Development of a Composite Layered Double Hydroxide-liposomal System for Gene Delivery

In recent years, gene therapy has attracted much attention in treating a wide range of severe diseases, including cancers. Unfortunately, naked genetic materials are rapidly degraded by the ubiquitous nucleases in the body and their negative charge property hinders crossing cell membrane to the cytoplasm. Thus, an efficient delivery system is urgently sought for desired gene therapeutic efficacy. Up to date, a wide variety of vectors have been examined for gene delivery, including viral and non-viral ones. Non-viral vectors, including polymers, cell penetrating peptides (CPPs), liposomes, and inorganic nanoparticles (NPs), are clinically promising due to their safety. It is well known that every delivery system has its own unique merits and inherent shortfalls, hence hybrid delivery systems are expected to merge their advantages while avoid their drawbacks. Inorganic layered double hydroxide (LDH) acts as an excellent drug/gene delivery vehicle due to its protection to the loaded drugs/genes and efficient cellular uptake by various mammalian cells. However, aggregation of LDH NPs in serum or culture medium limits the effective application in vivo. On the other hand, liposome is a promising lipid-based system which can protect the hydrophilic genes/drugs in the aqueous core or hydrophobic drugs between the lipid bilayers, and it is colloidally stable in the circulatory blood flow. Unfortunately, its disadvantages are also obvious, such as low transfection efficiency and inefficient endosome escape. This PhD project aims to develop a hybridised delivery system by forming small LDH (sLDH)-liposome composites. Considering encapsulating LDH NPs in the vesicles of lipid bilayers, small Mg-Al-LDH NPs were first prepared by a non-aqueous method with the Z-average diameter size of ~ 40 nm. This method requires co-precipitation of magnesium and aluminium nitrate solution with sodium hydroxide in methanol, followed by LDH slurry collection and re-suspension in methanol. The methanol suspension is then heated in an autoclave, followed by separation via centrifugation and thorough washing with deionised water. The NPs are finally dispersed in deionised water into homogeneous aqueous suspension after 4-6 day standing at room temperature. The prepared sLDH NPs have the Z-average size of 35-50 nm, the number-average size of 14-30 nm and the polydispersity index (PdI) of 0.19-0.25, with the colloidal suspension stable for at least 1 month when stored at fridge (2-8 °C) or ambient (22-25 °C) temperature. Engineered sLDH with the Z-average size of ~40 nm and normal LDH with the Z-average size of ~100 nm (large LDH, denoted as L-LDH in this thesis) were then compared in transfection efficiency to human colon cancer HCT-116 cells. Using fluorescein Isothiocyanate (FITC) to label LDH NPs and as a model anionic drug (where FITC is intercalated in the interlayers of LDH NPs), we found that 40 nm sLDH and 100 nm L-LDH have the similar cellular uptake rate based on the equivalent particle number concentration, which means in the size range of 35 to 100 nm, LDH NP size does not significantly affect the cellular uptake rate. Note worthily, a critical particle number concentration was found for both sLDH and L-LDH, below which the cellular uptake is in linear proportion to the concentration, while above which, the cellular uptake is not further improved. When delivering genetic materials to cancer cells (where DNA is adsorbed on the surfaces of LDH NPs), sLDH is far superior to L-LDH at low LDH:DNA mass ratio (e.g. 5:1). This is mainly attributed to full loading of DNA by sLDH and higher sLDH particle number concentration. At the high mass ratio (40:1), where both sLDH and L-LDH can completely bind and protect DNA, sLDHs are able to transport 2-fold DNA to the cells just because of the higher sLDH particle concentration. Finally, sLDH-liposome composites were prepared by the hydration of freeze-dried matrix (HFDM) method. This composite system exhibits good colloidal stability both in water and in cell culture medium, with the Z-average particle size ~ 200 nm, which is appropriate for cellular uptake. It is also proved for the composite system to completely bind/protect DNA at LDH:DNA mass ratio = 20:1, no matter how DNA is loaded in the composite system. About 3-time higher efficiency is observed in delivery of DNA to HCT-116 cells by the sLDH-liposome composite system compared to sLDH only. In general, the sLDH-liposome composite system shows higher cellular delivery efficiency than either sLDH or liposome only, good colloidal stability and low cytotoxicity, so it could be a promising gene delivery system

Particle size- and number-dependent delivery to cells by layered double hydroxide nanoparticles

It is well known that delivery efficiency to cells is highly dependent on particle size and the administered dose. However, there is a marked discrepancy in many reports, mainly due to the inconsistency in assessment of various parameters. In this particular research, we designed experiments using layered double hydroxide nanoparticles (LDH NPs) to specifically elucidate the effect of particle size, dose and dye loading manner on cellular uptake. Using the number of LDH NPs taken up by HCT-116 cells as the indicator of delivery efficiency, we found that (1) the size of sheet-like LDH in the range of 40–100 nm did not significantly affect their cellular uptake; (2) cellular uptake of 40 and 100 nm LDH NPs was increased proportionally to the number concentration below a critical value, but remained relatively constant beyond the critical value; and (3) the effect of the dye loading manner is mainly dependent on the loading capacity or yield. In particular, the loading capacity is determined by the NP specific surface area. This research may be extended to a larger size range to examine the size effect, but suggests that it is necessary to set up a protocol to evaluate the effects of NP’s physicochemical properties on the cellular delivery efficiency

Sequential Wnt Agonist then Antagonist Treatment Accelerates Tissue Repair and Minimizes Fibrosis

Tissue fibrosis compromises organ function and occurs as a potential long-term outcome in response to acute tissue injuries. Currently, lack of mechanistic understanding prevents effective prevention and treatment of the progression from acute injury to fibrosis. Here, we combined quantitative experimental studies with a mouse kidney injury model and a computational approach to determine how the physiological consequences are determined by the severity of ischemia injury, and to identify how to manipulate Wnt signaling to accelerate repair of ischemic tissue damage while minimizing fibrosis. The study reveals that Wnt-mediated memory of prior injury contributes to fibrosis progression, and ischemic preconditioning reduces the risk of death but increases the risk of fibrosis. Furthermore, we validated the prediction that sequential combination therapy of initial treatment with a Wnt agonist followed by treatment with a Wnt antagonist can reduce both the risk of death and fibrosis in response to acute injuries

Low Expression of DYRK2 (Dual Specificity Tyrosine Phosphorylation Regulated Kinase 2) Correlates with Poor Prognosis in Colorectal Cancer.

Dual-specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) is a member of dual-specificity kinase family, which could phosphorylate both Ser/Thr and Tyr substrates. The role of DYRK2 in human cancer remains controversial. For example, overexpression of DYRK2 predicts a better survival in human non-small cell lung cancer. In contrast, amplification of DYRK2 gene occurs in esophageal/lung adenocarcinoma, implying the role of DYRK2 as a potential oncogene. However, its clinical role in colorectal cancer (CRC) has not been explored. In this study, we analyzed the expression of DYRK2 from Oncomine database and found that DYRK2 level is lower in primary or metastatic CRC compared to adjacent normal colon tissue or non-metastatic CRC, respectively, in 6 colorectal carcinoma data sets. The correlation between DYRK2 expression and clinical outcome in 181 CRC patients was also investigated by real-time PCR and IHC. DYRK2 expression was significantly down-regulated in colorectal cancer tissues compared with adjacent non-tumorous tissues. Functional studies confirmed that DYRK2 inhibited cell invasion and migration in both HCT116 and SW480 cells and functioned as a tumor suppressor in CRC cells. Furthermore, the lower DYRK2 levels were correlated with tumor sites (P = 0.023), advanced clinical stages (P = 0.006) and shorter survival in the advanced clinical stages. Univariate and multivariate analyses indicated that DYRK2 expression was an independent prognostic factor (P < 0.001). Taking all, we concluded that DYRK2 a novel prognostic biomarker of human colorectal cancer

Overexpression of an isoform of AML1 in acute leukemia and its potential role in leukemogenesis

AML1/RUNX1 is a critical transcription factor in hematopoietic cell differentiation and proliferation. From the _AML1_ gene, at least three isoforms, _AML1a_, _AML1b_ and _AML1c_, are produced through alternative splicing. AML1a interferes with the function of AML1b/1c, which are often called AML1. In the current study, we found a higher expression level of _AML1a_ in ALL patients in comparison to the controls. Additionally, AML1a represses transcription from promotor of macrophage-colony simulating factor receptor (M-CSFR) mediated by AML1b, indicating that AML1a antagonized the effect of AML1b. In order to investigate the role of _AML1a_ in hematopoiesis and leukemogenesis _in vivo_, bone marrow mononuclear cells (BMMNCs) from mice were transduced with AML1a and transplanted into lethally irradiated mice, which develop lymphoblastic leukemia after transplantation. Taken together, these results indicate that overexpression of AML1a may be an important contributing factor to leukemogenesis

Tuberous Sclerosis complex protein 2-independent activation of mTORC1 by human cytomegalovirus pUL38

The mammalian target of rapamycin complex 1 (mTORC1) controls cell growth and anabolic metabolism and is a critical host factor activated by human cytomegalovirus (HCMV) for successful infection. The multifunctional HCMV protein pUL38 previously has been reported to activate mTORC1 by binding to and antagonizing tuberous sclerosis complex protein 2 (TSC2) (J. N. Moorman et al., Cell Host Microbe 3:253–262, 2008, http://dx.doi.org/10.1016/j.chom.2008.03.002). pUL38 also plays a role in blocking endoplasmic reticulum stress-induced cell death during HCMV infection. In this study, we showed that a mutant pUL38 lacking the N-terminal 24 amino acids (pHA-UL38(25–331)) was fully functional in suppressing cell death during infection. Interestingly, pHA-UL38(25–331) lost the ability to interact with TSC2 but retained the ability to activate mTORC1, although to a lesser extent than full-length pHA-UL38. Recombinant virus expressing pHA-UL38(25–331) replicated with ∼10-fold less efficiency than the wild-type virus at a low multiplicity of infection (MOI), but it grew similarly well at a high MOI, suggesting an MOI-dependent importance of pUL38-TSC2 interaction in supporting virus propagation. Site-directed mutational analysis identified a TQ motif at amino acid residues 23 and 24 as critical for pUL38 interaction with TSC2. Importantly, when expressed in isolation, the TQ/AA substitution mutant pHA-UL38 TQ/AA was capable of activating mTORC1 just like pHA-UL38(25–331). We also created TSC2-null U373-MG cell lines by CRISPR genome editing and showed that pUL38 was capable of further increasing mTORC1 activity in TSC2-null cells. Therefore, this study identified the residues important for pUL38-TSC2 interaction and demonstrated that pUL38 can activate mTORC1 in both TSC2-dependent and -independent manners. IMPORTANCE HCMV, like other viruses, depends exclusively on its host cell to propagate. Therefore, it has developed methods to protect against host stress responses and to usurp cellular processes to complete its life cycle. mTORC1 is believed to be important for virus replication, and HCMV maintains high mTORC1 activity despite the stressful cellular environment associated with infection. mTORC1 inhibitors suppressed HCMV replication in vitro and reduced the incidence of HCMV reactivation in transplant recipients. We demonstrated that mTORC1 was activated by HCMV protein pUL38 in both TSC2-dependent and TSC2-independent manners. The pUL38-independent mode of mTORC1 activation also has been reported. These novel findings suggest the evolution of sophisticated approaches whereby HCMV activates mTORC1, indicating its importance in the biology and pathogenesis of HCMV

Improving Model Drift for Robust Object Tracking

Discriminative correlation filters show excellent performance in object tracking. However, in complex scenes, the apparent characteristics of the tracked target are variable, which makes it easy to pollute the model and cause the model drift. In this paper, considering that the secondary peak has a greater impact on the model update, we propose a method for detecting the primary and secondary peaks of the response map. Secondly, a novel confidence function which uses the adaptive update discriminant mechanism is proposed, which yield good robustness. Thirdly, we propose a robust tracker with correlation filters, which uses hand-crafted features and can improve model drift in complex scenes. Finally, in order to cope with the current trackers' multi-feature response merge, we propose a simple exponential adaptive merge approach. Extensive experiments are performed on OTB2013, OTB100 and TC128 datasets. Our approach performs superiorly against several state-of-the-art trackers while runs at speed in real time.Comment: 7 pages, 6 figures, 4 table

Soil fungal community development in a high Arctic glacier foreland follows a directional replacement model, with a mid-successional diversity maximum.

International audienceDirectional replacement and directional non-replacement models are two alternative paradigms for community development in primary successional environments. The first model emphasizes turnover in species between early and late successional niches. The second emphasizes accumulation of additional diversity over time. To test whether the development of soil fungal communities in the foreland of an Arctic glacier conforms to either of these models, we collected samples from the Midtre Lovénbreen Glacier, Svalbard, along a soil successional series spanning >80 years. Soil DNA was extracted, and fungal ITS1 region was amplified and sequenced on an Illumina Miseq. There was a progressive change in community composition in the soil fungal community, with greatest fungal OTU richness in the Mid Stage (50-80 years). A nestedness analysis showed that the Early Stage (20-50 years) and the Late Stage (>80 years) fungal communities were nested within the Mid Stage communities. These results imply that fungal community development in this glacier succession follows a directional replacement model. Soil development processes may initially be important in facilitating arrival of additional fungal species, to give a mid-successional diversity maximum that contains both early- and late-successional fungi. Competition may then decrease the overall diversity due to the loss of early successional species