Abstract The genetic manipulation of basidiomycete mushrooms is notoriously difficult and immature, and there is a lack of research reports on clustered regularly interspaced short palindromic repeat (CRISPR) based gene editing of functional genes in mushrooms. In this work, Ganoderma lucidum, a famous traditional medicinal basidiomycete mushroom, which produces a type of unique triterpenoid-anti-tumor ganoderic acids (GAs), was used, and a CRISPR/CRISPR-associated protein-9 nuclease (Cas9) editing system for functional genes of GA biosynthesis was constructed in the mushroom. As proof of concept, the effect of different gRNA constructs with endogenous u6 promoter and self-cleaving ribozyme HDV on ura3 disruption efficiency was investigated at first. The established system was applied to edit a cytochrome P450 monooxygenase (CYP450) gene cyp5150l8, which is responsible for a three-step biotransformation of lanosterol at C-26 to ganoderic acid 3-hydroxy-lanosta-8, 24-dien-26 oic acid. As a result, precisely edited cyp5150l8 disruptants were obtained after sequencing confirmation. The fermentation products of the wild type (WT) and cyp5150l8 disruptant were analyzed, and a significant decrease in the titer of four identified GAs was found in the mutant compared to WT. Another CYP gene involved in the biosynthesis of squalene-type triterpenoid 2, 3; 22, 23-squalene dioxide, cyp505d13, was also disrupted using the established CRISPR-Cas9 based gene editing platform of G. lucidum. The work will be helpful to strain molecular breeding and biotechnological applications of G. lucidum and other basidiomycete mushrooms.

Microbial production of carotenoids has mainly focused towards a few products, such as β-carotene, lycopene and astaxanthin. However, other less explored carotenoids, like violaxanthin, have also shown unique properties and promissory applications. Violaxanthin is a plant-derived epoxidated carotenoid with strong antioxidant activity and a key precursor of valuable compounds, such as fucoxanthin and β-damascenone. In this study, we report for the first time the heterologous production of epoxycarotenoids in yeast. We engineered the yeast Saccharomyces cerevisiae following multi-level strategies for the efficient accumulation of violaxanthin. Starting from a β-carotenogenic yeast strain, we first evaluated the performance of several β-carotene hydroxylases (CrtZ), and zeaxanthin epoxidases (ZEP) from different species, together with their respective N-terminal truncated variants. The combined expression of CrtZ from Pantoea ananatis and truncated ZEP of Haematococcus lacustris showed the best performance and led to a yield of 1.6 mg/gDCW of violaxanthin. Further improvement of the epoxidase activity was achieved by promoting the transfer of reducing equivalents to ZEP by expressing several redox partner systems. The co-expression of the plant truncated ferredoxin-3, and truncated root ferredoxin oxidoreductase-1 resulted in a 2.2-fold increase in violaxanthin yield (3.2 mg/gDCW). Finally, increasing gene copy number of carotenogenic genes enabled reaching a final production of 7.3 mg/gDCW in shake flask cultures and batch bioreactors, which is the highest yield of microbially produced violaxanthin reported to date.

Urban treatment wetlands purify water and provide various ecological services such as food and habitat. We applied the sustainable structured wetland biotope (SSB) system, which considers site-specific ecological conditions and maximizes water purification efficiency, as a treatment wetland for non-point source pollution with short retention time and high biodiversity. This system is consisted of a forebay area (primary retention basin), a micro-pool area (secondary retention basin), and multi-cell structures of marshes and ponds. A wetland constructed with an SSB system for the treatment of non-point source pollution was completed at a site downstream from Jecheon city, located in the upper river basin of the Han River in the Republic of Korea. In addition to the SSB system, a rainwater detention tank for the inflow of the SSB was constructed. During the initial rainfall event, water through SSB were significantly purified (83–98% in BOD, 96–99% in SS, 41–90% in T-N, and 79–96% in T-P of removal efficiency). The flora in the SSB and its neighboring areas consisted of 125 species of 45 families. Three amphibian species, three reptile species, 17 benthic macroinvertebrate species, and 18 avian species were observed in the wetland. We recorded five mammals, including an endangered Eurasian otter species in Korea, Lutra lutra, present in the wetland. Eight fish species were also observed. The wetland showed high removal efficiency without a specific water-treatment device. In addition, local plant and animal species, including rare species, seemed to successfully settle in the SSB.

Ecological benefits and production of grassland on the Loess Plateau are limited by low soil N and P availability. Extraneous N and P fertilization is an efficient management measure to enhance grassland productivity and accelerate grassland restoration. However, biodiversity decline and species loss induced by N and P addition must be noticed. Two grassland communities dominated by tall clonal grass (TCG) and tall clonal forb (TCF) on Loess Plateau were selected. A two-year split-plot experiment (main-plot: 0, 25, 50, and 100 kgN ha−1 yr−1; subplot: 0, 20, 40 and 80 kg P2O5 ha−1 yr−1) was conducted to evaluate the effect of N and P addition on aboveground net primary production (ANPP), light availability, diversity and functional group composition. N and P addition slightly increased ANPP mainly benefiting from clonal species and had few effects on species composition and diversity due to the deficient precipitation during growing season in 2017. However, N or P addition alone resulted in significant ANPP increase, and adding N and P together had larger effects in 2018. The changes in ANPP and diversity after P alone addition were driven by legumes. The ANPP responses to 50 and 100 kg N ha−1 yr−1 combined with P addition in 2018 were mainly driven by pronounced increases in tall clonal or annual species. This large shift of species composition caused diversity decline only in TCF community. The extent of diversity decline was significantly correlated with the degree of light availability reduction. Diversity decline in TCF was a result of strong and uniform light availability reduction induced by increased tall annual forb. Tall clonal and annual species are the key functional groups that drive changes in productivity and diversity in the two communities. The optimum N and P amount to tradeoff productivity improvement and diversity decline were 50 kg N ha−1 yr−1 and 20 kg P2O5 ha−1 yr−1 combination for TCG and 25 kg N ha−1 yr−1 and 20 kg P2O5 ha−1 yr−1 combination for TCF.

The purpose of the present study was to evaluate the anti‐depressant effect of Deoxyschizandrin (DEO) in chronic unpredictable mild stress (CUMS)‐induced mice. The mice were subjected to CUMS paradigm for 8 weeks. From the sixth week, the mice were intragastrically treated with DEO once daily for continuous 3 weeks. The behavior tests including sucrose preference test (SPT), Forced swimming test (FST), Tail suspension test (TST), Open field test (OFT) were conducted. Additionally, the expressions of TLR4, MyD88, TRAF6, p‐NF‐κBp65, NLRP3, cleaved caspase‐1, cleaved IL‐1β, GluR and PSD95 in hippocampus were detected by western blot. The concentrations of IL‐6 and TNF‐α in hippocampus were determined by ELISA. The dendritic spine density was observed by Golgi‐Cox staining. As a result, the treatment with DEO relieved anhedonia in SPT, reduced immobile duration in FST and TST. DEO treatment effectively attenuated the CUMS‐caused alterations of TLR4, MyD88, TRAF6, p‐NF‐κBp65, NLRP3, cleaved caspase‐1, cleaved IL‐1β, GluR and PSD95. Furthermore, DEO could reduce the hippocampal inflammatory cytokine content and increase the density of dendritic spine. In conclusion, the present work indicated that DEO exhibited anti‐depressant effect on CUMS‐induced depressive mice, which was possibly due to the TLR4/NF‐κB/NLRP3 pathway and the amelioration of dendritic spine density through GluR/PSD95 cascade.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Stem cell-based therapy has been considered as a potential treatment to restore spinal cord injury (SCI) through reconstructing neural networks and providing a favorable microenvironment for neuronal survival, differentiation, and axonal outgrowth. Biomaterial scaffolds can promote cell attachment and survival, neuronal differentiation, and axonal outgrowth, therefore they were used to combine with stem cells for implantation in SCI treatment. In addition, a longitudinal scaffold can guide regenerated axons with orientated growth and axial extension. Both human umbilical cord-derived mesenchymal stem cells (hMSCs) and human fetal spinal cord-derived neural stem cells (hNSCs) have been applied in clinical trials worldwide. To our knowledge, a parallel comparison of the therapeutic effects of hMSC and hNSC implantations has not been conducted. Hence, in this study, we grafted hMSCs or hNSCs seeded on longitudinal collagen sponge scaffolds (LCSSs) into rats with completely transected SCI to examine differences in SCI repair. Both hMSCs and hNSCs had equivalent effects on reducing glial scar formation around the lesion gap. More neuronal class III β-tubulin-positive neurons and neurofilament-positive nerve fibers were found in the lesion cavity after hNSC implantation. In addition, hNSCs had better capabilities to improve motor function, attenuate inflammation, and promote cell survival than hMSCs. These encouraging results provide a clinical basis for future stem cell-based SCI therapies.

Protocatechuic acid (PCA) and hydroquinone (HQ) are two important phenolic molecules with recognized biological activities and industrial values. In the present study, we constructed a series of E. coli mono-cultures and co-cultures for the PCA and HQ biosynthesis from simple carbon substrate glucose. Metabolic engineering strategies, include over-expression of key pathway enzymes and utilization of biosensor-assisted cell selection, were adapted to improve the pathway intermediate supply and the overall biosynthesis. The comparative analysis showed that the adapted strategies had different degrees of impacts on the biosynthesis performance of the constructed mono-cultures and co-cultures. The highest PCA and HQ bioproduction (641 and 303 mg/L, respectively) was achieved using the optimized co-culture. Our findings show that, compared with mono-cultures, rationally designed co-cultures provide a new perspective for application of advanced metabolic engineering strategies.

Astaxanthin exhibiting strong antioxidant activity, is widely used as food and animal feed additives as well as in cosmetics, pharmaceutical and nutraceuticals industries. Microbial production of astaxanthin has received increasing concerns in recent years. A combined strategy consisting of physical mutagenesis by atmospheric and room temperature plasma (ARTP) and adaptive evolution driven by H2O2, was established to expand disturbance in genome of the engineered Saccharomyces cerevisiae. A titer of 65.9 mg/L astaxanthin at flask level was obtained via multiple rounds of crossed ARTP and H2O2 treatment, which was increased nearly 4-fold compared with the starting strain. Moreover, a trend of decreasing intracellular reactive oxygen species (ROS) and increasing chronological lifespan (CLS) was observed in cells with improved titer of astaxanthin. Eventually, a highest reported titer of 404.78 mg/L astaxanthin in S. cerevisiae was achieved in 5-L fermenter by further optimization of fermentation conditions. Our study not only highlights the iterative cycling of the integrated strategy combined by ARTP and adaptive evolution tightly based on the natural properties of the targets provides an efficient route to boost product accumulation, but also demonstrates it can serve as a general strategy for other microbial cell factories.

Cynomorium songaricum Rupr is widely known in China as a traditional herbal medicine. In this study, single‐factor experiments and response surface methodology were used to optimize the extraction of Cynomorium songaricum Rupr glycoprotein (CSG). The results show that a maximum glycoprotein yield of 6.39 ± 0.32% was achieved at a ratio of solid to liquid 32:1 for 4.2 h at 52 °C. Then the IR, monosaccharide composition, amino acid composition, type of glycopeptide linkage, and average molecular weight of CSG‐1 purified from CSG were characterized. The results indicate that CSG‐1 presented the characteristic absorption peak of polysaccharide and protein, including four monosaccharides and 17 amino acids, had O‐linked glycopeptide bonds, Mw, Wn, Mw/Mn, Mp, and the z‐average were 5.343 × 106, 3.203 × 106, 1.668, 8.911 × 106, and 6.948 × 106, respectively. Besides, CSG‐1 solution was described by the Herschel‐Bulkley model and it behaved as a shear‐thinning fluid. Also, under a frequency sweep the moduli G´ and G´´ both increased with increasing CSG‐1 concentration and the CSG‐1 dispersions had weak thermal stability over the temperature sweep. These results provide a scientific basis for the further study of Cynomorium songaricum Rupr.

Curcumin is extensively used in the prevention and treatment of various diseases. Recently, growing attention has been paid to the use of curcumin as a neurogenic and neuroprotective agent. This review study aimed to collect and categorize the recent findings regarding the effects of curcumin on various neurological diseases through the induction of neural stem cell proliferation and differentiation. In addition, we have discussed the molecular mechanisms modulated by curcumin that contribute to this efficacy and have summarized the recent advancements in the novel delivery strategies used to improve the induction of neural stem cells by curcumin.

Abstract Background Haematococcus pluvialis is the best source of natural astaxanthin, known as the king of antioxidants. H. pluvialis have four cell forms: spore, motile cell, non-motile cell and akinete. Spores and motile cells are susceptible to photoinhibition and would die under photoinduction conditions. Photoinduction using non-motile cells as seeds could result in a higher astaxanthin production than that using akinetes. However, the mechanism of this phenomenon has not been clarified. Results Transcriptome was sequenced and annotated to illustrate the mechanism of this phenomenon. All differentially expressed genes involved in astaxanthin biosynthesis were up-regulated. Particularly, chyb gene was up-regulated by 16-fold, improving the conversion of β-carotene into astaxanthin. Pyruvate was the precursor of carotenoids biosynthesis. Pyruvate kinase gene expression level was increased by 2.0-fold at the early stage of akinetes formation. More changes of gene transcription occurred at the early stage of akinetes formation, 52.7% and 51.9% of total DEGs in control group and treatment group, respectively. Conclusions Genes transcription network was constructed and the synthesis mechanism of astaxanthin was clarified. The results are expected to further guide the in-depth optimization of the astaxanthin production process in H. pluvialis by improving pyruvate metabolism.

Antibodies have been extensively used for the purpose of scientific research, clinical diagnosis and therapy. Combination of in vitro somatic hypermutation and mammalian cell surface display has been an efficient technology for antibody or other proteins optimization, in which the efficiency of activation‐induced cytidine deaminase (AID) mutations in genes is one of the most important key factors. Gene transcriptional level has been found to be positively proportional to AID‐induced mutation frequency. Thus, construction of the cell clone bearing a gene of interest (GOI) with high transcription level can increase AID‐induced mutations. In this study, we inserted a retargetable gene cassette to predetermined chromosome site (ywhae gene site) which is among the genes with the highest as well as stable transcription, and found that one sub‐site was suitable to be retargeted for efficient protein display in CHO cells. The resultant cell clone (T31) had higher and more stable transcription/expression than CHO‐puro clone which was previously established through the strategy of random insertion followed by a high‐throughput selection. It also possessed a significantly higher mutation frequency to GOI than CHO‐puro cells, thus it was a better clone for the in vitro improvement of antibody affinity, and probably other properties.

Wild type Escherichia coli usually does not accumulate L‐threonine, but E. coli strain TWF001 could produce 30.35 g/L L‐threonine after 23‐h fed‐batch fermentation. To understand the mechanism for the high yield of L‐threonine production in TWF001, transcriptomic analyses of the TWF001 cell samples collected at the logarithmic and stationary phases were performed, using the wild type E. coli strain W3110 as the control. Compared with W3110, 1739 and 2361 genes were differentially transcribed in the logarithmic and stationary phases, respectively. Most genes related to the biosynthesis of L‐threonine were significantly up‐regulated. Some key genes related to the NAD(P)H regeneration were up‐regulated. Many genes relevant to glycolysis and TCA cycle were down‐regulated. The key genes involved in the L‐threonine degradation were down‐regulated. The gene rhtA encoding the L‐threonine exporter was up‐regulated while the genes sstT and tdcC encoding the L‐threonine importer were down‐regulated. The up‐regulated genes in glutamate pathway might form an amino‐providing loop which is beneficial for high yield of L‐threonine production. Many genes encoding 30S and 50S subunits of ribosomes were also up‐regulated. The findings are useful for gene engineering to increase L‐threonine production in E. coli.

The chemokine CXCL10 is released by a plethora of cells, including immune and metastatic cancer cells, following stimulation with interferon-gamma. It acts via its GPC receptor on T-cells attracting them to various target tissues. Glycosaminoglycans (GAGs) are regarded as co-receptors of chemokines, which enable the establishment of a chemotactic gradient for target cell migration. We have engineered human CXCL10 towards improved T-cell mobilisation by implementing a single site-directed mutation N20K into the protein, which leads to a higher GAG binding affinity compared to the wild type. Interestingly, this mutation not only increased T-cell migration in a transendothelial migration assay, the mutant intensified T-cell chemotaxis also in a Boyden chamber set-up thereby indicating a strong role of T-cell-localised GAGs on leukocyte migration. A CXCL10 mutant with increased GAG-binding affinity could therefore potentially serve as a T-cell mobiliser in pathological conditions where the immune surveillance of the target tissue is impaired, as is the case for most solid tumors.

Metabolic engineering efforts that harness living organisms to produce natural products and other useful chemicals face inherent difficulties because the maintenance of life processes often runs counter to our desire to maximize important production metrics. These challenges are particularly problematic for commodity chemical manufacturing where cost is critical. A cell-free approach, where biochemical pathways are built by mixing desired enzyme activities outside of cells, can obviate problems associated with cell-based methods. Yet supplanting cell-based methods of chemical production will require the creation of self-sustaining, continuously operating systems where input biomass is converted into desired products at high yields, productivities, and titers. We call the field of designing and implementing reliable and efficient enzyme systems that replace cellular metabolism, synthetic biochemistry.

Collagen is the key protein of connective tissue (i.e., skin, tendons and ligaments, cartilage, among others) accounting for 25% to 35% of the whole-body protein content, and entitled of conferring mechanical stability. This protein is also a fundamental building block of bone due to its excellent mechanical properties together with carbonated hydroxyapatite minerals. While the mechanical resilience and viscoelasticity have been studied both in vitro and in vivo from the molecule to tissue level, wave propagation properties and energy dissipation have not yet been deeply explored, in spite of being crucial to understand the vibration dynamics of collagenous structures (e.g., eardrum, cochlear membranes) upon impulsive loads. By using a bottom-up atomistic modelling approach, here we study a collagen peptide under two distinct impulsive displacement loads, including longitudinal and transversal inputs. Using a one-dimensional string model as a model system, we investigate the roles of hydration and load direction on wave propagation along the collagen peptide and the related energy dissipation. We find that wave transmission and energy-dissipation strongly depend on the loading direction. Also, the hydrated collagen peptide can dissipate five times more energy than dehydrated one. Our work suggests a distinct role of collagen in term of wave transmission of different tissues such as tendon and eardrum. This study can step towards understanding the mechanical behaviour of collagen upon transient loads, impact loading and fatigue, and designing biomimetic and bio-inspired materials to replace specific native tissues such as the tympanic membrane.

Biodistribution of photosensitizer (PS) in photodynamic therapy (PDT) can be assessed by fluorescence imaging that visualizes the accumulation of PS in malignant tissue prior to PDT. At the same time, excitation of the PS during an assessment of its biodistribution results in premature photobleaching and can cause toxicity to healthy tissues. Combination of PS with a separate fluorescent moiety, which can be excited apart from PS activation, provides a possibility for fluorescence imaging (FI) guided delivery of PS to cancer site, followed by PDT. In this work, we report nanoformulations (NFs) of core–shell polymeric nanoparticles (NPs) co-loaded with PS [2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a, HPPH] and near infrared fluorescent organic dyes (NIRFDs) that can be excited in the first or second near-infrared windows of tissue optical transparency (NIR-I, ~ 700–950 nm and NIR-II, ~ 1000–1350 nm), where HPPH does not absorb and emit. After addition to nanoparticle suspensions, PS and NIRFDs are entrapped by the nanoparticle shell of co-polymer of N-isopropylacrylamide and acrylamide [poly(NIPAM-co-AA)], while do not bind with the polystyrene (polySt) core alone. Loading of the NIRFD and PS to the NPs shell precludes aggregation of these hydrophobic molecules in water, preventing fluorescence quenching and reduction of singlet oxygen generation. Moreover, shift of the absorption of NIRFD to longer wavelengths was found to strongly reduce an efficiency of the electronic excitation energy transfer between PS and NIRFD, increasing the efficacy of PDT with PS-NIRFD combination. As a result, use of the NFs of PS and NIR-II NIRFD enables fluorescence imaging guided PDT, as it was shown by confocal microscopy and PDT of the cancer cells in vitro. In vivo studies with subcutaneously tumored mice demonstrated a possibility to image biodistribution of tumor targeted NFs both using HPPH fluorescence with conventional imaging camera sensitive in visible and NIR-I ranges (~ 400–750 nm) and imaging camera for short-wave infrared (SWIR) region (~ 1000–1700 nm), which was recently shown to be beneficial for in vivo optical imaging. A combination of PS with fluorescence in visible and NIR-I spectral ranges and, NIR-II fluorescent dye allowed us to obtain PS nanoformulation promising for see-and-treat PDT guided with visible-NIR-SWIR fluorescence imaging.

Extracellular vesicles (EVs) have shown great potential for targeted therapy, as they have a natural ability to pass through biological barriers and, depending on their origin, can preferentially accumulate at defined sites, including tumors. Analyzing the potential of EVs to target specific cells remains challenging, considering the unspecific binding of lipophilic tracers to other proteins, the limitations of fluorescence for deep tissue imaging and the effect of external labeling strategies on their natural tropism. In this work, we determined the cell-type specific tropism of B16F10-EVs towards cancer cell and metastatic tumors by using fluorescence analysis and quantitative gold labeling measurements. Surface functionalization of plasmonic gold nanoparticles was used to promote indirect labeling of EVs without affecting size distribution, polydispersity, surface charge, protein markers, cell uptake or in vivo biodistribution. Double-labeled EVs with gold and fluorescent dyes were injected into animals developing metastatic lung nodules and analyzed by fluorescence/computer tomography imaging, quantitative neutron activation analysis and gold-enhanced optical microscopy. We determined that B16F10 cells preferentially take up their own EVs, when compared with colon adenocarcinoma, macrophage and kidney cell-derived EVs. In addition, we were able to detect the preferential accumulation of B16F10 EVs in small metastatic tumors located in lungs when compared with the rest of the organs, as well as their precise distribution between tumor vessels, alveolus and tumor nodules by histological analysis. Finally, we observed that tumor EVs can be used as effective vectors to increase gold nanoparticle delivery towards metastatic nodules. Our findings provide a valuable tool to study the distribution and interaction of EVs in mice and a novel strategy to improve the targeting of gold nanoparticles to cancer cells and metastatic nodules by using the natural properties of malignant EVs.

The ureolytic bacterium Sporosarcina pasteurii is well-known for its capability of microbially induced calcite precipitation (MICP), representing a great potential in constructional engineering and material applications. However, the molecular mechanism for its biomineralization remains unresolved, as few studies were carried out. The addition of urea into the culture medium provided an alkaline environment that is suitable for S. pasteurii. As compared to S. pasteurii cultivated without urea, S. pasteurii grown with urea showed faster growth and urease production, better shape, more negative surface charge and higher biomineralization ability. To survive the unfavorable growth environment due to the absence of urea, S. pasteurii up-regulated the expression of genes involved in urease production, ATPase synthesis and flagella, possibly occupying resources that can be deployed for MICP. As compared to non-mineralizing bacteria, S. pasteurii exhibited more negative cell surface charge for binding calcium ions and more robust cell structure as nucleation sites. During MICP process, the genes for ATPase synthesis in S. pasteurii was up-regulated while genes for urease production were unchanged. Interestingly, genes involved in flagella were down-regulated during MICP, which might lead to poor mobility of S. pasteurii. Meanwhile, genes in fatty acid degradation pathway were inhibited to maintain the intact cell structure found in calcite precipitation. Both weak mobility and intact cell structure are advantageous for S. pasteurii to serve as nucleation sites during MICP. Four factors are demonstrated to benefit the super performance of S. pasteurii in MICP. First, the good correlation of biomass growth and urease production of S. pasteurii provides sufficient biomass and urease simultaneously for improved biomineralization. Second, the highly negative cell surface charge of S. pasteurii is good for binding calcium ions. Third, the robust cell structure and fourth, the weak mobility, are key for S. pasteurii to be nucleation sites during MICP.

L-Asparaginase (L-ASNase) is a key component in the treatment of acute lymphoblastic leukemia (ALL), but several clinical disadvantages, such as immunogenicity and rapid clearance, are still present. We evaluated the possibility to synthesize a new L-ASNase from Escherichia coli with human-like glycosylation and study the glycosylation effect on the biochemical properties of the enzyme. Six L-ASNase mutants were also created in which L-ASNase glycosylation sites were removed through site-directed mutagenesis. The WT L-ASNase was successfully expressed, secreted and glycosylated by an engineered P. pastoris strain and presented predominantly Man5GlcNAc2 glycans on its structure, which were then able to decrease L-ASNase immunogenicity in vitro. The purified glycosylated L-ASNase has shown a 30-fold decrease in specific enzymatic activity compared to the non-glycosylated proteoform, but a triple mutant L-ASNase (3 M) was able to restore L-ASNase biological activity to significant levels. 3 M accumulated in the yeast periplasmic space and there presented a 28-fold increase in enzymatic activity when compared to the fully glycosylated proteoform. Both WT and 3 M L-ASNases presented increased stability in human serum compared to non-glycosylated L-ASNase. This study demonstrates the important effects of glycosylation on L-ASNase properties and opens up new possibilities to use glycosylated L-ASNases for the treatment of ALL.

Bioreactor design is a challenging endeavour that aims to provide the most ideal environment in which cells can grow and biological reactions can occur. The emergence of regenerative medicine and stem cell therapies has led to the need for more diverse environmental requirements in the bioreactor design space. The study presented uses an additive manufacturing approach for the initial design phase of a packed/fluidized bed bioreactor for mesenchymal stem cell expansion. Combining 3D-printing with CFD for precision control over the bioreactor flow dynamics. Novel flow distributors were developed to engender swirling particle fluidization. The design was simulated and optimised using CFD, demonstrating an increase from 0.01 m/s to 0.02 m/s in the radial velocity of 3.0 mm macrocarriers (1080 kg/m3) at the minimum fluidization velocity. An autoclavable prototype was constructed to illustrate proof-of-concept in the use of swirling flow distribution to enhance cell attachment efficiency (compared to static culture system). Commercial Cytodex 1 carriers were tested: an improvement in attachment efficiency after 24 hours from 50% to 95% was induced by the swirling flow distributor, with subsequent expansion of 2.4-fold after 6 days of culture. The computational design, modelling and 3D-printing of complex geometric architecture that control the flow dynamics within a bioreactor, provides a novel approach to bioprocess unit operation development for manufacturing novel ATMPs.

Competitive sustainable production in industry demands new and better biocatalysts, optimized bioprocesses and cost-effective product recovery. Our review sheds light on the progress made for the individual steps towards these goals, starting with the discovery of new enzymes and their corresponding genes. The enzymes are subsequently engineered to improve their performance, combined in reaction cascades to expand the reaction scope and integrated in whole cells to provide an optimal environment for the bioconversion. Strain engineering using synthetic biology methods tunes the host for production, reaction design optimizes the reaction conditions and downstream processing ensures the efficient recovery of commercially viable products. Selected examples illustrate how modified enzymes can revolutionize future-oriented applications ranging from the bioproduction of bulk-, specialty- and fine chemicals, active pharmaceutical ingredients and carbohydrates, over the conversion of the greenhouse-gas CO2 into valuable products and biocontrol in agriculture, to recycling of synthetic polymers and recovery of precious metals.

Cardiac tissue engineering holds great potential in regenerating functional cardiac tissues for various applications. The major strategy is to design scaffolds recapitulating the native cardiac microenvironment to enhance cell and tissue functionalities. Among various biomaterial systems, nanofibrous matrices with aligned morphologies and enhanced conductivity incline to induce the formation of oriented engineered cardiac tissues with enhanced functionalities. The challenge is to functionalize the scaffolds with conductive additives without influencing their biocompatibility. In this study, we developed a fully aqueous process for the fabrication of conductive carbon nanotube/silk fibroin (CNT/silk) electrospun scaffolds. The carbon nanotubes are well dispersed within the nanofibers, providing the scaffolds with enhanced conductivity and excellent biocompatibility for the culture of neonatal rat cardiomyocytes with improved cell spreading and enhanced expression of cardiac-specific proteins. Moreover, the aligned CNT/silk fibroin composite scaffolds exhibit abilities to guide the oriented organization of cardiac tissues and the biomimicking distribution of sarcomeres and gap junctions. The findings demonstrate the great potential of the CNT/silk scaffolds prepared through this aqueous processing method in supporting the formation of cardiac tissues with enhanced functionalities.

The vocal fold lamina propria (VFLP), one of the outermost layers of the vocal fold (VF), is composed of tissue-specific extracellular matrix (ECM) proteins and is highly susceptible to injury. Various biomaterials have been clinically tested to treat voice disorders (e.g., hydrogels, fat, hyaluronic acid), but satisfactory recovery of the VF functionality remains elusive. Fibrosis or scar formation in the VF is a major challenge, and the development and refinement of novel therapeutics that promote the healing and normal function of the VF are needed. Injectable hydrogels derived from native tissues have been previously reported with major advantages over synthetic hydrogels, including constructive tissue remodeling and reduced scar tissue formation. This study aims to characterize the composition of a decellularized porcine VFLP-ECM scaffold and the cytocompatibility and potential anti-fibrotic properties of a hydrogel derived from VFLP-ECM hydrogel. In addition, we isolated potential matrix-bound vesicles (MBVs) and macromolecules from the VFLP-ECM that also downregulated smooth muscle actin ACTA2 under TGF-β1 stimulation. The results provide evidence of the unique protein composition of VFLP-ECM and the potential link between the components of VFLP-ECM and the inhibition of transforming growth factor-beta 1 (TGF-β1) signaling observed in vitro when transformed into injectable forms.

Abstract Microbial community structure and metabolic activities have profound impacts on biogeochemical processes in marine sediments. Functional bacteria such as nitrate- and sulfate-reducing bacteria respond to redox gradients by coupling specific reactions amenable to relevant energy metabolisms. However, similar functional patterns have not been observed for sedimentary archaea (except for anaerobic methanotrophs and methanogens). We coupled taxonomic composition with comprehensive geochemical species to investigate the participation of distinct bacteria and archaea in sedimentary geochemical cycles in a sediment core (300 cm) from Pearl River Estuary (PRE). Geochemical properties (NO3−, dissolved Mn and Fe, SO42+, NH4+; dissolved inorganic carbon (DIC), δ13CDIC, dissolved organic carbon (DOC), total organic carbon (TOC), δ13CTOC, and fluorescent dissolved organic matter (FDOM)) exhibited strong depth variability of different trends. Bacterial 16S rRNA- and dsrB gene abundance decreased sharply with depth while archaeal and bathyarchaeotal 16S rRNA gene copies were relatively constant. This resulted in an increase in relative abundance of archaea from surface (11.6%) to bottom (42.8%). Network analysis showed that bacterial groups of Desulfobacterales, Syntrophobacterales and Gammaproteobacteria were significantly (P < 0.0001) associated with SO42− and dissolved Mn while archaeal groups of Bathyarchaeota, Group C3 and Marine Benthic Group D (MBGD) showed close positive correlations (P < 0.0001) with NH4+, δ13CTOC values and humic-like FDOM. Our study suggested that these bacterial groups dominated in redox processes relevant to sulfate or metal oxides, while the archaeal groups are more like to degrade recalcitrant organic compounds in anaerobic sediments.

The brain machine interface (BMI) describes a group of technologies capable of communicating with excitable nervous tissue within the central nervous system (CNS). BMI’s have seen major advances in recent years but these advances have been impeded due to a deterioration in the signal to noise ratio of recording electrodes over time following insertion into the CNS. This deterioration has been attributed to an intrinsic host tissue response, namely reactive gliosis. Gliosis involves a complex series of immune mediators resulting in encapsulation of implants via the synthesis of pro-inflammatory signaling molecules and the recruitment of glial cells. There is a clinical need to reduce tissue encapsulation in situ and improve long-term neuroelectrode functionality. Physical modification of the electrode surface at the nanoscale could satisfy these requirements by integrating electrochemical and topographical signals to modulate neural cell behaviour. In this study, commercially available platinum iridium (Pt/Ir) microelectrode probes and planar Pt/Ir substrates were nanotopographically (NT) functionalized using femto/picosecond laser processing to generate laser induced periodic surface structures (LIPSS). Three different topographies and their physical properties were assessed by scanning electron microscopy and atomic force microscopy. The electrochemical properties of these interfaces were investigated using electrochemical impedance spectroscopy and cyclic voltammetry. The in vitro response of mixed cortical cultures (embryonic rat E14/E17), was subsequently assessed by confocal microscopy, ELISA and multiplex protein array analysis. Overall LIPSS features improved electrochemical properties of the electrodes, promoted cell alignment and modulated ion channel expression all involved in key neuronal functions. Neuroelectrodes functionalized with nanotopographical LIPSS features were demonstrated on a microwire probe geometry for the first time. Results indicate that LIPSS can promote/enhance chronic electrode functionality whilst promoting an aligned cell network at the electrode interface.

Mesenchymal stem cells (MSCs) based regenerative medicine is widely considered as a promising approach for repairing tissue and re-establishing function in spinal cord injury (SCI). However, low survival rate, uncontrollable migration and differentiation of stem cells after implantation, represent major challenges towards the clinical deployment of this approach. In this study, we fabricated three-dimensional MSCs laden microfibers via electrospinning in a rotating cell culture to mimic nerve tissue, control stem cell behavior and promote integration with the host tissue. The hierarchically aligned fibrin hydrogel was used as the MSCs carrier though a rotating methods, and the aligned fiber structure induced the MSCs aligned adhesion on the surface of the hydrogel to form micro cell fibers. The MSCs laden microfiber implantation enhances the donor MSCs neural differentiation and encourages the migration of host neurons into the injury gap, and significantly promotes nerve fiber regeneration across the injury site. Abundant GAP-43 and NF positive nerve fibers were observed to regenerate in the caudal, rostral and middle sites of the injury position eight weeks after the surgery. The NF fiber density reached to 29±6 per 0.25 mm2 at the middle site, 82±13 per 0.25 mm2 at the adjacent caudal site, and 70±23 at the adjacent rostral site respectively. Similarly, motor axons labeled with 5-HT were significantly regenerated in the injury gap, which was 122±22 at the middle injury site that was beneficial for motor function recovery. Most remarkably, the transplantation of MSCs laden micro fibers significantly improves electrophysiological expression and re-establishes limb motor function. These findings highlight the combination of MSCs with micro hydrogel fibers, and may become a promising method for MSCs implantation and SCI repair.

The common treatment of epithelial ovarian cancer is aggressive surgery followed by platinum-based cytotoxic chemotherapy. However, residual tumor cells are resistant to chemotherapeutic drugs during postoperative recurrence. The treatment of ovarian cancer requires new breakthroughs and advances. In recent years, magnesium alloy has been widely developed as a new biodegradable material because of its great potential in the field of medical devices. From the degradation products of magnesium, biodegradable magnesium implants have great potential in anti-tumor. According to the disease characteristics of ovarian cancer, we choose it to study the anti-tumor characteristics of biodegradable magnesium. We tested the anti-ovarian tumor properties of Mg through both in vivo and in vitro experiments. According to the optical in vivo imaging and relative tumor volume statistics of mice, high-purity Mg wires significantly inhibited the growth of SKOV3 cells in vivo. We find that the degradation products of Mg, Mg2+ and H2, significantly inhibit the growth of SKOV3 cells and promote their apoptosis. Our study suggests a good promise for treatment of ovarian cancer.