Nanoparticles prepared from the water extract of Gusuibu (Drynaria fortunei J. Sm.) protects osteoblasts against insults and promotes cell maturation

Our previous study showed that Gusuibu (Drynaria fortunei J. Sm.) can stimulate osteoblast maturation. This study was further designed to evaluate the effects of nanoparticles prepared from the water extract of Gusuibu (WEG) on osteoblast survival and maturation. Primary osteoblasts were exposed to 1, 10, 100, and 1000 μg/mL nanoparticles of WEG (nWEG) for 24, 48, and 72 hours did not affect morphologies, viability, or apoptosis of osteoblasts. In comparison, treatment of osteoblasts with 1000 μg/mL WEG for 72 hours decreased cell viability and induced DNA fragmentation and cell apoptosis. nWEG had better antioxidant bioactivity in protecting osteoblasts from oxidative and nitrosative stress-induced apoptosis than WEG. In addition, nWEG stimulated greater osteoblast maturation than did WEG. Therefore, this study shows that WEG nanoparticles are safer to primary osteoblasts than are normal-sized products, and may promote better bone healing by protecting osteoblasts from apoptotic insults, and by promoting osteogenic maturation

Improvement of Carbon Tetrachloride-Induced Acute Hepatic Failure by Transplantation of Induced Pluripotent Stem Cells without Reprogramming Factor c-Myc

The only curative treatment for hepatic failure is liver transplantation. Unfortunately, this treatment has several major limitations, as for example donor organ shortage. A previous report demonstrated that transplantation of induced pluripotent stem cells without reprogramming factor c-Myc (3-genes iPSCs) attenuates thioacetamide-induced hepatic failure with minimal incidence of tumorigenicity. In this study, we investigated whether 3-genes iPSC transplantation is capable of rescuing carbon tetrachloride (CCl4)-induced fulminant hepatic failure and hepatic encephalopathy in mice. Firstly, we demonstrated that 3-genes iPSCs possess the capacity to differentiate into hepatocyte-like cells (iPSC-Heps) that exhibit biological functions and express various hepatic specific markers. 3-genes iPSCs also exhibited several antioxidant enzymes that prevented CCl4-induced reactive oxygen species production and cell death. Intraperitoneal transplantation of either 3-genes iPSCs or 3-genes iPSC-Heps significantly reduced hepatic necrotic areas, improved hepatic functions, and survival rate in CCl4-treated mice. CCl4-induced hepatic encephalopathy was also improved by 3-genes iPSC transplantation. Hoechst staining confirmed the successful engraftment of both 3-genes iPSCs and 3-genes iPSC-Heps, indicating the homing properties of these cells. The most pronounced hepatoprotective effect of iPSCs appeared to originate from the highest antioxidant activity of 3-gene iPSCs among all transplanted cells. In summary, our findings demonstrated that 3-genes iPSCs serve as an available cell source for the treatment of an experimental model of acute liver diseases

TRAF-6 Dependent Signaling Pathway Is Essential for TNF-Related Apoptosis-Inducing Ligand (TRAIL) Induces Osteoclast Differentiation

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Recent evidence indicates that in addition to triggering apoptosis, the TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. To understand TRAIL-mediated signal transduction mechanism in osteoclastogenesis, we demonstrated that TRAIL induces osteoclast differentiation via a Tumor necrosis factor receptor-associated factor 6 (TRAF-6)-dependent signaling pathway. TRAIL-induced osteoclast differentiation was significantly inhibited by treatment with TRAF-6 siRNA and TRAF6 decoy peptides in both human monocytes and murine RAW264.7 macrophage cell lines, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Moreover, TRAIL-induced osteoclast differentiation was also abolished in TRAF6 knockout bone marrow macrophages. In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation. Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway. This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Immunomodulatory effects of extracellular vesicles from mesenchymal stromal cells: Implication for therapeutic approach in autoimmune diseases

Abstract Autoimmune disease is characterized by the proliferation of harmful immune cells, inducing tissue inflammation and ultimately causing organ damage. Current treatments often lack specificity, necessitating high doses, prolonged usage, and high recurrence rates. Therefore, the identification of innovative and safe therapeutic strategies is urgently required. Recent preclinical studies and clinical trials on inflammatory and autoimmune diseases have evidenced the immunosuppressive properties of mesenchymal stromal cells (MSCs). Studies have demonstrated that extracellular vesicles (EV) derived from MSCs can mitigate abnormal autoinflammation while maintaining safety within the diseased microenvironment. This study conducted a systematic review to elucidate the crucial role of MSC‐EVs in alleviating autoimmune diseases, particularly focusing on their impact on the underlying mechanisms of autoimmune conditions such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and inflammatory bowel disease (IBD). By specifically examining the regulatory functions of microRNAs (miRNAs) derived from MSC‐EVs, the comprehensive study aimed to enhance the understanding related to disease mechanisms and identify potential diagnostic markers and therapeutic targets for these diseases

Increased neutrophil infiltration, IL-1 production and a SAPHO syndrome-like phenotype in PSTPIP2-deficient mice

Objective. Proline-serine-threonine-phosphatase-interacting protein 2 (PSTPIP2) is involved in macrophage activation, neutrophil motility and osteoclast differentiation. However, the role of PSTPIP2 in inflammation and autoinflammatory diseases is still not clear. In this study, we generated PSTPIP2 knockout (Pstpip2(-/-)) mice to investigate its phenotype and role in autoinflammatory diseases. Methods. We constructed a Pstpip2-targeting vector and generated Pstpip2(-/-) mice. The phenotype and immunopathology of Pstpip2(-/-) mice were analysed. Results. All Pstpip2(-/-) mice developed paw swelling, synovitis, hyperostosis and osteitis, resembling SAPHO syndrome, an inflammatory disorder of the bone, skin and joints. Multifocal osteomyelitis was found in inflamed paws, with increased macrophage and marked neutrophil infiltrations in the bone, joint and skin. Profound osteolytic lesions with markedly decreased bone volume density developed in paws and limbs. Neutrophil-attracting chemokines and IL-1 beta were markedly elevated in inflamed tissues. Conclusion. Our study suggests that PSTPIP2 could play a role in innate immunity and development of autoinflammatory bone disorders, and may be associated with the pathogenesis of human SAPHO syndrome

Regulation of osteoclast activation and inflammation signal transduction in inflammatory arthritis

蝕骨細胞是多核並具有蝕骨功能的細胞，屬於腫瘤壞死因子家族成員可與其接受體互相作用而促進蝕骨細胞分化，有許多研究已廣泛地指出RANKL的訊息機制可促進蝕骨細胞生成。RANKL活化蝕骨細胞分化中，TRAF6訊息路徑是不可或缺的，在之前的研究當中，我們的研究結果顯示TRAIL透過TRAF6訊息路徑活化蝕骨細胞分化，有趣的是，我們的結果也顯示TRAIL具有抑制RANKL以及M-CSF促進蝕骨細胞分化的現象。然而，TRAIL在RANKL或是其他腫瘤壞死因子中有無共同刺激，影響促進以及抑制蝕骨細胞分化，認為TRAIL可能在調控骨免疫學中蝕骨細胞分化中扮演重要的角色，很有可能是透過影響脂質閥以及TRAF6相關訊息，脂質閥對於聚集或排除接受器之特定訊息物質而活化免疫細胞中扮演重要的角色，我們的結果指出RANKL對於脂質閥之訊息分子傳遞而影響蝕骨細胞的功能，而TRAIL會干擾脂質閥中RANK訊息傳遞影響蝕骨細胞的分化。更進一步使用大鼠實驗性關節炎的動物模式，TRAIL不但可以改善骨密度以及骨侵蝕，更可以減少關節腫脹及發炎，然而，TRAIL抑制發炎的功能是透過抑制T細胞的活化而不是影響細胞凋亡，因此TRAIL有效地抑制實驗性關節炎的發炎以及關節炎的進展，TRAIL提供了關節炎具有潛力以及新的治療方針。 PSTPIP2參與巨噬細胞活化、噬中性白血球移動性以及蝕骨細胞分化，然而，PSTPIP2對於自體發炎以及蝕骨細胞生成的角色並不是很清楚。在我們的研究中，我們建立PSTPIP2基因剔除老鼠探討表型以及自體發炎疾病的角色，所有PSTPIP2基因剔除老鼠具有腳掌腫脹、滑膜炎、骨肥厚以及骨髓炎，類似SAPHO症候群（滑膜炎、痤瘡、膿皰病、骨肥厚、骨髓炎），發生在骨頭、皮膚以及關節的發炎疾病，多病灶性骨髓炎在發炎性腳掌中有較多的巨嗜細胞以及噬中性白血球浸潤在骨頭、關節以及皮膚，顯著性骨侵蝕的病灶部位腳掌以及肢體也發現明顯降低骨密度，在發炎的組織中也發現顯著增加趨化噬中性白血球的趨化素以及介白素-1β。我們的研究認為PSTPIP2在自體發炎骨疾病中，先天性免疫以及蝕骨細胞生成的發展扮演重要的角色，並且可能跟人類SAPHO症候群的病理具有相關性。 總而言之，我們指出TRAIL以及PSTPIP2參與蝕骨細胞活化以及發炎，認為與蝕骨細胞活化的訊息傳導路徑具有相關性，了解發炎性關節炎以及骨破壞的傳導路徑，對於骨免疫學是重要的並且可發展新的治療方式。Osteoclasts are bone resorbing multinucleated cells involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Accumulated evidences indicate that the signaling mechanism of RANKL in promoting osteoclasteogenesis has been extensively studied. RANKL activates TRAF6 signaling pathways, which are indispensable for the induction and activation of osteoclast differentiation. In previous study, our results have demonstrated that TRAIL induces osteoclast differentiation via a TRAF-6-dependent signaling pathway. Interestingly, our results also demonstrated that TRAIL suppressed osteoclastic differentiation induced by RANKL plus M-CSF. Therefore, TRAIL can both promote and suppress osteoclast differentiation, depending on the presence of RANKL or other TNF superfamily molecules, suggesting that TRAIL may play a role in regulating osteoclast differentiation in osteoimmunology. It is possible that the regulation is at the lipid raft and TRAF6 associated signaling. Membrane lipid rafts play a key role in immune cell activation by recruiting and excluding specific signaling components of immune cell surface receptors upon the receptor engagement. We demonstrated that rafts in osteoclasts may function as the platform for a network of signaling molecules that assemble in response to RANKL. TRAIL blocked osteoclastic differentiation was accompanied by interference the RANK signaling required the function of raft membrane microdomains. Furthermore, in collagen-induced arthritis rat animal model, treatment with TRAIL not only the improved the bone density and inhibited bone erosion, but also reduced the joint swelling and inflammation. However, the anti-inflammation effects of TRAIL inhibited T cell activation instead of increased apoptosis in T cells. Therefore, TRAIL administration is an effective anti-inflammatory treatment that prevents the development and progression of experimental arthritis. It may provide a potential and new therapeutic strategy of treatment in arthritis. PSTPIP2 is involved in macrophage activation, neutrophil motility, and osteoclast differentiation. However, the role of PSTPIP2 in autoinflammation and osteoclastogenesis is still not clear. In our study, we generated PSTPIP2 knockout (Pstpip2-/-) mice to investigate its phenotype and role in autoinflammatory diseases. All Pstpip2-/- mice developed paw swelling, synovitis, hyperostosis, and osteitis, resembling SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, osteitis), an inflammatory disorder of the bone, skin, and joints. Multifocal osteomyelitis was found in inflamed paws with increased macrophage and marked neutrophil infiltrations in the bone, joint, and skin. Profound osteolytic lesions with marked decreased bone volume density developed in paws and limbs. Neutrophil-attracting chemokines and IL-1s were markedly elevated in inflamed tissues. Our study suggests that PSTPIP2 could play a role in innate immunity and development of osteoclastogenesis in autoinflammatory bone disorders, and may associate with the pathogenesis of human SAPHO syndrome. Taken together, we demonstrated that both TRAIL and PSTPIP2 is involved in osteoclast activation and inflammation, suggesting the cross-talk between signaling transduction pathways in osteoclast activation and inflammation. Understanding of the regulation of these pathways is important to explore the critical steps in oseoimmunology, and help to development of new therapeutic approach in inflammatory arthritis and bone destruction

Potential of Using Infrapatellar–Fat–Pad–Derived Mesenchymal Stem Cells for Therapy in Degenerative Arthritis: Chondrogenesis, Exosomes, and Transcription Regulation

Infrapatellar fat pad–derived mesenchymal stem cells (IPFP-MSCs) are a type of adipose-derived stem cell (ADSC). They potentially contribute to cartilage regeneration and modulation of the immune microenvironment in patients with osteoarthritis (OA). The ability of IPFP-MSCs to increase chondrogenic capacity has been reported to be greater, less age dependent, and less affected by inflammatory changes than that of other MSCs. Transcription-regulatory factors strictly regulate the cartilage differentiation of MSCs. However, few studies have explored the effect of transcriptional factors on IPFP-MSC-based neocartilage formation, cartilage engineering, and tissue functionality during and after chondrogenesis. Instead of intact MSCs, MSC-derived extracellular vesicles could be used for the treatment of OA. Furthermore, exosomes are increasingly being considered the principal therapeutic agent in MSC secretions that is responsible for the regenerative and immunomodulatory functions of MSCs in cartilage repair. The present study provides an overview of advancements in enhancement strategies for IPFP-MSC chondrogenic differentiation, including the effects of transcriptional factors, the modulation of released exosomes, delivery mechanisms for MSCs, and ethical and regulatory points concerning the development of MSC products. This review will contribute to the understanding of the IPFP-MSC chondrogenic differentiation process and enable the improvement of IPFP-MSC-based cartilage tissue engineering

Biological functions of the disulfides in bovine pancreatic deoxyribonuclease

We characterized the biochemical functions of the small nonessential (C101–C104) and the large essential (C173–C209) disulfides in bovine pancreatic (bp) DNase using alanine mutants [brDNase(C101A)] and [brDNase(C173A) and brDNase(C209A)], respectively. We also characterized the effects of an additional third disulfide [brDNase(F192C/A217C)]. Without the Ca2+ protection, bpDNase and brDNase(C101A) were readily inactivated by trypsin, whereas brDNase(F192C/A217C) remained active. With Ca2+, all forms of DNase, except for brDNase(C101A), were protected against trypsin. All forms of DNase, after being dissolved in 6 M guanidine-HCl, were fully reactivated by diluting into a Ca2+-containing buffer. However, when diluted into a Ca2+-free buffer, bpDNase and brDNase(C101A) remained inactive, but 60% of the bpDNase activity was restored with brDNase(F192C/A217C). When heated, bpDNase was inactivated at a transition temperature of 65°C, brDNase(C101A) at 60°C, and brDNase(F192C/A217C) at 73°C, indicating that the small disulfide, albeit not essential for activity, is important for the structural integrity, and that the introduction of a third disulfide can further stabilize the enzyme. When pellets of brDNase(C173A) and brDNase(C209A) in inclusion bodies were dissolved in 6 M guanidine-HCl and then diluted into a Ca2+-containing buffer, 10%–18% of the bpDNase activity was restored, suggesting that the “essential” disulfide is not absolutely crucial for enzymatic catalysis. Owing to the structure-based sequence alignment revealing homology between the “nonessential” disulfide of bpDNase and the active-site motif of thioredoxin, we measured 39% of the thioredoxin-like activity for bpDNase based on the rate of insulin precipitation (ΔA650nm/min). Thus, the disulfides in bpDNase not only play the role of stabilizing the protein molecule but also may engage in biological functions such as the disulfide/dithiol exchange reaction