Response surface methodology mediated optimization of phytosulfokine and plant growth regulators for enhanced protoplast division, callus induction, and somatic embryogenesis in Angelica Gigas Nakai.

BACKGROUND: Angelica Gigas (Purple parsnip) is an important medicinal plant that is cultivated and utilized in Korea, Japan, and China. It contains bioactive substances especially coumarins with anti-inflammatory, anti-platelet aggregation, anti-cancer, anti-diabetic, antimicrobial, anti-obesity, anti-oxidant, immunomodulatory, and neuroprotective properties. This medicinal crop can be genetically improved, and the metabolites can be obtained by embryonic stem cells. In this context, we established the protoplast-to-plant regeneration methodology in Angelica gigas. RESULTS: In the present investigation, we isolated the protoplast from the embryogenic callus by applying methods that we have developed earlier and established protoplast cultures using Murashige and Skoog (MS) liquid medium and by embedding the protoplast in thin alginate layer (TAL) methods. We supplemented the culture medium with growth regulators namely 2,4-dichlorophenoxyaceticacid (2,4-D, 0, 0.75, 1.5 mg L- 1), kinetin (KN, 0, 0.5, and 1.0 mg L- 1) and phytosulfokine (PSK, 0, 50, 100 nM) to induce protoplast division, microcolony formation, and embryogenic callus regeneration. We applied central composite design (CCD) and response surface methodology (RSM) for the optimization of 2,4-D, KN, and PSK levels during protoplast division, micro-callus formation, and induction of embryogenic callus stages. The results revealed that 0.04 mg L- 1 2,4-D + 0.5 mg L- 1 KN + 2 nM PSK, 0.5 mg L- 1 2,4-D + 0.9 mg L- 1 KN and 90 nM PSK, and 1.5 mg L- 1 2,4-D and 1 mg L- 1 KN were optimum for protoplast division, micro-callus formation and induction embryogenic callus. MS basal semi-solid medium without growth regulators was good for the development of embryos and plant regeneration. CONCLUSIONS: This study demonstrated successful protoplast culture, protoplast division, micro-callus formation, induction embryogenic callus, somatic embryogenesis, and plant regeneration in A. gigas. The methodologies developed here are quite useful for the genetic improvement of this important medicinal plant.

Clinical application of both amniotic membranes and adipose derived mesenchymal stem cells in a cat with a large skin defect.

Surgical procedures on large skin defects can be challenging in the short term due to the size of the lesion, infection, and tissue defect. A regenerative therapy for skin wounds has been applied to promote the healing process. An 8-month-old, Korean domestic short-haired female cat, weighing 3 kg, was rescued with extensive defects on the right flank to right inguinal region caused by bite wounds. In this case, amniotic membranes and adipose-derived mesenchymal stem cells were used as the regenerative therapy to treat the large skin defect rather than a surgical intervention alone. To the best of our knowledge, this is the first report of a case with of a large skin defect treated by applying allogeneic amniotic membranes and allogeneic mesenchymal stem cells to a cat.

MLL1 histone methyltransferase and UTX histone demethylase functionally cooperate to regulate the expression of NRF2 in response to ROS-induced oxidative stress.

The transcription factor NRF2 plays a pivotal role in maintaining redox and metabolic homeostasis by orchestrating oxidative stress-dependent transcription programs. Despite growing evidence implicating various cellular components in the regulation of NRF2 activity at the posttranslational stage, relatively less is known about the factors dictating the transcriptional activation of NRF2 in response to oxidative stress. In this study, we report the crucial roles of MLL1, an H3K4-specific methyltransferase, and UTX, an H3K27-specific histone demethylase, in the NRF2-dependent transcription program under oxidative stress. We find that the depletion of MLL1 or UTX results in increased susceptibility to oxidative stress, accompanied by higher intracellular ROS and the failed activation of antioxidant genes, including NRF2. In addition, MLL1 and UTX selectively target the NRF2 promoter, and exogenous FLAG-NRF2 expression increases the viability of MLL1-or UTX-depleted cells upon exposure to hydrogen peroxide. RNA-seq analysis demonstrates that depletion of MLL1 or UTX affects the changes in NRF2-dependent transcriptome in response to oxidative stress. Furthermore, ChIP and ChIP-seq analyses find that MLL1 and UTX functionally cooperate to establish a chromatin environment that favors active transcription at the H3K4me3/H3K27me3 bivalent NRF2 promoter in response to ROS-induced oxidative stress. Collectively, these findings provide a molecular mechanism underlying the cellular response to oxidative stress and highlight the importance of the chromatin structure and function in maintaining redox homeostasis.

Cryo-EM structures of human DICER dicing a pre-miRNA substrate.

Dicer, a multi-domain ribonuclease III (RNase III) protein, is crucial for gene regulation via RNA interference. It processes hairpin-like precursors into microRNAs (miRNAs) and long double-stranded RNAs (dsRNAs) into small interfering RNAs (siRNAs). During the "dicing" process, the miRNA or siRNA substrate is stably anchored and cleaved by Dicer's RNase III domain. Although numerous studies have investigated long dsRNA cleavage by Dicer, the specific mechanism by which human Dicer (hDICER) processes pre-miRNA remains unelucidated. This review introduces the recently revealed hDICER structure bound to pre-miRNA uncovered through cryo-electron microscopy and compares it with previous reports describing Dicer. The domain-wise movements of the helicase and dsRNA-binding domain (dsRBD) and specific residues involved in substrate sequence recognition have been identified. During RNA substrate binding, the hDICER apical domains and dsRBD recognize the pre-miRNA termini and cleavage site, respectively. Residue rearrangements in positively charged pockets within the apical domain influence substrate recognition and cleavage site determination. The specific interactions between dsRBD positively charged residues and nucleotide bases near the cleavage site emphasize the significance of cis-acting elements in the hDICER processing mechanism. These findings provide valuable insights for understanding hDICER-related diseases.

Enhanced selective discrimination of point-mutated viral RNA through false amplification regulatory direct insertion in rolling circle amplification.

Coronaviruses are single-stranded RNA viruses with high mutation rates. Although a diagnostic method for coronaviruses has been developed, variants appear rapidly. Low test accuracy owing to single-point mutations is one of the main factors in the failure to prevent the early spread of coronavirus infection. Although reverse transcription-quantitative polymerase chain reaction can detect coronavirus infection, it cannot exclude the possibility of false positives, and an additional multiplexing kit is needed to discriminate single nucleotide polymorphism (SNP) variants. Therefore, in this study, we introduced a new nucleic acid amplification method to determine whether an infected person has a SNP mutation using a lateral flow assay (LFA) as a point-of-care test. Unlike traditional DNA amplification methods, direct insertion into rolling circle amplification amplifies the target genes without false amplification. After SNP-selective nucleic acid amplification, nuclease enzymes are used to make double-stranded DNA fragments that the LFA can detect, where specific mismatched DNA is found and cleaved to show different signals when a SNP-type is present. Therefore, wild- and SNP-type variants can be selectively detected. In this study, the limit of detection was 400 aM for viral RNA, and we successfully identified a dominant SNP variant selectively. Clinical tests were also conducted.

Interplay between MRI-based axon diameter and myelination estimates in macaque and human brain.

Axon diameter and myelin thickness are closely related microstructural tissue properties that affect the conduction velocity of action potentials in the nervous system. Imaging them non-invasively with MRI-based methods is thus valuable for studying brain microstructure and function. However, the relationship between MRI-based axon diameter and myelination measures has not been investigated across the brain, mainly due to methodological limitations in estimating axon diameters. In recent years, studies using ultra-high gradient strength diffusion MRI (dMRI) have demonstrated improved estimation of axon diameter across white-matter (WM) tracts in the human brain, making such investigations feasible. In this study, we aim to investigate relationships between tissue microstructure properties with MRI-based methods and compare the imaging findings to histological evidence from the literature. We collected dMRI with ultra-high gradient strength and multi-echo spin-echo MRI on ex vivo macaque and human brain samples on a preclinical scanner. From these data, we estimated axon diameter, intra-axonal signal fraction, myelin water fraction (MWF) and aggregate g-ratio and investigated their correlations. We found that the microstructural imaging parameters exhibited consistent patterns across WM tracts and species. Overall, the findings suggest that MRI-based axon geometry and myelination measures can provide complementary information about fiber morphology, and the relationships between these measures agree with prior histological evidence.

Intrinsic and extrinsic contributors to subregional thalamic volume loss in multiple sclerosis.

OBJECTIVE: To evaluate the intrinsic and extrinsic microstructural factors contributing to atrophy within individual thalamic subregions in multiple sclerosis using in vivo high-gradient diffusion MRI. METHODS: In this cross-sectional study, 41 people with multiple sclerosis and 34 age and sex-matched healthy controls underwent 3T MRI with up to 300 mT/m gradients using a multi-shell diffusion protocol consisting of eight b-values and diffusion time of 19 ms. Each thalamus was parcellated into 25 subregions for volume determination and diffusion metric estimation. The soma and neurite density imaging model was applied to obtain estimates of intra-neurite, intra-soma, and extra-cellular signal fractions for each subregion and within structurally connected white matter trajectories and cortex. RESULTS: Multiple sclerosis-related volume loss was more pronounced in posterior/medial subregions than anterior/ventral subregions. Intra-soma signal fraction was lower in multiple sclerosis, reflecting reduced cell body density, while the extra-cellular signal fraction was higher, reflecting greater extra-cellular space, both of which were observed more in posterior/medial subregions than anterior/ventral subregions. Lower intra-neurite signal fraction in connected normal-appearing white matter and lower intra-soma signal fraction of structurally connected cortex were associated with reduced subregional thalamic volumes. Intrinsic and extrinsic microstructural measures independently related to subregional volume with heterogeneity across atrophy-prone thalamic nuclei. Extrinsic microstructural alterations predicted left anteroventral, intrinsic microstructural alterations predicted bilateral medial pulvinar, and both intrinsic and extrinsic factors predicted lateral geniculate and medial mediodorsal volumes. INTERPRETATION: Our results might be reflective of the involvement of anterograde and retrograde degeneration from white matter demyelination and cerebrospinal fluid-mediated damage in subregional thalamic volume loss.

Age-related alterations in human cortical microstructure across the lifespan: Insights from high-gradient diffusion MRI.

The human brain undergoes age-related microstructural alterations across the lifespan. Soma and Neurite Density Imaging (SANDI), a novel biophysical model of diffusion MRI, provides estimates of cell body (soma) radius and density, and neurite density in gray matter. The goal of this cross-sectional study was to assess the sensitivity of high-gradient diffusion MRI toward age-related alterations in cortical microstructure across the adult lifespan using SANDI. Seventy-two cognitively unimpaired healthy subjects (ages 19-85 years; 40 females) were scanned on the 3T Connectome MRI scanner with a maximum gradient strength of 300mT/m using a multi-shell diffusion MRI protocol incorporating 8 b-values and diffusion time of 19 ms. Intra-soma signal fraction obtained from SANDI model-fitting to the data was strongly correlated with age in all major cortical lobes (r = -0.69 to -0.60, FDR-p < 0.001). Intra-soma signal fraction (r = 0.48-0.63, FDR-p < 0.001) and soma radius (r = 0.28-0.40, FDR-p < 0.04) were significantly correlated with cortical volume in the prefrontal cortex, frontal, parietal, and temporal lobes. The strength of the relationship between SANDI metrics and age was greater than or comparable to the relationship between cortical volume and age across the cortical regions, particularly in the occipital lobe and anterior cingulate gyrus. In contrast to the SANDI metrics, all associations between diffusion tensor imaging (DTI) and diffusion kurtosis imaging metrics and age were low to moderate. These results suggest that high-gradient diffusion MRI may be more sensitive to underlying substrates of neurodegeneration in the aging brain than DTI and traditional macroscopic measures of neurodegeneration such as cortical volume and thickness.

Automatic detection and classification of nasopalatine duct cyst and periapical cyst on panoramic radiographs using deep convolutional neural networks.

OBJECTIVE: The aim of this study was to evaluate a deep convolutional neural network (DCNN) method for the detection and classification of nasopalatine duct cysts (NPDC) and periapical cysts (PAC) on panoramic radiographs. STUDY DESIGN: A total of 1,209 panoramic radiographs with 606 NPDC and 603 PAC were labeled with a bounding box and divided into training, validation, and test sets with an 8:1:1 ratio. The networks used were EfficientDet-D3, Faster R-CNN, YOLO v5, RetinaNet, and SSD. Mean average precision (mAP) was used to assess performance. Sixty images with no lesion in the anterior maxilla were added to the previous test set and were tested on 2 dentists with no training in radiology (GP) and on EfficientDet-D3. The performances were comparatively examined. RESULTS: The mAP for each DCNN was EfficientDet-D3 93.8%, Faster R-CNN 90.8%, YOLO v5 89.5%, RetinaNet 79.4%, and SSD 60.9%. The classification performance of EfficientDet-D3 was higher than that of the GPs' with accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of 94.4%, 94.4%, 97.2%, 94.6%, and 97.2%, respectively. CONCLUSIONS: The proposed method achieved high performance for the detection and classification of NPDC and PAC compared with the GPs and presented promising prospects for clinical application.