Size-modulated synergy of cellulase clustering for enhanced cellulose hydrolysis

Immobilization of enzymes onto nanoparticles for enhanced biocatalytic activity via enzyme clustering is a growing field. In this paper, the effect of nanoparticle size on the hydrolytic activity of artificial cellulosomes was investigated. A simple method based on metal affinity coordination was employed to directly conjugate two enzymes, an endoglucanase CelA and an exoglucanase CelE, onto CdSe–ZnS core–shell quantum dots (QDs) without the use of any chemical modification or linker molecules such as streptavidin. Artificial cellulosomes were created by clustering the enzymes onto two different QDs (5 and 10 nm) to systematically study the influence of particle size and QD to enzyme ratio on the enhancement in cellulose hydrolysis. Our results indicate that enzyme proximity is the most important factor for activity enhancement while the influence of particle size is relatively modest. This detailed understanding will provide insights for the design of other artificial cellulosomes based on nanoclustering of multiple catalytic domains with significantly enhanced activities, and may be applicable for designing improved nanobiocatalysts for biofuel production, bioremediation, and drug design.     

Molecular characterization of human ninein protein: two distinct subdomains required for centrosomal targeting and regulating signals in cell cycle

The centrosomal protein ninein has been identified as a microtubules minus end capping, centriole position, and anchoring protein, but the true physiological function remains to be determined. In this report, using immunofluorescence analysis and GFP-fusions we show that coiled-coil II domain (CCII domain, 1303-2096) co-localized with gamma-tubulin and centrin at the centrosome. We further narrow down within 83 amino acids and classify a new centrosomal targeting signal. Interestingly, antibodies raised against CCII domain reveal that ninein protein declines from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Moreover, the data also suggest that fragment 1783-1866 may be attributed to declined signal of ninein. In kinase assay, we show that CCII domain could readily be phosphorylated by AIK and PKA. Taken together, our results suggest that ninein protein contains two distinct subdomains which are required for targeting and regulating asymmetry centrosomes. Importantly, the decline of ninein during mitosis implies that this centrosomal protein may play a role to regulate the process of chromosome segregation without discrimination. The model we propose here will foster a clearer picture of how two asymmetric centrosomes could direct and ensure the correct segregation of chromosomes during the mitotic stage.     

Cloning and characterization of a novel human ninein protein that interacts with the glycogen synthase kinase 3beta

Using human glycogen synthase kinase 3beta (GSK-3beta) as bait in the yeast two-hybrid system, we identified a novel human centrosome associated protein, hNinein. When the full length cDNA of hNinein was sequenced, it showed that an open reading frame encoded a protein consisting of 2047 amino acids with a predicted molecular mass of 239 kDa. The features of this protein include a potential GTP binding site, a large coiled-coil domain together with four leucine zipper domains and a GSK-3beta binding site. Fluorescence microscopy experiment showed that hNinein is localized in the pericentriolar matrix of the centrosome. In addition, hNinein also showed to react with centrosomal autoantibody sera. Our findings suggest that hNinein may be involved in the formation of centrosome matrix and interacts with the GSK-3beta, implying that it may also be regulated by GSK-3beta phosphorylation signaling.     

Involvement of the residues of GSKIP, AxinGID, and FRATtide in their binding with GSK3β to unravel a novel C-terminal scaffold-binding region

The specificity and regulation of GSK3β are thought to involve in the docking interactions at core kinase domain because of the particular amino acid residues. Recent X-ray diffraction studies illuminated the relative binding residues on AxinGID and FRATtide for GSK3β docking and appeared that GSK3β Val267Gly (V267G) and Tyr288Phe (Y288F) could distinguish the direct interaction between AxinGID and FRATtide. In order to explore the mode that involved the binding of GSKIP to GSK3β and compare it with that of AxinGID and FRATtide, we pinpointed the binding sites of GSKIP to GSK3β through the single-point mutation of four corresponding sites within GSK3β (residues 260–300) as scaffold-binding region I (designated SBR-I_260–300). Our data showed that these three binding proteins shared similar binding sites on GSK3β. We also found that the binding of GSK3β V267G mutant to GSKIP and AxinGID, but not that of Y288F mutant (effect on FRATtide), was affected. Further, based on the simulation data, the electron-density map of GSKIPtide bore closer similarity to the map AxinGID than to that of FRATtide. Interestingly, many C-terminal helix region point-mutants of GSK3β L359P, F362A, E366K, and L367P were able to eliminate the binding with FRATtide, but not AxinGID or GSKIP. In addition, CABYR exhibited a unique mode in binding to C-terminal helix region of GSK3β. Taken together, our data revealed that in addition to the core kinase domain, SBR-I_260–300, another novel C-terminus helix region, designated SBR-II_339–383, also appeared to participate in the recognition and specificity of GSK3β in binding to other specific proteins.     

17beta-estradiol inhibits endothelin-1 production and attenuates cerebral vasospasm after experimental subarachnoid hemorrhage

Though cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) has been recognized for over half a century, it remains a major complication in patients with SAH. Clinical studies have shown that elevated levels of endothelin-1 (ET-1) are present in the cerebrospinal fluid of patients with SAH, suggesting that ET-1-mediated vasoconstriction contributes to vascular constriction after SAH. Administration of estrogen promotes vasodilation in humans and in experimental animals, in part by decreasing the production of ET-1. This study evaluated the influence of 17beta-estradiol (E2) on the production of ET-1 and cerebrovasospasm in an experimental SAH 2-hemorrhage model in rat. A 30-mm Silastic tube filled with E2 in corn oil (0.3 mg/ml) was subcutaneously implanted in male rats just before SAH induction. The degree of vasospasm was determined by averaging the cross-sectional areas of basilar artery 7 days after first SAH. Plasma samples collected before death were assayed for ET-1. The protective effect of E2 in attenuating vasospasm achieved statistical significance when compared with the SAH only or SAH plus vehicle groups (P < 0.01). Concentrations of ET-1 were higher in the SAH only and SAH plus vehicle groups than in controls (P < 0.001). Serum levels of ET-1 in the SAH plus E2 and E2 only groups were significantly lower than those in the SAH only and SAH plus vehicle groups (P < 0.001). There was no significant difference between ET-1 levels in the healthy control and SAH plus E2 groups. A significant correlation was found between the cross-sectional areas of basilar artery and ET-1 levels (P < 0.001). The beneficial effect of E2 in attenuating SAH-induced vasospasm may be due in part to decreasing ET-1 production after SAH. The role of E2 in the treatment of cerebral vasospasm after SAH is promising and is worthy of further investigation.     

Understanding the Role of Dicer in Astrocyte Development

The Dicer1 allele is used to show that microRNAs (miRNAs) play important roles in astrocyte development and functions. While it is known that astrocytes that lack miRNAs are dysregulated, the in vivo phenotypes of these astrocytes are not well understood. In this study, we use Aldh1l1-EGFP transgene, a marker of astrocytes, to characterize mouse models with conditional Dicer1 ablation (via either human or mouse GFAP-Cre). This transgene revealed novel features of the defective astrocytes from the absence of miRNA. Although astrocyte miRNAs were depleted in both lines, we found histological and molecular differences in the Aldh1l1-EGFP cells between the two Cre lines. Aldh1l1-EGFP cells from hGFAP-Cre mutant lines displayed up-regulation of Aldh1l1-EGFP with increased proliferation and a genomic profile that acquired many features of wildtype primary astrocyte cultures. In the young mGFAP-Cre mutant lines we found that Aldh1l1-EGFP cells were disorganized and hyperproliferative in the developing cerebellum. Using the Aldh1l1-EGFP transgene, our work provides new insights into the roles of miRNAs in astrocyte development and the features of astrocytes in these two mouse models.     

Spatiotemporal Transmission Dynamics of Hemorrhagic Fever with Renal Syndrome in China, 2005–2012

BackgroundHemorrhagic fever with renal syndrome (HFRS) is a rodent-borne disease caused by many serotypes of hantaviruses. In China, HFRS has been recognized as a severe public health problem with 90% of the total reported cases in the world. This study describes the spatiotemporal dynamics of HFRS cases in China and identifies the regions, time, and populations at highest risk, which could help the planning and implementation of key preventative measures.MethodsData on all reported HFRS cases at the county level from January 2005 to December 2012 were collected from Chinese Center for Disease Control and Prevention. Geographic Information System-based spatiotemporal analyses including Local Indicators of Spatial Association and Kulldorff''s space-time scan statistic were performed to detect local high-risk space-time clusters of HFRS in China. In addition, cases from high-risk and low-risk counties were compared to identify significant demographic differences.ResultsA total of 100,868 cases were reported during 2005–2012 in mainland China. There were significant variations in the spatiotemporal dynamics of HFRS. HFRS cases occurred most frequently in June, November, and December. There was a significant positive spatial autocorrelation of HFRS incidence during the study periods, with Moran''s I values ranging from 0.46 to 0.56 (P<0.05). Several distinct HFRS cluster areas were identified, mainly concentrated in northeastern, central, and eastern of China. Compared with cases from low-risk areas, a higher proportion of cases were younger, non-farmer, and floating residents in high-risk counties.ConclusionsThis study identified significant space-time clusters of HFRS in China during 2005–2012 indicating that preventative strategies for HFRS should be particularly focused on the northeastern, central, and eastern of China to achieve the most cost-effective outcomes.