Dityrosine Crosslinking of Collagen and Amyloid-β Peptides Is Formed by Vitamin B12 Deficiency-Generated Oxidative Stress in Caenorhabditis elegans

(1) Background: Vitamin B₁₂ deficiency in Caenorhabditis elegans results in severe oxidative stress and induces morphological abnormality in mutants due to disordered cuticle collagen biosynthesis. We clarified the underlying mechanism leading to such mutant worms due to vitamin B₁₂ deficiency. (2) Results: The deficient worms exhibited decreased collagen levels of up to approximately 59% compared with the control. Although vitamin B₁₂ deficiency did not affect the mRNA expression of prolyl 4-hydroxylase, which catalyzes the formation of 4-hydroxyproline involved in intercellular collagen biosynthesis, the level of ascorbic acid, a prolyl 4-hydroxylase coenzyme, was markedly decreased. Dityrosine crosslinking is involved in the extracellular maturation of worm collagen. The dityrosine level of collagen significantly increased in the deficient worms compared with the control. However, vitamin B₁₂ deficiency hardly affected the mRNA expression levels of bli-3 and mlt-7, which are encoding crosslinking-related enzymes, suggesting that deficiency-induced oxidative stress leads to dityrosine crosslinking. Moreover, using GMC101 mutant worms that express the full-length human amyloid β, we found that vitamin B₁₂ deficiency did not affect the gene and protein expressions of amyloid β but increased the formation of dityrosine crosslinking in the amyloid β protein. (3) Conclusions: Vitamin B₁₂-deficient wild-type worms showed motility dysfunction due to decreased collagen levels and the formation of highly tyrosine-crosslinked collagen, potentially reducing their flexibility. In GMC101 mutant worms, vitamin B₁₂ deficiency-induced oxidative stress triggers dityrosine-crosslinked amyloid β formation, which might promote its stabilization and toxic oligomerization.     

Structure of N-Terminal Sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser of Aβ-Peptide with Phospholipase A2 from Venom of Andaman Cobra Sub-Species Naja naja sagittifera at 2.0 ? Resolution

Alzheimer’s disease (AD) is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ) are the prime player of AD’s neuropathology. Studies have implicated the varied role of phospholipase A₂ (PLA₂) in brain where it contributes to neuronal growth and inflammatory response. Overall contour and chemical nature of the substrate-binding channel in the low molecular weight PLA₂s are similar. This study involves the reductionist fragment-based approach to understand the structure adopted by N-terminal fragment of Alzheimer’s Aβ peptide in its complex with PLA₂. In the current communication, we report the structure determined by X-ray crystallography of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser (DAEFRHDS) of Aβ-peptide with a Group I PLA₂ purified from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution (Protein Data Bank (PDB) Code: 3JQ5). This is probably the first attempt to structurally establish interaction between amyloid-β peptide fragment and hydrophobic substrate binding site of PLA₂ involving H bond and van der Waals interactions. We speculate that higher affinity between Aβ and PLA₂ has the therapeutic potential of decreasing the Aβ–Aβ interaction, thereby reducing the amyloid aggregation and plaque formation in AD.     

CuInS2 quantum dot-sensitized TiO2 nanorod array photoelectrodes: synthesis and performance optimization

CuInS₂ quantum dots (QDs) were deposited onto TiO₂ nanorod arrays for different cycles by using successive ionic layer adsorption and reaction (SILAR) method. The effect of SILAR cycles on the light absorption and photoelectrochemical properties of the sensitized photoelectrodes was studied. With optimization of CuInS₂ SILAR cycles and introduction of In₂S₃ buffer layer, quantum dot-sensitized solar cells assembled with 3-μm thick TiO₂ nanorod film exhibited a short-circuit current density (I_sc) of 4.51 mA cm⁻², an open-circuit voltage (V_oc) of 0.56 V, a fill factor (FF) of 0.41, and a power conversion efficiency (η) of 1.06%, respectively. This study indicates that SILAR process is a very promising strategy for preparing directly anchored semiconductor QDs on TiO₂ nanorod surface in a straightforward but controllable way without any complicated fabrication procedures and introduction of a linker molecule.     

Differential Regulation of Ca2+-Activated Cl− Channel TMEM16A Splice Variants by Membrane PI(4,5)P2

TMEM16A is a Ca²⁺-activated Cl⁻ channel that controls broad cellular processes ranging from mucus secretion to signal transduction and neuronal excitability. Recent studies have reported that membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is an important cofactor that allosterically regulates TMEM16A channel activity. However, the detailed regulatory actions of PIP₂ in splice variants of TMEM16A remain unclear. Here, we demonstrated that the attenuation of membrane phosphoinositide levels selectively inhibited the current amplitude of the TMEM16A(ac) isoform by decreasing the slow, but not instantaneous, Cl⁻ currents, which are independent of the membrane potential and specific to PI(4,5)P₂ depletion. The attenuation of endogenous PI(4,5)P₂ levels by the activation of Danio rerio voltage-sensitive phosphatase (Dr-VSP) decreased the Cl⁻ currents of TMEM16A(ac) but not the TMEM16A(a) isoform, which was abolished by the co-expression of PIP 5-kinase type-1γ (PIPKIγ). Using the rapamycin-inducible dimerization of exogenous phosphoinositide phosphatases, we further revealed that the stimulatory effects of phosphoinositide on TMEM16A(ac) channels were similar in various membrane potentials and specific to PI(4,5)P₂, not PI4P and PI(3,4,5)P₃. Finally, we also confirmed that PI(4,5)P₂ resynthesis is essential for TMEM16A(ac) recovery from Dr-VSP-induced current inhibition. Our data demonstrate that membrane PI(4,5)P₂ selectively modulates the gating of the TMEM16A(ac) channel in an agonistic manner, which leads to the upregulation of TMEM16A(ac) functions in physiological conditions.     

Indomethacin Disrupts the Formation of β-Amyloid Plaques via an α2-Macroglobulin-Activating lrp1-Dependent Mechanism

Epidemiological studies have implied that the nonsteroidal anti-inflammatory drug (NSAID) indomethacin slows the development and progression of Alzheimer’s disease (AD). However, the underlying mechanisms are notably understudied. Using a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9) (APP/PS1) expressing transgenic (Tg) mice and neuroblastoma (N) 2a cells as in vivo and in vitro models, we revealed the mechanisms of indomethacin in ameliorating the cognitive decline of AD. By screening AD-associated genes, we observed that a marked increase in the expression of α₂-macroglobulin (A2M) was markedly induced after treatment with indomethacin. Mechanistically, upregulation of A2M was caused by the inhibition of cyclooxygenase-2 (COX-2) and lipocalin-type prostaglandin D synthase (L-PGDS), which are responsible for the synthesis of prostaglandin (PG)H₂ and PGD₂, respectively. The reduction in PGD₂ levels induced by indomethacin alleviated the suppression of A2M expression through a PGD₂ receptor 2 (CRTH2)-dependent mechanism. Highly activated A2M not only disrupted the production and aggregation of β-amyloid protein (Aβ) but also induced Aβ efflux from the brain. More interestingly, indomethacin decreased the degradation of the A2M receptor, low-density lipoprotein receptor-related protein 1 (LRP1), which facilitated the brain efflux of Aβ. Through the aforementioned mechanisms, indomethacin ameliorated cognitive decline in APP/PS1 Tg mice by decreasing Aβ production and clearing Aβ from the brains of AD mice.     

Nano SiO2 and MgO Improve the Properties of Porous β-TCP Scaffolds via Advanced Manufacturing Technology

Nano SiO₂ and MgO particles were incorporated into β-tricalcium phosphate (β-TCP) scaffolds to improve the mechanical and biological properties. The porous cylindrical β-TCP scaffolds doped with 0.5 wt % SiO₂, 1.0 wt % MgO, 0.5 wt % SiO₂ + 1.0 wt % MgO were fabricated via selective laser sintering respectively and undoped β-TCP scaffold was also prepared as control. The phase composition and mechanical strength of the scaffolds were evaluated. X-ray diffraction analysis indicated that the phase transformation from β-TCP to α-TCP was inhibited after the addition of MgO. The compressive strength of scaffold was improved from 3.12 ± 0.36 MPa (β-TCP) to 5.74 ± 0.62 MPa (β-TCP/SiO₂), 9.02 ± 0.55 MPa (β-TCP/MgO) and 10.43 ± 0.28 MPa (β-TCP/SiO₂/MgO), respectively. The weight loss and apatite-forming ability of the scaffolds were evaluated by soaking them in simulated body fluid. The results demonstrated that both SiO₂ and MgO dopings slowed down the degradation rate and improved the bioactivity of β-TCP scaffolds. In vitro cell culture studies indicated that SiO₂ and MgO dopings facilitated cell attachment and proliferation. Combined addition of SiO₂ and MgO were found optimal in enhancing both the mechanical and biological properties of β-TCP scaffold.     

Magnetic properties of fluffy Fe@α-Fe2O3 core-shell nanowires

Novel fluffy Fe@α-Fe₂O₃ core-shell nanowires have been synthesized using the chemical reaction of ferrous sulfate and sodium borohydride, as well as the post-annealing process in air. The coercivity of the as-synthesized nanowires is above 684 Oe in the temperature range of 5 to 300 K, which is significantly higher than that of the bulk Fe (approximately 0.9 Oe). Through the annealing process in air, the coercivity and the exchange field are evidently improved. Both the coercivity and the exchange field increase with increasing annealing time (T_A) and reach their maximum values of 1,042 and 78 Oe, respectively, at T_A = 4 h. The magnetic measurements show that the effective anisotropy is increased with increasing the thickness of theα-Fe₂O₃ by annealing. The large values of coercivity and exchange field, as well as the high surface area to volume ratio, may make the fluffy Fe@α-Fe₂O₃ core-shell nanowire a promising candidate for the applications of the magnetic drug delivery, electrochemical energy storage, gas sensors, photocatalysis, and so forth.     

Binding of Kingella kingae RtxA Toxin Depends on Cell Surface Oligosaccharides,but Not on β2 Integrins

The Gram-negative coccobacillus Kingella kingae is increasingly recognized as an important invasive pediatric pathogen that causes mostly bacteremia and skeletal system infections. K. kingae secretes an RtxA toxin that belongs to a broad family of the RTX (Repeats in ToXin) cytotoxins produced by bacterial pathogens. Recently, we demonstrated that membrane cholesterol facilitates interaction of RtxA with target cells, but other cell surface structures potentially involved in toxin binding to cells remain unknown. We show that deglycosylation of cell surface structures by glycosidase treatment, or inhibition of protein N- and O-glycosylation by chemical inhibitors substantially reduces RtxA binding to target cells. Consequently, the deglycosylated cells were more resistant to cytotoxic activity of RtxA. Moreover, experiments on cells expressing or lacking cell surface integrins of the β₂ family revealed that, unlike some other cytotoxins of the RTX family, K. kingae RtxA does not bind target cells via the β₂ integrins. Our results, hence, show that RtxA binds cell surface oligosaccharides present on all mammalian cells but not the leukocyte-restricted β₂ integrins. This explains the previously observed interaction of the toxin with a broad range of cell types of various mammalian species and reveals that RtxA belongs to the group of broadly cytolytic RTX hemolysins.     

Improving the performance of dye-sensitized solar cells with TiO2/graphene/TiO2 sandwich structure

This study investigates the extent to which the TiO₂/graphene/TiO₂ sandwich structure improves the performance of dye-sensitized solar cells (DSSCs) over that of DSSCs with the traditional structure. Studies have demonstrated that the TiO₂/graphene/TiO₂ sandwich structure effectively enhances the open circuit voltage (V_oc), short-circuit current density (J_sc), and photoelectrical conversion efficiency (η) of DSSCs. The enhanced performance of DSSCs with the sandwich structure can be attributed to an increase in electron transport efficiency and in the absorption of light in the visible range. The DSSC with the sandwich structure in this study exhibited a V_oc of 0.6 V, a high J_sc of 11.22 mA cm^-2, a fill factor (FF) of 0.58, and a calculated η of 3.93%, which is 60% higher than that of a DSSC with the traditional structure.     

Zinc Induced Aβ16 Aggregation Modeled by Molecular Dynamics

It is widely accepted that the addition of zinc leads to the formation of neurotoxic nonfibrillar aggregates of beta-amyloid peptides Aβ₄₀ and Aβ₄₂ and at the same time destabilizes amyloid fibrils. However, the mechanism of the effect of zinc on beta-amyloid is not fully understood. In this study, a fast zinc-induced aggregation of Aβ₁₆ (as compared to a system without zinc) via the formation of Aβ₁₆ dimers with one zinc ion coordinated in the metal-binding site ₁₁EVHH₁₄, followed by their polymerization, has been studied by molecular dynamics. The best aggregation was shown by the system composed of Aβ₁₆ dimers bound by one zinc ion, with no additional zinc in solution. The presence of Aβ₁₆ dimers was a major condition, sufficient for fast aggregation into larger complexes. It has been shown that the addition of zinc to a system with already formed dimers does not substantially affect the characteristics and rate of aggregation. At the same time, an excessive concentration of zinc at the early stages of the formation of conglomerates can negatively affect aggregation, since in systems where zinc ions occupied the ₁₁EVHH₁₄ coordination center and the His6 residue of every Aβ₁₆ monomer, the aggregation proceeded more slowly and the resulting complexes were not as large as in the zinc-free Aβ system. Thus, this study has shown that the formation of Aβ₁₆ dimers bound through zinc ions at the ₁₁EVHH₁₄ sites of the peptides plays an important role in the formation of neurotoxic non-fibrillar aggregates of beta-amyloid peptide Aβ₁₆. The best energetically favorable structure has been obtained for the complex of two Aβ₁₆ dimers with two zinc ions.     

Alzheimer’s Disease—A Panorama Glimpse

The single-mutation of genes associated with Alzheimer’s disease (AD) increases the production of Aβ peptides. An elevated concentration of Aβ peptides is prone to aggregation into oligomers and further deposition as plaque. Aβ plaques and neurofibrillary tangles are two hallmarks of AD. In this review, we provide a broad overview of the diverses sources that could lead to AD, which include genetic origins, Aβ peptides and tau protein. We shall discuss on tau protein and tau accumulation, which result in neurofibrillary tangles. We detail the mechanisms of Aβ aggregation, fibril formation and its polymorphism. We then show the possible links between Aβ and tau pathology. Furthermore, we summarize the structural data of Aβ and its precursor protein obtained via Nuclear Magnetic Resonance (NMR) or X-ray crystallography. At the end, we go through the C-terminal and N-terminal truncated Aβ variants. We wish to draw reader’s attention to two predominant and toxic Aβ species, namely Aβ₄₋₄₂/ and pyroglutamate amyloid-beta peptides, which have been neglected for more than a decade and may be crucial in Aβ pathogenesis due to their dominant presence in the AD brain.     

Simple fabrication of N-doped mesoporous TiO2 nanorods with the enhanced visible light photocatalytic activity

N-doped mesoporous TiO₂ nanorods were fabricated by a modified and facile sol–gel approach without any templates. Ammonium nitrate was used as a raw source of N dopants, which could produce a lot of gasses such as N₂, NO₂, and H₂O in the process of heating samples. These gasses were proved to be vitally important to form the special mesoporous structure. The samples were characterized by the powder X-ray diffraction, X-ray photoelectron spectrometer, nitrogen adsorption isotherms, scanning electron microscopy, transmission electron microscopy, and UV-visible absorption spectra. The average length and the cross section diameter of the as-prepared samples were ca. 1.5 μm and ca. 80 nm, respectively. The photocatalytic activity was evaluated by photodegradation of methylene blue (MB) in aqueous solution. The N-doped mesoporous TiO₂ nanorods showed an excellent photocatalytic activity, which may be attributed to the enlarged surface area (106.4 m² g^-1) and the narrowed band gap (2.05 eV). Besides, the rod-like photocatalyst was found to be easy to recycle.     

Single-crystalline δ-Ni2Si nanowires with excellent physical properties

In this article, we report the synthesis of single-crystalline nickel silicide nanowires (NWs) via chemical vapor deposition method using NiCl₂·6H₂O as a single-source precursor. Various morphologies of δ-Ni₂Si NWs were successfully acquired by controlling the growth conditions. The growth mechanism of the δ-Ni₂Si NWs was thoroughly discussed and identified with microscopy studies. Field emission measurements show a low turn-on field (4.12 V/μm), and magnetic property measurements show a classic ferromagnetic characteristic, which demonstrates promising potential applications for field emitters, magnetic storage, and biological cell separation.     

The Role of Melatonin on Behavioral Changes and Concomitant Oxidative Stress in icvAβ1-42 Rat Model with Pinealectomy

One of the pathological hallmarks of Alzheimer’s disease (AD) associated with its progression that contributes to β-amyloid (Aβ) generation is oxidative stress (OS). Clinical data suggest that melatonin is a potent antioxidant that might be effective in the adjunctive therapy of this neurodegenerative disease. The present study aimed to explore the role of melatonin on behavioral changes and markers of OS in three rat models, namely, pinealectomy (pin) model of melatonin deficit, intracerebroventricular (icv)Aβ_1-42 model of AD, and combination of both pin and Aβ_1-42 model (pin+icvAβ_1-42). The chronic injection with vehicle/melatonin (50 mg/kg, i.p. for 40 days) started on the same day of sham/pin and icv vehicle/Aβ_1-42 infusion procedures. Anxiety in the open field and the elevated plus-maze test and cognitive responses in the object recognition test were tested between the 30th–35th day after the surgical procedures. Markers of OS in the frontal cortex (FC) and hippocampus were detected by the ELISA method. Melatonin treatment corrected the exacerbated anxiety response only in the pin+icvAβ_1-42 model while it alleviated the cognitive impairment in the three models. Pinealectomy disturbed the antioxidant system via enhanced SOD activity and decreased GSH levels both in the FC and hippocampus. The Aβ_1-42 model decreased the SOD activity in the FC and elevated the MDA level in the two brain structures. The pin+icvAβ_1-42 model impaired the antioxidant system and elevated lipid peroxidation. Melatonin supplementation restored only the elevated MDA level of icvAβ_1-42 and pin+icvAβ_1-42 model in the hippocampus. In conclusion, our study reveals that the pin+icvAβ_1-42 rat model triggers more pronounced anxiety and alterations in markers of OS that may be associated with melatonin deficit concomitant to icvAβ_1-42-induced AD pathology.     

One-pot synthesis of α-Fe2O3 nanospheres by solvothermal method

We have successfully prepared α-Fe₂O₃ nanospheres by solvothermal method using 2-butanone and water mixture solvent for the first time, which were about 100 nm in diameter and composed of very small nanoparticles. The as-prepared samples were characterized using X-ray diffraction, scanning electron microscopy, and transmission electron microscopy. The results showed that the product was α-Fe₂O₃ nanosphere, and the temperature was an important factor on the formation of α-Fe₂O₃ nanospheres.     

Effect of TiO2 nanotubes with TiCl4 treatment on the photoelectrode of dye-sensitized solar cells

In this study, we used the electrochemical anodization to prepare TiO₂ nanotube arrays and applied them on the photoelectrode of dye-sensitized solar cells. In the field emission scanning electron microscopy analysis, the lengths of TiO₂ nanotube arrays prepared by electrochemical anodization can be obtained with approximately 10 to 30 μm. After titanium tetrachloride (TiCl₄) treatment, the walls of TiO₂ nanotubes were coated with TiO₂ nanoparticles. XRD patterns showed that the oxygen-annealed TiO₂ nanotubes have a better anatase phase. The conversion efficiency with different lengths of TiO₂ nanotube photoelectrodes is 3.21%, 4.35%, and 4.34% with 10, 20, and 30 μm, respectively. After TiCl₄ treatment, the efficiency of TiO₂ nanotube photoelectrode for dye-sensitized solar cell can be improved up to 6.58%. In the analysis of electrochemical impedance spectroscopy, the value of R_k (charge transfer resistance related to recombination of electrons) decreases from 26.1 to 17.4 Ω when TiO₂ nanotubes were treated with TiCl₄. These results indicate that TiO₂ nanotubes treated with TiCl₄ can increase the surface area of TiO₂ nanotubes, resulting in the increase of dye adsorption and have great help for the increase of the conversion efficiency of DSSCs.     

The Antithrombotic Agent Pterostilbene Interferes with Integrin αIIbβ3-Mediated Inside-Out and Outside-In Signals in Human Platelets

Platelets play a crucial role in the physiology of primary hemostasis and pathological processes such as arterial thrombosis; thus, developing a therapeutic target that prevents platelet activation can reduce arterial thrombosis. Pterostilbene (PTE) has remarkable pharmacological activities, including anticancer and neuroprotection. Few studies have reported the effects of pterostilbene on platelet activation. Thus, we examined the inhibitory mechanisms of pterostilbene in human platelets and its role in vascular thrombosis prevention in mice. At low concentrations (2–8 μM), pterostilbene strongly inhibited collagen-induced platelet aggregation. Furthermore, pterostilbene markedly diminished Lyn, Fyn, and Syk phosphorylation and hydroxyl radical formation stimulated by collagen. Moreover, PTE directly hindered integrin α_IIbβ₃ activation through interfering with PAC-1 binding stimulated by collagen. In addition, pterostilbene affected integrin α_IIbβ₃-mediated outside-in signaling, such as integrin β₃, Src, and FAK phosphorylation, and reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Furthermore, pterostilbene substantially prolonged the occlusion time of thrombotic platelet plug formation in mice. This study demonstrated that pterostilbene exhibits a strong activity against platelet activation through the inhibition of integrin α_IIbβ₃-mediated inside-out and outside-in signaling, suggesting that pterostilbene can serve as a therapeutic agent for thromboembolic disorders.     

The Effects of CoCl2 on HIF-1α Protein under Experimental Conditions of Autoprogressive Hypoxia Using Mouse Models

It is well known that cobalt chloride (CoCl₂) can enhance the stability of hypoxia-inducible factor (HIF)-1α. The aim of this study is to detect the effect of CoCl₂ on the hypoxia tolerance of mice which were repeatedly exposed to autoprogressive hypoxia. Balb/c mice were randomly divided into groups of chemical pretreatment and normal saline (NS), respectively injected with CoCl₂ and NS 3 h before exposure to hypoxia for 0 run (H0), 1 run (H1), and 4 runs (H4). Western Blot, electrophoretic mobility shift assay (EMSA), extracellular recordings population spikes in area cornus ammonis I (CA 1) of mouse hippocampal slices and real-time were used in this study. Our results demonstrated that the tolerance of mice to hypoxia, the changes of HIF-1α protein level and HIF-1 DNA binding activity in mice hippocampus, the mRNA level of erythropoietin (EPO) and vascular endothelial growth factor (VEGF), and the disappearance time of population spikes of hippocampal slices were substantially different between the control group and the CoCl₂ group. Over-induction of HIF-1α by pretreatment with CoCl₂ before hypoxia did not increase the hypoxia tolerance.     

Galectin-3 Contributes to the Inhibitory Effect of lα,25-(OH)2D3 on Osteoclastogenesis

The active form of vitamin D, 1α,25-(OH)₂D₃, not only promotes intestinal calcium absorption, but also regulates the formation of osteoclasts (OCs) and their capacity for bone mineral dissolution. Gal-3 is a newly discovered bone metabolic regulator involved in the proliferation, differentiation, and apoptosis of various cells. However, the role of galectin-3 (gal-3) in OC formation and the regulatory effects of 1α,25-(OH)₂D₃ have yet to be explored. To confirm whether gal-3 contributes to the regulatory effects of 1α,25-(OH)₂D₃ on osteoclastogenesis, osteoclast precursors (OCPs) were induced by macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). TRAP staining and bone resorption analyses were used to verify the formation and activation of OCs. qPCR, Western blotting, co-immunoprecipitation, and immunofluorescence assays were used to detect gene and protein expression. The regulatory effects of gal-3 in OC formation after treatment with 1α,25-(OH)₂D₃ were evaluated using gal-3 siRNA. The results showed that 1α,25-(OH)₂D₃ significantly increased gal-3 expression and inhibited OC formation and bone resorption. Expression levels of OC-related genes and proteins, matrix metalloproteinase 9 (MMP-9), nuclear factor of activated T cells 1 (NFATc1), and cathepsin K (Ctsk) were also inhibited by 1α,25-(OH)₂D₃. Gal-3 knockdown attenuated the inhibitory effects of 1α,25-(OH)₂D₃ on OC formation, activation, and gene and protein expression. In addition, gal-3 was co-localized with the vitamin D receptor (VDR). These data suggest that gal-3 contributes to the osteoclastogenesis inhibitory effect of lα,25-(OH)₂D₃, which is involved in bone and calcium homeostasis.