Determining the Optimal Administration Conditions under Which MIF Exerts Neuroprotective Effects by Inducing BDNF Expression and Inhibiting Apoptosis in an In Vitro Stroke Model.

Macrophage migration inhibitory factor (MIF) exerts neuroprotective effects against cerebral ischemia/reperfusion injury by inhibiting neuronal apoptosis and inducing the expression of brain-derived neurotrophic factor (BDNF). However, the optimal administration conditions of MIF are currently unknown. Here, we aimed to identify these conditions in an in vitro model. To determine the optimal concentration of MIF, human neuroblastoma cells were assigned to one of seven groups: control, oxygen and glucose deprivation/reperfusion (OGD/R), and OGD/R with different concentrations (1, 10, 30, 60, and 100 ng/mL) of MIF. Six groups were studied to investigate the optimal administration time: control, OGD/R, and OGD/R with MIF administered at different times (pre-OGD, OGD-treat, post-OGD, and whole-processing). Water-soluble tetrazolium salt-1 assay, Western blot analysis, and immunocytochemistry were used to analyze cell viability and protein expression. We found that 60 ng/mL was the optimal concentration of MIF. However, the effects of administration time were not significant; MIF elicited similar neuroprotective effects regardless of administration time. These findings correlated with the expression of BDNF and apoptosis-related proteins. This study provides detailed information on MIF administration, which offers a foundation for future in vivo studies and translation into novel therapeutic strategies for ischemic stroke.

Brain-derived neurotrophic factor mediates macrophage migration inhibitory factor to protect neurons against oxygen-glucose deprivation.

Macrophage migration inhibitory factor (MIF) is a chemokine that plays an essential role in immune system function. Previous studies suggested that MIF protects neurons in ischemic conditions. However, few studies are reported on the role of MIF in neurological recovery after ischemic stroke. The purpose of this study is to identify the molecular mechanism of neuroprotection mediated by MIF. Human neuroblastoma cells were incubated in Dulbecco's modified Eagle's medium under oxygen-glucose deprivation (OGD) for 4 hours and then returned to normal aerobic environment for reperfusion (OGD/R). 30 ng/mL MIF recombinant (30 ng/mL) or ISO-1 (MIF antagonist; 50 µM) was administered to human neuroblastoma cells. Then cell cultures were assigned to one of four groups: control, OGD/R, OGD/R with MIF, OGD/R with ISO-1. Cell viability was analyzed using WST-1 assay. Expression levels of brain-derived neurotrophic factor (BDNF), microtubule-associated protein 2 (MAP2), Caspase-3, Bcl2, and Bax were detected by western blot assay and immunocytochemistry in each group to measure apoptotic activity. WST-1 assay results revealed that compared to the OGD/R group, cell survival rate was significantly higher in the OGD/R with MIF group and lower in the OGD/R with ISO-1 group. Western blot assay and immunocytochemistry results revealed that expression levels of BDNF, Bcl2, and MAP2 were significantly higher, and expression levels of Caspase-3 and Bax were significantly lower in the MIF group than in the OGD/R group. Expression levels of BDNF, Bcl2, and MAP2 were significantly lower, and expression levels of Caspase-3 and Bax were significantly higher in the ISO-1 group than in the OGD/R group. MIF administration promoted neuronal cell survival and induced high expression levels of BDNF, MAP2, and Bcl2 (anti-apoptosis) and low expression levels of Caspase-3 and Bax (pro-apoptosis) in an OGD/R model. These results suggest that MIF administration is effective for inducing expression of BDNF and leads to neuroprotection of neuronal cells against hypoxic injury.

A novel dried blood spots analysis combined with on-spot reaction for determination of trimethylamine N-oxide and its related compounds.

The application of dried blood spots in clinical research is becoming increasingly popular owing to its convenient collection, storage, and transportation compared to that of conventional biological samples. The potential of trimethylamine N-oxide and its related compounds as biomarkers for various cardiovascular diseases, such as atherosclerosis, stroke, thrombosis, and heart failure, was recently highlighted, which was the driving force behind the development of an analytical method to identify trimethylamine N-oxide and eight related compounds in dried blood spots. In the proposed method, a novel "on-spot reaction" approach was introduced to overcome the low loading efficiency of trimethylamine in dried blood spots. Upon the addition of 50 µL of blood onto the filter paper pretreated with dilute HCl, an acid-base neutralization reaction in the blood spots transformed the volatile trimethylamine to a salt. Next, a punched disc with a diameter of 6.0 mm was eluted by agitation with 20 mM ammonium formate for 10 min and derivatized with 1.0 M ethyl bromoacetate at 80 °C for 60 min. A surrogate analyte approach was employed for quantification of these endogenous compounds in the complex matrix. Analysis was carried out using zwitterionic hydrophilic interaction liquid chromatography-high-resolution mass spectrometry. The established method was validated and applied to monitor real samples from 30 clinical cases. The proposed new methodology based on dried blood spots could greatly improve the convenience, analytical sensitivity, and selectivity of cardiovascular disease testing.

Development and investigation of a QuEChERS-based method for determination of phthalate metabolites in human milk.

Phthalates are commonly used as plasticizers and are known as risk factors toward several conditions such as cancer, birth defects, and endocrine disruption. Biomonitoring of phthalates is necessary to assess the potentially harmful effects of long-term exposure. In this work, we have developed a novel QuEChERS method to determine eight phthalate metabolites-mono-(3-carboxypropyl) phthalate, mono-(2-ethyl-5-carboxypentyl) phthalate, mono-(2-ethyl-5-hydroxyhexyl) phthalate, mono-(2-ethyl-5-oxohexyl) phthalate, mono-n-butyl phthalate, mono-benzyl phthalate, mono-(carboxyloctyl) phthalate, and mono-(carboxynonyl) phthalate-in human milk. The extraction process was optimized by comparing three different QuEChERS methods, and a further purification step was used to eliminate interferential lipid. In this process, several factors, such as the pH based on QuEChERS additive salts, acid dissociation constant, and distribution coefficient of the analyte, were found to have a significant effect on the extraction efficiency of the QuEChERS method. Target compounds were determined using liquid chromatography-tandem mass spectrometry equipped with electrospray ionization in the multiple-reaction monitoring mode. The developed method was verified by evaluating the selectivity, linearity, lower limit of quantification, accuracy, precision, and recovery, and applied to monitor real milk samples from 26 people. It is expected that the established method can be utilized not only to monitor phthalate metabolites in biological samples but also to identify the correlation between phthalate concentrations observed for the mother and the newborn.