WDR73 Depletion Destabilizes PIP4K2C Activity and Impairs Focal Adhesion Formation in Galloway-Mowat Syndrome.

(1) Background: Galloway-Mowat syndrome (GAMOS) is a rare genetic disease, classically characterized by a combination of various neurological symptoms and nephrotic syndrome. WDR73 is the pathogenic gene responsible for GAMOS1. However, the pathological and molecular mechanisms of GAMOS1, especially nephrotic syndrome caused by WDR73 deficiency, remain unknown. (2) Methods and Results: In this study, we first observed remarkable cellular morphological changes including impaired cell adhesion, decreased pseudopodia, and G2/M phase arrest in WDR73 knockout (KO) HEK 293 cells. The differentially expressed genes in WDR73 KO cells were enriched in the focal adhesion (FA) pathway. Additionally, PIP4K2C, a phospholipid kinase also involved in the FA pathway, was subsequently validated to interact with WDR73 via protein microarray and GST pulldown. WDR73 regulates PIP4K2C protein stability through the autophagy-lysosomal pathway. The stability of PIP4K2C was significantly disrupted by WDR73 KO, leading to a remarkable reduction in PIP2 and thus weakening the FA formation. In addition, we found that podocyte-specific conditional knockout (Wdr73 CKO) mice showed high levels of albuminuria and podocyte foot process injury in the ADR-induced model. FA formation was impaired in primary podocytes derived from Wdr73 CKO mice. (3) Conclusions: Since FA has been well known for its critical roles in maintaining podocyte structures and function, our study indicated that nephrotic syndrome in GAMOS1 is associated with disruption of FA caused by WDR73 deficiency.

Disordered farnesoid X receptor signaling is associated with liver carcinogenesis in Abcb11-deficient mice.

ABCB11 encodes the bile salt export pump (BSEP), a key regulator in maintaining bile acid (BA) homeostasis. Although inherited ABCB11 mutations have previously been linked to primary liver cancer, whether ABCB11 deficiency leads to liver cancer remains unknown. Here, we analyzed ABCB11 mRNA expression levels in liver tumor specimens [29 with hepatocellular carcinoma (HCC), one with intrahepatic cholangiocarcinoma (ICC), and one with mixed HCC/ICC] with adjacent normal specimens and published human datasets. Liver tissues obtained from Abcb11-deficient (Abcb11-/- ) mice and wild-type mice at different ages were compared by histologic, RNA-sequencing, and BA analyses. ABCB11 was significantly downregulated in human liver tumors compared with normal controls. Abcb11-/- mice demonstrated progressive intrahepatic cholestasis and liver fibrosis, and spontaneously developed HCC and ICC over 12 months of age. Abcb11 deficiency increased BAs in the liver and serum in mice, most of which are farnesoid X receptor (FXR) antagonists/non-agonists. Accordingly, the hepatic expression and transcriptional activity of FXR were downregulated in Abcb11-/- mouse livers. Administration of the FXR agonist obeticholic acid reduced liver injury and tumor incidence in Abcb11-/- mice. In conclusion, ABCB11 is aberrantly downregulated and plays a vital role in liver carcinogenesis. The cholestatic liver injury and liver tumors developed in Abcb11-/- mice are associated with increased FXR antagonist BAs and thereby decreased activation of FXR. FXR activation might be a therapeutic strategy in ABCB11 deficiency diseases. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pyrite/H2O2/hydroxylamine system for efficient decolorization of rhodamine B.

To improve the efficiency of the Fe(II)/Fe(III) cycle and continuous reactivity of pyrite, a pyrite/H2O2/hydroxylamine (HA) system was proposed to treat rhodamine B (RhB). The results showed that near-complete decolorization and 52.8% mineralization 50 mg L-1 RhB were achieved under its optimum conditions: HA 0.8 mM, H2O2 1.6 mM, pyrite 0.4 g L-1, and initial pH 4.0. The degradation reaction was dominated by an •OH radical produced by the reaction of Fe2+ with H2O2 in solution. HA primarily had two roles: in solution, HA could accelerate the Fe(II)/Fe(III) cycle through its strong reducibility to enhance RhB decolorization; on the pyrite surface, HA could improve the continuous reactivity of pyrite by inhibiting the oxidation of pyrite. In addition, the dosing manner of HA had a significant effect on RhB decolorization. In addition, the high decolorization and mineralization efficiency of other dye pollutants suggested that the pyrite/H2O2/HA system might be widely used in textile wastewater treatment.

The Function of Multidrug Resistance-associated Protein 3 in the Transport of Bile Acids under Normal Physiological and Lithocholic Acid-induced Cholestasis Conditions.

BACKGROUND: The role of multidrug resistance-associated protein 3 (Mrp3) in the transport of bile acid (BA) in drug-induced cholestasis has not been well studied. OBJECTIVE: In this study, wild type and Mrp3 knockout (Mrp3-/-) mice under normal physiological and lithocholic acid (LCA)-induced cholestatic conditions were employed to investigate the role of Mrp3 in BA transport. METHODS: The levels of BA in serum, liver, gallbladder, intestine, kidney, feces and urine were quantified in both wild type and Mrp3-/- mice via ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC-MS/MS). Quantitative real-time PCR (RT-PCR) analysis was used to measure the expression of genes related to the transport and synthesis of BA. RESULTS: The results showed that the liver did not suffer more serious damage as a result of cholestasis when Mrp3 was depleted. The level of some individual bile acids changed apparently in the compartments of enterohepatic circulation (EHC) between the two control and model groups, respectively, but the level of serum total bile acid was only slightly reduced for Mrp3-/- groups. In addition, the level of BA-related efflux transporters and synthases increased significantly when Mrp3 was knocked out under normal physiological conditions, but a negligible alteration appeared under cholestatic conditions. CONCLUSION: Our results indicated that Mrp3 could be responsible for the transport of some specific bile acids, and part of the Mrp3 role could be compensated for by other transporters. Moreover, Mrp3 deficiency has a direct effect on the expression of BA-related synthases and efflux transporters under normal physiological conditions, but this effect could be less prominent under cholestatic conditions. This study could provide much valuable insight into the physiological function of Mrp3 in the transport of bile acids.

Detection of Spinal Muscular Atrophy Using a Duplexed Real-Time PCR Approach With Locked Nucleic Acid-Modified Primers.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder mainly caused by homozygous deletions that include exon 7 of the survival motor neuron 1 (SMN1) gene. A nearby paralog gene, SMN2, obstructs the specific detection of SMN1. We optimized a duplexed real-time PCR approach using locked nucleic acid (LNA)-modified primers to specifically detect SMN1. METHODS: An LNA-modified primer pair with 3' ends targeting SMN1 specific sites c.835-44g and c.840C was designed, and its specificity was examined by real-time PCR and Sanger Sequencing. A duplexed real-time PCR approach for amplifying SMN1 and control gene albumin (ALB) was developed. A randomized double-blind trial with 97 fresh peripheral blood samples and 25 dried blood spots (DBS) was conducted to evaluate the clinical efficacy of the duplexed approach. This new approach was then used to screen 753 newborn DBS. RESULTS: The LNA-modified primers exhibited enhanced specificity and 6.8% increased efficiency for SMN1 amplification, compared with conventional primers. After stabilizing the SMN1 test by optimizing the duplexed real-time PCR approach, a clinical trial validated that the sensitivity and specificity of our new approach for detecting SMA patients and carriers was 100%. Using this new approach, 15 of the screened 753 newborns were identified as carriers via DBS, while the rest were identified as normal individuals. These data reveal a carrier rate of 1.99% in Hunan province, South Central China. CONCLUSIONS: We have developed a novel, specific SMN1 detection approach utilizing real-time PCR with LNA-modified primers, which could be applied to both prenatal carrier and newborn screening.

Changes and sex- and age-related differences in the expression of drug metabolizing enzymes in a KRAS-mutant mouse model of lung cancer.

BACKGROUND: This study aimed to systematically profile the alterations and sex- and age-related differences in the drug metabolizing enzymes (DMEs) in a KRAS-mutant mouse model of lung cancer (KRAS mice). METHODOLOGY: In this study, the LC-MS/MS approach and a probe substrate method were used to detect the alterations in 21 isoforms of DMEs, as well as the enzymatic activities of five isoforms, respectively. Western blotting was applied to study the protein expression of four related receptors. RESULTS: The proteins contents of CYP2C29 and CYP3A11, were significantly downregulated in the livers of male KRAS mice at 26 weeks (3.7- and 4.4-fold, respectively, p < 0.05). SULT1A1 and SULT1D1 were upregulated by 1.8- to 7.0- fold at 20 (p = 0.015 and 0.017, respectively) and 26 weeks (p = 0.055 and 0.031, respectively). There were positive correlations between protein expression and enzyme activity for CYP2E1, UGT1A9, SULT1A1 and SULT1D1 (r 2 ≥ 0.5, p < 0.001). Western blotting analysis revealed the downregulation of AHR, FXR and PPARα protein expression in male KRAS mice at 26 weeks. For sex-related differences, CYP2E1 was male-predominant and UGT1A2 was female-predominant in the kidney. UGT1A1 and UGT1A5 expression was female-predominant, whereas UGT2B1 exhibited male-predominant expression in liver tissue. For the tissue distribution of DMEs, 21 subtypes of DMEs were all expressed in liver tissue. In the intestine, the expression levels of CYP2C29, CYP27A1, UGT1A2, 1A5, 1A6a, 1A9, 2B1, 2B5 and 2B36 were under the limitation of quantification. The subtypes of CYP7A1, 1B1, 2E1 and UGT1A1, 2A3, 2B34 were detected in kidney tissue. CONCLUSIONS: This study, for the first time, unveils the variations and sex- and age-related differences in DMEs in C57 BL/6 (WT) mice and KRAS mice.

Maternal serine supply from late pregnancy to lactation improves offspring performance through modulation of metabolic pathways.

Maternal dietary serine affects free amino acid content in milk and the antioxidant ability of progeny. However, whether maternal dietary serine has any effects on offspring performance in pigs and related metabolic consequences remains unknown. This study was conducted to investigate the effects of different levels of maternal dietary serine from late pregnancy to lactation on sow reproductive performance and offspring performance, and on the metabolome of milk and the serum of sows and their offspring. The results showed that sows fed a diet supplemented with 25% serine of the basal diet (l-Ser) had a higher litter weight, and higher average piglet weight at birth and aged 21 days when compared with sows fed the basal diet (CON). We found a large number of metabolites in both colostrum and milk that differed significantly between sows in the CON and l-Ser groups. Additionally, twenty metabolites differed in the serum of piglets aged 21 days between the CON and l-Ser groups. Most of these metabolites are involved in purine and pyrimidine metabolism, glutathione and taurine metabolism, as well as phospholipid and sphingolipid metabolism, which may contribute to the growth-promoting effects of serine on offspring. Our results imply that maternal serine has the potential to improve offspring outcomes.

Melatonin in macrophage biology: Current understanding and future perspectives.

Melatonin is a ubiquitous hormone found in various organisms and highly affects the function of immune cells. In this review, we summarize the current understanding of the significance of melatonin in macrophage biology and the beneficial effects of melatonin in macrophage-associated diseases. Enzymes associated with synthesis of melatonin, as well as membrane receptors for melatonin, are found in macrophages. Indeed, melatonin influences the phenotype polarization of macrophages. Mechanistically, the roles of melatonin in macrophages are related to several cellular signaling pathways, such as NF-κB, STATs, and NLRP3/caspase-1. Notably, miRNAs (eg, miR-155/-34a/-23a), cellular metabolic pathways (eg, α-KG, HIF-1α, and ROS), and mitochondrial dynamics and mitophagy are also involved. Thus, melatonin modulates the development and progression of various macrophage-associated diseases, such as cancer and rheumatoid arthritis. This review provides a better understanding about the importance of melatonin in macrophage biology and macrophage-associated diseases.

An update on polyphenol disposition via coupled metabolic pathways.

INTRODUCTION: Polyphenols, which are widely distributed in plants and the human diets, are known to have numerous biological activities. However, the low bioavailability of polyphenols is mediated by coupled metabolic pathways. Areas covered: The key role of the interplay between drug metabolic enzymes (DMEs) and efflux transporters (ETs), nuclear receptors (NRs), and intestinal microflora in the disposition of polyphenols is summarized. Expert opinion: ETs are shown to act as a 'revolving door', facilitating and/or controlling cellular polyphenol glucuronide/sulfate excretion. Elucidating the mechanisms underlying the glucuronidation/sulfation-transport interplay and structure-activity relationships (SAR) of glucuronide/sulfate efflux by an ET is important. Some new physiologically based pharmacokinetic (PBPK) models could be developed to predict the interplay between glucuronides/sulfates and ETs. Additionally, the combined actions of uridine-5'-diphosphate glucuronosyltransferases, ETs, and intestinal microflora/enterocyte-derived ß-glucuronidase enable triple recycling (local, enteric, and enterohepatic recycling), thereby increasing the residence time of polyphenols and their glucuronides in the local intestine and liver. Further studies are necessary to explore these recycling mechanisms and interactions between polyphenols and the intestinal microbiota. Since NRs govern the inducible expression of target genes that encode DMEs and ETs. Determination of the regulation mechanism mediated by NRs using transgenic and knockout animals is still needed.

Jejunal Metabolic Responses to Escherichia coli Infection in Piglets.

This study aimed to investigate the jejunal metabolic variations in enterotoxigenic Escherichia coli (ETEC)-infected piglets. Piglets were infected with 1 × 1010 CFUs (colony-forming units) of ETEC W25K and assigned into diarrheal, recovered, control, and resistant groups. Jejunal samples were harvested at day 6 and metabolic profiles were analyzed via gas chromatography coupled to time-of-flight mass spectrometry (GC/TOFMS). The results showed that 33 metabolites in the jejunum were identified in ETEC-induced diarrhea, including amino acids, fatty acids, sugars, and organic acids. Compared with the control, resistant, and recovered piglets, diarrheal piglets showed higher concentrations of 4-aminobutyric acid (GABA) and glycine in the jejunum. Compared with the control and resistant piglets, six metabolites were markedly decreased in diarrheal piglets, including ornithine, asparagine, glutamine, citric acid, citrulline, and lysine. Collectively, this study provides insights into jejunal metabolic response to ETEC infection and ETEC induced diarrhea in piglets.

Age-related changes in hepatic expression and activity of drug metabolizing enzymes in male wild-type and breast cancer resistance protein knockout mice.

This study aimed to reveal age-related changes in the expression and activity of seven hepatic drug metabolizing enzymes (DMEs) in male wild-type and breast cancer resistance protein knockout (Bcrp1-/- ) FVB mice. The protein expression of four cytochrome P450 (Cyps) (Cyp3a11, 2d22, 2e1, and 1a2), and three UDP-glucuronosyltransferases (Ugts) (Ugt1a1, 1a6a, and 1a9) in liver microsomes of wild-type and Bcrp1-/- FVB mice at different ages were determined using a validated ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method. The activities and mRNA levels of these DMEs were measured using the probe substrates method and real-time PCR, respectively. In the liver of wild-type FVB mice, Cyp3a11, 2d22, 2e1, 1a2, Ugt1a1, and 1a6a displayed maximum protein levels at 6-9 weeks of age. Cyp1a2, Ugt1a1, 1a6a, and 1a9 showed maximum activities at 6-9 weeks of age, whereas Cyp3a11, 2d22, and 2e1 showed maximum activities in 1-3-week-old mice. Additionally, most of the DMEs showed maximum mRNA levels in 17-week-old mice liver. Compared with wild-type FVB mice, the protein levels of these DMEs showed no significant changes in Bcrp1-/- FVB mice liver. However, the activity of Cyp2e1 was increased and that of Cyp2d22 was decreased. In conclusion, the seven hepatic DMEs in FVB mice liver showed significant alterations in an isoform-specific manner with increased age. Although the protein levels of these DMEs showed no significant changes, the activities of Cyp2e1 and 2d22 were changed in Bcrp1-/- mice.

Tissue Distribution and Gender-Specific Protein Expression of Cytochrome P450 in five Mouse Genotypes with a Background of FVB.

PURPOSE: To systematically investigate tissue distribution and gender-specific protein expression of Cytochrome P450 (Cyps) in five mouse genotypes with a background of Friend virus B (FVB). METHODS: The Cyps were extracted from the tissue S9 fractions of the main metabolic organs and then absolutely quantified by applying the UHPLC-MS/MS method. RESULTS: The liver had the highest expression of Cyps, followed by the small intestine and kidney. In the liver, Cyp1a2, Cyp2c29, Cyp2c39, Cyp2d22, Cyp2e1, and Cyp3a11 were the main isoforms. Cyp1a2 and Cyp2c29 were male-specific, while Cyp2c39 was female-specific. Compared with the expression in Wild-type (WT) FVB mice, the expression of Cyp1a2, Cyp1b1, Cyp2d22, and Cyp3a25 significantly decreased in female efflux transporter (ET) knockout mice. In the small intestine, Cyp2c29 and Cyp3a11 were the major isoforms. Knockout of ET didn't alter the expression levels of most Cyps. However, female ET knockout mice presented higher Cyp2c29 expression than WT FVB mice. The Cyp7a1 expression was markedly decreased in ET knockout mice except Bcrp1-/- mice. In the kidney, Cyp2e1 was the main isoform and exhibited male specificity. Knockout of ET slightly affected the protein expression of Cyps in the brain, heart, lung, spleen and stomach. CONCLUSIONS: A comprehensive understanding of the distribution characteristics and gender-specific expression of Cyps in major metabolic organs of WT and ET knockout FVB mice should contribute to a better understanding of drug efficacy and toxicity, and drug-drug interactions.

Flavone protects HBE cells from DNA double-strand breaks caused by PM2.5.

Ambient air particulate matter 2.5 (PM2.5) contains many harmful components that can enter the circulatory system and produce reactive oxygen species (ROS) in body. Oxidative stress and DNA damage induced by ROS may affect any cellular macromolecule and lead to DNA double-strand breaks (DSBs). Flavonoids, widely distributed in some herbs and berries, have been proved having anti-oxidative or anti-cancer efficacy. In this study, we investigated whether Flavone, a kind of flavonoids, can protect human bronchial epithelial cells (HBE) from DSBs caused by PM2.5 and how this function is probably implemented. We found that cells exposed to PM2.5 obviously induced viability inhibition, DNA damage and part of apoptosis. However, Flavone treatment prior to PM2.5 apparently improved cell viability, and mitigated the formation of 8-hydroxy-2-deoxyguanosine, the expression of DNA damage-relative protein and cell apoptosis. Our studies demonstrated that PM2.5 induced oxidative DSBs while Flavone ameliorated the DNA damage and increased cell viability probably through influencing DNA repair mechanism of cells.

Profiles and Gender-Specifics of UDP-Glucuronosyltransferases and Sulfotransferases Expressions in the Major Metabolic Organs of Wild-Type and Efflux Transporter Knockout FVB Mice.

Hepatic and extrahepatic tissues participate in xenobiotic detoxication, carcinogen activation, prodrug processing, and estrogen regulation through UDP-glucuronosyltransferases (UGTs/Ugts) and sulfotransferases (SULTs/Sults). Wild-type (WT) and efflux transporter knockout (KO) FVB mice have been commonly used to perform the studies of pharmacokinetics, metabolism, and toxicity. We employed the developed UHPLC-MS/MS approach to gain systematic insight on gender-specific of Ugts and Sults in major metabolic organs. Results showed that the liver was the most abundant with Ugts/Sults, followed by the small intestine and the kidney. In the liver, Ugt2b5, Ugt2b1, Ugt1a6a, Ugt1a1, Sult1a1, and Sult1d1 were the major isoforms. The protein amounts of Ugt1a9 were significantly higher in male efflux transporter KO mice than in WT mice, whereas Ugt1a5 and Sult1a1 severely decreased in female efflux transporter KO mice. In WT and efflux transporter KO mice, the expression levels of Ugt1a1, Ugt1a5, Sult1a1, Sult1d1, and Sult3a1 were female-specific, whereas those of Ugt2b1, Ugt2b5, and Ugt2b36 were male-specific. In the small intestine, Ugt1a1, Sult1b1, and Sult2b1 were the major isoforms. The protein levels and gender differences of Ugts/Sults were obviously affected when KO of Mdr1a, and Bcrp1, Mrp1, Mrp2, and Mdr1a, respectively. The KO of efflux transporter affected the protein amounts of Ugts/Sults in the kidney, heart, and spleen. Therefore, a better understanding of the expression profiles and gender-specific of Ugts and Sults in major metabolic organs of WT and efflux transporter KO mice is useful for the evaluation of potential efficacy, and toxicity of corresponding substrates.

High-Throughput and Reliable Isotope Label-free Approach for Profiling 24 Metabolic Enzymes in FVB Mice and Sex Differences.

FVB mice are extensively used in transgenic and pharmacokinetic research. In this study, a validated isotope label-free method was constructed using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to quantify 24 drug-metabolizing enzymes (DMEs) in FVB mice. The DMEs include cytochrome P450s (CYP450s/Cyp450s), UDP-glucuronsyltransferases (UGTs/Ugts), and sulfotransferases (SULTs/Sults), which catalyze a variety of reactions to detoxify xenobiotics and endobiotics. The proposed UHPLC-MS/MS method exhibited good range and high sensitivity for signature peptides, as well as acceptable accuracy, precision, and recovery. The protein expression profiles of the DMEs were determined in male and female mice. Overall, the major Cyps, Ugts, and Sults were expressed in male mice followed the rank order: Cyp2c29 > 2e1 > 3a11 > 1a2 > 2d22 > 27a1 > 2c39; Ugt2b5 > 2b1 > 1a6a > 1a9 > 1a1 > 2a3 > 1a2 > 1a5; and Sult1a1 > 3a > 1d1. In contrast, the rank order in female mice was Cyp2c29 > 2e1 > 2c39 > 2d22 > 3a11 > 1a2 > 27a1; Ugt1a6a > 2b5 > 1a1 > 2b1 > 2a3 > 1a9 > 1a5 > 1a2; and Sult1a1 > 3a1 > 1d1. Cyp2c29, Cyp1a2, Cyp27a1, Ugt2b1, Ugt2b5 and Ugt2b36 were male predominant, whereas Cyp2c39, Cyp2d22, Cyp7a1, Ugt1a1, Ugt1a5, Sult1a1, Sult3a1, and Sult1d1 were female predominant. This work could serve as a useful reference for the metabolic study of new drugs and for elucidating the effectiveness and toxicity of drugs. The method is stable, simple, and rapid for determining the expression of DMEs in animals.