Walnut protein isolate based emulsion as a promising delivery system enhanced lutein bioaccessibility.

Lutein, a natural pigment with multiple beneficial bioactivities, faces limitations in food processing due to its instability. In this study, we constructed four modified walnut protein isolate (WNPI) based emulsions as emulsion-based delivery systems (EBDS) for lutein fortification. The modification treatments enhanced the encapsulation efficiency of the WNPI-based EBDS on lutein. The modified WNPI-based EBDS exhibited improved storage and digestive stability, as well as increased lutein delivery capability in simulated gastrointestinal conditions. After in vitro digestion, the lutein retention in the modified WNPI-based EBDS was higher than in the untreated WNPI-based EBDS, with a maximum retention of 49.67 ± 1.10 % achieved after ultrasonic modification. Furthermore, the modified WNPI-based EBDS exhibited an elevated lutein bioaccessibility, reaching a maximum value of 40.49 ± 1.29 % after ultrasonic modification, nearly twice as high as the untreated WNPI-based EBDS. Molecular docking analysis indicated a robust affinity between WNPI and lutein, involving hydrogen bonds and hydrophobic interactions. Collectively, this study broadens WNPI's application and provides a foundation for fortifying other fat-soluble bioactive substances.

Characteristics and risk factors for invasive fungal infection in hospitalized patients with acute-on-chronic hepatitis B liver failure: a retrospective cohort study from 2010 to 2023.

Background and aim: The global burden of invasive fungal infections (IFIs) is emerging in immunologic deficiency status from various disease. Patients with acute-on-chronic hepatitis B liver failure (ACHBLF) are prone to IFI and their conditions are commonly exacerbated by IFI. However, little is known about the characteristics and risk factors for IFI in hospitalized ACHBLF patients. Methods: A total of 243 hospitalized ACHBLF patients were retrospectively enrolled from January 2010 to July 2023. We performed restricted cubic spline analysis to determine the non-linear associations between independent variables and IFI. The risk factors for IFI were identified using logistic regression and the extreme gradient boosting (XGBoost) algorithm. The effect values of the risk factors were determined by the SHapley Additive exPlanations (SHAP) method. Results: There were 24 ACHBLF patients (9.84%) who developed IFI on average 17.5 (13.50, 23.00) days after admission. The serum creatinine level showed a non-linear association with the possibility of IFI. Multiple logistic regression revealed that length of hospitalization (OR = 1.05, 95% CI: 1.02-1.08, P = 0.002) and neutrophilic granulocyte percentage (OR = 1.04, 95% CI: 1.00-1.09, P = 0.042) were independent risk factors for IFI. The XGBoost algorithm showed that the use of antibiotics (SHAP value = 0.446), length of hospitalization (SHAP value = 0.406) and log (qHBV DNA) (SHAP value = 0.206) were the top three independent risk factors for IFI. Furthermore, interaction analysis revealed no multiplicative effects between the use of antibiotics and the use of glucocorticoids (P = 0.990). Conclusion: IFI is a rare complication that leads to high mortality in hospitalized ACHBLF patients, and a high neutrophilic granulocyte percentage and length of hospitalization are independent risk factors for the occurrence of IFI.

IL-22-producing CD3 + CD8- T cells increase in immune clearance stage of chronic HBV infection and correlate with the response of Peg-interferon treatment.

Interleukin (IL)-22 regulates host defense. This study investigated the predominant IL-22-producing cell subsets under HBV associated immune stages. We found circulating IL-22-producing CD3 + CD8- T cells were significantly increased in immune active (IA) stage than those in immunotolerant stage, inactive carrier and healthy controls (HCs). The plasma IL-22 level was higher in IA and HBeAg-negative CHB compared to HCs. Importantly, CD3 + CD8- T cells were identified as the predominant source of plasma IL-22 production. Up-regulated IL-22-producing CD3 + CD8- T cells obviously correlated with the grade of intrahepatic inflammation. The proportions of IL-22-producing CD3 + CD8- T cells were significantly down-regulated after 48 weeks of Peg-interferon treatment, and the differences were of great significance in patients with normalize ALT levels at 48 weeks, rather than those with elevated ALT levels. In conclusion, IL-22 might play a proinflammatory function in. chronic HBV infected patients with active inflammation and Peg-interferon treatment could attenuate the degree of liver inflammation through down-regulating IL-22-producing CD3 + CD8- T cells.

Structural modification induced by heat treatments improves the emulsifying attributes of lacquer seed protein isolate.

The lacquer seed oil has received extensive attention in the food industry due to its health function, such as regulating blood lipids. But its by-product, lacquer seed meal, is often used as a low-value-added product such as animal feed. Lacquer seed meal contains about 20 % protein, which has amphiphilic properties, and there is limited attention to its emulsifying properties. In this study, the impact of heat treatment on the emulsifying properties of lacquer seed protein isolate (LSPI) was investigated. The EAI and ESI of the 120 °C heated LSPI increased by 77.1 % and 55.2 %, respectively. The emulsions prepared using heat-modified LSPI (120 °C) further showed lower hydroperoxide, TBARS and protein carbonyl contents (only 61.3 %, 61.0 % and 58.6 % of control) after storage. This result indicates that heat-treated LSPI retarded lipid and protein oxidation in LSPI-stabilized emulsions during storage. Changes in protein structure showed that increasing heating temperature resulted in the depolymerization of tertiary structure, higher surface hydrophobicity and lower contents of α-helix of LSPI. These changes in protein structure made the heated LSPIs have better emulsifying properties. Therefore, these findings developed a new use of LSPI and greatly enhanced the potential of LSPI as a natural emulsifier in the food industry.

Allyl phenyl selenides as H2O2 acceptors to develop ROS-responsive theranostic prodrugs.

Reactive oxygen species (ROS)-responsive prodrugs have received significant attention due to their capacity to target tumors to relieve the side effects caused by chemotherapy. Herein, a series of novel H2O2-activated theranostic prodrugs (CPTSe1-CPTSe7) were developed containing allyl phenyl selenide moieties as H2O2 acceptors. Compared with conventional boronate ester-based prodrug CPT-B, CPTSe1 was more stable in human plasma and showed a more complete release of camptothecin (CPT) in H2O2 inducing experiment. The selectively activated fluorescence signals of CPTSe1 in tumor cells make it useful for real-time monitoring of CPT release and H2O2 detection. Furthermore, excellent selectivity of CPTSe1 was achieved for tumor cells over normal cells. Our results provide a new platform for the development of H2O2-responsive theranostic prodrugs.

Triazene as the Directing Group Achieving Highly Ortho-Selective Diborylation and Sequential Functionalization.

This study describes a regioselective ortho,ortho'-diborylation of aromatic triazenes catalyzed by [Ir(OMe)(cod)]2 in near-quantitative yields without an additional ligand. Aromatic triazenes act as both substrates and ligands. The X-ray structures of 2a and 2p indicate that the monoborylation products could promote the occurrence of diborylation. The synthesized triazene-substituted diboronate esters could undergo a variety of transformations including directing group removal. One-pot sequential modification provides a short entry to densely functionalized arenes.

Caprylic Acid Improves Lipid Metabolism, Suppresses the Inflammatory Response and Activates the ABCA1/p-JAK2/p-STAT3 Signaling Pathway in C57BL/6J Mice and RAW264.7 Cells.

OBJECTIVE: This study aimed to investigate the effects of caprylic acid (C8:0) on lipid metabolism and inflammation, and examine the mechanisms underlying these effects in mice and cells. METHODS: Fifty-six 6-week-old male C57BL/6J mice were randomly allocated to four groups fed a high-fat diet (HFD) without or with 2% C8:0, palmitic acid (C16:0) or eicosapentaenoic acid (EPA). RAW246.7 cells were randomly divided into five groups: normal, lipopolysaccharide (LPS), LPS+C8:0, LPS+EPA and LPS+cAMP. The serum lipid profiles, inflammatory biomolecules, and ABCA1 and JAK2/STAT3 mRNA and protein expression were measured. RESULTS: C8:0 decreased TC and LDL-C, and increased the HDL-C/LDL-C ratio after injection of LPS. Without LPS, it decreased TC in mice ( P < 0.05). Moreover, C8:0 decreased the inflammatory response after LPS treatment in both mice and cells ( P < 0.05). Mechanistic investigations in C57BL/6J mouse aortas after injection of LPS indicated that C8:0 resulted in higher ABCA1 and JAK2/STAT3 expression than that with HFD, C16:0 and EPA, and resulted in lower TNF-α, NF-κB mRNA expression than that with HFD ( P < 0.05). In RAW 264.7 cells, C8:0 resulted in lower expression of pNF-κBP65 than that in the LPS group, and higher protein expression of ABCA1, p-JAK2 and p-STAT3 than that in the LPS and LPS+cAMP groups ( P < 0.05). CONCLUSION: Our studies demonstrated that C8:0 may play an important role in lipid metabolism and the inflammatory response, and the mechanism may be associated with ABCA1 and the p-JAK2/p-STAT3 signaling pathway.

Synthesis and Biological Evaluation of 10-Substituted Camptothecin Derivatives with Improved Water Solubility and Activity.

Despite remarkable clinical achievements, camptothecin (CPT) still suffers from poor solubility and severe toxicity. Therefore, it is necessary to redevelop CPT derivatives as supplementary antitumor agents with good water solubility and small side effects. In this work, 27 camptothecin derivatives were synthesized and screened for their cytotoxicity against A549 (lung) and HCT-116 (colon) cancer cell lines. Among them, compound B7, 7-ethyl-10-(2-oxo-2-(4-methylpiperidin-1-yl)ethoxy)camptothecin,was demonstrated in vitro to be a more potent antitumor agent than SN-38 by comparison of their inhibitory activities against cell proliferation and colony formation and interference effect on process of cell cycle and cell apoptosis. Additionally, a molecular docking model revealed that B7 can interact with the topoisomerase I-DNA complex, and that the solubility of B7 reached 5.73 µg/mL in water. Moreover, B7 significantly inhibited tumor growth in an A549 xenograft model at dosages of 0.4 and 2.0 mg/kg, and exhibited minimum lethal doses comparable to those of irinotecan. These results indicated that B7, with improved solubility, enhanced activity and acceptable acute toxicity, can be used as a lead compound for the development of novel anticancer agents.

Discovery of potent small molecule PROTACs targeting mutant EGFR.

Epidermal growth factor receptor (EGFR) is an important therapeutic target for the treatment of non-small cell lung cancer. A number of efficacious EGFR tyrosine kinase inhibitors have been developed. However, acquired drug resistance largely encumbered their clinical practicability. Therefore, there is an urgent need to develop new therapeutic regime. Herein, we designed and synthesized a set of EGFR-targeting small molecule PROTACs which showed promising efficacy. In particular, VHL-recruiting compound P3 showed potent anti-proliferative activity against HCC827 and H1975 cell lines with IC50 values of 0.83 and 203.01 nM, respectively. Furthermore, both EGFRdel19 and EGFRL858R/T790M could be significantly induced to be degraded under treatment of P3 with DC50 values of 0.51 and 126.2 nM, respectively. Compound P3 was able to dramatically suppress EGFR pathway signal transduction. Moreover, compound P3 could significantly induce cell apoptosis, arrest cell cycle and suppress cell colony formation. In addition, we identified that ubiquitination was indispensable in the degradation process, and found that the degradation was related to autophagy. Our work would provide an alternative approach for development of potentially effective EGFR degraders and give a new clue for investigation of PROTAC-induced protein degradation.

F10, a new camptothecin derivative, was identified as a new orally-bioavailable, potent antitumor agent.

Topoisomerases are interesting targets for drug discovery. In the present study, we attached saturated carbon atoms to the 10-position of camptothecin and synthesized 10 new camptothecin derivatives from 10-HCPT or SN-38. The activities of new compounds were evaluated both in vitro and in vivo. The most promising compound F10, 7-ethyl-10-(2-oxo-2-(piperidin-1-yl)ethoxy)camptothecin, inhibited cancer cells growth with the IC50 of 0.002, 0.003, 0.011 and 0.081 µM on Raji, HCT116, A549 and Lovo cells, respectively. Meanwhile, oral administration of F10 remarkably suppressed the HCT116-xenograft tumor growth in the nude-mice model at the dosage of 0.5, 2.0 and 8.0 mg/kg in vivo. Intraperitoneal administration of F10 can completely inhibit Raji-xenograft tumor growth in established NPG mouse model at 2.0 and 4.0 mg/kg. In addition, the minimum lethal doses of F10 and SN-38 in mice by intravenous administration were 80 and 40 mg/kg (or 0.155, 0.102 mmol/kg), respectively. The solubility of F10 reached 9.86 µg/mL in a buffer solution of pH 4.5. The oral bioavailability of F10 achieved 22.4% in mice. The molecular docking model revealed that F10 can interact with topoisomerase I-DNA complex. Our findings indicate that F10 is a new orally-oavailable antitumor agent with potent anticancer effect. Furthermore, attaching a ring hydrophobic moiety to the 10-position of camptothecin provides a favorable approach in the optimization of camptothecin.

K2S2O8-promoted C-Se bond formation to construct α-phenylseleno carbonyl compounds and α,ß-unsaturated carbonyl compounds.

A novel K2S2O8-promoted C-Se bond formation from cross-coupling under neutral conditions has been developed. A variety of aldehydes and ketones react well using K2S2O8 as the oxidant in the absence of catalyst and afford desired products in moderate to excellent yields. This protocol provides a very simple route for the synthesis of α-phenylseleno carbonyl compounds and α,ß-unsaturated carbonyl compounds.

Novel mutations of AXIN2 identified in a Chinese Congenital Heart Disease Cohort.

Congenital heart defects (CHDs), the most common congenital human birth anomalies, involves complex genetic factors. Wnt/ß-catenin pathway is critical for cardiogenesis and proved to be associated with numerous congenital heart abnormities. AXIN2 has a unique role in Wnt/ß-catenin pathway, as it is not only an important inhibitor but also a direct target of Wnt/ß-catenin pathway. However, whether AXIN2 is associated with human CHDs has not been reported. In our present study, we found a differential expression of Axin2 mRNA during the development of mouse heart, indicating its importance in mouse cardiac development. Then using targeted next-generation sequencing, we found two novel case-specific rare mutations [c.28 C > T (p.L10F), c.395 A > G (p.K132R)] in the sequencing region of AXIN2. In vitro functional analysis suggested that L10F might be a loss-of-function mutation and K132R is a gain-of-function mutation. Both mutations disrupted Wnt/ß-catenin pathway and failed to rescue CHD phenotype caused by Axin2 knockdown in zebrafish model. Collectively, our study indicates that rare mutations in AXIN2 might contribute to the risk of human CHDs and a balanced canonical Wnt pathway is critical for cardiac development process. To our knowledge, it is the first study of AXIN2 mutations associated with human CHDs, providing new insights into CHD etiology.

Synthesis and biological evaluation of irreversible EGFR tyrosine kinase inhibitors containing pyrido[3,4-d]pyrimidine scaffold.

In the present study, a new class of compounds containing pyrido[3,4-d]pyrimidine scaffold with an acrylamide moiety was designed as irreversible EGFR-TKIs to overcome acquired EGFR-T790M resistance. The most promising compound 25h inhibited HCC827 and H1975 cells growth with the IC50 values of 0.025 µM and 0.49 µM, respectively. Meanwhile, 25h displayed potent inhibitory activity against the EGFRL858R (IC50 = 1.7 nM) and EGFRL858R/T790M (IC50 = 23.3 nM). 25h could suppress EGFR phosphorylation in HCC827 and H1975 cell lines and significantly induce the apoptosis of HCC827 cells. Additionally, compound 25h could remarkably inhibit cancer growth in established HCC827 xenograft mouse model at 50 mg/kg in vivo. These results indicated that the 2,4-disubstituted 6-(5-substituted pyridin-2-amino)pyrido[3,4-d]pyrimidine derivatives can serve as effective EGFR inhibitors and potent anticancer agents.

Handgrip Strength as a Predictor of Nutritional Status in Chinese Elderly Inpatients at Hospital Admission.

OBJECTIVE: To assess nutritional status and define gender- and age-specific handgrip strength (HGS) cut-point values for malnutrition or nutritional risk in elderly inpatients. METHODS: A cross-sectional study of 1,343 elderly inpatients was conducted in the Chinese PLA General Hospital. Nutrition Risk Screening (NRS 2002) and Subjective Global Assessment (SGA) were administered. Anthropometric measurements and blood biochemical indicators were obtained using standard techniques. The gender- and age-specific receiver operating characteristic (ROC) curves were constructed to evaluate the HGS for nutritional status by SGA and NRS 2002. Sensitivity, specificity, and areas under the curves (AUCs) were calculated. RESULTS: According to NRS 2002 and SGA, 63.81% of elderly inpatients were at nutritional risk and 28.22% were malnourished. Patients with higher HGS had an independently decreased risk of malnutrition and nutritional risk. The AUCs varied between 0.670 and 0.761. According to NRS 2002, the optimal HGS cut-points were 27.5 kg (65-74 years) and 21.0 kg (75-90 years) for men and 17.0 kg (65-74 years) and 14.6 kg (75-90 years) for women. According to SGA, the optimal HGS cut-points were 24.9 kg (65-74 years) and 20.8 kg (75-90 years) for men and 15.2 kg (65-74 years) and 13.5 kg (75-90 years) for women. CONCLUSION: Elderly inpatients had increased incidence of malnutrition or nutritional risk. HGS cut-points can be used for assessing nutritional status in elderly inpatients at hospital admission in China.

A TBX5 3'UTR variant increases the risk of congenital heart disease in the Han Chinese population.

TBX5 is a vital transcription factor involved in cardiac development in a dosage-dependent manner. But little is known about the potential association of TBX5 3' untranslated region (UTR) variations with congenital cardiac malformations. This study aimed to investigate the relationship between TBX5 3'UTR variants and risk for congenital heart disease (CHD) susceptibility in two Han Chinese populations, and to reveal its molecular mechanism. The relationship between TBX5 3'UTR variants and CHD susceptibility was examined in 1 177 CHD patients and 990 healthy controls in two independent case-control studies. Variant rs6489956 C>T was found to be associated with increased CHD susceptibility in both cohorts. The combined CHD risk for the CT and TT genotype carriers was 1.83 times higher than that of CC genotype, while the risk for CT or TT genotype was 1.94 times and 2.31 times higher than that of CC carriers, respectively. Quantitative real-time PCR and western blot analysis showed that T allele carriers exhibited reduced TBX5 mRNA and protein levels in CHDs tissues. Compared with C allele, T allele showed increased binding affinity to miR-9 and miR-30a in both luciferase assays and surface plasmon resonance analysis. Functional analysis confirmed that miR-9 and miR-30a downregulated TBX5 expression at the transcriptional and translational levels, respectively. The assays in zebrafish model were in support of the interaction of miR-9/30a and TBX5 3'UTR (C and T allele). We concluded that TBX5 3'UTR variant rs6489956 increased susceptibility of CHD in the Han Chinese population because it changes the binding affinity of two target miRNAs that specifically mediate TBX5 expression.

Flavonoids isolated from Sinopodophylli Fructus and their bioactivities against human breast cancer cells.

Four prenylated flavonoids compounds 1-4, named sinopodophyllines A-D, and a flavonoid glycoside (compound 13), sinopodophylliside A, together with 19 known compounds (compounds 5-12 and 14-24) were isolated from the fruits of Sinopodophyllum hexandrum. The structures of new compounds were elucidated by extensive spectroscopic analysis, including HRESIMS, 1D and 2D NMR. Compounds 1-6, 9-11, and 14-17 were tested for their cytotoxicity against human breast-cancer T47D, MCF-7 and MDA-MB-231 cells in vitro, and compounds 2, 5, 6, 10 and 11 showed significant cytotoxicity (IC50 values < 10 µmol·L-1) against T47D cells.

Lignans and diterpenes isolated from Tirpitzia ovoidea and their biological activities.

A new lignan, tirpitzin A (17) together with 20 known compounds (1-16, and 18-21) were isolated from the ethyl acetate soluble fraction of ethanol extract of the aerial parts of Tirpitzia ovoidea. The structure of new compound was elucidated by means of spectroscopic analysis. Of the known compounds, 7-21 were isolated from Linaceae family for the first time. The pharmacological activity of the crude extracts was tested using a mouse inflammation model induced by dimethyl benzene. The results demonstrated that the ethyl acetate soluble fraction had anti-inflammatory activity. Moreover, the cytotoxic and anti-inflammatory activities of some compounds were studied. The new compound 17 showed moderate cytotoxic effect against BxPC-3 cell line (IC50 = 19.51µmol·L-1) and Compound 10 showed significant cytotoxicity against HepG2, HL-60, U87 and BxPC-3 cell lines with IC50 values in the range 4.2-8.3µmol·L-1. Additionally, Compounds 2, 10, 11, and 13 exhibited potent inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages at the concentration of 50µmol·L-1.

Mild decrease in TBX20 promoter activity is a potentially protective factor against congenital heart defects in the Han Chinese population.

Congenital heart defects (CHDs) are one of the most common human birth defects worldwide. TBX20 is a crucial transcription factor for the development of embryonic cardiovascular system. Previous studies have demonstrated that mutations in the TBX20 coding region contribute to familial and sporadic CHD occurrence. However, it remains largely unknown whether variants in the TBX20 regulatory region are also related to CHDs. In this study, we sequenced the 2 kb region upstream of the TBX20 transcription start site in 228 CHD patients and 292 controls in a Han Chinese population. Among the 8 single nucleotide polymorphisms (SNPs) identified, six SNPs are in strong linkage disequilibrium and the minor alleles are associated with lower CHD risk (for rs10235849 chosen as tag SNP, p = 0.0069, OR (95% CI) = 0.68 (0.51-0.90)). Functional analysis showed that the minor alleles have lower transcriptional activity than major alleles in both human heart tissues and three cell lines. The electrophoretic mobility shift assay suggested that TBX20 minor alleles may exhibit higher binding affinity with certain transcription repressors. Our results indicate that a moderately lower TBX20 activity potentially reduces CHD risk in the Han Chinese population, providing new insight in the study of CHD etiology.

The Association of Maternal Body Composition and Dietary Intake with the Risk of Gestational Diabetes Mellitus during the Second Trimester in a Cohort of Chinese Pregnant Women.

OBJECTIVE: To investigate the association of maternal body composition and dietary intake with the risk of gestational diabetes mellitus (GDM). METHODS: A total 154 GDM subjects and 981 controls were enrolled in a prospective cohort study in 11 hospitals from May 20, 2012 to December 31, 2013. Bioelectrical impedance analysis and dietary surveys were used to determine body composition and to evaluate the intake of nutrients in subjects at 21-24 weeks' gestation (WG). Logistic regression analysis was applied to explore the relationships of maternal body composition and dietary intake with the risk of GDM morbidity. RESULTS: Age, pre-pregnant body weight (BW), and body mass index (BMI) were associated with increased risk of GDM. Fat mass (FM), fat mass percentage (FMP), extracellular water (ECW), BMI, BW, energy, protein, fat, and carbohydrates at 21-24 WG were associated with an increased risk of GDM. In contrast, fat free mass (FFM), muscular mass (MM), and intracellular water (ICW) were associated with a decreased risk of GDM. CONCLUSION: Maternal body composition and dietary intake during the second trimester of pregnancy were associated with the risk of GDM morbidity.

Medium-Chain Triglyceride Activated Brown Adipose Tissue and Induced Reduction of Fat Mass in C57BL/6J Mice Fed High-fat Diet.

OBJECTIVE: To investigate activation of brown adipose tissue (BAT) stimulated by medium-chain triglyceride (MCT). METHODS: 30 Male C57BL/6J obese mice induced by fed high fat diet (HFD) were divided into 2 groups, and fed another HFD with 2% MCT or long-chain triglyceride (LCT) respectively for 12 weeks. Body weight, blood biochemical variables, interscapular brown fat tissue (IBAT) mass, expressions of mRNA and protein of beta 3-adrenergic receptors (ß3-AR), uncoupling protein-1 (UCP1), hormone sensitive lipase (HSL), protein kinase A (PKA), and adipose triglyceride lipase (ATGL) in IBAT were measured. RESULTS: Significant decrease in body weight and body fat mass was observed in MCT group as compared with LCT group (P<0.05) after 12 weeks. Greater increases in IBAT mass was observed in MCT group than in LCT group (P<0.05). Blood TG, TC, LDL-C in MCT group were decreased significantly, meanwhile blood HDL-C, ratio of HDL-C/LDL-C and norepinephrine were increased markedly. Expressions of mRNA and protein of ß3-AR, UCP1, PKA, HSL, ATGL in BAT were greater in MCT group than in LCT group (P<0.05). CONCLUSION: Our results suggest that MCT stimulated the activation of BAT, possible via norepinephrine pathway, which might partially contribute to reduction of the body fat mass in obese mice fed high fat diet.