A programmable encapsulation system improves delivery of therapeutic bacteria in mice.

Living bacteria therapies have been proposed as an alternative approach to treating a broad array of cancers. In this study, we developed a genetically encoded microbial encapsulation system with tunable and dynamic expression of surface capsular polysaccharides that enhances systemic delivery. Based on a small RNA screen of capsular biosynthesis pathways, we constructed inducible synthetic gene circuits that regulate bacterial encapsulation in Escherichia coli Nissle 1917. These bacteria are capable of temporarily evading immune attack, whereas subsequent loss of encapsulation results in effective clearance in vivo. This dynamic delivery strategy enabled a ten-fold increase in maximum tolerated dose of bacteria and improved anti-tumor efficacy in murine models of cancer. Furthermore, in situ encapsulation increased the fraction of microbial translocation among mouse tumors, leading to efficacy in distal tumors. The programmable encapsulation system promises to enhance the therapeutic utility of living engineered bacteria for cancer.

Detection of Fe3+ and Hg2+ ions through photoluminescence quenching of carbon dots derived from urea and bitter tea oil residue.

In this study, we prepared nitrogen-doped carbon dots (xNCDs) using hydrothermally-treated bitter tea oil residue with urea for the detection of metal ions by monitoring the photoluminescence quenching. The quantum yields of the xNCDs increased from approximately 3.85% (CDs) to 5.5% (3NCDs) and 7.2% (1NCDs), revealing that nitrogen doping effectively increases the fluorescence emission. The increased emission of the xNCDs can be attributed to radiative recombination resulting from the π-π* transition of the C=C or the n-π* transition between the C=O or N=O of sp3 units. Moreover, the CDs have abundant surface-attached phenolic and hydroxyl groups that coordinate with Fe3+ ions and quench the fluorescence. Conversely, Hg2+ ions preferentially adsorb on nitrogen-containing groups, such as amide-carbonyl groups (O=C-NH2) and pyridinic and pyrrolic functionalities, on the surface of the NCDs owing to their strong affinity, quenching the substantial photoluminescence emissions. Our results suggest that bitter tea oil residue-derived carbon dots can be used to selectively detect metal ions, such as Fe3+ and Hg2+, by doping with nitrogen using urea as a nitrogen precursor.

Non-Conventional Fluorescence and Cytotoxicity of Two Aliphatic Hyperbranched Polymer Dots Having Poly(amic acid) Structures: Implications for Labeling Nanodrug Carriers.

In this study, we used one-pot A2 + B3 polymerizations to synthesize two aliphatic + alicyclic polymer dots (PDs) having non-conjugated hyperbranched structures, employing two types of dianhydrides as the A2 components, possessing bridged bicyclic alkene (PD-BT) and non-alkene (PD-ET) units, and Jeffamine T403 polyetheramine (T403) as the B3 components. We prepared PD-ET from commercially available ethylenediaminetetraacetic dianhydride (EDTAD, A2) and T403 (B3) and PD-BT from bicyclo[2.2.2]oct-7-ene-2,3,5,6-tetracarboxylic dianhydride (BCDA, A2) and T403 (B3). These two types of PDs possessed non-conjugated hyperbranched poly(amic acid) structures with terminal amino functional groups. PD-BT and PD-ET exhibited non-conventional fluorescence with emissions at 435 and 438 nm, respectively, and quantum yields of 12.8 and 14.0%, respectively. The fluorescence intensity of PD-ET was influenced by the pH, but PD-BT was less affected because of its rigid aliphatic bridged bicyclic structure. In aqueous solutions, the sizes of the PD-BT and PD-ET nanoparticles were 3-5 nm, and their net charges can be adjusted by varying the pH. These PDs were non-cytotoxic toward human MCF-7 breast cancer cells and human keratinocyte HaCaT cells at concentrations of 50 µg mL-1 for PD-BT and 500 µg mL-1 for PD-ET. Confocal microscopic bioimaging revealed that the PDs were located within the cells after treatment for 6 h. These PDs were easy to prepare, highly water-soluble, and possessed a large number of peripheral functional groups for further modification. Combined with their non-conventional fluorescence, they appear to have potential uses in bioimaging and as drug-labeling carriers.

Cytotoxicity and cell imaging of six types of carbon nanodots prepared through carbonization and hydrothermal processing of natural plant materials.

In this study we prepared six types of carbon nanodots (CNDs) from natural plant materials - through carbonization of two species of bamboo (Bamboo-I, Bamboo-II) and one type of wood (Wood), and through hydrothermal processing of the stem and root of the herb Mahonia oiwakensis Hayata (MO) and of the agricultural waste of two species of pineapple root (PA, PB). The resulting CNDs were spherical with dimensions on the nanoscale (3-7 nm); furthermore, CND-Bamboo I, CND-Wood, CND-Bamboo II, CND-MO, CND-PA, and CND-PB displayed fluorescence quantum yields of 9.63, 12.34, 0.90, 10.86, 0.35, and 0.71%, respectively. X-ray diffraction revealed that the carbon nanostructures possessed somewhat ordered and disordered lattices, as evidenced by broad signals at values of 2θ between 20 and 30°. CND-Bamboo I, CND-Wood, and CND-Bamboo II were obtained in yields of 2-3%; CND-MO, CND-PA, and CND-PB were obtained in yields of 17.64, 9.36, and 22.47%, respectively. Cytotoxicity assays for mouse macrophage RAW264.7 cells treated with the six types of CNDs and a commercial sample of Ag nanoparticles (NPs) revealed that each of our CNDs provided a cell viability of 90% at 2000 µg mL-1, whereas it was only 20% after treatment with the Ag NPs at 62.5 µg mL-1. The six types of CNDs also displayed low cytotoxicity toward human keratinocyte HacaT cells, human MCF-7 breast cancer cells, and HT-29 colon adenocarcinoma cells when treated at 500 µg mL-1. Moreover, confocal microscopic cell imaging revealed that the fluorescent CND-Bamboo I particles were located on the MCF-7 cell membrane and inside the cells after treatment for 6 and 24 h, respectively. We have thoroughly investigated the photoluminescence properties and carbon nanostructures of these highly dispersed CNDs. Because of the facile green synthesis of these six types of CNDs and their sourcing from abundant natural plants, herbs, and agriculture waste, these materials provide a cost-effective method, with low cytotoxicity and stable fluorescence, for biolabeling and for developing cell nanocarriers.

Wirelessly controlled, bioresorbable drug delivery device with active valves that exploit electrochemically triggered crevice corrosion.

Implantable drug release platforms that offer wirelessly programmable control over pharmacokinetics have potential in advanced treatment protocols for hormone imbalances, malignant cancers, diabetic conditions, and others. We present a system with this type of functionality in which the constituent materials undergo complete bioresorption to eliminate device load from the patient after completing the final stage of the release process. Here, bioresorbable polyanhydride reservoirs store drugs in defined reservoirs without leakage until wirelessly triggered valve structures open to allow release. These valves operate through an electrochemical mechanism of geometrically accelerated corrosion induced by passage of electrical current from a wireless, bioresorbable power-harvesting unit. Evaluations in cell cultures demonstrate the efficacy of this technology for the treatment of cancerous tissues by release of the drug doxorubicin. Complete in vivo studies of platforms with multiple, independently controlled release events in live-animal models illustrate capabilities for control of blood glucose levels by timed delivery of insulin.

Uncariitannin, a polyphenolic polymer from Uncaria gambier, attenuates Staphylococcus aureus virulence through an MgrA-mediated regulation of α-hemolysin.

A global transcriptional regulator, MgrA, was previously identified as a key determinant of virulence in Staphylococcus aureus. An 80% EtOH extract of Uncaria gambier was found to attenuate the virulence of S. aureus via its effects on MgrA. Using bioassay-guided fractionation, a polyphenolic polymer, uncariitannin, was found to be the main bioactive constituent of the extract, and its structure was characterized using spectral and chemical analysis. The molecular weight and polydispersity of uncariitannin were determined by gel permeation chromatography-refractive index-light scattering analysis. An electrophoretic mobility shift assay showed that uncariitannin could effectively inhibit the interaction of MgrA with DNA in a dose-dependent manner. Treatment with uncariitannin could decrease the mRNA and protein levels of Hla in both the S. aureus Newman and USA300 LAC strains. Further analysis of Hla expression levels in the Newman ΔmgrA and Newman ΔmgrA/pYJ335-mgrA strains indicated that uncariitannin altered Hla expression primarily in an MgrA-dependent manner. A mouse model of infection indicated that uncariitannin could attenuate MRSA virulence. In conclusion, uncariitannin may be a potential candidate for further development as an antivirulence agent for the treatment of S. aureus infection.

Cholest-4-en-3-one attenuates TGF-ß responsiveness by inducing TGF-ß receptors degradation in Mv1Lu cells and colorectal adenocarcinoma cells.

PURPOSE: The transforming growth factor-beta (TGF-ß) pathway is an important in the initiation and progression of cancer. Due to a strong association between an elevated colorectal cancer risk and increase fecal excretion of cholest-4-en-3-one, we aim to determine the effects of cholest-4-en-3-one on TGF-ß signaling in the mink lung epithelial cells (Mv1Lu) and colorectal cancer cells (HT29) in vitro. METHODS: The inhibitory effects of cholest-4-en-3-one on TGF-ß-induced Smad signaling, cell growth inhibition, and the subcellular localization of TGF-ß receptors were investigated in epithelial cells using a Western blot analysis, luciferase reporter assays, DNA synthesis assay, confocal microscopy, and subcellular fractionation. RESULTS: Cholest-4-en-3-one attenuated TGF-ß signaling in Mv1Lu cells and HT29 cells, as judged by a TGF-ß-specific reporter gene assay of plasminogen activator inhibitor-1 (PAI-1), Smad2/3 phosphorylation and nuclear translocation. We also discovered that cholest-4-en-3-one suppresses TGF-ß responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-ß receptors and facilitating rapid degradation of TGF-ß and thus suppressing TGF-ß-induced signaling. CONCLUSIONS: Our results suggest that cholest-4-en-3-one inhibits TGF-ß signaling may be due, in part to the translocation of TGF-ß receptor from non-lipid raft to lipid raft microdomain in plasma membranes. Our findings also implicate that cholest-4-en-3-one may be further explored for its potential role in colorectal cancer correlate to TGF-ß deficiency.

The efficacy of ultrasound-guided extracorporeal shockwave therapy in patients with cervical spondylosis and nuchal ligament calcification.

We investigated the effects of extracorporeal shockwave therapy (ESWT) on the rehabilitation of cervical spondylosis with nuchal ligament (NL) calcification under X-ray and ultrasound guidance. Sixty patients with cervical spondylosis and calcification of NL were selected and randomly assigned to three groups: A, B, and C. Patients in Group A received rehabilitation with 20 minutes of hot packs and underwent 15 minutes of intermittent cervical traction three times/week for 6 weeks. Patients in Group B received the same rehabilitation as those in Group A and ESWT (2000 impulses, 0.27 mJ/mm(2)) over the calcified NL guided by X-ray image. Patients in Group C received the same treatment as those in Group B, but the ESWT was guided by musculoskeletal sonography. The therapeutic effects were evaluated by: changes in range of motion (ROM) of the cervical spine including flexion, extension, lateral bending, and rotation; visual analog pain scale; and Neck Disability Index before and after treatment and at follow up 3 months later. We found a significant reduction in pain in each treated group after treatment and at follow up. However, patients in Groups B and C showed more improvements in ROM and neck pain relief after treatment and a decrease in Neck Disability Index. Furthermore, patients in Group C showed better cervical ROM at follow up than Group B. ESWT is an adjuvant treatment in the management of cervical spondylosis with calcification of NL and ultrasound-guided ESWT results in more functional improvements.

Imiquimod-induced AMPK activation causes translation attenuation and apoptosis but not autophagy.

BACKGROUND: AMP-activated protein kinase (AMPK), a principal intracellular energy sensor, plays a crucial role in cell growth, proliferation, apoptosis and autophagy. Imiquimod (IMQ) directly exhibits anti-tumor activity through the induction of apoptosis and autophagic cell death. OBJECTIVE: To evaluate the role of AMPK in IMQ-induced apoptosis and autophagy. METHODS: The phosphorylation of AMPK and its substrates was detected by immunoblotting. ATP contents were analyzed by an ATP bioluminescence assay. The upstream signaling for AMPK activation was dissected by examination of TLR7/8 expression, over-expression of TLR7/8, the addition of AMPK kinase inhibitors, and the genetic silencing of Myd88 and LKB1. The role of AMPK activation in IMQ-induced autophagy and apoptosis was assessed by inhibiting AMPK, genetically silencing AMPK and over-expressing AMPK dominant-negative mutants. Autophagy and apoptosis were evaluated by a DNA content assay, immunoblotting, EGFP-LC3 puncta detection and acridine orange staining. RESULTS: IMQ could activate AMPK and autophagy in cancer cells not expressing TLR7/8. IMQ caused ATP depletion and induced LKB1-mediated AMPK activation. The down-regulation of AMPK activity via pharmacological inhibition and genetic silencing resulted in reduced IMQ-induced apoptosis but did not influence autophagy, and this rescue effect was associated with the retention of translation factor activity and anti-apoptotic Bcl-2 family member Mcl-1 protein expression levels. CONCLUSION: IMQ induces AMPK activation independent of TLR7/8 expression, resulting in translation inhibition and subsequent apoptosis through ATP depletion and LKB1 signaling, in skin tumor cells.

p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells.

Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status.

[Induction of proinflammatory cytokines and cell apoptosis by Chlamydia pneumoniae Cpn0810].

AIM: To expresse the Chlamydia pneumoniae Cpn0810 in E.coli BL21, and to study weather could it inducing proinflamatory cytokines including TNF-α and IL-6 in human monocytic (THP-1) and cell apoptosis. METHODS: Polymerase chain reaction(PCR) was used to amplify the Cpn0810 gene, PCR products were purified and cloned into the prokaryotic expression vector pGEX6p-2. The restriction plasmids pGEX6p-2/Cpn0810 confirmed by PCR and sequencing was transformed into E.coli BL21. The recombinant protein was purified with glutathione S-transferase (GST) resin chromatography of Novagen after renaturation. THP-1 cells were stimulated by different concentrations of Cpn0810 and for various durations to test the production and the expression of TNF-α and IL-6 by ELISA. Cell apoptosis was detected in C.pneumoniae Cpn0810 cells by Hoechst33258 fluorescence staining and Cell apoptosis was detected in THP-1 cells by Annexin-V-FITC-propidiu-m iodide (PI) staining. RESULTS: The restriction enzymes cleavage analysis and nucleotide sequencing showed the target gene was successfully inserted into pGEX6p-2 prokaryotic expression vector. Cpn0810 stimulated THP-1 cell to produce proinflamatory cytokines including TNF-α and IL-6 in a dose and time-dependent manner. After THP-1 cells were treated with 10 mg/L Cpn0810 for 24 h, apoptosis with nuclear chromatin fragmentation as well as cell shrinkage was observed by fluorescent staining and microscopy; apoptosis of cell was detected after 24 h in THP-1 cells treated with Cpn0810. CONCLUSION: Cpn0810 recombinant protein could stimulate THP-1 cell to produce and express proinflamatory cytokines including TNF-α and IL-6; After THP-1 cells were treated with 10 mg/L Cpn0810 for 24 h, apoptosis of cell was detected after 24 h in THP-1 cells treated with Cpn0810.