Molecular characterization and functional analysis of peroxiredoxin 1 (Prx1) from roughskin sculpin (Trachidermus fasciatus).

Peroxiredoxin1(Prx1), also known as natural killer enhancing factor A (NKEF-A), is a crucial antioxidant involving in various cellular activities and immune response against bacterial and viral infection in fish. In the present study, a full-length Prx1 cDNA sequence (TfPrx1) was firstly cloned from roughskin sculpin (Trachidermus fasciatus), which was composed of 1044 bp nucleotides encoding a peptide of 199 amino acids with a molecular weight of 22.35 kDa and a theoretical pI of 6.42, respectively. The predicted peptide was a typical 2-cys Prx containing two conserved characteristic motifs 43FYPLDFTFVCPTEI56 and 170GEVCPA175 with the two conserved peroxidatic and resolving cysteine residuals forming disulfide bond. Quantitative real-time PCR analysis showed that TfPrx1 was ubiquitously expressed in all tested tissues with the highest expression in the intestine. It could be significantly induced following LPS injection and heavy metal exposure. Recombinant TfPrx1 (rTfPrx1) displayed insulin disulfide reduction and ROS-scavenging activity in a concentration-dependent manner, and further exhibited DNA and cytoprotective effects under oxidative stress. These results suggested that TfPrx1 protein may play an important role in fish immune protection from oxidative damage.

Germline-encoded amino acid-binding motifs drive immunodominant public antibody responses.

Despite the vast diversity of the antibody repertoire, infected individuals often mount antibody responses to precisely the same epitopes within antigens. The immunological mechanisms underpinning this phenomenon remain unknown. By mapping 376 immunodominant "public epitopes" at high resolution and characterizing several of their cognate antibodies, we concluded that germline-encoded sequences in antibodies drive recurrent recognition. Systematic analysis of antibody-antigen structures uncovered 18 human and 21 partially overlapping mouse germline-encoded amino acid-binding (GRAB) motifs within heavy and light V gene segments that in case studies proved critical for public epitope recognition. GRAB motifs represent a fundamental component of the immune system's architecture that promotes recognition of pathogens and leads to species-specific public antibody responses that can exert selective pressure on pathogens.

Low temperature response index for monitoring freezing injury of tea plant.

Freezing damage has been a common natural disaster for tea plantations. Quantitative detection of low temperature stress is significant for evaluating the degree of freezing injury to tea plants. Traditionally, the determination of physicochemical parameters of tea leaves and the investigation of freezing damage phenotype are the main approaches to detect the low temperature stress. However, these methods are time-consuming and laborious. In this study, different low temperature treatments were carried out on tea plants. The low temperature response index (LTRI) was established by measuring seven low temperature-induced components of tea leaves. The hyperspectral data of tea leaves was obtained by hyperspectral imaging and the feature bands were screened by successive projections algorithm (SPA), competitive adaptive reweighted sampling (CARS) and uninformative variable elimination (UVE). The LTRI and seven indexes of tea plant were modeled by partial least squares (PLS), support vector machine (SVM), random forests (RF), back propagation (BP) machine learning methods and convolutional neural networks (CNN), long short-term memory (LSTM) deep learning methods. The results indicated that: (1) the best prediction model for the seven indicators was LTRI-UVE-CNN (R2 = 0.890, RMSEP=0.325, RPD=2.904); (2) the feature bands screened by UVE algorithm were more abundant, and the later modeling effect was better than CARS and SPA algorithm; (3) comparing the effects of the six modeling algorithms, the overall modeling effect of the CNN model was better than other models. It can be concluded that out of all the combined models in this paper, the LTRI-UVE-CNN was a promising model for predicting the degree of low temperature stress in tea plants.

Antibody-mediated SARS-CoV-2 entry in cultured cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells by first engaging its cellular receptor angiotensin converting enzyme 2 (ACE2) to induce conformational changes in the virus-encoded spike protein and fusion between the viral and target cell membranes. Here, we report that certain monoclonal neutralizing antibodies against distinct epitopic regions of the receptor-binding domain of the spike can replace ACE2 to serve as a receptor and efficiently support membrane fusion and viral infectivity in vitro. These receptor-like antibodies can function in the form of a complex of their soluble immunoglobulin G with Fc-gamma receptor I, a chimera of their antigen-binding fragment with the transmembrane domain of ACE2 or a membrane-bound B cell receptor, indicating that ACE2 and its specific interaction with the spike protein are dispensable for SARS-CoV-2 entry. These results suggest that antibody responses against SARS-CoV-2 may help expand the viral tropism to otherwise nonpermissive cell types with potential implications for viral transmission and pathogenesis.

The application of enzymatic fermented soybean effectively regulates associated microbial communities in tea soil and positively affects lipid metabolites in tea new shoots.

Compared with traditional organic fertilizer, fermented soybean is a better fertilizer resource in tea plantations. The application of organic fertilizer is a feasible practice to mitigate the soil degradation caused by the overuse of chemical fertilizers, which can effectively regulate soil microbial communities in tea plantations. However, the effects of fermented soybean on soil microbial communities, soil metabolites and metabolites in tea new shoots have not been systematically demonstrated, and their interactions have never been studied. Here, we investigated the responses of the soil microbial community, soil metabolites and metabolites of tea new shoots to urea fertilization (UF), naturally fermented soybean fertilization (NFS) and enzymatic fermented soybean fertilization (EFS), and analyzed the relationships between soil microbes, soil metabolites and metabolites in tea new shoots. The results showed that soil bacterial communities were dominated by Pseudomonas, Romboutsia, Candidatus_Nitrosotalea and Helicobacter, and soil fungal communities were dominated by Peziza, Fusarium, Candida and Cheilymenia at the genus level. In EFS, bacterial genera (Glutamicibacter and Streptomyces) and fungal genera (Candida and Actinomucor) presented high abundances, which were correlated with soil carbohydrate and lipid including D-Mannitol, D-Sorbitol, 9,12-Octadecadienoic acid and (Z)-13-Docosenoic acid. Enzymatic fermented soybean fertilization also affected the lipid metabolites in tea new shoots. Glycerolipids and glycerophospholipids significantly increased in EFS, which positively correlated with some soil microbial communities. Besides, the application of fermented soybean fertilizer could increase the contents of TP, AP and AK, which were also important environmental factors affecting the structure of soil microbial community in tea plantation. It was concluded that fermented soybean fertilization could improve soil nutrition, regulate associated microbial communities, and positively affect lipid metabolites in tea new shoots. This study not only explores the relationships between soil microbes and metabolites in tea plants, but also provides feasible technical guidance to cultivate high-quality tea using soybean as high-grade fertilizer.

Dynamic changes in metabolic and lipidomic profiles of tea plants during drought stress and re-watering.

Tea (Camellia sinensis L.), as an evergreen plant, needs a humid environment. Water deficit would diminish tea yield and quality. We analyzed the dynamic changes in the metabolite and lipid profiling of tea leaves under various drought conditions and re-watering to determine the metabolic changes in tea leaves responding to drought challenges. In all, 119 metabolites showed substantial alterations in drought-stressed tea plants, including sugars and sugar alcohols, amino acids, and tricarboxylic acid cycle intermediates and lipids. We detected 29 lipids and they were classified into phosphatidylglycerol (PG), phosphatidic acid (PA), sulfoquinovosyl-diacylglycerol (SQDG), phosphatidylcholine (PC), lyso-phosphatidylcholine (LysoPC), and phosphatidylinositol (PI). The levels of sugar, sugar alcohol, and sugar precursors may change as a response to drought stress. Compared with these metabolites, the membrane lipids showed more dynamic changes in tea under drought stresses. Furthermore, metabolic recovery was only partial, with the majority of the examined metabolites exhibiting significantly different levels between samples from re-watered and well-watered tea plants. The findings also showed that comprehensive metabolomic and lipidomic approaches were efficient in elucidating the impacts of drought stress on tea plant metabolism. Our findings are valuable for understanding the mechanisms behind drought tolerance in tea plants from the metabolism perspective and utilizing the compounds to improve the drought tolerance of tea plants.

Immune recall improves antibody durability and breadth to SARS-CoV-2 variants.

Key features of immune memory are greater and faster antigen-specific antibody responses to repeat infection. In the setting of immune-evading viral evolution, it is important to understand how far antibody memory recognition stretches across viral variants when memory cells are recalled to action by repeat invasions. It is also important to understand how immune recall influences longevity of secreted antibody responses. We analyzed SARS-CoV-2 variant recognition; dynamics of memory B cells; and secreted antibody over time after infection, vaccination, and boosting. We find that a two-dose SARS-CoV-2 vaccination regimen given after natural infection generated greater longitudinal antibody stability and induced maximal antibody magnitudes with enhanced breadth across Beta, Gamma, Delta and Omicron variants. A homologous third messenger RNA vaccine dose in COVID-naïve individuals conferred greater cross-variant evenness of neutralization potency with stability that was equal to the hybrid immunity conferred by infection plus vaccination. Within unvaccinated individuals who recovered from COVID, enhanced antibody stability over time was observed within a subgroup of individuals who recovered more quickly from COVID and harbored significantly more memory B cells cross-reactive to endemic coronaviruses early after infection. These cross-reactive clones map to the conserved S2 region of SARS-CoV-2 spike with higher somatic hypermutation levels and greater target affinity. We conclude that SARS-CoV-2 antigen challenge histories in humans influence not only the speed and magnitude of antibody responses but also functional cross-variant antibody repertoire composition and longevity.

Diosmetin ameliorate type 2 diabetic mellitus by up-regulating Corynebacterium glutamicum to regulate IRS/PI3K/AKT-mediated glucose metabolism disorder in KK-Ay mice.

BACKGROUND AND PURPOSE: Diosmetin (Dios), a flavonoid compound with multiple pharmacological activities. However, fewer studies have reported its effects on type 2 diabetic mellitus (T2DM). Here, we address the effect of Dios on glucose metabolism and gut microbiota in KK-Ay diabetic mice. METHOD: Wild type C57BL/6 J mice or diabetic KK-Ay mice were treated with vehicle or Dios for one month. The ELISA kit and fluorescence microscope system were respectively employed to the evaluation of serum biochemical indicators and histopathological changes. Liver RNA-Seq and western blot were used to reveal the key signaling pathway. The effects of Dios on gut microbiota was investigated by the 16S rRNA gene sequencing, as well as the relationship between Dios and C. glu on glucose metabolism was explored with the C. glu transplantation. RESULTS: Dios treatment significantly decreased blood glucose and increased serum insulin concentrations. RNA-Seq analysis found that the underlying action mechanism of Dios on T2DM was via modulating glucose metabolism, which was proved by up-regulating IRS/PI3K/AKT signaling pathway to promote glycogen synthesis and GLUT4 translocation. Besides, Dios treatment reshaped the unbalanced gut microbiota by suppressing the ratio of Firmicutes/Bacteroidetes and markedly increasing the richness of C. glu. Moreover, treatment with C. glu and Dios together could markedly ameliorate glucose metabolism by up-regulating IRS/PI3K/AKT signaling pathway to promote glycogen synthesis and GLUT4 translocation. CONCLUSIONS: Dios treatment remarkably ameliorated glucose metabolism in KK-Ay diabetic mice by the regulation of C. glu via IRS/PI3K/AKT signaling pathway and reshaped the unbalanced gut microbiota. Our study provided evidence for the application of Dios to the treatment of T2DM.

Identification of novel epitopes targeting non-structural protein 2 of PRRSV using monoclonal antibodies.

Porcine reproductive and respiratory syndrome virus (PRRSV) is leading to huge losses in the swine industry worldwide. Its nonstructural protein 2 (Nsp2), with a cysteine protease domain (PL2), is crucial for virus replication and as a trigger to host innate immune regulation. In this study, three monoclonal antibodies (mAbs) to Nsp2, designated 4A12, 4G8, and 8H11, were generated. Subsequently, a sequence of recombinant peptides with partial overlap was utilized to determine the epitopes using these mAbs. We found three novel minimal linear Nsp2 B cell epitopes, 188ELSDDSNRPV197, 42HLKRYSPPAE51, and 54CGWHCISA61, which were identified by the antibodies 4A12, 4G8, and 8H11, respectively. Structure analysis indicates that 42HLKRYSPPAE51 and 188ELSDDSNRPV197 are located separately in hypervariable region 1 and hypervariable region 2 of Nsp2. Interestingly, 54CGWHCISA61 is located in the PL2 region, which is highly conserved in all arteriviruses, particularly at the expected conserved catalytic site at Cys54. Importantly, 54CGWHCISA61 is located in the inner region of the expected 3D structure of Nsp2, which reveals that the epitope is cryptic. These findings not only provide valuable insight for vaccine design and hold diagnostic potential for the identified epitopes, but also reveal a protective mechanism against variation under selective pressure in an important epitope.

DDX19 Inhibits Type I Interferon Production by Disrupting TBK1-IKKε-IRF3 Interactions and Promoting TBK1 and IKKε Degradation.

DExD/H-box helicase members are key receptors for recognizing viral nucleic acids, and they regulate retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-mediated type I interferon (IFN) production. Here, we report that the DExD/H-box helicase family member DExD/H-box RNA helicase 19 (DDX19) is a negative regulator of type I IFN production. Ectopic expression of DDX19 suppressed poly(I:C) (polyinosinic-polycytidylic acid)- and Sendai-virus-induced type I IFN production, whereas knockdown of DDX19 expression enhanced type I IFN production. Mechanistically, DDX19 inhibited TANK-binds kinase 1 (TBK1)- and inhibitor-κb kinase ε (IKKε)-mediated phosphorylation of interferon regulatory factor 3 (IRF3) by disrupting the interaction between TBK1 or IKKε and IRF3. Additionally, DDX19 recruited Lamtor2 and then formed the TBK1-IKKε-Lamtor2-DDX19-IRF3 complex to suppress IFN production by promoting TBK1 and IKKε degradation. We generated Ddx19 knockout mice using transcription activator-like effector nucleases (TALENs) and found that Ddx19 deficiency in vivo augmented type I IFN production, resulting in suppression of encephalomyocarditis virus replication. These data show that DDX19 is an important negative regulator of RLR-mediated type I IFN production.

Proteomic analysis of Camellia sinensis (L.) reveals a synergistic network in the response to drought stress and recovery.

Drought is a crucial limiting factor for tea yield and quality. To systematically characterize the molecular response of tea plants to drought stress and its capacity to recover, we used iTRAQ-based comparative proteomic approach to investigate the effects of drought on protein expression profiles in tea seedlings subjected to different drought treatments. A total of 3274 proteins were identified, of which 2169 and 2300 showed differential expressions during drought and recovery, respectively. Functional annotation showed that multiple biological processes were regulated, suggesting that tea plants probably employed multiple and synergistic resistance mechanisms in dealing with drought stress. Hierarchical clustering showed that chlorophyll a/b-binding proteins were up-regulated in DB and RE, suggesting that tea plants might regulate expression of chlorophyll a/b-binding proteins to maintain the photosystem II function during drought stress. Abundant proteins involved in sulfur-containing metabolite pathways, such as glutathione, taurine, hypotaurine, methionine, and cysteine, changed significantly during drought stress. Among them, TL29 interacted with LHCb6 to connect S-containing metabolites with chlorophyll a/b-binding proteins. This suggests that sulfur-containing compounds play important roles in the response to drought stress in tea plants. In addition, the expression of PAL was up-regulated in DA and down-regulated in DB. Cinnamyl alcohol dehydrogenase, caffeic acid O-methyltransferase, and 4-coumarate-CoA ligase also showed significant changes in expression levels, which regulated the biosynthesis of polyphenols. The results indicate that slight drought stress might promote polyphenol biosynthesis, while serious drought stress leads to inhibition. The expression of lipoxygenase and short-chain dehydrogenase increased during slight drought stress and some volatile metabolite pathways were enriched, indicating that drought stress might affect the tea aroma. The study provides valuable information that will lay the foundation for studies investigating the functions of drought response genes in tea leaves.

Secondary Haemophilus parasuis infection enhances highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) infection-mediated inflammatory responses.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) infection often predisposes pigs to secondary bacterial infection, which induces robust inflammatory responses. However, whether the secondary bacterial infection synergizes HP-PRRSV infection and enhances inflammatory responses is not fully understood. Here, we characterized HP-PRRSV infection-mediated secondary bacterial infection and robust inflammatory responses. HP-PRRSV infection induced higher levels of cytokines (IL-1ß, IL-18, IL-6 and TNF-α) in the sera in piglets and bacterial loads of 11 bacterial species in the lung were increased after HP-PRRSV infection, including Mycoplasma hyorhinis, Haemophilus parasuis and Escherichia coli. Concurrent infection with HP-PRRSV and H. parasuis model showed that inflammatory cytokines expression and secretion in porcine alveolar macrophages (PAMs) were increased in comparison with PAMs infected with HP-PRRSV or H. parasuis alone. Additionally, we found that H. parasuis RNA plays an important role in the robust inflammatory response enhancement in HP-PRRSV-infected PAMs. Taken together, our findings suggest that bacterial RNA transfection enhanced HP-PRRSV-mediated inflammatory responses in HP-PRRSV and H. parasuis (HPS) concurrent infection, which provides an important clue for comprehensive understanding of HP-PRRSV and bacterial coinfection-mediated pathology.

Identification of a linear B-cell epitope on non-structural protein 12 of porcine reproductive and respiratory syndrome virus, using a monoclonal antibody.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused tremendous economic losses and continues to be a serious problem to the swine industry worldwide. The structure and function of PRRSV nonstructural protein 12 (NSP12) is still unknown. In this study, we produced a monoclonal antibody, named as 1E5, against the NSP12 protein of HP (highly pathogenic) -PRRSV strain HuN4. A series of partially overlapping recombinant NSP12 truncations and synthesized peptides were used to define the epitope recognized by 1E5. We found that 130KANATSMRFH139 is the minimal linear epitope and that it is highly conserved among some HP-PRRSV isolates of type 2 PRRSV, but not the classical isolates of type 2 PRRSV or the isolates of type 1 PRRSV. Therefore, 1E5 can be used to establish a valuable tool to distinguish infections with HP-PRRSV isolates of type 2 PRRSV from the classical isolates of type 2 PRRSV and type 1 PRRSV.