Multiscale rheology from bulk to nano using a quartz tuning fork-atomic force microscope.

Rheological characteristics exhibit significant variations at nanoscale confinement or near interfaces, compared to bulk rheological properties. To bridge the gap between nano- and bulk-scale rheology, allowing for a better and holistic understanding of rheology, developing a single experimental platform that provides rheological measurements across different scales, from nano to bulk, is desirable. Here, we present the novel methodology for multiscale rheology using a highly sensitive atomic force microscope based on a quartz tuning fork (QTF) force sensor. We employ microscale and nanoscale shear probes attached to the QTF, oscillating parallel to a substrate surface for rheological measurements as a function of the tip-substrate distance with sub-nanometer resolution. Silicone oils with viscosities ranging from 5 cSt to 10 000 cSt are used as calibration samples, and we have successfully derived the bulk rheological moduli. Furthermore, an increase in modulus is observed within the regime of tribo-nanorheology at distances less than 50 nm from the surface. Through such multiscale measurements, it is confirmed that this increase is due to the formation of a layered structure of silicone oil polymers on the solid surface. These results provide a comprehensive understanding of the tribo-rheological properties of complex fluids across different scales.

LPS-induced systemic inflammation is suppressed by the PDZ motif peptide of ZO-1 via regulation of macrophage M1/M2 polarization.

The gram-negative bacterium lipopolysaccharide (LPS) is frequently administered to generate models of systemic inflammation. However, there are several side effects and no effective treatment for LPS-induced systemic inflammation. PEGylated PDZ peptide based on zonula occludens-1 (ZO-1) was analyzed for its effects on systemic inflammation induced by LPS. PDZ peptide administration led to the restoration of tissue injuries (kidney, liver, and lung) and prevented alterations in biochemical plasma markers. The production of pro-inflammatory cytokines was significantly decreased in the plasma and lung BALF in the PDZ-administered mice. Flow cytometry analysis revealed the PDZ peptide significantly inhibited inflammation, mainly by decreasing the population of M1 macrophages, and neutrophils (immature and mature), and increasing M2 macrophages. Using RNA sequencing analysis, the expression levels of the NF-κB-related proteins were lower in PDZ-treated cells than in LPS-treated cells. In addition, wild-type PDZ peptide significantly increased mitochondrial membrane integrity and decreased LPS-induced mitochondria fission. Interestingly, PDZ peptide dramatically could reduce LPS-induced NF-κB signaling, ROS production, and the expression of M1 macrophage marker proteins, but increased the expression of M2 macrophage marker proteins. These results indicated that PEGylated PDZ peptide inhibits LPS-induced systemic inflammation, reducing tissue injuries and reestablishing homeostasis, and may be a therapeutic candidate against systemic inflammation.

Autophagy controls neuronal differentiation by regulating the WNT-DVL signaling pathway.

Macroautophagy/autophagy dysregulation is associated with various neurological diseases, including Vici syndrome. We aimed to determine the role of autophagy in early brain development. We generated neurons from human embryonic stem cells and developed a Vici syndrome model by introducing a loss-of-function mutation in the EPG5 gene. Autophagy-related genes were upregulated at the neuronal progenitor cell stage. Inhibition of autolysosome formation with bafilomycin A1 treatment at the neuronal progenitor cell stage delayed neuronal differentiation. Notably, WNT (Wnt family member) signaling may be part of the underlying mechanism, which is negatively regulated by autophagy-mediated DVL2 (disheveled segment polarity protein 2) degradation. Disruption of autolysosome formation may lead to failure in the downregulation of WNT signaling, delaying neuronal differentiation. EPG5 mutations disturbed autolysosome formation, subsequently inducing defects in progenitor cell differentiation and cortical layer generation in organoids. Disrupted autophagy leads to smaller organoids, recapitulating Vici syndrome-associated microcephaly, and validating the disease relevance of our study.Abbreviations: BafA1: bafilomycin A1; co-IP: co-immunoprecipitation; DVL2: dishevelled segment polarity protein 2; EPG5: ectopic P-granules 5 autophagy tethering factor; gRNA, guide RNA; hESC: human embryonic stem cells; KO: knockout; mDA, midbrain dopamine; NIM: neural induction media; NPC: neuronal progenitor cell; qPCR: quantitative polymerase chain reaction; UPS: ubiquitin-proteasome system; WNT: Wnt family member; WT: wild type.

Advances in human pluripotent stem cell reporter systems.

The capability of human pluripotent stem cells (hPSCs) to self-renew and differentiate into any cell type has greatly contributed to the advancement of biomedicine. Reporter lines derived from hPSCs have played a crucial role in elucidating the mechanisms underlying human development and diseases by acting as an alternative reporter system that cannot be used in living humans. To bring hPSCs closer to clinical application in transplantation, scientists have generated reporter lines for isolating the desired cell populations, as well as improving graft quality and treatment outcomes. This review presents an overview of the applications of hPSC reporter lines and the important variables in designing a reporter system, including options for gene delivery and editing tools, design of reporter constructs, and selection of reporter genes. It also provides insights into the prospects of hPSC reporter lines and the challenges that must be overcome to maximize the potential of hPSC reporter lines.

PIEZO1 activation may serve as an early tissue biomarker for the prediction of irradiation-induced salivary gland dysfunction.

Irradiation (IR)-induced xerostomia is the most common side effect of radiation therapy in patients with head and neck cancer (HNC). Xerostomia diagnosis is mainly based on the patient's medical history and symptoms. Currently, no direct biomarkers are available for the early prediction of IR-induced xerostomia. Here, we identified PIEZO1 as a novel predictive tissue biomarker for xerostomia. Our data demonstrate that PIEZO1 is significantly upregulated at the gene and protein levels during IR-induced salivary gland (SG) hypofunction. Notably, PIEZO1 upregulation coincided with that of inflammatory (F4/80) and fibrotic markers (fibronectin and collagen fibers accumulation). These findings suggest that PIEZO1 upregulation in SG tissue may serve as a novel predictive marker for IR-induced xerostomia.

Reassessing the genetic lineage tracing of lingual Lgr5+ and Lgr6+ cells in vivo.

Taste buds, the neuroepithelial organs responsible for the detection of gustatory stimuli in the oral cavity, arise from stem/progenitor cells among nearby basal keratinocytes. Using genetic lineage tracing, Lgr5 and Lgr6 were suggested as the specific markers for the stem/progenitor cells of taste buds, but recent evidence implied that taste buds may arise even in the absence of these markers. Thus, we wanted to verify the genetic lineage tracing of lingual Lgr5- and Lgr6-expressing cells. Unexpectedly, we found that antibody staining revealed more diverse Lgr5-expressing cells inside and outside the taste buds of circumvallate papillae than was previously suggested. We also found that, while tamoxifen-induced genetic recombination occurred only in cells expressing the Lgr5 reporter GFP, we did not see any increase in the number of recombined daughter cells induced by consecutive injections of tamoxifen. Similarly, we found that cells expressing Lgr6, another stem/progenitor cell marker candidate and an analog of Lgr5, also do not generate recombined clones. In contrast, Lgr5-expressing cells in fungiform papillae can transform into Lgr5-negative progeny. Together, our data indicate that lingual Lgr5- and Lgr6-expressing cells exhibit diversity in their capacity to transform into Lgr5- and Lgr6-negative cells, depending on their location. Our results complement previous findings that did not distinguish this diversity.

Neuronal Cell Differentiation of iPSCs for the Clinical Treatment of Neurological Diseases.

Current chemical treatments for cerebrovascular disease and neurological disorders have limited efficacy in tissue repair and functional restoration. Induced pluripotent stem cells (iPSCs) present a promising avenue in regenerative medicine for addressing neurological conditions. iPSCs, which are capable of reprogramming adult cells to regain pluripotency, offer the potential for patient-specific, personalized therapies. The modulation of molecular mechanisms through specific growth factor inhibition and signaling pathways can direct iPSCs' differentiation into neural stem cells (NSCs). These include employing bone morphogenetic protein-4 (BMP-4), transforming growth factor-beta (TGFß), and Sma-and Mad-related protein (SMAD) signaling. iPSC-derived NSCs can subsequently differentiate into various neuron types, each performing distinct functions. Cell transplantation underscores the potential of iPSC-derived NSCs to treat neurodegenerative diseases such as Parkinson's disease and points to future research directions for optimizing differentiation protocols and enhancing clinical applications.

Correction: Choi et al. Genistin: A Novel Potent Anti-Adipogenic and Anti-Lipogenic Agent. Molecules 2020, 25, 2042.

Error in Figure [...].

In Vivo Effects of Crataegus pinnatifida Extract for Healthy Longevity.

Aging is a complex series of multi-organ processes that occur in various organisms. As such, an in vivo study using an animal model of aging is necessary to define its exact mechanisms and identify anti-aging substances. Using Drosophila as an in vivo model system, we identified Crataegus pinnatifida extract (CPE) as a novel anti-aging substance. Regardless of sex, Drosophila treated with CPE showed a significantly increased lifespan compared to those without CPE. In this study, we also evaluated the involvement of CPE in aging-related biochemical pathways, including TOR, stem cell generation, and antioxidative effects, and found that the representative genes of each pathway were induced by CPE administration. CPE administration did not result in significant differences in fecundity, locomotion, feeding amount, or TAG level. These conclusions suggest that CPE is a good candidate as an anti-aging food substance capable of promoting a healthy lifespan.

Identification and analysis of novel endometriosis biomarkers via integrative bioinformatics.

Endometriosis is a gynecological disease prevalent in women of reproductive age, and it is characterized by the ectopic presence and growth of the eutopic endometrium. The pathophysiology and diagnostic biomarkers of endometriosis have not yet been comprehensively determined. To discover molecular markers and pathways underlying the pathogenesis of endometriosis, we identified differentially expressed genes (DEGs) in three Gene Expression Omnibus microarray datasets (GSE11691, GSE23339, and GSE7305) and performed gene set enrichment analysis (GSEA) and protein-protein interaction (PPI) network analyses. We also validated the identified genes via immunohistochemical analysis of tissues obtained from patients with endometriosis or healthy volunteers. A total of 118 DEGs (79 upregulated and 39 downregulated) were detected in each dataset with a lower (fold change) FC cutoff (log2|FC| > 1), and 17 DEGs (11 upregulated and six downregulated) with a higher FC cutoff (log2|FC| > 2). KEGG and GO functional analyses revealed enrichment of signaling pathways associated with inflammation, complement activation, cell adhesion, and extracellular matrix in endometriotic tissues. Upregulation of seven genes (C7, CFH, FZD7, LY96, PDLIM3, PTGIS, and WISP2) out of 17 was validated via comparison with external gene sets, and protein expression of four genes (LY96, PDLIM3, PTGIS, and WISP2) was further analyzed by immunohistochemistry and western blot analysis. Based on these results, we suggest that TLR4/NF-κB and Wnt/frizzled signaling pathways, as well as estrogen receptors, regulate the progression of endometriosis. These pathways may be therapeutic and diagnostic targets for endometriosis.

Generation of caudal-type serotonin neurons and hindbrain-fate organoids from hPSCs.

Serotonin (5-HT) neurons, the major components of the raphe nuclei, arise from ventral hindbrain progenitors. Based on anatomical location and axonal projection, 5-HT neurons are coarsely divided into rostral and caudal groups. Here, we propose a novel strategy to generate hindbrain 5-HT neurons from human pluripotent stem cells (hPSCs), which involves the formation of ventral-type neural progenitor cells and stimulation of the hindbrain 5-HT neural development. A caudalizing agent, retinoid acid, was used to direct the cells into the hindbrain cell fate. Approximately 30%-40% of hPSCs successfully developed into 5-HT-expressing neurons using our protocol, with the majority acquiring a caudal rhombomere identity (r5-8). We further modified our monolayer differentiation system to generate 5-HT neuron-enriched hindbrain-like organoids. We also suggest downstream applications of our 5-HT monolayer and organoid cultures to study neuronal response to gut microbiota. Our methodology could become a powerful tool for future studies related to 5-HT neurotransmission.

Discovery of Orphan Olfactory Receptor 6M1 as a New Anticancer Target in MCF-7 Cells by a Combination of Surface Plasmon Resonance-Based and Cell-Based Systems.

Olfactory receptors (ORs) account for 49% of all G protein-coupled receptors (GPCRs), which are important targets for drug discovery, and hence ORs may also be potential drug targets. Various ORs are expressed in breast cancer cells; however, most of them are orphan receptors, and thus, their functions are unknown. Herein, we present an experimental strategy using a surface plasmon resonance (SPR) system and a cell-based assay that allowed the identification of orphan OR6M1 as a new anticancer target in the MCF-7 breast cancer cell line. After the construction of stable OR6M1-expressing cells, the SPR-based screening of 108 chemicals for ligand activity was performed against OR6M1-expressing whole cells (primary screening) or membrane fragments (secondary screening). As a result, anthraquinone (AQ) and rutin were discovered to be new OR6M1 ligands. Based on calcium imaging in OR6M1-expressing Hana3A cells, AQ and rutin were classified as an OR6M1 agonist and antagonist, respectively. Cell viability and live/dead assays showed that AQ induced the death of MCF-7 cells, which was inhibited by rutin. Therefore, OR6M1 may be considered an anticancer target, and AQ may be considered a chemotherapeutic agent. This combined method can be widely used to discover the ligands and functions of other orphan GPCRs.

Canonical WNT/ß-Catenin Signaling Activated by WNT9b and RSPO2 Cooperation Regulates Facial Morphogenesis in Mice.

The R-spondin (RSPO) family of proteins potentiate canonical WNT/ß-catenin signaling and may provide a mechanism to fine-tune the strength of canonical WNT signaling. Although several in vitro studies have clearly demonstrated the potentiation of canonical WNT signaling by RSPOs, whether this potentiation actually occurs in normal development and tissue function in vivo still remains poorly understood. Here, we provide clear evidence of the potentiation of canonical WNT signaling by RSPO during mouse facial development by analyzing compound Wnt9b and Rspo2 gene knockout mice and utilizing ex vivo facial explants. Wnt9b;Rspo2 double mutant mice display facial defects and dysregulated gene expression pattern that are significantly more severe than and different from those of Wnt9b or Rspo2 null mutant mice. Furthermore, we found suggestive evidence that the LGR4/5/6 family of the RSPO receptors may play less critical roles in WNT9b:RSPO2 cooperation. Our results suggest that RSPO-induced cooperation is a key mechanism for fine-tuning canonical WNT/ß-catenin signaling in mouse facial development.

Genistin: A Novel Potent Anti-Adipogenic and Anti-Lipogenic Agent.

Soy isoflavones are popular ingredients with anti-adipogenic and anti-lipogenic properties. The anti-adipogenic and anti-lipogenic properties of genistein are well-known, but those of genistin and glycitein remain unknown, and those of daidzein are characterized by contrasting data. Therefore, the purpose of our study was to investigate the effects of daidzein, glycitein, genistein, and genistin on adipogenesis and lipogenesis in 3T3-L1 cells. Proliferation of 3T3-L1 preadipocytes was unaffected by genistin and glycitein, but it was affected by 50 and 100 µM genistein and 100 µM daidzein for 48 h. Among the four isoflavones, only 50 and 100 µM genistin and genistein markedly suppressed lipid accumulation during adipogenesis in 3T3-L1 cells through a similar signaling pathway in a dose-dependent manner. Genistin and genistein suppress adipocyte-specific proteins and genes, such as peroxisome proliferator-activated receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), and adipocyte binding protein 2 (aP2)/fatty acid-binding protein 4 (FABP4), and lipogenic enzymes such as ATP citrate lyase (ACL), acetyl-CoA carboxylase 1 (ACC1), and fatty acid synthase (FAS). Both isoflavones also activate AMP-activated protein kinase α (AMPKα), an essential factor in adipocyte differentiation, and inhibited sterol regulatory element-binding transcription factor 1c (SREBP-1c). These results indicate that genistin is a potent anti-adipogenic and anti-lipogenic agent.

Suppressive Effect of Arctium Lappa L. Leaves on Retinal Damage Against A2E-Induced ARPE-19 Cells and Mice.

Age-related macular degeneration (AMD) is a major cause of irreversible loss of vision with 80-90% of patients demonstrating dry type AMD. Dry AMD could possibly be prevented by polyphenol-rich medicinal foods by the inhibition of N-retinylidene-N-retinylethanolamine (A2E)-induced oxidative stress and cell damage. Arctium lappa L. (AL) leaves are medicinal and have antioxidant activity. The purpose of this study was to elucidate the protective effects of the extract of AL leaves (ALE) on dry AMD models, including in vitro A2E-induced damage in ARPE-19 cells, a human retinal pigment epithelial cell line, and in vivo light-induced retinal damage in BALB/c mice. According to the total phenolic contents (TPCs), total flavonoid contents (TFCs) and antioxidant activities, ALE was rich in polyphenols and had antioxidant efficacies on 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and 2',7'-dichlorofluorescin diacetate (DCFDA) assays. The effects of ALE on A2E accumulation and A2E-induced cell death were also monitored. Despite continued exposure to A2E (10 µM), ALE attenuated A2E accumulation in APRE-19 cells with levels similar to lutein. A2E-induced cell death at high concentration (25 µM) was also suppressed by ALE by inhibiting the apoptotic signaling pathway. Furthermore, ALE could protect the outer nuclear layer (ONL) in the retina from light-induced AMD in BALB/c mice. In conclusion, ALE could be considered a potentially valuable medicinal food for dry AMD.

Microbial Metabolites Determine Host Health and the Status of Some Diseases.

The gastrointestinal (GI) tract is a highly complex organ composed of the intestinal epithelium layer, intestinal microbiota, and local immune system. Intestinal microbiota residing in the GI tract engages in a mutualistic relationship with the host. Different sections of the GI tract contain distinct proportions of the intestinal microbiota, resulting in the presence of unique bacterial products in each GI section. The intestinal microbiota converts ingested nutrients into metabolites that target either the intestinal microbiota population or host cells. Metabolites act as messengers of information between the intestinal microbiota and host cells. The intestinal microbiota composition and resulting metabolites thus impact host development, health, and pathogenesis. Many recent studies have focused on modulation of the gut microbiota and their metabolites to improve host health and prevent or treat diseases. In this review, we focus on the production of microbial metabolites, their biological impact on the intestinal microbiota composition and host cells, and the effect of microbial metabolites that contribute to improvements in inflammatory bowel diseases and metabolic diseases. Understanding the role of microbial metabolites in protection against disease might offer an intriguing approach to regulate disease.

Loss of SATB1 Induces p21-Dependent Cellular Senescence in Post-mitotic Dopaminergic Neurons.

Cellular senescence is a mechanism used by mitotic cells to prevent uncontrolled cell division. As senescent cells persist in tissues, they cause local inflammation and are harmful to surrounding cells, contributing to aging. Generally, neurodegenerative diseases, such as Parkinson's, are disorders of aging. The contribution of cellular senescence to neurodegeneration is still unclear. SATB1 is a DNA binding protein associated with Parkinson's disease. We report that SATB1 prevents cellular senescence in post-mitotic dopaminergic neurons. Loss of SATB1 causes activation of a cellular senescence transcriptional program in dopamine neurons both in human stem cell-derived dopaminergic neurons and in mice. We observed phenotypes that are central to cellular senescence in SATB1 knockout dopamine neurons in vitro and in vivo. Moreover, we found that SATB1 directly represses expression of the pro-senescence factor p21 in dopaminergic neurons. Our data implicate senescence of dopamine neurons as a contributing factor in the pathology of Parkinson's disease.

Analgesic Effects of Cnidium officinale Extracts on Postoperative, Neuropathic, and Menopausal Pain in Rat Models.

Cnidium officinale, widely cultivated in East Asia, has been reported to exhibit pharmacological efficacy in various disorders. However, little has been reported on its role as a pain killer. In this study, we reveal that the C. officinale extract (COE) has great efficacy as a novel analgesic in various in vivo pain models. Administration of COE attenuated hypersensitivity in all postoperative, neuropathic, and menopausal pain models. Decreased hyperalgesia was confirmed by a mechanical withdrawal threshold assay and ultrasonic vocalization call analysis. In addition, application of COE inhibited the induction of the proinflammatory cytokines and calpain-3 on dorsal root ganglion neurons in a spared nerve injury rat model. Treatment with ferulic acid, which was identified as one of the components of COE by HPLC analysis, alleviated nociceptive behaviors. Our findings suggest that ferulic acid is an active compound from COE, and COE is a potential phytomedical source for pain relief by inhibiting the process of inflammation.

Efficient Neural Differentiation of hPSCs by Extrinsic Signals Derived from Co-cultured Neural Stem or Precursor Cells.

In this study, we found that undifferentiated human pluripotent stem cells (hPSCs; up to 30% of total cells) present in the cultures of neural stem or precursor cells (NPCs) completely disappeared within several days when cultured under neural differentiation culture conditions. Intriguingly, the disappearance of undifferentiated cells was not due to cell death but was instead mediated by neural conversion of hPSCs. Based on these findings, we propose pre-conditioning of donor NPC cultures under terminal differentiation culture conditions as a simple but efficient method of eliminating undifferentiated cells to treat neurologic disorders. In addition, we could establish a new neural differentiation protocol, in which undifferentiated hPSCs co-cultured with NPCs become differentiated neurons or NPCs in an extremely efficient, fast, and reproducible manner across the hESC and human-induced pluripotent stem cell (hiPSC) lines.

Roles of oxides of nitrogen on quality enhancement of soybean sprout during hydroponic production using plasma discharged water recycling technology.

This study was performed to assess the effect of plasma-discharged water recycling technology as irrigation water on soybean sprout production. Two different types of irrigation water were used individually for cultivation, including plasma discharged water as a source of oxides of nitrogen and tap water, irrigation water was recycled for every 30 minutes. Plasma discharged irrigation water reduced overall 4.3 log CFU/ml aerobic microbe and 7.0 log CFU/ml of artificially inoculated S. Typhimurium within 5 minutes and 2 minutes, respectively, therefore sprout production occurs in a hygienic environment. Using of plasma-discharged water for cultivation, increases the amount of ascorbate, asparagine, and γ-aminobutyric acid (GABA) significantly (p < 0.05), in the part of cotyledon and hypocotyl of soybean sprout during 1 to 4 days of farming. A NO scavenger, 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (cPTIO), was added in irrigation water to elucidate the roles of the oxides of nitrogen such as NO3-, NO2- generated in plasma discharged water. It was observed that all three nutrients decreased in the cotyledon part, whereas ascorbate and GABA contents increased in the hypocotyl and radicle part of bean sprout for the same duration of farming. The addition of NO scavenger in the irrigation water also reduced growth and overall yield of the soybean sprouts. A recycling water system with plasma-discharged water helped to reduce the amount of water consumption and allowed soybean sprouts growth in a hygienic environment during the hydroponic production.