Investigation on the effect of roller compaction on paracetamol.

Roller compaction is a popular dry granulation method that has been associated with loss of tabletability. In this study, the effect of roller compaction on a model brittle elastic material, paracetamol, was examined. Roller compaction of paracetamol was carried out at three roll force to examine the effects of roll force on the tablet compaction properties. Paracetamol granules consisting of small fragmented crystals were created through the process of roller compaction. A compaction simulator was used to produce tablets from a sieved fraction of roller compacted paracetamol and non-roller compacted paracetamol. Despite the higher elastic energy to plastic energy ratio observed with tablets produced from roller compacted granules of higher forces, the table tensile strength obtained was higher with a lower capping coefficient. At the same time, tablet elastic recovery was found to be lower for tablets produced using roller compacted paracetamol granules. Prefragmentation during roller compaction process helped to reduce the energy required for fragmentation during tablet compaction, increasing the energy available for bond formation. Roller compaction of brittle elastic materials may be a viable option for improving tablet tensile strength and reducing tablet capping.

Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication.

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.

Identification of RFPL3 protein as a novel E3 ubiquitin ligase modulating the integration activity of human immunodeficiency virus, type 1 preintegration complex using a microtiter plate-based assay.

Integration, one of the hallmarks of retrovirus replication, is mediated by a nucleoprotein complex called the preintegration complex (PIC), in which viral DNA is associated with many protein components that are required for completion of the early phase of infection. A striking feature of the PIC is its powerful integration activity in vitro. The PICs from a freshly isolated cytoplasmic extract of infected cells are able to insert viral DNA into exogenously added target DNA in vitro. Therefore, a PIC-based in vitro assay is a reliable system for assessing protein factors influencing retroviral integration. In this study, we applied a microtiter plate-based in vitro assay to a screening study using a protein library that was produced by the wheat germ cell-free protein synthesis system. Using a library of human E3 ubiquitin ligases, we identified RFPL3 as a potential stimulator of human immunodeficiency virus, type 1 (HIV-1) PIC integration activity in vitro. This enhancement of PIC activity by RFPL3 was likely to be attributed to its N-terminal RING domain. To further understand the functional role of RFPL3 in HIV infection, we created a human cell line overexpressing RFPL3. Immunoprecipitation analysis revealed that RFPL3 was associated with the human immunodeficiency virus, type 1 PICs in infected cells. More importantly, single-round HIV-1 infection was enhanced significantly by RFPL3 expression. Our proteomic approach displays an advantage in the identification of new cellular proteins affecting the integration activity of the PIC and, therefore, contributes to the understanding of functional interaction between retroviral integration complexes and host factors.

Interferon-mediated ISG15 conjugation restricts dengue virus 2 replication.

ISGylation, an ubiquitin-like post-translational modification by ISG15, has been reported to participate in the interferon (IFN)-mediated antiviral response. In this study, we analyzed the functional role of ISGylation in dengue virus 2 (DENV-2) replication. Overexpression of ISG15 was found to significantly suppress the amount of extracellular infectious virus released, while intracellular viral RNA was unaffected. This effect was not observed with a conjugation-defective ISG15 mutant. In addition, extracellular virus infectivity was decreased by ISG15 overexpression. To further clarify the role of ISGylation in the anti-DENV-2 response, we depleted endogenous ISG15 by RNA interference and analyzed the virus production in the absence or presence of type-I IFN. Results showed a significant reduction in extracellular DENV-2 RNA levels for cells treated with IFN, and that these DENV-2 RNA levels could be partially restored by the ISG15 knockdown. Among various DENV-2 proteins, NS3 and NS5 were subjected to the ISGylation. These results demonstrate that IFN-inducible ISGylation suppresses DENV-2 particle release, and that ISG15 is one of the mediators of IFN-induced inhibition of DENV-2 replication. ISG15 therefore functions as a host antiviral factor against DENV-2 infection.