Preparation and characterization of antibody-drug conjugates acting on HER2-positive cancer cells.

Two systems of antibody-drug conjugates (ADCs), noncleavable H32-DM1 and cleavable H32-VCMMAE, were developed by using different linkers and drugs attached to the anti-HER2 antibody H32, which is capable of cell internalization. Activated functional groups, including an N-hydroxysuccinimidyl (NHS) ester and a maleimide, were utilized to make the ADCs. Mass spectrometry, hydrophobic interaction chromatography, polyacrylamide gel electrophoresis, and in vitro cell assays were performed to analyze and optimize the ADCs. Several H32-VCMMAE ADCs were established with higher DARs and greater synthetic yields without compromising potency. The anticancer efficacy of H32-DM1 was 2- to 8-fold greater than that of Kadcyla®. The efficacy of H32-VCMMAE was in turn better than that of H32-DM1. The anticancer efficacy of these ADCs against N87, SK-BR-3 and BT474 cells was in the following order: H32-VCMMAE series > H32-DM1 series > Kadcyla®. The optimal DAR for H32-VCMMAE was found to be 6.6, with desirable attributes including good cell penetration, a releasable payload in cancer cells, and high potency. Our results demonstrated the potential of H32-VCMMAE as a good ADC candidate.

Nε-carboxymethyllysine-mediated endoplasmic reticulum stress promotes endothelial cell injury through Nox4/MKP-3 interaction.

N(ε)-carboxymethyllysine (CML) is an important driver of diabetic vascular complications and endothelial cell dysfunction. However, how CML dictates specific cellular responses and the roles of protein tyrosine phosphatases and ERK phosphorylation remain unclear. We examined whether endoplasmic reticulum (ER) localization of MAPK phosphatase-3 (MKP-3) is critical in regulating ERK inactivation and promoting NADPH oxidase-4 (Nox4) activation in CML-induced endothelial cell injury. We demonstrated that serum CML levels were significantly increased in type 2 diabetes patients and diabetic animals. CML induced ER stress and apoptosis, reduced ERK activation, and increased MKP-3 protein activity in HUVECs and SVECs. MKP-3 siRNA transfection, but not that of MKP-1 or MKP-2, abolished the effects of CML on HUVECs. Nox4-mediated activation of MKP-3 regulated the switch to ERK dephosphorylation. CML also increased the integration of MKP-3 with ERK, which was blocked by silencing MKP-3. Exposure of antioxidants abolished CML-increased MKP-3 activity and protein expression. Furthermore, immunohistochemical staining of both MKP-3 and CML was increased, but phospho-ERK staining was decreased in the aortic endothelium of streptozotocin-induced and high-fat diet-induced diabetic mice. Our results indicate that an MKP-3 pathway might regulate ERK dephosphorylation through Nox4 during CML-triggered endothelial cell dysfunction/injury, suggesting that therapeutic strategies targeting the Nox4/MKP-3 interaction or MKP-3 activation may have clinical implications for diabetic vascular complications.

Advanced glycation end product Nε-carboxymethyllysine induces endothelial cell injury: the involvement of SHP-1-regulated VEGFR-2 dephosphorylation.

N(ε)-carboxymethyllysine (CML), a major advanced glycation end product, plays a crucial role in diabetes-induced vascular injury. The roles of protein tyrosine phosphatases and vascular endothelial growth factor (VEGF) receptors in CML-related endothelial cell injury are still unclear. Human umbilical vein endothelial cells (HUVECs) are a commonly used human EC type. Here, we tested the hypothesis that NADPH oxidase/reactive oxygen species (ROS)-mediated SH2 domain-containing tyrosine phosphatase-1 (SHP-1) activation by CML inhibits the VEGF receptor-2 (VEGFR-2, KDR/Flk-1) activation, resulting in HUVEC injury. CML significantly inhibited cell proliferation and induced apoptosis and reduced VEGFR-2 activation in parallel with the increased SHP-1 protein expression and activity in HUVECs. Adding recombinant VEGF increased forward biological effects, which were attenuated by CML. The effects of CML on HUVECs were abolished by SHP-1 siRNA transfection. Exposure of HUVECs to CML also remarkably escalated the integration of SHP-1 with VEGFR-2. Consistently, SHP-1 siRNA transfection and pharmacological inhibitors could block this interaction and elevating [(3)H]thymidine incorporation. CML also markedly activated the NADPH oxidase and ROS production. The CML-increased SHP-1 activity in HUVECs was effectively attenuated by antioxidants. Moreover, the immunohistochemical staining of SHP-1 and CML was increased, but phospho-VEGFR-2 staining was decreased in the aortic endothelium of streptozotocin-induced and high-fat diet-induced diabetic mice. We conclude that a pathway of tyrosine phosphatase SHP-1-regulated VEGFR-2 dephosphorylation through NADPH oxidase-derived ROS is involved in the CML-triggered endothelial cell dysfunction/injury. These findings suggest new insights into the development of therapeutic approaches to reduce diabetic vascular complications.

A novel method of ultrasound-assisted dispersive liquid-liquid microextraction coupled to liquid chromatography-mass spectrometry for the determination of trace organoarsenic compounds in edible oil.

A novel approach, ultrasound-assisted dispersive liquid-liquid microextraction combined with liquid chromatography-mass spectrometry (UA-DLLME with LC-MS) is demonstrated to be quite useful for the determination of trace amounts of organoarsenic compounds in edible oil. The organoarsenic compounds studied include dimethylarsinic acid (DMA), monomethylarsonic acid (MMA) and 3-nitro-4-hydroxyphenyl arsenic acid (Roxarsone). Orthogonal array experimental design (OAD) was utilized to investigate the parameter space of conditions for UA-DLLME. The optimum conditions were found to be 4 min of ultrasonic extraction using 1.25 mL of mixed solvent with 50 µL of buffer solution. Under these optimal conditions, the linear range was from 10 ng g(-1) to 500 ng g(-1) for DMA and Roxarsone, from 25 ng g(-1) to 500 ng g(-1) for MMA. Limits of detection of DMA, MMA and Roxarsone were 1.0 ng g(-1), 3.0 ng g(-1) and 5.8 ng g(-1), respectively. The precisions and recoveries also were investigated by spiking 3-level concentrations in edible oil. The recoveries obtained were over 89.9% with relative standard deviation (RSD) of 9.6%. The new approach was utilized to successfully detect trace amounts of organoarsenic compounds in various edible oil samples.

Electrically assisted solid-phase microextraction combined with liquid chromatography-mass spectrometry for determination of parathion in water.

A novel method for electrically assisted microextraction coupled to liquid chromatography-mass spectrometry was evaluated for determination of trace levels of parathion in water. A pencil lead electrode was used in a di-electrode system to extract parathion onto the electrode surface with a reductive potential applied. The optimum extraction conditions were found to be a potential of -600 mV for 60s in pH 2 phosphate buffer solution. The parathion was desorbed statically for 1 min and dynamically for 3 min in the commercial SPME-HPLC desorption chamber, then analyzed with LC-APCI-MS/MS. The detection limit (LOD) for parathion in water was found to be 0.3 ng/mL. The proposed technique was demonstrated to be fast, sensitive and not require a solvent sample pretreatment.

Determination of additives in cosmetics by supercritical fluid extraction on-line headspace solid-phase microextraction combined with gas chromatography-mass spectrometry.

A new hyphenated technique couples supercritical fluid extraction in situ derivatization and on-line headspace solid-phase microextraction to gas chromatography-mass spectrometry (SFE in situ derivatization on-line HS-SPME-GC-MS) for the determination of paraben preservatives and polyphenolic antioxidants in cosmetics. The preservatives and antioxidants were extracted from the cosmetic matrices with supercritical carbon dioxide at a pressure of 13,840 kPa. The supercritical fluid extraction was performed at 55 degrees C for 10 min of static extraction then 15 min of dynamic extraction. The extractant subsequently was derivatized in situ with the silylation reagent N,O-bis(trimethylsilyl)trifluoroacetamide with 0.1% trimethylchlorosilane. The product was then adsorbed on a polyacrylate solid-phase microextraction (SPME) fiber in the headspace. Sea sand was used as a dispersive material in the SFE step. The analytical linear ranges for the preservatives and antioxidants were found to be from 10 to 1000 ng g(-1) with RSD values below 7.8%. The detection limits ranged from 0.5 to 8.3 ng g(-1). These results are better than those obtained by using only SPME or SFE for trace preservatives and antioxidants analysis in cosmetic matrices. The new method was successfully utilized to determine the amounts of preservatives and antioxidants in real cosmetics without the need for tedious pretreatments.

Simultaneous production of trehalose, bioethanol, and high-protein product from rice by an enzymatic process.

Rice is a starch-rich raw material that can be used for trehalose production. It can be hydrolyzed with alpha-amylase, beta-amylase, and pullulanase to produce high-maltose content of rice saccharified solution for bioconversion of maltose into trehalose by trehalose synthase (TSase). For this purpose, an efficient enzymatic procedure has been successfully developed to simultaneously produce value-added trehalose, bioethanol, and high-protein product from rice as substrate. The highest maltose yield produced from the liquefied rice starch hydrolysate was 82.4 +/- 2.8% at 50 degrees C and pH 5.0 for 21-22 h. The trehalose conversion rate can reach at least 50% at 50 degrees C and pH 5.0 for 20-24 h by a novel thermostable recombinant Picrophilus torridus trehalose synthase (PTTS). All residual sugar, except trehalose, can be fully hydrolyzed by glucoamylase into glucose for further bioethanol production. The insoluble byproduct containing high yields of protein (75.99%) and dietary fiber (14.01%) can be processed as breakfast cereal product, health food, animal forage, etc. The conversion yield of bioethanol was about 98% after 64 h of fermentation time by Saccharomyces cerevisiae without any artificial culture solution addition. Ethanol can easily be separated from trehalose by distillation with a high recovery yield and purity of crystalline trehalose of 92.5 +/- 8.7% and 92.3%, respectively.

High throughput screening various abused drugs and metabolites in urine by liquid chromatography-heated electrospray ionization/tandem mass spectrometry.

An integrated method of liquid chromatography-heated electrospray ionization/tandem mass spectrometry was evaluated for high throughput screening of various abused drugs in urine. Chromatographic analysis was performed on a C18 reverse phase column using a linear gradient of 10mM ammonium acetate containing 0.1% formic acid-methanol as mobile phase and the total separation time was 7 min. A simple and rapid sample preparation method used was by passing urine samples through a 0.22 microm PVDF syringe filter. The detection limits of the studied abused drugs in urine were from 0.6 ng mL(-1) (ketamine) to 9.0 ng mL(-1) (norcodeine). According to the results, the linear range was from 1 to 1200 ng mL(-1) with relative standard deviation (R.S.D.s) value below 14.8% (intra-day) and 24.6% (inter-day). The feasibility of applying the proposed method to determine various abused drugs in real samples was examined by analyzing urine samples from drug-abused suspects. The abused drugs including ketamines and amphetamines were detected in suspected urine samples. The results demonstrate the suitability of LC-HESI-MS/MS for high throughput screening of the various abused drugs in urine.