Decellularized periosteum and sodium alginate-based photoresponsive dressing with copper selenide nanoparticles for infected-wound healing in diabetic mice.

Diabetes-related skin ulcers are of significant clinical concern. Although conventional dressings have been developed, their outcomes have not been adequate, indicating the need to investigate functional dressings for the treatment of diabetic ulcers. Copper selenide nanoparticles (Cu2Se NPs) demonstrate outstanding photoresponsiveness, which is critical to the healing process. However, their limited solubility in water restricts their application. To synthesize the ODT-PMMA@Cu2Se NP-doped decellularized periosteum­sodium alginate functional dressing-ODT-PMMA@Cu2Se/ECM-S (OP@Cu2Se/ECM-S), Cu2Se NPs were modified by n-octadecanethiol (ODT) end-functionalized poly (methacrylic acid) (PMAA) ligands homogeneously dispersed in a decellularized periosteum/sodium alginate matrix. This process improved the water solubility and stability. Moreover, under near-infrared irradiation (NIR), ODT-PMMA@Cu2Se demonstrated robust photo-responsiveness along with photothermal and photodynamic effects, leading to rapid heating and stimulation of reactive oxygen species (ROS) generation. These two processes work in concert to exhibit excellent antibacterial ability; at 20 µg/mL concentration of Cu2Se NPs, the bacterial activities of S. aureus and E. coli were 5.40 % and 0.96 %, respectively. Without the NIR laser irradiation, OP@Cu2Se/ECM-S rapidly increased the vascular endothelial growth factor (VEGF) expression, triggered the phosphatidylinositide 3-kinases (PI3K) and protein kinase B (AKT) signaling pathway, affected the expression of bFGF and CD31, and promoted neovascularization, proliferation, and cell migration. In a diabetic mouse wound model, OP@Cu2Se/ECM-S exhibited good biocompatibility and promoted epidermal regeneration, collagen deposition, and neovascularization. In a mouse model of subcutaneous abscesses, OP@Cu2Se/ECM-S also showed excellent antibacterial activity, in vivo experiments confirmed a decrease in bacterial activity to 1.97 %. Thus, OP@Cu2Se/ECM-S is a potentially useful approach for healing diabetic wounds.

Injectable silk fibroin peptide nanofiber hydrogel composite scaffolds for cartilage regeneration.

Transforming growth factor-ß1 (TGF-ß1) is essential for cartilage regeneration, but its susceptibility to enzymatic denaturation and high cost limit its application. Herein, we report Ac-LIANAKGFEFEFKFK-NH2 (LKP), a self-assembled peptide nanofiber hydrogel that can mimic the function of TGF-ß1. The LKP hydrogel is simple to synthesize, and in vitro experiments confirmed its good biocompatibility and cartilage-promoting ability. However, LKP hydrogels suffer from poor mechanical properties and are prone to fragmentation; therefore, we prepared a series of injectable hydrogel composite scaffolds (SF-GMA/LKP) by combining LKP with glycidyl methacrylate (GMA)-modified silk fibroin (SF). SF-GMA/LKP composite scaffolds instantaneously induced in-situ filling of cartilage defects and, at the same time, relied on the interaction between LKP and SF-GMA interaction to prolong the duration of action of LKP. The SF-GMA/LKP10 and SF-GMA/LKP20 composite scaffolds had the best effect on neocartilage and subchondral bone reconstruction. This composite hydrogel scaffold can be used for high-quality cartilage repair.

Inhibition of HOXD11 promotes cartilage degradation and induces osteoarthritis development.

The 5'-HOXD genes are important for chondrogenesis in vertebrates, but their roles in osteoarthritis (OA) are still ambiguous. In our study, 5'-HOXD genes involvement contributing to cartilage degradation and OA was investigated. In bioinformatics analysis of 5'-HOXD genes, we obtained the GSE169077 data set related to OA in the GEO and analyzed DEGs using the GEO2R tool attached to the GEO. Then, we screened the mRNA levels of 5'-HOXD genes by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). We discovered that OA chondrocyte proliferation was inhibited, and apoptosis was increased. Moreover, it was discovered that SOX9 and COL2A1 were downregulated at mRNA and protein levels, while matrix metalloproteinases (MMPs) and a disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTSs) were upregulated. According to the results of differentially expressed genes (DEGs) and qRT-PCR, we evaluated the protein level of HOXD11 and found that the expression of HOXD11 was downregulated, reversed to MMPs and ADAMTSs but consistent with the cartilage-specific factors, SOX9 and COL2A1. In the lentivirus transfection experiments, HOXD11 overexpression reversed the effects in OA chondrocytes. In human OA articular cartilage, aberrant subchondral bone was formed in hematoxylin-eosin (H&E) and Safranin O and fast green (SOFG) staining results. Furthermore, according to immunohistochemistry findings, SOX9 and HOXD11 expression was inhibited. The results of this study established that HOXD11 was downregulated in OA cartilage and that overexpression of HOXD11 could prevent cartilage degradation in OA.

Peptide-Based Hydrogel Scaffold Facilitates Articular Cartilage Damage Repair.

Articular cartilage injury is a common disease in clinical medicine. Because of its special physiological structure and lack of blood, lymph, and nerves, its ability to regenerate once damaged is very limited. In this study, we designed and synthesized a series of self- and coassembled cartilage-inducing functional peptide molecules and constructed a coassembled functional peptide hydrogel based on phenylboronic acid-o-dihydroxy "click chemistry" cross-linking to promote aggregation and signal transduction of mesenchymal stem cells (MSCs) in the early stage and differentiation toward cartilage, thereby promoting the repair of cartilage damage. Three functional peptide molecules were produced using solid-phase peptide synthesis technology, yielding a purity higher than 95%. DOPA-FEFEFEFEGHSNGLPL (DFP) and PBA-FKFKFKFKGHAVDI (BFP) were coassembled at near-neutral pH to form hydrogels (C Gels) based on phenylboronic acid-o-dihydroxy click chemistry cross-linking and effectively loaded transforming growth factor (TGF)-ß1 with a release period of up to 2 weeks. Furthermore, chondrocytes and bone marrow mesenchymal stem cells (BMSCs) were cocultured with functional peptide hydrogels, and the results displayed that the coassembled functional peptide hydrogel group C Gels significantly promoted the proliferation of chondrocytes and MSCs. The chondrocyte markers collagen type I, collagen type II, and glycosaminoglycan (GAG) in the coassembled functional peptide hydrogel group were significantly higher than those in the control group, indicating that it can induce the differentiation of MSCs into cartilage. In vivo experiments demonstrated that the size and thickness of the new cartilage in the compound gel group were the most beneficial to cartilage regeneration. These results indicated that peptide hydrogels are a promising therapeutic option for cartilage regeneration.

Engineering extracellular electron transfer to promote simultaneous brewing wastewater treatment and chromium reduction.

Microbial fuel cell (MFC) technology has been developed for wastewater treatment in the anodic chamber, and heavy metal reduction in the cathodic chamber. However, the limited extracellular electron transfer (EET) rate of exoelectrogens remained a constraint for practical applications of MFCs. Here, a MFC system that used the electricity derived from anodic wastewater treatment to drive cathodic Cr6+ reduction was developed, which enabled an energy self-sustained approach to efficiently address Cr6+ contamination. This MFC system was achieved by screening exoelectrogens with a superior EET rate, promoting the exoelectrogenic EET rate, and constructing a conductive bio-anode. Firstly, Shewanella algae-L3 was screened from brewing wastewater acclimatized sludge, which generated power density of 566.83 mW m-2. Secondly, to facilitate EET rate, flavin synthesis gene operon ribADEHC was overexpressed in engineered S. algae-L3F to increase flavins biosynthesis, which promoted the power density to 1233.21 mW m-2. Thirdly, to facilitate interface electron transfer, carbon nanotube (CNT) was employed to construct a S. algae-L3F-CNT bio-anode, which further enhanced power density to 3112.98 mW m-2. Lastly, S. algae-L3F-CNT bio-anode was used to harvest electrical energy from brewing wastewater to drive cathodic Cr6+ reduction in MFC, realizing 71.43% anodic COD removal and 98.14% cathodic Cr6+ reduction. This study demonstrated that enhanced exoelectrogenic EET could facilitate cathodic Cr6+ reduction in MFC.

Recent advances in enrichment, isolation, and bio-electrochemical activity evaluation of exoelectrogenic microorganisms.

Exoelectrogenic microorganisms (EEMs) catalyzed the conversion of chemical energy to electrical energy via extracellular electron transfer (EET) mechanisms, which underlay diverse bio-electrochemical systems (BES) applications in clean energy development, environment and health monitoring, wearable/implantable devices powering, and sustainable chemicals production, thereby attracting increasing attentions from academic and industrial communities in the recent decades. However, knowledge of EEMs is still in its infancy as only ∼100 EEMs of bacteria, archaea, and eukaryotes have been identified, motivating the screening and capture of new EEMs. This review presents a systematic summarization on EEM screening technologies in terms of enrichment, isolation, and bio-electrochemical activity evaluation. We first generalize the distribution characteristics of known EEMs, which provide a basis for EEM screening. Then, we summarize EET mechanisms and the principles underlying various technological approaches to the enrichment, isolation, and bio-electrochemical activity of EEMs, in which a comprehensive analysis of the applicability, accuracy, and efficiency of each technology is reviewed. Finally, we provide a future perspective on EEM screening and bio-electrochemical activity evaluation by focusing on (i) novel EET mechanisms for developing the next-generation EEM screening technologies, and (ii) integration of meta-omics approaches and bioinformatics analyses to explore nonculturable EEMs. This review promotes the development of advanced technologies to capture new EEMs.

Engineering Outer Membrane Vesicles to Increase Extracellular Electron Transfer of Shewanella oneidensis.

Outer membrane vesicles (OMVs) of Gram-negative bacteria play an essential role in cellular physiology. The underlying regulatory mechanism of OMV formation and its impact on extracellular electron transfer (EET) in the model exoelectrogenShewanella oneidensis MR-1 remain unclear and have not been reported. To explore the regulatory mechanism of OMV formation, we used the CRISPR-dCas9 gene repression technology to reduce the crosslink between the peptidoglycan (PG) layer and the outer membrane, thus promoting the OMV formation. We screened the target genes that were potentially beneficial to the outer membrane bulge, which were classified into two modules: PG integrity module (Module 1) and outer membrane component module (Module 2). We found that downregulation of the penicillin-binding protein-encoding gene pbpC for peptidoglycan integrity (Module 1) and the N-acetyl-d-mannosamine dehydrogenase-encoding gene wbpP involved in lipopolysaccharide synthesis (Module 2) exhibited the highest production of OMVs and enabled the highest output power density of 331.3 ± 1.2 and 363.8 ± 9.9 mW m-2, 6.33- and 6.96-fold higher than that of the wild-typeS. oneidensis MR-1 (52.3 ± 0.6 mW m-2), respectively. To elucidate the specific impacts of OMV formation on EET, OMVs were isolated and quantified for UV-visible spectroscopy and heme staining characterization. Our study showed that abundant outer membrane c-type cytochromes (c-Cyts) including MtrC and OmcA and periplasmic c-Cyts were exposed on the surface or inside of OMVs, which were the vital constituents responsible for EET. Meanwhile, we found that the overproduction of OMVs could facilitate biofilm formation and increase biofilm conductivity. To the best of our knowledge, this study is the first to explore the mechanism of OMV formation and its correlation with EET of S. oneidensis, which paves the way for further study of OMV-mediated EET.

Advances in mechanisms and engineering of electroactive biofilms.

Electroactive biofilms (EABs) are electroactive microorganisms (EAMs) encased in conductive polymers that are secreted by EAMs and formed by the accumulation and cross-linking of extracellular polysaccharides, proteins, nucleic acids, lipids, and other components. EABs are present in the form of multicellular aggregates and play a crucial role in bioelectrochemical systems (BESs) for diverse applications, including biosensors, microbial fuel cells for renewable bioelectricity production and remediation of wastewaters, and microbial electrosynthesis of valuable chemicals. However, naturally occurred EABs are severely limited owing to their low electrical conductivity that seriously restrict the electron transfer efficiency and practical applications. In the recent decade, synthetic biology strategies have been adopted to elucidate the regulatory mechanisms of EABs, and to enhance the formation and electrical conductivity of EABs. Based on the formation of EABs and extracellular electron transfer (EET) mechanisms, the synthetic biology-based engineering strategies of EABs are summarized and reviewed as follows: (i) Engineering the structural components of EABs, including strengthening the synthesis and secretion of structural elements such as polysaccharides, eDNA, and structural proteins, to improve the formation of biofilms; (ii) Enhancing the electron transfer efficiency of EAMs, including optimizing the distribution of c-type cytochromes and conducting nanowire assembly to promote contact-based EET, and enhancing electron shuttles' biosynthesis and secretion to promote shuttle-mediated EET; (iii) Incorporating intracellular signaling molecules in EAMs, including quorum sensing systems, secondary messenger systems, and global regulatory systems, to increase the electron transfer flux in EABs. This review lays a foundation for the design and construction of EABs for diverse BESs applications.

Engineering Shewanella oneidensis to efficiently harvest electricity power by co-utilizing glucose and lactate in thin stillage of liquor industry.

Thin stillage, rich in glucose and lactate, can seriously pollute water resources when directly discharged into the natural environment. Microbial fuel cells (MFC), as a green and sustainable technology, could utilize exoelectrogens to break down organics in wastewater and harvest electricity. Nevertheless, Shewanella oneidensis MR-1, cannot utilize thin stillage for efficient power generation. Here, to enable S. oneidensis to co-utilize glucose and lactate from thin stillage, an engineered S. oneidensis G7∆RSL1 was first created by constructing glucose metabolism pathway, promoting glucose and lactate co-utilization, and enhancing biofilm formation. Then, to enhance biofilm conductivity, we constructed a 3D self-assembled G7∆RSL1-rGO/CNT biohybrid with maximum power density of 560.4 mW m-2 and 373.7 mW m-2 in artificial and actual thin stillage, respectively, the highest among the reported genetically engineered S. oneidensis with thin stillage as carbon source. This study provides a new strategy to facilitate practical applications of MFC in wastewater remediation and efficient power recovery.

Construction of an Acetate Metabolic Pathway to Enhance Electron Generation of Engineered Shewanella oneidensis.

Background: Microbial fuel cells (MFCs) are a novel bioelectrochemical devices that can use exoelectrogens as biocatalyst to convert various organic wastes into electricity. Among them, acetate, a major component of industrial biological wastewater and by-product of lignocellulose degradation, could release eight electrons per mole when completely degraded into CO2 and H2O, which has been identified as a promising carbon source and electron donor. However, Shewanella oneidensis MR-1, a famous facultative anaerobic exoelectrogens, only preferentially uses lactate as carbon source and electron donor and could hardly metabolize acetate in MFCs, which greatly limited Coulombic efficiency of MFCs and the capacity of bio-catalysis. Results: Here, to enable acetate as the sole carbon source and electron donor for electricity production in S. oneidensis, we successfully constructed three engineered S. oneidensis (named AceU1, AceU2, and AceU3) by assembling the succinyl-CoA:acetate CoA-transferase (SCACT) metabolism pathways, including acetate coenzyme A transferase encoded by ato1 and ato2 gene from G. sulfurreducens and citrate synthase encoded by the gltA gene from S. oneidensis, which could successfully utilize acetate as carbon source under anaerobic and aerobic conditions. Then, biochemical characterizations showed the engineered strain AceU3 generated a maximum power density of 8.3 ± 1.2 mW/m2 with acetate as the sole electron donor in MFCs. In addition, when further using lactate as the electron donor, the maximum power density obtained by AceU3 was 51.1 ± 3.1 mW/m2, which approximately 2.4-fold higher than that of wild type (WT). Besides, the Coulombic efficiency of AceU3 strain could reach 12.4% increased by 2.0-fold compared that of WT, which demonstrated that the engineered strain AceU3 can further utilize acetate as an electron donor to continuously generate electricity. Conclusion: In the present study, we first rationally designed S. oneidensis for enhancing the electron generation by using acetate as sole carbon source and electron donor. Based on synthetic biology strategies, modular assembly of acetate metabolic pathways could be further extended to other exoelectrogens to improve the Coulombic efficiency and broaden the spectrum of available carbon sources in MFCs for bioelectricity production.

[Research progress in screening method of exoelectrogens].

Exoelectrogens are promising for a wide variety of potential applications in the areas of environment and energy, which convert chemical energy from organic matter into electrical energy by extracellular electrons transfer (EET). Microorganisms with different mechanisms and EET efficiencies have been elucidated. However, the practical applications of exoelectrogens are limited by their fundamental features. At present, it is difficult to realize the extensive application of exoelectrogens in complex and diverse environments by means of traditional engineering strategies such as rational design and directed evolution. The exoelectrogens with excellent performance in environments can be screened with efficient strain identification technologies, which promote the widespread applications of exoelectrogens. The aims of this review are to summarize the methods of screening based on different types of exoelectrogens, and to outline future research directions of strain screening.

RETRACTED: Silencing circANKRD36 protects H9c2 cells against lipopolysaccharide-induced injury via up-regulating miR-138.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Following the concerns raised about the background pattern of the Western Blots from Figures 1C and 3D, the corresponding author has contacted the journal to request the retraction of the article. Given the comments of Dr Elisabeth Bik regarding this article "This paper belongs to a set of over 400 papers (as per February 2020) that share very similar Western blots with tadpole-like shaped bands, the same background pattern, and striking similarities in title structures, paper layout, bar graph design, and - in a subset - flow cytometry panels", the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

Engineering Microbial Consortia for High-Performance Cellulosic Hydrolyzates-Fed Microbial Fuel Cells.

Microbial fuel cells (MFCs) are eco-friendly bio-electrochemical reactors that use exoelectrogens as biocatalyst for electricity harvest from organic biomass, which could also be used as biosensors for long-term environmental monitoring. Glucose and xylose, as the primary ingredients from cellulose hydrolyzates, is an appealing substrate for MFC. Nevertheless, neither xylose nor glucose can be utilized as carbon source by well-studied exoelectrogens such as Shewanella oneidensis. In this study, to harvest the electricity by rapidly harnessing xylose and glucose from corn stalk hydrolysate, we herein firstly designed glucose and xylose co-fed engineered Klebsiella pneumoniae-S. oneidensis microbial consortium, in which K. pneumoniae as the fermenter converted glucose and xylose into lactate to feed the exoelectrogens (S. oneidensis). To produce more lactate in K. pneumoniae, we eliminated the ethanol and acetate pathway via deleting pta (phosphotransacetylase gene) and adhE (alcohol dehydrogenase gene) and further constructed a synthesis and delivery system through expressing ldhD (lactate dehydrogenase gene) and lldP (lactate transporter gene). To facilitate extracellular electron transfer (EET) of S. oneidensis, a biosynthetic flavins pathway from Bacillus subtilis was expressed in a highly hydrophobic S. oneidensis CP-S1, which not only improved direct-contacted EET via enhancing S. oneidensis adhesion to the carbon electrode but also accelerated the flavins-mediated EET via increasing flavins synthesis. Furthermore, we optimized the ratio of glucose and xylose concentration to provide a stable carbon source supply in MFCs for higher power density. The glucose and xylose co-fed MFC inoculated with the recombinant consortium generated a maximum power density of 104.7 ± 10.0 mW/m2, which was 7.2-folds higher than that of the wild-type consortium (12.7 ± 8.0 mW/m2). Lastly, we used this synthetic microbial consortium in the corn straw hydrolyzates-fed MFC, obtaining a power density 23.5 ± 6.0 mW/m2.

Improved xylose tolerance and 2,3-butanediol production of Klebsiella pneumoniae by directed evolution of rpoD and the mechanisms revealed by transcriptomics.

BACKGROUND: The biological production of 2,3-butanediol from xylose-rich raw materials from Klebsiella pneumoniae is a low-cost process. RpoD, an encoding gene of the sigma factor, is the key element in global transcription machinery engineering and has been successfully used to improve the fermentation with Escherichia coli. However, whether it can regulate the tolerance in K. pneumoniae remains unclear. RESULTS: In this study, the kpC mutant strain was constructed by altering the expression quantity and genotype of the rpoD gene, and this exhibited high xylose tolerance and 2,3-butanediol production. The xylose tolerance of kpC strain was increased from 75 to 125 g/L, and the yield of 2,3-butanediol increased by 228.5% compared with the parent strain kpG, reaching 38.6 g/L at 62 h. The RNA sequencing results showed an upregulated expression level of 500 genes and downregulated expression level of 174 genes in the kpC mutant strain. The pathway analysis further showed that the differentially expressed genes were mainly related to signal transduction, membrane transport, carbohydrate metabolism, and energy metabolism. The nine most-promising genes were selected based on transcriptome sequencing, and were evaluated for their effects on xylose tolerance. The overexpression of the tktA encoding transketolase, pntA encoding NAD(P) transhydrogenase subunit alpha, and nuoF encoding NADH dehydrogenase subunit F conferred increased xylose consumption and increased 2,3-butanediol production to K. pneumoniae. CONCLUSIONS: These results suggest that the xylose tolerance and 2,3-butanediol production of K. pneumoniae can be greatly improved by the directed evolution of rpoD. By applying transcriptomic analysis, the upregulation of tktA, pntA, and nuoF that were coded are essential for the xylose consumption and 2,3-butanediol production. This study will provide reference for further research on improving the fermentation abilities by means of other organisms.

Reduction of biogenic amines production by eliminating the PEP4 gene in Saccharomyces cerevisiae during fermentation of Chinese rice wine.

Biogenic amines in Chinese rice wine have a potential threat of toxicity to human health. In this study, PEP4 gene in Saccharomyces cerevisiae was knocked out in order to evaluate its effect on biogenic amines production; the enzyme encodes proteinase A (PrA), an enzyme that is responsible for the production of free amino acids. It was found that compared to the wild type strain, the PrA activity and amino acid concentration decreased significantly, and the production of biogenic amines in this knockout strain decreased by 25.5%, from 180.1mg/L to 134.2mg/L. Especially, tyramine, cadaverine and histamine concentrations were also decreased by 57.5%, 24.6% and 54.3%, respectively. The main reason for the decrease of biogenic amines may be due to the low concentration of free amino acids. Our results provide a new strategy to minimize the biogenic amine production during fermentation of Chinese rice wine.

A rapid and efficient one-step site-directed deletion, insertion, and substitution mutagenesis protocol.

A rapid and efficient site-directed mutagenesis (SDM) protocol based on two separate polymerase chain reaction (PCR) amplifications and homologous recombination in Escherichia coli is described. This protocol can introduce deletions, substitutions, and insertions into any amplifiable site of the target genes by ligating two amplified DNA fragments into vectors. Compared with previously reported PCR-based SDM methods, our protocol significantly prevents primer dimerization even though partially complementary primers were used for PCR. The genome with the target gene was used directly as template, and DpnI was unnecessary. All of the procedures were performed within 24 h. The mutation frequencies of deletion, substitution, and insertion of the PEP4 (encode proteinase A) gene of Saccharomyces cerevisiae were nearly 100% using this new method. Thus, this method can potentially facilitate high-throughput genetic engineering and structure-function analyses and is useful for molecular biological research.