Treatment of murine colitis by Saccharomyces boulardii secreting atrial natriuretic peptide.

Inflammatory bowel disease is a lifelong disorder that involves chronic inflammation in the small and large intestines. Current therapies, including aminosalicylates, corticosteroids, and anti-inflammatory biologics, can only alleviate the symptoms and often cause adverse effects with long-term usage. Engineered probiotics provide an alternative approach to treat inflammatory bowel disease in a self-renewable and local delivery fashion. In this work, we utilized a yeast probiotic Saccharomyces boulardii for this purpose. We developed a robust method to integrate recombinant genes into the Ty elements of S. boulardii. Stable yeast cell lines that secreted various anti-inflammatory proteins, including IL-10, TNFR1-ECD, alkaline phosphatase, and atrial natriuretic peptide (ANP), were successfully created and investigated for their efficacies to the DSS-induced colitis in mice through oral administration. While IL-10, TNFR1-ECD, and alkaline phosphatase did not show therapeutic effects, the ANP-secreting S. boulardii effectively ameliorated the mouse conditions as reflected by the improvements in body weight, disease activity index, and survival rate. A post-mortem examination revealed that the ANP-treated mice exhibited significant downregulations of TNF-α and IL-1ß and an upregulation of IL-6 in colon tissues. This observation is consistent with the previous reports showing that TNF-α and IL-1ß are responsible for initiating the pathogenesis, whereas IL-6 plays a protective role in colitis. Overall, we demonstrated that S. boulardii is a safe and robust vehicle for recombinant protein delivery in the gastrointestinal tract, and ANP is a potential anti-inflammatory drug for colitis treatment. KEY MESSAGES: Recombinant genes can be robustly integrated into the transposable elements of S. boulardii. Oral administration of S. boulardii secreting IL-10 or TNF-α inhibitor did not exert therapeutic effects for DSS-induced colitis in mice. Atrial natriuretic peptide-secreting S. boulardii effectively ameliorated the murine colitis as reflected by improved body weight, disease activity index, and survival rate. The ANP-treated mice exhibited decreased mRNA levels of TNF-α and IL-1ß and an increased mRNA level of IL-6 in colon tissues.

SLC38A2 Overexpression Induces a Cancer-like Metabolic Profile and Cooperates with SLC1A5 in Pan-cancer Prognosis.

Cancer cells have dramatically increased demands for energy as well as biosynthetic precursors to fuel their restless growth. Enhanced glutaminolysis is a hallmark of cancer metabolism which fulfills these needs. Two glutamine transporters, SLC1A5 and SLC38A2, have been previously reported to promote glutaminolysis in cancer with controversial perspectives. In this study, we harnessed the proximity labeling reaction to map the protein interactome using mass spectrometry-based proteomics and discovered a potential protein-protein interaction between SLC1A5 and SLC38A2. The SLC1A5/SLC38A2 interaction was further confirmed by bimolecular fluorescence complementation assay. We further investigated the metabolic influence of SLC1A5 and SLC38A2 overexpression in human cells, respectively, and found that only SLC38A2, but not SLC1A5, resulted in a cancer-like metabolic profile, where the intracellular concentrations of essential amino acids and lactate were significantly increased as quantified by nuclear magnetic resonance spectroscopy. Finally, we analyzed the 5-year survival rates in a large pan-cancer cohort and found that the SLC1A5hi /SLC38A2lo group did not relate to a poor survival rate, whereas the SLC1A5lo /SLC38A2hi group significantly aggravated the lethality. Intriguingly, the SLC1A5hi /SLC38A2hi group resulted in an even worse prognosis, suggesting a cooperative effect between SLC1A5 and SCL38A2. Our data suggest that SLC38A2 plays a dominant role in reprogramming the cancer-like metabolism and promoting the cancer progression, whereas SLC1A5 may augment this effect when co-overexpressed with SLC38A2. We propose a model to explain the relationship between SLC1A5, SLC38A2 and SCL7A5, and discuss their impact on glutaminolysis and mTOR signaling.

Sleeping Beauty Transposon-Mediated Asparaginase Gene Delivery by a Nanoparticle Platform.

Transgenic genome integration using non-viral vehicles is a promising approach for gene therapy. Previous studies reported that asparagine is a key regulator of cancer cell amino acid homeostasis, anabolic metabolism and cell proliferation. The depletion of asparagine would inhibit the growth of many cancer cells. In this study, we develop a nanoparticle delivery system to permanently integrate the asparaginase gene into the genome of human lung adenocarcinoma cells. The asparaginase plasmid and the Sleeping Beauty plasmid were co-transfected using amine-functionalized mesoporous nanoparticles into the human lung adenocarcinoma cells. The intracellular asparaginase expression led to the cell cytotoxicity for PC9 and A549 cells. In addition, the combination of the chemotherapy and the asparaginase gene therapy additively enhanced the cell cytotoxicity of PC9 and A549 cells to 69% and 63%, respectively. Finally, we showed that the stable cell clones were successfully made by puromycin selection. The doxycycline-induced expression of asparaginase caused almost complete cell death of PC9 and A549 asparaginase-integrated stable cells. This work demonstrates that silica-based nanoparticles have great potential in gene delivery for therapeutic purposes.

The cysteine-free single mutant C32S of APEX2 is a highly expressed and active fusion tag for proximity labeling applications.

APEX2, an engineered ascorbate peroxidase for high activity, is a powerful tool for proximity labeling applications. Owing to its lack of disulfides and the calcium-independent activity, APEX2 can be applied intracellularly for targeted electron microscopy imaging or interactome mapping when fusing to a protein of interest. However, APEX2 fusion is often deleterious to the protein expression, which seriously hampers its wide utility. This problem is especially compelling when APEX2 is fused to structurally delicate proteins, such as multi-pass membrane proteins. In this study, we found that a cysteine-free single mutant C32S of APEX2 dramatically improved the expression of fusion proteins in mammalian cells without compromising the enzyme activity. We fused APEX2 and APEX2C32S to four multi-transmembrane solute carriers (SLCs), SLC1A5, SLC6A5, SLC6A14, and SLC7A1, and compared their expressions in stable HEK293T cell lines. Except the SLC6A5 fusions expressing at decent levels for both APEX2 (70%) and APEX2C32S (73%), other three SLC proteins showed significantly better expression when fusing to APEX2C32S (69 ± 13%) than APEX2 (29 ± 15%). Immunofluorescence and western blot experiments showed correct plasma membrane localization and strong proximity labeling efficiency in all four SLC-APEX2C32S cells. Enzyme kinetic experiments revealed that APEX2 and APEX2C32S have comparable activities in terms of oxidizing guaiacol. Overall, we believe APEX2C32S is a superior fusion tag to APEX2 for proximity labeling applications, especially when mismatched disulfide bonding or poor expression is a concern.

Dual Delivery of HNF4α and Cisplatin by Mesoporous Silica Nanoparticles Inhibits Cancer Pluripotency and Tumorigenicity in Hepatoma-Derived CD133-Expressing Stem Cells.

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly malignancies characterized by high rate of recurrence. Tumor recurrence is often attributed to the presence of a subpopulation of cells with stem cell properties, referred to as cancer stem cells (CSCs). Traditionally, cancer therapies target the entire bulk of tumor cells; however, they are poorly effective against CSCs, characterized by higher drug resistance. Therefore, approaches targeting CSCs may be required in addition to conventional chemotherapy to prevent tumor recurrence. In this study, we investigated an approach to target HCC by combining the conventional chemotherapeutic drug, cisplatin, to target the bulk of tumor cells, and differentiation therapy by delivering the gene encoding HNF4α, an important regulator of hepatocyte differentiation, to target CSCs. We used the Huh7 cell line as an in vitro model of HCC, which is characterized by a high proportion of CD133-expressing CSCs. By using flow cytometry, we separated CD133+ and CD133- Huh7 cell subpopulations and have shown that the former has highly pronounced in vivo tumorigenic capacity in contrast to the latter, which could not generate tumors in vivo. For the dual delivery of HNF4α-encoding plasmid and cisplatin, we used polyethyleneimine-modified mesoporous silica nanoparticles (PMSNs) as the nanocarriers. Here, we show that the treatment of CD133-expressing Huh7 cells with HNF4α-loaded PMSNs can suppress their proliferation rate, decrease the proportion of CSCs, downregulate stemness-associated genes, and increase the expression of mature hepatocyte-associated genes. At the same time, the treatment of Huh7 with PMSNs loaded with both HNF4α-encoding plasmid and cisplatin could block them in the S-phase of the cell cycle and cause apoptosis. In addition, dually loaded PMSNs were the most efficient formulation in suppressing tumor growth in vivo. To summarize, in this study, we tested the nanoparticle-based delivery system as both chemotherapy and gene-based therapy agents, which has great potential for development of effective treatment of HCC.

Bioinspired Pseudozwitterionic Hydrogels with Bioactive Enzyme Immobilization via pH-Responsive Regulation.

Hydrogels are hydrated networks of flexible polymers with versatile biomedical applications, and their resistance to nonspecific protein adsorption is critical. On the other hand, functionalization with other biomacromolecules would greatly enhance their biotechnological potential. The aim of this research is to prepare low fouling hydrogel polymers for selective protein immobilization. Initially, hydrogels were prepared by controlling the composition ratios of 2-carboxyethyl acrylate (CA) and 2-dimethylaminoethyl methacrylate (DMAEMA) monomers in an N, N-methylene-bis-acrylamide (NMBA) cross-linked free radical polymerization reaction. This series of hydrogels (C1D9 to C9D1) were then analyzed by X-ray photoelectron spectroscopy (XPS) and dynamic laser scattering to confirm the actual polymer ratios and surface charge. When the composition ratio was set at CA:6 vs DMEAMA:4 (C6D4), the hydrogel showed nearly neutral surface charge and an equivalent reaction ratio of CA vs DMAEMA in the hydrogel. Subsequent analysis showed excellent antifouling properties, low blood cell adhesion, hemocompatibility, and platelet deactivation. Moreover, this hydrogel exhibited pH responsiveness to protein adsorption and was then used to facilitate the immobilization of lipase as an indication of active protein functionalization while still maintaining a low fouling status. In summary, a mixed-charge nonfouling pseudozwitterionic hydrogel could be prepared, and its pH-responsive adsorption holds potential for designing a biocompatible tissue engineering matrix or membrane enzyme reactors.

Generation of Functional Dopaminergic Neurons from Reprogramming Fibroblasts by Nonviral-based Mesoporous Silica Nanoparticles.

Direct-lineage conversion of the somatic cell by reprogramming, in which mature cells were fully converted into a variety of other cell types bypassing an intermediate pluripotent state, is a promising regenerative medicine approach. Due to the risk of tumorigenesis by viral methods, a non-viral carrier for the delivery of reprogramming factors is very desirable. This study utilized the mesoporous silica nanoparticles (MSNs) as a non-viral delivery system for transduction of the three key factors to achieve conversion of mouse fibroblasts (MFs) into functional dopaminergic neuron-like cells (denoted as fDA-neurons). At the same time, a neurogenesis inducer, ISX-9, was co-delivered with the MSNs to promote the direct conversion of neuron-like cells. Good transfection efficiency of plasmid@MSN allowed repeated dosing to maintain high exogenous gene expression analyzed by qPCR and the changes in neural function markers were monitored. To further validate the dopaminergic function and the electrophysiological properties of fDA-neurons, the results of ELISA assay showed the high levels of secreted-dopamine in the conditional medium and rich Na+/K+-channels were observed in the fDA-neurons on Day 22. The results demonstrated that MSN nanocarrier is effective in delivering the reprogramming factors for the conversion of functional dopaminergic neurons from adult somatic cells.

Dual delivery of siRNA and plasmid DNA using mesoporous silica nanoparticles to differentiate induced pluripotent stem cells into dopaminergic neurons.

The cells of the central nervous system (CNS) show irreversible features after injury, and they could be re-established by stem cells. Induced pluripotent stem cells (iPSCs) may be generated from adult mammalian cells and gain pluripotency to differentiate into various lineages and functional cells. Non-viral carriers for delivering differentiation factors to transform iPSCs into neuronal cells are very desirable. In this study, mesoporous silica nanoparticles (MSNs) are used to co-deliver Nurr1 plasmid DNA (pNurr1) and Rex1 siRNA (siRex1) into iPSCs to achieve dopaminergic neuron differentiation. Sixty hours after treatment with siRex1 and pNurr1, the co-delivery of pNurr1 and siRex1 enhanced the Nurr1 gene expression three-fold, compared to the delivery of the plasmid pNurr1 only. The dopaminergic neuron-related protein level was identified using immunofluorescence staining and flow cytometry analysis, and there were 89.9% ± 0.5% tyrosine hydroxylase-expressing cells and 88.5% ± 2.0% dopamine transporter-expressing cells differentiated from iPSCs after transfection by MSNs. An ELISA was also used to determine the maturation of functional neurons, and there was 12.19 ng mL-1 dopamine released from cells after transfection by MSNs. These results demonstrate that MSNs are good non-viral nanocarriers for dual delivery of pNurr1 and siRex1 to significantly enhance the generation of dopaminergic neurons from iPSCs.

Formation of hollow silica nanospheres by reverse microemulsion.

Uniform hollow silica nanospheres (HSNs) synthesized with reverse microemulsion have great application potential as nanoreactors because enzymes or nanocatalysts can be easily encapsulated de novo in synthesis. Water-in-oil (w/o) reverse microemulsions comprising the polymeric surfactant polyoxyethylene (5) isooctylphenyl ether (Igepal CA-520), ammonia and water in a continuous oil phase (alkanes) coalesce into size-tunable silica nanoparticles via diffusion aggregation after the introduction of silica precursors. Here, we elucidate in detail the growth mechanism for silica nanoparticles via nucleation of ammonium-catalyzed silica oligomers from tetraethylorthosilicate (TEOS) and nanoporous aminopropyltrimethoxy silane (APTS) in the reverse microemulsion system. The formation pathway was studied in situ with small-angle X-ray scattering (SAXS). We find a four-stage process showing a sigmoidal growth behavior in time with a crossover from the induction period, early nucleation stage, coalescence growth and a final slowing down of growth. Various characterizations (TEM, N2 isotherm, dynamic light scattering, zeta potential, NMR, elemental analysis) reveal the diameters, scattering length density (SLD), mesoporosity, surface potentials and chemical compositions of the HSNs. Oil phases of alkanes with different alkyl chains are systematically employed to tune the sizes of HSNs by varying oil molar volumes, co-solvent amounts or surfactant mixture ratios. Silica condensation is incomplete in the core region, with the silica source of TEOS and APTS leading to the hollow silica nanosphere after etching with warm water.

Enzyme encapsulated hollow silica nanospheres for intracellular biocatalysis.

Hollow silica nanospheres (HSN) with low densities, large interior spaces and permeable silica shells are suitable for loading enzymes in the cavity to carry out intracellular biocatalysis. The porous shell can protect the encapsulated enzymes against proteolysis and attenuate immunological response. We developed a microemulsion-templating method for confining horseradish peroxidase (HRP) in the cavity of HSN. This simple one-pot enzyme encapsulation method allows entrapping of the enzyme, which retains high catalytic activity. Compared with HRP supported on solid silica spheres, HRP@HSN with thin porous silica shells displayed better enzyme activity. The small HRP@HSN (∼50 nm in diameter), giving satisfactory catalytic activity, can act as an intracellular catalyst for the oxidation of the prodrug indole-3-acetic acid to produce toxic free radicals for killing cancer cells. We envision this kind of hollow nanosystem could encapsulate multiple enzymes or other synergistic drugs and function as therapeutic nanoreactors.