Heparin-induced thrombocytopenia (HIT) is an autoimmune thrombotic disorder caused by immune complexes containing platelet factor 4 (PF4), antibodies to PF4 and heparin or cellular glycosaminoglycans (GAGs). Here we solve the crystal structures of the: (1) PF4 tetramer/fondaparinux complex, (2) PF4 tetramer/KKO-Fab complex (a murine monoclonal HIT-like antibody) and (3) PF4 monomer/RTO-Fab complex (a non-HIT anti-PF4 monoclonal antibody). Fondaparinux binds to the ‘closed' end of the PF4 tetramer and stabilizes its conformation. This interaction in turn stabilizes the epitope for KKO on the ‘open' end of the tetramer. Fondaparinux and KKO thereby collaborate to ‘stabilize' the ternary pathogenic immune complex. Binding of RTO to PF4 monomers prevents PF4 tetramerization and inhibits KKO and human HIT IgG-induced platelet activation and platelet aggregation in vitro, and thrombus progression in vivo. The atomic structures provide a basis to develop new diagnostics and non-anticoagulant therapeutics for HIT.

AIMTo determine whether circular RNAs (circRNAs) are involved in pathological processes of gastric cancer (GC).METHODSThree circRNAs with differential expression in GC and colorectal cancer were randomly selected for validation by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), using 20 pairs of gastric tissues and normal tissues. Based on the predicted circRNA-miRNA network, we then focused on hsa_circ_0000745, which was found to be down-regulated in 20 GC tissues compared with normal tissues. The hsa_circ_0000745 levels were further analyzed by qRT-PCR in 60 GC tissues and paired adjacent non-tumor tissues, as well as 60 plasma samples from GC patients and 60 plasma samples from healthy controls. The associations between the levels of hsa_circ_0000745 and the clinicopathological features of GC patients were statistically assessed. A receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of hsa_circ_0000745 in GC.RESULTSHsa_circ_0000745 was down-regulated in GC tissues vs non-tumorous tissues (P < 0.001) and in plasma samples from patients with GC vs healthy controls (P < 0.001). The expression level of hsa_circ_0000745 in GC tissues correlated with tumor differentiation, while the expression level in plasma correlated with tumor-node-metastasis stage. The area under the ROC curve (AUC) of hsa_circ_0000745 in plasma was 0.683, suggesting good diagnostic value. Plasma hsa_circ_0000745 level combined with carcinoembryogenic antigen (CEA) level increased the AUC to 0.775.CONCLUSIONHsa_circ_0000745 plays an important role in GC and its expression level in plasma in combination with CEA level is a promising diagnostic marker for this malignancy.

SUMMARY The human FOXP3 molecule is an oligomeric transcriptional factor able to mediate activities that characterize T regulatory cells, a class of lymphocytes central to the regulation of immune responses. The activity of FOXP3 is regulated at the post-translational level, in part by two histone acetyltransferases (HAT), TIP60 and p300. TIP60 and p300 work cooperatively to regulate FOXP3 activity. Initially p300 and TIP60 interactions lead to the activation of TIP60 and facilitate acetylation of K327 of TIP60, which functions as a molecular switch to allow TIP60 to change binding partners. Subsequently, p300 is released from this complex and TIP60 interacts with and acetylates FOXP3. Maximal induction of FOXP3 activities is observed when both p300 and TIP60 are able to undergo cooperative interactions. Conditional knockout of TIP60 in Treg cells significantly decreases the Treg population in the peripheral immune organs, leading to a scurfy-like fatal autoimmune disease.

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Vacuolar protein sorting protein 29 (Vps29p), which is involved in retrograde trafficking from prevacuolar endosomes to the trans-Golgi network, performs its biological functions by participating in the formation of a "retromer complex." In human cells, this complex comprises four conserved proteins: hVps35p, hVps29p, hVps26p, and sorting nexin 1 protein (SNX1). Here, we report the crystal structure of hVps29p at 2.1 Å resolution, the first three-dimensional structure of the retromer subunits. This novel structure adopts a four-layered ␣-␤-␤-␣ sandwich fold. hVps29p contains a metal-binding site that is very similar to the active sites of some proteins of the phosphodiesterase/nuclease protein family, indicating that hVps29p may carry out chemically similar functions. Structure and sequence conservation analysis suggests that hVps29p contains two proteinprotein interaction sites. One site, which potentially serves as the interface between hVps29p and hVps35p, comprises 5 conserved hydrophobic and 8 hydrophilic residues. The other site is relatively more hydrophilic and may serve as a binding interface with hVps26p, SNX1, or other target proteins.To function properly for the organelles in eukaryotic cells, it is necessary that specific sets of proteins and lipids are sorted and delivered to various intracellular compartments efficiently. The trafficking of proteins and lipids between intracellular organelles requires a series of proteins that are involved in the membrane transport pathways (1-3). Genetic screens in yeast have uncovered more than 50 Vps 1 genes. The products of these Vps genes are involved directly or indirectly in protein sorting or trafficking between the late Golgi and the vacuoles (4 -6). Disruption of Vps genes can cause disturbance and missorting of many proteins, such as CPY. As a vacuolar hydrolase, CPY is recognized by Vps10p in the late Golgi. Vps10p binds to CPY and conveys it via vesicle to the prevacuolar compartment where Vps10p releases it. After that, Vps10p returns to the Golgi for further rounds of transportation. Some yeast Vps gene products, Vps26p, Vps29p, Vps35p, Vps5p, and Vps17p, are necessary for the retrograde retrieval process of Vps10p from endosomes back to the trans-Golgi network (7-10). By forming a complex named "retromer complex," the five proteins are also involved in the proper sorting and transportation of dipetidyl aminopeptidase A, Kex2 protease (Kex2p), polymeric immunoglobulin receptor, and mannose 6-phosphate receptor (9 -12). This complex consists of two subcomplexes. One subcomplex, consisting of Vps35p, Vps29p, and Vps26p, is responsible for selecting cargoes retrieved from the prevacuolar compartment (10, 13). The other subcomplex of Vps5p and Vps17p provides structural components to complete the retromer complex and mechanical force for vesicle budding (10,14,15). The retromer complex is conserved and has homologs in mammalian cells. In human cells, the retromer complex comprises hVps26p, hVps29p, hVps35p, and SNX1 (the mammalian homolog of Vps5p) (16,...

Chiral N-heterocyclic carbene catalyzed annulations of ynals and enals with 1,3-dicarbonyls have been described. The two reactions provided direct and efficient methods for enantioselective synthesis of functionalized dihydropyranones. Comparatively, the reactions starting from ynals were atom-economical; furthermore the reactions of enals demonstrated broader substrate compatibility.

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1), as an epigenetic regulator, plays important roles in the tumorigenesis and cancer progression. KiSS1 functions as a metastasis suppressor in various cancers, and epigenetic silencing of KiSS1 increases the metastatic potential of cancer cells. We therefore investigated whether UHRF1 promotes bladder cancer cell invasion by inhibiting KiSS1. The expression levels of UHRF1 and KiSS1 were examined by quantitative real-time PCR assay in vitro and in vivo. The role of UHRF1 in regulating bladder cancer metastasis was evaluated in bladder cancer cell. We found that UHRF1 levels are upregulated in most clinical specimens of bladder cancer when compared with paired normal tissues, and UHRF1 expression levels are significantly increased in primary tumors that subsequently metastasized compared with non-metastatic tumors. Forced expression of UHRF1 promotes bladder cancer cell invasion, whereas UHRF1 knockdown decreases cell invasion. Overexpression of UHRF1 increases the methylation of CpG nucleotides and reduces the expression of KiSS1. UHRF1 and KiSS1 expression level is negatively correlated in vivo and in vitro. Knockdown of KiSS1 promotes bladder cancer cell invasion. Importantly, forced expression of KiSS1 partly abrogates UHRF1-induced cell invasion. These data demonstrated that upregulated UHRF1 increases bladder cancer cell invasion by epigenetic silencing of KiSS1.

We propose a simple but efficient multiplex coaxial flow focusing (MCFF) process for single-step fabrication of multicompartment Janus microcapsules (MJMs) in a wide range of operating parameters. The produced MJMs consist of a multicompartmental core-shell structure with material compositions tunable in individual shell and core compartments. Potential applications of such a MJM agent are demonstrated in both benchtop and in vitro experiments. For the benchtop experiment, magnetic nanoparticles are loaded into one of the shell compartments and photopolymerized under ultraviolet light for controlled alignment and rotation of the microcapsules in a magnetic field. For the in vitro experiment, four different types of cells are encapsulated in the desired compartments of sodium alginate MJMs and co-cultured for seven days. By increasing the number of coaxial needles, we are also able to produce MJMs with three or more compartments. Our studies have shown that the proposed MCFF process is able to produce MJMs with desired material compositions and narrow size distribution. This process is inexpensive and scalable for mass production of various MJMs in its potential applications in biomedical imaging, drug delivery, and regenerative medicine.