Search

Amodiaquine (AQ) is a commonly used antimalarial drug, and N-desethyl-AQ (N-DEAQ) is an active metabolite of AQ. Given the significance of drug quality in the management of malaria cases, this study aims to develop antibody-based assays for the detection and quantitation of AQ without the need for sophisticated equipment. Two monoclonal antibodies (mAbs) against AQ, designated as JUN7 and TE7, were selected, which showed 72.7% and 9.5% cross-reactivity to N-DEAQ, respectively. These mAbs showed <0.1% cross-reactivity to other commonly used antimalarial drugs. An indirect competitive enzyme-linked immunosorbent assay (icELISA) based on JUN7 showed a 50% inhibitory concentration (IC 50 ) of 0.16 ng/mL and a working range of 0.06-0.46 ng/mL. A lateral flow immunoassay (LFIA) based on JUN7 was also developed with a working range of 2.58-30.86 ng/mL. The icELISA and LFIA were applied for the quantification of AQ in commercial drugs, and the results were comparable to those determined using high-performance liquid chromatography. In addition, a combination dipstick for simultaneous, qualitative analysis of AQ and artesunate was developed. All immunoassays based on JUN7 can be applied for quality control of AQ-containing artemisinin-based combination therapies. As TE7 showed low cross-reactivity to N-DEAQ, an icELISA based on TE7 was developed with an IC 50 of 0.38 ng/mL and a working range of 0.14-1.67 ng/mL. The TE7 icELISA was applied for the study of pharmacokinetics of AQ in rat serum after intragastric administration, and the results were consistent with those of previous studies., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIβ (PI4KIIIβ). Molecular, biochemical, and cell biological studies show that PI4KIIIβ-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIβ-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIβ antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIβ-dependent secretion for cancer cell survival and tumor progression.

Modified Biejia Jianwan (M-BJJW), a Traditional Chinese Medicine (TCM) decoction, has exhibited great potential in treating hepatocellular carcinoma (HCC). However, its underlying functional mechanism still remains unknown. The study aimed to explore the anti-hepatocarcinogenic effects of M-BJJW, specifically its influence on PD-L1-mediated immune evasion in hypoxic conditions, and elucidate the related molecular mechanisms in HCC. To investigate the therapeutic efficacy and mechanisms underlying M-BJJW's effects on HCC, we employed a diethylnitrosamine (DEN)-induced rat model maintained for 120 days. Following model establishment, flow cytometry was utilized to assess the distribution of immune cell populations in peripheral blood, spleens, and tumor tissues after M-BJJW administration. Simultaneously, enzyme-linked immunosorbent assays (ELISA) were conducted to analyze cytokine profiles in serum samples. Immunohistochemistry was employed to determine the expression levels of crucial proteins within tumor tissues. Furthermore, HCC cells exposed to CoCl 2 underwent Western blot analysis to validate the expression levels of HIF-1α, PD-L1, STAT3, and nuclear factor kappa B (NF-κB) p65. The modulatory effects of STAT3 and NF-κB p65 were investigated using specific inhibitors and activators in wild-type cell lines. High-performance liquid chromatography coupled with mass spectrometry (HPLC/MS) was utilized to identify the chemical constituents present in M-BJJW-medicated serum. The immunomodulatory properties and the anti-tumor activities of M-BJJW were evaluated by co-culturing with peripheral blood mononuclear cells (PBMC) and the CCK-8 assay. Additionally, we assessed M-BJJW's impact on hypoxia-induced alterations in HCC cell lines using immunofluorescence and Western blot assessments. M-BJJW exhibited substantial therapeutic advantages by effectively alleviating pathological deterioration within the HCC microenvironment. In the DEN-induced rat model, M-BJJW administration notably reduced tumor growth. Flow cytometry analyses revealed an increased proportion of Cytotoxic T lymphocytes (CTLs) accompanied by a simultaneous decrease in regulatory T cells (Tregs). ELISA data supported a marked decrease in pro-inflammatory cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor α (TNF-α). Immunohistochemistry confirmed the suppressive effect of M-BJJW on the expression of HIF-1α and PD-L1. Notably, western blotting unveiled the role of HIF-1α in regulating PD-L1 expression via the STAT3 and NF-κB signaling pathways in HCC cell lines, which was validated using activators and inhibitors of STAT3 and NF-κB. The CCK-8 assay and co-culture techniques demonstrated the anti-tumor activity of M-BJJW. Immunofluorescence and western blotting further confirmed that M-BJJW-containing serum dose-dependently inhibited HIF-1α, PD-L1, p-STAT3, and p-p65 in hypoxic HCC cell lines. M-BJJW demonstrates significant therapeutic potential against HCC by influencing the hypoxic microenvironment, thereby regulating the immunosuppressive milieu. Specifically, M-BJJW modulates the HIF-1α/STAT3/NF-κB signaling pathway, leading to reduced PD-L1 expression and an elevated ratio of cytotoxic T lymphocytes (CTLs), while concurrently decreasing T regulatory cells (Tregs) and immunosuppressive factors. These synergistic effects aid in countering PD-L1-mediated immune evasion, presenting compelling pharmacological evidence supporting the clinical application of M-BJJW as a therapeutic approach for HCC. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • A lateral flow immunoassay (LFIA) was developed based on the obtained specific paraquat monoclonal antibody. • Herbicide products were tested after PBS dilution without other sample pretreatments. • Dipstick has been used to screen the illegal usage of paraquat in adulterated herbicides on-site. The use of the herbicide paraquat has been banned in many countries due to its high toxicity. However, there is an issue that illegal addition of paraquat in herbicide products. Therefore, a rapid, sensitive, and portable method will be useful to detect paraquat in adulterated herbicides for regulation purpose. In the present study, an indirect competition enzyme-linked immunosorbent assay (icELISA) was developed with a new paraquat-specific monoclonal antibody (mAb) for paraquat detection. The icELISA results showed a 50% inhibitory concentration (IC 50) of 7.63 ng/mL with a working range (IC 20 -IC 80) of 1.68–33.1 ng/mL. In addition, a lateral flow immunoassay (LFIA) with a cut-off value of 20.0 ng/mL was also developed. The LFIA showed no detection of the herbicides diquat, glyphosate and glufosinate up to 50 µg/mL. Nine herbicide samples were collected across Hainan province, China and analyzed by the LFIA dipsticks. One out of the nine samples was tested positive, which was confirmed with icELISA and HPLC. The accuracy of the LFIA was further verified through onsite tests in different places in China. Forty-seven commercial herbicide products were tested via the LFIA dipsticks, of which 9 samples were positive and confirmed with HPLC analysis. The paraquat LFIA exhibited the most promising potential as the point of care (POC) device for the rapid screening of paraquat in adulterated herbicide products. [ABSTRACT FROM AUTHOR]

[Display omitted] • The replicated CHF model of left anterior descending coronary artery ligation and exhaustive swimming is closer to the clinical manifestation than other models. • Fourteen potential biomarkers and eight metabolic pathways were identified by metabonomics, of which linoleic acid pathway is particularly closely related. • Moreover,quantitative real-time PCR (qRT-PCR), western blotting (WB), and Enzyme-linked immunosorbent assay (ELISA) were used to measure the RNA and protein expression levels in related pathways. The objective of this study was to investigate the protective effects of Shengmai Yin(SMY) on rats with chronic heart failure(CHF).Sprague‐Dawley rats were used to establish a CHF animal model via ligation of the left anterior descending branch of the coronary artery and exhaustive swimming.Echocardiography, serum biochemical indicators and histopathology were used to evaluate the pharmacodynamics of SMY in CHF rats.UPLC-Q-TOF/MS analysis based on serum was performed to identify the potential metabolites in the pathological process of CHF. Metabolic pathway analysis was carried out to elucidate the metabolic network associated with SMY treatment of CHF.Moreover,quantitative real-time PCR (qRT-PCR), Western blotting (WB), and Enzyme-linked immunosorbent assay (ELISA) were used to measure the RNA and protein expression levels in related pathways. Results revealed that SMY significantly restored the cardiac function of CHF rats, reduced the serum biochemical indicators, and alleviated cardiac histological damage. Metabolomics analysis shows that the therapeutic effect of SMY for CHF involves 14 biomarkers and 8 metabolic pathways, especially linoleic acid pathway, to be influenced, which implied the potential mechanism of SMY in treating CHF. Two key indicators Lipoxygenase arachidonic acid 15 lipoxygenase (ALOX15) and Cytochrome P450 1A2(CYP1A2) of linoleic acid metabolism pathway were verified by RT-PCR, WB and ELISA. Verification result showed that compared with the model group, expression levels of ALOX15 and CYP1A2 in SMY group were lower. In conclusion, SMY has cardioprotective effect on chronic heart failure rats, and its mechanism may be related to linoleic acid metabolism pathway. [ABSTRACT FROM AUTHOR]

• A highly specific monoclonal antibody against mefloquine was produced. • Two immunoassays dcELISA and dipsticks were developed for quantification of mefloquine commercial drugs. • The dipstick for simultaneously qualitative analysis of artesunate and mefloquine was developed. • The dipsticks have the potential for artesunate-mefloquine quality control as point-of-care devices. Artesunate-mefloquine is one of the commonly-used artemisinin-based combination therapies (ACTs). Given the significance of drug quality in the management of malaria cases, the objective of this study was to develop antibody-based assays as the point-of-care (POC) tests for monitoring the quality of this ACT. Using mefloquine conjugated to a carrier protein as the immunogen, we selected a specific monoclonal antibody (mAb) against mefloquine with no cross-reactivity to other antimalarial drugs. Using this mAb, we developed a direct competitive enzyme-linked immunosorbent assay (dcELISA) and a lateral flow immunoassay (LFIA) to measure the mefloquine contents. The dcELISA for mefloquine showed a 50% inhibitory concentration (IC 50) and a working range of 2.79 ng/mL and 0.58–16.37 ng/mL, respectively. With the aid of a portable optical scanner, the LFIA had a working range of 0.15–2.67 µg/mL for mefloquine. When used to measure mefloquine contents in commercial drugs, the dcELISA and LFIA results were compatible with those determined using high-performance liquid chromatography. Using the same LFIA format, we developed a combination LFIA, which correctly estimated the artesunate and mefloquine contents in commercial ACTs. Therefore, both LFIAs could be used as POC devices for rapid quality control of artesunate and mefloquine in ACTs. [ABSTRACT FROM AUTHOR]