Interaction between p21-activated kinase 4 and ß-catenin as a novel pathway for PTH-dependent osteoblast activation.

Parathyroid hormone (PTH) serves dual roles in bone metabolism, exhibiting both anabolic and catabolic effects. The anabolic properties of PTH have been utilized in the treatment of osteoporosis with proven efficacy in preventing fractures. Despite these benefits, PTH can be administered therapeutically for up to 2 years, and its use in patients with underlying malignancies remains a subject of ongoing debate. These considerations underscore the need for a more comprehensive understanding of the underlying mechanisms. p21-activated kinase 4 (PAK4) is involved in bone resorption and cancer-associated osteolysis; however, its role in osteoblast function and PTH action remains unknown. Therefore, in this study, we aimed to clarify the role of PAK4 in osteoblast function and its effects on PTH-induced anabolic activity. PAK4 enhanced MC3T3-E1 osteoblast viability and proliferation and upregulated cyclin D1 expression. PAK4 also augmented osteoblast differentiation, as indicated by increased mineralization found by alkaline phosphatase and Alizarin Red staining. Treatment with PTH (1-34), an active PTH fragment, stimulated PAK4 expression and phosphorylation in a protein kinase A-dependent manner. In addition, bone morphogenetic protein-2 (which is known to promote bone formation) increased phosphorylated PAK4 (p-PAK4) and PAK4 levels. PAK4 regulated the expression of both phosphorylated and total ß-catenin, which are critical for osteoblast proliferation and differentiation. Moreover, p-PAK4 directly interacted with ß-catenin, and disruption of ß-catenin's binding to T-cell factor impaired PAK4- and PTH-induced osteoblast differentiation. Our findings elucidate the effect of PAK4 on enhancing bone formation in osteoblasts and its pivotal role in the anabolic activity of PTH mediated through its interaction with ß-catenin. These insights improve the understanding of the mechanisms underlying PTH activity and should inform the development of more effective and safer osteoporosis treatments.

Host cell proteins in monoclonal antibody processing: Control, detection, and removal.

Host cell proteins (HCPs) are process-related impurities in a therapeutic protein expressed using cell culture technology. This review presents biopharmaceutical industry trends in terms of both HCPs in the bioprocessing of monoclonal antibodies (mAbs) and the capabilities for HCP clearance by downstream unit operations. A comprehensive assessment of currently implemented and emerging technologies in the manufacturing processes with extensive references was performed. Meta-analyses of published downstream data were conducted to identify trends. Improved analytical methods and understanding of "high-risk" HCPs lead to more robust manufacturing processes and higher-quality therapeutics. The trend of higher cell density cultures leads to both higher mAb expression and higher HCP levels. However, HCP levels can be significantly reduced with improvements in operations, resulting in similar concentrations of approx. 10 ppm HCPs. There are no differences in the performance of HCP clearance between recent enhanced downstream operations and traditional batch processing. This review includes best practices for developing improved processes.

Strong intramolecular charge-transfer effect strengthening naphthoquinone-based chemosensor: Experimental and theoretical evaluation.

An aminophenol-linked naphthoquinone-based fluorometric and colorimetric chemosensor 2-chloro-3-((3-hydroxyphenyl) amino) naphthalene-1,4-dione (2CAN-Dione) was synthesized for selective detection of Sn2+ ion in aqueous solution. The amine and conversion of carbonyl into carboxyl groups play a vital role in the sensing mechanism when Sn2+ is added to 2CAN-Dione. Comprehensive characterization of the sensor was carried out using standard spectral and analytical approaches. Because of the intramolecular charge transfer (ICT) effect and the turn-on sensing mode, the strong fluorometric emission towards Sn2+ was observed at about 435 nm. The chemosensor exhibited good selectivity for Sn2+ in the presence of coexisting metal ions. An improved linear connection was established with a low limit of detection (0.167 µM). FT-IR, 1H NMR, 13C NMR, and quantum chemistry methods were performed to verify the binding coordination mechanism. The chemosensing probe 2CAN-Dione was successfully employed in bioimaging investigations, demonstrating that it is a reliable fluorescent marker for Sn2+ in human cancer cells.

p21-activated kinase 4 counteracts PKA-dependent lipolysis by phosphorylating FABP4 and HSL.

Adipose tissue lipolysis is mediated by cAMP-protein kinase A (PKA)-dependent intracellular signalling. Here, we show that PKA targets p21-activated kinase 4 (PAK4), leading to its protein degradation. Adipose tissue-specific overexpression of PAK4 in mice attenuates lipolysis and exacerbates diet-induced obesity. Conversely, adipose tissue-specific knockout of Pak4 or the administration of a PAK4 inhibitor in mice ameliorates diet-induced obesity and insulin resistance while enhancing lipolysis. Pak4 knockout also increases energy expenditure and adipose tissue browning activity. Mechanistically, PAK4 directly phosphorylates fatty acid-binding protein 4 (FABP4) at T126 and hormone-sensitive lipase (HSL) at S565, impairing their interaction and thereby inhibiting lipolysis. Levels of PAK4 and the phosphorylation of FABP4-T126 and HSL-S565 are enhanced in the visceral fat of individuals with obesity compared to their lean counterparts. In summary, we have uncovered an important role for FABP4 phosphorylation in regulating adipose tissue lipolysis, and PAK4 inhibition may offer a therapeutic strategy for the treatment of obesity.

Cannabidiol Inhibits IgE-Mediated Mast Cell Degranulation and Anaphylaxis in Mice.

SCOPE: Cannabidiol (CBD), the most abundant non-psychoactive constituent of the plant Cannabis sativa, is known to possess immune modulatory properties. This study investigates the effects of CBD on mast cell degranulation in human and mouse primary mast cells and passive cutaneous anaphylaxis in mice. METHODS AND RESULTS: Mouse bone marrow-derived mast cells and human cord-blood derived mast cells are generated. CBD suppressed antigen-stimulated mast cell degranulation in a concentration-dependent manner. Mechanistically, CBD inhibited both the phosphorylation of FcεRI downstream signaling molecules and calcium mobilization in mast cells, while exerting no effect on FcεRI expression and IgE binding to FcεRI. These suppressive effects are preserved in the mast cells that are depleted of type 1 (CB1) and type 2 (CB2) cannabinoid receptors, as well as in the presence of CB1 agonist, CB2 agonist, CB1 inverse agonist, and CB2 inverse agonist. CBD also inhibited the development of mast cells in a long-term culture. The intraperitoneal administration of CBD suppressed passive cutaneous anaphylaxis in mice as evidenced by a reduction in ear swelling and decrease in the number of degranulated mast cells. CONCLUSION: Based on these results, the administration of CBD is a new therapeutic intervention in mast cell-associated anaphylactic diseases.

Synergistic and Additive Effects of Menadione in Combination with Antibiotics on Multidrug-Resistant Staphylococcus aureus: Insights from Structure-Function Analysis of Naphthoquinones.

Antimicrobial resistance (AMR) interferes with the effective treatment of infections and increases the risk of microbial spread and infection-related illness and death. The synergistic activities of combinations of antimicrobial compounds offer satisfactory approaches to some extent. Structurally diverse naphthoquinones (NQs) including menadione (-CH3 group at C2) exhibit substantial antimicrobial activities against multidrug-resistant (MDR) pathogens. We explored the combinations of menadione with antibiotic ciprofloxacin or ampicillin against Staphylococcus aureus and its biofilms. We found an additive (0.590 %) were also observed. However, preformed biofilms were not affected. Dent formation was also evident in S. aureus treated with the test compounds. The structure-function relationship (SFR) of NQs was used to determine and predict their activity pattern against pathogens. Analysis of 10 structurally distinct NQs revealed that the compounds with -Cl, -Br, -CH3 , or -OH groups displayed the lowest MICs (32-256 µg/mL). Furthermore, 1,4-NQs possessing a halogen or -CH3 moiety showed elevated ROS activity, whereas molecules with an -OH group affected cell integrity. Improved activity of antimicrobial combinations and SFR approaches are significant in antimicrobial therapies.

p21-activated kinase 4 suppresses fatty acid ß-oxidation and ketogenesis by phosphorylating NCoR1.

PPARα corepressor NCoR1 is a key regulator of fatty acid ß-oxidation and ketogenesis. However, its regulatory mechanism is largely unknown. Here, we report that oncoprotein p21-activated kinase 4 (PAK4) is an NCoR1 kinase. Specifically, PAK4 phosphorylates NCoR1 at T1619/T2124, resulting in an increase in its nuclear localization and interaction with PPARα, thereby repressing the transcriptional activity of PPARα. We observe impaired ketogenesis and increases in PAK4 protein and NCoR1 phosphorylation levels in liver tissues of high fat diet-fed mice, NAFLD patients, and hepatocellular carcinoma patients. Forced overexpression of PAK4 in mice represses ketogenesis and thereby increases hepatic fat accumulation, whereas genetic ablation or pharmacological inhibition of PAK4 exhibites an opposite phenotype. Interestingly, PAK4 protein levels are significantly suppressed by fasting, largely through either cAMP/PKA- or Sirt1-mediated ubiquitination and proteasome degradation. In this way, our findings provide evidence for a PAK4-NCoR1/PPARα signaling pathway that regulates fatty acid ß-oxidation and ketogenesis.

Sphingosylphosphorylcholine inhibits plasma cell differentiation and ameliorates experimental autoimmune encephalomyelitis.

Introduction: Multiple sclerosis (MS) is a potentially disabling disease that damages the brain and spinal cord, inducing paralysis of the body. While MS has been known as a T-cell mediated disease, recent attention has been drawn to the involvement of B cells in its pathogenesis. Autoantibodies from B cells are closely related with the damage lesion of central nervous system and worse prognosis. Therefore, regulating the activity of antibody secreting cell could be related with the severity of the MS symptoms. Methods: Total mouse B cells were stimulated with LPS to induce their differentiation into plasma cells. The differentiation of plasma cells was subsequently analyzed using flow cytometry and quantitative PCR analysis. To establish an experimental autoimmune encephalomyelitis (EAE) mouse model, mice were immunized with MOG35-55/CFA emulsion. Results: In this study, we found that plasma cell differentiation was accompanied by upregulation of autotaxin, which converts sphingosylphosphorylcholine (SPC) to sphingosine 1-phosphate in response to LPS. We observed that SPC strongly blocked plasma cell differentiation from B cells and antibody production in vitro. SPC downregulated LPS-stimulated IRF4 and Blimp 1, which are required for the generation of plasma cells. SPC-induced inhibitory effects on plasma cell differentiation were specifically blocked by VPC23019 (S1PR1/3 antagonist) or TY52159 (S1PR3 antagonist), but not by W146 (S1PR1 antagonist) and JTE013 (S1PR2 antagonist), suggesting a crucial role of S1PR3 but not S1PR1/2 in the process. Administration of SPC against an EAE mouse model significantly attenuated the symptoms of disease, showing decreased demyelinated areas of the spinal cord and decreased numbers of cells infiltrated into the spinal cord. SPC markedly decreased plasma cell generation in the EAE model, and SPC-induced therapeutic effects against EAE were not observed in µMT mice. Conclusion: Collectively, we demonstrate that SPC strongly inhibits plasma cell differentiation, which is mediated by S1PR3. SPC also elicits therapeutic outcomes against EAE, an experimental model of MS, suggesting SPC as a new material to control MS.

SIRT1 downregulation provokes immune-inflammatory responses in hair follicle outer root sheath cells and may contribute to development of alopecia areata.

BACKGROUND: Silent information regulator 1 (SIRT1), a type III histone deacetylase, is involved in various cutaneous and systemic autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and psoriasis. However, little is known about the role of SIRT1 in the development of alopecia areata (AA). OBJECTIVES: This study investigated whether SIRT1 regulates the hair follicle immune system and is involved in AA pathogenesis. METHODS: SIRT1 expression in human scalp tissue was analyzed using immunohistochemical staining, qPCR, and western blotting. The regulatory effect of SIRT1 was evaluated after stimulation with the double-stranded RNA mimic polyinosinic:polycytidylic acid (poly I:C) in hair follicle outer root sheath (ORS) cells and C3H/HeJ mice. RESULTS: SIRT1 expression was significantly reduced in the AA scalp compared to the normal scalp. SIRT1 inhibition upregulated MHC class I polypeptide-related sequence A and UL16 binding protein 3 in hair follicle ORS cells. SIRT1 inhibition also promoted the production of Th1 cytokines (IFN-γ and TNF-α), IFN-inducible chemokines (CXCL9 and CXCL10), and T cell migration in ORS cells. Conversely, SIRT1 activation suppressed the autoreactive inflammatory responses. The counteractive effect of the immune response by SIRT1 was mediated through the deacetylation of NF-κB and phosphorylation of STAT3. CONCLUSION: SIRT1 downregulation induces immune-inflammatory responses in hair follicle ORS cells and may contribute to AA development.

Multiple Roles of Sirtuin 6 in Adipose Tissue Inflammation.

Adipose tissue (AT) inflammation is strongly associated with obesity-induced insulin resistance. When subjected to metabolic stress, adipocytes become inflamed and secrete a plethora of cytokines and chemokines, which recruit circulating immune cells to AT. Although sirtuin 6 (Sirt6) is known to control genomic stabilization, aging, and cellular metabolism, it is now understood to also play a pivotal role in the regulation of AT inflammation. Sirt6 protein levels are reduced in the AT of obese humans and animals and increased by weight loss. In this review, we summarize the potential mechanism of AT inflammation caused by impaired action of Sirt6 from the immune cells' point of view. We first describe the properties and functions of immune cells in obese AT, with an emphasis on discrete macrophage subpopulations which are central to AT inflammation. We then highlight data that links Sirt6 to functional phenotypes of AT inflammation. Importantly, we discuss in detail the effects of Sirt6 deficiency in adipocytes, macrophages, and eosinophils on insulin resistance or AT browning. In our closing perspectives, we discuss emerging issues in this field that require further investigation.

Lycium chinense Miller fruit extract lowers liver enzyme levels in subjects with mild hepatic dysfunction: a randomized, double-blind, placebo-controlled clinical trial.

PURPOSE: In our previous study, we showed that Lycium chinense Miller fruit extract (LFE) exerted hepatoprotective effects in mice. In the current study, we examined the effect of LFE on liver enzyme levels in subjects with mild hepatic dysfunction. METHODS: A total of 90 subjects, aged 19 to 70 years old, with abnormal alanine aminotransferase (ALT) levels, were randomly placed into either an LFE (n = 45) treatment group or a placebo group (n = 45). During the 12-week clinical trial, subjects in each group received either LFE or placebo capsules, and were instructed to take four tablets per day (1760 mg/day). The primary outcome of the study was the changes of ALT and γ-glutamyltransferase (GGT) levels in each subject. The safety of LFE supplementation was assessed and adverse events were recorded. RESULTS: LFE supplementation for 12 weeks resulted in a significant reduction of ALT (P = 0.0498) and GGT (P = 0.0368) levels in comparison to the placebo. No clinically significant changes were observed in any safety parameters. CONCLUSION: These results suggest that LFE can be applied to subjects with mild hepatic dysfunction with no possible side effects. TRIAL REGISTRATION: This study was registered at the Clinical Research Information Service (CRIS) as no. KCT0003985.

LDHA Desuccinylase Sirtuin 5 as A Novel Cancer Metastatic Stimulator in Aggressive Prostate Cancer.

Prostate cancer (PCa) is the most commonly diagnosed genital cancer in men worldwide. Around 80% of the patients who developed advanced PCa suffered from bone metastasis, with a sharp drop in the survival rate. Despite great efforts, the detailed mechanisms underlying castration-resistant PCa (CRPC) remain unclear. Sirtuin 5 (SIRT5), an NAD+-dependent desuccinylase, is hypothesized to be a key regulator of various cancers. However, compared to other SIRTs, the role of SIRT5 in cancer has not been extensively studied. Here, we revealed significantly decreased SIRT5 levels in aggressive PCa cells relative to the PCa stages. The correlation between the decrease in the SIRT5 level and the patient's reduced survival rate was also confirmed. Using quantitative global succinylome analysis, we characterized a significant increase in the succinylation at lysine 118 (K118su) of lactate dehydrogenase A (LDHA), which plays a role in increasing LDH activity. As a substrate of SIRT5, LDHA-K118su significantly increased the migration and invasion of PCa cells and LDH activity in PCa patients. This study reveals the reduction of SIRT5 protein expression and LDHA-K118su as a novel mechanism involved in PCa progression, which could serve as a new target to prevent CPRC progression for PCa treatment.

SIRT1 Promotes Host Protective Immunity against Toxoplasma gondii by Controlling the FoxO-Autophagy Axis via the AMPK and PI3K/AKT Signalling Pathways.

Sirtuin 1 (SIRT1) regulates cellular processes by deacetylating non-histone targets, including transcription factors and intracellular signalling mediators; thus, its abnormal activation is closely linked to the pathophysiology of several diseases. However, its function in Toxoplasma gondii infection is unclear. We found that SIRT1 contributes to autophagy activation via the AMP-activated protein kinase (AMPK) and PI3K/AKT signalling pathways, promoting anti-Toxoplasma responses. Myeloid-specific Sirt1-/- mice exhibited an increased cyst burden in brain tissue compared to wild-type mice following infection with the avirulent ME49 strain. Consistently, the intracellular survival of T. gondii was markedly increased in Sirt1-deficient bone-marrow-derived macrophages (BMDMs). In contrast, the activation of SIRT1 by resveratrol resulted in not only the induction of autophagy but also a significantly increased anti-Toxoplasma effect. Notably, SIRT1 regulates the FoxO-autophagy axis in several human diseases. Importantly, the T. gondii-induced phosphorylation, acetylation, and cytosolic translocation of FoxO1 was enhanced in Sirt1-deficient BMDMs and the pharmacological inhibition of PI3K/AKT signalling reduced the cytosolic translocation of FoxO1 in BMDMs infected with T. gondii. Further, the CaMKK2-dependent AMPK signalling pathway is responsible for the effect of SIRT1 on the FoxO3a-autophagy axis and for its anti-Toxoplasma activity. Collectively, our findings reveal a previously unappreciated role for SIRT1 in Toxoplasma infection.

Limonium tetragonum Promotes Running Endurance in Mice through Mitochondrial Biogenesis and Oxidative Fiber Formation.

The purpose of this study was to examine whether Limonium tetragonum, cultivated in a smart-farming system with LED lamps, could increase exercise capacity in mice. C57BL/6 male mice were orally administered vehicle or Limonium tetragonum water extract (LTE), either 30 or 100 mg/kg, and were subjected to moderate intensity treadmill exercise for 4 weeks. Running distance markedly increased in the LTE group (100 mg/kg) by 80 ± 4% compared to the vehicle group, which was accompanied by a higher proportion of oxidative fibers (6 ± 6% vs. 10 ± 4%). Mitochondrial DNA content and gene expressions related to mitochondrial biogenesis were significantly increased in LTE-supplemented gastrocnemius muscles. At the molecular level, the expression of PGC-1α, a master regulator of fast-to-slow fiber-type transition, was increased downstream of the PKA/CREB signaling pathway. LTE induction of the PKA/CREB signaling pathway was also observed in C2C12 cells, which was effectively suppressed by PKA inhibitors H89 and Rp-cAMP. Altogether, these findings indicate that LTE treatment enhanced endurance exercise capacity via an improvement in mitochondrial biosynthesis and the increases in the formation of oxidative slow-twitch fibers. Future study is warranted to validate the exercise-enhancing effect of LTE in the human.

Supplementation with Vitis vinifera Jingzaojing Leaf and Shoot Extract Improves Exercise Endurance in Mice.

Switching myofibers from the fast-glycolytic type to the slow-oxidative type is associated with an alleviation of the symptoms associated with various cardiometabolic diseases. This study investigates the effect of Vitis vinifera Jingzaojing leaf and shoot extract (JLSE), which is rich in phenolic compounds, on the regulation of skeletal muscle fiber-type switching, as well as the associated underlying mechanism. Male C57BL/6N mice were supplemented orally with vehicle or JLSE (300 mg/kg) and subjected to treadmill exercise training. After four weeks, mice in the JLSE-supplemented group showed significantly improved exercise endurance and mitochondrial oxidative capacity. JLSE supplementation increased the expression of sirtuin 6 and decreased Sox6 expression, thereby elevating the number of mitochondria and encouraging fast-to-slow myofiber switching. The results of our experiments suggest that JLSE supplementation reprograms myofiber composition to favor the slow oxidative type, ultimately enhancing exercise endurance.

Metabolic improvement and liver regeneration by inhibiting CXXC5 function for non-alcoholic steatohepatitis treatment.

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease that results from multiple metabolic disorders. Considering the complexity of the pathogenesis, the identification of a factor mediating the multiple pathogenic phenotypes of NASH will be important for treatment. In this study, we found that CXXC5, a negative feedback regulator of the Wnt/ß-catenin pathway, was overexpressed with suppression of Wnt/ß-catenin signaling and its target genes involved in hepatic metabolism in obese-NASH patients. Cxxc5-/- mice were found to be resistant to NASH pathogenesis with metabolic improvements. KY19334, a small molecule that activates the Wnt/ß-catenin pathway via interference of the CXXC5-Dvl interaction, reversed the overall pathogenic features of NASH as Cxxc5-/- mice. The improvement in NASH by KY19334 is attributed to its regenerative effects through restorative activation of the suppressed Wnt/ß-catenin signaling. Overall, the pronounced metabolic improvements with the stimulation of liver regeneration by interfering with the CXXC5-Dvl interaction provide a therapeutic approach for NASH.

Targeting p21-activated kinase 4 (PAK4) with pyrazolo[3,4-d]pyrimidine derivative SPA7012 attenuates hepatic ischaemia-reperfusion injury in mice.

p21-Activated kinase 4 (PAK4), one of the serine/threonine kinases activated by Rho-family GTPases, has been widely studied as an oncogenic protein that is overexpressed in many types of cancers. In our recent study, PAK4 upregulation was observed in mice exhibiting hepatic ischaemia-reperfusion (I/R) and in liver transplantation patients. Liver I/R injury was also attenuated in Pak4 KO mice. Herein, we report a novel series of pyrazolo[3,4-d]pyrimidine derivatives of type I ½ PAK4 inhibitors. The most potent compound SPA7012 was evaluated to determine the pharmacological potential of PAK4 inhibitor in I/R injury in mice. Mice with I/R injury showed typical patterns of liver damage, as demonstrated by increases in serum levels of aminotransferases and proinflammatory cytokines, hepatocellular necrosis and apoptosis, and inflammatory cell infiltration, relative to sham mice. Conversely, intraperitoneal administration of SPA7012 dramatically attenuated biochemical and histopathologic changes. Mechanistically, stabilisation of nuclear factor-erythroid 2-related factor 2 (Nrf2), a master regulator of anti-oxidative response, was observed following SPA7012 treatment. SPA7012 treatment in primary hepatocytes also attenuated hypoxia-reoxygenation-induced apoptotic cell death and inflammation. Together, these results provide experimental evidence supporting the use of PAK4 inhibitors for alleviation of I/R-induced liver damage.

Therapeutic Effect of Acer tegmentosum Maxim Twig Extract in Bile Duct Ligation-Induced Acute Cholestasis in Mice.

Cholestatic liver disease, or cholestasis, is a condition characterized by liver inflammation and fibrosis following a bile duct obstruction and an intrahepatic accumulation of bile acids. Inhibiting inflammation is a promising therapeutic strategy for cholestatic liver diseases. Acer tegmentosum Maxim extract (ATE) is best known for its anti-inflammatory and antioxidative properties. In this study, we investigated the effects of ATE on liver injury and fibrosis in mice with bile duct ligation (BDL)-induced cholestasis through analysis of gene expression, cytokines, and histological examination. Oral administration of ATE (20 or 50 mg/kg) for 14 days significantly attenuated hepatocellular necrosis compared to vehicle-treated BDL mice, which was accompanied by the reduced level of serum bile acids and bilirubin. We determined that ATE treatment reduced liver inflammation, oxidative stress, and fibrosis. These beneficial effects of ATE were concurrent with the decreased expression of genes involved in the NF-κB pathway, suggesting that the anti-inflammatory effect of ATE could be a possible mechanism against cholestasis-associated liver injury. Our findings substantiate ATE's role as an alternative therapeutic agent for cholestasis-induced liver injury and fibrosis.