RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts.

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

Smart Contact Lens for Colorimetric Visualization of Glucose Levels in the Body Fluid.

Frequent blood glucose monitoring is a crucial routine for diabetic patients. Traditional invasive methods can cause discomfort and pain and even pose a risk of infection. As a result, researchers have been exploring noninvasive techniques. However, a limited number of products have been developed for the market due to their high cost. In this study, we developed a low-cost, highly accessible, and noninvasive contact lens-based glucose monitoring system. We functionalized the surface of the contact lens with boronic acid, which has a strong but reversible binding affinity to glucose. To achieve facile conjugation of boronic acid, we utilized a functional coating layer called poly(tannic acid). The functionalized contact lens binds to glucose in body fluids (e.g., tear) and releases it when soaked in an enzymatic cocktail, allowing for the glucose level to be quantified through a colorimetric assay. Importantly, the transparency and oxygen permeability of the contact lens, which are crucial for practical use, were maintained after functionalization, and the lenses showed high biocompatibility. Based on the analysis of colorimetric data generated by the smartphone application and ultraviolet-visible (UV-vis) spectra, we believe that this contact lens has a high potential to be used as a smart diagnostic tool for monitoring and managing blood glucose levels.

Simultaneous coupling of metal removal and visual detection by nature-inspired polyphenol-amine surface chemistry.

The interplay between polyphenols, amines, and metals has broad implications for surface chemistry, biomaterials, energy storage, and environmental science. Traditionally, polyphenol-amine combinations have been recognized for their ability to form adhesive, material-independent thin layers that offer a diverse range of surface functionalities. Herein, we demonstrate that a coating of tannic acid (TA) and polyethyleneimine (PEI) provides an efficient platform for capturing and monitoring metal ions in water. A unique feature of our PEI/TA-coated microbeads is the 'Detection-Capture' (Detec-Ture) mechanism. The galloyl groups in TA coordinate with Fe(III) ions (capture), initiating their oxidation to gallol-quinone. These oxidized groups subsequently react with PEI amines, leading to the formation of an Fe(II/III)-gallol-PEI network that produces a vivid purple color, thereby enabling visual detection. This mechanism couples metal capture directly with detection, distinguishing our approach from existing studies, which have either solely focused on metal removal or metal detection. The metal capturing capacity of our materials stands at 0.55 mg g-1, comparable to that of established materials like alginate and wollastonite. The detection sensitivity reaches down to 0.5 ppm. Our findings introduce a novel approach to the utility of metal-polyphenol-amine networks, presenting a new class of materials suited for simultaneous metal ion detection and capture in environmental applications.

Octyl gallate has potent anti-inflammasome activity by directly binding to NLRP3 LRR domain.

The NOD-, LRR-, and Pyrin domain-containing protein 3 (NLRP3) inflammasome plays key roles in regulating inflammation. Numerous studies show that the abnormal activation of NLRP3 associates with the initiation and progression of various diseases. Hence, the NLRP3 inflammasome may be a promising therapeutic target for these diseases. Octyl gallate (OG) is a small molecule with antioxidant, antimicrobial, antifungal, and anti-inflammatory activities; however, the mechanism underlying its anti-inflammatory activity is still unclear. Here, we developed a screening system for NLRP3-inflammasome inhibitors. A total of 3287 small molecules were screened for inhibitors of nigericin-induced NLRP3 oligomerization. OG was identified as a novel inhibitor. We show that OG directly targets the LRR domain of NLRP3 and thereby blocks the inflammatory cascade of the NLRP3 inflammasome. This contrasts with the mode-of-action of other direct NLRP3 inhibitors, which all bind to the NACHT domain of NLRP3. Interestingly, OG also inhibits the priming step by downregulating the Raf-MEK1/2-ERK1/2 axis. Thus, OG inhibits the NLRP3 inflammasome by two distinct mechanisms. Importantly, OG injection ameliorated the inflammation in mouse models of foot gout and sepsis. Our study identifies OG as a potential therapeutic agent for NLRP3-associated diseases.

Pim1 promotes IFN-ß production by interacting with IRF3.

The Pim (proviral integration site for Moloney murine leukemia virus) proteins compose a serine threonine kinase family whose members regulate cell proliferation, migration and cell survival. However, whether Pim kinases participate in innate immune responses is unclear. Here, we show for the first time that Pim1 plays an essential role in the production of interferon (IFN)-ß by macrophages after their Toll-like receptor (TLR) pathway is activated by pathogen-associated molecular patterns (PAMPs). Specifically, Pim1 was quickly upregulated in an NF-κB-dependent manner after TLR stimulation with PAMPs. Pim1 deficiency reduced TLR3- or TLR4-stimulated IFN-ß and IFN-stimulated gene (ISG) expression but not proinflammatory cytokine expression in macrophages. Mechanistically, Pim1 specifically upregulates IRF3 phosphorylation and nuclear translocation. However, this role is not dependent on Pim1 kinase activity. Rather, Pim1 appears to promote IRF3 phosphorylation by enhancing the formation of IFN-ß signaling complexes composed of TRIF, TRAF3, TBK1, and IRF3. Poly (I:C)-treated Pim1-/- mice produced less serum IFN-ß and were less likely to survive than wild-type mice. These findings show for the first time that Pim1 participates in TLR-mediated IFN-ß production, thus revealing a novel target for controlling antiviral innate immune responses.

Glycogen Synthase Kinase 3ß Regulates Antiviral Responses of TLR3 via TRAF2-Src Axis.

The protein tyrosine kinase Src regulates the synthesis of TLR3-mediated IFN-ß via the TBK1-IFN regulatory factor 3 axis. However, the molecular mechanisms regulating Src activity in TLR3 signaling remain unclear. In this study, we report that GSK3ß regulates Src phosphorylation via TNFR-associated factor 2 (TRAF2)-mediated Src ubiquitination. GSK3ß deficiency in mouse embryonic fibroblasts significantly reduces polyinosinic:polycytidylic acid-induced IFN-ß and IFN-stimulated gene expression, which is caused by diminished phosphorylation of Src at tyrosine 416. Src undergoes polyinosinic:polycytidylic acid-dependent lysine 63 chain ubiquitination, and TRAF2 is a direct E3 ligase for Src. Our study reveals novel mechanisms underlying TLR3-mediated antiviral responses mediated via the GSK3ß-TRAF2-Src axis.

Characterization and epitope mapping of two monoclonal antibodies against human CD99.

CD99 plays a critical role in the diapedesis of monocytes, T cell differentiation, and the transport of MHC molecules. Engagement of CD99 by agonistic monoclonal antibodies has been reported to trigger multifactorial events including T cell activation as well as cell-cell adhesion during hematopoietic cell differentiation. In this study, to identify the functional domains participating in the cellular events, we mapped the epitopes of CD99, which are recognized by two agonistic CD99 monoclonal antibodies, DN16 and YG32. Using recombinant fusion proteins of GST with whole or parts of CD99, we found that both antibodies interact with CD99 molecules independently of sugar moieties. DN16 mAb detected a linear epitope located in the amino terminal region of CD99 while YG32 mAb bound another linear epitope in the center of the extracellular domain. To confirm that the identified epitopes of CD99 are actually recognized by the two mAbs, we showed the presence of physical interaction between the mAbs and the fusion proteins or synthetic peptides containing the corresponding epitopes using surface plasmon resonance analyses. The dissociation constants of DN16 and YG32 mAbs for the antigen were calculated as 1.27 x 10(-7) and 7.08 x 10(-9) M, respectively. These studies will help understand the functional domains and the subsequent signaling mechanism of CD99.