-
BioRxiv : the Preprint Server For... Nov 2023Cnidarians have become valuable models for understanding many aspects of developmental biology including the evolution of body plan diversity, novel cell type...
UNLABELLED
Cnidarians have become valuable models for understanding many aspects of developmental biology including the evolution of body plan diversity, novel cell type specification, and regeneration. Most of our understanding of gene function during early development in cnidarians comes from a small number of experimental systems including the sea anemone, . Few molecular tools have been developed for use in hard corals, limiting our understanding of this diverse and ecologically important clade. Here, we report the development of a suite of tools for manipulating and analyzing gene expression during early development in the northern star coral, . We present methods for gene knockdown using short hairpin RNAs, gene overexpression using exogenous mRNAs, and endogenous gene tagging using CRISPR-mediated gene knock-in. Combined with our ability to control spawning in the laboratory, these tools make a tractable experimental system for investigative studies of coral development. Further application of these tools will enable functional analyses of embryonic patterning and morphogenesis across Anthozoa and open new frontiers in coral biology research.
SUMMARY STATEMENT
This study reports the development of the first transgenic knock-in coral, providing the opportunity to track the behavior of various cell types during early coral development.
PubMed: 38948709
DOI: 10.1101/2023.11.16.567385 -
Frontiers in Cellular and Infection... 2024Convergence of (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP....
BACKGROUND
Convergence of (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP. However, clinically used indicators for hypervirulent KP identification, such as hypermucoviscosity, appear to be differentially expressed in convergent KP, potential outbreak clones are difficult to identify. We aimed to fill such knowledge gaps by investigating the temperature dependence of hypermucoviscosity and virulence in a convergent KP strain isolated during a clonal outbreak and belonging to the high-risk sequence type (ST)307.
METHODS
Hypermucoviscosity, biofilm formation, and mortality rates in larvae were examined at different temperatures (room temperature, 28°C, 37°C, 40°C and 42°C) and with various phenotypic experiments including electron microscopy. The underlying mechanisms of the phenotypic changes were explored via qPCR analysis to evaluate plasmid copy numbers, and transcriptomics.
RESULTS
Our results show a temperature-dependent switch above 37°C towards a hypermucoviscous phenotype, consistent with increased biofilm formation and mortality, possibly reflecting a bacterial response to fever-like conditions. Furthermore, we observed an increase in plasmid copy number for a hybrid plasmid harboring carbapenemase and genes. However, transcriptomic analysis revealed no changes in expression at higher temperatures, suggesting alternative regulatory pathways.
CONCLUSION
This study not only elucidates the impact of elevated temperatures on hypermucoviscosity and virulence in convergent KP but also sheds light on previously unrecognized aspects of its adaptive behavior, underscoring its resilience to changing environments.
Topics: Klebsiella pneumoniae; Biofilms; Virulence; Animals; Klebsiella Infections; Temperature; Larva; Plasmids; Moths; Humans; Virulence Factors; Bacterial Proteins; Lepidoptera; Viscosity; Phenotype; Gene Expression Profiling
PubMed: 38947124
DOI: 10.3389/fcimb.2024.1411286 -
International Journal of Nanomedicine 2024Bone tissue engineering (BTE) is a promising alternative to autologous bone grafting for the clinical treatment of bone defects, and inorganic/organic composite...
BACKGROUND
Bone tissue engineering (BTE) is a promising alternative to autologous bone grafting for the clinical treatment of bone defects, and inorganic/organic composite hydrogels as BTE scaffolds are a hot spot in current research. The construction of nano-hydroxyapatite/gelatin methacrylate/oxidized sodium alginate (nHAP/GelMA/OSA), abbreviated as HGO, composite hydrogels loaded with bone morphogenetic protein 7 (BMP7) will provide a suitable 3D microenvironment to promote cell aggregation, proliferation, and differentiation, thus facilitating bone repair and regeneration.
METHODS
Dually-crosslinked hydrogels were fabricated by combining GelMA and OSA, while HGO hydrogels were formulated by incorporating varying amounts of nHAP. The hydrogels were physically and chemically characterized followed by the assessment of their biocompatibility. BMP7-HGO (BHGO) hydrogels were fabricated by incorporating suitable concentrations of BMP7 into HGO hydrogels. The osteogenic potential of BHGO hydrogels was then validated through in vitro experiments and using rat femoral defect models.
RESULTS
The addition of nHAP significantly improved the physical properties of the hydrogel, and the composite hydrogel with 10% nHAP demonstrated the best overall performance among all groups. The selected concentration of HGO hydrogel served as a carrier for BMP7 loading and was evaluated for its osteogenic potential both in vivo and in vitro. The BHGO hydrogel demonstrated superior in vitro osteogenic induction and in vivo potential for repairing bone tissue compared to the outcomes observed in the blank control, BMP7, and HGO groups.
CONCLUSION
Using hydrogel containing 10% HGO appears promising for bone tissue engineering scaffolds, especially when loaded with BMP7 to boost its osteogenic potential. However, further investigation is needed to optimize the GelMA, OSA, and nHAP ratios, along with the BMP7 concentration, to maximize the osteogenic potential.
Topics: Alginates; Animals; Bone Morphogenetic Protein 7; Gelatin; Tissue Engineering; Hydrogels; Durapatite; Osteogenesis; Rats; Bone Regeneration; Tissue Scaffolds; Rats, Sprague-Dawley; Methacrylates; Male; Humans; Bone and Bones
PubMed: 38946885
DOI: 10.2147/IJN.S461996 -
Analytical Cellular Pathology... 2024Osteochondral defects (OCDs) are localized areas of damaged cartilage and underlying subchondral bone that can produce pain and seriously impair joint function....
Synergistic Effects of Icariin and Extracellular Vesicles Derived from Rabbit Synovial Membrane-Derived Mesenchymal Stem Cells on Osteochondral Repair via the Wnt/-Catenin Pathway.
OBJECTIVES
Osteochondral defects (OCDs) are localized areas of damaged cartilage and underlying subchondral bone that can produce pain and seriously impair joint function. Literature reports indicated that icariin (ICA) has the effect of promoting cartilage repair. However, its mechanism remains unclear. Here, we explored the effects of icariin and extracellular vesicles (EVs) from rabbit synovial-derived mesenchymal stem cells (rSMSCs) on repairing of OCDs.
MATERIALS AND METHODS
Rabbit primary genicular chondrocytes (rPGCs), knee skeletal muscle cells (rSMCKs), and rSMSCs, and extracellular vesicles derived from the latter two cells (rSMCK-EVs and rSMSC-EVs) were isolated and identified. The rPGCs were stimulated with ICA, rSMSC-EVs either separately or in combination. The rSMCK-EVs were used as a control. After stimulation, chondrogenic-related markers were analyzed by quantitative RT-PCR and western blotting. Cell proliferation was determined by the CCK-8 assay. The preventative effects of ICA and SMSC-EVs were determined by H&E and toluidine blue staining. Immunohistochemical analyses were performed to evaluate the levels of COL2A1 and -catenin . , the proliferation of rPGCs was markedly increased by ICA treatment in a dose-dependent manner. When compared with ICA or rSMSC-EVs treatment alone, combined treatment with ICA and SMSC-EVs produced stronger stimulative effects on cell proliferation. Moreover, combined treatment with ICA and rSMSC-EVs promoted the expression of chondrogenic-related gene, including COL2A1, SOX-9, and RUNX2, which may be via the activation of the Wnt/-catenin pathway. , combined treatment with rSMSC-EVs and ICA promoted cartilage repair in joint bone defects. Results also showed that ICA or rSMSC-EVs both promoted the COL2A1 and -catenin protein accumulation in articular cartilage, and that was further enhanced by combined treatment with rSMSC-EVs and ICA.
CONCLUSION
Our findings highlight the promising potential of using combined treatment with ICA and rSMSC-EVs for promoting osteochondral repair.
Topics: Animals; Rabbits; Flavonoids; Mesenchymal Stem Cells; Wnt Signaling Pathway; Extracellular Vesicles; Chondrocytes; Synovial Membrane; Chondrogenesis; Cell Proliferation; beta Catenin; Cartilage, Articular
PubMed: 38946863
DOI: 10.1155/2024/1083143 -
Chemical & Pharmaceutical Bulletin 2024Lysine demethylase 5 (KDM5) proteins are involved in various neurological disorders, including Alzheimer's disease, and KDM5 inhibition is expected to be a therapeutic...
Lysine demethylase 5 (KDM5) proteins are involved in various neurological disorders, including Alzheimer's disease, and KDM5 inhibition is expected to be a therapeutic strategy for these diseases. However, the pharmacological effects of conventional KDM5 inhibitors are insufficient, as they only target the catalytic functionality of KDM5. To identify compounds that exhibit more potent pharmacological activity, we focused on proteolysis targeting chimeras (PROTACs), which degrade target proteins and thus inhibit their entire functionality. We designed and synthesized novel KDM5 PROTAC candidates based on previously identified KDM5 inhibitors. The results of cellular assays revealed that two compounds, 20b and 23b, exhibited significant neurite outgrowth-promoting activity through the degradation of KDM5A in neuroblastoma neuro 2a cells. These results suggest that KDM5 PROTACs are promising drug candidates for the treatment of neurological disorders.
Topics: Proteolysis; Humans; Neuronal Outgrowth; Structure-Activity Relationship; Enzyme Inhibitors; Cell Line, Tumor; Molecular Structure; Retinoblastoma-Binding Protein 2; Animals; Mice; Dose-Response Relationship, Drug; Proteolysis Targeting Chimera
PubMed: 38945940
DOI: 10.1248/cpb.c23-00026 -
Food Research International (Ottawa,... Aug 2024The antibiotic oxytetracycline (OTC) can be detected in contemporary natural aquatic environments and has been implicated in causing intestinal damage in humans exposed...
The protective effects of Kefir extract (KE) on intestinal damage in larval zebrafish induced by Oxytetracycline: Insights into intestinal function, morphology, and molecular mechanisms.
The antibiotic oxytetracycline (OTC) can be detected in contemporary natural aquatic environments and has been implicated in causing intestinal damage in humans exposed to OTC-contaminated food or water. The irreversible damage caused by high concentrations of OTC to the intestine suggests that treatment through dietary means could still be necessary. This study proved the effectiveness of kefir extract (KE) in reversing intestinal damage caused by oxytetracycline (OTC) exposure. Following a 24-hour KE treatment subsequent to OTC exposure from 3 to 8 days post-fertilization of zebrafish larvae, molecular-level and microbiomic assessments revealed significant improvements. These included reduced expression of proinflammatory factors (IL-8 and IL-1β), increased antioxidant levels, and reversed unhealthy distribution of intestinal microbiota. Furthermore, KE supplementation showed potential in enhancing intestinal motility in the experiment of Nile red staining and fluorescent microbead transit. However, histological analysis showed that this short-term treatment with KE only partially reversed the intestinal morphological changes induced by OTC, suggesting that a longer treatment period might be necessary for complete restoration.
Topics: Animals; Oxytetracycline; Zebrafish; Kefir; Larva; Intestines; Gastrointestinal Microbiome; Anti-Bacterial Agents; Antioxidants; Gastrointestinal Motility
PubMed: 38945628
DOI: 10.1016/j.foodres.2024.114642 -
Food Research International (Ottawa,... Aug 2024Sensory lexicons provide an important tool for describing the sensory properties of emerging, unfamiliar foods such as edible insects. This study sought to establish and...
Sensory lexicons provide an important tool for describing the sensory properties of emerging, unfamiliar foods such as edible insects. This study sought to establish and validate a sensory lexicon for the description and differentiation of edible insects commercially available in Australia and prepared using common preservation and cooking methods (freeze-drying, hot-air drying, roasting, sautéing and deep-frying). Five species were evaluated, including house crickets (Acheta domesticus), yellow mealworm larvae (Tenebrio molitor), king mealworm larvae (Zophobas morio), tyrant ants (Iridomyrmex spp.) and green tree ants (Oecophylla smaragdina). Following generic descriptive sensory analysis methods, a trained panel (n=8) developed a sensory lexicon of 29 aroma and flavour descriptors, and 16 texture descriptors. Vocabulary were then categorised and ordered to generate a sensory wheel. Due to a lack of cross-over in sensory attributes between species, sub-categories of species-specific vocabulary were also generated for each insect. The lexicon enabled sensory profiling of commercially available edible insect samples which revealed large variation in aroma, flavour, and texture attributes due to both species and preparation method. This work provides a platform for development of a globally relevant edible insect sensory lexicon. International collaboration will enable expansion of the lexicon for use with other insect species and preparation methods, insect-derived ingredients (such as insect powder, defatted insect powder and textured insect protein) and in different cultural settings. As the industry grows, the applicability of vocabulary for differentiating within species and between competitive products should also be assessed.
Topics: Animals; Edible Insects; Australia; Odorants; Tenebrio; Taste; Gryllidae; Humans; Cooking; Ants; Larva; Adult; Female; Male; Species Specificity; Middle Aged
PubMed: 38945565
DOI: 10.1016/j.foodres.2024.114574 -
The Bone & Joint Journal Jul 2024Given the possible radiation damage and inaccuracy of radiological investigations, particularly in children, ultrasound and superb microvascular imaging (SMI) may offer...
AIMS
Given the possible radiation damage and inaccuracy of radiological investigations, particularly in children, ultrasound and superb microvascular imaging (SMI) may offer alternative methods of evaluating new bone formation when limb lengthening is undertaken in paediatric patients. The aim of this study was to assess the use of ultrasound combined with SMI in monitoring new bone formation during limb lengthening in children.
METHODS
In this retrospective cohort study, ultrasound and radiograph examinations were performed every two weeks in 30 paediatric patients undergoing limb lengthening. Ultrasound was used to monitor new bone formation. The number of vertical vessels and the blood flow resistance index were compared with those from plain radiographs.
RESULTS
We categorized the new bone formation into three stages: stage I (early lengthening), in which there was no obvious callus formation on radiographs and ultrasound; stage II (lengthening), in which radiographs showed low-density callus formation with uneven distribution and three sub-stages could be identified on ultrasound: in Ia punctate callus was visible; in IIb there was linear callus formation which was not yet connected and in IIc there was continuous linear callus. In stage III (healing), the bone ends had united, the periosteum was intact, and the callus had disappeared, as confirmed on radiographs, indicating healed bone. A progressive increase in the number of vertical vessels was noted in the early stages, peaking during stages IIb and IIc, followed by a gradual decline (p < 0.001). Delayed healing involved patients with a prolonged stage IIa or those who regressed to stage IIa from stages IIb or IIc during lengthening.
CONCLUSION
We found that the formation of new bone in paediatric patients undergoing limb lengthening could be reliably evaluated using ultrasound when combined with the radiological findings. This combination enabled an improved assessment of the prognosis, and adjustments to the lengthening protocol. While SMI offered additional insights into angiogenesis within the new bone, its role primarily contributed to the understanding of the microvascular environment rather than directly informing adjustments of treatment.
Topics: Humans; Child; Retrospective Studies; Male; Female; Child, Preschool; Adolescent; Ultrasonography; Osteogenesis; Bone Lengthening; Bony Callus; Leg Length Inequality; Microvessels; Radiography
PubMed: 38945540
DOI: 10.1302/0301-620X.106B7.BJJ-2023-1019.R2 -
Journal of Microbiological Methods Jun 2024The ability to acquire three-dimensional (3D) information of cellular structures without the need for fluorescent tags or staining makes holotomographic imaging a...
The ability to acquire three-dimensional (3D) information of cellular structures without the need for fluorescent tags or staining makes holotomographic imaging a powerful tool in cellular biology. It provides valuable insights by measuring the refractive index (RI), an optical parameter describing the phase delay of light that passes through the living cell. Here, we demonstrate holotomographic imaging on industrial relevant ascomycete fungi and study their development and morphogenesis. This includes conidial germination, subcellular dynamics, and cytoplasmic flow during hyphal growth in Aspergillus niger. In addition, growth and budding of Aureobasidium pullulans cells are captured using holotomographic microscopy. Coupled to fluorescence imaging, lipid droplets, vacuoles, the mitochondrial network, and nuclei are targeted and analyzed in the 3D RI reconstructed images. While lipid droplets and vacuoles can be assigned to a specific RI pattern, mitochondria and nuclei were not pronounced. We show, that the lower sensitivity of RI measurements derives from the fungal cell wall that acts as an additional barrier for the illumination light of the microscope. After cell wall digest of hyphae and protoplast formation of A. niger expressing GFP-tagged histone H2A, location of nuclei could be determined by non-invasive RI measurements. Furthermore, we used coupled fluorescence microscopy to observe migration of nuclei in unperturbed hyphal segments and duplication during growth on a single-cell level. Detailed micromorphological studies in Saccharomyces cerevisiae and Trichoderma reesei are challenging due to cell size restrictions. Overall, holotomography opens up new avenues for exploring dynamic cellular processes in real time and enables the visualization of fungi from a new perspective.
PubMed: 38945304
DOI: 10.1016/j.mimet.2024.106983 -
Cell Reports Jun 2024The basement membrane (BM) is an extracellular matrix that plays important roles in animal development. A spatial heterogeneity in composition and structural properties...
The basement membrane (BM) is an extracellular matrix that plays important roles in animal development. A spatial heterogeneity in composition and structural properties of the BM provide cells with vital cues for morphogenetic processes such as cell migration or cell polarization. Here, using the Drosophila egg chamber as a model system, we show that the BM becomes heterogeneous during development, with a reduction in Collagen IV density at the posterior pole and differences in the micropattern of aligned fiber-like structures. We identified two AdamTS matrix proteases required for the proper elongated shape of the egg chamber, yet the molecular mechanisms by which they act are different. Stall is required to establish BM heterogeneity by locally limiting Collagen IV protein density, whereas AdamTS-A alters the micropattern of fiber-like structures within the BM at the posterior pole. Our results suggest that AdamTS proteases control BM heterogeneity required for organ shape.
PubMed: 38944833
DOI: 10.1016/j.celrep.2024.114399