Suppressing Wnt signaling of the blood‒tumor barrier to intensify drug delivery and inhibit lipogenesis of brain metastases.

Lipogenesis is often highly upregulated in breast cancer brain metastases to adapt to intracranial low lipid microenvironments. Lipase inhibitors hold therapeutic potential but their intra-tumoral distribution is often blocked by the blood‒tumor barrier (BTB). BTB activates its Wnt signaling to maintain barrier properties, e.g., Mfsd2a-mediated BTB low transcytosis. Here, we reported VCAM-1-targeting nano-wogonin (W@V-NPs) as an adjuvant of nano-orlistat (O@V-NPs) to intensify drug delivery and inhibit lipogenesis of brain metastases. W@V-NPs were proven to be able to inactivate BTB Wnt signaling, downregulate BTB Mfsd2a, accelerate BTB vesicular transport, and enhance tumor accumulation of O@V-NPs. With the ability to specifically kill cancer cells in a lipid-deprived environment with IC50 at 48 ng/mL, W@V-NPs plus O@V-NPs inhibited the progression of brain metastases with prolonged survival of model mice. The combination did not induce brain edema, cognitive impairment, and systemic toxicity in healthy mice. Targeting Wnt signaling could safely modulate the BTB to improve drug delivery and metabolic therapy against brain metastases.

G45 and V386 in Transmembrane Domains 1 and 8 Are Critical for the Activation of OATP1B3-Mediated E17ßG Uptake by Clotrimazole.

Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are two highly homologous transport proteins. However, OATP1B1- and 1B3-mediated estradiol-17ß-glucuronide (E17ßG) uptake can be differentially affected by clotrimazole. In this study, by functional characterization on chimeric transporters and single mutants, we find that G45 in transmembrane domain 1 (TM1) and V386 in TM8 are critical for the activation of OATP1B3-mediated E17ßG uptake by clotrimazole. However, the effect of clotrimazole on the function of OATP1B3 is substrate-dependent as clotrimazole does not stimulate OATP1B3-mediated uptake of 4',5'-dibromofluorescein (DBF) and rosuvastatin. In addition, clotrimazole is not transported by OATP1B3, but it can efficiently permeate the plasma membrane due to its lipophilic properties. Homology modeling and molecular docking indicate that E17ßG binds in a substrate binding pocket of OATP1B3 through hydrogen bonding and hydrophobic interactions, among which its sterol scaffold forms hydrophobic contacts with V386. In addition, a flexible glycine residue at position 45 is essential for the activation of OATP1B3. Finally, clotrimazole is predicted to bind at an allosteric site, which mainly consists of hydrophobic residues located at the cytoplasmic halves of TMs 4, 5, 10, and 11.

Importance of N-Glycosylation for the Expression and Function of Human Organic Anion Transporting Polypeptide 2B1.

Human organic anion transporting polypeptide 2B1 (OATP2B1) is a membrane transporter widely expressed in organs crucial for drug absorption and disposition such as the intestine, liver, and kidney. Evidence indicates that OATP2B1 is a glycoprotein. However, the sites of glycosylation and their contribution to the function and expression of OATP2B1 are largely unknown. In this study, by site-directed mutagenesis, we determined that two of four potential N-glycosylation sites in OATP2B1, N176 and N538, are indeed glycosylated. Functional studies revealed that the transport activities of mutants N176Q and N538Q were greatly reduced as compared to that of wild-type OATP2B1. However, the reduced activity was not due to the impairment of transport function per se but due to the decreased surface expression as the Km and normalized Vmax values of N176Q and N538Q were comparable to those of OATP2B1. Quantitative polymerase chain reaction (PCR) revealed that N176Q and N538Q mutations did not affect the expression of OATP2B1 at a transcriptional level. Immunofluorescence analysis showed that deglycosylated OATP2B1 was largely retained in the endoplasmic reticulum, which may activate the endoplasmic reticulum-associated degradation pathway, and the ubiquitin-proteasome system played a major role in the degradation of OATP2B1. Taken together, OATP2B1 is N-glycosylated, and N-glycosylation is essential for the surface expression of OATP2B1 but not critical for the transport function of OATP2B1 per se.

Effect of different chain lengths of monoglyceride on the O/W interfacial properties with high-melting and low-melting crystals in low-fat aerated emulsion.

The effect of different types of monoglycerides, including monopalmitin, capryl monoglyceride (GMB), and succinylated monoglyceride (GMSA) in combination with palm kernel stearin (PKS) and beeswax (BW), on the formation, crystal network structure, and partial coalescence properties of aerated emulsions (20 % w/w fat) was investigated. The stability of BW and PKS crystals with a 1 % concentration of GMSA and GMB, respectively, in the oil phase was lower than the other crystals. BW-GMSA and PKS-GMB crystals exhibited a lower crystallization rate, higher contact angles and no significant peak shift in the small-angle X-ray scattering results. The BW-GMSA and PKS-GMB emulsions had a lower nucleation rate in the bulk and a higher nucleation rate at the interface, resulting in a higher fraction of crystals adsorbed at the oil/water interface. This reduced the number of interfacial proteins and led to a high degree of partial coalescence and the formation of stable aerated networks.

Development of a sensitive LC-MS/MS method for the quantification of theranostic agent cypate in mouse plasma and application to a pharmacokinetic study.

Cypate, a heptamethine cyanine dye, is a prototypic near-infrared (NIR) theranostic agent for optical imaging and photothermal therapy. In the present study, a selective, sensitive, and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of cypate in mouse plasma. The chromatographic separation was achieved using a short C18 column (2.1 mm × 50 mm, 5 µm) with a run time of 5 min. The MS was operated in multiple reaction monitoring (MRM) mode via positive electrospray ionization. The ion transitions for cypate and internal standard IR-820 were m/z 626.3 â†’ 596.3 and m/z 827.4 â†’ 330.2, respectively. The method was linear over a concentration range of 1.0-500 ng/mL. The within-run and between-run precision was less than 14.4% with accuracy in the range of -13.4% ∼ 9.8%. The validated method was successfully applied to a pharmacokinetic study of cypate in mice following intravenous administration.

The Importance of Val386 in Transmembrane Domain 8 for the Activation of OATP1B3 by Epigallocatechin Gallate.

Estrone-3-sulfate (E3S) uptake mediated by organic anion transporting polypeptide 1B3 (OATP1B3) can be activated by epigallocatechin gallate (EGCG). In this study, by using chimeric transporters and site-directed mutagenesis, we found that Val386 in transmembrane domain 8 (TM8) is essential for OATP1B3's activation by EGCG. Kinetic studies showed that the loss of activation of 1B3-TM8 and 1B3-V386F in the presence of EGCG is due to their decreased substrate binding affinity and reduced maximal transport rate. The overall transport efficiencies of OATP1B3, 1B3-TM8, and 1B3-V386F in the absence and presence of EGCG are 8.6 ± 0.7 vs 15.9 ± 1.4 (p < 0.05), 11.2 ± 2.1 vs 2.7 ± 0.3 (p < 0.05), and 10.2 ± 1.0 vs 2.5 ± 0.3 (p < 0.05), respectively. While 1B3-V386F cannot be activated by EGCG, its transport activity for EGCG is also diminished. OATP1B3's activation by EGCG is substrate-dependent as EGCG inhibits OATP1B3-mediated pravastatin uptake. Furthermore, the activation of OATP1B3-mediated E3S uptake by quercetin 3-O-α-l-arabinopyranosyl(1 → 2)-α-l-rhamnopyranoside is not affected by TM8 and V386F. Taken together, the activation of OATP1B3 by small molecules is substrate- and modulator-dependent, and V386 in TM8 plays a critical role in the activation of OATP1B3-mediated E3S uptake by EGCG.

Crystal network structure and stability of beeswax-based oleogels with different polyunsaturated fatty acid oils.

The effect of different types of oils including camellia oil (CLO), sunflower oil (SFO), corn oil (CO) and linseed oil (LO) on the formation, crystal network structure and mechanical properties of 4%wt beeswax (BW) in oleogel was investigated. BW oleogels containing oils with higher contents of polyunsaturated fatty acids gelled first (1%wt), especially LO with higher contents of linolenic acid rather than CLO with higher contents of monounsaturated fatty acids. In comparison, oils with higher polyunsaturated fatty acid contents exhibited higher Db with more extensive microstructure at different cooling rates, which was related to shorter nucleation induction time of crystal and higher crystallinity. Stronger van der Waals forces were observed in oleogels with higher polyunsaturated fatty acid contents especially for LO oleogel. Rheology also showed that LO oleogel with higher content of linolenic acid had higher crystallinity and lower crystal melting interfacial tension, resulting in the formation of a more stable network structure.

Gelation behavior and crystal network of natural waxes and corresponding binary blends in high-oleic sunflower oil.

Wax-based oleogels attract considerable attention for their perfect gelation properties, but the waxy mouthfeel severely limits their implementation in food. Herein, we developed a novel strategy via designing the crystal network to produce wax-based oleogels with a suitable mouthfeel. Four natural waxes with different melting points were selected as oleogelators to investigate the gelation behavior. All waxes at 5 wt% concentrations could form stable oleogels with low-frequency dependence. Especially, rice bran wax (RBW) and beeswax (BW) with high oil-binding capacity indicated that the ordered crystal network with fiber or needle-like morphology is more suitable for trapping liquid oil. Interestingly, China lacquer wax (ZLW) presented satisfactory oral melting characteristics according to the melting properties. Subsequently, to enhance the structure of ZLW-oleogel, RBW and BW with desirable crystal networks were added at varying mass ratios (100:0, 75:25, 50:50, 25:75, and 0:100). The binary oleogels exhibited monotectic behavior from thermodynamic phase diagrams. The polarization microscope indicated that similar needle-like crystals in BW/ZLW system enhanced the order of network structure, while long fiber-like crystals by RBW dominated the crystallization of RBW/ZLW binary oleogels. Finally, the BW/ZLW binary oleogels with ratios of 25:75 and 50:50 showed no-waxy mouthfeels in sensory analysis. These findings provide strong theoretical support for the application of wax-based oleogels in plastic fats replacement. PRACTICAL APPLICATION: Natural wax-based oleogel has been widely investigated due to the high oil binding capacity and perfect gelation properties. But its waxy mouthfeel severely limits the application in the food industry. In this study, oleogels with no-waxy an mouthfeel were obtained by designing wax-blend crystalline network. These findings provide strong theoretical support for the application of wax-based oleogels in plastic fats replacement.

Human Organic Anion Transporting Polypeptides 1B1, 1B3, and 2B1 Are Involved in the Hepatic Uptake of Phenolsulfonphthalein.

Phenolsulfonphthalein (PSP or phenol red), a sulfonphthalein dye, has been used as a diagnostic agent and a pH indicator in cell culture medium. After administered into the body, PSP is excreted into urine and bile. The urinary excretion of PSP is mediated by organic anion transporter 1/3 (OAT1/3) and multidrug resistance protein 2 (MRP2). In biliary excretion, PSP is effluxed from hepatocytes into the bile via MRP2. However, so far, the molecular mechanism for PSP transport from the blood into hepatocytes is unclear. In the present study, six human major hepatic uptake transporters expressed on the basolateral membrane of hepatocytes, namely, organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, OATP2B1, Na+/taurocholate cotransporting polypeptide (NTCP), organic cation transporter 1 (OCT1), and OAT2, have been investigated to see whether they are involved in the hepatic uptake of PSP. An in vitro cell-based study demonstrated that PSP is a substrate for OATP1B1, OATP1B3, and OATP2B1, with OATP1B3 showing the highest transport efficiency. The K m values for OATP1B1-, OATP1B3-, and OATP2B1-mediated PSP uptake were 11.3 ± 1.5, 7.0 ± 1.5, and 5.1 ± 1.0 µM, respectively. PSP interacts with known OATP substrates/inhibitors. However, the presence of PSP in cell culture medium has no significant effect on OATP's function. In vivo pharmacokinetic study in wild-type and Oatp1b2-knockout mice showed that Oatp1b2-knockout led to elevated plasma concentration and decreased liver accumulation of PSP. Taken together, the present study showed that in the liver, OATP1B1, OATP1B3, and OATP2B1 are involved in the uptake of PSP from the blood into hepatocytes, which, along with MRP2-mediated efflux of PSP from hepatocytes into the bile, constitute the vectorial transport of PSP from the blood to the bile and may play a critical role in the biliary excretion of PSP.

Analysis of the monitoring status of residual nitrite in meat products in China from 2000 to 2011.

The aim of this article was to analyze the monitoring status of nitrite in meat products consumed from 2000 to 2011 in 24 provinces, autonomous regions or direct-controlled municipalities in China. Statistical analyses were performed on the monitoring status including number, proportion, and distribution of 13,316 samples, of which 11,320 (85%) contained up to 2808.2mg/kg nitrite and 1996 (15%) contained no nitrite. A total of 10,299 samples (77%) qualified for GB/T 5009.33-2003, 2003; however, 3017 samples (23%) contained nitrite at levels higher than the national standard. The districts with high percentage of samples with no nitrite were Shanghai (49%), Beijing (47%), and Liaoning (30%). While the districts with high percentage of meat products containing nitrite at levels exceeding the national standard were Jiangxi (49%), Jiangsu (33%), Shandong (29%) and Sichuan (29%). Therefore, the status of residue nitrite in meat products is of concern.

Clinical Study of NeuroRegen Scaffold Combined With Human Mesenchymal Stem Cells for the Repair of Chronic Complete Spinal Cord Injury.

Regeneration of damaged neurons and recovery of sensation and motor function after complete spinal cord injury (SCI) are challenging. We previously developed a collagen scaffold, NeuroRegen, to promote axonal growth along collagen fibers and inhibit glial scar formation after SCI. When functionalized with multiple biomolecules, this scaffold promoted neurological regeneration and functional recovery in animals with SCI. In this study, eight patients with chronic complete SCI were enrolled to examine the safety and efficacy of implanting NeuroRegen scaffold with human umbilical cord mesenchymal stem cells (hUCB-MSCs). Using intraoperative neurophysiological monitoring, we identified and surgically resected scar tissues to eliminate the inhibitory effect of glial scarring on nerve regeneration. We then implanted NeuroRegen scaffold loaded with hUCB-MSCs into the resection sites. No adverse events (infection, fever, headache, allergic reaction, shock, perioperative complications, aggravation of neurological status, or cancer) were observed during 1 year of follow-up. Primary efficacy outcomes, including expansion of sensation level and motor-evoked potential (MEP)-responsive area, increased finger activity, enhanced trunk stability, defecation sensation, and autonomic neural function recovery, were observed in some patients. Our findings suggest that combined application of NeuroRegen scaffold and hUCB-MSCs is safe and feasible for clinical therapy in patients with chronic SCI. Our study suggests that construction of a regenerative microenvironment using a scaffold-based strategy may be a possible future approach to SCI repair.

[Study on biological characteristics and stability of the linear derivative Bac2a from bactenecin].

The objective of the study is to analyze the biological characteristics and stability of the linear derivative Bac2a from bactenecin, compared with the control peptide melittin. The secondary structure, antibacterial activity, hemolytic activity, cell toxicity and stability of the Bac2a were determined by circular dichroism spectroscopy, broth micro-dilution method and MTT assay. The results showed that Bac2a was a nonregular curl in aqueous solution, however, it was an α-helix structure in the hydrophobic environment. The minimal inhibitory concentration (MIC) of Bac2a ranged from 2 to 32 µmol/L, so the bacteriostatic activity of Bac2a was strong. The hemolytic rate was only 14.81% when the concentration of Bac2a was 64 µmol/L, which showed that the hemolytic rate of Bac2a was low. The therapy index of Bac2a was 3.26, and the cytotoxicity was relatively low, thus the cell selectivity was relatively high. In addition, with the heating treatment of 100℃ for 1 h, Bac2a still possessed rather a high antibacterial activity and showed a good heating stability. In a word, Bac2a has good application prospects in food, medicine and other fields, and is expected as a substitute for traditional antibiotics.

[Therapeutic bloodletting at Jing-well points combine hypothermia attenuated acute cerebral edema after traumatic brain injury in rats].

OBJECTIVE: To investigate the influence of therapeutic bloodletting at Jing-well points and hypothermia on acute cerebral edema after traumatic brain injury (TBI) in rats. METHODS: Seventy-five SD rats were randomly divided into sham-operation group (Sham), TBI group (TBI), bloodletting group (BL), mild-induced hypothermia group (MIH), and bloodletting plus MIH group (BL + MIH) (n = 15). The model of TBI was established by electric controlled cortical impactor (eCCI). The rats of BL group were bloodletting at Jing-well points immediately after injury, twice daily. While the MIH group was settled on a hypothermia blanket promptly after TBI for 6 hours, so that the temperature dropped to 32 degrees. Each of measurement was performed after 48 hours. Magnetic resonance imaging (MRI) was used to evaluate the dynamic impairment of cerebral edema after TBI (n = 3). In addition, mNSS score, measurements of wet and dry brain weight, and Evans Blue assay were performed to investigate the neurologic deficit, cerebral water content (n = 8), and blood-brain barrier permeability (BBB), (n = 4), respectively. RESULTS: MRI analysis showed that the cerebral edema, hematoma and midline shifting of rats in TBI group was more serious than other treatment group. Meanwhile compared with TBI group, the mNSS scores of every treatment group were meaningfully lower (all P < 0.05). Furthermore, treatment with BL+ MIH group was superior to the separated BL and MIH group (all P < 0.01). In addition, brain water content of each intervention group reduced to varying degrees (all P < 0.05), especially that of MIH group and BL + MIH group (P <0.01). BBB permeability of each treatment group was also significantly improved (all P < 0.01), and the improvement in MIH group and BL + MIH group was much better than the BL alone group (P < 0.05, P < 0.01). CONCLUSION: Our major finding is that bloodletting at Jing-well points and MIH can reduce cerebral edema and BBB dysfunction and exert neuroprotective effects after TBI. The results suggest that the combination of BL and MIH is more effective than other treatment being used alone.