Functional viromic screens uncover regulatory RNA elements.

The number of sequenced viral genomes has surged recently, presenting an opportunity to understand viral diversity and uncover unknown regulatory mechanisms. Here, we conducted a screening of 30,367 viral segments from 143 species representing 96 genera and 37 families. Using a library of viral segments in 3' UTR, we identified hundreds of elements impacting RNA abundance, translation, and nucleocytoplasmic distribution. To illustrate the power of this approach, we investigated K5, an element conserved in kobuviruses, and found its potent ability to enhance mRNA stability and translation in various contexts, including adeno-associated viral vectors and synthetic mRNAs. Moreover, we identified a previously uncharacterized protein, ZCCHC2, as a critical host factor for K5. ZCCHC2 recruits the terminal nucleotidyl transferase TENT4 to elongate poly(A) tails with mixed sequences, delaying deadenylation. This study provides a unique resource for virus and RNA research and highlights the potential of the virosphere for biological discoveries.

Role of the proline-rich disordered domain of DROSHA in intronic microRNA processing.

DROSHA serves as a gatekeeper of the microRNA (miRNA) pathway by processing primary transcripts (pri-miRNAs). While the functions of structured domains of DROSHA have been well documented, the contribution of N-terminal proline-rich disordered domain (PRD) remains elusive. Here we show that the PRD promotes the processing of miRNA hairpins located within introns. We identified a DROSHA isoform (p140) lacking the PRD, which is produced by proteolytic cleavage. Small RNA sequencing revealed that p140 is significantly impaired in the maturation of intronic miRNAs. Consistently, our minigene constructs demonstrated that PRD enhances the processing of intronic hairpins, but not those in exons. Splice site mutations did not affect the PRD's enhancing effect on intronic constructs, suggesting that the PRD acts independently of splicing reaction by interacting with sequences residing within introns. The N-terminal regions from zebrafish and Xenopus DROSHA can replace the human counterpart, indicating functional conservation despite poor sequence alignment. Moreover, we found that rapidly evolving intronic miRNAs are generally more dependent on PRD than conserved ones, suggesting a role of PRD in miRNA evolution. Our study reveals a new layer of miRNA regulation mediated by a low-complexity disordered domain that senses the genomic contexts of miRNA loci.

Molecular Basis for the Single-Nucleotide Precision of Primary microRNA Processing.

Microprocessor, composed of DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) biogenesis by processing the primary transcripts of miRNA (pri-miRNAs). Here we investigate the mechanism by which Microprocessor selects the cleavage site with single-nucleotide precision, which is crucial for the specificity and functionality of miRNAs. By testing ∼40,000 pri-miRNA variants, we find that for some pri-miRNAs the cleavage site is dictated mainly by the mGHG motif embedded in the lower stem region of pri-miRNA. Structural modeling and deep-sequencing-based complementation experiments show that the double-stranded RNA-binding domain (dsRBD) of DROSHA recognizes mGHG to place the catalytic center in the appropriate position. The mGHG motif as well as the mGHG-recognizing residues in DROSHA dsRBD are conserved across eumetazoans, suggesting that this mechanism emerged in an early ancestor of the animal lineage. Our findings provide a basis for the understanding of miRNA biogenesis and rational design of accurate small-RNA-based gene silencing.

Adenylation of maternally inherited microRNAs by Wispy.

Early development depends heavily on accurate control of maternally inherited mRNAs, and yet it remains unknown how maternal microRNAs are regulated during maternal-to-zygotic transition (MZT). We here find that maternal microRNAs are highly adenylated at their 3' ends in mature oocytes and early embryos. Maternal microRNA adenylation is widely conserved in fly, sea urchin, and mouse. We identify Wispy, a noncanonical poly(A) polymerase, as the enzyme responsible for microRNA adenylation in flies. Knockout of wispy abrogates adenylation and results in microRNA accumulation in eggs, whereas overexpression of Wispy increases adenylation and reduces microRNA levels in S2 cells. Wispy interacts with Ago1 through protein-protein interaction, which may allow the effective and selective adenylation of microRNAs. Thus, adenylation may contribute to the clearance of maternally deposited microRNAs during MZT. Our work provides mechanistic insights into the regulation of maternal microRNAs and illustrates the importance of RNA tailing in development.

ERH facilitates microRNA maturation through the interaction with the N-terminus of DGCR8.

The microprocessor complex cleaves the primary transcript of microRNA (pri-miRNA) to initiate miRNA maturation. Microprocessor is known to consist of RNase III DROSHA and dsRNA-binding DGCR8. Here, we identify Enhancer of Rudimentary Homolog (ERH) as a new component of Microprocessor. Through a crystal structure and biochemical experiments, we reveal that ERH uses its hydrophobic groove to bind to a conserved region in the N-terminus of DGCR8, in a 2:2 stoichiometry. Knock-down of ERH or deletion of the DGCR8 N-terminus results in a reduced processing of suboptimal pri-miRNAs in polycistronic miRNA clusters. ERH increases the processing of suboptimal pri-miR-451 in a manner dependent on its neighboring pri-miR-144. Thus, the ERH dimer may mediate 'cluster assistance' in which Microprocessor is loaded onto a poor substrate with help from a high-affinity substrate in the same cluster. Our study reveals a role of ERH in the miRNA biogenesis pathway.

Differentiation and Establishment of Dental Epithelial-Like Stem Cells Derived from Human ESCs and iPSCs.

Tooth development and regeneration occur through reciprocal interactions between epithelial and ectodermal mesenchymal stem cells. However, the current studies on tooth development are limited, since epithelial stem cells are relatively difficult to obtain and maintain. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be alternative options for epithelial cell sources. To differentiate hESCs/hiPSCs into dental epithelial-like stem cells, this study investigated the hypothesis that direct interactions between pluripotent stem cells, such as hESCs or hiPSCs, and Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cell line may induce epithelial differentiation. Epithelial-like stem cells derived from hES (EPI-ES) and hiPSC (EPI-iPSC) had morphological and immunophenotypic characteristics of HERS/ERM cells, as well as similar gene expression. To overcome a rare population and insufficient expansion of primary cells, EPI-iPSC was immortalized with the SV40 large T antigen. The immortalized EPI-iPSC cell line had a normal karyotype, and a short tandem repeat (STR) analysis verified that it was derived from hiPSCs. The EPI-iPSC cell line co-cultured with dental pulp stem cells displayed increased amelogenic and odontogenic gene expression, exhibited higher dentin sialoprotein (DSPP) protein expression, and promoted mineralized nodule formation. These results indicated that the direct co-culture of hESCs/hiPSCs with HERS/ERM successfully established dental epithelial-like stem cells. Moreover, this differentiation protocol could help with understanding the functional roles of cell-to-cell communication and tissue engineering of teeth.

Relationship between Athletes' Big Five Model of Personality and Athletic Performance: Meta-Analysis.

Academic interest in athletic performance is ongoing. To examine the correlation between athletic performance and athletes' personality types, data extraction in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines was completed in October 2021, and a meta-analysis was performed using 180 data from 18 selected papers using the "meta" package version 4.8-4 of R Studio 3.3.3. As a result, these selected studies proved to have reliable quality in proceeding with this study via quality assessment. The overall effect of personality on athletic performance (AP) was ESr = 0.124, p < 0.01. Furthermore, only conscientiousness (ESr = 0.178, p < 0.001) and extroversion (ESr = 0.145, p < 0.01), among the five personality types, showed statistically significant results, and these two personality types had a positive correlation with performance. In the publication bias test, this study found that (a) agreeableness had a publication bias; but, with an additional test using trim-and-fill, (b) the effect was not significant enough to be considered. In addition, the analysis of the moderating effects was conducted in four aspects, and all moderating effect analyses showed statistically significant differences between the groups, demonstrating the heterogeneity of this study. Therefore, this study found a significant relationship between personality and athletic performance and showed the importance of conscientiousness and extroversion.

Implication of Amyloid Precursor-like Protein 2 Expression in Cutaneous Squamous Cell Carcinoma Pathogenesis.

BACKGROUND/AIM: Regulatory functions of amyloid precursor-like protein 2 (APLP2) expression in intracellular trafficking of major histocompatibility complex class I (MHC-I) and biological behavior of tumor cells have been reported in various types of malignancies but not in cutaneous squamous cell carcinoma (CSCC). This study aimed to investigate the role of APLP2 expression in the pathogenesis of CSCC. PATIENTS AND METHODS: The expression of APLP2 and a key modulator of cancer immune escape, MHC-I, were determined in CSCC tissue samples obtained from 141 patients using immunohistochemistry. The regulatory effects of APLP2 expression on the biological behavior and surface expression of MHC-I in CSCC cells were investigated by trypan blue assay, Matrigel invasion assay, and in vivo xenograft analysis. RESULTS: APLP2 immunoreactivity was high in 73 (51.8%) tissue samples from patients with CSCC and was significantly related to subcutaneous fat invasion and poor prognosis in our cohort. Moreover, proliferation of and invasion by CSCC cells were significantly reduced after APLP2 knockdown in CSCC cells both in vitro and in vivo. A significant association was found between APLP2 and membrane MHC-I expression in patients with CSCC. In vivo xenograft analysis showed that APLP2 knockdown increased membrane MHC-I expression in CSCC cells. CONCLUSION: APLP2 not only acts as an oncogene in CSCC progression but also as a possible modulator of cancer immune escape by influencing MHC-I expression on the cell surface. APLP2 may serve as a novel molecular biomarker and therapeutic target for patients with CSCC.

Effects of golf instructors' professional certification levels on amateur golfers' perception of instructor expertise, instructor credibility, and lesson participation intention: testing placebo and nocebo effects.

This study investigated the differences in amateur golfers' perceptions of instructor expertise, instructor credibility, and lesson participation intention depending on the golf instructor's certification level to investigate whether placebo and nocebo effects exist depending on the certification level. Accordingly, the study analyzed 153 amateur golfers with at least 1 year of playing experience, and the results were as follows: First, there was a difference in the perception of instructor expertise among amateur golfers depending on the golf instructor's certification level. Specifically, there were significant differences in perceived performance and teaching skills but no differences in personality and emphasis on basic principles. Second, the participants reported significant differences in their perceptions of instructor credibility depending on the instructor's certification level. Instructor credibility of the tournament professional group was the highest, whereas that of the amateur group was the lowest. Third, the results showed differences in lesson participation intention among amateur golfers depending on the instructor's certification level. Lesson participation intention was higher for semi-professional and tournament professional instructors than for amateur instructors. These results verified the presence of psychological biases, such as placebo/nocebo effects, that result in differences in the perception of instructor expertise, instructor credibility, and lesson participation intention depending on the certification level of instructors. Additionally, based on the data obtained from this study, further research is required to improve the performance of golf instructors and create an efficient teaching environment.

Differentiation of Human-induced Pluripotent Stem Cell-derived Dental Stem Cells through Epithelial-Mesenchymal Interaction.

Research on tooth regeneration using human-induced pluripotent stem cells (hiPSCs) is valuable for autologous dental regeneration. Acquiring mesenchymal and epithelial cells as a resource for dental regeneration is necessary because mesenchymal-epithelial interactions play an essential role in dental development. We reported the establishment of hiPSCs-derived dental epithelial-like cell (EPI-iPSCs), but hiPSCs-derived dental mesenchymal stem cells (MSCs) have not yet been reported. This study was conducted to establish hiPSCs-derived MSCs and to differentiate them into dental cells with EPI-iPSCs. Considering that dental MSCs are derived from the neural crest, hiPSCs were induced to differentiate into MSCs through neural crest formation to acquire the properties of dental MSCs. To differentiate hiPSCs into MSCs through neural crest formation, established hiPSCs were cultured and differentiated with PA6 stromal cells and differentiated hiPSCs formed neurospheres on ultralow-attachment plates. Neurospheres were differentiated into MSCs in serum-supplemented medium. Neural crest-mediated MSCs (NC-MSCs) continuously showed typical MSC morphology and expressed MSC markers. After 8 days of odontogenic induction, the expression levels of odontogenic/mineralization-related genes and dentin sialophosphoprotein (DSPP) proteins were increased in the NC-MSCs alone group in the absence of coculturing with dental epithelial cells. The NC-MSCs and EPI-iPSCs coculture groups showed high expression levels of amelogenesis/odontogenic/mineralization-related genes and DSPP proteins. Furthermore, the NC-MSCs and EPI-iPSCs coculture group yielded calcium deposits earlier than the NC-MSCs alone group. These results indicated that established NC-MSCs from hiPSCs have dental differentiation capacity with dental epithelial cells. In addition, it was confirmed that hiPSCs-derived dental stem cells could be a novel cell source for autologous dental regeneration.

Proline-Hinged α-Helical Peptides Sensitize Gram-Positive Antibiotics, Expanding Their Physicochemical Properties to Be Used as Gram-Negative Antibiotics.

The outer membrane (OM) of Gram-negative bacteria is the most difficult obstacle for small-molecule antibiotics to reach their targets in the cytosol. The molecular features of Gram-negative antibiotics required for passing through the OM are that they should be positively charged rather than neutral, flat rather than globular, less flexible, or more increased amphiphilic moment. Because of these specific molecular characteristics, developing Gram-negative antibiotics is difficult. We focused on sensitizer peptides to facilitate the passage of hydrophobic Gram-positive antibiotics through the OM. We explored ways of improving the sensitizing ability of proline-hinged α-helical peptides by adjusting their length, hydrophobicity, and N-terminal groups. A novel peptide, 1403, improves the potentiation of rifampicin in vitro and in vivo and potentiates most Gram-positive antibiotics. The "sensitizer" approach is more plausible than those that rely on conventional drug discovery methods concerning drug development costs and the development of drug resistance.

Blue Light Sensing BlsA-Mediated Modulation of Meropenem Resistance and Biofilm Formation in Acinetobacter baumannii.

The presence or absence of BlsA, a protein with a blue light-sensing flavin domain in the genomes of Acinetobacter species has aroused curiosity about its roles in the regulation of bacterial lifestyle under light. Genomic and transcriptomic analyses revealed the loss of BlsA in several multidrug-resistant (MDR) A. baumannii strains as well as the light-mediated induction of blsA, along with a possible BlsA-interacting partner BipA. Their direct in vivo interactions were verified using a bacterial two-hybrid system. The results demonstrated that the C-terminal region of BipA could bind to the C-terminal residues of BlsA under blue light at 23°C but not at 37°C. Genetic manipulations of blsA and bipA revealed that the coexistence of BlsA and BipA was required to induce the light-dependent expression of ompA in A. baumannii ATCC 17978 at 23°C. The same phenomenon occurred in the BlsA-deficient MDR strain in our functional complementation assay; however, the underlying molecular mechanism remains poorly understood. BlsA-modulated amounts of OmpA, the most abundant porin, in the outer membrane affected the membrane integrity and permeability of small molecules. Dark conditions or the deletion of ompA made the membrane more permeable to lipophilic ethidium bromide (EtBr) but not to meropenem. Interestingly, light illumination and low temperature conditions made the cells more sensitive to meropenem; however, this bactericidal effect was not noted in the blsA mutant or in the BlsA-deficient MDR strains. Light-mediated cell death and the reduction of biofilm formation at 23°C were abolished in the blsA mutant strain, suggesting multifaceted roles of BlsA in A. baumannii strains. IMPORTANCE Little is known about the functional roles of BlsA and its interacting partners in Acinetobacter species. Intriguingly, no BlsA homolog was found in several clinical isolates, suggesting that BlsA was not required inside the host because of the lack of blue light and the warm temperature conditions. As many chromophore-harboring proteins interact with various partners to control light-dependent cellular behaviors, the maintenance of blsA in the genomes of many Acinetobacter species during their evolution may be beneficial when fluctuations occur in two important environmental factors: light and temperature. Our study is the first to report the novel protein partner of BlsA, namely, BipA, and its contribution to multiple phenotypic changes, including meropenem resistance and biofilm formation. Rapid physiological acclimation to changing light or temperature conditions may be possible in the presence of the light-sensing BlsA protein, which may have more interacting partners than expected.

The ubiquitin-proteasome system links NADPH metabolism to ferroptosis.

Ferroptosis is the type of cell death arising from uncontrolled and excessive lipid peroxidation. NADPH is essential for ferroptosis regulation because it supplies reducing equivalents for antioxidant defense systems and contributes to the generation of reactive oxygen species. Moreover, NADPH level serves as a biomarker for predicting the sensitivity of cells to ferroptosis. The ubiquitin-proteasome system governs the stability of many ferroptosis effectors. Recent research has revealed MARCHF6, the endoplasmic reticulum ubiquitin ligase, as an unprecedented NADPH sensor in the ubiquitin system and a critical regulator of ferroptosis involved in tumorigenesis and fetal development. This review summarizes the current understanding of NADPH metabolism and the ubiquitin-proteasome system in regulating ferroptosis and highlights the emerging importance of MARCHF6 as a vital connector between NADPH metabolism and ferroptosis.

Improvement of crAssphage detection/quantification method and its extensive application for food safety.

Water-borne diseases are usually caused by the fecal-oral transmission of human fecal pathogens. Traditionally, coliforms and enterococci are widely used as indicator bacteria, but they do not allow to differentiate between human and animal fecal contamination. Owing to its presence only in the human gut environment, crAssphage has been suggested as an alternative indicator of human fecal contamination to overcome the above challenges. In this study, 139 human and 89 animal fecal samples (e.g., chicken, cow, dog, pig, pigeon, and mouse) were collected. For the rapid detection of human crAssphage in fecal samples, quantitative real-time PCR (qPCR) was performed using five different oligonucleotide primer/probe combinations. These included three previously reported oligonucleotide primer/probe combinations (RQ, CPQ056, and CrAssBP) and two newly developed combinations (ORF00018-targeting CrAssPFL1 and ORF00044-targeting CrAssPFL2). The detection rate (crAssphage-positive rate) in human fecal samples were 23.0, 30.2, 28.8, 20.1, and 30.9%, respectively, suggesting CrAssPFL2 showed the highest detection rate. Furthermore, the lowest copy numbers (436.16 copy numbers) could be detected using the CrAssPFL2 combination. Interestingly, no difference in crAssphage detection rates was found between healthy people and intestinal inflammatory patients. As expected, no crAssphage was detected in any animal fecal samples, indicating its human specificity. Furthermore, qPCR analysis of sewage samples collected from five different sewage treatment plants revealed that they were all contaminated with 105.71 copy numbers/mL of crAssphage on average. The simulation test of crAssphage-contaminated food samples also confirmed that the detection limit was from 107.55 copy numbers of crAssphage in foods. Therefore, the newly developed and optimized qPCR would be useful for the sensitive detection of crAssphage while identifying the source of human fecal contamination.

AamA-mediated epigenetic control of genome-wide gene expression and phenotypic traits in Acinetobacter baumannii ATCC 17978.

Individual deletions of three genes encoding orphan DNA methyltransferases resulted in the occurrence of growth defect only in the aamA (encoding AcinetobacterAdenine Methylase A) mutant of A. baumannii strain ATCC 17978. Our single-molecule real-time sequencing-based methylome analysis revealed multiple AamA-mediated DNA methylation sites and proposed a potent census target motif (TTTRAATTYAAA). Loss of Dam led to modulation of genome-wide gene expression, and several Dam-target sites including the promoter region of the trmD operon (rpsP, rimM, trmD, and rplS) were identified through our methylome and transcriptome analyses. AamA methylation also appeared to control the expression of many genes linked to membrane functions (lolAB, lpxO), replication (dnaA) and protein synthesis (trmD operon) in the strain ATCC 17978. Interestingly, cellular resistance against several antibiotics and ethidium bromide through functions of efflux pumps diminished in the absence of the aamA gene, and the complementation of aamA gene restored the wild-type phenotypes. Other tested phenotypic traits such as outer-membrane vesicle production, biofilm formation and virulence were also affected in the aamA mutant. Collectively, our data indicated that epigenetic regulation through AamA-mediated DNA methylation of novel target sites mostly in the regulatory regions could contribute significantly to changes in multiple phenotypic traits in A. baumannii ATCC 17978.

The Relationship between MZ Generation Screen Golf Participants' Participation Motivation, Self-Esteem, and Psychological Happiness.

The purpose of this study is to analyze and clarify the relationship between the MZ generation's participation motivation in screen golf, self-esteem, and psychological happiness. To reach the goals of this study, 300 MZ generation screen golf participants were selected for this study. Accordingly, a questionnaire was distributed and 275 questionnaires were used for this study, excluding the answers that were omitted or unfaithful. SPSS Version 29.0 was used to show the frequency analysis, exploratory factor analysis, correlation analysis, and multiple regression analysis of the research. The results of this study are as follows. First, it was found that the participation motivation of MZ generation screen golf participants had significant effects on positive self-esteem. Second, it was found that the participation motivation of MZ generation screen golf participants had significant effects on negative self-esteem. Third, it was found that the participation motivation of MZ generation screen golf participants had significant effects on psychological happiness. Fourth, it was found that the self-esteem of MZ generation screen golf participants had significant effects on psychological happiness. This study shows how to screen golf as part of a healthy leisure culture for the MZ generation and can enhance its psychological factors.

Systematic Review and Meta-Analysis on Burnout Owing to Perfectionism in Elite Athletes Based on the Multidimensional Perfectionism Scale (MPS) and Athlete Burnout Questionnaire (ABQ).

Previous studies have shown that burnout negatively affects athletes' mental health. To further explore this subject, we conducted a systematic review and meta-analysis by combining data from previous studies. This study followed the PRISMA guidelines for systematic and reliable research and completed data extraction using 10 databases and 8 keywords in December 2021. There were 93 cases of initially extracted data from the selected articles (n = 14) and the meta-analysis was conducted using the "meta" package, version 4.8-4 of R Studio 3.3.3, with data (k = 77) excluding other-oriented perfectionism data (k = 16). The results showed that self-oriented perfectionism had a negative effect on sports devaluation (SD) (ESr = -0.246, p < 0.001), and socially prescribed perfectionism had a positive effect on emotional/physical exhaustion (ESr = 0.150, p < 0.05) and SD (ESr = 0.138, p < 0.05). Furthermore, the test for publication bias showed that no groups had asymmetrical data, and four moderator analyses were conducted to prove the heterogeneity (I2) of the total effect size; however, there was no difference among groups (QB), thereby resulting in unexplained variance. Consequently, this study presents variable data that determine the effects of perfectionism and burnout on elite athletes.

SIRT1 ubiquitination is regulated by opposing activities of APC/C-Cdh1 and AROS during stress-induced premature senescence.

SIRT1, a member of the mammalian sirtuin family, is a nicotinamide adenosine dinucleotide (NAD)-dependent deacetylase with key roles in aging-related diseases and cellular senescence. However, the mechanism by which SIRT1 protein homeostasis is controlled under senescent conditions remains elusive. Here, we revealed that SIRT1 protein is significantly downregulated due to ubiquitin-mediated proteasomal degradation during stress-induced premature senescence (SIPS) and that SIRT1 physically associates with anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase. Ubiquitin-dependent SIRT1 degradation is stimulated by the APC/C coactivator Cdh1 and not by the coactivator Cdc20. We found that Cdh1 depletion impaired the SIPS-promoted downregulation of SIRT1 expression and reduced cellular senescence, likely through SIRT1-driven p53 inactivation. In contrast, AROS, a SIRT1 activator, reversed the SIRT1 degradation induced by diverse stressors and antagonized Cdh1 function through competitive interactions with SIRT1. Furthermore, our data indicate opposite roles for Cdh1 and AROS in the epigenetic regulation of the senescence-associated secretory phenotype genes IL-6 and IL-8. Finally, we demonstrated that pinosylvin restores downregulated AROS (and SIRT1) expression levels in bleomycin-induced mouse pulmonary senescent tissue while repressing bleomycin-promoted Cdh1 expression. Overall, our study provides the first evidence of the reciprocal regulation of SIRT1 stability by APC/C-Cdh1 and AROS during stress-induced premature senescence, and our findings suggest pinosylvin as a potential senolytic agent for pulmonary fibrosis.

Analgesic effects and metabolome analyses of laser- and electro-acupuncture combined therapies in paclitaxel-induced neuropathic pain model.

Introduction: Allodynia, which can be induced by paclitaxel administration, is the presence of pain as a result of a stimulus that does not usually provoke pain. Many studies have investigated the analgesic efficacy of acupuncture, including laser acupuncture (LA) and electroacupuncture (EA). Although pain-related diseases are relatively common, few studies have analyzed the analgesic effects and mechanisms of LA combined with EA. The purpose of this study was to investigate the therapeutic effect and mechanism of manual acupuncture (MA), EA, LA, and combined therapy (LA + EA) in a paclitaxel-induced allodynia rat model. Methods: A total of 56 rats were classified into eight groups: a normal (Nor, n = 7), a control (Con, n = 7), an MA (n = 7), an EA (n = 7), a 650-nm LA (650LA, n = 7), an 830-nm LA (830LA, n = 7), a 650-nm LA combined with EA (650LA + EA, n = 7), and an 830-nm LA combined with EA group (830LA + EA, n = 7). Allodynia was induced by intraperitoneal injection of 2 mg/kg of paclitaxel every other day for a total of four times except the Nor group. Acupuncture treatments were conducted at the points of Jungwan (CV12) and Joksamni (ST36) once every other day for 6 min, for a total of nine times. Withdrawal response reaction times and force intensity of the foot were measured before the start of the experiment, after the 4th paclitaxel administration (day 8), and after the 9th and last treatment (day 15). On the 16th day, mRNA and protein expression in the spinal nerves was assessed, and a metabolome analysis of the animals' feces was performed. Results and discussion: Our analyses show that 650LA + EA treatment resulted in an upregulation of protein expression related to pain relief and nerve regeneration, whereas 830LA + EA treatment led to significant changes in metabolomes. This study demonstrates that a combination treatment of EA and LA can suppress allodynia and promote upregulation of protein expression related to nerve regeneration and is effective in changing the intestinal microbiome. Further large-scale research is required to assess the exact mechanism underlying the therapeutic effect of this combination treatment in pain-related diseases.

Marchf6 E3 ubiquitin ligase critically regulates endoplasmic reticulum stress, ferroptosis, and metabolic homeostasis in POMC neurons.

The metabolic prohormone pro-opiomelanocortin (POMC) is generally translocated into the endoplasmic reticulum (ER) for entry into the secretory pathway. Patients with mutations within the signal peptide (SP) of POMC or its adjoining segment develop metabolic disorders. However, the existence, metabolic fate, and functional outcomes of cytosol-retained POMC remain unclear. Here, we show that SP-uncleaved POMC is produced in the cytosol of POMC neuronal cells, thus inducing ER stress and ferroptotic cell death. Mechanistically, the cytosol-retained POMC sequesters the chaperone Hspa5 and subsequently accelerates degradation of the glutathione peroxidase Gpx4, a core regulator of ferroptosis, via the chaperone-mediated autophagy. We also show that the Marchf6 E3 ubiquitin ligase mediates the degradation of cytosol-retained POMC, thereby preventing ER stress and ferroptosis. Furthermore, POMC-Cre-mediated Marchf6-deficient mice exhibit hyperphagia, reduced energy expenditure, and weight gain. These findings suggest that Marchf6 is a critical regulator of ER stress, ferroptosis, and metabolic homeostasis in POMC neurons.