Overexpression of Corin Ameliorates Kidney Fibrosis through Inhibition of Wnt/ß-Catenin Signaling in Mice.

The Wnt/ß-catenin pathway represents a promising therapeutic target for mitigating kidney fibrosis. Corin possesses the homologous ligand binding site [Frizzled-cysteine-rich domain (Fz-CRD)] similar to Frizzled proteins, which act as receptors for Wnt. The Fz-CRD has been found in eight different proteins, all of which, except for corin, are known to bind Wnt and regulate its signal transmission. We hypothesized that corin may inhibit the Wnt/ß-catenin signaling pathway and thereby reduce fibrogenesis. Reduced expression of corin along with the increased activity of Wnt/ß-catenin signaling was found in unilateral ureteral obstruction (UUO) and ureteral ischemia/reperfusion injury (UIRI) models. In vitro, corin bound to the Wnt1 through its Fz-CRDs and inhibit the Wnt1 function responsible for activating ß-catenin. Transforming growth factor-ß1 inhibited corin expression, accompanied by activation of ß-catenin; conversely, overexpression of corin attenuated the fibrotic effects of transforming growth factor-ß1. In vivo, adenovirus-mediated overexpression of corin attenuated the progression of fibrosis, which was potentially associated with the inhibition of Wnt/ß-catenin signaling and the down-regulation of its target genes after UUO and UIRI. These results suggest that corin acts as an antagonist that protects the kidney from pathogenic Wnt/ß-catenin signaling and from fibrosis following UUO and UIRI.

Effects of resveratrol on DLD and NDUFB9 decrease in frozen semen of Mongolian sheep.

Mongolian sheep are a breed of sheep in China known for their excellent cold and drought resistance. Sperm from Mongolian sheep are often cryopreserved to improve breeding outcomes. However, cryopreservation of sperm often results in issues such as reduced vitality and altered morphology. Therefore, the objective of this study was to investigate the impact of the cryoprotectant resveratrol on frozen sperm from Mongolian sheep, specifically examining its effects on key proteins during cryopreservation. In this study, sperm samples were obtained from three adult Mongolian rams and processed through semen centrifugation. The sperm motility parameters of Fresh Sperm Group (FR), Resveratrol added before freezing group (FF-Res), Resveratrol-free frozen sperm group (FT), and Resveratrol added after freeze-thawing group (FA-Res) were determined. The tandem mass tags (TMT) peptide labeling combined with LC-MS/MS was used for proteomic analysis of the total proteins in FR and FT groups. A total of 2651 proteins were identified, among which 41 proteins were upregulated and 48 proteins were downregulated after freezing. In-depth bioinformatics analysis of differentially abundant proteins (DAPs) revealed their close association with the tricarboxylic acid cycle (TCA) and oxidative phosphorylation pathway. The energy-related protein dihydrolipoamide dehydrogenase (DLD) and the reactive oxygen species (ROS)-related protein NADH dehydrogenase 1 beta subcomplex subunit 9 (NDUFB9) exhibited significant decreases, indicating their potential role as key proteins contributing to reduced sperm vitality. The study demonstrated that the addition of resveratrol (RES) to semen could elevate the expression levels of DLD and NDUFB9 proteins. This study represents the pioneering proteomic analysis of Mongolian ram sperm before and after cryopreservation, establishing the significance of DLD and NDUFB9 as key proteins influencing the decline in vitality following cryopreservation of Mongolian ram sperm. These findings clarify that resveratrol can enhance the levels of DLD and NDUFB9 proteins in cryopreserved Mongolian ram sperm, consequently enhancing their vitality.

Paternal high-fat diet altered H3K36me3 pattern of pre-implantation embryos.

The global transition towards diets high in calories has contributed to 2.1 billion people becoming overweight, or obese, which damages male reproduction and harms offspring. Recently, more and more studies have shown that paternal exposure to stress closely affects the health of offspring in an intergenerational and transgenerational way. SET Domain Containing 2 (SETD2), a key epigenetic gene, is highly conserved among species, is a crucial methyltransferase for converting histone 3 lysine 36 dimethylation (H3K36me2) into histone 3 lysine 36 trimethylation (H3K36me3), and plays an important regulator in the response to stress. In this study, we compared patterns of SETD2 expression and the H3K36me3 pattern in pre-implantation embryos derived from normal or obese mice induced by high diet. The results showed that SETD2 mRNA was significantly higher in the high-fat diet (HFD) group than the control diet (CD) group at the 2-cell, 4-cell, 8-cell, and 16-cell stages, and at the morula and blastocyst stages. The relative levels of H3K36me3 in the HFD group at the 2-cell, 4-cell, 8-cell, 16-cell, morula stage, and blastocyst stage were significantly higher than in the CD group. These results indicated that dietary changes in parental generation (F0) male mice fed a HFD were traceable in SETD2/H3K36me3 in embryos, and that a paternal high-fat diet brings about adverse effects for offspring that might be related to SETD2/H3K36me3, which throws new light on the effect of paternal obesity on offspring from an epigenetic perspective.

A near-infrared fluorescent probe for detecting hydrazine metabolized from isoniazid in living cells.

Isoniazid is a drug for treating tuberculosis, but hydrazine (N2 H4 ), the major metabolite of isoniazid, can cause hepatotoxicity. Therefore, monitoring the content of N2 H4 in time is of great significance for studying the hepatotoxicity induced by isoniazid. In this study, a near-infrared fluorescent probe (BC-N) was designed and synthesized based on the specific reaction of acetyl ester with N2 H4 . BC-N exhibits excellent selectivity, sensitivity, and biocompatibility. In addition, BC-N is applied in the visualization of N2 H4 produced from isoniazid in living cells and is a potential tool for monitoring hepatotoxicity induced by isoniazid.

A ratiometric fluorescent probe based on a novel fluorophore with high selectivity for imaging cysteine in living cells.

As a biothiol, cysteine (Cys) is essential to both physiological and pathological processes and has been associated with many diseases, including neurological disorders, rheumatoid arthritis, and renal dysfunction. Therefore, the development of a high-performance probe for detecting Cys levels can help prevent and diagnose disease. In this study, a ratiometric fluorescent probe based on a novel fluorophore was developed for detecting Cys, and it showed high specificity and a rapid response time toward Cys. This probe demonstrates excellent biocompatibility and has been utilized effectively for the imaging of Cys in living cells.

Bacillus spore germination: mechanisms, identification, and antibacterial strategies.

Bacterial spores are metabolically inactive and highly resistant to harsh environmental conditions in nature and during decontamination processes in food and related industries. However, inducing germination using specific germinants in dormant spores can convert them into vegetative cells which are metabolically active and fragile. The potential utility of a "germinate to eradicate" strategy, also known as germination-inactivation, has been validated in foods. Meanwhile, the strategy has sparked much interest in triggering and maximizing spore germination. Although many details of the spore germination process have been identified over the past decades, there remain many uncertainties, including some signal transduction mechanisms involved in germination. In addition, the successful implementation of the germination-inactivation strategy relies on the sensitive detection of germinative biomarkers within minutes of germination initiation and the optimal timing for the subsequent inactivation step. Meanwhile, the emergence of biomarkers has renewed attention to the practical application of the spore germination process. Here, this review presents the current knowledge of the germination mechanisms of Bacillus spore, influencing factors, and germination biomarkers. It also covers a detailed discussion on the development of germination-inactivation as a spore eradication strategy.

SNAI2 Attenuated the Stem-like Phenotype by Reducing the Expansion of EPCAMhigh Cells in Cervical Cancer Cells.

SNAI2 (Snai2) is a zinc-finger transcriptional repressor that belongs to the Snail family. The accumulated evidence suggests that SNAI2 exhibits biphasic effects on regulating a stem-like phenotype in various types of cells, both normal and malignant. In this study, by exogenously expressing SNAI2 in SiHa cells, SNAI2 exhibited the capacity to inhibit a stem-like phenotype in cervical cancer cells. The SNAI2-overexpressing cells inhibited cell growth, tumorsphere formation, tumor growth, enhanced sensitivity to cisplatin, reduced stem cell-related factors' expression, and lowered tumor initiating frequency. In addition, the EPCAMhigh cells sorted from SiHa cells exhibited an enhanced capacity to maintain a stem-like phenotype. Further study demonstrated that the trans-suppression of EPCAM expression by SNAI2 led to blockage of the nuclear translocation of ß-catenin, as well as reduction in SOX2 and c-Myc expression in SiHa and HeLa cells, but induction in SNAI2 knockdown cells (CaSki), which would be responsible for the attenuation of the stem-like phenotype in cervical cancer cells mediated by SNAI2. All of these results demonstrated that SNAI2 could attenuate the stem-like phenotype in cervical cancer cells through the EPCAM/ß-catenin axis.

Accelerating carbon neutrality could help China's energy system align with below 1.5 °C.

To support the achievement of the Paris Agreement's 1.5 °C global warming threshold, China aims to peak CO2 emissions before 2030 and achieve carbon neutrality before 2060. However, the specific carbon neutrality pathway remains to be designed. By applying a refined Chinese version of Global Change Analysis Model, this study examines implications of four illustrative carbon neutrality scenarios for aligning China's energy system with below 1.5 °C by 2100. The results feature a trade-off between China's ambition to transform its energy system toward mid-century and its reliance on carbon dioxide removal (CDR) after carbon neutrality. From a full time perspective until 2100, accelerating carbon neutrality could help China's energy system align with below 1.5 °C. Compared to a 2060 carbon neutrality scenario, a 2050 carbon neutrality scenario reduces China's total mitigation costs between 2021 and 2100 by 1.04% of GDP, reduces reliance on CDR by 36%, and provides some additional co-benefits, such as reduced air pollutants. However, special attention needs to be paid to the fact that accelerating carbon neutrality poses greater challenges and costs to China in overcoming development inertia and restructuring its energy system over the next 30-40 years. Compared to a 2060 carbon neutrality scenario, a 2050 scenario increases China's mitigation costs by a factor of 1.13 between 2021 and 2050. This study suggests through quantitative evidence that China could accelerate emissions reductions and energy system transformation to achieve carbon neutrality, based on its national circumstances and capabilities and international support.

Magnetic biochar promotes the risk of mobile genetic elements propagation in sludge anaerobic digestion.

Mobile genetic elements (MGEs) mediated horizontal gene transfer is the primary reason for the propagation of antibiotic resistance genes in environment. The behavior of MGEs under magnetic biochar pressure in sludge anaerobic digestion (AD) is still unknown. This study evaluated the effects of different dosage magnetic biochar on the MGEs in AD reactors. The results showed that the biogas yield was highest (106.68 ± 1.16 mL g-1 VSadded) with adding optimal dosage of magnetic biochar (25 mg g-1 TSadded), due to it increased the microorganism's abundance involved in hydrolysis and methanogenesis. While, the total absolute abundance of MGEs in the reactors with magnetic biochar addition increased by 11.58%-77.37% compared with the blank reactor. When the dosage of magnetic biochar was 12.5 mg g-1 TSadded, the relative abundance of most MGEs was the highest. The enrichment effect on ISCR1 was the most significant, and the enrichment rate reached 158.90-214.16%. Only the intI1 abundance was reduced and the removal rates yield 14.38-40.00%, which was inversely proportional to the dosage of magnetic biochar. Co-occurrence network explored that Proteobacteria (35.64%), Firmicutes (19.80%) and Actinobacteriota (15.84%) were the main potential host of MGEs. Magnetic biochar changed MGEs abundance by affecting the potential MGEs-host community structure and abundance. Redundancy analysis and variation partitioning analysis showed that the combined effect of polysaccharides, protein and sCOD exhibited the greatest contribution (accounted for 34.08%) on MGEs variation. These findings demonstrated that magnetic biochar increases the risk of MGEs proliferation in AD system.

Sperm-borne proteins improve rabbit cloning efficiency via regulating embryonic cleavage and epigenetics.

Somatic cell nuclear transfer (SCNT) shows great application value in the generation of transgenic animals, protection of endangered species, and therapeutic cloning. However, the cloning efficiency is still very low, which greatly restricts its application. Compared to fertilized embryos, cloned embryos lack the sperm proteins, which are considered to play an important role in embryonic development. Here, we compared the sperm proteome, with that of donor fibroblasts and oocytes, and identified 342 proteins unique to sperm, with 42 being highly expressed. The 384 proteins were mainly enriched in the categories of post-translational modification and cytoskeletal arrangement. Extracts of soluble sperm or fibroblast proteins were injected into cloned embryos, and the result showed that injection of sperm protein significantly inhibited abnormal embryonic cleavage, significantly decreased the level of trimethylated histone H3 Lys9 (H3K9me3) and the apoptotic index, and increased the inner cell mass (ICM)-to-trophectoderm (TE) ratio. More importantly, the sperm proteins also significantly enhanced the birthrate. The results of in vitro and in vivo experiments demonstrate that sperm-derived proteins improve embryo cloning efficiency. Our findings not only provide new insights into ways to overcome low cloning efficiency, but also add to the understanding of sperm protein function.

Potato (Solanum tuberosum L.) non-specific lipid transfer protein StLTP6 promotes viral infection by inhibiting virus-induced RNA silencing.

MAIN CONCLUSION: For the first time it is reported that members of the nsLTP protein family could promote viral infection by inhibiting virus-induced RNA silencing. Non-specific lipid transfer proteins (nsLTPs) are a class of soluble proteins with low relative molecular weight and widely present in higher plants. The role of nsLTPs in biotic and abiotic stresses has been studied, but no report has shown that nsLTPs play a role in the process of viral infection. We report the function and mechanism of the classical nsLTP protein StLTP6 in viral infection. We found that StLTP6 expression was remarkably upregulated in potato infected with potato virus Y and potato virus S. The infection efficiency and virus content of StLTP6-overexpressed potato and Nicotiana benthamiana were remarkable increased. Further study found that the overexpression of StLTP6 inhibited the expression of multiple genes in the RNA silencing pathway, thereby inhibiting virus-induced RNA silencing. This result indicated that StLTP6 expression was induced during viral infection to inhibit the resistance of virus-induced RNA silencing and promote viral infection. In summary, we reported the role of StLTP6 in viral infection, broadening the biological function range of the nsLTP family and providing valuable information for the study of viral infection mechanism.

Infectious hematopoietic necrosis virus (IHNV) nucleoprotein amino acid residues affect viral virulence and immunogenicity in rainbow trout (Oncorhynchus mykiss).

This study compared the N protein sequences of genotype J with other genotypes of IHNV to select amino acid residues that may be related to the change in viral virulence. The recombinant viruses containing different mutation sites were rescued by alanine scanning mutagenesis and the reverse genetic system. The nine recombinant virus strains obtained in this work were named rIHNV-N85, rIHNV-N102, rIHNV-N146, rIHNV-N380, rIHNV-N85-102-146, rIHNV-N85-102-380, rIHNV-N85-146-380, rIHNV-N102-146-380, and rIHNV-N85-102-146-380. Pathogenicity and immunity assays were performed to determine the role of virulence sites. The result of the pathogenicity test showed that the survival rates of rIHNV-N85, rIHNV-N102, rIHNV-N85-102-146, and rIHNV-N85-102-380 groups were 52.5%, 55%, 67.5%, and 57.5%, while the survival rate of wild-type (wt) IHNV HLJ-09 group was only 10%. The replication ability of recombinant viruses with substitutions at positions 85 and 102 was significantly inhibited in vivo and in vitro. The qRT-PCR result indicated that the cytokines of IFN1, IL-8, and IL-1ß expression levels were increased in rIHNV-N85, rIHNV-N102, rIHNV-N85-102-146, and rIHNV-N85-102-380 groups. In addition, these four recombinant viruses could cause the rainbow trout to produce anti-IHNV-specific antibodies immunoglobulin M (IgM) earlier, confirming that 85 and 102 amino acid residues of N protein affected the virulence and immunogenicity of IHNV. All these results suggest that mutations of the N protein virulence sites reduce virulence while retaining immunogenicity. This also provides a new idea for studying the virulence mechanism of rhabdoviruses and preparing attenuated vaccines.

Recent advances in enterovirus A71 pathogenesis: a focus on fatal human enterovirus A71 infection.

Enterovirus A71 (EV-A71) is one of the major pathogens responsible for hand, foot, and mouth disease (HFMD). Many HFMD outbreaks have been reported throughout the world in the past decades. Compared with other viruses, EV-A71 infection is more frequently associated with severe neurological complications and even death in children. EV-A71 can also infect adults and cause severe complications and death, although such cases are very uncommon. Although fatal cases of EV-A71 infection have been reported, the underlying mechanisms of EV-A71 infection, especially the mode of viral spread into the central nervous system (CNS) and mechanisms of pulmonary edema, which is considered to be the direct cause of death, have not yet been fully clarified, and more studies are needed. Here, we first summarize the pathological findings in various systems of patients with fatal EV-A71 infections, focussing in detail on gross changes, histopathological examination, tissue distribution of viral antigens and nucleic acids, systemic inflammatory cell infiltration, and tissue distribution of viral receptors and their co-localization with viral antigens. We then present our conclusions about viral dissemination, neuropathogenesis, and the mechanism of pulmonary edema in EV-A71 infection, based on pathological findings.

Identification of Prognostic Gene Signatures by Developing a scRNA-Seq-Based Integration Approach to Predict Recurrence and Chemotherapy Benefit in Stage II-III Colorectal Cancer.

Prospective identification of robust biomarkers related to prognosis and adjuvant chemotherapy has become a necessary and critical step to predict the benefits of adjuvant therapy for patients with stage II-III colorectal cancer (CRC) before clinical treatment. We proposed a single-cell-based prognostic biomarker recognition approach to identify and construct CRC up- and down-regulated prognostic signatures (CUPsig and CDPsig) by integrating scRNA-seq and bulk datasets. We found that most genes in CUPsig and CDPsig were known disease genes, and they had good prognostic abilities in CRC validation datasets. Multivariate analysis confirmed that they were two independent prognostic factors of disease-free survival (DFS). Significantly, CUPsig and CDPsig could effectively predict adjuvant chemotherapy benefits in drug-treated validation datasets. Additionally, they also performed well in patients with CMS4 subtype. Subsequent analysis of drug sensitivity showed that expressions of these two signatures were significantly associated with the sensitivities of CRC cell lines to multiple drugs. In summary, we proposed a novel prognostic biomarker identification approach, which could be used to identify novel prognostic markers for stage II-III CRC patients who will undergo adjuvant chemotherapy and facilitate their further personalized treatments.

Absence of EpCAM in cervical cancer cells is involved in sluginduced epithelial-mesenchymal transition.

BACKGROUND: Slug (Snai2) is a pivotal player in initiating epithelial-mesenchymal transition (EMT) through its trans-suppression effect on E-cadherin in various normal and malignant cells. In this study, the positive effect of Slug on promoting cell motility and metastasis in cervical cancer was further confirmed in this study. METHODS: RNA-Seq was performed to explore the potential molecules that participate in Slug-mediated EMT in cervical cancer cells. The negative correlation between Slug and EpCAM expression in cervical cancer cells was detected in this study, and linked them with in vitro migration and invasion assay, in vivo metastasis experiments, luciferase reporter assay and Chromatin immunoprecipitation. RESULTS: Transcriptome sequencing analysis revealed that epithelial cell adhesion molecule (EpCAM) was significantly decreased in Slug-overexpressing SiHa cells. Simultaneously, an absence of EpCAM expression was observed in Slug-overexpressing cells. Further studies revealed the trans-suppression effect of Slug on EpCAM through its binding to the E-boxes in the proximal promoter region of EpCAM in cervical cancer cells. Restoring EpCAM in Slug-overexpressing cells by transiently transfecting an EpCAM recombinant plasmid attenuated cell motility and promoted cell growth. Moreover, the negative correlation between Slug and EpCAM expression in human squamous cervical carcinoma (SCC) samples was verified by using Pearson correlation analysis. CONCLUSIONS: These results demonstrated that the absence of EpCAM under Slug expression in cervical cancer cells probably participated in Slug-regulated EMT and further promoted tumor metastasis. Additionally, this study supports a potential way for Slug to initiate EMT progression in cervical cancer cells in addition to inhibiting E-cadherin.

Hexokinases 2 promoted cell motility and distant metastasis by elevating fibronectin through Akt1/p-Akt1 in cervical cancer cells.

BACKGROUND: Hexokinases 2 (HK2) is a member of the hexokinases, linking with malignant tumor growth and distant metastasis. However, evidence regarding the potential role of HK2 in regulating cell motility and tumor metastasis during the cervical cancer malignant progression remains limited. METHODS: In vitro migration and invasion assay, in vivo metastasis experiments were performed to detect the effective of HK2 on regulating cell motility and tumor metastasis in cervical cancer cells. RNA-Seq was performed to explore the potential molecules that participate in HK2-mediated cell motility and tumor metastasis in cervical cancer cells. The correlation between HK2 and Akt1, p-Akt1, FN1 expression in cervical cancer cells and human squamous cervical carcinoma (SCC) samples was verified in this study. RESULTS: In this study, cervical cancer cells with exogenous HK2 expression exhibited enhanced cell motility and distant metastasis. Transcriptome sequencing analysis revealed that fibronectin (FN1) was significantly increased in HK2-overexpressing HeLa cells, and the PI3K/Akt signaling pathway was identified by KEGG pathway enrichment analysis. Further studies demonstrated that this promotion of cell motility by HK2 was probably a result of it inducing FN1, MMP2 and MMP9 expression by activating Akt1 in cervical cancer cells. Additionally, HK2 expression was altered with the changing of Akt1/p-Akt1 expression, implying that HK2 expression is also modulated by Akt1/p-Akt1. Moreover, the positive correlation between HK2 and Akt1, p-Akt1, FN1 expression in human squamous cervical carcinoma (SCC) samples was verified by using Pearson correlation analysis. CONCLUSIONS: This study demonstrated that HK2 could activate Akt1 in cervical cancer cells, subsequently enhancing cell motility and tumor metastasis by inducing FN1, MMP2 and MMP9 expression. There likely exists an interactive regulatory mechanism between HK2 and Akt1 during the malignant process of cervical cancer.

The Mpro structure-based modifications of ebselen derivatives for improved antiviral activity against SARS-CoV-2 virus.

The main protease (Mpro or 3CLpro) of SARS-CoV-2 virus is a cysteine enzyme critical for viral replication and transcription, thus indicating a potential target for antiviral therapy. A recent repurposing effort has identified ebselen, a multifunctional drug candidate as an inhibitor of Mpro. Our docking of ebselen to the binding pocket of Mpro crystal structure suggests a noncovalent interaction for improvement of potency, antiviral activity and selectivity. To test this hypothesis, we designed and synthesized ebselen derivatives aimed at enhancing their non-covalent bonds within Mpro. The inhibition of Mpro by ebselen derivatives (0.3 µM) was screened in both HPLC and FRET assays. Nine ebselen derivatives (EBs) exhibited stronger inhibitory effect on Mpro with IC50 of 0.07-0.38 µM. Further evaluation of three derivatives showed that EB2-7 exhibited the most potent inhibition of SARS-CoV-2 viral replication with an IC50 value of 4.08 µM in HPAepiC cells, as compared to the prototype ebselen at 24.61 µM. Mechanistically, EB2-7 functions as a noncovalent Mpro inhibitor in LC-MS/MS assay. Taken together, our identification of ebselen derivatives with improved antiviral activity may lead to developmental potential for treatment of COVID-19 and SARS-CoV-2 infection.

Sperm-borne small RNAs improve the developmental competence of pre-implantation cloned embryos in rabbit.

The low efficiency of somatic cell nuclear transfer (SCNT) greatly limits its application. Compared with the fertilized embryo, cloned embryos display abnormal epigenetic modification and other inferior developmental properties. In this study, small RNAs were isolated, and miR-34c and miR-125b were quantified by real-time PCR; results showed that these micro-RNAs were highly expressed in sperm. The test sample was divided into three groups: one was the fertilized group, one was the SCNT control group (NT-C group), and the third group consisted of SCNT embryos injected with sperm-borne small RNA (NT-T group). The level of tri-methylation of lysine 9 on histone H3 (H3K9me3) at the 8-cell stage was determined by immunofluorescence staining, and the cleavage ratio, blastocyst ratio, apoptotic cell index of the blastocyst and total cell number of blastocysts in each group were analyzed. Results showed that the H3K9me3 level was significantly higher in the NT-C group than in the fertilized group and the NT-T group. The apoptosis index of blastocysts in the NT-C group was significantly higher than that in the fertilized group and the NT-T group. The total cell number of SCNT embryos was significantly lower than that of fertilized embryos, and injecting sperm-borne small RNAs could significantly increase the total cell number of SCNT blastocysts. Our study not only demonstrates that sperm-borne small RNAs have an important role in embryo development, but also provides a new strategy for improving the efficiency of SCNT in rabbit.

Establishment of an anti-inflammation-based bioassay for the quality control of the 13-component TCM formula (Lianhua Qingwen).

CONTEXT: Owing to the complexity of chemical ingredients in traditional Chinese medicine (TCM), it is difficult to maintain quality and efficacy by relying only on chemical markers. OBJECTIVE: Lianhua Qingwen capsule (LHQW) was selected as an example to discuss the feasibility of a bioassay for quality control. MATERIALS AND METHODS: Network pharmacology was used to screen potential targets in LHQW with respect to its anti-inflammatory effects. An in vitro cell model was used to validate the prediction. An anti-inflammatory bioassay was established for the quality evaluation of LHQW in 40 batches of marketed products and three batches of destructed samples. RESULTS: The tumor necrosis factor/interleukin-6 (TNF/IL-6) pathway via macrophage was selected as the potential target of LHQW. The IC50 value of LHQW on RAW 264.7 was 799.8 µg/mL. LHQW had significant inhibitory effects on the expression of IL-6 in a dose-dependent manner (p < 0.05). The anti-inflammatory biopotency of LHQW was calculated based on the inhibitory bioactivity on IL-6. The biopotency of 40 marketed samples ranged from 404 U/µg to 2171 U/µg, with a coefficient of variation (CV) of 37.91%. By contrast, the contents of forsythin indicated lower CV (28.05%) than the value of biopotency. Moreover, the biopotencies of destructed samples declined approximate 50%, while the contents of forsythin did not change. This newly established bioassay revealed a better ability to discriminate the quality variations of LHQW as compared to the routine chemical determination. CONCLUSIONS: A well-established bioassay may have promising ability to reveal the variance in quality of TCM.

Visualizing TRPA1 in the Plasma Membrane for Rapidly Screening Optical Control Agonists via a Photochromic Ligand Based Fluorescent Probe.

Fluorescent probes have been used as effective methods for profiling proteins in biological systems because of their high selectivity, sensitivity, and temporal-spatial resolution. A specific fluorescent probe for understanding the function of the transient receptor potential ankyrin 1 (TRPA1) channel that is closely related with various diseases like persistent pain, respiratory, and chronic itch syndromes, however, is still lacking. Here, we report a "turn-on" fluorescent probe (A1CA) for visualizing TRPA1 channels in the plasma membrane of live cells based on a photochromic ligand derived from 4-(phenylazo)benzenamine. Evaluating the specificity and sensitivity of A1CA by electrophysiology and confocal imaging showed that the A1CA probe displays higher affinity and selectivity to TRPA1 channel versus all other ion channels including TRPV1, TRPV3, Nav1.4, Nav1.5, and hERG. Based on the supporting evidence, A1CA has great potential as a molecular imaging probe for high-throughput screening of novel TRPA1 agonists.