Regeneration System Research Articles

Rational design of a zinc-dependence secondary alcohol dehydrogenase to boost its oxidation activity for α-hydroxyketones biosynthesis

α-Hydroxyketones, which have significant industrial applications, can be sustainably synthesized through the oxidation of secondary alcohols using secondary alcohol dehydrogenase (SADH). However, the activity of the SADH oxidation reaction is generally low, making it unsuitable for large-scale production. In this study, a rational design approach was employed to computationally engineer SADH derived from Ogataea parapolymorpha (OpSADH), significantly enhancing its oxidation activity towards (R)-1,2-propanediol (PDO). The mutant M2 (S222T/S316A) exhibited a 14.2-fold increase in specific enzyme activity compared to the wild type (WT) and was employed as a catalyst for high-concentration hydroxyacetone production, facilitated by an NAD+ regeneration system. By applying an appropriate Zn2+ ion force field in molecular dynamics (MD) simulations, it was found that two mutation sites could stabilize the conformations of NAD+ and PDO, thereby revealing the molecular mechanism behind the enhanced activity of this metalloenzyme mutant. Notably, we first uncovered the dynamic mechanism by which four key residues (Cys39, His75, Glu76, and Glu175) and PDO within the active pocket contribute to the formation of coordination bonds with Zn2+. The findings of this study provide robust support for researching the catalytic mechanisms and dynamics processes of metalloenzymes.

Establishment of callus induction and plantlet regeneration systems of Peucedanum Praeruptorum dunn based on the tissue culture method.

Peucedanum praeruptorum Dunn has typical stacked umbels and medicinal value; however, the lack of an effective tissue culture system for P. praeruptorum has limited the large-scale propagation of its seedlings. We systematically established an in vitro regeneration system for P. praeruptorum using young leaves and stems as explants. Tissue culture plantlets were successfully obtained within 123 and 90 d of somatic embryogenesis and organogenesis, respectively. Combined plant growth regulators (PGRs) were optimized to promote efficient plant regeneration at each stage of the culture process. Specifically, embryogenic callus induction was superior in Murashige and Skoog (MS) medium supplemented with 0.5mg/L 6-benzyladenine (BA) and 2.0mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). For somatic embryonic development, the highest differentiation rates were achieved using BA, 2,4-D, and 6-furfuryl aminopurine (6-KT). Induction of organogenesis resulted in the highest differentiation rates and proliferation coefficients of buds in MS medium supplemented with BA and α-naphthaleneacetic acid (NAA). Moreover, regeneration of P. praeruptorum seedlings was achieved by adjusting the BA and indole-3-butyric acid (IBA) concentrations in 1/2 MS medium. Our results provide a technical system for the rapid propagation of P. praeruptorum, which can facilitate germplasm improvement, resource conservation, and further genetic transformation of Peucedanum species.

Multi-omics analysis reveals the positive impact of differential chloroplast activity during in vitro regeneration of barley.

Existence of potent in vitro regeneration system is a prerequisite for efficient genetic transformation and functional genomics of crop plants. In this study, two contrasting cultivars differencing in their in vitro regeneration efficiency were identified. Tissue culture friendly cultivar Golden Promise (GP) and tissue culture resistant DWRB91(D91) were selected as contrasting cultivars to investigate the molecular basis of regeneration efficiency through multiomics analysis. Transcriptomics analysis revealed 1487 differentially expressed genes (DEGs), in which 795 DEGs were upregulated and 692 DEGs were downregulated in the GP-D91 transcriptome. Genes encoding proteins localized in chloroplast and involved in ROS generation were upregulated in the embryogenic calli of GP. Moreover, proteome analysis by LC-MS/MS revealed 3062 protein groups and 16,989 peptide groups, out of these 1586 protein groups were differentially expressed proteins (DEPs). Eventually, GC-MS based metabolomics analysis revealed the higher activity of plastids and alterations in key metabolic processes such as sugar metabolism, fatty acid biosynthesis, and secondary metabolism. TEM analysis also revealed differential plastid development. Higher accumulation of sugars, amino acids and metabolites corresponding to lignin biosynthesis were observed in GP as compared to D91. A comprehensive examination of gene expression, protein profiling and metabolite patterns unveiled a significant increase in the genes encompassing various functions, such as ion homeostasis, chlorophyll metabolic process, ROS regulation, and the secondary metabolic pathway.

Climate Justice Strategies Implemented by Public Health Nurses and Their Community Partners.

To describe nurses' and community-based organization representatives' collaborative strategies for advancing climate justice with communities. This study used a descriptive, qualitative research design. Data were gathered from August 2022 to February 2023 with nurses (n = 8) and their community partners (n = 5) in the United States. Community partners were representatives of community-based organizations. Photovoice provided greater context for the thematic analysis of collaborative strategies discussed in semi-structured interviews. Nurse participants worked in academic or non-profit settings. Nurse-community partnerships addressed corporate pollution and promoted Indigenous sovereignty and multispecies justice. Themes included investigating disease and health events, identifying at-risk populations and connecting them with resources, providing health teaching and counseling, organizing communities and coalitions, and advocating for policy development and enforcement. Self-care supported resilience and well-being in the long struggle for climate justice. Findings from this study indicated that nurses and their community partners strategize to transition communities away from systems of extraction towards local and regenerative systems that support resilience. Nurses and their community partners recognized the importance of applying an expansive understanding of climate justice, including intersections of pollution and multispecies justice, to promoting planetary health. Findings from this study support nurse-community collaboration in policy work to advance planetary health. This study also supports nurses' collective action with their community partners to address the effects of white supremacy and colonization. Future research is needed to evaluate the outcomes of nurse-community partnerships for planetary health. Nurses have called for action on climate justice; however, evidence of effective nursing strategies that advance climate justice is sparse. This study is the first to describe the collaborative strategies nurses implement with community partners to support the transition from injustice to justice in communities most burdened by climate change and industrial pollution.

Establishment and application of highly efficient regeneration, genetic transformation and genome editing system for cucurbitacins biosynthesis in Hemsleya chinensis

Background Hemsleya Chinensis is a perennial plant in the Cucurbitaceae family containing antibacterial and anti-inflammatory compounds. The lack of genetic transformation systems makes it difficult to verify the functions of genes controlling important traits and conduct molecular breeding in H. chinensis.ResultsHighly efficient calli were induced on MS medium added 1.5 mg·L− 1 6-benzylaminopurine (6-BA) and 0.02 mg·L− 1 1-naphthylacetic acid (NAA) with high efficiency (> 95%). The frequency of shoot induction was increased to 90% with a plant growth regulator combination of 1.5 mg·L− 1 6-BA and 0.1 mg·L− 1 NAA. Our results also showed that 100% of shoot regeneration was achieved in a shoot regeneration medium. Additionally, more than 92% of kanamycin-resistant plants were confirmed. Furthermore, we achieved 42% genome editing efficiency by applying this transformation method to HcOSC6, a gene that catalyzes the formation of cucurbitadienol. HPLC analysis showed OE-HcOSC6 lines exhibited significantly higher cucurbitadienol levels than the genome-edited lines. Transcriptomic analysis revealed that some downstream genes related to cucurbitadienol biosynthesis, such as HcCYP87D20, HcCYP81Q58, and HcSDR34, were up-regulated in OE lines and down-regulated in mutants.ConclusionsHere, we established a process for regeneration, transformation, and genome editing of H. chinensis using stem segments. This provides valuable insight into the underlying molecular mechanisms of medicinal compound production. By combining high-efficiency tissue culture, transformation, and genome editing systems, we provide a powerful platform that supports functional research on molecular mechanisms of secondary metabolism.

Biosynthesis of Sialyllacto-N-tetraose c in Engineered Escherichia coli.

Human milk oligosaccharides (HMOs) have attracted considerable interest for their vital role in supporting infant health. Among these, sialyllacto-N-tetraose c (LST c), a pentasaccharide with the structure Neu5Ac(α2,6)Gal(β1,4)GlcNAc(β1,3)Gal(β1,4)Glc, stands out due to its critical importance in the development and application of complex HMOs. In this study, we employed multivariate modular metabolic engineering (MMME) to screen for efficient sialyltransferases and balance metabolic fluxes, successfully constructing strains capable of LST c biosynthesis. Additionally, by blocking competing pathway genes, enhancing the supply of UDP-GlcNAc and UDP-Gal precursors, and establishing a CTP cofactor regeneration system, we developed a high-yielding Escherichia coli strain, W15. This strain achieved an LST c titer of 220.9 mg/L in shake flask cultures. In a 3-L fed-batch fermentation, the LST c concentration reached 922.2 mg/L, with a productivity of 10.25 mg/L/h and a specific yield of 38.70 mg/g DCW. This research provides an effective strategy for producing LST c in microbial cell factories.

Establishment of an efficient regeneration system for microspores in non-heading Chinese cabbage

Establishment of an efficient regeneration system for microspores in non-heading Chinese cabbage

The Deposition of Hydroxyapatite Particles Within an Organic Matrix on the Surface of Poly(lactic acid).

Hydroxyapatite (HAP) is a well-established material in biomedical applications, especially for bone tissue regeneration, dental implants, and drug delivery systems. Recent research emphasizes enhancing the biocompatibility and osteoconductivity of orthopedic implants using HAP. This study explores the potential of combining HAP with a lipid matrix to improve the surface properties and biocompatibility of poly(lactic acid) (PLA)-based, 3D-printed, resorbable bone implants. We utilized the Langmuir-Blodgett method to deposit HAP within a dihexadecyl phosphate (DHP) matrix onto PLA substrates. This study demonstrates that DHP and HAP form stable monolayers at the air/water interface with HAP particles distributed within a homogeneous lipid matrix. The presence of HAP and the resulting changes in surface free energy (SFE) are hypothesized to enhance the biocompatibility of PLA implants. Our findings indicate that films composed of DHP + HAP 5:1 are particularly effective in altering PLA surface characteristics, potentially improving osteointegration, and reducing microbial adherence. Overall, this work highlights that surface modification of PLA with HAP and lipid matrices is the first step towards new, promising, and cost-effective strategies for developing advanced biomaterials for bone regeneration.

Haploid regeneration system suitable for various genotypes of thin-skinned melons

Haploid regeneration system suitable for various genotypes of thin-skinned melons

Development and implementation of a simulated microgravity setup for edible cyanobacteria

Regenerative life support systems for space crews recycle waste into water, food, and oxygen using different organisms. The European Space Agency’s MELiSSA program uses the cyanobacterium Limnospira indica PCC8005 for air revitalization and food production. Before space use, components’ compatibility with reduced gravity was tested. This study introduced a ground analog for microgravity experiments with oxygenic cyanobacteria under continuous illumination, using a random positioning machine (RPM) setup. L. indica PCC8005 grew slower under low-shear simulated microgravity, with proteome analysis revealing downregulation of ribosomal proteins, glutamine synthase, and nitrate uptake transporters, and upregulation of gas vesicle, photosystem I and II, and carboxysome proteins. Results suggested inhibition due to high oxygen partial pressure, causing carbon limitation when cultivated in low-shear simulated microgravity. A thicker stagnant fluid boundary layer reducing oxygen release in simulated microgravity was observed. These findings validate this RPM setup for testing the effects of non-terrestrial gravity on photosynthetic microorganisms.

Engineering the Substrate Specificity of UDP-Glycosyltransferases for Synthesizing Triterpenoid Glycosides with a Linear Trisaccharide as Aided by Ancestral Sequence Reconstruction.

Triterpenoids have wide applications in the pharmaceutical and agricultural industries. The glycosylation of triterpenoids catalyzed by UDP-glycosyltransferases (UGTs) is a crucial method for producing valuable derivatives with enhanced functions. However, only a few UDP-glucosyltransferases have been reported to synthesize the rare triterpenoids with linear-chain trisaccharide at C3-OH. This study revealed that the UGT91H subfamily primarily contributed to the 2"-O-glycosylation of triterpenoids with high regioselectivity, then the substrate scope was further expanded by ancestral sequence reconstruction (ASR). With ancestral enzyme UGT91H_A1 as a model, the sequence-structure-function relationship was explored. A RTAS loop (R212/T213/A214/S215) was identified to affect the substrate specificity of UGT91H_A1. Transferring this RTAS loop to the corresponding position of UGT91H enzymes successfully expanded their substrate spectra. The functional role of RTAS loop was further elucidated by molecular dynamics simulation and quantum mechanical computation. UGT91H_A1 was applied to the low-cost synthesis of terpenoid rhamnosides with a linear trisaccharide in combining with a self-sufficient UDP-rhamnose regeneration system. Finally, we developed a phylogeny-based platform to efficiently mining new UGT91Hs from plant genomic data. This study provided robust biocatalysts for synthesizing various triterpenoid glycosides with a linear trisaccharide and demonstrated ASR as an efficient tool in engineering the function of UDP-glycosyltransferases.

Inborn Errors of Immunity-related Immunological Mechanisms and Pharmacological Therapy Alternatives in Periodontitis.

Periodontitis is a frequent local inflammatory disease. The microbiota and repeated exposure to bacterial endotoxins triggers excessive inflammation through oral mucosal immunity, sometimes leading to a destructive effect on the supportive mucosal tissues around the teeth. Elimination of the pathogens and increasing the tolerance of the cellular immune response is crucial in addition to standard dental therapies like mechanical debridement. Based on our experience on immune-mediated diseases, especially primary immunodeficiency diseases (PIDs), we wrote this review to discuss the treatment alternatives for severe periodontal disease. Risk factors are malnutrition, vitamin deficiencies, smoking, systemic inherited and acquired immune-mediated diseases, infections, endocrinological diseases, and pharmacological agents may accompany periodontitis. The diagnosis and treatment of dietary deficiencies, as well as the addition of nutritional supplements, may aid in epithelial regeneration and immune system function. Recently, modifications to the therapeutic option for severe periodontitis have been made depending on the fact that the immune response against bacteria may modify the severity of periodontal inflammation. The anti-inflammatory therapies support or inhibit the host's immune response. The clinical approach to severe periodontitis should extend beyond classical therapies. There is a need for a diverse therapeutic strategy that supports the epithelial barrier, which is the crucial component of innate immunity against microbiota. Leukocytes are the main cellular component in periodontal inflammation. Anti-inflammatory therapeutic options directed at leukocytes, such as IL-17 and IL-23-targeted therapies, could be the candidates for the treatment of severe periodontitis. Therapy against other inflammatory cytokines, IL-1, IL-6, IL12, IL23, TNF-alpha, PGE2, and cytokine receptors, could also be used in periodontal inflammation control.

Highly efficient synthesis of the chiral ACE inhibitor intermediate (R)-2-hydroxy-4-phenylbutyrate ethyl ester via engineered bi-enzyme coupled systems

(R)-2-Hydroxy-4-phenylbutyric acid ethyl ester ((R)-HPBE) is an essential chiral intermediate in the synthesis of angiotensin-converting enzyme (ACE) inhibitors. Its production involves the highly selective asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE), catalyzed by carbonyl reductase (CpCR), with efficient cofactor regeneration playing a crucial role. In this study, an in-situ coenzyme regeneration system was developed by coupling carbonyl reductase (CpCR) with glucose dehydrogenase (GDH), resulting in the construction of five recombinant strains capable of NADPH regeneration. Among these, the recombinant strain E. coli BL21-pETDuet-1-GDH-L-CpCR, where CpCR is fused to the C-terminus of GDH, demonstrated the highest catalytic activity. This strain exhibited an enzyme activity of 69.78 U/mg and achieved a conversion rate of 98.3%, with an enantiomeric excess (ee) of 99.9% during the conversion of 30 mM OPBE to (R)-HPBE. High-density fermentation further enhanced enzyme yield, achieving an enzyme activity of 1960 U/mL in the fermentation broth, which is 16.2 times higher than the volumetric activity obtained from shake flask fermentation. Additionally, the implementation of a substrate feeding strategy enabled continuous processing, allowing the strain to efficiently convert a final OPBE concentration of 920 mM, producing 912 mM of (R)-HPBE. These findings highlight the system’s improved catalytic efficiency, stability, and scalability, making it highly suitable for industrial-scale biocatalytic production.

Unveiling the Molecular Mechanisms of Browning in Camellia hainanica Callus through Transcriptomic and Metabolomic Analysis.

Camellia hainanica is one of the camellia plants distributed in tropical regions, and its regeneration system and genetic transformation are affected by callus browning. However, the underlying mechanism of Camellia hainanica callus browning formation remains largely unknown. To investigate the metabolic basis and molecular mechanism of the callus browning of Camellia hainanica, histological staining, high-throughput metabolomics, and transcriptomic assays were performed on calli with different browning degrees (T1, T2, and T3). The results of histological staining revealed that the brown callus cells had obvious lignification and accumulation of polyphenols. Widely targeted metabolomics revealed 1190 differentially accumulated metabolites (DAMs), with 53 DAMs annotated as phenylpropanoids and flavonoids. Comparative transcriptomics revealed differentially expressed genes (DEGs) of the T2 vs. T1 associated with the biosynthesis and regulation of flavonoids and transcription factors in Camellia hainanica. Among them, forty-four enzyme genes associated with flavonoid biosynthesis were identified, including phenylalaninase (PAL), 4-coumaroyl CoA ligase (4CL), naringenin via flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), Chalcone synthase (CHS), Chalcone isomerase (CHI), hydroxycinnamoyl-CoA shikimate transferase (HCT), Dihydroflavonol reductase (DFR), anthocyanin reductase (LAR), anthocyanin synthetase (ANS), and anthocyanin reductase (ANR). Related transcription factors R2R3-MYB, basic helix-loop-helix (bHLH), and WRKY genes also presented different expression patterns in T2 vs. T1. These results indicate that the browning of calli in Camellia hainanica is regulated at both the transcriptional and metabolic levels. The oxidation of flavonoids and the regulation of related structural genes and transcription factors are crucial decisive factors. This study preliminarily revealed the molecular mechanism of the browning of the callus of Camellia hainanensis, and the results can provide a reference for the anti-browning culture of Camellia hainanica callus.

Using CRISPR-Cas9 genome editing to enhance disease resistance in banana

Abstract Bananas and plantains ( Musa spp.) are leading global fruit crops, cultivated in over 150 countries and several island nations and serving as fruit and staple food in tropical and subtropical regions. Notwithstanding their economic importance, banana production is hindered by pests, diseases, and climatic challenges, resulting in significant yield gaps and post-harvest losses. Conventional breeding methods are time-consuming and labor-intensive due to the 15–18 months long life cycle and seedless nature of the edible crop, while transgenic approaches face regulatory, public acceptance, and sociopolitical hurdles. The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) technology offers a precise, targeted, and efficient tool for genetic mutation derived from the bacterial antiviral defense system. Researchers are exploring CRISPR-Cas9 to develop banana varieties resistant to diseases and pests, exhibit higher yields, and possess enhanced nutritional profiles. The well-annotated complete banana genome, combined with advanced regeneration and transformation systems, facilitates the application of CRISPR-Cas9 technology. This innovative approach promises to create superior banana cultivars that benefit consumers and farmers, addressing critical challenges in banana production and contributing to global food security. This review article discusses the challenges in banana production, including biotic and abiotic constraints, and explores recent advancements in genome editing.

Regeneration and Genetic Transformation in Eucalyptus Species, Current Research and Future Perspectives

Eucalyptus is an important plantation tree with a high economic value in China. The tree contributes significantly to China’s timber production. The stable and efficient Eucalyptus regeneration system and genetic transformation system are of great significance for exploring the regulatory function and possible genetic breeding capacity of important genes in the species. However, as a woody plant, Eucalyptus has problems, such as a long generation cycle, strong specificity of the regeneration system, and a low genetic conversion rate, which seriously limit the rapid development of Eucalyptus genetics and breeding programs. The present review summarizes the status of research on Eucalyptus regeneration and genetic transformation, with a focus on the effects of explants, media, plant growth regulators (PGRs), and concentrations in the Eucalyptus regeneration process. In addition, the effects of genotype, Agrobacterium, antibiotics, preculture, and co-culture on the genetic transformation efficiency of Eucalyptus are discussed. Furthermore, the study also summarizes the problems encountered in Eucalyptus regeneration and genetic transformation, with reference to previous studies, and it outlines future developments and prospects. The aim was to provide a reference for solving the problems of genetic instability and the low transformation efficiency of eucalyptus, and to establish an efficient and stable eucalyptus regeneration and transformation system to accelerate the process of its genetic improvement.

Application of Nanomaterials in the Repair and Regeneration of Lymphatic Organs and Corresponding Biophysical Simulation Strategies

Immune system diseases, malignant tumors, and traumatic injuries can directly damage the structure and function of lymphoid organs, while subsequent radiotherapy, chemotherapy, and lymph node dissection further damage the patient's immune system, leading to immune dysfunction, metabolic disorders, and increased susceptibility to infection, which seriously affect the patient's prognosis and quality of life. In this context, nanotechnology plays a key role in lymphoid organ regeneration and immune function recovery, including improving the therapeutic effect through targeted drug delivery systems, using targeted imaging probes to achieve tumor prediction and early detection, combining nanoplatforms with immunotherapy and photodynamic therapy to achieve synergistic therapeutic effects, and using nanomaterials to regulate the tumor microenvironment to enhance the sensitivity of traditional treatments. In addition, biophysical simulation strategies that simulate the microenvironment of lymphoid organs have also attracted widespread attention, aiming to construct a native cell environment to support the regeneration and functional recovery of damaged lymphoid tissues, or to simulate immune cells to regulate lymphocytes and induce specific immune responses. The multifaceted application of nanotechnology provides promising prospects for lymphoid organ regeneration and immune system repair.

Establishment of regeneration system for tissue culture of Euphorbia milii

Establishment of regeneration system for tissue culture of Euphorbia milii

Effective in vitro regeneration systems from leaf explants for commercially important pear cultivars (Pyrus communis L.)

Effective in vitro regeneration systems from leaf explants for commercially important pear cultivars (Pyrus communis L.)

Use of endogenous acetate kinase for ATP regeneration system of whole-cell L-theanine production catalyzed by γ-glutamylmethylamide synthetase

Use of endogenous acetate kinase for ATP regeneration system of whole-cell L-theanine production catalyzed by γ-glutamylmethylamide synthetase