Dual regulation of cytochrome P450 gene expression by two distinct small RNAs, a novel tasiRNA and miRNA, in Marchantia polymorpha.

The miR390-derived TAS3 trans-acting short-interfering RNAs (tasiRNAs) module represents a conserved RNA silencing pathway in the plant kingdom; however, its characterization in the bryophyte Marchantia polymorpha is limited. This study elucidated that MpDCL4 processes MpTAS3 double-stranded RNA (dsRNA) to generate tasiRNAs, primarily from the 5'- and 3'-ends of dsRNA. Notably, we discovered a novel tasiRNA, tasi78A, can negatively regulate a cytochrome P450 gene, MpCYP78A101. Additionally, tasi78A was abundant in MpAGO1, and transient expression assays underscored the role of tasi78A in repressing MpCYP78A101. A microRNA, miR11700, also regulates MpCYP78A101 expression. This coordinate regulation suggests a role in modulating auxin signaling at apical notches of gemma, influencing the growth and sexual organ development of M. polymorpha and emphasizing the significance of RNA silencing in MpCYP78A101 regulation. However, phylogenetic analysis identified another paralog of the CYP78 family, Mp1g14150, which may have a redundant role with MpCYP78A101, explaining the absence of noticeable morphological changes in loss-of-function plants. Taken together, our findings provide new insights into the combined regulatory roles of miR390/MpTAS3/miR11700 in controlling MpCYP78A101 and expand our knowledge about the biogenesis and regulation of tasiRNAs in M. polymorpha.

A novel computer-assisted tool for 3D imaging of programmed death-ligand 1 expression in immunofluorescence-stained and optically cleared breast cancer specimens.

BACKGROUND: Programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) are the two most common immune checkpoints targeted in triple-negative breast cancer (BC). Refining patient selection for immunotherapy is non-trivial and finding an appropriate digital pathology framework for spatial analysis of theranostic biomarkers for PD-1/PD-L1 inhibitors remains an unmet clinical need. METHODS: We describe a novel computer-assisted tool for three-dimensional (3D) imaging of PD-L1 expression in immunofluorescence-stained and optically cleared BC specimens (n = 20). The proposed 3D framework appeared to be feasible and showed a high overall agreement with traditional, clinical-grade two-dimensional (2D) staining techniques. Additionally, the results obtained for automated immune cell detection and analysis of PD-L1 expression were satisfactory. RESULTS: The spatial distribution of PD-L1 expression was heterogeneous across various BC tissue layers in the 3D space. Notably, there were six cases (30%) wherein PD-L1 expression levels along different layers crossed the 1% threshold for admitting patients to PD-1/PD-L1 inhibitors. The average PD-L1 expression in 3D space was different from that of traditional immunohistochemistry (IHC) in eight cases (40%). Pending further standardization and optimization, we expect that our technology will become a valuable addition for assessing PD-L1 expression in patients with BC. CONCLUSION: Via a single round of immunofluorescence imaging, our approach may provide a considerable improvement in patient stratification for cancer immunotherapy as compared with standard techniques.

Panoramic Tissue Examination That Integrates 3-Dimensional Pathology Imaging and Gene Mutation: Potential Utility in Non-Small Cell Lung Cancer.

Novel therapeutics have significantly improved the survival and quality of life of patients with malignancies in this century. Versatile precision diagnostic data were used to formulate personalized therapeutic strategies for patients. However, the cost of extensive information depends on the consumption of the specimen, raising the challenges of effective specimen utilization, particularly in small biopsies. In this study, we proposed a tissue-processing cascaded protocol that obtains 3-dimensional (3D) protein expression spatial distribution and mutation analysis from an identical specimen. In order to reuse the thick section tissue evaluated after the 3D pathology technique, we designed a novel high-flatness agarose-embedded method that could improve tissue utilization rate by 1.52 fold, whereas it reduced the tissue-processing time by 80% compared with the traditional paraffin-embedding method. In animal studies, we demonstrated that the protocol would not affect the results of DNA mutation analysis. Furthermore, we explored the utility of this approach in non-small cell lung cancer because it is a compelling application for this innovation. We used 35 cases including 7 cases of biopsy specimens of non-small cell lung cancer to simulate future clinical application. The cascaded protocol consumed 150-µm thickness of formalin-fixed, paraffin-embedded specimens, providing 3D histologic and immunohistochemical information approximately 38 times that of the current paraffin-embedding protocol, and 3 rounds of DNA mutation analysis, offering both essential guidance for routine diagnostic evaluation and advanced information for precision medicine. Our designed integrated workflow provides an alternative way for pathological examination and paves the way for multidimensional tumor tissue assessment.

Development of an assay system for the analysis of host RISC activity in the presence of a potyvirus RNA silencing suppressor, HC-Pro.

BACKGROUND: To investigate the mechanism of RNA silencing suppression, the genetic transformation of viral suppressors of RNA silencing (VSRs) in Arabidopsis integrates ectopic VSR expression at steady state, which overcomes the VSR variations caused by different virus infections or limitations of host range. Moreover, identifying the insertion of the transgenic VSR gene is necessary to establish a model transgenic plant for the functional study of VSR. METHODS: Developing an endogenous AGO1-based in vitro RNA-inducing silencing complex (RISC) assay prompts further investigation into VSR-mediated suppression. Three P1/HC-Pro plants from turnip mosaic virus (TuMV) (P1/HC-ProTu), zucchini yellow mosaic virus (ZYMV) (P1/HC-ProZy), and tobacco etch virus (TEV) (P1/HC-ProTe) were identified by T-DNA Finder and used as materials for investigations of the RISC cleavage efficiency. RESULTS: Our results indicated that the P1/HC-ProTu plant has slightly lower RISC activity than P1/HC-ProZy plants. In addition, the phenomena are consistent with those observed in TuMV-infected Arabidopsis plants, which implies that HC-ProTu could directly interfere with RISC activity. CONCLUSIONS: In this study, we demonstrated the application of various plant materials in an in vitro RISC assay of VSR-mediated RNA silencing suppression.

Changes in group counseling engagement and conflict and growth in emotional cultivation for children and adolescents.

Group climate is an important factor in group counseling and psychotherapy process and outcome research. The current investigation examined group climate changes (from early to late sessions) at the within-group (i.e., group members) and between-group (i.e., group-as-a-whole) levels in predicting changes in group members' emotional cultivation in group counseling. A total of 236 Taiwanese children and adolescents across 41 groups participated in this study. Members' ratings of group climate (i.e., engagement and conflict) were partitioned into within-group and between-group components, and polynomial regression with response surface analysis was used to examine the association between changes in group engagement and conflict (at the member- and group-level) from early to late group sessions on changes in emotional cultivation. Results supported the theoretical hypothesis that when a group-as-a-whole reported increasing engagement from early to late group sessions, relative to other groups (i.e., between-group effect), members of that group experienced greater growth in emotional cultivation. Results also indicated that group members reported greater growth in emotional cultivation when there was consistent and high engagement or consistent and low conflict from early to late group sessions. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Rapid Histological Assessment of Prostate Specimens in the Three-dimensional Space by Hydrophilic Tissue Clearing and Confocal Microscopy.

Microscopic examination of biopsied and resected prostatic specimens is the mainstay in the diagnosis of prostate cancer. However, conventional analysis of hematoxylin and eosin (H&E)-stained tissue is time-consuming and offers limited two-dimensional (2D) information. In the current study, we devised a method-termed Prostate Rapid Optical examination for cancer STATus (proSTAT)-for rapid screening of prostate cancer using high-resolution 2D and three-dimensional (3D) confocal images obtained after hydrophilic tissue clearing of 100-µm-thick tissue slices. The results of the proSTAT method were compared with those of traditional H&E stains for the analysis of cores (n=15) obtained from radical prostatectomy specimens (n=5). Gland lumen formation, consistent with Gleason pattern 3, was evident following tracking of multiple optical imaging sections. In addition, 3D rendering allowed visualizing a tubular network of interconnecting branches. Rapid 3D fluorescent labeling of tumor protein p63 accurately distinguished prostate adenocarcinoma from normal tissue and benign lesions. Compared with conventional stains, the 3D spatial and molecular information extracted from proSTAT may significantly increase the amount of available data for pathological assessment of prostate specimens. Our approach is amenable to automation and-subject to independent validation-can find a wide spectrum of clinical and research applications.

Development of a petal protoplast transfection system for Sinningia speciosa.

Premise: Transient gene expression systems are powerful tools for studying gene interactions in plant species without available or stable genetic transformation protocols. We optimized a petal protoplast transformation protocol for Sinningia speciosa, a model plant, to study the development of floral symmetry. Methods and Results: A high yield of petal protoplasts was obtained using a 6-h enzyme digestion in a solution of 1.5% cellulase and 0.4% macerozyme. Modest transfection efficiency (average 41.4%) was achieved. The viability of the transfected protoplasts remained at more than 90%. A fusion of green fluorescent protein and CYCLOIDEA (SsCYC), the Teosinte branched 1/Cincinnata/Proliferating cell factor transcription factor responsible for floral symmetry, was subcellularly localized inside the nuclei of the protoplasts. Transiently overexpressing SsCYC indicates the success of this system, which resulted in the predicted increased (but nonsignificant) expression of its known target RADIALIS (SsRAD1), consistent with gene network expectations. Conclusions: The transient transfection system presented herein can be effectively used to study gene-regulatory interactions in Gesneriaceae species.

Computer-assisted three-dimensional quantitation of programmed death-ligand 1 in non-small cell lung cancer using tissue clearing technology.

Immune checkpoint blockade therapy has revolutionized non-small cell lung cancer treatment. However, not all patients respond to this therapy. Assessing the tumor expression of immune checkpoint molecules, including programmed death-ligand 1 (PD-L1), is the current standard in predicting treatment response. However, the correlation between PD-L1 expression and anti-PD-1/PD-L1 treatment response is not perfect. This is partly caused by tumor heterogeneity and the common practice of assessing PD-L1 expression based on limited biopsy material. To overcome this problem, we developed a novel method that can make formalin-fixed, paraffin-embedded tissue translucent, allowing three-dimensional (3D) imaging. Our protocol can process tissues up to 150 µm in thickness, allowing anti-PD-L1 staining of the entire tissue and producing high resolution 3D images. Compared to a traditional 4 µm section, our 3D image provides 30 times more coverage of the specimen, assessing PD-L1 expression of approximately 10 times more cells. We further developed a computer-assisted PD-L1 quantitation method to analyze these images, and we found marked variation of PD-L1 expression in 3D. In 5 of 33 needle-biopsy-sized specimens (15.2%), the PD-L1 tumor proportion score (TPS) varied by greater than 10% at different depth levels. In 14 cases (42.4%), the TPS at different depth levels fell into different categories (< 1%, 1-49%, or ≥ 50%), which can potentially influence treatment decisions. Importantly, our technology permits recovery of the processed tissue for subsequent analysis, including histology examination, immunohistochemistry, and mutation analysis. In conclusion, our novel method has the potential to increase the accuracy of tumor PD-L1 expression assessment and enable precise deployment of cancer immunotherapy.

Investigation of P1/HC-Pro-Mediated ABA/Calcium Signaling Responses via Gene Silencing through High- and Low-Throughput RNA-seq Approaches.

The P1/HC-Pro viral suppressor of potyvirus suppresses posttranscriptional gene silencing (PTGS). The fusion protein of P1/HC-Pro can be cleaved into P1 and HC-Pro through the P1 self-cleavage activity, and P1 is necessary and sufficient to enhance PTGS suppression of HC-Pro. To address the modulation of gene regulatory relationships induced by turnip mosaic virus (TuMV) P1/HC-Pro (P1/HC-ProTu), a comparative transcriptome analysis of three types of transgenic plants (P1Tu, HC-ProTu, and P1/HC-ProTu) were conducted using both high-throughput (HTP) and low-throughput (LTP) RNA-Seq strategies. The results showed that P1/HC-ProTu disturbed the endogenous abscisic acid (ABA) accumulation and genes in the signaling pathway. Additionally, the integrated responses of stress-related genes, in particular to drought stress, cold stress, senescence, and stomatal dynamics, altered the expressions by the ABA/calcium signaling. Crosstalk among the ABA, jasmonic acid, and salicylic acid pathways might simultaneously modulate the stress responses triggered by P1/HC-ProTu. Furthermore, the LTP network analysis revealed crucial genes in common with those identified by the HTP network in this study, demonstrating the effectiveness of the miniaturization of the HTP profile. Overall, our findings indicate that P1/HC-ProTu-mediated suppression in RNA silencing altered the ABA/calcium signaling and a wide range of stress responses.

FOXM1 is required for small cell lung cancer tumorigenesis and associated with poor clinical prognosis.

Small cell lung cancer (SCLC) continues to cause poor clinical outcomes due to limited advances in sustained treatments for rapid cancer cell proliferation and progression. The transcriptional factor Forkhead Box M1 (FOXM1) regulates cell proliferation, tumor initiation, and progression in multiple cancer types. However, its biological function and clinical significance in SCLC remain unestablished. Analysis of the Cancer Cell Line Encyclopedia and SCLC datasets in the present study disclosed significant upregulation of FOXM1 mRNA in SCLC cell lines and tissues. Gene set enrichment analysis (GSEA) revealed that FOXM1 is positively correlated with pathways regulating cell proliferation and DNA damage repair, as evident from sensitization of FOXM1-depleted SCLC cells to chemotherapy. Furthermore, Foxm1 knockout inhibited SCLC formation in the Rb1fl/flTrp53fl/flMycLSL/LSL (RPM) mouse model associated with increased levels of neuroendocrine markers, Ascl1 and Cgrp, and decrease in Yap1. Consistently, FOXM1 depletion in NCI-H1688 SCLC cells reduced migration and enhanced apoptosis and sensitivity to cisplatin and etoposide. SCLC with high FOXM1 expression (N = 30, 57.7%) was significantly correlated with advanced clinical stage, extrathoracic metastases, and decrease in overall survival (OS), compared with the low-FOXM1 group (7.90 vs. 12.46 months). Moreover, the high-FOXM1 group showed shorter progression-free survival after standard chemotherapy, compared with the low-FOXM1 group (3.90 vs. 8.69 months). Our collective findings support the utility of FOXM1 as a prognostic biomarker and potential molecular target for SCLC.

Auricular acupressure combined with an internet-based intervention or alone for primary dysmenorrhea: a control study.

Background. Primary dysmenorrhea is prevalent in adolescents and young women. Menstrual pain and distress causes poor school performance and physiological damage. Auricular acupressure can be used to treat these symptoms, and Internet-based systems are a flexible way of communicating and delivering the relevant information. Objective. This study investigates the effects of auricular acupressure (AA) alone and combined with an interactive Internet-based (II) intervention for the management of menstrual pain and self-care of adolescents with primary dysmenorrhea. Design. This study adopts a pretest/posttest control research design with a convenience sample of 107 participants. Results. The outcomes were measured using the short-form McGill pain questionnaire (SF-MPQ), visual analogue scale (VAS), menstrual distress questionnaire (MDQ), and adolescent dysmenorrheic self-care scale (ADSCS). Significant differences were found in ADSCS scores between the groups, and in SF-MPQ, VAS, MDQ, and ADSCS scores for each group. Conclusion. Auricular acupressure alone and a combination of auricular acupressure and interactive Internet both reduced menstrual pain and distress for primary dysmenorrhea. Auricular acupressure combined with interactive Internet instruction is better than auricular acupuncture alone in improving self-care behaviors.

Auricular acupressure for pain relief in adolescents with dysmenorrhea: a placebo-controlled study.

OBJECTIVES: Primary dysmenorrhea is a common problem among menstruating adolescents and young women. It may cause physical distress and result in school absenteeism and reduced physical activity. This study aimed to evaluate the effects of auricular acupressure on menstrual pain and distress in adolescents with dysmenorrhea. DESIGN: A single-blind, placebo-controlled design was used. SETTING/LOCATION: Participants were obtained from one senior high school in northern Taiwan. SUBJECTS: One hundred and thirteen (113) adolescent participants with primary dysmenorrhea were recruited and assigned to the experimental or control group by a coin toss. INTERVENTION: The experimental group received auricular acupressure applied to six true acupoints (shenmen, Kidney, Liver, Internal Genitals, Central Rim, and Endocrine). The control group received six sham acupoints without effects on dysmenorrhea. All participants were instructed to press each acupoint for 1 minute, 4 times a day for 2 days. OUTCOME MEASURES: The outcomes were assessed by rating dysmenorrhea severity on a visual analogue scale (VAS) and using the Short-Form McGill Pain Questionnaire (SF-MPQ) and Menstrual Distress Questionnaire (MDQ). RESULTS: Between-group differences were found in VAS and MDQ after the interventions. Within-group differences were found in the score changes of VAS, MDQ, and SF-MPQ during the interventions for both groups. CONCLUSIONS: Auricular acupressure relieves menstrual pain and distress in high-school adolescents. The findings may serve as a basis for using auricular acupressure to treat dysmenorrhea in adolescents. There was pain reduction with sham as well as with true acupoint acupressure, but the latter was significantly greater. The sham acupoint may not be used as a control for auricular acupoint and qualitative evaluation of dysmenorrhea should be added to the evaluation by SF-MPQ in future studies.

[Factors related to health status in mothers of children with cancer].

BACKGROUND: Mothers are typically the primary caretakers of children with cancer, and the burden of caring responsibilities may influence their health status. Few studies have previously explored factors related to the health status of mothers with children suffering from cancer. PURPOSE: The purpose of this study was to examine factors related to health status in mothers of children with cancer. METHODS: A total of 120 mothers of children with cancer were recruited from two medical centers in southern Taiwan. Associations among demographic data, the Social Support: Personal Resource Questionnaire (PRQ85) part 2, and the Duke Health Profile (DUKE) were examined using one-way ANOVA and stepwise regression. RESULTS: A significant, positive relationship was found between social support and mother health status. Marital status was related to physical health, social health, general health, anxiety, depression, anxiety-depression and total health score. The mother's age and family monthly income were correlated with depression. Stage of child's disease was correlated with mother's mental health, general health, anxiety, depression, anxiety-depression and general health. Mother's social support and stage of child's disease were found to be significant predictors, accounting for 39% of variance in mother's health status. CONCLUSIONS/IMPLICATIONS FOR PRACTICE: Clinical staff should be aware that stage of (child's) disease may influence the health status of the mother care-giver. Clinical staff should also further enhance social support to promote the health of mothers with children who have cancer.

[Exploration of social support available to mothers of children with cancer, their health status, and other factors related to their family function].

The mother is typically the primary caregiver for children suffering from cancer. The long-term care responsibilities involved with caring for a cancer-stricken child impacts upon the mother's family role and functions and causes lifestyle changes or imbalances. The aim of this study was to explore social support, health status, and related factors in families of children with cancer. This study adopted a cross-sectional descriptive design. Study subjects consisted of 120 mothers of children with cancer recruited from the outpatient and inpatient departments of two medical centers in southern Taiwan between January and April 2006. Research instruments included demographic characterizations of the children with cancer, their mothers, and families; the Social Support: Personal Resource Questionnaire (PRQ85) part 2, the Duke Health Profile (DUKE), and the Family Assessment Device (FAD). Results showed a mean score of social support for mothers of children with cancer was 131.21 out of a total possible 175. The standard score for social support was 75.0. The standard score for health was 67.15. The mean score of family functioning was 128.48 out of a total possible 240. The standard score of family function was 53.53. Mother's age, marriage, family type, family socioeconomic stage, disease stage, and relapse of child's disease all had a statistically significant relationship to family function. Significant positive relationships were identified between the two variables social support and health status and family function. Mother's health, social support, disease relapse, and mother's age were significant predictors of mother's family function, explaining 42% of variance in family function for mothers of children with cancer. We hope findings will help clinical health professionals identify and implement nursing strategies to increase the health of mothers and their children as well as promote social support to strengthen family functions.