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Atopic dermatitis (AD) is a relapsing inflammatory skin disorder, with characteristic T helper 2 (Th2)-based immune dysregulation and epidermal barrier function defect. In AD, keratinocyte plays a critical role in maintaining skin integrity and immune homeostasis. Interleukin (IL)-19 is a member of the IL-10 cytokine family. It can be secreted by and act on keratinocytes. Although it has been demonstrated that increased IL-19 in AD patients’ lesions and serum is positively correlated with the severity of AD, the role of IL-19 in the regulation of the epidermal barrier and immune homeostasis of keratinocytes remains unclear. Thus, we aim to investigate the effect of IL-19 on the production of epidermal barrier related proteins and proinflammatory cytokines in keratinocytes, and the synergistic effect of IL-19 with IL-4/IL-13 in vitro experiment. In this study, barrier related proteins (filaggrin/FLG, loricrin/LOR, keratin-10), epithelium-derived cytokines (thymic stromal lymphopoietin/TSLP, IL-33, IL-25), IL-19 and the phosphorylation level of STAT3 and STAT6 were measured in HaCaT cells by RT-PCR and/or western blot before and after stimulated with IL-4/IL-13 with or without different concentrations of IL-19. The changes of IL-19 levels in HaCaT cells after stimulation with house dust mite (HDM) or staphylococcal enterotoxin type B (SEB) were also examined. We found that IL-19 could promote the production of TSLP in keratinocytes, but reduce the expression levels of LOR and keratin-10. Meanwhile, IL-19 significantly enhanced the effects of IL-4/IL-13 on keratinocytes, including inducing the expression of IL-19 and epithelial derived cytokines and inhibiting the expression of barrier proteins. These enhanced effects were accompanied by increased phosphorylation of STAT3 and STAT6, but no change in the expression level of IL-4/13 receptor. In addition, HDM but not SEB can induce keratinocytes to secrete IL-19. Taken together, IL-19 can enhance the effect of IL-4 / IL-13 on keratinocytes in vitro, and may play an important role in the pathogenesis and progression of AD.

Backgroud: Progression of acute-to-chronic atopic dermatitis is accompanied by multiple helper T cell cytokine responses, but the mechanisms and relative importance of these changes remains unclear. There is no animal model for atopic dermatitis that recapitulates these cytokine responses.Objective: We sought to build a novel mouse model for Atopic Dermatitis(AD) that recapitulates these helper T cell responses and some dynamic changes of cytokine responses in the progression of AD.Methods: Female BALB/c mice were subjected to the application of Dinitrofluorobenzene(DNFB) and Ovalbumin(OVA) to induce AD-like dermatitis. Skin lesions and serum were collected from mice of acute and chronic phase to detect the changes of cytokine responses and other features of AD.Results: Combined application of DNFB and OVA successfully induced AD-like dermatitis and histological changes as well as epidermal barrier dysfunction. In acute phase of AD-like dermatitis, Th2-associated cytokines were mainly increased in serum and skin lesions. In chronic phase of AD-like dermatitis,Th2-associated cytokines were still highly expressed, while Th1- and Th17-associated cytokines were also gradually increased. Compared with acute phase, the JAK-STAT signaling pathway was highly expressed in chronic phase of AD-like dermatitis.Conclusion: The combined application of DNFB and OVA could be used to build a new mouse model for atopic dermatitis. This mouse model recapitulates the helper T cell responses and some dynamic changes of cytokine responses in the progression of acute-to-chronic in human AD. JAK-STAT signaling pathway plays a pivotal role in the chronicity of AD.

Long-term and frequent observation of rainwater chemistry provides an opportunity to explore the evolution of air pollutant emissions and the effectiveness of the emission control measures. This study presents variations in rainwater chemistry, possible sources of air pollutant emissions, and wet deposition fluxes of ionic species in the rainfall samples during 2018–2020 at Mt. Lushan located in South China. Results showed that the annual volume-weighted mean (VWM) pH of rainwater was 5.8. Moreover, the VWM of 87.7% rainwater samples had pH values above 5.6, which indicated that the atmospheric pollution at Mt. Lushan had less effect during the study period. The polytonic nitrate ion (NO3−), the doubly charged positive calcium ion (Ca2+) and the ammonium ion (NH4+) were the dominant ions in rainwater where Ca2+ and NH4+ were the main neutralizing ionic species for rainwater acidity. The highly seasonal variability of ionic concentrations in rainwater was observed among different ionic species, which is closely associated with meteorological conditions and anthropogenic emissions present in the various seasons. Annual wet deposition flux showed an increasing trend during the entire periods. Hence, the wet flux of NO3− (76.3 kg ha−1 yr−1) was three times higher than the annual wet deposition flux of sulfate (SO42−, 21.7 kg ha−1 yr−1), which points to the fact that acid deposition is still a severe environmental issue in the Mt. Lushan region. Positive matrix factorization (PMF) and correlation matrix were used to identify five sources for ionic species, including aging sea salt, fossil fuel combustion, agriculture, dust, and biomass burning. These findings could be helpful in establishing emission control policies to protect and manage pollution control in the eco-environment in South China.

HAT tag, a natural His affinity tag, is one of the most popular fusion tags. However, HAT tag containing three positive charges limited its self-assembly with quantum dots (QDs). Herein, ATTO 590-labeled HAT peptide was synthesized to self-assemble with QDs inside the capillary. QDs and ATTO 590-labeled HAT peptide were sequentially injected into the capillary and probed by fluorescence-coupled CE (CE-FL), showing an obvious Forster resonance energy transfer signal. Online self-assembly, separation, and detection were achieved within 10 min. CE-FL further revealed that imidazole and H6 G6 peptide could partially outcompete with HAT tag on the QDs surface inside the capillary. The displacement intermediates were separated clearly using CE-FL. Our study demonstrates the power of online CE-FL in analyzing the binding interaction between ligands containing positive charges and QDs.

Confocal fluorescence demonstrates that single molecules of dye-labelled Cytochrome C or B5 containing paramagnetic Fe(III) can be magnetically placed into the interstices of super-crystal which is composed of three dimensional regular arrays of Fe(3)O(4) nanoparticles.

Filamin, a large modular protein composed mainly of many immunoglobulin-like domains, is a potent cross-linker of actin filaments. The region containing immunoglobulin type modules 19-21 makes up the binding site for the cytoplasmic tails of the integrin adhesion receptors. Here we investigate the stability of the Ig-like filamin domains using NMR studies over a range of pH and temperature. We show that the 21st Ig-like module (FLNa21) is partly unfolded even under physiological conditions and when attached to FLNa20. It is, however, appreciably stabilized upon binding to integrins. FLNa21 is noticeably less stable than neighboring homologous modules, such as FLNa19 and FLNa17. This variability in stability could be related to the known sensitivity of filamin to cell-mediated mechanical forces.

Filamins are large proteins that cross-link actin filaments and connect to other cellular components. The C-terminal rod 2 region of FLNa (filamin A) mediates dimerization and interacts with several transmembrane receptors and intracellular signalling adaptors. SAXS (small-angle X-ray scattering) experiments were used to make a model of a six immunoglobulin-like domain fragment of the FLNa rod 2 (domains 16–21). This fragment had a surprising three-branched structural arrangement, where each branch was made of a tightly packed two-domain pair. Peptides derived from transmembrane receptors and intracellular signalling proteins induced a more open structure of the six domain fragment. Mutagenesis studies suggested that these changes are caused by peptides binding to the CD faces on domains 19 and 21 which displace the preceding domain A-strands (18 and 20 respectively), thus opening the individual domain pairs. A single particle cryo-EM map of a nine domain rod 2 fragment (domains 16–24), showed a relatively compact dimeric particle and confirmed the three-branched arrangement as well as the peptide-induced conformation changes. These findings reveal features of filamin structure that are important for its interactions and mechanical properties.

Filamins are scaffold proteins that bind to various proteins, including the actin cytoskeleton, integrin adhesion receptors, and adaptor proteins such as migfilin. Alternative splicing of filamin, largely constructed from 24 Ig-like domains, is thought to have a role in regulating its interactions with other proteins. The filamin A splice variant-1 (FLNa var-1) lacks 41 amino acids, including the last β-strand of domain 19, FLNa(19), and the first β-strand of FLNa(20) that was previously shown to mask a key binding site on FLNa(21). Here, we present a structural characterization of domains 18-21, FLNa(18-21), in the FLNa var-1 as well as its nonspliced counterpart. A model of nonspliced FLNa(18-21), obtained from small angle x-ray scattering data, shows that these four domains form an L-shaped structure, with one arm composed of a pair of domains. NMR spectroscopy reveals that in the splice variant, FLNa(19) is unstructured whereas the other domains retain the same fold as in their canonical counterparts. The maximum dimensions predicted by small angle x-ray scattering data are increased upon migfilin binding in the FLNa(18-21) but not in the splice variant, suggesting that migfilin binding is able to displace the masking β-strand and cause a rearrangement of the structure. Possible function roles for the spliced variants are discussed.

We describe a novel pulse sequence, MQ-HNCO-TROSY, for the measurement of scalar and residual dipolar couplings between amide proton and nitrogen in larger proteins. The experiment utilizes the whole 2T(N) polarization transfer delay for labeling of (15)N chemical shift in a constant time manner, which efficiently doubles the attainable resolution in (15)N dimension with respect to the conventional HNCO-TROSY experiment. In addition, the accordion principle is employed for measuring (J + D)(NH)s, and the multiplet components are selected with the generalized version of the TROSY scheme introduced by Nietlispach (J Biomol NMR 31:161-166, 2005). Therefore, cross peak overlap is diminished while the time period during which the (15)N spin is susceptible to fast transverse relaxation associated with the anti-TROSY transition is minimized per attainable resolution unit. The proposed MQ-HNCO-TROSY scheme was employed for measuring RDCs in high molecular weight protein IgFLNa16-21 of 557 residues, resulting in 431 experimental RDCs. Correlations between experimental and back-calculated RDCs in individual domains gave relatively low Q-factors (0.19-0.39), indicative of sufficient accuracy that can be obtained with the proposed MQ-HNCO-TROSY experiment in high molecular weight proteins.

Human filamins are large actin-crosslinking proteins composed of an N-terminal actin-binding domain followed by 24 Ig-like domains (IgFLNs), which interact with numerous transmembrane receptors and cytosolic signaling proteins. Here we report the 2.5 A resolution structure of a three-domain fragment of human filamin A (IgFLNa19-21). The structure reveals an unexpected domain arrangement, with IgFLNa20 partially unfolded bringing IgFLNa21 into close proximity to IgFLNa19. Notably the N-terminus of IgFLNa20 forms a beta-strand that associates with the CD face of IgFLNa21 and occupies the binding site for integrin adhesion receptors. Disruption of this IgFLNa20-IgFLNa21 interaction enhances filamin binding to integrin beta-tails. Structural and functional analysis of other IgFLN domains suggests that auto-inhibition by adjacent IgFLN domains may be a general mechanism controlling filamin-ligand interactions. This can explain the increased integrin binding of filamin splice variants and provides a mechanism by which ligand binding might impact filamin structure.

In this report, fluorescence detection coupled capillary electrophoresis (CE-FL) was used to detect Protein A. Antibody was first labeled with Cy5 and then mixed with quantum dots (QDs) to form QDs-antibody bioprobe. Further, we observed fluorescence resonance energy transfer (FRET) from QDs donor to Cy5 acceptor. The bioprobe was formed and brought QDs and Cy5 close enough to allow FRET to occur. After adding protein A, the FRET system was broken and caused the FRET signal to decrease. Thus, a new method for the determination of protein A was proposed based on the FRET signal changes. This study provides a new trail of thought for the detection of protein.

In this report, the use of fluorescence detection coupled capillary electrophoresis (CE-FL) allowed us to fully characterize the antigen-antibody interaction. CE-FL allowed separation of unbound quantum dots (QDs) and ligand bound QDs and also revealed an ordered assembly of biomolecules on QDs. Further, we observed FRET from QDs donor to DyLight acceptor, which were covalently conjugated with human IgG and goat anti-human IgG, respectively. The immunocomplex was formed and the mutual affinity of the antigen and antibody brought QDs and DyLight close enough to allow FRET to occur. This novel CE-based technique can be easily extended to other FRET systems based on QDs and may have potential application in the detection of antibodies.

Filamins are actin-binding proteins that participate in a wide range of cell functions, including cell morphology, locomotion, membrane protein localization, and intracellular signaling. The three filamin isoforms found in humans, filamins A, B, and C, are highly homologous, and their roles are partly complementary. In addition to actin, filamins interact with dozens of other proteins that have roles as membrane receptors and channels, enzymes, signaling intermediates, and transcription factors. Filamins are composed of an N-terminal actin-binding domain and 24 filamin-type immunoglobulin-like domains (FLN) that form tail-to-tail dimers with their C-terminal FLN domain. Many of the filamin interactions including those for glycoprotein Ibα and integrins have been mapped to the region comprising FLN domains 16-21. Traditionally, FLN domains have been viewed as independent folding units, arranged in a linear chain joined with flexible linkers. Recent structural findings have shown that consecutive FLNs form more intricate superstructures. The crystal structure of filamin A domains 19-21 (FLNa19-21) revealed that domains 20 and 21 fold together and that the domain interaction can be autoregulatory. The solution structure of domains 18-19 showed a similar domain interaction, whereas domain pair 16-17 has a completely different domain packing mode. In this study, we characterize the domain organization of the FLNa domain sextet 16-21 using NMR spectroscopy. A structure model of this 60-kDa protein has been built using residual dipolar coupling restraints. RDCs and (15)N relaxation data have been used to characterize interdomain motions.

A link between sites of cell adhesion and the cytoskeleton is essential for regulation of cell shape, motility, and signaling. Migfilin is a recently identified adaptor protein that localizes at cell-cell and cell-extracellular matrix adhesion sites, where it is thought to provide a link to the cytoskeleton by interacting with the actin cross-linking protein filamin. Here we have used x-ray crystallography, NMR spectroscopy, and protein-protein interaction studies to investigate the molecular basis of migfilin binding to filamin. We report that the N-terminal portion of migfilin can bind all three human filamins (FLNa, -b, or -c) and that there are multiple migfilin-binding sites in FLNa. Human filamins are composed of an N-terminal actin-binding domain followed by 24 immunoglobulin-like (IgFLN) domains and we find that migfilin binds preferentially to IgFLNa21 and more weakly to IgFLNa19 and -22. The filamin-binding site in migfilin is localized between Pro5 and Pro19 and binds to the CD face of the IgFLNa21 β-sandwich. This interaction is similar to the previously characterized β7 integrin-IgFLNa21 interaction and migfilin and integrin β tails can compete with one another for binding to IgFLNa21. This suggests that competition between filamin ligands for common binding sites on IgFLN domains may provide a general means of modulating filamin interactions and signaling. In this specific case, displacement of integrin tails from filamin by migfilin may provide a mechanism for switching between different integrin-cytoskeleton linkages.

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. Here, we describe the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and we suggest it is a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin, but not talin, binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions.