Anti-inflammatory Effects of Proteolytic Peptides from Different Ginseng Concoctions on LPS-induced RAW264.7 Cells

The purpose of this study was to conduct a screening and comparative analysis of proteolytic peptides with anti-inflammatory properties derived from three different ginseng concoctions: Sundried ginseng, red ginseng and black ginseng. Ginseng proteins were extracted from three different types of ginseng products using a low-temperature leaching method. Subsequently, the extracted proteins underwent enzymatic digestion using alkaline protease, neutral protease, and pepsin through a stepwise enzyme digestion method. This process yielded three distinct enzyme digestion products, namely BGP (black ginseng proteolytic peptide), RGP (red ginseng proteolytic peptide), and SGP (sundried ginseng proteolytic peptide). The samples were subjected to separation using ultrafiltration membranes, resulting in the acquisition of ultrafiltration fractions with distinct molecular weights. Subsequently, the ultrafiltration fractions were further separated utilizing ultrafiltration membranes to obtain fractions with varying molecular weights. The fraction exhibiting the most potent anti-inflammatory activity was determined through the application of a lipopolysaccharide (LPS)-induced RAW264.7 inflammation model. The impact of the active fractions on the secretion of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β ((IL-1β), and interleukin-6 (IL-6) by RAW264.7 cells was assessed using enzyme immunoassay. The amino acid composition and content of the three proteolytic peptides were examined. Multivariate statistical analysis was employed to identify the distinct amino acids in the three ginseng concoctions and investigate their correlation with the inhibition of cytokine secretion by RAW264.7 cells. The findings of the study indicated that the proteolytic peptide fraction with a molecular weight of less than 1 kDa in the three ginseng products exhibited the most pronounced impact on the proliferation of RAW264.7 cells compared to the other fractions. Additionally, this fraction significantly suppressed the secretion of NO, TNF-α, IL-6, and IL-1β at concentrations ranging from 50~200 μg/mL (P<0.05). Notably, at a concentration of 200 μg/mL, the three groups receiving proteolytic peptide administration demonstrated the most potent inhibitory effect on cytokine release. Furthermore, the inhibitory effect of BGP-4 on cytokine release surpassed that of RGP-4 and SGP-4, exhibiting a statistically significant difference (P<0.05). All three peptides consisted of 17 amino acids, however, their compositions exhibited significant variations. Notably, phenylalanine exhibited the highest content, and the differential amino acids present in the three ginseng concoctions were closely associated with the inhibition of inflammatory factor secretion. This study represents an initial exploration into the impact of concoctions on the anti-inflammatory properties of ginseng, identifying distinct amino acids among different concoctions. These findings offer a valuable reference for the formulation of ginseng concoctions

Spatiotemporal Genotype Replacement of H5N8 Avian Influenza Viruses Contributed to H5N1 Emergence in 2021/2022 Panzootic

Since 2020, clade 2.3.4.4b highly pathogenic avian influenza H5N8 and H5N1 viruses have swept through continents, posing serious threats to the world. Through comprehensive analyses of epidemiological, genetic, and bird migration data, we found that the dominant genotype replacement of the H5N8 viruses in 2020 contributed to the H5N1 outbreak in the 2021/2022 wave. The 2020 outbreak of the H5N8 G1 genotype instead of the G0 genotype produced reassortment opportunities and led to the emergence of a new H5N1 virus with G1's HA and MP genes. Despite extensive reassortments in the 2021/2022 wave, the H5N1 virus retained the HA and MP genes, causing a significant outbreak in Europe and North America. Furtherly, through the wild bird migration flyways investigation, we found that the temporal-spatial coincidence between the outbreak of the H5N8 G1 virus and the bird autumn migration may have expanded the H5 viral spread, which may be one of the main drivers of the emergence of the 2020-2022 H5 panzootic.IMPORTANCESince 2020, highly pathogenic avian influenza (HPAI) H5 subtype variants of clade 2.3.4.4b have spread across continents, posing unprecedented threats globally. However, the factors promoting the genesis and spread of H5 HPAI viruses remain unclear. Here, we found that the spatiotemporal genotype replacement of H5N8 HPAI viruses contributed to the emergence of the H5N1 variant that caused the 2021/2022 panzootic, and the viral evolution in poultry of Egypt and surrounding area and autumn bird migration from the Russia-Kazakhstan region to Europe are important drivers of the emergence of the 2020-2022 H5 panzootic. These findings provide important targets for early warning and could help control the current and future HPAI epidemics.</p

Optimization of the Preparation Process of Oyster Peptide by Enzymatic Hydrolysis and Its Effects on Testosterone Secretion and Oxidative Stress in Mouse Testicular Interstitial Cells

In order to explore the research and development of oyster in the field of medicine and food homology, response surface methodology was used to optimize the preparation process of oyster protease-depeptidase, and its effects on testosterone secretion and oxidative stress in mouse leydig cells were studied. Based on the investigation of hydrolysis degree as the evaluation index, a biomimetic enzymatic hydrolysis method was employed to optimize the preparation process of oyster protein enzymolysis peptides using response surface analysis, building upon the foundation of single-factor experiments. Simultaneously, a hydrogen peroxide (H2O2)-induced oxidative damage model was established using mouse testicular interstitial cells (TM3), and the effects of oyster protein enzymolysis peptides on testosterone (T) secretion and oxidative stress were investigated through assessments of cell viability, DAPI staining, testosterone secretion level, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content in TM3 cells. The results showed that the optimal enzymatic hydrolysis conditions for oyster protein enzymolysis peptides were as follows: Substrate-to-solvent ratio of 1:10 g/mL, gastric protease concentration of 1.1%, hydrolysis time of 1.0 h, pancreatic protease concentration of 2.1%, and hydrolysis time of 3.1 h. Under these conditions, the degree of hydrolysis was determined to be 39.43%±0.42%. Oyster protein enzymolysis peptides exhibited varying degrees of proliferative activity on H2O2-induced TM3 cells, significantly (P<0.05) increasing testosterone secretion, SOD enzyme activity, and reducing MDA levels in TM3 cells. The most pronounced effects were observed at a concentration of 200 μg/mL of oyster protein enzymolysis peptides. In conclusion, the optimization of enzymatic hydrolysis process using response surface methodology proved to be effective and feasible. Oyster protein enzymolysis peptides was found to extremely significant promote TM3 cell proliferation, increase testosterone secretion, enhance SOD enzyme activity, and reduce MDA levels (P<0.01)

Nanoparticles of Block Ionomer Complexes from Double Hydrophilic Poly(acrylic acid)-b-poly(ethylene oxide)-b-poly(acrylic acid) Triblock Copolymer and Oppositely Charged Surfactant

The novel water-dispersible nanoparticles from the double hydrophilic poly(acrylic acid)-b-poly(ethylene oxide)-b-poly(acrylic acid) (PAA-b-PEO-b-PAA) triblock copolymer and oppositely charged surfactant dodecyltrimethyl ammonium bromide (DTAB) were prepared by mixing the individual aqueous solutions. The structure of the nanoparticles was investigated as a function of the degree of neutralization (DN) by turbidimetry, dynamic light scattering (DSL),ζ-potential measurement, and atomic force microscope (AFM). The neutralization of the anionic PAA blocks with cationic DTAB accompanied with the hydrophobic interaction of alkyl tails of DTAB led to formation of core–shell nanoparticles with the core of the DTAB neutralized PAA blocks and the shell of the looped PEO blocks. The water-dispersible nanoparticles with negative ζ-potential were obtained over the DN range from 0.4 to 2.0 and their sizes depended on the DN. The looped PEO blocks hindered the further neutralization of the PAA blocks with cationic DTAB, resulting in existence of some negative charged PAA-b-PEO-b-PAA backbones even when DN > 1.0. The spherical and ellipsoidal nature of these nanoparticles was observed with AFM

Generation, Characterization and Epitope Mapping of Two Neutralizing and Protective Human Recombinant Antibodies against Influenza A H5N1 Viruses

The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development.We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs), AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model.Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines

Adaption of Seasonal H1N1 Influenza Virus in Mice

The experimental infection of a mouse lung with influenza A virus has proven to be an invaluable model for studying the mechanisms of viral adaptation and virulence. The mouse adaption of human influenza A virus can result in mutations in the HA and other proteins, which is associated with increased virulence in mouse lungs. In this study, a mouse-adapted seasonal H1N1 virus was obtained through serial lung-to-lung passages and had significantly increased virulence and pathogenicity in mice. Genetic analysis indicated that the increased virulence of the mouse-adapted virus was attributed to incremental acquisition of three mutations in the HA protein (T89I, N125T, and D221G). However, the mouse adaption of influenza A virus did not change the specificity and affinity of receptor binding and the pH-dependent membrane fusion of HA, as well as the in vitro replication in MDCK cells. Notably, infection with the mouse adapted virus induced severe lymphopenia and modulated cytokine and chemokine responses in mice. Apparently, mouse adaption of human influenza A virus may change the ability to replicate in mouse lungs, which induces strong immune responses and inflammation in mice. Therefore, our findings may provide new insights into understanding the mechanisms underlying the mouse adaption and pathogenicity of highly virulent influenza viruses

Mutations in Polymerase Genes Enhanced the Virulence of 2009 Pandemic H1N1 Influenza Virus in Mice

Influenza A virus can infect a wide variety of animal species with illness ranging from mild to severe, and is a continual cause for concern. Genetic mutations that occur either naturally or during viral adaptation in a poorly susceptible host are key mechanisms underlying the evolution and virulence of influenza A virus. Here, the variants containing PA-A36T or PB2-H357N observed in the mouse-adapted descendants of 2009 pandemic H1N1 virus (pH1N1), A/Sichuan/1/2009 (SC), were characterized. Both mutations enhanced polymerase activity in mammalian cells. These effects were confirmed using recombinant SC virus containing polymerase genes with wild type (WT) or mutant PA or PB2. The PA-A36T mutant showed enhanced growth property compared to the WT in both human A549 cells and porcine PK15 cells in vitro, without significant effect on viral propagation in murine LA-4 cells and pathogenicity in mice; however, it did enhance the lung virus titer. PB2-H357N variant demonstrated growth ability comparable to the WT in A549 cells, but replicated well in PK15, LA-4 cells and in mice with an enhanced pathogenic phenotype. Despite such mutations are rare in nature, they could be observed in avian H5 and H7 subtype viruses which were currently recognized to pose potential threat to human. Our findings indicated that pH1N1 may adapt well in mammals when acquiring these mutations. Therefore, future molecular epidemiological surveillance should include scrutiny of both markers because of their potential impact on pathogenesis

Genomic Polymorphism of the Pandemic A (H1N1) Influenza Viruses Correlates with Viral Replication, Virulence, and Pathogenicity In Vitro and In Vivo

The novel pandemic A (H1N1) virus was first identified in Mexico in April 2009 and quickly spread worldwide. Like all influenzas, the H1N1 strain-specific properties of replication, virulence, and pathogenicity are a result of the particular genomic sequence and concerted expression of multiple genes. Thus, specific mutations may support increased virulence and may be useful as biomarkers of potential threat to human health. We performed comparative genomic analysis of ten strains of the 2009 pandemic A (H1N1) influenza viruses to determine whether genotypes associated with clinical phenotypes, which ranged from mild to severe illness and up to lethal. Virus replication capacity was tested for each strain in vitro using cultured epithelial cells, while virulence and pathogenicity were investigated in vivo using the BALB/c mouse model. The results indicated that A/Sichuan/1/2009 strain had significantly higher replication ability and virulence than the other strains, and five unique non-synonymous mutations were identified in important gene-encoding sequences. These mutations led to amino acid substitutions in HA (L32I), PA (A343T), PB1 (K353R and T566A), and PB2 (T471M), and may be critical molecular determinants for replication, virulence, and pathogenicity. Our results suggested that the replication capacity in vitro and virulence in vivo of the 2009 pandemic A (H1N1) viruses were not associated with the clinical phenotypes. This study offers new insights into the transmission and evolution of the 2009 pandemic A (H1N1) virus

Network Analysis Measuring the Impact of Volcanic Eruptions

Volcanoes can be extremely damaging to the environment, human society, and also impact climate change. During volcanic eruption, massive amounts of gases and dust particles are thrown into the atmosphere and propagated instantaneously by the stratospheric circulation, resulting in a huge impact on the interactive pattern of the atmosphere. Here, we develop a climate network-based framework to study the temporal evolution of lower stratospheric atmosphere conditions in relation to a volcanic eruption, the Hunga Tonga-Hunga Ha’apai (HTHH) volcano, which erupted on 20 December 2021. Various spatial-temporal topological features of the climate network are introduced to analyze the nature of the HTHH. We show that our framework has the potential to identify the dominant eruption events of the HTHH and reveal the impact of the HTHH eruption. We find that during the eruption periods of the HTHH, the correlation behaviors in the lower stratosphere became much stronger than during normal periods. Both the degree and clustering coefficients increased significantly during the dominant eruption periods, and could be used as indications for the eruption of HTHH. The underlying mechanism for the observed cooperative mode is related to the impact of a volcanic eruption on global mass circulations. The study on the network topology of the atmospheric structure during a volcanic eruption provides a fresh perspective to investigate the impact of volcanic eruptions. It can also reveal how the interactive patterns of the atmosphere respond to volcanic eruptions and improve our understanding regarding the global impacts of volcanic eruptions