Tissue Culture System Research Articles

The mechanical environment regulates the extent of aortic valve calcification and thickness: an ex vivo whole mouse heart study

Abstract Background Aortic stenosis (AS) is a narrowing of the aortic valve opening due to calcification and thickening of the leaflets. Calcifications are mostly located at regions with high mechanical stress and disturbed flow suggesting that mechanical environment is important in the regulation of calcification. However, direct evidence linking valve calcification and mechanical stress is still lacking. Recently, we established an ex vivo calcification model for whole mouse hearts (Miniature Tissue Culture System MTCS) which allows the study of the mechanical environment underlying aortic valve calcification. Purpose Aim of this study was to investigate the role of mechanical stress on aortic valve calcification and thickening using the MTCS. Methods Whole mouse hearts were cultured in the MTCS with 2 distinct configurations: 1) medium flowing from the aorta towards the aortic valve to create a continuous closed aortic valve (n=28) and 2) medium flowing from the left ventricle through the aortic valve towards the aorta to create a continuous open aortic valve (n=17) (Figure1). The hearts were subjected to low (300 ul/min) or high (3000 ul/min) flow speed in the presence of calcifying medium (3mM phosphate buffer). After culture for 1 week, the hearts were isolated and fixed overnight with 4% PFA/PBS. Alizarin red staining was performed to determine calcification presence. Quantifications were performed using Caseviewer software. Results Aortic valves in closed configuration exposed to a high flow speed, exhibited significant increase in calcification compared to aortic valves in the closed configuration exposed to low flow speed or aortic valve cultured in the open position (p<0.01, Figure2A). Of interest, the extent of calcification in the aortic valve in closed position exposed to high flow speed strongly correlated with the diameter of the aortic annulus (R²=0.86, p<0.0001, Figure2B). Conversely, leaflet thickness was significantly lower in the closed aortic valve configuration as compared with the open aortic valves configuration (p<0.05, p<0.001, Figure2C). Conclusion Our study demonstrates the influence of mechanical environment on aortic valve calcification and thickening. The presence of calcification in the closed but not the open aortic valve configuration exposed to high flow suggests that increased pressure promotes calcification. The correlation of the extent of aortic valve calcification with the diameter of the aortic annulus implies that configuration of the valve is an important determinator of calcification, with potential implications for the results of aortic valve repair. The presence of a minimal amount of mechanical stress as experienced by aortic valves in the open position, however, resulted in thicker aortic valves, indicating that the right balance of mechanical stresses is required to maintain normal valve morphology. These findings provide valuable insights into AS pathogenesis, guiding future therapeutic approaches.ex vivo cultured aortic valvesCalcification in ex vivo aortic valves

Silver Nanoparticles (AgNPs) Act as Nanoelicitors in Melissa officinalis to Enhance the Production of Some Important Phenolic Compounds and Essential Oils

ABSTRACTNanoparticles (NPs) are well‐known biostimulants in plant biotechnology, utilised to enhance the physical properties of plants and exhibit positive effects on them. The important key role is the most suitable type, effective dose and size of NP to be used in plant tissue culture systems. In this study, various concentrations of silver nanoparticles (AgNPs; 0, 25, 50, 75 and 100 μg L−1) were tested as elicitors in callus culture with the aim of enhancing secondary metabolite production in lemon balm (Melissa officinalis L.). According to the results obtained, callus formation rates have shown an increase in all applications compared to the control group. The highest callus formation, weight and diameter were observed in 50 μg L−1 application. In this application, the callus structure was compact and its colour was green. However, the aromatic compounds, neral and geranial increased significantly in 25 μg L−1 application. The maximum increase in phenolic compounds such as caffeic acid, chlorogenic acid, proto‐catechic acid, hesperidin and p‐coumaric acid was observed in the 75 μg L−1 AgNP and the highest increase in rosmarinic acid compound was determined in the 50 μg L−1 application. The study found that AgNP applications are an effective method for increasing the production of secondary metabolites in medicinal and aromatic plants, such as lemon balm, in vitro.

The Efficacy of Orange Terpene and Bacillus mycoides Strain BM103 on the Control of Periwinkle Leaf Yellowing Phytoplasma.

Phytoplasmas are obligate phytopathogenic bacteria belonging to the class Mollicutes. The pathogens, transmitted by insect vectors, associated with hundreds of plant diseases worldwide. Due to the regulation on banning use of antibiotics and limited efficacy of the traditional disease management manners, an eco-friendly alternative is needed. Given that terpene and probiotics have antibiotic activity and the ability to induce systemic resistance, in this study, the effectiveness of orange terpene and a Bacillus mycoides strain, BM103, was evaluated in periwinkle plants infected with periwinkle leaf yellowing (PLY) phytoplasma derived from a shoot-tip tissue culture system. Weekly drenching of 1,000 ppm diluted orange terpene emulsion or pre-activated strain BM103 liquid culture dilution exhibited the ability to inhibit PLY phytoplasma accumulation. The expression of the genes associated with plant defense response and flower development was upregulated after treatment. Moreover, pre-treatment of orange terpene or strain BM103 delayed PLY infection via cleft-grafting inoculation. While orange terpene did not suppress the symptoms, strain BM103 did result in a milder symptom expression that might partially attribute to its plant growth-promoting characteristics. Additionally, the pre-activation of strain BM103 may contribute to its efficacy. Taken together, this research indicates that orange terpene and B. mycoides BM103, with the ability to rapidly induce plant defense responses, could potentially be developed into biological control materials as preventive agents or biofertilizers.

Plant Growth Regulator-based Tissue Culture System Optimization for Cymbidium faberi Rolfe

Cymbidium faberi, a member of the Cymbidium genus known for its fragrant blooms and graceful foliage, has recently become endangered in the wild due to reproductive challenges. This study aimed to establish systematically a tissue culture system for Cymbidium faberi Rolfe (wild species) by evaluating the effects of various plant growth regulators its propagation stages, including rhizome proliferation, differentiation, shoot strengthening, and rooting. The results showed that 0.5 mg·L−1 thidiazuron significantly promoted rhizome proliferation, achieving a proliferation coefficient of 6.08 after 60 days of culture. For adventitious bud induction, 1.92 mg·L−1 brassinolide was most effective, inducing 6.43 buds per rhizome with an average bud height of 5.25 mm after 90 days of culture. The optimal strategy for shoot growth was using 3.0 mg·L−1 1-naphthaleneacetic acid, resulting in an average shoot height of 6.47 cm after 60 days. The highest rooting rate of 87.5% was achieved with 0.5 mg·L−1 zeatin, producing an average of 3.5 roots per shoot with an average root length of 3.06 cm. This study successfully developed a propagation system for C. faberi and highlighted the significant role of BL in promoting rhizome differentiation. In conclusion, this study provides a robust propagation method to support the conservation and industrial development of C. faberi.

OS01-02 New strategy of genotoxicity test using organoids in the 3-dimensional tissue culture system

OS01-02 New strategy of genotoxicity test using organoids in the 3-dimensional tissue culture system

The ester-containing prodrug NT-0796 enhances delivery of the NLRP3 inflammasome inhibitor NDT-19795 to monocytic cells expressing carboxylesterase-1

NT-0796 is an ester prodrug which is metabolized by carboxylesterase-1 (CES1) to yield the carboxylic acid NDT-19795, an inhibitor of the NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome. When applied to human monocytes/macrophages which express CES1, however, NT-0796 is much more potent at inhibiting NLRP3 inflammasome activation than is NDT-19795. Comparison of the binding of NDT-19795 and NT-0796 in a cell-based NLRP3 target engagement assay confirms that NDT-19795 is the active species. Moreover, microsomes expressing CES1 efficiently convert NT-0796 to NDT-19795, confirming CES1-dependent activation. To understand the basis for the enhanced potency of the ester prodrug species in human monocytes, we analyzed the accumulation and de-esterification of NT-0796 in cultured cells. Our studies reveal NT-0796 rapidly accumulates in cells, achieving estimated cellular concentrations above those applied to the medium, with concomitant metabolism to NDT-19795 in CES1-expressing cells. Using cells lacking CES1 or a poorly hydrolysable NT-0796 analog demonstrated that de-esterification is not required for NT-0796 to achieve high cellular levels. As a result of a dynamic equilibrium whereby NDT-19795 formed intracellularly is subsequently released to the medium, concentrations of NT-0796 sufficient to inhibit NLRP3 can be completely metabolized to NDT-19795 resulting in a transient pharmacodynamic response. In contrast, when NDT-19795 is applied directly to cells, observed cell-associated levels are below those present in the medium and remain stable over time. Dynamics observed within the context of a closed tissue culture system highlight the utility of NT-0796 as a vehicle for delivering the NDT-19795 acid payload to CES1 expressing cells.

Transwell‐Based Microfluidic Platform for High‐Resolution Imaging of Airway Tissues

AbstractTranswell‐based airway models have become increasingly important in studying the effects of respiratory diseases and drug treatment at the air–liquid interface of the lung epithelial barrier. However, the underlying mechanisms at the tissue and cell level often remain unclear, as transwell inserts feature limited live‐cell imaging compatibility. Here, a novel microfluidic platform is reported for the cultivation of transwell‐based lung tissues providing the possibility to alternate between air–liquid and liquid–liquid interfaces. While the air–liquid interface recapitulates physiological conditions for the lung model, the liquid–liquid interface enables live imaging of the tissue at high spatiotemporal resolution. The plastics‐based microfluidic platform enables the insertion and recuperation of the transwell inserts, which allows for tissue cultivation and analysis under standardized well plate conditions. The device is used to monitor infections of Pseudomonas aeruginosa in human stem‐cell‐derived bronchial epithelial tissue. The progression of a P. aeruginosa infection in real‐time at high resolution is continuously imaged, which provides insights into bacterial spreading and invasion on the apical tissue surface, as well as insights into tissue breaching and destruction over time. The airway tissue culture system is a powerful tool to visualize and elucidate key processes of developing respiratory diseases and to facilitate drug testing and development.

Towards Pathogen-Free Coconut Germplasm Exchange.

Coconut (Cocos nucifera L.) is an important palm species that serves as the mainstay of several industries and contributes to the livelihoods of millions of smallholder farmers. International exchange of coconut germplasm has been undertaken for several decades to facilitate the conservation of selected varieties within global genebanks and for the distribution to farmers and scientists. In vitro systems are a convenient and an efficient method for the exchange of coconut germplasm. However, it is possible that these tissue culture systems can transfer lethal pathogens causing a threat to the importing countries. In this review, the following topics are discussed: the major disease-causing agents of concern, the various tissues that could be used for coconut germplasm exchange, and the techniques available for the detection and elimination of disease-causing agents from various transmission systems. Additionally, the lack of clear, science-backed guidelines to facilitate the exchange of in vitro coconut materials is raised, along with recommendations for future studies to ensure the safe movement of coconut germplasm without biosecurity risks.

Evaluation of bacteriophages for the alleviation of potential bacterial contamination risks in developmental engineering.

This research aimed to address the potential bacterial contamination risks in developmental engineering (DE) using bacteriophages. To compare and contrast the exemplar Escherichia coli T4 and M13 bacteriophages, human dermal fibroblasts cultivated on culture plates, natural cellulosic scaffolds, and poly(methyl methacrylate) (PMMA) particles were utilized as two-dimensional (2D) cell, three-dimensional (3D) tissue, and modular tissue culture models, respectively. When directly introduced into these distinct culture systems, both phages survived, exhibited no significant effects on the cultured cells or tissues, yet displayed their potentials to alleviate the infections caused by corresponding bacterial host cells. Apart from direct addition into the culture medium, both phages were also coated on PMMA, polystyrene, poly(lactic acid) particles with different diameters (5, 10, 30, and 100 µm) and cellulosic scaffolds. The coated phages endured the coating processes and demonstrated their viabilities in plaque assays. Further testing indicated that the phages coated on the PMMA particles tolerated multiple deliberate rinses and centrifugations, but not thermal treatment at 60-80°C. In summary, T4 and M13 bacteriophages not only manifested their antibacterial functions in diverse 2D cell, 3D tissue, and modular tissue culture systems, but also demonstrated their potentials of coating modular scaffolds to alleviate the bacterial contamination risks in DE.

Natural biosynthesis, pharmacological applications, and sustainable biotechnological production of ornamental plant-derived anthocyanin: beyond colorants and aesthetics.

Flowers have long been admired for their aesthetic qualities and have even found their way to be included in the human diet. Among the many chemical compounds found in flowers, anthocyanins stand out for their versatile applications in the food, cosmetic, and nutraceutical industries. The biosynthetic pathway of anthocyanins has been thoroughly studied in certain flower species, leading to the detection of key regulatory genes that can be controlled to enhance the production of anthocyanins via biotechnological methods. Nevertheless, the quantity and form of anthocyanins found in natural sources differ, bothqualitativelyandquantitatively, depending on the ornamental plant species. For this reason, research on in vitroplantcultures has been conducted for years in an attempt to comprehend how these essential substances are produced. Different biotechnological systems, like in vitro plant cell, organ, and tissue cultures, and transgenic approaches, have been employed to produce anthocyanins under controlled conditions. However, multiple factors influence the production of anthocyanins and create challenges during large-scale production. Metabolic engineering techniques have also been utilized for anthocyanin production in microorganisms and recombinant plants. Although these techniques are primarily tested at lab- and pilot-scale, limited studies have focused on scaling up the production. This review analyses the chemistry and biosynthesis of anthocyanin along with the factors that influence the biosynthetic pathway. Further emphasis has been given on strategies for conventional and non-conventional anthocyanin production along with their quantification, addressing the prevailing challenges, and exploring ways to ameliorate the production using the in vitro plant cell and tissue culture systems and metabolic engineering to open up new possibilities for the cosmetic, pharmaceutical, and food industries.

Efficient In Vitro Propagation of Turpinia arguta and Quantitative Analysis of Its Ligustroflavone and Rhoifolin Content

Turpinia arguta is an excellent medicinal plant mainly used for the treatment of pharyngitis, tonsillitis, and tonsillar abscesses. However, an efficient regeneration protocol using tissue cultures for T. arguta does not exist. Its main medicinal constituents are flavonoids, particularly ligustroflavone and rhoifolin. Here, we aimed to establish a tissue culture system for T. arguta for the first time using annual stem segments with axillary buds harvested from the field of the Jiangxi Academy of Forestry as explants by dynamically determining the accumulation of effective functional components in the tissue culture plantlets. Orthogonal tests were conducted to compare the effects of different explant disinfection times, media, and exogenous hormone ratios on the induction of the axillary bud growth, successional proliferation, and rooting of T. arguta stem segments. The best explant disinfection effect was achieved by disinfecting the T. arguta explant with 75% ethanol for 50 s, followed by 0.1% mercuric chloride (HgCl2) for 6 min, and the optimal media for successional proliferation and rooting were Murashige and Skoog (MS) + 0.2 mg/L of 6-benzyladenine (6-BA), + 0.03 mg/L of naphthaleneacetic acid (NAA), and ½ MS + 2.5 mg/L of indole-3-butyric acid + 0.5 mg/L of NAA, respectively. The detection of ligustroflavone and rhoifolin in tissue culture plantlets 0, 3, and 5 months after transplanting showed a significant increasing trend and eventually exceeded the content requirements of the 2020 Edition ofChinese Pharmacopoeia for T. arguta. Our findings provide, for the first time, an effective tissue culture system for T. arguta, thereby providing important information to support the germplasm preservation, innovation, and application of T. arguta in the future.

Resveralogues protect HepG2 cells against cellular senescence induced by hepatotoxic metabolites

Progressive liver disease and dysfunction cause toxic metabolites including ammonia and unconjugated bilirubin to accumulate in plasma. As the population ages alternatives to liver transplantation become increasingly important. One approach for use as a bridge to transplant or recovery is the use of bioartificial liver systems (BALS) containing primary or immortalised hepatocytes as ex-vivo replacements or supports for endogenous liver function. However, exposure to the hepatotoxic metabolites present in plasma causes the rapid failure of these cells to carry out their primary metabolic functions despite remaining viable. Hypothesizing that this loss of core hepatocyte phenotypes was caused by cell senescence we exposed HepG2 cell populations, grown in both standard two-dimensional tissue culture systems and in three dimensional cultures on novel alginate modified HEMA-MBA cryogels, to physiologically reflective concentrations of hepatotoxic metabolites and cytokines. HepG2 cells are forced into senescence by the toxic metabolites in under six hours (as measured by loss of thymidine analog incorporation or detectable Ki67 staining) which is associated with a ten to twenty-fold reduction in the capacity of the cultures to synthesise albumin or urea. This state of senescence induced by liver toxins (SILT) can be prevented by preincubation with either 2–5 µM resveratrol, its major in vivo metabolite dihydroresveratrol or a series of novel resveralogues with differential capacities to scavenge radicals and activate SIRT1 (including V29 which does not interact with the protein). SILT appears to be a previously unrecognised barrier to the development of BALS which can now be overcome using small molecules that are safe for human use at concentrations readily achievable in vivo.

Tissue culture and Agrobacterium-mediated genetic transformation of the oil crop sunflower

Sunflower is one of the four major oil crops in the world. ‘Zaoaidatou’ (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.

Retinoic acid modulation guides human-induced pluripotent stem cell differentiation towards left or right ventricle-like cardiomyocytes

Background: Cardiomyocytes (CMs) induced from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and other non-cardiomyocytes. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart. Aim: Create engineered heart tissue (EHT) with left and right ventricular phenotype from hiPSCs by intervening with specific concentrations of RA during hiPSC differentiation towards CM. Methods: hiPSC were derived by reprogramming skin fibroblasts and erythroid progenitors. Different concentrations of RA (Control without RA, LowRA with 0.05 μM and HighRA with 0.1 μM) were administered during third to sixth days of the differentiation process. EHTs were generated by assembling CMs derived from hiPSC (hiPSC-CM) at high cell density in a low collagen hydrogel. EHTs were further matured and grown in a customized biomimetic tissue culture system, that provides continuous electrical stimulation, medium agitation and stretch. Finally, RNA extraction and tissue fixation were performed on CMs and EHTs for RT-qPCR and immunofluorescence staining analysis, while RNA sequencing was conducted on EHTs to examine how RA affects gene expression associated with function and structure of EHTs. Results: HighRA-hiPSC-CMs exhibited highest expression of maturity genes MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are transcription factors associated with left ventricular development, was also the highest in the HighRA-hiPSC-CM. The expression of TBX5 and NKX2.5 was also found to be highest in the HighRA-EHTs, while expression of right ventricular transcrition factors TBX20 and ISL1 was highest in control-EHTs. RNAseq of EHTs showed augmented extracellular matrix gene expression in HighRA-EHTs. The HighRA-EHTs were functionally most similar to left ventricle with the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline. Conclusion: By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHT with a phenotype similar to that of the left ventricle or right ventricle. Our research paves the way for future studies on in vitro left ventricular or right ventricular function, personalized drug screening, and precision medicine. German Center for Cardiovascular Research (DZHK81Z0600207 to D.M. and P.S.) China Scholarship Council (CSC202108410141) and Henan Provincial Medical Science and Technology Research Project. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Establishment of Tissue Culture and Regeneration System in Hippophae gyantsensis Lian

Hippophae gyantsensis Lian is a pioneering tree species in Chinese forestry ecological engineering, known for its robust stress tolerance, water retention capacity, and soil improvement qualities. However, the lack of rapid nursery technology has been a significant impediment to the development of the H. gyantsensis industry. In the present study, we have successfully established a tissue culture regeneration system for H. gyantsensis. The most effective methods for seed disinfection, ensuring sterility in seedlings, were found to be 75% alcohol disinfection for 40 s and 10% sodium hypochlorite disinfection for 10 min. The best media tested for callus induction in cotyledons and hypocotyls of sterile seedlings were 1/3 MS + 0.5 mg/L KT + 0.75 mg/L NAA and 1/3 MS + 0.3 mg/L 6-BA + 1.5 mg/L IBA, respectively. As the explants, cotyledons yielded larger calli with a greater size and differentiation ability than hypocotyls. For the induction of indeterminate shoots and proliferation, the most suitable media were 1/3 MS + 0.5 mg/L IAA + 0.75 mg/L 6-BA and 1/3 MS + 1.0 mg/L 6-BA + 0.05 mg/L IBA + 0.1 mg/L KT, respectively. Lastly, the best worked rooting formulation was 1/4 MS + 0.3 mg/L IBA. This study marks a significant milestone in the establishment of a systematic tissue culture regeneration system for H. gyantsensis, which will facilitate the industrial rapid propagation of high-quality seedlings and provide the foundation for improvement through genetic transformation.

Myeloid cell expression of CD200R is modulated in active TB disease and regulates Mycobacterium tuberculosis infection in a biomimetic model.

A robust immune response is required for resistance to pulmonary tuberculosis (TB), the primary disease caused by Mycobacterium tuberculosis (Mtb). However, pharmaceutical inhibition of T cell immune checkpoint molecules can result in the rapid development of active disease in latently infected individuals, indicating the importance of T cell immune regulation. In this study, we investigated the potential role of CD200R during Mtb infection, a key immune checkpoint for myeloid cells. Expression of CD200R was consistently downregulated on CD14+ monocytes in the blood of subjects with active TB compared to healthy controls, suggesting potential modulation of this important anti-inflammatory pathway. In homogenized TB-diseased lung tissue, CD200R expression was highly variable on monocytes and CD11b+HLA-DR+ macrophages but tended to be lowest in the most diseased lung tissue sections. This observation was confirmed by fluorescent microscopy, which showed the expression of CD200R on CD68+ macrophages surrounding TB lung granuloma and found expression levels tended to be lower in macrophages closest to the granuloma core and inversely correlated with lesion size. Antibody blockade of CD200R in a biomimetic 3D granuloma-like tissue culture system led to significantly increased Mtb growth. In addition, Mtb infection in this system reduced gene expression of CD200R. These findings indicate that regulation of myeloid cells via CD200R is likely to play an important part in the immune response to TB and may represent a potential target for novel therapeutic intervention.

Development of indirect somatic embryogenesis and plant regeneration system with immature embryos of the cultivated traditional Chinese medicinal herb Polygonatum cyrtonema

The dried rhizome of Polygonatum cyrtonema represents a highly valued traditional Chinese medicine. With a growing demand, the imperative arises for the establishment of an efficient tissue culture system for large-scale propagation. Here, we successfully developed a tissue culture system capable of yielding P. cyrtonema seedlings through somatic embryogenesis using immature embryos as explants within a 120-day timeframe. An optimum callus induction rate was achieved through a combination of 1.0 mg/L 6-BA and 0.5 mg/L 2,4-D in MS medium, resulting in the formation of numerous highly active callus. Subsequent transfer of primary callus to a medium containing 1.0 mg/L 6-BA and 1.0 mg/L 2,4-D in MS medium for 30 days yielded induction rates of 97.78%, 89.62%, 73.33%, and 66.67% for globular, heart, torpedo, and cotyledonary embryos, respectively. The highest rate of somatic embryo normal germination (73.33%) was observed in the MS+6-BA 2.0 mg/L+NAA 1.0 mg/L medium. Subsequent transfer of germinated somatic embryos to MS+IAA 0.5 mg/L+IBA 1.0 mg/L medium for 20 days resulted in robust tissue culture seedlings, with a survival rate of 93.33% when transplanted into a seedling substrate consisting of soil: perlite (3:1, v/v). This study not only marks the inaugural instance of somatic embryogenesis in Polygonatum species but also paves a foundation for the advent of breeding traditional Chinese medicinal herb P. cyrtonema via recent advanced biotechnological strategies.

Characterization of three novel stem rot pathogens and their antagonistic endophytic bacteria associated with Cistanche deserticola.

Cistanche deserticola is a precious Chinese medicinal material with extremely high health care and medicinal value. In recent years, the frequent occurrence of stem rot has led to reduced or even no harvests of C. deserticola. The unstandardized use of farm chemicals in the prevention and control processes has resulted in excessive chemical residues, threatening the fragile desert ecological environment. Therefore, it is urgent to explore safe and efficient prevention and control technologies. Biocontrol agents, with the advantages of safety and environment-friendliness, would be an important idea. The isolation, screening and identification of pathogens and antagonistic endophytic bacteria are always the primary basis. In this study, three novel pathogens causing C. deserticola stem rot were isolated, identified and pathogenicity tested, namely Fusarium solani CPF1, F. proliferatum CPF2, and F. oxysporum CPF3. For the first time, the endophytic bacteria in C. deserticola were isolated and identified, of which 37 strains were obtained. Through dual culture assay, evaluation experiment and tissue culture verification, a biocontrol candidate strain Bacillus atrophaeus CE6 with outstanding control effect on the stem rot was screened out. In the tissue culture system, CE6 showed excellent control effect against F. solani and F. oxysporum, with the control efficacies reaching 97.2% and 95.8%, respectively, indicating its great potential for application in the production. This study is of great significance for the biocontrol of plant stem rot and improvement of the yield and quality of C. deserticola.

Anti-IL-8 monoclonal antibodies inhibits the autophagic activity and cancer stem cells maintenance within breast cancer tumor microenvironment.

Breast cancer tumor microenvironment (TME) is a promising target for immunotherapy. Autophagy, and cancer stem cells (CSCs) maintenance are essential processes involved in tumorigenesis, tumor survival, invasion, and treatment resistance. Overexpression of angiogenic chemokine interleukin-8 (IL-8) in breast cancer TME is associated with oncogenic signaling pathways, increased tumor growth, metastasis, and poor prognosis. Thus, we aimed to investigate the possible anti-tumor effect of neutralizing antibodies against IL-8 by evaluating its efficacy on autophagic activity and breast CSC maintenance. IL-8 monoclonal antibody supplemented tumor tissue culture systems from 15 females undergoing mastectomy were used to evaluate the expression of LC3B as a specific biomarker of autophagy and CD44, CD24 as cell surface markers of breast CSCs using immunofluorescence technique. Our results revealed that anti-IL-8 mAb significantly decreased the level of LC3B in the cultured tumor tissues compared to its non-significant decrease in the normal breast tissues.Anti-IL-8 mAb also significantly decreased the CD44 expression in either breast tumors or normal cultured tissues. While it caused a non-significant decrease in CD24 expression in cultured breast tumor tissue and a significant decrease in its expression in the corresponding normal ones. Anti-IL-8 monoclonal antibody exhibits promising immunotherapeutic properties through targeting both autophagy and CSCs maintenance within breast cancer TME.

Treatments of Coleus forskholii that increase rooting to get larger crude forskolin extract

The aim is study of effect of some phytohormones and microelements in a tissue culture system to extract forskolin by different solvents from hairy roots in Plectranthus barbatus Andr. (Syn.Coleus forskohlii Briq.). Our results indicated that, the best parameters for increasing the number and length of roots were by indole butyric acid. Low concentrations of boron and silicon also promote rooting of Coleus cuttings. Methanol is a more efficient solvent than ethanol and ethyl acetate in forskolin extraction. Root hairs of the terminal cuttings of Coleus forskohlii appear after treatment with bacterial inoculum Agrobacterium rhizogenes. Forskolin levels increased to 13 and 5.3 mg/L in hair roots after 4 weeks of incubation. Forskolin extraction from tissue culture media is more efficient (40 mg/L per g wet weight) compared to extraction from root cuttings (13 mg/10 g wet weight).