CRISPR-Hg: Rapid and visual detection of Hg2+ based on PCR coupled with CRISPR/Cas12a.

Mercury (Hg) is a notorious toxic heavy metal, causing neurotoxicity and liver damage, posing grave threats to human health and environmental safety. There is an urgent imperative for developing novel Hg2+ detection methods. In this work, we developed a CRISPR-based method for Hg2+ detection named CRISPR-Hg. A CRISPR/Cas12a system was employed and could be activated by the PCR product, generating fluorescence signals based on the trans-cleavage activity. CRISPR-Hg exhibited remarkable selectivity and specificity, achieving a detection limit of 10 pM and minimal interference with background signals. This approach has been successfully applied to detect Hg2+ in real samples, including water, soil, and mushroom. Ulteriorly, a portable device was devised to streamline the readout of fluorescence signals by a smartphone within 30 min. We offer an affordable, highly selective and visually interpretable method for Hg2+ detection, with the potential for broad application in Hg2+ monitoring for food safety and public health.

Label-free Colorimetric Detection of Viral RNA Based on Clustered Regularly Interspaced Short Palindromic Repeats and Gold Nanoparticles with a Portable Device.

Viral infections, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are some of the most dangerous threats to humans. SARS-CoV-2 has caused a global pandemic, highlighting the unprecedented demand for rapid and portable diagnostic methods. To meet these requirements, we designed a label-free colorimetric platform that combines the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas) 12a system for naked-eye detection (named LFP). This method utilizes reverse transcription loop-mediated isothermal amplification (RT-LAMP) and the trans-cleavage activity of the CRISPR/Cas12a system to increase the sensitivity and specificity of the reaction. This platform can detect as few as 4 copies/µL of RNA and produces no false positive results when tested against the influenza virus. To better meet the requirements of point-of-care (POC) detection, we developed a portable device that can be applied in resource-poor and densely populated regions. The LFP assay holds great potential for application in resource-limited settings, and the label-free gold nanoparticle (AuNPs) probe can reduce costs, making it suitable for large-scale screening. We expect that the LFP assay will be promising for the POC screening of COVID-19.

The amelioration of a purified Pleurotus abieticola polysaccharide on atherosclerosis in ApoE-/- mice.

In this study, a polysaccharide known as PAPS2 was eluted from Pleurotus abieticola fruiting bodies using 0.1 M NaCl solutions. PAPS2 has a Mw of 19.64 kDa and its backbone is mainly composed of →6)-α-D-Galp-(1→, →6)-ß-D-Glcp-(1→ and →2,6)-α-D-Galp-(1→ residues, and its branches mainly end with ß-D-Manp-(1→, which is attached at C2 of →2,6)-α-D-Galp-(1→. PAPS2 elicited several effects in high-fat diet (HFD)-fed ApoE-/- mice. It significantly reduced the body weight, liver index, and serum levels of total cholesterol (TC) and triglycerides (TGs), and it alleviated lipid accumulation in the aorta. Intestinal microflora analysis showed that PAPS2 suppressed the abundances of Adlercreutzia, Turicibacter, and Helicobacter and enriched that of Roseburia. It also influenced lipid metabolism, suggesting that it reduced the levels of TGs, lysophosphatidylcholine (LPC), phosphatidylcholine (PC), and ceramide (Cer). Moreover, it suppressed oxidative response by increasing nuclear factor erythroid 2 (Nrf2)-related factor expression and activating the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) to reduce the level of reactive oxygen species (ROS). Meanwhile, it showed anti-inflammatory effects partially related to the inhibition of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling induced by lipopolysaccharide (LPS) in RAW 264.7 cells, as well as in the aorta of HFD-fed ApoE-/- mice. This study provides experimental evidence of the auxiliary applicability of PAPS2 in atherosclerosis treatment.

Exosomes as a modulator of immune resistance in human cancers.

In the tumor microenvironment (TME), exosomes secreted by cells form interactive networks between the tumor cells and immune cells, thereby regulating immune signaling cascades in the TME. As key messengers of cell-to-cell communication in the TME, exosomes not only take charge of tumor cell antigen presentation to the immune cells, but also regulate the activities of immune cells, inhibit immune function, and, especially, promote immune resistance, all of which affects the therapeutic outcomes of tumors. Exosomes, which are small-sized vesicles, possess some remarkable advantages, including strong biological activity, a lack of immunogenicity and toxicity, and a strong targeting ability. Based on these characteristics, research on exosomes as biomarkers or carriers of tumor therapeutic drugs has become a research hotspot in related fields. This review describes the role of exosomes in cell communications in the TME, summarizes the effectiveness of exosome-based immunotherapy in overcoming immune resistance in cancer treatment, and systematically summarizes and discusses the characteristics of exosomes from different cell sources. Furthermore, the prospects and challenges of exosome-related therapies are discussed.

A Cas12a-based platform combined with gold nanoparticles for sensitive and visual detection of Alternaria solani.

Alternaria solani (A. solani), the causal agent of early blight in potatoes, poses a serious and persistent threat to potato production worldwide. Therefore, developing a method that can accurately detect A. solani in the early stage to avoid further spread is urgent. However, the conventional PCR-based method is not appropriate for application in the fields. Recently, the CRISPR-Cas system has been developed for nucleic acids analysis at point-of-care. Here, we propose a gold nanoparticles-based visual assay combining loop-mediated isothermal amplification with CRISPR-Cas12a to detect A. solani. After optimization, the method could detect 10-3 ng/µL genomic gene of A. solani. The specificity of the method was confirmed by discriminating A. solani from other three highly homologous pathogens. We also developed a portable device that could be used in the fields. By integrating with the smartphone readout, this platform holds significant potential in high-throughput detection of multiple pathogens in the fields.

Study of anti-fatigue activity of polysaccharide from fruiting bodies of Armillaria gallica.

Fatigue is a common physiological response that is closely related to energy metabolism. Polysaccharides, as excellent dietary supplements, have been proven to have a variety of pharmacological activities. In this study, A 23.007 kDa polysaccharide from Armillaria gallica (AGP) was purified and performed structural characterization, including analysis of homogeneity, molecular weight and monosaccharide composition. Methylation analysis is used to analyze the glycosidic bond composition of AGP. The mouse model of acute fatigue was used to evaluate the anti-fatigue effect of AGP. AGP-treatment improved exercise endurance in mice and reduced fatigue symptoms caused by acute exercise. AGP regulated the levels of adenosine triphosphate, lactic acid, blood urea nitrogen and lactate dehydrogenase, muscle glycogen and liver glycogen of acute fatigue mice. AGP affected the composition of intestinal microbiota, the changes of some intestinal microorganisms are correlated with fatigue and oxidative stress indicators. Meanwhile, AGP reduced oxidative stress levels, increased antioxidant enzyme activity and regulated the AMP-dependent protein kinase/nuclear factor erythroid 2-related factor 2 signaling pathway. AGP exerted an anti-fatigue effect through modulation of oxidative stress, which is related to intestinal microbiota.

Anti-Obesity Effect of Auricularia delicate Involves Intestinal-Microbiota-Mediated Oxidative Stress Regulation in High-Fat-Diet-Fed Mice.

Auricularia delicate (ADe), an edible fungus belonging to the family Auriculariaceae and order Auriculariales, possesses antimicrobial, hepatoprotective, and antioxidant effects. In this study, after systematic analysis of its composition, ADe was administered to high-fat-diet (HFD)-fed mice to investigate its anti-obesity effect. ADe significantly controlled body weight; alleviated hepatic steatosis and adipocyte hypertrophy; reduced aspartate aminotransferase, total cholesterol, insulin, and resistin; and increased adiponectin levels in HFD-fed mice serum. Based on intestinal microbiota and lipidomics analysis, ADe treatment regulated the composition and abundance of 49 intestinal microorganisms and influenced the abundance of 8 lipid species compared with HFD-fed mice. Based on a correlation analysis of the intestinal microbiota and lipids, Coprococcus showed significant negative associations with ceramide (d18:0 20:0+O), phosphatidylserine (39:4), sphingomyelin (d38:4), and zymosterol (20:2). Moreover, ADe treatment decreased the levels of ROS and MDA and increased the levels of Nrf2, HO-1, and three antioxidant enzymes in HFD-fed mice livers. Collectively, the anti-obesity effect of ADe involves the regulation of oxidative stress and is mediated by the intestinal microbiota. Hence, this study provides a reference for the application of ADe as a candidate food for obesity.

The Structural Characterization and Immunomodulatory Activity of Polysaccharides from Pleurotus abieticola Fruiting Bodies.

Polysaccharides obtained from mushrooms have been reported to possess immunomodulatory properties. In this study, a water-soluble polysaccharide was purified from the fruiting bodies of Pleurotus abieticola, entitled PAPS1. After its composition and structural analysis, the immunomodulatory activity was investigated in immunosuppressed mice induced by cyclophosphamide (CTX) at a dosage of 70 mg/kg by intraperitoneal injection for 7 days. After 28 days of intragastric administration, PAPS1 alleviated cyclophosphamide (CTX)-induced histopathological damage and increased the expressions of splenic CD4, CD8, CD56 and IgM in the serums of immunosuppressed mice. PAPS1 suppressed the oxidative stress indicated by preventing the increases in ROS and MDA levels. According to the intestinal microflora analysis, PAPS1 regulated 11 bacteria at the gene level, including Helicobacter and Paraprevotella, which are related to immunity and oxidative capacity. Compared with CTX-treated mice, significant increases in immune-related cytokines, such as interleukin (IL)-2, IL-6 and IL-12 in the serums of mice treated with PAPS1, were observed. Finally, PAPS1 can strongly increase the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream proteins. In conclusion, PAPS1-boosted immunity may be related to its suppression on oxidative stress via enhancing the activity of Nrf2 signaling. Thus, PAPS1 can be investigated as a candidate for immunomodulatory therapy.

Calf Thymus Polypeptide Restrains the Growth of Colorectal Tumor via Regulating the Intestinal Microbiota-Mediated Immune Function.

Calf thymus polypeptide (CTP), with a molecular mass of <10 kDa, is prepared from the thymus of less than 30-day-old newborn cattle. In the present study, the inhibitory function of CTP in colorectal cancer (CRC) was investigated in B6/JGpt-Apc em1Cin(MinC) /Gpt (Apc Min/+) mice. CTP hampered tumor development and enhanced the ratio of CD3e-NK1.1+ cells by 113.0% and CD3e+CD28+ cells by 84.7% in the peripheral blood of Apc Min/+ mice. CTP improved the richness, diversity, and evenness of the intestinal microbiota of Apc Min/+ mice, particularly by regulating the abundance of immune-related microorganisms. CTP effectively regulated the expression of immune-related cytokines, such as interleukin (IL)-2 (15.19% increment), IL-12 (17.47% increment), and transforming growth factor (TGF)-ß (11.19% reduction). Additionally, it enhanced the levels of CD4 and CD8, as well as the ratio of helper T lymphocytes (Th)1/Th2 in the spleen and tumors of Apc Min/+ mice. In CTP-treated mice, reduced levels of programmed death-1 (PD-1), programmed cell death-ligand 1 (PD-L1), cytotoxic T lymphocyte-associated antigen 4 (CTLA4), activated nuclear factor of activated T cells 1 (NFAT1), and nuclear factor κB (NF-κB) p65 signaling were noted. Collectively, the anti-CRC effect of CTP is related to the modulation of intestinal microbiota-mediated immune function, which provides a reference for CTP as a therapeutic drug or a combination drug used in CRC treatment in a clinical setting.

Forsythoside B attenuates memory impairment and neuroinflammation via inhibition on NF-κB signaling in Alzheimer's disease.

BACKGROUND: Neuroinflammation is a principal element in Alzheimer's disease (AD) pathogenesis, so anti-inflammation may be a promising therapeutic strategy. Forsythoside B (FTS•B), a phenylethanoid glycoside isolated from Forsythiae fructus, has been reported to exert anti-inflammatory effects. However, no studies have reported whether the anti-inflammatory properties of FTS•B have a neuroprotective effect in AD. In the present study, these effects of FTS•B were investigated using amyloid precursor protein/presenilin 1 (APP/PS1) mice, BV-2 cells, and HT22 cells. METHODS: APP/PS1 mice were administered FTS•B intragastrically for 36 days. Behavioral tests were then carried out to examine cognitive functions, including the Morris water maze, Y maze, and open field experiment. Immunohistochemistry was used to analyze the deposition of amyloid-beta (Aß), the phosphorylation of tau protein, and the levels of 4-hydroxynonenal, glial fibrillary acidic protein, and ionized calcium-binding adapter molecule 1 in the hippocampus. Proteins that showed marked changes in levels related to neuroinflammation were identified using proteomics and verified using enzyme-linked immunosorbent assay and western blot. BV-2 and HT22 cells were also used to confirm the anti-neuroinflammatory effects of FTS•B. RESULTS: In APP/PS1 mice, FTS•B counteracted cognitive decline, ameliorated the deposition of Aß and the phosphorylation of tau protein, and attenuated the activation of microglia and astrocytes in the cortex and hippocampus. FTS•B affected vital signaling, particularly by decreasing the activation of JNK-interacting protein 3/C-Jun NH2-terminal kinase and suppressing WD-repeat and FYVE-domain-containing protein 1/toll-like receptor 3 (WDFY1/TLR3), further suppressing the activation of nuclear factor-κB (NF-κB) signaling. In BV-2 and HT22 cells, FTS•B prevented lipopolysaccharide-induced neuroinflammation and reduced the microglia-mediated neurotoxicity. CONCLUSIONS: FTS•B effectively counteracted cognitive decline by regulating neuroinflammation via NF-κB signaling in APP/PS1 mice, providing preliminary experimental evidence that FTS•B is a promising therapeutic agent in AD treatment.

Triterpenoids Extracted From Antrodia cinnamomea Mycelia Attenuate Acute Alcohol-Induced Liver Injury in C57BL/6 Mice via Suppression Inflammatory Response.

Excessive alcohol consumption causes liver injury-induced mortality. Here we systematically analyzed the structure of triterpenoids extracted from Antrodia cinnamomea mycelia (ACT) and investigated their protective effects against acute alcohol-induced liver injury in mice. Liquid chromatography-mass spectrometry and liquid chromatography with tandem mass spectrometry were performed to determine the structures of ACT constituents. Alcohol-induced liver injury was generated in C57BL/6 mice by oral gavage of 13 g/kg white spirit (a wine at 56% ABV). Mice were treated with either silibinin or ACT for 2 weeks. Liver injury markers and pathological signaling were then quantified with enzyme-linked immunosorbent assays, antibody array assays, and Western blots, and pathological examinations were performed using hematoxylin-eosin staining and periodic acid-Schiff staining. Triterpenoids extracted from A. cinnamomea mycelia contain 25 types of triterpenoid compounds. A 2-weeks alcohol consumption treatment caused significant weight loss, liver dyslipidemia, and elevation of alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, and alkaline phosphatase activities in the serum and/or liver. These effects were markedly reversed after 2-weeks ACT administration. Triterpenoids extracted from A. cinnamomea mycelia alleviated the organ structural changes and inflammatory infiltration of alcohol-damaged tissues. Triterpenoids extracted from A. cinnamomea mycelia inhibited proinflammatory cytokine levels and enhanced anti-inflammatory cytokine levels. Acute alcohol treatment promoted inflammation with significant correlations to hypoxia-inducible factor 1α (HIF-1α), which was reduced by ACT and was partially related to modulation of the protein kinase B (Akt)/70-kDa ribosomal protein S6 kinase phosphorylation (p70S6K) and Wnt/ß-catenin signaling pathways. In conclusion, ACT protected against acute alcohol-induced liver damage in mice mainly through its suppression of the inflammatory response, which may be related to HIF-1α signaling.

Possible mechanisms of the antimicrobial effects of polypeptide­enriched Gastrodia elata Blume extracts.

The present study aimed to evaluate the antimicrobial activity and the possible mechanisms of activity of polypeptide­enriched Gastrodia elata extracts (GEP) against the gram­negative bacteria Escherichia coli and Pseudomonas aeruginosa, the gram­positive bacterium Staphylococcus aureus and the fungus Candida albicans. The antimicrobial activity of GEP was first confirmed by determining the minimum inhibitory concentration by growth curve analysis. GEP was found to damage the cell wall and membrane of the microorganisms tested, as revealed by the morphological changes visible through scanning electron microscopy, and by the observed leakage of alkaline phosphatase and ß­galactosidase from cells. GEP was demonstrated to perturb the metabolism of the microorganisms, especially the tricarboxylic acid cycle, as indicated by the reduced intracellular activity of succinate dehydrogenase, malate dehydrogenase and ATPases, including the Na+/K+­ATPase and the Ca2+­ATPase. In addition, GEP caused the leakage of the genetic material of the bacteria and the fungus, as indicated by the increased OD260. The results of the present study indicated that GEP may exert its antimicrobial activity by damaging cell walls and membranes, causing the leakage of genetic material, and by perturbing cellular metabolism.

Candida albicans vaginitis in a murine model is reduced by polypeptide-enriched Gastrodia elata extracts.

Aim: The primary objective of this study was to evaluate the effects of polypeptide-enriched Gastrodia elata extracts (GE) on vulvovaginal candidiasis (VVC). Materials & methods: A VVC model induced by Candida albicans (C. albicans) infection was successfully developed in BALB/c mice. After treatment, the colony-forming unit (CFU) of vaginal lavage was measured by plating. The extent of the inflammatory response was assessed by hematoxylin-eosin (H&E) staining and enzyme-linked immunosorbent assay (ELISA). Results: GE had an inhibitory effect on the proliferation of C. albicans and inflammatory reaction. Meanwhile, it had a potentially beneficial effect on the growth of Lactobacillus. Conclusion: These results showed the potential application of GE as an antifungal agent in VVC treatment.

Investigation of the neuroprotective effects of crocin via antioxidant activities in HT22 cells and in mice with Alzheimer's disease.

Due to its complex pathogenesis, the prevention and therapization of Alzheimer's disease (AD) remains a serious challenge. Crocin, the main compound isolated from Crocus sativus L., demonstrates various pharmacological activities including anti­apoptotic properties. The present study investigated the neuroprotective effect of crocin and the underlying mechanisms. In l­glutamate­damaged HT22 cells, 3­h crocin pretreatment strongly enhanced the HT22 cell viability, reduced the apoptotic rate, mitigated mitochondrial dysfunction, suppressed intracellular reactive oxygen species (ROS) accumulation and Ca2+ overload compared with untreated cells. Additionally, crocin significantly decreased the expression levels of Bax, Bad and cleaved caspase­3 and increased the expression levels of B­cell lymphoma­extra large, phosphorylated (P­) protein kinase B and P­mammalian target of rapamycin compared with untreated cells. In mice with AD induced by d­galactose and aluminum trichloride, crocin substantially improved the cognition and memory abilities of the mice as measured by their coordination of movement in an open field test, and reduced their escape time in the Morris water maze test compared with untreated mice. Biochemical analysis confirmed that crocin was able to reduce the Aß1­42 content in the mouse brains, increase the levels of glutathione peroxidase, superoxide dismutase, acetylcholine and choline acetyltransferase, and reduce the levels of ROS and acetylcholinesterase in the serum, cerebral cortex and hypothalamus compared with untreated mice. Immunohistochemical analysis demonstrated that crocin reduced Aß1­42 deposition in the hippocampus of the brains of treated mice compared with untreated mice. In conclusion, crocin demonstrates good prospects in the treatment of AD through the oxidative stress­associated apoptosis signaling pathway.

Investigation of the immunomodulatory activity of Tricholoma matsutake mycelium in cyclophosphamide-induced immunosuppressed mice.

Tricholoma matsutake, a popular food and biopharmaceutical resource in Asia, possesses various pharmacological activities. Although T. matsutake mycelium (TM) may enhance immunity, previous studies, to the best of our knowledge, have been performed on normal animals or cells alone. The present study aimed to evaluate the immunomodulatory activity of TM at doses of 0.3, 1.0 and 2.0 g/kg in cyclophosphamide (CTX)­induced immunosuppressed mouse models. TM treatment for 2 weeks markedly improved the gain in bodyweight, increased organ indices, reduced hind paw swelling and positively regulated the cytotoxicity of natural killer cells and the proliferation of lymphocytes. These effects are similar to that of thymosin α1 (0.16 mg/kg) which served as the positive control. In CTX­induced immunosuppressed mice, TM demonstrated marked effects on the modulation of the production of immunoglobulin (Ig)G and IgA, and the levels of interleukin­2, 6, 10 and 12, interferon­α and γ and tumor necrosis factor­α in serum. Compared with CTX mice, the reduced activity of nuclear factor (NF)­κB in serum and spleen, and phosphorylation of inhibitor of NF­κB kinase α/ß in spleen were observed in TM­treated mice. Taken together, TM effectively improved immune function in immunosuppressed mice via modulation of ILs and inflammatory factors associated with the NF­κB signaling pathway.

Antifatigue Activity of Liquid Cultured Tricholoma matsutake Mycelium Partially via Regulation of Antioxidant Pathway in Mouse.

Tricholoma matsutake has been popular as food and biopharmaceutical materials in Asian countries for its various pharmacological activities. The present study aims to analyze the antifatigue effects on enhancing exercise performance of Tricholoma matsutake fruit body (ABM) and liquid cultured mycelia (TM) in mouse model. Two-week Tricholoma matsutake treatment significantly enhances the exercise performance in weight-loaded swimming, rotating rod, and forced running test. In TM- and ABM-treated mice, some factors were observed at 60 min after swimming compared with nontreated mice, such as the increased levels of adenosine triphosphate (ATP), antioxidative enzymes, and glycogen and the reduced levels of malondialdehyde and reactive oxygen species in muscle, liver, and/or serum. Further data obtained from western blot show that CM and ABM have strongly enhanced the activation of 5'-AMP-activated protein kinase (AMPK), and the expressions of peroxisome proliferator have activated receptor γ coactivator-1α (PGC-1α) and phosphofructokinase-1 (PFK-1) in liver. Our data suggest that both Tricholoma matsutake fruit body and liquid cultured mycelia possess antifatigue effects related to AMPK-linked antioxidative pathway. The information uncovered in our study may serve as a valuable resource for further identification and provide experimental evidence for clinical trials of Tricholoma matsutake as an effective agent against fatigue related diseases.