iRhom2 Promotes Hepatic Steatosis by Activating MAP3K7-Dependent Pathway.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) has been widely recognized as a precursor to metabolic complications. Elevated inflammation levels are predictive of NAFLD-associated metabolic disorder. Inactive rhomboid-like protein 2 (iRhom2) is regarded as a key regulator in inflammation. However, the precise mechanisms by which iRhom2-regulated inflammation promotes NAFLD progression remain to be elucidated. APPROACH AND RESULTS: Here, we report that insulin resistance, hepatic steatosis, and specific macrophage inflammatory activation are significantly alleviated in iRhom2-deficient (knockout [KO]) mice, but aggravated in iRhom2 overexpressing mice. We further show that, mechanistically, in response to a high-fat diet (HFD), iRhom2 KO mice and mice with iRhom2 deficiency in myeloid cells only showed less severe hepatic steatosis and insulin resistance than controls. Inversely, transplantation of bone marrow cells from healthy mice to iRhom2 KO mice expedited the severity of insulin resistance and hepatic dyslipidemia. Of note, in response to HFD, hepatic iRhom2 binds to mitogen-activated protein kinase kinase kinase 7 (MAP3K7) to facilitate MAP3K7 phosphorylation and nuclear factor kappa B cascade activation, thereby promoting the activation of c-Jun N-terminal kinase/insulin receptor substrate 1 signaling, but disturbing AKT/glycogen synthase kinase 3ß-associated insulin signaling. The iRhom2/MAP3K7 axis is essential for iRhom2-regulated liver steatosis. CONCLUSIONS: iRhom2 may represent a therapeutic target for the treatment of HFD-induced hepatic steatosis and insulin resistance.

Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK.

Nonalcoholic fatty liver disease (NAFLD) is featured by hepatic steatosis, insulin resistance, lipid deposition and inflammation. However, the pathogenic mechanism of NAFLD is still poorly understood. Dual-specificity phosphatase 16 (DUSP16), a c-Jun N-terminal kinase-specific phosphatase, has been reported to negatively modulate the mitogen-activated protein kinases (MAPKs) signaling, and it has never been investigated in NAFLD progression. In the study, we identified that DUSP16 could directly interact with TAK1 in human hepatocytes. DUSP16 knockdown in the isolated primary hepatocytes stimulated by palmitate (PA) showed accelerated lipid deposition and inflammatory response, along with the exacerbated activation of c-Jun NH2-terminal kinase (JNK), Transforming growth factor ß (TGF-ß)-activated kinase (TAK1) and nuclear factor-κB (NF-κB) signaling pathways; however, the opposite results were detected in PA-treated hepatocytes with DUSP16 over-expression. The in vivo experiments confirmed that DUSP16 knockout significantly aggravated the metabolic disorder and insulin resistance in high fat diet (HFD)-challenged mice. In addition, HFD-provoked hepatic lipid accumulation and inflammation were further promoted in mice with DUSP16 knockout through the same molecular mechanism as detected in vitro. Herein, these findings demonstrated that DUSP16 could directly interact with TAK1 and negatively regulate JNK signaling to alleviate metabolic stress-induced hepatic steatosis, and thus could be considered as a promising new molecular target for NAFLD treatment.

iRhom2 deficiency relieves TNF-α associated hepatic dyslipidemia in long-term PM2.5-exposed mice.

Accumulating researches reported that particulate matter (PM2.5) is a risk factor for developing various diseases, including metabolic syndrome. Recently, inactive rhomboid protein 2 (iRhom2) was considered as a necessary modulator for shedding of tumor necrosis factor-α (TNF-α) in immune cells. TNF-α, a major pro-inflammatory cytokine, was linked to various pathogenesis of diseases, including dyslipidemia. Here, wild type (WT) and iRhom2-knockout (iRhom2-/-) mice were used to investigate the effects of iRhom2 on PM2.5-induced hepatic dyslipidemia. The hepatic histology, inflammatory response, glucose tolerance, serum parameters and gene expressions were analyzed. We found that long-term inhalation of PM2.5 resulted in hepatic steatosis. And a significant up-regulation of iRhom2 in liver tissues was observed, accompanied with elevated TNF-α, TNF-α converting enzyme (TACE), TNFα receptor (TNFR)2 and various inflammatory cytokines expressions. Additionally, PM2.5 treatment caused TG and TC accumulation in serum and liver, probably attributed to changes of genes modulating lipid metabolism. Intriguingly, hepatic injury and dyslipidemia were attenuated by iRhom2-/- in mice with PM2.5 challenge. In vitro, iRhom2-knockdwon reduced TNF-α expressions and its associated inflammatory cytokines in Kupffer cells, implying that liver-resident macrophages played an important role in regulating hepatic inflammation and lipid metabolism in cells treated with PM2.5. The findings indicated that long-term PM2.5 exposure caused hepatic steatosis and dyslipidemia through triggering inflammation, which was, at least partly, dependent on iRhom2/TNF-α pathway in liver-resident macrophages.

Comparative analysis of the gut microbiota of black bears in China using high-throughput sequencing.

The Asiatic black bear (Ursus thibetanus) is a protected species from eastern Asia. In China, the Asiatic black bear occurs in 17 provinces from northeast to southwest regions. To date, information on microbial diversity in the gut of the Asiatic black bears from different populations remains limited. To determine the species composition and community structure of the gut microbiota in the Asiatic black bear, we characterized 36 fecal samples from Sichuan, Yunnan, and Heilongjiang provinces, China, by pyrosequencing the 16S V3-V4 hypervariable regions using the Illumina Miseq platform. Results showed that Firmicutes and Proteobacteria were the predominant phyla in the samples, which were largely comprised Escherichia-Shigella, Peptostreptococcaceae_incertae_sedis, Turicibacter, Streptococcus, and Clostridium. By analyzing the community structure from these 36 samples, we found that there were significant differences in the species diversity and richness between Sichuan, Yunnan, and Heilongjiang populations. In conclusion, our results reveal the species composition and structure of the gut microbiota in captive black bears in China, and suggest that biogeography could affect the black bear' gut microbiota.

Engineering the cofactor binding site of 7α-hydroxysteroid dehydrogenase for improvement of catalytic activity, thermostability, and alteration of substrate preference.

Hydroxysteroid dehydrogenases (HSDHs) are crucial for bile acid metabolism and influence the size of the bile acid pool and gut microbiota composition. HSDHs with high activity, thermostability, and substrate selectivity are the basis for constructing engineered bacteria for disease treatment. In this study, we designed mutations at the cofactor binding site involving Thr15 and Arg16 residues of HSDH St-2-2. The T15A, R16A, and R16Q mutants exhibited 7.85-, 2.50-, and 4.35-fold higher catalytic activity than the wild type, respectively, while also displaying an altered substrate preference (from taurocholic acid (TCA) to taurochenodeoxycholic acid (TCDCA)). These mutants showed lower Km and higher kcat values, indicating stronger binding to the substrate and resulting in 3190-, 3123-, and 3093-fold higher kcat/Km values for TCDCA oxidation. Furthermore, the Tm values of the T15A, R16A, and R16Q mutants were found to increase by 4.3 °C, 6.0 °C, and 7.0 °C, respectively. Molecular structure analysis indicated that reshaped internal hydrogens and surface mutations could improve catalytic activity and thermostability, and altered interactions among the catalytic triad, cofactor binding sites, and substrates could change substrate preference. This work provides valuable insights into modifying substrate preference as well as enhancing the catalytic activity and thermostability of HSDHs by targeting the cofactor binding site.

Characterization of a novel 3-quinuclidinone reductase possessing remarkable thermostability.

The 3-quinuclidinone reductase plays an irreplaceable role in the biopreparation of (R)-3-quinuclidinol, an intermediate vital for synthesis of various pharmaceuticals. Thermal robustness is a critical factor for enzymatic synthesis in industrial applications. This study characterized a new 3-quinuclidinone reductase, named SaQR, with significant thermal stability. The SaQR was overexpressed in a GST-fused state, and substrate and cofactor screening were conducted. Additionally, three-dimensional structure prediction using AlphaFold and analysis were performed, along with relevant thermostability tests, and the evaluation of factors influencing enzyme activity. The findings highlight the remarkable thermostability of SaQR, retaining over 90% of its activity after 72 h at 50°C, with an optimal operational temperature of 85°C. SaQR showed typical structural traits of the SDR superfamily, with its cofactor-determining residue being aspartic acid, conferring nicotinamide adenine dinucleotide (NAD(H)) preference. Moreover, K+ and Na+, at a concentration of 400 mM, could significantly enhance the activity, while Mg2+ and Mn2+ only display inhibitory effects within the tested concentration range. The findings of molecular dynamics simulations suggest that high temperatures may disrupt the binding of enzyme to substrate by increasing the flexibility of residues 205-215. In conclusion, this study reports a novel 3-quinuclidinone reductase with remarkable thermostability.

Characterization of a Novel Thermostable 7α-Hydroxysteroid Dehydrogenase.

BACKGROUND: 7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays a pivotal role in vivo in the biotransformation of secondary bile acids and has great potential in industrial biosynthesis due to its broad substrate specificity. In this study, we expressed and characterized a novel thermostable 7α-HSDH (named Sa 7α-HSDH). METHODS: The DNA sequence was derived from the black bear gut microbiome metagenomic sequencing data, and the coding sequence of Sa 7α-HSDH was chemically synthesized. The heterologous expression of the enzyme was carried out using the pGEX-6p-1 vector. Subsequently, the activity of the purified enzyme was studied by measuring the absorbance change at 340 nm. Finally, the three-dimensional structure was predicted with AlphaFold2. RESULTS: Coenzyme screening results confirmed it to be NAD(H) dependent. Substrate specificity test revealed that Sa 7α-HSDH could catalyze taurochenodeoxycholic acid (TCDCA) with catalytic efficiency (kcat/Km) 3.81 S-1 mM-1. The optimum temperature of Sa 7α-HSDH was measured to be 75°C, confirming that it belongs to thermophilic enzymes. Additionally, its thermostability was assessed using an accelerated stability test over 32 hours. The catalytic activity of Sa 7α-HSDH remained largely unchanged for the first 24 hours and retained over 90% of its functionality after 32 hours at 50°C. Sa 7α-HSDH exhibited maximal activity at pH 10. The effect of metal ions-K+, Na+, Mg2+ and Cu2+-on the enzymatic activity of Sa 7α-HSDH was investigated. Only Mg2+ was observed to enhance the enzyme's activity by 27% at a concentration of 300 mM. Neither K+ nor Na+ had a significant influence on activity. Only Cu2+ was found to reduce enzyme activity. CONCLUSION: We characterized the thermostable 7α-HSDH, which provides a promising biocatalyst for bioconversion of steroids at high reaction temperatures.

A novel crosstalk between Nrf2 and Smad2/3 bridged by two nuanced Keap1 isoforms with their divergent effects on these distinct family transcription factors.

The Keap1-Nrf2 signalling to transcriptionally regulate antioxidant response element (ARE)-driven target genes has been accepted as key redox-sensitive pathway governing a vast variety of cellular stresses during healthy survival and disease development. Herein, we identified two nuanced isoforms α and ß of Keap1 in HepG2 cells, arising from its first and another in-frame translation starting codons, respectively. In identifying those differential expression genes monitored by Keap1α and/or Keap1ß, an unusual interaction of Keap1 with Smad2/3 was discovered by parsing transcriptome sequencing, Keap1-interacting protein profiling and relevant immunoprecipitation data. Further examination validated that Smad2/3 enable physical interaction with Keap1, as well as its isoforms α and ß, by both EDGETSD and DLG motifs in the linker regions between their MH1 and MH2 domains, such that the stability of Smad2/3 and transcriptional activity are enhanced with their prolonged half-lives and relevant signalling responses from the cytoplasmic to nuclear compartments. The activation of Smad2/3 by Keap1, Keap1α or Keap1ß was much likely contributable to a coordinative or another competitive effect of Nrf2, particularly in distinct Keap1-based cellular responses to its cognate growth factor (i.e. TGF-ß1) or redox stress (e.g. stimulated by tBHQ and DTT). Overall, this discovery presents a novel functional bridge crossing the Keap1-Nrf2 redox signalling and the TGF-ß1-Smad2/3 pathways so as to coordinately regulate the healthy growth and development.

The deubiquitinating enzyme 13 retards non-alcoholic steatohepatitis via blocking inactive rhomboid protein 2-dependent pathway.

Nowadays potential preclinical drugs for the treatment of nonalcoholic steatohepatitis (NASH) have failed to achieve expected therapeutic efficacy because the pathogenic mechanisms are underestimated. Inactive rhomboid protein 2 (IRHOM2), a promising target for treatment of inflammation-related diseases, contributes to deregulated hepatocyte metabolism-associated nonalcoholic steatohepatitis (NASH) progression. However, the molecular mechanism underlying Irhom2 regulation is still not completely understood. In this work, we identify the ubiquitin-specific protease 13 (USP13) as a critical and novel endogenous blocker of IRHOM2, and we also indicate that USP13 is an IRHOM2-interacting protein that catalyzes deubiquitination of Irhom2 in hepatocytes. Hepatocyte-specific loss of the Usp13 disrupts liver metabolic homeostasis, followed by glycometabolic disorder, lipid deposition, increased inflammation, and markedly promotes NASH development. Conversely, transgenic mice with Usp13 overexpression, lentivirus (LV)- or adeno-associated virus (AAV)-driven Usp13 gene therapeutics mitigates NASH in 3 models of rodent. Mechanistically, in response to metabolic stresses, USP13 directly interacts with IRHOM2 and removes its K63-linked ubiquitination induced by ubiquitin-conjugating enzyme E2N (UBC13), a ubiquitin E2 conjugating enzyme, and thus prevents its activation of downstream cascade pathway. USP13 is a potential treatment target for NASH therapy by targeting the Irhom2 signaling pathway.

Tripartite motif containing 26 prevents steatohepatitis progression by suppressing C/EBPδ signalling activation.

Currently potential preclinical drugs for the treatment of nonalcoholic steatohepatitis (NASH) and NASH-related pathopoiesis have failed to achieve expected therapeutic efficacy due to the complexity of the pathogenic mechanisms. Here we show Tripartite motif containing 26 (TRIM26) as a critical endogenous suppressor of CCAAT/enhancer binding protein delta (C/EBPδ), and we also confirm that TRIM26 is an C/EBPδ-interacting partner protein that catalyses the ubiquitination degradation of C/EBPδ in hepatocytes. Hepatocyte-specific loss of Trim26 disrupts liver metabolic homeostasis, followed by glucose metabolic disorder, lipid accumulation, increased hepatic inflammation, and fibrosis, and dramatically facilitates NASH-related phenotype progression. Inversely, transgenic Trim26 overexpression attenuates the NASH-associated phenotype in a rodent or rabbit model. We provide mechanistic evidence that, in response to metabolic insults, TRIM26 directly interacts with C/EBPδ and promotes its ubiquitin proteasome degradation. Taken together, our present findings identify TRIM26 as a key suppressor over the course of NASH development.

A PEGylation technology of L-asparaginase with monomethoxy polyethylene glycol-propionaldehyde.

Polyethylene glycol (PEG) conjugation technology has been successfully applied to improve the performance of protein drugs. In this study, L-asparaginase was N-terminal site-specifically modified by alkylating PEG with monomethoxy polyethylene glycol-propionaldehyde (mPEG-ALD20000). The optimum reaction parameters were determined as pH 5.0, a molar ratio of mPEG-ALD2000 to L-asparaginase of 10:1, a reaction time of 16 h and temperature of 25 degrees C. PEG-L-asparaginase (PEG-L-ASNase) was isolated and purified with consecutive anion-exchange (XK, 16 x 20 cm, Q Sepharose FF) and gel-filtration (Tricorn, 10 x 600 cm, Sephacryl S-300 HR) chromatography, respectively. PEG-L-ASNase retained 43.5% of its activity and the N-terminal amino groups were modified to an extent of 3.67%.

A novel NADP(H)-dependent 3α-HSDH from the intestinal microbiome of Ursus thibetanus.

3α-HSDHs have a crucial role in the bioconversion of steroids, and have been widely applied in the detection of total bile acid (TBA). In this study, we report a novel NADP(H)-dependent 3α-HSDH (named Sc 3α-HSDH) cloned from the intestinal microbiome of Ursus thibetanus. Sc 3α-HSDH was solubly expressed in E. coli (BL21) as a recombinant glutathione-S-transferase (GST)-tagged protein and freed from its GST-fusion by cleavage using the PreScission protease. Sc 3α-HSDH is a new member of the short-chain dehydrogenases/reductase superfamily (SDRs) with a typical α/ß folding pattern, based on protein three-dimensional models predicted by AlphaFold. The best activity of Sc 3α-HSDH occurred at pH 8.5 and the temperature optima was 55 °C, indicating that Sc 3α-HSDH is not an extremozyme. The catalytic efficiencies (kcat/Km) of Sc 3α-HSDH catalyzing the oxidation reaction with the substrates, glycochenodeoxycholic acid (GCDCA) and glycoursodeoxycholic acid (GUDCA), were 183.617 and 34.458 s-1 mM-1, respectively. In addition, multiple metal ions can enhance the activity of Sc 3α-HSDH when used at concentrations ranging from 2 % to 42 %. The results also suggest that the metagenomic approach is an efficient method for identifying novel enzymes.

Expression and Functional Characterization of a Novel NAD(H)- dependent 3α-HSDH.

BACKGROUND: 3α-Hydroxysteroid dehydrogenase (3α-HSDH) reversibly catalyzes the oxidation of the C3-hydroxyl group of steroids, and has been used in clinical applications to detect serum total bile acid (TBA). In this study, A novel 3α-HSDH (called Sb 3α-HSDH) was expressed and characterized. METHODS: Plasmid pGEX-6p-1 was used for the expression of Sb 3α-HSDH in Escherichia coli (BL21), and activities were determined by recording the change in absorbance at 340 nm with/without adding of ions. A prediction of its three-dimensional structure was performed with AlphaFold. RESULTS: The substrate specificity test indicated that Sb 3α-HSDH is NAD(H)-dependent and has no activity with NADP(H). We also showed that Sb 3α-HSDH can catalyze the oxidation reaction of GCDCA and GUDCA with catalytic efficiencies (kcat/Km) of 29.060 and 45.839 s-1mM-1, respectively. The temperature dependence of catalysis suggests that Sb 3α-HSDH is a member of the mesophilic enzymes with its best activity at about 45 °C. The optimum pH of Sb 3α-HSDH was found to be between pH 8.0 and 9.0. The effect of ions, including K+, Mg2+, Na+, Cu2+, Mn2+, Fe2+, and Fe3+ on enzyme activity was evaluated and K+ and Mg2+ were found to enhance the activity of Sb 3α-HSDH by about 20% at concentrations of 200 mM and 50 mM, respectively. The well-conserved GIG motif, the active sites, and the Rossmann fold in the threedimensional structure indicate that Sb 3α-HSDH belongs to the "classical" type of SDR superfamily. CONCLUSION: We expressed and characterized a novel NAD(H)-dependent 3α-HSDH with typical threedimensional characteristics of the SDRs that exhibited substrate specificity to GCDCA and GUDCA.

A novel NAD(H)-dependent 3alpha-HSDH with enhanced activity by magnesium or manganese ions.

3α-Hydroxysteroid dehydrogenase (3α-HSDH) plays a crucial role in the metabolism of sex hormones and bile acids. In this study, we heterologously expressed and characterized a novel 3α-HSDH (named Sa 3α-HSDH). Substrate specificity tests showed that Sa 3α-HSDH could catalyze Glycochenodeoxycholic acid (GCDCA) and Glycoursodeoxycholic acid (GUDCA) with catalytic efficiency (kcat/Km) 40.815 and 14.616 s-1 mM-1, respectively. Sa 3α-HSDH is NAD(H) dependent according to the results of coenzyme screening, and one of mesophilic enzymes with optimum temperature 40 °C. Additionally, Sa 3α-HSDH displayed the highest activity at pH 8.5. In this study, effect of metal ions on activity was investigated, and the results showed Mn2+ (10 mM) and Mg2+ (50 mM) could significantly enhance the activity by nearly 140% and 100%, respectively. Fe2+, Cu2+, Fe3+ and K+ could enhance the activity of Sa 3α-HSDH at different levels. Meanwhile, Na+ only displayed activity-declining effect. The three-dimensional structure of Sa 3α-HSDH was predicted and displayed the well-conserved α/ß folding patterns (Rossman-fold) with a central ß-sheet. These results indicated that Sa 3α-HSDH would contribute to the quantitative determination of serum total bile acids and associated bioconversion.

Mulberrin confers protection against hepatic fibrosis by Trim31/Nrf2 signaling.

Mulberrin (Mul) is a key component of the traditional Chinese medicine Romulus Mori with various biological functions. However, the effects of Mul on liver fibrosis have not been addressed, and thus were investigated in our present study, as well as the underlying mechanisms. Here, we found that Mul administration significantly ameliorated carbon tetrachloride (CCl4)-induced liver injury and dysfunction in mice. Furthermore, CCl4-triggerd collagen deposition and liver fibrosis were remarkably attenuated in mice with Mul supplementation through suppressing transforming growth factor ß1 (TGF-ß1)/SMAD2/3 signaling pathway. Additionally, Mul treatments strongly restrained the hepatic inflammation in CCl4-challenged mice via blocking nuclear factor-κB (NF-κB) signaling. Importantly, we found that Mul markedly increased liver TRIM31 expression in CCl4-treated mice, accompanied with the inactivation of NOD-like receptor protein 3 (NLRP3) inflammasome. CCl4-triggered hepatic oxidative stress was also efficiently mitigated by Mul consumption via improving nuclear factor E2-related factor 2 (Nrf2) activation. Our in vitro studies confirmed that Mul reduced the activation of human and mouse primary hepatic stellate cells (HSCs) stimulated by TGF-ß1. Consistently, Mul remarkably retarded the inflammatory response and reactive oxygen species (ROS) accumulation both in human and murine hepatocytes. More importantly, by using hepatocyte-specific TRIM31 knockout mice (TRIM31Hep-cKO) and mouse primary hepatocytes with Nrf2-knockout (Nrf2KO), we identified that the anti-fibrotic and hepatic protective effects of Mul were TRIM31/Nrf2 signaling-dependent, relieving HSCs activation and liver fibrosis. Therefore, Mul-ameliorated hepatocyte injury contributed to the suppression of HSCs activation by improving TRIM31/Nrf2 axis, thus providing a novel therapeutic strategy for hepatic fibrosis treatment.

The E3 ubiquitin-protein ligase Trim31 alleviates non-alcoholic fatty liver disease by targeting Rhbdf2 in mouse hepatocytes.

Systemic metabolic syndrome significantly increases the risk of morbidity and mortality in patients with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). However, no effective therapeutic strategies are available, practically because our understanding of its complicated pathogenesis is poor. Here we identify the tripartite motif-containing protein 31 (Trim31) as an endogenous inhibitor of rhomboid 5 homolog 2 (Rhbdf2), and we further determine that Trim31 directly binds to Rhbdf2 and facilitates its proteasomal degradation. Hepatocyte-specific Trim31 ablation facilitates NAFLD-associated phenotypes in mice. Inversely, transgenic or ex vivo gene therapy-mediated Trim31 gain-of-function in mice with NAFLD phenotypes virtually alleviates severe deterioration and progression of steatohepatitis. The current findings suggest that Trim31 is an endogenous inhibitor of Rhbdf2 and downstream cascades in the pathogenic process of steatohepatitis and that it may serve as a feasible therapeutical target for the treatment of NAFLD/NASH and associated metabolic disorders.

Expression profiles of glutathione S-transferase genes in larval midgut of Bombyx mori exposed to insect hormones.

Glutathione S-transferases (GSTs) are believed to play a role in the detoxification of xenobiotics, resistance to insect viruses and pesticides, intracellular transport, biosynthesis of hormones and protection against oxidative stress. In this study, we used quantitative real time RT-PCR to examine expression profiles of the silkworm Bombyx mori GST-Sigma (BmGSTS2) and GST-Delta (BmGSTD2) genes in the larval midgut of the silkworm after exposure to 2-hydroxyecdysone (20E) and juvenile hormone analog (JHA). In concentration-course study, 20E at higher concentrations (1.0 and 2.0 µg/µl) caused significant upregulation of BmGSTD2, and all concentrations (0.5-2.0 µg/µl) of 20E caused significant upregulation of BmGSTS2. However, JHA in all concentrations downregulated the expression of BmGSTD2 and BmGSTS2. When exposed to either 20E (2.0 µg/µl) or JHA (2.0 µg/µl) on the third day of the fifth instar, the silkworm had higher BmGSTD2 at later time points: 15, 18, and 24 h for 20E and 24 h for JHA. BmGSTS2 expression was downregulated within 24 h after exposure to JHA and showed a time-dependent response after exposure to 20E. We also did a stage-dependent study, in which JHA downregulated BmGSTD2 expression and upregulated BmGSTS2 expression significantly at both day 1 and day 3 of the fifth instar. 20E upregulated the expression of BmGSTD2 and BmGSTS2 at the two stages. These findings imply that hormones have an important role in the regulation of basal GST expression. However, further validation and field trials should be carried out on the regulatory elements relevant to BmGSTD2 and BmGSTS2 gene expression.

Nanoscale delivery systems for multiple drug combinations in cancer.

Although drug-delivery systems have been developed to improve drug biodistribution and efficiency in cancer therapy, some limitations still hinder successful drug targeting and delivery. Multiple drugs in combination seems a promising strategy for cancer therapy. It enables drugs to be delivered to multiple targets and exhibits the additive or synergistic effects of drugs. Physiological barriers are known to be the main obstacles of insufficient drug efficacy and delivery in tumors, but they are likely to be potential targets in combination therapy as well. This article discusses some general considerations for optimizing multiply drug delivery, including drug-release profiles and loading strategies.

An Overview of 7α- and 7ß-Hydroxysteroid Dehydrogenases: Structure, Specificity and Practical Application.

7α-Hydroxysteroid dehydrogenase and 7ß-hydroxysteroid dehydrogenase are key enzymes involved in bile acid metabolism. They catalyze the epimerization of a hydroxyl group through 7-keto bile acid intermediates. Basic research of the two enzymes has focused on exploring new enzymes and the structure-function relationship. The application research focused on the in vitro biosynthesis of bile acid drugs and the exploration and improvement of their catalytic ability based on molecular engineering. This article summarized the primary and advanced structural characteristics, specificities, biochemical properties, and applications of the two enzymes. The emphasis is also given to obtaining novel 7α-hydroxysteroid dehydrogenase and 7ß-hydroxysteroid dehydrogenase that are thermally stable and active in the presence of organic solvents, high substrate concentration, and extreme pH values. To achieve these goals, enzyme redesigning based on protein engineering and genomics may be the most useful approaches.

A novel 7α-hydroxysteroid dehydrogenase: Magnesium ion significantly enhances its activity and thermostability.

7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays an important role in the efficient biotransformation of taurochenodeoxycholic acid (TCDCA) to tauroursodeoxycholic acid (TUDCA). In this paper, a novel NADP(H)-dependent 7α-HSDH (named J-1-1) was discovered, heterologously expressed in Escherichia coli and biochemically characterized. J-1-1 exhibited high enzymatic activities. The specific activities of J-1-1 toward TCDCA, glycochenodeoxycholic acid (GCDCA) and ethyl benzoylacetate (EBA) were 188.3 ± 0.2, 217.6 ± 0.4, and 20.0 ± 0.2 U·mg-1, respectively, in 50 mM Glycine-NaOH, pH 10.5. Simultaneously, J-1-1 showed high thermostability; 73% of its activity maintained after heat treatment at 40 °C for 100 h. Particularly noteworthy is that magnesium ion could stabilize the structure of J-1-1, resulting in the enhancement of its enzymatic activity and thermostability. The enzymatic activity of J-1-1 increased 40-fold in the presence of 50 mM Mg2+, and T0.5 increased by approximately 6 °C. Furthermore, after heat treatment at 40 °C for 20 min, the control group only retained 52% of the residual enzyme activity, while the residual enzyme activity of the experimental group was still 77% of the J-1-1 enzyme activity with Mg2+ and without heat treatment. These properties of 7α-HSDH would be expected to contribute to more extensive applications in the biotransformation of related substrates.