The aim of this study is to confirm the hepatocellular protective functions of apigenin and the molecular mechanism on liver fibrosis in mice. 4) and bile duct ligature (BDL) mouse fibrosis models were used to investigate the effects of apigenin on liver fibrosis. Sixty-six male C57 mice were randomly divided into eight groups, including the vehicle group, CCl4 group, CCl4+L-apigenin (20 mg/kg) group, CCl4+H-apigenin (40 mg/kg) group, sham group, BDL group, BDL+L-apigenin(20 mg/kg) group, and BDL+H-apigenin(40 mg/kg) group. Serum liver enzymes (ALT and AST), proteins associated with autophagy, and indicators linked with the TGF-β1/Smad3 and p38/PPARα pathways were detected using qRT-PCR, immunohistochemical staining, and western blotting.  Carbon tetrachloride (CClβ1/Smad3 and p38/PPARα pathways.  Our findings confirmed that apigenin could decrease the levels of ALT and AST, suppress the generation of ECM, inhibit the activation of HSCs, regulate the balance of MMP2 and TIMP1, reduce the expression of autophagy-linked protein, and restrain the TGF-β1/Smad3 and p38/PPARα pathways. Apigenin could alleviate liver fibrosis by inhibiting hepatic stellate cell activation and autophagy via TGF- Liver fibrosis is a chronic pathological change caused by a variety of reasons, such as chronic infection by hepatotropic viruses, excess alcohol consumption, nonalcoholic fatty liver disease, autoimmune liver diseases, and hereditary disease, and is a necessary stage for the development of many liver diseases to liver cirrhosis and even liver cancer , 2. Liveβ1 (TGF-β1) will contribute to the excessive accumulation of ECM which promotes liver fibrosis ) for in vitro treatment.Apigenin (HPLC ≥ 98% CAS:520-36-5) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. . When used, it is dissolved into dimethyl sulfoxide (DMSO) at 2 mg/ml and 4 mg/ml concentrations. Carbon tetrachloride  for 24 hours. Subsequently, 10 μM BrdU was added to each well, and samples were incubated for 12 h at 37°C. BrdU signaling was determined by measuring the absorbance at 450 nm.Proliferation of the cells was evaluated using the BrdU Cell Proliferation ELISA Kit  according to the manufacturer's instruction. Briefly, cells were cultured in 96-well plates and exposed to apigenin  were obtained from Shanghai SLAC Laboratory Animal  and housed in a standard animal laboratory with free access to food and water. All experimental procedures involving mice were approved by the Animal Care and Use Committee of Shanghai Tongji University. Handling and care of mice conformed to the National Institutes of Health Guidelines.4-induced liver fibrosis model, mice were injected with 10% CCl4  intraperitoneally three times a week for 8 weeks. In the bile duct ligation- (BDL-) induced liver fibrosis model, all mice were fasted for 12 h and anesthetized intraperitoneally by 1.25% pentobarbital sodium salt (40 mg/kg). After opening the abdomen via the linea alba, the bile duct was exposed and isolated over a certain length. Two surgical knots were tied in the isolated bile duct, which was then cut between the knots. The abdomen was then closed.We established two different mouse liver fibrosis models. To create the CCln = 6): no treatmentNormal control (NC) (n = 6): mice were injected intraperitoneally with DMSO three times a weekVehicle group (n = 6): apigenin (40 mg/kg) was given to mice by intragastric administration three times a week.API (40 mg/kg) group (In order to verify whether the apigenin dose (20 mg/kg and 40 mg/kg) could cause damage to the structure and function of the liver and other internal organs, we designed a preliminary experiment. The eighteen mice were randomly divided into the following 3 groups. 4-induced liver fibrosis model, 24 mice were randomly divided into the following 4 groups. n = 6): mice were injected intraperitoneally with DMSO three times a week for 8 weeksVehicle group (4 group (n = 6): mice were injected with CCl4 intraperitoneally three times a week for 8 weeksCCl4+L-API group (n = 6): mice were injected with CCl4 intraperitoneally and gavaged with 20 mg/kg apigenin three times a week for 8 weeksCCl4+H-API group (n = 6): mice were injected with CCl4 intraperitoneally and gavaged with 40 mg/kg apigenin three times a week for 8 weeksCClIn the CCln = 6): all mice underwent laparotomy without BDLSham group (n = 6): all mice underwent BDL surgeryBDL group (n = 6): all mice were gavaged with 20 mg/kg apigenin once a day for 14 days after BDLBDL+L-API group (n = 6): all mice were gavaged with 40 mg/kg apigenin once a day for 14 days after BDLBDL+H-API group  and kept at −80°C. Liver tissues were stored at −80°C.The blood sample collected from the mouse orbit was placed at 4°C for 5 hours. And then, the serum sample was separated from the blood by centrifuging at 4,600 × g at 4°C for 10 minutes. Serum levels of ALT and AST were detected by microplate test kits.μm thick sections and stained with hematoxylin and eosin (H&E) to determine the severity of injury.A part of the fresh left liver lobe was excised and then fixed in 4% paraformaldehyde for 24 h. The tissues were dehydrated with ethanol and embedded in paraffin. Next, the liver tissues were cut into 3 The total RNA was extracted from 100 mg liver tissue by TRIzol . Then, the purified RNA was reverse-transcribed into cDNA. The levels of mRNA were determined by SYBR Premix EX Taq through a 7900HT fast PCR system . The primers used for qRT-PCR are listed in β, anti-α-SMA, anti-Col, anti-LC3, anti-Beclin-1, anti-p62-, anti-TGF-β1-, anti-p-Smad3-, anti-p-38-, and anti-PPARα . Then, the primary antibodies in the liver sections were incubated with secondary antibodies using a diaminobenzidine (DAB) kit. Final evaluations were performed with Image-Pro Plus software 6.0 to calculate the mean of integrated optical densities (MIOD = sum IOD/sum area) of the positive staining area.Paraffin sections were baked in a 60°C oven for 1 hour and then dewaxed and rehydrated. Antigen was placed into a citrate buffer, which was then heated to 95°C for 10 minutes and cooled to room temperature. Next, the sections were covered in 3% hydrogen peroxide for 20 minutes to block endogenous peroxidase activity, and then 5% BSA was added to block nonspecific binding for 15 minutes (both at room temperature). Slices were then incubated overnight at 4°C with the following antibodies: anti-IL-1Firstly, liver tissues were ground (100 mg) into powder in liquid nitrogen, and then the powder was homogenized in RIPA lysis containing phenylmethanesulfonyl fluoride (PMSF) and protease inhibitors (PI). The protein concentrations were detected using the bicinchoninic acid method before being mixed with a 6x loading buffer and boiled at 100°C for 10 minutes. Secondly, protein samples were electrophoresed by 10% or 12.5% SDS-PAGE and transferred onto polyvinylidene fluoride or nitrocellulose membranes. Next, membranes were blocked with 5% skimmed milk for at least 1 hour and subsequently incubated overnight at 4°C with the primary antibodies . Thirdlyn = 6; ∗P < 0.05 for CCl4 (BDL) vs. control; #P < 0.05 for CCl4 (BDL)+API (20 mg/kg) vs. CCl4 (BDL); +P < 0.05 for CCl4 (BDL)+API (40 mg/kg) vs. CCl4 (BDL); !P < 0.05 for CCl4 (BDL)+API (40 mg/kg) vs. CCl4 (BDL)+API(20 mg/kg)). One-way ANOVA using the Student–Newman–Keuls method was used to compare statistical differences among three or four groups using SPSS version 20.0 software . P < 0.05 was regarded as statistically significant.Experimental data which was repeated at least three times was presented as mean ± SD  was used in this study to investigate the effect of apigenin on HSCs. The CCK8 assay was used to measure the toxicity of apigenin in LX2 cells . Apigeni4 and BDL surgery in a dose-dependent manner. Next, HE and Masson staining were used to evaluate the pathological changes of liver tissues. HE staining showed that the morphology and structure of mouse liver cells in the control groups were normal, with normal arrangement, normal hepatic lobules and portal area, and no inflammatory cell exudation. Compared with the control groups, the disordered arrangement of liver cells, the damaged normal structure, the exudation of many inflammatory cells, and the proliferation of collagen fibers were significantly observed in the CCl4 and BDL groups. When apigenin was given at the same time, the disordered arrangement of liver cells was significantly reduced, the structure of portal area was almost normalized, fibrous tissue hyperplasia and inflammatory cell infiltration were significantly decreased, and the morphological structure was close to normal liver tissue , fibroblast growth factor (FGF), epidermal growth factor (EGF), and VEGF [The first step to try to stop and reverse liver fibrosis is to explore the molecular mechanisms of this disease. The formation of liver fibrosis is a complex pathophysiological process involving many cells, molecules, and signaling pathways. The accumulation of ECM is regarded as the important character, and the activation of HSCs is considered as the initial process of liver fibrosis , 7, 10. and VEGF . The synand VEGF , 13, 47.β is generally considered to be the strongest fibrogenic factor. The activation of the TGF-β1/Smad3 signaling pathway plays an important role in liver fibrosis [β1 [β can also activate the p38 MAPK signaling pathway to promote the transcription of collagen which is the main ingredient of ECM [β1/Smad pathway could efficiently reduce the injury of liver fibrosis [β1 and Smad3 expressed much more in fibrosis model groups than in control groups, and at the same time, apigenin groups obviously reduced the expression of TGF-β1, Smad3, and the other related proteins. Thus, we concluded that the protective effect of apigenin was closely related to the inhibition of the TGF-β1/Smad3 pathway.TGF-fibrosis . Smad3 iosis [β1 . In addit of ECM . Besidesfibrosis , 51–53. β1 could exert a positive effect on liver fibrosis [α to protect liver from concanavalin A-induced injury [α, PPAR β/δ, and PPARγ have many biological functions such as liver protection, antitumor, antiasthma, antidiabetes, and antineuropathic pain [α could reverse fibrosis by reducing lipid peroxides and inhibiting the activation of HSCs and Kupffer cells (KCs) [α and proved that apigenin could inhibit the phosphorylation of p38 which further increased PPARα to protect the liver from fibrosis.p38 MAPK belongs to the family of MAPKs that affects a variety of intracellular responses including cell-cycle regulation, inflammation, cell death, and tumorigenesis . p38 MAPfibrosis . In addid injury . PPARs whic pain –62. It wls (KCs) , 64. So,β1/Smad3 pathway, which could increase the expression of Bechin1 and LC3 and decrease the generation of p62 [α pathway could exert positive effects in liver injury [β1/Smad3 and p38/PPARα pathways.Autophagy is a self-selective mode of cell death which contributes a lot to the basic liver functions . Hernandn of p62 . In addir injury . Our cur4- and BDL-induced liver fibrosis models. Apigenin could inhibit the activation of HSCs which promote the accumulation of ECM and the secretion of many fibrogenic factors such as α-SMA and collagen 1. In addition, the TGF-β1/Smad3 and p38/PPARα pathways are proven to be the main signaling pathways through which apigenin exerts its function (In general, our study illustrated the liver-protective effect of apigenin in CClfunction . Therefo4- and BDL-induced liver fibrosis models. Inhibiting the TGF-β1/Smad3 and p38/PPARα pathways, reducing autophagy, and decreasing ECM formation are the major mechanism of the antifibrotic effects of apigenin.Our study illustrated the liver-protective effect of apigenin in CCl


Owing to the importance of GABAergic system in impulsivity and memory, the present study examines whether testosterone-mediated impulsivity is associated with changes in the expression of Gamma-Aminobutyric Acid (GABA) A and B receptor subunit transcripts  in rat PFC, and whether testosterone influences GABAA receptor subunit organization. We studied GABA receptor functions by examining GABA receptor-mediated calcium/calmodulin-dependent kinase signaling genes  in the testosterone-induced impulsivity model. Rats were left untreated as controls (C), gonadectomized (GDX), or GDX and injected with supraphysiological doses of testosterone (T). Impulsive behavior was examined using the go/no-go paradigm. Gene expression was studied using qRT-PCR and GABAA subunit reorganization using cross correlation. Our findings show that expressions of select GABAA receptor subunits  were significantly upregulated in PFC of T group compared to GDX or C groups. GABAA receptor subunit organization was different in C, T, and GDX groups. Additionally, Camk4 expression was significantly downregulated in T compared to C group. Our findings suggest that specific GABAA receptor subunit expression, their reorganization, and Camk4-mediated functions may be associated with testosterone-mediated impulsivity.Testosterone can induce impulsivity, a behavioral impairment associated with various psychiatric illnesses. The molecular mechanisms associated with testosterone-induced impulsivity are unclear. Our earlier studies showed that supraphysiological doses of testosterone to rats induced impulsive behavior, impacted hypothalamic-pituitary-adrenal axis (HPA) and hypothalamic-pituitary-gonadal axis interactions, and altered α In conjunction with excitatory glutamate, GABA is involved in balancing excitatory and inhibitory response, critical in proper brain functioning  GABAB receptor subtype, which are G-protein-coupled receptors  group compared to control (C) and gonadectomized (GDX) groups. GABA studied . We also suicide . Additio suicide . Further suicide  decision suicide , and emo suicide . Our pre suicide .ad libitum. The rats were acclimatized for 5 days before starting the experiment. They were divided into a control (C) group and two experimental groups, both of which received gonadectomies to eliminate testosterone. The age of rats was ∼6 weeks at the time of gonadectomy. One of the experimental groups was given daily injections of excessive testosterone and is known as the testosterone (T) group. The group with the gonadectomy only is known as the gonadectomized (GDX) group. Experimental procedures were approved by the IACUC of the University of Alabama at Birmingham and all procedures were conducted in strict adherence to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.A total of 30 Male Long Evans rats were received from Envigo Laboratories and maintained as described in our previous studies . They weRats receiving a GDX or testosterone injections were subjected to the same protocol used previously in this lab . To summA schematic diagram showing the experimental protocol is mentioned in After 24 weeks of testosterone administration, rats were subjected to the go/no-go task paradigm to test their impulsive behavior. This paradigm is described in our previous study . Rats pl® software ran on a computer. Impulsive behavior was defined as pressing the lever during the “no-go” phase. Impulsive behavior was indexed by the go/no-go ratio, which was the number of times the lever was pressed during the “go” phase compared to the number of times it was pressed during the “no-go” phase. A high ratio indicated low impulsive behavior, while a low ratio indicated high impulsive behavior. If variance in this ratio between the 3 sessions was <10% for any individual rat, results were recorded. Individual responses were averaged based on experimental group .Rats that continued testing moved to the “go/no go” task phase of the experiment. Rats were to press the lever to dispense sucrose pellets during the “go” phase, which was signaled by a bright white light and continued with a house light. They were not to press the lever during the “no-go” phase, which was signaled by a red light and continued with no light. Each phase was 10 min, with 10 s timeouts between phases. In total, there were 4 “go” and 4 “no-go” phases which alternated for a total of an 80 m session. Rats underwent 3 sessions that were included in results, and they completed one warm-up session of this paradigm that was not included in results. The sessions were controlled by Med-PC IVTwenty-four hours after the last session, rats were decapitated, trunk blood was collected, and brains were flash frozen and stored at −80°C. The PFC was dissected out from 300 μm sections prepared from the frozen brain on a cryostat  and PFC was carved out precisely by scalpel using the Rat Brain Atlas and stored at −80°C until further analyses.t-tests were performed between groups to determine variance and statistical significance. Data are presented as testosterone concentration values in ng/mL.Serum isolated from trunk blood was tested for testosterone levels using an ELISA kit . One-way ANOVA and Student’s Primers were designed using Rat Genomic Database (RGD) and NCBI BLAST search tool. Primer sequences were designed to target the 3′ end of the gene and accounted for all the transcript variants for each gene. Primers were designed to have low self-complementarity, low self-3′ complementarity, low GC%, and melt temperatures within 2°C of each other. A list of all primers used in the gene expression assay can be found in ®  method as described earlier , GABAA receptor subunits β1–3 , GABAA receptor subunits γ1–3 , GABAB receptor subunits 1–2 , calmodulin genes 1–3 , calcium/calmodulin-dependent protein kinase type II subunits α, β, γ, and δ , and calcium/calmodulin-dependent protein kinase type IV (Camk4). The possibility of primer dimer formation and secondary product amplification was ruled out by running template-free samples. Gapdh normalized gene expression values were used to determine the relative gene expression levels of individual transcripts following Livak’s ΔΔCt calculation method  in C, T, and GDX groups independently using Pearson Product Moment Analysis.GABAn = 5) was 11.6 ng/mL (SEM = 4.67), compared to the average concentration for the control (C) group (n = 6) of 1.63 ng/mL (SEM = 0.4), and the average concentration for the gonadectomized (GDX) group (n = 6) of 0 ng/mL (SEM = 0.0061). T group concentration was significantly higher than both C group (p = 0.04) and GDX group (p = 0.02). GDX group concentration was significantly lower than C group (p = 0.001).As can be seen in our prior study , averagen = 5) had a ratio of 0.54 (SEM = 0.035), C group (n = 6) had a ratio of 0.87 (SEM = 0.073), and GDX group (n = 6) had a ratio of 0.76 (SEM = 0.075). T group’s ratio was significantly lower than the ratio for C group (p = 0.004) and GDX group (p = 0.03). There was no significant difference between the ratios of C group and GDX group (p = 0.32).As detailed in our previous study, T group rats had significantly lower go/no-go ratios than those in the GDX and C groups . T groupGene expression analysis was done in rat PFC to determine the effect of a supraphysiological dose of testosterone (T) on GABA receptor subunit genes and genes associated with calcium-dependent calmodulin kinase signaling pathway compared to rats with normal levels of testosterone (C) and rats with no testosterone due to gonadectomy (GDX).A and GABAB receptor subunit transcripts in PFC are presented in Gabra3 fold change was 1.29 (SEM = 0.058) for T group and 1.10 (SEM = 0.035) for GDX group, compared to 1 (SEM = 0.28) for C group. T group’s Gabra3 expression was significantly upregulated compared to GDX (p = 0.008), but not significant compared to C group (p = 0.244). Gabra5 fold change was 1.71 (SEM = 0.18) for T group and 1.39 (SEM = 0.15) for GDX group, compared to 1 (SEM = 0.22) for C group. T group’s Gabra5 expression was significantly upregulated compared to C (p = 0.038), but not significant compared to GDX group (p = 0.24). Gabra6 fold change was 2.14 (SEM = 0.17) for T group and 1.10 (SEM = 0.19) for GDX group, compared to 1 (SEM = 0.40) for C group. T group’s Gabra6 expression was significantly upregulated compared to both GDX (p = 0.009) and C (p = 0.047).Expression level changes of all GABAA receptor subunits  were not significantly altered between groups. Neither of the GABAB receptor subunits (Gabbr1 and Gabbr2) were significantly different between groups.Other GABAin Camk4. Camk4 fold change was 0.55 (SEM = 0.222) for T group and 0.55 (SEM = 0.227) for GDX group, compared to 1 (SEM = 0.173) for C group. Camk4 expression was significantly downregulated in T group compared to C group (p = 0.02). Camk4 expression was also significantly downregulated in GDX group compared to C group (p = 0.03). No significant differences were found in Camk4 expression between T and GDX groups. Additionally, Calm3 expression was nearly significantly downregulated in T group compared to C group (p = 0.069), but not to GDX (p = 0.233). No significant differences were found in the expression levels of Calm1, Calm2, Camk2a, Camk2b, Camk2g, and Camk2d.mRNA expression changes of all calmodulin and CaMK subunit genes in PFC are presented in A receptor subunit gene expression in the control group. Significant correlations were found in 17 different subunit combinations: Gabra3 and Gabra4, Gabra3 and Gabra5, Gabra4 and Gabra5, Gabra5 and Gabrb1, Gabra3 and Gabrb2, Gabra4 and Gabrb2, Gabra5 and Gabrb2, Gabrb1 and Gabrb2, Gabra3 and Gabrb3, Gabra4 and Gabrb3, Gabra2 and Gabrg1, Gabra2 and Gabrg2, Gabrg1 and Gabrg2, Gabra2 and Gabrg3, Gabra3 and Gabrg3, Gabrg1 and Gabrg3, and Gabrg2 and Gabrg3.A receptor subunit gene expression. Significant correlations were found in 9 different subunit combinations: Gabra1 and Gabra4, Gabra2-transcript variant 2 and Gabra6, Gabra2-transcript variant 2 and Gabrb3, Gabra6 and Gabrb3, Gabra2-transcript variant 2 and Gabrg1, Gabra6 and Gabrg1, Gabrb2 and Gabrg2, Gabra6 and Gabrg3, and Gabrg1 and Gabrg3.A receptor subunit gene expression. Significant correlations were found in 5 different subunit combinations: Gabra3 and Gabra4, Gabra4 and Gabra6, Gabra3 and Gabrb3, Gabra2 and Gabrg1, and Gabrb1 and Gabrg2.A and GABAB receptor subunit mRNA levels in rat PFC. As indicated in the results section, we found that only GABAA, but not GABAB receptor transcript levels, showed changes. Within GABAA receptors, testosterone had a subunit-specific effect on Gabra3, Gabra5, and Gabra6 transcript levels. We predict that changes in only GABAA but not in GABAB could be due to the underlying differences in receptor composition between these two receptors and their distinct mechanisms of interaction with specific ligands and consequent downstream functions , testosterone (T), and gonadectomized (GDX) groups. We found that while C group rats had 17 significant correlations, T rats had 9, and GDX had only 5. These differences in subunit composition indicate a loss of co-regulated genes in GDX group and testosterone partially restored this loss. Interestingly, studies have examined the organization of various GABA receptor subunits and found that in certain psychiatric conditions, such as depression and suicide, not only are mRNA expression altered in specific GABA receptor subunits, but inter-relations between various subunits differ  has been found to play a role in integrating Ca2+ signals in dendritic spines (Camk2b is found to be more prevalent in sympathetic neurons (Camk2 protein complex to dendritic spines (Camk2a and Camk2b are primarily found in the nervous system, Camk2g and Camk2d are found throughout the body (Camk2g functions to transport calmodulin from the cell surface to the nucleus (Camk2d is predominantly found in cardiac tissue, and its expression alters during cardiomyocyte differentiation, heart failure, and ischemia (Camk2 inhibitor can block the potentiating effect of Ca2+ on Cl– current gated by GABAA receptors (A receptors by inhibiting glutamate release via Ca2+/calmodulin-dependent signaling (A receptors and CaMKIV is not well established, a histochemical study shows CaMKIV is expressed in a subgroup of GABAergic neurons in all layers of cortical interneurons of adult monkey area V1 in which parvalbumin was present (Calm1, Calm2, Calm3, Camk2a, Camk2b, Camk2g, Camk2d, and Camk4. It was observed that Camk4 gene expression was significantly lower when comparing T rats to C rats and GDX rats to C rats. Additionally, Calm3 was lower when comparing T rats to C rats, but it could not reach significance level. It appears that Camk4 might be involved in differential regulation of Gabra3, Gabra5, and Gabra6 receptors. However, changes in Camk4 are rather surprising given that this kinase is not very well linked to GABAA receptor functioning (Camk4 cannot be ruled out.Protein phosphorylation is one of the main mechanisms of regulating GABA receptor functions. It has been shown that CamKII-dependent phosphorylation can increase GABA receptor binding, and thus modulate GABA receptor-mediated chloride ion channel activity . It has c spines , and incc spines . Camk2b  neurons , and to c spines . While Cthe body . Camk2g  nucleus . Camk2d ischemia . Also, beceptors . Moreoveignaling . Althoug present . In thisctioning . The posA and GABAB subunit gene expression analysis in an animal model of testosterone-induced impulsivity. We show that the impulsivity response may be GABAA subunit-specific when involving supraphysiological concentrations of testosterone. Also, the organization of GABAA receptor subunits is quite different between control, testosterone, and GDX rats. Further, this study shows that subunit-specific effects of testosterone may be associated with calmodulin/calcium-dependent kinases. Our study thus provides an interplay between testosterone, impulsivity and GABAergic functions. From a clinical perspective, GABAA receptors are more frequently used as therapeutic targets when treating disorders such as anxiety and epilepsy (A receptor subunits and CaM kinases and not from protein levels. Thus, there is a possibility that changes in transcription may not necessarily reflect changes in protein. Similarly, CaM kinase activity was not studied. However, as mentioned above, based on mRNA expression levels, A receptor organization was altered in the brain of depressed subjects. These investigators convincingly argued that the relative mRNA abundance of GABAA receptor subunits would be a mechanism that ensured proportional abundance of protein. In addition, A receptors were chosen to study, other subunits such as δ and ρ could also be studied in the future.Altogether, this is the first GABAepilepsy . In the epilepsy . There wThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The experimental procedures were approved by the IACUC of the University of Alabama at Birmingham.JA performed the experiments and analyzed the data. YD conceptualized the idea. JA and YD co-wrote the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Exf-GO) as a filler. Exf-GO was first obtained by the strong oxidation and exfoliation of graphite. The structural, morphological and mechanical properties of the composites (CMCx/Exf-GO) were evaluated, and the obtained composites were homogenous, transparent and brownish in color. The results confirmed that Exf-GO may be homogeneously dispersed in CMC. It was found that the composite has an inhibitory activity against the Gram-positive Staphylococcus aureus, but not against Gram-negative Pseudomonas aeruginosa. At the same time, it does not exhibit any cytotoxic effect on normal fibroblasts.Sore, infected wounds are a major clinical issue, and there is thus an urgent need for novel biomaterials as multifunctional constituents for dressings. A set of biocomposites was prepared by solvent casting using different concentrations of carboxymethylcellulose (CMC) and exfoliated graphene oxide ( Nowadays, there is an urgent need for new innovative solutions in the field of biomedicine, since non-healing wounds remain a major clinical issue and are a significant burden to the medical system ,2. Chron2 hybridized carbon atoms creating a hexagonal lattice, and possessing unique properties, i.e., high specific surface area (~2630 m2/g), high electron mobility [−1, [Recently, the study of graphene and its derivatives as a promising material for biomedical application—and particularly for wound dressing—has been considered ,7,8,9,10mobility ,12, therity [−1, ,14 and tity [−1, ,16. Moreity [−1, ,18,19,20ity [−1, . GO can ce area ~30 m2/g, ity [−1, ,28,29,30ity [−1, . Thus, city [−1, , endotoxity [−1, .S. aureus and P. aeruginosa-associated with infected wounds [Escherichia coli and S. aureus [Carboxymethylcellulose (CMC) is an anionic water-soluble biopolymer, which due to its characteristics such as hydrophilicity, bioadhesivity, pH-sensitivity, non-toxicity, high water solubility, non-allergenicity, low immunogenicity and gel-forming properties, is used in applications such as coating fluids, binders, textiles, paper, food, drug delivery systems and cosmetics . CMC is d wounds . Rasoulzd wounds . Justin d wounds . Shao et. aureus .Exf-GO), also focusing on the evaluation of its biological activity, in terms of both cytotoxicity and antibacterial properties.In this work, we report the preparation and the characterization of CMC composites containing exfoliated GO , H2O2 , KMnO4 (p.a.), P2O5 (p.a.) and K2S2O8 (p.a.) were purchased from Chempur, , while HCl  was purchased from POCH , Gliwice, Poland)Graphite powder, 11 microns of 99% purity, was purchased from AlfaAesar . Sodium carboxymethyl cellulose (CMC) was purchased from Sigma-Aldrich , average Mw ~250,000, degree of substitution 0.7. HExf-GO was prepared through oxidation of graphite powder in a two-step synthesis , according to the modified Hummers’ method [2SO4 (1.5 mL), K2S2O8 (0.5 g) and P2O5 (0.5 g) was mixed at 80 °C for 30 min. Then, 1 g of graphite was added. When the color of the mixture became dark blue, it was thermally isolated by coating with aluminum foil and being left to cool at room temperature (6 h). The mixture was then carefully diluted with distilled water, and then filtered and washed with water until the pH became neutral. The solution was air-dried at room temperature for 16 h on top of a Petri Dish, in order to obtain pre-treated graphite.’ method ,36. In t2SO4 (23 mL) cooled in an ice-water bath. KMnO4 (3 g) was then gradually added to the mixture, which was maintained at a temperature below 20 °C. After this, the reaction mixture was heated up to 35–40 °C and stirred for 2 h. The mixture was then diluted by adding 46 mL of distilled water. The rapid reaction was stopped after 15 min by adding 140 mL of water and 2.5 mL of 30% H2O2 solution. The product was then filtered and washed with 1:10 water:HCl solution (250 mL), in order to remove metal ions. The mixture was suspended in distilled water to give a viscous brown 2 wt% dispersion.In the second step, the pre-treated graphite was oxidized. For this purpose, 1 g of the pre-treated graphite powder was added to HExf-GO was obtained by liquid-phase exfoliation assisted by ultrasonication. Fully oxidized graphene oxide (GO) (0.1 g) was dispersed in distilled water (20 mL). The suspension was sonicated  and purified by centrifugation at 3000 rpm for 30 min .Exf-GO aqueous dispersion, which were maintained in agitation at room temperature for 3 h and then sonicated for 1 h. The total volume was 30 mL. The pH of each dispersion was 7. The dispersions were then transferred into Petri dishes and kept in an oven at 50 °C to facilitate the evaporation of the solvent, which occurred within 24 h. All the obtained nanocomposites were homogeneous and brown. The colorless and transparent sample of Exf-GO free polymer has been also prepared as a reference , a thermoelectrically cooled CCD as a detector, and an argon laser operating at 514 nm, power 5% (~0.5 mW), acquisition time 10 s and 10 accumulations.Raman Spectroscopy. Raman spectra were recorded in the 1000–1800 cm−1 range, with a step of 2 cm−1, by using a FT-IR Bruker Vertex 70 Advanced Research Fourier Transform Infrared Spectrometer  equipped with Platinum ATR .Fourier Transform Infrared Spectroscopy . The Attenuated Total Reflectance (ATR) spectra  were recorded in the 40–4000 cmAtomic Force Microscopy (AFM). Topography of the produced materials  was investigated in contact mode by using an Atomic Force Microscope . The deflection of the cantilever in the horizontal direction was also measured with the help of a Lateral Force Microscope . All measurements were performed for the following scanning area 45 µm × 45 µm, 20 µm × 20 µm, 10 µm × 10 µm, 5 µm × 5 µm, 2.5 µm × 2.5 µm and 1 µm × 1 µm. The data were analyzed using XEI Software (Version 4.3.4) provided by the microscope’s manufacturer.X-ray Diffraction (XRD). XRD patterns were acquired by a PANalytical X’Pert pro X-ray powder diffractometer  using nickel-filtered Cu Kα1 radiation operating at 40 keV and 30 mA.Mechanical properties. Tensile mechanical measurements were carried out using a dynamometer  on rectangular-shaped specimens (10 × 90 mm) that were cut off from films. The grip distance was in all cases 30 mm, and the crosshead speed 5 mm/min. Five samples were tested for each material.Pseudomonas aeruginosa ATCC® 10145™ and Staphylococcus aureus ATCC® 25923™ were used as tester strains of Gram-negative and Gram-positive bacteria, respectively. Bacterial cells were pre-cultured for 16 h in sterile glassware tubes containing 3 mL of Luria Bertani (LB) broth  at 37 °C with shaking (160 rpm). The day after, bacterial cells were inoculated (1% v/v) in 3 mL of LB broth amended with 2 × 2 cm of different films , as well as 10% GO powder in an aqueous solution. Bacterial cells were challenged for 24 h at 37 °C with shaking (160 rpm). The effect of films and Exf-GO on bacterial growth was evaluated through the spot plate count method [10 CFU mL−1) for each biological trial (n = 3) with standard deviation.Antibacterial tests. P. aeruginosa and S. aureus cells were grown for 24 h in the presence of CMC, CMC6/Exf-GO and Exf-GO, prior to their imaging by means of a fluorescence microscope , as described by [v/v) mixture of acridine orange (100 μg/mL) and ethidium bromide (100 μg/mL), and were then immediately imaged at 1000× magnification by using excitation and emission wavelength of 488 nm and 550 nm, respectively.ribed by . Briefly® CCL-186™) cell line, derived from the normal lungs of a 16-week female fetus and used as a prototype of normal fibroblasts cells, was cultured in Eagle’s Minimum Essential Medium (EMEM), supplemented with 10% fetal bovine serum, 100 U mL−1 penicillin and 100 mg mL−1 streptomycin. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2, as previously described [2 incubator. 300 µl of Exf-GO, diluted in culture medium, and 4 × 4 cm of each composite were added to the wells for 48 h. At the end of treatment, the cells were observed under inverted microscopy, and then detached from the plates and counted after staining with trypan blue dye.IMR-90  and carboxymethylcellulose (CMC) composites containing exfoliated graphene oxide (GO) (CMCx/Exf-GO) nanocomposites are reported in −1.The obtained nanocomposites were analyzed to investigate their structure, antibacterial properties and biocompatibility. Raman spectra of exfoliated graphene oxide  spectra and X-ray diffraction (XRD) patterns were recorded to investigate possible interactions between reported A,B.−1, due to the –OH group stretching. The bands at 2924 and 2856 cm−1 are due to C–H asymmetric and symmetric stretching of the CH2 vibration. The bands at 1590, 1415, 1324 and 1053 cm−1 are due to bending modes of the –COO group, the scissoring of –CH2, the bending vibration of –OH and the stretching of the CH–O–CH2 group, respectively. No significant differences, in terms of IR bands (both shape and position), are observed in the IR spectra of the nanocomposites, suggesting that Exf-GO does not interact with CMC through intermolecular hydrogen bonds. By adding Exf-GO, however, the C–H stretching modes of CH2 vibration became sharper as two partially overlapped bands, indicating that there should be good compatibility between CMC and GO, as evident by the transparency of the material.The typical IR spectrum of CMC shows a broad absorption band at 3440 cmExf-GO shows a strong peak at 2 = 10.35° and a large band at 2 = 20.18°, corresponding to d-spacing of 8.54 and 4.40 Å respectively, which are related to the inter-layer distance between the GO layers and to a short-range order in stacked graphene layers. The XRD patterns of CMC films show two large bands , indicating a relative ordered structure characterized for the crystalline form of cellulose II [Exf-GO does not cause any change in the nanocomposite. Alternately, when combined with CMC 6%, the disappearing of the band can be observed at 2 = 11.96°, while the second band at 20.18° remains almost unvaried  can provide spatial information both parallel and perpendicular to the surface, thus allowing a 3D reconstruction of the sample surface. In doing so, we investigated both the topographical properties and the quality of the materials. 2D/3D AFM topography images of all CMC films and nanocomposites are reported in the  6% film .Exf-GO samples. The same conclusion was obtained from simultaneous analysis of the topographies during left-to-right and right-to-left scanning, with the help of later force microscopy (LFM). Roughness Rq, Ra and Rz for the AFM measured topographies of CMC and CMCx/Exf-GO composites , since this formulation, among those investigated, showed the lower value ID/IG, and is also the lesser organized.The antibacterial and the cytotoxic activities were evaluated on CMC 6%-based composites, the composite constituted by CMC 6%, and on 2.5 mL S. aureus cell growth was inhibited by the presence of either CMC or CMC6/Exf-GO in the culture broth, which determined a decrease in the number of viable cells, corresponding to 0.8 and 1 log units  [Exf-GO upon S. aureus and P. aeruginosa cells was corroborated by fluorescence microscopy imaging, after dual staining with acridine orange and ethidium bromide  ,55, whices (LPS) ,58,59,60 bromide . S. aureExf-GO nanocomposite were also evaluated for their biocompatibility, performing cytotoxicity tests on normal fibroblasts cell line (IMR-90). As a result, no cytotoxic effect was observed on normal fibroblasts after 48 h of treatment. CMC6/Exf-GO was more resistant to solubilization than CMC, although its structural change in terms of softening and delamination was observed  were prepared and characterized by using microstructural techniques and biological tests. Raman, infrared (IR) spectra and X-ray diffraction (XRD) patterns showed that there is an increasing of disorder in the Exf-GO into polymer structure, as well as in the CMC chain structure. Atomic force microscopy (AFM) images evidenced no changes in the morphology of the CMC in presence of Exf-GO. The AFM and later force microscopy (LFM) investigations of the topography show a good quality of both CMC and Exf-GO samples. Moreover, the fabrication process of CMCx/Exf-GO materials does not change topography-obtained materials. On the other hand, the increase in tensile stress was observed in the composite compared to CMC.Nanocomposites consisting of carboxymethylcellulose (CMC) and exfoliated graphene oxide (Exf-GO exhibits antimicrobial activity against Gram-positive S. aureus. At the same time, the same composite does not exhibit any cytotoxic effect on normal fibroblasts and haemolytic activity, which makes it a promising candidate for use in medical materials such as wound dressing.It was demonstrated that the composite constituted by CMC 6% and on 2.5 mL


The effect of sotagliflozin  on intestinal glucose absorption has not been investigated in humans.aO) using a dual glucose tracer method following standardized mixed meals taken after single sotagliflozin or canagliflozin doses.To measure rate of appearance of oral glucose , 24 healthy participants were randomized to 2 cohorts of 12 participants. Within each cohort, participants were randomly assigned single oral doses of either sotagliflozin 400 mg, canagliflozin 300 mg, or placebo on each of test days 1, 8, and 15. On test days, Cohort 1 had breakfast containing [6,6-aO, postprandial glucose (PPG), and postprandial insulin.R0–1 hour and/or AUC0–2 hours for RaO, PPG, and insulin after breakfast and/or the 4.25-hour postdose lunch (P < .05 versus placebo). After the 5.25-hour postdose lunch, sotagliflozin lowered RaO AUC0–1 hour and PPG AUC0–5 hours versus both placebo and canagliflozin (P < .05).Sotagliflozin and canagliflozin decreased area under the curve (AUC)Sotagliflozin delayed and blunted intestinal glucose absorption after meals, resulting in lower PPG and insulin levels, likely due to prolonged local inhibition of intestinal SGLT1 that persisted for ≥5 hours after dosing. Sodium–glucose cotransporter (SGLT) 2 is primarily responsible for the reabsorption of filtered glucose from the lumen of the renal proximal tubule, while SGLT1 is responsible for the absorption of oral glucose from the lumen of the small intestine ,4. In pa50] = 1.8 nM) and SGLT1 (IC50 = 36 nM) (aO). In animals, the sotagliflozin-mediated decrease in gastric inhibitory polypeptide (GIP) levels and increases in glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) levels after oral glucose challenge are consistent with local inhibition of intestinal SGLT1-mediated glucose absorption  . In huma= 36 nM) ,19. Thissorption . However50 = 4.2 nM) that also inhibits SGLT1 (IC50 = 663 nM) with much lower potency than sotagliflozin, also decreases incremental PPG excursions during the first meal after a dose of more than 200 mg (aO rather than increased UGE (50 = 1.2 nM] than SGLT1 [IC50 = 1400 nM]), which had no effect on PPG  using a dual glucose tracer method after single doses of sotagliflozin or canagliflozin in healthy adults.This single-center, partially double-blind, randomized, single-dose, 3-period crossover study was conducted from August 6, 2013, to October 14, 2013, at ProSciento Inc, Chula Vista, CA, USA, to assess RTwenty-four eligible males and females were randomly assigned to 2 cohorts of 12 participants each. Within each cohort, participants were randomly assigned to receive single oral doses of either 400 mg sotagliflozin, 300 mg canagliflozin (INVOKANA®), or sotagliflozin-identical placebo on each of 3 successive treatment days separated by 7-day washout periods: on days 1, 8, and 15, respectively . The 3 t2. Exclusion criteria included receipt of any protein or antibody-based therapeutic agents within 3 months before screening; prior exposure to sotagliflozin or canagliflozin; and any surgical or medical condition that might have interfered with the absorption, distribution, metabolism, or excretion of study treatment.Eligible participants were aged 18 to 55 years and had normal blood pressure, heart rate and electrocardiogram, kidney and liver function, and a body mass index of 18 to 35 kg/mAll participants provided written informed consent before enrollment. The protocol and amendment were reviewed by a centralized Institutional Review Board. The study was conducted in accordance with Good Clinical Practice as required by the International Council on Harmonisation guidelines and in accordance with US laws and the ethical principles of the Declaration of Helsinki.Before each treatment day, all participants fasted overnight for a minimum of 10 hours, then took their assigned study drug orally with water approximately 15 minutes before breakfast . The bre2H2] glucose) and lunch  MMTTs  MMTTs . In Coho13C6] glucose,  glucose, and [1-2H1] glucose enrichment profiles using the dual-isotope tracer method. Glucose fluxes were calculated using nonsteady state equations of Steele as previously described  was calculated from the infused glucose tracer ([U-13C6] glucose). RaO was calculated from the rate of appearance of ingested glucose tracer  glucose or [1-2H1] glucose, and the glucose tracer enrichment of the meals. Endogenous glucose production (EGP) was calculated by subtracting RaO from the rate of appearance of total glucose (RaT), and total glucose disposal (RdT) was calculated by subtracting the change in glucose mass from RaT.Glucose fluxes during the MMTT were determined from the measured plasma glucose concentration and plasma  glucose, many of the blood samples were used to measure glucose by enzymatic assay, insulin and connecting peptide (C-peptide) by electrochemiluminescence, active GLP-1 (aGLP-1) and total GLP-1 (tGLP-1) by electrochemiluminescent immunoassay, total PYY and GIP by enzyme-linked immunosorbent assay, and glucagon by radioimmunoassay ; in addition, acetaminophen was measured by enzymatic assay . The 24-hour urine collections were obtained to evaluate UGE for each treatment period.Blood samples were collected before the start of the stable isotope infusions and then every 10 to 30 minutes during the 10 hours after breakfast in Cohort 1, and during the 5 hours after lunch in Cohort 2, to measure plasma glucose enrichments to calculate glucose fluxes (RaO following breakfast (Cohort 1) and lunch (Cohort 2) MMTTs in healthy adults. The primary endpoint was the total area under the (rate–time) curve over the intervals of 0 to 1 hour (AUC0–1 hour) and 0 to 2 hours (AUC0–2 hours) for RaO for both the breakfast (Cohort 1) and lunch (Cohort 2) MMTTs.The primary objective was to evaluate the effect of single doses of sotagliflozin or canagliflozin on R0–4 hours); insulin and C-peptide ; RaT ; RdT ; EGP ; and tGLP-1, aGLP-1, PYY, GIP, and glucagon . Other secondary endpoints included PPG for Cohort 1 during the lunch MMTT  and UGE for Cohorts 1 and 2 (grams/24 hours).Secondary objectives and endpoints that applied to both breakfast of Cohort 1 and lunch of Cohort 2 were to evaluate the effects of single doses of sotagliflozin and canagliflozin on PPG ; RaO (AUC0–5 hours and AUC2–5 hours); RaO (AUC5–10 hours) for the lunch MMTT of Cohort 1; and PPG, insulin, C-peptide, tGLP-1, aGLP-1, PYY, GIP, glucagon, and gastric emptying (AUC0–1 hour and AUC0–2 hours). For Cohort 1, PPG and RaO AUC5–10 hours were equivalent to, and presented as, Cohort 1 lunch AUC0–5 hours so that for all MMTTs 0 hour represented the time of meal initiation.Exploratory objectives included the effects of single doses of sotagliflozin and canagliflozin on gastric emptying rate . Separate models were used for each cohort. Least squares mean differences with 2-sided 90% confidence intervals were calculated for canagliflozin versus placebo, sotagliflozin versus canagliflozin, and sotagliflozin versus placebo. Statistical assessment of treatment differences for the primary endpoint comparisons of each active drug versus placebo involved 1-sided tests with α = .05. Tests between the 2 active arms and for all nonprimary endpoint comparisons were based on 2-sided statistics. Point estimates of treatment effects used maximum likelihood methods. Tests of treatment effects for the primary and secondary endpoints are prespecified. Due to the exploratory nature of the study, adjustments for multiplicity were not performed. Hence, nominal P values and confidence intervals were provided as descriptive statistical summaries of the statistical data. Interpretation of pharmacodynamic effects due to findings from active study treatment comparisons should largely be based on the point estimates of effect and the confidence intervals derived from these point estimates; P values may be useful to guide inferences.Continuous variables were summarized descriptively. Categorical variables were summarized descriptively by their participant counts and associated percentages. For the primary pharmacodynamic parameters of RThe demographic characteristics of the 24 study participants who were enrolled and randomly assigned into the 2 cohorts are summarized in aO data are presented in aO compared with placebo: the AUC0–1 hour decreased an insignificant 19% with sotagliflozin (P = .06) and 29% with canagliflozin (P = .005), while RaO AUC0–2 hours decreased 15% with sotagliflozin (P = .01) and 17% with canagliflozin (P = .005). In contrast, RaO AUC2–5 hours increased 20% with sotagliflozin (P = .003) and 14% with canagliflozin (P = .023); this compensated for the decrease in RaO over the first 2 hours, resulting in insignificant RaO AUC0–5 hours decreases of 3% for sotagliflozin (P = .31) and 5% for canagliflozin (P = .05), all relative to placebo.RaO AUC0–1 hour decreased 32% with sotagliflozin (P < .001) and 20% with canagliflozin (P < .001) relative to placebo, with sotagliflozin showing a greater decrease relative to canagliflozin (P = .01), while RaO AUC0–2 hours decreased with both sotagliflozin  and canagliflozin  relative to placebo. RaO AUC2–5 hours again increased with sotagliflozin  and canagliflozin , resulting in RaO AUC0–5 hours that was not significantly different between either compound and placebo.After the lunch MMTT starting 4.25 hours postdose  and 3D, aO AUC0–5 hours did not differ significantly between either compound and placebo. A post hoc analysis, performed to evaluate the same AUC time points as the first 2 MMTTs, showed that RaO AUC0–1 hour decreased 19% for sotagliflozin (P = .013) but only 4% for canagliflozin (P = .63), relative to placebo. Also, the decrease in RaO AUC0–1 hour was greater with sotagliflozin relative to canagliflozin (P = .036). RaO AUC2–5 hours was increased 23% by sotagliflozin (P = .033), but only 2% by canagliflozin (P = .81), relative to placebo, providing further evidence that sotagliflozin, but not canagliflozin, delayed RaO during this MMTT.After the lunch MMTT starting 5.25 hours postdose  and 3F, aO  for sotagliflozin and decreases of 43% (P = .016) and 36% (P = .026), respectively, for canagliflozin, all relative to placebo. Consistent with these results, total PPG AUC0–1 hour and AUC0–2 hours decreased significantly with canagliflozin relative to placebo .Both sotagliflozin and canagliflozin blunted the increase in PPG concentrations after meals in a manner that mirrored their effect on RaO . After baO , increme0–1 hour decreased 47% (P < .001) with sotagliflozin and 36% (P = .009) with canagliflozin, and PPG AUC0–2 hours decreased 31% (P = .033) with sotagliflozin and 30% (P = .044) with canagliflozin, all relative to placebo. Total PPG AUC0–1 hour and AUC0–2 hours were also decreased by sotagliflozin and canagliflozin relative to placebo .After the 4.25-hour postdose lunch , increme0–5 hours was lower for sotagliflozin relative to both placebo (P = .018) and canagliflozin (P = .041), consistent with the RaO results. A post hoc analysis showed that sotagliflozin decreased incremental PPG AUC0–1 hour by 27% (P = .014) relative to placebo and decreased total PPG AUC0–1 hour and AUC0–2 hours relative to placebo and to canagliflozin .After the 5.25-hour postdose lunch , total P0–1 hour, AUC0–2 hours, and AUC0–4 hours decreased 26%, 23%, and 20%, respectively, with sotagliflozin and 32%, 32%, and 31%, respectively, with canagliflozin, all P < .05 relative to placebo. Consistent with these results, incremental C-peptide AUC0–1 hour, AUC0–2 hours, and AUC0–4 hours decreased 22%, 18%, and 13%, respectively, with sotagliflozin and 24%, 21%, and 18%, respectively, with canagliflozin, all P < .05 relative to placebo  with canagliflozin, relative to placebo  with sotagliflozin and 14% (P = .002) with canagliflozin, relative to placebo. The increases were similar after the 5.25-hour postdose lunch (0–5 hours increased 9% (P = .1) with sotagliflozin and 13% (P = .021) with canagliflozin, relative to placebo.At baseline, the EGP rate was ~2 mg/kg fat-free mass (FFM)/min before breakfast in Cohort 1 and ~3 mg/kg FFM/min before lunch in Cohort 2, with no difference among treatment groups in either cohort. After each of the 3 MMTTs, the EGP rate was initially suppressed for 2 hours and then slowly rose through hour 5  and 6B. se lunch  total EGse lunch : total E0–4 hours was not increased by either compound after breakfast (0–4 hours increased 6% (P = .026) with sotagliflozin and 7% (P = .011) with canagliflozin, relative to placebo.The effects of both compounds on glucagon mirrored their effects on EGP  and 6D. reakfast ; howeverreakfast , total gdT mirrored their effects on RaO (dT AUC0–2 hours decreased 12% with sotagliflozin (P = .014) and 15% with canagliflozin (P < .001), while RdT AUC2–5 hours increased 10% for both sotagliflozin (P < .001) and canagliflozin (P < .001), relative to placebo. This late compensation resulted in RdT AUC0–5 hours that did not differ between placebo and either compound. Findings after the 4.25-hour postdose lunch showed a similar pattern  and by 23% with canagliflozin (P = .08), and AUC0–5 hours decreased 14% by sotagliflozin (P = .013), all relative to placebo.Gastric emptying was determined indirectly by measuring the absorption of acetaminophen administered just after breakfast or lunch . NeitherP < .001 vs placebo), and 73.3 ± 20.6 g for canagliflozin (P < .001 vs placebo and vs sotagliflozin). For Cohort 2, the 24-hour UGE was 0.5 ± 1.2 g for placebo, 44.4 ± 24.3 g for sotagliflozin (P < .001 vs placebo), and 60 ± 14.5 g for canagliflozin (P < .001 vs placebo and vs sotagliflozin).All participants provided 24-hour urine collections. For Cohort 1, the 24-hour UGE (mean ± standard deviation) was 0.9 ± 2.1 g for placebo, 42.5 ± 15.3 g for sotagliflozin  from the Phase 3 inTandem1 study presented at the US Food and Drug Administration Endocrinologic and Metabolic Drugs Advisory Committee Meeting on sotagliflozin  suggesteaO and PPG data. Also, because adjustments for multiple comparisons were not performed due to the exploratory nature of the study, nominal P values and confidence intervals were provided as descriptive statistical summaries of the statistical data to guide inferences, rather than as rigorous proof of the conclusions. Accordingly, the results are hypothesis generating for the effects of sotagliflozin in patients with diabetes.Study limitations included the small number of participants, the absence of MMTTs conducted more than 5.25 hours after treatment and the post hoc analysis of some RIn conclusion, sotagliflozin blunted and delayed intestinal glucose absorption in healthy adults, resulting in lower PPG and insulin levels. The underlying mechanism was likely prolonged local inhibition of intestinal SGLT1 that persisted for at least 5 hours after sotagliflozin dosing. The sotagliflozin-mediated decrease in GIP and increases in GLP-1 and PYY levels after oral challenge with a glucose-containing meal are consistent with local inhibition of intestinal SGLT1.The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.


To subjectively and quantitatively compare the quality of 3 Tesla magnetic resonance imaging of the prostate acquired with a novel flexible surface coil (FSC) and with a conventional endorectal coil (ERC).Six radiologists independently reviewed 200 pairs of axial, high-resolution T2-weighted and diffusion-weighted image data sets, each containing one examination acquired with the FSC and one with the ERC, respectively. Readers selected their preferred examination from each pair and assessed every single examination using six quality criteria on 4-point scales. Signal-to-noise ratios were measured and compared.p = 0.04). SNR and CNR were significantly higher in the ERC acquisitions (p<0.001) except CNR differentiating tumor lesions from benign prostate (p=0.1).Two readers preferred FSC acquisition (36.5–45%) over ERC acquisition (13.5–15%) for both sequences combined, and four readers preferred ERC acquisition (41–46%). Analysis of pooled responses for both sequences from all readers shows no significant preference for FSC or ERC. Analysis of the individual sequences revealed a pooled preference for the FSC in T2WI (38.7% vs 17.8%) and for the ERC in DWI (50.9% vs 19.6%). Patients’ weight was the only weak predictor of a preference for the ERC acquisition  contains supplementary material, which is available to authorized users. Multiparametric magnetic resonance (MR) imaging of the prostate has become integral to management of patients with suspected or known prostate cancer (PCA) , 2. ProsHowever, ERCs entail several disadvantages such as increased costs, examination time, and discomfort for patients , 10, posERCs were previously considered indispensable when patients were scanned using 1.5 T MR scanners. However, several recent studies revealed comparable image quality and similar prostate cancer detection rates with or without ERCs using modern 3 T MR scanners and surface pelvic phased-array receiver coils , 12–17. Recently, novel surface coil array designs have been developed that include flexible surface coils (FSC) that can conform to the perineum, thus minimizing the distance to the prostate.In this multicenter study, we quantitatively and qualitatively compared the quality of 3 Tesla magnetic resonance imaging of the prostate acquired with the novel FSC and with a conventional ERC in T2-weighted images (T2WI) and diffusion-weighted images (DWI), since these are generally considered to be the most important sequences for prostate MR image interpretation.This retrospective, cross-sectional, HIPAA-compliant study was approved by the institutional review board with a waiver for signed consent.Images from 150 men were selected from a pool of 1200 consecutive patients who underwent multiparametric MR imaging of the prostate between June 15, 2017 and April 14, 2019. The original pool of 1200 patients was divided into two groups. Group A (300 patients) consisted of patients who underwent MR imaging using the FSC. Group B (900 patients) received routine scans with an ERC.A smaller, matched subset of these groups was chosen for study as follows: First, 50 men were randomly selected from group A. These patients were sequentially matched with 100 patients from group B at a 1:2 ratio by bodyweight , age , prostate volume , and prostate-specific antigen (PSA) range  in that particular order, to ensure similar conditions. These matched parameters were chosen to minimize potential confounding factors, for example, distance between the surface coils and the prostate. Previous biopsy status, histopathological results, and treatment history were also retrieved from the electronic medical records. All data was collected by one of the authors (TU).Imaging protocols were in accordance to the PI-RADS v2 guidelines.Images were acquired on a 3T MR scanner  using a body coil for excitation and a flexible surface phased-array coil  for reception.Thin-section high-spatial resolution axial T2-weighted 2D Fast Spin Echo (FSE) MR images of the prostate and seminal vesicles were obtained using the following parameters: Field-of-view (FOV), 18 × 10 cm; repetition time (TR)/effective echo time (TE), 9225/120; echo-train length, 30; section thickness, 3 mm; no intersection gap; acquisition matrix, 320 × 300 (512 × 512 interpolated reconstruction); frequency direction, anteroposterior, flip angle, 160°; and 1 signal excitation. The final voxel size was 0.56 mm × 0.6 mm × 3 mm (interpolated voxel size 0.35 mm × 0.35 mm × 3 mm).Diffusion-weighted images (DWI) were obtained using a spin-echo echo planar imaging (SE-EPI) acquisition using b values:  s/mm^2. Other acquisition parameters were as follows: FOV, 18 cm; TR/TE, 4500–4621 ms/minimum; thickness/gap, 3 mm/0 mm; acquisition matri× 100 × 64; acceleration factor, 2; number of excitations, 6.Images were acquired on a 3 T MR scanner  using a body coil for excitation and a pelvic phased-array coil together with an inflatable endorectal coil  filled with air for reception.Thin-section high-spatial-resolution axial T2-weighted 2D FSE MR images of the prostate and seminal vesicles were obtained using the following parameters: FOV, 18 cm; TR/effective TE, 5600–7400/99–114; echo-train length, 16; section thickness, 3 mm; no intersection gap; acquisition matrix, 384 × 384 (512 × 512 interpolated reconstruction); frequency direction, anteroposterior, flip angle, 111; and 1 signal excitation. The final voxel size was 0. 47 mm × 0. 47 mm × 3 mm (interpolated voxel size 0.35 × 0.35 × 3 mm).b values:  s/mm2. Other acquisition parameters were as follows: FOV, 18 × 10 cm; TR/TE, 4725 ms/minimum; thickness/gap, 3 mm/0 mm; acquisition matrix 128 × 64; acceleration factor, 2; number of excitations, 6. Signal non-uniformity due to the presence of an ERC was corrected using the available coil-correction software [Diffusion-weighted images (DWI) were obtained using a spin-echo echo planar imaging (SE-EPI) acquisition using b value DWI , extrapolated high b value images (1400 s/mm2 and 2000 s/mm2), and calculated apparent diffusion coefficient (ADC) maps, all of which were not reviewed in this study. All T2-weighted images were obtained prior to the injection of gadolinium.Both protocols also included sagittal and coronal T2-weighted 2D FSE images, T1-weighted images, dynamic contrast-enhanced spoiled gradient echo images, and, in some cases, spectroscopic images, T2-weighted 2D PROPELLER images, and T2-weighted 3D FSE images, but these were not reviewed in this study. Both protocols also included higher The SNR and contrast-to-noise ratios (CNR) were measured using our institutional Picture Archiving and Communication System .Regions of interest (ROI) were systematically drawn on T2WI at approximately the same location in all patients. This was done to minimize the impact of the distance between the ERC and the ROI in group B. The signal intensities (SI) of the whole prostate, peripheral zone (PZ), and transition zone (TZ), as well as of urine in the bladder were measured in all patients. The standard deviation (SD) of the SI of the urine was defined as image noise, assuming a homogenous composition and, therefore, SI of the urine.In patients with histologically proven prostate cancer, the signal intensities of the MRI lesions that were positive on MR-TRUS guided fusion biopsy were also measured.Calculation of SNR and CNR was performed as follows , 20:\docNote – SI = signal intensity, SD = standard deviation, BT = benign tissue in the respective intraprostatic zone, PCA = prostate cancer, PZ = peripheral zone, TZ = transitional zone.De-identified T2-weighted and diffusion-weighted MRI sequences were retrieved from PACS and transferred to a web-based annotation platform  that was utilized to show images to readers . Aside fSix radiologists from different imaging centers in four countries and at least 6 years of experience reading prostate MR images independently assessed multiple pairs of images. 3/6 readers evaluated all cases, whereas the other three readers assessed half of the cases due to time constraints. Each pair consisted of images of an examination from group A (FSC) and one of its matched cases from group B (ERC) combined into a single patient with two series so that they could be compared side-by-side in a single viewer. Further, paired images consisted of either T2WI or DWI. Thus, 200 pairs of sequences were available for comparison. Furthermore, each group A (FSC) examination was compared to two different group B (ERC) examination. As readers were not aware that each group A image was going to be shown twice, these were used as an internal control of consistency of assessment. The order in which pairs were presented was randomly determined. For each pair, readers chose which set of images (right or left) was generally preferred . Next, readers assessed each individual sequence’s general image quality, as well as its delineation of the prostate boundary and differentiation of the peripheral from the transition zone using a 4-point scale: 1=excellent, no need to rescan; 2=adequate, good to interpret but could rescan to improve quality if easy; 3=marginally acceptable, best to rescan but would interpret if rescanning is difficult; 4=not acceptable, must rescan.Readers also evaluated image distortion, motion artifacts, and other artifacts on 4-point scales: 1=none or minimal, no impact on interpretation; 2=moderate, minimal impact on interpretation; 3=pronounced, limits interpretation; and 4=marked, precludes interpretation.Readers did not have access to any medical history. All answers were collected using a web-based survey platform .U test (MWU) was used to test non-parametric data. To clearly separate the two sources of variation in the subjective image analysis in this study, (a) the slides presented to the radiologists and (b) the individual radiologists who rated the slides, we used a two-level analysis. First the data for each radiologist were summarized, then we summarized the data across radiologists using a meta-analysis with a random-effects model. Cohens’ kappa coefficient was calculated as measure of intrareader agreement, i.e. consistency of the given single scores for duplicate MRIs. Agreement was defined as almost perfect (k > 0.81), substantial (k = 0.61–0.80), moderate (k = 0.41–0.60), fair (k = 0.21–0.40), and poor (k ≤ 0.20) [U test. We used multivariate logistic regression to determine if patients’ age (continuous variable), prostate volume (continuous variable), bodyweight (continuous variable), PSA value (continuous variable), prior treatment (categoric variable), or readers’ number of years of experience , and experience with ERC (categoric variable) were predictors of the choice for FSC or ERC images. Statistical analysis was performed using IBM SPSS® Statistics . All tests were two tailed, and a 5% level of confidence was considered statistically significant.Descriptive statistics and the corresponding measures of dispersion were used to summarize the population characteristics. For the objective image assessment, the Mann–Whitney  ≤ 0.20) . To compApproximately half of patients in group A  and about two-thirds of patients in group B  had biopsy-proven PCA at the time of the scan, whereas PCA was suspected in the remaining cases due to elevated PSA. Gleason scores (GS) of men with known PCA in group A were 3+3 , 3+4 , 4+3 , 4+4 , and 4+5 . Gleason scores of men with known PCA in group B were 3+3 , 3+4 , 4+3 , and 4+4 . Follow-up biopsy revealed another 10 PCA cases in group A  and another 14 PCA cases in group B .In group A, 20/26 (77%) men with known disease were on active surveillance, six had received prior treatment as follows: permanent prostatic implant brachytherapy , external beam radiation therapy , and focal cryoablation . In group B, 52/69 (75%) men with known disease were on active surveillance, 17 had received prior treatment as follows: permanent prostatic implant brachytherapy , external beam radiation therapy , focal cryoablation , and androgen deprivation therapy .Patients in group A and group B did not differ significantly in bodyweight, age, prostate volume, and PSA range (Supplementary Table 1).p<0.001). The CNR discerning PZ from TZ was also significantly higher when images were acquired with an ERC (p<0.001). The CNR discerning PCA lesions from benign prostatic tissue were also higher in T2WI acquired with an ERC, but the difference was not statistically significant (p=0.1).Table Figure Table Figure P=0.11) and in T2WI when ERC was preferred (P = 0.47), and “distortion” in T2WI when ERC was preferred (P=0.58). By contrast, when readers did not have a general preference for one of the coil techniques, the differences in the individual scores were generally not statistically significant, either. The exceptions were better scores for “motion artifacts” (P<0.001) and “other artifacts” (P=0.03) for the FSC in T2WI and a better score for “motion artifacts” for the ERC in DWI (P<0.001). Supplementary Table 3 shows the pooled given mean scores for both sequences and all readers combined.Figure k: 0.42, 0.42, 0.48, and 0.53), substantial for one reader (k = 0.62), and almost perfect for one reader (k = 0.90).The intrareader agreement, i.e. the consistency of given scores for duplicate FSC cases Table , was modp = 0.04). No associations were found between coil preference and patients’ age, PSA, prostate volume, treatment, prior experience with ERC acquisition, or years of MR imaging experience in multivariate logistic regression analysis Below is the link to the electronic supplementary material.


Upon challenge with BLM, the pulmonary capillary permeability, immune cell infiltration, inflammatory cytokine production, and collagen deposition were enhanced in PKCδ–/– mice compared to that in PKCδ+/+ mice. In response to poly(I:C) stimulation, PKCδ deficient macrophages displayed an increased production of IL-1β, IL-6, TNF-α, and IL-33, which were associated with an enhanced NF-κB activation. Furthermore, we found that PKCδ could directly bind to and phosphorylate A20, an inhibitory protein of NF-κB signal. These results suggested that PKCδ may inhibit the NF-κB signaling pathway via enhancing the stability and activity of A20, which in turn attenuates pulmonary fibrosis, suggesting that PKCδ is a promising target for treating pulmonary fibrosis.Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and lethal interstitial lung disease characterized by consistent pulmonary inflammation. Although protein kinase C delta (PKCδ) is involved in broad scope cellular response, the role of PKCδ in IPF is complicated and has not been fully defined yet. Here, we reported that PKCδ deficiency (PKCδ Idiopathic pulmonary fibrosis (IPF) is a progressive, devastating, and lethal interstitial lung disease. It has a prevalence of 7∼10 per 100,000 people worldwide and a mean survival of only 3∼4 years since diagnosis . CharactAlthough the etiology of pulmonary fibrosis is complicated and unclear, inflammation is definitively involved in pathogenesis of IPF . Damagedin vitro and kinetic analysis, they demonstrated that the Tyr311 phosphorylation enhances the PKCδ basal enzymatic activity and elevates its maximal velocity in the presence of diacylglycerol. The mutation of Tyr311 to phenylalanine prevents an increase in this maximal activity -stimulated macrophages. In addition, PKCδ attenuates NF-κB activation through inducing phosphorylation and stability of A20. Our results indicated that PKCδ inhibits BLM-induced pulmonary fibrosis and is a promising drug target for IPF.–/–) mice in C57BL/6 × 129 were kindly provided by Dr. Robert Messing  were administered BLM  dissolved in saline via a single intratracheal instillation under anesthesia at a dose of 1.6 U/kg body weight to induce pulmonary fibrosis  for 1 h. Following being rinsed with PBS, the slides were covered with chromogen of final developmental DAB. Stained and differentiated slides in hematoxylin. After the dehydration, transparency and mounting of the slides, images of the tissues sections were captured by digital microscope.The lung tissue specimens were taken from six healthy donors  and six recipients  with pulmonary fibrosis during lung transplantation in Wuxi People’s Hospital , using procedures approved by the Institutional Review Board of Wuxi People’s Hospital. Sections (5 μm) were cut from the paraffin-embedded blocks prepared from the human lung tissues. Washed the slides with the specific reagents in the following order: xylene, two times, 5 min each; 100% ethanol, two times, 5 min each; 95% ethanol, two times, 5 min each; 80% ethanol, once, 5 min; 70% ethanol, once, 5 min; 50% ethanol, once, 5 min; ddHOn days 3, 7, 14, and 21 post-BLM treatment, the lungs from mice in each treatment group were collected. The right lungs were frozen until further use, while the left lung was collected after inflating with 1 ml of 4% paraformaldehyde  under constant pressure and placed in 4% paraformaldehyde. The left lung tissues were then embedded in paraffin blocks and cut into 5 μm sections for hematoxylin and eosin (H&E) staining or Masson trichrome staining to observe the inflammatory cell infiltration and collagen deposition respectively .Hydroxyproline is an indicator of the level of collagen present in a sample. The hydroxyproline content was assayed in lung hydrolyzate according to the manufacturer’s protocol for the hydroxyproline assay kit . The absorbance of the colored product was measured at 550 nm using a microplate reader to evaluate collagen deposition. In addition, myeloperoxidase (MPO) activity in lung tissue was assessed using tetramethylbenzidine (TMB), as previously described .Mice were sacrificed on days 3 and 7 after treatment with saline or BLM. Following blood collection by cardiac puncture, the trachea was exposed and intubated with a polyethylene catheter. Lungs were lavaged three times with 0.6 ml PBS (PH 7.4). All resulting BALF was collected and centrifuged at 500 g for 5 min. The supernatant was used to detect total protein concentration using a BCA Protein Assay Kit  and the pellet was resuspended to determine the number of infiltrating cells using a hemocytometer .–ΔΔCt method and was normalized to the GAPDH mRNA level.Frozen right lung lobes were homogenized with the TissueLyser system (Qiagen). Total cellular RNA in lung tissue homogenates or bone marrow derived macrophages (BMDM) as well as Raw264.7 cells were extracted by Trizol reagent (Thermo Fisher Scientific) . The cDN+/+ and PKCδ–/– mice as previously described  or PBS for different lengths of time. After platting THP1 cells in six-well plates at 1 × 106 cells per well, the cells were pretreated by rottlerin for 30 min and then challenged with 100 μg/ml poly(I:C) or PBS for 2 h. The cells were lysed by addition of 1 x loading buffer and analyzed by SDS-PAGE followed by immunoblotting using antibodies against phospho-p65, p65, phospho-p38, p38, phospho-JNK, JNK, phospho-ERK, ERK, phospho-PKCδ, PKCδ, IκBα, A20, β-actin, phospho-hA20, Myc, and Flag. The IL-33 antibody was purchased from R&D Company and other antibody reagents were obtained from Cell Signaling Technology. Quantification of Western blots was performed with ImageJ software.Three days after BLM administration, the right lungs were collected and lysed by RIPA Lysis buffer . The tissue lysate was analyzed by immunoblotting using an antibody against mouse IL-33. BMDMs were plated in six-well plates at 1 × 10BMDM cells were washed three times with PBS, fixed on coverslips with 2.5% paraformaldehyde (Sigma) for 10 min at room temperature, rinsed twice with PBS, and treated with 0.5% Triton X-100  for 10 min at room temperature. Cells were then blocked with 100 μl of 3% BSA for 1 h followed by addition of 100 μl of NF-κB antibody diluted 1:500 in 3% BSA overnight at 4°C, and then washed with PBS (3 × 5 min). The FITC-conjugated goat anti-rabbit antibody was diluted 1:1000 and 100 μl of the antibody solution was placed on each coverslip for 1 h at room temperature, followed by washing with PBS (3 × 5 min). Nuclei were stained with 0.1 μg/ml propidium iodide. The coverslips were mounted face down on microscope slides with mounting medium  and viewed on a Zeiss 410 confocal microscope . The slides were stored in a lightproof black box. The fluorescence intensity in the nucleus and cytoplasm were analyzed and calculated by Laser software.HEK-293T cells were obtained from ATCC and cultured in DMEM supplemented with 10% FBS. Raw264.7 cells were obtained from ATCC and grown in RPMI containing 10% FBS. Since commercial anti-mouse phosphorylated A20 antibodies are not available, we constructed eukaryotic expression plasmids for human PKCδ and human A20 which respectively been fused with Flag tag and Myc tag in order to detect their interaction. Flag-PKCδ and Myc-A20 were generated by PCR and were respectively cloned into the BamH1, Xbal1 and EcoR1, Xbal1 sites of pcDNA3.1. The full-length A20 protein was divided into three truncated regions, A20-1(1-383aa) included OTU domain, A20-2 (384-790aa) included Zn fingers domain and A20-3 (258-491aa) was an overlap region between A20-1 with A20-2. HEK-293T cells were transfected with pcDNA3.1-Flag-PKCδ, pcDNA3.1-Myc-A20 or three A20 truncates expression vectors by PEI method. We detected the phosphorylation level of human A20 in cells that were co-transfected with a different quantity of pcDNA3.1-Flag-PKCδ by WB. The co-IP assays were performed as previously described to detect the binding interaction between PKCδ and A20 both in 293T cells and macrophages . After at-test. One-way ANOVA, followed by Dunnett’s post hoc test, was used for multiple comparisons. Prism 5.0 software  was used for statistical analyses. A value P < 0.05 was considered statistically significant.All data are presented as means ± SEM. Differences between 2-experimental groups were analyzed using Student’s –/– mice. Fourteen and twenty one days after BLM treatment, lung tissue of PKCδ–/– mice displayed more aggravated multifocal fibrotic pulmonary lesions and inflammatory cell accumulation  staining. As shown in mulation . By usin+/+ mice . In addi+/+ mice , fibrone+/+ mice , and alp+/+ mice  was up-r–/– mice displayed more aggravated lung injury and inflammatory cell infiltration than that of PKCδ+/+ mice  was used to inhibit the PKCδ activity in poly(I:C) stimulated Raw264.7 cells  is a chronic and fatal interstitial lung disease for which current therapies have limited efficacy . It is iIn this study, we found that PKCδ deficient mice displayed severe inflammation and pulmonary fibrosis in BLM-induced pulmonary fibrosis model, which was controversial in the field. PKCδ has been reported to enhance the expression of fibronectin, collagen, and α-SMA in fibroblasts, indirectly suggesting that PKCδ promotes IPF . HoweverMacrophages, as a central contributor to pulmonary fibrosis, have many effects on regulating fibrotic response . Howeverin vitro and in vivo  ubiquitinating enzyme A20, which can be induced by tumor necrosis factor receptor (TNFR) and Toll-like receptor (TLR) pathways. Generally, A20 serves as a (de)ubiquitinating enzyme to deactivate the NF-κB signal pathway . The A20Collectively, our study defined an important role for PKCδ in BLM-induced pulmonary fibrosis and inflammation. We demonstrated that PKCδ inhibits the activation of the NF-κB signal pathway by binding and phosphorylating A20, which in turn reduces the expression of IL-33 and alleviates IPF, suggesting that PKCδ is a potential drug target for treating IPF.All datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.JW, YN, SD, TZ, and WW performed the experiments. JW and LS analyzed the data and wrote the manuscript. JW and FQ designed the study. RY, SH, and FQ provided critical review of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In vitro cultures of primary human airway epithelial cells (hAECs) grown at air–liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions, and for drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number of different differentiation media, are now available, making comparison of data between studies difficult. Here, we investigated the impact of two common differentiation media on phenotypic, transcriptomic, and physiological features of CF and non-CF epithelia. Cellular architecture and density were strongly impacted by the choice of medium. RNA-sequencing revealed a shift in airway cell lineage; one medium promoting differentiation into club and goblet cells whilst the other enriched the growth of ionocytes and multiciliated cells. Pathway analysis identified differential expression of genes involved in ion and fluid transport. Physiological assays  specifically showed that ATP12A and CFTR function were altered, impacting pH and transepithelial ion transport in CF hAECs. Importantly, the two media differentially affected functional responses to CFTR modulators. We argue that the effect of growth conditions should be appropriately determined depending on the scientific question and that our study can act as a guide for choosing the optimal growth medium for specific applications. In the respiratory tract, cells differentiate into over 40 different cell types with well-defined characteristics ,2. In ad+ absorption and Cl− secretion, key ion transport mechanisms in airway epithelia.Primary airway cell cultures are developed from intact airways, nasal, tracheo-bronchial, or alveolar cells, and are usually obtained from explanted lungs, biopsies, or brushings . The firThe ALI cell culture method is now considered the gold standard for studies on airway biology under normal or pathologic conditions ,26. It iOne of the strengths of the ALI method is that it has been shown to be predictive of the outcomes following therapies . HoweverThe aim of this study was therefore to characterize how two commonly used airway differentiation media affected the phenotypes of fully differentiated primary human airway epithelial cells (hAECs) from healthy (NCF for non-cystic fibrosis) and CF donors. Here, we show that although the epithelia displayed some degree of functional similarity, the choice of differentiation media profoundly affected histological, transcriptomic, and physiological features. Particularly relevant in the context of CF, the response to CFTR modulators was affected by growth conditions. This study provides an improved understanding of the specific characteristics of epithelia differentiated using different, but commonly used, growth conditions, and can act as a guide for choosing the optimal growth medium for a specific cell biological application.Materials and chemicals were from Alfa-Aesar , Sigma-Aldrich , Thermo Fisher Scientific , CFF Therapeutics  and Tocris Bioscence . See 3− Krebs (HCO3− KRB) contained, in mM: 25 NaHCO3, 115 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 5 D-glucose, pH 7.4 at 37 °C, 5% CO2. Standard NaCl HEPES solution, hereafter called HEPES solution, contained, in mM: 130 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES , 5 D-glucose, pH 7.4 at 37 °C. Zero K+ HCO3− KRB solution, hereafter called 0K+ solution contained, in mM: 25 NaHCO3, 120 NaCl, 1 CaCl2, 1 MgCl2, 5 D-glucose. High K+ HEPES/Nigericin  intracellular pH calibration solutions contained, in mM: 5 NaCl, 130 KCl, 1 CaCl2, 1 MgCl2, 10 NaHEPES, 5 D-glucose. ASL pH standard calibration solutions were modified Ringer solutions containing, in mM, 86 NaCl, 5 KCl, 1.2 CaCl2, 1.2 MgCl2, and either 50 MES , 50 HEPES (pH 7.0 and 7.5), or 50 Tris (pH 8.0) at 37 °C.The following buffered solutions were used in the physiological assays: HCOn = 3 donors) and CF  human airway epithelial cells (hAECs) were a kind gift from Dr. Scott H. Randell . The cells were obtained under protocol #03-1396 approved by the University of North Carolina at Chapel Hill Biomedical Institutional Review Board. Additional primary cells from 3 different CF donors  were obtained via the CFFT Biorepository. Cells were expanded using the conditionally reprogrammed cell (CRC) culture method as previously described [Primary NCF (escribed . Brieflyi) measurements, whereas cells seeded on 6.5-mm transwells were used for phenotypic analysis (histology and immunofluorescence), ion transport measurements in Ussing chambers, and airway surface liquid (ASL) pH measurements.The protocol for comparing the effect of the differentiation media is presented in v/v).In some experiments, differentiated CF epithelial cells were additionally treated for 48 h with the CFTR corrector VX-809  or vehicle control , following the manufacturer’s instructions. Briefly, lysates were mixed with 70% ethanol and loaded onto a silica-membrane column. Columns were washed and total RNA was eluted in nuclease-free water and stored at −80 °C until use.RNA isolation from fully differentiated hAECs was performed using the PureLinkThe quality of all samples was assessed with a TapeStation 4200  to verify RNA integrity. All samples had an RNA integrity number >8. Sequencing libraries were prepared using a TruSeq stranded mRNA sample preparation kit  following manufacturer’s instructions. Pooled libraries were sequenced at >15 million (75 bp) single reads per sample using a NextSeq 500 and high output kit (75 cycles) (Illumina). The RNA-sequencing data are accessible through Gene Expression Omnibus (GEO) series accession number GSE154905.® 480 SYBR Green I Master , 1.5 µL of cDNA, 0.2 μM forward primer, and 0.2 μM reverse primer  in a 96-well plate  was also performed to further validate results from the RNA-sequencing (RNA-seq) data. Prior to reverse transcription, DNase treatment was performed on 300 ng RNA using RNAse-free DNAse I  at 37 °C for 10 min. Reaction was then stopped by increasing the temperature to 70 °C for 10 min. Complementary DNA (cDNA) was synthesized from DNase I-treated total RNA (300 ng) using M-MLV reverse transcriptase  as per supplier’s protocol (1 h at 37 °C followed by 10 min at 70 °C). Real-time quantitative PCR (qPCR) was performed in a total volume of 15 µL using 2× LightCyclerll plate . PCR wasDifferentiated epithelia were washed twice with cold PBS and then lysed in RIPA (Radioimmunoprecipitation assay) buffer containing proteases inhibitors. The extracted proteins were quantified with BCA protein assay  according to the manufacturer’s protocol. ATP12A and CFTR protein levels were evaluated by Western blot (WB) analysis. Thirty µl of proteins (containing 30 µg of proteins) were incubated at 37 °C for 15 min and then loaded onto 8% SDS-PAGE gels. Proteins were electrophoretically separated and transferred onto activated PVDF membranes using a semi-dry transfer method . Membranes were blocked for one hour at room temperature with 5% skimmed milk in TBS-T  and further incubated with primary antibodies against CFTR, ATP12A, or β-actin . After the last wash, 4% paraformaldehyde , reconstituted in sterile PBS and filtered, was added to both apical and basolateral compartments and incubated for 20 min at room temperature. PFA was then removed and cells were further washed 3 times with PBS. Fixed cells were then stored at 4 °C in PBS until use.2O. Alcian blue-periodic acid–Schiff (AB-PAS) staining was then performed by incubating the slides for 5 min in Alcian blue , periodic acid solution  for 10 min and Schiff’s reagent (Reagent 2 of the PAS staining kit) for 15 min (slides were rinsed in running water and then dH20 between each incubation). Following this, slides were dehydrated by sequential incubation in solutions containing increasing ethanol concentrations and cleared in xylene for 5 min before being mounted using DPX mountant  and a 22-mm square coverslip. Slides were left to dry overnight before imaging using a Zeiss Axio Imager 2 . Epithelial thickness was later measured using Fiji imaging software [Fixed cells on semi-permeable supports were detached from the plastic holder using a scalpel, carefully cut in 2, and placed in a histological cassette kept in 70% EtOH, followed by exposure to solutions of increasing EtOH concentration, to allow for sample dehydration. Epithelia were further processed and embedded in paraffin, blocks were cut (5-µm thick) with a rotating microtome and sections collected onto slides . Slides were then incubated at 60 °C for 2 h to allow for complete adhesion and removal of extra paraffin. They were then rehydrated by sequential immersion in xylene  for 5 min and then for 1 min in solutions of decreasing EtOH concentrations (99 to 70%) and finally rinsed with dHsoftware .PFA-fixed epithelia were washed with PBS for 5 min at RT with gentle shaking before being permeabilized using a solution of 0.3% Triton in PBS  washed again for 5 min with PBS, blocked in 5% BSA in PBS , washed for 5 min in PBS, and incubated over night at 4 °C with the primary antibodies diluted in 1% BSA in PBS . The ins3− KRB solution gassed with 5% (v/v) CO2/95% (v/v) O2. Solutions at 37 °C were perfused across the apical and basolateral surfaces at a flow rate of 3 mL.min−1  and 6 mL.min−1 , respectively. Intracellular pH (pHi) was measured using a Life Sciences microfluorimeter system  in which cells were alternately excited at 490 and 440 nm wavelengths every 1.024 s with emitted light collected at 510 nm. The ratio of 490 to 440 nm emission was recorded using PhoCal 1.6 b software  and calibrated to pHi using the high K+/nigericin technique [+ solutions containing 10 µM nigericin , set to a desired pH, ranging from 6 to 7.5. For analysis of pHi measurements, ΔpHi was determined by calculating the mean pHi over 60 s resulting from treatment. The initial rate of pHi change (ΔpHi/Δt) was determined by performing a linear regression over a period of at least 40 s.Primary airway epithelial cells grown on 12-mm supports were loaded with the pH-sensitive, fluorescent dye BCECF-AM (10 µM) for 1 h in a HEPES solution at 37 °C. Cells were mounted onto the stage of a Nikon fluor inverted microscope and perfused with a modified HCO3−KRB continuously gassed with 95% O2− 5% CO2 and maintained at 37 °C. Monolayers were voltage-clamped to 0 mV and cells were left to equilibrate for at least 20 min before ion transport agonists and inhibitors were added. These were added in the following sequence: amiloride , forskolin , potentiator 5, P5 , and CFTRinh172 . Changes in short-circuit current (ΔIsc) were monitored using Ag/AgCl reference electrodes. The transepithelial short-circuit current (Isc) and the transepithelial electrical resistance (TEER) were recorded using Ag-AgCl electrodes in 3 M KCl agar bridges and the Acquire & Analyze software , as previously described [2 and expressed as Isc (µAmp.cm−2).Cells grown on 6.5 mm-inserts were mounted into the EasyMount Ussing Chamber Systems (VCC MC8 Physiologic Instrument) and bathed in bilateral HCOescribed , and res3−KRB for 15 min at 37 °C, 5% CO2. The ASL was stained using 3 µL of a mixture of dextran-coupled pH-sensitive pHrodo Red  and dextran-coupled pH-insensitive Alexa Fluor® 488  diluted in glucose-free HCO3− KRB, overnight at 37 °C, 5% CO2. Alexa Fluor® 488 was used as a loading control as pHrodo is not a ratiometric dye. The next day, fluorescence was recorded using a temperature and CO2-controlled plate reader  and agonists (Forskolin and P5) were added to the basolateral compartment at the indicated times (see figures). The data analysis was performed as previously described [® 488, ratios were generated for each time point and pH was calculated from a standard curve where pH was clamped using highly buffered solutions between 5.5 and 8. To reduce inter-experiment variability, the standard curve calibration was performed at the end of each independent experiment.Cells grown on 6.5-mm transwells were washed apically with 120 µL glucose-free HCOescribed : after s2FC) were tested for significance using a Wald test, in which the estimate of Log2FC was divided by its standard error to obtain a test statistic, which was then compared to a standard normal distribution.Quality of FASTQ files was assessed with FastQC, Version 11.7. No read trimming was necessary, and all samples were retained for further analysis. Reads were quantified against transcripts using Salmon, a program for quantifying expression of transcripts from RNA-Seq data. Salmon quantifies reads against transcripts. To obtain gene-level counts, the R package ‘tximport’ was used. Gene annotation was obtained from Ensembl gene ids using the R package ‘biomaRt’. Differential expression analysis was carried out using the R package DESeq2 . DESeq2 https://cells.ucsc.edu) [2FC values obtained from our RNA-seq dataset were then matched to these genes when present.The 50 most expressed markers for each airway cell subtype were extracted from the “Lung Atlas: Epithelial Cells” dataset from the “Teichmann Lung and Asthma Atlases—available from UCSC Cell Browser (csc.edu) . The Loghttps://www.networkanalyst.ca, [H. sapiens) and ID type (ensemble gene ID) were specified and lists of upregulated and downregulated genes were uploaded along with the corresponding Log2FC. The list enrichment network of the assorted visual analytics was then selected and enrichment analysis performed using the KEGG, Reactome, PANTHER BP , MF (Molecular Function), and CC (Cellular Component) databases. Lists were extracted and resulting Gene counts and Log plotted using GraphPad Prism8.NetworkAnalyst  was useAll other analyses were performed using GraphPad Prism8 . Where applicable, results are shown as mean ± sem. Parametric and non-parametric data distributions were assessed with the D’Agostino and Pearson normality test. Multiple group and two-group comparisons were performed using appropriate statistical tests for specific data sets .2 vs. UNC, 801 ± 45 Ω·cm2, p < 0.0001; NCF SC, 156 ± 5 Ω·cm2 vs. UNC, 629 ± 42 Ω·cm2, p < 0.0001). Interestingly, CF hAECs had a higher TEER than NCF cells for both differentiation media . Further phenotypic characterization was then performed by immunofluorescence  and adjusted p-value (p-adj), respectively, were used to determine the significantly up- or down-regulated transcripts  to which the Log2FC values obtained from our RNA-Seq dataset were matched. The 15 most expressed markers for each cell subtype are presented in The RNA-seq dataset was next used to investigate the impact of the two media on epithelial cell subtype markers of CF cultures. The 50 most expressed markers for each airway cell subtype were extracted from the “Lung Atlas: Epithelial Cells” dataset from the “Teichmann Lung and Asthma Atlases—available from UCSC Cell Browser  were not affected by differentiation media but that genes associated with club, goblet 1, and goblet 2 subtypes were significantly increased in UNC-grown epithelial cells compared to SC by 1.6 fold (Log2FC = 0.6596 ± 0.2134), 2.1 fold (Log2FC = 1.060 ± 0.1698), and 1.9 fold (Log2FC = 0.9173 ± 0.2001), respectively. Conversely, markers of ciliated 1 and 2 and ionocytes were increased in SC-grown cells compared to UNC-grown cultures by 2.2 fold (Log2FC = −1.106 ± 0.0387), 2 fold (Log2FC = −0.9802 ± 0.0549) and 2.1 fold (Log2FC = −1.070 ± 0.2491).On average, for each set of 50 genes, the results show that basal cell markers  between CF and NCF cells, a similar pattern of pathways and functions specific to epithelial cells was observed. Therefore, the main differences observed in CF cells are reported here . ATP12A is a H+/K+ ATPase that has previously been shown to contribute to a decreased ASL pH in CF [The most differentially regulated gene, involved in ion transport, was ATP12A in both CF and NCF cells (CF: LogpH in CF  and therpH in CF . In ordei) experiments [+-free modified Krebs solution (0K+) induced an intracellular acidification of −0.06 ± 0.01 at a rate of −0.04 ± 0.01 pH·min−1, due to the inhibition of H+ secretion by ATP12A, which accumulated in the cytosol . Fold changes in mRNA, protein, and levels of activity are reported in + transport  that was confirmed by RT-qPCR  and ASL pH, respectively. In CF hAECs Cl−, secretion was measured by the Isc response to forskolin (Fsk) and the CFTR potentiator, P5, as well as the total Isc inhibited by the specific CFTR inhibitor, CFTRInh172 (172), after first inhibiting sodium transport by ENaC with amiloride (Inh172-induced changes in short-circuit currents (ΔIsc) compared to UNC-grown cells, consistent with more F508del-CFTR at the cell surface. The capacity of CFTR to secrete bicarbonate was also investigated by ASL pH experiments. Although CFTR mRNA and protein levels were increased by SC medium compared to UNC, there was no difference in immunofluorescence staining of the CFTR channel C. To invv/v) for 48 h and then CFTR-dependent ion transport evaluated  or vehicle  UNC = 2.14 ± 1.71, SC = 0.35 ± 0.41, p = 0.539). Taken together, these results strongly suggest that both differentiation media affected the Cl− and HCO3− conductance of CFTR differently, particularly after treatment with VX-809, which is important in the context of CFTR modulator therapies.There have been reports of CFTR modulators impacting the chloride and bicarbonate conductance of CFTR differently ,60, and,Fully differentiated cultures of primary human airway cells grown at ALI are now considered the gold standard for in vitro studies on airway biology under normal or disease conditions ,26. SpecAlthough a reduced TEER was observed in SC- compared to UNC-grown epithelia, this was not because the cells formed a leakier epithelium containing fewer tight junctions, as SC cultures demonstrated robust tight junctional protein expression with an increased cell density. Tight junctions help the development of cell polarity, provide for cell–cell adhesion, act as a permeability barrier in well-differentiated epithelia, and also regulate the paracellular flux of ions by creating charge-selective pores, whose selectivity is mainly determined by claudin expression ,66. Our 3− secretion via dysfunctional CFTR [+ absorption [2FC = 1.54, p-adj = 7.36.10−5), which therefore provides a potential explanation for the higher ASL pH in UNC-grown cells, despite these cells having active secretion of protons. The higher resting ASL pH in UNC-grown cells could also be due to differences in paracellular flux of anions, particularly HCO3−, across the tight junctions. Recently, Thornell et al. studied the HCO3− permeability of the paracellular pathway in CF and NCF bronchial epithelia. They showed that at a steady state, HCO3− flux was weakly secretory and could be modulated by cytokines, suggesting that paracellular HCO3− transport could offset changes in ASL pH induced by transcellular movement of ions [3− permeability of the tight junctions in UNC-grown cells is consistent with previous results by Coakley et al., which also used cells grown in UNC media [3− flux across the tight junctions [+ transporters (or base-importers) must be responsible. Potential candidates include H+ channels and sodium-hydrogen exchangers (NHEs), both of which have been shown to be active in airway cells [+ channels’ expression . MVB12B has been shown to physically interact with immature band B F508del-CFTR, and is the most relevant gene involved in unconventional CFTR secretion [Our study also showed that the two culture media had distinct effects on the expression and trafficking of F508del-CFTR ; indeed ecretion . This isecretion . Misfoldecretion . AlthougInh172) was unable to block all the Fsk+P5-induced current . However, current evidence suggests that SLC26A9 protein expression is reduced in CF cells because of its interaction with F508del-CFTR at the level of the ER, and thus it also fails to traffic to the cells’ surface [Finally, one interesting observation from short-circuit current measurements was the finding that the CFTR-specific inhibitor (CFTR current  in CF cecurrents , and was surface ,92. None surface , particu surface  and more surface , in whicSignificant progress has recently been made in the characterization of the different cell types that compose the conducting airways through advances in single cell RNA-seq  and a be