Semi-quantitative IHC is a powerful method for investigating protein expression and localization within tissues. The semi-quantitative immunohistochemistry (IHC) involves using software such as free software ImageJ Fiji to conduct deconvolution and downstream analysis. Currently, there is lack of an integrated protocol that includes a detailed procedure of how to measure or compare protein expression. Publications that use semi-quantification methods to quantify protein expression often don't provide enough details in their methods section, which makes it difficult for the reader to reproduce their data. The current protocol for the first time provides a detailed, stepby-step instruction of conducting semi-quantitative analysis of IHC images using ImageJ Fiji software so that researchers would be able to follow this single protocol to conduct their research. The protocol uses semi-quantitative IHC of organic anion transporting polypeptide (OATP1B1) as an example, and gives detailed steps on how to deconvolute IHC images stained with hematoxylin and 3, 3-diaminobenzidine (DAB) and how to quantify their expression using ImageJ Fiji. The protocol includes clear steps for a reader so that this method can be applied to many different proteins. We anticipate this method will provide a practical guidance to the reader and make semiquantification of proteins an easier task to include in publications.

Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP(+)RFP(+) puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 μM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.

Previous reports have indicated that in vitro biliary clearance (Cl biliary ) determined in sandwich-cultured hepatocytes correlates well with in vivo Cl biliary for limited sets of compounds. This study was designed to estimate the in vitro Cl biliary in sandwich-cultured rat hepatocytes (SCRHs) of angiotensin II receptor blockers and HMG-CoA reductase inhibitors that undergo limited metabolism, to compare the estimated Cl biliary values with published in vivo Cl biliary data in rats, and to characterize the mechanism(s) of basolateral uptake and canalicular excretion of these drugs in rats. The average biliary excretion index (BEI) and in vitro Cl biliary values of olmesartan, valsartan, pravastatin, rosuvastatin, and pitavastatin were 15, 19, 43, 45, and 20%, respectively, and 1.7, 3.2, 4.4, 46.1, and 34.6 ml/min/ kg, respectively. Cl biliary predicted from SCRHs, accounting for plasma unbound fraction, correlated with reported in vivo Cl biliary for these drugs. The rank order of Cl biliary values predicted from SCRHs was consistent with in vivo Cl biliary values. Bromosulfophthalein inhibited the uptake of all drugs. BEI and Cl biliary values of olmesartan, valsartan, pravastatin, and rosuvastatin, known multidrug resistance-associated protein (Mrp) 2 substrates, were reduced in SCRHs from Mrp2-deficient (TR Ϫ ) compared with wild-type (WT) rats. Although Mrp2 plays a minor role in pitavastatin biliary excretion, pitavastatin BEI and Cl biliary were reduced in TR Ϫ compared with WT SCRHs; Bcrp expression in SCRHs from TR Ϫ rats was decreased. In conclusion, in vitro Cl biliary determined in SCRHs can be used to estimate and compare in vivo Cl biliary of compounds in rats and to characterize transport proteins responsible for their hepatic uptake and excretion.

The present study reports on the extraction of phenolic compounds from sparganii rhizome. Box-Behnken Design (BBD), a widely used form of response surface methodology (RSM), was used to investigate the effect of process variables on the ultrasound-assisted extraction (UAE). Three independent variables including ethanol concentration (%), extraction time (min) and solvent-to-material ratio (mL/g) were studied. The results showed that the optimal UAE condition was obtained with an ethanol concentration of 75.3%, an extraction time of 40min and a solvent-to-material ratio of 19.21mL/g for total phenols, and an ethanol concentration of 80%, an extraction time of 33.54min and solvent-to-material ratio of 22.72mL/g for combination of ρ-hydroxybenzaldehyde, ρ-coumaric acid, vanillic acid, ferulic acid, rutin and kaempferol. The experimental values under optimal conditions were in good consistent with the predicted values, which suggested UAE is more efficient process as compared to solvent extraction.

Organic anion transporting polypeptides (OATP) 1B1 and OATP1B3 are important hepatic transporters that mediate the uptake of many clinically important drugs, including statins from the blood into the liver. Reduced transport function of OATP1B1 and OATP1B3 can lead to clinically relevant drug-drug interactions (DDIs). Considering the importance of OATP1B1 and OATP1B3 in hepatic drug disposition, substantial efforts have been given on evaluating OATP1B1/1B3-mediated DDIs in order to avoid unwanted adverse effects of drugs that are OATP substrates due to their altered pharmacokinetics. Growing evidences suggest that the transport function of OATP1B1 and OATP1B3 can be regulated at various levels such as genetic variation, transcriptional and post-translational regulation. The present review summarizes the up to date information on the regulation of OATP1B1 and OATP1B3 transport function at different levels with a focus on potential impact on OATP-mediated DDIs.

Aim: To systematically examine the effectiveness of music therapy on preterm infants in neonatal intensive care unit. Background: In recent years, the application of music therapy for preterm infants in neonatal intensive care unit has attracted more and more attention because of its clinical effects. However, there still exist disputes among different studies. Design: A systematic review and meta-analysis. Data sources: Eleven databases were searched over the period from 1910-4 November 2019. Review methods: Papers were selected for analysis in accordance with the PRISMA guidelines. The meta-analysis was carried out by using Review Manager 5.3 software. Results: A total of 13 trials involving 1,093 participants were included. Meta-analysis showed music therapy had a significant influence on preterm infant's heart rate, respiratory rate, oral feeding volume, stress level, and maternal anxiety with moderate-to-high heterogeneity among studies. Also, music therapy had no influences on oxygen saturation and behavioural state. Conclusions: Music therapy can not only effectively improve preterm infant's heart rate, stable respiratory rate, and attenuate stress level but also exert positive impact on oral feeding volume. In addition, music therapy also plays a role in reducing maternal anxiety. However, due to the heterogeneity across studies in some outcomes, further studies with larger sample size and more stringent design should be conducted before recommendation. Impact: Music therapy can significantly improve preterm infant's heart rate, respiratory rate, and stress level, as well as increase oral feeding volume. These results may exert a positive impact on well-being and quality of life in preterm infants in the neonatal intensive care unit. Hospitals can apply music therapy which has been considered a non-pharmacological and no-invasive treatment to preterm infants in the neonatal intensive care unit.

BCRP transports numerous drugs/derived metabolites and toxins, and exhibits overlapping substrate specificity with P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2). Assessing the contribution of BCRP to drug/metabolite biliary excretion in the intact hepatocytes remains a challenge. Current studies were designed to develop a novel in vitro tool to specifically assess the contribution of Bcrp to drug biliary excretion. Adenoviral vectors expressing short hairpin (sh) RNA targeting Bcrp (Ad-si01Bcrp) or a non-target control (Ad-siNT) were packaged and infected into sandwich-cultured rat hepatocytes (SCRH). Protein levels were detected by immunoblot. Biliary excretion index (BEI) and in vitro biliary clearance (Clbiliary) of nitrofurantoin (BCRP substrate) and digoxin (P-gp substrate) were compared among non-infected, Ad-siNT- and Ad-si01Bcrp-infected cells in SCRH. shRNA targeting Bcrp efficiently knocked down Bcrp in SCRH, while levels of other transport proteins (P-gp, Mrp2, Bsep, Mrp4 and Oatp1a1) were unaffected. In SCRH exhibiting Bcrp knockdown, cellular accumulation of nitrofurantoin was increased markedly and nitrofurantoin BEI and in vitro Clbiliary were decreased to 11% and 14% of control, respectively. Digoxin values were unaffected by knockdown of Bcrp. Results indicated that Bcrp contributed predominantly to nitrofurantoin biliary excretion, but played a negligible role in digoxin biliary excretion. In summary, Bcrp knockdown in SCRH is the first in vitro model utilizing intact hepatocytes to assess the contribution of Bcrp to the biliary excretion of drugs. This approach may be useful in predicting drug-drug interactions in biliary excretion and the consequence of impaired BCRP function on the hepatic exposure of drugs/derived metabolites.

The organic anion-transporting polypeptide (OATP) 1B3 is a membrane transport protein that mediates hepatic uptake of many drugs and endogenous compounds. Currently, determination of OATP-mediated drug-drug interactions in vitro is focused primarily on direct substrate inhibition. Indirect inhibition of OATP1B3 activity is under-appreciated. OATP1B3 has putative protein kinase C (PKC) phosphorylation sites. Studies were designed to determine the effect of PKC activation on OATP1B3-mediated transport in human hepatocytes using cholecystokinin-8 (CCK-8), a specific OATP1B3 substrate, as the probe. A PKC activator, phorbol-12-myristate-13-acetate (PMA), did not directly inhibit [ 3 H]CCK-8 accumulation in human sandwich-cultured hepatocytes (SCH). However, pretreatment with PMA for as little as 10 minutes rapidly decreased [ 3 H]CCK-8 accumulation. Treatment with a PKC inhibitor bisindolylmaleimide (BIM) I prior to PMA treatment blocked the inhibitory effect of PMA, indicating PKC activation is essential for downregulating OATP1B3 activity. PMA pretreatment did not affect OATP1B3 mRNA or total protein levels. To determine the mechanism(s) underlying the indirect inhibition of OATP1B3 activity upon PKC activation, adenoviral vectors expressing FLAG-Myc-tagged OATP1B3 (Ad-OATP1B3) were transduced into human hepatocytes; surface expression and phosphorylation of OATP1B3 were determined by biotinylation and by an anti-phosphor-Ser/Thr/Tyr antibody, respectively. PMA pretreatment markedly increased OATP1B3 phosphorylation without affecting surface or total OATP1B3 protein levels. In conclusion, PKC activation rapidly decreases OATP1B3 transport activity by posttranslational regulation of OATP1B3. These studies elucidate a novel indirect inhibitory mechanism affecting hepatic uptake mediated by OATP1B3, and provide new insights into predicting OATP-mediated drug interactions between OATP substrates and kinase modulator drugs/endogenous compounds.