Potential role of ice-binding protein in mitochondria-lipid and ATP mechanisms during freezing of plant callus.

Plant calli, a perpetually undifferentiated cell culture, have defects in maintaining their genetic fidelity during prolonged tissue culture. Cryopreservation using ice-binding proteins (IBP) is a potential solution. Despite a few studies on cryopreservation using IBPs in plant calli, detailed insights into the intracellular metabolism during freezing, thawing, and re-induction remain sparse. This study investigated and employed IBP from polar yeast Leucosporidium sp. (LeIBP) in the cryopreservation process across diverse taxa, including gymnosperms, monocots, dicots, and woody plants. Molecular-level analyses encompassing reactive oxygen species levels, mitochondrial function, and ATP and lipophilic compounds content were conducted. The results across nine plant species revealed the effects of LeIBP on callus competency post-thawing, along with enhanced survival rates, reactive oxygen species reduction, and restored metabolic activities to the level of those of fresh calli. Moreover, species-specific survival optimization with LeIBP treatments and morphological assessments revealed intriguing extracellular matrix structural changes post-cryopreservation, suggesting a morphological strategy for maintaining the original cellular states and paracrine signaling. This study pioneered the comprehensive application of LeIBP in plant callus cryopreservation, alleviating cellular stress and enhancing competence. Therefore, our findings provide new insights into the identification of optimal LeIBP concentrations, confirmation of genetic conformity post-thawing, and the intracellular metabolic mechanisms of cryopreservation advancements in plant research, thereby addressing the challenges associated with long-term preservation and reducing labor-intensive cultivation processes. This study urges a shift towards molecular-level assessments in cryopreservation protocols for plant calli, advocating a deeper understanding of callus re-induction mechanisms and genetic fidelity post-thawing.

Comparative Transcriptome Profiles of Human HaCaT Cells in Response to Gynostemma pentaphyllum Extracts Obtained Using Three Independent Methods by RNA Sequencing.

Gynostemma pentaphyllum (GP) is widely used in herbal medicine. In this study, we developed a method for the large-scale production of GP cells using plant tissue culture techniques combined with bioreactors. Six metabolites (uridine, adenosine, guanosine, tyrosine, phenylalanine, and tryptophan) were identified in GP extracts. Transcriptome analyses of HaCaT cells treated with GP extracts using three independent methods were conducted. Most differentially expressed genes (DEGs) from the GP-all condition (combination of three GP extracts) showed similar gene expression on treatment with the three individual GP extracts. The most significantly upregulated gene was LTBP1. Additionally, 125 and 51 genes were upregulated and downregulated, respectively, in response to the GP extracts. The upregulated genes were associated with the response to growth factors and heart development. Some of these genes encode components of elastic fibers and the extracellular matrix and are associated with many cancers. Genes related to folate biosynthesis and vitamin D metabolism were also upregulated. In contrast, many downregulated genes were associated with cell adhesion. Moreover, many DEGs were targeted to the synaptic and neuronal projections. Our study has revealed the functional mechanisms of GP extracts' anti-aging and photoprotective effects on the skin using RNA sequencing.

Giant Y79 retinoblastoma cells contain functionally active T-type calcium channels.

Retinoblastoma is the most common malignant intraocular tumor in children. Y79 human retinoblastoma cells are in vitro models of retinal tumors used for drug screening. Undifferentiated Y79 cells originate from a primitive multi-potential neuroectodermal cell and express neuronal and glial properties. However, the nature of cellular heterogeneity in Y79 cells is unclear because functional methods to characterize neurons or glial cells have not been employed to Y79 cells. Here, we perform patch-clamp recordings to characterize electrophysiological properties in retinoblastoma cells. We identified a population of large-sized Y79 cells (i.e., giant cells, ~ 40-µm diameter), hyperpolarized resting membrane potential (-54 mV), and low input resistance (~ 600 MΩ), indicating electrically mature cells. We also found that giant Y79 cells contain increased density of T-type calcium channels. Finally, we found that T-type calcium channels are active only in giant cells suggesting that cancer treatments aimed to prevent calcium influx in retinoblastomas should be tested in giant cells.

Enhancement of dendritic persistent Na+ currents by mGluR5 leads to an advancement of spike timing with an increase in temporal precision.

Timing and temporal precision of action potential generation are thought to be important for encoding of information in the brain. The ability of single neurons to transform their input into output action potential is primarily determined by intrinsic excitability. Particularly, plastic changes in intrinsic excitability represent the cellular substrate for spatial memory formation in CA1 pyramidal neurons (CA1-PNs). Here, we report that synaptically activated mGluR5-signaling can modulate the intrinsic excitability of CA1-PNs. Specifically, high-frequency stimulation at CA3-CA1 synapses increased firing rate and advanced spike onset with an improvement of temporal precision. These changes are mediated by mGluR5 activation that induces cADPR/RyR-dependent Ca2+ release in the dendrites of CA1-PNs, which in turn causes an increase in persistent Na+ currents (INa,P) in the dendrites. When group I mGluRs in CA1-PNs are globally activated pharmacologically, afterdepolarization (ADP) generation as well as increased firing rate are observed. These effects are abolished by inhibiting mGluR5/cADPR/RyR-dependent Ca2+ release. However, the increase in firing rate, but not the generation of ADP is affected by inhibiting INa,P. The differences between local and global activation of mGluR5-signaling in CA1-PNs indicates that mGluR5-dependent modulation of intrinsic excitability is highly compartmentalized and a variety of ion channels are recruited upon their differential subcellular localizations. As mGluR5 activation is induced by physiologically plausible brief high-frequency stimulation at CA3-CA1 synapses, our results suggest that mGluR5-induced enhancement of dendritic INa,P in CA1-PNs may provide important implications for our understanding about place field formation in the hippocampus.

The Influence of an Isthmus on the Outcomes of Surgically Treated Molars: A Retrospective Study.

INTRODUCTION: The purpose of this study was to investigate the effects of an isthmus on the success rate of surgically treated molars. METHODS: The study included 106 maxillary and mandibular first molars with endodontic lesions limited to the periapical area. Endodontic microsurgical procedures were performed according to the Yonsei protocol reported in a previous study. When an isthmus was observed after a high-magnification inspection, it was included in the retrograde preparation design. When an isthmus was not observed, only the main root canal space was prepared. The patients were followed up periodically every year after treatment. Success was defined as the absence of clinical signs and symptoms and radiographic evidence of complete or incomplete healing. RESULTS: Of the 106 teeth included in the study, 72 teeth had an isthmus, and 34 did not. Kaplan-Meier analysis revealed that the cumulative survival rate after surgery was 61.5% and 87.4% for 4 years when an isthmus was present and absent, respectively. A multivariate Cox proportional hazards regression analysis showed that the adjusted hazard ratio for failure was 6.01 times higher for the isthmus-present teeth than for the isthmus-absent teeth (P < .05). CONCLUSIONS: In this study, the success rate for endodontic microsurgery on isthmus-absent teeth was higher than that for isthmus-present teeth. Considering the success rate and potential risk of weakening the root after preparation, the techniques of isthmus preparation need to be improved.