Climate impacts of alternative beef production systems depend on the functional unit used: Weight or monetary value.

Beef production has been identified as a significant source of anthropogenic greenhouse gas (GHG) emissions in the agricultural sector. United States and Canada account for about a quarter of the world's beef supply. To compare the GHG emission contributions of alternative beef production systems, we conducted a meta-analysis of 32 studies that were conducted between 2001 and 2023. Results indicated that GHG emissions from beef production in North America varied almost fourfold from 10.2 to 37.6 with an average of 21.4 kg CO2e/kg carcass weight (CW). Studies that considered soil C sequestration (C-seq) reported the highest mitigation potential in GHG emissions (80%), followed by growth enhancement technology (16%), diet modification (6%), and grazing management improvement (7%). Our study highlights the implications of using carbon intensity per economic activity (i.e., GHG emissions per monetary unit), compared to the more common metric of intensity on per weight of product basis (GHG emissions per kg CW) for comparisons across differentiated beef cattle products. While a positive association was found between the proportion of lifespan on grassland and the conventional weight-based indicator, grass-finished beef was found to have lower carbon intensity per economic activity than feedlot-finished beef. Our study emphasizes the need to incorporate land use and management effects and soil C-seq as fundamental aspects of beef GHG emissions and mitigation assessments.

GCRV-II invades monocytes/macrophages and induces macrophage polarization and apoptosis in tissues to facilitate viral replication and dissemination.

Grass carp reovirus (GCRV), particularly the highly prevalent type II GCRV (GCRV-II), causes huge losses in the aquaculture industry. However, little is known about the mechanisms by which GCRV-II invades grass carp and further disseminates among tissues. In the present study, monocytes/macrophages (Mo/Mφs) were isolated from the peripheral blood of grass carp and infected with GCRV-II. The results of indirect immunofluorescent microscopy, transmission electron microscopy, real-time quantitative RT-PCR (qRT-PCR), western blot (WB), and flow cytometry analysis collectively demonstrated that GCRV-II invaded Mo/Mφs and replicated in them. Additionally, we observed that GCRV-II induced different types (M1 and M2) of polarization of Mo/Mφs in multiple tissues, especially in the brain, head kidney, and intestine. To assess the impact of different types of polarization on GCRV-II replication, we recombinantly expressed and purified the intact cytokines CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B and successfully induced M1 and M2 type polarization of macrophages using these cytokines through in vitro experiments. qRT-PCR, WB, and flow cytometry analyses showed that M2 macrophages had higher susceptibility to GCRV-II infection than other types of Mo/Mφs. In addition, we found GCRV-II induced apoptosis of Mo/Mφs to facilitate virus replication and dissemination and also detected the presence of GCRV-II virus in plasma. Collectively, our findings indicated that GCRV-II could invade immune cells Mo/Mφs and induce apoptosis and polarization of Mo/Mφs for efficient infection and dissemination, emphasizing the crucial role of Mo/Mφs as a vector for GCRV-II infection.IMPORTANCEType II grass carp reovirus (GCRV) is a prevalent viral strain and causes huge losses in aquaculture. However, the related dissemination pathway and mechanism remain largely unclear. Here, our study focused on phagocytic immune cells, monocytes/macrophages (Mo/Mφs) in blood and tissues, and explored whether GCRV-II can invade Mo/Mφs and replicate and disseminate via Mo/Mφs with their differentiated type M1 and M2 macrophages. Our findings demonstrated that GCRV-II infected Mo/Mφs and replicated in them. Furthermore, GCRV-II infection induces an increased number of M1 and M2 macrophages in grass carp tissues and a higher viral load in M2 macrophages. Furthermore, GCRV-II induced Mo/Mφs apoptosis to release viruses, eventually infecting more cells. Our study identified Mo/Mφs as crucial components in the pathway of GCRV-II dissemination and provides a solid foundation for the development of treatment strategies for GCRV-II infection.

Liquid-liquid phase separation is essential for reovirus viroplasm formation and immune evasion.

Grass carp reovirus (GCRV) is the most virulent pathogen in the genus Aquareovirus, belonging to the family Spinareoviridae. Members of the Spinareoviridae family are known to replicate and assemble in cytoplasmic inclusion bodies termed viroplasms; however, the detailed mechanism underlying GCRV viroplasm formation and its specific roles in virus infection remains largely unknown. Here, we demonstrate that GCRV viroplasms form through liquid-liquid phase separation (LLPS) of the nonstructural protein NS80 and elucidate the specific role of LLPS during reovirus infection and immune evasion. We observe that viroplasms coalesce within the cytoplasm of GCRV-infected cells. Immunofluorescence and transmission electron microscopy indicate that GCRV viroplasms are membraneless structures. Live-cell imaging and fluorescence recovery after photobleaching assay reveal that GCRV viroplasms exhibit liquid-like properties and are highly dynamic structures undergoing fusion and fission. Furthermore, by using a reagent to inhibit the LLPS process and constructing an NS80 mutant defective in LLPS, we confirm that the liquid-like properties of viroplasms are essential for recruiting viral dsRNA, viral RdRp, and viral proteins to participate in viral genome replication and virion assembly, as well as for sequestering host antiviral factors for immune evasion. Collectively, our findings provide detailed insights into reovirus viroplasm formation and reveal the specific functions of LLPS during virus infection and immune evasion, identifying potential targets for the prevention and control of this virus. IMPORTANCE: Grass carp reovirus (GCRV) poses a significant threat to the aquaculture industry, particularly in China, where grass carp is a vital commercial fish species. However, detailed information regarding how GCRV viroplasms form and their specific roles in GCRV infection remains largely unknown. We discovered that GCRV viroplasms exhibit liquid-like properties and are formed through a physico-chemical biological phenomenon known as liquid-liquid phase separation (LLPS), primarily driven by the nonstructural protein NS80. Furthermore, we confirmed that the liquid-like properties of viroplasms are essential for virus replication, assembly, and immune evasion. Our study not only contributes to a deeper understanding of GCRV infection but also sheds light on broader aspects of viroplasm biology. Given that viroplasms are a universal feature of reovirus infection, inhibiting LLPS and then blocking viroplasms formation may serve as a potential pan-reovirus inhibition strategy.

CRISPR-based genome editing of a diurnal rodent, Nile grass rat (Arvicanthis niloticus).

BACKGROUND: Diurnal and nocturnal mammals have evolved distinct pathways to optimize survival for their chronotype-specific lifestyles. Conventional rodent models, being nocturnal, may not sufficiently recapitulate the biology of diurnal humans in health and disease. Although diurnal rodents are potentially advantageous for translational research, until recently, they have not been genetically tractable. The present study aims to address this major limitation by developing experimental procedures necessary for genome editing in a well-established diurnal rodent model, the Nile grass rat (Arvicanthis niloticus). RESULTS: A superovulation protocol was established, which yielded nearly 30 eggs per female grass rat. Fertilized eggs were cultured in a modified rat 1-cell embryo culture medium (mR1ECM), in which grass rat embryos developed from the 1-cell stage into blastocysts. A CRISPR-based approach was then used for gene editing in vivo and in vitro, targeting Retinoic acid-induced 1 (Rai1), the causal gene for Smith-Magenis Syndrome, a neurodevelopmental disorder. The CRISPR reagents were delivered in vivo by electroporation using an improved Genome-editing via Oviductal Nucleic Acids Delivery (i-GONAD) method. The in vivo approach produced several edited founder grass rats with Rai1 null mutations, which showed stable transmission of the targeted allele to the next generation. CRISPR reagents were also microinjected into 2-cell embryos in vitro. Large deletion of the Rai1 gene was confirmed in 70% of the embryos injected, demonstrating high-efficiency genome editing in vitro. CONCLUSION: We have established a set of methods that enabled the first successful CRISPR-based genome editing in Nile grass rats. The methods developed will guide future genome editing of this and other diurnal rodent species, which will promote greater utility of these models in basic and translational research.

Grass xylan structural variation suggests functional specialization and distinctive interaction with cellulose and lignin.

Xylan is the most abundant non-cellulosic polysaccharide in grass cell walls, and it has important structural roles. The name glucuronoarabinoxylan (GAX) is used to describe this variable hemicellulose. It has a linear backbone of ß-1,4-xylose (Xyl) residues that may be substituted with α-1,2-linked (4-O-methyl)-glucuronic acid (GlcA), α-1,3-linked arabinofuranose (Araf), and sometimes acetylation at the O-2 and/or O-3 positions. The role of these substitutions remains unclear, although there is increasing evidence that they affect the way xylan interacts with other cell wall components, particularly cellulose and lignin. Here, we used substitution-dependent endo-xylanase enzymes to investigate the variability of xylan substitution in grass culm cell walls. We show that there are at least three different types of xylan: (i) an arabinoxylan with evenly distributed Araf substitutions without GlcA (AXe); (ii) a glucuronoarabinoxylan with clustered GlcA modifications (GAXc); and (iii) a highly substituted glucuronoarabinoxylan (hsGAX). Immunolocalization of AXe and GAXc in Brachypodium distachyon culms revealed that these xylan types are not restricted to a few cell types but are instead widely detected in Brachypodium cell walls. We hypothesize that there are functionally specialized xylan types within the grass cell wall. The even substitutions of AXe may permit folding and binding on the surface of cellulose fibrils, whereas the more complex substitutions of the other xylans may support a role in the matrix and interaction with other cell wall components.

Preliminary study of BF/C2 on immune mechanism of grass carp against GCRV infection.

BF/C2 is a crucial molecule in the coagulation complement cascade pathway and plays a significant role in the immune response of grass carp through the classical, alternative, and lectin pathways during GCRV infection. In vivo experiments demonstrated that the mRNA expression levels of BF/C2 (A, B) in grass carp positively correlated with GCRV viral replication at various stages of infection. Excessive inflammation leading to death coincided with peak levels of BF/C2 (A, B) mRNA expression and GCRV viral replication. Correspondingly, BF/C2 (A, B) recombinant protein, CIK cells and GCRV co-incubation experiments yielded similar findings. Therefore, 3 h (incubation period) and 9 h (death period) were selected as critical points for this study. Transcriptome sequencing analysis revealed significant differences in the expression of BF/C2A and BF/C2B during different stages of CIK infection with GCRV and compared to the blank control group (PBS). Specifically, the BF/C2A_3 and BF/C2A_9 groups exhibited 2729 and 2228 differentially expressed genes (DEGs), respectively, with 1436 upregulated and 1293 downregulated in the former, and 1324 upregulated and 904 downregulated in the latter. The BF/C2B_3 and BF/C2B_9 groups showed 2303 and 1547 DEGs, respectively, with 1368 upregulated and 935 downregulated in the former, and 818 upregulated and 729 downregulated in the latter. KEGG functional enrichment analysis of these DEGs identified shared pathways between BF/C2A and PBS groups at 3 and 9 h, including the C-type lectin receptor signaling pathway, protein processing in the endoplasmic reticulum, Toll-like receptor signaling pathway, Salmonella infection, apoptosis, tight junction, and adipocytokine signaling pathway. Additionally, the BF/C2B groups at 3 and 9 h shared pathways related to protein processing in the endoplasmic reticulum, glycolysis/gluconeogenesis, and biosynthesis of amino acids. The mRNA levels of these DEGs were validated in cellular models, confirming consistency with the sequencing results. In addition, the mRNA expression levels of these candidate genes (mapk1, il1b, rela, nfkbiab, akt3a, hyou1, hsp90b1, dnajc3a et al.) in the head kidney, kidney, liver and spleen of grass carp immune tissue were significantly different from those of the control group by BF/C2 (A, B) protein injection in vivo. These candidate genes play an important role in the response of BF/C2 (A, B) to GCRV infection and it also further confirmed that BF/C2 (A, B) of grass carp plays an important role in coping with GCRV infection.

Genome-wide analysis of the WRKY gene family and their response to low-temperature stress in elephant grass.

BACKGROUD: Elephant grass (Cenchrus purpureus) is a perennial forage grass characterized by tall plants, high biomass and wide adaptability. Low-temperature stress severely limits elephant grass biomass and geographic distribution. WRKY is one of the largest families of plant-specific transcription factors and plays important roles in plant resistance to low-temperature. However, the understanding of the WRKY family in grasses is limited. In this study, we conducted a genome-wide characterization of WRKY proteins in elephant grass, including gene structure, phylogeny, expression, conserved motif organization, and functional annotation, to identify key CpWRKY candidates involved in cold tolerance. RESULTS: In this study, a total of 176 WRKY genes were identified in elephant grass. It was found that 172 were unevenly distributed across its 14 chromosomes, while the remaining 4 genes were not anchored to any chromosome. The genes were classified into three groups based on their WRKY conserved domains and zinc finger motifs. There were 12, 8, 19, 27, 12, 18 and 80 CpWRKYs belonging to group I, group IIa, group IIb, group IIc, group IId, group IIe and group III, respectively. We hypothesized that the ancient subgroup IIc WRKY gene is the ancestor of all WRKY genes in elephant grass. Most CpWRKYs in the same group have similar structure and motif composition. A total of 169 duplicate gene pairs were identified, suggesting that segmental duplication might have contributed to the expansion of the CpWRKY gene family. Ka/Ks analysis revealed that most of the CpWRKYs were subjected to purifying selection during the evolution. It was also found that six genes (CpWRKY51, CpWRKY81, CpWRKY100, CpWRKY101, CpWRKY140 and CpWRKY143) exhibited higher expression in roots compare to leaves, and were significantly induced by low temperature stress. Among them, CpWRKY81 had the highest expression under low-temperature stress, and its over-expression significantly enhanced the cold tolerance in yeast. CONLUSIONS: In this study, we characterized WRKY genes in elephant grass and further investigated their physicochemical properties, evolution, and expression patterns under low-temperature stress. This research provides valuable resources for identifying key CpWRKY genes that contribute to cold tolerance in elephant grass.

Genome-wide identification of Gα family in grass carp (Ctenopharyngodon idella) and reproductive regulation functional characteristics of Cignaq.

BACKGROUND: The Gα family plays a crucial role in the complex reproductive regulatory network of teleosts. However, the characterization and function of Gα family members, especially Gαq, remain poorly understood in teleosts. To analyze the characterization, expression, and function of grass carp (Ctenopharyngodon idella) Gαq, we identified the Gα family members in grass carp genome, and analyzed the expression, distribution, and signal transduction of Gαq/gnaq. We also explored the role of Gαq in the reproductive regulation of grass carp. RESULTS: Our results showed that the grass carp genome contains 27 Gα genes with 46 isoforms, which are divided into four subfamilies: Gαs, Gαi/o, Gαq/11, and Gα12/13. The expression level of Cignaq in the testis was the highest and significantly higher than in other tissues, followed by the hypothalamus and brain. The luteinizing hormone receptor (LHR) was mainly localized to the nucleus in grass carp oocytes, with signals also present in follicular cells. In contrast, Gαq signal was mainly found in the cytoplasm of oocytes, with no signal in follicular cells. In the testis, Gαq and LHR were co-localized in the cytoplasm. Furthermore, the grass carp Gαq recombinant protein significantly promoted Cipgr expression. CONCLUSIONS: These results provided preliminary evidence for understanding the role of Gαq in the reproductive regulation of teleosts.

The Potential of CRISPR/Cas9 to Circumvent the Risk Factor Neurotoxin ß-N-oxalyl-L-α, ß-diaminopropionic acid Limiting Wide Acceptance of the Underutilized Grass Pea (Lathyrus sativus L.).

Grass pea (Lathyrus sativus L.) is a protein-rich crop that is resilient to various abiotic stresses, including drought. However, it is not cultivated widely for human consumption due to the neurotoxin ß-N-oxalyl-L-α, ß-diaminopropionic acid (ß-ODAP) and its association with neurolathyrism. Though some varieties with low ß-ODAP have been developed through classical breeding, the ß-ODAP content is increasing due to genotype x environment interactions. This review covers grass pea nutritional quality, ß-ODAP biosynthesis, mechanism of paralysis, traditional ways to reduce ß-ODAP, candidate genes for boosting sulfur-containing amino acids, and the potential and targets of gene editing to reduce ß-ODAP content. Recently, two key enzymes (ß-ODAP synthase and ß-cyanoalanine synthase) have been identified in the biosynthetic pathway of ß-ODAP. We proposed four strategies through which the genes encoding these enzymes can be targeted and suppressed using CRISPR/Cas9 gene editing. Compared to its homology in Medicago truncatula, the grass pea ß-ODAP synthase gene sequence and ß-cyanoalanine synthase showed 62.9% and 95% similarity, respectively. The ß-ODAP synthase converts the final intermediate L-DAPA into toxic ß-ODAP, whist ß-cyanoalanine synthase converts O-Acetylserine into ß-isoxazolin-5-on-2-yl-alanine. Since grass pea is low in methionine and cysteine amino acids, improvement of these amino acids is also needed to boost its protein content. This review contains useful resources for grass pea improvement while also offering potential gene editing strategies to lower ß-ODAP levels.

Duplicated antagonistic EPF peptides optimize grass stomatal initiation.

Peptide signaling has emerged as a key component of plant growth and development, including stomatal patterning, which is crucial for plant productivity and survival. Although exciting progress has been made in understanding EPIDERMAL PATTERNING FACTOR (EPF) signaling in Arabidopsis, the mechanisms by which EPF peptides control different stomatal patterns and morphologies in grasses are poorly understood. Here, by examining expression patterns, overexpression transgenics and cross-species complementation, the antagonistic stomatal ligands orthologous to Arabidopsis AtEPF2 and AtSTOMAGEN/AtEPFL9 peptides were identified in Triticum aestivum (wheat) and the grass model organism Brachypodium distachyon. Application of bioactive BdEPF2 peptides inhibited stomatal initiation, but not the progression or differentiation of stomatal precursors in Brachypodium. Additionally, the inhibitory roles of these EPF peptides during grass stomatal development were suppressed by the contrasting positive action of the BdSTOMAGEN peptide in a dose-dependent manner. These results not only demonstrate how conserved EPF peptides that control different stomatal patterns exist in nature, but also suggest new strategies to improve crop yield through the use of plant-derived antagonistic peptides that optimize stomatal density on the plant epidermis.

Microarray-based evaluation of selected recombinant timothy grass allergens expressed in E. Coli and N. Benthamiana.

BACKGROUND: Timothy grass (Phleum pratense) is a significant source of allergens, and recombinant allergens are increasingly used for diagnostic purposes. However, the performance of different recombinant allergen production systems in diagnostic assays needs further investigation to optimize their use in clinical settings. OBJECTIVE: The main objective of this study was to analyze and compare the diagnostic performance of recombinant timothy grass allergens produced in E. coli and N. benthamiana using a custom-made microarray chip. METHODS: Recombinant timothy grass allergens Phl p 1, Phl p 2, Phl p 5, Phl p 6, Phl p 11, and Phl p 12 were produced in E. coli and/or N. benthamiana. A total of 113 patient serum samples were tested to evaluate the diagnostic sensitivity, specificity, inter-assay variability, and correlation of allergen-specific IgE detection compared to commercial multiplex tests (ALEX and ISAC). Additionally, the prevalence of sIgE to these allergens was assessed. RESULTS: Phl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl p 11 showed high or very high positive correlation in immunoreactivity with other commercial multiplex tests. Notably, Phl p 11 fused with maltose-binding protein (MBP) demonstrated high diagnostic specificity and sensitivity, with a 0.3 arbitrary cut-off value. However, a high intra-assay variation was observed. The study also assessed specific IgE prevalence to timothy grass allergens within the tested patient cohort. CONCLUSIONS: Recombinant allergens from both E. coli and N. benthamiana demonstrated strong diagnostic potential on the microarray platform, with Phl p 11 (MBP-fused) showing particularly high performance. High intra-assay variation highlights the need for further optimization in allergen formulation and microarray storage conditions. These results highlight the potential of recombinant allergens for diagnostic applications, despite challenges with allergen stability in microarray formats. Specific IgE prevalence to timothy allergens revealed a sensitization profile consistent with findings from multiple studies.

The molecular regulatory mechanism of reed canary grass under salt, waterlogging, and combined stress was analyzed by transcriptomic analysis.

BACKGROUND: Reed canary grass has been identified as a suitable species for restoring plateau wetlands and understanding plant adaptation mechanisms in wetland environments. In this study, we subjected a reed canary grass cultivar 'Chuanxi' to waterlogging, salt, and combined stresses to investigate its phenotypic characteristics, physiological indices, and transcriptome changes under these conditions. RESULTS: The results revealed that the growth rate was slower under salt stress than under waterlogging stress. The chlorophyll content and energy capture efficiency of the PS II reaction center decreased with prolonged exposure to each stress. Conversely, while the activities of enzymes associated with respiratory metabolism, as well as MDA, PRO, Na+, and K+-ATPase, increased. The formation of distinct aerenchyma was observed under waterlogging stress and combined stress. Transcriptome sequencing analysis identified 5,379, 4,169, and 14,993 DEGs under CK vs. W, CK vs. S, and CK vs. SW conditions, respectively. The WRKY was found to be the most abundant under waterlogging stress, whereas the MYB predominated under salt stress and combined stress. Glutathione metabolic pathways and Plant hormone signal transduction have also been found to play important roles in stress. CONCLUSION: By integrating phenotypic, physiological, anatomical, and transcriptomic, this research provides valuable insights into how reed canary grass responds to salt, waterlogging, and combined stresses. These findings may inform the ecological application of reed canary grass in high-altitude wetlands and for breeding purposes.

Glutathione S-transferase activity facilitates rice tolerance to the barnyard grass root exudate DIMBOA.

BACKGROUND: In paddy fields, the noxious weed barnyard grass secretes 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) to interfere with rice growth. Rice is unable to synthesize DIMBOA. Rice cultivars with high or low levels of allelopathy may respond differently to DIMBOA. RESULTS: In this study, we found that low concentrations of DIMBOA (≤ 0.06 mM) promoted seedling growth in allelopathic rice PI312777, while DIMBOA (≤ 0.08 mM) had no significant influence on the nonallelopathic rice Lemont. DIMBOA treatment caused changes in the expression of a large number of glutathione S-transferase (GST) proteins, which resulting in enrichment of the glutathione metabolic pathway. This pathway facilitates plant detoxification of heterologous substances. The basal levels of GST activity in Lemont were significantly higher than those in PI312777, while GST activity in PI312777 was slightly induced by increasing DIMBOA concentrations. Overexpression of GST genes (Os09g0367700 and Os01g0949800) in these two cultivars enhanced rice resistance to DIMBOA. CONCLUSIONS: Taken together, our results indicated that different rice accessions with different levels of allelopathy have variable tolerance to DIMBOA. Lemont had higher GST activity, which helped it tolerate DIMBOA, while PI312777 had lower GST activity that was more inducible. The enhancement of GST expression facilitates rice tolerance to DIMBOA toxins from barnyard grass root exudates.

Unravelling the molecular mechanism underlying drought stress tolerance in Dinanath (Pennisetum pedicellatum Trin.) grass via integrated transcriptomic and metabolomic analyses.

Dinanath grass (Pennisetum pedicellatum Trin.) is an extensively grown forage grass known for its significant drought resilience. In order to comprehensively grasp the adaptive mechanism of Dinanath grass in response to water deficient conditions, transcriptomic and metabolomics were applied in the leaves of Dinanath grass exposed to two distinct drought intensities (48-hour and 96-hour). Transcriptomic analysis of Dinanath grass leaves revealed that a total of 218 and 704 genes were differentially expressed under 48- and 96-hour drought conditions, respectively. The genes that were expressed differently (DEGs) and the metabolites that accumulated in response to 48-hour drought stress mainly showed enrichment in the biosynthesis of secondary metabolites, particularly phenolics and flavonoids. Conversely, under 96-hour drought conditions, the enriched pathways predominantly involved lipid metabolism, specifically sterol lipids. In particular, phenylpropanoid pathway and brassinosteroid signaling played a crucial role in drought response to 48- and 96-hour water deficit conditions, respectively. This variation in drought response indicates that the adaptation mechanism in Dinanath grass is highly dependent on the intensity of drought stress. In addition, different genes associated with phenylpropanoid and fatty acid biosynthesis, as well as signal transduction pathways namely phenylalanine ammonia-lyase, putrescine hydroxycinnamoyl transferase, abscisic acid 8'-hydroxylase 2, syntaxin-61, lipoxygenase 5, calcium-dependent protein kinase and phospholipase D alpha one, positively regulated with drought tolerance. Combined transcriptomic and metabolomic analyses highlights the outstanding involvement of regulatory pathways related to secondary cell wall thickening and lignin biosynthesis in imparting drought tolerance to Dinanath grass leaves. These findings collectively contribute to an enhanced understanding of candidate genes and key metabolites relevant to drought response in Dinanath grass. Furthermore, they establish a groundwork for the creation of a transcriptome database aimed at developing abiotic stress-tolerant grasses and major crop varieties through both transgenic and genome editing approaches.

A rice GT61 glycosyltransferase possesses dual activities mediating 2-O-xylosyl and 2-O-arabinosyl substitutions of xylan.

MAIN CONCLUSION: A member of the rice GT61 clade B is capable of transferring both 2-O-xylosyl and 2-O-arabinosyl residues onto xylan and another member specifically catalyses addition of 2-O-xylosyl residue onto xylan. Grass xylan is substituted predominantly with 3-O-arabinofuranose (Araf) as well as with some minor side chains, such as 2-O-Araf and 2-O-(methyl)glucuronic acid [(Me)GlcA]. 3-O-Arabinosylation of grass xylan has been shown to be catalysed by grass-expanded clade A members of the glycosyltransferase family 61. However, glycosyltransferases mediating 2-O-arabinosylation of grass xylan remain elusive. Here, we performed biochemical studies of two rice GT61 clade B members and found that one of them was capable of transferring both xylosyl (Xyl) and Araf residues from UDP-Xyl and UDP-Araf, respectively, onto xylooligomer acceptors, whereas the other specifically catalysed Xyl transfer onto xylooligomers, indicating that the former is a xylan xylosyl/arabinosyl transferase (named OsXXAT1 herein) and the latter is a xylan xylosyltransferase (named OsXYXT2). Structural analysis of the OsXXAT1- and OsXYXT2-catalysed reaction products revealed that the Xyl and Araf residues were transferred onto O-2 positions of xylooligomers. Furthermore, we demonstrated that OsXXAT1 and OsXYXT2 were able to substitute acetylated xylooligomers, but only OsXXAT1 could xylosylate GlcA-substituted xylooligomers. OsXXAT1 and OsXYXT2 were predicted to adopt a GT-B fold structure and molecular docking revealed candidate amino acid residues at the predicted active site involved in binding of the nucleotide sugar donor and the xylohexaose acceptor substrates. Together, our results establish that OsXXAT1 is a xylan 2-O-xylosyl/2-O-arabinosyl transferase and OsXYXT2 is a xylan 2-O-xylosyltransferase, which expands our knowledge of roles of the GT61 family in grass xylan synthesis.

Peripheral blood mononuclear cell transcriptome profile in a clinical trial with subcutaneous, grass pollen allergoid immunotherapy.

INTRODUCTION: Allergen-specific immunotherapy (AIT) is the only disease-modifying treatment in allergic airway diseases. Underlying immunological mechanisms and candidate biomarkers, which may be translated into predictive/surrogate measures of clinical efficacy, remain an active area of research. The aim of this study was to evaluate Pollinex Quattro (PQ) Grass AIT induced immunomodulatory mechanisms, based on transcriptome profiling of peripheral blood mononuclear cells. METHODS: 119 subjects with grass pollen induced seasonal allergic rhinitis (SAR) were randomized in a 2:2:1:1 ratio to receive a cumulative dose of PQ Grass as a conventional or extended pre-seasonal regimen, placebo, or placebo with MicroCrystalline Tyrosine. Gene expression analysis was an exploratory endpoint evaluated in a subgroup of 30 subjects randomly selected from the four treatment arms. Samples were collected at three time points: screening (baseline), before the start of the grass pollen season and at the end of the season. This study was funded by the manufacturer of PQ. RESULTS: Transcriptome analysis demonstrated that the most significant changes in gene expression, for both treatment regimens, were at the end of the grass pollen season, with the main Th1 candidate molecules (IL-12A, IFNγ) upregulated and Th2 signature cytokines downregulated (IL-4, IL-13, IL-9) (p < .05). Canonical pathways analysis demonstrated Th1, Th2, Th17 and IL-17 as the most significantly enriched pathways based on absolute value of activation z-score (IzI score ≥ 2, p < .05). Upstream regulator analysis showed pronounced inhibition of pro-inflammatory allergic molecules IgE, IL-17A, IL-17F, IL-25 (IL-17E) (IzI score ≥ 2, FDR < 0.05) and activation of pro-tolerogenic molecules IL-12A, IL-27, IL-35 (EBI3) at the end of the grass pollen season. CONCLUSION: Peripheral blood mononuclear cells transcriptome profile showed an inhibition of Th2, Th17 pro-inflammatory allergic responses and immune deviation towards Th1 responses. PQ Grass extended regimen exhibited a superior mechanistic efficacy profile in comparison with PQ conventional regimen.

Clinical validation of grass pollen exposure chamber in patients with allergic rhinitis triggered by timothy grass.

BACKGROUND: The fluctuation in concentrations of airborne allergens frequently presents a challenge to assessing the efficacy of allergen immunotherapy (AIT) in 'field' studies. Allergen exposure chambers (AECs) are specialized medical installations developed to expose individuals to allergens at defined and consistent concentrations under a controlled environment. The aim of the study was to validate the provocation test with timothy grass pollen as well as to assess its safety in the AEC in patients with allergic rhinitis. METHODS: In the ALLEC® AEC, varying concentrations of timothy grass pollen were dispersed. Allergic symptoms were measured by total nasal symptom score (TNSS), acoustic rhinometry, peak nasal inspiratory flow (PNIF) and nasal discharge volume. Lung function, assessed through peak expiratory flow rate (PEFR) and forced expiratory volume in the first second (FEV1), was used to evaluate safety. RESULTS: The consistency of the test was proved by the stability of environmental conditions, including temperature, humidity and CO2 levels, as well as constant concentrations of grass pollen at predetermined levels ranging from 1000 to 10,000 particles per cubic meter (p/m3). Allergic individuals developed symptoms at concentrations of 3000 p/m3 and above, across all measured endpoints. Lung function was not affected throughout all the challenges. The reproducibility of symptoms was confirmed throughout the tests. The concentration of 8000 p/m3 together with a challenge duration of 120 min was found to be optimal. CONCLUSION: The study demonstrates that the ALLEC® grass pollen exposure chamber provides a reliable and safe method for inducing repeatable symptoms in patients with allergic rhinitis. This approach can be effectively applied for allergy diagnostics and clinical endpoint determination during AIT.

IgE to cross-reactive carbohydrate determinants (CCD) in childhood: Prevalence, risk factors, putative origins.

BACKGROUND: IgE antibodies to cross-reactive carbohydrate determinants (CCD) are usually clinically irrelevant but they can be a cause of false positive outcomes of allergen-specific IgE tests in vitro. Their prevalence and levels have been so far cross-sectionally examined among adult allergic patients and much less is known about their origins and relevance in childhood. METHODS: We examined CCD with a cross-sectional approach in 1263 Italian pollen allergic children (Panallergen in Paediatrics, PAN-PED), as well as with a longitudinal approach in 612 German children (Multicenter Allergy Study, MAS), whose cutaneous and IgE sensitization profile to a broad panel of allergen extracts and molecules was already known. The presence and levels of IgE to CCD were examined in the sera of both cohorts using bromelain (MUXF3) as reagent and a novel chemiluminescence detection system, operating in a solid phase of fluorescently labelled and streptavidin-coated paramagnetic microparticles (NOVEOS, HYCOR, USA). RESULTS: IgE to CCD was found in 22% of the Italian pollen allergic children, mainly in association with an IgE response to grass pollen. Children with IgE to CCD had higher total IgE levels and were sensitized to more allergenic molecules of Phleum pratense than those with no IgE to CCD. Among participants of the German MAS birth cohort study, IgE to CCD emerged early in life (even at pre-school age), with IgE sensitization to group 1 and 4 allergen molecules of grasses, and almost invariably persisted over the full observation period. CONCLUSIONS: Our results contribute to dissect the immunological origins, onset, evolution and risk factors of CCD-sIgE response in childhood, and raise the hypothesis that group 1 and/or 4 allergen molecules of grass pollen are major inducers of these antibodies through an antigen-specific, T-B cell cognate interaction.

Successional changes in bacterial phyllosphere communities are plant-host species dependent.

Phyllosphere microbial communities are increasingly experiencing intense pulse disturbance events such as drought. It is currently unknown how phyllosphere communities respond to such disturbances and if they are able to recover. We explored the stability of phyllosphere communities over time, in response to drought stress, and under recovery from drought on temperate forage grasses. Compositional or functional changes were observed during the disturbance period and whether communities returned to non-stressed levels following recovery. Here, we found that phyllosphere community composition shifts as a result of simulated drought but does not fully recover after irrigation is resumed and that the degree of community response to drought is host species dependent. However, while community composition had changed, we found a high level of functional stability (resistance) over time and in the water deficit treatment. Ecological modeling enabled us to understand community assembly processes over a growing season and to determine if they were disrupted during a disturbance event. Phyllosphere community succession was characterized by a strong level of ecological drift, but drought disturbance resulted in variable selection, or, in other words, communities were diverging due to differences in selective pressures. This successional divergence of communities with drought was unique for each host species. Understanding phyllosphere responses to environmental stresses is important as climate change-induced stresses are expected to reduce crop productivity and phyllosphere functioning. IMPORTANCE: Leaf surface microbiomes have the potential to influence agricultural and ecosystem productivity. We assessed their stability by determining composition, functional resistance, and resilience. Resistance is the degree to which communities remain unchanged as a result of disturbance, and resilience is the ability of a community to recover to pre-disturbance conditions. By understanding the mechanisms of community assembly and how they relate to the resistance and resilience of microbial communities under common environmental stresses such as drought, we can better understand how communities will adapt to a changing environment and how we can promote healthy agricultural microbiomes. In this study, phyllosphere compositional stability was highly related to plant host species phylogeny and, to a lesser extent, known stress tolerances. Phyllosphere community assembly and stability are a result of complex interactions of ecological processes that are differentially imposed by host species.

Interactions between Balantidium ctenopharyngodoni and microbiota reveal its low pathogenicity in the hindgut of grass carp.

BACKGROUND: Hosts, parasites, and microbiota interact with each other, forming a complex ecosystem. Alterations to the microbial structure have been observed in various enteric parasitic infections (e.g. parasitic protists and helminths). Interestingly, some parasites are associated with healthy gut microbiota linked to the intestinal eubiosis state. So the changes in bacteria and metabolites induced by parasite infection may offer benefits to the host, including protection from other parasitesand promotion of intestinal health. The only ciliate known to inhabit the hindgut of grass carp, Balantidium ctenopharyngodoni, does not cause obvious damage to the intestinal mucosa. To date, its impact on intestinal microbiota composition remains unknown. In this study, we investigated the microbial composition in the hindgut of grass carp infected with B. ctenopharyngodoni, as well as the changes of metabolites in intestinal contents resulting from infection. RESULTS: Colonization by B. ctenopharyngodoni was associated with an increase in bacterial diversity, a higher relative abundance of Clostridium, and a lower abundance of Enterobacteriaceae. The family Aeromonadaceae and the genus Citrobacter had significantly lower relative abundance in infected fish. Additionally, grass carp infected with B. ctenopharyngodoni exhibited a significant increase in creatine content in the hindgut. This suggested that the presence of B. ctenopharyngodoni may improve intestinal health through changes in microbiota and metabolites. CONCLUSIONS: We found that grass carp infected with B. ctenopharyngodoni exhibit a healthy microbiota with an increased bacterial diversity. The results suggested that B. ctenopharyngodoni reshaped the composition of hindgut microbiota similarly to other protists with low pathogenicity. The shifts in the microbiota and metabolites during the colonization and proliferation of B. ctenopharyngodoni indicated that it may provide positive effects in the hindgut of grass carp.