The Acetic Acid Produced by Lactobacillus Species Regulates Immune Function to Alleviate PEDV Infection in Piglets.

Porcine epidemic diarrhea virus (PEDV) infection results in significant mortality among newborn piglets, leading to substantial economic setbacks in the pig industry. Short-chain fatty acids (SCFA), the metabolites of intestinal probiotics, play pivotal roles in modulating intestinal function, enhancing the intestinal barrier, and bolstering immune responses through diverse mechanisms. The protective potential of Lactobacillus delbrueckii, Lactobacillus johnsonii, and Lactococcus lactis was first noted when administered to PEDV-infected piglets. Histological evaluations, combined with immunofluorescence studies, indicated that piglets receiving L. lactis displayed less intestinal damage, with diminished epithelial cell necrosis and milder injury levels. Differences in immunofluorescence intensity revealed a significant disparity in antigen content between the L. lactis and PEDV groups, suggesting that L. lactis might suppress PEDV replication, the intestine. We then assessed short-chain fatty acid content through targeted metabolomics, finding that acetate levels markedly varied from other groups. This protective impact was confirmed by administering acetate to PEDV-infected piglets. Data suggested that piglets receiving acetate exhibited resistance to PEDV. Flow cytometry analyses were conducted to evaluate the expression of innate and adaptive immune cells in piglets. Sodium acetate appeared to bolster innate immune defenses against PEDV, marked by elevated NK cell and macrophage counts in mesenteric lymph nodes, along with increased NK cells in the spleen and macrophages in the bloodstream. Acetic acid was also found to enhance the populations of CD8+ IFN-γ T cells in the blood, spleen, and mesenteric lymph, CD4+ IFN-γ T cells in mesenteric lymph nodes and spleen, and CD4+ IL-4+T cells in the bloodstream. Transcriptome analyses were carried out on the jejunal mucosa from piglets with PEDV-induced intestinal damage and from healthy counterparts with intact barriers. Through bioinformatics analysis, we pinpointed 189 significantly upregulated genes and 333 downregulated ones, with the PI3K-AKT, ECM-receptor interaction, and pancreatic secretion pathways being notably enriched. This transcriptomic evidence was further corroborated by western blot and qPCR. Short-chain fatty acids (SCFA) were found to modulate G protein-coupled receptor 41 (GPR41) and 43 (GPR43) in porcine intestinal epithelial cells (IPEC-J2). Post-acetic acid exposure, there was a notable upsurge in the ZO-1 barrier protein expression in IPEC-J2 compared to the unexposed control group (WT), while GPR43 knockdown inversely affected ZO-1 expression. Acetic acid amplified the concentrations of phosphorylated PI3K and AKT pivotal components of the PI3K/AKT pathway. Concurrently, the co-administration of AKT agonist SC79 and PI3K inhibitor LY294002 revealed acetic acid's role in augmenting ZO-1 expression via the P13K/AKT signaling pathway. This study demonstrates that acetic acid produced by Lactobacillus strains regulates intestinal barrier and immune functions to alleviate PEDV infection. These findings provide valuable insights for mitigating the impact of PEDV in the pig industry.

[18F]AlF-ND-bisFAPI PET imaging of fibroblast activation protein as a biomarker to monitor the progression of liver fibrosis.

BACKGROUND: Hepatic fibrosis is a progressive disease, which is reversible in the early stages. The current monitoring methods have notable limitations that pose a challenge to early detection. In this study, we evaluated the utility of [18F]AlF-ND-bisFAPI positron emission tomography imaging of fibroblast activation protein (FAP) to monitor the progression of liver fibrosis. METHODS: Two mouse models of liver fibrosis were established by bile duct ligation and carbon tetrachloride administration, respectively. Positron emission tomography imaging was performed with the FAP-specific radiotracer [18F]AlF-ND-bisFAPI for the evaluation of rat HSCs and mouse models of fibrosis and combined with histopathology, immunohistochemical staining, and immunoblotting to elucidate the relationships among radioactivity uptake, FAP levels, and liver fibrosis progression. Furthermore, [18F]AlF-ND-bisFAPI autoradiography was performed to assess tracer binding in liver sections from patients with varying degrees of liver fibrosis. RESULTS: Cell experiments demonstrated that [18F]AlF-ND-bisFAPI uptake was specific in activated HSCs. Compared with control mice, [18F]AlF-ND-bisFAPI uptake in livers increased in the early stages of fibrosis and increased significantly further with disease progression. Immunohistochemistry and western blot analyses demonstrated that FAP expression increased with fibrosis severity. In accordance with the findings in animal models, ex vivo autoradiography on human fibrotic liver sections showed that radioactivity increased as fibrosis progressed from mild to severe. CONCLUSIONS: [18F]AlF-ND-bisFAPI positron emission tomography imaging is a promising noninvasive method for monitoring the progression of liver fibrosis.

A Turn-On Fluorescent Probe for Highly Selective Detection and Visualization of Hydrogen Sulfide in Fungi.

Hydrogen sulfide (H2S) is a significant actor in the virulence and pathogenicity of fungi. The analysis of endogenous H2S in fungi benefits the prevention and treatment of pathogenic infections. Herein, a H2S-activated turn-on fluorescent probe named DDX-DNP was developed for the sensitive and selective detection of H2S. Using DDX-DNP, the ability of several oral fungi strains to produce H2S was identified, which was also validated using a typical chromogenic medium. In addition, DDX-DNP was successfully used for the visual sensing of endogenous H2S in fungal cells via microscope, flow cytometry, and colony imaging, along with a specific validation with the co-incubation of H2S production inhibitors in living cells. Above all, DDX-DNP could be used for H2S detection, the fluorescent imaging of fungi, and even the identification of related fungi.

Gut microbiota-derived LCA mediates the protective effect of PEDV infection in piglets.

BACKGROUND: The gut microbiota is a critical factor in the regulation of host health, but the relationship between the differential resistance of hosts to pathogens and the interaction of gut microbes is not yet clear. Herein, we investigated the potential correlation between the gut microbiota of piglets and their disease resistance using single-cell transcriptomics, 16S amplicon sequencing, metagenomics, and untargeted metabolomics. RESULTS: Porcine epidemic diarrhea virus (PEDV) infection leads to significant changes in the gut microbiota of piglets. Notably, Landrace pigs lose their resistance quickly after being infected with PEDV, but transplanting the fecal microbiota of Min pigs to Landrace pigs alleviated the infection status. Macrogenomic and animal protection models identified Lactobacillus reuteri and Lactobacillus amylovorus in the gut microbiota as playing an anti-infective role. Moreover, metabolomic screening of the secondary bile acids' deoxycholic acid (DCA) and lithocholic acid (LCA) correlated significantly with Lactobacillus reuteri and Lactobacillus amylovorus, but only LCA exerted a protective function in the animal model. In addition, LCA supplementation altered the distribution of intestinal T-cell populations and resulted in significantly enriched CD8+ CTLs, and in vivo and in vitro experiments showed that LCA increased SLA-I expression in porcine intestinal epithelial cells via FXR receptors, thereby recruiting CD8+ CTLs to exert antiviral effects. CONCLUSIONS: Overall, our findings indicate that the diversity of gut microbiota influences the development of the disease, and manipulating Lactobacillus reuteri and Lactobacillus amylovorus, as well as LCA, represents a promising strategy to improve PEDV infection in piglets. Video Abstract.

Synthesis and preclinical evaluation of a heterodimeric radioligand targeting fibroblast activation protein and integrin-αvß3.

Tumor progression is accompanied by intrinsic heterogeneity and different phenotypes, which implies a different expression of cell surface receptors. Fibroblast activation protein (FAP) and integrin αvß3 are highly expressed in the cell surface of cancer-associated cells or cancer cells compared with normal cells. Therefore, a FAP/integrin αvß3 bispecific heterodimer was developed for positron emission tomography (PET) diagnostic imaging and radiotherapy. The heterodimer DOTA-FAPI-RGD was labeled with the diagnostic radionuclide gallium-68 or the therapeutic radionuclide lutetium-177, with yields >80%, and high stability. The competitive displacement binding assay showed an IC50 = 6.8 ± 0.6 nM for DOTA-FAPI-RGD towards FAP and IC50 = 2.1 ± 0.4 nM towards integrin αvß3. Radionuclide labeled DOTA-FAPI-RGD showed high specificity and rapid internalization into U87MG cells (FAP/αvß3-positive) in vitro. Micro-PET and biodistribution studies of [68Ga]Ga-DOTA-FAPI-RGD in tumor-bearing mice demonstrated that a high and specific tumor uptake of the tracer and a fast body clearance, resulting in high contrast images. In addition to the imaging applications demonstrated in this study, the labeling of the heterodimeric ligand with the radionuclide lutetium-177 used in cancer treatment might allow the therapeutic application of this ligand.

18F- or 177Lu-labeled bivalent ligand of fibroblast activation protein with high tumor uptake and retention.

PURPOSE: Fibroblast activation protein (FAP) has become a promising cancer-related target for diagnosis and therapy. The aim of this study was to develop a bivalent FAP ligand for both diagnostic PET imaging and endoradiotherapy. METHODS: We synthesized a bivalent FAP ligand (ND-bisFAP) and labeled it with 18F or 177Lu. FAP-positive A549-FAP cells were used to study competitive binding to FAP, cellular internalization, and efflux properties in vitro. Micro-PET imaging with [18F]AlF-ND-bisFAPI was conducted in mice bearing A549-FAP or U87MG tumors. Biodistribution and therapeutic efficacy of [177Lu]Lu-ND-bisFAPI were conducted in mice bearing A549-FAP tumors. RESULTS: The FAP binding affinity of ND-bisFAPI is 0.25 ± 0.05 nM, eightfold higher in potency than the monomeric DOTA-FAPI-04 (IC50 = 2.0 ± 0.18 nM). In A549-FAP cells, ND-bisFAPI showed specific uptake, a high internalized fraction, and slow cellular efflux. Compared to the monomeric [18F]AlF-FAPI-42, micro-PET imaging with [18F]AlF-ND-bisFAPI showed higher specific tumor uptake and retention for at least 6 h. Biodistribution studies showed that [177Lu]Lu-ND-bisFAPI had higher tumor uptake than [177Lu]Lu-FAPI-04 at the 24, 72, 120, and 168 h time points (all P < 0.01). [177Lu]Lu-ND-bisFAPI delivered fourfold higher radiation than [177Lu]Lu-FAPI-04 to A549-FAP tumors. For the endoradiotherapy study, 37 MBq of [177Lu]Lu-ND-bisFAPI significantly reduced tumor growth compared to the same dose of [177Lu]Lu-FAPI-04. Half of the dose of [177Lu]Lu-ND-bisFAPI (18.5 MBq) has comparable median survival as 37 MBq of [177Lu]Lu-FAPI-04 (37 vs 36 days). CONCLUSION: The novel bivalent FAP ligand was developed as a theranostic radiopharmaceutical and showed promising properties including higher tumor uptake and retention compared to the established radioligands [18F]AlF-FAPI-42 and [177Lu]Lu-FAPI-04. Preliminary experiments with 18F- or 177Lu-labeled ND-bisFAPI showed promising imaging properties and favorable anti-tumor responses.

Radiosynthesis and Preclinical Evaluation of Bispecific PSMA/FAP Heterodimers for Tumor Imaging.

Due to tumor heterogeneity and complex tumor-stromal interactions in multicellular systems, the efficiency of monospecific tracers for tumor diagnosis and therapy is currently limited. In light of the evidence of prostate-specific membrane antigen (PSMA) overexpression in tumor cells and fibroblast activation protein (FAP) upregulation in the tumor stroma, heterodimer dual targeting PSMA and FAP may have the potential to improve tumor diagnosis. Herein, we described the radiosynthesis, in vitro characterization, and micro-PET/CT imaging of two novel 18F-labeled bispecific PSMA/FAP heterodimers. 18F-labeled heterodimers showed high specificity and affinity targeting to PSMA and FAP in vitro and in vivo. Compared with the monospecific tracers [18F]AlF-PSMA-BCH and [18F]FAPI-42, both 18F-labeled heterodimers exhibited better tumor uptake in tumor-bearing mice. Their favorable characterizations such as convenient synthesis, high tumor uptake, and favorable pharmacokinetic profile could lead to their future applications as bispecific radiotracers for clinical cancer imaging.

Radiosynthesis and biological evaluation of 18F-labeled bispecific heterodimer targeted dual gastrin-releasing peptide receptor and prostate-specific membrane antigen for prostate cancer imaging.

OBJECTIVE: Approximately 5% of prostatic primary tumors and 15% of metastatic tumors were found to be prostate-specific membrane antigen (PSMA)-negative. Targeting gastrin-releasing peptide receptor (GRPR) has been shown to complement patients with PSMA-negative prostate cancer (PCa). Based on previous findings, simultaneously targeting PSMA and GRPR imaging may improve the diagnosis of PCa. In this study, we report the radiosynthesis and biological evaluation of a bispecific heterodimer of NOTA-GRPR-PSMA that targeted both PSMA and GRPR for extended PCa imaging. METHODS: NOTA-GRPR-PSMA was labeled using the Al18F-chelating one-step method. The competitive combination experiment and specific binding assay were performed in vitro using 22Rv1 (PSMA+) and PC-3 (GRPR+) cells. To determine the distribution and specificity in vivo, biodistribution and micro-PET/computed tomography of [18F]AlF-GRPR-PSMA were performed on mice bearing 22Rv1 or PC-3 tumors. RESULTS: [18F]AlF-GRPR-PSMA had a radiochemical purity of over 98% and demonstrated high stability in vivo and in vitro, with a LogD of -1.2 ± 0.05. Cell uptake and inhibition studies of [18F]AlF-GRPR-PSMA in 22Rv1 and PC-3 cells revealed bispecific GRPR and PSMA bindings. According to the biodistribution study and PET imaging, [18F]AlF-GRPR-PSMA was mainly excreted through the kidney. Tumor uptake was high in 22Rv1 tumor (10.1 ± 0.4 %ID/g) and moderate in PC-3 tumor (2.1 ± 0.6 %ID/g) 2 h p.i., whereas blocking studies significantly decreased the tumor uptake of 22Rv1 and PC-3. CONCLUSION: [18F]AlF-GRPR-PSMA has the potential to simultaneously target PSMA and GRPR for PCa imaging.

Optimizing the Microstructure and Mechanical Properties of Vacuum Counter-Pressure Casting ZL114A Aluminum Alloy via Ultrasonic Treatment.

The effect of ultrasonic temperature on density, microstructure and mechanical properties of vacuum counter-pressure casting ZL114A alloy during solidification was investigated by optical microscopy (OM), scanning electron microscope (SEM) and a tensile test. The results show that compared with the traditional vacuum counter-pressure casting aluminum alloy, the primary phase and eutectic silicon of the alloy with ultrasonic treatment has been greatly refined due to the dendrites broken by ultrasonic vibration. However, the refining effect of ultrasonic treatment on vacuum counter-pressure casting aluminum alloy will be significantly affected by ultrasonic temperature. When the ultrasonic temperature increases from 680 °C to 720 °C, the primary phase is gradually refined, and the morphology of eutectic silicon also changes from coarse needle-like flakes to fine short rods. With a further increase in the ultrasonic temperature, the microstructure will coarse again. The tensile strength and elongation of vacuum counter-pressure casting ZL114A alloy increases first and then decreases with the increase of ultrasonic temperature. The optimal mechanical properties were achieved with tensile strength of 327 MPa and the elongation of 5.57% at ultrasonic temperature of 720 °C, which is 6.3% and 8.2%, respectively, higher than that of alloy without ultrasonic treatment.

Effects of Continuous and Pulsed Ultrasonic Treatment on Microstructure and Microhardness in Different Vertical Depth of ZL205A Castings.

In this paper, in order to improve the performance of the ZL205A castings, continuous ultrasonic and pulsed ultrasonic treatments were applied to the melted alloy to study the effect of ultrasound propagation distance on microstructure and microhardness. The results indicated that ZL205A grains were significantly refined by ultrasonic vibration, but the refinement effect became weak gradually with the increase of sampling depth. The minimum grain sizes were 103.2 µm and 122.5 µm respectively in continuous and pulsed ultrasonic treatment. Grain boundary segregation also became more serious and coarser with the increase of vertical depth. In addition, microhardness and vertical depth are not positively correlated linearly. As the vertical depth increased, microhardness first decreased and then increased, the maximum hardnesses were 73.9 HV and 72.84 HV, respectively, in the two process modes. According to the experiment results and available studies, the mechanism of ultrasonic treatment maybe that: the cooling rate of solidification interface front increased by cavitation and streaming, thus changing the solute redistribution behavior of the ZL205A melt.

Effect of Ceramic and Nylon Fiber Content on Composite Silica Sol Slurry Properties and Bending Strength of Investment Casting Shell.

In order to improve the performance of the investment casting shell, ceramic and nylon fiber was added to the silica sol slurry to study the effect of ceramic and nylon fiber on the liquidity of the silica sol slurry and the bending strength of the investment casting shell. Meanwhile, the fracture surface of shell sample was observed by SEM. The experiment results indicate that the movement viscosity of slurry increases with ceramic and nylon fiber content, increasing from 0 to 0.75 wt.%. The movement viscosity of ceramic fiber composite slurry is higher than nylon fiber composite slurry. The wet and high strength of shell firstly increases then decreases, with an increase of ceramic and nylon fiber content. When the ceramic and nylon fibers' contents are 0.6 wt.%, the maximum wet strengths are 3.56 MPa and 3.84 Mpa respectively, increasing by approximately 38% and 43%. Moreover, the high strength of shell achieves its highest value, 5.08 Mpa, when the ceramic fiber content is 0.6 wt.%; however, when the nylon fiber content was more than 0.3%, the high strength of the nylon fiber reinforced shell was decreased sharply. Therefore, an addition of ceramic and nylon fiber to silica sol slurry distinctly influences the wet and high strength of investment casting shell.

Ratiometric fluorescent probe for sensing Streptococcus mutans glucosyltransferase, a key factor in the formation of dental caries.

We report on a naphthalimide ratiometric fluorescent probe for the real-time sensing and imaging of pathogenic bacterial glucosyltransferases, which are associated with the development of dental caries. Using a high-throughput screening method, we identified that several natural polyphenols from green tea were GTFs inhibitors that could eventually lead to suitable oral treatments to prevent the development of dental caries.

A far-red fluorescent probe for sensing laccase in fungi and its application in developing an effective biocatalyst for the biosynthesis of antituberculous dicoumarin.

A far-red fluorescent probe has been developed for sensing fungal laccase. The probe was used to determine that Rhizopus oryzae had a high level endogenous laccase amongst 24 fungal strains. The Rhizopus oryzae was then used as a biocatalyst for the preparation of dicoumarin resulting in significant inhibition of Mycobacterium tuberculosis H37Ra.

Ebracpenes A and B, Unusual Ring C- seco and Ring D-aromatic Nor-Triterpenoids, from Euphorbia ebracteolata and Lipase Inhibitory Evaluation.

Two unusual ring C- seco and ring D-aromatic nor-triterpenoids, ebracpenes A (1) and B (2), were obtained from Euphorbia ebracteolata roots and elucidated by widely spectroscopic data analyses, such as 1D, 2D NMR, HRESIMS, and X-ray crystallography analysis. Compound 2 showed a significant inhibitory activity on lipase (IC50 = 0.89 µM as well as the Ki determined to be 0.69 µM). Enzyme kinetics analysis and molecular docking experiments revealed that compound 2 was a competitive inhibitor, binding to the active site of lipase.

Biocatalytic oxidation of flavone analogues mediated by general biocatalysts: horseradish peroxidase and laccase.

Horseradish peroxidase (HRP) and laccase are well known oxidases, which have been widely applied for the biosynthesis of organic compounds. In the present work, flavone analogues as an important type of bioactive natural product could be oxidized by HRP or laccase, which afforded dimeric and oxidative flavones. All of the flavone analogues usually possessing phenolic groups could be transformed using HRP. However, only flavonols, isoflavones and chalcones with phenolic groups and dihydroxylflavones were effective substrates of laccase. The radical reaction mechanism with the B-ring of flavone analogues as the radical reaction trigger was proposed for the oxidation of flavones. In silico molecular docking analyses for assaying the interaction between flavone analogues and oxidases indicated that the phenolic groups at the B rings of flavones docked into the HEME active pocket of HRP well. Kinetic behaviors of the oxidation for various flavone analogues mediated by HRP or laccase displayed Hill and substrate inhibition kinetic models. Therefore, in the present work, the oxidation of various flavone analogues mediated by HRP or laccase has been successfully characterized, which would be helpful for the preparation of flavone derivatives.

α-Pyrones, secondary metabolites from fungus Cephalotrichum microsporum and their bioactivities.

Cephalotrichum microsporum (SYP-F 7763) was a fungus isolated from the rhizosphere soil of traditional Chinese medicine Panax notoginseng. The EtOAc extract of Cephalotrichum microsporum cultivated on sterilized moistened-rice medium was separated by various chromatographic techniques, which yielded 11 metabolites (1-11) of this fungus. On the basis of the widely spectroscopic data, the chemical structures of isolated metabolites were determined, most of which were α-pyrones, including 5 compounds (4-7, and 10) unreported. In the anti-bacterial bioassay, compound 1 displayed significant inhibitory effects on three pathogenic bacteria, MR S. aureus, S. aureus, and B. cereus. α-Pyrones 2, 3, and 5-7 also displayed moderate inhibitory effects on MR S. aureus, S. aureus, and B. subtilis, which could be the major anti-bacterial constituents of Cephalotrichum microsporum. Additionally, compounds 1, 4, and 5 displayed significant cytotoxicity on five human cancer cell lines, with the IC50 values < 20 µM, which are more effective than positive control 5-fluorouracil. Therefore, α-pyrones were important secondary metabolites of Cephalotrichum microsporum, which displayed anti-bacterial and anti-tumor activities.

Diterpenoids from the roots of Euphorbia ebracteolata and their anti-tuberculosis effects.

Euphorbia ebracteolata was a natural medicine for the treatment of tuberculosis. The present work has performed the investigation of bioactive chemical substances from the roots of E. ebracteolata. Using various chromatographic techniques, 15 compounds were obtained from the roots of E. ebracteolata. On the basis of widely spectroscopic data analyses, the isolated compounds were determined to be diterpenoids, including rosane derivatives (1-12), isopimarane (13), abietane (14), and lathyrane (15), among which compounds 1-4, and 9 were undescribed previously. The inhibitory effects of isolated diterpenoids against Mycobacterium tuberculosis were evaluated using an Alamar blue cell viability assay. And two rosane-type diterpenoids 3 and 8 displayed moderate inhibitory effects on with the MIC values of 18 µg/mL and 25 µg/mL, respectively. For the potential inhibitor 3, the inhibitory effect against the target enzyme GlmU was evaluated, which displayed a moderate inhibitory effect with the IC50 12.5 µg/mL. Therefore, the diterpenoids from the roots of E. ebracteolata displayed anti-tuberculosis effects, which would be pay more attentions for the anti-tuberculosis agents.