) α-interacting protein that possesses actin-binding domains. We showed that Phactr-1 depletion decreased PP1 activity, disrupted the fine-tuning of actin polymerization and impaired lamellipodial dynamics. Taken together our results strongly suggest that Phactr-1 is a key component in the angiogenic process.

Impairment by Mucosal Adjuvants and Cross-Reactivity with Variant Peptides of the Mucosal Immunity Induced by Injection of the Fusion Peptide PADRE-ELDKWA

ABSTRACT Secretory immunity protects against mucosal transmission of viruses, as demonstrated with the oral poliovirus vaccine. In a previous study we showed that this immunity could be induced in mice by injection of a fusion peptide consisting of an unnatural peptide-like sequence (PADRE) and a viral epitope (ELDKWASLW). PADRE is a T-helper-cell epitope able to bind most major histocompatibility complex class II molecules of different haplotypes in mice and humans and to increase antibody responses. ELDKWA is a well-known consensual sequence of gp41 involved in a key structure of human immunodeficiency virus (HIV) type 1. Here, the antibody response to the native form of ELDKWA was mainly of the immunoglobulin A isotype and selectively occurred in mucosa. Adjuvants, such as cholera toxin and cytosine polyguanine, were useless and even competed with PADRE for the response. Interestingly, these antibodies were cross-reactive with the three major variants of the epitope, as shown both by direct enzyme-linked immunosorbent assay and by inhibition. This unconventional route of mucosal immunization allows control of the administered dose. The lack of adjuvant and the cross-reactivity of the antibodies increase the safety and the spectrum of the candidate vaccine, respectively. The drug-like nature of the construct suggests further improvements by synthesis of more antigenic sequences. The reasonable cost of short peptides at the industrial level and their purity make this approach of interest for future vaccines against mucosal transmission of HIV or other pathogens.

Induction of a Mucosal Immune Response to the Streptococcal M Protein by Intramuscular Administration of a PADRE-ASREAK Peptide

In a previous study, it was shown that an intramuscular administration of amino acid PADRE‐ELDKWA sequence induced a mucosal immune response to a conserved epitope of human immunodeficiency virus in mice. In the same model, here it is shown that this method can be used with a selected peptide from the M protein of group A streptococci. The PADRE‐ASREAK sequence was injected in mice by the intramuscular route. Antibodies against M protein were detected in extracts of mucosal tissues and in serum. The repertoire isotypes of serum immunoglobulin G (IgG) and mucosal IgA and IgG antibodies varied, according to the dose of injected peptide. The highest mucosal IgA antibody response was obtained with 0.01u2003µg of antigen per injection, whereas the systemic IgG antibody response increased with 10u2003µg of antigen. Mucosal antibody production against streptococci was confirmed by immunofluorescence analysis. These results provide evidence that this novel approach of mucosal vaccination may be of advantage for bacterial systems and suggest a new field of investigation based on synthetic peptide analogues.

Blended Learning for Improving Flexibility of Learning Structure Query Language (SQL)

The Structural Query Language (SQL) is the popular language used for managing and retrieving data and information from relational databases. To support the decision making for healthcare professionals including pharmacists, learning SQL is one of an important topic in a database course. The objective of this research was to introduce blended learning for improving flexibility of learning SQL. The learning process was discussed and planned. Contents in this topic and examples of relational databases were designed and implemented. The SQLite was selected as a relational database management system due to its flexibility of using and managing. After a face-to-face class, students could practice their exercises with any of their computing devices, i.e., computers, tablets and smartphones. Moreover, these devices could operate with or without Internet connection. Students could practice and manage their exercises at any time and from anywhere. With evaluations from students, the results showed that the flexibility of the system enhanced their ability for learning SQL.

Correction for Tun-Yhong et al., “Tenofovir Disoproxil Fumarate Is a New Substrate of ATP-Binding Cassette Subfamily C Member 11”

Volume 61, no. 4, e01725-16, 2017, https://doi.org/10.1128/AAC.01725-16. Page 5, Figure 4: the order of magnitude of TDF in Fig. 4C should read 1.75 104 instead of 1.75 103 and that of MTX in Fig. 4D should read 1.6 104 instead of 1.6 103. The correct figure is shown below. Citation Tun-Yhong W, Chinpaisal C, Pamonsinlapatham P, Kaewkitichai S. 2017. Correction for Tun-Yhong et al., “Tenofovir disoproxil fumarate is a new substrate of ATPbinding cassette subfamily C member 11.” Antimicrob Agents Chemother 61:e01753-17. https://doi.org/10.1128/AAC.01753-17. Copyright

Tenofovir Disoproxil Fumarate Is a New Substrate of ATP-Binding Cassette Subfamily C Member 11

ABSTRACT Tenofovir disoproxil fumarate (TDF), a nucleotide reverse transcriptase inhibitor, after conversion to tenofovir (TFV), is mainly eliminated by glomerular filtration and active tubular secretion. The major adverse effect of tenofovir is nephrotoxicity; however, the exact mechanism remains poorly understood. In this study, the ATP-binding cassette subfamily C member 11 (ABCC11; multidrug resistance protein 8 [MRP8]) transporter, which is abundant in proximal tubular cells, was demonstrated to act as an efflux transporter of tenofovir. Real-time PCR (RT-PCR) and indirect immunofluorescence assays were used to determine MRP8 overexpression in a continuous cell line. Tenofovir accumulations were assessed by cytotoxicity, cellular transport, and vesicular uptake assays. Substrate specificity was confirmed using MK-571, an MRP-specific inhibitor, and methotrexate, which served as a known substrate. Intracellular and intravesicular concentrations of tenofovir were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The 50% cytotoxic concentration (CC50) of TDF in MRP8-overexpressing cells was 4.78 times higher than that of parental cells. Transport assays also showed that the intracellular accumulation of tenofovir in MRP8-overexpressing cells was 55 times lower than that in parental cells and was partly reversed by MK-571. Similarly, an “inside-out” vesicular uptake assay, using Sf9 inverted membrane vesicles to allow measuring of accumulation of the substrates into the vesicles, demonstrated a higher intravesicular concentration of tenofovir in MRP8-overexpressing vesicles than in Sf9 insect control vesicles. These effects were effectively reversed by increasing concentrations of the specific inhibitor MK-571. In conclusion, tenofovir is a new substrate of the MRP8 transporter. An alteration in the activity of this efflux pump may increase the intracellular accumulation of tenofovir in proximal renal tubular cells.

Mechanisms of Cellular Uptake with Chitosan/DNA Complex in Hepatoma Cell Line

Chitosan (CS) has a high potential for gene delivery into mammalian cells. However, its uptake mechanism is not well clarified. We investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine), caveolae-mediated endocytosis (genistein), macropinocytosis (LY 29004 and wortmannin), microtubuli polymerization (nocodazole) and of membrane cholesterol recycle (methyl-β-cyclodextrin) on the transfection efficiency with CS/pEGFP complexes and on the internalization of CS/rhodamine-labeled pEGFP complexes by hepatoma cell line (Huh 7 cells). The transfection was blocked by nocodazole, genistein, and methyl-β-cyclodextrin, respectively. CS/DNA complexes internalization was clearly inhibited by genistein. We conclude that the complexes uptake predominantly by caveolin-mediated pathways. In addition, fluorescence colocalization studies with acidotropic probes, LysoSensor dye, illustrated that CS/DNA complexes are targeted to lysosomes for the degradation after internalization.