Innate immunity to intracellular LPS.

Monitoring of the cytosolic compartment by the innate immune system for pathogen-encoded products or pathogen activities often enables the activation of a subset of caspases. In most cases, the cytosolic surveillance pathways are coupled to activation of caspase-1 via canonical inflammasome complexes. A related set of caspases, caspase-11 in rodents and caspase-4 and caspase-5 in humans, monitors the cytosol for bacterial lipopolysaccharide (LPS). Direct activation of caspase-11, caspase-4 and caspase-5 by intracellular LPS elicits the lytic cell death called 'pyroptosis', which occurs in multiple cell types. The pyroptosis is executed by the pore-forming protein GSDMD, which is activated by cleavage mediated by caspase-11, caspase-4 or caspase-5. In monocytes, formation of GSDMD pores can induce activation of the NLRP3 inflammasome for maturation of the cytokines IL-1ß and IL-18. Caspase-11-mediated pyroptosis in response to cytosolic LPS is critical for antibacterial defense and septic shock. Here we review the emerging literature on the sensing of cytosolic LPS and its regulation and pathophysiological functions.

A light-driven enzymatic enantioselective radical acylation.

Enzymes are recognized as exceptional catalysts for achieving high stereoselectivities1-3, but their ability to control the reactivity and stereoinduction of free radicals lags behind that of chemical catalysts4. Thiamine diphosphate (ThDP)-dependent enzymes5 are well-characterized systems that inspired the development of N-heterocyclic carbenes (NHCs)6-8 but have not yet been proved viable in asymmetric radical transformations. There is a lack of a biocompatible and general radical-generation mechanism, as nature prefers to avoid radicals that may be harmful to biological systems9. Here we repurpose a ThDP-dependent lyase as a stereoselective radical acyl transferase (RAT) through protein engineering and combination with organophotoredox catalysis10. Enzyme-bound ThDP-derived ketyl radicals are selectively generated through single-electron oxidation by a photoexcited organic dye and then cross-coupled with prochiral alkyl radicals with high enantioselectivity. Diverse chiral ketones are prepared from aldehydes and redox-active esters (35 examples, up to 97% enantiomeric excess (e.e.)) by this method. Mechanistic studies reveal that this previously elusive dual-enzyme catalysis/photocatalysis directs radicals with the unique ThDP cofactor and evolvable active site. This work not only expands the repertoire of biocatalysis but also provides a unique strategy for controlling radicals with enzymes, complementing existing chemical tools.

Spatiotemporal imaging of charge transfer in photocatalyst particles.

The water-splitting reaction using photocatalyst particles is a promising route for solar fuel production1-4. Photo-induced charge transfer from a photocatalyst to catalytic surface sites is key in ensuring photocatalytic efficiency5; however, it is challenging to understand this process, which spans a wide spatiotemporal range from nanometres to micrometres and from femtoseconds to seconds6-8. Although the steady-state charge distribution on single photocatalyst particles has been mapped by microscopic techniques9-11, and the charge transfer dynamics in photocatalyst aggregations have been revealed by time-resolved spectroscopy12,13, spatiotemporally evolving charge transfer processes in single photocatalyst particles cannot be tracked, and their exact mechanism is unknown. Here we perform spatiotemporally resolved surface photovoltage measurements on cuprous oxide photocatalyst particles to map holistic charge transfer processes on the femtosecond to second timescale at the single-particle level. We find that photogenerated electrons are transferred to the catalytic surface quasi-ballistically through inter-facet hot electron transfer on a subpicosecond timescale, whereas photogenerated holes are transferred to a spatially separated surface and stabilized through selective trapping on a microsecond timescale. We demonstrate that these ultrafast-hot-electron-transfer and anisotropic-trapping regimes, which challenge the classical perception of a drift-diffusion model, contribute to the efficient charge separation in photocatalysis and improve photocatalytic performance. We anticipate that our findings will be used to illustrate the universality of other photoelectronic devices and facilitate the rational design of photocatalysts.

SLKB: synthetic lethality knowledge base.

Emerging CRISPR-Cas9 technology permits synthetic lethality (SL) screening of large number of gene pairs from gene combination double knockout (CDKO) experiments. However, the poor integration and annotation of CDKO SL data in current SL databases limit their utility, and diverse methods of calculating SL scores prohibit their comparison. To overcome these shortcomings, we have developed SL knowledge base (SLKB) that incorporates data of 11 CDKO experiments in 22 cell lines, 16,059 SL gene pairs and 264,424 non-SL gene pairs. Additionally, within SLKB, we have implemented five SL calculation methods: median score with and without background control normalization (Median-B/NB), sgRNA-derived score (sgRNA-B/NB), Horlbeck score, GEMINI score and MAGeCK score. The five scores have demonstrated a mere 1.21% overlap among their top 10% SL gene pairs, reflecting high diversity. Users can browse SL networks and assess the impact of scoring methods using Venn diagrams. The SL network generated from all data in SLKB shows a greater likelihood of SL gene pair connectivity with other SL gene pairs than non-SL pairs. Comparison of SL networks between two cell lines demonstrated greater likelihood to share SL hub genes than SL gene pairs. SLKB website and pipeline can be freely accessed at https://slkb.osubmi.org and https://slkb.docs.osubmi.org/, respectively.

Cell type-specific cytonuclear coevolution in three allopolyploid plant species.

Cytonuclear disruption may accompany allopolyploid evolution as a consequence of the merger of different nuclear genomes in a cellular environment having only one set of progenitor organellar genomes. One path to reconcile potential cytonuclear mismatch is biased expression for maternal gene duplicates (homoeologs) encoding proteins that target to plastids and/or mitochondria. Assessment of this transcriptional form of cytonuclear coevolution at the level of individual cells or cell types remains unexplored. Using single-cell (sc-) and single-nucleus (sn-) RNAseq data from eight tissues in three allopolyploid species, we characterized cell type-specific variations of cytonuclear coevolutionary homoeologous expression and demonstrated the temporal dynamics of expression patterns across development stages during cotton fiber development. Our results provide unique insights into transcriptional cytonuclear coevolution in plant allopolyploids at the single-cell level.

A serine carboxypeptidase-like acyltransferase catalyzes consecutive four-step reactions of hydrolyzable tannin biosynthesis in Camellia oleifera.

Hydrolyzable tannins (HTs), a class of polyphenolic compounds found in dicotyledonous plants, are widely used in food and pharmaceutical industries because of their beneficial effects on human health. Although the biosynthesis of simple HTs has been verified at the enzymatic level, relevant genes have not yet been identified. Here, based on the parent ion-fragment ion pairs in the feature fragment data obtained using UPLC-Q-TOF-/MS/MS, galloyl phenolic compounds in the leaves of Camellia sinensis and C. oleifera were analyzed qualitatively and quantitatively. Correlation analysis between the transcript abundance of serine carboxypeptidase-like acyltransferases (SCPL-ATs) and the peak area of galloyl products in Camellia species showed that SCPL3 expression was highly correlated with HT biosynthesis. Enzymatic verification of the recombinant protein showed that CoSCPL3 from C. oleifera catalyzed the four consecutive steps involved in the conversion of digalloylglucose to pentagalloylglucose. We also identified the residues affecting the enzymatic activity of CoSCPL3 and determined that SCPL-AT catalyzes the synthesis of galloyl glycosides. The findings of this study provide a target gene for germplasm innovation of important cash crops that are rich in HTs, such as C. oleifera, strawberry, and walnut.

Mendelian randomization analyses for PCOS: evidence, opportunities, and challenges.

Identifying etiological risk factors is significant for preventing and treating patients with polycystic ovary syndrome (PCOS). Through genetic variation, Mendelian randomization (MR) assesses causal associations between PCOS risk and related exposure factors. This emerging technology has provided evidence of causal associations of anti-Müllerian hormone (AMH) levels, sex hormone-binding globulin (SHBG) levels, menopause age, adiposity, insulin resistance (IR), depression, breast cancer, ovarian cancer, obsessive-compulsive disorder (OCD), and forced vital capacity (FVC) with PCOS, while lacking associations of type 2 diabetes mellitus (T2DM), coronary heart disease (CHD), stroke, anxiety disorder (AD), schizophrenia (SCZ), bipolar disorder (BIP), and offspring birth weight with PCOS. In this review, we briefly introduce the concept and methodology of MR in terms of the opportunities and challenges in this field based on recent results obtained from MR analyses involving PCOS.

VP2 residue N142 of coxsackievirus A10 is critical for the interaction with KREMEN1 receptor and neutralizing antibodies and the pathogenicity in mice.

Coxsackievirus A10 (CVA10) has recently emerged as one of the major causative agents of hand, foot, and mouth disease. CVA10 may also cause a variety of complications. No approved vaccine or drug is currently available for CVA10. The residues of CVA10 critical for viral attachment, infectivity and in vivo pathogenicity have not been identified by experiment. Here, we report the identification of CVA10 residues important for binding to cellular receptor KREMEN1. We identified VP2 N142 as a key receptor-binding residue by screening of CVA10 mutants resistant to neutralization by soluble KREMEN1 protein. The receptor-binding residue N142 is exposed on the canyon rim but highly conserved in all naturally occurring CVA10 strains, which provides a counterexample to the canyon hypothesis. Residue N142 when mutated drastically reduced receptor-binding activity, resulting in decreased viral attachment and infection in cell culture. More importantly, residue N142 when mutated reduced viral replication in limb muscle and spinal cord of infected mice, leading to lower mortality and less severe clinical symptoms. Additionally, residue N142 when mutated could decrease viral binding affinity to anti-CVA10 polyclonal antibodies and a neutralizing monoclonal antibody and render CVA10 resistant to neutralization by the anti-CVA10 antibodies. Overall, our study highlights the essential role of VP2 residue N142 of CVA10 in the interactions with KREMEN1 receptor and neutralizing antibodies and viral virulence in mice, facilitating the understanding of the molecular mechanisms of CVA10 infection and immunity. Our study also provides important information for rational development of antibody-based treatment and vaccines against CVA10 infection.

Oncogenic role and target properties of the lysine-specific demethylase KDM1A in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), epigenetic alterations are considered to centrally shape the transcriptional signatures that drive disease evolution and underlie its biological and clinical subsets. Characterizations of epigenetic regulators, particularly histone-modifying enzymes, are very rudimentary in CLL. In efforts to establish effectors of the CLL-associated oncogene T-cell leukemia 1A (TCL1A), we identified here the lysine-specific histone demethylase KDM1A to interact with the TCL1A protein in B cells in conjunction with an increased catalytic activity of KDM1A. We demonstrate that KDM1A is upregulated in malignant B cells. Elevated KDM1A and associated gene expression signatures correlated with aggressive disease features and adverse clinical outcomes in a large prospective CLL trial cohort. Genetic Kdm1a knockdown in Eµ-TCL1A mice reduced leukemic burden and prolonged animal survival, accompanied by upregulated p53 and proapoptotic pathways. Genetic KDM1A depletion also affected milieu components (T, stromal, and monocytic cells), resulting in significant reductions in their capacity to support CLL-cell survival and proliferation. Integrated analyses of differential global transcriptomes (RNA sequencing) and H3K4me3 marks (chromatin immunoprecipitation sequencing) in Eµ-TCL1A vs iKdm1aKD;Eµ-TCL1A mice (confirmed in human CLL) implicate KDM1A as an oncogenic transcriptional repressor in CLL which alters histone methylation patterns with pronounced effects on defined cell death and motility pathways. Finally, pharmacologic KDM1A inhibition altered H3K4/9 target methylation and revealed marked anti-B-cell leukemic synergisms. Overall, we established the pathogenic role and effector networks of KDM1A in CLL via tumor-cell intrinsic mechanisms and its impacts in cells of the microenvironment. Our data also provide rationales to further investigate therapeutic KDM1A targeting in CLL.

Organic Peroxides in Aerosol: Key Reactive Intermediates for Multiphase Processes in the Atmosphere.

Organic peroxides (POs) are organic molecules with one or more peroxide (-O-O-) functional groups. POs are commonly regarded as chemically labile termination products from gas-phase radical chemistry and therefore serve as temporary reservoirs for oxidative radicals (HOx and ROx) in the atmosphere. Owing to their ubiquity, active gas-particle partitioning behavior, and reactivity, POs are key reactive intermediates in atmospheric multiphase processes determining the life cycle (formation, growth, and aging), climate, and health impacts of aerosol. However, there remain substantial gaps in the origin, molecular diversity, and fate of POs due to their complex nature and dynamic behavior. Here, we summarize the current understanding on atmospheric POs, with a focus on their identification and quantification, state-of-the-art analytical developments, molecular-level formation mechanisms, multiphase chemical transformation pathways, as well as environmental and health impacts. We find that interactions with SO2 and transition metal ions are generally the fast PO transformation pathways in atmospheric liquid water, with lifetimes estimated to be minutes to hours, while hydrolysis is particularly important for α-substituted hydroperoxides. Meanwhile, photolysis and thermolysis are likely minor sinks for POs. These multiphase PO transformation pathways are distinctly different from their gas-phase fates, such as photolysis and reaction with OH radicals, which highlights the need to understand the multiphase partitioning of POs. By summarizing the current advances and remaining challenges for the investigation of POs, we propose future research priorities regarding their origin, fate, and impacts in the atmosphere.

Metal-free directed sp2-C-H borylation.

Organoboron reagents are important synthetic intermediates that have a key role in the construction of natural products, pharmaceuticals and organic materials1. The discovery of simpler, milder and more efficient approaches to organoborons can open additional routes to diverse substances2-5. Here we show a general method for the directed C-H borylation of arenes and heteroarenes without the use of metal catalysts. C7- and C4-borylated indoles are produced by a mild approach that is compatible with a broad range of functional groups. The mechanism, which is established by density functional theory calculations, involves BBr3 acting as both a reagent and a catalyst. The potential utility of this strategy is highlighted by the downstream transformation of the formed boron species into natural products and drug scaffolds.

Lipid signalling drives proteolytic rewiring of mitochondria by YME1L.

Reprogramming of mitochondria provides cells with the metabolic flexibility required to adapt to various developmental transitions such as stem cell activation or immune cell reprogramming, and to respond to environmental challenges such as those encountered under hypoxic conditions or during tumorigenesis1-3. Here we show that the i-AAA protease YME1L rewires the proteome of pre-existing mitochondria in response to hypoxia or nutrient starvation. Inhibition of mTORC1 induces a lipid signalling cascade via the phosphatidic acid phosphatase LIPIN1, which decreases phosphatidylethanolamine levels in mitochondrial membranes and promotes proteolysis. YME1L degrades mitochondrial protein translocases, lipid transfer proteins and metabolic enzymes to acutely limit mitochondrial biogenesis and support cell growth. YME1L-mediated mitochondrial reshaping supports the growth of pancreatic ductal adenocarcinoma (PDAC) cells as spheroids or xenografts. Similar changes to the mitochondrial proteome occur in the tumour tissues of patients with PDAC, suggesting that YME1L is relevant to the pathophysiology of these tumours. Our results identify the mTORC1-LIPIN1-YME1L axis as a post-translational regulator of mitochondrial proteostasis at the interface between metabolism and mitochondrial dynamics.

Hierarchical DNA branch assembly-encoded fluorescent nanoladders for single-cell transcripts imaging.

Spatial visualization of single-cell transcripts is limited by signal specificity and multiplexing. Here, we report hierarchical DNA branch assembly-encoded fluorescent nanoladders, which achieve denoised and highly multiplexed signal amplification for single-molecule transcript imaging. This method first offers independent RNA-primed rolling circle amplification without nonspecific amplification based on circular DNAzyme. It then executes programmable DNA branch assembly on these amplicons to encode virtual signals for visualizing numbers of targets by FISH. In theory, more virtual signals can be encoded via the increase of detection spectral channels and repeats of the same sequences on barcode. Our method almost eliminates nonspecific amplification in fixed cells (reducing nonspecific spots of single cells from 16 to nearly zero), and achieves simultaneous quantitation of nine transcripts by using only two detection spectral channels. We demonstrate accurate RNA profiling in different cancer cells, and reveal diverse localization patterns for spatial regulation of transcripts.

Rearrangement of a unique Kv1.3 selectivity filter conformation upon binding of a drug.

We report two structures of the human voltage-gated potassium channel (Kv) Kv1.3 in immune cells alone (apo-Kv1.3) and bound to an immunomodulatory drug called dalazatide (dalazatide-Kv1.3). Both the apo-Kv1.3 and dalazatide-Kv1.3 structures are in an activated state based on their depolarized voltage sensor and open inner gate. In apo-Kv1.3, the aromatic residue in the signature sequence (Y447) adopts a position that diverges 11 Å from other K+ channels. The outer pore is significantly rearranged, causing widening of the selectivity filter and perturbation of ion binding within the filter. This conformation is stabilized by a network of intrasubunit hydrogen bonds. In dalazatide-Kv1.3, binding of dalazatide to the channel's outer vestibule narrows the selectivity filter, Y447 occupies a position seen in other K+ channels, and this conformation is stabilized by a network of intersubunit hydrogen bonds. These remarkable rearrangements in the selectivity filter underlie Kv1.3's transition into the drug-blocked state.

Ubiquitin-specific peptidase 14 maintains estrogen receptor α stability via its deubiquitination activity in endometrial cancer.

USP14 deubiquitinates ERα to maintain its stability in ECEndometrial cancer (EC) is one of the common gynecological malignancies of which the incidence has been rising for decades. It is considered that continuously unopposed estrogen exposure is the main risk factor for EC initiation. Thus, exploring the modulation of estrogen/estrogen receptor α (ERα) signaling pathway in EC would be helpful to well understand the mechanism of EC development and find the potential target for EC therapy. Ubiquitin-specific peptidase 14 (USP14), a member of the proteasome-associated deubiquitinating enzyme family, plays a crucial role in a series of tumors. However, the function of USP14 in EC is still elusive. Here, our results have demonstrated that USP14 is highly expressed in EC tissues compared with that in normal endometrial tissues, and higher expression of USP14 is positively correlated with poor prognosis. Moreover, USP14 maintains ERα stability through its deubiquitination activity. Our results further demonstrate that USP14 depletion decreases the expression of ERα-regulated genes in EC-derived cell lines. Moreover, knockdown of USP14 or USP14-specific inhibitor treatment significantly suppresses cell growth and migration in EC cell lines or in mice. We further provide the evidence to show that the effect of USP14 on EC cell growth, if not all, at least is partially related to ERα pathway. Our study provides new sights for USP14 to be a potential therapeutic target for the treatment of EC, especially for EC patients with fertility preservation needs.

New insights into the function of plant tannase with promiscuous acyltransferase activity.

Plant tannases (TAs) or tannin acyl hydrolases, a class of recently reported carboxylesterases in tannin-rich plants, are involved in the degalloylation of two important groups of secondary metabolites: flavan-3-ol gallates and hydrolyzable tannins. In this paper, we have made new progress in studying the function of tea (Camellia sinensis) (Cs) TA-it is a hydrolase with promiscuous acyltransferase activity in vitro and in vivo and promotes the synthesis of simple galloyl glucoses and flavan-3-ol gallates in plants. We studied the functions of CsTA through enzyme analysis, protein mass spectrometry, and metabolic analysis of genetically modified plants. Firstly, CsTA was found to be not only a hydrolase but also an acyltransferase. In the two-step catalytic reaction where CsTA hydrolyzes the galloylated compounds epigallocatechin-3-gallate or 1,2,3,4,6-penta-O-galloyl-ß-d-glucose into their degalloylated forms, a long-lived covalently bound Ser159-linked galloyl-enzyme intermediate is also formed. Under nucleophilic attack, the galloyl group on the intermediate is transferred to the nucleophilic acyl acceptor (such as water, methanol, flavan-3-ols, and simple galloyl glucoses). Then, metabolic analysis suggested that transient overexpression of TAs in young strawberry (Fragaria × ananassa) fruits, young leaves of tea plants, and young leaves of Chinese bayberry (Myrica rubra) actually increased the total contents of simple galloyl glucoses and flavan-3-ol gallates. Overall, these findings provide new insights into the promiscuous acyltransferase activity of plant TA.

Metabolic differences in MSTN and FGF5 dual-gene edited sheep muscle cells during myogenesis.

Dynamic metabolic reprogramming occurs at different stages of myogenesis and contributes to the fate determination of skeletal muscle satellite cells (MuSCs). Accumulating evidence suggests that mutations in myostatin (MSTN) have a vital role in regulating muscle energy metabolism. Here, we explored the metabolic reprogramming in MuSCs and myotube cells in MSTN and FGF5 dual-gene edited sheep models prepared previously, and also focused on the metabolic alterations during myogenic differentiation of MuSCs. Our study revealed that the pathways of nucleotide metabolism, pantothenate and CoA biosynthesis were weakened, while the unsaturated fatty acids biosynthesis were strengthened during myogenic differentiation of sheep MuSCs. The MSTN and FGF5 dual-gene editing mainly inhibited nucleotide metabolism and biosynthesis of unsaturated fatty acids in sheep MuSCs, reduced the number of lipid droplets in per satellite cell, and promoted the pentose phosphate pathway, and the interconversion of pentose and glucuronate. The MSTN and FGF5 dual-gene editing also resulted in the inhibition of nucleotide metabolism and TCA cycle pathway in differentiated myotube cells. The differential metabolites we identified can be characterized as biomarkers of different cellular states, and providing a new reference for MSTN and FGF5 dual-gene editing in regulation of muscle development. It may also provide a reference for the development of muscle regeneration drugs targeting biomarkers.

P(III)-Directed Asymmetric C-H Arylation toward Planar Chiral Ferrocenes by Palladium Catalysis.

Planar chiral ferrocenyl phosphines have been employed as highly valuable ligands in metal-catalyzed asymmetric reactions. However, their preparation remains a formidable challenge due to the requirement for intricate, multistep synthetic sequences. In addressing this issue, we have developed a groundbreaking enantioselective C-H activation strategy facilitated by P(III) directing groups, enabling the efficient construction of planar chiral ferrocenyl phosphines in a single step. Our innovative approach entails the combination of a palladium catalyst, a parent ferrocenyl phosphine, and a chiral phosphoramidite ligand, leading to exceptional reactivity and enantioselectivity. Remarkably, these novel ligands exhibit remarkable efficacy in silver-catalyzed asymmetric 1,3-dipolar cycloadditions. We carried out a combination of experimental and computational studies to obtain a more comprehensive understanding of the reaction pathway and the factors contributing to enantioselectivity.

Electronic Delocalization and σ-Aromaticity in Heterometallic Cluster with Multiple Thorium-Palladium Bonds.

Research on metal-metal bonds involving f-block actinides, such as thorium, lags far behind the well-studied metal-metal bonds of d-block transition metals. The complexes with Th-TM bonds are extremely rare; all previously identified examples have only a single Th-TM bond with the Th center at an invariably +IV oxidation state. Herein, we report a series of Th2Pdn (n = 2, 3, and 6) clusters (complexes 3, 4, and 7) with multiple Th(III)-Pd bonds. Theoretical studies reveal that the Th2Pdn unit allows electronic delocalization and σ aromaticity, leading to unexpected closed-shell singlet structures for these Th(III) species. This electronic delocalization is evident in the highest occupied molecular orbital of Th(III) complexes and facilitates a 2e reduction of alkyne by complex 7, resulting in the formation of 8. Complexes 7 and 8 are distinctive in featuring a Th2Pd6 core with six and eight Th-Pd bonds, respectively, making them the largest known d-f heterometallic clusters exhibiting metal-metal bonding.

Modular Access to Chiral Amines via Imine Reductase-Based Photoenzymatic Catalysis.

The development of catalysts serves as the cornerstone of innovation in synthesis, as exemplified by the recent discovery of photoenzymes. However, the repertoire of naturally occurring enzymes repurposed by direct light excitation to catalyze new-to-nature photobiotransformations is currently limited to flavoproteins and keto-reductases. Herein, we shed light on imine reductases (IREDs) that catalyze the remote C(sp3)-C(sp3) bond formation, providing a previously elusive radical hydroalkylation of enamides for accessing chiral amines (45 examples with up to 99% enantiomeric excess). Beyond their natural function in catalyzing two-electron reductive amination reactions, upon direct visible-light excitation or in synergy with a synthetic photoredox catalyst, IREDs are repurposed to tune the non-natural photoinduced single-electron radical processes. By conducting wet mechanistic experiments and computational simulations, we unravel how engineered IREDs direct radical intermediates toward the productive and enantioselective pathway. This work represents a promising paradigm for harnessing nature's catalysts for new-to-nature asymmetric transformations that remain challenging through traditional chemocatalytic methods.