| 1. |
- Feng, Dawei, et al.
(författare)
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Stable metal-organic frameworks containing single-molecule traps for enzyme encapsulation
- 2015
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 6, s. 5979-
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Tidskriftsartikel (refereegranskat)abstract
- Enzymatic catalytic processes possess great potential in chemical manufacturing, including pharmaceuticals, fuel production and food processing. However, the engineering of enzymes is severely hampered due to their low operational stability and difficulty of reuse. Here, we develop a series of stable metal-organic frameworks with rationally designed ultra-large mesoporous cages as single-molecule traps (SMTs) for enzyme encapsulation. With a high concentration of mesoporous cages as SMTs, PCN-333(Al) encapsulates three enzymes with record-high loadings and recyclability. Immobilized enzymes that most likely undergo single-enzyme encapsulation (SEE) show smaller Km than free enzymes while maintaining comparable catalytic efficiency. Under harsh conditions, the enzyme in SEE exhibits better performance than free enzyme, showing the effectiveness of SEE in preventing enzyme aggregation or denaturation. With extraordinarily large pore size and excellent chemical stability, PCN-333 may be of interest not only for enzyme encapsulation, but also for entrapment of other nanoscaled functional moieties.
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| 2. |
- Li, Qin, et al.
(författare)
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Dynamics inside the cancer cell attractor reveal cell heterogeneity, limits of stability, and escape
- 2016
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 113:10, s. 2672-2677
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Tidskriftsartikel (refereegranskat)abstract
- The observed intercellular heterogeneity within a clonal cell population can be mapped as dynamical states clustered around an attractor point in gene expression space, owing to a balance between homeostatic forces and stochastic fluctuations. These dynamics have led to the cancer cell attractor conceptual model, with implications for both carcinogenesis and new therapeutic concepts. Immortalized and malignant EBV-carrying B-cell lines were used to explore this model and characterize the detailed structure of cell attractors. Any subpopulation selected from a population of cells repopulated the whole original basin of attraction within days to weeks. Cells at the basin edges were unstable and prone to apoptosis. Cells continuously changed states within their own attractor, thus driving the repopulation, as shown by fluorescent dye tracing. Perturbations of key regulatory genes induced a jump to a nearby attractor. Using the Fokker-Planck equation, this cell population behavior could be described as two virtual, opposing influences on the cells: one attracting toward the center and the other promoting diffusion in state space (noise). Transcriptome analysis suggests that these forces result from high-dimensional dynamics of the gene regulatory network. We propose that they can be generalized to all cancer cell populations and represent intrinsic behaviors of tumors, offering a previously unidentified characteristic for studying cancer.
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| 3. |
- Zou, Jie-Zhi
(författare)
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Epstein-Barr virus latency in transplant patients and healthy carriers
- 2005
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis, I studied the latency situation of Epstein-Barr virus (EBV) in bone marrow transplanted (BMT) patients and healthy virus carriers. Paper 1: Epstein-Barr virus genomes are found predominantly in lgA-positive B cells in the blood of healthy carriers. B lymphocytes have been identified as the main reservoir of latent Epstein-Barr virus (EBV) in healthy virus carriers. We have established a semi-quantitative PCR method (SQ-PCR) to estimate the EBV genome load in the blood B-cell subpopulation in healthy individuals. EBV DNA was detected in subfractionated IgM-, IgG- and IgA-positive B cells. Between 80% and 90% of the viral DNA was found in the lgA-positive compared with the IgA-negative fraction. Paper 2: Epstein-Barr virus (EBV) load in bone marrow transplant (BMT) recipients at risk to develop posttransplant lymphoproliferative disease: prophylactic infusion of EBV-specific cytotoxic T cells. We used same SQ-PCR as reported in paper 1 to monitor the blood levels of EBVDNA in 9 patients receiving allogeneic BMT. Four of 5 recipients of HLA-mismatched T-cell-depleted grafts showed a 4- to 5-log increase of EBV-DNA within 1 to 3 months after BMT. Administration of 2 to 4 infusions of 107 EBV-specific cytotoxic T-lymphocytes (CTLS)/M2 starting from the time of maximal virus load resulted in a 2- to 3-log decrease of virus titers in 3 patients. One patient, who received a T-cell culture lacking a major EBV-specific component, progressed to fatal EBV-positive lymphoma. Administration of EBVCTLs before the onset of the EBV-DNA peak resulted in stabilization of the virus titers within 2 to 3 logs above the normal levels in the fifth patient. A moderate increase of virus titers was also detected in 3 of 4 patients receiving unmanipulated HLA-matched grafts, whereas one patient with Wiskott-Aldrich syndrome (WAS) reached a 5-log increase of EBV-DNA load within 70 days after BMT. Our results suggest that a rapid increase of circulating EBV-DNA occurs in the absence of EBV-specific T-cell precursors or in the presence of congenital immune defects that prevent the reestablishment of virus-specific immunity. Prophylactic administration of EBV-CTLs early after BMT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease. Paper 3: Circulating Epstein-Barr virus infected "resting" B lymphocytes in bone marrow transplant recipients at risk to develop post-transplant lymphoproliferative disease. EBV establishes a life long infection of humans where the proliferative potential of latently infected B blasts is kept in cheek by strong T-cell mediated rejection responses. A key feature of this virus host relationship is the capacity of the virus to establish a restricted latent infection in resting B cells that are insensitive to rejection and provide a reservoir for reactivation and spread to susceptible hosts. In immunosuppressed individuals the EBV-infected blasts may give rise to lymphoproliferative disorders that are preceded by a dramatic increase of virus load in blood. We have used SQ-PCR assays and reverse transcriptase assisted (RT)-PCRs to investigate the EBV-DNA load and the pattern of viral gene expression in peripheral blood of immunosuppressed patients receiving T cell depleted or unmanipulated bone marrow grafts from healthy EBV carriers. Patients in both groups showed a significant increase of EBV-DNA load compared to healthy controls. Virus titers exceeding the normal levels by more than 4 logs were detected in recipients of T-cell depleted marrow and in one patient with Wiskott-Aldrich syndrome. Measurement of EBV-DNA in serum and limiting dilution analysis of EBV-DNA in PBMC demonstrated that the increased virus load is due to expansion of a latently infected cell compartment that contains less than 10 EBV genomes copies per cell and expresses EBERs, LMP-2A and occasionally LMP 1 and EBNA 1 but not other latency associated viral proteins that serve as targets for virus-specific immune responses. This is compatible with latency forms I-II in non proliferating B-cells, but not latency Ill. Administration of EBV-specific CTLs correlated with a slow decrease of EBV-DNA load followed by stabilization at levels significantly higher then in healthy controls. The results suggest that suppression of EBV-specific T cell responses allows the proliferation infected B blasts in lymphoid tissues and that these cells retain the capacity to differentiate into resting B cells that enter the circulation. Reconstitution of T cell immunity results in the establishment of a new virus host-balance characterised by a significant expansion of the latent viral reservoir. The following main conclusions could be drawn from our results: · EBV can infect IgM-, IgG- and IgA-positive B cells. Between 80% and 90% of the viral DNA was found in the IgA-positive subfraction; · HLA-mismatched T-cell-depleted grafts showed a 4- to 5-log increase of EBV-DNA within 1 to 3 months after BMT; · Administration of EBV-CTLs early after BMT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease; · In BMT patients, EBV is in latency forms I-II in non proliferating B-cells, neither latency 111 nor infected B cells in lytic cycle .
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| 4. |
- Zou, Jie-Zhi
(författare)
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Epstein-Barr virus latency in vivo and in vitro
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis, I studied 1) The latency situation of Epstein- Barr virus (EBV) in bone marrow transplanted (BMT) patients and healthy virus carriers. 2) The role of EBNA1 and cellular transcriptional factors Oct and Grg/TLE family in EBV latency switch. B lymphocytes have been identified as the main reservoir of latent Epstein-Barr virus (EBV) in healthy virus carriers. We have established a semi-quantitative PCR method (SQ-PCR) to estimate the EBV genome load in the blood B-cell subpopulation in healthy individuals. EBV DNA was detected in subfractionated IgM-, IgG- and IgA-positive B cells. Between 80%. and 90% of the viral DNA was found in the lgA-positive compared with the lgA-negative fraction. We used SQ-PCR to monitor the blood levels of EBV-DNA in 9 patients receiving allogeneic BMT. Four of 5 recipients of HLA-mismatched T-cell-depleted grafts showed a 4- to 5-log increase of EBV-DNA within 1 to 3 months after BMT. Administration of 2 to 4 infusions of 107 EBV-specific cytotoxic T-lymphocytes (CTLs)/M2 starting from the time of maximal virus load resulted in a 2- to 3-log decrease of virus titers in 3 patients. A moderate increase of virus titers was also detected in 3 of 4 patients receiving unmanipulated HLA-matched grafts, whereas one patient with Wiskott-Aldrich syndrome (WAS) reached a 5-log increase of EBVDNA load within 70 days after BMT. Our results suggest that a rapid increase of circulating EBV-DNA occurs in the absence of EBV-specific T-cell precursors or in the presence of congenital immune defects that prevent the reestablishment of virus-specific immunity. Prophylactic administration of EBV-CTI-s early after 13MT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease. We identified that Cp could also can be activated by octamer-binding factor (OCT) proteins. Physical binding to the FR by the cellular transcription factors OCT1 and OCT2 was demonstrated by using electrophoretic mobility shift assay (EMSA). Furthermore, OCT2 alone or OCTI in combination with co-regulator Bob-1could drive transcription of a heterologous thymidine kinase promoter liked to the FR both in B cells and epithelial cells. Cp controlled by the FR also activated by binding of OCT2 to FR. OCT proteins can recruit the Grg/TLE - farmly of transcription factor regulatory proteins (co-factors) and those proteins repress OCT2 promoter activity through FR. Although all Grg/TLE variants could repress the OCT2 induced activity from a FR-luciferase report vector, Grg-3 was most efficient. Grg/TLE proteins did not inhibit the EBNA1 activity on their own but when cotransfected with OCT2 also the EBNA I-induced FR enhancer effect was repressed. Addition of EBNA1 efficiently counteracted the transcriptional repression evoked by OCT-2 and Grg/TLE3. We also demonstrate that the levels of EBNA1 and OCT2 differ dramatically between latency III and I cells. Using competition and base substitutions of oligo-probes in EMSA1 we mapped in detail the binding of OCT-proteins to all octamer sequence three bp away from the EBNA1 core binding site. Different FR repeats vary in their ability to form complexes with OCTI, OCT2 and their cofactors. Direct physical binding of OCT and Grg/TLE to FR repeats was demonstrated in vitro by affinity adsorption with FR specific DNA as bait. Both OCT2 and EBNA I were shown to bind FR in vivo in EBV-positive cells representing latency I or III utilizing Chromatin immunoprecipitation assay (ChIP). Based on our results, a model is suggested that the on-off switch of the EBV C promoter in latency is controlled by competition between EBNA1 and OCT-proteins together with their co regulators.
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