Mouse Serum Research Articles

Improvement of glucose metabolism disorder by wheat dietary fibre depended on the intake mode and regulated through TLRs/NF-κB/TNF pathway in db/db mice

ABSTRACT To study the effect of wheat dietary fibre (WDF) on glucose metabolism disorder and its mechanisms, 6-week-old db/db mice were given WDF by adding to diet or oral-gavage. The results showed that WDF by oral-gavage instead of adding to the feed can significantly reduce blood glucose levels. The levels of insulin and HOMA-IR were both decreased in the serum of WDF-gavaged mice as well as several inflammatory cytokines including TNF-α, IL-6 and IL-1β. WDF intake by gavage could better regulate the intestinal flora, thus inhibiting diabetes. The relative expressions of genes and proteins including TLR, P65, P50 and TNF-α were all significantly decreased by oral-gavaged WDF. Moreover, the ratio of phosphorylated p65 to total p65 was significantly decreased. In conclusion, ingestion of WDF by gavage can effectively improve glucose metabolism disorder in db/db mice by improving intestinal microbiota and regulating the TLRs/NF-κB/TNF pathway.

Study of the Immunologic Value of Trichinella spiralis recombinant Protein Tsp-LE

Background and Aim. Trichinellosis is a serious zoonotic disease, with frequent outbreaks reported across various regions in Europe and Asia. However, current diagnostic tests based on excretory-secretory antigens (ES-Ag) and somatic antigens (S-Ag) lack adequate specificity and sensitivity. Moreover, the production of these antigens is both labor-intensive and costly.Materials and Methods. The immunodiagnostic potential of a recombinant 11 kDa serine protease protein, previously characterized in earlier studies was assessed. This study detected infection in Balb/c mice as early as 14 days post infection with T. spiralis larvae, yielding no false-positive results and an average infection level of 61±5.3 larvae per mouse.Results. Comparative indirect ELISA analysis on mouse sera after the 3rd and 5th immunizations, alongside tests on S Ag and S-Ag using sera from infected rabbits and pigs, demonstrated the high diagnostic accuracy of the recombinant protein. The protein revealed strong specificity for trichinellosis detection, showing no cross reactivity with echinococcosis.Conclusion. These findings support using rTsp-LE as a reliable component in developing ELISA test systems for trichinellosis detection.

Metalomics Revealed that Changes of Serum Elements were Associated with Oxidative Stress-Induced Inflammation of Cortex in a Mouse Model of Autism.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder emerging during early childhood. However, the mechanism underlying the pathogenesis of ASD remains unclear. This study investigated the alterations of elements in serum and prefrontal cortex of BTBR T + tf/J (BTBR) mice and potential mechanisms. The male BTBR mice were used for experimental group and C57BL/6J (C57) mice were used for control group (n = 15). After behavioral tests were monitored, serum and prefrontal cortex of mice were analyzed by ICP-MS. The results demonstrated that the level of copper (Cu) was increased, and the levels of calcium (Ca), magnesium (Mg), selenium (Se), cobalt (Co), iron (Fe) and zinc (Zn) were decreased in BTBR mice compared to C57 mice (p < 0.01). The levels of above differential elements in serum demonstrated positive correlations with those in prefrontal cortex. Meanwhile, differential elements in prefrontal cortex had correlations with the total distance traveled (open field test) and the number of marbles buried (marble burying test) in BTBR mice (p < 0.05 or p < 0.01). The abnormally changed elements in serum might cross blood-brain-barrier into the brain and lead to oxidative stress, causing inflammation. Furtherly, the levels of inflammation-related indicators including tumor necrosis factor-alpha (TNF-α), nuclear factor kappa-B (NF-κB), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were increased in prefrontal cortex of BTBR mice (p < 0.01), which were consistent with the aforementioned results. Our study suggested that the abnormal elements in the serum of BTBR mice may cause oxidative stress and inflammation in prefrontal cortex, which might contribute to increase the understanding of ASD pathogenesis.

Exploratory miRNA profiling from serum and bone tissue of mice with T1D-induced bone loss.

Type 1 diabetes (T1D) represents a significant health burden worldwide, with associated complications including bone fragility. Current clinical methods and biomarkers for assessing bone health and predicting fracture risk in T1D are limited and lack accuracy. MicroRNAs (miRNAs) have emerged as potential biomarkers for predicting T1D-induced bone loss, although comprehensive profiling studies are lacking. Previous investigations have indicated a link between dysregulated miRNA expression levels and impaired bone health in T1D. Therefore, in this study, we explored differential miRNA expression levels in serum and bone tissue of mice with T1D-induced bone loss using Next Generation Sequencing (NGS). T1D was induced using streptozotocin in male wild-type mice. Serum and bone tissues were analyzed at 14 weeks of age, following the prior characterization of bone loss in this mouse model. MiRNA profiling was conducted using two-independent NGS analyses and validated through quantitative RT-PCR. NGS profiling identified differential expression of miRNAs in serum and bone tissue of T1D mice compared to controls. The first NGS analysis revealed 24 differentially expressed miRNAs in serum and 13 in bone tissue. Especially, miR-136-3p was consistently downregulated in both serum and bone tissue. However, the second NGS analysis presented a distinct set of dysregulated miRNAs, with miR-206-3p overlapping in both tissues but exhibiting differential expression patterns. Surprisingly, miR-144-5p, miR-19a-3p, and miR-21a-5p displayed contrasting regulatory patterns between NGS and qPCR analyses. Finally, gene network analysis identified associations between dysregulated miRNAs and pathways involved in bone physiology, including TGF-beta, PI3-Akt signaling, and osteoclast differentiation in humans. In conclusion, our study offers initial insights into dysregulated miRNAs associated with T1D-induced bone loss, but also highlights the lack of consistency in the results obtained from miRNA sequencing in different cohorts. Thus, further investigation is needed to better understand the complexities of miRNA analyses before they can be established as reproducible biomarkers for predicting bone health in T1D.

β-Asarone regulates microglia polarization to alleviate TBI-induced nerve damage via Fas/FasL signaling axis.

Acute injury and secondary injury caused by traumatic brain injury (TBI) seriously threaten the health of patients. The purpose of this study was to investigate the role of β-Asarone in TBI-induced neuroinflammation and injury. In this work, the effects of β-Asarone on nerve injury and neuronal apoptosis were investigated in mice with TBI by controlled cortical impingement. The results of this research implied that β-Asarone dose-dependently decreased the mNSS score, brain water content and neuronal apoptosis, but increased the levels of the axonal markers Nrp-1 and Tau in TBI mice. In addition, β-Asarone caused a decrease in the levels of Fas, FasL, and inflammatory factors in cerebrospinal fluid and serum of TBI mice. Therefore, β-Asarone inhibited neuroinflammation and promoted axon regeneration in TBI mice. Besides, β-Asarone treatment inhibited M1 phenotype polarization but promoted M2 phenotype polarization in microglia of TBI mice. Overexpression of Fas and FasL reversed the above effects of β-Asarone. Thus, β-Asarone regulated microglial M1/M2 polarization balance in TBI mice by suppressing Fas/FasL signaling axis. In conclusion, β-Asarone inhibited Fas/FasL signaling pathway to promote the M1/M2 polarization balance of microglia toward M2 polarization, thus alleviating TBI-induced nerve injury.

In vitro potentiation of tetracyclines in Pseudomonas aeruginosa by RW01, a new cyclic peptide.

The pipeline for new drugs against multidrug-resistant Pseudomonas aeruginosa remains limited, highlighting the urgent need for innovative treatments. New strategies, such as membrane-targeting molecules acting as adjuvants, aim to enhance antibiotic effectiveness and combat resistance. RW01, a cyclic peptide with low antimicrobial activity, was selected as an adjuvant to enhance drug efficacy through membrane permeabilization. RW01's activity was evaluated via antimicrobial susceptibility testing in combination with existing antibiotics on 10 P. aeruginosa strains and analog synthesis. Synergy was assessed using checkerboard assays, and one-step mutants were generated to identify altered pathways through whole-genome sequencing and variant analysis. Permeabilizing activity was studied using flow cytometry and real-time fluorescence measurement. In vivo toxicity was assessed in female C57BL/6J mice, and possible interaction with mouse serum was also evaluated. Susceptibility testing revealed specific synergy with tetracyclines, with up to a 16-fold reduction in minimum inhibitory concentrations. Sequencing revealed that resistance to the RW01-minocycline combination involved mutations in the pmrB gene, affecting outer membrane lipopolysaccharide composition. This was further confirmed by the identification of cross-resistance to colistin in these mutants. RW01 reduced the mutant prevention concentration of minocycline from 64 to 8 mg/L. RW01 was demonstrated to enhance membrane permeabilization and therefore minocycline uptake with statistical significance. Synthetic derivatives of RW01 showed a complete loss of activity, highlighting the importance of RW01's D-proline(NH2) residue. No acute or cumulative in vivo toxicity was observed in mice. These findings suggest that RW01 could revitalize obsolete antimicrobials and potentially expand therapeutic options against multidrug-resistant P. aeruginosa.

Tryptophan metabolite atlas uncovers organ, age, and sex-specific variations.

Although tryptophan (Trp) is the largest and most structurally complex amino acid, it is the least abundant in the proteome. Its distinct indole ring and high carbon content enable it to generate various biologically active metabolites such as serotonin, kynurenine (Kyn), and indole-3-pyruvate (I3P). Dysregulation of Trp metabolism has been implicated in diseases ranging from depression to cancer. Investigating Trp and its metabolites in healthy tissues offers pathways to target disease-associated disruptions selectively, while preserving essential functions. In this study, we comprehensively mapped Trp metabolites across the Kyn, serotonin, and I3P pathways, as well as the microbiome-derived metabolite tryptamine, in C57BL/6 mice. Our comprehensive analysis covered 12 peripheral organs, the central nervous system, and serum in both male and female mice at three life stages: young (3 weeks), adult (54 weeks), and aged (74 weeks). We found significant tissue-, sex-, and age-specific variations in Trp metabolism, with notably higher levels of the oncometabolites I3P and Kyn in aging males. These findings emphasize the value of organ-specific analysis of Trp metabolism for understanding its role in disease progression and identifying targeted therapeutic opportunities.

Selenium-Enriched Aspergillus oryzae A02 Enhances Testicular Antioxidant Capacity in Mice by Regulating Intestinal Microbiota and Serum Metabolite.

Selenium (Se) is a trace element that is essential for health. Organic Se created by Se-enriched microorganisms has the characteristics of low toxicity, high bioavailability, and regulation of physiological functions. Here, the regulatory effect of Se-enriched Aspergillus oryzae A02 on the reproductive function of male mice and its potential molecular mechanism was studied. Specifically, twenty-four male mice were randomly divided into a control group and a Se-enriched A. oryzae A02 (Nano-Se) (daily gavage of 0.5mg/kg, dissolved in saline) for an 8-week experiment. The results showed that Nano-Se intervention did not affect body weight and testicular index, but increased sperm concentration and seminiferous epithelium height in experimental mice, indicating that Nano-Se has the potential to improve the reproductive performance of male mice. Mechanistically, Nano-Se intervention increased the levels of antioxidant-related indicators catalase (CAT) and glutathione peroxidase (GSH-Px) in mouse serum, and increased the relative mRNA expression of GSH-Px, heme oxygenase-1 (HO-1), and NADPH quinine oxidoreductase-1 (NQO-1) in testicular tissues. We identified 9,10,13-trihydroxyoctadecenoic acids (TriHOMEs), stearidonic acid and selenomethionine linked with alpha-linolenic acid metabolism, selenocompound metabolism, folate biosynthesis, ubiquinone, and other terpenoid-quinone biosynthesis and biosynthesis of cofactors. In addition, Nano-Se did not influence the fecal bacterial alpha and beta diversity (P > 0.05), but increased the abundance of the Actinobacteriota and Proteobacteria phyla and the Staphylococcus and Corynebacterium genera, and lowered the abundance of the Bacteroidota phylum and the Lactobacillus and norank_f_Muribaculaceae genera. Nano-Se is considered a novel and promising nutritional regulator to improve reproductive function.

Antigenic features of the strains SARS-CoV-2 of omicron sublines assessed by hyperimmune mouse serum neutralisation

Introduction. The emergence and spread of new genetic variants of SARS-CoV-2 underlies periodic upsurge in COVID-19 incidence. It has been shown that the most rapidly spreading genetic variants of SARS-CoV-2 are resistant to antibodies specific to the previous variant of the SARS-CoV-2, thereby necessitating to analyze the antibody evasion ability of previously circulating variants for newly emerging subvariants. The aim of this work was to assess SARS-CoV-2 cross-reactivity of coronavirus strains belonging to different genetic subvariants of Omicron isolated in the territory of the Russian Federation in the period 2020–2023 in microneutralization reaction using hyperimmune mouse sera. Materials and methods. Mouse hyperimmune sera were obtained against 10 SARS-CoV-2 strains belonging to subvariants BA.1, BA.2, CH.1.1, BN.1, BA.5.1, CL.1.2, BA.5.2, BQ.1.2.1 XBB.1.5 and XBB.3. BALB/c mice were immunized with inactivated concentrated antigen mixed at 1:1 ratio with an adjuvant representing Quillaja saponaria saponin-based virus-like immunostimulatory complex. The antibody titer was determined by neutralization test. The neutralizing activity of the hyperimmune sera was analyzed against the relevant viruses as well as against previous genetic variants of SARS-CoV-2 (Wuhan, Alpha, Beta, Gamma, Delta). Results. Cross-reactivity for all Omicron-variant strains analyzed here was shown; the degree of cross-reactivity depended on the degree of inter-strain relatedness. A prominent cross-reactivity was observed for subvariants of BA.5 so that their neutralizing activity against recombinant SARS-CoV-2 lineages was markedly reduced. Neutralizing serum titers obtained for subvariants of BA.5 against genetic variants of SARS-CoV-2 isolated during the early periods of the pandemic are reduced more than 60-fold. Conclusion. The presented method for obtaining and using hyperimmune mouse sera for neutralization reaction allows the assessment of cross-reactivity for strains belonging to different SARS-CoV-2 subvariants.

Effects of ALA-PDT on the murine footpad model of Fonsecaea monophora infection and its related mechanisms in vivo.

5-aminolevulinic acid photodynamic therapy (ALA-PDT) has received growing attention for treating chromoblastomycosis (CBM) and has shown efficacy in a handful of clinical case reports. However, there is insufficient information regarding the effects of ALA-PDT on Fonsecaea monophora in mouse infection model and the related mechanisms. This study investigated these issues in vivo. A F. monophora infection mouse model inoculated in footpads was used. Changes in the footpad volume, tissue fungal burden, and histopathological characteristics were investigated to determine the efficacy of ALA-PDT. Scavenger receptor MARCO (Macrophage receptor with collagenous structure) was further evaluated at the gene and protein levels. Serum cytokines TNF-α, GM-CSF, IL-4, and IL-10 were measured using enzyme-linked immunosorbent assay to indicate changes in the immune microenvironment after PDT. ALA-PDT reduced infected footpad volume, fungal burden, and pathological inflammatory infiltration in vivo. It also increased the expression of Marco in the murine infection model. Furthermore, PDT upregulated the anti-inflammatory cytokines IL-4 and IL-10 while downregulated the pro-inflammatory cytokines TNF-α and GM-CSF in mouse serum. ALA-PDT demonstrated fungicidal effects in a mouse footpad infection model with F. monophora and attenuated the inflammatory reactions. It may also assist against the intracellular fungi by the host through macrophage receptor MARCO and regulation of the immune microenvironment. This study provides scientific evidence for the protocol selection of ALA-PDT as a promising adjunctive modality for treating chromoblastomycosis.

Roles of gut microbiome-associated metabolites in pulmonary fibrosis by integrated analysis

Lung diseases often coincide with imbalances in gut microbiota, but the role of gut microbiota in pulmonary fibrosis (PF) remains unclear. This study investigates the impact of gut microbiota and their metabolites on PF. Serum and lung tissues of normal, bleomycin (BLM)- and silica-induced mice showed significant differences in gut microbiota. l-Tryptophan was upregulated within pulmonary tissue and serum metabolites both in the BLM and Silica groups. The dominant gut microbiota associated with l-tryptophan metabolism included Lachnospiraceae_NK4A136_Group, Allobaculum, Alistipes, and Candidatus_Saccharimonas. l-Tryptophan promoted BLM- and silica-induced pathological damage in PF mice. l-Tryptophan promoted TGF-β1-induced EMT and fibroblast activation in vitro via activating the mTOR/S6 pathway. In conclusion, PF mice exhibited alterations in gut microbiota and serum and lung tissue metabolites. l-Tryptophan level was associated with changes in gut microbiota, and l-tryptophan promoted PF progression in both in vivo and in vitro models, potentially through activation of the mTOR/S6 pathway.

Scutellariae Radix Ameliorates Prenatal Stress Induced Anxiety-like and Depression-like Behavior in the Offspring via Reversing HPA Axis Hyperfunction and Ameliorating Neurodevelopmental Dysfunction.

This study aimed to explore the impact and mechanism of Scutellariae radix (SR), dried root of Scutellaria baicalensis Georgi of Labiatae, on prenatal stress (PS) induced anxiety-like and depression-like behavior in the offspring in a mouse prenatal stress model. The open field test (OFT), tail suspension test (TST), and forced swimming test (FST) were utilized to assess the behavior of the offspring. Histological changes were evaluated using HE staining and Nissl staining. ELISA was employed to detect the levels of related factors in the serum and fetal brains of offspring mice. Immunohistochemistry was used to determine the expressions of doublecortin and neurotrophic factors in the hippocampus, and RT-PCR reflected the expression of factors in the hippocampus and placenta of offspring mice. These various techniques collectively provided insight into the neurodevelopmental status by detecting indicators related to neurodevelopmental status. LC-MS/MS and molecular docking were used to clarify the chemical constituents and the pharmacodynamic components in Scutellariae radix. Scutellariae radix ameliorated prenatal stress-induced anxiety-like and depression-like behavior in the offspring. It also alleviated hippocampal neurogenesis impairment caused by prenatal stress and restored abnormal expression of hippocampal glutamate (Glu) and brain-derived neurotrophic factor in the offspring. Additionally, Scutellariae radix maintained normal 11β-HSD1 expression in the placenta of prenatal stress mice, ensuring a normal level of glucocorticoid (GC) and glucocorticoid receptors (GR) in the fetus. Furthermore, Scutellariae radix increased the mRNA expression of GR and 11β-HSD2 while decreasing the mRNA expression of 11β-HSD1, thereby normalizing levels of serum CRH, ACTH, and GC in the offspring. Finally, docking results indicated that baicalein, wogonin, wogonoside, and baicalin exhibited stronger binding ability with the target. The results of our study indicate that Scutellariae radix may have the potential to alleviate prenatal stress-induced anxiety-like and depression-like behaviors in offspring, at least partially through protecting placental barrier function, reversing HPA axis hyperfunction, and ameliorating neurodevelopmental dysfunction.

[89Zr]Zr-DFO-TOC: a novel radiopharmaceutical for PET imaging of somatostatin receptor positive neuroendocrine tumors

BackgroundNeuroendocrine tumors (NETs) are clinically diverse types of tumors that can arise anywhere in the body. Previous studies have shown that somatostatin receptors (SSTRs) are overexpressed on NET cell membranes relative to healthy tissue, allowing for tumor targeting through radiolabeled somatostatin analogs (SSAs). This work aims to develop a novel 89Zr-labeled tracer incorporating the SSA, octreotide (TOC), for positron emission tomography (PET) imaging of SSTR + NETs and predictive dosimetry calculations, leveraging the excellent nuclear (t½ = 3.27 days, β+ = 22.3%, β+avg = 395.5 keV) and chemical characteristics (+ 4 oxidation state, preferential coordination number of 7/8, favorable aqueous chemistry) of 89Zr. In combination with 89Zr, the known radiochemistry with the chelator deferoxamine (DFO) gives reason to believe that this radiopharmaceutical incorporating an octreotide conjugate will be successful in studying the suitability of detecting SSTR + NETs.ResultsRadiochemical tracer assessment indicated that amounts as low as 0.1 nmol DFO-TOC can be effectively radiolabeled with 89Zr, while maintaining ≥ 95% radiochemical yield. The stability of the compound was found to maintain radiochemical yields of 89.6% and 88.7% on the benchtop and in mouse serum, respectively, after 9 days. Receptor binding and competitive receptor blocking assays compared AR42J (high SSTR expression), PC-3 (moderate SSTR expression), and PANC-1 (minimal SSTR expression) cell lines at time points up to 6 days. In vitro studies demonstrated highest uptake in AR42J cells, and statistically significant differences in tracer uptake were seen after 1 h. Internalization assays showed maximum internalization after 3 h for all cell lines.ConclusionsIn this work, [89Zr]Zr-DFO-TOC was synthesized with radiochemical yields ≥ 95% and was found to remain stable in vitro at extended time points. In vitro cell studies demonstrated a statistically significant difference between receptor binding and blocking experiments. The development of this work shows potential to positively impact patient care through the predictive dosimetry calculations for the FDA-approved therapeutic agent [177Lu]Lu-DOTA-TATE, while allowing for imaging at extended timepoints and should be studied further.

First preclinical SPECT/CT imaging and biodistribution of [165Er]ErCl3 and [165Er]Er-PSMA-617

Background165Er (t1/2 = 10.4 h, Ex-ray = 47.1 keV (59.4%) and 54.3 keV (14.3%)) is a promising radionuclide suitable for targeted Auger electron therapy of cancer. 165Er can be produced at a relatively low cost, high yield, and high purity using small medical cyclotrons. As a late lanthanide, 165Er is easy to label and can be used as a surrogate for other lanthanides or Ac in proof-of-concept studies. In this report, we explore the radiochemistry, in vitro, and in vivo behavior of [165Er]ErCl3 and [165Er]Er-PSMA-617 to showcase the application of this radionuclide. Particularly, we report the first phantom and preclinical SPECT imaging of this radionuclide leveraging its characteristic X-ray photon emissions.ResultsThe 165Ho(p,n)165Er nuclear reaction using a 13 MeV cyclotron demonstrated production yields of up to 25 ± 5 MBq. µA−1 h−1 at the end of the bombardment. After the purification (4.0 ± 0.5 h) using a sequential combination of cation exchange and extraction chromatography, 4-h irradiation produced up to 1.5 GBq of [165Er]ErCl3. High molar activity [165Er]Er-PSMA-617 was prepared (~ 200 MBq/nmol). [165Er]Er-PSMA-617 showed a LogD7.4 value of -2.34 ± 0.24 meaning high hydrophilicity of the complex as expected. The stability of [165Er]Er-PSMA-617 in saline, human, and mouse serum was studied and showed intact tracer after 12 h in all three cases. [165Er]ErCl3 and [165Er]Er-PSMA-617 were both taken up by LNCaP cells. PSMA-617 has IC50 at nanomolar range for [165Er]Er-PSMA-617 in LNCaP cells. SPECT images with preclinical phantoms showed good uniformity, spatial resolution, and quantitative accuracy. SPECT/CT imaging in LNCaP tumor-bearing mice injected with [165Er]Er-PSMA-617 showed high tumor uptake and quantitative accuracy when comparing the results to ex vivo biodistribution %IA/g values. Mice injected with [165Er]ErCl3 showed uptake in bone structures and excretion through both liver and kidneys.ConclusionsThis study demonstrated the preclinical use of 165Er for the first time. Using [165Er]ErCl3 and [165Er]Er-PSMA-617 as examples, the radiochemistry, cell, and animal studies showed that 165Er can be used as a tool for evaluating targeted radiopharmaceuticals. The X-ray emission from 165Er can be used for quantitative SPECT imaging in mice.

Immunoregulatory Effects of Codonopsis pilosula Polysaccharide Modified Selenium Nanoparticles on H22 Tumor-Bearing Mice.

Codonopsis pilosula polysaccharide (CPP) and rare element selenium (Se) have been proved to exert various biological activities, and our previous study demonstrated that selenium nanoparticles modified with CPP (CPP-SeNPs) possessed significantly enhanced tumor cytotoxicity in vitro. This study aimed to investigated the inhibitory effects of CPP-SeNPs complex on H22 solid tumors via immune enhancement. In this study, the H22 tumor-bearing mice model was constructed, and the potential mechanisms of CPP-SeNPs antitumor effects were further explored by evaluating cytokines expression levels, immune cells activities and tumor cells apoptotic indicators in each group. The results demonstrated that CPP-SeNPs effectively exerted dose-dependent protective effects on the immune organs of tumor-bearing mice in vivo, leading to increase in peripheral white blood cell counts and inhibition of solid tumor growth with inhibitory rate of 47.18% in high-dose group (1.5 mg/kg). Furthermore, CPP-SeNPs treatment significantly elevated the levels of TNF-α, IFN-γ, and IL-2 in mice sera, enhanced NK cell cytotoxicity, augmented macrophage phagocytosis capacity, as well as increased both the amounts and proliferation activity of lymphocyte subsets. CPP-SeNPs improved the immune system's ability to clear tumor cells by up-regulating Bax expression while down-regulating Bcl-2 expression within solid tumors, indicating the potential activation of mitochondrial apoptosis pathway. Therefore, CPP-SeNPs administration can effectively inhibit tumor growth by enhancing immune response in tumor-bearing mice, which might be relevant to the regulation of gut microbiota short-chain fatty acids metabolisms. These findings could provide theoretical support and data foundation for further development of CPP-SeNPs as functional food and drug adjuvants.

Efficient, Low-Cost, and High-Throughput Sodium Dodecyl Sulfate (SDS) Removal from Protein Digests Using Weak-Anion Exchange.

Sodium dodecyl sulfate (SDS) plays a pivotal role in protein denaturation, tissue extraction, and protein mass-based electrophoretic separations. However, even modest concentrations of SDS can cause column overpressure, retention time shifts, and ionization signal suppression during liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Thus, SDS removal is a critical step for LC-MS/MS analysis of protein digests containing SDS. This study describes an inexpensive and high-throughput method to remove SDS from protein digests using weak-anion exchange (WAX) resins in 96-well filter plates. Requiring less than 3 min, this method can reduce SDS concentrations from 0.1-0.4% to less than 5 ppm and from 0.6-1% to less than 100 ppm. After SDS removal, the recoveries of unmodified tryptic peptides and phosphorylated peptides (at 94.3 nM) were ∼90% and ∼70%, respectively. Additionally, when using aqueous 1% SDS to solubilize trastuzumab-spiked mouse serum and subsequently removing the SDS using the WAX resin, quantitation of trastuzumab exhibited excellent linearity (R2 = 0.9996) together with a low coefficient of variation (<10%). Calculated concentrations were within 20% of the expected value for spiked standard samples (0.5, 1, and 2 μg/mL trastuzumab in mouse serum). The method is about 20× more cost-effective versus commercialized SDS removal kits and both the resin and filter plate are readily available, so the method should easily transfer to other laboratories.

Comparative study of two Rift Valley fever virus field strains originating from Mauritania.

Rift Valley fever (RVF) is one of the major viral arthropod-borne diseases in Africa. In recent decades, RVF virus (RVFV), the causative agent of RVF, has been responsible for multiple outbreaks in West Africa with important consequences on human and animal health. In particular, an outbreak occurred in 2010 after heavy rains in the desertic region of Adrar, Mauritania. It was characterized by the appearance of severe clinical signs among dromedary camels. Another one occurred in 2013-2014 across Senegal and the southern part of Mauritania. In this study, we characterized two RVFV field strains isolated during these two outbreaks. The first strain, MRU25010-30, was isolated from a camel (2010) while the second, MRU2687-3, was isolated from a goat (2013). By deep-sequencing and rapid amplification of cDNA-ends by polymerase chain reaction, we successfully sequenced the complete genome of these two RVFV strains as well as the reference laboratory strain ZH548. Phylogenetic analysis showed that the two field viruses belong to two different RVFV genetic lineages. Moreover, we showed that MRU25010-30 replicates more efficiently in various in vitro cell culture models than MRU2687-3 and ZH548. In vivo, MRU25010-30 caused rapid death of BALB/c mice and proved to be more virulent than MRU2687-3, regardless of the route of inoculation (subcutaneous or intranasal). The virulence of MRU25010-30 is associated with a high viral load in the liver and serum of infected mice, while the death of mice infected with MRU2687-3 and ZH548 correlated with a high viral load in the brain. Altogether, the data presented in this study provide new avenues to unveil the molecular viral determinants that modulate RVFV virulence and replication capacity.

The role of FOXM1 in acetylcysteine improving diabetic periodontitis.

Diabetic periodontitis (DP) stems from hyperglycemia-driven oxidative stress amplification and chronic inflammation, leading to periodontal tissue breakdown. Misregulated forkhead box protein M1 (FOXM1) play key roles in this process, exacerbating both inflammation and oxidative stress. In light of N-Acetylcysteine (NAC)'s potent anti-oxidative capacity and anti-inflammatory potential, understanding how it modulates these central pathways to alleviate DP holds high scientific and clinical importance. An animal model of diabetic mice periodontitis was established, and the model mice were injected with FOXM 1 adenovirus to enrich FOXM 1, and the periodontal pathological histology of each group was evaluated by HE staining. Western blotting and RT-PCR evaluated the expression levels of factors involved in bone destruction. ELISA evaluated the amount of inflammatory factors in mice serum. FOXM 1 over-expression and NAC were treated in murine macrophages, and the intracellular reactive oxygen species(ROS) levels in macrophages were measured using a DCFH-DA probe. Receptor activator of NF-κB ligand (RANKL) and lipopolysaccharide (LPS) were used to establish the macrophage osteoclast differentiation model and test the expression level of osteoclast differentiation factors after giving NAC. Hydrogen peroxide was used to establish a peroxidation environment, the plasmid silenced C-JUN, and the DNA binding activity of activating protein-1(AP1) was detected by EMSA. The effect of peroxidation on the osteoclast differentiation level was determined by WB. Mice with DP model had epithelial damage and inflammatory infiltration in periodontal tissues, and in the FOXM1 enriched group, the periodontal epithelial damage was repaired and inflammation was alleviated. FOXM1 enrichment resulted in DP model lower expression of RANKL (P < 0.01), macrophage colony-stimulating factor (M-CSF) (P < 0.01) and elevated expression of osteoprotegerin (OPG) (P < 0.001). Serum levels of pro-inflammatory factors interleukin (IL)-1β, tumor necrosis factor (TNF-α), and inducible nitric oxide synthase (iNOS) were elevated in DP mice (P < 0.001), and anti-inflammatory factor IL-10 was reduced(P < 0.001),, and FOXM1 enrichment significantly reversed inflammatory factor levels (P < 0.01). Overexpression of FOXM1 reduced ROS content in macrophages (P < 0.001), and NAC was performed to further reduce ROS content (P < 0.01). Silencing of FOXM1 elevated the expression of osteoclast-specific genes NFATc1, TRAP and OSCAR (P < 0.01), and the addition of NAC on top of silencing of FOXM1 markedly suppressed the expression level of osteoclast-specific genes (P < 0.01). ROS increased the transcriptional activity of AP1 (P < 0.001), which promoted osteoclast-specific gene expression (P < 0.001), and osteoclast-specific gene expression was decreased after silencing C-JUN (P < 0.01). FOXM1 relieve diabetic periodontitis inflammation and promote bone formation, regulates ROS production and ROS increases the transcriptional activity of AP1 and affects the osteoclastic differentiation of macrophages, which plays a positive role in bone protection in diabetic periodontitis.

SON-1010: an albumin-binding IL-12 fusion protein that improves cytokine half-life, targets tumors, and enhances therapeutic efficacy.

Cytokines have been promising cancer immunotherapeutics for decades, yet only two are licensed to date. Interleukin-12 (IL-12) is a potent regulator of cell-mediated immunity that activates NK cells and interferon-γ (IFNγ) production. It plays a central role in multiple pathways that can enhance cancer cell death and modify the tumor microenvironment (TME). Attempts to dose rIL-12 were initially successful but IFNγ toxicity in Phase 2 complicated further development in the late 1990s. Since then, better dosing strategies have been developed, but none have achieved the level of cancer control seen in preclinical models. We set out to develop a novel strategy to deliver fully functional IL-12 and other biologics to the TME by binding albumin, taking advantage of its ability to be concentrated and retained in the tumor. Single-chain variable fragments (scFv) were identified from a human phage display library that bound human, mouse, and cynomolgus macaque serum albumin, both at physiologic and acidic conditions. These were taken through a series of steps to identify strongly binding molecules that don't interfere with the normal physiology of albumin to bind FcRn, giving it prolonged half-life in serum, along with SPARC/GP60, which allows albumin to target the TME. A final molecule was chosen and a single mutation was made that minimizes the potential for immunogenicity. This fully human albumin-binding (FHAB®) domain was characterized and manufacturing processes were developed to bring the first drug candidate into the clinic. Once identified, the murine form of mIL12-FHAB was studied preclinically to understand its mechanism of action and biodistribution. It was found to be much more efficient at blocking tumor growth compared to murine IL-12, while stimulating significant IFNγ production with minimal toxicity. SON-1010, which uses the human IL-12 sequence, passed through all of the characterization and required toxicology and is currently being studied in the clinic. We identified and developed a platform technology with prolonged half-life that can target IL-12 and other immune modulators to the TME. Safety and efficacy are being studied using SON-1010 as monotherapy and in combination with checkpoint blockade strategies.

Tanshinone Ⅱ_A exerts anti-hepatocellular carcinoma effects by inhibiting oxidative stress via PI3K/Akt and Nrf2/HO-1 signaling pathway

This study aims to investigate the mechanism of tanshinone Ⅱ_A(Tan Ⅱ_A) in protecting mice from diethylinitrosamine(DEN)/carbon tetrachloride(CCl_4)/ethanol(C_2H_5OH)-induced hepatocellular carcinoma(HCC) and HepG2 cells from hydrogen peroxide(H_2O_2)-induced oxidative damage via the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt) and nuclear factor E2-related factor 2(Nrf2)/heme oxygenase 1(HO-1) signaling pathways. Sixty male C57BL/6J mice were grouped as follows: control, model, low, medium, and high-dose(10, 20, 40 mg·kg~(-1), respectively) Tan Ⅱ_A, and colchicine(0.2 mg·kg~(-1)), with 10 mice in each group. The HCC model was established with DEN/CCl_4/C_2H_5OH for 20 weeks, and the mice were then euthanized for collection of blood and liver specimens. A protein-protein interaction(PPI) network of the targets of Tan Ⅱ_A in the prevention of HCC was constructed. HepG2 cells were treated with 150 μmol·L~(-1) H_2O_2 for the modeling of oxidative stress. The cell counting kit-8(CCK-8) was used to assess the effects of different concentrations(1, 2, 4 μmol·L~(-1)) of Tan Ⅱ_A on the relative viability of cells. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in the liver tissue, and an automatic biochemical analyzer was used to measure the levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) in the mice serum. Relevant assay kits were used to measure the levels of reactive oxygen species(ROS), malondialdehyde(MDA), superoxide dismutase(SOD), glutathione(GSH), and glutathione peroxidase(GSH-Px) in the liver and cells. Immunohistochemistry, immunofluorescence assay, and Western blot were employed to determine the expression of phosphorylated PI3K(p-PI3K), PI3K, phosphorylated Akt(p-Akt), Akt, Nrf2, and HO-1. Compared with the control group, the model group exhibited typical pathological manifestations of liver cancer in the liver tissue, with elevated levels of ALT and AST in the serum, risen levels of ROS and MDA in the liver, and lowered levels of SOD and GSH in the liver. Compared with the control group, the HepG2 cells treated with H_2O_2 showed significantly decreased activities of SOD and GSH-Px and increased ROS and MDA levels. In the liver tissue and HepG2 cells, the modeling up-regulated the expression of p-PI3K and p-Akt while down-regulating the expression of Nrf2 and HO-1. Compared with the model group, different doses of Tan Ⅱ_A reduced the levels of ALT and AST in the serum, elevated the levels of SOD, GSH, and GSH-Px in the liver tissue and cells, and lowered the ROS and MDA levels, which indicated significant alleviation of oxidative stress. The PPI network showed that Akt was a core target of the PI3K/Akt and Nrf2/HO-1 signaling pathways. Western blot, immunohistochemistry, and immunofluorescence assay results indicated that Tan Ⅱ_A promoted the expression of Nrf2 and HO-1 while inhibiting the phosphorylation of PI3K and Akt. In conclusion, Tan Ⅱ_A may delay the progression of HCC by inhibiting oxidative stress via the PI3K/Akt and Nrf2/HO-1 signaling pathways.