Engineering Escherichia coli to assimilate ß-alanine as a major carbon source.

The threat of global plastic waste accumulation has spurred the exploration of plastics derived from biological sources. A well-known example is polyester made of 1,3-propanediol (1,3-PDO). However, there is no known pathway to assimilate 1,3-PDO into the central carbon metabolism, posing a potential challenge to upcycling such plastic wastes. Here, we proposed that the 1,3-PDO assimilation pathway could pass through malonate semialdehyde (MSA) as an intermediate. Since MSA is a toxic aldehyde, ß-alanine was chosen as a surrogate substrate in this study to construct the lower part of the proposed pathway. To this end, we successfully engineered E. coli MG1655 to assimilate ß-alanine as the major carbon source. ß-alanine could be easily converted into MSA using a ß-alanine/pyruvate transaminase from Pseudomonas aeruginosa (PaBapt). However, the subsequent step to generate acetyl-CoA from MSA was unknown. After a series of phenotype screenings, adaptive laboratory evolution and transcriptomic analysis, two CoA-acylating MSA dehydrogenases from Vibrio natriegens (VnMmsD), were found to be able to complete the metabolic pathway. Optical density at 600 nm (OD600) of the resulting strain E. coli BA02 could reach 4.5 after 96 h. Two approaches were subsequently used to improve its performance. First, PaBapt and both VnMmsDs were expressed from a single plasmid to mitigate antibiotic stress. Second, a native 3-hydroxy acid dehydrogenase (EcYdfG) was disrupted to address the carbon loss to 3-hydroxypropionate (3-HP) production from MSA. OD600 of the best-performing strain E. coli BA07∆ could reach 6 within 24 h using 5 g/L ß-alanine. The construction of E. coli BA07∆ lays a solid foundation to establishing a 1,3-PDO assimilation pathway. KEYPOINTS: • This study demonstrates the implementation of a metabolic pathway to assimilate ß-alanine as the major carbon source in E. coli MG1655. • Two V. natriegens CoA-acylating methyl malonate semialdehyde dehydrogenases were used to complete the pathway in E. coli BA02. • The construction of E. coli BA02 also revealed the plasmid fusion event between two plasmids with the same replication origin.

Matrix metalloprotein-triggered, cell penetrating peptide-modified star-shaped nanoparticles for tumor targeting and cancer therapy.

BACKGROUND: Specific targeting ability and good cell penetration are two critical requirements of tumor-targeted delivery systems. In the present work, we developed a novel matrix metalloprotein-triggered, cell-penetrating, peptide-modified, star-shaped nanoparticle (NP) based on a functionalized copolymer (MePEG-Peptide-Tri-CL), with the peptide composed of GPLGIAG (matrix metalloprotein-triggered peptide for targeted delivery) and r9 (cell-penetrating peptide for penetration improvement) to enhance its biological specificity and therapeutic effect. RESULTS: Based on the in vitro release study, a sustained release profile was achieved for curcumin (Cur) release from the Cur-P-NPs at pH 7.4. Furthermore, the release rate of Cur was accelerated in the enzymatic reaction. MTT assay results indicated that the biocompatibility of polymer NPs (P-NPs) was inversely related to the NP concentration, while the efficiency toward tumor cell inhibition was positively related to the Cur-P-NP concentration. In addition, Cur-P-NPs showed higher fluorescence intensity than Cur-NPs in tumor cells, indicating improved penetration of tumor cells. An in vivo biodistribution study further demonstrated that Cur-P-NPs exhibited stronger targeting to A549 xenografts than to normal tissue. Furthermore, the strongest tumor growth inhibition (76.95%) was observed in Cur-P-NP-treated A549 tumor xenograft nude mice, with slight pulmonary toxicity. CONCLUSION: All results demonstrated that Cur-P-NP is a promising drug delivery system that possesses specific enzyme responsiveness for use in anti-tumor therapy.

Liposomes for effective drug delivery to the ocular posterior chamber.

BACKGROUND: Age-related macular degeneration (AMD) is a leading cause of severe visual deficits and blindness. Meanwhile, there is convincing evidence implicating oxidative stress, inflammation, and neovascularization in the onset and progression of AMD. Several studies have identified berberine hydrochloride and chrysophanol as potential treatments for ocular diseases based on their antioxidative, antiangiogenic, and anti-inflammatory effects. Unfortunately, their poor stability and bioavailability have limited their application. In order to overcome these disadvantages, we prepared a compound liposome system that can entrap these drugs simultaneously using the third polyamidoamine dendrimer (PAMAM G3.0) as a carrier. RESULTS: PAMAM G3.0-coated compound liposomes exhibited appreciable cellular permeability in human corneal epithelial cells and enhanced bio-adhesion on rabbit corneal epithelium. Moreover, coated liposomes greatly improved BBH bioavailability. Further, coated liposomes exhibited obviously protective effects in human retinal pigment epithelial cells and rat retinas after photooxidative retinal injury. Finally, administration of P-CBLs showed no sign of side effects on ocular surface structure in rabbits model. CONCLUSIONS: The PAMAM G3.0-liposome system thus displayed a potential use for treating various ocular diseases.

Structural and enzymatic characterization of the sialidase SiaPG from Porphyromonas gingivalis.

The sialidases, which catalyze the hydrolysis of sialic acid from extracellular glycoconjugates, are a group of major virulence factors in various pathogenic bacteria. In Porphyromonas gingivalis, which causes human periodontal disease, sialidase contributes to bacterial pathogenesis via promoting the formation of biofilms and capsules, reducing the ability for macrophage clearance, and providing nutrients for bacterial colonization. Here, the crystal structure of the P. gingivalis sialidase SiaPG is reported at 2.1 Šresolution, revealing an N-terminal carbohydrate-binding domain followed by a canonical C-terminal catalytic domain. Simulation of the product sialic acid in the active-site pocket together with functional analysis enables clear identification of the key residues that are required for substrate binding and catalysis. Moreover, structural comparison with other sialidases reveals distinct features of the active-site pocket which might confer substrate specificity. These findings provide the structural basis for the further design and optimization of effective inhibitors to target SiaPG to fight against P. gingivalis-derived oral diseases.

Engineering microbes to synthesize functionalized biopolymers.

Engineering the metabolism of microbes has allowed simultaneous co-production of functional small molecules and biopolymers. This perspective briefly introduces its working principles and summarizes my views on how this approach could lead to consolidated fermentation processes for producing functionalized biopolymers. The potential applications of the polymer products and the foreseen challenges are also discussed.

Star polyester-based folate acid-targeting nanoparticles for doxorubicin and curcumin co-delivery to combat multidrug-resistant breast cancer.

Chemotherapeutic treatments are indispensable in the treatment of breast cancer. However, the emergence of multidrug-resistance, strong cell toxicity, and poor targeting selection has inhibited their clinical application. In this study, two synergistic drugs, doxorubicin (DOX) and curcumin (CUR), were co-administered to overcome multidrug resistance (MDR). Based on the characteristics of the tumor microenvironment, we developed folic acid-modified nanoparticles ((DOX + CUR)-FA-NPs) based on a star-shaped polyester (FA-TRI-CL) to enhance the tumor targeting selectivity and drug loading (DL) capacity. The (DOX + CUR)-FA-NPs displayed a characteristic spheroid morphology with an ideal diameter (186.52 nm), polydispersity index (0.024), zeta potential (-18.87 mV), and good entrapment efficiency (97.64%/78.13%, DOX/CUR) and DL (20.27%/11.29%, DOX/CUR) values. In vitro pharmacokinetic and pharmacodynamic experiments demonstrated that the (DOX + CUR)-FA-NPs were gradually released, and they displayed the highest cell apoptosis and cellular uptake in MCF-7/ADR cells. Additionally, in vivo results illustrated that (DOX + CUR)-FA-NPs not only displayed significant tumor targeting and anticancer efficacy, but also induced less pathological damage to the normal tissue. In summary, co-administered DOX and CUR appeared to reverse MDR, and this targeted combinational nanoscale delivery system could thus be a promising carrier for tumor therapies in the future.

Discovery of Pyrrole-imidazole Polyamides as PD-L1 Expression Inhibitors and Their Anticancer Activity via Immune and Nonimmune Pathways.

In recent years, PD-1 immune checkpoint inhibitors based on monoclonal antibodies have revolutionized cancer therapy, but there still exist unresolved issues, such as the high cost, the relatively low response rates, and so on, compared with small-molecule drugs. Herein a type of pyrrole-imidazole (Py-Im) polyamide as a small-molecule DNA binder was designed and synthesized, which could competitively bind to the same double-stranded DNA stretch in the PD-L1 promoter region as the STAT3 binding site and thus downregulate PD-L1 expression. It was demonstrated that the Py-Im polyamides directly caused apoptosis in tumor cells and retarded cell migration in the absence of T cells through inhibiting the Akt/caspase-3 pathway. Also, in a coculture system, they enhanced the T-cell-mediated killing of tumor cells by the reversal of immune escape. Because such polyamides induced antitumor effects via both immune and nonimmune pathways, they could be further developed as promising PD-L1 gene-targeting antitumor drugs.

Studies of poly(ethylene glycol) modification of HM-3 polypeptides.

An RGD modified endostatin-derived synthetic peptide, named HM-3, is a polypeptide angiogenesis inhibitor previously synthesized in our laboratory. Its robust inhibitory effects on endothelial cell migration and tumor growth have been demonstrated by in vivo and in vitro activity assays. The RGD integrin recognition sequence enables the selective binding of HM-3 and its specific targeting to tumor cells that express high levels of integrin. However, the drug has relatively short half-life in vivo, thus requiring administration twice a day to achieve its optimal in vivo antitumor efficacy. In the current study designed to prolong HM-3 half-life, we used methoxy-poly(ethylene glycol)-aldehyde (mPEG-ALD) to specifically modify its N terminus and optimized the reaction condition via monitoring the modification by reverse-phase high-performance liquid chromatograph (RP-HPLC) under varying stoichiometric ratios (n(mPEG10k-ALD):n(HM-3)), reaction times, and pH values. The maximal modification rate was achieved in a reaction when substrates mPEG10k-ALD and HM-3 were mixed at the molar ratio of 2:1 in a pH 6 phosphate buffer after 4 h incubation at room temperature. The reaction product of this optimal reaction was purified to 96% purity by RP-HPLC. Compared with HM-3, the newly modified PEG(10k)-HM-3 was shown to be more active in the inhibition of angiogenesis in the chorioallantoic membrane of chick embryos (CAM), its rate of in vitro degradation in serum was markedly reduced, and its in vivo half-life was prolonged by 5.86-fold relative to unmodified HM-3 after intravenous injection into male SD rats.

A Novel Delivery System of Cyclovirobuxine D for Brain Targeting: Angiopep-Conjugated Polysorbate 80-Coated Liposomes via Intranasal Administration.

The blood-brain barrier (BBB) poses a challenge for the treatment of cerebrovascular diseases including cerebral ischemia-reperfusion injury, Parkinson's syndrome, and cerebral tumors. Nanotechnology has developed as a promising strategy for drug delivery applications to the brain, especially liposomes (Lps) that have shown an intrinsic ability to cross the BBB. Angiopep-2 (ANG), a ligand for low-density lipoprotein receptor-related protein-1 (LRP1), is a good prospect for use as a targeting ligand for brain delivery using Lps. It was also reported that Polysorbate 80 (Tween 80, T80) plays a special role in brain targeting. Moreover, the nasal drug delivery method has attracted increased attention with its brain targeting capability in the clinical treatment of cerebrovascular diseases. The aim of this work was to evaluate the capability of Angiopep-conjugated Polysorbate 80-Coated Liposomes in the delivery of cyclovirobuxine D across the BBB in vitro and in vivo. For this purpose, we first synthesized DSPE-PEG2000-Angiopep-2 then cyclovirobuxine D was encapsulated in Angiopep-conjugated Polysorbate 80-Coated Liposomes (T80-An2-CVB-D-Lps) prepared by thin film evaporation and an ultrasonic technique. Formulations were characterized in terms of encapsulation efficiency, transmission electron microscope (TEM) morphology, size distribution, and zeta potential. Angiopep-conjugated Polysorbate 80-Coated Liposomes enhanced in vitro BBB transport of CVB-D compared to the nontargeted liposomes and the CVB-D solution in the BBB model consisting of brain microvascular endothelial (bEnd.3) cells. To evaluate the brain targeting of T80-An2-CVB-D-Lps in vivo, microdialysis samples were collected from the striatum and blood simultaneously. Rats were dosed with brain-targeting liposomes, CVB-D liposomes and CVB-D solution by intranasal administration and with brain-targeting liposomes by intravenous injection. The results showed that T80-An2-CVB-D-Lps were spherical, small (approximately 80 nm), homogeneously dispersed, negatively charged and possessed a high encapsulation efficiency. T80-An2-CVB-D-Lps crossed the BBB model better than the other treatments did. In addition, in a pharmacodynamic study, there was a higher AUC in the brain after T80-An2-CVB-D-Lps by intranasal administration. In conclusion, T80-An2-Lps can enhance the BBB permeability and improve the transport of CVB-D to the brain. This coadministration strategy can be utilized to enhance the brain accumulation in other cerebrovascular diseases.

Harnessing the hygroscopic and biofluorescent behaviors of genetically tractable microbial cells to design biohybrid wearables.

Cells' biomechanical responses to external stimuli have been intensively studied but rarely implemented into devices that interact with the human body. We demonstrate that the hygroscopic and biofluorescent behaviors of living cells can be engineered to design biohybrid wearables, which give multifunctional responsiveness to human sweat. By depositing genetically tractable microbes on a humidity-inert material to form a heterogeneous multilayered structure, we obtained biohybrid films that can reversibly change shape and biofluorescence intensity within a few seconds in response to environmental humidity gradients. Experimental characterization and mechanical modeling of the film were performed to guide the design of a wearable running suit and a fluorescent shoe prototype with bio-flaps that dynamically modulates ventilation in synergy with the body's need for cooling.

[Detection of anaerobes and drug sensitivity from the periodontal pockets of patients with combined periodontal-endodontic lesions].

PURPOSE: To investigate the correlation between the composition of bacterial flora isolated from deep periodontal pockets with combined periodontal-endodontic lesions, and to test the antibiotic susceptibility of anaerobes. METHODS: Microbial samples were taken from 82 deep periodontal pockets with combined periodontal-endodontic lesions using adequate techniques, and divided into two groups: endo-perio group and perio-endo group. Antibiotic susceptibility of cultivable anaerobes were tested with agar dilution method. RESULTS: Eighty-six cultivable anaerobes were isolated, including Streptococcus spp, Prevotella spp, Actinomycetes spp, Veillonella spp, Fusobacterium spp and etc. 53.4% were gram-positive anaerobes, while 46.51% were gram-negative anaerobes. The detection rate of anaerobes in endo-perio group and that in perio-endo group had significant difference. Most isolated anaerobes were sensitive to six drugs. The resistance rates of penicillin, clindamycin, metronidazole were 6.8%, 13.9% and 2.2%. CONCLUSIONS: There is a close relationship between anaerobes and combined periodontal-endodontic lesions. Drug sensitive susceptibility should be detected prior to clinical treatment of combined periodontal-endodontic lesions.

A safety study of inactivated Enterovirus 71 vaccine.

Enterovirus 71 (EV71) is one of the major causative agents for hand, foot and mouth disease (HFMD) in childhood. Nowadays, HFMD or EV71 infections have already become an important public health issue throughout the world. Vaccination may be the most effective measure to control the transmission of the virus. Therefore, to pave EV71 vaccine into human clinical trial, in the present study a comprehensive preclinical safety assessment of inactivated EV71 vaccine including single- and repeat-dose toxicity studies were conducted in rats and cynomolgus monkeys. No abnormal findings were observed in rats following single intramuscular administration with EV71 vaccine (640 U). The results also showed no obvious systemic toxicities from four repetitive intramuscular injections, with a 14-d interval, of two dosages of EV71 vaccine in the two animal species. Antinuclear antibody response was not detected after the repeated administrations. Histopathological examination demonstrated the minimal to severe inflammatory changes in muscle tissues of the injection sites in EV71 vaccine-injected animals and most of findings have been improved over time. Furthermore, test article could induce highly EV71-specfic neutralizing antibody response in both animal species. Taken together, these data suggested a favorable safety profile for inactivated EV71 vaccine and supported this product to enter human phase I clinical trial.

A series of novel zinc(II) entangled coordination polymers based on carboxyphenyl-terpyridine ligands.

Hydrothermal synthesis has afforded five divalent zinc coordination polymers containing 4-(4-carboxyphenyl)-2,2':6',2''-terpyridine (HL1) or its isomer 4-(4-carboxyphenyl)-2,2':4',4''-terpyridine (HL2), with or without the addition of auxiliary ligands, 1,3,5-benzenetricarboxylic acid (H3btc) and 1,4-benzenedicarboxylic acid (H2bdc). Their structures have been characterized by single crystal X-ray analyses and further characterized by infrared spectra, elemental analyses, powder X-ray diffraction, thermogravimetric analyses and photoluminescent spectra. Across this series, the π···π interactions have a dramatic impact on the self-assembly of these entanglement structures, in either case it can exert an important structure-directing role. In addition, the disposition of pyridine nitrogen atoms in ligands also plays a large role in structure direction in this system. Complex 1 is a 2D + 2D→3D inclined polycatenated coordination polymer based on the resulting array of 2D (6,3) layers constructed by 1D→2D π···π directed self-assembly. Complex 2 is assembled into a 3D framework by means of 1D + 1D→3D mutual interdigitation based on 1D→1D self-assembly driven by π···π stacking interactions. Complex 3 shows a 2D + 2D→3D interdigital network involving 2D + 2D→2D parallel interpenetrated and 2D + 2D→2D interdigital (4,4) layer motifs. Complex 4 displays a 2D + 2D→3D polythreaded framework based on a 2D (4,4) network comprised of alternating rings and rods. Complex 5 is a (3,4)-connected 3D framework with topology (4.8(2).10(3))(4.8(2)). In comparison with covalently connected entanglements, such π···π directing self-assembly of entanglements are far less explored, especially, polycatenane based on 1D chain motifs and polythread based on 2D layer motifs are rarely reported. Furthermore, the luminescent properties of complexes 1-5 at room temperature have also been studied in detail herein.

[The influence of active calcium ions solution on the ultrastructure of the enamel and cementum of extracted teeth and the evaluation of its cytotoxicity].

PURPOSE: To detect the effect of active calcium ions solution on the ultrastructure of enamel and cementum of the extracted teeth and observe the cytotoxicity of the solution on NIH3T3 cell through cell culture. METHODS: Active calcium ions solution composition was detected with a Quanta200FEG field emission scanning electron microscopy, and the ultrastructure of teeth enamel and cementum was observed before and after the application of calcium ions solution. After diluting the calcium ions solution, MTT assay method was used to observe the impact of active calcium ions solution of different concentrations on the NIH3T3 cells growth. Based on cell relative growth rate, the cytotoxicity grade was rated. RESULTS: Active calcium ions solution was composed of calcium, carbon, oxygen, and chlorine. And there was no change in the enamel and cementum before and after soaking the teeth with active calcium ions solution. The maximum concentration of safe calcium ions solution on cytotoxicity grade was 0.344 mg/mL. CONCLUSIONS: Active calcium ions solution can be used in the oral cavity,but appropriate concentration should be selected.