Regeneration System Research Articles

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

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.

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

Analysis of the plant hormone expression profile during somatic embryogenesis induction in teak (Tectona grandis).

Plant somatic embryogenesis (SE) is an efficient regeneration system for propagation. It involves the regulation of a complex molecular regulatory network encompassing endogenous hormone synthesis, metabolism, and signal transduction processes, induced through exogenous plant growth regulators. Previous studies have focused primarily on traditional propagation methods for Tectona grandis, but there is limited knowledge on SE and its hormonal regulatory mechanisms. In our study, different SE stages, including the nonembryogenic callus (NEC), embryogenic callus (EC), and globular and heart-shaped embryo (E-SEs) stages, were induced in teak cotyledons incubated on MS medium supplemented with 0.1 mg/L thidiazuron (TDZ). Morphological and histological observations indicated that EC primarily originates from the development of embryogenic cell clusters. During SE induction, the levels of six classes of endogenous hormones, IAA, CTK, ETH, ABA, SA, and JA, changed significantly. Transcriptome analysis revealed that endogenous hormones participate in SE induction in teak through various biological processes, such as biosynthesis, metabolism, and signal transduction pathways. We found that IAA biosynthesis primarily occurs through the IAM pathway during these three stages. The ETH receptor kinase gene SERF1 exhibited the highest expression levels in E-SEs. The ABA-, SA-, and JA-related signal transduction genes ABI3, NPR1, and JAZ exhibited no differential expression during different stages. Moreover, key encoding genes of SE regulators, including WUS, WOX and SERK, were differentially expressed during SE. In conclusion, this study offers insights into the roles of endogenous hormones and their interactions during SE induction.

Injectable double-crosslinked bone cement with enhanced bone adhesion and improved osteoporotic pathophysiological microenvironment for osteoregeneration in osteoporosis

The osteoporotic bone defect caused by excessive activity of osteoclasts has posed a challenge for public healthcare. However, most existing bioinert bone cement fails to effectively regulate the pathological bone microenvironment and reconstruct bone homeostasis in the presence of osteoclast overactivity and osteoblast suppression. Herein, inspired by natural bone tissue, an in-situ modulation system for osteoporotic bone regeneration is developed by fabricating an injectable double-crosslinked PEGylated poly(glycerol sebacate) (PEGS)/calcium phosphate cement (CPC) loaded with sodium alendronate (ALN) (PEGS/CPC@ALN) adhesive bone cement. By incorporating ALN, the organic-inorganic interconnection within PEGS/CPC@ALN results in a 100 % increase in compression modulus and energy dissipation efficiency. Additionally, PEGS/CPC@ALN effectively adheres to the bone by bonding with amine and calcium ions present on the bone surface. Moreover, this in-situ regulation system comprehensively mitigates excessive bone resorption through the buffering effect of CPC to improve the acidic microenvironment of osteoporotic bone and the release of ALN to inhibit hyperactive osteoclasts, and facilitates stem cell proliferation and differentiation into osteoblasts through calcium ion release. Overall, the PEGS/CPC@ALN effectively regulates the pathological microenvironment of osteoporosis while promoting bone regeneration through synergistic effects of drugs and materials, thereby improving bone homeostasis and enabling minimally invasive treatment for osteoporotic defects.

Mini Review: Challenges and Promising Advances in Hydrogel‐Based Scaffolds for Breast Tissue Regeneration

ABSTRACTThe loss of breast tissue is a problem that plagues many women. Total mastectomy procedures after breast cancer or extensive tissue loss by accident generate permanent aesthetic‐functional lesions that are correlated with the loss of breastfeeding capacity, loss of self‐esteem, anxiety, depression, reduced sexual attraction, hopelessness, and often, suicide. Although current clinical practices try to restore aesthetics, functional improvements are not readily resolved by these maneuvers. Those who undergo such procedures often end up with permanent aesthetic and functional injuries. The difficulty in regenerating breast tissue stems from its high structural complexity. In cases of extensive loss of breast tissue, the use of regeneration strategies becomes necessary for proper healing and tissue reconstruction. In this context, the use of regeneration scaffolds may open new possibilities for the regeneration of various body tissues, especially breast tissue, enabling the adequate healing of large injuries in different tissues. Hydrogels are examples of highly versatile materials for this application, given the range of methodologies for their preparation and composition components. The results of various studies indicate that the application of hydrogel‐based scaffolds for breast regeneration is highly promising. Among the materials and systems most applied for this purpose, biodegradable polymers stand out due to their high biocompatibility and the ability for spontaneous removal from the organism. In this sector, systems obtained through 3D printing also stand out due to the increased predictability of geometry and pore distribution. However, it is noteworthy that the evaluated regeneration systems are primarily focused on regenerating adipose tissue, with no specific systems for the epithelial regeneration of the breasts or the areas of glandular tissue. These areas represent sectors that still need further exploration.

Scaffold-free 3D culture systems for stem cell-based tissue regeneration.

Recent advances in scaffold-free three-dimensional (3D) culture methods have significantly enhanced the potential of stem cell-based therapies in regenerative medicine. This cutting-edge technology circumvents the use of exogenous biomaterial and prevents its associated complications. The 3D culture system preserves crucial intercellular interactions and extracellular matrix support, closely mimicking natural biological niches. Therefore, stem cells cultured in 3D formats exhibit distinct characteristics, showcasing their capabilities in promoting angiogenesis and immunomodulation. This review aims to elucidate foundational technologies and recent breakthroughs in 3D scaffold-free stem cell engineering, offering comprehensive guidance for researchers to advance this technology across various clinical applications. We first introduce the various sources of stem cells and provide a comparative analysis of two-dimensional (2D) and 3D culture systems. Given the advantages of 3D culture systems, we delve into the specific fabrication and harvesting techniques for cell sheets and spheroids. Furthermore, we explore their applications in pre-clinical studies, particularly in large animal models and clinical trials. We also discuss multidisciplinary strategies to overcome existing limitations such as insufficient efficacy, hostile microenvironments, and the need for scalability and standardization of stem cell-based products.

In vitro cluster buds regeneration and control of shoot tip necrosis in tissue cultures of Trichosanthes cucumerina L.

Trichosanthes cucumerina L. is a medicinal vine with great potential that can be used as a vegetable. In this study, an efficient in vitro regeneration system of T. cucumerina was established by inducing adventitious buds and controlling Shoot Tip Necrosis (STN) in the in vitro culture. The results of hormone single factor and orthogonal experiments showed that 6-BA had a significant effect on inducing adventitious buds. The best medium for bud proliferation was MS + 6-BA 3.0 mg·L− 1 + KIN 2.0 mg·L− 1 + NAA 0.5 mg·L− 1, and the proliferation capacity was 14.68. However, after three consecutive subcultures, a higher incidence of STN (78.86%) was observed. The results of the STN control experiment showed that reducing the concentration of inorganic salt, shortening the subculture interval, and increasing the Ca2+ in the medium could effectively control the occurrence of STN. However, reducing the concentration of inorganic salt and shortening the subculture interval excessively reduces the proliferation capacity significantly. Considering both the incidence of STN and proliferation capacity, the measures proposed in this study to alleviate STN were to keep the concentration of inorganic salt unchanged, adjust the subculture interval from 45 days to 40 days, and increase the Ca2+ concentration in the medium to 480 mg·L− 1. When the subculture interval was 40 d, the stem segments obtained the lowest STN incidence (8.3%) and higher shoot proliferation capacity (10.02) on the medium of MS + 3.0 mg·L− 1 6-BA + 2.0 mg·L− 1 KIN + 0.5 mg·L− 1 NAA + 360.36 mg·L− 1 Ca2+ (360 mg·L− 1 is the concentration of additional calcium to be added, and after this addition, the total calcium concentration in the MS medium was 480.48 mg·L− 1). The best rooting medium for stem segments was MS + 0.50 mg·L− 1 NAA; the rooting rate was 100%, and no STN occurred. After acclimation, the survival rate of the rooted plantlet reached 95% after 40 days in the soil matrix of coconut husk: humus soil: perlite = 2: 2: 1 (v/v). The results of this study not only reveal the mechanism of STN in T. cucumerina, but also provide technical support and a theoretical basis for its further breeding and cultivation.