Most mammalian genes produce multiple distinct messenger RNAs through alternative splicing, but the extent of splicing conservation is not clear. To assess tissue-specific transcriptome variation across mammals, we sequenced complementary DNA from nine tissues from four mammals and one bird in biological triplicate, at unprecedented depth. We find that while tissue-specific gene expression programs are largely conserved, alternative splicing is well conserved in only a subset of tissues and is frequently lineage-specific. Thousands of previously unknown, lineage-specific, and conserved alternative exons were identified; widely conserved alternative exons had signatures of binding by MBNL, PTB, RBFOX, STAR, and TIA family splicing factors, implicating them as ancestral mammalian splicing regulators. Our data also indicate that alternative splicing often alters protein phosphorylatability, delimiting the scope of kinase signaling.

Evolutionary Dynamics of Gene and Isoform Regulation in Mammalian Tissues

iTRAQ (isobaric tags for relative or absolute quantitation) is a mass spectrometry technology that allows quantitative comparison of protein abundance by measuring peak intensities of reporter ions released from iTRAQ-tagged peptides by fragmentation during MS/MS. However, current data analysis techniques for iTRAQ struggle to report reliable relative protein abundance estimates and suffer with problems of precision and accuracy. The precision of the data is affected by variance heterogeneity: low signal data have higher relative variability; however, low abundance peptides dominate data sets. Accuracy is compromised as ratios are compressed toward 1, leading to underestimation of the ratio. This study investigated both issues and proposed a methodology that combines the peptide measurements to give a robust protein estimate even when the data for the protein are sparse or at low intensity. Our data indicated that ratio compression arises from contamination during precursor ion selection, which occurs at a consistent proportion within an experiment and thus results in a linear relationship between expected and observed ratios. We proposed that a correction factor can be calculated from spiked proteins at known ratios. Then we demonstrated that variance heterogeneity is present in iTRAQ data sets irrespective of the analytical packages, LC-MS/MS instrumentation, and iTRAQ labeling kit (4-plex or 8-plex) used. We proposed using an additive-multiplicative error model for peak intensities in MS/MS quantitation and demonstrated that a variance-stabilizing normalization is able to address the error structure and stabilize the variance across the entire intensity range. The resulting uniform variance structure simplifies the downstream analysis. Heterogeneity of variance consistent with an additive-multiplicative model has been reported in other MS-based quantitation including fields outside of proteomics; consequently the variance-stabilizing normalization methodology has the potential to increase the capabilities of MS in quantitation across diverse areas of biology and chemistry.

Addressing accuracy and precision issues in iTRAQ quantitation

Aflatoxin B1, a potently carcinogenic fungal metabolite, is converted to the biologically active form by chemical oxidation using dimethyldioxirane and enzymatically by cytochrome P450 mixed-function oxidases. Both processes give rise to mixtures of the exo- and endo-8,9-epoxides. Methanolysis studies reveal exclusive trans opening of both epoxides under neutral conditions in CH3OH and CH3OH/H2O mixtures; an SN2 mechanism is postulated. Under acidic conditions, the exo isomer gives mixtures of trans and cis solvolysis products, suggesting that the reaction is, at least in part, SN1; the endo isomer gives only the trans product. The exo isomer reacts with DNA by attack of the nitrogen atom at the 7 position of guanine on C8 of the epoxide to give the trans adduct; the endo epoxide fails to form an adduct at this or any other site in DNA. The exo isomer is strongly mutagenic in a base-pair reversion assay employing Salmonella typhimurium; the endo isomer is essentially nonmutagenic. Aflatoxin B1 and its derivatives intercalate in DNA. These results are consistent with a mechanism in which intercalation of the exo epoxide optimally orients the epoxide for an SN2 reaction with guanine but intercalation of the endo isomer places the epoxide in an orientation which precludes reaction. Thus, while the exo epoxide is a potent mutagen, the endo epoxide fails to react with DNA.

DNA adduction by the potent carcinogen aflatoxin B1 : mechanistic studies

Mycotoxins are highly diverse secondary metabolites produced in nature by a wide variety of fungi. Mycotoxins cause animal feed and food contamination, resulting in mycotoxicosis. T-2 toxin is one of the most common and toxic trichothecene mycotoxins. For the last decade, it has garnered considerable attention due to its potent neurotoxicity. Worryingly, T-2 toxin can cross the blood-brain barrier and accumulate in the central nervous system (CNS) to cause neurotoxicity. This review covers the current knowledge base on the molecular mechanisms of T-2 toxin-induced oxidative stress and mitochondrial dysfunction in the CNS. In vitro and animal data have shown that induction of reactive oxygen species (ROS) and oxidative stress plays a critical role during T-2 toxin-induced neurotoxicity. Mitochondrial dysfunction and cascade signaling pathways including p53, MAPK, Akt/mTOR, PKA/CREB and NF-κB contribute to T-2 toxin-induced neuronal cell death. T-2 toxin exposure can also result in perturbations of mitochondrial respiratory chain complex and mitochondrial biogenesis. T-2 toxin exposure decreases the mitochondria unfolded protein response and dampens mitochondrial energy metabolism. Antioxidants such as N-acetylcysteine (NAC), activation of Nrf2/HO-1 and autophagy have been shown to provide a protective effect against these detrimental effects. Clearly, translational research and the discovery of effective treatment strategies are urgently required against this common food-borne threat to human health and livestock.

T-2 toxin neurotoxicity: role of oxidative stress and mitochondrial dysfunction.

Oral administration has been the most common therapeutic regimen in various diseases because of its high safety, convenience, lower costs, and high compliance of patients. However, susceptible in hostile gastrointestinal (GI) environment, many drugs show poor permeability across GI tract mucus and intestinal epithelium with poor oral absorption and limited therapeutic efficacy. In recent years, nanoparticulate drug delivery systems (NDDS) have become a hot research spot because of their unique advantages including protecting drug from premature degrading and interacting with the physiological environment, increasing intracellular penetration, and enhancing drug absorption. However, a slight change in physicochemistry of nanoparticles can significantly impact their interaction with biological pathways and alter the oral bioavailability of drugs. Hence, this review focuses on the factors affecting oral bioavailability from two aspects. On the one hand, the factors are the biochemical and physiological barriers in oral drugs delivery. On the other hand, the factors are the nanoparticle properties including size, surface properties, and shape of nanoparticles.

/pdf/the-influence-of-nanoparticle-properties-on-oral-4lgt4lfxon.pdf

The Influence of Nanoparticle Properties on Oral Bioavailability of Drugs

Cytochrome P450 1A1 (CYP1A1) is abundant in the kidney, liver, and intestine and is involved in the phase I metabolism of numerous endogenous and exogenous compounds. Therefore, exploring the regulatory mechanism of its basal expression in humans is particularly important to understand the bioactivation of several procarcinogens to their carcinogenic derivatives. Site-specific mutagenesis and deletion of the transcription factor binding site determined the core cis-acting elements in the human CYP1A1 proximal and distal promoter regions. The proximal promoter region [overlapping xenobiotic-responsive element (XRE) and GC box sequences] determined the basal expression of CYP1A1. In human hepatocellular carcinoma cells (HepG2) with aryl hydrocarbon receptor (AhR) or specificity protein 1 (Sp1) knockdown, we confirmed that AhR and Sp1 are involved in basal CYP1A1 expression. In HepG2 cells overexpressing either AhR or Sp1, AhR determined the proximal transactivation of basal CYP1A1 expression. Via DNA affinity precipitation assays and ChIP, we found that AhR bound to the promoter and recruited Sp1 to transactivate CYP1A1 expression. The coordinated interaction between Sp1 and AhR was identified to be DNA mediated. Our work revealed a basal regulatory mechanism of an interesting human gene by which AhR interacts with Sp1 through DNA and recruits Sp1 to regulate basal CYP1A1 expression.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/febs.14956

AhR regulates the expression of human cytochrome P450 1A1 (CYP1A1) by recruiting Sp1.

Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B1 (AFB1) and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB1 in pigs remains to be clarified, especially the principal cytochrome P450 oxidases responsible for its activation. In this study, we cloned CYP3A29 from pig liver and expressed it in Escherichia coli, and its activity has been confirmed with the typical P450 CO-reduced spectral characteristic and nifedipine-oxidizing activity. The reconstituted membrane incubation proved that the recombinant CYP3A29 was able to oxidize AFB1 to form AFB1-exo-8,9-epoxide in vitro. The structural basis for the regioselective epoxidation of AFB1 by CYP3A29 was further addressed. The T309A mutation significantly decreased the production of AFBO, whereas F304A exhibited an enhanced activation towards AFB1. In agreement with the mutagenesis study, the molecular docking simulation suggested that Thr309 played a significant role in stabilization of AFB1 binding in the active center through a hydrogen bond. In addition, the bulk phenyl group of Phe304 potentially imposed steric hindrance on the binding of AFB1. Our study demonstrates the bioactivation of pig CYP3A29 towards AFB1 in vitro, and provides the insight for understanding regioselectivity of CYP3A29 to AFB1.

https://www.mdpi.com/2072-6651/8/9/267/pdf

Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B1

Diabetic nephropathy (DN) is the most important cause of middle and late-stage chronic kidney disease. Green tea polypeptides are extracted from tea pomace, and exhibit various pharmacological effects. In this study, we analyzed the reno-protective effects of green tea peptides in diabetic db/db mice, and explored the underlying mechanisms. Peptide treatment for 5 weeks significantly reduced the blood glucose levels and other indices of diabetes, and alleviated renal injury measured in terms of blood creatinine, urea nitrogen and urinary albumin/urinary creatinine levels. Mechanistically, the green tea peptides downregulated p-Smad2/3, α-SMA, ZO-1 and vimentin proteins in the kidney tissues, and elevated Smad7. Thus, green tea peptides inhibited the deposition of ECM proteins by suppressing excessive activation of the TGF-β/Smad signaling pathway and reducing fibronectin levels. On the other hand, tea peptides ameliorated renal injury by inhibiting the production of inflammatory factors (iNOS and TNF-α) by suppressing the NF-κB signaling pathway. In addition, we confirmed the inhibitory effect of green tea peptides on the TGF-β/Smad signaling pathway in TGF-β1-stimulated HK-2 cells. Therefore, tea peptides can be considered as an effective candidate for alleviating DN.

Green tea peptides ameliorate diabetic nephropathy by inhibiting the TGF-β/Smad signaling pathway in mice.

Background The health benefits of tea are as diverse including the reduction of uric acid levels. Xanthine oxidase is the most directly mediated enzyme in the production of uric acid. Objective To explore the inhibitory effects of different teas and its main bioactive components on the production of uric acid. Design Experimental study. The experiments were conducted in vitro using human immortalized normal liver cell line HL-7702 (L-02). Results The inhibition of the xanthine oxidase activities and the expression level of xanthine dehydrogenase mRNA stimulated in the hyperuric hepatocyte cell model showed that the unfermented green tea and th1e lightly fermented yellow tea, white tea, and oolong tea significantly stronger than the highly fermented black tea and dark tea. The main bioactive compound, gallic acid, showed the strongest inhibitory effect on uric acid production, followed by tea polyphenols and theaflavins. Discussion All teas exhibited significant inhibition of xanthine oxidase activities, and the degree of fermentation of tea may be inversely proportional to its ability to inhibit the production of uric acid. Compared with tea polyphenols rich in tea, gallic acid may be a more potential uric acid-lowering component. Conclusion In this article, we first compared the effects of six traditional Chinese tea made from a single variety in stabilizing the synthesis of uric acid and found that the lighter the fermentation, the greater the potential for inhibiting the production of uric acid. Furthermore, we analyzed the inhibitory effects of its main biochemical active ingredients and found that the inhibitory effects of polyphenols rich in lightly fermented tea were significantly stronger than caffeine rich in highly fermented tea. Our findings will be helpful for people to choose a proper tea for alleviating hyperuricemia and provide a scientific basis for uric acid-lowering tea processing.

https://foodandnutritionresearch.net/index.php/fnr/article/download/8239/14679

Tea and its components reduce the production of uric acid by inhibiting xanthine oxidase

Liver injury is a life-threatening condition that is usually caused by excessive alcohol consumption, improperdiet, and stressful lifestyle and can even progress to liver cancer. Tea is a popular beverage with proven health benefits and is known to exert a protective effect on the liver, intestines, and stomach. In this study, we analyzed the therapeutic effects of six kinds of tea on carbon tetrachloride (CCl4)-induced liver injury in a mouse model. The mice were injected with 10 mL/kg 5% CCl4 to induce liver injury and then given oral gavage of green tea, yellow tea, oolong tea, white tea, black tea, and dark tea, respectively. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, and the expression levels of inflammation and oxidative stress-related proteins in the liver tissues were quantified. All six kinds of tea partly reduced the liver index, restored the size of the enlarged liver in the CCl4 model, and decreased the serum levels of ALT and AST. Furthermore, the highly fermented dark tea significantly reduced the expression levels of NF-κB and the downstream inflammatory factors, whereas the unfermented green tea inhibited oxidative stress by activating the antioxidant Nrf2 pathway. Taken together, tea can protect against liver inflammation, and unfermented tea can improve antioxidant levels. Further studies are needed on the bioactive components of tea to develop drugs against liver injury.

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Ruohong Chen

Papers

AhR regulates the expression of human cytochrome P450 1A1 (CYP1A1) by recruiting Sp1.

Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B1

Green tea peptides ameliorate diabetic nephropathy by inhibiting the TGF-β/Smad signaling pathway in mice.

Tea and its components reduce the production of uric acid by inhibiting xanthine oxidase

Chinese Tea Alleviates CCl4-Induced Liver Injury through the NF-κBorNrf2Signaling Pathway in C57BL-6J Mice