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Introduction: Ferroptosis plays a crucial role in the progression of postmenopausal osteoporosis. Anemarrhena asphodeloides Bunge/ Phellodendron chinense C.K. Schneid (AA/PC) is the core herb pair in traditional Chinese medicines formulae for postmenopausal osteoporosis treatment. However, the synergistic effects, and mechanisms, of AA/PC on alleviating ferroptosis and postmenopausal osteoporosis remain unclear. Methods: The goal herein was to analyze the effective ingredients and molecular mechanisms of AA/PC in the treatment of osteoporosis through serum pharmacochemistry, network pharmacology, metabolomics analysis, and pharmacodynamics evaluation. A bilateral ovariectomized (OVX) mouse model was established. Results and Discussion: Micron-scale computed tomography analysis showed that AA/PC increased bone mineral density in OVX mice. The effects of AA/PC were better than AA or PC alone on inhibiting the bone resorption marker nuclear factor of activated T-cells 1. Furthermore, five absorbable compounds were detected in serum: mangiferin, magnoflorine, berberine, timosaponin BIII, and timosaponin AIII. Network pharmacology showed these compounds had close relationship with seven ferroptosis targets. Importantly, compared with AA or PC alone, the AA/PC herb pair exerted better effects on regulating crucial ferroptosis pathways, including the system xc-/glutathione/glutathione peroxidase 4, transferrin receptor/ferritin, and acyl-CoA synthetase long chain family member 4/polyunsaturated fatty acids signaling pathways. These results indicate that AA/PC exerts synergistic effects on regulating glutathione synthesis, iron homeostasis, and lipid metabolism in ferroptosis. This work lays the foundation for further development and use of AA/PC herb pair for preventing and treating postmenopausal osteoporosis. [ABSTRACT FROM AUTHOR]

High salt conditions and subsequent hyperosmolarity are injurious cellular stresses that can activate immune signaling. Nuclear factor of activated T-cells 5 (NFAT5) is an essential transcription factor that induces osmoprotective genes such as aldose reductase (AR) and betaine-GABA transporter 1 (BGT1). High salt stress-mediated NFAT5 activation is also reported to accelerate the inflammatory response and autoimmune diseases. However, the systemic regulation of NFAT5 remains unclear. Here, we performed a genome-wide siRNA screen to comprehensively identify the regulators of NFAT5. We monitored NFAT5 nuclear translocation and identified one of the Notch signaling effectors, Hairy and enhancer of split-1 (HES1), as a positive regulator of NFAT5. HES1 was induced by high salinity via ERK signaling and facilitated NFAT5 recruitment to its target promoter region, resulting in the proper induction of osmoprotective genes and cytoprotection under high salt stress. These findings suggest that, though HES1 is well known as a transcriptional repressor, it positively regulates NFAT5-dependent transcription in the context of a high salinity/hyperosmotic response. HES1, a well-known transcriptional repressor, promotes NFAT5-dependent transcription in the context of a high salinity/hyperosmotic environment and contributes to high salt stress response. [ABSTRACT FROM AUTHOR]

Aims Chronic hypoxia causes detrimental structural alterations in the lung, which may cause pulmonary hypertension and are partially mediated by the endothelium. While its relevance for the development of hypoxia-associated lung diseases is well known, determinants controlling the initial adaptation of the lung endothelium to hypoxia remain largely unexplored. Methods and results We revealed that hypoxia activates the transcription factor nuclear factor of activated T-cells 5 (NFAT5) and studied its regulatory function in murine lung endothelial cells (MLECs). EC-specific knockout of Nfat5 (Nfat5(EC)−/−) in mice exposed to normobaric hypoxia (10% O2) for 21 days promoted vascular fibrosis and aggravated the increase in pulmonary right ventricular systolic pressure as well as right ventricular dysfunction as compared with control mice. Microarray- and single-cell RNA-sequencing-based analyses revealed an impaired growth factor-, energy-, and protein–metabolism-associated gene expression in Nfat5 -deficient MLEC after exposure to hypoxia for 7 days. Specifically, loss of NFAT5 boosted the expression and release of platelet-derived growth factor B (Pdgfb)—a hypoxia-inducible factor 1 alpha (HIF1α)-regulated driver of vascular smooth muscle cell (VSMC) growth—in capillary MLEC of hypoxia-exposed Nfat5(EC)−/− mice, which was accompanied by intensified VSMC coverage of distal pulmonary arteries. Conclusion Collectively, our study shows that early and transient subpopulation-specific responses of MLEC to hypoxia may determine the degree of organ dysfunction in later stages. In this context, NFAT5 acts as a protective transcription factor required to rapidly adjust the endothelial transcriptome to cope with hypoxia. Specifically, NFAT5 restricts HIF1α-mediated Pdgfb expression and consequently limits muscularization and resistance of the pulmonary vasculature. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To compare the efficacy of bortezomib and pamilophosphate in the rat model of disuse osteoporosis (DOP). METHODS: Forty 16-week-old male SD rats were randomly divided into the control group, sham surgery group, model group, pamilphosphate treatment group and bortezomib treatment group, with 8 rats in each group. DOP rat model was established. The microstructural parameters of hind limb tibia were determined by micro-CT. Serological indexes receptor activator of nuclear factor-κB ligand (RANKL) / osteoprotegerin (OPG) concentration ratio, serum Sclerostin and alkaline phosphatase (ALP) levels were determined by enzyme linked immunosorbent assay. The mRNA expression levels of bone resorption-related factors type I procollagen amino-terminal prepeptide (PINP), C-terminal crosslinking telopeptide of type I collagen (CTX)-1, matrix metalloproteinase (MMP)-3 were determined by quantitative polymerase chain reaction. Signaling axis of Calcineurin/ nuclear factor of activated T-cells isoform (NFAT) and signaling pathway of glycogen synthase kinase-3β (GSK3β) / protein kinase B (Akt), a key transcription factor for osteogenic differentiation, were determined by Western blotting. RESULTS: Compared with sham surgery group, bone volume fraction (BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th), connectivity density (Conn. D), structural model index (SMI), bone mineral density ( BMD) and trabecular separation (Tb. Sp) of the posterior limb tibia in model group rats decreased, trabecular separation ( Tb. Sp) increased; serum RANKL/OPG concentration ratio and serum sclerostin level increased, serum ALP level decreased; the relative expression levels of PINP and CTX-1 mRNA in bone tissues decreased, while the relative expression levels of MMP-3 and MMP-9 mRNA increased, the relative expression levels of Calcineurin decreased; the relative expression levels of p-Akt decreased, and the relative expression levels of p-GSK3 increased, with statistically significant differences (P < 0. 05). Compared with the model group, the pamilphosphate treatment group and bortezomib treatment group partially reversed the above indicators ( including BV/ TV, Tb. N, Tb. Th, Tb. Sp, Conn. D, SMI and BMD in posterior limb tibia, serum RANKL/OPG concentration ratio, levels of Sclerostin and ALP, the relative expression levels of MMP-3 and MMP-9 mRNA in bone tissues, the relative expression levels of Calcineurin in bone tissues, and the relative expression levels of p-Akt and p-GSK3β in bone tissues), with statistically significant differences (P <0. 05). The relative expression levels of PINP and CTX-1 mRNA and the relative expression levels of NFAT in bone tissues of rats in bortezomib treatment group increased, with statistically significant difference (P <0. 05). Compared with the pamilophosphate treatment group, bortezomib treatment group had a more significant effect on reversing the above indicators, the differences were statistically significant (P<0. 05). CONCLUSIONS: The efficacy of bortezomib for DOP rat model is better than that of pamilophosphate. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play important roles in the regulation of cellular function and fate via post-transcriptional regulation of gene expression. Although several miRNAs are associated with physiological processes and kidney diseases, not much is known about changes in miRNAs in aging kidneys. We previously demonstrated that sodium hydrogen exchanger 1 (NHERF1) expression regulates cellular responses to cisplatin, age-dependent salt-sensitive hypertension, and sodium-phosphate cotransporter trafficking. However, the mechanisms driving these regulatory effects of NHERF1 on cellular processes are unknown. Here, we hypothesize that dysregulation of miRNA-mediated gene regulatory networks that induce fibrosis and cytokines may depend on NHERF1 expression. To address this hypothesis, we compared miRNA expression in kidneys from both male and female old (12–18-month-old) and young (4–7-month-old) wild-type (WT) and NHERF1 knockout (NHERF1−/−) mice. Our results identified that miRNAs significantly decreased in NHERF1−/− mice included miR-669m, miR-590-3p, miR-153, miR-673-3p, and miR-127. Only miR-702 significantly decreased in aged WT mice, while miR-678 decreased in both WT and NHERF1−/− old versus young mice. miR-153 was shown to downregulate transcription factors NFATc2 and NFATc3 which regulate the transcription of several cytokines. Immunohistochemistry and western blotting revealed a significant increase in nuclear NFATc2 and NFATc3 in old NHERF1−/− mice compared to old WT mice. Our data further show that expression of the cytokines IL-1β, IL-6, IL-17A, MCP1, and TNF-α significantly increased in the old NHERF1−/− mice compared to the WT mice. We conclude that loss of NHERF1 expression induces cytokine expression in the kidney through interactive regulation between miR-153 and NFATc2/NFATc3 expression. [ABSTRACT FROM AUTHOR]

Euryhaline fish experience variable osmotic environments requiring physiological adjustments to tolerate elevated salinity. Mozambique tilapia (Oreochromis mossambicus) possess one of the highest salinity tolerance limits of any fish. In tilapia and other euryhaline fish species, the myo-inositol biosynthesis (MIB) pathway enzymes, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1), are among the most upregulated mRNAs and proteins indicating the high importance of this pathway for hyperosmotic (HO) stress tolerance. These abundance changes must be precluded by HO perception and signaling mechanism activation to regulate the expression of MIPS and IMPA1.1 genes. In previous work using a O. mossambicus cell line (OmB), a reoccurring osmosensitive enhancer element (OSRE1) in both MIPS and IMPA1.1 was shown to transcriptionally upregulate these enzymes in response to HO stress. The OSRE1 core consensus (5′-GGAAA-3′) matches the core binding sequence of the predominant mammalian HO response transcription factor, nuclear factor of activated T-cells (NFAT5). HO-challenged OmB cells showed an increase in NFAT5 mRNA suggesting NFAT5 may contribute to MIB pathway regulation in euryhaline fish. Ectopic expression of wild-type NFAT5 induced an IMPA1.1 promoter-driven reporter by 5.1-fold (P < 0.01). Moreover, expression of dominant negative NFAT5 in HO media resulted in a 47% suppression of the reporter signal (P < 0.005). Furthermore, reductions of IMPA1.1 (37–49%) and MIPS (6–37%) mRNA abundance were observed in HO-challenged NFAT5 knockout cells relative to control cells. Collectively, these multiple lines of experimental evidence establish NFAT5 as a tilapia transcription factor contributing to HO-induced activation of the MIB pathway. NEW & NOTEWORTHY: In our study, we use a multi-pronged synthetic biology approach to demonstrate that the fish homolog of the predominant mammalian osmotic stress transcription factor nuclear factor of activated T-cells (NFAT5) also contributes to the activation of hyperosmolality inducible genes in cells of extremely euryhaline fish. However, in addition to NFAT5 the presence of other strong osmotically inducible signaling mechanisms is required for full activation of osmoregulated tilapia genes. [ABSTRACT FROM AUTHOR]

The CACNA1C gene encodes the alpha-1c subunit of the Ca v 1.2 calcium channel, a regulator of neuronal calcium influx involved in neurotransmitter release and synaptic plasticity. Genetic data show a role for CACNA1C in depressive symptoms underlying different psychiatric diagnoses. However, the mechanisms involved still require further exploration. This study aimed to investigate sex and region-specific changes in the Cacna1c gene and behavioral outcomes in mice exposed to chronic stress. Moreover, we evaluated the Nuclear factor of activated T-cells 5 (Nfat5) and the Brain-derived neurotrophic factor (Bdnf) as potential upstream and downstream Cacna1c targets and their correlation in stressed mice and humans with depression. Male and female Swiss mice were exposed to chronic unpredictable stress (CUS) for 21 days. Animal-integrated emotionality was assessed using the sucrose splash test, the tail suspension, the open-field test, and the elevated-plus-maze. Gene expression analysis was performed in the amygdala, prefrontal cortex, and hippocampus. Human data for in silico analysis was obtained from the Gene Expression Omnibus. CUS-induced impairment in integrated emotional regulation was observed in males. Gene expression analysis showed decreased levels of Cacna1c and Nfat5 and increased levels of Bdnf transcripts in the amygdala of stressed male mice. In contrast, there were no major changes in behavioral responses or gene expression in female mice after stress. The expression of the three genes was significantly correlated in the amygdala of mice and humans. The strong and positive correlation between Canac1c and Nfat5 suggests a potential role for this transcription factor in Canac1c expression. These changes could impact amygdala reactivity and emotional responses, making them a potential target for psychiatric intervention. [ABSTRACT FROM AUTHOR]

Osteoclast differentiation is crucial for orchestrating both tooth movement and the maintenance of bone density. Therefore, the current study sought to explore the impact of low-level laser therapy (LLLT) on osteoclast differentiation, functional gene expression, molecular signaling pathways, and orthodontic tooth movement in clinical settings. The RAW 264.7 cell line served as the precursor for osteoclasts, and these cells underwent irradiation using a 808-nm LLLT. Osteoclast differentiation was assessed through tartrate-resistant acid phosphatase (TRAP) staining. Functional gene expression levels were evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) while signaling molecules were examined through Western blot analysis. In the clinical study, 12 participants were enrolled. Their tooth movement was monitored using a TRIOS desktop scanner. Bone density measurements were conducted using Mimics software, which processed cone-beam computed tomography (CBCT) images exported in Digital Imaging and Communications in Medicine (DICOM) format. We found that LLLT effectively promoted receptor activator of nuclear factor-κB ligand (RANKL)-dependent osteoclast differentiation and the expression of osteoclast functional genes, including matrix metallopeptidase 9 (MMP9), nuclear factor of activated T-cells cytoplasmic 1(NFATc1), tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK) in RAW264.7 cells. Clinically, the cumulative tooth movement over 90 days was significantly higher in the laser group than in the control group. Our research demonstrates that LLLT not only significantly promotes osteoclast differentiation but is also a valuable adjunct in orthodontic therapy. [ABSTRACT FROM AUTHOR]

The editorial in Frontiers in Immunology explores the complex interplay between cell death, oxidative stress, and immune regulation, focusing on their roles in immune-mediated diseases. Various types of cell death, such as apoptosis and necroptosis, are discussed, along with therapeutic strategies targeting these pathways. Research articles highlight the potential of repurposed drugs and stem cell-derived extracellular vesicles in treating diseases related to oxidative stress. The study emphasizes the importance of understanding these mechanisms for the diagnosis and treatment of immune-related conditions. [Extracted from the article]

Exogenous polyamines, including putrescine (PUT), spermidine (SPD), and spermine (SPM), and the irreversible inhibitor of the rate-limiting enzyme ornithine decarboxylase (ODC) of polyamine biosynthesis, α-difluoromethylornithine (DFMO), are implicated as stimulants for bone formation. We demonstrate in this study the osteogenic potential of exogenous polyamines and DFMO in human osteoblasts (hOBs), murine monocyte cell line RAW 264.7, and an ovariectomized rat model. The effect of polyamines and DFMO on hOBs and RAW 264.7 cells was studied by analyzing gene expression, alkaline phosphatase (ALP) activity, tartrate-resistant acid phosphatase (TRAP) activity, and matrix mineralization. Ovariectomized rats were treated with polyamines and DFMO and analyzed by micro computed tomography (micro CT). The mRNA level of the early onset genes of osteogenic differentiation, Runt-related transcription factor 2 (Runx2) and ALP, was significantly elevated in hOBs under osteogenic conditions, while both ALP activity and matrix mineralization were enhanced by exogenous polyamines and DFMO. Under osteoclastogenic conditions, the gene expression of both receptor activator of nuclear factor-κB (RANK) and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) was reduced, and TRAP activity was suppressed by exogenous polyamines and DFMO in RAW 264.7 cells. In an osteoporotic animal model of ovariectomized rats, SPM and DFMO were found to improve bone volume in rat femurs, while trabecular thickness was increased in all treatment groups. Results from this study provide in vitro and in vivo evidence indicating that polyamines and DFMO act as stimulants for bone formation, and their osteogenic effect may be associated with the suppression of osteoclastogenesis. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons. LncRNA small nucleolar RNA host gene 14 (SNHG14) was found to promote neuron injury in PD. Here, we investigated the mechanisms of SNHG14 in PD process. In vivo or in vitro PD model was established by using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice or 1-methyl-4-phenylpyridinium (MPP +)-stimulated SK-N-SH cells. The expression of genes and proteins was measured by qRT-PCR and Western blot. In vitro assays were conducted using ELISA, CCK-8, colony formation, EdU, flow cytometry, and Western blot assays, respectively. The oxidative stress was evaluated by determining the production of superoxide dismutase (SOD) and malondialdehyde (MDA). The direct interactions between miR-375-3p and NFAT5 (Nuclear factor of activated T-cells 5) or SNHG14 was verified using dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. SNHG14 and NFAT5 were elevated, while miR-375-3p was decreased in MPTP-mediated PD mouse model and MPP + -induced SK-N-SH cells. Knockdown of SNHG14 or NFAT5, or overexpression of miR-375-3p reversed MPP + -induced neuronal apoptosis, inflammation, and oxidative stress. Mechanistically, SNHG14 directly bound to miR-375, which targeted NFAT5. Inhibition of miR-375-3p abolished the inhibitory activity of SNHG14 knockdown on MPP + -evoked neuronal damage. Besides that, NFAT5 up-regulation counteracted the effects of miR-375-3p on MPP + -mediated neuronal damage. SNHG14 contributed to MPP + -induced neuronal injury by miR-375/NFAT5 axis, suggesting a new insight into the pathogenesis of PD. MPP + elevated SNHG14, which then increased NFAT5 by sequestering miR-375, thereby contributing to MPP + -induced inflammation, apoptosis, oxidative stress, and arrest of proliferation in neurons. [ABSTRACT FROM AUTHOR]

Astrocyte swelling is implicated in various neurological disorders. However, whether astrocyte swelling contributes to neuropathic pain remains elusive. This study elucidates the pivotal role of the nuclear factor of activated T‐cells 5 (NFAT5) emerges as a master regulator of astrocyte swelling in the spinal dorsal horn (SDH) during neuropathic pain. Despite the ubiquitous expression of NFAT5 protein in SDH cell types, it selectively induces swelling specifically in astrocytes, not in microglia. Mechanistically, NFAT5 directly controls the expression of the water channel aquaporin‐4 (AQP4), a key regulator exclusive to astrocytes. Additionally, aurora kinase B (AURKB) orchestrates NFAT5 phosphorylation, enhancing its protein stability and nuclear translocation, thereby regulating AQP4 expression. The findings establish NFAT5 as a crucial regulator for neuropathic pain through the modulation of astrocyte swelling. The AURKB‐NFAT5‐AQP4 pathway in astrocytes emerges as a potential therapeutic target to combat neuropathic pain. [ABSTRACT FROM AUTHOR]

Nuclear factor of activated T-cells 5 (NFAT5), an osmo-sensitive transcription factor, can be activated by isotonic stimuli, such as infection. It remains unclear, however, whether NFAT5 is required for damage-associated molecular pattern--triggered (DAMP-triggered) inflammation and immunity. Here, we found that several DAMPs increased NFAT5 expression in macrophages. In particular, serum amyloid A (SAA), primarily generated by the liver, substantially upregulated NFAT5 expression and activity through TLR2/4-JNK signalling pathway. Moreover, the SAA-TLR2/4-NFAT5 axis promoted migration and chemotaxis of macrophages in an IL-6--and chemokine ligand 2--dependent (CCL2-dependent) manner in vitro. Intraarticular injection of SAA markedly accelerated macrophage infiltration and arthritis progression in mice. By contrast, genetic ablation of NFAT5 or TLR2/4 rescued the pathology induced by SAA, confirming the SAA-TLR2/4-NFAT5 axis in vivo. Myeloid-specific depletion of NFAT5 also attenuated SAA-accelerated arthritis. Of note, inflammatory arthritis in mice strikingly induced SAA overexpression in the liver. Conversely, forced overexpression of the SAA gene in the liver accelerated joint damage, indicating that the liver contributes to bolstering chronic inflammation at remote sites by secreting SAA. Collectively, this study underscores the importance of the SAA-TLR2/4-NFAT5 axis in innate immunity, suggesting that acute phase reactant SAA mediates mutual interactions between liver and joints and ultimately aggravates chronic arthritis by enhancing macrophage activation. [ABSTRACT FROM AUTHOR]

Interferon regulatory factor 8 (IRF8), a transcription factor expressed in immune cells, functions as a negative regulator of osteoclasts and helps maintain dental and skeletal homeostasis. Previously, we reported that a novel mutation in the IRF8 gene increases susceptibility to multiple idiopathic cervical root resorption (MICRR), a form of tooth root resorption mediated by increased osteoclast activity. The IRF8 G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. To investigate the molecular basis of MICRR and IRF8 function in osteoclastogenesis, we generated Irf8 knock-in (KI) mice using CRISPR/Cas9 technique modeling the human IRF8 G388S mutation. The heterozygous (Het) and homozygous (Homo) Irf8 KI mice showed no gross morphological defects, and the development of hematopoietic cells was unaffected and similar to wild-type (WT) mice. The Irf8 KI Het and Homo mice showed no difference in macrophage gene signatures important for antimicrobial defenses and inflammatory cytokine production. Consistent with the phenotype observed in MICRR patients, Irf8 KI Het and Homo mice demonstrated significantly increased osteoclast formation and resorption activity in vivo and in vitro when compared to WT mice. The oral ligature–inserted Het and Homo mice displayed significantly increased root resorption and osteoclast-mediated alveolar bone loss compared to WT mice. The increased osteoclastogenesis noted in KI mice is due to the inability of IRF8 G388S mutation to inhibit NFATc1-dependent transcriptional activation and downstream osteoclast specific transcripts, as well as its impact on autophagy-related pathways of osteoclast differentiation. This translational study delineates the IRF8 domain important for osteoclast function and provides novel insights into the IRF8 mutation associated with MICRR. IRF8 G388S mutation mainly affects osteoclastogenesis while sparing immune cell development and function. These insights extend beyond oral health and significantly advance our understanding of skeletal disorders mediated by increased osteoclast activity and IRF8's role in osteoclastogenesis. [ABSTRACT FROM AUTHOR]

Aims Physiological cardiac hypertrophy occurs in response to exercise and can protect against pathological stress. In contrast, pathological hypertrophy occurs in disease and often precedes heart failure. The cardiac pathways activated in physiological and pathological hypertrophy are largely distinct. Our prior work demonstrated that miR-222 increases in exercised hearts and is required for exercise-induced cardiac hypertrophy and cardiomyogenesis. Here, we sought to define the role of miR-222 in pathological hypertrophy. Methods and results We found that miR-222 also increased in pathological hypertrophy induced by pressure overload. To assess its functional significance in this setting, we generated a miR-222 gain-of-function model through cardiac-specific constitutive transgenic miR-222 expression (TgC-miR-222) and used locked nucleic acid anti-miR specific for miR-222 to inhibit its effects. Both gain- and loss-of-function models manifested normal cardiac structure and function at baseline. However, after transverse aortic constriction (TAC), miR-222 inhibition accelerated the development of pathological hypertrophy, cardiac dysfunction, and heart failure. Conversely, miR-222-overexpressing mice had less pathological hypertrophy after TAC, as well as better cardiac function and survival. We identified p53-up-regulated modulator of apoptosis, a pro-apoptotic Bcl-2 family member, and the transcription factors, Hmbox1 and nuclear factor of activated T-cells 3, as direct miR-222 targets contributing to its roles in this context. Conclusion While miR-222 is necessary for physiological cardiac growth, it inhibits cardiac growth in response to pressure overload and reduces adverse remodelling and cardiac dysfunction. These findings support the model that physiological and pathological hypertrophy are fundamentally different. Further, they suggest that miR-222 may hold promise as a therapeutic target in pathological cardiac hypertrophy and heart failure. [ABSTRACT FROM AUTHOR]

Chronic lymphocytic leukemia (CLL) mainly affects the health of older adults and is difficult to cure. Upstream stimulatory factor 2 (USF2) has been implicated in several diseases and conditions including cancers. However, the effect of USF2 on CLL has not been elucidated. To investigate the effect of USP2 on proliferation and autophagy of CLL, and to explore the underlying mechanism. The mRNA of USF2 and STIP1 homology and U-Box containing protein 1 (STUB1) was analyzed using qRT-PCR. Western blots were used to evaluate the expression level of USF2, LC3II, Beclin-1, P62, STUB1, and NFAT5. The cell proliferation was evaluated using CCK-8 and EdU assays. The cell apoptosis was evaluated using flow cytometry. Indirect fluorescent assay (IFA) was performed to analyze LC3 signal. Nuclear factor of activated T-cells 5 (NFAT5) ubiquitination was detected using immunoprecipitation (IP) assay. The CLL progression was evaluated in xenotransplantation model of nude mice. USF2 was highly expressed in CLL tissues and cell lines. USF2 knockdown suppressed the cell viability and EdU incorporation, while promoting cell apoptosis. Meanwhile, USF2 knockdown reduced the level of LC3II and Beclin-1, but increased P62, illustrating USF2 knockdown inhibiting autophagy. USF2 induced NFAT5 ubiquitination and promoted NFAT5 protein level via repressing STUB1. The downregulation of USF2 weakened CLL progression in xenotransplantation model of nude mice. CLL survival and autophagy was dependent on highly expressed USF2 which promoted the expression and ubiquitination of NFAT5 through inhibiting the transcription of STUB1, which makes USF2 a promising therapeutic candidate for CLL treatment. [ABSTRACT FROM AUTHOR]

In primary Sjögren's syndrome (pSS) patients, salivary gland (SG) epithelial cells (SGECs) could be exposed to chronic hyperosmotic stress (HOS), consecutive to their destruction and deregulation, that exacerbates an inflammatory response. The aims of this study were to assess the mechanism accounting for C-C motif chemokine ligand 2 (CCL2) expression in an immortalized human salivary gland epithelial acinar cell line (NS-SV-AC) subjected to HOS, as well as the involvement of CCL2 in pSS. CCL2 mRNA and protein levels were determined via RT-qPCR and ELISA. Reporter plasmids and a promoter pull-down assay were used to identify transcription factors associated with CCL2 mRNA increase. Our data showed that HOS-induced CCL2 mRNA increase was independent of the nuclear factor of activated T-cells 5 (NFAT5) and nuclear factor-kappa B (NFkB) but involved Kruppel-like factor 5 (KLF5). CCL2 protein levels, quantified by enzyme-linked immunosorbent assay (ELISA) in sera samples from pSS patients, correlated with the European Alliance of Associations for Rheumatology's Sjogren's syndrome disease activity index (ESSDAI) score for systemic activity. In addition, CCL2 protein levels were higher in patients with biological activity, cutaneous manifestations, and ESSDAI score superior or equal to five. Our data suggest that chronic HOS could exacerbate pSS disease by contributing to the inflammatory process induced by the expression and secretion of CCL2. [ABSTRACT FROM AUTHOR]

Background & Objectives: Breast cancer is the most lethal malignancy in women. miRNAs function as epigenetic regulators and contribute to the pathogenesis of breast cancer. Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and 3 (NFATc3), are targeted by microRNA-93 (miR-93). This study aims to evaluate the expression of these genes in peripheral blood mononuclear cells (PBMCs) of healthy women and women with breast cancer. Materials & Methods: In this cross-sectional study, blood samples were collected from 20 healthy women and 20 women with early-stage breast cancer. After isolating peripheral blood mononuclear cells (PBMCs), RNA extraction and cDNA synthesis were performed. The expression of the desired genes was then examined by Real-Time Polymerase Chain Reaction (RT-PCR). Statistical analysis was conducted. A p-value less than 0.05 was considered statistically significant, and Student's t-test was used to evaluate the relative changes in gene expression. Results: The results demonstrated that the expression of NFATc1 and NFATc3 genes in peripheral blood mononuclear cells (PBMCs) of breast cancer patients was significantly reduced compared to their expression in healthy individuals. Conversely, the expression of the miR-93-3p gene was significantly lower in healthy women than in breast cancer patients (p < 0.05). Conclusions: This study investigated the expression of miR-93-3p and its downstream targets, the NFATc1 and NFATc3 genes, for the first time in peripheral blood mononuclear cells. The expression levels were shown to be significantly different in patients with breast cancer compared to healthy women. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive functions. While the exact causes of this debilitating disorder remain elusive, numerous investigations have characterized its two core pathologies: the presence of β-amyloid plaques and tau tangles. Additionally, multiple studies of postmortem brain tissue, as well as results from AD preclinical models, have consistently demonstrated the presence of a sustained inflammatory response. As the persistent immune response is associated with neurodegeneration, it became clear that it may also exacerbate other AD pathologies, providing a link between the initial deposition of β-amyloid plaques and the later development of neurofibrillary tangles. Initially discovered in T cells, the nuclear factor of activated T-cells (NFAT) is one of the main transcription factors driving the expression of inflammatory genes and thus regulating immune responses. NFAT-dependent production of inflammatory mediators is controlled by Ca2+-dependent protein phosphatase calcineurin (CaN), which dephosphorylates NFAT and promotes its transcriptional activity. A substantial body of evidence has demonstrated that aberrant CaN/NFAT signaling is linked to several pathologies observed in AD, including neuronal apoptosis, synaptic deficits, and glia activation. In view of this, the role of NFAT isoforms in AD has been linked to disease progression at different stages, some of which are paralleled to diminished cognitive status. The use of classical inhibitors of CaN/NFAT signaling, such as tacrolimus or cyclosporine, or adeno-associated viruses to specifically inhibit astrocytic NFAT activation, has alleviated some symptoms of AD by diminishing β-amyloid neurotoxicity and neuroinflammation. In this article, we discuss the recent findings related to the contribution of CaN/NFAT signaling to the progression of AD and highlight the possible benefits of targeting this pathway in AD treatment. [ABSTRACT FROM AUTHOR]

T-cell receptor stimulation triggers cytosolic Ca2+ signaling by inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum (ER) and Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels gated by ER-located stromal-interacting molecules (STIM1/2). Physiologically, cytosolic Ca2+ signaling manifests as regenerative Ca2+ oscillations, which are critical for nuclear factor of activated T-cells-mediated transcription. In most cells, Ca2+ oscillations are thought to originate from IP3 receptor-mediated Ca2+ release, with CRAC channels indirectly sustaining them through ER refilling. Here, experimental and computational evidence support a multipleoscillator mechanism in Jurkat T-cells whereby both IP3 receptor and CRAC channel activities oscillate and directly fuel antigen-evoked Ca2+ oscillations, with the CRAC channel being the major contributor. KO of either STIM1 or STIM2 significantly reduces CRAC channel activity. As such, STIM1 and STIM2 synergize for optimal Ca2+ oscillations and activation of nuclear factor of activated T-cells 1 and are essential for ER refilling. The loss of both STIM proteins abrogates CRAC channel activity, drastically reduces ER Ca2+ content, severely hampers cell proliferation and enhances cell death. These results clarify the mechanism and the contribution of STIM proteins to Ca2+ oscillations in T-cells. [ABSTRACT FROM AUTHOR]

Streptococcus pyogenes is an important human pathogen, commonly spread by airborne droplets but also by ingestion of contaminated food. Apart from causing infection, this pathogen produces 13 distinct types of streptococcal pyrogenic exotoxins (SPE). The current method for detection cannot distinguish between the biologically active form of SPE that has been reported to cause foodborne outbreaks and the inactivated toxin that poses no health risk. To measure the biological activity of SPE type C (SPE-C), one such toxin that was linked to foodborne outbreaks associated with milk and milk products, we developed a cell-based assay that can discern between biologically active and inactive SPE-C. To the best of our knowledge, this is the first showing that SPE-C activates T-cells expressing Vβ8. With this finding, we used a T-cell line natively expressing Vβ8 that was genetically engineered to also express the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element in combination with a B-cell line to present the recombinant SPE-C (rSPE-C) toxin via major histocompatibility complex (MHC) class II to the Vβ8 T-cell receptor (TCR) in an assay to detect and to discern between biologically active and inactive rSPE-C. By using this system, we demonstrated that SPE-C induced significant IL-2 secretion after 72 h and visible light emission after only 5 h, doubling by 24 h. We utilize this finding to assess the specificity of the assay and the effect of pasteurization on SPE-C activity. We observed no cross-reactivity with SPE-B and significant loss of SPE-C biological activity in spiked phosphate-buffered saline while SPE-C spiked into milk is heat stable. Once SPE-C has formed, it is infeasible to eliminate it from milk by thermal treatment. [ABSTRACT FROM AUTHOR]

The hyperproliferation and hyperactivation of CD4 + T cells in salivary gland tissues are hallmarks of Sjögren's syndrome (SS). Fangchinoline (Fan) is extracted from the root of Stephania tetrandra Moore, which is used for treating rheumatic diseases in many studies. This study aimed to identify the mechanism underlying the inhibition of CD4 + T cells by Fan in the SS model NOD/ShiLtj mice. In vivo, Fan alleviated the dry mouth and lymphocyte infiltration in the salivary gland tissues of the NOD/ShiLtj mice and inhibited the number of CD4 + T cells in the infiltrating focus. In vitro, Fan's inhibitory effect on the proliferation of mouse primary CD4 + T cells was verified by CFSE and EdU tests. Furthermore, qRT-PCR and WB analysis confirmed that Fan could inhibit the expression of NFATc1 (Nuclear factor of activated T-cells, cytoplasmic 1) by upregulating miR-506-3p. Dual luciferase reporter gene assay suggested that miR-506-3p interacted with NFATc1. CFSE and EdU tests showed that Fan could inhibit the proliferation of CD4 + T cells through miR-506-3p/NFATc1. The key role of NFATc1 in the activation of CD4 + T cells and the high expression of NFATc1 in samples from SS patients suggested that NFATc1 might become a therapeutic target for SS. In vivo, 11R-VIVIT (NFATc1 inhibitor) alleviated SS-like symptoms. This study not only explained the new mechanism of Fan inhibiting proliferation of CD4 + T cells and alleviating SS-like symptoms but also provided NFATc1 as a potential target for the subsequent research and treatment of SS. [ABSTRACT FROM AUTHOR]

Angelica dahurica radix has a long history of traditional use in China and Korea for treating headaches, cold-damp pain and skin diseases. Despite various pharmacological studies on A. dahurica, its impact on bones remains unclear. Hence, this study investigated the inhibitory effect of A. dahurica's radix water extract (WEAD) on osteoclast differentiation. In vitro experiments showed that WEAD effectively suppresses osteoclast differentiation. Treatment of an osteoclast precursor with WEAD significantly suppressed the expression of nuclear factor of activated T-cells 1 (NFATc1), essential transcription factor for osteoclastogenesis, while increasing the expression of negative regulators, interferon regulatory factor 8 (Irf8) and v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MafB). Consistent with the in vitro findings, the oral administration of WEAD (100 and 300 mg/kg/day) to mice subjected to surgical ovariectomy for a duration of six weeks alleviated bone loss, while also mitigating weight gain and liver fat accumulation. In addition, we also identified phytochemicals present in WEAD, known to regulate osteoclastogenesis and/or bone loss. These results suggest the potential use of WEAD for treating various bone disorders caused by excessive bone resorption. [ABSTRACT FROM AUTHOR]

The effect of urotensin II (U-II), a powerful endogenous vasoconstrictor substance, on the immune system and its mediators is very important. It was herein aimed to demonstrate the possible relationship between the calcineurin/nuclear factor of activated T-cells cytoplasmic 1/interleukin-2 (CaN/NFATc/IL-2) pathway and urotensin receptors (UTRs) in inflammatory response due to lipopolysaccharide (LPS). An LPS-induced inflammation model was used on the human umbilical vein endothelial cells (HUVEC) cell line and drugs were applied accordingly, forming the following groups: Control Group, LPS Group, Agonist Group (10−8 ​M U-II), Antagonist Group (10−6 ​M palosuran), Tacrolimus (TAC) Group (10 ​ng/mL FK-506), Agonist ​+ ​TAC Group, and Antagonist ​+ ​TAC Group. Gene expression analyses were performed using real-time polymerase chain reaction (RT-PCR). In the analysis of the cell viability at 48 and 72 ​h, there was a decrease in the Agonist Group, while in the Agonist ​+ ​TAC Group, the cell viability increased. In the Antagonist Group, cell viability was maintained when compared to the LPS Group, while in the TAC Group, this effect was reduced. The mRNA expression levels of UTR, CaN, NFATc, IL-2 receptor (IL-2R), IL-6 and nuclear factor kappa B (NF-κB) were higher in the LPS Group than in the Control Group, and even the UTR, CaN, NFATc, IL-2R were higher with agonist administration. This effect of the agonist was shown to be completely mitigated in the presence of the CaN inhibitor. U-II and its receptors can perform key functions regarding the endothelial cell damage via the CaN/NFATc/IL-2 pathway. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection is a global health problem and lacks effective therapies in clinic. This study attempted to investigate the role of histone deacetylase 3 (HDAC3) in HBV replication. Cells were treated with 1.3 folds of HBV genome. The expression patterns of HDAC3, miR-29a-3p, and nuclear factor of activated T-cells 5 (NFAT5) in cells were determined by real-time quantitative polymerase chain reaction and Western blot analysis. HBV replication was assessed by measurements of HBV DNA, HBV RNA, hepatitis B surface antigen, and hepatitis B E antigen. After chromatin immunoprecipitation and RNA pull-down assays to testify gene interactions, rescue experiments and animal experiments were performed to assess the role of miR-29a-3p/NFAT5 in HBV replication and the role of HDAC3 in vivo. HDAC3 level was decreased by pHBV1.3 plasmid in a concentration-dependent manner. HDAC3 overexpression can inhibit HBV replication, which was neutralized by miR-29a-3p overexpression or NFAT5 downregulation. Mechanically, HDAC3 overexpression reduced the enrichment of histone 3 lysine 9 acetylation on the miR-29a-3p promoter to inhibit miR-29a-3p expression and then promote NFAT5 transcription. In vivo , HDAC3 restrained HBV replication through the miR-29a-3p/NFAT5 axis. Overall, HDAC3 downregulation was associated with HBV replication and HDAC3 overexpression inhibited HBV replication through H3K9ac/miR-29a-3p/NFAT5. • HDAC3 is decreased in HBV replication. • HDAC3 overexpression inhibits HBV replication. • HDAC3 inhibits miR-29a-3p by reducing H3K9ac enrichment on the promoter. • NFAT5 is a downstream target gene of miR-29a-3p. • HDAC3 attenuates HBV replication via the miR-29a-3p/NFAT5 axis. [ABSTRACT FROM AUTHOR]

Gene signature related to cancer stem cells and fibroblasts of stem-like gastric cancer predicts immunotherapy response Moreover, we identified 110 genes with significantly high expression in stem-like tumours through DEG analysis of stem-like tumour cells and fibroblasts at the single-cell level. (E) Gene ontology network for differentially expressed genes between stem-like tumour cells and others at the single-cell level. gl Based on five molecular subtypes, we identified 276 up-regulated genes between stem-like and other tumour cells at the single-cell level in the bulk Y497 cohort (Figure 2A). We found stem-like tumour cells and other types of tumour cells (mixed stroma, gastric, inflammatory, intestinal), except for common genes among the 276 signatures and 110 genes (Table S3) highly expressed in stem-like tumour cells that differed between stem-like tumour cells and fibroblasts. [Extracted from the article]

Endocannabinoid anandamide (AEA) has a physiological role in regulating renal blood flow, whereas its analogs ameliorated renal ischemia/reperfusion injury. Nonetheless, the role of AEA against mercuric chloride (HgCl2)-induced renal toxicity has not been unraveled. Rats were allocated into control, HgCl2, and HgCl2/AEA treated groups. The administration of AEA quelled the HgCl2-mediated increase in inositol trisphosphate (IP3) and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). The endocannabinoid also signified its anti-inflammatory potential by turning off the inflammatory cascade evidenced by the suppression of high mobility group box protein-1 (HMGB1), receptor of glycated end products (RAGE), nuclear factor-κB p65 (NF-κB), and unexpectedly PPAR-γ. Additionally, the aptitude of AEA to inhibit malondialdehyde and boost glutathione points to its antioxidant capacity. Moreover, AEA by enhancing the depleted renal WNT-5A and reducing cystatin-C and KIM-1 (two kidney function parameters) partly verified its anti-apoptotic capacity, confirmed by inhibiting caspase-3 and increasing B-cell lymphoma-2 (BCL-2). The beneficial effect of AEA was mirrored by the improved architecture and kidney function evidenced by the reduction in cystatin-C, KIM-1, creatinine, BUN, and caspase1-induced activated IL-18. In conclusion, our results verify the reno-protective potential of AEA against HgCl2-induced kidney injury by its anti-inflammatory, antioxidant, and anti-apoptotic capacities by modulating WNT-5A/BCL-2, IP3/NFATC1, HMGB-1/RAGE/NF-κB, caspase-1/IL-18, and caspase-3/BCL-2 cues. [ABSTRACT FROM AUTHOR]

Through dystrophin protein fragments, the authors were able to distinguish DMD patients from healthy individuals and from people affected by other neuromuscular diseases. According to Virchow's insights, we strongly believe that the reported advancements in molecular pathology will open up new landscapes in diagnosis, treatment and monitoring of diseases. Comparing the immune profile of 30 patients with 20 healthy controls, Lioulios et al. showed that exhausted T lymphocyte subpopulations predominated within LN patients, while the T cell phenotype was related to the disease stage. Indeed, combined spectrometric and chromatographic methods are reshaping the detection of disease markers, opening new landscapes in complex disease diagnosis and follow-up of genetic disorders, metabolic alterations and infectious diseases. [Extracted from the article]

This article discusses the role of CRM1-mediated nuclear export in the conversion of the tumor suppressor gene BATF2 in colorectal cancer (CRC). The dysregulation of nuclear-cytoplasmic translocation (NCT) has been linked to the initiation and progression of CRC. The study demonstrates that CRM1 is responsible for the NCT of BATF2, leading to its cytoplasmic translocation and activation of the AP-1/cyclin D1/pRb signaling pathway, which promotes tumor growth and progression. Inhibiting the nuclear export of BATF2 through targeting CRM1 may be a promising therapeutic strategy for CRC. [Extracted from the article]

The B cell receptor (BCR) is a membrane receptor specific to B cells and involved in the selection, activation, maturation, survival and proliferation of these cells. Its activation involves a large number of proteins, triggering a series of signalling pathways which, if they become constitutive, particularly via mutations, may be at the origin of and/or be key in many B cancers. The BCR associates with various co-receptors such as CD19, CD20 or CD22, thus positively or negatively regulating the signalling cascades induced by its activation. These include mitogen-activated protein kinases (MAPK), nuclear factor- κ B (NF- κ B), nuclear factor of activated T-cells (NF-AT) and protein kinase B (AKT); their activation is particularly dependent on BCR-related proteins such as Bruton's agammaglobulinemia tyrosine kinase (BTK) or phospho-inositide 3-kinase (PI3K). Targeting these proteins with inhibitors such as ibrutinib or idelasib has revolutionised the treatment of certain B cancers for which constitutive BCR hyperactivation could not, until now, be effectively attenuated. A great deal of research, both basic and clinical, is underway to identify other therapeutics that can specifically target (i) the different signalling pathways mentioned above, (ii) proteins directly linked to the BCR, or (iii) co-receptors associated with it. This review presents all existing therapies to date, innovative treatments currently in clinical trials and innovative treatments proposed by fundamental research. These include therapies using the most innovative technologies, such as conjugated monoclonal antibodies, chimeric antigen receptor T cells (CAR-T cells) and inhibitors capable of reversible covalent binding. In addition, this review accurately describes the therapeutic effects of alternative targets in the context of resistance to prolonged treatments. Such resistance, particularly to ibrutinib, represents a major public health issue and needs to be studied regularly in order to keep pace with the development of cancers in society. [ABSTRACT FROM AUTHOR]

CD8+ T cells are crucial for the clearance of viral infections. During the acute phase, proinflammatory conditions increase the amount of circulating phosphatidylserine+ (PS) extracellular vesicles (EVs). These EVs interact especially with CD8+ T cells; however, it remains unclear whether they can actively modulate CD8+ T cell responses. In this study, we have developed a method to analyze cell-bound PS+ EVs and their target cells in vivo. We show that EV+ cell abundance increases during viral infection and that EVs preferentially bind to activated, but not naive, CD8+ T cells. Superresolution imaging revealed that PS+ EVs attach to clusters of CD8 molecules on the T cell surface. Furthermore, EV-binding induces antigen (Ag)-specific TCR signaling and increased nuclear translocation of the transcription factor Nuclear factor of activated T-cells (NFATc1) in vivo. EV-decorated but not EV-free CD8+ T cells are enriched for gene signatures associated with T-cell receptor signaling, early effector differentiation, and proliferation. Our data thus demonstrate that PS+ EVs provide Ag-specific adjuvant effects to activated CD8+ T cells in vivo. [ABSTRACT FROM AUTHOR]

In this study, we aimed to analyze the role of the Homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 (Herpud1) gene in the development of cardiomyocyte hypertrophy in association with Calmodulin (CaM) nuclear translocation and cytosolic Ca2+ levels. To observe the mobilization of CaM in cardiomyocytes, we stably expressed eGFP-CaM in rat myocardium-derived H9C2 cells. These cells were then treated with Angiotensin II (Ang II), which stimulates a cardiac hypertrophic response, or dantrolene (DAN), which blocks the release of intracellular Ca2+. To observe intracellular Ca2+ in the presence of eGFP fluorescence, a Rohd-3 Ca2+ sensing dye was used. To examine the effect of suppressing Herpud1 expression, Herpud1 small interfering RNA (siRNA) were transfected into H9C2 cells. To examine whether hypertrophy induced by Ang II could be suppressed by Herpud1 overexpression, a Herpud1-expressing vector was introduced into H9C2 cells. CaM translocation was observed using eGFP fluorescence. Nuclear translocation of Nuclear factor of activated T-cells, cytoplasmic 4 (NFATc4) and nuclear export of Histone deacetylase 4 (HDAC4) were also examined. First, Ang II induced H9C2 hypertrophy with nuclear translocation of CaM and elevation of cytosolic Ca2+, which were inhibited by DAN treatment. We also found that Herpud1 overexpression suppressed Ang II-induced cellular hypertrophy without preventing nuclear translocation of CaM or elevation of cytosolic Ca2+. Additionally, Herpud1 knockdown induced hypertrophy without the nuclear translocation of CaM, which was not inhibited by DAN treatment. Finally, Herpud1 overexpression suppressed Ang II-induced NFATc4 nuclear translocation but did not suppress Ang II-induced CaM nuclear translocation or HDAC4 nuclear export. Ultimately, this study lays the groundwork for elucidating the anti-hypertrophic effects of Herpud1 and the underlying mechanism of pathological hypertrophy. • Dantrolene inhibits Ang II-induced cardiac hypertrophy. • Effect of Dantrolene is due to inhibiting an increase of cytosolic Ca2+ and the nuclear translocation of CaM. • Overexpression of Herpud1 inhibits Ang II-induced cardiac hypertrophy. • Effect of Herpud 1 is shown without inhibiting the increase of cytosolic Ca2+ or the nuclear translocation of CaM. [ABSTRACT FROM AUTHOR]

The origin and differentiation mechanism of articular chondrocytes remain poorly understood. Broadly, the difference in developmental mechanisms of articular and growth-plate cartilage is still less elucidated. Here, we identified that the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) is a crucial regulator of articular, but not growth-plate, chondrocyte differentiation during development. At the early stage of mouse knee development (embryonic day 13.5), NFATc1-expressing cells were mainly located in the flanking region of the joint interzone. With development, NFATc1-expressing cells generated almost all articular chondrocytes but not chondrocytes in limb growth-plate primordium. NFATc1-expressing cells displayed prominent capacities for colony formation and multipotent differentiation. Transcriptome analyses revealed a set of characteristic genes in NFATc1-enriched articular cartilage progenitors. Strikingly, the expression of NFATc1 was diminished with articular chondrocyte differentiation, and suppressing NFATc1 expression in articular cartilage progenitors was sufficient to induce spontaneous chondrogenesis while overex-pressing NFATc1 suppresses chondrogenesis. Mechanistically, NFATc1 negatively regulated the transcriptional activity of the Col2a1 gene. Thus, our results reveal that NFATc1 characterizes articular, but not growth-plate, cartilage progenitors during development and negatively determines articular chondrocyte differentiation at least partly through regulating COL2A1 gene transcription. [ABSTRACT FROM AUTHOR]

Store operated Ca2+ entry (SOCE) is a cornerstone for the maintenance of intracellular Ca2+ homeostasis and the regulation of a variety of cellular functions. SOCE is mediated by STIM and Orai proteins following the activation of inositol 1,4,5‐trisphosphate receptors. Then, a reduction of the endoplasmic reticulum intraluminal Ca2+ concentration is sensed by STIM proteins, which undergo a conformational change and activate plasma membrane Ca2+ channels comprised by Orai proteins. STIM1/Orai‐mediated Ca2+ signals are finely regulated and modulate the activity of different transcription factors, including certain isoforms of the nuclear factor of activated T‐cells, the cAMP‐response element binding protein, the nuclear factor κ‐light chain‐enhancer of activated B cells, c‐fos, and c‐myc. These transcription factors associate SOCE with a plethora of signaling events and cellular functions. Here we provide an overview of the current knowledge about the role of Orai channels in the regulation of transcription factors through Ca2+‐dependent signaling pathways. [ABSTRACT FROM AUTHOR]

BACKGROUND: Preliminary studies found that calcium phosphate cement/poly(lactic-co-glycolic acid) composite can accelerate the process of bone remodeling, and the effect of its degradation products on the differentiation of mononuclear osteoclasts needs further study. OBJECTIVE: To observe the effect of calcium phosphate cement/poly(lactic-co-glycolic acid) degradation products on the differentiation of mouse RAW264.7 monocytes and osteoclasts. METHODS: The experiment was divided into blank control group, glycolic acid group, and calcium phosphate cement/poly(lactic-co-glycolic acid) group. According to the experimental group assignment, the prepared solution and 50 μg/L ligand of nuclear factor κB receptor activator were added to complete medium to prepare media with different conditions. Mouse RAW264.7 cells were cultured for 5 days to induce their differentiation. Cell counting kit-8 assay was used to analyze the effect of different culture media on cell proliferation. RT-PCR and western blot assay were used to detect the changes of gene and protein expression levels of nuclear factor of activated T-cells cytoplasmic 1, matrix metalloproteinase-9, nuclear factor κB receptor activator and tartrate-resistant acid phosphatase activated by osteoclast differentiation-related factors in mouse RAW264.7 monocytes. The osteoclast-like cells were identified by tartrate-resistant acid phosphatase staining. RESULTS AND CONCLUSION: Cell counting kit-8 assay results showed that the cells were in a rapid growth period within 72 hours, and then began to decline after 96 hours. The results of RT-PCR and western blot assay showed that the expression levels of nuclear factor of activated T-cells cytoplasmic 1, nuclear factor-κB receptor activator and tartrate-resistant acid phosphatase mRNA and protein expression levels of nuclear factor-1 and nuclear factor-κB receptor activator in activated T cells in calcium phosphate cement/poly(lactic-co-glycolic acid) group were significantly higher than those in glycolic acid group and control group (P < 0.05). There was no significant difference in gene and protein expression levels of matrix metalloproteinase-9 between glycolic acid group and calcium phosphate cement/poly(lactic-co-glycolic acid) group, but it was higher than that of the control group (P < 0.01). Tartrate-resistant acid phosphatase staining results showed that the number of osteoclast-like cells in the calcium phosphate cement/poly(lactic-co-glycolic acid) group was significantly higher than that in the control group and the glycolic acid group (P < 0.05). Overall, these results indicate that the microenvironment formed by the degradation products of calcium phosphate cement/poly(lactic-co-glycolic acid) can promote the osteoclast differentiation of mouse RAW264.7 monocytes. [ABSTRACT FROM AUTHOR]

Ca2+ release-activated Ca2+ (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca2+-sensor STIM1 in the ER membrane and the Ca2+ ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca2+ imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation. In particular, photoactivation at A137 using Bpa in Orai1 activates Ca2+ currents that best match the biophysical properties of CRAC channels and are capable of triggering downstream signaling pathways such as nuclear factor of activated T-cells (NFAT) translocation into the nucleus without the need for the physiological activator STIM1. The Ca2+ ion channel Orai1 is crucial in immune cells. Here, the authors applied genetic code expansion to transfer light-sensitivity to the Orai1 channel and achieved precise control over its function. [ABSTRACT FROM AUTHOR]

Histone deacetylase (HDAC) has been found to play a crucial role in the regulation of osteoclast differentiation and formation. This study was designed to identify the effect of the HDAC6 inhibitor CKD-WID on the receptor for the activation of nuclear factor-κB ligand (RANKL)-mediated osteoclast formation in the presence of monosodium urate (MSU) in RAW 264.7 murine macrophage cells. The expression of osteoclast-specific target genes, calcineurin, and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) was evaluated in RAW 264.7 murine macrophages treated with MSU, RANKL, or CKD-WID by real-time quantitative polymerase chain reaction and Western blot assay. The effect of CKD-WID on osteoclast formation was measured by tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring formation staining, and assays for bone resorption activity. RANKL in the presence of MSU significantly induced HDAC6 gene and protein expression in RAW 264.7 cells. CKD-WID markedly suppressed the expression of osteoclast-related markers such as c-Fos, TRAP, cathepsin K, and carbonic anhydrase II induced by co-stimulation with RANKL and MSU in RAW 264.7 cells. Transcription factor NFATc1 mRNA expression and nuclear NFATc1 protein expression induced by co-stimulation with RANKL and MSU were significantly inhibited by CKD-WID treatment. CKD-WID also decreased the number of TRAP-positive multinuclear cells and F-actin ring-positive cells and attenuated bone resorption activity. Co-stimulation with RANKL and MSU increased calcineurin gene and protein expression, which was significantly blocked by CKD-WID treatment. The HDAC6 inhibitor CKD-WID suppressed MSU-induced osteoclast formation through blocking the calcineurin-NFAT pathway in RAW 264.7 cells. This suggests that HDAC6 is considered a therapeutic target in uric acid-mediated osteoclastogenesis. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor (FGF)23 is a bone-derived phosphotropic hormone that regulates phosphate and mineral homeostasis. Recent studies have provided evidence that a high plasma concentration of FGF23 is associated with cardiac disease, including left ventricular hypertrophy (LVH), heart failure, atrial fibrillation, and cardiac death. Experimental studies have shown that FGF23 activates fibroblast growth factor receptor 4 (FGFR4)/phospholipase Cγ/calcineurin/nuclear factor of activated T-cells signaling in cardiomyocytes and induces cardiac hypertrophy in rodents. Activation of FGFR4 by FGF23 normally requires the co-receptor α-klotho, and klotho-independent signaling occurs only under conditions characterized by extremely high FGF23 concentrations. Recent studies have demonstrated that FGF23 activates the renin-angiotensin-aldosterone system (RAAS) and induces LVH, at least in part as a result of lower vitamin D activation. Moreover, crosstalk between FGF23 and RAAS results in the induction of cardiac hypertrophy and fibrosis. In this review, we summarize the results of studies regarding the relationships between FGF23 and cardiac events, and describe the potential direct and indirect mechanisms whereby FGF23 induces LVH. [ABSTRACT FROM AUTHOR]

Regulation of osteoclastogenesis and bone-resorbing activity can be an efficacious strategy for treating bone loss diseases because excessive osteoclastic bone resorption leads to the development of such diseases. Here, we investigated the role of (-)-tubaic acid, a thermal degradation product of rotenone, in osteoclast formation and function in an attempt to identify alternative natural compounds. (-)-Tubaic acid significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclast differentiation at both the early and late stages, suggesting that (-)-tubaic acid affects the commitment and differentiation of osteoclast progenitors as well as the cell-cell fusion of mononuclear osteoclasts. (-)-Tubaic acid attenuated the activation of extracellular signal-regulated kinase (ERK) and expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and its target genes in response to RANKL. Furthermore, a pit-formation assay revealed that (-)-tubaic acid significantly impaired the bone-resorbing activity of osteoclasts. Our results demonstrated that (-)-tubaic acid exhibits anti-osteoclastogenic and anti-resorptive effects, indicating its therapeutic potential in the management of osteoclast-related bone diseases. [ABSTRACT FROM AUTHOR]

Inflammation is an important risk factor for bone-destroying diseases. Our preliminary research found that Zanthoxylum bungeanum seed oil (ZBSO) is abundant in unsaturated fatty acids and could inhibit osteoclastogenesis in receptor activator of nuclear factor κB ligand (RANKL)-induced RAW264.7 cells. However, the key constituents in ZBSO in the prevention of osteoclastogenesis and its possible mechanism related to inflammation are still unclear. Therefore, in this study, oleic acid (OA), linoleic acid (LA), palmitoleic acid (PLA), and alpha-linolenic acid (ALA) in ZBSO, havingthe strongest effect on RANKL-induced osteoclastogenesis, were selected by a tartrate-resistant acid phosphatase (TRAP) staining method. Furthermore, the effects of the selected fatty acids on anti-inflammation and anti-osteoclastogenesis in vitro and in vivo were assessed using RT-qPCR. Among the four major unsaturated fatty acids we tested, ALA displayed the strongest inhibitory effect on osteoclastogenesis. The increased expression of free fatty acid receptor 4 (FFAR4) and β-arrestin2 (βarr2), as well as the decreased expression of nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), nuclear factor of activated T-cells c1 (NFATc1), and tartrate-resistant acid phosphatase (TRAP) in RAW264.7 cells after ALA treatment were observed. Moreover, in ovariectomized osteoporotic rats with ALA preventive intervention, we found that the expression of TNF-α, interleukin-6 (IL-6), interleukin-1β (IL-1β), NFATc1, and TRAP were decreased, while with the ALA therapeutic intervention, downregulated expression of NF-κB, NFATc1, TRAP, and transforming growth factor beta-activated kinase 1 (TAK1) were noticed. These results indicate that ALA, as the major unsaturated fatty acid in ZBSO, could inhibit RANKL-induced osteoclastogenesis via the FFAR4/βarr2 signaling pathway and could prevent inflammation, suggesting that ZBSO may be a promising potential natural product of unsaturated fatty acids and a dietary supplement for the prevention of osteoclastogenesis and inflammatory diseases. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the anti-osteoporosis effects of Osmanthus fragrans leaf ethanol extract (OFLEE) in bone marrow-derived macrophages (BMM) and animals with osteoporosis. OFLEE not only suppressed tartrate-resistant acid phosphatase (TRAP)-positive cells with multiple nuclei but also decreased TRAP activity in BMM treated with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). The formation of F-actin rings and the expression and activation of matrix metalloproteinases were decreased by OFLEE in BMM treated with M-CSF and RANKL. OFLEE suppressed M-CSF- and RANKL-induced osteoclastogenesis by inhibiting NF-κB phosphorylation, tumor necrosis factor receptor-associated factor 6, c-fos, the nuclear factor of activated T-cells, cytoplasmic 1, and cathepsin K in BMM. OFLEE downregulated reactive oxygen species, cyclooxygenase-2, inducible nitric oxide synthase, prostaglandin E2, tumor necrosis factor α, interleukin (IL)-1β, IL-6, IL-17, and RANKL in BMM treated with M-CSF and RANKL. Oral administration of OFLEE suppressed osteoporotic bone loss without hepatotoxicity in ovariectomy-induced osteoporosis animals. Our findings suggest that OFLEE, with anti-inflammatory effects, prevents osteoporotic bone loss through the suppression of osteoclastic differentiation in BMM and animals with osteoporosis. [ABSTRACT FROM AUTHOR]

YIV-906 is a systems biology botanical cancer drug, inspired by a traditional Chinese herbal formulation. Results from eight Phase I/II to II clinical studies demonstrated the potential of YIV-906 to prolong survival and improve the quality of life of cancer patients. As an immunomodulator in the tumor microenvironment, YIV-906 can turn cold tumors hot and potentiate anti-tumor activity for different classes of anticancer agents; and as a cytoprotector in the GI, YIV-906 can reduce non-hematological side effects and speed up damaged tissue recovery. YIV-906 enhanced anti-PD1 action against hepatoma in mice by stimulating both innate and adaptive immunity. In a Jurkat cell-staphylococcal superantigen E (SEE)-Raji cell culture model, YIV-906 promoted T cell activation with upregulation of CD69 by enhancing NFAT activity, with or without PD1-PD-L1 interaction. YIV-906 could trigger the phosphorylation of TCR downstream signaling cascades without the involvement of TCR. YIV-906 could inhibit SHP1 and SHP2 activities, which dephosphorylates TCR downstream proteins due to the PD1-PD-L1 interaction. Therefore, YIV-906 could enhance anti-PD1 action to rescue the depressed NFAT activity of Jurkat cells due to the PD1-PDL1 interaction. In addition, YIV-906 enhanced the NFAT activity and killing capability of Jurkat cells expressing chimeric antigen receptor (CAR-CD19-CD3z) toward CD19 expressing cells, such as Raji cells, with or without PD1-PDL1 overexpression. Ingredient herb S (Scutellaria baicalensis Georgi) of YIV-906 and some S compounds were found to play key roles in these activities. In conclusion, YIV-906 modulates adaptive immunity by activating T effector cells mainly through its action on SHP1/2. YIV-906 could also facilitate immune checkpoint blockade therapy or CAR-T cell therapy for cancer treatment. [ABSTRACT FROM AUTHOR]

Histone acetylation orchestrates a complex symphony of gene expression that controls cellular fate and activities, including the intricate processes of bone remodeling. Despite its proven significance, a systematic illustration of this process has been lacking due to its complexity, impeding clinical application. In this review, we delve into the central regulators of histone acetylation, unveiling their multifaceted roles in modulating bone physiology. We explore both contradictory and overlapping roles among these regulators and assess their potential as therapeutic targets for various bone disorders. Furthermore, we highlight current applications and discuss looming questions for a more effective use of epigenetic therapy in bone diseases, aiming to address gaps in knowledge and clinical practice. By providing a panoramic view of histone acetylation’s impact on bone health and disease, this review unveils promising avenues for therapeutic intervention and enhances our understanding of skeletal physiology, crucial for improving therapeutical outcomes and quality of patients’ life.The regulatory network of histone acetylation and deacetylation in bone remodeling. Osteoblasts and osteoclasts are the central functioning cells in bone remodeling. Osteoblasts derive from MSCs and require two-stage differentiation, while macrophages must undergo differentiation, fusion, and activation to form osteoclasts. In osteoblast formation, the direct acetylation changes of BMP2/4, Wnt, RUNX2 and CCN1, can indirectly alter the expression of osteoblastic genes. In osteoclast formation, HATs and HDACs mainly modify RANKL, NFATc1, and M-CSF to influence osteoclastic genes' expression. Abbreviation: RUNX2 (Runt-related transcription factor 2); OSX (Osterix); COL1a1 (Collagen, type I, alpha 1); RANKL (Receptor activator of nuclear kappa-b ligand); OPG (Osteoprotegerin); OCN (Osteocalcin); CCN1 (Recombinant human cyr61); Nrf2 (Nuclear factor erythroid 2-related factor 2); TGF-β (Transforming growth factor beta); BMP (Bone morphogenetic protein); HAT (Histone acetyltransferase); HDAC (Histone deacetylase); M-CSF (43 Macrophagecolony stimulating factor); NFATC1 (Nuclear factor of activated T-cells c1); MITF (Microphthalmia-associated transcription factor); CXCL12 (Chemokine (C-X-C motif) ligand 12)The regulatory network of histone acetylation and deacetylation in bone remodeling. Osteoblasts and osteoclasts are the central functioning cells in bone remodeling. Osteoblasts derive from MSCs and require two-stage differentiation, while macrophages must undergo differentiation, fusion, and activation to form osteoclasts. In osteoblast formation, the direct acetylation changes of BMP2/4, Wnt, RUNX2 and CCN1, can indirectly alter the expression of osteoblastic genes. In osteoclast formation, HATs and HDACs mainly modify RANKL, NFATc1, and M-CSF to influence osteoclastic genes' expression. Abbreviation: RUNX2 (Runt-related transcription factor 2); OSX (Osterix); COL1a1 (Collagen, type I, alpha 1); RANKL (Receptor activator of nuclear kappa-b ligand); OPG (Osteoprotegerin); OCN (Osteocalcin); CCN1 (Recombinant human cyr61); Nrf2 (Nuclear factor erythroid 2-related factor 2); TGF-β (Transforming growth factor beta); BMP (Bone morphogenetic protein); HAT (Histone acetyltransferase); HDAC (Histone deacetylase); M-CSF (43 Macrophagecolony stimulating factor); NFATC1 (Nuclear factor of activated T-cells c1); MITF (Microphthalmia-associated transcription factor); CXCL12 (Chemokine (C-X-C motif) ligand 12)Graphical Abstract: Histone acetylation orchestrates a complex symphony of gene expression that controls cellular fate and activities, including the intricate processes of bone remodeling. Despite its proven significance, a systematic illustration of this process has been lacking due to its complexity, impeding clinical application. In this review, we delve into the central regulators of histone acetylation, unveiling their multifaceted roles in modulating bone physiology. We explore both contradictory and overlapping roles among these regulators and assess their potential as therapeutic targets for various bone disorders. Furthermore, we highlight current applications and discuss looming questions for a more effective use of epigenetic therapy in bone diseases, aiming to address gaps in knowledge and clinical practice. By providing a panoramic view of histone acetylation’s impact on bone health and disease, this review unveils promising avenues for therapeutic intervention and enhances our understanding of skeletal physiology, crucial for improving therapeutical outcomes and quality of patients’ life.The regulatory network of histone acetylation and deacetylation in bone remodeling. Osteoblasts and osteoclasts are the central functioning cells in bone remodeling. Osteoblasts derive from MSCs and require two-stage differentiation, while macrophages must undergo differentiation, fusion, and activation to form osteoclasts. In osteoblast formation, the direct acetylation changes of BMP2/4, Wnt, RUNX2 and CCN1, can indirectly alter the expression of osteoblastic genes. In osteoclast formation, HATs and HDACs mainly modify RANKL, NFATc1, and M-CSF to influence osteoclastic genes' expression. Abbreviation: RUNX2 (Runt-related transcription factor 2); OSX (Osterix); COL1a1 (Collagen, type I, alpha 1); RANKL (Receptor activator of nuclear kappa-b ligand); OPG (Osteoprotegerin); OCN (Osteocalcin); CCN1 (Recombinant human cyr61); Nrf2 (Nuclear factor erythroid 2-related factor 2); TGF-β (Transforming growth factor beta); BMP (Bone morphogenetic protein); HAT (Histone acetyltransferase); HDAC (Histone deacetylase); M-CSF (43 Macrophagecolony stimulating factor); NFATC1 (Nuclear factor of activated T-cells c1); MITF (Microphthalmia-associated transcription factor); CXCL12 (Chemokine (C-X-C motif) ligand 12)The regulatory network of histone acetylation and deacetylation in bone remodeling. Osteoblasts and osteoclasts are the central functioning cells in bone remodeling. Osteoblasts derive from MSCs and require two-stage differentiation, while macrophages must undergo differentiation, fusion, and activation to form osteoclasts. In osteoblast formation, the direct acetylation changes of BMP2/4, Wnt, RUNX2 and CCN1, can indirectly alter the expression of osteoblastic genes. In osteoclast formation, HATs and HDACs mainly modify RANKL, NFATc1, and M-CSF to influence osteoclastic genes' expression. Abbreviation: RUNX2 (Runt-related transcription factor 2); OSX (Osterix); COL1a1 (Collagen, type I, alpha 1); RANKL (Receptor activator of nuclear kappa-b ligand); OPG (Osteoprotegerin); OCN (Osteocalcin); CCN1 (Recombinant human cyr61); Nrf2 (Nuclear factor erythroid 2-related factor 2); TGF-β (Transforming growth factor beta); BMP (Bone morphogenetic protein); HAT (Histone acetyltransferase); HDAC (Histone deacetylase); M-CSF (43 Macrophagecolony stimulating factor); NFATC1 (Nuclear factor of activated T-cells c1); MITF (Microphthalmia-associated transcription factor); CXCL12 (Chemokine (C-X-C motif) ligand 12) [ABSTRACT FROM AUTHOR]

The article focuses on the Systemic lupus erythematosus is an autoimmune disease that involves all organ systems and affects young women. Topics include examines despite available treatments, SLE is responsible for substantial morbidity and mortality worldwide; and considered current treatment relies on the use of immunosuppressive drugs that control the immune dysregulation at the price of increased risk of infections and cardiovascular disease.

Keywords: melanocortin 4 receptor; MC4R; stop mutation; PTC; translational readthrough; G418 EN melanocortin 4 receptor MC4R stop mutation PTC translational readthrough G418 1793 17 11/17/22 20221101 NES 221101 1. On the other hand, the W258X mutant, in which the mutation is part of the highly conserved and important CWxP motif in I MC4R i and other GPCRs [[37]], in transmembrane helix (TMH) 6, and the Q307X mutant with a PTC in the beginning of helix 8 showed a cell surface expression that was similar to the WT. Thus, in this study, we evaluated the hypothesis of a readthrough of stop mutations in combination with the synthetic I MC4R i ligand setmelanotide (SM) being a treatment option for obese patients carrying I MC4R i stop mutations and performed our investigation in a human cell model for the first time. Thus, we here evaluated whether treatment with the aminoglycoside G418 can rescue I MC4R i stop mutations and increase receptor expression as well as signaling in human MC4R-transfected HEK-293 cells. We conducted research on human HEK-293 cells transiently transfected with different I MC4R i stop mutations, as so far, a rescue of I MC4R i stop mutations has only been investigated in nonhuman COS-7 cells, focusing on G SB s sb signaling only, and the results were auspicious [[30]]. [Extracted from the article]

Benzyl isothiocyanate (BITC), derived from cruciferous vegetables, is an organosulfur compound exerting antiproliferative effects in several human cancer cells. In this study, we assessed BITC as a potential osteoclastogenesis inhibitor and investigated its underlying mechanism. BITC at 5 μM significantly decreased the viability of the osteoclast‐like differentiating RAW264.7 cells, coinciding with the downregulation of the primary biomarkers for osteoclast differentiation, such as the tartrate‐resistant acid phosphatase activity and nuclear factor of activated T‐cells gene expression. Not only BITC but also its metabolites, inhibited cell proliferation in the normal RAW264.7 cells, suggesting that BITC shows an anti‐osteoclastogenesis effect in vivo after its ingestion and metabolism, possibly through an antiproliferative action. Both BITC and its metabolites also enhanced the DNA fragmentation and the caspase‐3 activity, whereas their higher concentrations tended to suppress these effects. BITC was intracellularly accumulated when the cells were treated with its metabolites via their degradation into the free form. A quantitative experiment using the proteolysis/high performance liquid chromatography technique showed that the amount of BITC‐lysine thiourea in the cells was also increased in a time‐dependent manner, suggesting that lysine modification of the cellular proteins actually took place in the cells treated by BITC. Among the cellular proteins, the cleaved caspase‐3 was identified as a potential target for lysine modification by BITC. Taken together, BITC dissociated from its metabolites as well as its free form might modulate osteoclastogenesis, possibly through inhibition of cell proliferation by protein modification. [ABSTRACT FROM AUTHOR]

Disturbances in the excitatory/inhibitory balance of brain neural circuits are the main source of encephalopathy during neurodevelopment. Changes in the function of neural circuits can lead to depolarization or repeat rhythmic firing of neurons in a manner similar to epilepsy. GABAergic neurons are inhibitory neurons found in all the main domains of the CNS. Previous studies suggested that DjCamkII and DjCaln play a crucial role in the regulation of GABAergic neurons during planarian regeneration. However, the mechanisms behind the regeneration of GABAergic neurons have not been fully explained. Herein, we demonstrated that DjCamkII and DjCaln were mutual negative regulation during planarian head regeneration. DjNFAT exerted feedback positive regulation on both DjCaln and DjCamkII. Whole-mount in situ hybridization (WISH) and fluorescence in situ hybridization (FISH) revealed that DjNFAT was predominantly expressed in the pharynx and parenchymal cells in intact planarian. Interestingly, during planarian head regeneration, DjNFAT was predominantly located in the newborn brain. Down-regulation of DjNFAT led to regeneration defects in the brain including regenerative brain became small and the lateral nerves cannot be regenerated completely, and a decreasein the number of GABAergic neurons during planarian head regeneration. These findings suggest that the feedback loop between DjCaln, DjCamkII, and DjNFAT is crucial for the formation of GABAergic neurons during planarian head regeneration. [ABSTRACT FROM AUTHOR]