Optimization of the Zhou Tian Formula extraction technology based on AHP-CRITIC method and analysis of transfer efficiency of key components based on HPLC fingerprinting.

INTRODUCTION: Zhou Tian Formula (ZTF) is an antidepressant traditional Chinese medicine utilized widely in clinical settings for the treatment of patients with depression. However, shortcomings persist in its extraction technology and quality control. OBJECTIVE: This study aimed to propose a methodology for ZTF extraction technology based on the analytic hierarchy process (AHP)-criteria importance through intercriteria correlation (CRITIC) method and to establish a quality control framework for the efficient transfer of index components. METHOD: Firstly, we analyzed the chemical components of ZTF and determined the optimal extraction technology. Secondly, we calculated the transfer efficiency of the index components during the conversion of water decoction to extract powder and subsequently to granules. Thirdly, we established HPLC fingerprints for 15 batches of ZTF water decoction, extract powder, and granules. We employed SIMCA software to analyze the chemicals responsible for variations in quality among different batches of ZTF granules. RESULTS: We determined the optimal extraction process. The average transfer efficiency of ferulic acid, puerarin, mirificin, isoferulic acid, and calycosin during the conversion of water decoction to extract powder and subsequently to granules exceeded 41%. The HPLC fingerprints of ZTF exhibited a similarity exceeding 0.890. Variable importance in projection values indicated that calycosin, ferulic acid, and puerarin were the primary contributors to quality variations. CONCLUSIONS: The AHP-CRITIC method, coupled with an orthogonal array design, could be used for exploring extraction technology. In addition, the rules governing the transfer of index components from water decoction to extract powder, and subsequently to granules, could be applied for the evaluation and quality assessment of ZTF.

In Vitro and Ex Planta Gold-Bonded and Gold-Mineralized Tobacco Mosaic Virus.

Biotemplated mineralization is a promising and ecofriendly approach to manufacture metal nanoparticles and composites with precise size control. Plant viruses are suitable templates for biomineralization because they are chemically robust and highly scalable through molecular farming. Here, we report a gold-nanoparticle-coated tobacco mosaic virus (TMV) synthesized in a test tube or in plant extracts making use of a TMV displaying a gold-binding peptide (GBP). The methods developed are a step toward engineered living materials, where gold nanowires could be formed in plant tissues for sensing or energy harvest applications.

The Effects of Auditory Working Memory Task on Situation Awareness in Complex Dynamic Environments: An Eye-movement Study.

OBJECTIVE: This study investigated the effect of auditory working memory task on situation awareness (SA) and eye-movement patterns in complex dynamic environments. BACKGROUND: Many human errors in aviation are caused by a lack of SA, and distraction from auditory secondary tasks is a serious threat to SA. However, it remains unclear how auditory working memory tasks affect SA and eye-movement patterns. METHOD: Participants (n = 28) were randomly allocated to two groups and received different periods of visual search training (short versus long). They subsequently completed a situation awareness measurement task in three auditory secondary task conditions (without secondary task, auditory calculation task, and auditory 2-back task). Eye-movement data were collected during the situation awareness measurement task. RESULTS: The auditory 2-back task significantly reduced overall SA, Level 1 SA, dwell times, and total percentage of fixation time on task-related areas of interests in the SA measurement task. Overall SA and Level 3 SA were not reduced by the auditory 2-back task in individuals in the longer visual search training time condition. CONCLUSION: Auditory working memory load impairs SA in the perception and projection stage; however, greater experience can overcome impairment of SA in the projection stage. APPLICATION: This study provided possible approaches to preventing loss of SA: (1) improving crew members' communication skills to ensure the accurate and clear transmission of information, reducing the difficulty of processing information, and (2) providing targeted cognitive training tailored to each pilot's level of experience.

Ferrocene as a potential electrochemical reporting surrogate of abasic sites in DNA.

Methods for the real-time monitoring of the substrate acceptance of modified nucleotides by DNA polymerases are in high demand. In a step towards this aim, we have incorporated ferrocene-based abasic nucleotides into DNA templates and evaluated their compatibility with enzymatic synthesis of unmodified and modified DNA. All canonical nucleotides can be incorporated opposite ferrocene sites with a strong preference for purines. DNA polymerases with lesion-bypass capacity such as Dpo4 allow DNA synthesis to be resumed beyond the site of incorporation. Modified purine nucleotides can readily be incorporated opposite ferrocene basic site analogs, while pyrimidine nucleotides decorated with simple side-chains are also readily tolerated. These findings open up directions for the design of electrochemical sensing devices for the monitoring of enzymatic synthesis of natural or modified DNA.

Overexpression of Peroxisome-Localized GmABCA7 Promotes Seed Germination in Arabidopsis thaliana.

Peroxisome is one of the important organelles for intracellular lipid metabolism in plant cells and ß-oxidation of fatty acids in peroxisomes provides the energy for oil-containing seed germination. In this study, we identified an ATP-binding cassette (ABC) transporter gene, GmABCA7 from soybean, which is highly expressed in the different developmental stages of seeds. Transient expression of GmABCA7 in tobacco epidermal cells showed that GmABCA7 was specifically localized at the peroxisomes. Overexpression of GmABCA7 in Arabidopsis does not change seed phenotypes, or the overall levels of lipid, protein and sugar stored in the seeds; however, the transgenic seeds produced more gluconeogenic pathway precursors such as succinate and malate and germinated earlier compared to the wild type seeds. These results suggest that GmABCA7 may affect the ß-oxidation of fatty acids and play an important role in seed germination.

In Vitro Susceptibility Testing of GSK656 against Mycobacterium Species.

In this study, we aimed to assess the in vitro susceptibility to GSK656 among multiple mycobacterial species and to investigate the correlation between leucyl-tRNA synthetase (LeuRS) sequence variations and in vitro susceptibility to GSK656 among mycobacterial species. A total of 187 mycobacterial isolates, comprising 105 Mycobacterium tuberculosis isolates and 82 nontuberculous mycobacteria (NTM) isolates, were randomly selected for the determination of in vitro susceptibility. For M. tuberculosis, 102 of 105 isolates had MICs of ≤0.5 mg/liter, demonstrating a MIC50 of 0.063 mg/liter and a MIC90 of 0.25 mg/liter. An epidemiological cutoff value of 0.5 mg/liter was proposed for identification of GSK656-resistant M. tuberculosis strains. For NTM, the MIC50 and MIC90 values were >8.0 mg/liter for both Mycobacterium intracellulare and Mycobacterium avium In contrast, all Mycobacterium abscessus isolates had MICs of ≤0.25 mg/liter, yielding a MIC90 of 0.063 mg/liter. LeuRS from M. abscessus showed greater sequence similarity to M. tuberculosis LeuRS than to LeuRSs from M. avium and M. intracellulare Sequence alignment revealed 28 residues differing between LeuRSs from M. avium and M. intracellulare and LeuRSs from M. tuberculosis and M. abscessus; among them, 15 residues were in the drug binding domain. Structure modeling revealed that several different residues were close to the tRNA-LeuRS interface or the entrance of the drug-tRNA binding pocket. In conclusion, our data demonstrate significant species diversity in in vitro susceptibility to GSK656 among various mycobacterial species. GSK656 has potent efficacy against M. tuberculosis and M. abscessus, whereas inherent resistance was noted for M. intracellulare and M. avium.

In Vitro Activities of Bedaquiline and Delamanid against Nontuberculous Mycobacteria Isolated in Beijing, China.

Due to the natural resistance of nontuberculous mycobacteria (NTM) against multiple antibiotics, treatment of infections caused by them is often long-course and less successful. The main objective of our study was the evaluation of in vitro susceptibility of 209 isolates consisting of different NTM species against bedaquiline and delamanid. Furthermore, reference strains of 33 rapidly growing mycobacterium (RGM) species and 19 slowly growing mycobacterium (SGM) species were also tested. Bedaquiline exhibited strong in vitro activity against both reference strains and clinical isolates of different SGM species, as the majority of the strains demonstrated MICs far below 1 µg/ml. Bedaquiline (Bdq) also exhibited potent activity against the recruited RGM species. A total of 29 out of 33 reference RGM strains had MICs lower than 1 µg/ml. According to the MIC distributions, the tentative epidemiological cutoff (ECOFF) values, and the pharmacokinetic data, a uniform breakpoint of 2 µg/ml was temporarily proposed for NTM's Bdq susceptibility testing. Although delamanid (Dlm) was not active against most of the tested reference strains and clinical isolates of RGM species, it exhibited highly variable antimicrobial activities against the 19 tested SGM species. Eleven species had MICs lower than 0.25 µg/ml, and 7 species had MICs greater than 32 µg/ml. Large numbers of M. kansasii (39/45) and M. gordonae (6/10) clinical isolates had MICs of ≤0.125 µg/ml. This study demonstrated that bedaquiline had potent activity against different NTM species in vitro, and delamanid had moderate activity against certain species of SGM. The data provided important insights on the possible clinical application of Bdq and Dlm to treat NTM infections.

GeneXpert of stool versus gastric lavage fluid for the diagnosis of pulmonary tuberculosis in severely ill adults.

PURPOSE: Stool is an alternative specimen matrix for tuberculosis (TB) tests, because Mycobacterium tuberculosis (MTB) can be swallowed and detected in the samples from digestive tract. We aimed to assess the performance of GeneXpert on stool and gastric lavage fluid (GALF) in diagnosing TB among patients with severe pulmonary TB. METHODS: We enrolled adults with suspected pulmonary TB who were unable to produce sputum at visit between January 2016 and June 2018. Bacteriological samples consisted of one transtracheal aspirate sputum specimen, one stool specimen and/or one gastric lavage fluid specimen. Bacterial culture of transtracheal aspirate sputum provided the gold standard. RESULTS: Of 65 individuals recruited for analysis, MGIT culture identified the presence of MTB in 32 samples. Overall, 29 of 32 stool samples from culture-positive cases were detected by the GeneXpert test, demonstrating a sensitivity of 90.6%. For GALF, 13 patients were detected as infected with MTB by GeneXpert, yielding a sensitivity of 56.5%. The statistical analysis revealed that GeneXpert showed significantly better sensitivity in detecting MTB from stool samples than GALF samples (P = 0.003). Among individuals with GeneXpert-positive stool, the percentage of individuals with comorbid diabetes was significantly higher than among individuals with GeneXpert-negative stool (19.4% vs. 2.9%, P = 0.034). CONCLUSIONS: In conclusion, our data reveal that GeneXpert provides a higher detection rate on stool compared to GALF, indicating stool should be considered as an alternative for adult TB patients unable to produce sputum. Individuals with diabetes are more likely to have positive GeneXpert stool than nondiabetic individuals.

Change in prevalence and molecular characteristics of isoniazid-resistant tuberculosis over a 10-year period in China.

BACKGROUND: Isoniazid (INH) represents the cornerstone for the treatment of cases infected with Mycobacterium tuberculosis (MTB) strains. Several molecular mechanisms have been shown to be the major causes for INH resistance, while the dynamic change of mutations conferring INH resistance among MTB strains during the past decade is still unknown in China. METHODS: In this study, we carried out a comparative analysis of the INH minimal inhibitory concentration (MIC) distribution, and investigate the dynamic change of molecular characteristics among INH-resistant MTB strains between 2005 and 2015. RESULTS: The proportion of INH resistance (39.0%, 105/269) in 2015 was significantly higher than in 2005 (30.0%, 82/273; P = 0.03). Among 269 isolates collected in 2015, 76 (28.3%, 76/269) exhibited high-level INH-resistance (MIC≥32 mg/L), which was significantly higher than that in 2005 (20.5%, 56/273, P = 0.04). In addition, a significantly higher percentage of INH-resistant isolates carried inhA promoter mutations in 2015 (26.7%) versus that in 2005 (14.6%, P = 0.04), while no significant difference was observed in the rates of isolates containing katG mutations between 2005 (76.8%) and 2015 (70.5%, P = 0.33). Notably, the proportion of MTB isolates with inhA mutations (26.7%, 28/105) for patients who had previous exposure to protionamide (PTH) was higher than that for patients who had no previous exposure to PTH (21.4%, 6/28). CONCLUSIONS: In conclusion, our results demonstrated that the proportion of INH-resistant MTB isolates significantly increased during the last decade, which was mainly attributed to an increase of high-level INH-resistant MTB. In addition, prior exposure to PTH may be associated with the increased frequency of INH-resistant tuberculosis strains with inhA mutations in China.

A 10-Year Comparative Analysis Shows that Increasing Prevalence of Rifampin-Resistant Mycobacterium tuberculosis in China Is Associated with the Transmission of Strains Harboring Compensatory Mutations.

In this work, we conducted bacterial population profile studies to assess trends of rifampin (RIF) resistance of Mycobacterium tuberculosis isolates collected across China from 2005 to 2015. Totals of 273 and 269 randomly selected M. tuberculosis isolates from 2005 and 2015, respectively, were analyzed. The rates of RIF resistance (36.4%), isoniazid resistance (39.0%), and levofloxacin resistance (25.7%) in 2015 were significantly higher than those in 2005 (28.2%, 30.0%, and 15.4%, respectively; P < 0.05). Genotypic data revealed 256 (95.2%) Beijing-type isolates in 2015, a rate significantly higher than that in 2005 (86.4%) (P < 0.01). A higher proportion of mutations was identified within the rifampin resistance-determining region (RRDR) of rpoB in isolates from 2015 (99.0%) than in 2005 isolates (85.7%, P < 0.01). In addition, a significantly higher proportion of RIF-resistant isolates carrying compensatory mutations was observed in 2015 (31.6%) than in 2005 (7.8%). Notably, the great majority of these compensatory mutations (91.9%) were observed in isolates that harbored a mutation of codon 531 of the rpoB gene. In conclusion, our data demonstrate that resistance to RIF, isoniazid, and levofloxacin has become significantly more prevalent during the past decade. In addition, the prevalence of the Beijing genotype significantly increased from 2005 to 2015. Notably, a significantly increased frequency of strains with mutations in rpoC or rpoA is observed among those that have codon 531 mutations, which suggests that they may be compensatory and may play a role in facilitating transmission.

In Vitro Activity of Clofazimine against Nontuberculous Mycobacteria Isolated in Beijing, China.

Due to the natural resistance of nontuberculous mycobacteria (NTM) to many antibiotics, the treatment of diseases caused by NTM is often long-term but unsuccessful. The main goal of this study was to evaluate the in vitro susceptibilities to clofazimine of 209 isolates consisting of different NTM species isolated in Beijing, China. Furthermore, 47 reference strains were also tested, including 30 rapidly growing mycobacterium (RGM) species and 17 slowly growing mycobacterium (SGM) species. The potential molecular mechanism contributing to clofazimine resistance of NTM was investigated as well. Clofazimine exhibited excellent activity against both reference strains and clinical isolates of different SGM species, and most of the strains had MICs far below 1 µg/ml. Although the majority of the clinical isolates of Mycobacterium abscessus and Mycobacterium fortuitum had MICs higher than 2 µg/ml, 17 out of the 30 reference strains of different RGM species had MICs below 1 µg/ml in vitro According to the MIC distributions, the tentative epidemiological cutoff (ECOFF) values for Mycobacterium kansasii, Mycobacterium avium, and Mycobacterium intracellulare were defined at 0.5 µg/ml, 1 µg/ml, and 2 µg/ml, respectively. Intriguingly, single-direction cross-resistance between bedaquiline- and clofazimine (Cfz)-resistant isolates was observed among the tested NTM species. This study demonstrates that clofazimine had strong activity against most SGM species in vitro, as well as some RGM species. The data provide important insights into the possible clinical application of Cfz to treat NTM infections.

Overexpression of PtABCC1 contributes to mercury tolerance and accumulation in Arabidopsis and poplar.

Mercury (Hg) is a highly biotoxic heavy metal that contaminates the environment. Phytoremediation is a green technology for environmental remediation and is used to clean up Hg contaminated soil in recent years. In this study, we isolated an ATP-binding cassette (ABC) transporter gene PtABCC1 from Populus trichocarpa and overexpressed it in Arabidopsis and poplar. The transgenic plants conferred higher Hg tolerance than wild type (WT) plants, and overexpression of PtABCC1 could lead to 26-72% or 7-160% increase of Hg accumulation in Arabidopsis or poplar plants, respectively. These results demonstrated that PtABCC1 plays a crucial role in enhancing tolerance and accumulation to Hg in plants, which provides a promising way for phytoremediation of Hg contamination.

In Vitro Drug Susceptibility of Bedaquiline, Delamanid, Linezolid, Clofazimine, Moxifloxacin, and Gatifloxacin against Extensively Drug-Resistant Tuberculosis in Beijing, China.

Extensively drug-resistant tuberculosis (XDR-TB) is a deadly form of TB that can be incurable due to its extreme drug resistance. In this study, we aimed to explore the in vitro susceptibility to bedaquiline (BDQ), delamanid (DMD), linezolid (LZD), clofazimine (CLO), moxifloxacin (MFX), and gatifloxacin (GAT) of 90 XDR-TB strains isolated from patients in China. We also describe the genetic characteristics of XDR-TB isolates with acquired drug resistance. Resistance to MFX, GAT, LZD, CLO, DMD, and BDQ was found in 82 (91.1%), 76 (84.4%), 5 (5.6%), 5 (5.6%), 4 (4.4%), and 3 (3.3%) isolates among the XDR-TB strains, respectively. The most frequent mutations conferring fluoroquinolone resistance occurred in codon 94 of the gyrA gene (57.8%), and the strains with these mutations (69.2%) were associated with high-level MFX resistance compared to strains with mutations in codon 90 (25.0%) (P < 0.01). All 5 CLO-resistant isolates exhibited ≥4-fold upward shifts in the BDQ MIC, which were attributed to mutations of codons 53 (60.0%) and 157 (20.0%) in the Rv0678 gene. Additionally, mutation in codon 318 of the fbiC gene was identified as the sole mutation related to DMD resistance. In conclusion, our data demonstrate that the XDR-TB strains exhibit a strikingly high proportion of resistance to the current anti-TB drugs, whereas BDQ, DMD, LZD, and CLO exhibit excellent in vitro activity against XDR-TB in the National Clinical Center on TB of China. The extensive cross-resistance between OFX and later-generation fluoroquinolones indicates that MFX and GAT may have difficulty in producing the desired effect for XDR-TB patients.

Transmitted Extended-Spectrum Extensively Drug-Resistant Tuberculosis in Beijing, China, with Discordant Whole-Genome Sequencing Analysis Results.

Drug resistance to tuberculosis remains a major public health threat. Here, we report two cases of extended-spectrum extensively drug-resistant (XXDR) tuberculosis showing resistance to most first- and second-line agents. The results of a correlation of whole-genome sequencing (WGS) and phenotypic testing were discordant, suggesting that overreliance on WGS may miss clinically relevant resistance in extensively drug-resistant disease.

[Detection of mutations in the gyrA and gyrB genes associated with fluoroquinolone-resistance among clinical isolates of Mycobacterium abscessus in China].

OBJECTIVE: To determine molecular mutations in gyrA and gyrB genes and their association with fluoroquinone-resistance among Mycobacterium abscessus isolates from China. METHODS: Antimicrobial susceptibility profile of Mycobacterium abscessus isolates was evaluated for resistance to levofloxacin (LFX) and moxifloxacin (MFX). The quinolone resistant determing regions (QRDR) of gyrA and gyrB genes of all the isolates was sequenced and analyzed. RESULTS: A total 70 Mycobacterium abscessus isolates were analyzed, including 45 (64%) M. abscessus subsp. abscessus and 25 (36%) M. abscessus subsp. massiliense. Analysis of antimicrobial susceptibility profile showed 97% (n=68) of isolates were resistance to LFX and 14.3% (n=10) of isolates were resistance to MFX. All the MFX-resistant strains were resistant to LFX. Cross-resistance to LFX and MFX was noted in 14.3% (n=10) of isolates. Sequencing analysis revealed that the Ser83Ala substitution in the gyrA gene and Lys447Arg and Ser464Asn substitutions in the gyrB gene were found in all the isolates from both M. abscessus subsp. abscessus and M. abscessus subsp. massiliense. No mutation was found to be associated with cross-resistance of M. abscessus to LFX and MFX in the entire gyrA and gyrB gene. CONCLUSIONS: Our data suggest that there is no clear correlation between the type of mutation in the gene gyrA and gyrB, and the MIC levels of LFX and MFX for resistant M. abscessus strains.

StyleTalk++: A Unified Framework for Controlling the Speaking Styles of Talking Heads.

Individuals have unique facial expression and head pose styles that reflect their personalized speaking styles. Existing one-shot talking head methods cannot capture such personalized characteristics and therefore fail to produce diverse speaking styles in the final videos. To address this challenge, we propose a one-shot style-controllable talking face generation method that can obtain speaking styles from reference speaking videos and drive the one-shot portrait to speak with the reference speaking styles and another piece of audio. Our method aims to synthesize the style-controllable coefficients of a 3D Morphable Model (3DMM), including facial expressions and head movements, in a unified framework. Specifically, the proposed framework first leverages a style encoder to extract the desired speaking styles from the reference videos and transform them into style codes. Then, the framework uses a style-aware decoder to synthesize the coefficients of 3DMM from the audio input and style codes. During decoding, our framework adopts a two-branch architecture, which generates the stylized facial expression coefficients and stylized head movement coefficients, respectively. After obtaining the coefficients of 3DMM, an image renderer renders the expression coefficients into a specific person's talking-head video. Extensive experiments demonstrate that our method generates visually authentic talking head videos with diverse speaking styles from only one portrait image and an audio clip.

Potato Virus X Inactivation and Characterization.

The nucleoprotein components of plant viruses self-assemble into monodisperse, nanoscale structures with a high degree of symmetry and polyvalency. Of particular interest are the filamentous plant viruses which provide uniform high aspect ratio nanostructures-such structures remain challenging to obtain using purely synthetic approaches. Potato virus X (PVX) has drawn interest by the materials science community because of its filamentous structure measuring 515 × 13 nm; and both genetic engineering and chemical conjugation methods have been reported to impart new functionalities and develop PVX-based nanomaterials for applications in the health and materials sector. Toward environmentally safe materials-i.e., materials that are not infectious toward crops, such as potato, we reported methods to inactivate PVX. In this chapter, we describe the three methods to inactivate PVX and render it non-infectious toward plants, while maintaining structure and function.

Biological interpretation of the sporadic sputum smear-positive-culture-negative outcome for patients with tuberculosis undertaking treatments.

Introduction: The objective of the study was to identify the causes of smear-positive-culture-negative (S+/C-) outcomes of patients with tuberculosis during the treatment course. Methods: A laboratory-based retrospective study was performed at the Beijing Chest Hospital in China. Within the study period, all patients with pulmonary tuberculosis (PTB) who undertook anti-TB treatments and yielded smear positive outcomes with simultaneous culture outcomes on sputa were considered. Patients were classified into three groups: (I) performed LJ medium culture only; (II) performed BACTEC MGIT960 liquid culture only; and (III) performed both LJ culture and MGIT960 culture. The S+/C- rates of each group were analyzed. The clinical medical records regarding patient category, follow-up bacteriologic examination data, and treatment response were investigated. Results: In total, 1,200 eligible patients were enrolled, and the overall S+/C- rate was 17.5% (210/1,200). Group I had obviously higher S+/C- rate (37%) than group II (18.5%) and group III (9.5%). When solid and liquid cultures were considered independently, the S+/C- outcome was observed more frequently in the solid culture group than in the liquid culture group (30.4%, 345/1,135 vs. 11.5%, 100/873; p < 0.001, χ2 = 102.64). Among the 102 S+/C- patients who had follow-up cultures performed, 35 (34.3%) had positive culture outcomes. Whereas among the 67 patients with follow-up information for more than 3 months but without supportive bacteriological evidence, 45 (67.2%, 45/67) had unfavorable prognosis (including relapse and unimproved conditions), and only 22 (32.8%, 22/67) patients had improved conditions. Compared with new cases, retreated cases produced S+/C- outcomes more frequently and had more chances to be cultivated bacilli successfully afterward. Conclusions: Among our patients, the sporadic smear positive and culture negative outcomes for sputa are more likely associated with the technical failures of culture than with dead bacilli, and this is especially noteworthy for LJ medium culture.

Effects of fertilization gradient on the production performance and nutritional quality of cultivated grasslands in karst areas.

Optimal fertilization is an important measure for managing cultivated grasslands, and a necessary means for maintaining the nutrient balance, yield, and quality of grassland ecosystems. This study aimed to explore the effects of organic fertilizers on the production performance and nutritional quality of cultivated grasslands in karst areas. Two types of monocultured cultivated grasslands (i.e., Medicago sativa and Dactylis glomerata) were employed as the research objects, and a randomized block design was adopted to investigate the effects of five fertilization gradients on the forage height, coverage, yield, and nutritional quality of the cultivated grasslands. According to the results, the plant height, coverage, and yield of M. sativa first presented an increasing trend, then decreased with increasing fertilization gradient, with a peak at 20-30 t/hm2 fertilization gradient. The height, coverage, and yield of D. glomerata increased gradually with increase in fertilization gradient, and peaked at 40 t/hm2. Meanwhile, the crude protein (CP) and ether extract (EE) contents of both grassland types displayed first presented an increasing trend, then decreased with increasing fertilization gradient, and peaked at 10-30 t/hm2 fertilization gradient. The neutral detergent fiber (NDF) and acidic detergent fiber (ADF) contents of M. sativa presented "N-shaped" and "M-shaped" change trends with increasing fertilization gradient, while those of D. glomerata showed "V-shaped" and "M-shaped" change trends, reaching minimum values under fertilization gradients of 30 and 20 t/hm2, respectively. Year, fertilization, and year × fertilization (Y×F) significantly affected the plant heights, coverages, dry/fresh weight ratios, and yields of M. sativa and D. glomerata. The contribution of coverage to the subordinate function of M. sativa was greatest at a fertilization gradient of 20 t/hm2. Meanwhile, the subordinate function values of the height and coverage of D. glomerata increased gradually with increasing fertilization gradient, but the difference in the subordinate function value of height was only 0.09%-0.18% under the fertilization gradient of 20-40 t/hm2. Evaluation of forage nutrition revealed 10-30 t/hm2 and 20-30 t/hm2 as the optimal organic fertilizer application rates for M. sativa and D. glomerata, respectively.

Toward Plant Cyborgs: Hydrogels Incorporated onto Plant Tissues Enable Programmable Shape Control.

Engineered living materials (ELMs) that incorporate living organisms and synthetic materials enable advanced functional properties. Here, we seek to create plant cyborgs by combining plants or plant tissues with stimuli-responsive polymeric materials. Plant tissues with integrated shape control may find applications in regenerative medicine, and the shape control of living plants enables another dimension of adaptability and response to environmental threats, which can be applied to next-generation precision farming. In this work, we develop chemistry to integrate stimuli-responsive poly(N-isopropylacrylamide) (PNIPAM) hydrogels with decellularized plant tissues assisted by 3D printing. We demonstrate programmable shape morphing in response to thermal cues and ultraviolet (UV) light. Specifically, by taking advantage of the extrusion-based 3D printing method, we deposit nanocomposite PNIPAM precursors onto silane-treated decellularized leaf surface with prescribed shapes and spatial control. When subjected to external stimuli, the strain mismatch generated between the swellable nanocomposite PNIPAM and nonswellable decellularized leaf enables folding and bending to occur. This strategy to integrate the plant tissues with stimuli-responsive hydrogels allows the control of leaf morphology, opening avenues for plant-based biosensors and soft actuators to enhance food security; such materials also may find applications in biomedicine as tissue-engineering scaffolds.