Decorating a Blank Slate Protein Hydrogel: A General and Robust Approach for Functionalizing Protein Hydrogels.

Protein hydrogels constructed from recombinant proteins have attracted increasing interests for fundamental biological studies as well as applications in biomedical engineering field. In such protein hydrogels, biochemical and physical properties of protein hydrogels are often coupled to each other, making it challenging to investigate the individual effect of chemical and physical cues on cells. Moreover, laborious engineering is often required to incorporate different protein ligands into such hydrogels. To address these challenges, functionalizing a blank slate protein hydrogel is an attractive approach. However, conjugating ligands to such a blank slate protein hydrogel is challenging, as existing bioconjugation methods developed in synthetic polymer hydrogels cannot be readily adapted for protein hydrogels, significantly impeding the use of this approach in the field. Here we report a facile, general, and robust method, which is based on the SpyCatcher-SpyTag chemistry, to covalently functionalize the "blank slate" of protein hydrogels using genetically encoded interacting partners. We demonstrate that this novel method enables covalent conjugation of a wide variety of ligands, including full-length intact folded proteins, to a blank slate protein hydrogel, and allows for the decoupling of biochemical and physical properties of hydrogels from each other and investigating the individual effect of biochemical and mechanical cues on cell behaviors. To our best knowledge, this is the first general approach enabling functionalization of protein hydrogels, and we anticipate that this novel approach will find a broad range of uses in protein-based biomaterials for applications in biomedical engineering.

Engineering Protein Hydrogels Using SpyCatcher-SpyTag Chemistry.

Constructing hydrogels from engineered proteins has attracted significant attention within the material sciences, owing to their myriad potential applications in biomedical engineering. Developing efficient methods to cross-link tailored protein building blocks into hydrogels with desirable mechanical, physical, and functional properties is of paramount importance. By making use of the recently developed SpyCatcher-SpyTag chemistry, we successfully engineered protein hydrogels on the basis of engineered tandem modular elastomeric proteins. Our resultant protein hydrogels are soft but stable, and show excellent biocompatibility. As the first step, we tested the use of these hydrogels as a drug carrier, as well as in encapsulating human lung fibroblast cells. Our results demonstrate the robustness of the SpyCatcher-SpyTag chemistry, even when the SpyTag (or SpyCatcher) is flanked by folded globular domains. These results demonstrate that SpyCatcher-SpyTag chemistry can be used to engineer protein hydrogels from tandem modular elastomeric proteins that can find applications in tissue engineering, in fundamental mechano-biological studies, and as a controlled drug release vehicle.

Dry-wet cycling area enhances soil ecosystem multifunctionality in the aquatic-terrestrial ecotones of the Caohai Lake in China.

The microbial need for nutrient resources can be assessed by soil extracellular enzymes and their stoichiometry. Changes in lake water levels affect land use and nutrient management in the aquatic-terrestrial ecotones of the lakeshore. However, the drivers of changes in microbial nutrient limitation under different inundation gradients in the lake's aquatic-terrestrial ecotones remain unclear. Here, based on vector analysis, we assessed microbial nutrient limitation by studying soil enzyme activities in four different inundation zones (heavy, moderate, mild, and non-inundation) in the aquatic-terrestrial ecotones of Caohai Lake. The findings indicate that inundation conditions significantly influenced the soil properties and enzyme activities. The mean nitrogen and phosphorus acquisition enzymes were higher in both moderate inundation (Mod-inu) and mild inundation (Mil-inu) zone soils, indicating rapid N and P turnover rates in these two zones. However, microorganisms had higher carbon requirements and higher enzyme C:N and vector lengths in heavily inundated compared to lightly inundated. Compared to the non-inundation zone, the microbial phosphorus limitation was found to be most severe in heavy inundation (Hea-inu) and Mod-inu zones. Decreased phosphorus limitation following the inundation weakens could be contributed to improving soil ecosystem multifunctionality. The alterations in the soil extracellular enzymes and stoichiometric characteristics in various inundation zones were primarily influenced by factors such as soil moisture content, available phosphorus, and nitrate nitrogen. Overall, the Mod-inu and Mil-inu zones can better maintain the multifunctionality of the aquatic and terrestrial ecosystems; special attention should be given to the microbial phosphorus limitation in the Hea-inu zone in order to effectively manage nutrients and restore soil ecosystems in the aquatic-terrestrial ecotones.

The Evaluation of Physiological Index Changes and Safety Work of Female Medical Staff With Different Medical Protection Standards in the Ward of COVID-19.

Background: Effective personal protective equipment (PPE) contribute to the prevention of COVID-19 infection. However, it is necessary to evaluate the potential risk of different medical protections in the isolation ward of COVID-19. Objectives: We aimed to explore the dynamics in physiological indexes of medical staff under primary and secondary PPE in the isolation ward of COVID-19 and provide the scientific basis for determining the safe work strategy. Materials and Methods: In this study, 30 female nurses were selected to simulate medical work under the primary or secondary PPE, respectively. The oral temperature, axillary temperature, heart rate, respiratory rate, blood oxygen saturation, and blood pressure were measured and recorded every 20 min. The subjective adverse symptoms were recorded every 30 min. The blood glucose and weight of the individuals were measured and recorded before and after the trial. Results: The results indicated that the median trial persistence time in the participants with moderate-intensity work wearing the secondary PPE (70.0 min) was much lower than that with moderate-intensity work wearing the primary PPE (180 min) and with light-intensity work wearing the primary PPE (110 min; p < 0.05). Importantly, the heart rate, oral/axillary temperature, and respiratory rate of physiological indexes of the participants under moderate-intensity work wearing the secondary PPE increased significantly faster than the primary PPE (p < 0.001), while blood oxygen saturation decreased significantly faster than the primary PPE (p < 0.001). In addition, the proportions of subjective adverse symptoms (such as dry mouth, dizziness, palpitations, and anhelation) were much higher than primary PPE (p < 0.001). The average sweat volume and blood glucose consumption of participants under moderate-intensity work wearing primary PPE were higher than secondary PPE (p < 0.001). Conclusion: The combination of an exacerbated workload and secondary PPE worn by COVID-19 healthcare workers increases the change in physiological indicators, and in some cases the adverse symptoms, which can affect and even suspend their medical work. For any medical institution, there is room for improvement in terms of bioethics of a "Job Well Done" to reduce the risks of medical activities under secondary PPE.

Seasonal Variation in Soil and Herbage CO2 Efflux for a Sheep-Grazed Alpine Meadow on the North-East Qinghai-Tibetan Plateau and Estimated Net Annual CO2 Exchange.

The Qinghai-Tibetan Plateau is a vast geographic area currently subject to climate warming. Improved knowledge of the CO2 respiration dynamics of the Plateau alpine meadows and of the impact of grazing on CO2 fluxes is highly desirable. Such information will assist land use planning. We measured soil and vegetation CO2 efflux of alpine meadows using a closed chamber technique over diurnal cycles in winter, spring and summer. The annual, combined soil and plant respiration on ungrazed plots was 28.0 t CO2 ha-1 a-1, of which 3.7 t ha-1 a-1occurred in winter, when plant respiration was undetectable. This suggests winter respiration was driven mainly by microbial oxidation of soil organic matter. The winter respiration observed in this study was sufficient to offset the growing season CO2 sink reported for similar alpine meadows in other studies. Grazing increased herbage respiration in summer, presumably through stimulation of gross photosynthesis. From limited herbage production data, we estimate the sustainable yield of these meadows for grazing purposes to be about 500 kg herbage dry matter ha-1 a-1. Addition of photosynthesis data and understanding of factors affecting soil carbon sequestration to more precisely determine the CO2 balance of these grasslands is recommended.

Key Metabolite Differences Between Korean Pine (Pinus koraiensis) Seeds With Primary Physiological Dormancy and No-Dormancy.

Pinus Koraiensis seeds have physiological dormancy. Cold stratification releases seed dormancy. The changes in metabolite profiles of dormant seeds and cold stratified seeds during shorter incubation time in a favorable condition for seed germination have been studied. However, a more-long-term detection of the changes in metabolites in dormant seeds can identify the real metabolic pathways responsible for dormancy. Metabolite composition was investigated in embryo and megagametophyte of primary physiological dormant seeds (DS) of P. Koraiensis collected at 0, 1, 2, 4, and 6 weeks of incubation and of non-primary physiological dormant seeds (NDS) sampled at 0 and 1 week of incubation, seed coat rupture stage, and radicle protrusion stage. Embryos contained higher levels of most metabolites than megagametophyte. Strong accumulation of most metabolites in DS occurred at 1 and 4 weeks of incubation. A larger reduction in the relative levels of most phosphorylated sugars and amino acids in NDS was found between 1-week-incubation and seed coat rupture stage. The relative levels of metabolites involved in carbohydrate metabolism, especially the pentose phosphate pathway (PPP) and tricarboxylic acid (TCA) cycle, were higher in the embryos of 4-week-incubated DS, but the relative contents of intermediate metabolites of most amino acid metabolism were lower compared to 1-week-incubated NDS. We suggested that the disturbed carbohydrate metabolism and amino acid metabolism in the embryos of DS after 4 weeks of incubation maybe related to primary dormancy. Our study provides information for a better understanding of the mechanism of seed dormancy.

Effect of Land Use Conversion on Surface Soil Heavy Metal Contamination in a Typical Karst Plateau Lakeshore Wetland of Southwest China.

Land use conversion could directly or indirectly influence heavy metal geochemistry by changing soil properties. The aim of this study was to explore the effect of land use conversion on surface soil heavy metal contamination in the karst plateau lakeshore wetlands of Southwest China. Based on this, a total of 120 soil samples were collected from 30 sites from different types of land uses (farmlands, grasslands and woodlands) around a lake in Suohuangcang National Wetland Park in August 2017. Contents of As, Cd, Cu, Cr, Hg, Pb and Zn were analyzed, and soil heavy metal contamination was assessed in all three land use types. Results showed that land use transformation from farmland to grassland or woodland was not conducive to the release of soil heavy metal. Surface soil of all three land use types have been moderately polluted by As, Cr, Pb, and Zn, and grassland and woodland also had moderate Cd contamination. The pollution load index (PLI) results revealed low heavy metal contamination in grassland and woodland but no contamination in farmland. Although the integrated contamination in the studied region did not pose a serious potential ecological risk (RI < 150), it might affect human health through the water supply and food chain. Therefore, it is necessary to monitor and control As, Cd, Cr, Pb, and Zn concentrations of surface soil through controlling pollutants, improving waste treatment, as well as strengthening supervision and management in the vicinity of the Suohuangcang National Wetland Park.

Increase phosphorus availability from the use of alfalfa (Medicago sativa L) green manure in rice (Oryza sativa L.) agroecosystem.

Alfalfa is a good green manure source, but its effect on rice growth has not been fully elucidated. Two green manure species, alfalfa and broad bean (Vicia faba L.), and two N fertilizer levels, alone or combination, were applied to a rice field. The results indicated that alfalfa had more pronounced effects on increasing soil labile phosphorus (P) fractions (including NaHCO3-Pi, NaOH-Pi), P uptake and soil enzyme activities (dehydrogenase, urease, acid phosphatase and ß-glucosidase) than broad bean and N fertilizer. The transformation of NaHCO3-Po to labile P regulated by alfalfa played a significant direct and indirect effect on grain yield. Although a much lower N input from alfalfa addition, a similar grain yield with N fertilizer treatment was achieved, and the integration of alfalfa with N fertilizer produced the highest grain yield and P availability, which was associated with the highest urease, acid phosphatase and ß-glucosidase activity in soil. These results indicate that alfalfa green manure had a great ability of increasing grain yield through enhancing P availability in rice paddy, which could give us a way to reduce N fertilizer application by enhancing P availability.

pH-Responsive Poly(ethylene glycol)/Poly(L-lactide) Supramolecular Micelles Based on Host-Guest Interaction.

pH-responsive supramolecular amphiphilic micelles based on benzimidazole-terminated poly(ethylene glycol) (PEG-BM) and ß-cyclodextrin-modified poly(L-lactide) (CD-PLLA) were developed by exploiting the host-guest interaction between benzimidazole (BM) and ß-cyclodextrin (ß-CD). The dissociation of the supramolecular micelles was triggered in acidic environments. An antineoplastic drug, doxorubicin (DOX), was loaded into the supramolecular micelles as a model drug. The release of DOX from the supramolecular micelles was clearly accelerated as the pH was reduced from 7.4 to 5.5. The DOX-loaded PEG-BM/CD-PLLA supramolecular micelles displayed an enhanced intracellular drug-release rate in HepG2 cells compared to the pH-insensitive DOX-loaded PEG-b-PLLA counterpart. After intravenous injection into nude mice bearing HepG2 xenografts by the tail vein, the DOX-loaded supramolecular micelles exhibited significantly higher tumor inhibition efficacy and reduced systemic toxicity compared to free DOX. Furthermore, the DOX-loaded supramolecular micelles showed a blood clearance rate markedly lower than that of free DOX and comparable to that of the DOX-loaded PEG-b-PLLA micelles after intravenous injection into rats. Therefore, the pH-responsive PEG-BM/CD-PLLA supramolecular micelles hold potential as a smart nanocarrier for anticancer drug delivery.

Biodegradable, pH-responsive carboxymethyl cellulose/poly(acrylic acid) hydrogels for oral insulin delivery.

Biodegradable and pH-responsive carboxymethyl cellulose/poly(acrylic acid) hybrid hydrogels are synthesized. The hydrogels deswell in acidic artificial gastric fluid (AGF) but rapidly swell in neutral artificial intestinal fluid (AIF), rendering selective enzymatic degradation of the gels as well as accelerated drug release from insulin-loaded hydrogels in AIF. Oral administration of insulin-loaded hydrogels to streptozotocin-induced diabetic rats leads to a continuous decline in the fasting blood glucose level within 6 h post-administration, and the relative pharmacological availability increases more than 10 times compared to oral administration of free insulin solution. The relative bioavailability of hydrogel-encapsulated insulin after oral administration to healthy rabbits is 6.6%.

pH- and thermo-responsive poly(N-isopropylacrylamide-co-acrylic acid derivative) copolymers and hydrogels with LCST dependent on pH and alkyl side groups.

A series of pH- and temperature-responsive poly(N-isopropylacrylamide-co-acrylic acid derivative) (P(NIPAM-co-AAD)) copolymers and hydrogels were prepared. The lower critical solution temperatures (LCSTs) of the copolymers exhibited a dependence on both pH and the hydrophobicity of the AAD unit. The influence of pH and temperature on the equilibrium swelling ratio of the hydrogels was investigated. The hydrogels displayed a unique thermo-induced swelling-deswelling transition that can be self-regulated to occur at above or below the physiological temperature in response to the environmental pH. Scanning electron microscopic (SEM) analysis revealed porous sponge-like microstructures of the hydrogels. Insulin was loaded into the hydrogels as a model protein, and the in vitro release profiles indicated that the loaded protein could be protected within the hydrogels in an acidic environment and selectively released in neutral medium. MTT assay proved that both the copolymers and hydrogels are nontoxic. After oral administration of the insulin-loaded hydrogels to streptozotocin-induced diabetic rats at 60 IU per kg, the fasting plasma glucose level was reduced continuously to 72.1% within 6 h. The bioavailability of hydrogel-encapsulated insulin via the oral administration to healthy rabbits reached 5.24%, which is much higher than that of pure insulin solution given orally. These results showed that the smart copolymers and hydrogels may hold great promise for pH-triggered drug delivery systems.

Forced protein unfolding leads to highly elastic and tough protein hydrogels.

Protein-based hydrogels usually do not exhibit high stretchability or toughness, significantly limiting the scope of their potential biomedical applications. Here we report the engineering of a chemically cross-linked, highly elastic and tough protein hydrogel using a mechanically extremely labile, de novo-designed protein that assumes the classical ferredoxin-like fold structure. Due to the low mechanical stability of the ferredoxin-like fold structure, swelling of hydrogels causes a significant fraction of the folded domains to unfold. Subsequent collapse and aggregation of unfolded ferredoxin-like domains leads to intertwining of physically and chemically cross-linked networks, entailing hydrogels with unusual physical and mechanical properties: a negative swelling ratio, high stretchability and toughness. These hydrogels can withstand an average strain of 450% before breaking and show massive energy dissipation. Upon relaxation, refolding of the ferredoxin-like domains enables the hydrogel to recover its massive hysteresis. This novel biomaterial may expand the scope of hydrogel applications in tissue engineering.

Synthesis of temperature and pH-responsive crosslinked micelles from polypeptide-based graft copolymer.

A polypeptide-based double hydrophilic graft copolymer was synthesized by the sequential grafting of poly(N-isopropylacrylamide) (PNIPAM) and 2-hydroxyethyl methacrylate (HEMA) onto poly(l-glutamic acid) (PGA) backbone. The copolymers were sensitive to both temperature and pH. The phase transition and aggregation behaviors of the graft copolymers in aqueous solutions were investigated by the turbidity measurements and dynamic laser scattering (DLS). The light transmittance decrease of the copolymers at temperature above lower critical solution temperature (LCST) was remarkably weakened at pH around 6.5 due to the coil to α helix change of PGA chain induced by pH. The copolymers can self-assembly into micelles with PNIPAM cores in the aqueous solution at pH 8.0 and 60°C. Subsequently, polymerization of HEMA led to the facile preparation of crosslinked micelles, which were observed directly by transmission electron microscopy (TEM). The temperature controlled shrinkage behaviors of crosslinked micelles highly depended on the pH values of the solution. The crosslinked micelles aggregated at pH 5.0 due to the increased hydrophobic interactions among them induced by the protonation of PGA component. These crosslinked micelles have promising applications as intelligent drug delivery vehicles.