Integrative pharmacology reveals the mechanisms of Erzhi pills, A traditional Chinese formulation, stimulating melanogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Erzhi pills (EZP), a traditional Chinese medicine formula prescribed for the treatment of vitiligo, has shown promising efficacy. However, the oral bioactive components and mechanisms underlying the promotion of melanogenesis by EZP remain unclear. AIM OF THE STUDY: This study aimed to investigate the pharmacological basis and mechanism of EZP in promoting melanogenesis. MATERIALS AND METHODS: UHPLC-TOF-MS analysis was used to identify absorbed phytochemicals in serum after oral administration of EZP. Network pharmacology methods were used to predict potential targets and pathways involved in the melanogenic activity of EZP, resulting in the construction of a "compound-target-pathway" network. Zebrafish and B16F10 cells were used to evaluate the effects of EZP on tyrosinase activity and melanin content. Western blot and ELISA analyses were used to validate the effects of EZP on melanogenesis-related proteins, including MITF, TYR, CREB, p-CREB, and cAMP. RESULTS: UHPLC-TOF-MS analysis identified 36 compounds derived from EZP in serum samples. Network pharmacology predictions revealed 89 target proteins associated with the identified compounds and closely related to vitiligo. GO and KEGG analyses indicated the involvement of the cAMP/PKA signaling pathway in the promotion of melanogenesis by EZP. Experimental results showed that EZP increased tyrosinase activity and melanin content in zebrafish and B16F10 cells without inducing toxicity. Western blot and ELISA results suggested that the melanogenic effect of EZP may be related to the activation of the cAMP/PKA signaling pathway. These results confirm the feasibility of combining serum pharmacological and network pharmacological approaches. CONCLUSIONS: EZP have the potential to increase tyrosinase activity and melanin content in zebrafish and cells possibly through activation of the cAMP/PKA pathway.

[Anti-depression mechanism of Zuojin Pills:based on UHPLC-TOF-MS, network pharmacology, and experimental verification].

This study aims to explore the anti-depression mechanism of Zuojin Pills based on the plasma constituents, network pharmacology, and experimental verification. UHPLC-TOF-MS was used for qualitative analysis of Zuojin Pills-containing serum. Targets of the plasma constituents and the disease were retrieved from PharmMapper and GeneCards. Then the protein-protein interaction(PPI) network was constructed and core targets were screened for GO term enrichment and KEGG pathway enrichment. Cytoscape 3.7.2 was employed construct the "compound-target-pathway" network and the targets and signaling pathways of Zuojin Pills against depression were predicted. CUMS-induced depression mouse model was established to verify the key targets. The results showed that a total of 21 constituents migrating to blood of Zuojin Pills were identified, which were mainly alkaloids. A total of 155 common targets of the constituents and the disease and 67 core targets were screened out. KEGG enrichment and PPI network analysis showed that Zuojin Pills may play a role in the treatment of depression through AMPK/SIRT1, NLRP3, insulin and other targets and pathways. Furthermore, the results of animal experiments showed that Zuojin Pills could significantly improve the depression behaviors of depression, reduce the levels of IL-1ß, IL-6 and TNF-α in hippocampus and serum, activate AMPK/SIRT1 signaling, and reduce the protein expression of NLRP3. In conclusion, Zuojin Pills may play a role in the treatment of depression by activating AMPK/SIRT1 signaling pathway, and inhibiting NLRP3 activation and neuroinflammation in the hippocampus of mice.

Clinical analysis of deceased donor liver transplantation in the treatment of hepatocellular carcinoma with segmental portal vein tumor thrombus: A long-term real-world study.

Background: Hepatocellular carcinoma (HCC) patients with portal vein tumor thrombus (PVTT) have conventionally been regarded as a contraindication for liver transplantation (LT). However, the outcomes of deceased donor liver transplantation (DDLT) in patients with segmental PVTT remain unknown. The aim of this study is to evaluate the feasibility and effectiveness of DDLT in the treatment of HCC with segmental PVTT. Methods: We retrospectively analyzed 254 patients who underwent DDLT for HCC in our institution from January 2015 to November 2019. To assess the risks of PVTT, various clinicopathological variables were evaluated. Overall (OS) and recurrence-free survival (RFS) analyses based on different PVTT types were performed in HCC patients. Results: Of the 254 patients, a total of 46 patients had PVTT, of whom 35 had lobar PVTT and 11 had segmental PVTT in second-order branches or below. Alpha-fetoprotein (AFP) level, tumor maximal diameter, histological grade, micro-vascular invasion (MVI), RFS, and OS were significantly different between the control and PVTT groups. Lobar PVTT was associated with unfavorable 5-year RFS and OS compared with MVI group (28.6% and 17.1%, respectively). Instead, no significant difference was observed between the segmental PVTT and MVI group in terms of 5-year RFS and OS (RFS: 36.4% vs. 40.4%, p=0.667; OS: 54.5% vs. 45.1%, p=0.395). Further subgroup analysis showed segmental PVTT with AFP levels ≤100 ng/ml presented significantly favorable RFS and OS rates than those with AFP level >100 ng/ml (p=0.050 and 0.035, respectively). Conclusions: In summary, lobar PVTT remains a contraindication to DDLT. HCC patients with segmental PVTT and AFP level ≤100 ng/ml may be acceptable candidates for DDLT.

Risk factors and outcomes of early infection in liver transplant recipients with acute-on-chronic liver failure.

OBJECTIVES: Patients with acute-on-chronic liver failure (ACLF) have a high risk of infection after liver transplantation (LT). In this study, we aimed to evaluate the prevalence of early post-LT infection (within one month after LT) in recipients with ACLF, and to compare the survival rate between patients with or without post-LT infection. METHODS: Patients with ACLF who underwent LT between January 2015 and December 2017 were retrospectively included. Characteristics of the patients, prevalence, site and pathogen of post-LT infection, and its risk factors were evaluated. RESULTS: A total of 62 patients with ACLF developed bacterial or fungal infection after LT. The 30-day, 90-day, and 1-year survival rates in the infected group were found to be significantly lower than those in the non-infected group (67.7% vs 98.5%, 64.5% vs 97.7%, and 48.4% vs 95.4%; all P < 0.001). The most common pathogens involved were carbapenem-resistant gram-negative organisms, including Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter lwoffi. Multivariate analysis demonstrated that reoperation and length of intensive care unit stay were independently associated with post-LT infection. In addition, living donor LT and early allograft dysfunction were independently associated with 30-day all-cause mortality, whereas red blood cell transfusion and post-LT infection were independently associated with all-cause 30-day and 90-day mortality after LT. CONCLUSIONS: Early infection after LT is a major prognostic factor in patients with ACLF. Constant vigilance for the risk factors of early infection after LT is needed for timely diagnosis and prompt intervention.

B2M gene expression shapes the immune landscape of lung adenocarcinoma and determines the response to immunotherapy.

Loss of the B2M gene is associated with tumour immune escape and resistance to immunotherapy. However, genetic alterations of the B2M gene are rare. We performed an integrative analysis of the mutational and transcriptional profiles of large cohorts of non-small-cell lung cancer (NSCLC) patients and found that epigenetic downregulation of B2M is common. B2M-low tumours exhibit a suppressive immune microenvironment characterized by reduced infiltration of immune cells of various lineages; in B2M-high tumours, more T and natural killer cells are present, but their activities are constrained by immune checkpoint molecules, indicating the diverse mechanisms of immune evasion. High levels of B2M mRNA, but not PD-L1, are correlated with an enhanced response to PD-1-based immunotherapy, suggesting its value for immunotherapy response prediction in solid tumours. Notably, a high tumour mutation burden (TMB) is associated with low B2M expression, which may explain the poor predictive value of the TMB in some situations. In syngeneic mouse models, genetic ablation of B2M in tumour cells causes resistance to PD-1-based immunotherapy, and B2M knockdown also diminishes the therapeutic efficacy. Moreover, forced expression of B2M in tumour models improves the response to immunotherapy, suggesting that B2M levels have significant impacts on treatment outcomes. Finally, we provide insight into the roles of transcription factors and KRAS mutations in B2M expression and the anticancer immune response. In conclusion, genetic and epigenetic regulation of B2M fundamentally shapes the NSCLC immune microenvironment and may determine the response to checkpoint blockade-based immunotherapy.

Bacteriophage biocontrol of Shiga toxigenic Escherichia coli (STEC) O145 biofilms on stainless steel reduces the contamination of beef.

Shiga toxigenic Escherichia coli (STEC) can form biofilms and frequently cause serious foodborne illnesses. A strain of STEC O145:H25 (EC19990166) known to be a strong biofilm former was used to evaluate the efficacy of bacteriophage AZO145A against biofilms formed on stainless steel (SS) coupons. Exposure of STEC O145:H25 to phage AZO145A (1010 PFU/mL) for 2 h resulted in a 4.0 log10 reduction (P < 0.01) of planktonic cells grown in M9 broth at 24 °C for 24 h, while reductions were 2.0 log10 CFU/mL if these cells were grown for 48 h or 72 h prior to phage treatment. STEC O145 biofilms formed on SS coupons for 24, 48 and 72 h were reduced (P < 0.01) 2.9, 1.9 and 1.9 log10 CFU/coupon by phages. STEC O145 cells in biofilms were readily transferred from the surface of the SS coupon to beef (3.6 log10 CFU/coupon) even with as little as 10 s of contact with the meat surface. However, transfer of STEC O145 cells from biofilms that formed on SS coupons for 48 h to beef was reduced (P < 0.01) by 3.1 log10 CFU by phage (2 × 1010 PFU/mL) at 24 °C. Scanning electron microscopy revealed that bacterial cells within indentations on the surface of SS coupons were reduced by phage. These results suggest that bacteriophage AZO145A could be effective in reducing the viability of biofilm-adherent STEC O145 on stainless steel in food industry environments.

Hedgehog promotes cell proliferation in the midgut of silkworm, Bombyx mori.

The Hedgehog (Hh) signaling pathway is one of the major regulators of embryonic development and tissue homeostasis in multicellular organisms. However, the role of this pathway in the silkworm, especially in the silkworm midgut, remains poorly understood. Here, we report that Bombyx mori Hedgehog (BmHh) is expressed in most tissues of silkworm larvae and that its functions are well-conserved throughout evolution. We further demonstrate that the messenger RNA of four Hh signaling components, BmHh ligand, BmPtch receptor, signal transducer BmSmo and transcription factor BmCi, are all upregulated following Escherichia coli or Bacillus thuringiensis infection, indicating the activation of the Hh pathway. Simultaneously, midgut cell proliferation is strongly promoted. Conversely, the repression of Hh signal transduction with double-stranded RNA or cyclopamine inhibits the expression of BmHh and BmCi and reduces cell proliferation. Overall, these findings provide new insights into the Hh signaling pathway in the silkworm, B. mori.

Resistance mechanisms adopted by a Salmonella Typhimurium mutant against bacteriophage.

Bacteriophages have key roles in regulating bacterial populations in most habitats. A Salmonella Typhimurium mutant (N18) with impaired sensitivity to phage fmb-p1 was obtained and examined, the adsorption efficiency of fmb-p1 to N18 was reduced to 6%, compared to more than 97% for wild type S. Typhimurium CMCC50115. Reduced adsorption was accompanied by a reduction of 90% in the LPS content compared to wild type. Electron microscopy showed phage scattered around N18 with minimal engagement, while the phage were efficiently adsorbed to the wild type with tails oriented towards the bacterial surface. Evidence suggests fmb-p1 can slightly infect N18 and this does not give rise to an increase of phage titer. RT-qPCR data show that several Salmonella genes involved in lipopolysaccharide synthesis and five virulence related genes were down-regulated upon exposure of N18 to phage fmb-p1. In contrast, phage resistance related genes such as the SOS response, restriction-modification (RM), and Cas1 gene were up-regulated in N18. These data suggest that although inefficient adsorption and entry is the primary mechanism of resistance, transcriptional responses to phage exposure indicate that alternative resistance mechanisms against phage infection are also brought to bear, including digestion of phage nucleic acids and activation of the SOS. These findings may help develop strategies for biocontrol of Salmonella where multi-resistant bacteria are encountered or emerge in applications for food production, bioremediation or wastewater treatment.

Design and Properties of an Immobilization Enzyme System for Inulin Conversion.

A commercial inulinase could convert inulin into fructose, which was optimized to be entrapped in the calcium alginate-gelatin beads with the immobilization yield of 86% for free inulinase activities. The optimum pH values and temperatures were 4.5 and 40 °C for the free enzyme and 5.0-5.5 and 45-50 °C for the immobilized enzyme. The kinetic parameters of V max and K m were 5.24 µmol/min and 57.6 mg/mL for the free inulinase and 4.32 µmol/min and 65.8 mg/mL for the immobilized inulinase, respectively. The immobilized enzyme retained 80% of its initial activities at 45 °C for 4 days, which could exhibit better thermal stability. The reuse of immobilized inulinase throughout the continuous batch operations was explored, which had better reusability of the immobilized biocatalyst. At the same time, the stability of immobilized enzyme in the continuous packed-bed bioreactor was estimated, which showed the better results and had its potential scale-up fructose production for inulin conversion.

Recent advances on the difructose anhydride IV preparation from levan conversion.

Difructose anhydride IV (DFA IV) is a cyclic disaccharide consisting of two fructose residues, which is obtained from levan conversion with levan fructotransferase (LFTase) and rarely found in nature as a low-calorie sugar substitute. Some beneficial effects of DFA IV connected with its consumption have been described. The article reviews the properties and physiological functions of DFA IV, levan conversion, resources and properties of LFTase and the produced methods of DFA IV. LFTase as a relatively novel enzyme and its molecular evolution are discussed as well. The aim is to better understand a novel sugar-substituting sweetener of DFA IV as a food additive.

Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival.

Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of ß-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote ß-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF.

Overexpression of hepatocyte nuclear factor 4α in human mesenchymal stem cells suppresses hepatocellular carcinoma development through Wnt/ß-catenin signaling pathway downregulation.

Mesenchymal stem cells (MSCs) hold promise as cellular vehicles for the delivery of therapeutic gene products because they can be isolated, expanded, and genetically modified in vitro and possess tumor-oriented homing capacity in vivo. (1) Hepatocyte nuclear factor 4α (HNF4α) is a dominant transcriptional regulator of hepatocyte differentiation and hepatocellular carcinogenesis (HCC). (2,3) We have previously demonstrated that overexpression of HNF4α activates various hepatic-specific genes and enhances MSC differentiation. (4) However, the extent that overexpression of HNF4α in MSCs influences HCC progression has yet to be examined. Here we sought to investigate what effect MSCs overexpressing HNF4α (MSC-HNF4α) have on human hepatoma cells in vitro and in vivo. Conditioned medium collected from in vitro MSC-HNF4α cultures significantly inhibited hepatoma cell growth and metastasis compared with controls. Additionally, nude mice administered MSC-HNF4α exhibited significantly smaller tumors compared with controls in vivo. Immunoblot analysis of HCC cells treated with MSC-HNF4α displayed downregulated ß-catenin, cyclinD1, c-Myc, MMP2 and MMP9. Taken together, our results demonstrate that MSC-HNF4α inhibits HCC progression by reducing hepatoma cell growth and metastasis through downregulation of the Wnt/ß-catenin signaling pathway.

Role of BMSCs in liver regeneration and metastasis after hepatectomy.

Hepatocellular carcinoma (HCC), which develops from liver cirrhosis, is highly prevalent worldwide and is a malignancy that leads to liver failure and systemic metastasis. While surgery is the preferred treatment for HCC, intervention and liver transplantation are also treatment options for end-stage liver disease. However, the success of partial hepatectomy and intervention is hindered by the decompensation of liver function. Conversely, liver transplantation is difficult to carry out due to its high cost and the lack of donor organs. Fortunately, research into bone-marrow stromal cells (BMSCs) has opened a new door in this field. BMSCs are a type of stem cell with powerful proliferative and differential potential that represent an attractive tool for the establishment of successful stem cell-based therapy for liver diseases. A number of different stromal cells contribute to the therapeutic effects exerted by BMSCs because BMSCs can differentiate into functional hepatic cells and can produce a series of growth factors and cytokines capable of suppressing inflammatory responses, reducing hepatocyte apoptosis, reversing liver fibrosis and enhancing hepatocyte functionality. Additionally, it has been shown that BMSCs can increase the apoptosis rate of cancer cells and inhibit tumor metastasis in some microenvironments. This review focuses on BMSCs and their possible applications in liver regeneration and metastasis after hepatectomy.

Dry powder preparation of inulin fructotransferase from Arthrobacter aurescens SK 8.001 fermented liquor.

Difructosan anhydrides III (DFA III) are usually obtained by inulin conversion with inulin fructotransferase (IFTase). IFTase liquor is difficult to store for a long time, which could greatly restrict its application and DFA III production. To meet DFA III scale-up preparation, this work was explored to research dry powder preparation of IFTase from Arthrobacter aurescens SK 8.001 fermented liquor by ultrafiltration concentration, ammonium sulfate precipitation and freeze drying. IFTase powder (10.2g) was obtained from IFTase precipitation (126.4 g) and its specific activity determined was 16.4 U/mg. Dry powder of IFTase could maintain over 120 days at different temperatures. These results showed that it is easy to scale up DFA III preparation for industrial capacity.

Difructosan anhydrides III preparation from sucrose by coupled enzyme reaction.

Difructosan anhydrides III (DFA III) preparation was usually obtained by inulin hydrolysis with inulin fructotransferase (IFTase). The fructofuranosidic linkages of inulin were the same as fructooligosaccharides (FOS), which was synthesized by sucrose with fructosyltransferase (FTase). FOS was mainly composed of 1-kestose (GF(2)), nystose (GF(3)) and fructofuranosylnystose (GF(4)), and nystose was observed to be the smallest substrate for IFTase to synthesize DFA III. So sucrose, much cheaper than inulin, was considered to produce DFA III by coupled FTase and IFTase reaction. DFA III yield was obtained about 100mg/g (DFA III weight/sucrose weight) through this method. The results demonstrated the high potential of the coupled enzyme reaction as a novel DFA III producing method.

DFA III production from inulin with inulin fructotransferase in ultrafiltration membrane bioreactor.

An ultrafiltration membrane bioreactor was used for the production of DFA III from enzymatic conversion of inulin. Compared with the traditional batch reactor, the productivity and purity of DFA III could be markedly enhanced and product inhibition was removed and IFTase could be continuously used for six runs in the UF membrane bioreactor. When the substrate concentration was 100 g/L, the concentration of DFA III was about 78.4 g/L, while the productivity and purity of DFA III could attain about 2385 and 92%, respectively.

Cloning and extracellular expression of inulin fructotransferase from Arthrobacter aurescens SK 8.001 in E. coli.

BACKGROUND: Difructose anhydride (DFA) III is a natural and low-calorie sweetener. It stimulates the absorption of calcium and other minerals. Inulin fructotransferase (IFTase; EC 4.2.2.18), catalysing inulin hydrolysis to DFA III, is considered to be the most promising enzyme for the production of DFA III. RESULTS: IFTase gene from Arthrobacter aurescens SK 8.001 was cloned and sequenced. Transformant with native IFTase signal peptide was a useful system for extracellular over-expression of IFTase, and its extracellular IFTase activity reached 81.0 U mL(-1) . This value was 4.1-fold of that obtained with A. aurescens SK 8.001 for IFTase production. The recombinant IFTase was purified to electrophoretical homogeneity and characterized. The enzyme showed maximum activity at pH 6.0 and 55 °C, and retained 81.3% of its initial activity after incubation at 60 °C for 4 h. CONCLUSION: IFTase gene from A. aurescens SK 8.001 was cloned, sequenced and over-expressed in E. coli. IFTase was reported for the first time to be over-expressed extracellularly. The recombinant IFTase was purified and characterized, and shown to be a good candidate for potential application in DFA III production.

Recent advances on biological difructose anhydride III production using inulase II from inulin.

Difructose anhydride III (DFA III), the smallest cyclic disaccharide, consists of two fructose residues. DFA III is a hydrolysate of inulin and is rarely found in nature. Industrial interest in DFA III as a low-calorie sugar substitute is increasing. The present review describes the properties and physiological functions of DFA III as well as its commercial importance. Focus is also given on the biological production of DFA III from inulin, which contains enzyme resources, inulase II properties, and the capacity for mass DFA III production. Inulase II as an industrial enzyme and its molecular evolution are discussed as well. The aim is to better understand commercial-scale DFA III production as a food product.

[Impact of muscle strength on knee joint stability in static loading].

OBJECTIVE: To study the in vivo stability of normal and anterior cruciate ligament (ACL)-injured knee joint before and after epidural anesthesia under 134 N pre-loading and evaluate the influence of muscular tension on the knee stability. METHODS: Eight volunteers with unilateral ACL rupture and normal contralateral knee were enrolled in this study. CT (3D) images and 2 orthogonal images of the knee were captured at 0°, 30°, 60°, and 90° under 134 N pre-loading. The orthogonal images were used to recreate the in vivo knee positions at each of the targeted flexion angles by 2D/3D registration to analyze the tibial translation data. RESULTS: The anterior tibia translation of both the intact and ACL-injured knees after anesthesia was significantly different from that before anesthesia at all the angles (P<0.05). The anterior tibial translation of the intact knee after anesthesia increased by 1.7 mm at 0°, 2.7 mm at 30°, 2.6 mm at 60°, and 2.3 mm at 90°, as compare to the increase of ACL-injured knee by 4.2 mm, 2.6 mm, 1.2 mm, and 1.6 mm, respectively. CONCLUSION: The muscular tension has evident influence on the knee stability in static loading.