Metabolic engineering of Halomonas bluephagenesis for production of five carbon molecular chemicals derived from L-lysine.

5-Aminovaleric acid (5-AVA), 5-hydroxyvalerate (5HV), copolymer P(3HB-co-5HV) of 3-hydroxybutyrate (3HB) and 5HV were produced from L-lysine as a substrate by recombinant Halomonas bluephagenesis constructed based on codon optimization, deletions of competitive pathway and L-lysine export protein, and three copies of davBA genes encoding L-lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) inserted into its genome to form H. bluephagenesis YF117ΔgabT1+2, which produced 16.4 g L-1 and 67.4 g L-1 5-AVA in flask cultures and in 7 L bioreactor, respectively. It was able to de novo synthesize 5-AVA from glucose by L-lysine-overproducing H. bluephagenesis TD226. Corn steep liquor was used instead of yeast extract for cost reduction during the 5-AVA production. Using promoter engineering based on Pporin mutant library for downstream genes, H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC produced 6 g L-1 5HV in shake flask growth, while H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC-Pporin278-phaCRE-abfT synthesized 42 wt% P(3HB-co-4.8 mol% 5HV) under the same condition. Thus, H. bluephagenesis was successfully engineered to produce 5-AVA and 5HV in supernatant and intracellular P(3HB-co-5HV) utilizing L-lysine as the substrate.

Flux optimization using multiple promoters in Halomonas bluephagenesis as a model chassis of the next generation industrial biotechnology.

Predictability and robustness are challenges for bioproduction because of the unstable intracellular synthetic activities. With the deeper understanding of the gene expression process, fine-tuning has become a meaningful tool for biosynthesis optimization. This study characterized several gene expression elements and constructed a multiple inducible system that responds to ten different small chemical inducers in halophile bacterium Halomonas bluephagenesis. Genome insertion of regulators was conducted for the purpose of gene cluster stabilization and regulatory plasmid simplification. Additionally, dynamic ranges of the multiple inducible systems were tuned by promoter sequence mutations to achieve diverse scopes for high-resolution gene expression control. The multiple inducible system was successfully employed to precisely control chromoprotein expression, lycopene and poly-3-hydroxybutyrate (PHB) biosynthesis, resulting in colorful bacterial pictures, optimized cell growth, lycopene and PHB accumulation. This study demonstrates a desirable approach for fine-tuning of rational and efficient gene expressions, displaying the significance for metabolic pathway optimization.

Nitric Oxide-Releasing Porous Coating with Antibacterial Activity and Blood Compatibility.

Nitric oxide (NO)-releasing coating is promising to enhance the biocompatibility of medical devices. In this study, polyurethane (PU) and S-nitrosated keratin (KSNO) were dissolved with dimethyl sulfoxide (DMSO) and tetrahydrofuran (THF) to prepare a coating solution. This solution is facile to form a porous coating on various substrates based on solvent-evaporation-induced phase separation (SEIPS). The coating could continuously release NO up to 200 h in the presence of ascorbic acid (Asc). In addition, the coating could accelerate endothelialization by promoting the viability of human umbilical vein endothelial cells (HUVECs) while inhibiting the proliferation of human umbilical artery smooth muscle cells (HUASMCs). Furthermore, the coating had good antibacterial activity and blood compatibility. Taken together, this universal coating provides wider potential applications in the field of cardiovascular implants.

Dual-targeting liposomes modified with BTP-7 and pHA for combined delivery of TCPP and TMZ to enhance the anti-tumour effect in glioblastoma cells.

AIM: To construct a novel nano-carrier with dual ligands to achieve superior anti-tumour efficacy and lower toxic side effects. METHODS: Liposomes were prepared by thin film hydration method. Ultraviolet, high performance liquid chromatography, nano-size analyser, ultrafiltration centrifugation, dialysis, transmission electron microscope, flow cytometry, Cell Counting Kit-8, confocal laser scanning microscopy, transwell, and tumorsphere assay were used to study the characterisations, cytotoxicity, and in vitro targeting of dg-Bcan targeting peptide (BTP-7)/pHA-temozolomide (TMZ)/tetra(4-carboxyphenyl)porphyrin (TCPP)-Lip. RESULTS: BTP-7/pHA-TMZ/TCPP-Lip was a spheroid with a mean diameters of 143 ± 3.214 nm, a polydispersity index of 0.203 ± 0.025 and a surface charge of -22.8 ± 0.425 mV. The drug loadings (TMZ and TCPP) are 7.40 ± 0.23% and 2.05 ± 0.03% (mg/mg); and the encapsulation efficiencies are 81.43 ± 0.51% and 84.28 ± 1.64% (mg/mg). The results showed that BTP-7/pHA-TMZ/TCPP-Lip presented enhanced targeting and cytotoxicity. CONCLUSION: BTP-7/pHA-TMZ/TCPP-Lip can specifically target the tumour cells to achieve efficient drug delivery, and improve the anti-tumour efficacy and reduces the systemic toxicity.

Efficacy of a mouthwash containing ε-poly-L-lysine, funme peptides and domiphen in reducing halitosis and supragingival plaque: a randomized clinical trial.

OBJECTIVE: To evaluate the antibacterial effectiveness of a combination of ε-poly-L-lysine (ε-PL), funme peptide (FP) as well as domiphen against oral pathogens, and assess the efficacy of a BOP® mouthwash supplemented with this combination in reducing halitosis and supragingival plaque in a clinical trial. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the compound against Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, and Aggregatibacter actinomycetemcomitans were determined by the gradient dilution method. Subsequently, the CCK-8 assay was used to detect the toxicity of mouthwash on human gingival fibroblastst, and the effectiveness in reducing halitosis and supragingival plaque of the mouthwash supplemented with the combination was analyzed by a randomized, double-blind, parallel-controlled clinical trial. RESULTS: The combination exhibited significant inhibitory effects on tested oral pathogens with the MIC < 1.56% (v/v) and the MBC < 3.13% (v/v), and the mouthwash containing this combination did not inhibit the viability of human gingival fibroblasts at the test concentrations. The clinical trial showed that the test group displayed notably lower volatile sulfur compounds (VSCs) at 0, 10, 24 h, and 7 d post-mouthwash (P < 0.05), compared with the baseline. After 7 days, the VSC levels of the and control groups were reduced by 50.27% and 32.12%, respectively, and notably cutting severe halitosis by 57.03% in the test group. Additionally, the Plaque Index (PLI) of the test and control group decreased by 54.55% and 8.38%, respectively, and there was a significant difference in PLI between the two groups after 7 days (P < 0.01). CONCLUSIONS: The combination of ε-PL, FP and domiphen demonstrated potent inhibitory and bactericidal effects against the tested oral pathogens, and the newly formulated mouthwash added with the combination exhibited anti-dental plaque and anti-halitosis properties in a clinical trial and was safe. TRIAL REGISTRATION: The randomized controlled clinical trial was registered on Chinese Clinical Trial Registry (No. ChiCTR2300073816, Date: 21/07/2023).

Macrophage-derived apoptotic bodies impair the osteogenic ability of osteoblasts in periodontitis.

OBJECTIVES: Periodontitis is induced by the imbalance between osteoblast and osteoclast activity, which leads to periodontal tissue destruction. Macrophages play a vital role in periodontitis. However, the hypoxic periodontal environment will also induce macrophage apoptosis within a short time. Apoptotic bodies (ABs) are the major products generated from apoptotic cells, but whether macrophage-derived ABs play a regulatory role as their mother cells in periodontitis remains unknown. In the present study, we aimed to investigate the effects of ABs on osteoblasts. METHOD: ABs derived from hypoxia-induced macrophages were co-cultured with osteoblasts and the impact of ABs on osteoblast differentiation in vitro was assessed. In vivo, periodontitis model was established and macrophages-derived ABs were injected into the gingival sulcus. The effects of ABs on periodontal bone resorption were determined. RESULTS: The results showed that ABs significantly inhibit osteoblast differentiation and promoted alveolar bone resorption in periodontitis. MicroRNA (miRNAs) array analysis was performed and revealed that miR-483-5p is the key miRNA in ABs. Dual luciferase reporter assays were performed and confirmed that miR-483-5p targeted Col1A1 mRNA and attenuated its expression. CONCLUSION: Macrophage-derived ABs inhibit osteoblast differentiation via the transfer of miR-483-5p, which downregulates Col1A1 expression and finally suppresses osteogenic activity.

Modified minimally invasive surgery in reconstructing the temporomandibular joint disk by transplantation of the temporalis myofascial flap.

OBJECTIVE: The transplantation of temporalis myofascial flap (TMF) is an indispensable method to treat severe temporomandibular joint disorders with disk failure. How to select the indication and the ways of transplantation is still a challenging topic for achieving the ideal effect. We reported the new methods and follow-up results of the patients treated with pedicled TMF transplantation. METHODS: Retrospective case series was performed at Temporomandibular Joint Specialist Clinic, the First Affiliated Hospital of Xinjiang Medical University, from December 2014 to August 2022. 39 patients (50 sides) included anterior disk displacement without reduction and disk perforation who underwent discectomy and immediate reconstruction with pedicled TMF. The initial and postoperative maximum mouth opening (MMO), and pain visual analogue scale (VAS) were compared via paired t-test and Wilcoxon signed-rank sum test, respectively. RESULTS: The average of follow-up time was 30.07 months. The reconstructed temporomandibular joints basically achieved stable occlusion, good function, and satisfactory effect. The patients displayed a remarkable reduction of VAS score of pain, and improvement of the MMO compared with that before operation (P <  0.001). MR scanning revealed the grafts in joint space showed band-shaped soft tissue density of medium signal and had the clear edges, without interruption and fragment. CONCLUSION: Reconstruction of the temporomandibular joint disk by transplantation of the TMF applying modified minimally invasive surgery was a feasible method, which could improve the function of joints and prevent adhesion, without obvious complications in donor region.

A pilot investigation of condylar position and asymmetry in patients with unilateral posterior scissors-bite malocclusion based on three-dimensional reconstructive imaging technique.

OBJECTIVE: Unilateral posterior scissors-bite (uPSB) malocclusion is common clinically. This study aimed to investigate the condylar morphological alterations and condyle-fossa relationship in patients with uPSB, through cone beam computed tomography (CBCT) and three-dimensional reconstructive imaging technique. METHODS: A retrospective study was designed to comparatively analyze 95 patients with uPSB between July 2016 and December 2021. They were divided into three subgroups: 12 to 20, 21 to 30, and ≥ 31 years, according the age distribution. The morphological parameters regarding condyle, fossa, and joint space after three-dimensional reconstruction were measured and analyzed by a series of digital software. SPSS 26.0 software package was performed for statistical analysis on data sets, using paired t-test, one-way analysis of variance, Wilcoxon signed-rank sun test, Kruskal-Wallis H test, and Bonferroni correction. RESULTS: The condylar volume (CV) of scissors-bite side was greater than that of the non-scissors-bite side (CV A = 1740.68 ± 559.80 mm3 > CV N = 1662.25 ± 524.88 mm3, P = 0.027). So was the condylar superficial area (CSA) (CSA A = 818.71 ± 186.82 mm2 > CSA N = 792.63 ± 173.44 mm2, P = 0.030), and the superior joint space (SJS) [SJS A = 2.46 (1.61, 3.68) mm) > SJS N = 2.01 (1.55, 2.87) mm), P = 0.018], and the anterior joint space (AJS) (AJS A = 3.94 ± 1.46 mm > AJS N = 3.57 ± 1.30 mm, P = 0.017). The constituent ratios of the different parts of the bilateral condyles were 23% on the posterior slope, 21% on the top, 20% on the anterior slope, 19% on the lateral slope and 17% on the medial slope, respectively. CONCLUSION: Due to long-term abnormal occlusion of uPSB, the pathological bite force in temporomandibular joint would cause changes in the shape of the condyle. Among them, CV, CSA, SJS and AJS had significant changes in the scissors-bite status, which has the greatest damage to the posterior slope of the condyloid process.

Effects of osseous structure based on three-dimensional reconstructive imaging evaluation in the assessment of temporomandibular joint disc position.

OBJECTIVES: This pilot morphological study aimed to investigate the association between anterior disc displacement (ADD) and the status of the mandibular condyle and articular fossa. MATERIALS AND METHODS: Thirty-four patients were divided into a normal articular disc position group and an ADD with and without reduction group. Images reconstructed were used to determine multiple group comparisons of these three different types of disc position, and the diagnostic efficacy for the morphological parameters with significant group difference was analyzed to assess. RESULTS: The condylar volume (CV), condylar superficial area (CSA), superior joint space (SJS), and medial joint space (MJS) all exhibited obvious changes (P < 0.05). Additionally, they all had a reliable diagnostic accuracy in differentiating between normal disc position and ADD with an AUC value from 0.723 to 0.858. Among them, the CV, SJS, and MJS (P < 0.05) had a significantly positive impact on the groups by analysis of multivariate logistic ordinal regression model. CONCLUSIONS: The CV, CSA, SJS, and MJS are significantly associated with different disc displacement types. The condyle in ADD exhibited altered dimensions. These could be promising biometric markers for assessing ADD. CLINICAL RELEVANCE: The morphological changes of mandibular condyle and glenoid fossa were significantly influenced by the status of disc displacement, and condyles with disc displacement had three-dimensionally altered condylar dimensions, irrespective of age and sex.

Investigation of Morphologic Changes in Temporomandibular Joint With Anterior Disk Displacement Based on 3-Dimensional Reconstructive Imaging.

The objective of this study was to measure the association between the disk position and condylar alteration in patients diagnosed with anterior disk displacement (ADD) of temporomandibular joint (TMJ). A retrospective cross-sectional study was designed, dividing into 4 groups: normal articular disk position (NADP) of unilateral ADD patients (n=10), ADD with reduction (ADDwR, n=16), ADD without reduction (ADDwoR, n=24), and healthy volunteers (HV, n=30) based on magnetic resonance imaging and cone-beam computed tomography. After morphologic parameters were calculated from 3-dimensional reconstructive images, differences in parameters with respect to ADD status were tested with analysis of variance and Fisher least significant difference multiple comparisons were performed. Condylar volume of ADDwR, ADDwoR, NADP, and HVs were 1768.29±404.19, 1467.13±438.20, 1814.48±753.60, and 1914.66±476.48 mm, respectively, showing a significant downward trend from healthy disk to a displaced one ( P <0.05). Same trend also found in condylar superficial area, with the condylar superficial area of the ADDwR, ADDwoR, NADP, and HVs were 842.56±138.78 mm, 748.52±157.42 mm, 842.87±263.00 mm, and 892.73±164.19 mm, respectively. From NADP to ADDwR to ADDwoR, superior joint space (SJS) was declined, [SJS (NADP) (2.10±0.91 mm) > SJS (ADDwR) (1.85±0.61 mm) > SJS (ADDwoR) (1.50±0.50 mm), P =0.034]; medial joint space was significantly associated with the different disk displacement types ( P(HV-ADDwR) =0.001; P(HV-ADDwoR) =0.021; P(NADP-ADDwR) =0.022; P(ADDwR-ADDwoR) =0.001). Our findings suggest that condylar volume and superficial area, and superior and medial joint space, albeit with a small sample size, are significantly associated with different disk displacement types, detection of which might therefore be worth exploring for assessing ADD.

Cellulose-Acetate Coating by Integrating Ester Group with Zinc Salt for Dendrite-Free Zn Metal Anodes.

Zinc (Zn) metal is considered a potential anode owing to its high theoretical capacity, safety, and low cost. However, the dendrites and corresponding side reactions in aqueous electrolytes hinder their further development in environmentally-friendly energy storage. Herein, ion-affiliative cellulose acetate (CA) coating with Zn(CF3 SO3 )2 is constructed on Zn anode (CAZ@Zn). Owing to the complexation effect between the polar ester group (CO) and Zn salt (Zn2+ ), the CAZ polymer coating enhances the hydrophilicity of the Zn anode and reduces the interfacial resistance, allowing the rapid Zn2+ diffusion and homogenizing the Zn deposition in an aqueous electrolyte to suppress zinc dendrite formation and growth. Therefore, the symmetric CAZ@Zn//CAZ@Zn battery achieves reversible plating/stripping over 2800 h at 1 mA cm-2 with 1 mAh cm-2 , about sevenfold higher than bare Zn. The full cell fabricated with an optimized Zn anode and the NH4 V4 O10 cathode achieves substantially stable performance, superior to that of bare Zn. This work provides a straightforward, effective, and scalable method to suppress the zinc dendrites and corresponding side reactions for aqueous Zn-ions batteries.

Biosynthesis of diverse α,ω-diol-derived polyhydroxyalkanoates by engineered Halomonas bluephagenesis.

Polyhydroxyalkanoates (PHA) are a family of biodegradable and biocompatible plastics with potential to replace petroleum based plastics. Diversity of PHA monomer structures provides flexibility in material properties to suit more applications. In this study, 5-hydroxyvalerate (5HV) synthesis pathway was established based on intrinsic alcohol/aldehyde dehydrogenases. The PHA polymerase cloned from Cupriavidus necator functions to polymerize 5HV into its copolymers in ratios ranging from 8% to 32%. Elastic copolymer P(85% 3HB-co-15% 5HV) was generated with an elongation at break and a Young's modulus of 1283% and 73.1 MPa, respectively. The recombinant H. bluephagenesis was able to convert various diols including 1, 3-propanediol, 1, 4-butanediol and 1, 5-pentanediol into PHA, leading to 13 PHA polymers including transparent P(53% 3HB-co-20% 4HB-co-27% 5HV) and sticky P(3HB-co-3HP-co-4HB-co-5HV). The engineered H. bluephagenesis was successfully grown in a 7-L bioreactor to produce the highly elastic P(85% 3HB-co-15% 5HV) and the sticky P(3HB-co-3HP-co-4HB-co-5HV), demonstrating their potential for industrial scale-up.

Rapid Quantification of Polyhydroxyalkanoates Accumulated in Living Cells Based on Green Fluorescence Protein-Labeled Phasins: The qPHA Method.

Polyhydroxyalkanoates (PHAs) are microbial polyesters that have the potential to replace nonbiodegradable petroplastics. A real-time in situ PHA quantification method has long been awaited to replace the traditional method, which is time- and labor-consuming. Quantification of PHA in living cells was finally developed from fluorescence intensities generated from the green fluorescence protein (GFP) fused with the Halomonas bluephagenesis phasin proteins. Phasins PhaP1 and PhaP2 were used to fuse with GFP, which reflected PHA accumulation with an R-square of over 0.9. Also, a standard correlation was established to calculate PHA contents based on the fluorescence and cell density recorded via a microplate reader with an R-square of over 0.95 when grown on various substrates. The PhaP2-GFP containing H. bluephagenesis was applied successfully to quantify PHA synthesis in a 7.5 L fermenter with high precision. Moreover, the method was found to be feasible in non-natural PHA producers such as Escherichia coli, demonstrating its broad applicability.

Silencing integrin α6 enhances the pluripotency-differentiation transition in human dental pulp stem cells.

OBJECTIVES: Although integrins have been shown to be associated with proliferation and differentiation in some stem cells, the regulatory effect of integrin α6 (ITGα6) on the human dental pulp stem cells (hDPSCs) has not been reported. Here, we detected the roles of ITGα6 in hDPSCs. MATERIALS AND METHODS: Attached to Cytodex 3 microcarriers, hDPSCs grown under stimulated microgravity (SMG) or conventional culture conditions were measured the proliferation and different gene expression. Further, ITGα6 was silenced in hDPSCs, and its effect on proliferation, differentiation, and cytoskeletal organization was analyzed. RESULTS: SMG conditions increased the number of Ki67-positive hDPSCs and progression into S phase of cell cycle. WB analysis showed the expression of ITGα6 was upregulated in hDPSCs under SMG conditions. Knockdown of ITGα6 decreased the expression of stemness markers, CD105 and STRO-1 in hDPSCs, but promoted the osteogenic and odontogenic differentiation by increased ALP expression and Alizarin Red nodules. Moreover, RNA-seq demonstrated that RHO/ROCK signaling pathway upregulated silencing ITGα6-hDPSCs. Treatment with Y-27632 inhibited the effect of ITGα6 depletion on hDPSCs stemness, rearranged the cytoskeleton, promoted the pluripotency, proliferation ability, and inhibited the differentiation. CONCLUSION: ITGα6 promotes hDPSCs stemness via inhibiting RHO/ROCK and restoring cytoskeleton.

Chemiluminescence sensors based on molecularly imprinted polymers for the determination of organophosphorus in milk.

As a food adapted to all kinds of people, milk has a high nutritional value. Because milk is a complex biological matrix, detecting illegal compounds is often difficult. As a common pesticide, organophosphorus (OP) residues caused by nonstandard use may be ignored, which is a threat to milk quality. In this study, using coumaphos as template molecule, the synthesized molecularly imprinted polymer (MIP) can specifically recognize 7 kinds of OP. Then, the MIP was used as an identification element to prepare a chemiluminescence sensor on a 96-well microplate for the determination of OP residues in milk samples. Due to the 4-(imidazol-1-yl)phenol-enhanced luminol-H2O2 system, the sensitivity of the system is very high; the detection limits of 7 OP including coumaphos, fenthion, chlorpyrifos, parathion, diazinon, fenchlorphos, and fenitrothion were 1 to 3 pg/mL, and the half maximal inhibitory concentrations were 1 to 20 ng/mL. The intraday recoveries of 7 OP were in the range of 86.1 to 86.5%, and the interday recoveries were in the range of 83.6 to 94.2%. Furthermore, the sensor can be reused up to 5 times. Therefore, the MIP-based chemiluminescence sensor can be used as a routine tool to detect OP residues in milk samples.

The impact of family doctor system on patients' utilisation of general practitioner in primary care facilities-Evidence from Hangzhou, China.

OBJECTIVE: This study aims to examine whether participating in the contracted family doctor system increases patients' utilisation of primary care general practitioner for multiple disease outcomes in China. METHODS: Binary logistic regression models were estimated using data collected from 372 community residents in nine selected districts of Hangzhou, China. RESULTS: Findings revealed that (1) for patients with influenza, diabetes, upper respiratory infection, and gingivitis, those who participated in the contracted family doctor system were approximately 4.3 times, 98.4%, 92.5%, and 52.8% more likely to choose primary care general practitioners (GP) for their initial diagnosis, respectively, as compared with their counterparts who did not have contracted family doctors; (2) For patients with stroke or cerebrovascular disease and cholecystitis or cholelithiasis, those who had contracted family doctors were 1.111 times and 80.6% more likely to choose primary care GP for their subsequent disease maintenance, respectively, as compared to their counterparts without contracted family doctors. CONCLUSION: Our findings indicate that the contracted family doctor system not only increases the utilisation of primary care GP for patients with many chronic conditions but also promotes the overall completion of China's hierarchical medical system in the long run. Policy implications were provided to help policymakers actively construct and develop the contracted family doctor system to promote the hierarchical medical system in China.

Morphologic Analysis of Condyle among Different Disc Status in the Temporomandibular Joints by Three-dimensional Reconstructive Imaging: A Preliminary Study.

OBJECTIVES: Morphological study is a common approach in the field of anterior disc displacement (ADD) pathology; however, analysis based on three-dimensional reconstructive imaging has not been investigated. This study investigated the association between ADD and the status of the mandibular condyle and articular fossa. METHODS: Thirty-four patients were divided into three groups: normal articular disc position (NADP), anterior disc displacement with reduction (ADDwR), and anterior disc displacement without reduction (ADDwoR). Multiple grouped comparisons of three different disc statuses were performed by Kruskal-Wallis H test and variance analysis respectively. Receiver-operating characteristic curve was plotted to assess the diagnostic efficacy of the morphological parameters. Multivariate logistic regression analysis was used to investigate the interfering factors of ADD. RESULTS: The condylar volume (CV) and condylar superficial area (CSA) in the NADP, ADDwR, and ADDwoR groups exhibited obvious changes (P < 0.05). Both CV and superior joint space (SJS) presented a good diagnostic accuracy for NADP-ADDwoR [area under the curve (AUC)CV = 0.813; AUCSJS = 0.855)], and ADDwR-ADDwoR (AUCCV = 0.858; AUCSJS = 0.801). CSA presented a good diagnostic accuracy for ADDwR-ADDwoR (AUC = 0.813). A multivariate logistic ordinal regression model showed that the CV [odds ratio (OR) = 1.011; regression coefficient (RC) = 0.011, P = 0.018], SJS (OR, 8.817; RC = 2.177; P < 0.001), and medial joint space (MJS) (OR, 1.492; RC = 0.400; P = 0.047) had a significantly impact on the groups. CONCLUSION: CV, CSA, SJS, and MJS were significantly associated with the different disc status, and the condyle in ADD exhibited 3-dimensionally altered dimensions. They could be considered as promising biometric markers to assess the ADD.

Degradable cationic polyesters via ring-opening copolymerization of valerolactones as nanocarriers for the gene delivery.

The development of cationic polymers as non-viral gene vectors has been hurdled by their high toxicity, thus degradable and biocompatible polymers are urgently demanded. Herein, five polyesters (B3a-B3e) were synthesized based on the ring-opening copolymerization between α-allyl-δ-valerolactone and δ-valerolactone derivatives decorated with alkyl or alkoxyl chains of different lengths, followed by the modification with 1,5,9-triazacyclododecyl ([12]aneN3) through thiol-ene click reactions. The five polyesters effectively condensed DNA into nanoparticles. Of them, B3a with a shorter alkyl chain and B3d with more positive charged units showed stronger DNA condensing performance and can completely retard the migration of DNA at N/P = 1.6 in the presence of DOPE. B3b/DOPE with a longer alkyl chain exhibited the highest transfection efficiency in HeLa cells with 1.8 times of 25 kDa PEI, while B3d/DOPE with more positive charged units exhibited highest transfection efficiency in A549 cells with 2.3 times of 25 kDa PEI. B3b/DOPE and B3d/DOPE successfully delivered pEGFP into zebrafish, which was superior to 25 kDa PEI (1.5 folds and 1.1 folds, respectively). The cytotoxicity measurements proved that the biocompatibility of these polyesters was better than 25 kDa PEI, due to their degradable property in acid environment. The results indicated that these cationic polyesters can be developed as potential non-viral gene vectors for DNA delivery.

Oxygen nanocarrier broke the hypoxia trap of solid tumors and rescued transfection efficiency for gene therapy.

BACKGROUND: Gene therapy shows great promise for a broad array of diseases. However, we found that hypoxic tumor microenvironment (TME) exerted significant inhibitory effects on transfection efficiency of a variety of gene vectors (such as Lipo 2000 and PEI) in an oxygen-dependent manner. Solid tumors inevitably resulted in acute hypoxic areas due to the rapid proliferation of tumor cells and the aberrant structure of blood vessels. Thus, the hypoxic TME severely limited the efficiency and application of gene therapy. METHODS: In our previous study, we constructed endoplasmic reticulum-targeted cationic liposomes, PAR-Lipo, which could effectively deliver genes and ensure high transfection efficiency under normoxia. Unsatisfactorily, the transfection efficiency of PAR-Lipo was rather poor under hypoxia. We believed that reoxygenation was the most direct and effective means to rescue the low transfection under hypoxia. Hence, we fabricated liposomes modified with perfluorooctyl bromide (PFOB@Lipo) to load oxygen and deliver it to tumor sites, which effectively alleviated the hypoxic nature of tumor. Then PAR-Lipo were applied to mediate high-efficiency delivery of tumor suppressor gene pTP53 to inhibit tumor progression. RESULTS: The results showed that such staged strategy augmented the expression of P53 protein in tumors and extremely suppressed tumor growth. CONCLUSION: This work was the first attempt to utilize an oxygen nanocarrier to assist the therapeutic effect of gene therapy under hypoxia, providing a new reference for gene therapy in malignant tumors. GRAPHICAL ABSTARCT.

Engineering cell wall synthesis mechanism for enhanced PHB accumulation in E. coli.

The rigidity of bacterial cell walls synthesized by a complicated pathway limit the cell shapes as coccus, bar or ellipse or even fibers. A less rigid bacterium could be beneficial for intracellular accumulation of poly-3-hydroxybutyrate (PHB) as granular inclusion bodies. To understand how cell rigidity affects PHB accumulation, E. coli cell wall synthesis pathway was reinforced and weakened, respectively. Cell rigidity was achieved by thickening the cell walls via insertion of a constitutive gltA (encoding citrate synthase) promoter in front of a series of cell wall synthesis genes on the chromosome of several E. coli derivatives, resulting in 1.32-1.60 folds increase of Young's modulus in mechanical strength for longer E. coli cells over-expressing fission ring FtsZ protein inhibiting gene sulA. Cell rigidity was weakened by down regulating expressions of ten genes in the cell wall synthesis pathway using CRISPRi, leading to elastic cells with more spaces for PHB accumulation. The regulation on cell wall synthesis changes the cell rigidity: E. coli with thickened cell walls accumulated only 25% PHB while cell wall weakened E. coli produced 93% PHB. Manipulation on cell wall synthesis mechanism adds another possibility to morphology engineering of microorganisms.