Nanobody-Nanoluciferase Fusion Protein-Enabled Immunoassay for Ochratoxin A in Coffee with Enhanced Specificity and Sensitivity.

Ochratoxin A (OTA), one of the best-known mycotoxins, causes problems concerning food safety with potential toxic effects in humans and animals. So, it is crucial to develop simple and sensitive methods for the detection of OTA. Herein, a nanoluciferase-nanobody fusion protein (Nb28-Nluc)-retaining antibody recognition and enzymatic activity was first prepared, which was then applied as a bifunctional tracer to construct a one-step bioluminescent enzyme-linked immunosorbent assay (BLEIA) for OTA in coffee samples. On the basis of Nb28-Nluc, the BLEIA can be completed with a one-step incubation and detection, with only a substrate replacement from 3,3',5,5'-tetramethylbenzidine (TMB) to a Nluc assay reagent (Furimazine). Under the optimal experimental conditions, the proposed one-step BLEIA achieved a detection limit of 3.7 ng/mL (IC10) within 3 h. Moreover, the BLEIA method showed good repeatability and accuracy in the spike recovery experiments with recoveries of 83.88% to 120.23% and relative standard deviations (RSDs) of 5.2% to 24.7%, respectively. Particularly, the BLEIA displayed superior performances, such as fewer operations and more rapid and sensitive detection as compared with Nb28-based enzyme-linked immunosorbent assay. Therefore, the proposed one-step BLEIA has great potential for the sensitive and accurate screening of OTA in food samples.

Relative increase in cerebellar gray matter in young onset essential tremor: Evidence from voxel-based morphometry analysis.

This study is to investigate the presence of brain gray matter abnormalities in young onset essential tremor (ET) patients with arm tremor. Thirty ET patients together with 30 healthy volunteers were taken as candidates. Magnetic resonance imaging (MRI) was performed and voxel-based morphometry was used to compare gray matter density between the patients and volunteers. Washington Heights-Inwood Genetic Study of Essential Tremor (WHIGET) rating scale was applied to assess tremor severity in the patients. Eventually Twenty-Seven ET patients and Twenty-Seven volunteers were enrolled in the study. Voxel-based morphometry showed significant expansion of the bilateral cerebellum, occipital fusiform cortices, right inferior temporal gyrus, and precentral lobes (P < 0.05, TFCE corrected). Decrease in gray matter was detected only in the left parietal lobe. Region of interest analysis showed volume enlargement in thalamus, midbrain, and precuneus (P < 0.05, TFCE corrected). Importantly, significant negative correlation was found between the lateralized index of cerebellum and the tremor score which might implicate that the altered rightward lateralization in the cerebellum is possibly a response of the tremor effects in ET patients. Cerebellar gray matter expansion in young onset ET patients with arm tremor might be the result of compensation towards decline of cerebellar function.

Electroacupuncture improves neurovascular unit reconstruction by promoting collateral circulation and angiogenesis.

Acupuncture at Shuigou (GV26) shows good clinical efficacy for treating stroke, but its mechanism remains poorly understood. In this study, a cerebral infarction model of ischemia/reperfusion injury received electroacupuncture at GV26 (15 Hz and 1 mA, continuous wave [biphasic pulses], for 5 minutes). Electroacupuncture effectively promoted regional cerebral blood flow on the infarct and non-infarct sides, increased infarct lesions, lectin, and number of blood vessels, upregulated von Willebrand factor and cell proliferation marker Ki67 expression, and diminished neurological severity score. These findings confirm that electroacupuncture at GV26 promotes establishment of collateral circulation and angiogenesis, and improves neurological function.

The protective effects of paeonol against epirubicin-induced hepatotoxicity in 4T1-tumor bearing mice via inhibition of the PI3K/Akt/NF-kB pathway.

Epirubicin is widely used for the treatment of various breast cancers; however, it has serious adverse side effects, such as hepatotoxicity, which require dose-adjustment or therapy substitution. Paeonol, an active component from Moutan Cortex, has a variety of biological activities, including preventing or reducing various toxicities induced by antineoplastics. Protection by paeonol against hepatotoxicity induced by epirubicin and the underlying mechanism of action were investigated in this study. Cytosolic enzymes in the serum and oxidative stress indices in the liver were determined. The protective effects were determined using the MTT assay in vitro or by evaluating the expression of apoptotic factors and crucial proteins in the PI3K/Akt/NF-kB pathway using western blot analysis. It is concluded that paeonol alleviates epirubicin-induced hepatotoxicity in 4T1-tumor bearing mice by inhibiting the PI3K/Akt/NF-kB pathway.

Discovery of cyclooxygenase inhibitors from medicinal plants used to treat inflammation.

Eleven authenticated botanicals used in the traditional Chinese medicine Huo-Luo-Xiao-Ling Dan were screened for ligands to cyclooxygenase (COX) using pulsed ultrafiltration liquid chromatography-mass spectrometry, and a mass spectrometry-based enzyme assay was used to determine the concentration of each of 17 ligands that inhibited COX-1 or COX-2 by 50% (IC(50)). Acetyl-11-keto-beta-boswellic acid, beta-boswellic acid, acetyl-alpha-boswellic acid, acetyl-beta-boswellic acid, and betulinic acid were COX-1 selective inhibitors with IC(50) values of approximately 10 microM. Senkyunolide O and cryptotanshinone were COX-2 selective inhibitors with IC(50) values of 5 microM and 22 microM, respectively. Roburic acid and phenethyl-trans-ferulate inhibited COX-1 and COX-2 equally. COX inhibition and the IC(50) values of most of these natural product ligands have not been reported previously.

Isolation and evaluation of kaempferol glycosides from the fern Neocheiropteris palmatopedata.

Kaempferol glycosides, named palmatosides A (1), B (2) and C (3), together with three known kaempferol glycosides, multiflorins A (4) and B (5), and afzelin (6), were isolated from the roots of the fern Neocheiropteris palmatopedata. Palmatosides A (1) and B (2) each possessed an unusual sugar moiety containing a 4,4-dimethyl-3-oxo-butoxy substituent group. The isolated compounds were evaluated for their cancer chemopreventive potential based on their ability to inhibit tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activity, nitric oxide (NO) production, aromatase, quinone reductase 2 (QR2) and COX-1/-2 activities. Palmatosides B (2) and C (3) inhibited TNF-alpha-induced NF-kappaB activity with IC(50) values of 15.7 and 24.1 microM, respectively; multiflorin A (4) inhibited aromatase enzyme with an IC(50) value of 15.5 microM; afzelin (6) showed 68.3% inhibition against QR2 at a concentration of 11.5 microg/ml; palmatoside A (1) showed 52% inhibition against COX-1 enzyme at a concentration of 10 microg/ml; and multiflorin B (5) showed 52% inhibition against nitric oxide production at a concentration of 20 microg/ml. In addition, compounds 3-6 were shown to bind QR2 enzyme using LC-MS ultrafiltration binding assay.