NF-κB-inducing kinase maintains T cell metabolic fitness in antitumor immunity.

Metabolic reprograming toward aerobic glycolysis is a pivotal mechanism shaping immune responses. Here we show that deficiency in NF-κB-inducing kinase (NIK) impairs glycolysis induction, rendering CD8+ effector T cells hypofunctional in the tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8+ T cell metabolism and effector function, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. NIK regulates T cell metabolism via a NF-κB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK prevents autophagic degradation of HK2 through controlling cellular reactive oxygen species levels, which in turn involves modulation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that mediates production of the antioxidant NADPH. We show that the G6PD-NADPH redox system is important for HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a post-translational mechanism of metabolic regulation.

Structural Basis for Transcript Elongation Control by NusG Family Universal Regulators.

NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. Bacterial NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most genes, enhancing elongation by suppressing RNAP backtracking and coordinating ρ-dependent termination and translation. The NusG paralog RfaH engages the EC only at operon polarity suppressor (ops) sites and suppresses both backtrack and hairpin-stabilized pausing. We used single-particle cryoelectron microscopy (cryo-EM) to determine structures of ECs at ops with NusG or RfaH. Both factors chaperone base-pairing of the upstream duplex DNA to suppress backtracking, explaining stimulation of elongation genome-wide. The RfaH-opsEC structure reveals how RfaH confers operon specificity through specific recognition of an ops hairpin in the single-stranded nontemplate DNA and tighter binding to the EC to exclude NusG. Tight EC binding by RfaH sterically blocks the swiveled RNAP conformation necessary for hairpin-stabilized pausing. The universal conservation of NusG/RfaH/Spt5 suggests that the molecular mechanisms uncovered here are widespread.

NIK signaling axis regulates dendritic cell function in intestinal immunity and homeostasis.

Dendritic cells (DCs) play an integral role in regulating mucosal immunity and homeostasis, but the signaling network mediating this function of DCs is poorly defined. We identified the noncanonical NF-κB-inducing kinase (NIK) as a crucial mediator of mucosal DC function. DC-specific NIK deletion impaired intestinal immunoglobulin A (IgA) secretion and microbiota homeostasis, rendering mice sensitive to an intestinal pathogen, Citrobacter rodentium. DC-specific NIK was required for expression of the IgA transporter polymeric immunoglobulin receptor (pIgR) in intestinal epithelial cells, which in turn relied on the cytokine IL-17 produced by TH17 cells and innate lymphoid cells (ILCs). NIK-activated noncanonical NF-κB induced expression of IL-23 in DCs, contributing to the maintenance of TH17 cells and type 3 ILCs. Consistent with the dual functions of IL-23 and IL-17 in mucosal immunity and inflammation, NIK deficiency also ameliorated colitis induction. Thus, our data suggest a pivotal role for the NIK signaling axis in regulating DC functions in intestinal immunity and homeostasis.

PD-1-Expressing SARS-CoV-2-Specific CD8+ T Cells Are Not Exhausted, but Functional in Patients with COVID-19.

Memory T cell responses have been demonstrated in COVID-19 convalescents, but ex vivo phenotypes of SARS-CoV-2-specific T cells have been unclear. We detected SARS-CoV-2-specific CD8+ T cells by MHC class I multimer staining and examined their phenotypes and functions in acute and convalescent COVID-19. Multimer+ cells exhibited early differentiated effector-memory phenotypes in the early convalescent phase. The frequency of stem-like memory cells was increased among multimer+ cells in the late convalescent phase. Cytokine secretion assays combined with MHC class I multimer staining revealed that the proportion of interferon-γ (IFN-γ)-producing cells was significantly lower among SARS-CoV-2-specific CD8+ T cells than those specific to influenza A virus. Importantly, the proportion of IFN-γ-producing cells was higher in PD-1+ cells than PD-1- cells among multimer+ cells, indicating that PD-1-expressing, SARS-CoV-2-specific CD8+ T cells are not exhausted, but functional. Our current findings provide information for understanding of SARS-CoV-2-specific CD8+ T cells elicited by infection or vaccination.

TREK-1 and Best1 channels mediate fast and slow glutamate release in astrocytes upon GPCR activation.

Astrocytes release glutamate upon activation of various GPCRs to exert important roles in synaptic functions. However, the molecular mechanism of release has been controversial. Here, we report two kinetically distinct modes of nonvesicular, channel-mediated glutamate release. The fast mode requires activation of G(αi), dissociation of G(ßγ), and subsequent opening of glutamate-permeable, two-pore domain potassium channel TREK-1 through direct interaction between G(ßγ) and N terminus of TREK-1. The slow mode is Ca(2+) dependent and requires G(αq) activation and opening of glutamate-permeable, Ca(2+)-activated anion channel Best1. Ultrastructural analyses demonstrate that TREK-1 is preferentially localized at cell body and processes, whereas Best1 is mostly found in microdomains of astrocytes near synapses. Diffusion modeling predicts that the fast mode can target neuronal mGluR with peak glutamate concentration of 100 µM, whereas slow mode targets neuronal NMDA receptors at around 1 µM. Our results reveal two distinct sources of astrocytic glutamate that can differentially influence neighboring neurons.

Pseudo-halide anion engineering for α-FAPbI3 perovskite solar cells.

Metal halide perovskites of the general formula ABX3-where A is a monovalent cation such as caesium, methylammonium or formamidinium; B is divalent lead, tin or germanium; and X is a halide anion-have shown great potential as light harvesters for thin-film photovoltaics1-5. Among a large number of compositions investigated, the cubic α-phase of formamidinium lead triiodide (FAPbI3) has emerged as the most promising semiconductor for highly efficient and stable perovskite solar cells6-9, and maximizing the performance of this material in such devices is of vital importance for the perovskite research community. Here we introduce an anion engineering concept that uses the pseudo-halide anion formate (HCOO-) to suppress anion-vacancy defects that are present at grain boundaries and at the surface of the perovskite films and to augment the crystallinity of the films. The resulting solar cell devices attain a power conversion efficiency of 25.6 per cent (certified 25.2 per cent), have long-term operational stability (450 hours) and show intense electroluminescence with external quantum efficiencies of more than 10 per cent. Our findings provide a direct route to eliminate the most abundant and deleterious lattice defects present in metal halide perovskites, providing a facile access to solution-processable films with improved optoelectronic performance.

Structural biology of the Toll-like receptor family.

Innate immune receptors respond to common structural patterns in microbial molecules and are called pattern recognition receptors. Toll-like receptors (TLRs) play critical roles in the innate immune system by recognizing microbial lipids, carbohydrates, nucleic acids, and proteins. Precise definition of the ligand "pattern" of TLRs has been difficult to determine primarily owing to a lack of high-resolution structures. Recently, the structures of several TLR-ligand complexes and the intracellular signaling domains have been determined by X-ray crystallography. This new structural information, combined with extensive biochemical and immunological data accumulated over decades, sheds new light on ligand-recognition and -activation mechanisms. In this review, we summarize the TLR structures and discuss proposed ligand-recognition and -activation mechanisms.

Activation of invariant natural killer T cells stimulates adipose tissue remodeling via adipocyte death and birth in obesity.

In obesity, adipose tissue undergoes dynamic remodeling processes such as adipocyte hypertrophy, hypoxia, immune responses, and adipocyte death. However, whether and how invariant natural killer T (iNKT) cells contribute to adipose tissue remodeling are elusive. In this study, we demonstrate that iNKT cells remove unhealthy adipocytes and stimulate the differentiation of healthy adipocytes. In obese adipose tissue, iNKT cells were abundantly found nearby dead adipocytes. FasL-positive adipose iNKT cells exerted cytotoxic effects to eliminate hypertrophic and pro-inflammatory Fas-positive adipocytes. Furthermore, in vivo adipocyte-lineage tracing mice model showed that activation of iNKT cells by alpha-galactosylceramide promoted adipocyte turnover, eventually leading to potentiation of the insulin-dependent glucose uptake ability in adipose tissue. Collectively, our data propose a novel role of adipose iNKT cells in the regulation of adipocyte turnover in obesity.

Neurodevelopmental disorder-associated CYFIP2 regulates membraneless organelles and eIF2α phosphorylation via protein interactors and actin cytoskeleton.

De novo variants in the Cytoplasmic FMR1-interacting protein 2 (CYFIP2) have been repeatedly associated with neurodevelopmental disorders and epilepsy, underscoring its critical role in brain development and function. While CYFIP2's role in regulating actin polymerization as part of the WAVE regulatory complex (WRC) is well-established, its additional molecular functions remain relatively unexplored. In this study, we performed unbiased quantitative proteomic analysis, revealing 278 differentially expressed proteins (DEPs) in the forebrain of Cyfip2 knock-out embryonic mice compared to wild-type mice. Unexpectedly, these DEPs, in conjunction with previously identified CYFIP2 brain interactors, included not only other WRC components but also numerous proteins associated with membraneless organelles (MLOs) involved in mRNA processing and translation within cells, including the nucleolus, stress granules, and processing bodies. Additionally, single-cell transcriptomic analysis of the Cyfip2 knock-out forebrain revealed gene expression changes linked to cellular stress responses and MLOs. We also observed morphological changes in MLOs in Cyfip2 knock-out brains and CYFIP2 knock-down cells under basal and stress conditions. Lastly, we demonstrated that CYFIP2 knock-down in cells, potentially through WRC-dependent actin regulation, suppressed the phosphorylation levels of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α), thereby enhancing protein synthesis. These results suggest a physical and functional connection between CYFIP2 and various MLO proteins and also extend CYFIP2's role within the WRC from actin regulation to influencing eIF2α phosphorylation and protein synthesis. With these dual functions, CYFIP2 may fine-tune the balance between MLO formation/dynamics and protein synthesis, a crucial aspect of proper mRNA processing and translation.

A single-component, light-assisted uncaging switch for endoproteolytic release.

Proteases function as pivotal molecular switches, initiating numerous biological events. Notably, potyviral protease, derived from plant viruses, has emerged as a trusted proteolytic switch in synthetic biological circuits. To harness their capabilities, we have developed a single-component photocleavable switch, termed LAUNCHER (Light-Assisted UNcaging switCH for Endoproteolytic Release), by employing a circularly permutated tobacco etch virus protease and a blue-light-gated substrate, which are connected by fine-tuned intermodular linkers. As a single-component system, LAUNCHER exhibits a superior signal-to-noise ratio compared with multi-component systems, enabling precise and user-controllable release of payloads. This characteristic renders LAUNCHER highly suitable for diverse cellular applications, including transgene expression, tailored subcellular translocation and optochemogenetics. Additionally, the plug-and-play integration of LAUNCHER into existing synthetic circuits facilitates the enhancement of circuit performance. The demonstrated efficacy of LAUNCHER in improving existing circuitry underscores its significant potential for expanding its utilization in various applications.

RNA Polymerase Accommodates a Pause RNA Hairpin by Global Conformational Rearrangements that Prolong Pausing.

Sequence-specific pausing by RNA polymerase (RNAP) during transcription plays crucial and diverse roles in gene expression. In bacteria, RNA structures are thought to fold within the RNA exit channel of the RNAP and can increase pause lifetimes significantly. The biophysical mechanism of pausing is uncertain. We used single-particle cryo-EM to determine structures of paused complexes, including a 3.8-Å structure of an RNA hairpin-stabilized, paused RNAP that coordinates RNA folding in the his operon attenuation control region of E. coli. The structures revealed a half-translocated pause state (RNA post-translocated, DNA pre-translocated) that can explain transcriptional pausing and a global conformational change of RNAP that allosterically inhibits trigger loop folding and can explain pause hairpin action. Pause hairpin interactions with the RNAP RNA exit channel suggest how RNAP guides the formation of nascent RNA structures.

An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options.

Transcriptional pausing underpins the regulation of cellular RNA synthesis, but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, multidomain RNA polymerase (RNAP) trigger reversible conformational changes at pause sites that temporarily interrupt the nucleotide addition cycle. These interactions initially rearrange the elongation complex (EC) into an elemental paused EC (ePEC). ePECs can form longer-lived PECs by further rearrangements or interactions of diffusible regulators. For both bacterial and mammalian RNAPs, a half-translocated state in which the next DNA template base fails to load into the active site appears central to the ePEC. Some RNAPs also swivel interconnected modules that may stabilize the ePEC. However, it is unclear whether swiveling and half-translocation are requisite features of a single ePEC state or if multiple ePEC states exist. Here, we use cryo-electron microscopy (cryo-EM) analysis of ePECs with different RNA-DNA sequences combined with biochemical probes of ePEC structure to define an interconverting ensemble of ePEC states. ePECs occupy either pre- or half-translocated states but do not always swivel, indicating that difficulty in forming the posttranslocated state at certain RNA-DNA sequences may be the essence of the ePEC. The existence of multiple ePEC conformations has broad implications for transcriptional regulation.

A selective ER-phagy exerts neuroprotective effects via modulation of α-synuclein clearance in parkinsonian models.

The endoplasmic reticulum (ER) is selectively degraded by ER-phagy to maintain cell homeostasis. α-synuclein accumulates in the ER, causing ER stress that contributes to neurodegeneration in Parkinson's disease (PD), but the role of ER-phagy in α-synuclein modulation is largely unknown. Here, we investigated the mechanisms by which ER-phagy selectively recognizes α-synuclein for degradation in the ER. We found that ER-phagy played an important role in the degradation of α-synuclein and recovery of ER function through interaction with FAM134B, where calnexin is required for the selective FAM134B-mediated α-synuclein clearance via ER-phagy. Overexpression of α-synuclein in the ER of the substantia nigra (SN) resulted in marked loss of dopaminergic neurons and motor deficits, mimicking PD characteristics. However, enhancement of ER-phagy using FAM134B overexpression in the SN exerted neuroprotective effects on dopaminergic neurons and recovered motor performance. These data suggest that ER-phagy represents a specific ER clearance mechanism for the degradation of α-synuclein.

Mosaic RBD nanoparticles induce intergenus cross-reactive antibodies and protect against SARS-CoV-2 challenge.

Recurrent spillovers of α- and ß-coronaviruses (CoV) such as severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome-CoV, SARS-CoV-2, and possibly human CoV have caused serious morbidity and mortality worldwide. In this study, six receptor-binding domains (RBDs) derived from α- and ß-CoV that are considered to have originated from animals and cross-infected humans were linked to a heterotrimeric scaffold, proliferating cell nuclear antigen (PCNA) subunits, PCNA1, PCNA2, and PCNA3. They assemble to create a stable mosaic multivalent nanoparticle, 6RBD-np, displaying a ring-shaped disk with six protruding antigens, like jewels in a crown. Prime-boost immunizations with 6RBD-np in mice induced significantly high Ab titers against RBD antigens derived from α- and ß-CoV and increased interferon (IFN-γ) production, with full protection against the SARS-CoV-2 wild type and Delta challenges. The mosaic 6RBD-np has the potential to induce intergenus cross-reactivity and to be developed as a pan-CoV vaccine against future CoV spillovers.

The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses.

Metabolic fitness of T cells is crucial for immune responses against infections and tumorigenesis. Both the T cell receptor (TCR) signal and environmental cues contribute to the induction of T cell metabolic reprogramming, but the underlying mechanism is incompletely understood. Here, we identified the E3 ubiquitin ligase Peli1 as an important regulator of T cell metabolism and antitumor immunity. Peli1 ablation profoundly promotes tumor rejection, associated with increased tumor-infiltrating CD4 and CD8 T cells. The Peli1-deficient T cells display markedly stronger metabolic activities, particularly glycolysis, than wild-type T cells. Peli1 controls the activation of a metabolic kinase, mTORC1, stimulated by both the TCR signal and growth factors, and this function of Peli1 is mediated through regulation of the mTORC1-inhibitory proteins, TSC1 and TSC2. Peli1 mediates non-degradative ubiquitination of TSC1, thereby promoting TSC1-TSC2 dimerization and TSC2 stabilization. These results establish Peli1 as a novel regulator of mTORC1 and downstream mTORC1-mediated actions on T cell metabolism and antitumor immunity.

The Effect of Nationwide Organized Cancer Screening Programs on Gastric Cancer Mortality: A Synthetic Control Study.

BACKGROUND & AIMS: Nationwide organized gastric cancer (GC) screening programs have been running for decades in South Korea and Japan. This study conducted a quasi-experimental analysis to assess the population impact of these programs on GC mortality. METHODS: We used the flexible synthetic control method (SCM) to estimate the effect of the screening programs on age-standardized GC mortality and other upper gastrointestinal (UGI) diseases (esophageal cancer and peptic ulcer) among people aged ≥40 years. World Health Organization mortality data and country-level covariates from the World Bank and the Global Burden of Diseases study were used for the analyses. We compared postintervention trends in outcome with the counterfactual trend of the synthetic control and estimated average postintervention rate ratios (RRs) with associated 95% confidence intervals (CIs). A series of sensitivity analyses were conducted. RESULTS: The preintervention fits were acceptable for the analyses of South Korea and Japan's GC mortality but poor for Japan's other UGI disease mortality. The average postintervention RRs were 0.83 (95% CI, 0.71-0.96) for GC mortality and 0.72 (95% CI, 0.57-0.90) for other UGI disease mortality in South Korea. The RR reached 0.59 by the 15th year after the initiation of nationwide screening. For Japan, the average RRs were 0.97 (95% CI, 0.88-1.07) for GC mortality and 0.93 (95% CI, 0.68-1.28) for other UGI disease mortality. Sensitivity analysis reveals the result for Japan may potentially be biased. CONCLUSIONS: South Korea's nationwide GC screening has apparent benefits, whereas the Japanese program's effectiveness is uncertain. The experiences of South Korea and Japan could serve as a reference for other countries.

Chronic hepatitis B baseline viral load and on-treatment liver cancer risk: A multinational cohort study of HBeAg-positive patients.

BACKGROUND AND AIMS: A single-nation study reported that pretreatment HBV viral load is associated with on-treatment risk of HCC in patients who are HBeAg-positive without cirrhosis and with chronic hepatitis B initiating antiviral treatment. We aimed to validate the association between baseline HBV viral load and on-treatment HCC risk in a larger, multinational cohort. APPROACH AND RESULTS: Using a multinational cohort from Korea, Hong Kong, and Taiwan involving 7545 adult patients with HBeAg-positive, without cirrhosis and with chronic hepatitis B who started entecavir or tenofovir treatment with baseline HBV viral load ≥5.00 log 10 IU/mL, HCC risk was estimated by baseline viral load. HBV viral load was analyzed as a categorical variable. During continuous antiviral treatment (median, 4.28 y), HCC developed in 200 patients (incidence rate, 0.61 per 100 person-years). Baseline HBV DNA level was independently associated with on-treatment HCC risk in a nonlinear pattern. HCC risk was lowest with the highest baseline viral load (≥8.00 log 10 IU/mL; incidence rate, 0.10 per 100 person-years), but increased sharply as baseline viral load decreased. The adjusted HCC risk was 8.05 times higher (95% CI, 3.34-19.35) with baseline viral load ≥6.00 and <7.00 log 10 IU/mL (incidence rate, 1.38 per 100 person-years) compared with high (≥8.00 log 10 IU/mL) baseline viral load ( p <0.001). CONCLUSIONS: In a multinational cohort of adult patients with HBeAg-positive without cirrhosis and with chronic hepatitis B, baseline HBV viral load was significantly associated with HCC risk despite antiviral treatment. Patients with the highest viral load who initiated treatment had the lowest long-term risk of HCC development.

Enteric Viruses Ameliorate Gut Inflammation via Toll-like Receptor 3 and Toll-like Receptor 7-Mediated Interferon-ß Production.

Metagenomic studies show that diverse resident viruses inhabit the healthy gut; however, little is known about the role of these viruses in the maintenance of gut homeostasis. We found that mice treated with antiviral cocktail displayed more severe dextran sulfate sodium (DSS)-induced colitis compared with untreated mice. DSS-induced colitis was associated with altered enteric viral abundance and composition. When wild-type mice were reconstituted with Toll-like receptor 3 (TLR3) or TLR7 agonists or inactivated rotavirus, colitis symptoms were significantly ameliorated. Mice deficient in both TLR3 and TLR7 were more susceptible to DSS-induced experimental colitis. In humans, combined TLR3 and TLR7 genetic variations significantly influenced the severity of ulcerative colitis. Plasmacytoid dendritic cells isolated from inflamed mouse colon produced interferon-ß in a TLR3 and TLR7-dependent manner. These results imply that recognition of resident viruses by TLR3 and TLR7 is required for protective immunity during gut inflammation.

Loss of Smooth Muscle Tenascin-X Inhibits Vascular Remodeling Through Increased TGF-ß Signaling.

BACKGROUND: Vascular smooth muscle cells (VSMCs) are highly plastic. Vessel injury induces a phenotypic transformation from differentiated to dedifferentiated VSMCs, which involves reduced expression of contractile proteins and increased production of extracellular matrix and inflammatory cytokines. This transition plays an important role in several cardiovascular diseases such as atherosclerosis, hypertension, and aortic aneurysm. TGF-ß (transforming growth factor-ß) is critical for VSMC differentiation and to counterbalance the effect of dedifferentiating factors. However, the mechanisms controlling TGF-ß activity and VSMC phenotypic regulation under in vivo conditions are poorly understood. The extracellular matrix protein TN-X (tenascin-X) has recently been shown to bind TGF-ß and to prevent it from activating its receptor. METHODS: We studied the role of TN-X in VSMCs in various murine disease models using tamoxifen-inducible SMC-specific knockout and adeno-associated virus-mediated knockdown. RESULTS: In hypertensive and high-fat diet-fed mice, after carotid artery ligation as well as in human aneurysmal aortae, expression of Tnxb, the gene encoding TN-X, was increased in VSMCs. Mice with smooth muscle cell-specific loss of TN-X (SMC-Tnxb-KO) showed increased TGF-ß signaling in VSMCs, as well as upregulated expression of VSMC differentiation marker genes during vascular remodeling compared with controls. SMC-specific TN-X deficiency decreased neointima formation after carotid artery ligation and reduced vessel wall thickening during Ang II (angiotensin II)-induced hypertension. SMC-Tnxb-KO mice lacking ApoE showed reduced atherosclerosis and Ang II-induced aneurysm formation under high-fat diet. Adeno-associated virus-mediated SMC-specific expression of short hairpin RNA against Tnxb showed similar beneficial effects. Treatment with an anti-TGF-ß antibody or additional SMC-specific loss of the TGF-ß receptor reverted the effects of SMC-specific TN-X deficiency. CONCLUSIONS: In summary, TN-X critically regulates VSMC plasticity during vascular injury by inhibiting TGF-ß signaling. Our data indicate that inhibition of vascular smooth muscle TN-X may represent a strategy to prevent and treat pathological vascular remodeling.