Replication-Deficient Lymphocytic Choriomeningitis Virus-Vectored Vaccine Candidate for the Induction of T Cell Immunity against Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) represents a major burden to global health, and refined vaccines are needed. Replication-deficient lymphocytic choriomeningitis virus (rLCMV)-based vaccine vectors against cytomegalovirus have proven safe for human use and elicited robust T cell responses in a large proportion of vaccine recipients. Here, we developed an rLCMV vaccine expressing the Mtb antigens TB10.4 and Ag85B. In mice, rLCMV elicited high frequencies of polyfunctional Mtb-specific CD8 and CD4 T cell responses. CD8 but not CD4 T cells were efficiently boosted upon vector re-vaccination. High-frequency responses were also observed in neonatally vaccinated mice, and co-administration of rLCMV with Expanded Program of Immunization (EPI) vaccines did not result in substantial reciprocal interference. Importantly, rLCMV immunization significantly reduced the lung Mtb burden upon aerosol challenge, resulting in improved lung ventilation. Protection was associated with increased CD8 T cell recruitment but reduced CD4 T cell infiltration upon Mtb challenge. When combining rLCMV with BCG vaccination in a heterologous prime-boost regimen, responses to the rLCMV-encoded Mtb antigens were further augmented, but protection was not significantly different from rLCMV or BCG vaccination alone. This work suggests that rLCMV may show utility for neonatal and/or adult vaccination efforts against pulmonary tuberculosis

Unique domain appended to vertebrate tRNA synthetase is essential for vascular development

New domains were progressively added to cytoplasmic aminoacyl transfer RNA (tRNA) synthetases during evolution. One example is the UNE-S domain, appended to seryl-tRNA synthetase (SerRS) in species that developed closed circulatory systems. Here we show using solution and crystal structure analyses and in vitro and in vivo functional studies that UNE-S harbours a robust nuclear localization signal (NLS) directing SerRS to the nucleus where it attenuates vascular endothelial growth factor A expression. We also show that SerRS mutants previously linked to vasculature abnormalities either deleted the NLS or have the NLS sequestered in an alternative conformation. A structure-based second-site mutation, designed to release the sequestered NLS, restored normal vasculature. Thus, the essential function of SerRS in vascular development depends on UNE-S. These results are the first to show an essential role for a tRNA synthetase-associated appended domain at the organism level, and suggest that acquisition of UNE-S has a role in the establishment of the closed circulatory systems of vertebrates

Elements for European logistics policy - A discussion paper

Following a Finnish initiative the European Commission is preparing a communication on logistics in 2006. To support the preparations Finland arranged the so called EULOC-process in which logistics experts from different countries were invited to participate. First, an industry foresight of European logistics in 2015 was created. Next, the mission, vision and policy priorities of the European logistics policy were discussed. The mission for European logistics policy was created from the viewpoint of citizens, companies, states and Europe. Seven vision elements were created. The driving visions are “Seamless systems” and “Intelligent regulation”. The guiding and enabling visions are “Resources” and “Cost efficiency”. The outcome visions are “Europe’s competitiveness”, “Equal business opportunity” and “Sustainability”. According to the experts’ views the priority areas of European logistics policy are Infrastructure – Seamless systems require investments Research, development and training – Strengthen the competitiveness of the European Union Enterprises – The reinforcement of logistic industry Regulation – Innovative and intelligent Cost Efficiency – Effective logistics Sustainability – From environmental, social and economic viewpoint, a must in modern logistics Co-Operation – A strategic issue in network society Public-Private Partnership – Agile solutions for investments

An Essential Role for the Proximal but Not the Distal Cytoplasmic Tail of Glycoprotein M in Murid Herpesvirus 4 Infection

Murid herpesvirus-4 (MuHV-4) provides a tractable model with which to define common, conserved features of gamma-herpesvirus biology. The multi-membrane spanning glycoprotein M (gM) is one of only 4 glycoproteins that are essential for MuHV-4 lytic replication. gM binds to gN and is thought to function mainly secondary envelopment and virion egress, for which several predicted trafficking motifs in its C-terminal cytoplasmic tail could be important. We tested the contribution of the gM cytoplasmic tail to MuHV-4 lytic replication by making recombinant viruses with varying C-terminal deletions. Removing an acidic cluster and a distal YXXΦ motif altered the capsid distribution somewhat in infected cells but had little effect on virus replication, either in vitro or in vivo. In contrast, removing a proximal YXXΦ motif as well completely prevented productive replication. gM was still expressed, but unlike its longer forms showed only limited colocalization with co-transfected gN, and in the context of whole virus appeared to support gN expression less well. We conclude that some elements of the gM cytoplasmic tail are dispensible for MuHV-4 replication, but the tail as a whole is not

Tumor Susceptibility Gene 101 (TSG101) Is a Novel Binding-Partner for the Class II Rab11-FIPs

The Rab11-FIPs (Rab11-family interacting proteins; henceforth, FIPs) are a family of Rab11a/Rab11b/Rab25 GTPase effector proteins implicated in an assortment of intracellular trafficking processes. Through proteomic screening, we have identified TSG101 (tumor susceptibility gene 101), a component of the ESCRT-I (endosomal sorting complex required for transport) complex, as a novel FIP4-binding protein, which we find can also bind FIP3. We show that α-helical coiled-coil regions of both TSG101 and FIP4 mediate the interaction with the cognate protein, and that point mutations in the coiled-coil regions of both TSG101 and FIP4 abrogate the interaction. We find that expression of TSG101 and FIP4 mutants cause cytokinesis defects, but that the TSG101-FIP4 interaction is not required for localisation of TSG101 to the midbody/Flemming body during abscission. Together, these data suggest functional overlap between Rab11-controlled processes and components of the ESCRT pathway

Understanding the role of hospice pharmacists: a qualitative study

Background Pharmacists are important members of multidisciplinary teams but, despite surveys of provision, the role of the hospice pharmacist is not well described. Objective To explore the role of the hospice pharmacist and identify barriers and facilitators to the role. Setting Hospices offering in-patient services caring for adults towards the end of life in one geographical area of northern England. Method Pharmacists providing services to hospices were invited to take part in qualitative semi-structured interviews asking about experience, patient contact, team working and barriers and facilitators to the role. These were recorded verbatim and data were analysed thematically using framework analysis. Main outcome measure The hospice pharmacist’s perceptions of their role and barriers and facilitators to it. Results Fifteen pharmacists took part. Two themes and ten subthemes were identified focused on tasks and communication. Practise was varied and time limited the quantity and depth of services carried out but was often spent navigating complex drug supply routes. Participants found methods of communication suited to the hours they spent in the hospice although communication of data was a barrier to effective clinical service provision. Participants identified the need for appropriate training and standards of practice for hospice pharmacists would enable better use of their skills. Conclusion Barriers to the role of hospice pharmacist include time, access to role specific training, access to clinical information and complex medicines supply chains. The role would benefit from definition to ensure that hospices are able to use hospice pharmacists to their greatest potential

Comparative analysis of co-processed starches prepared by three different methods

Co-processing is currently of interest in the generation of high-functionality excipients for tablet formulation. In the present study, comparative analysis of the powder and tableting properties of three co-processed starches prepared by three different methods was carried out. The co-processed excipients consisting of maize starch (90%), acacia gum (7.5%) and colloidal silicon dioxide (2.5%) were prepared by co-dispersion (SAS-CD), co-fusion (SAS-CF) and co-granulation (SAS-CG). Powder properties of each co-processed excipient were characterized by measuring particle size, flow indices, particle density, dilution potential and lubricant sensitivity ratio. Heckel and Walker models were used to evaluate the compaction behaviour of the three co-processed starches. Tablets were produced with paracetamol as the model drug by direct compression on an eccentric Tablet Press fitted with 12 mm flat-faced punches and compressed at 216 MPa. The tablets were stored at room temperature for 24 h prior to evaluation. The results revealed that co-granulated co-processed excipient (SAS-CG) gave relatively better properties in terms of flow, compressibility, dilution potential, deformation, disintegration, crushing strength and friability. This study has shown that the method of co-processing influences the powder and tableting properties of the co-processed excipient

Comparative analysis of co-processed starches prepared by three different methods

Co-processing is currently of interest in the generation of high-functionality excipients for tablet formulation. In the present study, comparative analysis of the powder and tableting properties of three co-processed starches prepared by three different methods was carried out. The co-processed excipients consisting of maize starch (90%), acacia gum (7.5%) and colloidal silicon dioxide (2.5%) were prepared by co-dispersion (SAS-CD), co-fusion (SAS-CF) and co-granulation (SAS-CG). Powder properties of each co-processed excipient were characterized by measuring particle size, flow indices, particle density, dilution potential and lubricant sensitivity ratio. Heckel and Walker models were used to evaluate the compaction behaviour of the three co-processed starches. Tablets were produced with paracetamol as the model drug by direct compression on an eccentric Tablet Press fitted with 12 mm flat-faced punches and compressed at 216 MPa. The tablets were stored at room temperature for 24 h prior to evaluation. The results revealed that co-granulated co-processed excipient (SAS-CG) gave relatively better properties in terms of flow, compressibility, dilution potential, deformation, disintegration, crushing strength and friability. This study has shown that the method of co-processing influences the powder and tableting properties of the co-processed excipient

EGFR interacts with the fusion protein of respiratory syncytial virus strain 2-20 and mediates infection and mucin expression.

Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2-20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore, we hypothesized that the RSV 2-20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2-20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2-20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from "mucogenic" strains. RSV 2-20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease

Apical Transport of Influenza A Virus Ribonucleoprotein Requires Rab11-positive Recycling Endosome

Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs). Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM). However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE) in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD) of Rab11 family interacting proteins (Rab11-FIPs). Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking