Intracellular Localization of HBV Capsid in Hepatocyte Line After Transfected by The Entire HBV Genome = Lokalisasi Intraseluler Kapsid HBV Pada Sel Line Hepatosit Setelah Ditransfeksi Dengan Genom Utuh HBV.

HBV replicates within the nucleus of hepatocyte using cellular transport machinery for the import of their genomes into the nucleus. Genome of HBV has to transported through the cytoplasm towards the nuclear pore complex (NPC) followed by subsequent passage through the pore. HBV capsid is involved in a number of important functions in the replication cycle of HBV. It can be detected in the nucleus, cytoplasm or both within infected hepatocytes. Nuclear localization of HBV capsid protein, which is karyophilic, depends on the cell cycle. The objective of the present study was to analyzes the intracellular localization of HBV capsid protein after transfected by entire HBV genome into hepatocyte cell lines (HuH-7) and to determine the predominantly localization of the capsid into cell compartment. In this work we analysed the intracellular localization of the HBV capsid in human hepatocyte cell lines liuH-7 by transfection using entire HBV genome and transient expression. The transfected cells were fixed and an indirect immune staining against the HBV capsid was performed to detect the capsid. To verify the location within the cell, an additional co-staining against the nuclear pore complexes was performed. The Intracellular localization of the HBV capsid and NPC were analyzed by a confocal laser scan microscope. The observed of localizations into the transfected cells were classified to be predominantly as nuclear localization, cytoplasmic localization or distributed within both of these compartments. Result of this study indicated that Staining of HBV capsid found predominantly within the nucleus (71%). Less frequently, the HBV capsid localized within the cytoplasm (26%). Only in a minority of cases, the capsids were localized within cytoplasm and nucleus (3%). This low frequency indicate that the capsids were not diffusing within the cells being in accordance to the in vivo situation in which the nuclear membrane was impermeable for the capsid. Key words: HBV capsid, hepatocyte, NPC, HuH-7, confocal laser scan microscope Replikasi HBV terjadi di dalam nukleus hepatosit dengan mempergunakan mekanisme transport seluler untuk mengirimkan genomnya ke dalam nukleus. Genom HBV hams ditransportasikan ke dalam nukleus melalui nuclear pore complex (NPC). Kapsid HBV terlibat dalam sejumlah fungsi panting dalam siklus replikasi HBV. Protein kapsid HBV dapat dideteksi didalam nukleus, sitoplasm ataupun di kedua kompartemen tersebut di dalam sel hepatosit yang terinfeksi HBV. Lokalisasi protein kapsid HBV yang bersifat karyophilik tergantung pada siklus sel. Adapun tujuan dari penelitian ini adalah untuk menganalisis lokalisasi intraseluler protein kapsid HBV pada sel line hepatosit HuH-7 pasca ditransfeksi dengan genom HBV utuh serta untuk menentukan lokalisasi kapsid HBV yang dominan dalam kompartemen sel. Pada penelitian telah dilakukan analisis lokalisasi intraseluler kapsid HBV dengan mentransfeksikan genome HBV utuh dan mengekspresikannya di dalam sel line HuH-7. Sel yang sudah ditransfeksi selanjumya difiksasi dan dilakukan penwarnaan secara tidak langsung terhadap kapsid HBV. Untuk menentukan lokalisasi dalam sel, pewarnaan lanjutan dilakukan terhadap NPC. Selanjutnya, dengan menggunakan mikroskop scan laser konfokal dianalisis lokalisasi intraseluler kapsid HBV dan NPC. Hasil pengamatan menunjukan bahwa distribusi HBV di dalam nukleus lebih dominan dibandingkan dengan distribusi di dalam sitoplasma dan distribusi pada kedua kompartemen tersebut. Secara kuantitatif menunjukkan bahwa distribusi protein kapsid HBV dalam nukleus adalah dominan dengan 71%, sedangkan dalam sitoplasma adalah 26%, sangat minoritas dijurnpai distribusi protein kapsid HBV pada kedua kompartemen sel, yaitu hanya 3%. Hal ini menunjukkan bahwa masuknya kapsid HBV kedalam nukleus tidak terdifusi secara pasif, namun secara aktif melalui mekanisme protein transport melalui NPC. Membran nukleus bersifat permeable terhadap protein HBV kapsid. Kata kunci: kapsid HBV, hepatosit, NPC, HuH-7, mikroskop scan laser konfoca

Safety Grand Challenge: Safe Ship Boarding and Thames Safest River 2030

This report describes the first Lloyd’s Register Foundation Safety Grand Challenge and details how a collaborative, cross disciplinary design research and teaching methodology can provide a platform for a broad variety of participants to develop projects in a complex design safety environment, encourage collaboration and industrial involvement in design education and contribute to a balance between technological developments and the needs of people in the future. The Royal College of Art, generously supported by the Lloyd’s Register Foundation and working with a group of industry stakeholders, investigated two major areas of risk within the maritime context: Sea Safe transfers from ship to ship, and making the Thames the safest city river by the year 2030. In a four month project, thirty two post graduate participants from eleven disciplines and six researcher-tutors at the Royal College of Art worked together and tackle these complex and wicked design challenges using a number of novel design methods. With a focus on finding cutting edge innovative design solution that would reduce risk on the ship to ship transfer and on increasing safety on the River Thames, the research project explored a wide range of approaches that encouraged collaboration, innovation and risk taking in design research practice. The different cultures, practices and knowledge bases led to an array of eight pioneering design solutions, ranging from product-focused innovations through to systemic solutions, material innovations and educational strategies. This report makes a case for the culture of design engaging with risk on water in the context of the wicked problems (Rittel & Webber, 1973; Buchanan, 1992) we identified, the methods and techniques used to tackle these challenges, how cross disciplinary projects can lead to novel insights, and how design education can be used to engage with industry and users to bridge the gap between technological innovation and user needs. Our conclusions support the view that this approach can develop implementable new design for safety solutions, incorporate the social, cultural and psychological human factors into safety design and balances users’ needs by engagement through an appropriate use of technology. Furthermore, we uncover insights into training designers for safety critical environments and the implications this has in terms of projects, cross disciplinarity and practices in the role of design thinking in general and relating to the context of risk and safety at sea and on rivers

Effect of Nuclear Export Inhibitor Leptomycin B on the Intracellular Localization of HBV Core Protein into Hepatocytes Cell Line Huh-7 and HepG2 Cells

Leptomycin B (LMB) was originally discovered as a potent anti-fungal antibiotic from Streptomyces species. The cellular target of LMB has been identified as the nuclear export receptor CRM-1 or exportin-1, which is involved in nuclear trafficking of cellular RNAs or proteins containing the nuclear export sequence (NES). CRM-1 is the main mediator of nuclear export in many cell types including hepatocyte cell lines. The ability of LMB to inhibit nuclear export has made it a useful tool in the study of the intracellular localization of manyregulatory proteins. In this study, we evaluated the effect of nuclear export inhibitor LMB treatment on the intracellular localization of HBV core protein into the hepatocyte cell lines, Huh-7 and HepG2 cells. We also reported the quantification of the distribution of EGFP-Core fusion protein with redundant core NLS as well as SV-40 NLS into cell compartments. Results shown that in Huh-7 cells treatment of LMB caused retention of EGFP-Core fusion protein into the nucleus, so increased the nuclear localization of EGFP-Core and all variants.In HepG2 cells, although not significantly, treatment of LMB increased a number of nuclear localization in all EGFP-Core constructions, even the nuclear localization in HepG2 cells is not so high as in Huh-7 cells. Keywords: Leptomycin B, HBV, core protein, intracellular localization, NLS, Huh-7, HepG2 cel

Effect of Staurosporine on the Intracellular Localization of Hepatitis B Virus Core Protein

protein is also including in the HBV genome targeting into the nucleus through modulating carboxyl residues byphosphorylation. Nuclear localication Signal (NLS) in HBV core protein is inside the virion structure and it must beunmasked in order to function, perhaps by phosphorylation. Phosphorylation of of HBV core protein in turn couldbegin to alter capsid conformation. Staurosporine is a natural product originally isolated from bacteriumStreptomyces staurosporeus. Staurosporine was discovered to have biological activities ranging from anti-fungal toanti-hypertensive. The interest in these activities resulted in a large investigative effort in chemistry and biology andthe discovery of the potential for anti-cancer treatment. The main biological activity of Staurosporine is the inhibitionof protein kinases through the prevention of ATP binding to the kinase. In the present study, we have studied theintracellular localization of EGFP-Core fusion protein with triple HBV core and SV-40 nuclear localization signal atits carboxyl terminal in presence and absence of Staurosporine. We also to study the effect of Staurosporine treatmenton the intracellular localization of EGFP-Core fusion protein in the hepatocyte cells line of HepG2 cell. Resultsshowed that effect of Staurosporine is prevent the nuclear localization of EGFP-Core fusion protein into nucleusthrough an inhibition of the phosphorylation of core protein. Stauroporine also prevents cell division so that passivetrapping of core protein is inhibited

Effect of Nuclear Export Inhibitor Leptomycin Bon the Intracellular Localization of HBV Core Protein into Hepatocytes Cell Line Huh-7 and HepG2 Cells

Leptomycin B (LMB) was originally discovered as a potent anti-fungal antibiotic from Streptomyces species. The cellular target of LMBhas been identified as the nuclear export receptor CRM-1 or exportin-1, which is involved in nuclear trafficking of cellular RNAs or proteins containing the nuclear export sequence (NES). CRM-1 is the main mediator of nuclear export in many cell types including hepatocyte cell lines. The ability of LMB to inhibit nuclear export has made it a useful tool in the study of the intracellular localization of many regulatory proteins. In this study, we evaluated the effect of nuclear export inhibitor LMB treatment on the intracellular localization of HBV core protein into the hepatocyte cell lines, Huh-7 and HepG2 cells. We also reported the quantification of the distribution of EGFP-Core fusion protein with redundant core NLS as well as SV-40 NLS into cell compartments. Results shown that in Huh-7 cells treatment of LMBcaused retention of EGFP-Core fusion protein into the nucleus, so increased the nuclear localization of EGFP-Core and all variants. In HepG2 cells, although not significantly, treatment of LMB increased a number of nuclear localization in all EGFP-Core constructions, even the nuclear localization in HepG2 cells is not so high as in Huh-7 cells

Designing the domestic Internet of Things using a practice-orientated perspective

The Internet of Things (IoT) is a system of sensing, actuating and networked objects, often discussed as delivering efficiency through machine determined, automated decision making and action to achieve ‘Smartness’ in a logistically based paradigm. When applied to the domestic space these values are touted as beneficially controlling lighting, heating and entertainment to improve efficiency and comfort, while reducing costs. This approach follows the external goods of effectiveness, reducing everything to an objective value/cost proposition; however, the home is a subjectively experienced space incorporating differing values, so this reductive perspective overlooks a wider spectrum of inhabitant’s concerns relating to their daily activities and the domestic space. Furthermore, this approach can supplant involvement in domestic activities by treating these as computable problems to solve, alienating users through automation, a lack of transparency and poor understanding of the reasoning behind machine decision making. Existing attempts to address this topic indicate Techno-Centric approaches impact on understanding and engagement with the domestic space; Human-Centric perspectives focus on supporting people’s subjective experiences by prioritising their activities, sense-making and sensory experiences within the design process; Beyond Human-Centric IoT perspectives broaden this understanding to propose non-hierarchical, flat ontologies for the IoT and the implications this has on integrating human/non-human agency in the IoT, generally and domestically. This supported an approach utilising Practice Theory, a development of organising concepts for theorising social life, with sociality dependent on activities conducted with materials to develop a coherent sense of self and which understands place as a meshwork of human/non-human agency. Practice Theory is applied within a Design Research approach using a synergistic Participatory Action Research (PAR) / Participatory Design (PD) process. Exploring Domestic Practices contextualised the IoT through a range of methods including interactive installations, interviews and design workshops, uncovering participant attitudes towards the IoT, generating Practice Themes and specific examples of practices and constituent elements. These acted as User Generated Values (UGV) in a Values-Led PD process to inform the project pathway and the conceptualisation of a Practice-Oriented IoT through PAR’s Action-Reflection spirals. Additionally, a parallel PD process explored the effective communication of UGV within Professional Design Practice (PDP) workshops with the intent of reducing communicative distance between end-users and developers, supporting communication of user’s attitudes towards the IoT and Practice within PDP through inclusion as guiding values. Models of the IoT balancing Practice and technical concerns, workshops and toolkit were developed iteratively, leading to an outcome modelling the IoT and Practice within a flat ontology. Through this, and by embedding Practice within the IoT itself, IoT agency was reframed from automation towards assistiveness in Practice and IoT values shifted from efficiency in external goods of effectiveness towards internally derived goods of excellence, supporting skill development, engagement and reflection on action. This identifies the value of using PAR and PD to consider people’s values, goals and existing practices when developing the domestic IoT. This was particularly valuable in exploring Practice to understand people’s activities in the home and contextualise attitudes towards the IoT. This informed the development of a framework balancing the IoT’s technological nature with people’s activities and values, a system guided by Practice elements reciprocally informing and supporting participant engagement in dynamically developing domestic practices

Collaborating Design Risk

The “Safety Grand Challenge” is a collaborative research project between the Royal College of Art (RCA) School of Design, and the Lloyd's Register Foundation (LRF). The maritime industry is dominated by “grandfathering” leading to a slow-pace of adopting innovations that can reduce risk and save lives at sea. We describe how impact was achieved through collaboration and design innovations that bridged the risk gap between technologies and human behaviours. Starting from the project brief we designed a collaborative platform that supported a constructive dialogue between academia and partner organisations that aimed to foster innovative design approaches to risk and safety. The project generated an engaged community with diverse expertise that influenced the outcomes which included seven prototypes designed by a group of thirty postgraduates from across the RCA. Throughout the course of the project the network extended to other partners beyond the initial ones that included the RCA, LRF and Royal National Lifeboat Institution. The “Safety Grand Challenge” demonstrates how research can be an explorative platform that offers opportunities to analyse and design solutions to real life safety problems in mature industries through the prototypes that reflect the sophistication of the project’s collaborations. Our conclusions support how design research helped identify the value of design for safety in tackling complex issues that intertwine human, environmental and commercial views and can shape new forms of collaborative research between academia and industrial partners

Nuclear Import Analysis of Two Different Fluorescent Marker Proteins into Hepatocyte Cell Lines (HuH-7 Cell)

The application of fluorescent proteins as expression markers and protein fusion partners has proved immensely valuable for resolving the organization of biological events in living cells. EGFP and DsRed2 are commonly fluorescent marker protein which is used for biotechnology and cell biology research. The present study was designed to identify the expression vector that suitable to ligate with DNA encoding HBV core protein for intracellular localization study in hepatocyte cell, which were expressed as fusion proteins. We also compared and quantified the expressed fluorescent protein which predominantly localized in the cell compartment. The results indicated that DsRed2 shown as less than ideal for intracellular localization study of than EGFP, because of its tetrameric structure of the fluorescent protein and when fused to a protein of interest, the fusion protein often forms aggregates in the living cells. In contrast, EGFP fluorescent protein shown a much higher proportion of cytoplasmic localization, thus being more suitable for analysis of intracellular localization than DsRed2 fluorescent protein. EGFP fluorescent protein is also capable to produce a strong green fluorescence when excited by blue light, without any exogenously added substrate or cofactor, events inside living cell can thus be visualized in a non-invasive way. Based on our present quantitative data and some reasons above shown that EGFP is more suitable than DsRed2 as a fluorescent marker protein for intracellular localization study into HuH-7 cell. Keywords: EGFP, DsRed2 fluorescent protein , HuH-7 cell, HBV, intracellular localizatio

Viruses

Hepatitis B virus (HBV) is an enveloped pararetrovirus with a DNA genome, which is found in an up to 36 nm-measuring capsid. Replication of the genome occurs via an RNA intermediate, which is synthesized in the nucleus. The virus must have thus ways of transporting its DNA genome into this compartment. This review summarizes the data on hepatitis B virus genome transport and correlates the finding to those from other viruses