[Protective effects of galacto-oligosaccharide and polydextrose on Caco-2 intestinal cell barrier injury model].

OBJECTIVE: To explore the protective effect of different ratios of galactose oligosaccharide(GOS) and polydextrose(PDX) on intestinal cell barrier damage model of Caco-2. METHODS: The same batch of Caco-2 cells were cultured to form a cell barrier model and randomly divided into damaged model group without calcium, calcium-containing blank control group(1.8 mmol/L Ca~(2+)), low-ratio/low-dose group(1.8 mmol/L Ca~(2+)+2 mg/mL GOS+2 mg/mL PDX) and low-ratio/medium-dose group(1.8 mmol/L Ca~(2+)+4 mg/mL GOS+4 mg/mL PDX), low-ratio/high-dose group(1.8 mmol/L Ca~(2+)+8 mg/mL GOS+8 mg/mL PDX) and high-ratio/low-dose group(1.8 mmol/L Ca~(2+)+0.8 mg/mL GOS+3.2mg/mL PDX), high-ratio/medium-dose group(1.8 mmol/L Ca~(2+)+1.6 mg/mL GOS+6.4 mg/mL PDX), high-ratio/high-dose group(1.8 mmol/L Ca~(2+)+3.2mg/mL GOS+12.8 mg/mL PDX), a total of 8 groups, three parallel groups were performed in each group. The Trans Epithelial Electrical Resistance value and apparent permeability coefficient value of each group were determined after 4 d culture, and the morphology of tight junction proteins ZO-1, Occludin and Claudin-1 were observed by immunofluorescence method, and the expression levels of inflammatory related factors in each group were determined by protein microarray method. RESULTS: Compared with damaged model group, TEER ratio in calcium-containing blank control group was significantly increased(P<0.05), while Papp value was significantly decreased(P<0.05);Compared with calcium-containing blank control group, TEER ratio in low-ratio/medium-dose group and high-ratio/high-dose group was significantly increased(P<0.05) while Papp value was significantly decreased(P<0.05), and they could significantly down-regulate some inflammatory response related cytokines. The cell barrier was intact in all groups except for the compact junction protein structure in the model group. CONCLUSION: Compared with Ca~(2+) alone, the combination of two prebiotics can enhance the density of Caco-2 cell barrier and reduced the permeability of cell bypass. And it can significantly reduce the expression level of some inflammatory cytokines and effectively protect the intestinal cell barrier.

Transcription factor EB-mediated autophagy affects cell migration and inhibits apoptosis to promote endometriosis.

Autophagy has emerged as an important process of cell metabolism. With continuous in-depth research on autophagy, TFEB has been a key transcription factor regulating autophagy levels in recent years. Studies have established that TFEB regulates autophagy and apoptosis in various diseases. However, the relationship between TFEB and the pathogenesis of endometriosis remains unclear. This study aimed to investigate the effect of TFEB on the mechanism of endometriosis progression. The results showed that TFEB and autophagy-related protein LC3 are highly expressed in ectopic endometrium of patients with endometriosis, overexpression of TFEB in cultured human endometrial stromal cells (HESCs) by lentivirus not only promoted autophagy but also inhibited apoptosis. In addition, the migration and invasion ability of HESCs were enhanced by TFEB overexpression. Furthermore, inhibiting autophagy with specific inhibitors can attenuate migration and invasion of HESCs induced by TFEB. The rat models of endometriosis show that TFEB knockdown can suppress lesion growth in vivo. Our results suggest that autophagy may be involved in the progression mechanism of endometriosis, and the mechanism of autophagy disorder in endometriosis is probably related to TFEB. TFEB may be a key molecule in promoting endometriosis.

Genotoxic mode of action and threshold exploration of 2-methyl furan under 120-day sub-chronic exposure in male Sprague-Dawley rats.

2-Methylfuran (2-MF) is an important member of the furan family generated during food thermal processing. An in-vivo multiple endpoint genotoxicity assessment system was applied to explore the genotoxic mode of action and threshold of 2-MF. Male Sprague-Dawley rats received 2-MF by oral gavage at doses of 0.16, 0.625, 2.5, and 10 mg/kg.bw/day for 120 days. An additional 15 days were granted for recovery. The Pig-a gene mutation frequency of RET and RBC showed significant increases among the 2-MF groups on day 120. After a 15-day recovery period, the Pig-a gene mutation frequency returned to levels similar to those in the vehicle control. The tail intensity (TI) values of peripheral blood cells at a dose of 10 mg/kg.bw/day significantly increased from day 4 and remained at a high level after the recovery period. No statistical difference was found in the micronucleus frequency of peripheral blood between any 2-MF dose group and the corn oil group at any timepoint. 2-MF may not induce the production of micronuclei, but it could cause DNA breakage. It could not be ruled out that 2-MF may accumulate in vivo and cause gene mutations. Hence, DNA, other than the spindle, may be directly targeted. The mode of action of 2-MF may be that it was metabolized by EPHX1 to more DNA-active metabolites, thus leading to oxidative and direct DNA damage. The point of departure (PoD) of 2-MF-induced genotoxicity was derived as 0.506 mg/kg bw/day.

Mode of action exploration of reproductive toxicity induced by bisphenol S using human normal ovarian epithelial cells through ERß-MAPK signaling pathway.

BACKGROUND: In the plastics production sector, bisphenol S (BPS) has gained popularity as a replacement for bisphenol A (BPA). However, the mode of action (MOA) of female reproductive toxicity caused by BPS remains unclear and the safety of BPS is controversial. METHODS: Human normal ovarian epithelial cell line, IOSE80, were exposed to BPS at human-relevant levels for short-term exposure at 24 h or 48 h, or for long-term exposure at 28 days, either alone or together with five signaling pathway inhibitors: ICI 18,2780 (estrogen receptor [ER] antagonist), G15 (GPR30 specific inhibitor), U0126 (extracellular regulated protein kinase [ERK] 1/2 inhibitor), SP600125 (c-Jun N-terminal kinase [JNK] inhibitor) or SB203580 (p38 mitogen­activated protein kinase [p38MAPK] inhibitor). MOA through ERß-MAPK signaling pathway interruption was explored, and potential thresholds were estimated by the benchmark dose method. RESULTS: For short-term exposure, BPS exposure at human-relevant levels elevated the ESR2 and MAPK8 mRNA levels, along with the percentage of the G0/G1 phase. For long-term exposure, BPS raised the MAPK1 and EGFR mRNA levels, the ERß, p-ERK, and p-JNK protein levels, and the percentage of the G0/G1 phase, which was partly suppressed by U0126. The benchmark dose lower confidence limit (BMDL) of the percentage of the S phase after 24 h exposure was the lowest among all the BMDLs of a good fit, with BMDL5 of 9.55 µM. CONCLUSIONS: The MOA of female reproductive toxicity caused by BPS at human-relevant levels might involve: molecular initiating event (MIE)-BPS binding to ERß receptor, key event (KE)1-the interrupted expression of GnRH, KE2-the activation of JNK (for short-term exposure) and ERK pathway (for long-term exposure), KE3-cell cycle arrest (the increased percentage of the G0/G1 phase), and KE4-interruption of cell proliferation (only for short-term exposure). The BMDL of the percentage of the S phase after 24 h exposure was the lowest among all the BMDLs of a good fit, with BMDL5 of 9.55 µM.

Milk proteomic analysis reveals differentially expressed proteins in high-yielding and low-yielding Guanzhong dairy goats at peak lactation.

The aim of this experiment was to investigate the differential proteomic characteristics of milk from high- and low-yielding Guanzhong dairy goats during the peak lactation period under the same feeding conditions. Nine Guanzhong dairy goats with high yield (H: 3.5 ± 0.17 kg/d) and nine with low yield (L:1.2 ± 0.25 kg/d) were selected for milk proteomic analysis using tandem mass tag technology. A total of 78 differentially expressed proteins were identified. Compared with L, 50 proteins including HK3, HSPB1 and ANXA2 were significantly upregulated in H milk, while 28 proteins including LALBA and XDH were significantly downregulated. Bioinformatics analysis of the differentially expressed proteins showed that galactose metabolism, purine metabolism, glycolysis/gluconeogenesis, MAPK signaling pathway, regulation of actin cytoskeleton and other pathways were closely related to milk yield. HK3, HSPB1, ANXA2, LALBA and XDH were important candidate proteins associated with the milk production characteristics of Guanzhong dairy goats. Our data provide relevant biomarkers and a theoretical basis for improving milk production in Guanzhong dairy goats.

The cGAS-STING pathway promotes the development of preeclampsia by upregulating autophagy: Mechanisms and implications.

OBJECTIVE: To investigate the influence and significance of cGAS-STING signaling pathway and autophagy on the occurrence and development of preeclampsia. DESIGN: A case-control experimental study, in vitro cell culture study, and in vivo animal research. METHODS: Human placenta tissue was collected and the differences in HE staining were observed. Immunohistochemistry and Western blot were used to verify differences in cGAS, STING and autophagy associated proteins. The PE rat model was established, the pathological changes of placenta and kidney were observed by HE staining, and the expression levels of related proteins were detected. In the lv-STING transfected HTR-8/SVneo trophoblast cell model, the expressions of autophagy indexes such as P62 and LC3 were verified by RT-PCR, Western blot and cell fluorescence experiments, and then the invasion and migration ability of cells were detected by Transwell and scrape tests. As an effective STING antagonist, C176 was administered to PE rats to observe whether it was effective in the treatment of PE disease. RESULTS: The expression levels of cGAS, STING and autophagy related proteins were increased in human and rat placental tissues. In the HTR-8/SVneo cell model which transfected by lv-STING, the expression levels of autophagy related indicators such as P62 and LC3 were increased. The invasion and migration ability of HTR-8/SVneo cells were significantly inhibited, which was improved by the autophagy inhibitor chloroquine. Acting as an effective STING antagonist in vivo, C176 significantly reversed the outcome of PE, alleviated and prevented the occurrence and development of PE. CONCLUSION: Our study proved that the cGAS-STING signaling pathway and autophagy levels are elevated in preeclampsia disease, and the cGAS-STING signaling pathway promotes the occurrence and development of preeclampsia through up-regulation of autophagy. This finding provides new insights into the pathogenesis of preeclampsia. Targeting this pathway may provide a potential therapeutic strategy for the treatment of preeclampsia.

Proof-of-Concept in Developing a 45% Drug Loaded Amorphous Nanoparticle Formulation.

Amorphous solid dispersion (ASD) is an enabling approach utilized to deliver poorly soluble compounds. ASDs can spontaneously generate drug-rich amorphous nanoparticles upon dissolution, which can act as a reservoir for maintaining supersaturation during oral absorption. But, conventional ASDs are often limited in drug loadings to < 20 %. For indications where the dose is high, this can translate into a significant pill burden. The aim of this research was to develop a high drug loading (DL) amorphous nanoparticle (ANP) formulation that can release the drug-rich nanoparticles into solution upon contact with aqueous environment. Nanoparticles were directly engineered using solvent/anti-solvent precipitation. The obtained nanoparticle suspension was then concentrated followed by solidification to a re-dispersible amorphous dosage form using spray drying or lyophilization. The impact of process variables was studied using dynamic light scattering (DLS), scanning electron microscopy (SEM), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC). It was observed that spray drying led to a non-re-dispersible formulation. Sucrose and trehalose containing lyocakes resulted in re-dispersible formulations. The trehalose containing lyocakes, in a dog study, gave comparable performance to the reference tablet in the fasted state but lower area under the curve (AUC) in fed state.

Design of Redispersible High-Drug-Load Amorphous Formulations: Impact of Ionic vs Nonionic Surfactants on Processing and Performance.

Amorphous solid dispersions (ASDs) are an enabling formulation approach used to enhance bioavailability of poorly water-soluble molecules in oral drug products. Drug-rich amorphous nanoparticles generated in situ during ASD dissolution maintain supersaturation that drives enhanced absorption. However, in situ formation of nanoparticles requires large quantities of polymers to release drugs rapidly, resulting in an ASD drug load <25%. Delivering directly engineered drug-rich amorphous nanoparticles can reduce the quantities of polymers significantly without sacrificing bioavailability. Preparation of 90% drug-load amorphous nanoparticles (ANPs) of <300 nm diameter using solvent/antisolvent nanoprecipitation, organic solvent removal, and spray drying was demonstrated previously on model compound ABT-530 with Copovidone and sodium dodecyl sulfate (anionic). In this work, nonionic surfactant d-α-tocopheryl polyethylene glycol succinate (Vitamin E TPGS, or TPGS) was used to prepare ANPs as a comparison. Characterization of ANPs by dynamic light scattering, filtrate potency assay, scanning electron microscopy, and differential scanning calorimetry revealed differences in surface properties of nanoparticles afforded by surfactants. This work demonstrates the importance of understanding the impact of the stabilizing agents on nanoparticle behavior when designing a high-drug-load amorphous formulation for poorly water-soluble compounds as well as the impact on redispersion.

Mitochonic acid 5 rescues cardiomyocytes from doxorubicin-induced toxicity via repressing the TNF-α/NF-κB/NLRP3-mediated pyroptosis.

AIMS: Doxorubicin (DOX) is an effective anti-tumor drug, but the cardiotoxicity severely limits its clinical use. Interestingly, a hypothesis has emerged suggesting an association between DOX-induced cardiotoxicity and mitochondrial disorders and oxidative stress. The mitochonic acid 5 (MA5) shows promise in alleviating mitochondrial dysfunction by promoting mitochondrial ATP synthesis and reducing reactive oxygen species (ROS) accumulation, though its potential in ameliorating DOX-induced cardiotoxicity remains elusive. METHODS: Network pharmacology approach, molecular docking techniques, and molecular dynamics simulation (MDS) were used to reveal the specific drug targets and pharmaceutical mechanisms involved in the treatment of DOX-induced cardiotoxicity using MA5. For experimental verification, cardiomyocytes (H9c2) and mice were exposed to DOX in the presence or absence of MA5. Our investigation involved the assessment of echocardiographic parameters, cardiac enzymes, inflammatory factors, mitochondrial function, myocardial structure, and cardiomyocyte pyroptosis. RESULTS: Among the 100 core targets identified in network pharmacology, MA5 was pharmacologically active against DOX-induced cardiotoxicity via pathways implicated in cancer, prostate cancer, lipids and atherosclerosis. Molecular docking analysis confirmed that MA5 docked well with TNF-α, interleukin-6 (IL-6), and caspase-3. Furthermore, MA5 exhibited a stronger affinity toward TNF-α than IL-6 and caspase-3. Subsequent MDS revealed the stability of binding between MA5 and TNF-α. The DOX-challenged mice also displayed abnormal myocardial enzymogram, disrupted systolic and diastolic function, and elevated inflammation and cardiomyocyte pyroptosis, which could be mitigated by the administration of MA5. Similarly, H9c2 cells exposed to DOX showed increased intracellular ROS production and impaired mitochondrial function, which were relieved by MA5 treatment. CONCLUSION: Our findings suggest that MA5 attenuates DOX-induced cardiac anomalies through the TNF-α-mediated regulation of inflammation and pyroptosis. These insights offer a potential therapeutic strategy for managing DOX-induced cardiac complications, thereby improving the safety and efficacy of cancer treatments.

Comparative analysis of different metabolites in semen of Guanzhong dairy goats with different motility rates.

In order to explore the different metabolites of buck semen with different motility stored at 4 °C, the semen of bucks was collected by artificial vagina. The collected semen was divided into high motility group and low motility group after treatment, with 6 replicates set for each group. The semen metabolites of high motility group and low motility group were detected by Liquid Chromatography-Mass Spectrometry (LC-MS). The results showed that 101 different metabolites were detected in the high and low motility groups of bucks, of which 48 metabolites were significantly up-regulated (P < 0.05) and 53 metabolites were significantly down regulated (P < 0.05). Most of these metabolites belonged to lipids and lipid-like molecules, organic acids and their derivatives, and organic oxygen compounds, which were mainly related to energy metabolism. According to the functional enrichment analysis of the former differential metabolites in KEGG database, the top 20 most representative metabolic pathways were detected, among which the glycerophospholipid metabolic pathways changed significantly. From the perspective of metabolomics, this study revealed the differences of metabolites and characteristic compounds of semen with different motility of bucks under low temperature preservation, which provided a scientific basis for the preservation and utilization of semen of Guanzhong dairy goats in the future.

Comprehensive analysis of the key amino acid substitutions in the polymerase and NP of avian influenza virus that enhance polymerase activity and affect adaptation to mammalian hosts.

Accumulation of adaptive mutations in the polymerase and NP genes is crucial for the adaptation of avian influenza A viruses (IAV) to a new host. Here, we identified residues in the polymerase and NP proteins for which the percentages were substantially different between avian and human influenza viruses, to screen for key mammalian adaptive markers. The top 10 human virus-like residues in each gene segment were then selected for analysis of polymerase activity. Our research revealed that the PA-M311I and PA-A343S mutations increased the polymerase activity among the 40 individual mutations that augmented viral transcription and genomic replication, leading to increased virus yields, pro-inflammatory cytokine/chemokine levels and pathogenicity in mice. We also investigated the accumulative mutations in multiple polymerase genes and discovered that a combination of PB2-E120D/V227I, PB1-K52R/L212V/R486K/V709I, PA-R204K/M311I, and NP-E18D/R65K (hereafter referred to as the ten-sites joint mutations) has been identified to generate the highest polymerase activity, which can to some extent make up for the highest polymerase activity caused by the PB2-627 K mutation. When the ten-sites joint mutations co-occur with 627 K, the polymerase activity was further enhanced, potentially resulting in a virus with an improved phenotype that can infect a broader range of hosts, including mammals. This could lead to a greater public health concern than the current epidemic, highlighting that continuous surveillance of the variations of these sites is utmost important.

The cGAS-STING pathway promotes endometriosis by up-regulating autophagy.

OBJECTIVE: To investigate the roles of the cGAS-STING signal pathway and autophagy in the disease progression of endometriosis and to explore the regulatory mechanism of the cGAS-STING signal pathway on autophagy. DESIGN: A case-control experimental study, in vitro primary cell culture study, and in vivo animal research. MAIN OUTCOME MEASURES: Immunohistochemistry, RT-PCR and Western Blot were used to detect cGAS-STING signal pathway and autophagy expression differences in human and rat models. The lentivirus was used to overexpress STING in cells. The expression level of autophagy in human endometrial stromal cells (HESCs) transfected with lv-STING was detected by Western Blot, RT-PCR, and immunofluorescence. Transwell migration and invasion assays were conducted to assess cellular motility. The STING antagonist was applicated in vivo to investigate the therapeutic effects. RESULTS: The expression levels of the cGAS-STING signal pathway and autophagy in Human and Rat ectopic endometrium were increased. STING overexpression promotes the expression of autophagy in human endometrial stromal cells (HESCs). STING overexpression enhances the migration and invasion of the human endometrial stromal cells (HESCs), but the addition of autophagy antagonists could significantly reverse this. STING antagonists inhibited the expression of autophagy in vivo and reduced the volume of ectopic lesions. CONCLUSION: The expression levels of the cGAS-STING signal pathway and autophagy were increased in endometriosis. cGAS-STING signal pathway promotes the development of endometriosis by upregulating autophagy.

Engineered Toll-like Receptor Nanoagonist Binding to Extracellular Matrix Elicits Safe and Robust Antitumor Immunity.

Cancer immunotherapy, such as the Toll-like receptor (TLR) agonist including CpG oligodeoxynucleotide, has shown potency in clinical settings. However, it is still confronted with multiple challenges, which include the limited efficacy and severe adverse events caused by the rapid clearance and systemic diffusion of CpG. Here we report an improved CpG-based immunotherapy approach composed of a synthetic extracellular matrix (ECM)-anchored DNA/peptide hybrid nanoagonist (EaCpG) via (1) a tailor designed DNA template that encodes tetramer CpG and additional short DNA moieties, (2) generation of elongated multimeric CpG through rolling circle amplification (RCA), (3) self-assembly of densely packaged CpG particles composed of tandem CpG building blocks and magnesium pyrophosphate, and (4) incorporation of multiple copies of ECM binding peptide through hybridization to short DNA moieties. The structurally well-defined EaCpG shows dramatically increased intratumoral retention and marginal systemic dissemination through peritumoral administration, leading to potent antitumor immune response and subsequent tumor elimination, with minimal treatment-related toxicity. Combined with conventional standard-of-care therapies, peritumor administration of EaCpG generates systemic immune responses that lead to a curative abscopal effect on distant untreated tumors in multiple cancer models, which is superior to the unmodified CpG. Taken together, EaCpG provides a facile and generalizable strategy to simultaneously potentiate the potency and safety of CpG for combinational cancer immunotherapies.

Study on comparative analysis of differential metabolites in Guanzhong dairy goat semen before and after freezing.

In order to explore the differential metabolites between fresh and frozen-thawed semen of Guanzhong dairy goats, semen samples were collected by artificial vagina method, and divided into fresh and frozen-thawed semen groups, with six replicates in each group. Liquid Chromatography-mass spectrometry (LC-MS) technology was used to detect semen metabolites in both groups. The metabolites were analyzed and identified by the combination of multidimensional statistical analysis, namely principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA) and univariate statistical analysis, Differential metabolites were identified according to One-step Solution for Identification of Small Molecules in Metabolomics Studies (OSI/SMMS) combined with Human Metabolome Database (HMDB), Lipidmaps and Metlin and the metabolic pathways of different metabolites were enriched and analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The results showed that a total of 53 differential metabolites were detected in fresh and frozen-thawed semen groups, of which 10 metabolites were significantly up-regulated (P < 0.05) and 43 were significantly down-regulated (P < 0.05). Most of the metabolites belonged to lipids and lipid-like molecules, organic acids and their derivatives, organic oxygen compounds, etc. According to the functional enrichment analysis of the top twenty differential metabolites in the KEGG database, significant changes occurred in linoleic acid metabolism pathway out of total eleven pathways observed. These differential metabolites can be used as metabolic markers of sperm cryo-injury in dairy goats.

Design of a Re-Dispersible High Drug Load Amorphous Formulation.

Amorphous solid dispersions (ASD) are a commonly used enabling formulation technology to drive oral absorption of poorly soluble drugs. To ensure adequate solid-state stability and dissolution characteristics, the ASD formulation design typically has ≤ 25% drug loading. Exposed to aqueous media, ASD formulations can produce drug-rich colloidal dispersion with particle size < 500 nm. This in situ formation of colloidal particles requires incorporation of excess excipients in the formulation. The concept of using engineered drug-rich particles having comparable size as those generated by ASDs in aqueous media is explored with the goal of increasing drug loading in the solid dosage form. Utilizing ABT-530 as model compound, a controlled solvent-antisolvent precipitation method resulted in a dilute suspension that contained drug-rich (90% (w/w)) amorphous nanoparticles (ANP). The precipitation process was optimized to yield a suspension containing < 300 nm ANP. A systematic evaluation of formulation properties and process variables resulted in the generation of dry powders composed of 1-8 µm agglomerates of nanoparticles which in contact with water regenerated the colloidal suspension having particle size comparable to primary particles. Thus, this work demonstrates an approach to designing a re-dispersible ANP based powder containing ≥90% w/w ABT-530 that could be used in preparation of a high drug load solid dosage form.

Segregation patterns in three-dimensional granular flows.

Flow of size-bidisperse particle mixtures in a spherical tumbler rotating alternately about two perpendicular axes produces segregation patterns that track the location of nonmixing islands predicted by a dynamical systems approach. To better understand the paradoxical accumulation of large particles in regions defined by barriers to transport, we perform discrete element method (DEM) simulations to visualize the three-dimensional structure of the segregation patterns and track individual particles. Our DEM simulations and modeling results indicate that segregation pattern formation in the biaxial spherical tumbler is due to the interaction of size-driven radial segregation with the weak spanwise component of the advective surface flow. Specifically, we find that after large particles segregate to the surface, slow axial drift in the flowing layer, which is inherent to spherical tumblers, is sufficient to drive large particles across nominal transport barriers and into nonmixing islands predicted by an advective flow model in the absence of axial drift. Axial drift alters the periodic dynamics of nonmixing islands, turning them into "sinks" where large particles accumulate even in the presence of collisional diffusion. Overall, our results indicate that weak perturbation of chaotic flow has the potential to alter key dynamical system features (e.g., transport barriers), which ultimately can result in unexpected physical phenomena.

Multiple Fingerprints and Spectrum-Effect Relationship of Polysaccharides from Saposhnikoviae Radix.

PMP-HPLC, FT-IR, and HPSEC fingerprints of 10 batches of polysaccharides from Saposhnikoviae Radix with different production areas and harvest times have been prepared, and the chemometrics analysis was performed. The anti-allergic activity of 10 batches of Saposhnikoviae Radix polysaccharide (SP) was evaluated, and the spectrum-effect relationship of the 10 batches of SP was analyzed by gray correlation degree with the chromatographic fingerprint as the independent variable. The results showed that the PMP-HPLC, HPSEC, and FT-IR fingerprints of 10 batches of SP had a high similarity. Two monosaccharides (rhamnose and galactose), the polysaccharide fragment Mn = 8.67 × 106~9.56 × 106 Da, and the FT-IR absorption peak of 892 cm-1 can be used as the quality control markers of SPs. All 10 batches of SP could significantly inhibit the release of ß-HEX in RBL-231 cells, and the polysaccharides harvested from Inner Mongolia in the winter had the best anti-allergic activity. The spectrum-effect relationship model showed that the monosaccharide composition and molecular weight were related to the anti-allergic activity of the SPs. Multiple fingerprints combined with spectrum-effect relationship analysis can evaluate and control the quality of SPs from the aspects of overall quality and efficacy, which has more application value.

D2I and F9Y Mutations in the NS1 Protein of Influenza A Virus Affect Viral Replication via Regulating Host Innate Immune Responses.

Influenza A viruses (IAV) modulate host antiviral responses to promote viral growth and pathogenicity. The non-structural (NS1) protein of influenza A virus has played an indispensable role in the inhibition of host immune responses, especially in limiting interferon (IFN) production. In this study, random site mutations were introduced into the NS1 gene of A/WSN/1933 (WSN, H1N1) via an error prone PCR to construct a random mutant plasmid library. The NS1 random mutant virus library was generated by reverse genetics. To screen out the unidentified NS1 functional mutants, the library viruses were lung-to-lung passaged in mice and individual plaques were picked from the fourth passage in mice lungs. Sanger sequencing revealed that eight different kinds of mutations in the NS1 gene were obtained from the passaged library virus. We found that the NS1 F9Y mutation significantly enhanced viral growth in vitro (MDCK and A549 cells) and in vivo (BALB/c mice) as well as increased virulence in mice. The NS1 D2I mutation attenuated the viral replication and pathogenicity in both in vitro and in vivo models. Further studies demonstrated that the NS1 F9Y mutant virus exhibited systematic and selective inhibition of cytokine responses as well as inhibited the expression of IFN. In addition, the expression levels of innate immunity-related cytokines were significantly up-regulated after the rNS1 D2I virus infected A549 cells. Collectively, our results revealed that the two mutations in the N-terminal of the NS1 protein could alter the viral properties of IAV and provide additional evidence that the NS1 protein is a critical virulence factor. The two characterized NS1 mutations may serve as potential targets for antiviral drugs as well as attenuated vaccine development.

Antigenic Evolution Characteristics and Immunological Evaluation of H9N2 Avian Influenza Viruses from 1994-2019 in China.

The H9N2 subtype avian influenza viruses (AIVs) have been circulating in China for more than 20 years, attracting more and more attention due to the potential threat of them. At present, vaccination is a common prevention and control strategy in poultry farms, but as virus antigenicity evolves, the immune protection efficiency of vaccines has constantly been challenged. In this study, we downloaded the hemagglutinin (HA) protein sequences of the H9N2 subtype AIVs from 1994 to 2019 in China-with a total of 5138 sequences. The above sequences were analyzed in terms of time and space, and it was found that h9.4.2.5 was the most popular in various regions of China. Furthermore, the prevalence of H9N2 subtype AIVs in China around 2006 was different. The domestic epidemic branch was relatively diversified from 1994 to 2006. After 2006, the epidemic branch each year was h9.4.2.5. We compared the sequences around 2006 as a whole and screened out 15 different amino acid positions. Based on the HA protein of A/chicken/Guangxi/55/2005 (GX55), the abovementioned amino acid mutations were completed. According to the 12-plasmid reverse genetic system, the rescue of the mutant virus was completed using A/PuertoRico/8/1934 (H1N1) (PR8) as the backbone. The cross hemagglutination inhibition test showed that these mutant sites could transform the parental strain from the old to the new antigenic region. Animal experiments indicated that the mutant virus provided significant protection against the virus from the new antigenic region. This study revealed the antigenic evolution of H9N2 subtype AIVs in China. At the same time, it provided an experimental basis for the development of new vaccines.

Integration of a miniaturized DMMB assay with high-throughput screening for identifying regulators of proteoglycan metabolism.

Defective biosynthesis or function of proteoglycans causes pathological conditions in a variety of tissue systems. Osteoarthritis (OA) is a prevalent degenerative joint disorder characterized by progressive cartilage destruction caused by imbalanced proteoglycan synthesis and degradation. Identifying agents that regulate proteoglycan metabolism may benefit the development of OA-modifying therapeutics. High-throughput screening (HTS) of chemical libraries has paved the way for achieving this goal. However, the implementation and adaptation of HTS assays based on proteoglycan measurement remain underexploited. Using primary porcine chondrocytes as a model, we report a miniaturized dimethyl-methylene blue (DMMB) assay, which is commonly used to quantitatively evaluate sulfated glycosaminoglycan (GAG) content, with an optimized detection range and reproducibility and its integration with HTS. Treatment with TGF-ß1 and IL1-α, known as positive and negative proteoglycan regulators, respectively, supported the assay specificity. A pre-test of chemical screening of 960 compounds identified both stimulators (4.48%) and inhibitors (6.04%) of GAG production. Fluorophore-assisted carbohydrate electrophoresis validated the activity of selected hits on chondroitin sulfate expression in an alginate culture system. Our findings support the implementation of this simple colorimetric assay in HTS to discover modifiers of OA or other diseases related to dysregulated proteoglycan metabolism.