Next-Generation Glycan Microarray Enabled by DNA-Coded Glycan Library and Next-Generation Sequencing Technology.

Interactions of glycans with proteins, cells, and microorganisms play important roles in cell-cell adhesion and host-pathogen interaction. Glycan microarray technology, in which multiple glycan structures are immobilized on a single glass slide and interrogated with glycan-binding proteins (GBPs), has become an indispensable tool in the study of protein-glycan interactions. Despite its great success, the current format of the glycan microarray requires expensive, specialized instrumentation and labor-intensive assay and image processing procedures, which limit automation and possibilities for high-throughput analyses. Furthermore, the current microarray is not suitable for assaying interaction with intact cells due to their large size compared to the two-dimensional microarray surface. To address these limitations, we developed the next-generation glycan microarray (NGGM) based on artificial DNA coding of glycan structures. In this novel approach, a glycan library is presented as a mixture of glycans and glycoconjugates, each of which is coded with a unique oligonucleotide sequence (code). The glycan mixture is interrogated by GBPs followed by the separation of unbound coded glycans. The DNA sequences that identify individual bound glycans are quantitatively sequenced (decoded) by powerful next-generation sequencing (NGS) technology, and copied numbers of the DNA codes represent relative binding specificities of corresponding glycan structures to GBPs. We demonstrate that NGGM generates glycan-GBP binding data that are consistent with that generated in a slide-based glycan microarray. More importantly, the solution phase binding assay is directly applicable to identifying glycan binding to intact cells, which is often challenging using glass slide-based glycan microarrays.

PIK3CA mutation sensitizes breast cancer cells to synergistic therapy of PI3K inhibition and AMPK activation.

Breast cancer has been emerging as a most common threat among women, thus many efforts were made to find drugs for fighting breast cancer. So far, PI3K (Phosphatidylinositol-4,5-bisphosphate 3-kinase) inhibitors have been believed to be effective drugs until frequent resistance emerged. Recently, PI3K H1047R mutation has been reported to sensitize breast cancer cells to PI3K inhibition by aspirin. Considering aspirin activates AMPK (AMP-activated protein kinase) simultaneously, it is possible that AMPK activators and PI3K inhibitors can synergistically inhibit breast cancers. Here we clearly observed synergistic suppression of cell growth in all three breast cancer cell lines (MCF-7, MDA-MB-361 and HCC38) when co-treating cells with PI3K inhibitor GDC-0941 and AMPK activator AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide). What is more, it is rather remarkable that the synergistic effect was much more dramatic in PIK3CA (PI3K catalytic subunit alpha) mutated (E545K) cells (MCF-7 and MDA-MB-361) than in PIK3CA wild-type cells (HCC38), which implied there is a relationship between PI3K genetic status and the efficacy of combination therapy. By using PIK3CA wild-type isogenic MCF-7 cell line, which exhibited attenuated cell proliferation compared with the parental MCF-7 cell line, we found endogenous reverse mutation of PIK3CA E545K alleles to wild-type sequence in MCF-7 cells dramatically impaired the synergy of PI3Ki&Ka (combinatorial PI3K inhibition and AMPK activation). Furthermore, PI3Ki&Ka significantly attenuated tumorigenesis of parental MCF-7 cells but not PIK3CA wild-type isogenic MCF-7 cells in tumor xenograft models. Taken together, our results suggest a promising precision therapy of PI3Ki&Ka in PIK3CA mutant breast cancers.

Phosphorylation of PP1 Regulator Sds22 by PLK1 Ensures Accurate Chromosome Segregation.

During cell division, accurate chromosome segregation is tightly regulated by Polo-like kinase 1 (PLK1) and opposing activities of Aurora B kinase and protein phosphatase 1 (PP1). However, the regulatory mechanisms underlying the aforementioned hierarchical signaling cascade during mitotic chromosome segregation have remained elusive. Sds22 is a conserved regulator of PP1 activity, but how it regulates PP1 activity in space and time during mitosis remains elusive. Here we show that Sds22 is a novel and cognate substrate of PLK1 in mitosis, and the phosphorylation of Sds22 by PLK1 elicited an inhibition of PP1-mediated dephosphorylation of Aurora B at threonine 232 (Thr232) in a dose-dependent manner. Overexpression of a phosphomimetic mutant of Sds22 causes a dramatic increase in mitotic delay, whereas overexpression of a non-phosphorylatable mutant of Sds22 results in mitotic arrest. Mechanistically, the phosphorylation of Sds22 by PLK1 strengthens the binding of Sds22 to PP1 and inhibits the dephosphorylation of Thr232 of Aurora B to ensure a robust, error-free metaphase-anaphase transition. These findings delineate a conserved signaling hierarchy that orchestrates dynamic protein phosphorylation and dephosphorylation of critical mitotic regulators during chromosome segregation to guard chromosome stability.

Regulation of a dynamic interaction between two microtubule-binding proteins, EB1 and TIP150, by the mitotic p300/CBP-associated factor (PCAF) orchestrates kinetochore microtubule plasticity and chromosome stability during mitosis.

The microtubule cytoskeleton network orchestrates cellular dynamics and chromosome stability in mitosis. Although tubulin acetylation is essential for cellular plasticity, it has remained elusive how kinetochore microtubule plus-end dynamics are regulated by p300/CBP-associated factor (PCAF) acetylation in mitosis. Here, we demonstrate that the plus-end tracking protein, TIP150, regulates dynamic kinetochore-microtubule attachments by promoting the stability of spindle microtubule plus-ends. Suppression of TIP150 by siRNA results in metaphase alignment delays and perturbations in chromosome biorientation. TIP150 is a tetramer that binds an end-binding protein (EB1) dimer through the C-terminal domains, and overexpression of the C-terminal TIP150 or disruption of the TIP150-EB1 interface by a membrane-permeable peptide perturbs chromosome segregation. Acetylation of EB1-PCAF regulates the TIP150 interaction, and persistent acetylation perturbs EB1-TIP150 interaction and accurate metaphase alignment, resulting in spindle checkpoint activation. Suppression of the mitotic checkpoint serine/threonine protein kinase, BubR1, overrides mitotic arrest induced by impaired EB1-TIP150 interaction, but cells exhibit whole chromosome aneuploidy. Thus, the results identify a mechanism by which the TIP150-EB1 interaction governs kinetochore microtubule plus-end plasticity and establish that the temporal control of the TIP150-EB1 interaction by PCAF acetylation ensures chromosome stability in mitosis.

Optimization of the extract from flower of Citrus aurantium L. var. amara Engl. and its inhibition of lipid accumulation.

Flower of Citrus aurantium L. var. amara Engl. (CAVA) has been confirmed to have promising anti-obesity effects. However, the regulation of alkaloid extracts from flower of CAVA (Al) on lipid metabolism remain unknown. In this study, Al was optimized by ultrasound-assisted extraction using response surface methodology. The optimal conditions were ultrasonic time 72 min, ethanol concentration 78% and liquid/solid ratio 30 ml/g with the maximum alkaloid yield 5.66%. LC-MS assay indicated that the alkaloid compounds were enriched in Al after optimization. Nine alkaloid compounds were identified in Al by LC-MS assay and stachydrine, caffeine and cathine appeared as the major alkaloid compounds. Bioactivity assay showed that Al treatment significantly increased superoxide dismutase (SOD) activity, and reduced malonaldehyde (MDA) and reactive oxygen species (ROS) levels. Al administration also reversed oleic acid-induced hepatic steatosis in Hep G2 cells by inhibiting the expression of lipogenesis-signaling genes including fatty acid synthase (FAS), peroxisome proliferator-activated receptor subtype γ (PPARγ), uncoupling protein 2 (UCP2), and retinol binding protein (RBP4). However, OA-induced reduction of lipolysis-related gene carnitine palmitoyl transferase 1A (CPT1A) in Hep G2 cells was not improved by Al supplementation. Moreover, the increased SOD activity and decreased MDA and ROS contents were also observed in Caenorhabditis elegans by Al addition. Al intervention exhibited the ability to inhibit lipid accumulation in C. elegans by suppressing expression of lipid metabolism-related genes. These results suggested that the alkaloid extracts from the flower of CAVA showed great potential to regulate lipid metabolism. PRACTICAL APPLICATIONS: The extraction of alkaloid extracts from the flower of CAVA was optimized with a maximum yield of 5.66%. The regulatory effects and mechanisms of Al on lipid metabolism of Hep G2 cells and Caenorhabditis elegans were also investigated. More clinical studies are required to evaluate the potential of using alkaloids from the flower of CAVA as therapeutic agents against lipid metabolic disorders.

Large scale preparation of high mannose and paucimannose N-glycans from soybean proteins by oxidative release of natural glycans (ORNG).

Despite the important advances in chemical and chemoenzymatic synthesis of glycans, access to large quantities of complex natural glycans remains a major impediment to progress in Glycoscience. Here we report a large-scale preparation of N-glycans from a kilogram of commercial soy proteins using oxidative release of natural glycans (ORNG). The high mannose and paucimannose N-glycans were labeled with a fluorescent tag and purified by size exclusion and multidimensional preparative HPLC. Side products are identified and potential mechanisms for the oxidative release of natural N-glycans from glycoproteins are proposed. This study demonstrates the potential for using the ORNG approach as a complementary route to synthetic approaches for the preparation of multi-milligram quantities of biomedically relevant complex glycans.

Regulation of NDR1 activity by PLK1 ensures proper spindle orientation in mitosis.

Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids which depends on correct position of mitotic spindle relative to membrane cortex. Although recent work has identified the role of PLK1 in spindle orientation, the mechanisms underlying PLK1 signaling in spindle positioning and orientation have not been fully illustrated. Here, we identified a conserved signaling axis in which NDR1 kinase activity is regulated by PLK1 in mitosis. PLK1 phosphorylates NDR1 at three putative threonine residues (T7, T183 and T407) at mitotic entry, which elicits PLK1-dependent suppression of NDR1 activity and ensures correct spindle orientation in mitosis. Importantly, persistent expression of non-phosphorylatable NDR1 mutant perturbs spindle orientation. Mechanistically, PLK1-mediated phosphorylation protects the binding of Mob1 to NDR1 and subsequent NDR1 activation. These findings define a conserved signaling axis that integrates dynamic kinetochore-microtubule interaction and spindle orientation control to genomic stability maintenance.

Genetic introgression and species boundary of two geographically overlapping pine species revealed by molecular markers.

Gene introgression and hybrid barriers have long been a major focus of studies of geographically overlapping species. Two pine species, Pinus massoniana and P. hwangshanensis, are frequently observed growing adjacent to each other, where they overlap in a narrow hybrid zone. As a consequence, these species constitute an ideal system for studying genetic introgression and reproductive barriers between naturally hybridizing, adjacently distributed species. In this study, we sampled 270 pine trees along an elevation gradient in Anhui Province, China and analyzed these samples using EST-SSR markers. The molecular data revealed that direct gene flow between the two species was fairly low, and that the majority of gene introgression was intermediated by backcrossing. On the basis of empirical observation, the on-site distribution of pines was divided into a P. massoniana zone, a hybrid zone, and a P. hwangshanensis zone. STRUCTURE analysis revealed the existence of a distinct species boundary between the two pine species. The genetic boundary of the hybrid zone, on the other hand, was indistinct owing to intensive backcrossing with parental species. Compared with P. massoniana, P. hwangshanensis was found to backcross with the hybrids more intensively, consistent with the observation that morphological and anatomical characteristics of trees in the contact zone were biased towards P. hwangshanensis. The introgression ability of amplified alleles varied across species, with some being completely blocked from interspecific introgression. Our study has provided a living example to help explain the persistence of adjacently distributed species coexisting with their interfertile hybrids.

DDA3 associates with microtubule plus ends and orchestrates microtubule dynamics and directional cell migration.

Cell motility and adhesion involve orchestrated interaction of microtubules (MTs) with their plus-end tracking proteins (+TIPs). However, the mechanisms underlying regulations of MT dynamics and directional cell migration are still elusive. Here, we show that DDA3-EB1 interaction orchestrates MT plus-end dynamics and facilitates directional cell migration. Biochemical characterizations reveal that DDA3 interacts with EB1 via its SxIP motif within the C-terminal Pro/Ser-rich region. Time-lapse and total internal reflection fluorescence (TIRF) microscopic assays demonstrate that DDA3 exhibits EB1-dependent, MT plus-end loading and tracking. The EB1-based loading of DDA3 is responsible for MT plus-ends stabilization at the cell cortex, which in turn orchestrates directional cell migration. Interestingly, the DDA3-EB1 interaction is potentially regulated by EB1 acetylation, which may account for physiological regulation underlying EGF-elicited cell migration. Thus, the EB1-based function of DDA3 links MT dynamics to directional cell migration.