Emodin alleviates cholestatic liver injury by modulating Sirt1/Fxr signaling pathways.

Emodin (EMO) has the effect of anti-cholestasis induced by alpha-naphthylisothiocyanate (ANIT). But its mechanism is still unclear. The farnesoid X receptor (Fxr) is the master bile acid nuclear receptor. Recent studies have reported that Sirtuin 1 (Sirt1) can regulate the activities of Fxr. The purpose of the current study was to investigate the mechanism of EMO against ANIT-induced liver injury based on Sirt1/Fxr signaling pathway. The ANIT-induced cholestatic rats were used with or without EMO treatment. Serum biochemical indicators, as well as liver histopathological changes were examined. The genes expressions of Sirt1, Fxr, Shp, Bsep and Mrp2 were detected. The expressions of Sirt1, Fxr and their downstream related genes were investigated in vitro. The results showed that EMO significantly alleviated ANIT-induced liver injury in rats, and increased Sirt1, Fxr, Shp, Bsep and Mrp2 gene expression in liver, while decreased the expression of Cyp7a1. EMO significantly activated Fxr, while Sirt1 inhibitor and Sirt1 gene silencing significantly reduced Fxr activity in vitro. Collectively, EMO in the right dose has a protective effect on liver injury induced by ANIT, and the mechanism may be through activation of Fxr by Sirt1, thus regulating bile acid metabolism, and reducing bile acid load in hepatocytes.

Cardiovascular toxicities following the use of tyrosine kinase inhibitors in hepatocellular cancer patients: a retrospective, pharmacovigilance study.

BACKGROUND: Cardiac adverse events (AEs) are common in tyrosine kinase inhibitors(TKIs). This study explored the cardiac AEs of TKIs through the Food and Drug Administration's Adverse Event Reporting System (FAERS). METHODS: Disproportionality analysis and Bayesian analysis were utilized for data mining of the suspected cardiac AEs of TKIs, based on FAERS data from January 2004 to December 2021. RESULTS: A total of 4708 cardiac AEs reports of sorafenib, regorafenib, lenvatinib, and cabozantinib were identified. Hypertension accounts for the most reported cardiac AE. Lenvatinib appears to induce cardiac failure with the highest signals strength [ROR = 7.7 (3.46,17.17)]. Acute myocardial infarction was detected in lenvatinib [ROR = 7.91 (5.64,11.09)] and sorafenib [ROR = 2.22 (1.74, 2.84)]. Acute coronary syndrome was detected in lenvatinib [ROR = 11.57 (6.84, 19.58)] and sorafenib [ROR = 2.81 (1.87,4.24)]. Atrial fibrillation was detected in sorafenib [ROR = 1.82 (1.55,2.14)] and regorafenib [ROR = 1.36 (1.03,1.81)]. Meanwhile, aortic dissections were detected in sorafenib [ROR = 5.08 (3.31,7.8)] and regorafenib [ROR = 3.39 (1.52,7.56)]. Most patients developed hypertension and cardiac failure within 30 days of initiating TKI treatments. Patients taking lenvatinib had an increased incidence of developing acute coronary syndrome after 180 days of treatment. CONCLUSION: Analysis of FAERS data provides a precise profile on the characteristics of cardiac AEs associated with different TKI regimens. Distinct monitoring and appropriate management are needed in the care of TKI recipients.

Anti-inflammatory effect and component analysis of Chaihu Qingwen granules.

ETHNOPHARMACOLOGICAL RELEVANCE: As prevalent acute respiratory condition in clinical practice, acute lung injury has a quick start and severe symptoms which can harm patients physically. Chaihu Qingwen granules (CHQW) is a classic formula for the treatment of respiratory diseases. Clinical observation shows that CHQW has good efficacy in treating colds, coughs, and fevers. AIM OF THE STUDY: The aim of this study was to investigate the anti-inflammatory effect of CHQW on lipopolysaccharide (LPS)-induced acute lung injury (ALI) model in rats and to explore its potential mechanism, as well as to clarify its substance composition. MATERIALS AND METHODS: Male SD rats were randomly divided into the blank group, the model group, the ibuprofen group, the Lianhua Qingwen capsule group and the CHQW group (2, 4 and 8 g/kg, respectively). The LPS-induced acute lung injury (ALI) model in rats was established after pre-administration. The histopathological changes in the lung and the levels of inflammatory factors in bronchoalveolar lavage fluid (BALF) and serum of ALI rats were observed. The inflammation-related proteins toll-like receptor 4 (TLR4), inhibitory kappa B alpha (IκBα), phospho-IκBα (p-IκBα), nuclear-factor-kappa B (NF-κB), and NLR family pyrin domain containing 3(NLRP3) expression levels were measured by western blotting analysis and immunohistochemical analysis. The chemical composition of CHQW was identified by liquid chromatography-quadrupole-time of flight-mass spectrometry (LC-Q-TOF-MS). RESULTS: CHQW significantly ameliorated lung tissue pathological injury in LPS-induced ALI rats and decreased the release of inflammatory cytokines (interleukin-1ß, interleukin-17 and tumor necrosis factor-α) in BALF and serum. In addition, CHQW decreased the expression of TLR4, p-IκBα and NF-κB proteins, increased the level of IκBα, regulated the TLR4/NF-κB signaling pathway, and inhibited the activation of NLRP3. The chemical components of CHQW were analyzed by LC-Q-TOF-MS, and a total of 48 components were identified by combining information from the literature, mainly flavonoids, organic acids, lignans, iridoids and phenylethanoid glycosides. CONCLUSION: The results of this study showed that the pretreatment of CHQW had a strong protective effect on LPS-induced ALI in rats, reducing lung tissue lesions and decreasing inflammatory cytokines released in BALF and serum. The protective mechanism of CHQW may be related to the inhibition of the TLR4/NF-κB signaling pathway and NLRP3 activation. The main active ingredients of CHQW are flavonoids, organic acids, lignans, iridoids and phenylethanoid glycosides.

Structural characterization and anti-lipotoxicity effects of a pectin from okra (Abelmoschus esculentus (L.) Moench).

Okra (Abelmoschus esculentus (L.) Moench) is rich in various bioactive ingredients and used as a medicinal plant in traditional medicine. In the present study, to find the polysaccharide with anti-lipotoxicity effects from okra and clarify its structure, a pectin OP-1 was purified from okra, which had a backbone containing →4)-α-GalpA-(1 â†’ residues, and 1,5-Ara linked the main chain through the O-3 of the residue →3,4)-α-GalpA-(1→, and the C-6 of residue 1, 4-α-GalpA replaced by methyl ester. In vitro experiments showed that OP-1 pretreatment alleviated oleic acid (OA)-induced lipid accumulation, ROS generation, apoptosis, transaminase leakage, and inflammatory cytokine secretion in HepG2 cells, resulting in reduced lipotoxicity. Further molecular results revealed that OP-1 increased Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and affected the expression of AMPK downstream targets, including inhibit SREBP1c and FAS, as well as activate CPT-1A. Impressively, AMPK inhibitor dorsomorphin (Compound C) blocked the effects of OP-1 against lipotoxicity. The effects of OP-1 on lipid metabolism were also diminished by dorsomorphin. Our results demonstrated that OP-1 possesses a potent function in preventing lipotoxicity via regulating AMPK-mediated lipid metabolism and provide a novel insight into the future utilization of okra polysaccharide.

Nor-triterpenoids from the fruiting bodies of Ganoderma lucidum and their inhibitory activity against FAAH.

Two new nor-triterpenoids ganodrenol A (1), B (2), and a new natural product ganodrenol C (3), along with three known nor-triterpenoids (4-6) were isolated from the fruiting bodies of Ganoderma lucidum. The chemical structures of these isolates were determined by 1 D and 2 D NMR, HRESIMS, and X-ray crystallography analysis. The inhibitory effects of isolated triterpenoids (1-6) against FAAH were evaluated by an in vitro assay, and compound 4 showed an inhibition rate of 70.27%. In addition, the cytotoxic effect of compounds (1-6) was evaluated against LOVO, MCF-7, and RAW264.7 cells, which displayed no significant cytotoxicity.

Data mining and safety analysis of BTK inhibitors: A pharmacovigilance investigation based on the FAERS database.

Objective: The introduction of Bruton's tyrosine kinase (BTK) inhibitors was a milestone in the treatment of B-cell malignancies in recent years owing to its desired efficacy against chronic lymphocytic leukaemia and small cell lymphocytic lymphoma. However, safety issues have hindered its application in clinical practice. The current study aimed to explore the safety warning signals of BTK inhibitors in a real-world setting using the FDA Adverse Event Reporting System (FAERS) to provide reference for clinical rational drug use. Methods: Owing to the short marketing time of other drugs (zanbrutinib and orelabrutinib), we only analysed ibrutinib and acalabrutinib in this study. All data were obtained from the FAERS database from January 2004 to December 2021. Disproportionality analysis and Bayesian analysis were utilised to detect and assess the adverse event (AE) signals of BTK inhibitors. Results: In total, 43,429 reports of ibrutinib were extracted and 1527 AEs were identified, whereas 1742 reports of acalabrutinib were extracted and 220 AEs were identified by disproportionality analysis and Bayesian analysis. Among reports, males were more prone to develop AEs (58.2% for males vs. 35.6% for females treated with ibrutinib, and 55.9% vs. 31.9%, respectively, for acalabrutinib), and more than 30% of patients that suffered from AEs were over 65 years of age. Subsequently, we investigated the top 20 preferred terms (PTs) associated with the signal strength of ibrutinib and acalabrutinib, and our results identified 25 (13 vs. 12, respectively) novel risk signals. Among the top 20 PTs related to death reports, the terms infectious, pneumonia, pleural effusion, fall, asthenia, diarrhoea, and fatigue were all ranked high for these two BTK inhibitors. Further, cardiac disorders were also an important cause of death with ibrutinib. Conclusion: Patients treated with ibrutinib were more prone to develop AEs than those treated with acalabrutinib. Importantly, infection-related adverse reactions, such as pneumonia and pleural effusion, were the most common risk signals related to high mortality associated with both BTK inhibitors, especially in elderly patients. Moreover, cardiovascular-related adverse reactions, such as atrial fibrillation and cardiac failure, were fatal AEs associated with ibrutinib. Our results provide a rationale for physicians to choose suitable BTK inhibitors for different patients and provide appropriate monitoring to achieve safer therapy and longer survival.

Lanostane triterpenoids from Ganoderma lucidum and their inhibitory effects against FAAH.

Ganoderma lucidum is a famous edible and medicinal fungus. Through a bioactive phytochemical investigation of the ethanolic extracts of the fruiting bodies of G. lucidum, twenty-nine triterpenoids, including eleven previously undescribed triterpenoids, were isolated and characterized based on spectroscopic data. The inhibitory effects of all the triterpenes against fatty acid amide hydrolase (FAAH) were found to be in the range of 30-60% at 100 µM. Methyl ganoderate A displayed the strongest inhibitory activity (61%) against FAAH. Furthermore, all compounds displayed no cytotoxicity against LOVO and MCF-7 human cancer cells. Hence, our present study provides information about G. lucidum as a functional food or pharmaceutical supplement for the treatment of neuroinflammation.

Epifriedelanol enhances adriamycin-induced cytotoxicity towards K562/ADM cells by down regulating of P-gp and MRP2.

1. Multidrug resistance (MDR) is a critical issue during chemotherapy of cancers. Epifriedelanol (Epi) is the effective compounds from the Root Bark of Ulmus davidiana. This study aims to investigate the effect of Epi on MDR and its potential mechanism in the adriamycin (Adr)-resistant K562/ADM cells.2. The effect of Epi on MDR, P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs) were investigated in the adriamycin (Adr)-resistant K562/ADM cells. In addition, the alterations of nuclear receptor pregnane X receptor (PXR) and constitutive androstane receptor (CAR) mRNA expression levels in K562/ADM cells after Epi treatment were also examined.3. Epi significantly enhanced Adr-induced cytotoxicity towards K562/ADM cells. Combination of Epi and Adr can significantly reduce the 50% inhibitory concentration (IC50) of K562/ADM cells to Adr. The reversal fold was 1.83 and 3.64 after treated with Epi at 10 and 20 µM, respectively. The intracellular accumulation of Adr was significant increased after exposure to Epi at 5-20 µM compared with the control group. Furthermore, Epi treatment significantly decreased the mRNA and protein expression of P-gp and MRP2 in K562/ADM cells.4. The present study demonstrated that Epi could enhance Adr-induced cytotoxicity towards K562/ADM cells accompanied by the down-regulation of P-gp and MRP2.

Interactions of the major effective components in Shengmai formula with breast cancer resistance protein at the cellular and vesicular levels.

Shengmai Formula (SMF) is one of the traditional Chinese medicine representative formulas and is widely used for the treatment of cardio- and cerebrovascular disease. Previous studies demonstrated that the major effective ingredients in SMF can interact with each other based on some uptake transporters. However, the role of the efflux transporter breast cancer resistance protein (BCRP) in these interactions involving SMF remains unclear. The purpose of this study was to investigate the interactions of the major active components of SMF with BCRP and the compatibility mechanism of these complex components in SMF based on BCRP. We selected 4 main fractions, including ginseng total saponins (GTS), ophiopogon total saponins (OTS), ophiopogon total flavonoids (OTF), and fructus schisandrae total lignans (STL), and 12 bioactive components, including ginsenosides Re, Rd, Rb1, and Rg1, ophiopogonins D and D', methylophiopogonanones A and B, schizandrins A and B, and schizandrols A and B to explore the interactions of SMF with BCRP in LLC-PK1 and LLC-PK1/BCRP cells and BCRP membrane vesicles. The results showed that ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A can be transported by BCRP into LLC-PK1/BCRP cells. Schisandrol B exhibited a markedly inhibitory effect on the transport function of BCRP and can significantly inhibit the uptake of methylophiopogonanone B and schizandrin A into LLC-PK1/BCRP cells. In "Inside-Out" BCRP membrane vesicles, BCRP mediated the transport of ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A, with Km values of 111.9 ±â€¯31.26 µM, 82.01 ±â€¯16.72 µM, 57.06 ±â€¯8.789 µM, and 37.19 ±â€¯6.512 µM, respectively. GTS, STL, ginsenosides Rd and Rb1, and schisandrol B were potent inhibitors of BCRP and showed different degrees of inhibition on the transport of ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A via BCRP. In conclusion, GTS, STL, ginsenosides Rd and Rb1, and schizandrol B are potential inhibitors of BCRP. Ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A are potential substrates of BCRP, and their transport, which is mediated by BCRP, may be inhibited by potential inhibitors in SMF. There are potential interactions of these main effective components of SMF at the cellular and vesicular levels that are mediated by BCRP. The interplay of these bioactive components based on BCRP may be an important compatibility mechanism in SMF.

LncRNA HOTAIR modulates the expression of OATP1B1 in HepG2 cells by sponging miR-206/miR-613.

OATP1B1 is an important drug transporter with a complex regulatory mechanism. In this study, we wanted to investigate how LncRNA HOTAIR regulates the expression of OATP1B1 through its action on miR-206/miR-613 in HepG2 cells. The expression level of LncRNA HOTAIR, miR-206/miR-613, and OATP1B1 mRNA was detected by RT-qPCR, and the OATP1B1 protein level was detected by Western blot. The competitive endogenous RNA mechanism was validated by bioinformatics analysis and a dual-luciferase reporter gene assay. Our results showed that over- or under-expression of LncRNA HOTAIR correspondingly significantly increased or decreased the protein level of OATP1B1 in HepG2 cells, while no significant change in OATP1B1 mRNA level was observed. In addition, the stimulatory or inhibitory effect of LncRNA HOTAIR on OATP1B1 protein expression was correspondingly reversed by miR-206/miR-613 mimic or miR-206/miR-613 inhibitor. Finally, the reporter gene assay revealed that LncRNA HOTAIR can sponge miR-206/miR-613, which breaks the binding site of miR-206/miR-613 and OATP1B1 mRNA 3'-UTR, eliminating the stimulatory effect of LncRNA HOTAIR on OATP1B1 protein. Thus, we conclude that LncRNA HOTAIR can affect the expression of OATP1B1 in HepG2 cells by sponging miR-206/miR-613, which, in turn, prevents the binding of miR-206/miR-613 and OATP1B1 mRNA 3'-UTR.

Inhibition of the PD-1/PD-L1 signaling pathway enhances innate immune response of alveolar macrophages to mycobacterium tuberculosis in mice.

BACKGROUND: Mycobacterium tuberculosis (TB) is a pathogen that consequently leads to TB infection, which remains a significant global health concern. Programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) signaling pathway is critical for terminating immune responses. The present study aimed to elucidate the regulatory role of the PD-1/PD-L1 signaling pathway in alveolar macrophages against Mycobacterium TB in mice. METHODS: Specific pathogen free mice were initially prepared for Mycobacterium TB model establishment. The alveolar macrophages of the successfully modeled rats were evaluated to determine the levels of PD-1, PD-L1, AKT, mTOR, TNF-α, NF-κB, IL-2, IL-4, IL-6, IL-10, IL-17, IL-17A, and IFN-γ. The surface makers of macrophages (CD11c, CD16, CD86, CD163, CD206, CX3CR-1 and CSF-1R), level of ROS, apoptosis and cell cycle, were all assessed. RESULTS: Elevated levels of PD-1 and PD-L1, decreased levels of AKT and mTOR, along with elevated levels of TNF-α, NF-κB, IL-17, IL-2, IL-6, IL-17A and IFN-γ were identified in the alveolar macrophages infected with Mycobacterium TB, while an opposite trend was observed when PD-1/PD-L1 signaling pathway was inhibited. Additionally, elevated protein levels of CD11c, CD16 and CD86, as well as an increased rate of positive ROS and cell apoptosis, levels of Bax, and a diminished percentage of alveolar macrophages at the S and G2/M stages were detected in the event of Mycobacterium TB infection. A contrasting trend to the aforementioned findings was detected when the PD-1/PD-L1 signaling pathway was inhibited. CONCLUSION: Taken together, these results suggested that inhibition of the PD-1/PD-L1 signaling pathway enhanced the innate immune response of alveolar macrophages to Mycobacterium TB in mice.

Effect of panaxytriol on cytochrome P450 3A4 via the pregnane X receptor regulatory pathway.

Panaxytriol (PXT) is one of the major effective components of red ginseng and Shenmai injection. The present study aimed to explore the effect of PXT on cytochrome P450 3A4 (CYP3A4) based on the pregnane X receptor (PXR)-CYP3A4 regulatory pathway in HepG2 cells and hPXR-overexpressing HepG2 cells treated with PXT for different time periods using quantitative polymerase chain reaction, Western blot, and dual-luciferase reporter gene assays. PXT could upregulate the levels of PXR and CYP3A4 mRNA in HepG2 cells treated with PXT for 1 hr, with no impact on the expression of their protein levels. The expression levels of both PXR and CYP3A4 mRNA and protein in HepG2 cells treated with PXT for 24 hr increased in a concentration-dependent manner. The effects of PXT on the expression of PXR and CYP3A4 mRNA and protein in hPXR-overexpressing HepG2 cells were similar to those in HepG2 cells. Moreover, the influence trend of PXT on CYP3A4 was consistent with that of PXR in HepG2 cells and hPXR-overexpressing HepG2 cells. The dual-luciferase reporter gene assay in HepG2 cells further demonstrated that PXT treatment for specific time periods could significantly induce the expression of CYP3A4 mediated by the PXR regulatory pathway.

High expression of RAD51 promotes DNA damage repair and survival in KRAS-mutant lung cancer cells.

RAD51 recombinase plays a critical role in homologous recombination and DNA damage repair. Here we showed that expression of RAD51 is frequently upregulated in lung cancer tumors compared with normal tissues and is associated with poor survival (hazard ratio (HR) = 2, P = 0.0009). Systematic investigation of lung cancer cell lines revealed higher expression of RAD51 in KRAS mutant (MT) cells compared to wildtype (WT) cells. We further showed that MT KRAS, but not WT KRAS, played a critical role in RAD51 overexpression via MYC. Moreover, our results revealed that KRAS MT cells are highly dependent on RAD51 for survival and depletion of RAD51 resulted in enhanced DNA double strand breaks, defective colony formation and cell death. Together, our results suggest that mutant KRAS promotes RAD51 expression to enhance DNA damage repair and lung cancer cell survival, suggesting that RAD51 may be an effective therapeutic target to overcome chemo/radioresistance in KRAS mutant cancers. [BMB Reports 2019; 52(2): 151-156].

A novel Babesia orientalis 135-kilodalton spherical body protein like: identification of its secretion into cytoplasm of infected erythrocytes.

BACKGROUND: The spherical body is a distinct organelle only existing in Babesia and Theileria. Spherical body proteins (SBPs) are secreted from spherical bodies and incorporated into the cytoplasm of infected erythrocytes during invasion and post-invasion stages. Four different SBP homologues (SBP1, SBP2, SBP3 and SBP4) have been identified in Babesia bovis and Babesia bigemina. So far, there has been no report available about the identification of SBPs in Babesia orientalis. METHODS: The SBP3-like in B. orientalis (BoSBP3-like) was cloned, sequenced, characterized and compared to the SBP3 sequences of B. bovis and B. bigemina by bioinformatics analyses. The BoSBP3-like gene was truncated into three fragments: BoSBP3-like-1 (915 bp), BoSBP3-like-2 (1311 bp) and BoSBP3-like-3 (1011 bp), which were amplified and cloned into the expression vector pET-28a and expressed as three truncated recombinant (His-fusion) proteins. The immunogenicity, native forms and localization of BoSBP3-like were identified by western blot and indirect immunofluorescence assay (IFA). RESULTS: The BoSBP3-like gene was intronless with an open reading frame (ORF) of 3237 bp, encoded a 1079 amino acid polypeptide with a predicted size of 135 kDa, and contained a cysteine-rich region, three dispersing FAINT domains and a signal peptide (1-16 aa) at the N-terminus. The amino acid sequence of BoSBP3-like was 61.6 and 35.0% identical to that of B. bovis and B. bigemina, respectively. BoSBP3-like was identified as 135 kDa in the parasite lysate by rabbit antiserum against the truncated recombinant BoSBP3-like-1 (rBoSBP3-like-1). Three specific bands corresponding to rBoSBP3-like-1 (1-305 aa, 43 kDa), rBoSBP3-like-2 (306-742 aa, 58 kDa) and rBoSBP3-like-3 (743-1079 aa, 52 kDa) were detected by reaction with serum from B. orientalis-infected buffalo. The BoSBP3-like was not only localized in the spherical body of B. orientalis but also in the cytoplasm of infected erythrocytes of buffalo as puncta-like protein specks at both single and paired parasite development stages. CONCLUSIONS: Through secretion into the cytoplasm of infected erythrocytes, BoSBP3-like may play a significant role in adaptation, interaction, and modification related to the host environment to benefit the growth and survival of Babesia. BoSBP3-like could react with the serum from B. orientalis-infected buffalo, but not healthy buffalo, implicating that BoSBP3-like is highly antigenic and may serve as a candidate diagnostic antigen for the detection of B. orientalis.

Establishment and evaluation of four different types of patient-derived xenograft models.

BACKGROUND: Patient-derived xenografts (PDX) have a biologically stable in tumor architecture, drug responsiveness, mutational status and global gene-expression patterns. Numerous PDX models have been established to date, however their thorough characterization regarding the tumor formation and rates of tumor growth in the established models remains a challenging task. Our study aimed to provide more detailed information for establishing the PDX models successfully and effectively. METHODS: We transplanted four different types of solid tumors from 108 Chinese patients, including 21 glioblastoma (GBM), 11 lung cancers (LC), 54 gastric cancers (GC) and 21 colorectal cancers (CRC), and took tumor tissues passaged for three successive generations. Here we report the rate of tumor formation, tumor-forming times, tumor growth curves and mortality of mice in PDX model. We also report H&E staining and immunohistochemistry for HLA-A, CD45, Ki67, GFAP, and CEA protein expression between patient cancer tissues and PDX models. RESULTS: Tumor formation rate increased significantly in subsequent tumor generations. Also, the survival rates of GC and CRC were remarkably higher than GBM and LC. As for the time required for the formation of tumors, which reflects the tumor growth rate, indicated that tumor growth rate always increased as the generation number increased. The tumor growth curves also illustrate this law. Similarly, the survival rate of PDX mice gradually improved with the increased generation number in GC and CRC. And generally, there was more proliferation (Ki67+) in the PDX models than in the patient tumors, which was in accordance with the results of tumor growth rate. The histological findings confirm similar histological architecture and degrees of differentiation between patient cancer tissues and PDX models with statistical analysis by GraphPad Prism 5.0. CONCLUSION: We established four different types of PDX models successfully, and our results add to the current understanding of the establishment of PDX models and may contribute to the extension of application of different types of PDX models.

Identification and characterization of a novel 34 kDa merozoite protein in Babesia orientalis.

A novel Babesia orientalis 34 kDa protein (designated BoP34) was obtained by immunoscreening of a cDNA expression library using B. orientalis infected water buffalo serum. The complete nucleotide sequence of the BoP34 was 1088 bp, which contained one open reading frame (ORF), two untranslated regions (UTRs) and a poly (A) tail. The length of ORF was 933 bp, encoding a polypeptide of 310 aa with a predicted size of 34 kDa. BLAST analysis showed that the nucleotide sequence of BoP34 had 71% similarity with that of the Babesia bovis gene XM_001611335, which encodes a nuclear movement family protein. This suggested that BoP34 is a homologous of the movement family protein. Structural analysis of the BoP34 protein indicated a CS domain which may interact with the ATPase domain of the heat shock protein 90. A truncated version of BoP34 was cloned into the expression vector pET-32a and subsequently expressed and purified from the Escherichia coli Rosetta™ (DE3) pLysS stain as a Trx-fusion (designated rBoP34-T). Antibodies in the serum of a B. orientalis-infected water buffalo were able to recognize this protein in immune-bloting analysis. Rabbit antibodies raised against rBoP34-T could detecte native BoP34 (34 kDa) in B. orientalis-infected water buffalo erythrocytes. These results suggested that BoP34 might be a good diagnostic antigen for the specific detection of anti-B. orientalis antibody in water buffalo. Further research is required to explore the biological function and diagnostic potential of this molecule.

Characterisation of a Babesia orientalis apical membrane antigen, and comparison of its orthologues among selected apicomplexans.

In the present study, we identified and characterised the complete coding sequence of Babesia orientalis apical membrane antigen 1 (designated Bo-ama1); it is 1803bp in length and encodes a polypeptide of 601 amino acids (aa). The Bo-ama-1 gene product (Bo-AMA1) is predicted to be 67kDa in size and contains a signal peptide. Mature Bo-AMA1 is predicted to have one transmembrane region and a short cytoplasmic tail (C-terminal domain). The extracellular part of Bo-AMA1 has three functional domains (DI, DII and DIII) with 14 conserved cysteine residues. A Bo-AMA1 fragment containing all three of these domains (designated Bo-AMA1-DI/II/III) was cloned into the plasmid vector pET-28a and expressed as a recombinant (His-fusion) protein of 53kDa. Antibodies in the serum from a B. orientalis-infected water buffalo specifically recognised this protein in immunoblotting analysis. Rabbit antibodies raised against the recombinant protein were able to detect native Bo-AMA1 (67kDa) from erythrocytes of B. orientalis-infected water buffalo. Bo-AMA1 is a new member of the AMA1 family and might be a good antigen for the specific detection of antibodies produced in B. orientalis infected cattle. This protein is likely to play critical roles during host cell adherence and invasion by B. orientalis, as the AMA1s reported in other organisms such as Plasmodium falciparum and Toxoplasma gondii. Further research is required to explore the biological functions of this protein and to determine whether its immunisation can induce protective effects in water buffalo against B. orientalis infection.

Molecular cloning and characterization of Babesia orientalis rhoptry-associated protein 1.

The rhoptry-associated protein 1 (RAP-1) gene of Babesia orientalis was obtained from a cDNA expression library by immunoscreening with B. orientalis-infected water buffalo sera. The nucleotide sequence of the cDNA was 1732 bp with an open reading frame (ORF) of 1434 bp, encoding a polypeptide of 478 amino acid residues with a predicted size of 52.5 kDa. The ORF was cloned into a pGEX-KG plasmid and subsequently expressed as a GST-fusion protein. The recombinant RAP-1 of B. orientalis (rBoRAP-1) was purified and evaluated as an antigen using Western blotting. The native BoRAP-1 was recognized by the antibodies raised in rabbits against rBoRAP-1. Strong immunofluorescence signals were observed in erythrocytes infected with B. orientalis. Phylogentic analysis revealed that B. orientalis fell into a Babesia clade and most closely related to Babesia bovis and Babesia ovis, which was similar to the previous reported trees based on 18S rRNA and HSP70 genes. The present study suggests that the BoRAP-1 might be a potential diagnostic antigen, and the RAP-1 genes can aid in the classification of Babesia and Theileria species.

Identification of two novel HSP90 proteins in Babesia orientalis: molecular characterization, and computational analyses of their structure, function, antigenicity and inhibitor interaction.

BACKGROUND: HSP90 protects the cells from heat stress and facilitates protein maturation and stability. The full genome sequences of piroplasms contain two putative HSP90 proteins, which are yet uncharacterized. To this end, the two putative HSP90 proteins of Babesia orientalis were identified and characterized by molecular and in silico methods. METHODS: The two putative proteins in B. orientalis genome showing homology with putative HSP90 of other piroplasms were cloned and sequenced. A computational analysis was carried out to predict the antigenic determinants, structure and function of these proteins. The interactions of two HSP90 isoforms with respective inhibitors were also examined through docking analysis. RESULTS: The length of BoHSP90-A gene (amplified from gDNA) was 2706 bp with one intron from position 997 to 1299 bp. This gene amplified from cDNA corresponded to full length CDS with an open reading frame (ORF) of 2403 bp encoding a 800 amino acid (AA) polypeptide with a predicted size of 91.02 kDa. The HSP90-B gene was intronless with an ORF of 2349 bp, and predicted polypeptide comprised of 797 AA with a size of 90.59 kDa. The AA sequences of these two proteins of B. orientalis were the most identical to those of B. bovis. The BoHSP90-A and BoHSP90-B were recognized as 90 kDa in the parasite lysate by the rabbit antisera raised against the recombinant BoHSP90 proteins. The anti-B. orientalis buffalo serum reacted with the rBoHSP90s expressed in E. coli, indicating that these proteins might be secreted by the parasite before entry into host cells. The overall structure and functional analyses showed several domains involved in ATPase activity, client protein binding and HSP90 dimerization. Likewise, several HSP90 inhibitors showed binding to ATP binding pockets of BoHSP90-A and BoHSP90-B, as observed through protein structure-ligand interaction analysis. CONCLUSIONS: The two putative HSP90 proteins in B. orientalis were recognized as 90 kDa. The rBoHSP90-A and rBoHSP90-B were reacted with the B. orientalis infected buffalo serum. The computational structure and functional analyses revealed that these two proteins may have chaperonic activity. The protein structure-ligand interaction analyses indicated that these two proteins had many drug target sites.