The effects of epigenetic modifications on bone remodeling in age-related osteoporosis.

PURPOSE: As the population ages, there is an increased risk of fracture and morbidity diseases associated with aging, such as age-related osteoporosis and other bone diseases linked to aging skeletons. RESULTS: Several bone-related cells, including multipotent bone mesenchymal stem cells, osteoblasts that form bone tissue, and osteoclasts that break it down, are in symbiotic relationships throughout life. Growing evidence indicates that epigenetic modifications of cells caused by aging contribute to compromised bone remodeling and lead to osteoporosis. A number of epigenetic mechanisms are at play, including DNA/RNA modifications, histone modifications, microRNAs (miRNAs), and long noncoding RNAs (lncRNAs), as well as chromatin remodeling. CONCLUSION: In this review, we summarized the epigenetic modifications of different bone-related cells during the development and progression of osteoporosis associated with aging. Additionally, we described a compensatory recovery mechanism under epigenetic regulation that may lead to new strategies for regulating bone remodeling in age-related osteoporosis.

Mannose-doped metal-organic frameworks induce tumor cell pyroptosis via the PERK pathway.

BACKGROUND: The implementation of pyroptosis exhibits significant potential as a tactic to enhance tumor immune microenvironments. Previous applications of pyroptosis inducers have encountered various limitations, such as the development of drug resistance, manifestation of toxic side effects, and a deficiency in targeting capabilities. As a result, there is a growing demand for tumor therapeutic molecules that can overcome these obstacles. Therefore, the objective of this study is to develop a multifunctional nanospheres that addresses these challenges by enabling high-precision targeting of tumor cells and inducing effective pyroptosis. RESULTS: We prepared a mannose-modified MOF called mannose-doped Fe3O4@NH2-MIL-100 (M-FNM). M-FNM could enter CAL27 cells through MR-mediated endocytosis, which caused in a significant increase in the level of intracellular ROS. This increase subsequently triggered ER stress and activated the PERK-eIF2α-ATF4-CHOP signaling pathway. CHOP then mediated the downstream cascade of Caspase-1, inducing pyroptosis. In in vivo experiments, M-FNM demonstrated excellent targeting ability and exhibited anti-tumor effects. Additionally, M-FNM reshaped the immune microenvironment by promoting the infiltration of anti-tumor immune cells, primarily T lymphocytes. CONCLUSIONS: M-FNM significantly decreased tumor growth. This novel approach to induce pyroptosis in tumor cells using M-FNM may offer new avenues for the development of effective immunotherapies against cancer.

Exogenous adenosine activates A2A adenosine receptor to inhibit RANKL-induced osteoclastogenesis via AP-1 pathway to facilitate bone repair.

BACKGROUND: Adenosine is a purine nucleoside involved in regulating bone homeostasis through binding to A1, A2A, A2B, and A3 adenosine receptors (A1R, A2AR, A2BR, and A3R, respectively). However, the underlying mechanisms by which adenosine and receptor subtypes regulate osteoclast differentiation remain uncertain. This study aims to assess the role of exogenous adenosine and receptor subtypes in receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation and explore the underlying molecular mechanisms. METHODS AND RESULTS: The nanofibrous mats incorporated with adenosine exhibited robust ability to facilitate rat critical-size calvarial defect healing with decreased number of osteoclasts. Moreover, exogenous adenosine substantially enhanced the expression of A2AR and suppressed tartrate-resistant acid phosphatase-positive osteoclast formation and expression of osteoclast-related genes Ctsk, NFATc1, MMP9, and ACP5. This enhancement and suppression could be reversed by adding an A2AR antagonist, ZM241385, in RAW264.7 cells. Finally, RNA sequencing showed that the expression of Fos-related antigen 2 (Fra2) was distinctly downregulated through stimulation of adenosine in RAW264.7 cells treated with RANKL. This downregulation was reversed by ZM241385 according to real-time PCR, Western blot, and immunofluorescence analyses. CONCLUSIONS: These findings demonstrated that exogenous adenosine binding to A2AR attenuated osteoclast differentiation via the inhibition of activating protein-1 (AP-1, including Fra2 subunit) pathway both in vitro and in vivo.

Fe3O4@TiO2-Laden Neutrophils Activate Innate Immunity via Photosensitive Reactive Oxygen Species Release.

Although a variety of advanced sterilization materials and treatments have emerged, the complete elimination of bacterial infection, especially drug-resistant bacterial infection, remains an immense challenge. Here, we demonstrate the use of neutrophils loaded with photocatalytic nanoparticles to reduce bacterial infection. This method activates the immune system to achieve an anti-infection response. We prepared the photocatalytic nanoparticle-laden neutrophils in vivo through neutrophil phagocytosis. The resulting loaded cells retained the cell membrane functionality of the source cell, as well as the complete immune cell function of neutrophils, particularly the ability to recruit macrophages to the target area. Photocatalytic nanoparticle-laden neutrophils can target infection sites and release reactive oxygen species to induce the secretion of chemokines, leading to the targeted recruitment of macrophages and enhancing a powerful immune cascade. In a severe mouse infection model induced by pathogenic bacteria, small doses of photocatalytic nanoparticle-laden neutrophils showed a remarkable therapeutic effect by enhancing macrophage recruitment and the immune cascade.

Near-Infrared Light-Sensitive Nano Neuro-Immune Blocker Capsule Relieves Pain and Enhances the Innate Immune Response for Necrotizing Infection.

Sensory neurons promote profound suppressive effects on neutrophils during Streptococcus pyogenes infection and contribute to the pathogenesis of necrotizing infection ("flesh-eating disease"). Thus, the development of new antibacterial agents for necrotizing infection is promising because of the clear streptococcal neuro-immune communication. Herein, based on the immune escape membrane exterior and competitive membrane functions of the glioma cell membrane, a novel nano neuro-immune blocker capsule was designed to prevent neuronal activation and improve neutrophil immune responses for necrotizing infection. These nano neuro-immune blockers could neutralize streptolysin S, suppress neuron pain conduction and calcitonin gene-related peptide release, and recruit neutrophils to the infection site, providing a strong therapeutic effect against necrotizing infection. Furthermore, nano neuro-immune blockers could serve as an effective inflammatory regulator and antibacterial agent via photothermal effects under near-infrared irradiation. In the Streptococcus pyogenes-induced necrotizing fasciitis mouse model, nano neuro-immune blockers showed significant therapeutic efficacy by ameliorating sensitivity to pain and promoting the antibacterial effect of neutrophils.

HnRNPL inhibits the osteogenic differentiation of PDLCs stimulated by SrCl2 through repressing Setd2.

Osteoporosis has been shown to intensify bone loss caused by periodontitis and both share common risk factors. One strategy utilized to manage the disease has been via the release of Sr ions by Strontium Ranelate having a direct effect on preventing osteoclast activation and promoting osteoblast differentiation. Previously we have developed and characterized porous Sr-mesoporous bioactive glass (Sr-MBG) scaffolds and demonstrated their ability to promote periodontal regeneration when compared to MBG alone. Our group further discovered a splicing factor, heterogeneous nuclear ribonucleoprotein L (hnRNPL), was drastically down-regulated in periodontal ligament stem cells (PDLCs) stimulated by Sr through the activation of AKT pathway. Furthermore, hnRNPL restrained the osteogenic differentiation of PDLCs through down-regulating H3K36me3-specific methyltransferase Setd2. The goal of the present study was to investigate the mechanism of periodontal regeneration stimulated by Sr It was first found that the epigenetic mechanism of splicing factor hnRNPL participated in the osteogenesis processing of PDLCs stimulated by SrCl2 . Meanwhile, the different role of hnRNPL and SET domain containing 2 (Setd2) may provide some implication of the treatment of periodontitis patients simultaneously suffering from osteoporosis.

Transcription factor 7-like 2-associated signaling mechanism in regulating cementum generation by the NF-κB pathway.

Cementum regeneration is an important and challenging stage in periodontal tissue engineering and regeneration. Pathosis of the periodontium, including cementum, is important in precision diagnosis and obstinate treatment of systemic diseases, such as diabetes, leukemia, and Acquired Immune Deficiency Syndrome. Here, we found that during periodontium development, transcription factor 7-like 2 (Tcf7l2) was widely expressed in the periodontium and dental sac. In mouse cementoblast cell line (OCCM-30), the activation of NF-κB and cementoblast mineralization was significantly reduced when Tcf7l2 gene was silenced. Moreover, Tcf7l2 has a positive effect on NF-κB and cementoblast mineralization. Therefore, Tcf7l2 promotes cementum formation through the NF-κB pathway. In addition, we found a decreased expression of phosphorylated p65 and a thin layer of cementum in Tcf7l2fl/fl mice. These results suggest that Tcf7l2, which accelerates cementum formation by activating NF-κB, has great potential in the treatment of periodontitis and provide guidance for periodontal tissue regeneration.

Occurrence of contaminants of emerging concern in surface and waste water from the Yangtze River chemical contiguous zone, China: Distribution, sources and ecological risk assessment.

Limited knowledge exists regarding the occurrence, potential sources, and risks of contaminants of emerging concern (CECs) in surface and waste water from chemical contiguous zones. A total of 136 CECs were detected at 32 sampling sites along the Yangtze River, with concentrations ranging from 0.55 to 4.21 × 104 ng/L. Hydrocortisonacetate, cortisone, prednisone, enalapril and medroxyprogesterone were detected across all sampling sites. Hierarchical cluster analysis based on 47 core CECs yielded similar results compared with principal components analysis and identified two major clusters: wastewater sites and surface water sites. Distinct patterns of CECs were observed in wastewater from three industrial parks owing to variations in the industrial facilities and products within each park. Nineteen CECs were initially classified as presenting a high or medium risk to aquatic organisms. Further quantitative probabilistic risk assessment revealed that caffeine, trenbolone and norethindrone posed a threat to the most vulnerable aquatic species while high-risk sites mainly occurred downstream of the chemical industrial park. The joint ecological risk of high-risk CECs was evaluated using potentially affected fractions, which ranged from 0.44 % to 47.9 % with concentration addition and 0.33 % to 45.1 % with response addition. This suggests the need to consider the joint ecological risk of the detected compounds in future studies.

Novel Carbon Quantum Dots Precisely Trigger Ferroptosis in Cancer Cells through Antioxidant Inhibition Synergistic Nanocatalytic Activity.

High levels of glutathione (GSH) are an important characteristic of malignant tumors and a significant cause of ineffective treatment and multidrug resistance. Although reactive oxygen species (ROS) therapy has been shown to induce tumor cell death, the strong clearance effect of GSH on ROS significantly reduces its therapeutic efficacy. Therefore, there is a need to develop new strategies for targeting GSH. In this study, novel carbon quantum dots derived from gentamycin (GM-CQDs) were designed and synthesized. On the basis of the results obtained, GM-CQDs contain sp2 and sp3 carbon atoms as well as nitrogen oxygen groups, which decrease the intracellular levels of GSH by downregulating SLC7A11, thereby disrupting redox balance, mediating lipid peroxidation, and inducing ferroptosis. Transcriptome analysis demonstrated that GM-CQDs downregulated the expression of molecules related to GSH metabolism while significantly increasing the expression of molecules related to ferroptosis. The in vivo results showed that the GM-CQDs exhibited excellent antitumor activity and immune activation ability. Furthermore, because of their ideal biological safety, GM-CQDs are highly promising for application as drugs targeting GSH in the treatment of malignant tumors.

Effects of biochar in combination with varied N inputs on grain yield, N uptake, NH3 volatilization, and N2O emission in paddy soil.

Biochar application can improve crop yield, reduce ammonia (NH3) volatilization and nitrous oxide (N2O) emission from farmland. We here conducted a pot experiment to compare the effects of biochar application on rice yield, nitrogen (N) uptake, NH3 and N2O losses in paddy soil with low, medium, and high N inputs at 160 kg/ha, 200 kg/ha and 240 kg/ha, respectively. The results showed that: (1) Biochar significantly increased the rice grain yield at medium (200 kg/ha) and high (240 kg/ha) N inputs by 56.4 and 70.5%, respectively. The way to increase yield was to increase the rice N uptake, rice panicle number per pot and 1,000 grain weight by 78.5-96.5%, 6-16% and 4.4-6.1%, respectively; (2) Under low (160 kg/ha) N input, adding biochar effectively reduced the NH3 volatilization by 31.6% in rice season. The decreases of pH value and NH4+-N content in surface water, and the increases of the abundance of NH4+-N oxidizing archaea and bacteria (AOA and AOB) communities contributed to the reduction of NH3 volatilization following the biochar application; (3) Under same N input levels, the total N2O emission in rice season decreased by 43.3-73.9% after biochar addition. The decreases of nirK and nirS gene abundances but the increases of nosZ gene abundance are the main mechanisms for biochar application to reduce N2O emissions. Based on the results of the current study, adding biochar at medium (200 kg/ha) N level (N200 + BC) is the best treatment to synchronically reduce NH3 and N2O losses, improve grain yield, and reduce fertilizer application in rice production system.

Micro/nano-modified titanium surfaces accelerate osseointegration via Rab7-dependent mitophagy.

To achieve rapid and successful osseointegration of titanium (Ti) implants, the underlying mechanisms of surface modification-mediated bone metabolism need to be clarified. Given that the microenvironment surrounding Ti implants may be altered after implant insertion, mitophagy as a key control system for cellular homeostasis is most likely to regulate osseointegration. Recent findings suggest that PTEN-induced putative kinase 1 (Pink1)/Parkin-mediated mitophagy plays a key role in bone metabolism. Since the micro/nano-modified surfaces of Ti implants have been widely appreciated for osseointegration acceleration, we used two common micro/nano-modified techniques and demonstrated elevations of both the osteo-differentiation potential and Pink1/Parkin pathway of osteoblasts. Moreover, the Pink1/Parkin pathway exhibited an upward trend during osteoblast differentiation. However, when osteoblasts were treated with CCCP, a Pink1/Parkin inducer, the osteo-differentiation potential decreased. Our further study showed that the small GTPase Rab7, which was inhibited by CCCP, was essential for the Pink1/Parkin pathway. Upon Pink1 or Rab7 knockdown, the pro-osteogenic effect of micro/nano-modified Ti surfaces was significantly weakened. The present results demonstrated that Rab7 activation was essential for active mitophagy and osteogenesis. In addition, Rab7 was confirmed to mediate the process of autophagosome formation. Our findings provide novel insights into new targets for osseointegration promotion, regardless of Ti surface characteristics.

Toxicological effects of tris(1,3-dichloro-2-propyl) phosphate in oyster Crassostrea gigas using proteomic and phosphoproteomic analyses.

As a typical organophosphorus pollutant, tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been widely detected in aquatic environment. Previous studies showed that protein phosphorylation might be a vital way of TDCIPP to exert multiple toxic effects. However, there is a lack of high-throughput investigations on how TDCIPP affected protein phosphorylation. In this study, the toxicological effects of TDCIPP were explored by proteomic and phosphoproteomic analyses together with traditional means in oysters Crassostrea gigas treated with 0.5, 5 and 50 µg/L TDCIPP for 28 days. Integration of omic analyses revealed that TDCIPP dysregulated transcription, energy metabolism, and apoptosis and cell proliferation by either directly phosphorylating pivotal proteins or phosphorylating their upstream signaling pathways. The U-shaped response of acetylcholinesterase activities suggested the neurotoxicity of TDCIPP in a hormesis manner. What's more, the increase in caspase-9 activity as well as the expression or phosphorylation alterations in eukaryotic translation initiation factor 4E, cell division control protein 42 and transforming growth factor-ß1-induced protein indicated the disruption of homeostasis between apoptosis and cell proliferation, which was consistent with the observation of shedding of digestive cells. Overall, combination of proteomic and phosphoproteomic analyses showed the capability of identifying molecular events, which provided new insights into the toxicological mechanisms of TDCIPP.

Porphyromonas gingivalis lipopolysaccharide induced RIPK3/MLKL-mediated necroptosis of oral epithelial cells and the further regulation in macrophage activation.

Necroptosis, a new type of regulated cell death with massive release of damage-associated molecular patterns (DAMPs), is involved in the pathogenesis of periodontitis. However, the role of necroptosis in oral epithelial cells and the following effect on macrophages activation remain unknown. Human immortalized oral epithelial cells were stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). Cell death was assessed while expressions of RIPK3/MLKL and toll-like receptors (TLRs) were evaluated. Necrosulfonamide (NSA), an inhibitor of MLKL was applied to block necroptosis. The expression of DAMPs and the epithelial connection protein were evaluated by qPCR and immunofluorescence, respectively. Immortalized human monocytes U937 were induced into the M0 or M2 subset, and influences of HIOECs-derived DAMPs on macrophage polarization as well as activation of the Mincle/SYK axis were assessed. P. gingivalis LPS could be recognized by TLR2 and regulates necroptosis of HIOECs by activating RIPK3/MLKL. NSA inhibited cell death of HIOECs, alleviated impaired epithelial connection, and inhibited expressions of DAMPs. Low dose of DAMPs derived from HIOECs promoted M2-like polarization by activating the Mincle/SYK axis, which was significantly suppressed with increased doses of DAMPs. P. gingivalis LPS destructed oral epithelial cells via RIPK3/MLKL-mediated necroptosis, which further regulated macrophage activation via DAMPs from oral epithelial cells.

Erratum: Minimally invasive implantation and decreased inflammation reduce osteoinduction of biomaterial: Erratum.

[This corrects the article DOI: 10.7150/thno.39507.].

Individualized plasticity autograft mimic with efficient bioactivity inducing osteogenesis.

Mineralized tissue regeneration is an important and challenging part of the field of tissue engineering and regeneration. At present, autograft harvest procedures may cause secondary trauma to patients, while bone scaffold materials lack osteogenic activity, resulting in a limited application. Loaded with osteogenic induction growth factor can improve the osteoinductive performance of bone graft, but the explosive release of growth factor may also cause side effects. In this study, we innovatively used platelet-rich fibrin (PRF)-modified bone scaffolds (Bio-Oss®) to replace autograft, and used cytokine (BMP-2) to enhance osteogenesis. Encouragingly, this mixture, which we named "Autograft Mimic (AGM)", has multiple functions and advantages. (1) The fiber network provided by PRF binds the entire bone scaffold together, thereby shaping the bone grafts and maintaining the space of the defect area. (2) The sustained release of BMP-2 from bone graft promoted bone regeneration continuously. (3) AGM recruited bone marrow mesenchymal stem cells (BMSCs) and promote their proliferation, migration, and osteogenic differentiation. Thus, AGM developed in this study can improve osteogenesis, and provide new guidance for the development of clinical bone grafts.

Biomimetic Silk Fibroin Hydrogels Strengthened by Silica Nanoparticles Distributed Nanofibers Facilitate Bone Repair.

Various materials are utilized as artificial substitutes for bone repair. In this study, a silk fibroin (SF) hydrogel reinforced by short silica nanoparticles (SiNPs)-distributed-silk fibroin nanofibers (SiNPs@NFs), which exhibits a superior osteoinductive property, is fabricated for treating bone defects. SF acts as the base part of the composite scaffold to mimic the extracellular matrix (ECM), which is the organic component of a native bone. The distribution of SiNPs clusters within the composite hydrogel partially mimics the distribution of mineral crystals within the ECM. Incorporation of SiNPs@NFs enhances the mechanical properties of the composite hydrogel. In addition, the composite hydrogel provides a biocompatible microenvironment for cell adhesion, proliferation, and osteogenic differentiation in vitro. In vivo studies confirm that the successful repair is achieved with the formation of a large amount of new bone in the large-sized cranial defects that are treated with the composite hydrogel. In conclusion, the SiNPs@NFs-reinforced-hydrogel fabricated in this study has the potential for use in bone tissue engineering.

Sustained calcium ion release from bioceramics promotes CaSR-mediated M2 macrophage polarization for osteoinduction.

Innate immune cells, especially macrophages, play a dual role in tissue repair and the defense against foreign bodies. Although biphasic calcium phosphate (BCP) ceramics have been confirmed as an excellent osteoimmunoregulatory biomaterial, it is unclear whether the ions release of BCP directly affects macrophage polarization and the mechanism by which the ions release is involved in osteoimmunomodulation. Herein, we verified the superior osteoinductive capacity of BCP in wild-type mice and showed its inability to promote this process in macrophage-deficient (LysM-/- ) mice. Moreover, scanning electron microscopy, ion release curve, and calcein AM-staining results confirmed that BCP-released Ca2+ in a sustained manner, thereby maintaining the long-term induction of M2 macrophage polarization and promoting the differentiation of mesenchymal stem cells into osteoblasts during osteogenesis. Furthermore, Ca2+ targeted the Wnt/ß-catenin signaling pathway and activated Arg1 and IL-10 (M2 marker genes) transcription through the calcium-sensing receptor (CaSR) in macrophages. Under treatment with a CaSR antagonist, macrophages cultured with the BCP fluid extract exhibited lower Ca2+ intake and weaker M2 macrophage polarization. These findings underscore the critical role of macrophages in bone regeneration and clarify the molecular mechanisms of Ca2+ -mediated osteoinduction by biomaterials, which is of great significance for the future design of biomaterial-oriented tissue regeneration engineering.

Activation of Autophagy Through the NLRP3/mTOR Pathway: A Potential Mechanism for Alleviation of Pneumonia by QingFei Yin.

QingFei Yin (QFY), a Chinese traditional medicine recipe, is known for its excellent therapeutic pharmacological effects for the treatment of bacterial lung infections, although its molecular mechanism of action remains unknown. Here, QFY chemical composition was determined using a High-Performance Liquid Chromatography-Mass (HPLC-MS/MS)-based method then QFY was evaluated for protective pharmacological effects against pneumonia using two models: a Streptococcus pneumoniae-induced in vivo mouse model and an in vitro pneumolysin (PLY)-induced murine lung alveolar-derived MH-S cell line-based model. Notably, QFY exerted prominent anti-pneumonia effects both in vivo and in vitro. To further explore QFY protective effects, 4D label-free proteomics analysis, pathologic evaluation, and immunohistochemical (IHC) analysis were conducted to identify cellular pathways involved in QFY protection. Notably, our results indicated that NF-κB/NLRP3 and autophagy pathways may contribute to pharmacological effects associated with QFY-based protection. Briefly, QFY triggered autophagy via down-regulation of upstream NLRP3/mTOR signaling pathway events, resulting in the amelioration of inflammatory injury. Collectively, our results revealed molecular mechanisms underlying QFY protection against pneumonia as a foundation for the future development of novel treatments to combat this disease and reduce antibiotic abuse.

Electrospinning Nanofiber-Reinforced Aerogels for the Treatment of Bone Defects.

Objective: Application of aerogels in bone tissue engineering is an emerging field, while the reports of electrospinning nanofiber-reinforced aerogels are limited. This research aimed at fabricating the nanofiber-reinforced aerogels and evaluating their physiochemical and biological properties. Approach: The chitosan (CS) aerogels incorporated with cellulose acetate (CA) and poly (ɛ-caprolactone) (PCL) nanofibers were fabricated via ball milling and freeze-drying techniques. Scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectrum, X-ray photoelectron spectroscopy (XPS), compressive experiment, and in vitro experiment were conducted to assess their physiochemical properties and biological behavior. Results: The SEM examination showed that satisfying morphology was attained in the CA/PCL/CS aerogels with incorporation of CA/PCL nanofibers and CS solution. The results of FT-IR and XPS indicated the perfect incorporation of CA, PCL, and CS. A compressive experiment confirmed that the CA/PCL/CS aerogels enhanced the compressive modulus of the pure CS aerogel. For in vitro experiment, the CA/PCL/CS composite scaffolds were proven to possess better cytocompatibility compared with the pure CS. Also, cells on the CA/PCL/CS showed well-extended morphology and could infiltrate into a porous scaffold. Furthermore, confocal experiment revealed that the CA/PCL/CS could also promote the osteogenic differentiation of MC3T3-E1 cells. Innovation: This study fabricated the nanofiber-reinforced aerogels mainly to optimize the cell/material interaction of the pure CS scaffold. Conclusion: The CA/PCL nanofibers not only improved the mechanical property of the CS aerogel to some extent but also facilitated cell adhesion and osteogenic differentiation. Thus, it could be considered a promising candidate for bone tissue engineering.

Confirmation of brand identification in infant formulas by using near-infrared spectroscopy fingerprints.

In this study, a near infrared (NIR) spectroscopy fingerprinting method coupled with principal component analysis (PCA) was developed for the confirmation of brand identification in infant formulas. The NIR spectroscopy fingerprints of the Brand A infant formula were acquired in 12 000-4000 cm-1 at a sample temperature of 20 °C without pressing the sample. The contents of major nutrients of Stage 1, 2, and 3 infant formulas were compared within Brand A. The NIR spectroscopy fingerprints of Brand A Stage 1 samples were compared with those of four other brand-named Stage 1 samples, whereas the fingerprints of Brand A Stage 2 and 3 were compared with those of two of the four brands, to distinguish the differences between brands. The NIR spectroscopy fingerprinting results showed that the Brand A formula can be completely differentiated from the other brands at each stage. The combination of NIR spectroscopy fingerprinting and PCA is an effective method for the purpose of confirmation of brand identification and brand protection in infant formulas.