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The circular economy is essential to combat climate change. As part of the circular economy, supply chains are converted into closed-loop supply chains as this prevents non-recoverable waste and is thus more sustainable than traditional linear supply chains. The reverse supply chain is the part of the closed-loop supply chain that represents the phase whereby goods, at the end of their life span, are sent to a recycling or recovery facility. It is essential that goods in reverse supply chains reach their destination instead of being discarded along the way. In this article the authors presume that the current compensation systems in the international transport conventions discourage carriers or participants to close the material loop. The reason for this is that the compensation systems in international transport conventions are primarily based on the value of the transported goods. Because an inherent characteristic of goods in a reverse supply chain is that they have a negligible or low value, a financial incentive is lacking for the carrier to take care of the goods and make sure they reach their destination. The authors found several solutions to overcome these flaws. One is including a financial incentive in the transport conventions by making use of the principles for determining the customs value of imported goods. Taking a bottom-up or top-down approach are two other solutions explored in this article. In the case of the bottom-up approach, private parties include different contract models to facilitate the successful closed material loop, whereas the top-down approach introduces legal instruments to governments to intervene if the value for the compensation between the parties is too low.

The high-resolution bistatic lidar developed at the Physikalisch-Technische Bundesanstalt (PTB) aims to overcome the limitations of conventional monostatic lidar technology which is widely used for wind velocity measurements in wind energy and meteorology applications. Due to the large measurement volume of a combined optical transmitter and receiver tilting in multiple directions, monostatic lidar generally has poor spatial and temporal resolution. It also exhibits large measurement uncertainty when operated in inhomogeneous flow, for instance, over complex terrain. In contrast, PTB’s bistatic lidar uses three dedicated receivers arranged around a central transmitter, resulting in an exceptionally small measurement volume. The coherent detection and modulation schemes used allow the detection of backscattered, Doppler shifted light down to the scale of single aerosols, realising the simultaneous measurement of all three wind velocity components. This paper outlines design details and the theory of operation of PTB’s bistatic lidar and provides an overview of selected comparative measurements. The results of these measurements have shown that the measurement uncertainty of PTB’s bistatic lidar is well within the measurement uncertainty of traditional cup anemometers, while being fully independent of its site and traceable to the SI units. This allows its use as a transfer standard for the calibration of other remote sensing devices. Overall, PTB’s bistatic lidar shows great potential to universally improve the capability and accuracy of wind velocity measurements, such as for the investigation of highly dynamic flow processes upstream and in the wake of wind turbines.

The guinea pig is the only small animal model for congenital cytomegalovirus (CMV) but requires species-specific guinea pig cytomegalovirus (GPCMV). Tegument protein GP83 is the presumed homolog of HCMV pp65, but gene duplication in the UL82-UL84 homolog locus in various animal CMVs made it unclear if GP83 was a functional homolog. A GP83 null deletion mutant GPCMV (GP83dPC+) generated in the backdrop of glycoprotein pentamer complex (PC)-positive virus, required for nonfibroblast infection, had normal growth kinetics on fibroblasts but was highly impaired on epithelial and trophoblast cells. GP83dPC+ virus was highly sensitive to type I interferon (IFN-I), suggesting that GP83 had an innate immune evasion function. GP83 interacted with cellular DNA sensors guinea pig IFI16 and cGAS, indicating a role in the cGAS/STING pathway. Ectopically expressed GP83 in trophoblast cells restored GP83dPC+ virus growth. Additionally, mutant virus growth was restored in epithelial cells by expression of bovine viral diarrhea virus (BVDV) N(pro) protein targeting IRF3 as part of the cGAS/STING pathway, or alternatively, by expression of the fibroblast cell receptor platelet-derived growth factor receptor alpha (PDGFRA). HCMV pp65 is a T cell target antigen, and a recombinant adenovirus encoding GP83 was evaluated as a vaccine. In GPCMV challenge studies, vaccinated animals had various levels of protection against wild-type virus, with a protective response against the 22122 prototype strain but little protection against a novel clinical strain of GPCMV (TAMYC), despite 100% identity in GP83 protein sequences. Overall, GP83 is a functional pp65 homolog with novel importance for epithelial cell infection, but a GP83 T cell response provides limited vaccine efficacy. IMPORTANCE Congenital CMV (cCMV) is a leading cause of cognitive impairment and deafness in newborns, and a vaccine is a high priority. The guinea pig is the only small animal model for cCMV but requires guinea pig cytomegalovirus (GPCMV). The translational impact of GPCMV research is potentially reduced if the virus does not encode functional HCMV homolog proteins. This study demonstrates that tegument protein GP83 (pp65 homolog) is involved in innate immune evasion and highly important for infection of nonfibroblast cells via the viral glycoprotein pentamer complex (PC)-dependent endocytic entry pathway. The PC pathway is highly significant for virus dissemination and disease in the host, including cCMV. A GP83 candidate adenovirus (Ad) vaccine strategy in animals induced a cell-mediated response but failed to provide cross-strain protection against a novel clinical strain of GPCMV. Results suggest that the pp65 antigen provides very limited efficacy as a stand-alone vaccine, especially in cross-strain protection.

The high-resolution bistatic lidar developed at the Physikalisch-Technische Bundesanstalt (PTB) aims to overcome the limitations of conventional monostatic lidar technology, which is widely used for wind velocity measurements in wind energy and meteorology applications. Due to the large measurement volume of a combined optical transmitter and receiver tilting in multiple directions, monostatic lidar generally has poor spatial and temporal resolution. It also exhibits large measurement uncertainty when operated in inhomogeneous flow, for instance, over complex terrain. In contrast, PTB’s bistatic lidar uses three dedicated receivers arranged around a central transmitter, resulting in an exceptionally small measurement volume. The coherent detection and modulation schemes used allow the detection of backscattered, Doppler shifted light down to the scale of single aerosols, realising the simultaneous measurement of all three wind velocity components. This paper outlines the design details and theory of operation of PTB’s bistatic lidar and provides an overview of selected comparative measurements. The results of these measurements show that the measurement uncertainty of PTB’s bistatic lidar is well within the measurement uncertainty of traditional cup anemometers while being fully independent of its site and traceable to the SI units. This allows its use as a transfer standard for the calibration of other remote sensing devices. Overall, PTB’s bistatic lidar shows great potential to improve the capability and accuracy of wind velocity measurements, such as for the investigation of highly dynamic flow processes upstream and in the wake of wind turbines.

Guinea pig cytomegalovirus (GPCMV) immediate early proteins, IE1 and IE2, demonstrated structural and functional homologies with human cytomegalovirus (HCMV). GPCMV IE1 and IE2 co-localized in the nucleus with each other, the viral polymerase and guinea pig ND10 components (gpPML, gpDaxx, gpSp100, gpATRX). IE1 showed direct interaction with ND10 components by immunoprecipitation unlike IE2. Additionally, IE1 protein disrupted ND10 bodies. IE1 mutagenesis mapped the nuclear localization signal to the C-terminus and identified the core domain for gpPML interaction. Individual knockout of GPCMV GP122 or GP123 (IE2 and IE1 unique exons respectively) was lethal to the virus. However, an IE1 mutant (codons 234–474 deleted), was viable with attenuated viral growth kinetics and increased susceptibility to type I interferon (IFN-I). In HCMV, the IE proteins are important T cell target antigens. Consequently, characterization of the homologs in GPCMV provides a basis for their evaluation in candidate vaccines against congenital infection.

Although a number of antiviral agents are licensed for treatment of some human herpesvirus (HHV) infections, effective antiviral therapy is not available for all HHVs. Additional complications are associated with approved drugs, such as toxicity and side effects, and rise in drug-resistant strains is a driving force for new drug development. Success in HHV vaccine development is limited with only vaccines against varicella-zoster virus currently in use in the clinic. In vitro, in vivo and in silico high-throughput (HTP) approaches and innovative microfluidic systems will provide novel technologies to efficiently identify and evaluate new targets and antiherpetic compounds. Coupled with HTP strategies for manipulation of herpesvirus viral genomes, these strategies will greatly accelerate the development of future antivirals as well as candidate vaccine intervention strategies.The authors provide a brief overview of the herpesvirus family and associated diseases. Further, the authors discuss the approved and investigational antiherpetic drugs in the context of current HTP technologies.HTP technology such as microfluidic systems is crucial for the identification and validation of novel drug targets and next-generation antivirals. Current drug development is limited by the unavailability of HTP preclinical model systems. Specific advancement in the development of HTP animal-specific technology, applied in parallel, allows a more rapid evaluation of drugs at the preclinical stage. The advancement of HTP combinatorial drug therapy, especially 'Organ-on-a-Chip' approaches, will aid in the evaluation of future antiviral compounds and intervention strategies.

Dendritic cell (DC) subsets can mediate diverse responses, but little is known about the Toll-like receptor (TLR) signalling pathways in different human DC subsets. Despite expressing many TLRs in common, we found that in vitro-derived Langerhans cells (LCs) and monocyte-derived DCs (moDCs) undergo differential signalling events following TLR stimulation. TLR-stimulated LCs did not secrete interleukin (IL)-12p70 and thus induced a T helper type 2 (Th2)-biased response. moDCs secrete high levels of IL-12p70 and induce a Th1 response. Stimulation of moDCs through TLR2 or TLR7/8 was able to induce phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular-signal-regulated kinase (ERK). However, phosphorylated ERK was not induced in TLR-stimulated LCs, suggesting an ERK-independent method of Th2 cell induction. Inhibition of p38 MAPK suppressed moDC maturation, but was much less effective at inhibiting LC maturation. Phosphatidylinositol-3 kinase (PI3K) was also found to play a greater role in moDC survival compared with the LCs. Polymerase chain reaction (PCR) arrays to compare the expression of signalling molecules in LCs and moDCs identified differences in TLR recognition molecules and cytokine response genes, suggesting that differential functional responses are probably mediated at the post-transcriptional level. Thus we have described differences in LC and moDC responses to TLR stimulation, and have identified key differences in ERK phosphorylation and the involvement of MAPK and PI3K.

Dendritic cells (DCs) are important in linking innate and adaptive immune responses by priming and polarizing naive CD4+ Th cells, but little is known about the effect of different human DC subsets on Th cells, particularly Th17 cells. We have investigated the ability of TLR-stimulated human Langerhans cells (LC), dermal DCs (dDC), and monocyte-derived DCs (moDC) to affect naive and memory Th17 and Th1 responses. MoDCs stimulated greater memory T cell proliferation while LCs and dDCs more potently stimulated naive T cell proliferation, indicating functionally distinct subsets of DCs. TLR stimulation of all three DC types was unable to induce Th17 polarization from naive T cell precursors, despite inducing Th1 polarization. Dectin stimulation of DCs in IMDM was however able to produce Th17 cells. TLR-stimulated DCs were capable of inducing IL-17A and IFN-γ production from memory T cells, although the mechanism used by each DC subset differed. MoDCs partially mediated this effect on memory Th1 and Th17 cells by the production of soluble factors, which correlated with their ability to secrete IL-12p70 and IL-23. In contrast, LCs and dDCs were able to elicit a similar memory response to moDCs, but in a contact dependent manner. Additionally, the influence of microbial stimulation was demonstrated with TLR3 and TLR7/8 agonists inducing a Th1 response, whereas TLR2 or dectin stimulation of moDCs enhanced the IL-17 response. This study emphasizes the differences between human DC subsets and demonstrates that both the DC subset and the microbial stimulus influence the Th cell response.

Development of a cytomegalovirus (CMV) vaccine is a major public health priority due to the risk of congenital infection. A key component of a vaccine is thought to be an effective neutralizing antibody response against the viral glycoproteins necessary for cell entry. Species specificity of human CMV (HCMV) precludes direct studies in an animal model. The guinea pig is the only small animal model for congenital cytomegalovirus infection. Analysis of the guinea pig CMV (GPCMV) genome indicates that it potentially encodes homologs to the HCMV glycoproteins (including gB, gH, gL, gM, gN and gO) that form various cell entry complexes on the outside of the virus: gCI (gB); gCII (gH/gL/gO); gCIII (gM/gN). The gB homolog (GP55) has been investigated as a candidate subunit vaccine but little is known about the other homolog proteins. GPCMV glycoproteins were investigated by transient expression studies which indicated that homolog glycoproteins to gN and gM, or gH, gL and gO were able to co-localize in cells and generate respective homolog complexes which could be verified by immunoprecipitation assays. ELISA studies demonstrated that the individual complexes were highly immunogenic in guinea pigs. The gO (GP74) homolog protein has 13 conserved N-glycosylation sites found in HCMV gO. In transient expression studies, only the glycosylated protein is detected but in virus infected cells both N-glycosylated and non-glycosylated gO protein were detected. In protein interaction studies, a mutant gO that lacked N-glycosylation sites had no impact on the ability of the protein to interact with gH/gL which indicated a potential alternative function associated with these sites. Knockout GPCMV BAC mutagenesis of the respective glycoprotein genes (GP55 for gB, GP75 for gH, GP115 for gL, GP100 for gM, GP73 for gN and GP74 for gO) in separate reactions was lethal for virus regeneration on fibroblast cells which demonstrated the essential nature of the GPCMV glycoproteins. The gene knockout results were similar to HCMV, except in the case of the gO homolog, which was non-essential in epithelial tropic virus but essential in lab adapted GPCMV. Overall, the findings demonstrate the similarity between HCMV and GPCMV glycoproteins and strengthen the relevance of this model for development of CMV intervention strategies.

Following the publication of this article (Journal of Translational Medicine, 8:72), it was noted that a co-author had been omitted from the authors list. The submitting authors would like to apologise to Hannes Uchtenhagen for this error. The Competing interests and Authors' contributions sections have now been updated to reflect this amendment.

Dendritic cell (DC) subsets can mediate diverse responses, but little is known about the Toll-like receptor (TLR) signalling pathways in different human DC subsets. Despite expressing many TLRs in common, we found that in vitro-derived Langerhans cells (LCs) and monocyte-derived DCs (moDCs) undergo differential signalling events following TLR stimulation. TLR-stimulated LCs did not secrete interleukin (IL)-12p70 and thus induced a T helper type 2 (Th2)-biased response. moDCs secrete high levels of IL-12p70 and induce a Th1 response. Stimulation of moDCs through TLR2 or TLR7/8 was able to induce phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular-signal-regulated kinase (ERK). However, phosphorylated ERK was not induced in TLR-stimulated LCs, suggesting an ERK-independent method of Th2 cell induction. Inhibition of p38 MAPK suppressed moDC maturation, but was much less effective at inhibiting LC maturation. Phosphatidylinositol-3 kinase (PI3K) was also found to play a greater role in moDC survival compared with the LCs. Polymerase chain reaction (PCR) arrays to compare the expression of signalling molecules in LCs and moDCs identified differences in TLR recognition molecules and cytokine response genes, suggesting that differential functional responses are probably mediated at the post-transcriptional level. Thus we have described differences in LC and moDC responses to TLR stimulation, and have identified key differences in ERK phosphorylation and the involvement of MAPK and PI3K.

Abscisic acid (ABA) and sugars regulate many aspects of plant growth and development, and we are only just beginning to understand the complex interactions between ABA and sugar signaling networks. Here, we show that ABA-dependent transcription factors bind to the promoter of the Arabidopsis thaliana AtSUC1 (At1g71880) sucrose transporter gene in vitro. We present the characterization of a cis-regulatory element by truncation of the AtSUC1 promoter and by electrophoretic mobility shift assays that is identical to a previously characterized ABA-responsive element (ABRE). In yeast 1-hybrid analyses we identified ABI5 (AtbZIP39; At2g36270) and AREB3 (AtbZIP66; At3g56850) as potential interactors. Analyses of plants expressing the beta-glucuronidase reporter gene under the control of ABI5 or AREB3 promoter sequences demonstrated that both transcription factor genes are co-expressed with AtSUC1 in pollen and seedlings, the primary sites of AtSUC1 action. Mutational analyses of the identified cis-regulatory element verified its importance for AtSUC1 expression in young seedlings. In abi5-4 seedlings, we observed an increase of sucrose-dependent anthocyanin accumulation and AtSUC1 mRNA levels. This suggests that ABI5 prevents an overshoot of sucrose-induced AtSUC1 expression and confirmed a novel cross-link between sugar and ABA signaling.

EUROPRISE is a Network of Excellence sponsored from 2007 to 2011 by the European Commission within the 6th Framework Program. The Network encompasses a wide portfolio of activities ranging from an integrated research program in the field of HIV vaccines and microbicides to training, dissemination and advocacy. The research program covers the whole pipeline of vaccine and microbicide development from discovery to early clinical trials. The Network is composed of 58 partners representing more than 65 institutions from 13 European countries; it also includes three major pharmaceutical companies (GlaxoSmithKline, Novartis and Sanofi-Pasteur) involved in HIV microbicide and vaccine research. The Network displays a dedicated and informative web page: http://www.europrise.org. Finally, a distinguishing trait of EUROPRISE is its PhD School of students from across Europe, a unique example in the world of science aimed at spreading excellence through training. EUROPRISE held its second annual conference in Budapest in November, 2009. The conference had 143 participants and their presentations covered aspects of vaccine and microbicide research, development and discovery. Since training is a major task of the Network, the students of the EUROPRISE PhD program summarized certain presentations and their view of the conference in this paper.

In the recently halted HIV type 1 (HIV-1) vaccine STEP trial, individuals that were seropositive for adenovirus serotype 5 (Ad5) showed increased rates of HIV-1 infection on vaccination with an Ad5 vaccine. We propose that this was due to activation and expansion of Ad5-specific mucosal-homing memory CD4 T cells. To test this hypothesis, Ad5 and Ad11 antibody titers were measured in 20 healthy volunteers. Dendritic cells (DCs) from these individuals were pulsed with replication defective Ad5 or Ad11 and co-cultured with autologous lymphocytes. Cytokine profiles, proliferative capacity, mucosal migration potential, and susceptibility to HIV infection of the adenovirus-stimulated memory CD4 T cells were measured. Stimulation of T cells from healthy Ad5-seropositive but Ad11-seronegative individuals with Ad5, or serologically distinct Ad11 vectors induced preferential expansion of adenovirus memory CD4 T cells expressing α 4 β 7 integrins and CCR9, indicating a mucosal-homing phenotype. CD4 T-cell proliferation and IFN-γ production in response to Ad stimulation correlated with Ad5 antibody titers. However, Ad5 serostatus did not correlate with total cytokine production upon challenge with Ad5 or Ad11. Expanded Ad5 and Ad11 memory CD4 T cells showed an increase in CCR5 expression and higher susceptibility to infection by R5 tropic HIV-1. This suggests that adenoviral-based vaccination against HIV-1 in individuals with preexisting immunity against Ad5 results in preferential expansion of HIV-susceptible activated CD4 T cells that home to mucosal tissues, increases the number of virus targets, and leads to a higher susceptibility to HIV acquisition.

Open Access Oral presentation OA07-02. Adenovirus vectors induce expansion of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to HIV-1 infection A Benlahrech*1, J Harris2, A Meiser1, T Papagatsias1, J Hornig1, P Hayes1, A Lieber3, T Athanasopoulos2, V Bachy4, R Daniels5, K Fisher6, F Gotch1, L Klavinskis4, L Seymour6, K Logan1, R Barbagallo1, G Dickson2 and S Patterson1

Neural crest cells and oligodendrocytes as the myelinating glia of the central nervous system exist only in vertebrates. Their development is regulated by complex regulatory networks, of which the SoxE-type high-mobility-group domain transcription factors Sox8, Sox9 and Sox10 are essential components. Here we analyzed by in ovo electroporation in chicken and by gene replacement in the mouse whether the Drosophila ortholog Sox100B can functionally substitute for vertebrate SoxE proteins. Sox100B overexpression in the chicken neural tube led to the induction of neural crest cells as previously observed for vertebrate SoxE proteins. Furthermore, many aspects of neural crest and oligodendrocyte development were surprisingly normal in mice in which the Sox10 coding information was replaced by Sox100B arguing that Sox100B integrates well into the gene-regulatory networks that drive these processes. Our results therefore provide strong evidence for a model in which SoxE proteins were co-opted to these gene-regulatory networks mainly through the acquisition of novel expression patterns. However, later developmental defects in several neural crest derived lineages in mice homozygous for the Sox100B replacement allele indicate that some degree of functional specialization and adaptation of SoxE protein properties have taken place in addition to the co-option event.

In maart 2021 bleef het Suezkanaal gedurende 6 dagen volledig versperd voor alle scheepvaartverkeer door het vastlopen van het containerschip Ever Given. Een 150-tal andere schepen hebben hierdoor aanzienlijke vertraging opgelopen. In deze bijdrage worden een aantal van de mogelijke juridische en verzekeringsrechtelijke gevolgen van dit incident besproken.