Multiomics Analysis of Interleukin-21 as a Potential Immunologic and Biomarker in Hepatocellular Carcinoma.

Interleukin-21 (IL-21) is an important antitumor cytokine that contributes to the proliferation and differentiation of CD8+ T cells. It has been proven to enhance the response to immune checkpoint inhibitors (ICIs) in various solid tumors. However, its role in hepatocellular carcinoma (HCC) has not yet been clarified. In this research, we aimed to investigate the antitumor effect of IL-21 in HCC and its effect on ICI treatment. Through transcriptome sequencing analysis and immunohistochemistry validation, we found that patients with high IL-21 expression had a better prognosis. HCCs with high expression of IL-21 had higher infiltration of CD8+ T cells, increased expression of immune checkpoints, and an improved response to ICI treatment. In conclusion, IL-21 can enhance the efficacy of ICI treatment and improve the prognosis of patients by promoting the infiltration of CD8+ T cells and the expression of immune checkpoint-related genes.

Gut-lung axis and asthma: A historical review on mechanism and future perspective.

BACKGROUND: Gut microbiota are closely related to the development and regulation of the host immune system by regulating the maturation of immune cells and the resistance to pathogens, which affects the host immunity. Early use of antibiotics disrupts the homeostasis of gut microbiota and increases the risk of asthma. Gut microbiota actively interact with the host immune system via the gut-lung axis, a bidirectional communication pathway between the gut and lung. The manipulation of gut microbiota through probiotics, helminth therapy, and fecal microbiota transplantation (FMT) to combat asthma has become a hot research topic. BODY: This review mainly describes the current immune pathogenesis of asthma, gut microbiota and the role of the gut-lung axis in asthma. Moreover, the potential of manipulating the gut microbiota and its metabolites as a treatment strategy for asthma has been discussed. CONCLUSION: The gut-lung axis has a bidirectional effect on asthma. Gut microecology imbalance contributes to asthma through bacterial structural components and metabolites. Asthma, in turn, can also cause intestinal damage through inflammation throughout the body. The manipulation of gut microbiota through probiotics, helminth therapy, and FMT can inform the treatment strategies for asthma by regulating the maturation of immune cells and the resistance to pathogens.

Pancreatobiliary reflux increases macrophage-secreted IL-8 and activates the PI3K/NFκB pathway to promote cholangiocarcinoma progression.

BACKGROUND: Persistent pancreaticobiliary reflux (PBR) is associated with a high risk of biliary malignancy. This study aimed to evaluate the proportion of PBR in biliary tract diseases and mechanisms by which PBR promoted cholangiocarcinoma progression. METHODS: Overall 227 consecutive patients with primary biliary tract disease participated in this study. The amylase levels in the collected bile were analyzed. The mechanisms underlying the effect of high-amylase bile on bile duct epithelial and cholangiocarcinoma cells progression were analyzed. The source of interleukin-8 (IL-8) and its effects on the biological functions of cholangiocarcinoma cells were investigated. RESULTS: The bile amylase levels in 148 of 227 patients were higher than the upper serum amylase limit of 135 IU/L. PBR was significantly correlated with sex, pyrexia, and serum gamma-glutamyl transferase (GGT) levels in the patient cohort. High-amylase bile-induced DNA damage and genetic differences in the transcript levels of the gallbladder mucosa and facilitated the proliferation and migration of bile duct cancer cells (HUCCT1 and QBC939 cells). The concentration of many cytokines increased in high-amylase bile. IL-8 is secreted primarily by macrophages via the mitogen-activated protein kinase pathway and partially by bile duct epithelial cells. IL-8 promotes the progression of HUCCT1 and QBC939 cells by regulating the expression of epithelial-mesenchymal transition-associated proteins and activating the phosphatidylinositol 3-kinase/nuclear factor kappa-B pathway. CONCLUSIONS: PBR is one of the primary causes of biliary disease. IL-8 secreted by macrophages or bile duct epithelial cells stimulated by high-amylase bile promotes cholangiocarcinoma progression.

Solid-liquid distribution of SARS-CoV-2 in primary effluent of a wastewater treatment plant.

Distributions of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and fecal viral biomarkers between solid and liquid phases of wastewater are largely unknown. Herein, distributions of SARS-CoV-2, Pepper Mild Mottle Virus (PMMoV), and F-RNA bacteriophage group II (FRNAPH-II) were determined by viral RNA RT-qPCR. Comparison of viral recovery using three conventional fractionation methods included membrane filtration, a combination of mid-speed centrifugation and membrane filtration, and high-speed centrifugation. SARS-CoV-2 partitioned to the solids fraction in greater abundance compared to liquid fractions in a combination of mid-speed centrifugation and membrane filtration and high-speed centrifugation, but not in membrane filtration method in a particular assay, while fecal biomarkers (PMMoV and FRNAPH-II) exhibited the reciprocal relationship. The wastewater fractionation method had minimal effects on the solids-liquids distribution for all viral and phage markers tested; however, viral RNA load was significantly greater in solid-liquid fractions viral RNA loads compared with the than whole-wastewater PEG precipitation. A RNeasy PowerWater Kit with PCR inhibitor removal resulted in greater viral RNA loads and lesser PCR inhibition compared to a QIAamp Viral RNA Mini Kit without PCR inhibitor removal. These results support the development of improved methods and interpretation of WBE of SARS-CoV-2. •Distribution of SARS-CoV-2 to liquid and solid portions was addressed.•Addressing PCR inhibition is important in wastewater-based epidemiology.•Fraction methods have minimal effect.

Simulated sunlight/periodate-triggered formation of toxic halogenated bisphenols in highly saline water.

Periodate (PI)-based oxidation using the activators, such as metal ions and light irradiation, has emerged as a feasible treatment strategy for the effective remediation of contaminated water and wastewater. Given the pervasive nature of PI residues and solar exposure during application, the role of solar light in remediating the challenging highly saline water matrices needs to be elucidated. In this study, bisphenol A (BPA) was selected as the targeted micropollutant, which can be efficiently eliminated by the simulated sunlight (SSL)/PI system in the presence of high-level Cl- (up to 846.0 mM) at pH 7.0. The presence of different background constituents of water, such as halides, nitrate, and dissolved organic matter, had no effect, or even accelerated BPA abatement. Particularly, the ubiquitous Br- or I- appreciably enhanced the BPA transformation efficiency, which may be ascribed to the generation of high-selective reactive HOBr or HOI. The in silico predictions suggested that the transformation products generated by halide-mediated SSL/PI systems via halogen substitutions showed greater persistence, bioaccumulation, and aquatic toxicity than BPA itself. These findings highlighted a widespread phenomenon during PI-based oxidative treatment of highly saline water, which needs special attention under solar light illumination.

Fe(IV)/Fe(V)-mediated polyferric sulfate/periodate system: A novel coagulant/oxidant strategy in promoting micropollutant abatement.

Strategic modulation of the advanced oxidation processes for the selective oxidation of micropollutants has attracted accumulating attention in water decontamination. This study first reported the combination of the coagulant polyferric sulfate (PFS) and oxidant periodate (PI) to accomplish synergistic abatement of the antibiotic sulfamethoxazole (SMX). The oxidizing performance of SMX by this system was almost unaffected by coexisting water constituents, indicating the great promise of selective oxidation. Different from the current hydroxyl radicals (•OH)-mediated coagulant/oxidant systems (e.g., PFS/H2O2 and PFS/ozone), the dominance of high-valent Fe(IV)/Fe(V) intermediates was unambiguously verified in the PFS/PI treatment. The PFS colloids before and after the oxidation were characterized and the iron speciation was analyzed. The transformation of monomeric iron configurations (Fe(a)) to oligomeric iron configurations (Fe(b)) could maintain the homeostasis of surface-bound Fe(III) and Fe(II). The interaction mechanisms included the production of reactive species and dynamic reaction equilibrium for micropollutant degradation. Finally, the transformation pathways of SMX and carbamazepine (CMZ) in the PFS/PI system were postulated. Overall, this study provided a novel coagulant/oxidant strategy to achieve selective and sustainable water purification.

Emergence and spread of SARS-CoV-2 variants of concern in Canada: a retrospective analysis from clinical and wastewater data.

BACKGROUND: The spread of SARS-CoV-2 has been studied at unprecedented levels worldwide. In jurisdictions where molecular analysis was performed on large scales, the emergence and competition of numerous SARS-CoV-2lineages have been observed in near real-time. Lineage identification, traditionally performed from clinical samples, can also be determined by sampling wastewater from sewersheds serving populations of interest. Variants of concern (VOCs) and SARS-CoV-2 lineages associated with increased transmissibility and/or severity are of particular interest. METHOD: Here, we consider clinical and wastewater data sources to assess the emergence and spread of VOCs in Canada retrospectively. RESULTS: We show that, overall, wastewater-based VOC identification provides similar insights to the surveillance based on clinical samples. Based on clinical data, we observed synchrony in VOC introduction as well as similar emergence speeds across most Canadian provinces despite the large geographical size of the country and differences in provincial public health measures. CONCLUSION: In particular, it took approximately four months for VOC Alpha and Delta to contribute to half of the incidence. In contrast, VOC Omicron achieved the same contribution in less than one month. This study provides significant benchmarks to enhance planning for future VOCs, and to some extent for future pandemics caused by other pathogens, by quantifying the rate of SARS-CoV-2 VOCs invasion in Canada.

Degradation of methotrexate by unactivated and solar-activated peroxymonosulfate in water: Moiety-specific reaction kinetics and transformation product-associated risks.

Anticancer drugs have raised worldwide concern owing to their ubiquitous occurrence and ecological risks, necessitating the development of efficient removal strategies in water and wastewater treatment. Although peroxymonosulfate (PMS) is known to be a promising chemical in water decontamination, limited information is available regarding the removal efficiency of anticancer drugs by PMS and solar/PMS systems. This study first reports the moiety-specific reaction kinetics and mechanisms of methotrexate (MTX), an anticancer drug with widespread attention, by PMS (unactivated) and solar-activated PMS in water. It was found that MTX abatement by the direct PMS oxidation followed second-order kinetics, and the pH-dependent rate constants increased from 0.4 M-1 s-1 (pH 5.0) to 1.3 M-1 s-1 (pH 8.0), with a slight decrease to 1.1 M-1 s-1 at pH 9.0. The presence of chloride and bromide exerted no obvious influence on the removal of MTX by PMS. Furthermore, the chemical reactivity of MTX and its seven substructures with different reactive species was evaluated, and the degradation contributions of the reactive species involved were quantitatively analyzed in the solar/PMS system. The product analysis suggested similar reaction pathways of MTX by PMS and solar/PMS systems. The persistence, bioaccumulation, and toxicity of the transformation products were investigated, indicating treatment-driven risks. Notably, MTX can be removed efficiently from both municipal and hospital wastewater effluents by the solar/PMS system, suggesting its great potential in wastewater treatment applications. Overall, this study systematically evaluated the elimination of MTX by the unactivated PMS and solar/PMS treatment processes in water. The obtained findings may have implications for the mechanistic understanding and development of PMS-based processes for the degradation of such micropollutants in wastewater.

A comparative analysis of the partitioning behaviour of SARS-CoV-2 RNA in liquid and solid fractions of wastewater.

As fragments of SARS-CoV-2 RNA can be quantified and measured temporally in wastewater, surveillance of concentrations of SARS-CoV-2 in wastewater has become a vital resource for tracking the spread of COVID-19 in and among communities. However, the absence of standardized methods has affected the interpretation of data for public health efforts. In particular, analyzing either the liquid or solid fraction has implications for the interpretation of how viral RNA is quantified. Characterizing how SARS-CoV-2 or its RNA fragments partition in wastewater is a central part of understanding fate and behaviour in wastewater. In this study, partitioning of SARS-CoV-2 was investigated by use of centrifugation with varied durations of spin and centrifugal force, polyethylene glycol (PEG) precipitation followed by centrifugation, and ultrafiltration of wastewater. Partitioning of the endogenous pepper mild mottled virus (PMMoV), used to normalize the SARS-CoV-2 signal for fecal load in trend analysis, was also examined. Additionally, two surrogates for coronavirus, human coronavirus 229E and murine hepatitis virus, were analyzed as process controls. Even though SARS-CoV-2 has an affinity for solids, the total RNA copies of SARS-CoV-2 per wastewater sample, after centrifugation (12,000 g, 1.5 h, no brake), were partitioned evenly between the liquid and solid fractions. Centrifugation at greater speeds for longer durations resulted in a shift in partitioning for all viruses toward the solid fraction except for PMMoV, which remained mostly in the liquid fraction. The surrogates more closely reflected the partitioning of SARS-CoV-2 under high centrifugation speed and duration while PMMoV did not. Interestingly, ultrafiltration devices were inconsistent in estimating RNA copies in wastewater, which can influence the interpretation of partitioning. Developing a better understanding of the fate of SARS-CoV-2 in wastewater and creating a foundation of best practices is the key to supporting the current pandemic response and preparing for future potential infectious diseases.

A nitrocellulose/cotton fiber hybrid composite membrane for paper-based biosensor.

Nitrocellulose (NC) membrane was fabricated and tested for its potential use in various paper-based biosensors for use in point-of-care testing. However, contemporary technologies are complex, expensive, non-scalable, limited by conditions, and beset with potentially adverse effects on the environment. Herein, we proposed a simple, cost-effective, scalable technology to prepare nitrocellulose/cotton fiber (NC/CF) composite membranes. The NC/CF composite membranes with a diameter of 20 cm were fabricated in 15 min using papermaking technology, which contributes to scalability in the large-scale production of these composites. Compared with existing commercial NC membranes, the NC/CF composite membrane is characterized by small pore size (3.59 ± 0.19 µm), low flow rate (156 ± 55 s/40 mm), high dry strength (up to 4.04 MPa), and wet strength (up to 0.13 MPa), adjustable hydrophilic-hydrophobic (contact angles ranged from 29 ± 4.6 to 82.8 ± 2.4°), the good adsorption capacity of protein (up to 91.92 ± 0.07 µg). After lateral flow assays (LFAs) detection, the limit of detection is 1 nM, which is similar to commercial NC membrane (Sartorius CN 140). We envision the NC/CF composite membrane as a promising material for paper-based biosensors of point-of-care testing applications.

Understanding common population markers for SARS-CoV-2 RNA normalization in wastewater - A review.

Wastewater monitoring and epidemiology have seen renewed interest during the recent COVID-19 pandemic. As a result, there is an increasing need to normalize wastewater-derived viral loads in local populations. Chemical tracers, both exogenous and endogenous compounds, have proven to be more stable and reliable for normalization than biological indicators. However, differing instrumentation and extraction methods can make it difficult to compare results. This review examines current extraction and quantification methods for ten common population indicators: creatinine, coprostanol, nicotine, cotinine, sucralose, acesulfame, androstenedione 5-hydroindoleacetic acid (5-HIAA), caffeine, and 1,7-dimethyluric acid. Some wastewater parameters such as ammonia, total nitrogen, total phosphorus, and daily flowrate were also evaluated. The analytical methods included direct injection, dilute and shoot, liquid/liquid, and solid phase extraction (SPE). Creatine, acesulfame, nicotine, 5-HIAA and androstenedione have been analysed by direct injection into LC-MS; however, most authors prefer to include SPE steps to avoid matrix effects. Both LC-MS and GC-MS have been successfully used to quantify coprostanol in wastewater, and the other selected indicators have been quantified successfully with LC-MS. Acidification to stabilize the sample before freezing to maintain the integrity of samples has been reported to be beneficial. However, there are arguments both for and against working at acidic pHs. Wastewater parameters mentioned earlier are quick and easy to quantify, but the data does not always represent the human population effectively. A preference for population indicators originating solely from humans is apparent. This review summarises methods employed for chemical indicators in wastewater, provides a basis for choosing an appropriate extraction and analysis method, and highlights the utility of accurate chemical tracer data for wastewater-based epidemiology.

Wastewater discharges alter microbial community composition in surface waters of the canadian prairies.

Microbial communities are an important component of freshwater biodiversity that is threatened by anthropogenic impacts. Wastewater discharges pose a particular concern by being major sources of anthropogenic contaminants and microorganisms that may influence the composition of natural microbial communities. Nevertheless, the effects of wastewater treatment plant (WWTP) effluents on microbial communities remain largely unexplored. In this study, the effects of wastewater discharges on microbial communities from five different WWTPs in Southern Saskatchewan were investigated using rRNA gene metabarcoding. In parallel, nutrient levels and the presence of environmentally relevant organic pollutants were analyzed. Higher nutrient loads and pollutant concentrations resulted in significant changes in microbial community composition. The greatest changes were observed in Wascana Creek (Regina), which was found to be heavily polluted by wastewater discharges. Several taxa occurred in greater relative abundance in the wastewater-influenced stream segments, indicating anthropogenic pollution and eutrophication, especially taxa belonging to Proteobacteria, Bacteroidota, and Chlorophyta. Strong decreases were measured within the taxa Ciliphora, Diatomea, Dinoflagellata, Nematozoa, Ochrophyta, Protalveolata, and Rotifera. Across all sample types, a significant decline in sulfur bacteria was measured, implying changes in functional biodiversity. In addition, downstream of the Regina WWTP, an increase in cyanotoxins was detected which was correlated with a significant change in cyanobacterial community composition. Overall, these data suggest a causal relationship between anthropogenic pollution and changes in microbial communities, possibly reflecting an impairment of ecosystem health.

Circulating the HLA-DR+ T Cell Ratio Is a Prognostic Factor for Recurrence of Patients with Hepatocellular Carcinoma after Curative Surgery.

Background: HLA-DR+ T cell, accounting for 1.2%-5.8% of peripheral lymphocyte, is a type of activated T lymphocyte. This retrospective study aimed to evaluate the prognostic value of HLA-DR+ T cell for progression-free survival (PFS) and overall survival (OS) in hepatocellular carcinoma (HCC) patients after curative surgery. Patients and Methods. Clinicopathological data of 192 patients who underwent curative resection for hepatocellular carcinoma in the affiliated hospital of Qingdao University between January 2013 and December 2021 were collected and analyzed. Statistical tests used in this study were the chi-square test and Fisher's exact test. The prognostic value of the HLA-DR+ T cell ratio was analyzed using univariate and multivariate Cox regression analyses. The Kaplan-Meier curves were drawn by the R programming language. Results: HCC patients were divided into high (≥5.8%) and low (<5.8%) HLADR+ T cell ratio groups. Cox regression analysis indicated that a high HLA-DR+ T cell ratio was positively related to the PFS in HCC patients (P=0.003) and AFP-positive (≥20 ng/ml) HCC patients (P=0.020). HCC patients and AFP-positive HCC patients in the high HLA-DR+ T cell ratio group were prone to have a higher T cell ratio, a higher CD8+T cell ratio, and a lower B cell ratio than the low HLA-DR+ T cell ratio group. However, the HLA-DR+ T cell ratio was not a statistically significant predictor for OS in HCC patients (P=0.57) as well as PFS (P=0.088) and OS (P=0.63) in AFP-negative HCC patients. Conclusions: This study confirmed that the HLA-DR+ T cell ratio was a significant predictor of PFS in HCC patients and AFP-positive HCC patients after curative surgery. This association may have guiding significance for the follow-up work of HCC patients after surgery.

Assessment of the benefits of essential fatty acids and risks associated with antimicrobial residues in aquatic products: A case study of Chinese mitten crab (Eriocheir sinensis).

Much attention has been given to the safety and quality of aquatic products, including consuming Chinese mitten crab (Eriocheir sinensis), which offers both nutritional benefits and toxicological risks. Eighteen sulfonamides, 9 quinolones and 37 fatty acids were analyzed in 92 crab samples from primary aquaculture provinces in China. Enrofloxacin and ciprofloxacin have been mentioned as typical antimicrobials occurring at the greatest concentrations (>100 µg/kg, wm). By use of an in vitro method, the proportions of enrofloxacin, ciprofloxacin and essential fatty acids (EFAs, DHA and EPA) in ingested nutrients were determined to be 12 %, none and 95 %, respectively. The risk-benefit quotient (HQ) between the adverse effects of antimicrobials and nutritional benefits of EFAs in crabs found that HQs based on data after digestion were significantly less (HQ = 0.0086) than that of the control group where no digestion occurred (HQ = 0.055). This result suggested that (1) there was less risk posed by antimicrobials due to the consumption of crab, and (2) ignoring the bioaccessible fraction of antimicrobials in crabs might overestimate risks to the health of humans due to dietary exposure. Meaning bioaccessibility can improve the accuracy of the risk assessment process. Realistic risk evaluation should be recommended to achieve a quantified assessment of the dietary risks and benefits of aquatic products.

A wastewater-based risk index for SARS-CoV-2 infections among three cities on the Canadian Prairie.

Wastewater surveillance (WWS) is useful to better understand the spreading of coronavirus disease 2019 (COVID-19) in communities, which can help design and implement suitable mitigation measures. The main objective of this study was to develop the Wastewater Viral Load Risk Index (WWVLRI) for three Saskatchewan cities to offer a simple metric to interpret WWS. The index was developed by considering relationships between reproduction number, clinical data, daily per capita concentrations of virus particles in wastewater, and weekly viral load change rate. Trends of daily per capita concentrations of SARS-CoV-2 in wastewater for Saskatoon, Prince Albert, and North Battleford were similar during the pandemic, suggesting that per capita viral load can be useful to quantitatively compare wastewater signals among cities and develop an effective and comprehensible WWVLRI. The effective reproduction number (Rt) and the daily per capita efficiency adjusted viral load thresholds of 85 × 106 and 200 × 106 N2 gene counts (gc)/population day (pd) were determined. These values with rates of change were used to categorize the potential for COVID-19 outbreaks and subsequent declines. The weekly average was considered 'low risk' when the per capita viral load was 85 × 106 N2 gc/pd. A 'medium risk' occurs when the per capita copies were between 85 × 106 and 200 × 106 N2 gc/pd. with a rate of change <100 %. The start of an outbreak is indicated by a 'medium-high' risk classification when the week-over-week rate of change was >100 %, and the absolute magnitude of concentrations of viral particles was >85 × 106 N2 gc/pd. Lastly, a 'high risk' occurs when the viral load exceeds 200 × 106 N2 gc/pd. This methodology provides a valuable resource for decision-makers and health authorities, specifically given the limitation of COVID-19 surveillance based on clinical data.

Acute Exposure of Zebrafish (Danio rerio) to the Next-Generation Perfluoroalkyl Substance, Perfluoroethylcyclohexanesulfonate, Shows Similar Effects as Legacy Substances.

Perfluoroethylcyclohexanesulfonate (PFECHS) is an emerging perfluoroalkyl substance (PFAS) that has been considered a potential replacement for perfluorooctanesulfonic acid (PFOS). However, there is little information characterizing the toxic potency of PFECHS to zebrafish embryos and its potential for effects in aquatic environments. This study assessed toxic potency of PFECHS in vivo during both acute (96-hour postfertilization) and chronic (21-day posthatch) exposures and tested concentrations of PFECHS from 500 ng/L to 2 mg/L. PFECHS was less likely to cause mortalities than PFOS for both the acute and chronic experiments based on previously published values for PFOS exposure, but exposure resulted in a similar incidence of deformities. Exposure to PFECHS also resulted in significantly increased abundance of transcripts of peroxisome proliferator activated receptor alpha (pparα), cytochrome p450 1a1 (cyp1a1), and apolipoprotein IV (apoaIV) at concentrations nearing those of environmental relevance. Overall, these results provide further insight into the safety of an emerging PFAS alternative in the aquatic environment and raise awareness that previously considered "safer" alternatives may show similar effects as legacy PFASs.

Perfluoroethylcyclohexane sulphonate, an emerging perfluoroalkyl substance, disrupts mitochondrial membranes and the expression of key molecular targets in vitro.

Perfluoroethylcyclohexane sulphonate (PFECHS) is an emerging, replacement perfluoroalkyl substance (PFAS) with little information available on the toxic effects or potencies with which to characterize its potential impacts on aquatic environments. This study aimed to characterize effects of PFECHS using in vitro systems, including rainbow trout liver cells (RTL-W1 cell line) and lymphocytes separated from whole blood. It was determined that exposure to PFECHS caused minor acute toxic effects for most endpoints and that little PFECHS was concentrated into cells with a mean in vitro bioconcentration factor of 81 ± 25 L/kg. However, PFECHS was observed to affect the mitochondrial membrane and key molecular receptors, such as the peroxisome proliferator receptor, cytochrome p450-dependent monooxygenases, and receptors involved in oxidative stress. Also, glutathione-S-transferase was significantly down-regulated at a near environmentally relevant exposure concentration of 400 ng/L. These results are the first to report bioconcentration of PFECHS, as well as its effects on the peroxisome proliferator and glutathione-S-transferase receptors, suggesting that even with little bioconcentration, PFECHS has potential to cause adverse effects.

[Preparation of monoclonal antibodies against Pinellia ternata lectin protein and establishment of double-antibody sandwich ELISA].

To determine the content of endogenous toxic substance Pinellia ternata lectin(PTL) protein in Pinelliae Rhizoma and the related processed products, this study prepared specific monoclonal antibodies against PTL by hybridoma cell technology, and established a quantitative double-antibody sandwich enzyme linked immunosorbent assay(ELISA) for PTL antigen. The detection conditions were 2.5 µg·mL~(-1) working concentration of the captured antibody and 1∶450 of the dilution multiple of detected antibody. The coating condition was staying overnight at 4 ℃. The blocking time and incubation times of antigen and detected antibody were all 90 minutes. The incubation time of horseradish peroxidase conjugated streptavidin-horseradish peroxidase(SA-HRP) was 15 minutes. The quantitative limit of the method for PTL antigen was 0.375 ng·mL~(-1). The linear range was 75.000-4 800.000 pg·mL~(-1), and R~2=0.997 1. The recovery rate was 90.0%-110.0%, and the variation coefficients of intra-test and inter-test precision were 2.0%-3.0% and 2.0%-8.5%.The content of PTL in three batches of Pinelliae Rhizoma and the related processed products was determined by the method, and the average content of PTL in Pinelliae Rhizoma was 35.42 mg·g~(-1). The average content of PTL in Pinelliae Rhizoma Praeparatum Cum Alumine, Pinelliae Rhizoma Praeparatum, and Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumine were 1.15 mg·g~(-1), 16.53 µg·g~(-1), and 122.63 ng·g~(-1), respectively, indicating that the content of PTL decreased significantly after processing. The quantitative double-antibody sandwich ELISA for PTL antigen established in this paper had good linearity, sensitive response, and high accuracy, which provided a simple and effective monitoring method for the detection of PTL content in the processing of Pinelliae Rhizoma.

[Irritant toxicity and lectin content of different processed products of Pinelliae Rhizoma].

The present study aims to investigate the correlation between irritant toxicity variation and lectin content variation during the processing of Pinelliae Rhizoma products and to explore the feasibility of Western blot as a method for the detection of lectin. We processed Pinelliae Rhizoma Praeparatum Cum Alumine, Pinelliae Rhizoma Praeparatum, and Pinelliae Rhizoma Praeparatumcum Zingibere et Alumine to different degrees and then analyzed their irritant toxicity via Draize rabbit eye test. Western blot was employed to determine the lectin content in Pinelliae Rhizoma products processed with different methods. The correlation between toxicity variation and lectin content variation was then analyzed. Different decoction pieces of Pinelliae Rhizoma were collected for the determination of lectin content. The three processed products of Pinelliae Rhizoma showed gradually decreased toxicity and lectin content as the processing continued. The decreasing trend of lectin content was consistent with that of irritant toxicity during processing, which indicated that the change in lectin content could reflect the trend of irritant toxicity. No band of lectin appeared in the Western blot of processed products of Pinelliae Rhizoma, which suggested that western blotting can be used for the detection of toxic lectin in the processed products of Pinelliae Rhizoma. Lectin should not be detected in the Pinelliae Rhizoma products processed according to the methods in the Chinese Pharmacopoeia.