Assessing Aptamer-Analyte Binding Kinetics by Microfluidic Fluorescence Microscopy.

Kinetic measurement plays a crucial role in understanding aptamer binding mechanisms and identifying appropriate aptamers for clinical and research applications. Current techniques, while well established, generally require large sample volumes, bulky and expensive instruments operated by trained personnel, and are hence not readily accessible to resource-limited research laboratories. This paper presents a fluorescence microscopy-based microfluidic assay for measuring aptamer-analyte binding kinetics in a simple and cost-effective manner. Kinetic measurements are achieved by monitoring time-course fluorescence of fluorescently labeled aptamers as they bind to the targets trapped in a microfluidic chip. Fluorescence measurements are performed on a standard fluorescence microscope and are accessible to laboratories with only modest resources. Moreover, microfluidic technology allows efficient and cost-effective immobilization of small amounts of target molecules or live cells as well as flow-based manipulation of aptamers for the measurements. Kinetic measurements of aptamer binding to immunoglobulin E protein and CCRF-CEM cells have yielded results consistent with those obtained from established methods, demonstrating the potential utility of our method for exploring aptamer-target interactions and identifying aptamers that best suit specific given biomedical applications.

Fabrication of hyaluronic acid-altered gold complex delivery for head and neck squamous cell carcinoma therapy with high antitumor efficacy and low in vivo toxicity.

The use of multifunctional nanomedicines in the treatment of tumors is gaining popularity. Here, we constructed a nanodrug delivery system (HA/Au-PDA@CZT) that targets tumors and responds to pH and near-infrared (NIR) dual stimuli. By precisely interacting with an overexpressed CD44 receptor in specific cancer cells, hyaluronic acid (HA) is coated on the Au-PDA NP surface for tumor-targeting abilities. When exposed to NIR radiation, polydopamine (PDA) and gold nanoshells exhibit exceptional photothermal performance that has the potential to both accelerate and kill HLAC 78 head and neck squamous cell carcinoma cells. Antitumor investigations conducted in vivo and in vitro demonstrated that nanomedicine had remarkable synergistic benefits with chemotherapy and photothermal treatment. Only 25.2% of the cells in the HA/Au-PDA@CZT with a NIR irradiation group were viable. Any group's lowest tumor volume was shown in the tumor mice subjected to HA/Au-PDA@CZT with NIR at 0.3 ± 0.1. Consequently, for synergistic chemo-photothermal therapy, our logically designed nanoplatform would be the potential for a head and neck squamous tumor-targeting drug delivery system.

The syndrome of inappropriate antidiuretic hormone associated with nasal and paranasal malignant tumors.

PURPOSE: To investigate the clinical characteristics of the syndrome of inappropriate antidiuretic hormone (SIADH) associated with nasal and paranasal malignant tumors. METHODS: Patients with locally advanced or recurrence/metastatic malignant tumors of the nasal and paranasal sinuses were included. The SIADH was diagnosed according to the diagnostic criteria. The clinical characteristics of SIADH patients were retrospectively analyzed. RESULTS: Six patients (6/188, 3.2%) met the diagnostic criteria of SIADH, including four olfactory neuroblastoma (4/26, 15.4%), one neuroendocrine carcinoma (1/9, 11.1%), and one squamous cell carcinoma (1/63, 1.6%). Five patients (83.3%) had severe hyponatremia; however, the hyponatremia could be improved by fluid restriction or tolvaptan. Three patients' SIADH were recovered during the chemotherapy and the other three were recovered after the surgery. CONCLUSION: The incidence of SIADH associated with nasal and paranasal malignant tumors is relatively more common in olfactory neuroblastoma and neuroendocrine carcinoma. The hyponatremia caused by SIADH may be corrected by fluid restriction or tolvaptan, and the SIADH may be recovered through anti-tumor therapy.

Efficacy and safety of immune checkpoint inhibitors in recurrent or metastatic head and neck squamous cell carcinoma: A systematic review and meta-analysis of randomized clinical trials.

BACKGROUND: Immune checkpoint inhibitors (ICIs) showed antitumor activity for recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC). However, the results from different studies were controversial. METHODS: Online databases were searched for randomized clinical trials (RCTs) evaluating ICIs for R/M HNSCC. The characteristics of the studies and the results of overall survival (OS), progression-free survival (PFS), objective response rate (ORR), treatment-related adverse events (TRAEs) were extracted. RESULTS: A total of 4936 patients from eight studies were included. Anti-PD1/PDL1 monotherapy significantly improved OS in total population (hazard ratio, HR, 0.87, 95% CI, 0.79-0.95, p = 0.003) and PD-L1 high expression patients (HR, 0.71, 95% CI, 0.55-0.90, p = 0.006) with significant lower incidence of any grade TRAEs (odds ratio, OR, 0.16, 95% CI, 0.07-0.37, p < 0.00001) and Grades 3-5 TRAEs (OR, 0.18, 95% CI, 0.10-0.33, p < 0.0001) compared with standard of care (SOC); however, the pooled results of PFS and ORR were not significant different. PD1/PDL1 inhibitors plus CTLA4 inhibitors did not improve OS, PFS, ORR compared with SOC or ICIs monotherapy; however, the incidence of Grades 3-5 TRAEs was significant higher compared with ICIs monotherapy (OR, 1.80, 95% CI, 1.34-2.41, p = 0.0001). CONCLUSIONS: Anti-PD1/PDL1 monotherapy could improve OS for R/M HNSCC with significant lower incidence of TRAEs compared with SOC. PD1/PDL1 inhibitors plus CTLA4 inhibitors showed no more benefit compared with both SOC and ICIs monotherapy, but the incidence of Grades 3-5 TRAEs was significant higher compared with ICIs monotherapy.

Three-dimensional porous wood monolithic columns for efficient purification of spike glycoprotein of SARS-CoV-2.

Considerable research has been devoted to finding a cost-effective chromatographic matrix with efficient adsorption and high throughput. Wood exhibits complex micro-network structures that make it a powerful contender for a novel environment-friendly chromatographic matrix material. We demonstrate a novel strategy to manufacture a wood monolithic column, which chemically modified the wood and imported diethyl aminoethyl, diethylamine, and amino groups. This wood monolithic column can maintain fully monolithic column performances and highly selective to spike glycoprotein of SARS-CoV-2 by ion exchange force. The wood monolithic column was evaluated by static adsorption, dynamic adsorption, and frontal analysis. The results showed that the static adsorption capacity of the wood monolithic column with 2-diethylaminoethylchloride hydrochloride for bovine serum albumin was 14.72 mg/g, and the adsorption process was chemisorption. In addition, it retained 80 % adsorption capacity after 110 repeated adsorption-elution cycles.

Endothelial cell protein C receptor regulates neutrophil extracellular trap-mediated rheumatoid arthritis disease progression.

Endothelial cell protein C receptor (EPCR) is a 46 kDa transmembrane protein receptor, expressed in most immune cells (T cells, monocytes, dendritic cells, polymorphonuclear neutrophils [PMN]). EPCR reportedly plays a vital role in rheumatoid arthritis (RA). Our results confirmed that EPCR expression exists in the PMN of RA patients, and animal experiments demonstrated that down-regulation of EPCR expression affects disease progression in collagen-induced arthritis (CIA) mice. PMN is the immune cell type that first enters the site of inflammation in the early stages of inflammation. In the early stage of RA, PMN cells migrate into the joint cavity and function in the process of RA synovial inflammation, aggravating the bone destruction found in RA and mediating the progression of RA disease progression. We verified the differences in EPCR expression in PMN cells between RA and osteoarthritis (OA) patients by Western blot and then confirmed this difference in animals. We found that CIA mice treated with PMN-neutralizing antibody intervention had reduced disease performance. On this basis, EPCR was knocked down at the same time. The therapeutic effect of PMN-neutralizing antibody treatment was subsequently diminished. To explore the relationship between EPCR and PMN in RA, we used immunofluorescence to detect the expression of PMN-neutrophil extracellular traps (NETs) in RA patients and used EPCR neutralizing antibodies as an intervention. The results showed that the formation of PMN-NETs in RA patients increased. Finally, through in vitro intervention experiments involving EPCR and PMN transcriptome analysis of the peripheral blood of RA patients, we concluded that EPCR may regulate the formation of PMN-NETs in RA patients through the activated protein C (APC)-EPCR signaling pathway, thereby affecting the progression of disease in RA patients.

Enhanced proton conductivity of Nafion membrane induced by incorporation of MOF-anchored 3D microspheres: a superior and promising membrane for fuel cell applications.

A novel 3D core-shell material composed of polyacrylate carboxyl microspheres (PCMs) and ZIF-8 nanoparticles was used to promote proton conduction in the Nafion matrix. The Nafion/ZIF-8@PCM membranes displayed excellent proton conductivity (0.24 S cm-1) and physicochemical properties due to special structural characteristics. More importantly, this new concept has a strong practical guiding significance for the fabrication of novel PEMs.

A novel green lignosulfonic acid/Nafion composite membrane with reduced cost and enhanced thermal stability.

A green biopolymer, lignosulfonate acid (LSA), was first used as an additive in the Nafion membrane for fuel cell applications. The Nafion/LSA composite membrane displayed enhanced thermal stability and other satisfactory properties due to the stable aromatic groups and multiple active sites of LSA. More importantly, the cost-effectiveness and simple fabrication of such novel composite PEMs make their use in PEMFCs very attractive and economical.

Self-Assembled pH-Sensitive Nanoparticles Based on Ganoderma lucidum Polysaccharide-Methotrexate Conjugates for the Co-delivery of Anti-tumor Drugs.

In tumor therapy, polymer nanoparticles are ideal drug delivery materials because they can mask the disadvantages of anti-tumor drugs such as poor solubility in water, high toxicity, and side effects. However, most polymer-based nanoparticles do not themselves have anti-tumor properties. Herein, a novel pH-sensitive nanoparticle drug delivery system based on Ganoderma lucidum polysaccharides (GLPs), which have demonstrated anti-tumor activities, was designed to enable the delivery of methotrexate (MTX) and 10-hydroxycamptothecin (HCPT) to tumor cells, where they could exert synergistic anti-tumor effects. The prepared nanoparticles were irregularly spherical in shape with a uniform particle size of ∼190 nm, and they exhibited a high drug-loading capacity (MTX 21.5% and HCPT 22.6%) and excellent biocompatibility. Moreover, the loaded MTX and HCPT units were rapidly released under acidic conditions within the tumor cells while remaining stable under normal physiological conditions. Meanwhile, compared to free MTX and HCPT, the GLP-APBA-MTX/HCPT nanoparticles presented exhibited better tumor suppressive effects and fewer side effects in vivo, which indicates that they may be an effective anti-tumor treatment strategy.

Self-Assembling pH-Responsive Nanoparticle Platform Based on Pectin-Doxorubicin Conjugates for Codelivery of Anticancer Drugs.

Pharmaceutical science based on biological nanotechnology is developing rapidly in parallel with the development of nanomaterials and nanotechnology in general. Pectin is a natural polysaccharide obtainable from a wide range of sources. Here, we show that doxorubicin (DOX)-conjugated hydrophilic pectin (PET) comprising an amphiphilic polymer loaded with hydrophobic dihydroartemisinin (DHA) self-assemble into nanoparticles. Importantly, conjugated DOX and DHA could be released quickly in a weakly acidic environment by cleavage of the acid-sensitive acyl hydrazone bond. Confocal microscopy and flow cytometry confirmed that these PET-DOX/DHA nanoparticles efficiently delivered DOX into the nuclei of MCF-7 cells. Significant tumor growth reduction was monitored in a female C57BL/6 mouse model, showing that the PET-DOX/DHA nanoparticle-mediated drug delivery system inhibited tumor growth and may improve therapy. Thus, we have demonstrated that pectin may be useful in the design of materials for biomedical applications.

Machine learning-based cytokine microarray digital immunoassay analysis.

Serial measurement of a large panel of protein biomarkers near the bedside could provide a promising pathway to transform the critical care of acutely ill patients. However, attaining the combination of high sensitivity and multiplexity with a short assay turnaround poses a formidable technological challenge. Here, the authors develop a rapid, accurate, and highly multiplexed microfluidic digital immunoassay by incorporating machine learning-based autonomous image analysis. The assay has achieved 12-plexed biomarker detection in sample volume <15 µL at concentrations < 5 pg/mL while only requiring a 5-min assay incubation, allowing for all processes from sampling to result to be completed within 40 min. The assay procedure applies both a spatial-spectral microfluidic encoding scheme and an image data analysis algorithm based on machine learning with a convolutional neural network (CNN) for pre-equilibrated single-molecule protein digital counting. This unique approach remarkably reduces errors facing the high-capacity multiplexing of digital immunoassay at low protein concentrations. Longitudinal data obtained for a panel of 12 serum cytokines in human patients receiving chimeric antigen receptor-T (CAR-T) cell therapy reveals the powerful biomarker profiling capability. The assay could also be deployed for near-real-time immune status monitoring of critically ill COVID-19 patients developing cytokine storm syndrome.

Targeted Delivery of Dual Anticancer Drugs Based on Self-Assembled iRGD-Modified Soluble Drug-Polymer Pattern Conjugate Nanoparticles.

A tumor-penetrating peptide, iRGD (a tumor-homing peptide, CRGDKGPDC), could enhance the penetration of drugs via the specific receptor-binding affinity to αvß3 and NRP-1 that overexpressed on tumor vasculature and tumor cells. Considering the side effects of traditional chemotherapy, here, poly(ethylene glycol) (PEG, Mw = 7500)-based and iRGD-modified poly(ethylene glycol)-based nanoparticles were successfully prepared. iRGD, as a tumor-targeting and tumor-penetrating agent, was combined with PEG after the esterification reaction between PEG and diosgenin (DGN). After the efficient loading of 10-hydroxycamptothecin (HCPT), the iRGD-PEG-DGN/HCPT NPs of chemotherapy were established. The characteristics of iRGD-PEG-DGN/HCPT NPs were evaluated. This nano-delivery system possessed high drug loading efficiency (∼17.34 wt % HCPT), controlled release rate, good pH response, and iRGD active targeting and passive targeting with an appropriate size (∼140 nm). All these features forcefully indicated that the iRGD-modified drug delivery system could markedly ameliorate the tumor therapy efficacy compared to the nontargeted nanoparticles through enhancing the tumor accumulation and penetration.

HSCCC Separation of Three Main Compounds from the Crude Extract of Dracocephalum Tanguticum by Using Dimethyl Sulfoxide as Cosolvent.

Separation of natural compounds directly from the crude extract is a challenging work for traditional column chromatography. In the present study, an efficient method for separation of three main compounds from the crude extract of Dracocephalum tanguticum has been successfully established by high-speed counter-current chromatography (HSCCC). The crude extract was directly introduced into HSCCC by using dimethyl sulfoxide as cosolvent. Ethyl acetate/n-butyl alcohol/0.3% glacial acetic acid (4: 1: 5, v/v) system was used and three target compounds with purity higher than 80% were obtained. Preparative HPLC was used for further purification and three target compounds with purity higher than 98% were obtained. The compounds were identified as chlorogenic acid, pedaliin and pedaliin-6″-acetate.

Procyanidin B2 mitigates endothelial endoplasmic reticulum stress through a PPARδ-Dependent mechanism.

Hyperglycemia-induced endothelial endoplasmic reticulum (ER) stress is implicated in the pathophysiology of diabetes and its vascular complications. Procyanidins are enriched in many plant foods and have been demonstrated to exert several beneficial effects on diabetes, cardiovascular and other metabolic diseases. In the present study, we investigated the effect of procyanidin B2 (PCB2), the most widely distributed natural procyanidin, on ER stress evoked by high glucose in endothelial cells (ECs) and the underlying mechanisms. We showed that PCB2 mitigated the high glucose-activated ER stress pathways (PERK, IRE1α and ATF6) in human vascular ECs. In addition, we found that PCB2 attenuated endothelial ER stress via the activation of peroxisome proliferator-activated receptor δ (PPARδ). We demonstrated that PCB2 directly bound to and activated PPARδ. Conversely, GSK0660, a selective PPARδ antagonist, attenuated the suppressive effect of PCB2 on the ER stress signal pathway. Functionally, PCB2 ameliorated the high glucose-impaired endothelium-dependent relaxation in mouse aortas. The protective effect of PCB2 on vasodilation was abolished in the aortas pretreated with GSK0660 or those from the EC-specific PPARδ knockout mice. Moreover, the protective effects of PCB2 on ER stress and endothelial dysfunction required the inter-dependent actions of PPARδ and AMPK. Collectively, we demonstrated that PCB2 mitigated ER stress and ameliorated vasodilation via a PPARδ-mediated mechanism beyond its classic action as a scavenger of free radicals. These findings further highlighted the novel roles of procyanidins in intervening the ER stress and metabolic disorders related to endothelial dysfunction.

Separation of five flavonoids with similar polarity from Caragana korshinskii Kom. by preparative high speed counter-current chromatography with recycling and heart cut mode.

Five flavonoids with similar polarity were obtained from Caragana korshinskii Kom. by preparative high speed counter-current chromatography. Due to their similar polarity, it is difficult to select a suitable solvent system for one-step separation. Three fractions including one pure compound were only obtained with a relatively suitable solvent system of ethyl acetate: n-butanol:water (0.5% glacial acetic acid) (4:6:10, v:v:v). In order to improve the separation efficiency, recycling and heart cut modes were introduced. As a result, two flavonoids with separation factor value at 1.13 and the other two with separation factor value at 1.20 were successfully separated. Finally, five flavonoids with purity higher than 98% were obtained. Fortunately, a new compound named myricetin 3'-methyl ether 3-O-glucoside-7-O-rhamnoside was obtained. The results indicated that the recycling combined with heart-cut mode could be effective for the preparation of natural compounds with similar polarity.

[Research and development of certified reference material of swertioside].

The standard sample of natural products is an essential standard reference to determine the quality of the product in the quality control of natural products. To develop a certified reference material(CRM) of swertioside according to the Work Guideline for Reference Materials(3): Reference Material-General Principles and Statistical Method for Certification(GB/T 15000.3-2008), swertioside was purified from whole plant of Swertia mussotii by extraction, isolation and Prep-HPLC to obtain certified reference material of swertioside. The structure of swertioside was identified by IR, UV, high-resolution MS, NMR. Thin layer chromatography, optical rotation, elemental analysis and melting point was carried out for the identification. The purity of the prepared sample was tested from different chromatographic elution conditions, thin layer chromatography and HPLC-MS. Swertioside was divided into 140 bottles, with 10 mg per bottle after homogeneity test, stability test and quantitative analysis. This CRM is 7-O-[α-L-rhamnopyranosyl-(1→2)-ß-D-xylopyranosyl]; the homogeneity of the 95% confidence interval was good; the certified purity value was 98.66%, with a relative expanded uncertainty of 0.38%; the storage period was 36 months at 0-8 ℃. Therefore, the CRM of sakuranetin reached the technical requirements of CRM, and was accepted by SAC. Swertioside is successfully developed and can be used for determining content, evaluating test methods, detecting relevant products and controlling quality.

Separation of 3-Hydroxy-3-Methylglutaryl Flavonoid Glycosides from Oxytropis falcata Using 2D Preparative Chromatography.

Oxytropis falcata Bunge, known as "the King of Herbs" in Tibetan medicine, is used for treatment of hyperpyrexia, pain, wounds, inflammation and anthrax. However, it is difficult to isolate compound with high-purity from O. falcata because of its complexity. In this work, an efficient method was successfully established for the separation of 3-hydroxy-3-methylglutaryl (HMG) flavonoid glycosides from O. falcata by 2D preparative chromatography (2D-HPLC). An ODS C18 preparative column was used for the first-dimension preparation, and the XCharge C18 preparative column with different separation selectivity to the ODS C18 stationary phase was used for the second-dimension preparation. Four HMG flavonoid glycosides (oxytroflavosides A-D) and two flavonoid glycosides (oxytroflavosides F and G) were separated with purities over 98%. Their structures were elucidated by nuclear magnetic resonance spectroscopy. The 2D-HPLC method used in this study is effective for preparative separation of HMG flavonoid glycosides from O. falcata. Additionally, this method shows great potential for the separation of flavonoid glycosides from other plant extracts.

Combined chromatographic strategy based on macroporous resin, high-speed counter-current chromatography and preparative HPLC for systematic separation of seven antioxidants from the fruit of Terminalia billerica.

In the present study, combined chromatographic strategy based on macroporous resin, high-speed counter-current chromatography and preparative high-performance liquid chromatography for systematic separation of antioxidants from crude samples guided by high-performance liquid chromatography with 1,1-diphenyl-2-picrylhydrazyl has been successfully established. Based on this strategy, seven antioxidants including isorugosin A, ß -1,2,3,6-tetragalloyl-D-glucose, chebulinic acid, 1,2,3,4,6-penta-O-galloyl-ß -D-glucose, chebulagic acid, ethyl gallate, and gallic acid were obtained from the fruit of Terminalia billerica. First, high-performance liquid chromatography with 1,1-diphenyl-2-picrylhydrazyl experiment showed the presence of seven main antioxidants in the crude extract of the fruit of Terminalia billerica. Then, a macroporous resin column chromatography method was developed for the enrichment of these seven antioxidants. Finally, an efficient method based on high-speed counter-current chromatography and preparative high-performance liquid chromatography was developed for the separation of these antioxidants. In the selection of solvent systems, it was found that acetic acid could be a good regulator for modifying the partition coefficient values of tannins. The present study provides a reference for systematic separation of antioxidants from crude samples. Considering the general existence of antioxidants in crude samples, this combined chromatographic strategy might lead to broader application prospects.

An efficient prodrug-based nanoscale delivery platform constructed by water soluble eight-arm-polyethylene glycol-diosgenin conjugate.

Drug resistance in tumors is one of the reasons result in the low anticancer efficiency of numerous drugs. Combination therapy has been proven to be a valid way against drug-resistant cancers. However, simply mix the drugs will not only cause many side efforts but also decrease anticancer effect. Herein, a self-assembled nanoparticle platform based on eight-arm-polyethylene glycol-diosgenin (8armPEG-DGN) conjugate was produced for encapsulating another hydrophobic anticancer drug. The 8armPEG-DGN/HCPT NPs were prepared through a simple nanoprecipitation method. The 8armPEG-DGN/HCPT NPs possess suitable size (~107 nm) and high binary drug loading capacity (15.67 wt% of DGN and 14.72 wt% of HCPT). Laser confocal scanning microscopy revealed that 8armPEG-DGN/HCPT NPs significantly increased intracellular uptake toward B16 cells compared with free drugs. Cytotoxicity assay showed the IC50 of 8armPEG-DGN/HCPT NPs were lower than simply mixing DGN and HCPT. In vivo tumor transplantation assay indicated that 8armPEG-DGN/HCPT NPs exhibited superior tumor grown inhibition compared with free drugs and HCPT/DGN Mix. These studies showed that the prepared 8armPEG-DGN/HCPT NPs drug delivery system could serve as a promising candidate for cancer therapy.

A practicable strategy for enrichment and separation of four minor flavonoids including two isomers from barley seedlings by macroporous resin column chromatography, medium-pressure LC, and high-speed countercurrent chromatography.

Separation of minor compounds especially with similar polarities and structures from complex samples is a challenging work. In the present study, an efficient method was successfully established by macroporous resin column chromatography, medium-pressure liquid chromatography, and high-speed countercurrent chromatography for separation of four minor flavonoids from barley seedlings. Macroporous resin column chromatography and medium-pressure liquid chromatography were used for enrichment of these four flavonoids. High-pressure liquid chromatography analysis showed the total content of these four flavonoids increased from 2.2% in the crude extract to 95.3% in the medium-pressure liquid chromatography fraction. It was indicated that the combination of macroporous resin column chromatography and medium-pressure liquid chromatography could be a practicable strategy for enrichment of minor compounds from complex sample. Then, high-speed countercurrent chromatography was employed for separation of these four flavonoids using ethyl acetate/n-butanol/water (0.1% glacial acetic acid) (4:1:5, v/v/v) as solvent system. As a result, four flavonoids including two isomers with purities higher than 98% were obtained. Interestingly, two flavonoids existing in one high-pressure liquid chromatography peak were also successfully separated. All these indicated high-speed countercurrent chromatography had great potential for separation of compounds with similar structures and polarities. This study provides a reference for efficient enrichment and separation of minor compounds from complex sample.