Long-term exposure to microplastics induces intestinal function dysbiosis in rare minnow (Gobiocypris rarus).

Microplastics are ubiquitous in the natural environment, especially in waters, and their potential impact is also a key issue of concern. In this study, we used 1 µm, 1000 µg/L, polystyrene (PS-MPs) particles to analyze the effects after exposure for 14 and 28 days in rare minnow (Gobiocypris rarus). Results indicated that PS-MPs induce structural alterations in the intestinal tissue, including epithelial damage, villi damage and the inflammatory cell infiltration, while the changes were severer after exposure for 28 days. Polystyrene microplastics also significantly increased the activities of catalase (CAT, increased 142 % and 385 % in 14d and 28d), superoxide dismutase (SOD, increased 17.76 % and 23.43 % in the 14d and 28d) and the content of malondialdehyde (MDA, increased 14.5 % and 442 % in the 14d and 28d), glutathione (GSH, increased 146 % and 298 % in the 14d and 28d). The results not only showed the characterization of gut microbial communities in rare minnow, but also indicated that microbial diversity and composition were altered in gut of fish exposed to PS-MPs. In the control groups, Proteobacteria (31.36-54.54 %), Actinobacteriota (39.99-52.54 %), Fusobacteriota (1.43-1.78 %), Bacteriadota (0.31-0.57 %) were the four dominant bacterial phyla in the intestinal of rare minnow. After exposure to microplastics, In the gut microbiota, the proportion of Proteobacteria increased 9.27 % and 30 % with exposure time, while Actinobacteria decreased 37.89 % and significantly different after 28 days. In addition, metabolomic analysis suggested that exposure to PS-MPs induced alterations of metabolic profiles in rare minnow and differential metabolites were involved in energy metabolism, inflammatory responsible secretion, oxidative stress, nucleotide and its metabolomics. In conclusion, our findings suggest that long-term exposure to microplastics could induce intestinal inflammation, oxidative stress, microbiota dysbiosis and metabolic disorder in rare minnow, and the alterations and severity were exacerbated by prolonged exposure. This study has extended our cognition of the toxicity of polystyrene, and enriched theoretical data for exploring the toxicological mechanism of microplastics.

Hybrid elastin-like polypeptide-polyethylene glycol (ELP-PEG) hydrogels with improved transparency and independent control of matrix mechanics and cell ligand density.

Hydrogels have been developed as extracellular matrix (ECM) mimics both for therapeutic applications and basic biological studies. In particular, elastin-like polypeptide (ELP) hydrogels, which can be tuned to mimic several biochemical and physical characteristics of native ECM, have been constructed to encapsulate various types of cells to create in vitro mimics of in vivo tissues. However, ELP hydrogels become opaque at body temperature because of ELP's lower critical solution temperature behavior. This opacity obstructs light-based observation of the morphology and behavior of encapsulated cells. In order to improve the transparency of ELP hydrogels for better imaging, we have designed a hybrid ELP-polyethylene glycol (PEG) hydrogel system that rapidly cross-links with tris(hydroxymethyl) phosphine (THP) in aqueous solution via Mannich-type condensation. As expected, addition of the hydrophilic PEG component significantly improves the light transmittance. Coherent anti-Stokes Raman scattering (CARS) microscopy reveals that the hybrid ELP-PEG hydrogels have smaller hydrophobic ELP aggregates at 37 °C. Importantly, this hydrogel platform enables independent tuning of adhesion ligand density and matrix stiffness, which is desirable for studies of cell-matrix interactions. Human fibroblasts encapsulated in these hydrogels show high viability (>98%) after 7 days of culture. High-resolution confocal microscopy of encapsulated fibroblasts reveals that the cells adopt a more spread morphology in response to higher RGD ligand concentrations and softer gel mechanics.

Characterization of genes for chitin catabolism in Haloferax mediterranei.

Chitin is the second most abundant natural polysaccharide after cellulose. But degradation of chitin has never been reported in haloarchaea. In this study, we revealed that Haloferax mediterranei, a metabolically versatile haloarchaeon, could utilize colloidal or powdered chitin for growth and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) accumulation, and the gene cluster (HFX_5025-5039) for the chitin catabolism pathway was experimentally identified. First, reverse transcription polymerase chain reaction results showed that the expression of the genes encoding the four putative chitinases (ChiAHme, ChiBHme, ChiCHme, and ChiDHme, HFX_5036-5039), the LmbE-like deacetylase (DacHme, HFX_5027), and the glycosidase (GlyAHme, HFX_5029) was induced by colloidal or powdered chitin, and chiA Hme, chiB Hme, and chiC Hme were cotranscribed. Knockout of chiABC Hme or chiD Hme had a significant effect on cell growth and PHBV production when chitin was used as the sole carbon source, and the chiABCD Hme knockout mutant lost the capability to utilize chitin. Knockout of dac Hme or glyA Hme also decreased PHBV accumulation on chitin. These results suggested that ChiABCDHme, DacHme, and GlyAHme were indeed involved in chitin degradation in H. mediterranei. Additionally, the chitinase assay showed that each chitinase possessed hydrolytic activity toward colloidal or powdered chitin, and the major product of colloidal chitin hydrolysis by ChiABCDHme was diacetylchitobiose, which was likely further degraded to monosaccharides by DacHme, GlyAHme, and other related enzymes for both cell growth and PHBV biosynthesis. Taken together, this study revealed the genes and enzymes involved in chitin catabolism in haloarchaea for the first time and indicated the potential of H. mediterranei as a whole-cell biocatalyst in chitin bioconversion.

Multiple propionyl coenzyme A-supplying pathways for production of the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Haloferax mediterranei.

Haloferax mediterranei is able to accumulate the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with more than 10 mol% 3-hydroxyvalerate (3HV) from unrelated carbon sources. However, the pathways that produce propionyl coenzyme A (propionyl-CoA), an important precursor of 3HV monomer, have not yet been determined. Bioinformatic analysis of H. mediterranei genome indicated that this strain uses multiple pathways for propionyl-CoA biosynthesis, including the citramalate/2-oxobutyrate pathway, the aspartate/2-oxobutyrate pathway, the methylmalonyl-CoA pathway, and a novel 3-hydroxypropionate pathway. Cofeeding of pathway intermediates and inactivating pathway-specific genes supported that these four pathways were indeed involved in the biosynthesis of 3HV monomer. The novel 3-hydroxypropionate pathway that couples CO2 assimilation with PHBV biosynthesis was further confirmed by analysis of (13)C positional enrichment in 3HV. Notably, (13)C metabolic flux analysis showed that the citramalate/2-oxobutyrate pathway (53.0% flux) and the 3-hydroxypropionate pathway (30.6% flux) were the two main generators of propionyl-CoA from glucose. In addition, genetic perturbation on the transcriptome of the ΔphaEC mutant (deficient in PHBV accumulation) revealed that a considerable number of genes in the four propionyl-CoA synthetic pathways were significantly downregulated. We determined for the first time four propionyl-CoA-supplying pathways for PHBV production in haloarchaea, particularly including a new 3-hydroxypropionate pathway. These results would provide novel strategies for the production of PHBV with controllable 3HV molar fraction.

Antimicrobial and Pro-Osteogenic Coaxially Electrospun Magnesium Oxide Nanoparticles-Polycaprolactone /Parathyroid Hormone-Polycaprolactone Composite Barrier Membrane for Guided Bone Regeneration.

Introduction: An antibacterial and pro-osteogenic coaxially electrospun nanofiber guided bone regeneration (GBR) membrane was fabricated to satisfy the complicated and phased requirements of GBR process. Methods: In this study, we synthesize dual-functional coaxially electrospun nanofiber GBR membranes by encapsulating parathyroid hormone (PTH) in the core layer and magnesium oxide nanoparticles (MgONPs) in the shell layer (MgONPs-PCL/PTH-PCL). Herein, the physicochemical characterization of MgONPs-PCL/PTH-PCL, the release rates of MgONPs and PTH, and antibacterial efficiency of the new membrane were evaluated. Furthermore, the pro-osteogenicity of the membranes was assessed both in-vitro and in-vivo. Results: We successfully fabricated a coaxially electrospun nanofiber MgONPs-PCL/PTH-PCL membrane with the majority of nanofibers (>65%) ranged from 0.40~0.60µm in diameter. MgONPs-PCL/PTH-PCL showed outstanding antibacterial potential against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) through the release of MgONPs. We also discovered that the incorporation of MgONPs significantly prolonged the release of PTH. Furthermore, both the in-vivo and in-vitro studies demonstrated that high dosage of PTH promoted pro-osteogenicity of the membrane to improve bone regeneration efficacy with the presence of MgONPs. Conclusion: The new composite membrane is a promising approach to enhance bone regeneration in periodontitis or peri-implantitis patients with large-volume bone defects.

Complete genome sequence of the metabolically versatile halophilic archaeon Haloferax mediterranei, a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) producer.

Haloferax mediterranei, an extremely halophilic archaeon, has shown promise for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated cheap carbon sources. Here we report the complete genome (3,904,707 bp) of H. mediterranei CGMCC 1.2087, consisting of one chromosome and three megaplasmids.

Photocured biodegradable polymer substrates of varying stiffness and microgroove dimensions for promoting nerve cell guidance and differentiation.

Photocross-linkable and biodegradable polymers have great promise in fabricating nerve conduits for guiding axonal growth in peripheral nerve regeneration. Here, we photocross-linked two poly(ε-caprolactone) triacrylates (PCLTAs) with number-average molecular weights of ~7000 and ~10,000 g mol(-1) into substrates with parallel microgrooves. Cross-linked PCLTA7k was amorphous and soft, while cross-linked PCLTA10k was semicrystalline with a stiffer surface. We employed different dimensions of interests for the parallel microgrooves, that is, groove widths of 5, 15, 45, and 90 µm and groove depths of 0.4, 1, 5, and 12 µm. The behaviors of rat Schwann cell precursor line (SpL201) cells with the glial nature and pheochromocytoma (PC12) cells with the neuronal nature were studied on these microgrooved substrates, showing distinct preference to the substrates with different mechanical properties. We found different threshold sensitivities of the two nerve cell types to topographical features when their cytoskeleton and nuclei were altered by varying the groove depth and width. Almost all of the cells were aligned in the narrowest and deepest microgrooves or around the edge of microgrooves. Oriented SpL201 cell movement had a higher motility as compared to unaligned ones. After forskolin treatment, SpL201 cells demonstrated significantly upregulated S-100 and O4 on stiffer substrates or narrower microgrooves, suggesting more differentiation toward early Schwann cells (SCs). PC12 neurites were oriented with enhanced extension in narrower microgrooves. The present results not only improve our fundamental understanding on nerve cell-substrate interactions, but also offer useful conduit materials and appropriate feature dimensions to foster guidance for axonal growth in peripheral nerve regeneration.

Poly(ε-caprolactone)-banded spherulites and interaction with MC3T3-E1 cells.

We report that protein adsorption, cell attachment, and cell proliferation were enhanced on spherulites-roughened polymer surfaces. Banded spherulites with concentric alternating succession of ridges and valleys were observed on spin-coated thin films of poly(ε-caprolactone) (PCL) and two series of PCL binary homoblends composed of high- and low-molecular-weight components when they were isothermally crystallized at 25-52 °C. Their thermal properties, crystallization kinetics, and surface morphology were examined. The melting temperature (T(m)), crystallinity (χ(c)), crystallization rate, and spherulitic patterns showed strong dependence on the crystallization temperature (T(c)) and the blend composition. The surface roughness of the spherulites was higher when T(c) was higher; thus, the larger surface area formed in banded spherulites could adsorb more serum proteins from cell culture media. In vitro mouse preosteoblastic MC3T3-E1 cell attachment, proliferation, and nuclear localization were assessed on the hot-compressed flat disks and spherulites-roughened films of the high-molecular-weight PCL and one of its homoblends. The number of attached MC3T3-E1 cells and the proliferation rate were greater on the rougher surfaces than those on the flat ones. It is interesting to note that cell nuclei were preferentially, though not absolutely, located in or close to the valleys of the banded spherulites. The percentage of cell nuclei in the valleys was higher than 78% when the ridge height and adjacent ridge distance were ~350 and ~35 nm, respectively. This preference was weaker when the ridge height was lower or at a higher cell density. These results suggest that isothermal crystallization of semicrystalline polymers can be an effective thermal treatment method to achieve controllable surface roughness and pattern for regulating cell behaviors in tissue-engineering applications.

Lubricated biodegradable polymer networks for regulating nerve cell behavior and fabricating nerve conduits with a compositional gradient.

We present a method of tuning surface chemistry and nerve cell behavior by photo-cross-linking methoxy poly(ethylene glycol) monoacrylate (mPEGA) with hydrophobic, semicrystalline poly(ε-caprolactone) diacrylate (PCLDA) at various weight compositions of mPEGA (ø(m)) from 2 to 30%. Improved surface wettability is achieved with corresponding decreases in friction, water contact angle, and capability of adsorbing proteins from cell culture media because of repulsive PEG chains tethered in the network. The responses of rat Schwann cell precursor line (SpL201), rat pheochromocytoma (PC12), and E14 mouse neural progenitor cells (NPCs) to the modified surfaces are evaluated. Nonmonotonic or parabolic dependence of cell attachment, spreading, proliferation, and differentiation on ø(m) is identified for these cell types with maximal values at ø(m) of 5-7%. In addition, NPCs demonstrate enhanced neuronal differentiated lineages on the mPEGA/PCLDA network at ø(m) of 5% with intermediate wettability and surface energy. This approach lays the foundation for fabricating heterogeneous nerve conduits with a compositional gradient along the wall thickness, which are able to promote nerve cell functions within the conduit while inhibiting cell attachment on the outer wall to prevent potential fibrous tissue formation following implantation.

Promoting nerve cell functions on hydrogels grafted with poly(L-lysine).

We present a novel photopolymerizable poly(L-lysine) (PLL) and use it to modify polyethylene glycol diacrylate (PEGDA) hydrogels for creating a better, permissive nerve cell niche. Compared with their neutral counterparts, these PLL-grafted hydrogels greatly enhance pheochromocytoma (PC12) cell survival in encapsulation, proliferation, and neurite growth and also promote neural progenitor cell proliferation and differentiation capacity, represented by percentages of both differentiated neurons and astrocytes. The role of efficiently controlled substrate stiffness in regulating nerve cell behavior is also investigated and a polymerizable cationic small molecule, [2-(methacryloyloxy)ethyl]-trimethylammonium chloride (MTAC), is used to compare with this newly developed PLL. The results indicate that these PLL-grafted hydrogels are promising biomaterials for nerve repair and regeneration.

Optimal poly(L-lysine) grafting density in hydrogels for promoting neural progenitor cell functions.

Recently, we have developed a photopolymerizable poly(L-lysine) (PLL) that can be covalently incorporated into poly(ethylene glycol) diacrylate (PEGDA) hydrogels to improve their bioactivity by providing positive charges. To explore the potential of these PLL-grafted PEGDA hydrogels as a cell delivery vehicle and luminal filler in nerve guidance conduits for peripheral and central nerve regeneration, we varied the number of pendent PLL chains in the hydrogels by photo-cross-linking PEGDA with weight compositions of PLL (φ(PLL)) of 0, 1, 2, 3, and 5%. We further investigated the effect of PLL grafting density on E14 mouse neural progenitor cell (NPC) behavior including cell viability, attachment, proliferation, differentiation, and gene expression. The amount of actually grafted PLL and charge densities were characterized, showing a proportional increase with the feed composition φ(PLL). NPC viability in 3D hydrogels was significantly improved in a PLL grafting density-dependent manner at days 7 and 14 postencapsulation. Similarly, NPC attachment and proliferation were promoted on the PLL-grafted hydrogels with increasing φ(PLL) up to 2%. More intriguingly, NPC lineage commitment was dramatically altered by the amount of grafted PLL chains in the hydrogels. NPC differentiation demonstrated a parabolic or nonmonotonic dependence on φ(PLL), resulting in cells mostly differentiated toward mature neurons with extensive neurite formation and astrocytes rather than oligodendrocytes on the PLL-grafted hydrogels with φ(PLL) of 2%, whereas the neutral hydrogels and PLL-grafted hydrogels with higher φ(PLL) of 5% support NPC differentiation less. Gene expression of lineage markers further illustrated this trend, indicating that PLL-grafted hydrogels with an optimal φ(PLL) of 2% could be a promising cell carrier that promoted NPC functions for treatment of nerve injuries.

[Recent progress in the study of reduction-sensitive drug carriers].

With rapid and efficient drug release, few side effects and excellent biodegradable properties, the reduction-sensitive carriers is not only the new hot point in the field of pharmaceutical research, but also the most promising intelligent drug carrier on clinical application. This paper reviews the latest research of reduction-sensitive drug and gene carriers, including the mechanisms of drug release and the synthesis of the reduction-sensitive conjugates, reduction-sensitive nano polymer micelles, nano vesicles, nano hollow microspheres, nano liposomes, as well as the characteristics and advantages of various kinds of carrier system. It will provide a theoretical basis for its further application.

Green Fluorescent Protein GFP-Chromophore-Based Probe for the Detection of Mitochondrial Viscosity in Living Cells.

Viscosity is a pivotal factor for indicating the dysfunction of the mitochondria. To date, most of the fluorescent probes developed for mitochondrial viscosity have been designed using BODIPY, hemicyanine, or pyridine-based molecular rotors as part of the core structure. Our aim with this research was to extend the range of suitable fluorophores available for the construction of such fluorescent molecular rotors for evaluating the viscosity of mitocondria. Herein, we have developed a green fluorescent protein (GFP)-chromophore-based fluorescent probe (MIT-V) for the detection of mitochondrial viscosity in live cells. MIT-V exhibited a high sensitivity toward viscosity (from 7.9 cP to 438.4 cP). The "off-on" sensing mechanism of MIT-V was ascribed to the restricted rotation of single bonds and excited-state C═C double bonds of MIT-V. Cell studies indicated that MIT-V targets the mitochondria and that it was able to monitor real-time changes in the viscosity of live HeLa cell mitochondria. Therefore, we propose that MIT-V can be used as an effective chemosensor for the real-time imaging of mitochondrial viscosity in live cells. Our results clearly demonstrate the utility of such GFP-chromophore-based derivatives for the development of viscosity-sensitive systems.

JK-2 loaded electrospun membrane for promoting bone regeneration.

Hydrogen sulfide (H2S) has been as an essential gasotransmitter and a potential therapeutic approach for several biomedical treatments such as cardiovascular disorders, hypertension, and other diseases. The endogenous and exogenous H2S also plays a crucial role in the bone anabolic process and a protective mechanism in cell signalling. In this study, we have utilized two types of polymers, polycaprolactone (PCL) and gelatin (Gel), for the fabrication of JK-2 (H2S donor) loaded nanofibrous scaffold via electrospinning process for bone healing and bone tissue engineering. Comparing the PCL/Gel and PCL/Gel-JK-2 scaffolds, the latter demonstrated enhanced cell adhesion and proliferation capabilities. Furthermore, both experimental scaffolds have been subjected to an in vivo experiment for 4 and 8 weeks in a bone-defect model of a rabbit to determine their biological responses under physiological conditions. There was an obvious increase in bone regeneration in the PCL/Gel-JK-2 group compared to the control and PCL/Gel groups. These results indicate the use of PCL/Gel scaffolds loaded with JK-2 should be considered for possible bone regeneration.

Preparation of Zirconium Hydrogen Phosphate Coatings on Sandblasted/Acid-Etched Titanium for Enhancing Its Osteoinductivity and Friction/Corrosion Resistance.

BACKGROUND: Sandblasted/acid-etched titanium (SLA-Ti) implants are widely used for dental implant restoration in edentulous patients. However, the poor osteoinductivity and the large amount of Ti particles/ions released due to friction or corrosion will affect its long-term success rate. PURPOSE: Various zirconium hydrogen phosphate (ZrP) coatings were prepared on SLA-Ti surface to enhance its friction/corrosion resistance and osteoinduction. METHODS: The mixture of ZrCl4 and H3PO4 was first coated on SLA-Ti and then calcined at 450°C for 5 min to form ZrP coatings. In addition to a series of physiochemical characterization such as morphology, roughness, wettability, and chemical composition, their capability of anti-friction and anti-corrosion were further evaluated by friction-wear test and by potential scanning. The viability and osteogenic differentiation of MC3T3-E1 cells on different substrates were investigated via MTT, mineralization and PCR assays. RESULTS: The characterization results showed that there were no significant changes in the morphology, roughness and wettability of ZrP-modified samples (SLA-ZrP0.5 and SLA-ZrP0.7) compared with SLA group. The results of electrochemical corrosion displayed that both SLA-ZrP0.5 and SLA-ZrP0.7 (especially the latter) had better corrosion resistance than SLA in normal saline and serum-containing medium. SLA-ZrP0.7 also exhibited the best friction resistance and great potential to enhance the spreading, proliferation and osteogenic differentiation of MC3T3-E1 cells. CONCLUSION: We determined that SLA-ZrP0.7 had excellent comprehensive properties including anti-corrosion, anti-friction and osteoinduction, which made it have a promising clinical application in dental implant restoration.

A glucose biosensor based on glucose oxidase fused to a carbohydrate binding module family 2 tag that specifically binds to the cellulose-modified electrode.

The method of immobilization of glucose oxidase (GOD) on electrodes is especially important for the fabrication and performance of glucose biosensors. In this study, a carbohydrate binding module family 2 (CBM2) was successfully fused to the C terminal of GOD with a natural linker (NL) in endo-ß-xylanase by genetic recombination, and a fusion GOD (GOD-NL-CBM2) was obtained. The CBM2 was used as an affinity adsorption tag for immobilization of the GOD-NL-CBM2 on a cellulose modified electrode. The specific activity of GOD-NL-CBM2 was comparable to that of the wild type GOD. In addition, the CBM2 tag of fusion GOD almost maintained its highest binding capacity under optimal catalytic conditions (pH 5.0, 50 °C). The morphology and composition analysis of the cellulose film reacted with and without GOD or GOD-NL-CBM2 confirmed the immobilization of GOD-NL-CBM2. The electrochemical properties of the GOD-NL-CBM2/cellulose film bioelectrode, with a characteristic peak of H2O2 at +0.6 V in the presence of glucose, revealed the capability of the immobilized GOD-NL-CBM2 to efficiently catalyze glucose and produce H2O2. Additionally, the current signal response of the biosensor to glucose was linear in the concentration range from 1.25 to 40 mM (r2 ≥ 0.99). The sensitivity and detection limit of the GOD-NL-CBM2/cellulose film bioelectrode were 466.7 µA mol-1 L cm-2 and 0.475 mM (S/N = 3), respectively. Moreover, the glucose biosensor exhibited a rapid current change (< 5 s), high reproducibility (Relative standard deviation, RSD < 5%), substrate selectivity and stability, and retained about 80 % of the original current response after 2 months. The affinity adsorption-based immobilization strategy for GOD provides a promising approach to develop a high performance glucose biosensor.

Distinct cell responses to substrates consisting of poly(ε-caprolactone) and poly(propylene fumarate) in the presence or absence of cross-links.

To investigate the role of chemical cross-links in regulating biomaterial properties and cell behavior, we have prepared and characterized a series of biodegradable polymer blends in both un-cross-linked and photo-cross-linked forms. In this comparative study, these blends consisted of an oligomeric, cross-linkable, amorphous poly(propylene fumarate) (PPF) and a high-molecular-weight, semicrystalline poly(ε-caprolactone) (PCL). After cross-linking, semi-interpenetrating polymer networks (semi-IPNs) were formed by combining PPF chemical network and PCL physical network that was associated by the crystallites. The material design strategy presented here was different from previously studied semicrystalline polymer networks, in which crystallizable segments participated covalently in the chemical network and were significantly suppressed by the network. For these PPF/PCL blends, thermal properties such as melting temperature (T(m)) and crystallinity have been correlated with their rheological and mechanical properties to demonstrate the effects of cross-linking density and crystallinity. Surface morphology, hydrophilicity, and the capability of adsorbing proteins from cell culture media have also been determined. For potential applications in bone and vascular tissue engineering and demonstration of regulating cell behavior on polymer substrates with controllable physicochemical characteristics, in vitro cell studies that included cell viability, attachment, spreading, and proliferation have been performed using mouse MC3T3 cells and primary rat aortic smooth muscle cells (SMCs). In a similar manner, these two cell types have been found to show distinct cell responses to the polymer substrates in the presence or absence of cross-links.

Overexpression of NAD kinase in recombinant Escherichia coli harboring the phbCAB operon improves poly(3-hydroxybutyrate) production.

NAD kinase was overexpressed to enhance the accumulation of poly(3-hydroxybutyrate) (PHB) in recombinant Escherichia coli harboring PHB synthesis pathway via an accelerated supply of NADPH, which is one of the most crucial factors influencing PHB production. A high copy number expression plasmid pE76 led to a stronger NAD kinase activity than that brought about by the low copy number plasmid pELRY. Overexpressing NAD kinase in recombinant E. coli was found not to have a negative effect on cell growth in the absence of PHB synthesis. Shake flask experiments demonstrated that excess NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16-35 wt.% compared with the controls; meanwhile, NADP concentration was enhanced threefold to sixfold. Although the two NAD kinase overexpression recombinants exhibited large disparity on NAD kinase activity, their influence on cell growth and PHB accumulation was not proportional. Under the same growth conditions without process optimization, the NAD kinase-overexpressing recombinant produced 14 g/L PHB compared with 7 g/L produced by the control in a 28-h fermentor study. In addition, substrate to PHB yield Y (PHB/glucose) showed an increase from 0.08 g PHB/g glucose for the control to 0.15 g PHB/g glucose for the NAD kinase-overexpressing strain, a 76% increase for the Y (PHB/glucose). These results clearly showed that the overexpression of NAD kinase could be used to enhance the PHB synthesis.

Bone Marrow Mononuclear Cells Combined with Beta-Tricalcium Phosphate Granules for Alveolar Cleft Repair: A 12-Month Clinical Study.

Alveolar cleft is the most common congenital bone defect. Autologous iliac crest bone graft (ICBG) is the most widely adopted procedure for alveolar cleft repair, but the condition is associated with door-site morbidities. For the first time, this study used bone marrow mononuclear cells (BMMNCs) combined with beta-tricalcium phosphate (ß-TCP) granules to repair alveolar bone defect. The effectiveness of this technique was compared with autologous ICBG after 12 months of follow-up. The bone formation volume was quantitatively evaluated by three-dimensional computed tomography and computer aided engineering technology. BMMNCs/ß-TCP granule grafting was radiographically equivalent to ICBG in alveolar cleft repair. Although considerable resorption was observed up to 6 months after surgery, no significant differences were noted in the Chelsea score and bone formation volume between groups. These finding indicate that BMMNCs/ß-TCP grafting is a safe and effective approach for alveolar bone regeneration.

A High-Level Fungal Diversity in the Intertidal Sediment of Chinese Seas Presents the Spatial Variation of Community Composition.

The intertidal region is one of the most dynamic environments in the biosphere, which potentially supports vast biodiversity. Fungi have been found to play important roles in marine ecosystems, e.g., as parasites or symbionts of plants and animals, and as decomposers of organic materials. The fungal diversity in intertidal region, however, remains poorly understood. In this study, sediment samples from various intertidal habitats of Chinese seas were collected and investigated for determination of fungal community and spatial distribution. Through ribosomal RNA internal transcribed spacer-2 (ITS2) metabarcoding, a high-level fungal diversity was revealed, as represented by 6,013 OTUs that spanned six phyla, 23 classes, 84 orders and 526 genera. The presence of typical decomposers (e.g., Corollospora in Ascomycota and Lepiota in Basidiomycota) and pathogens (e.g., Olpidium in Chytriomycota, Actinomucor in Zygomycota and unidentified Rozellomycota spp.), and even mycorrhizal fungi (e.g., Glomus in Glomeromycota) indicated a complicated origin of intertidal fungi. Interestingly, a small proportion of sequences were classified to obligate marine fungi (e.g., Corollospora, Lignincola, Remispora, Sigmoidea). Our data also showed that the East China Sea significantly differed from other regions in terms of species richness and community composition, indicating a profound effect of the huge discharge of the Yangtze River. No significant difference in fungal communities was detected, however, among habitat types (i.e., aquaculture, dock, plant, river mouth and tourism). These observations raise further questions on adaptation of these members to environments and the ecological functions they probably perform.