Molecular evolution of the hemoglobin gene family across vertebrates.

Adaptation to various altitudes and oxygen levels is a major aspect of vertebrate evolution. Hemoglobin is an erythrocyte protein belonging to the globin superfamily, and the α-, ß-globin genes of jawed vertebrates encode tetrameric ((α2ß2) hemoglobin, which contributes to aerobic metabolism by delivering oxygen from the respiratory exchange surfaces into cells. However, there are various gaps in knowledge regarding hemoglobin gene evolution, including patterns in cartilaginous fish and the roles of gene conversion in various taxa. Hence, we evaluated the evolutionary history of the vertebrate hemoglobin gene family by analyses of 97 species representing all classes of vertebrates. By genome-wide analyses, we extracted 879 hemoglobin sequences. Members of the hemoglobin gene family were conserved in birds and reptiles but variable in mammals, amphibians, and teleosts. Gene motifs, structures, and synteny were relatively well-conserved among vertebrates. Our results revealed that purifying selection contributed substantially to the evolution of all vertebrate hemoglobin genes, with mean dN/dS (ω) values ranging from 0.057 in teleosts to 0.359 in reptiles. In general, after the fish-specific genome duplication, the teleost hemoglobin genes showed variation in rates of evolution, and the ß-globin genes showed relatively high ω values after a gene transposition event in amniotes. We also observed that the frequency of gene conversion was high in amniotes, with fewer hemoglobin genes and higher rates of evolution. Collectively, our findings provide detail insight into complex evolutionary processes shaping the vertebrate hemoglobin gene family, involving gene duplication, gene loss, purifying selection, and gene conversion.

Effect of rare coding variants of charged amino acid residues on the function of human organic anion transporting polypeptide 1B3 (SLCO1B3).

Human organic anion transporting polypeptide 1B3 (OATP1B3, gene symbol SLCO1B3) is a liver-specific uptake transporter. Its function was reported to be largely affected by some positively charged amino acid residues. However, so far the effect of naturally occurring genetic variants of charged residues on OATP1B3's function has not been explored yet. Therefore, in the present study nonsynonymous single nucleotide variants that led to the replacement of charged residues of OATP1B3 were investigated. Our results demonstrated that rare coding variants c.542G > A (p.R181H) and c.592G > A (p.D198N) had a great effect on the function of OATP1B3 mainly due to their influence on protein's surface expression. Further mutation studies showed that a negatively charged residue at position 198 was indispensable to the proper expression of OATP1B3 on the plasma membrane, while a positively charged reside at position 181 was not a must. Structural modeling indicated that R181 is located at the center of putative transmembrane domain 4 (TM4) and its side chain faces towards TM2 instead of towards the substrate translocation pathway, whereas D198 is located at the border of TM4 and intracellular loop 2 and may electrostatically repulse negatively charged phospholipid head groups. In conclusion, our results indicated that rare coding variants that cause changes of charged amino acid residues might have large influence on the function and expression of OATP1B3.

Development of predictive QSAR models for the substrates/inhibitors of OATP1B1 by deep neural networks.

The organic anion transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter. Inhibition of its normal function could lead to drug-drug interactions. In silico prediction is an effective means to identify potential OATP1B1 inhibitors and quantitative structure-activity relationship (QSAR) modeling is extensively used. As the structures of OATP1B1 substrates/inhibitors are quite diverse, machine learning based methods should be a good option for their QSAR analysis. In the present study, deep neural networks (DNNs) were employed to develop QSAR models for the substrates/inhibitors of OATP1B1 with different molecular fingerprints. Our results showed that QSAR models based on 4-hidden layer DNNs and ECFP4/FCFP4 fingerprints had the best generalization performance. The correlation coefficients (R2) of test set for ECFP4 and FCFP4 models were 0.641 and 0.653, respectively. Model application domain (AD) was calculated with Euclidean distance-based method, and AD could improve the performance of ECFP4 model but has little effect on FCFP4 model. Finally, the prediction of additional 8 compounds that not included in the data set further demonstrated that our QSAR models had a good predictive ability (averaged prediction accuracy >92%). The developed QSAR models could be used to screen large data sets and discover novel inhibitors for OATP1B1.

Investigating the interactions of flavonoids with human OATP2B1: inhibition assay, IC50 determination, and structure-activity relationship analysis.

Human organic anion transporting polypeptide 2B1 (OATP2B1) is a crucial transporter for the absorption and disposition of many drugs. Its inhibition by small molecules may alter the pharmacokinetic profile of its substrate drugs. In this study, the interactions of 29 common flavonoids with OATP2B1 were explored using the fluorescent substrate 4',5'-dibromofluorescein and structure-activity relationship analysis. Our results showed that flavonoid aglycones interact with OATP2B1 more strongly than their 3-O- and 7-O-glycoside counterparts, as hydrophilic and bulky groups at these two sites are detrimental to flavonoids' binding with OATP2B1. In contrast, hydrogen-bond forming groups at the C-6 position of ring A and the C-3' and C-4' positions of ring B could strengthen the interaction of flavonoids with OATP2B1. However, a hydroxyl or sugar moiety at the C-8 position of ring A is unfavorable. Our results also indicated that flavones usually interact more strongly with OATP2B1 than their 3-hydroxyflavones (flavonols). The obtained information could be useful for the prediction of additional flavonoids for their interaction with OATP2B1.

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.

Ubiquinol-cytochrome c reductase core protein 1 contributes to cardiac tolerance to acute exhaustive exercise.

Ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) is an indispensable component of mitochondrial complex III. It plays a key role in cardioprotection and maintaining mitochondrion function. However, the exact role of UQCRC1 in maintaining cardiac function has not been reported by in vivo models. Also, the exact biological functions of UQCRC1 are far from fully understood. UQCRC1+/- mice had decreased both mRNA and protein expression of UQCRC1 in the left ventricular myocardia, and these mice had reduced tolerance to acute exhaustive exercise including decreased time and distance with higher apoptosis rate, higher expression level of cleaved CASPASE 3, and higher ratio of cleaved PARP1 to full-length PARP1. Moreover, UQCRC1 knockdown led to increased LV interventricular septal thicknesses both at systole and diastole, as well as decreased LV volume both at end-systole and end-diastole. Finally, UQCRC1 gene disruption resulted in mitochondrial vacuolation, fibril disarrangement, and more severe morphological and structural changes in mitochondria after acute exhaustive exercise. In conclusion, UQCRC1 contributes to cardiac tolerance to acute exhaustive exercise in mice, and it may be an essential component of complex III, playing a crucial role in maintaining cardiac functions.

Naringin Relieves Diabetic Cardiac Autonomic Neuropathy Mediated by P2Y14 Receptor in Superior Cervical Ganglion.

Diabetes mellitus (DM), an emerging chronic epidemic, contributes to mortality and morbidity around the world. Diabetic cardiac autonomic neuropathy (DCAN) is one of the most common complications associated with DM. Previous studies have shown that satellite glial cells (SGCs) in the superior cervical ganglia (SCG) play an indispensable role in DCAN progression. In addition, it has been shown that purinergic neurotransmitters, as well as metabotropic GPCRs, are involved in the pathophysiological process of DCAN. Furthermore, one traditional Chinese medicine, naringin may potently alleviate the effects of DCAN. Ferroptosis may be involved in DCAN progression. However, the role of naringin in DCAN as well as its detailed mechanism requires further investigation. In this research, we attempted to identify the effect and relevant mechanism of naringin in DCAN mitigation. We observed that compared with those of normal subjects, there were significantly elevated expression levels of P2Y14 and IL-1ß in diabetic rats, both of which were remarkably diminished by treatment with either P2Y14 shRNA or naringin. In addition, abnormalities in blood pressure (BP), heart rate (HR), heart rate variability (HRV), sympathetic nerve discharge (SND), and cardiac structure in the diabetic model can also be partially returned to normal through the use of those treatments. Furthermore, a reduced expression of NRF2 and GPX4, as well as an elevated level of ROS, were detected in diabetic cases, which can also be improved with those treatments. Our results showed that naringin can effectively relieve DCAN mediated by the P2Y14 receptor of SGCs in the SCG. Moreover, the NRF2/GPX4 pathway involved in ferroptosis may become one of the principal mechanisms participating in DCAN progression, which can be modulated by P2Y14-targeted naringin and thus relieve DCAN. Hopefully, our research can supply one novel therapeutic target and provide a brilliant perspective for the treatment of DCAN.

LncRNA-UC.25 + shRNA Alleviates P2Y14 Receptor-Mediated Diabetic Neuropathic Pain via STAT1.

Diabetic neuropathic pain (DNP) is a common complication of diabetes, and its complicated pathogenesis, as well as clinical manifestations, has brought great trouble to clinical treatment. The spinal cord is an important part of regulating the occurrence and development of DNP. Spinal microglia can regulate the activity of spinal cord neurons and have a regulatory effect on chronic pain. P2Y12 receptor is involved in DNP. P2Y14 and P2Y12 receptors belong to the Gi subtype of P2Y receptors, but there is no report that the P2Y14 receptor is involved in DNP. Closely related to many human diseases, the dysregulation of long noncoding RNA (lncRNA) has the effect of promoting or inhibiting the occurrence and development of diseases. The aim of this research is to investigate the function of the spinal cord P2Y14 receptor in type 2 DNP and to understand the function as well as the possible mechanism of lncRNA-UC.25 + (UC.25 +) in rat spinal cord P2Y14 receptor-mediated DNP. Our results showed that P2Y14 shRNA can reduce the expression of P2Y14 in DNP rats, thereby restraining the activation of microglia, decreasing the expression of inflammatory factors and the level of p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. At the same time, UC.25 + shRNA can downregulate the expression of the P2Y14 receptor, reduce the release of inflammatory factors, and diminish the p38 MAPK phosphorylation, indicating that UC.25 + can alleviate spinal cord P2Y14 receptor-mediated DNP. The RNA immunoprecipitation result showed that UC.25 + enriched signal transducers and activators of transcription1 (STAT1) and positively regulated its expression. The chromatin immunoprecipitation result indicated that STAT1 combined with the promoter region of the P2Y14 receptor and positively regulated the expression of the P2Y14 receptor. Therefore, we infer that UC.25 + may alleviate DNP in rats by regulating the expression of the P2Y14 receptor in spinal microglia via STAT1.

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.

The Effects of Intraoperative Dexmedetomidine Use and Its Different Dose on Postoperative Sleep Disturbance in Patients Who Have Undergone Non-Cardiac Major Surgery: A Real-World Cohort Study.

OBJECTIVE: The study aimed to investigate the effects of intraoperative dexmedetomidine on postoperative sleep disturbance for different surgical patients and compare such effects between different dose of dexmedetomidine. METHODS: A total of 7418 patients undergoing nine types of non-cardiac major surgeries were retrospectively studied. Patients were separated into DEX (dexmedetomidine) or Non-DEX (Non-dexmedetomidine) groups based on the use of dexmedetomidine during surgery. The patients who reported they could not fall asleep during the night or woke up repeatedly during the most of the night at the day of the surgery and whose NRS were >6 were defined as cases with severe sleep disturbance. Propensity score matched analysis based on all preoperative baseline data was performed along with logistic regression analysis including different surgery types and dosage of dexmedetomidine use. RESULTS: In both of the unmatched cohort (OR, 0.49 [95% CI: 0.43-0.56]) and matched cohort (0.49 [95% CI: 0.42-0.58]), the DEX group had a significantly lower incidence of severe sleep disturbance than the Non-DEX group. In the subgroup analysis, for gynecological and urological surgery population, the ORs for DEX-group reached 0.21 (95% CI, 0.13-0.33; P<0.0001) and 0.30 (95% CI,0.19-0.47; P<0.0001), respectively. In addition, low-dose dexmedetomidine (0.2-0.4 µg·kg-1·h-1) showed the greatest effect with an odds ratio of 0.38 (95% CI: 0.31-0.44; P<0.0001), and the incidence of severe sleep disturbance in the low-dose group was significantly lower (11.5% vs. 17.7% vs. 16.5%, P<0.0001) than that in the medium- (0.4-0.6 µg·kg-1·h-1) and high-dose (0.6-0.8 µg·kg-1·h-1) groups. CONCLUSION: Intraoperative dexmedetomidine use can significantly decrease the incidence of severe sleep disturbance on the day of surgery for patients undergoing non-cardiac major surgery, and the effects were most significant in patients receiving gynecological and urological surgery. Furthermore, low-dose dexmedetomidine (0.2-0.4 µg·kg-1·h-1) is most effective for prevention of postoperative sleep disturbance.