Y-27632 enables long-term expansion of mouse submandibular gland epithelial cells via inactivation of TGF-ß1/CTGF/p38 and ROCK2/JNK signaling pathway.

OBJECTIVES: This study aimed to investigate the effects of Y-27632 on the long-term maintainence of mouse submandibular epithelial cells (SG-Epis) in vitro and to elucidate the underlying mechanisms. METHODS: The role of the Rho-associated kinase (ROCK) inhibitor Y-27632 in maintaining SG-Epis and its underlying mechanisms were evaluated by examining the in vitro expansion of mouse SG-Epis. Changes in key cellular characteristics, such as proliferation, long-term expansion, and mRNA and protein expression, were assessed in the presence or absence of Y-27632. RESULTS: Treatment with Y-27632 significantly enhanced the proliferative potential of SG-Epis, preserving Krt8 and Krt14 expression over 17 passages. In the absence of Y-27632, SG-Epis lost their epithelial morphology. However, Y-27632 treatment maintained the epithelial morphology and downregulated mRNA levels of Tgf-ß1, Ctgf, and Rock2. Treatment with TGF-ß1 indicated that TGF-ß/CTGF/p38 signaling is responsible for the maintenance of SG-Epis, while RNA interference studies revealed that ROCK2/c-Jun N-terminal kinase (JNK) signaling is also crucial for SG-Epis proliferation and maintenance. CONCLUSIONS: The TGF-ß1/CTGF/p38 and ROCK2/JNK signaling pathways are responsible for SG-Epis proliferation, and Y-27632 treatment effectively inactivates these pathways, enabling long-term in vitro maintenance of SG-Epis. The culture method utilizing Y-27632 provides an effective approach for the in vitro expansion of SG-Epis.

Global burden of vaccine-associated hepatobiliary and gastrointestinal adverse drug reactions, 1967-2023: A comprehensive analysis of the international pharmacovigilance database.

Although previous studies have focused on hepatobiliary and gastrointestinal adverse drug reactions (ADRs) associated with COVID-19 vaccines, literature on such ADRs with other vaccines is limited, particularly on a global scale. Therefore, we aimed to investigate the global burden of vaccine-associated hepatobiliary and gastrointestinal ADRs and identify the vaccines implicated in these occurrences. This study utilized data from the World Health Organization (WHO) international pharmacovigilance database to extract reports of vaccine-associated hepatobiliary and gastrointestinal ADRs from 1967 to 2023 (total reports = 131 255 418). Through global reporting counts, reported odds ratios (ROR) with 95% confidence interval (CI), and information components (IC) with IC0.25, the study examined the association between 16 vaccines and the incidence of hepatobiliary and gastrointestinal ADRs across 156 countries. Of the 6 842 303 reports in the vaccine-associated ADRs, 10 786 reports of liver injury, 927 870 reports of gastrointestinal symptoms, 2978 reports of pancreas and bile duct injury, and 96 reports of intra-abdominal hemorrhage between 1967 and 2023 were identified. Most hepatobiliary and gastrointestinal ADRs surged after 2020, with the majority of reports attributed to COVID-19 messenger RNA (mRNA) vaccines. Hepatitis A vaccines exhibited the highest association with liver injury (ROR [95% CI]: 10.30 [9.65-10.99]; IC [IC0.25]: 3.33 [3.22]), followed by hepatitis B, typhoid, and rotavirus. Specifically, ischemic hepatitis had a significant association with both Ad5-vectored and mRNA COVID-19 vaccines. Gastrointestinal symptoms were associated with all vaccines except for tuberculosis vaccines, particularly with rotavirus (11.62 [11.45-11.80]; 3.05 [3.03]) and typhoid (11.02 [10.66-11.39]; 3.00 [2.96]). Pancreas and bile duct injury were associated with COVID-19 mRNA (1.99 [1.89-2.09]; 0.90 [0.83]), MMR (measles, mumps, and rubella), and papillomavirus vaccines. For intra-abdominal hemorrhage, inactivated whole-virus COVID-19 vaccines (3.93 [1.86-8.27]; 1.71 [0.41]) had the highest association, followed by COVID-19 mRNA (1.81 [1.42-2.29]; 0.77 [0.39]). Most of these ADRs had a short time to onset, within 1 day, and low mortality rate. Through a global scale database, the majority of ADRs occurred within 1 day, emphasizing the importance of healthcare workers' vigilant monitoring and timely management.

Brain connectivity during social exclusion differs depending on the closeness within a triad among older adults living in a village.

Social exclusion occurs in various types of social relationships, from anonymous others to close friends. However, the role that social relationships play in social exclusion is less well known because most paradigms investigating social exclusion have been done in laboratory contexts, without considering the features of individuals' real-world social relationships. Here, we addressed this gap by examining how pre-existing social relationships with rejecters may influence the brain response of individuals experiencing social exclusion. Eighty-eight older adults living in a rural village visited the laboratory with two other participants living in the same village and played Cyberball in an Magnetic Resonance Imaging scanner. Utilizing whole-brain connectome-based predictive modeling, we analyzed functional connectivity (FC) data obtained during the social exclusion task. First, we found that the level of self-reported distress during social exclusion was significantly related to sparsity, i.e. lack of closeness, within a triad. Furthermore, the sparsity was significantly predicted by the FC model, demonstrating that a sparse triadic relationship was associated with stronger connectivity patterns in brain regions previously implicated in social pain and mentalizing during Cyberball. These findings extend our understanding of how real-world social intimacy and relationships with excluders affect neural and emotional responses to social exclusion.

Psychological well-being and salivary markers of inflammation: The moderating effect of age.

Increasing evidence suggests a significant impact of higher psychological well-being (PWB) on health outcomes; however, such associations have been studied exclusively in middle-aged to older adults. This study examined the aging effect on PWB measures as well as the moderating effect of age on the link between PWB and inflammation, using salivary markers by comparing the younger adults (n = 127; Mage = 22.98 years) versus older adults (n = 75; Mage = 75.60 years). Older adults showed significantly lower levels of PWB, particularly regarding purpose in life and personal growth. Moreover, higher purpose in life was associated with lower salivary IL-1ß and IL-6 (b = 0.83, p < .001; b = 0.81, p < .01) only in the older adult group but not in younger adults. These findings highlight the potential buffering effect of the sense of living well on physiological pathways in later life.

Exopolysaccharide of Enterococcus faecium L15 promotes the osteogenic differentiation of human dental pulp stem cells via p38 MAPK pathway.

BACKGROUND: Bone has important functions in the body. Several researchers have reported that the polysaccharides and lipopolysaccharide derived from microbes can promote osteogenic differentiation of stem cells. Enterococcus faecium, a lactic acid bacterium (LAB), produces several bioactive metabolites and has been widely applied in the food and nutraceutical industries. The exopolysaccharide (EPS) from LAB has also been extensively examined for its postbiotic effects and for its in vivo and in vitro functionalities. However, studies on promoting bone differentiation using polysaccharides from LAB are lacking. Therefore, the purpose of this study was to investigate the effect of E. faecium L15 extract and EPS on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and to identify the underlying mechanisms. METHODS: hDPSCs were obtained from dental pulp tissue, and L15 extract and EPS were isolated from L15. Gene and protein expression of the osteogenic differentiation markers were analyzed with qPCR and western blotting and the possible signaling pathways were also investigated using western blotting. Osteogenic differentiation potential was examined by alkaline phosphatase (ALP) staining and alizarin red s (ARS) staining. In addition, osteogenic differentiation potential of L15 EPS was explored in ex vivo culture of neonate murine calvaria. RESULTS: The calcium deposition and ALP activity were enhanced by addition of L15 extract or EPS. The expression levels of RUNX2, ALP, and COL1A1 mRNA and the protein expression levels of RUNX2, ALP, and BMP4 were increased in hDPSCs treated with the L15 extract or EPS. The L15 EPS treatment enhanced phosphorylation of the p38 mitogen-activated protein kinase (MAPK). The L15 EPS-induced increases in RUNX2, ALP, and BMP4 expression were suppressed by the p38 MAPK inhibitor SB203580. The promoting effect of L15 EPS on osteogenic differentiation was not only seen in hDPSCs, but also in osteoblast precursors. ALP activity and the expression of RUNX2, ALP, and COL1A1 increased in the L15 EPS-treated osteoblast precursors. In addition, L15 EPS increased bone thickness of neonate murine calvaria in ex vivo culture. CONCLUSIONS: The stimulatory effect of L15 extract and EPS on osteogenic differentiation occurred through the p38 MAPK pathway, and L15 EPS enhanced new bone formation in neonate murine calvaria. These data suggest that L15 EPS has therapeutic potential applicable to bone regeneration.

Comparison of Cell-Free Extracts from Three Newly Identified Lactobacillus plantarum Strains on the Inhibitory Effect of Adipogenic Differentiation and Insulin Resistance in 3T3-L1 Adipocytes.

Obesity and associated metabolic disorders, including cardiovascular disease and diabetes, are rapidly becoming serious global health problems. It has been reported that Lactobacillus plantarum (L. plantarum) extracts have the beneficial activities of antiobesity and antidiabetes, although few studies have compared the beneficial effects among various L. plantarum extracts. In this study, three new L. plantarum (named LP, LS, and L14) strains were identified, and the antiobesogenic and diabetic effects of their extracts were investigated and compared using 3T3-L1 cells in vitro. Lipid accumulation in maturing 3T3-L1 cells was significantly decreased by the addition of LS and L14 extracts. The mRNA expression levels of Pparγ, C/ebpα, Fabp4, Fas, and Dgat1 were significantly decreased by the addition of LP, LS, and L14 extracts. Interestingly, the protein expression levels of PPARγ, C/EBPα, FABP4, and FAS were downregulated in mature 3T3-L1 cells with the addition of the L14 extract. Moreover, the LS and L14 extract treatments stimulated glucose uptake in maturing adipocytes. The L14 extract treatments exhibited a significant reduction in TNF-α protein expression, which is a key factor of insulin resistance in adipocytes. Of the three extracts, L14 extract markedly reduced adipogenic differentiation and insulin resistance in vitro, suggesting that the L14 extract may be used as a therapeutic agent for obesity-associated metabolic disorders.

Oral intake of Lactobacillus plantarum L-14 extract alleviates TLR2- and AMPK-mediated obesity-associated disorders in high-fat-diet-induced obese C57BL/6J mice.

OBJECTIVES: Whether periodic oral intake of postbiotics positively affects weight regulation and prevents obesity-associated diseases in vivo is unclear. This study evaluated the action mechanism of Lactobacillus plantarum L-14 (KTCT13497BP) extract and the effects of its periodic oral intake in a high-fat-diet (HFD) mouse model. MATERIALS AND METHODS: Mouse pre-adipocyte 3T3-L1 cells and human bone marrow mesenchymal stem cells (hBM-MSC) were treated with L-14 extract every 2 days during adipogenic differentiation, and the mechanism underlying anti-adipogenic effects was analysed at cellular and molecular levels. L-14 extract was orally administrated to HFD-feeding C57BL/6J mice every 2 days for 7 weeks. White adipose tissue was collected and weighed, and liver and blood serum were analysed. The anti-adipogenic mechanism of exopolysaccharide (EPS) isolated from L-14 extract was also analysed using Toll-like receptor 2 (TLR2) inhibitor C29. RESULTS: L-14 extract inhibited 3T3-L1 and hBM-MSC differentiation into mature adipocytes by upregulating AMPK signalling pathway in the early stage of adipogenic differentiation. The weight of the HFD + L-14 group (31.51 ± 1.96 g) was significantly different from that of the HFD group (35.14 ± 3.18 g). L-14 extract also significantly decreased the serum triacylglycerol/high-density lipoprotein cholesterol ratio (an insulin resistance marker) and steatohepatitis. In addition, EPS activated the AMPK signalling pathway by interacting with TLR2, consequently inhibiting adipogenesis. CONCLUSIONS: EPS from L-14 extract inhibits adipogenesis via TLR2 and AMPK signalling pathways, and oral intake of L-14 extract improves obesity and obesity-associated diseases in vivo. Therefore, EPS can be used to prevent and treat obesity and metabolic disorders.

Cell-Penetrating Peptides Enhance the Activity of Human Fibroblast Growth Factor 2 by Prolonging the Retention Time: A New Vision for Drug-Delivery Systems.

Cell-penetrating peptides (CPPs) are defined by their ability to deliver cargo into cells and have been studied and developed as a promising drug-delivery system (DDS). However, the issue of whether the CPPs that have already entered the cells can be re-released or reused has not been studied. The purpose of this research was to construct CPP-conjugated human fibroblast growth factor 2 (hFGF2) and investigate whether they can be re-released from the cell membrane for reuse. This study combined hFGF2 with Tat or Ara27, a newly developed CPP derived from the zinc knuckle (CCHC-type) family protein of Arabidopsis. Human dermal fibroblast (HDF) was treated with Tat-conjugated hFGF2 (tFGF2) and Ara27-conjugated hFGF2 (NR-FGF2) for both long and short durations, and the effects on cell growth were compared. Furthermore, tFGF2 and NR-FGF2 re-released from the cells were quantified and the effects were evaluated by culturing HDF in a conditioned medium. Interestingly, the proliferation of HDF increased only when NR-FGF2 was treated for 1 h in endocytosis-independent manner. After 1 h, NR-FGF2 was significantly re-released, reaching a maximum concentration at 5 h. Furthermore, increased proliferation of HDF cultured in the conditioned medium containing re-released NR-FGF2 was discovered. While previous studies have focused on the delivery of cargo and its associated applications, this study has revealed that combinations of superior CPPs and therapeutics can be expected to prolong both the retention time and the cell-penetrating capacity, even in the presence of external factors. Therefore, CPPs can be applied in the context of topical drugs and cosmetics as a new DDS approach.

Contribution of actin filaments and microtubules to cell elongation and alignment depends on the grating depth of microgratings.

BACKGROUND: It has been reported that both chemical and physical surface patterns influence cellular behaviors, such as cell alignment and elongation. However, it still remains unclear how actin filament and microtubules (MTs) differentially respond to these patterns. RESULTS: We examined the effects of chemical and physical patterns on cell elongation and alignment by observing actin filament and MTs of retinal pigment epithelium-1(RPE-1) cells, which were cultured on either fibronectin (FN)-line pattern (line width and spacing: 1 µm) or FN-coated 1 µm gratings with two different depths (0.35 or 1 µm). On the surface with either FN-line pattern or micrograting structure, the cell aspect ratios were at least two times higher than those on the surface with no pattern. Cell elongation on the gratings depended on the depth of the gratings. Cell elongation and alignment on both FN-line pattern and 1 µm gratings with 0.35 µm depth were perturbed either by inhibition of actin polymerization or MT depletion, while cell elongation and alignment on 1 µm gratings with 1 µm depth were perturbed only by MT depletion. CONCLUSIONS: Our results suggest that the contribution of actin filaments and MTs to the elongation and alignment of epithelial cells on microgratings depends on the groove depth of these gratings.