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Helminth parasites secrete a wide variety of immunomodulatory proteins and lipids to dampen host immune responses. Many of these immunomodulatory compounds are modified with complex sugar structures (or glycans), which play an important role at the host–parasite interface. As an example, the human blood fluke Schistosoma mansoni produces highly fucosylated glycan structures on glycoproteins and glycolipids. Up to 20 different S. mansoni fucosyltransferase (SmFucT) genes can be found in genome databases, but thus far only one enzyme has been functionally characterized. To unravel the synthesis of highly fucosylated N-glycans by S. mansoni , we examined the ability of ten selected SmFucTs to modify N-glycans upon transient expression in Nicotiana benthamiana plants. All enzymes were localized in the plant Golgi apparatus, which allowed us to identify the SmFucTs involved in core fucosylation and the synthesis of complex antennary glycan motifs. This knowledge provides a starting point for investigations into the role of specific fucosylated glycan motifs of schistosomes in parasite-host interactions. The functionally characterized SmFucTs can also be applied to synthesize complex N-glycan structures on recombinant proteins to study their contribution to immunomodulation. Furthermore, this plant expression system will fuel the development of helminth glycoproteins for pharmaceutical applications or novel anti-helminth vaccines.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ornithine carbamoyltransferases (OTCs) are involved in the arginine deiminase (ADI) pathway and in arginine biosynthesis. Two OTCs in a pair are named catalytic OTC (cOTC) and anabolic OTC (aOTC). The cOTC is responsible for catalyzing the third step of the ADI pathway to catabolize citrulline into carbamoyl phosphate (CP), as well as ornithine, and displays CP cooperativity. In contrast, aOTC catalyzes the biosynthesis of citrulline from CP and ornithine in vivo and is thus involved in arginine biosynthesis. Structural and biochemical analyses were employed to investigate the CP cooperativity and unidirectional function of two sequentially similar OTCs (32.4% identity) named Ps _cOTC and Ps _aOTC from Psychrobacter sp. PAMC 21119. Comparison of the trimeric structure of these two OTCs indicated that the 80s loop of Ps _cOTC has a unique conformation that may influence cooperativity by connecting the CP binding site and the center of the trimer. The corresponding 80s loop region of in Ps _aOTC was neither close to the CP binding site nor connected to the trimer center. In addition, results from the thermal shift assay indicate that each OTC prefers the substrate for the unidirectional process. The active site exhibited a blocked binding site for CP in the Ps _cOTC structure, whereas residues at the active site in Ps _aOTC established a binding site to facilitate CP binding. Our data provide novel insights into the unidirectional catalysis of OTCs and cooperativity, which are distinguishable features of two metabolically specialized proteins.

Parasitic nematodes responsible for filarial diseases cause chronic disablement in humans worldwide. Elimination programs have substantially reduced the rate of infection in certain areas, but limitations of current diagnostics for population surveillance have been pointed out and improved assays are needed to reach the elimination targets. While serological tests detecting antibodies to parasite antigens are convenient tools, those currently available are compromised by the occurrence of antibodies cross-reactive between nematodes, as well as by the presence of residual antibodies in sera years after treatment and clearance of the infection. We recently characterized the N-linked and glycosphingolipid derived glycans of the parasitic nematode Brugia malayi and revealed the presence of various antigenic structures that triggered immunoglobulin G (IgG) responses in infected individuals. To address the specificity of IgG binding to these glycan antigens, we screened microarrays containing Brugia malayi glycans with plasma from uninfected individuals and from individuals infected with Loa loa , Onchocerca volvulus , Mansonella perstans and Wuchereria bancrofti , four closely related filarial nematodes. IgG to a restricted subset of cross-reactive glycans was observed in infection plasmas from all four species. In plasma from Onchocerca volvulus and Mansonella perstans infected individuals, IgG binding to many more glycans was additionally detected, resulting in total IgG responses similar to the ones of Brugia malayi infected individuals. For these infection groups, Brugia malayi , Onchocerca volvulus and Mansonella perstans , we further studied the different IgG subclasses to Brugia malayi glycans. In all three infections, IgG1 and IgG2 appeared to be the major subclasses involved in response to glycan antigens. Interestingly, in Brugia malayi infected individuals, we observed a marked reduction in particular in IgG2 to parasite glycans post-treatment with anthelminthic, suggesting a promising potential for diagnostic applications. Thus, we compared the IgG response to a broad repertoire of Brugia malayi glycans in individuals infected with various filarial nematodes. We identified broadly cross-reactive and more specific glycan targets, extending the currently scarce knowledge of filarial nematode glycosylation and host anti-glycan antibody response. We believe that our initial findings could be further exploited to develop disease-specific diagnostics as part of an integrated approach for filarial disease control.

Secretions of parasitic worms (helminths) contain a wide collection of immunomodulatory glycoproteins with the potential to treat inflammatory disorders, like autoimmune diseases. Yet, the identification of single molecules that can be developed into novel biopharmaceuticals is hampered by the limited availability of native parasite-derived proteins. Recently, pioneering work has shown that helminth glycoproteins can be produced transiently in Nicotiana benthamiana plants while simultaneously mimicking their native helminth N-glycan composition by co-expression of desired glycosyltransferases. However, efficient “helminthization” of N-glycans in plants by glyco-engineering seems to be hampered by the undesired truncation of complex N-glycans by β- N -acetyl-hexosaminidases, in particular when aiming for the synthesis of N-glycans with antennary GalNAcβ1-4GlcNAc (LacdiNAc or LDN). In this study, we cloned novel β-hexosaminidase open reading frames from N. benthamiana and characterized the biochemical activity of these enzymes. We identified HEXO2 and HEXO3 as enzymes responsible for the cleavage of antennary GalNAc residues of N-glycans on the model helminth glycoprotein kappa-5. Furthermore, we reveal that each member of the HEXO family has a distinct specificity for N-glycan substrates, where HEXO2 has strict β-galactosaminidase activity, whereas HEXO3 cleaves both GlcNAc and GalNAc. The identification of HEXO2 and HEXO3 as major targets for LDN cleavage will enable a targeted genome editing approach to reduce undesired processing of these N-glycans. Effective knockout of these enzymes could allow the production of therapeutically relevant glycoproteins with tailor-made helminth N-glycans in plants.

Human interleukin‐22 (IL‐22) is a member of the IL‐10 cytokine family that has recently been shown to have major therapeutic potential. IL‐22 is an unusual cytokine as it does not act directly on immune cells. Instead, IL‐22 controls the differentiation, proliferation and antimicrobial protein expression of epithelial cells, thereby maintaining epithelial barrier function. In this study, we transiently expressed human IL‐22 in Nicotiana benthamiana plants and investigated the role of N‐glycosylation on protein folding and biological activity. Expression levels of IL‐22 were up to 5.4 μg/mg TSP, and N‐glycan analysis revealed the presence of the atypical Lewis A structure. Surprisingly, upon engineering of human‐like N‐glycans on IL‐22 by co‐expressing mouse FUT8 in ΔXT/FT plants a strong reduction in Lewis A was observed. Also, core α1,6‐fucoylation did not improve the biological activity of IL‐22. The combination of site‐directed mutagenesis of Asn54 and in vivo deglycosylation with PNGase F also revealed that N‐glycosylation at this position is not required for proper protein folding. However, we do show that the presence of a N‐glycan on Asn54 contributes to the atypical N‐glycan composition of plant‐produced IL‐22 and influences the N‐glycan composition of N‐glycans on other positions. Altogether, our data demonstrate that plants offer an excellent tool to investigate the role of N‐glycosylation on folding and activity of recombinant glycoproteins, such as IL‐22.

Using panel data of twenty Asian countries over a period of six years (2011-2016), this research investigated the key determinants of financial inclusion among Asian countries via the Random Effects Model (REM). Financial inclusion has been considered as enabler for seven of the seventeen sustainable development goals, which brings access to financial services to all and directly contributes to poverty reduction, capacity buildings and equality. The main findings are that: (i) the countries with stronger economic growth and higher income have a significantly higher financial inclusion index, as people have more resources/incomes and better chances to utilize financial services; (ii) the higher the literacy, the better the financial inclusion as people with higher literacy understand the pros and cons of financial services and providers, better knowledge of using financial services wisely; (iii) unemployment rates had a negative impact on the financial inclusion index; (iv) surprisingly differing from previous studies, inflation, population density, network and deposit interest rate were not statistically significantly correlated with financial inclusion.

This study analyzes the factors that influence the choice of saltwater intrusion adaptation strategies of farmers living in Vietnam’s Central Coastal region, using a multinomial choice model fitted to data from a cross-sectional survey of 414 farmers. The adaptation strategies identified include cultivating new varieties of rice or switching to papyrus planting, vegetable planting, shrimp production, or lotus–fish production. The results reveal that family size decreases the likelihood of applying new varieties of rice and plant papyrus but increases the probability of applying shrimp production. The level of education of the household’s head negatively impacts the probability of switching to new varieties of rice and shrimp production but positively affects the odds of implementing papyrus and vegetable production. The older, more experienced farmers are more likely to cultivate new varieties of rice. Regarding the farm’s characteristics, the higher the percentage of salted land and the less serious the saltwater intrusion level, the higher the probability of switching to shrimp or lotus–fish production. Access to information from the local authorities, taking part in training courses, and being a member of Vietnamese Women Union all promote adaptation to saltwater intrusion. From a policy perspective, we recommend that the government develop official media channels to promote saltwater intrusion adaptation, organize more training courses for farmers, support the activities of the Woman Union, and relax constraints on accessing public credit.

Artificial Intelligence (AI) when incorporated with machine learning and deep learning algorithms can have a wide variety of applications today. This book focuses on the implementation of various elementary and advanced approaches in AI that can be used in various domains to solve real-time decision-making problems. The book focuses on concepts and techniques used to run tasks in an automated manner. It discusses computational intelligence in the detection and diagnosis of clinical and biomedical images, it covers the automation of a system through machine learning and deep learning approaches, presents data analytics and mining for decision support applications, includes case-based reasoning, natural language processing, computer vision, and AI approaches in real-time applications. Academic scientists, researchers and students in the various domains of computer science engineering, electronics and communication engineering, information technology, industrial engineers, biomedical engineers, and management will find this book useful. By the end of this book, you will have understood the fundamentals of AI and various case studies that will develop your adaptive thinking to solve real time AI problems.

Many polar organisms produce antifreeze proteins (AFPs) and ice-binding proteins (IBPs) to protect themselves from ice formation. As IBPs protect cells and organisms, the potential of IBPs as natural or biological cryoprotective agents (CPAs) for the cryopreservation of animal cells, such as oocytes and sperm, has been explored to increase the recovery rate after freezing–thawing. However, only a few IBPs have shown success in cryopreservation, possibly because of the presence of protein denaturants, such as dimethyl sulfoxide, alcohols, or ethylene glycol, in freezing buffer conditions, rendering the IBPs inactive. Therefore, we investigated the thermal and chemical stability of Ff IBP isolated from Antarctic bacteria to assess its suitability as a protein-based impermeable cryoprotectant. A molecular dynamics (MD) simulation identified and generated stability-enhanced mutants ( Ff IBP_CC1). The results indicated that Ff IBP_CC1 displayed enhanced resistance to denaturation at elevated temperatures and chemical concentrations, compared to wildtype Ff IBP, and was functional in known CPAs while retaining ice-binding properties. Given that Ff IBP shares an overall structure similar to DUF3494 IBPs, which are recognized as the most widespread IBP family, these findings provide important structural information on thermal and chemical stability, which could potentially be applied to other DUF3494 IBPs for future protein engineering.