An Interspecific Nicotiana Hybrid as a Useful and Cost-Effective Platform for Production of Animal Vaccines

The use of transgenic plants to produce novel products has great biotechnological potential as the relatively inexpensive inputs of light, water, and nutrients are utilised in return for potentially valuable bioactive metabolites, diagnostic proteins and vaccines. Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines of importance for both animals and humans. Vaccine purification is generally regarded as being integral to the preparation of safe and effective vaccines for use in humans. However, the use of crude plant extracts for animal immunisation may enable plant-made vaccines to become a cost-effective and efficacious approach to safely immunise large numbers of farm animals against diseases such as avian influenza. Since the technology associated with genetic transformation and large-scale propagation is very well established in Nicotiana, the genus has attributes well-suited for the production of plant-made vaccines. However the presence of potentially toxic alkaloids in Nicotiana extracts impedes their use as crude vaccine preparations. In the current study we describe a Nicotiana tabacum and N. glauca hybrid that expresses the HA glycoprotein of influenza A in its leaves but does not synthesize alkaloids. We demonstrate that injection with crude leaf extracts from these interspecific hybrid plants is a safe and effective approach for immunising mice. Moreover, this antigen-producing alkaloid-free, transgenic interspecific hybrid is vigorous, with a high capacity for vegetative shoot regeneration after harvesting. These plants are easily propagated by vegetative cuttings and have the added benefit of not producing viable pollen, thus reducing potential problems associated with bio-containment. Hence, these Nicotiana hybrids provide an advantageous production platform for partially purified, plant-made vaccines which may be particularly well suited for use in veterinary immunization programs

Transient Expression of Hemagglutinin Antigen from Low Pathogenic Avian Influenza A (H7N7) in Nicotiana benthamiana

The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species

O2-Filled Swimbladder Employs Monocarboxylate Transporters for the Generation of O2 by Lactate-Induced Root Effect Hemoglobin

The swimbladder volume is regulated by O2 transfer between the luminal space and the blood In the swimbladder, lactic acid generation by anaerobic glycolysis in the gas gland epithelial cells and its recycling through the rete mirabile bundles of countercurrent capillaries are essential for local blood acidification and oxygen liberation from hemoglobin by the “Root effect.” While O2 generation is critical for fish flotation, the molecular mechanism of the secretion and recycling of lactic acid in this critical process is not clear. To clarify molecules that are involved in the blood acidification and visualize the route of lactic acid movement, we analyzed the expression of 17 members of the H+/monocarboxylate transporter (MCT) family in the fugu genome and found that only MCT1b and MCT4b are highly expressed in the fugu swimbladder. Electrophysiological analyses demonstrated that MCT1b is a high-affinity lactate transporter whereas MCT4b is a low-affinity/high-conductance lactate transporter. Immunohistochemistry demonstrated that (i) MCT4b expresses in gas gland cells together with the glycolytic enzyme GAPDH at high level and mediate lactic acid secretion by gas gland cells, and (ii) MCT1b expresses in arterial, but not venous, capillary endothelial cells in rete mirabile and mediates recycling of lactic acid in the rete mirabile by solute-specific transcellular transport. These results clarified the mechanism of the blood acidification in the swimbladder by spatially organized two lactic acid transporters MCT4b and MCT1b

Wastewater to clinical case (WC) ratio of COVID-19 identifies insufficient clinical testing, onset of new variants of concern and population immunity in urban communities

Clinical testing has been the cornerstone of public health monitoring and infection control efforts in communities throughout the COVID-19 pandemic. With the extant and anticipated reduction of clinical testing as the disease moves into an endemic state, SARS-CoV-2 wastewater surveillance (WWS) is likely to have greater value as an important diagnostic tool to inform public health. As the widespread adoption of WWS is relatively new at the scale employed for COVID-19, interpretation of data, including the relationship to clinical cases, has yet to be standardized. An in-depth analysis of the metrics derived from WWS is required for public health units/agencies to interpret and utilize WWS-acquired data effectively and efficiently. In this study, the SARS-CoV-2 wastewater signal to clinical cases (WC) ratio was investigated across seven different cities in Canada over periods ranging from 8 to 21 months. Significant increases in the WC ratio occurred when clinical testing eligibility was modified to appointment-only testing, identifying a period of insufficient clinical testing in these communities. The WC ratio decreased significantly during the emergence of the Alpha variant of concern (VOC) in a relatively non-immunized community’s wastewater (40-60% allelic proportion), while a more muted decrease in the WC ratio signaled the emergence of the Delta VOC in a relatively well-immunized community’s wastewater (40-60% allelic proportion). Finally, a rapid and significant decrease in the WC ratio signaled the emergence of the Omicron VOC, likely because of the variant’s greater effectiveness at evading immunity, leading to a significant number of new reported clinical cases, even when vaccine-induced community immunity was high. The WC ratio, used as an additional monitoring metric, complements clinical case counts and wastewater signals as individual metrics in its ability to identify important epidemiological occurrences, adding value to WWS as a diagnostic technology during the COVID-19 pandemic and likely for future pandemics.Ontario's Ministry of Environment, Conservation and Parks||Alberta Healt

Recent advances quantifying the large wood dynamics in river basins: New methods and remaining challenges

Citation: Ruiz-Villanueva, V., Piégay, H., Gurnell, A. A., Marston, R. A., & Stoffel, M. (2016). Recent advances quantifying the large wood dynamics in river basins: New methods and remaining challenges. Reviews of Geophysics. doi:10.1002/2015RG000514Large wood is an important physical component of woodland rivers and significantly influences river morphology. It is also a key component of stream ecosystems. However, large wood is also a source of risk for human activities as it may damage infrastructure, block river channels, and induce flooding. Therefore, the analysis and quantification of large wood and its mobility are crucial for understanding and managing wood in rivers. As the amount of large-wood-related studies by researchers, river managers, and stakeholders increases, documentation of commonly used and newly available techniques and their effectiveness has also become increasingly relevant as well. Important data and knowledge have been obtained from the application of very different approaches and have generated a significant body of valuable information representative of different environments. This review brings a comprehensive qualitative and quantitative summary of recent advances regarding the different processes involved in large wood dynamics in fluvial systems including wood budgeting and wood mechanics. First, some key definitions and concepts are introduced. Second, advances in quantifying large wood dynamics are reviewed; in particular, how measurements and modeling can be combined to integrate our understanding of how large wood moves through and is retained within river systems. Throughout, we present a quantitative and integrated meta-analysis compiled from different studies and geographical regions. Finally, we conclude by highlighting areas of particular research importance and their likely future trajectories, and we consider a particularly underresearched area so as to stress the future challenges for large wood research. ©2016. American Geophysical Union