GSDMB is increased in IBD and regulates epithelial restitution/repair independent of pyroptosis.

Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.

Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

Caspase-1 Engages Full-Length Gasdermin D through Two Distinct Interfaces That Mediate Caspase Recruitment and Substrate Cleavage.

The recognition and cleavage of gasdermin D (GSDMD) by inflammatory caspases-1, 4, 5, and 11 are essential steps in initiating pyroptosis after inflammasome activation. Previous work has identified cleavage site signatures in substrates such as GSDMD, but it is unclear whether these are the sole determinants for caspase engagement. Here we report the crystal structure of a complex between human caspase-1 and the full-length murine GSDMD. In addition to engagement of the GSDMD N- and C-domain linker by the caspase-1 active site, an anti-parallel ß sheet at the caspase-1 L2 and L2' loops bound a hydrophobic pocket within the GSDMD C-terminal domain distal to its N-terminal domain. This "exosite" interface endows an additional function for the GSDMD C-terminal domain as a caspase-recruitment module besides its role in autoinhibition. Our study thus reveals dual-interface engagement of GSDMD by caspase-1, which may be applicable to other physiological substrates of caspases.

Crystal Structures of the Full-Length Murine and Human Gasdermin D Reveal Mechanisms of Autoinhibition, Lipid Binding, and Oligomerization.

Gasdermin D (GSDMD) is an effector molecule for pyroptosis downstream of canonical and noncanonical inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases triggers the oligomerization and lipid binding by its N-terminal domain, which assembles membrane pores, whereas its C-terminal domain binds the N-terminal domain to inhibit pyroptosis. Despite recent progress in our understanding of the structure and function of the murine gasdermin A3 (mGSDMA3), the molecular mechanisms of GSDMD activation and regulation remain poorly characterized. Here, we report the crystal structures of the full-length murine and human GSDMDs, which reveal the architecture of the GSDMD N-terminal domains and demonstrate distinct and common features of autoinhibition among gasdermin family members utilizing their ß1-ß2 loops. Disruption of the intramolecular domain interface enhanced pyroptosis, whereas mutations at the predicted lipid-binding or oligomerization surface reduced cytolysis. Our study provides a framework for understanding the autoinhibition, lipid binding, and oligomerization of GSDMD by using overlapping interfaces.

Chemical modulation of gasdermin D activity: Therapeutic implications and consequences.

The gasdermin family of proteins are central effectors of the inflammatory, lytic cell death modality known as pyroptosis. Characterized in 2015, the most well-studied member gasdermin D can be proteolyzed, typically by caspases, to generate an active pore-forming N-terminal domain. At least well-studied three pharmacological inhibitors (necrosulfonamide, disulfiram, dimethyl fumarate) since 2018 have been shown to affect gasdermin D activity either through modulation of processing or interference with pore formation. A multitude of murine in vivo studies have since followed. Here, we discuss the current state of research surrounding these three inhibitors, caveats to their use, and a set of guiding principles that researchers should consider when pursuing further studies of gasdermin D inhibition.

Post-translational control of NLRP3 inflammasome signaling.

Inflammasomes serve as critical sensors for disruptions to cellular homeostasis, with inflammasome assembly leading to inflammatory caspase activation, gasdermin cleavage, and cytokine release. While the canonical pathways leading to priming, assembly, and pyroptosis are well characterized, recent work has begun to focus on the role of post-translational modifications (PTMs) in regulating inflammasome activity. A diverse array of PTMs, including phosphorylation, ubiquitination, SUMOylation, acetylation, and glycosylation, exert both activating and inhibitory influences on members of the inflammasome cascade through effects on protein-protein interactions, stability, and localization. Dysregulation of inflammasome activation is associated with a number of inflammatory diseases, and evidence is emerging that aberrant modification of inflammasome components contributes to this dysregulation. This review provides insight into PTMs within the NLRP3 inflammasome pathway and their functional consequences on the signaling cascade and highlights outstanding questions that remain regarding the complex web of signals at play.

Protein engineering reveals that gasdermin A preferentially targets mitochondrial membranes over the plasma membrane during pyroptosis.

When activated, gasdermin family members are thought to be pore-forming proteins that cause lytic cell death. Despite this, numerous studies have suggested that the threshold for lytic cell death is dependent on which gasdermin family member is activated. Determination of the propensity of various gasdermin family members to cause pyroptosis has been handicapped by the fact that for many of them, the mechanisms and timing of their activation are uncertain. In this article, we exploit the recently discovered exosite-mediated recognition of gasdermin D (GSDMD) by the inflammatory caspases to develop a system that activates gasdermin family members in an efficient and equivalent manner. We leverage this system to show that upon activation, GSDMD and gasdermin A (GSDMA) exhibit differential subcellular localization, differential plasma membrane permeabilization, and differential lytic cell death. While GSDMD localizes rapidly to both the plasma membrane and organelle membranes, GSDMA preferentially localizes to the mitochondria with delayed and diminished accumulation at the plasma membrane. As a consequence of this differential kinetics of subcellular localization, N-terminal GSDMA results in early mitochondrial dysfunction relative to plasma membrane permeabilization. This study thus challenges the assumption that gasdermin family members effect cell death through identical mechanisms and establishes that their activation in their respective tissues of expression likely results in different immunological outcomes.

The kinase IKKα inhibits activation of the transcription factor NF-κB by phosphorylating the regulatory molecule TAX1BP1.

In response to stimulation with proinflammatory cytokines, the deubiquitinase A20 inducibly interacts with the regulatory molecules TAX1BP1, Itch and RNF11 to form the A20 ubiquitin-editing complex. However, the molecular signal that coordinates the assembly of this complex has remained elusive. Here we demonstrate that TAX1BP1 was inducibly phosphorylated on Ser593 and Ser624 in response to proinflammatory stimuli. The kinase IKKα, but not IKKß, was required for phosphorylation of TAX1BP1 and directly phosphorylated TAX1BP1 in response to stimulation with tumor necrosis factor (TNF) or interleukin 1 (IL-1). TAX1BP1 phosphorylation was pivotal for cytokine-dependent interactions among TAX1BP1, A20, Itch and RNF11 and downregulation of signaling by the transcription factor NF-κB. IKKα therefore serves a key role in the negative feedback of NF-κB canonical signaling by orchestrating assembly of the A20 ubiquitin-editing complex to limit inflammatory gene activation.

Discovery of a Redox Thiol Switch: Implications for Cellular Energy Metabolism.

The redox-based modifications of cysteine residues in proteins regulate their function in many biological processes. The gas molecule H2S has been shown to persulfidate redox sensitive cysteine residues resulting in an H2S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent in detecting cysteine modifications. Here we developed a TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. We revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as a novel substrate in pancreatic beta cells. Moreover, we showed that under oxidative stress conditions, increased H2S can target enzymes involved in energy metabolism by switching specific cysteine modifications to persulfides. Specifically, we discovered a Redox Thiol Switch, from protein S-glutathioinylation to S-persulfidation (RTSGS). We propose that the RTSGS from S-glutathioinylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

Human polymorphisms in GSDMD alter the inflammatory response.

Exomic studies have demonstrated that innate immune genes exhibit an even higher degree of variation than the majority of other gene families. However, the phenotypic implications of this genetic variation are not well understood, with effects ranging from hypomorphic to silent to hyperfunctioning. In this work, we study the functional consequences of this variation by investigating polymorphisms in gasdermin D, the key pyroptotic effector protein. We find that, although SNPs affecting potential posttranslational modifications did not affect gasdermin D function or pyroptosis, polymorphisms disrupting sites predicted to be structurally important dramatically alter gasdermin D function. The manner in which these polymorphisms alter function varies from conserving normal pyroptotic function to inhibiting caspase cleavage to disrupting oligomerization and pore formation. Further, downstream of inflammasome activation, polymorphisms that cause loss of gasdermin D function convert inflammatory pyroptotic cell death into immunologically silent apoptotic cell death. These findings suggest that human genetic variation can alter mechanisms of cell death in inflammation.

Study of vibrational resonance in nonlinear signal processing.

Vibrational resonance (VR) intentionally applies high-frequency periodic vibrations to a nonlinear system, in order to obtain enhanced efficiency for a number of information processing tasks. Note that VR is analogous to stochastic resonance where enhanced processing is sought via purposeful addition of a random noise instead of deterministic high-frequency vibrations. Comparatively, due to its ease of implementation, VR provides a valuable approach for nonlinear signal processing, through detailed modalities that are still under investigation. In this paper, VR is investigated in arrays of nonlinear processing devices, where a range of high-frequency sinusoidal vibrations of the same amplitude at different frequencies are injected and shown capable of enhancing the efficiency for estimating unknown signal parameters or for detecting weak signals in noise. In addition, it is observed that high-frequency vibrations with differing frequencies can be considered, at the sampling times, as independent random variables. This property allows us here to develop a probabilistic analysis-much like in stochastic resonance-and to obtain a theoretical basis for the VR effect and its optimization for signal processing. These results provide additional insight for controlling the capabilities of VR for nonlinear signal processing. This article is part of the theme issue 'Vibrational and stochastic resonance in driven nonlinear systems (part 1)'.

Faster, reduced cost calibration method development methods for the analysis of fermentation product using near-infrared spectroscopy (NIRS).

Recent innovations in synthetic biology, fermentation, and process development have decreased time to market by reducing strain construction cycle time and effort. Faster analytical methods are required to keep pace with these innovations, but current methods of measuring fermentation titers often involve manual intervention and are slow, time-consuming, and difficult to scale. Spectroscopic methods like near-infrared (NIR) spectroscopy address this shortcoming; however, NIR methods require calibration model development that is often costly and time-consuming. Here, we introduce two approaches that speed up calibration model development. First, generalized calibration modeling (GCM) or sibling modeling, which reduces calibration modeling time and cost by up to 50% by reducing the number of samples required. Instead of constructing analyte-specific models, GCM combines a reduced number of spectra from several individual analytes to produce a large pool of spectra for a generalized model predicting all analyte levels. Second, randomized multicomponent multivariate modeling (RMMM) reduces modeling time by mixing multiple analytes into one sample matrix and then taking the spectral measurements. Afterward, individual calibration methods are developed for the various components in the mixture. Time saved from the use of RMMM is proportional to the number of components or analytes in the mixture. When combined, the two methods effectively reduce the associated cost and time for calibration model development by a factor of 10.

Mechanism of gasdermin D recognition by inflammatory caspases and their inhibition by a gasdermin D-derived peptide inhibitor.

The inflammasomes are signaling platforms that promote the activation of inflammatory caspases such as caspases-1, -4, -5, and -11. Recent studies identified gasdermin D (GSDMD) as an effector for pyroptosis downstream of the inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases allows its N-terminal domain to associate with membrane lipids and form pores that induce pyroptotic cell death. Despite the important role of GSDMD in pyroptosis, the molecular mechanisms of GSDMD recognition and cleavage by inflammatory caspases that trigger pyroptosis are poorly understood. Here, we demonstrate that the catalytic domains of inflammatory caspases can directly bind to both the full-length GSDMD and its cleavage site peptide, FLTD. A GSDMD-derived inhibitor, N-acetyl-Phe-Leu-Thr-Asp-chloromethylketone (Ac-FLTD-CMK), inhibits GSDMD cleavage by caspases-1, -4, -5, and -11 in vitro, suppresses pyroptosis downstream of both canonical and noncanonical inflammasomes, as well as reduces IL-1ß release following activation of the NLRP3 inflammasome in macrophages. By contrast, the inhibitor does not target caspase-3 or apoptotic cell death, suggesting that Ac-FLTD-CMK is a specific inhibitor for inflammatory caspases. Crystal structure of caspase-1 in complex with Ac-FLTD-CMK reveals extensive enzyme-inhibitor interactions involving both hydrogen bonds and hydrophobic contacts. Comparison with other caspase-1 structures demonstrates drastic conformational changes at the four active-site loops that assemble the catalytic groove. The present study not only contributes to our understanding of GSDMD recognition by inflammatory caspases but also reports a specific inhibitor for these caspases that can serve as a tool for investigating inflammasome signaling.

IL-33 promotes recovery from acute colitis by inducing miR-320 to stimulate epithelial restitution and repair.

Defective and/or delayed wound healing has been implicated in the pathogenesis of several chronic inflammatory disorders, including inflammatory bowel disease (IBD). The resolution of inflammation is particularly important in mucosal organs, such as the gut, where restoration of epithelial barrier function is critical to reestablish homeostasis with the interfacing microenvironment. Although IL-33 and its receptor ST2/ILRL1 are known to be increased and associated with IBD, studies using animal models of colitis to address the mechanism have yielded ambiguous results, suggesting both pathogenic and protective functions. Unlike those previously published studies, we focused on the functional role of IL-33/ST2 during an extended (2-wk) recovery period after initial challenge in dextran sodium sulfate (DSS)-induced colitic mice. Our results show that during acute, resolving colitis the normal function of endogenous IL-33 is protection, and the lack of either IL-33 or ST2 impedes the overall recovery process, while exogenous IL-33 administration during recovery dramatically accelerates epithelial restitution and repair, with concomitant improvement of colonic inflammation. Mechanistically, we show that IL-33 stimulates the expression of a network of microRNAs (miRs) in the Caco2 colonic intestinal epithelial cell (IEC) line, especially miR-320, which is increased by >16-fold in IECs isolated from IL-33-treated vs. vehicle-treated DSS colitic mice. Finally, IL-33-dependent in vitro proliferation and wound closure of Caco-2 IECs is significantly abrogated after specific inhibition of miR-320A. Together, our data indicate that during acute, resolving colitis, IL-33/ST2 plays a crucial role in gut mucosal healing by inducing epithelial-derived miR-320 that promotes epithelial repair/restitution and the resolution of inflammation.

Mid-infrared hybrid Si/VO2 modulator electrically driven by graphene electrodes.

Silicon photonic platforms are of significant interest for a variety of applications that operate in the mid-infrared regime. However, the realization of efficient mid-IR modulators, key components in any integrated optics platform, is still a challenging topic. Here, an ultra-compact high-speed hybrid Si/VO2 modulator operating at a mid-IR wavelength of 3.8 µm is presented. Electrical properties of graphene are employed to achieve a reversible insulating-metal phase transition in VO2 by electrical actuation. The thermal characteristics of graphene are employed to improve the response time of the VO2 phase transition through speed up heating and dissipation processes, thus enhancing the modulation speed. Optical and thermal simulations show an extinction ratio of 4.4 dB/µm, an insertion loss of 0.1 dB/µm, and high modulation speed of 23 ns. A larger modulation depth as high as 10 dB/µm can be achieved at the cost of lower modulation speed.

Terahertz optical fibers [Invited].

Lying between optical and microwave ranges, the terahertz band in the electromagnetic spectrum is attracting increased attention. Optical fibers are essential for developing the full potential of complex terahertz systems. In this manuscript, we review the optimal materials, the guiding mechanisms, the fabrication methodologies, the characterization methods and the applications of such terahertz waveguides. We examine various optical fiber types including tube fibers, solid core fiber, hollow-core photonic bandgap, anti-resonant fibers, porous-core fibers, metamaterial-based fibers, and their guiding mechanisms. The optimal materials for terahertz applications are discussed. The past and present trends of fabrication methods, including drilling, stacking, extrusion and 3D printing, are elaborated. Fiber characterization methods including different optics for terahertz time-domain spectroscopy (THz-TDS) setups are reviewed and application areas including short-distance data transmission, imaging, sensing, and spectroscopy are discussed.

Phosphorylation of the E3 ubiquitin protein ligase ITCH diminishes binding to its cognate E2 ubiquitin ligase.

Heightened and extended inflammation underlies the pathogenesis of many disorders, including inflammatory bowel disease, sepsis, and inflammatory arthritis. Ubiquitin networks help dictate the strength and duration of inflammatory signaling. In innate immunity, the itchy E3 ubiquitin protein ligase (ITCH)-A20 ubiquitin-editing complex inhibits receptor-interacting Ser/Thr kinase (RIPK) activation by removing Lys-63-linked polyubiquitinated chains from key proteins in the nuclear factor kappa B (NF-κB) signaling pathway. The complex then attaches polyubiquitinated chains to these proteins to target them for lysosomal or proteasomal destruction. ITCH is phosphorylated and thereby inhibited by inhibitor of nuclear factor kappa B kinase subunit beta (IKKß) to fine-tune the inflammatory response to the strength of the offending signal. However, the biochemical mechanism by which E3 ubiquitination is impaired by IKK-driven phosphorylation remains unclear. Here, we report that this phosphorylation impedes ITCH binding to its cognate E2 ubiquitin ligase, UbcH7. Using CRISPR-Cas9 genetic knockout to mimic the ITCH-UbcH7-inhibited state, we further show that genetic UbcH7 deficiency phenocopies ITCH phosphorylation in regulating RIPK2 ubiquitination. We conclude that phosphorylation can disrupt the binding of an E3 ubiquitin ligase to an E2-conjugating enzyme, leading to prolonged inflammatory signaling. To our knowledge, this is the first report of E3 ubiquitin ligase phosphorylation inhibiting E3 ligase activity by impairing E2-E3 complex formation.

Neural evidence for long-term marriage shaping the functional brain network organization between couples.

Long-term married couples have been reported to share personality and behavioural similarities, but whether long-term marriage would shape the brain is hitherto unknown. In this study, 35 pairs of long-term married couples, who have married and living together at least 30 years, were recruited, and resting state functional magnetic resonance imaging was used to examine the neural correlates of long-term marriage between couples. Seven intrinsic connectivity networks were extracted using spatially constrained group independent component analysis, and the spatial similarity of each network as well as functional connectome similarity between couples were investigated respectively. The significant spatial similarities in the salience and frontoparietal networks as well as marginally significant connectome similarity were observed in long-term married couples. In addition, the marital duration showed a significantly positive correlation with the spatial similarity in the frontoparietal network and connectome similarity. The results provide objective evidence that long-term marriage would shape brain network organization, and the combination of initial personality traits and long-term common experience of the couples may be potential factors that account for similar brain network organizations between couples.

Localized surface plasmon resonance biosensor: an improved technique for SERS response intensification.

As technology continues to advance, the development of novel sensing systems opens new possibilities for low-cost, practical biosensing applications. In this Letter, we demonstrate a localized surface plasmon resonance system that combines both wave-guiding and plasmonic resonance sensing with a single microstructured polymeric structure. Characterizing the sensor using the finite element method simulation shows, to the best of our knowledge, a record wavelength sensitivity (WS) of 111000 nm/refractive index unit (RIU), high amplitude sensitivity (AS) of 2050 RIU-1, high sensor resolution and limit of detection of 9×10-7 RIU and 8.12×10-12 RIU2/nm, respectively. Furthermore, these sensors have the capability to detect an analyte within the refractive index range of 1.33-1.43 in the visible to mid-IR, therefore being potentially suitable for applications in biomolecular and chemical analyte detection.