Search Results (151)

Genetic transformation is an essential tool for investigating gene function and editing genomes. Kiwifruit, recognized as a significant global fresh fruit crop, holds considerable economic and nutritional importance. However, current genetic transformation techniques for kiwifruit are impeded by low efficiency, lengthy culture durations (a minimum of six months), and substantial labor requirements. In this research, we established an efficient system for shoot regeneration and the stable genetic transformation of the ‘Hayward’ cultivar, utilizing leaf explants in conjunction with two strains of Agrobacterium that harbor the expression vector pBI121-35S::GFP, which contains the green fluorescent protein (GFP) gene as a visible marker within the T-DNA region. Our results show that 93.3% of leaf explants responded positively to the regeneration medium, producing multiple independent adventitious shoots around the explants within a six-week period. Furthermore, over 71% of kanamycin-resistant plantlets exhibited robust GFP expression, and the entire transformation process was completed within four months of culture. Southern blot analysis confirmed the stable integration of GFP into the genome, while RT-PCR and fluorescence microscopy validated the sustained expression of GFP in mature plants. This efficient protocol for regeneration and transformation provides a solid foundation for micropropagation and the enhancement of desirable traits in kiwifruit through overexpression and gene silencing techniques. Full article

Introduction: Two-stage revision with an antibiotic-loaded, temporary static cement spacer is a common treatment for periprosthetic joint infection (PJI) of the knee. However, limited data exists on in vivo antibiotic elution kinetics after spacer implantation. This pilot study uses the technique of microdialysis (MD) to collect intra-articular knee samples. The aim was to evaluate MD as an intra-articular sampling method to detect spacer-eluted antibiotics within 72 h after surgery and to determine whether they show specific elution kinetics. Methods: Ten patients (six male, four female; age median 71.5 years) undergoing two-stage revision for knee PJI were included. A MD catheter was inserted into the joint during explantation of the infected inlying implant and implantation of a custom-made static spacer coated with COPAL cement (0.5 g gentamicin (G) and 2 g vancomycin (V)). Over 72 h postoperatively, samples were collected and analyzed for spacer-eluted antibiotics, intravenously administered antibiotics (e.g., cefazolin and cefuroxime), metabolic markers (glucose and lactate), and Interleukin-6 (IL-6). Local and systemic levels were compared. Results: All catheters were positioned successfully and well tolerated for 72 h. Antibiotic concentrations in MD samples peaked within the first 24 h (G: median 9.55 µg/mL [95% CI: 0.4–17.36]; V: 37.57 µg/mL [95% CI: 3.26–81.6]) and decreased significantly over 72 h (for both p < 0.05, G: 4.27 µg/mL [95% CI: 2.26–7.2]; V: 9.69 µg/mL [95% CI: 3.86–24]). MD concentrations consistently exceeded blood levels (p < 0.05), while intravenously administered antibiotics showed higher blood concentrations. Glucose in MD samples decreased from 17.71 mg/dL to 0.89 mg/dL (p < 0.05). IL-6 and lactate concentrations showed no difference between MD and blood samples. Conclusions: Monitoring antibiotics eluted by a static spacer with intra-articular MD for 72 h is feasible. Gentamicin and vancomycin levels remained above the minimal inhibitory concentration. Differentiating infection from surgical response using metabolic and immunological markers remains challenging. Prolonged in vivo studies with MD are required to evaluate extended antibiotic release in two-stage exchanges. Full article

Bananas and plantains, belonging to the Musa genus, are important food crops that sustain the livelihoods of countless smallholder farmers globally. However, their production is hindered by various challenges, including abiotic and biotic stresses, climate change, and poor access to clean planting materials, which negatively impact their yields. Addressing these constraints is essential for improving production and ensuring food security. This study investigated the influence of triploid genome background and exogenous growth regulators on the regeneration of Musa cultivars [Gros Michel (AAA genome), Obino l’Ewai and Silk (AAB genome), and Poteau Naine (ABB genome)]. Shoot tip explants of the AAA, AAB, and ABB triploid genomes were cultured in Murashige and Skoog (MS) media supplemented with varying 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), or naphthaleneacetic acid (NAA) hormones. Shoot induction was successfully achieved within 21.50 ± 2.00 days, with AAA exhibiting the highest shoot induction frequencies ranging from 30.00 ± 1.57% to 100% and shoot numbers per explant ranging from 3.00 ± 0.50 to 8.80 ± 0.80, followed by the ABB genome ranging from 20.00 ± 3.45% to 100% and from 2.00 ± 0.55 to 5.60 ± 0.50 shoots, and the AAB genome ranging from 17.50 ± 5.01% to 100% and from 2.00 ± 0.04 to 6.60 ± 0.25 shoots, respectively, in media amended with 1.2 to 6.0 mg.L⁻¹ BAP and 0.1 mg.L⁻¹ IAA. The highest rooting rate of 100% was recorded in all three genomes in media containing 1.4 mg.L⁻¹ IBA and 0.5 mg.L⁻¹ IAA, with the AAA genome producing the maximum number of 14.8 roots per explant. The results indicate the positive influence of the AAA genome background on in vitro regeneration and its potential utilization for genomic editing transformation protocols Full article

Zinc oxide nanoparticles (ZnONPs) are increasingly used in agriculture to stimulate plant growth and development, including under in vitro culture conditions. However, there is limited data on the effects of ZnONPs on the micropropagation of Scutellaria baicalensis Georgi. The pharmacological properties of this species make it a valuable medicinal plant. In Poland, it does not occur naturally but is cultivated for the production of herbal material. In vitro micropropagation is an effective method for obtaining genetically uniform plantlets. The aim of this study was to evaluate the effects of various concentrations of ZnONPs on growth parameters and the content of mineral nutrients, phenolic compounds, antioxidants, and photosynthetic pigments in Scutellaria baicalensis cultured in vitro. Shoot tip explants were cultured on MS medium supplemented with 1.0 mg dm⁻³ BA and 0.1 mg dm⁻³ IBA, together with ZnONPs at concentrations of 0 (control), 10, 20, 30, and 40 mg dm⁻³. The results showed that ZnONPs at concentrations of 10–20 mg dm⁻³ had no statistically significant effect on shoot or root development or on fresh weight gain. However, higher concentrations (30 and 40 mg dm⁻³) had a significantly negative impact on the number and length of shoots and roots, as well as on biomass accumulation. ZnONPs at 10–20 mg dm⁻³ significantly increased the content of potassium, calcium, magnesium, iron, and zinc in regenerated multi-shoot plantlets. A strong positive correlation (r = 0.951) was observed between ZnONP concentration and zinc accumulation in the plantlets. The levels of manganese and copper were not significantly different from the control. Plantlets treated with 30–40 mg dm⁻³ ZnONPs had significantly lower levels of calcium, iron, manganese, and copper. Those grown at 30 mg dm⁻³ had the highest potassium and magnesium levels, while plantlets exposed to 40 mg dm⁻³ had the highest zinc content. The total phenolic content and antioxidant activity (measured using ABTS and DPPH assays) were significantly higher in ZnONP-treated plantlets compared to the control. In contrast, the levels of chlorophyll a, chlorophyll b, total chlorophyll (a + b), and carotenoids were significantly lower in plants treated with ZnONPs. A strong negative correlation was found between ZnONP concentration and photosynthetic pigment content, while the ZnONP concentration was positively correlated with total phenolic content and antioxidant activity (ABTS⁺ and DPPH). Full article

Background: Silicone breast implants have been linked to autoimmune/inflammatory syndrome induced by adjuvants (ASIA). This study evaluates the role of ^99mTc-HYNIC-TOC somatostatin receptor scintigraphy in assessing somatostatin-mediated inflammation and the impact of explantation on inflammatory activity. Methods: Fifty patients with silicone breast implants and symptoms suggestive of ASIA were evaluated. Pre- and postexplantation imaging was performed using ^99mTc-HYNIC-TOC scintigraphy. Matthews correlation coefficients quantified associations between clinical symptoms and imaging findings, and autoantibody profiles were analysed. Results: Scintigraphy identified a significant uptake in organs associated with autoimmune symptoms, particularly joints and salivary glands. Strong correlations were found between imaging findings and symptoms, including knee pain (MCC = 0.81) and sicca syndrome (MCC = 0.96). Explantation resolved abnormal uptake in the surgical bed, though variable uptake persisted in other organs, reflecting systemic inflammatory heterogeneity. Autoantibody analysis revealed positivity in 66% of patients, with antinuclear antibodies being most frequent (30%). Conclusions: ^99mTc-HYNIC-TOC scintigraphy effectively evaluates organ-specific inflammation in ASIA. Explantation reduces localized inflammation but does not consistently address systemic autoimmune responses. Larger prospective studies are needed to validate these findings and improve management strategies for ASIA. Full article

Nanobiotechnology applications in plant tissue culture have improved the development and physiology of explants, resulting in plants with high genetic homogeneity and phytosanitary quality. Silver nanoparticles (AgNPs) are well-known for their microbicidal properties, but their biochemical effects on plants require further exploration. In this work, green-synthesized AgNPs were evaluated in strawberry in vitro culture, photosynthetic pigment production, and acclimatization. AgNPs produced by Lysinibacillus fusiformis were characterized. Strawberry explants were grown in vitro on MS medium with 0, 100, 200, and 300 mg L⁻¹ AgNPs at 24 ± 2 °C and a photoperiod of 16:8 h light/dark. Shoot height and number, number of leaves, number of roots, and root length were evaluated, and chlorophyll (a, b, and total) was quantified. Rooted shoots were acclimatized ex vitro on substrates containing 0 and 200 mg L⁻¹ AgNPs. The results showed that low AgNPs concentrations had a positive impact on shoot multiplication, development, and rooting, but at higher concentrations, the effects decayed. However, chlorophyll production improved with increasing AgNP concentration. Shoots treated with AgNPs showed higher ex vitro survival. Our study has direct implications for the profitability and sustainability of commercial strawberry production. Full article

Cartilage-derived migratory cells show great potential for autologous use in cartilage repair surgery. However, their collection through arthroscopic biopsy has not been previously reported in individuals without osteoarthritis. This study aimed to characterize migratory cartilage cells isolated from arthroscopic biopsies of volunteers without osteoarthritis and compare them with cells obtained by enzymatic digestion. Cell cultures were successfully established using both methods—enzymatic digestion and cell migration—from cartilage explants, with no significant differences observed in stem cell markers or plasticity between the cell lines. Cells derived from both procedures exhibited characteristics of mesenchymal stem cell, including fibroblast-like morphology, expression of CD29, CD90, and CD105 markers, absence of hematopoietic and endothelial cell markers, and the ability to differentiate into adipocytes, chondrocytes, and osteoblasts under appropriate conditions. Cells obtained by migration showed lower expression of collagen I and II, along with reduce collagen II/collagen I ratio, both positively associated with chondral matrix production, as well as lower RUNX2 expression. However, no differences were found in the levels of SOX9, essential for chondrogenic differentiation, or in the expression of perlecan gene. Syndecan-1 expression was lower in cells obtained by migration. In conclusion, this study demonstrates that cartilage-derived migratory cells can be successfully obtained from arthroscopic biopsies of individuals without osteoarthritis, presenting comparable dedifferentiation and plasticity profiles. Furthermore, these cells express essential chondrogenic markers and proteins. Although further in vivo studies are needed to determine their effective regenerative potential, cartilage-derived migratory cells represent a promising avenue for cartilage repair strategies. Full article

Background: Implant-based reconstruction (IBR) is the most common method of breast reconstruction, but complications lead to patient distress and delays in cancer treatment. Management of implant complications is varied with no defined guidelines. The inability to salvage IBR is associated with infection, but the impact of antibiotics remains controversial. We aimed to analyze factors affecting salvage rates of threatened IBR requiring operative intervention. The primary outcomes were the rates of unplanned re-operation for threatened IBR for wound closure, exchange, or explant. We hypothesized antibiotic administration would improve salvage rates. Methods: A retrospective review of patients undergoing mastectomy with IBR from 2012 to 2023 was performed. Threatened IBR was defined as implant exposure, infection, skin necrosis, hematoma, seroma, or wound dehiscence without implant exposure. Management options for patients with implant infection included implant removal and antibiotic treatment, antibiotic treatment alone, implant replacement, washout and implant replacement, or implant removal without a salvage attempt. Results: In total, 6901 patients underwent post-mastectomy IBR, and 184 (2.7%) patients had an unplanned re-operation. A total of 166/184 patients (90.2%) underwent explantation, and 18/184 (9.8%) patients had implant salvage. Between the explant and salvage groups, there were no differences in patient demographics, oncologic treatments, or operative characteristics. The explant group had a higher rate of infection (77.7% vs. 22.2%, p < 0.0001). There was no difference in culture positivity or antibiotic administration history. Conclusions: Implant salvage is feasible but limited by infection. Antibiotic administration does not improve salvage rates. Patient factors, oncologic treatment factors, or operative factors do not impact the ability to salvage. Full article

Hemerocallis middendorffii is widely used in the landscaping of Northern China for its exceptional ornamental and ecological attributes. It is also the focus of a substantial body of germplasm development and stress tolerance research. However, the absence of an efficient regeneration and genetic transformation system has been a critical barrier to conducting gene function studies on this species. In this research, the aerial parts of seed-derived H. middendorffii plantlets were used as explants, and the callus induction, proliferation, subculture, differentiation, and rooting conditions in the in vitro regeneration process were optimized. A callus induction rate of 95.6% was achieved, with a regeneration rate of 84.4%. Based on this procedure, a simple and effective Agrobacterium-mediated genetic transformation system was preliminarily developed using a hygromycin-based selection system. The system comprised an Agrobacterium tumefaciens culture solution optical density at 600 nm (OD₆₀₀) of 0.6, an acetosyringone concentration of 100 μmol·L⁻¹ in both the A. tumefaciens infection solution and the co-cultivation medium, a sterilization culture with Timentin at 300 mg·L⁻¹, and a selection culture with hygromycin at 9 mg·L⁻¹. Transgenic H. middendorffii T0 rooted plants were produced within a 5-month period, with a transformation rate of 11.9% and positive rate of 32.8%. The regeneration and genetic transformation system established in this study should help advance functional gene research and genetic improvement in H. middendorffii. However, the genetic transformation was only validated in the T0 plants. To confirm stable integration and long-term transgene stability, future research on the phenotypic and molecular characterization of T1 progeny, including segregation analysis and Southern blot verification, will be conducted. Full article

Porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV), a porcine herpesvirus, has been shown to significantly reduce the survival time of porcine xenotransplants in non-human primates. The virus was detected in all the examined organs of baboons transplanted with PCMV/PRV-positive organs and it was also transmitted to the first human recipient of a pig heart, contributing to the patient’s death. PCMV/PRV induces consumptive coagulopathy and thrombocytopenia in xenotransplant recipients. Initial studies in baboons revealed that the virus triggered increased release of tumor necrosis factor α (TNFα) and interleukin 6 (IL-6), along with elevated levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) complexes. Since there is no evidence that PCMV/PRV infects primate cells, including human cells, the virus appears to directly interact with immune and endothelial cells, disrupting cytokine signaling and coagulation pathways. The highest viral load was detected in the explanted pig heart, suggesting active replication at this site. Additionally, cells expressing PCMV/PRV proteins were identified in all the examined baboon organs, where pig cells were also found. Since PCMV/PRV affects only xenotransplant recipients and not healthy humans, this condition should be classified as a xenozoonosis. Interestingly, antibodies against human herpesvirus 6 (HHV-6) cross-react with PCMV/PRV and may contribute to protection against infection in humans. Further research is needed to uncover the molecular mechanisms underlying this xenozoonotic disease. Full article

Biofilm formation on orthopedic joint implants complicates diagnosis of periprosthetic joint infections (PJIs). Sonication of explanted orthopedic implants for diagnostic enhances pathogen detection, but it shows limitations in sensitivity and handling. We investigated whether the biosurfactant saponin could improve bacterial recovery from orthopaedic implants and thereby enhance infection diagnosis ex vivo. Orthopaedic material discs of 1 cm diameter were contaminated with different clinical bacterial PJI isolates. Biofilms of Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Cutibacterium avidum, and Cutibacterium acnes were grown on the discs, which were then treated with either saline solution or various concentrations of saponin. Next, the discs were vortexed or sonicated. Colony-forming units (CFUs) enumeration and time-to-positivity of liquid cultures were determined. Additionally, a novel 3D PJI soft tissue in vitro model was established to validate these findings in a more representative scenario. Median CFU enumeration showed that 0.001% (w/v) saponin as compared to saline solution increased CFUs recovery by 2.2 log₁₀ for S. epidermidis, 0.6 log₁₀ for S. aureus, 0.6 log₁₀ for C. avidum, 1.1 log₁₀ for C. acnes, and 0.01 log₁₀ for E. coli. Furthermore, saponin treatment resulted in a >1 log₁₀ increase in S. epidermidis CFU recovery from implants in the 3D tissue model compared to standard saline sonication. With that, we propose a novel two-component kit, consisting of a saponin solution and a specialized transportation box, for the efficient collection, transportation, and processing of potentially infected implants. Our data suggest that biosurfactants can enhance bacterial recovery from artificially contaminated orthopedic implants, potentially improving the diagnosis of PJIs. Full article

Background/Objectives: Dental pulp stem cells (DPSCs) are of significant interest due to their mesenchymal lineage and relative availability from extracted teeth. This study aims to examine the relationship between fibronectin-adherent, non-fibronectin-adherent, and explant-derived DPSC populations in terms of the population doubling rate in culture and the expression of mesenchymal cell surface markers and their capacity for osteodifferentiation. Methods: Human pulp tissue was removed from healthy extracted human teeth, enzymatically digested prior to seeding onto fibronectin-coated plates, and left to adhere for 20 min, yielding a fibronectin-adherent population. The remaining non-adherent cells were transferred and designated ‘non-fibronectin-adherent.’ Intact pulp was placed on uncoated plastic for 5 days, with the migrated cells designated ‘explant-derived’. DPSCs from these populations were examined in terms of population doubling rates, the expression of CD90, CD44, CD105, and CD73, and the expression of RUNX2, SPP1, and BGLAP after 7 days in osteoinductive media. Results: The fibronectin-adherent cells had the greatest population doubling over time. All populations demonstrated comparable percentages of cells positive for mesenchymal markers, though individual marker expression varied slightly. The explant-derived cells showed increased expression of RUNX2 after 7 days in osteoinductive media, while the treated single-cell-suspension-derived populations showed increased expression of SPP1 mRNA. Conclusions: Fibronectin enrichment resulted in a population with the greatest rate of population doubling over extended culture compared to the other two populations. The proportion of cells positive for all four mesenchymal surface markers was the same between populations. The fibronectin-adherent and non-adherent cultures may have responded more rapidly to osteoinductive media than the explant-derived cells. Full article

In Coffea canephora, a direct somatic embryogenesis (SE) protocol has been established by pretreating plants with NAA and kinetin, followed by the induction of leaf explants in a liquid medium with BA. Through a transcriptomic analysis of 10 key moments of the induction of SE in C. canephora, we were able to establish that the transcriptional responses of this process are divided into four stages. These stages correspond to the pretreatment, characterized by the positive regulation of genes associated with cell wall remodeling, flavonoid biosynthesis, and antioxidant activity that prepare the explants for the intense cellular activity that represents the induction of SE. During the first few hours, the early response to induction occurs, characterized by the highest number of differentially expressed genes, most related to the response to multiple stimuli. At 24 h, a late response begins with the upregulation of genes related to energy production and the biosynthesis of amino acids. Finally, WOX, BBM, and ABI3 genes are upregulated during the embryogenic response. The downregulation of genes related to the circadian cycle, photomorphogenesis, photosynthesis, and chloroplast components were observed throughout the process. The detailed analysis of the primary transcriptional responses that occur during the SE of C. canephora helps us to clarify how auxins and cytokinins orchestrate the integration of different networks of plant metabolism and lead to the development of somatic embryos. Full article

The ‘Yuluxiang’ pear is a key cultivated variety in China, celebrated for its high quality. However, it exhibits a low leaf regeneration frequency of only 35.0%, which hinders its transgenic breeding process. To establish an efficient regeneration system, we utilized tissue culture seedlings of the ‘Yuluxiang’ pear and investigated various factors influencing leaf regeneration: plant growth regulators, natural organic materials, leaf wounding and positioning methods, duration of dark culture, ages and lines of plantlets, as well as culture containers. Our results indicated that the optimal medium for leaf regeneration consisted of MS supplemented with 6-BA (6-Benzyl Aminopurine) 1.5 mg/L, NAA (α-Naphthalene acetic acid) 0.4 mg/L along with 10% (v/v) coconut water. Suitable wounding involved ensuring no damage to leaves while placing the abaxial side facing down on the medium; the ideal duration for dark culture was determined to be 21 days; optimal plantlet age was found to be 20 days; both plantlet line 1 and line 2 demonstrated effectiveness; triangle bottles were identified as appropriate culture containers. In summary, we successfully established an efficient leaf regeneration system for the ‘Yuluxiang’ pear that achieved a maximum regeneration frequency of 96.70% with an average bud number of 5.15 per explant. This system also proved effective for the ‘Qiuyue’ pear, yielding a regeneration frequency of 88.89% and an average bud number of 3.44 per explant. After investigating the germination methods of 200 leaves from this screened leaf regeneration system of ‘Yuluxiang’, it was found that there were both direct and indirect regeneration methods, and the germination rate of direct and indirect regeneration was 68.00% and 70.50%, respectively. Therefore, this study also laid a solid foundation for the future genetic transformation of ‘Yuluxiang’ pear. Full article

Adventitious root (AR) formation in plants originates from non-root organs such as leaves and hypocotyls. Auxin signaling is essential for AR formation, but the roles of other phytohormones are less clear. In Arabidopsis, at least two distinct mechanisms can produce ARs, either from hypocotyls as part of the general root architecture or from wounded organs during de novo root regeneration (DNRR). In previous reports, gibberellin acid (GA) appeared to play reverse roles in both types of ARs, since GA treatment blocks etiolation-induced AR formation from hypocotyls, whereas GA synthesis and signaling mutants apparently displayed reduced DNRR from detached leaves. In order to clarify this contradiction, we employed the GA biosynthesis inhibitor paclobutrazol (PBZ) and found that PBZ had positive effects on both types of AR formation in Arabidopsis. Consistently, GA treatment had negative effects on both AR formation mechanisms, while loss of GA synthesis and signaling promoted DNRR under our conditions. Our results show that PBZ treatment can rescue declined AR formation in difficult-to-root leaf explants such as erecta receptor mutants. Furthermore, transcriptional profiling revealed that PBZ treatment altered GA, brassinosteroids, and auxin responses, which included the up-regulation of LBD16 that is well known for its pivotal role in AR initiation. Full article