The Eotaxin-1/CCR3 Axis and Matrix Metalloproteinase-9 Are Critical in Anti-NC16A IgE-Induced Bullous Pemphigoid.

Bullous pemphigoid (BP) is the most common autoimmune bullous skin disease of humans and is characterized by eosinophilic inflammation and circulating and tissue-bound IgG and IgE autoantibodies directed against two hemidesmosomal proteins: BP180 and BP230. The noncollagenous 16A domain (NC16A) of BP180 has been found to contain major epitopes recognized by autoantibodies in BP. We recently established the pathogenicity of anti-NC16A IgE through passive transfer of patient-derived autoantibodies to double-humanized mice that express the human high-affinity IgE receptor, FcεRI, and human NC16A domain (FcεRI/NC16A). In this model, anti-NC16A IgEs recruit eosinophils to mediate tissue injury and clinical disease in FcεRI/NC16A mice. The objective of this study was to characterize the molecular and cellular events that underlie eosinophil recruitment and eosinophil-dependent tissue injury in anti-NC16A IgE-induced BP. We show that anti-NC16A IgEs significantly increase levels of key eosinophil chemoattractants, eotaxin-1 and eotaxin-2, as well as the proteolytic enzyme matrix metalloproteinase-9 (MMP-9) in the lesional skin of FcεRI/NC16A mice. Importantly, neutralization of eotaxin-1, but not eotaxin-2, and blockade of the main eotaxin receptor, CCR3, drastically reduce anti-NC16A IgE-induced disease activity. We further show that anti-NC16A IgE/NC16A immune complexes induce the release of MMP-9 from eosinophils, and that MMP-9-deficient mice are resistant to anti-NC16A IgE-induced BP. Lastly, we find significantly increased levels of eotaxin-1, eotaxin-2, and MMP-9 in blister fluids of BP patients. Taken together, this study establishes the eotaxin-1/CCR3 axis and MMP-9 as key players in anti-NC16A IgE-induced BP and candidate therapeutic targets for future drug development and testing.

Grape seed proanthocyanidins regulate mitophagy of endothelial cells and promote wound healing in mice through p-JNK/FOXO3a/ROS signal pathway.

Skin wound healing is a dynamic and complex process that involves multiple physiological and cellular events. Grape seed proanthocyanidins (GSP) have strong anti-oxidation and elimination of oxygen free radicals, and have been shown to significantly promote wound healing, but the underlying mechanism remains unclear. Studies have indicated that reactive oxygen species (ROS) acts as an upstream signal to induce mitophagy, suggesting that GSP can regulate mitophagy through the signal pathway. This study aimed to investigate whether GSP regulates mitophagy by down-regulating oxidative stress to promote wound healing. In vivo, GSP treatment accelerated wound healing, granulation tissue formation, collagen deposition, and angiogenesis in mice. Moreover, GSP down-regulated ROS levels and promoted the expression of antioxidant proteins by up-regulating the expression of p-JNK/FOXO3a protein, thereby regulating the expression of mitophagy-related proteins. In vitro, 4 µg/mL GSP showed no apparent toxic effects on cells and effectively reduce the oxidative stress damage of cells induced by H2O2. Western blot and superoxide anion fluorescence probe further confirmed that GSP effectively reduced Dihydroethidium content and up-regulated the expression of antioxidant proteins by activation of p-JNK/FOXO3a protein expression, thereby regulating mitophagy. Taken together, the findings from in vitro and in vivo experiments provide new insights into the promotion of wound healing by GSP.

Counterintuitive Lanthanide Hydrolysis-Induced Assembly Mechanism.

The understanding of the hydrolysis mechanism of lanthanide ions is limited by their elusive coordination configuration and undeveloped technology. A potential solution by high-resolution mass spectroscopy studies is hindered by the lack of a stable model under electrospray ionization (ESI) conditions and the complexity of the spectra. Herein, it is demonstrated that diketonate ligands can efficiently stabilize the hydrolyzed intermediate cluster of Ln3+ under ESI conditions, and an effective mass difference fingerprint of isomorphism (MDFI) method is proposed, which can allow the determination of the nuclearity-number of the species without depth resolution. Thus, the hydrolysis of Ln3+ into an atomically precise hydroxide cluster is observed at the level of precise formulae. The species evolution upon hydrolysis is along the dominant path of {Eu3}-{Eu4}-{Eu9}-{Eu10}-{Eu11}-{Eu15}-{Eu16} and a nondominant path of {Eu3}-{Eu4}-{Eu8-1}-{Eu8-2} under the investigated conditions. The crystal of the {Eu16} species was obtained via low-temperature crystallization, and single-crystal X-ray diffraction studies show that its structure contains three octahedral {o-Ln6} units. The contradiction between multiple {o-Ln6} units in the structure and the absence in the formation process indicates that the repetitive subunit observed in the structure does not necessarily correspond to the construction units of high-nuclearity clusters. Photophysical measurements indicate that Eu16 cluster has a high total emission quantum efficacy of 12.8% in the solid state. This study provides fundamental insights into the formation, evolution, and assembly of small lanthanide hydroxide units upon hydrolysis, which is vital for the goal of directional synthesis of lanthanide hydroxide clusters.

Bioglass promotes wound healing by inhibiting endothelial cell pyroptosis through regulation of the connexin 43/reactive oxygen species (ROS) signaling pathway.

Bioactive glass (BG) has recently shown great promise in soft tissue repair, especially in wound healing; however, the underlying mechanism remains unclear. Pyroptosis is a novel type of programmed cell death that is involved in various traumatic injury diseases. Here, we hypothesized that BG may promote wound healing through suppression of pyroptosis. To test this scenario, we investigated the possible effect of BG on pyroptosis in wound healing both in vivo and in vitro. This study showed that BG can accelerate wound closure, granulation formation, collagen deposition, and angiogenesis. Moreover, western blot analysis and immunofluorescence staining revealed that BG inhibited the expression of pyroptosis-related proteins in vivo and in vitro. In addition, while BG regulated the expression of connexin43 (Cx43), it inhibited reactive oxygen species (ROS) production. Cx43 activation and inhibition experiments further indicate that BG inhibited pyroptosis in endothelial cells by decreasing Cx43 expression and ROS levels. Taken together, these studies suggest that BG promotes wound healing by inhibiting pyroptosis via Cx43/ROS signaling pathway.

An Argonaute from Thermus parvatiensis exhibits endonuclease activity mediated by 5' chemically modified DNA guides.

Prokaryotic Argonaute (pAgo) nucleases with precise DNA cleavage activity show great potential for gene manipulation. Extensive biochemical studies have revealed that recognition of guides with different 5' groups by Ago is important for biocatalysis. Here, we identified an Ago from the thermophilic Thermus parvatiensis ( TpsAgo) and analyzed the regulatory effect of 5'-modified guides on TpsAgo cleavage activity. Recombinant TpsAgo cleaves single-stranded DNA and RNA targets at 65-90°C, which is mediated by a 5' hydroxyl or phosphate DNA guide. Notably, TpsAgo can utilize various 5'-modified DNA guides for catalysis, including 5'-NH 2C 6, 5'-Biotin, 5'-FAM and 5'-SHC 6 guides. Moreover, TpsAgo performs programmable cleavage of double-stranded DNA at temperatures over 80°C and strongly tolerates NaCl concentrations up to 3.2 M. These results provide insight into the catalytic performance of Agos by guide regulation, which may facilitate their biotechnological applications.

Doped Luminescent Lanthanide Coordination Polymers Exhibiting both White-Light Emission and Thermal Sensitivity.

Multifunctional lanthanide coordination polymers (CPs) have the advantages of acting in two or more fields simultaneously. Herein, two single lanthanide CPs, formulated as LnL(D/L-Hlac)(H2O)2·0.5H2O (Ln = Eu (1), Tb (2); H2L = 4,4'-(pyridine-3,5-diyl)dibenzoic acid) and their doped lanthanide analogue Tb0.9373Eu0.0627L(D/L-Hlac)(H2O)2·0.5H2O (3) were prepared through hydrothermal methods. Luminescence measurements reveal that 1 displays red photoluminescence and its Commission International ed'Eclairage (CIE) coordinates are almost invariant in the temperature range from 80 to 300 K, while the emission color of 2 changes from yellow to green and its CIE coordinates change from (0.36132, 0.56365) at 80 K to (0.30448, 0.45566) at 300 K. Significantly, 3 not only displays white-light emission with CIE coordinates of (0.32999, 0.33406) but also exhibits a thermal sensitivity of 2.27% K-1 at 230-300 K. The obviously larger thermal sensitivity in 3 in comparison to that of 1.07% K-1 for 2 demonstrates that lanthanide CPs with both a heat-sensitive fluorescent thermometer and high-efficiency white-light emission can be expected by doping Eu(III) ions into Tb(III)-based CPs.

Therapy of paraneoplastic pemphigus with Rituximab: A case report.

We report a case of 39-year-old female patient with paraneoplastic pemphigus (PNP) associated with thymoma treated with rituximab plus corticosteroids and methotrexate. After rituximab therapy for 8 weeks, oral ulcerations had cured, lesions on the trunk and limbs improved. Rituximab may be useful for PNP therapy, but further studies are necessary to confirm this hypothesis.

Structure-guided reshaping of the acyl binding pocket of 'TesA thioesterase enhances octanoic acid production in E. coli.

Medium-chain fatty acids (C6-C10) have attracted much attention recently for their unique properties compared to their long-chain counterparts, including low melting points and relatively higher carbon conversion yield. Thioesterase enzymes, which can catalyze the hydrolysis of acyl-ACP (acyl carrier protein) to release free fatty acids (FAs), regulate both overall FA yields and acyl chain length distributions in bacterial and yeast fermentation cultures. These enzymes typically prefer longer chain substrates. Herein, seeking to increase bacterial production of MCFAs, we conducted structure-guided mutational screening of multiple residues in the substrate-binding pocket of the E. coli thioesterase enzyme 'TesA. Confirming our hypothesis that enhancing substrate selectivity for medium-chain acyl substrates would promote overall MCFA production, we found that replacement of residues lining the bottom of the pocket with more hydrophobic residues strongly promoted the C8 substrate selectivity of 'TesA. Specifically, two rounds of saturation mutagenesis led to the identification of the 'TesARD-2 variant that exhibited a 133-fold increase in selectivity for the C8-ACP substrate as compared to C16-ACP substrate. Moreover, the recombinant expression of this variant in an E. coli strain with a blocked ß-oxidation pathway led to a 1030% increase in the in vivo octanoic acid (C8) production titer. When this strain was fermented in a 5-L fed-batch bioreactor, it produced 2.7 g/L of free C8 (45%, molar fraction) and 7.9 g/L of total free FAs, which is the highest-to-date free C8 titer to date reported using the E. coli type II fatty acid synthetic pathway. Thus, reshaping the substrate binding pocket of a bacterial thioesterase enzyme by manipulating the hydrophobicity of multiple residues altered the substrate selectivity and therefore fatty acid product distributions in cells. Our study demonstrates the relevance of this strategy for increasing titers of industrially attractive MCFAs as fermentation products.

Consensus of Chinese experts on protection of skin and mucous membrane barrier for health-care workers fighting against coronavirus disease 2019.

Health professions preventing and controlling Coronavirus Disease 2019 are prone to skin and mucous membrane injury, which may cause acute and chronic dermatitis, secondary infection and aggravation of underlying skin diseases. This is a consensus of Chinese experts on protective measures and advice on hand-cleaning- and medical-glove-related hand protection, mask- and goggles-related face protection, UV-related protection, eye protection, nasal and oral mucosa protection, outer ear, and hair protection. It is necessary to strictly follow standards of wearing protective equipment and specification of sterilizing and cleaning. Insufficient and excessive protection will have adverse effects on the skin and mucous membrane barrier. At the same time, using moisturizing products is highly recommended to achieve better protection.

Study on laryngopharyngeal and esophageal reflux characteristics using 24-h multichannel intraluminal impedance-pH monitoring in healthy volunteers.

OBJECTIVE: We aimed to analyze the results of 24-h multichannel intraluminal impedance and pH-monitoring (MII-pH) of the laryngopharynx and esophagus in asymptomatic volunteers. Moreover, we also aimed to gain insight into and establish a baseline for laryngopharyngeal reflux in the healthy population by quantitatively and qualitatively comparing the reflux and pH distribution in both the laryngopharynx and the esophagus. METHODS: Healthy volunteers were recruited and observed; they underwent 24-h ambulatory combined MII-pH monitoring. The proximal sensor (pH1) was positioned approximately 1 cm above the upper esophageal sphincter with the aid of a solid-state high-resolution esophageal manometer. Laryngopharyngeal reflux events were detected and characterized by the incidence and property of reflux both in the laryngopharynx and the esophagus. RESULTS: Thirty-eight asymptomatic volunteers who completed all the examinations were included in this study. The median pH detected by the proximal sensor was 6.6 (6.2, 7.0), with an average of 6.58 ± 0.74. A total of 814 laryngopharyngeal reflux events were detected, including 722 (89%) in the upright position and 92 (11%) in the supine position with incidence (0%) in the liquid state, 44 (5%) in the mixture, and 769 (95%) in the gaseous state. Furthermore, 5 incidences (1%) of acid reflux and 809 incidences of non-acid reflux (99%) were noted. A total of 5779 esophageal reflux events were detected, including 5020 (87%) in the upright position, 759 (13%) in the supine position, with 2051 (36%) in the liquid state, 2050 (35%) in the mixed condition, and 1678 (29%) in the gaseous state; adding up to 805 incidences (14%) of acid reflux and 4974 incidences (86%) of non-acid reflux. CONCLUSION: Non-acid reflux in the upright position is characteristic of laryngopharyngeal reflux. Acid reflux is almost undetectable in healthy subjects. Hence, the diagnostic indicators of gastroesophageal reflux disease are not suitable for laryngopharyngeal reflux disease.

Structure of an endogalactosylceramidase from Rhodococcus hoagii 103S reveals the molecular basis of its substrate specificity.

Endoglycoceramidases (EGCs) are family 5 glycoside hydrolases that catalyze hydrolysis of the glycosidic linkages between the oligosaccharide and ceramide moieties of glycosphingolipids. Three orthologs of EGCs, each with distinct substrate specificity, have been identified to date, including EGC-I, EGC-II, and EGALC. Although the structures of EGC-I and EGC-II have been reported, the substrate preference mechanism of EGC enzymes remains unclear. Here, we determined the crystal structure of EGALC from Rhodococcus hoagii 103S at a resolution of 1.20 Å. Distinct from EGC-I and EGC-II, which both have a tunnel-like substrate binding site, the structure of EGALC accommodates substrates in a long groove. Further, the oligosaccharide-binding region of groove could be divided into two small pockets that separately bind to the Gal1 and to the Gal3/Gla3 present in 6-gala series substrates. Structural and sequence comparisons of EGC enzymes revealed that the conformation and length of their Nß8-Lα1 regions are crucial in determining the architectures of their specific substrate binding sites. Importantly, molecular docking analyses indicate that the substrate specificity of each EGC is mainly derived from the complementarity of its active site groove/tunnel with substrates adopting particular conformations. Our study provide insights for understanding the catalytic mechanism of EGALC, which will help protein engineering for improving the substrate preference and catalytic efficiency of EGC enzymes toward important glycosphingolipid substrates.

The Effect on the Luminescent Properties in Lanthanide-Titanium OXO Clusters.

Two lanthanide-titanium oxo clusters (LTOCs), formulated as [Eu3Ti3(µ3-O)2(µ3-OH)(CH3O)2(Ac)2(CH3OH)(tbba)12]·CH3OH (1) and [Eu6Ti8(µ3-O)13(µ2-OH)(µ3-OCH3)2(µ2-OCH3)2(H2O)(CH3OH)2(tbba)19]·Htbba·10H2O (2), were synthesized through the solvothermal reaction of 4-tert-butylbenzoate ligand, Eu(Ac)3·3H2O, and Ti(OiPr)4 in methanol. Single-crystal structural analysis reveals that 1 crystallized in the orthorhombic space group Pnn2, and 2 crystallized in the triclinic space group P1. Structurally, the core of 1 can be viewed as a coplanar unit of [Eu3Ti3(µ3-O)2(µ3-OH)]16+ formed through each µ3-O2- and µ3-OH- bridging one Ti4+ and two Eu3+ ions, while that of 2 can be viewed as two units of [Eu3Ti3(µ3-O)3(µ2-O)4(µ2-OCH3)2(CH3OH)]5+ and [Eu3Ti5(µ3-O)6(µ2-OH)(µ3-OCH3)2(H2O)(CH3OH)]14+ connected through four µ2-O units from the [Eu3Ti3(µ3-O)3(µ2-O)4(µ2-OCH3)2(CH3OH)]5+ unit to respectively coordinate two Eu3+ and two Ti4+ ions from the [Eu3Ti5(µ3-O)6(µ2-OH)(µ3-OCH3)2(H2O)(CH3OH)]14+ unit. Measurement of their luminescence properties shows that the luminescence lifetime and quantum yield are 1212 µs and 69.6% for 1 and 857 µs and 56.2% for 2. When F- was introduced into 1 in a molar ratio of F- to 1 of 1:3, its quantum yield was increased 1.3 times, and the lifetime increased from 1.2 to 1.4 ms. However, no obvious enhancement in the emission intensity was observed in 2; even the molar ratio of F- to 2 is in the range from 2 to 1/4. Investigation on the structures of 1 and 2 reveals that the luminescence lifetime and quantum yield in 1 is significantly larger than that in 2 are attributed to the vibration of the nonradiative O-H vibration groups in 1 being significantly smaller than that in 2.

High-Nuclearity Chiral 3 d-4 f Heterometallic Clusters Ln6Cu24 and Ln6Cu12.

Based on the anion template and chiral ligand inducting role, two series of high-nuclearity 3 d-4 f heterometallic clusters with formulas [NO3@Ln6Cu24(µ3-OH)30(µ2-OH)3(OAc)6( R/ S-L)12(H2O)24](NO3)14· x(H2O) (Ln = Dy, x = 30 for 1a( R-L) and 1b( S-L); Ln = Tb, x = 40 for 2a( R-L) and 2b( S-L)) and (Et3NH)4[Ln6Cu12(µ3-OH)14(µ2-Cl)6Cl12( R/ S-L)12]Cl2· x(H2O) (Ln = Dy, x = 28 for 3a( R-L) and 3b( S-L); Ln = Tb, x = 33 for 4a ( R-L) and 4b( S-L); HL = ( R/ S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), have been synthesized and characterized. Structural analysis reveals that the metal skeleton of compounds 1 and 2 display a Ln6Cu12 octahedral inner core encapsulated by six outer Cu2 units. In the Ln6Cu12 octahedron, 6 Ln3+ ions located at the six vertices and 12 inner Cu2+ ions located at the 12 edges of octahedron, and one NO3- locates in the center of the octahedron. The metal core of compounds 3 and 4 can be viewed as a Ln6 octahedron encapsulated by six Cu2 units. It is interesting that the different inorganic anions involved in the reaction result in the difference in the structures of 1 to 2 and 3 to 4. Circular dichroism spectra of 1-4 display obvious mirror symmetry effect at 600-800 nm of d-d transition of Cu2+, suggesting that the chirality transferred from chiral R- and S-ligand to Cu2+ ions in this system. Notably, the CD peak at the Cu2+ d-d transition position of Ln6Cu12 cluster is obviously blue-shifted compared with that of Ln6Cu24 due to the different coordinated environments of Cu2+. Magnetic studies indicate that 1a and 2a show weak ferromagnetic interactions, while 3a and 4a display antiferromagnetic interactions.

Structural dissection of sterol glycosyltransferase UGT51 from Saccharomyces cerevisiae for substrate specificity.

Sterol glycosyltransferases catalyze the formation of a variety of glycosylated sterol derivatives and are involved in producing a plethora of bioactive natural products. To understand the molecular mechanism of sterol glycosyltransferases, we determined crystal structures of a sterol glycosyltransferase UGT51 from Saccharomyces cerevisiae. The structures of the UGT51 and its complex with uridine diphosphate glucose (UDPG) were solved at resolutions of 2.77 Šand 1.9 Å, respectively. The structural analysis revealed that a long hydrophobic cavity, 9.2 Šin width and 17.6 Šin length located at the N-terminal domain of UGT51, is suitable for the accommodation of sterol acceptor substrates. Furthermore, a short, conserved sequence of S847-M851 was identified at the bottom of the hydrophobic cavity, which might be the steroid binding site and play an important role for the UGT51 catalytic specificity towards sterols. Molecular docking simulations indicated that changed unique interaction network in mutant M7_1 (S801A/L802A/V804A/K812A/E816K/S849A/N892D), with an 1800-fold activity improvement toward an unnatural substrate protopanaxadiol (PPD), might influence its substrate preference. This study reported the first sterol glycosyltransferase structure, providing a molecular blueprint for generating tailored sterol glycosyltransferases as potential catalytic elements in synthetic biology.

Structural Insights into the Broad Substrate Specificity of a Novel Endoglycoceramidase I Belonging to a New Subfamily of GH5 Glycosidases.

Endoglycoceramidases (EGCases) specifically hydrolyze the glycosidic linkage between the oligosaccharide and the ceramide moieties of various glycosphingolipids, and they have received substantial attention in the emerging field of glycosphingolipidology. However, the mechanism regulating the strict substrate specificity of these GH5 glycosidases has not been identified. In this study, we report a novel EGCase I from Rhodococcus equi 103S (103S_EGCase I) with remarkably broad substrate specificity. Based on phylogenetic analyses, the enzyme may represent a new subfamily of GH5 glycosidases. The X-ray crystal structures of 103S_EGCase I alone and in complex with its substrates monosialodihexosylganglioside (GM3) and monosialotetrahexosylganglioside (GM1) enabled us to identify several structural features that may account for its broad specificity. Compared with EGCase II from Rhodococcus sp. M-777 (M777_EGCase II), which possesses strict substrate specificity, 103S_EGCase I possesses a longer α7-helix and a shorter loop 4, which forms a larger substrate-binding pocket that could accommodate more extended oligosaccharides. In addition, loop 2 and loop 8 of the enzyme adopt a more open conformation, which also enlarges the oligosaccharide-binding cavity. Based on this knowledge, a rationally designed experiment was performed to examine the substrate specificity of EGCase II. The truncation of loop 4 in M777_EGCase II increased its activity toward GM1 (163%). Remarkably, the S63G mutant of M777_EGCase II showed a broader substrate spectra and significantly increased activity toward bulky substrates (up to >1370-fold for fucosyl-GM1). Collectively, the results presented here reveal the exquisite substrate recognition mechanism of EGCases and provide an opportunity for further engineering of these enzymes.

Expression of NLPR3 in Psoriasis Is Associated with Enhancement of Interleukin-1ß and Caspase-1.

BACKGROUND NLPR3 is an important gene that belongs to the family of NOD-like receptors and is thought to play an important role in psoriasis. The aim of the present study was to investigate the expression of NLRP3 in psoriasis biopsy samples and to assess the possible correlation of its expression with that of interleukin IL-1ß and Caspase-1. MATERIAL AND METHODS The mRNA expression was checked by qRT-PCR. The expression of the proteins was checked by Western blotting. The distribution of the proteins was determined by immunohistochemistry. RESULTS The results of our study indicate that the expression of NLRP3 was significantly upregulated in all the psoriatic biopsy samples as indicated by qRT-PCR and Western blotting. The expression of NLRP3 in psoriatic samples was 3.5 to 4.3 times higher than the expression of NLRP3 in normal skin biopsy samples. Moreover, our results indicated that the expression levels of IL-1ß were higher as compared to the normal skin biopsy samples. Relative to the expression of IL-1ß in normal skin biopsy samples, the expression of IL-1b was about 2.7-4.6 times higher. Additionally, the expression of caspase-1 was considerably upregulated in the psoriatic samples. Caspase-1 gene expression was 2.2-3.4 times higher than in normal skin biopsy samples. CONCLUSIONS NLPR3 may prove to be an important therapeutic target for psoriasis.