TFPI is a colonic crypt receptor for TcdB from hypervirulent clade 2 C. difficile.

The emergence of hypervirulent clade 2 Clostridioides difficile is associated with severe symptoms and accounts for >20% of global infections. TcdB is a dominant virulence factor of C. difficile, and clade 2 strains exclusively express two TcdB variants (TcdB2 and TcdB4) that use unknown receptors distinct from the classic TcdB. Here, we performed CRISPR/Cas9 screens for TcdB4 and identified tissue factor pathway inhibitor (TFPI) as its receptor. Using cryo-EM, we determined a complex structure of the full-length TcdB4 with TFPI, defining a common receptor-binding region for TcdB. Residue variations within this region divide major TcdB variants into 2 classes: one recognizes Frizzled (FZD), and the other recognizes TFPI. TFPI is highly expressed in the intestinal glands, and recombinant TFPI protects the colonic epithelium from TcdB2/4. These findings establish TFPI as a colonic crypt receptor for TcdB from clade 2 C. difficile and reveal new mechanisms for CDI pathogenesis.

Engineered CD47 protects T cells for enhanced antitumour immunity.

Adoptively transferred T cells and agents designed to block the CD47-SIRPα axis are promising cancer therapeutics that activate distinct arms of the immune system1,2. Here we administered anti-CD47 antibodies in combination with adoptively transferred T cells with the goal of enhancing antitumour efficacy but observed abrogated therapeutic benefit due to rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors. Anti-CD47-antibody-mediated CAR T cell clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered the CD47 variant CD47(Q31P) (47E), which engages SIRPα and provides a 'don't eat me' signal that is not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47E are resistant to clearance by macrophages after treatment with anti-CD47 antibodies, and mediate substantial, sustained macrophage recruitment to the tumour microenvironment. Although many of the recruited macrophages manifested an M2-like profile3, the combined therapy synergistically enhanced antitumour efficacy. Our study identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T-cell-directed therapeutics with those designed to activate macrophages. It delivers a therapeutic approach that is capable of simultaneously harnessing the antitumour effects of T cells and macrophages, offering enhanced potency against solid tumours.

Complete integration of carbene-transfer chemistry into biosynthesis.

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

High-κ perovskite membranes as insulators for two-dimensional transistors.

The scaling of silicon metal-oxide-semiconductor field-effect transistors has followed Moore's law for decades, but the physical thinning of silicon at sub-ten-nanometre technology nodes introduces issues such as leakage currents1. Two-dimensional (2D) layered semiconductors, with an atomic thickness that allows superior gate-field penetration, are of interest as channel materials for future transistors2,3. However, the integration of high-dielectric-constant (κ) materials with 2D materials, while scaling their capacitance equivalent thickness (CET), has proved challenging. Here we explore transferrable ultrahigh-κ single-crystalline perovskite strontium-titanium-oxide membranes as a gate dielectric for 2D field-effect transistors. Our perovskite membranes exhibit a desirable sub-one-nanometre CET with a low leakage current (less than 10-2 amperes per square centimetre at 2.5 megavolts per centimetre). We find that the van der Waals gap between strontium-titanium-oxide dielectrics and 2D semiconductors mitigates the unfavourable fringing-induced barrier-lowering effect resulting from the use of ultrahigh-κ dielectrics4. Typical short-channel transistors made of scalable molybdenum-disulfide films by chemical vapour deposition and strontium-titanium-oxide dielectrics exhibit steep subthreshold swings down to about 70 millivolts per decade and on/off current ratios up to 107, which matches the low-power specifications suggested by the latest International Roadmap for Devices and Systems5.

Physical immune escape: Weakened mechanical communication leads to escape of metastatic colorectal carcinoma cells from macrophages.

The significance of biochemical cues in the tumor immune microenvironment in affecting cancer metastasis is well established, but the role of physical factors in the microenvironment remains largely unexplored. In this article, we investigated how the mechanical interaction between cancer cells and immune cells, mediated by extracellular matrix (ECM), influences immune escape of cancer cells. We focus on the mechanical regulation of macrophages' targeting ability on two distinct types of colorectal carcinoma (CRC) cells with different metastatic potentials. Our results show that macrophages can effectively target CRC cells with low metastatic potential, due to the strong contraction exhibited by the cancer cells on the ECM, and that cancer cells with high metastatic potential demonstrated weakened contractions on the ECM and can thus evade macrophage attack to achieve immune escape. Our findings regarding the intricate mechanical interactions between immune cells and cancer cells can serve as a crucial reference for further exploration of cancer immunotherapy strategies.

Trichinella spiralis -induced immunomodulation signatures on gut microbiota and metabolic pathways in mice.

The hygiene hypothesis proposes that decreased exposure to infectious agents in developed countries may contribute to the development of allergic and autoimmune diseases. Trichinella spiralis, a parasitic roundworm, causes trichinellosis, also known as trichinosis, in humans. T. spiralis had many hosts, and almost any mammal could become infected. Adult worms lived in the small intestine, while the larvae lived in muscle cells of the same mammal. T. spiralis was a significant public health threat because it could cause severe illness and even death in humans who eat undercooked or raw meat containing the parasite. The complex interactions between gastrointestinal helminths, gut microbiota, and the host immune system present a challenge for researchers. Two groups of mice were infected with T. spiralis vs uninfected control, and the experiment was conducted over 60 days. The 16S rRNA gene sequences and untargeted LC/MS-based metabolomics of fecal and serum samples, respectively, from different stages of development of the Trichinella spiralis-mouse model, were examined in this study. Gut microbiota alterations and metabolic activity accompanied by parasite-induced immunomodulation were detected. The inflammation parameters of the duodenum (villus/crypt ratio, goblet cell number and size, and histological score) were involved in active inflammation and oxidative metabolite profiles. These profiles included increased biosynthesis of phenylalanine, tyrosine, and tryptophan while decreasing cholesterol metabolism and primary and secondary bile acid biosynthesis. These disrupted metabolisms adapted to infection stress during the enteral and parenteral phases and then return to homeostasis during the encapsulated phase. There was a shift from an abundance of Bacteroides in the parenteral phase to an abundance of probiotic Lactobacillus and Treg-associated-Clostridia in the encapsulated phase. Th2 immune response (IL-4/IL-5/IL-13), lamina propria Treg, and immune hyporesponsiveness metabolic pathways (decreased tropane, piperidine and pyridine alkaloid biosynthesis and biosynthesis of alkaloids derived from ornithine, lysine, and nicotinic acid) were all altered. These findings enhanced our understanding of gut microbiota and metabolic profiles of Trichinella -infected mice, which could be a driving force in parasite-shaping immune system maintenance.

An unexpected hydratase synthesizes the green light-absorbing pigment fucoxanthin.

The ketocarotenoid fucoxanthin and its derivatives can absorb blue-green light enriched in marine environments. Fucoxanthin is widely adopted by phytoplankton species as a main light-harvesting pigment, in contrast to land plants that primarily employ chlorophylls. Despite its supreme abundance in the oceans, the last steps of fucoxanthin biosynthesis have remained elusive. Here, we identified the carotenoid isomerase-like protein CRTISO5 as the diatom fucoxanthin synthase that is related to the carotenoid cis-trans isomerase CRTISO from land plants but harbors unexpected enzymatic activity. A crtiso5 knockout mutant in the model diatom Phaeodactylum tricornutum completely lacked fucoxanthin and accumulated the acetylenic carotenoid phaneroxanthin. Recombinant CRTISO5 converted phaneroxanthin into fucoxanthin in vitro by hydrating its carbon-carbon triple bond, instead of functioning as an isomerase. Molecular docking and mutational analyses revealed residues essential for this activity. Furthermore, a photophysiological characterization of the crtiso5 mutant revealed a major structural and functional role of fucoxanthin in photosynthetic pigment-protein complexes of diatoms. As CRTISO5 hydrates an internal alkyne physiologically, the enzyme has unique potential for biocatalytic applications. The discovery of CRTISO5 illustrates how neofunctionalization leads to major diversification events in evolution of photosynthetic mechanisms and the prominent brown coloration of most marine photosynthetic eukaryotes.

Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling.

In the Drosophila immune response, bacterial derived diaminopimelic acid-type peptidoglycan binds the receptors PGRP-LC and PGRP-LE, which through interaction with the adaptor protein Imd leads to activation of the NF-κB homolog Relish and robust antimicrobial peptide gene expression. PGRP-LC, PGRP-LE, and Imd each contain a motif with some resemblance to the RIP Homotypic Interaction Motif (RHIM), a domain found in mammalian RIPK proteins forming functional amyloids during necroptosis. Here we found that despite sequence divergence, these Drosophila cryptic RHIMs formed amyloid fibrils in vitro and in cells. Amyloid formation was required for signaling downstream of Imd, and in contrast to the mammalian RHIMs, was not associated with cell death. Furthermore, amyloid formation constituted a regulatable step and could be inhibited by Pirk, an endogenous feedback regulator of this pathway. Thus, diverse sequence motifs are capable of forming amyloidal signaling platforms, and the formation of these platforms may present a regulatory point in multiple biological processes.

Suppression of distracting inputs by visual-spatial cues is driven by anticipatory alpha activity.

A growing body of research demonstrates that distracting inputs can be proactively suppressed via spatial cues, nonspatial cues, or experience, which are governed by more than one top-down mechanism of attention. However, how the neural mechanisms underlying spatial distractor cues guide proactive suppression of distracting inputs remains unresolved. Here, we recorded electroencephalography signals from 110 participants in 3 experiments to identify the role of alpha activity in proactive distractor suppression induced by spatial cues and its influence on subsequent distractor inhibition. Behaviorally, we found novel changes in the spatial proximity of the distractor: Cueing distractors far away from the target improves search performance for the target, while cueing distractors close to the target hampers performance. Crucially, we found dynamic characteristics of spatial representation for distractor suppression during anticipation. This result was further verified by alpha power increased relatively contralateral to the cued distractor. At both the between- and within-subjects levels, we found that these activities further predicted the decrement of the subsequent PD component, which was indicative of reduced distractor interference. Moreover, anticipatory alpha activity and its link with the subsequent PD component were specific to the high predictive validity of distractor cue. Together, our results reveal the underlying neural mechanisms by which cueing the spatial distractor may contribute to reduced distractor interference. These results also provide evidence supporting the role of alpha activity as gating by proactive suppression.

G-quadruplexes on chromosomal DNA negatively regulates topoisomerase 1 activity.

Human DNA topoisomerase 1 (Top1) is a crucial enzyme responsible for alleviating torsional stress on DNA during transcription and replication, thereby maintaining genome stability. Previous researches had found that non-working Top1 interacted extensively with chromosomal DNA in human cells. However, the reason for its retention on chromosomal DNA remained unclear. In this study, we discovered a close association between Top1 and chromosomal DNA, specifically linked to the presence of G-quadruplex (G4) structures. G4 structures, formed during transcription, trap Top1 and hinder its ability to relax neighboring DNAs. Disruption of the Top1-G4 interaction using G4 ligand relieved the inhibitory effect of G4 on Top1 activity, resulting in a further reduction of R-loop levels in cells. Additionally, the activation of Top1 through the use of a G4 ligand enhanced the toxicity of Top1 inhibitors towards cancer cells. Our study uncovers a negative regulation mechanism of human Top1 and highlights a novel pathway for activating Top1.

The Drosophila NPY-like system protects against chronic stress-induced learning deficit by preventing the disruption of autophagic flux.

Chronic stress may induce learning and memory deficits that are associated with a depression-like state in Drosophila melanogaster. The molecular and neural mechanisms underlying the etiology of chronic stress-induced learning deficit (CSLD) remain elusive. Here, we show that the autophagy-lysosomal pathway, a conserved cellular signaling mechanism, is associated with chronic stress in Drosophila, as indicated by time-series transcriptome profiling. Our findings demonstrate that chronic stress induces the disruption of autophagic flux, and chronic disruption of autophagic flux could lead to a learning deficit. Remarkably, preventing the disruption of autophagic flux by up-regulating the basal autophagy level is sufficient to protect against CSLD. Consistent with the essential role of the dopaminergic system in modulating susceptibility to CSLD, dopamine neuronal activity is also indispensable for chronic stress to induce the disruption of autophagic flux. By screening knockout mutants, we found that neuropeptide F, the Drosophila homolog of neuropeptide Y, is necessary for normal autophagic flux and promotes resilience to CSLD. Moreover, neuropeptide F signaling during chronic stress treatment promotes resilience to CSLD by preventing the disruption of autophagic flux. Importantly, neuropeptide F receptor activity in dopamine neurons also promotes resilience to CSLD. Together, our data elucidate a mechanism by which stress-induced excessive dopaminergic activity precipitates the disruption of autophagic flux, and chronic disruption of autophagic flux leads to CSLD, while inhibitory neuropeptide F signaling to dopamine neurons promotes resilience to CSLD by preventing the disruption of autophagic flux.

Structure, dynamics, and stability of the smallest and most complex 71 protein knot.

Proteins can spontaneously tie a variety of intricate topological knots through twisting and threading of the polypeptide chains. Recently developed artificial intelligence algorithms have predicted several new classes of topological knotted proteins, but the predictions remain to be authenticated experimentally. Here, we showed by X-ray crystallography and solution-state NMR spectroscopy that Q9PR55, an 89-residue protein from Ureaplasma urealyticum, possesses a novel 71 knotted topology that is accurately predicted by AlphaFold 2, except for the flexible N terminus. Q9PR55 is monomeric in solution, making it the smallest and most complex knotted protein known to date. In addition to its exceptional chemical stability against urea-induced unfolding, Q9PR55 is remarkably robust to resist the mechanical unfolding-coupled proteolysis by a bacterial proteasome, ClpXP. Our results suggest that the mechanical resistance against pulling-induced unfolding is determined by the complexity of the knotted topology rather than the size of the molecule.

Induction-concurrent chemoradiotherapy with or without sintilimab in patients with locoregionally advanced nasopharyngeal carcinoma in China (CONTINUUM): a multicentre, open-label, parallel-group, randomised, controlled, phase 3 trial.

BACKGROUND: Anti-PD-1 therapy and chemotherapy is a recommended first-line treatment for recurrent or metastatic nasopharyngeal carcinoma, but the role of PD-1 blockade remains unknown in patients with locoregionally advanced nasopharyngeal carcinoma. We assessed the addition of sintilimab, a PD-1 inhibitor, to standard chemoradiotherapy in this patient population. METHODS: This multicentre, open-label, parallel-group, randomised, controlled, phase 3 trial was conducted at nine hospitals in China. Adults aged 18-65 years with newly diagnosed high-risk non-metastatic stage III-IVa locoregionally advanced nasopharyngeal carcinoma (excluding T3-4N0 and T3N1) were eligible. Patients were randomly assigned (1:1) using blocks of four to receive gemcitabine and cisplatin induction chemotherapy followed by concurrent cisplatin radiotherapy (standard therapy group) or standard therapy with 200 mg sintilimab intravenously once every 3 weeks for 12 cycles (comprising three induction, three concurrent, and six adjuvant cycles to radiotherapy; sintilimab group). The primary endpoint was event-free survival from randomisation to disease recurrence (locoregional or distant) or death from any cause in the intention-to-treat population. Secondary endpoints included adverse events. This trial is registered with ClinicalTrials.gov (NCT03700476) and is now completed; follow-up is ongoing. FINDINGS: Between Dec 21, 2018, and March 31, 2020, 425 patients were enrolled and randomly assigned to the sintilimab (n=210) or standard therapy groups (n=215). At median follow-up of 41·9 months (IQR 38·0-44·8; 389 alive at primary data cutoff [Feb 28, 2023] and 366 [94%] had at least 36 months of follow-up), event-free survival was higher in the sintilimab group compared with the standard therapy group (36-month rates 86% [95% CI 81-90] vs 76% [70-81]; stratified hazard ratio 0·59 [0·38-0·92]; p=0·019). Grade 3-4 adverse events occurred in 155 (74%) in the sintilimab group versus 140 (65%) in the standard therapy group, with the most common being stomatitis (68 [33%] vs 64 [30%]), leukopenia (54 [26%] vs 48 [22%]), and neutropenia (50 [24%] vs 46 [21%]). Two (1%) patients died in the sintilimab group (both considered to be immune-related) and one (<1%) in the standard therapy group. Grade 3-4 immune-related adverse events occurred in 20 (10%) patients in the sintilimab group. INTERPRETATION: Addition of sintilimab to chemoradiotherapy improved event-free survival, albeit with higher but manageable adverse events. Longer follow-up is necessary to determine whether this regimen can be considered as the standard of care for patients with high-risk locoregionally advanced nasopharyngeal carcinoma. FUNDING: National Natural Science Foundation of China, Key-Area Research and Development Program of Guangdong Province, Natural Science Foundation of Guangdong Province, Overseas Expertise Introduction Project for Discipline Innovation, Guangzhou Municipal Health Commission, and Cancer Innovative Research Program of Sun Yat-sen University Cancer Center. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

iDRPro-SC: identifying DNA-binding proteins and RNA-binding proteins based on subfunction classifiers.

Nucleic acid-binding proteins are proteins that interact with DNA and RNA to regulate gene expression and transcriptional control. The pathogenesis of many human diseases is related to abnormal gene expression. Therefore, recognizing nucleic acid-binding proteins accurately and efficiently has important implications for disease research. To address this question, some scientists have proposed the method of using sequence information to identify nucleic acid-binding proteins. However, different types of nucleic acid-binding proteins have different subfunctions, and these methods ignore their internal differences, so the performance of the predictor can be further improved. In this study, we proposed a new method, called iDRPro-SC, to predict the type of nucleic acid-binding proteins based on the sequence information. iDRPro-SC considers the internal differences of nucleic acid-binding proteins and combines their subfunctions to build a complete dataset. Additionally, we used an ensemble learning to characterize and predict nucleic acid-binding proteins. The results of the test dataset showed that iDRPro-SC achieved the best prediction performance and was superior to the other existing nucleic acid-binding protein prediction methods. We have established a web server that can be accessed online: http://bliulab.net/iDRPro-SC.

Advances in new target molecules against schistosomiasis: A comprehensive discussion of physiological structure and nutrient intake.

Schistosomiasis, a severe parasitic disease, is primarily caused by Schistosoma mansoni, Schistosoma japonicum, or Schistosoma haematobium. Currently, praziquantel is the only recommended drug for human schistosome infection. However, the lack of efficacy of praziquantel against juvenile worms and concerns about the emergence of drug resistance are driving forces behind the research for an alternative medication. Schistosomes are obligatory parasites that survive on nutrients obtained from their host. The ability of nutrient uptake depends on their physiological structure. In short, the formation and maintenance of the structure and nutrient supply are mutually reinforcing and interdependent. In this review, we focus on the structural features of the tegument, esophagus, and intestine of schistosomes and their roles in nutrient acquisition. Moreover, we introduce the significance and modes of glucose, lipids, proteins, and amino acids intake in schistosomes. We linked the schistosome structure and nutrient supply, introduced the currently emerging targets, and analyzed the current bottlenecks in the research and development of drugs and vaccines, in the hope of providing new strategies for the prevention and control of schistosomiasis.

A NAC transcription factor represses a module associated with xyloglucan content and regulates aluminum tolerance.

The transcriptional regulation of aluminum (Al) tolerance in plants is largely unknown, although Al toxicity restricts agricultural yields in acidic soils.. Here, we identified a NAM, ATAF1/2, and cup-shaped cotyledon 2 (NAC) transcription factor that participates in Al tolerance in Arabidopsis (Arabidopsis thaliana). Al substantially induced the transcript and protein levels of ANAC070, and loss-of-function anan070 mutants showed remarkably increased Al sensitivity, implying a beneficial role of ANAC070 in plant tolerance to Al toxicity. Further investigation revealed that more Al accumulated in the roots of anac070 mutants, especially in root cell walls, accompanied by a higher hemicellulose and xyloglucan level, implying a possible interaction between ANAC070 and genes that encode proteins responsible for the modification of xyloglucan, including xyloglucan endo-transglycosylases/hydrolase (XTH) or ANAC017. Yeast one hybrid analysis revealed a potential interaction between ANAC070 and ANAC017, but not for other XTHs. Furthermore, dual-luciferase reporter assay, RT-qPCR, and GUS analysis revealed that ANAC070 could directly repress the transcript levels of ANAC017, and knockout of ANAC017 in the anac070 mutant partially restored its Al sensitivity phenotype, indicating that ANAC070 contributes to Al tolerance mechanisms other than suppression of ANAC017 expression. Further analysis revealed that the core transcription factor SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) and its target genes, which control Al tolerance in Arabidopsis, may also be involved in ANAC070-regulated Al tolerance. In summary, we identified a transcription factor, ANAC070, that represses the ANAC017-XTH31 module to regulate Al tolerance in Arabidopsis.

GRK5 promoted renal fibrosis via HDAC5/Smad3 signaling pathway.

Renal fibrosis is a common pathological feature of chronic kidney diseases (CKD), poses a significant burden in the aging population, and is a major cause of end-stage renal disease (ESRD). In this study, we investigated the role of G protein-coupled receptor kinases (GRKs) 5 in the pathogenesis of renal fibrosis. GRK5 is a serine/threonine kinase that regulates G protein-coupled receptor (GPCR) signaling. GRK5 has been shown to play a role in various diseases including cardiac disorders and cancer. However, the role of GRK5 in renal fibrosis remains largely unknown. Our finding revealed that GRK5 was significantly overexpressed in renal fibrosis. Specifically, GRK5 was transferred into the nucleus via its nuclear localization sequence to regulate histone deacetylases (HDAC) 5 expression under renal fibrosis. GRK5 acted as an upstream regulator of HDAC5/Smad3 signaling pathway. HDAC5 regulated and prevented the transcriptional activity of myocyte enhancer factor 2A (MEF2A) to repress the transcription of Smad7 which leading to the activation of Smad3. These findings first revealed that GRK5 may be a potential therapeutic target for the treatment of renal fibrosis. Inhibition of GRK5 activity may be a promising strategy to attenuate the progression of renal fibrosis.

TRIM26 restricts Epstein-Barr virus infection in nasopharyngeal epithelial cells through K48-linked ubiquitination of HSP-90ß.

The tripartite interaction motif (TRIM) family of proteins is known for their antiviral activity through different mechanisms, such as interfering with viral components, regulating immune responses, and participating in autophagy-mediated defense pathways. In this study, we investigated the role of tripartite interaction motif 26 (TRIM26), which is encoded by a major histocompatibility complex (MHC) gene, in regulating Epstein-Barr virus (EBV) infection of nasopharyngeal epithelial cells. We found that TRIM26 expression was induced upon EBV infection and that it indirectly targeted EphA2, a crucial epithelial receptor for EBV entry. Our results showed that TRIM26 interacted with heat shock protein 90-beta (HSP-90ß) and promoted its polyubiquitination, which led to its degradation via the proteasome pathway. This, in turn, affected EphA2 integrity and suppressed EBV infection. These findings suggest that TRIM26 could be a valuable target for developing therapeutic interventions against EBV infection and its associated pathogenesis.

Structural basis of nucleosome recognition and modification by MLL methyltransferases.

Methyltransferases of the mixed-lineage leukaemia (MLL) family-which include MLL1, MLL2, MLL3, MLL4, SET1A and SET1B-implement methylation of histone H3 on lysine 4 (H3K4), and have critical and distinct roles in the regulation of transcription in haematopoiesis, adipogenesis and development1-6. The C-terminal catalytic SET (Su(var.)3-9, enhancer of zeste and trithorax) domains of MLL proteins are associated with a common set of regulatory factors (WDR5, RBBP5, ASH2L and DPY30) to achieve specific activities7-9. Current knowledge of the regulation of MLL activity is limited to the catalysis of histone H3 peptides, and how H3K4 methyl marks are deposited on nucleosomes is poorly understood. H3K4 methylation is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120ub1), a prevalent histone H2B mark that disrupts chromatin compaction and favours open chromatin structures, but the underlying mechanism remains unknown10-12. Here we report cryo-electron microscopy structures of human MLL1 and MLL3 catalytic modules associated with nucleosome core particles that contain H2BK120ub1 or unmodified H2BK120. These structures demonstrate that the MLL1 and MLL3 complexes both make extensive contacts with the histone-fold and DNA regions of the nucleosome; this allows ease of access to the histone H3 tail, which is essential for the efficient methylation of H3K4. The H2B-conjugated ubiquitin binds directly to RBBP5, orienting the association between MLL1 or MLL3 and the nucleosome. The MLL1 and MLL3 complexes display different structural organizations at the interface between the WDR5, RBBP5 and MLL1 (or the corresponding MLL3) subunits, which accounts for the opposite roles of WDR5 in regulating the activity of the two enzymes. These findings transform our understanding of the structural basis for the regulation of MLL activity at the nucleosome level, and highlight the pivotal role of nucleosome regulation in histone-tail modification.

Building a novel TRUCK by harnessing the endogenous IFN-gamma promoter for cytokine expression.

Despite the remarkable success of chimeric antigen receptor (CAR) T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous interferon (IFN)-γ promoter for transgenic interleukin (IL)-15 expression. We demonstrate that IFN-γ expression is tightly regulated by T cell receptor signaling. By introducing an internal ribosome entry site IL15 into the 3' UTR of the IFN-γ gene via homology directed repair-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Last, as a part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.