Clinicopathological and prognostic values of ErbB receptor family amplification in primary osteosarcoma.

Osteosarcoma is a malignant bone tumor with extremely high invasion, metastasis and mortality. The prognosis of patients with osteosarcoma remains poor. The ErbB receptor family was found to be overexpressed in human cancers and associated with poor prognosis. However, the role of ErbB receptor family in osteosarcoma has not been fully understood. The present study aimed to investigate the clinicopathological and prognostic significances of ErbB receptors in primary osteosarcoma. Western blot (WB), reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and fluorescence in situ hybridization (FISH) were used to detect the protein and gene expression of ErbB receptors in 60 primary osteosarcoma specimens and 30 non-neoplastic bone tissues. WB and RT-qPCR analyses showed that the protein and mRNA expression levels of EGFR, ErbB3 and ErbB4 in osteosarcoma specimens were significantly higher than those in non-neoplastic bone tissues. Seventeen (28.33%), 15 (25.00%) and 15 (25.00%) osteosarcoma specimens presented with amplification of EGFR, ErbB3 and ErbB4 gene, respectively, which were significantly higher compared with non-neoplastic bone tissues. The amplification of ErbB3 and ErbB4 in osteosarcoma was associated with advanced surgical stage. The amplification of EGFR, ErbB3, ErbB4 and the co-amplification of EGFR-ErbB3, EGFR-ErbB4, ErbB3-ErbB4 was linked with poor response to chemotherapy and distant metastasis. The amplification of EGFR, ErbB3 and ErbB4, as well as their co-amplification demonstrated independent prognostic values for reduced survival time of osteosarcoma patients and may serve as potential therapeutic targets for osteosarcoma patients in the future.

Controlled release by novel lysostaphin-loaded hydroxyapatite/chitosan composites.

Lysostaphin is highly effective on eliminating methicillin resistant Staphylococcus aureus (MRSA). In order to achieve controlled release of lysostaphin, a biocompatible drug carrier is needed. Hydroxyapatite/chitosan (HA/CS) composites were chosen to carry lysostaphin and sample composites with different weight ratios of HA to CS, including 80/20, 70/30, 60/40, and 40/60, were prepared. Multiple analyses were performed to determine the structural and physicochemical properties of the composites, including scanning electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. We immersed HA/CS composites loaded with 1 wt% lysostaphin to test in vitro release activity and cultured MC3T3-E1 cells to carry out biocompatibility test. The result of the release behavior of the composites revealed that the controlled release of lysostaphin from 60/40 HA/CS composites was the highest release rate of (87.4 ± 2.8)%, which lasted for 120 hours. In biocompatibility testing, MC3T3-E1 cells were able to proliferate on the surface of these composites, and the extract liquid from the composites could increase the growth of the cells. These results demonstrate the controlled release of lysostaphin from HA/CS composites and their biocompatibility, suggesting the potential application of these composites to bone injury and infection applications.

Hydrogen/deuterium exchange mass spectrometry and site-directed disulfide cross-linking suggest an important dynamic interface between the two lysostaphin domains.

Lysostaphin is a peptidoglycan hydrolase secreted by Staphylococcus simulans. It can specifically lyse Staphylococcus aureus and is being tested as a novel antibacterial agent. The protein contains an N-terminal catalytic domain and a C-terminal cell wall targeting domain. Although the two domains from homologous enzymes were structurally determined, the structural organization of lysostaphin domains remains unknown. We used hydrogen/deuterium exchange mass spectrometry (H/DX-MS) and site-directed disulfide cross-linking to probe the interface between the lysostaphin catalytic and targeting domains. H/DX-MS-mediated comparison of peptides from full-length lysostaphin and the separated domains identified four peptides of lower solvent accessibility in the full-length protein. Cross-linking analysis using cysteine pair substitutions within those peptides showed that two pairs of cysteines can form disulfide bonds, supporting the domain association role of the targeted peptides. The cross-linked mutant exhibited a binding capacity to S. aureus that was similar to that of the wild-type protein but reduced bacteriolytic activity probably because of restraint in conformation. The diminished activity was further reduced with increasing NaCl concentrations that can cause contractions of bacterial peptidoglycan. The lytic activity, however, could be fully recovered by reducing the disulfide bonds. These results suggest that lysostaphin may require dynamic association of the two domains for coordinating substrate binding and target cleavage on the elastic peptidoglycan. Our study will help develop site-specific PEGylated lysostaphin to treat systemic S. aureus infections.

Effects of remote ischaemic preconditioning on myocardial injury after major abdominal surgery in patients at high risk for cardiovascular adverse events in China (RIPC-MAS): protocol for a randomised, sham-controlled, observer-blinded trial.

INTRODUCTION: Myocardial injury after non-cardiac surgery (MINS) caused by an ischaemic mechanism is common and is associated with adverse short-term and long-term prognoses. However, MINS is a recent concept, and few studies have prospectively used it as a primary outcome. Remote ischaemic preconditioning (RIPC) is a non-invasive procedure that induces innate cardioprotection and may reduce MINS. METHODS AND ANALYSIS: This is a multicentre, randomised, sham-controlled, observer-blinded trial. Patients with a high clinical risk of cardiovascular events who are scheduled to undergo major abdominal surgery will be enrolled. A total of 766 participants will be randomised (1:1 ratio) to receive RIPC or control treatment before anaesthesia. RIPC will comprise four cycles of cuff inflation for 5 min to 200 mm Hg and deflation for 5 min. In the controls, an identical-looking cuff will be placed around the arm but will not be actually inflated. The primary outcome will be MINS, defined as at least one postoperative cardiac troponin (cTn) concentration above the 99th percentile upper reference limit of the cTn assay as a result of a presumed ischaemic mechanism. This trial will test the concentration of high-sensitivity cardiac troponin T (hs-cTnT). The secondary outcomes will be hs-cTnT levels reaching/above the prognostically important thresholds, peak hs-cTnT and total hs-cTnT release during the initial 3 days after surgery, length of hospital stay after surgery, length of stay in the intensive care unit, myocardial infarction, major adverse cardiovascular events, cardiac-related death, all-cause death within 30 days, 6 months, 1 year and 2 years after surgery, and postoperative complications and adverse events within 30 days after surgery. ETHICS AND DISSEMINATION: This study protocol (version 5.0 on 7 April 2023) was approved by the Ethics Committee of Sixth Affiliated Hospital of Sun Yat-sen University. The findings will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT05733208.

Clinicopathological value of ErbB2 gene and protein expression in osteochondroma.

OBJECTIVE: The aim of this study was to investigate ErbB2 expression in osteochondroma and its relationship with clinicopathologic features of osteochondroma, so as to identify a new biomarker for the malignant transformation potential of osteochondroma. METHODS: Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were used to investigate the expression status of ErbB2 protein and gene in 30 osteochondroma tissues and 20 non-neoplastic bone tissues. The association of ErbB2 gene and protein expression with clinicopathological parameters of osteochondroma was analyzed by using the χ2 test and Fishers exact test. RESULTS: ErbB2 protein was found to be over-expressed in 4 of 30 (13.3%) osteochondromas and 1 of 20 (5%) non-neoplastic bone samples, which were not statistically significant (p=0.336). However, 13 of the 30 (43.3%) osteochondromas showed ErbB2 gene amplification, which was failed to be observed in any of the non-neoplastic bone tissue. ErbB2 gene amplification in osteochondroma was significantly higher compared with that in non-neoplastic bone tissue (p=0.001). In addition, the ErbB2 gene amplification was closely associated with clinical pathological parameters of osteochondroma, including high expression of cellularity (p=0.001), presence of binucleated cells (p=0.001), nuclear pleomorphism (p=0.003), calcification (p=0.002), nodularity (p=0.002), necrosis (p=0.009) and cartilage thickness (p=0.026). The association of the gene amplification with other clinicopathological parameters of osteochondroma, including permeation of trabecular bone, cystic/mucoid changes, mitosis, radiographic appearance, cap volume and subtype of osteochondroma was not observed. The over-expression of ErbB2 protein was not found to be associated with the above stated clinical pathological parameters of osteochondroma. CONCLUSION: ErbB2 gene amplification was associated with adverse clinicopathological status of osteochondroma and could serve as an index for malignant conversion of osteochondroma. Further research is required to verify the predictive values of ErbB2 for osteochondroma. LEVEL OF EVIDENCE: Level IV, Diagnostic Study.

MicroRNA-885-5p promotes osteosarcoma proliferation and migration by downregulation of cell division cycle protein 73 homolog expression.

Osteosarcoma (OS) is the most common primary malignant bone tumor. Numerous studies have strongly implicated the ectopic expression of microRNAs (miRNAs/miRs), including miR-885-5p, which is aberrantly expressed in several cancer types, in multiple cancer-related processes. However, the role of miR-885-5p in OS remains unknown. In the present study, it was found that the expression of miR-885-5p was markedly upregulated in OS cell lines and clinical tissues. Moreover, high expression of miR-885-5p was significantly associated with the development of OS. The human OS MG-63 cell line was transfected with recombinant lentivirus to regulate miR-885-5p expression. Overexpressed miR-885-5p significantly promoted the proliferation and migration of MG-63 cells in vitro, while downregulating miR-885-5p expression reversed these effects. Furthermore, bioinformatic analysis was used to predict the potential target genes of miR-885-5p, and cell division cycle protein 73 homolog (CDC73) was identified as a novel and direct target of miR-885-5p. This interaction was further confirmed using reverse transcription-quantitative polymerase chain reaction, western blotting and luciferase activity assays. These findings suggest that miR-885-5p serves a critical role in facilitating OS proliferation and migration, and can regulate CDC73 expression in OS cells and tissues. Thus, miR-885-5p could be a promising novel therapeutic biomarker for OS.

In vitro activity of recombinant lysostaphin against Staphylococcus aureus isolates from hospitals in Beijing, China.

Lysostaphin is a glycylglycine endopeptidase. It cleaves the pentaglycine cross-bridge structure unique to the staphylococcal cell wall and is considered to be a potential drug for Staphylococcus aureus. In the present study, the in vitro activity of recombinant lysostaphin was investigated in 257 S. aureus isolates collected from hospital patients in Beijing, China, by determination of MIC and minimum bactericidal concentration (MBC) and a time-kill curve test. An agar dilution method was used for MIC determination in all of the isolates and a macrobroth dilution method was employed to verify MIC values for a subset of the isolates. All of the S. aureus strains were sensitive to the recombinant lysostaphin with MICs ranging from 0.03 to 2 microg ml(-1) in the agar dilution assay. The antibacterial activity of lysostaphin was greater than that of vancomycin and other reference agents. For most of the isolates, the MICs from the agar dilution method were higher than those from the broth dilution method. The MBCs of lysostaphin in the test isolates were between 1- and 8-fold higher than their MIC values. Bactericidal activity (>99.9 % reduction) was observed after 2 h exposure of the isolates to lysostaphin at concentrations of > or =0.5 MIC. Lysostaphin showed a rapid bactericidal activity against the test strains of meticillin-susceptible S. aureus and meticillin-resistant S. aureus. Its activity at > or =0.5 MIC was sustained for at least 6 h. These results will be informative for the clinical application and evaluation of lysostaphin.

[A group of synthetic antimicrobial peptides].

Cecropin A1 (CA), first found in the diapause pupa of Hyalophora cecropia, is an alpha-helix antimicrobial peptide. It has suppressive effect on several strains of bacteria but its effect is moderate. Therefore it is imperative to find new peptides of high lethality to pathogenic bacteria. The structure of CA was modified by increasing its alpha-helix number to improve the antimicrobial activity on the basis of previous works that there is a close relationship between the alpha-helix status of peptide and its antimicrobial activity. Fifteen peptides (GK1-GK15) containing the 1st to 8th amino acids (KWKLFKKI) at N terminus of CA linking with a typical alpha-helix structure by GIG hinge were synthesized. Purity (97%) of GK-8 was confirmed by HPLC and molecular weight (2934.0 Da) was measured by mass spectrometry. Minimum inhibitory concentration (MIC, microg/mL) was used to compare their killing efficiency on various bacteria both Grams-positive and Grams-negative. The results showed the killing efficiency of the modified peptides (GK-1, GK-2, GK-8, GK-10) on various bacteria were much more effective than the original and their MICs were only one percent (0.25 microg/ mL) of CA and magainin. It indicates that typical core alpha-helix of the peptides is essential for their lethality to pathogens and some of modified peptides can serve as attractive candidates for antimicrobial drug discovery. The patent number is PCT/ CN 03/00522.

[Effect of Tripterygium wilfordii polyglycoside on serum IL-2 and TNF-alpha in patients with acute anterior U-veitis].

OBJECTIVE: To explore the effect of Tripterygium wilfordii polyglycoside (TWP) on level of serum interleukin 2 (IL-2), and tumor necrosis factor alpha (TNF-alpha) in patients of acute anterior uveitis (AAU). METHODS: Patients of AAU were randomly divided into two groups. The treated group (n = 50) was mainly treated with TWP and the control group (n = 50) treated with bimolani. The level of IL-2 and TNF-alpha before and after treatment were determined and compared between the two groups and also compared between the treated group and normal group consisted of 50 healthy subjects. RESULTS: Levels of IL-2 and TNF-alpha in the treated group before treatment were 1.31 +/- 0.27 micrograms/L and 1.20 +/- 0.65 micrograms/L, and after treatment 1.19 +/- 0.27 micrograms/L and 0.96 +/- 0.54 microgram/L, those in the control group were 1.31 +/- 0.26 micrograms/L and 1.22 +/- 0.66 micrograms/L before treatment and 1.20 +/- 0.27 micrograms/L and 0.98 +/- 0.51 microgram/L after treatment respectively. Comparisons of the two parameters before and after treatment in both groups showed significant difference (P < 0.05 in TWP group and P < 0.01 in bimolani group). The two parameters in both treated groups were all higher than those in the normal group before treatment (P < 0.05), but showed insignificant difference after treatment (P > 0.05). CONCLUSION: Abnormal changes of IL-2 and TNF-alpha exist in AAU patients, TWP could suppress both parameters markedly therefore has a reliable effect in treatment of AAU.

[Reduction of tumorigenicity of SMMC7721 hepatoma cell line by VEGF siRNA].

In this study the small interfering RNA(siRNA) targeting human VEGF effectively inhibited the expression of VEGF in human hepatoma cell line, SMMC7721, and could dramatically decrease the tumorigenicity of SMMC7721 s.c. xenograft tumor. Chemically synthesized siRNA targeting VEGF was transiently transfected into SMMC7721 cells by lipofectamine, RT-PCR and Elisa analysis suggested that the expression of VEGF mRNA and secreted protein in SMMC7721 cells with VEGF siRNA transfection were suppressed with inhibition rates of 76.16% and 96.28% respectively compared with negative control, but the growth rate of SMMC 7721 cells with VEGF siRNA transfection was the same as the control cells. In vivo test, siRNA was injected directly into implanted tumors and the tumors volume were calculated at different time interval. Result showed that VEGF siRNA greatly inhibited the growth of tumors tissues, which was consistent with decrease of VEGF mRNA and protein compared with control. In addition, the VEGF siRNA-treated group exhibited obvious signs of necrosis compared with control.

[Detection of recombinant lysostaphin using antibody sandwish enzyme-linked immunoadsorbent assay].

The double-antibody-sandwich enzyme-linked immunoadsorbent assay (ELISA) for detection of rLysostaphin in humans had been developed and established through this study. rLysostaphin of high purity ( > 95 % ) produced in Shanghai Hi-Tech United Bio-Technological Research & Development Co., Ltd (SHUBRD) was used to produce a rabbit anti-rLysostaphin polyclonal antibody. The standard curve of rLysostaphin polyclonal antibody that was constructed showed that the lowest range of detection was found at 0. 98 ng of rLysostaphin/mL, and the curve exhibited linearity preferably from 0. 98 to 500 ng of rLysostaphin/mL. When three serum samples of the same batch were assayed for 6 replicates, and more 3 samples from different batches for 6 replicates, the average intra-assay and inter-assay coefficient variances ( CV) were 6. 4% and 6. 5%, respectively. The relative recovery rate was 98.6% when quantitative standard antigens were added to the serum. The present method for detection of rLysostaphin in serum is specific, highly sensitive, highly precise, and exhibited a low CV and will be helpful in the further study of rLysostaphin pharmacokinetics and holds promise in clinical applications.

Analysis of gene expression profiles in human HL-60 cell exposed to cantharidin using cDNA microarray.

Cantharidin is a natural toxin that has antitumor properties and causes leukocytosis as well as increasing sensitivity of tumor cells resistant to other chemotherapeutic agents. There is limited information, however, on the molecular pharmacological mechanisms of cantharidin on human cancer cells. We have used cDNA microarrays to identify gene expression changes in HL-60 promyeloid leukemia cells exposed to cantharidin. Cantharidin-treated cells not only decreased expression of genes coding for proteins involved in DNA replication (e.g., DNA polymerase delta), DNA repair (e.g., FANCG, ERCC), energy metabolism (e.g., isocitrate dehydrogenase alpha, ADP/ATP translocase), but also decreased expression of genes coding for proteins that have oncogenic activity (e.g., c-myc, GTPase) or show tumor-specific expression (e.g., phosphatidylinositol 3-kinase). In contrast, these treated cells overexpressed several genes that encode intracellular and secreted growth-inhibitory proteins (e.g., BTG2, MCP-3) as well as proapoptotic genes (e.g., ATL-derived PMA-responsive peptide). Our findings suggest that alterations in specific genes functionally related to cell proliferation or apoptosis may be responsible for cantharidin-mediated cytotoxicity. We also found that exposure of HL-60 cells to cantharidin resulted in the decreased expression of multidrug resistance-associated protein genes (e.g., ABCA3, MOAT-B), suggesting that cantharidin may be used as an oncotherapy sensitizer, and the increased expression of genes in modulating cytokine production and inflammatory response (e.g., NFIL-3, N-formylpeptide receptor), which may partly explain the stimulating effects on leukocytosis. Our data provide new insight into the molecular mechanisms of cantharidin.