Identification of potential miRNA biomarkers for traumatic osteonecrosis of femoral head

Traumatic osteonecrosis of femoral head (TONFH) is a common orthopedic disease caused by physical injury in hip. However, the unclear pathogenesis mechanism of TONFH and lacking of simple noninvasive early diagnosis method cause the necessity of hip replacement for most patients with TONFH. In this study, we aimed to identify circulating microRNAs (miRNAs) by integrated bioinformatics analyses as potential biomarker of TONFH. mRNA expression profiles were downloaded from the Gene Expression Omnibus database. Then we combined two miRNA screen methods: Weighted gene co-expression network analysis and fold change based differentially expressed miRNAs analysis. As a result, we identified 14 key miRNAs as potential biomarkers for TONFH. Besides, 302 target genes of these miRNAs were obtained and the miRNA–mRNA interaction network was constructed. Furthermore, the results of Kyoto Encyclopedia of Gene and Genome pathway analysis, Gene Ontology function analysis, protein–protein interaction (PPI) network analysis and PPI network module analysis showed close correlation between these 14 key miRNAs and TONFH. Then we established receiver operating characteristic curves and identified 6-miRNA signature with highly diagnosis value including miR-93-5p (area under the curve [AUC] = 0.93), miR-1324 (AUC = 0.92), miR-4666a-3p (AUC = 0.92), miR-5011-3p (AUC = 0.92), and miR-320a (AUC = 0.89), miR-185-5p (AUC = 0.89). Finally, the results of quantitative real-time polymerase chain reaction confirmed the significantly higher expression of miR-93-5p and miR-320a in the serum of patients with ONFH. These circulating miRNAs could serve as candidate early diagnosis markers and potential treatment targets of TONFH.     

Structure of a bacterial type IV secretion core complex at subnanometre resolution

Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1‐11 and VirD4. VirB7, 9 and 10 assemble into a 1.07 MegaDalton membrane‐spanning core complex (CC), around which all other components assemble. This complex is made of two parts, the O‐layer inserted in the outer membrane and the I‐layer inserted in the inner membrane. While the structure of the O‐layer has been solved by X‐ray crystallography, there is no detailed structural information on the I‐layer. Using high‐resolution cryo‐electron microscopy and molecular modelling combined with biochemical approaches, we determined the I‐layer structure and located its various components in the electron density. Our results provide new structural insights on the CC, from which the essential features of T4S system mechanisms can be derived.     

Ubc9 acetylation modulates distinct SUMO target modification and hypoxia response

While numerous small ubiquitin‐like modifier (SUMO) conjugated substrates have been identified, very little is known about the cellular signalling mechanisms that differentially regulate substrate sumoylation. Here, we show that acetylation of SUMO E2 conjugase Ubc9 selectively downregulates the sumoylation of substrates with negatively charged amino acid‐dependent sumoylation motif (NDSM) consisting of clustered acidic residues located downstream from the core ψ‐K‐X‐E/D consensus motif, such as CBP and Elk‐1, but not substrates with core ψ‐K‐X‐E/D motif alone or SUMO‐interacting motif. Ubc9 is acetylated at residue K65 and K65 acetylation attenuates Ubc9 binding to NDSM substrates, causing a reduction in NDSM substrate sumoylation. Furthermore, Ubc9 K65 acetylation can be downregulated by hypoxia via SIRT1, and is correlated with hypoxia‐elicited modulation of sumoylation and target gene expression of CBP and Elk‐1 and cell survival. Our data suggest that Ubc9 acetylation/deacetylation serves as a dynamic switch for NDSM substrate sumoylation and we report a previously undescribed SIRT1/Ubc9 regulatory axis in the modulation of protein sumoylation and the hypoxia response.     

π–π Stacking mediated drug–drug interactions in human CYP2E1

Because of having many low molecular mass substrates, CYP2E1 is of particular interests to the pharmaceutical industry. Many evidences showed that this enzyme can adopt multiple substrates to significantly reduce the oxidation rate of the substrates. The detailed mechanism for this observation is still unclear. In the current study, we employed GPU‐accelerated molecular dynamics simulations to study the multiple‐binding mode of human CYP2E1, with an aim of offering a mechanistic explanation for the unexplained multiple‐substrate binding. Our results showed that Thr303 and Phe478 were key factors for the substrate recognition and multiple‐substrate binding. The former can form a significant hydrogen bond to recognize and position the substrate in the productive binding orientation in the active site. The latter acted as a mediator for the substrate communications via π–π stacking interactions. In the multiple‐binding mode, the aforementioned π–π stacking interactions formed by the aromatic rings of both substrates and Phe478 drove the first substrate far away from the catalytic center, orienting in an additional binding position and going against the substrate metabolism. All these findings could give atomic insights into the detailed mechanism for the multiple‐substrate binding in human CYP2E1, providing useful information for the drug metabolism mechanism and personalized use of clinical drugs. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

Combined effect of cyprid age and lipid content on larval attachment and metamorphosis of Balanus amphitrite darwin

Various algal diets of different lipid content and composition were used to rear batches of naupliar larvae of Balanus amphitrite. The cyprids in these larval batches differed in lipid content and were used to investigate the combined effect of cyprid lipid content and cyprid age on attachment and metamorphosis. For this purpose, cyprids were aged for 0,3 and 6 d at 8°C prior to utilization in laboratory attachment assays. The percentage attachment of cyprids with similar lipid content differed significantly among the three age categories. A strong and a weak positive relationship between cyprid lipid content and attachment were monitored in young and old cyprids, respectively. A significant interaction (two‐way ANOVA) between cyprid age and lipid content was observed, indicating that these factors jointly affect larval attachment and metamorphosis in B. amphitrite from the beginning of the cyprid stage.     

Effect of cyprid age on the settlement of balanus amphitrite darwin in response to natural biofilms

Larval settlement of the barnacle Balanus amphitrite Darwin (Cirripedia, Balanidae) is influenced by natural biofilms. In previous work by others, discriminatory settlement of aged cyprids has been observed in response to biofilms of different age. This study extends prior work by considering the effect of the age of cyprids on the outcome of settlement assays. Settlement was investigated with 0‐day‐old (newly metamorphosed) and 5‐day‐old cyprids. Biofilms under investigation were developed in the field for periods of 5 d and 1 month, and were subsequently included in laboratory settlement assays with a choice between a filmed and an unfilmed substratum. The bioassay was modified from the conventional horizontal dish design in order to generate a low water surface‐to‐volume ratio, which served to suppress larval entrapment in an organic layer on the water surface. Irrespective of cyprid age, a clear discrimination between a filmed and an unfilmed substrata was observed, and the preference for filmed or unfilmed substratum was dependent on the age of the cyprids. Settlement of 0‐day‐old cyprids was inhibited by a biofilmed substratum whereas induction occurred with aged cyprids. This pattern of settlement was independent of biofilm age. Bacterial abundance on unfilmed substrata in treatments and controls was significantly lower than that on biofilmed surfaces, confirming that bacterial contamination did not change the qualitative option during the assay.     

Proteomic-based identification of Apg-2 as a therapeutic target for chronic myeloid leukemia

The oncogenic BCR/ABL tyrosine kinase induces constitutive enhanced “spontaneous” DNA damage and unfaithful repair in Philadelphia chromosome positive leukemia cells. Here, we investigated the changes of protein profile in H₂O₂-induced DNA damage/repair in BaF3-MIGR1 and BaF3-BCR/ABL cells through a proteomic strategy consisting of two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry. In total, 41 spots were differentially expressed and 13 proteins were identified with further MS analysis. Two essential proteins, Proto-oncogene tyrosine–protein kinase ABL1 (c-ABL) and Heat shock 70 kDa protein 4 (Apg-2), were confirmed by Western blot and showed consistent changes with proteomic results. Moreover, functional analysis demonstrated that inhibition of Apg-2 not only decreased cell proliferation, but also induced cell apoptosis in BCR/ABL positive cells (BaF3-BCR/ABL, BaF3-BCR/ABL^T315I). We also proved that Apg-2 inhibition aggravated H₂O₂ induced damage in BCR/ABL positive cells, and enhanced the sensitivity of BaF3-BCR/ABL^T315I to STI571. Taken together, the findings in this work provide us with some clues to a better understanding of the molecular mechanisms underlying BCR/ABL in the DNA damage/repair processes and demonstrated that Apg-2 would be a valid target for anti-leukemia drug development.