5' UTR m(6)A Promotes Cap-Independent Translation.

Protein translation typically begins with the recruitment of the 43S ribosomal complex to the 5' cap of mRNAs by a cap-binding complex. However, some transcripts are translated in a cap-independent manner through poorly understood mechanisms. Here, we show that mRNAs containing N(6)-methyladenosine (m(6)A) in their 5' UTR can be translated in a cap-independent manner. A single 5' UTR m(6)A directly binds eukaryotic initiation factor 3 (eIF3), which is sufficient to recruit the 43S complex to initiate translation in the absence of the cap-binding factor eIF4E. Inhibition of adenosine methylation selectively reduces translation of mRNAs containing 5'UTR m(6)A. Additionally, increased m(6)A levels in the Hsp70 mRNA regulate its cap-independent translation following heat shock. Notably, we find that diverse cellular stresses induce a transcriptome-wide redistribution of m(6)A, resulting in increased numbers of mRNAs with 5' UTR m(6)A. These data show that 5' UTR m(6)A bypasses 5' cap-binding proteins to promote translation under stresses.

XIAP-mediated degradation of IFT88 disrupts HSC cilia to stimulate HSC activation and liver fibrosis.

Activation of hepatic stellate cells (HSCs) plays a critical role in liver fibrosis. However, the molecular basis for HSC activation remains poorly understood. Herein, we demonstrate that primary cilia are present on quiescent HSCs but exhibit a significant loss upon HSC activation which correlates with decreased levels of the ciliary protein intraflagellar transport 88 (IFT88). Ift88-knockout mice are more susceptible to chronic carbon tetrachloride-induced liver fibrosis. Mechanistic studies show that the X-linked inhibitor of apoptosis (XIAP) functions as an E3 ubiquitin ligase for IFT88. Transforming growth factor-ß (TGF-ß), a profibrotic factor, enhances XIAP-mediated ubiquitination of IFT88, promoting its proteasomal degradation. Blocking XIAP-mediated IFT88 degradation ablates TGF-ß-induced HSC activation and liver fibrosis. These findings reveal a previously unrecognized role for ciliary homeostasis in regulating HSC activation and identify the XIAP-IFT88 axis as a potential therapeutic target for liver fibrosis.

N6-Methyladenosine Guides mRNA Alternative Translation during Integrated Stress Response.

The integrated stress response (ISR) facilitates cellular adaptation to stress conditions via the common target eIF2α. During ISR, the selective translation of stress-related mRNAs often relies on alternative mechanisms, such as leaky scanning or reinitiation, but the underlying mechanism remains incompletely understood. Here we report that, in response to amino acid starvation, the reinitiation of ATF4 is not only governed by the eIF2α signaling pathway, but is also subjected to regulation by mRNA methylation in the form of N6-methyladenosine (m6A). While depleting m6A demethylases represses ATF4 reinitiation, knocking down m6A methyltransferases promotes ATF4 translation. We demonstrate that m6A in the 5' UTR controls ribosome scanning and subsequent start codon selection. Global profiling of initiating ribosomes reveals widespread alternative translation events influenced by dynamic mRNA methylation. Consistently, Fto transgenic mice manifest enhanced ATF4 expression, highlighting the critical role of m6A in translational regulation of ISR at cellular and organismal levels.

A plant-unique protein BLISTER coordinates with core retromer to modulate endosomal sorting of plasma membrane and vacuolar proteins.

The retromer is a heteromeric protein complex that localizes to endosomal membranes and drives the formation of endosomal tubules that recycle membrane protein cargoes. In plants, the retromer plays essential and canonical functions in regulating the transport of vacuolar storage proteins and the recycle of endocytosed plasma membrane proteins (PM); however, the mechanisms underlying the regulation of assembly, protein stability, and membrane recruitment of the plant retromer complex remain to be elucidated. In this study, we identify a plant-unique endosomal regulator termed BLISTER (BLI), which colocalizes and associates with the retromer complex by interacting with the retromer core subunits VPS35 and VPS29. Depletion of BLI perturbs the assembly and membrane recruitment of the retromer core VPS26-VPS35-VPS29 trimer. Consequently, depletion of BLI disrupts retromer-regulated endosomal trafficking function, including transport of soluble vacuolar proteins and recycling of endocytosed PIN-FORMED (PIN) proteins from the endosomes back to the PM. Moreover, genetic analysis in Arabidopsis thaliana mutants reveals BLI and core retromer interact genetically in the regulation of endosomal trafficking. Taken together, we identified BLI as a plant-specific endosomal regulator, which functions in retromer pathway to modulate the recycling of endocytosed PM proteins and the trafficking of soluble vacuolar cargoes.

Ciliary mechanosensation - roles of polycystins and mastigonemes.

Cilia are surface-exposed organelles that provide motility and sensory functions for cells, and it is widely believed that mechanosensation can be mediated through cilia. Polycystin-1 and -2 (PC-1 and PC-2, respectively) are transmembrane proteins that can localize to cilia; however, the molecular mechanisms by which polycystins contribute to mechanosensation are still controversial. Studies detail two prevailing models for the molecular roles of polycystins on cilia; one stresses the mechanosensation capabilities and the other unveils their ligand-receptor nature. The discovery that polycystins interact with mastigonemes, the 'hair-like' protrusions of flagella, is a novel finding in identifying the interactors of polycystins in cilia. While the functions of polycystins proposed by both models may coexist in cilia, it is hoped that a precise understanding of the mechanism of action of polycystins can be achieved by uncovering their distribution and interacting factors inside cilia. This will hopefully provide a satisfying answer to the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD), which is caused by mutations in PC-1 and PC-2. In this Review, we discuss the characteristics of polycystins in the context of cilia and summarize the functions of mastigonemes in unicellular ciliates. Finally, we compare flagella and molecular features of PC-2 between unicellular and multicellular organisms, with the aim of providing new insights into the ciliary roles of polycystins in general.

Decoding CRISPR-Cas PAM recognition with UniDesign.

The critical first step in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (CRISPR-Cas) protein-mediated gene editing is recognizing a preferred protospacer adjacent motif (PAM) on target DNAs by the protein's PAM-interacting amino acids (PIAAs). Thus, accurate computational modeling of PAM recognition is useful in assisting CRISPR-Cas engineering to relax or tighten PAM requirements for subsequent applications. Here, we describe a universal computational protein design framework (UniDesign) for designing protein-nucleic acid interactions. As a proof of concept, we applied UniDesign to decode the PAM-PIAA interactions for eight Cas9 and two Cas12a proteins. We show that, given native PIAAs, the UniDesign-predicted PAMs are largely identical to the natural PAMs of all Cas proteins. In turn, given natural PAMs, the computationally redesigned PIAA residues largely recapitulated the native PIAAs (74% and 86% in terms of identity and similarity, respectively). These results demonstrate that UniDesign faithfully captures the mutual preference between natural PAMs and native PIAAs, suggesting it is a useful tool for engineering CRISPR-Cas and other nucleic acid-interacting proteins. UniDesign is open-sourced at https://github.com/tommyhuangthu/UniDesign.

HDAC6 deacetylates IDH1 to promote the homeostasis of hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are cells mainly present in the bone marrow and capable of forming mature blood cells. However, the epigenetic mechanisms governing the homeostasis of HSPCs remain elusive. Here, we demonstrate an important role for histone deacetylase 6 (HDAC6) in regulating this process. Our data show that the percentage of HSPCs in Hdac6 knockout mice is lower than in wild-type mice due to decreased HSPC proliferation. HDAC6 interacts with isocitrate dehydrogenase 1 (IDH1) and deacetylates IDH1 at lysine 233. The deacetylation of IDH1 inhibits its catalytic activity and thereby decreases the 5-hydroxymethylcytosine level of ten-eleven translocation 2 (TET2) target genes, changing gene expression patterns to promote the proliferation of HSPCs. These findings uncover a role for HDAC6 and IDH1 in regulating the homeostasis of HSPCs and may have implications for the treatment of hematological diseases.

m6A Facilitates eIF4F-Independent mRNA Translation.

In eukaryotic cells, protein synthesis typically begins with the binding of eIF4F to the 7-methylguanylate (m7G) cap found on the 5' end of the majority of mRNAs. Surprisingly, overall translational output remains robust under eIF4F inhibition. The broad spectrum of eIF4F-resistant translatomes is incompatible with cap-independent translation mediated by internal ribosome entry sites (IRESs). Here, we report that N6-methyladenosine (m6A) facilitates mRNA translation that is resistant to eIF4F inactivation. Depletion of the methyltransferase METTL3 selectively inhibits translation of mRNAs bearing 5' UTR methylation, but not mRNAs with 5' terminal oligopyrimidine (TOP) elements. We identify ABCF1 as a critical mediator of m6A-promoted translation under both stress and physiological conditions. Supporting the role of ABCF1 in m6A-facilitated mRNA translation, ABCF1-sensitive transcripts largely overlap with METTL3-dependent mRNA targets. By illustrating the scope and mechanism of eIF4F-independent mRNA translation, these findings reshape our current perceptions of cellular translational pathways.

Base-Resolution Mapping Reveals Distinct m1A Methylome in Nuclear- and Mitochondrial-Encoded Transcripts.

Gene expression can be post-transcriptionally regulated via dynamic and reversible RNA modifications. N1-methyladenosine (m1A) is a recently identified mRNA modification; however, little is known about its precise location and biogenesis. Here, we develop a base-resolution m1A profiling method, based on m1A-induced misincorporation during reverse transcription, and report distinct classes of m1A methylome in the human transcriptome. m1A in 5' UTR, particularly those at the mRNA cap, associate with increased translation efficiency. A different, small subset of m1A exhibit a GUUCRA tRNA-like motif, are evenly distributed in the transcriptome, and are dependent on the methyltransferase TRMT6/61A. Additionally, we show that m1A is prevalent in the mitochondrial-encoded transcripts. Manipulation of m1A level via TRMT61B, a mitochondria-localizing m1A methyltransferase, demonstrates that m1A in mitochondrial mRNA interferes with translation. Collectively, our approaches reveal distinct classes of m1A methylome and provide a resource for functional studies of m1A-mediated epitranscriptomic regulation.

Kurarinone alleviated Parkinson's disease via stabilization of epoxyeicosatrienoic acids in animal model.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders and is characterized by loss of dopaminergic neurons in the substantia nigra (SN), causing bradykinesia and rest tremors. Although the molecular mechanism of PD is still not fully understood, neuroinflammation has a key role in the damage of dopaminergic neurons. Herein, we found that kurarinone, a unique natural product from Sophora flavescens, alleviated the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced behavioral deficits and dopaminergic neurotoxicity, including the losses of neurotransmitters and tyrosine hydroxylase (TH)-positive cells (SN and striatum [STR]). Furthermore, kurarinone attenuated the MPTP-mediated neuroinflammation via suppressing the activation of microglia involved in the nuclear factor kappa B signaling pathway. The proteomics result of the solvent-induced protein precipitation and thermal proteome profiling suggest that the soluble epoxide hydrolase (sEH) enzyme, which is associated with the neuroinflammation of PD, is a promising target of kurarinone. This is supported by the increase of plasma epoxyeicosatrienoic acids (sEH substrates) and the decrease of dihydroxyeicosatrienoic acids (sEH products), and the results of in vitro inhibition kinetics, surface plasmon resonance, and cocrystallization of kurarinone with sEH revealed that this natural compound is an uncompetitive inhibitor. In addition, sEH knockout (KO) attenuated the progression of PD, and sEH KO plus kurarinone did not further reduce the protection of PD in MPTP-induced PD mice. These findings suggest that kurarinone could be a potential natural candidate for the treatment of PD, possibly through sEH inhibition.

Correction: Bacterial recognition by PGRP-SA and downstream signalling by Toll/DIF sustain commensal gut bacteria in Drosophila.

[This corrects the article DOI: 10.1371/journal.pgen.1009992.].

Bacterial recognition by PGRP-SA and downstream signalling by Toll/DIF sustain commensal gut bacteria in Drosophila.

The gut sets the immune and metabolic parameters for the survival of commensal bacteria. We report that in Drosophila, deficiency in bacterial recognition upstream of Toll/NF-κB signalling resulted in reduced density and diversity of gut bacteria. Translational regulation factor 4E-BP, a transcriptional target of Toll/NF-κB, mediated this host-bacteriome interaction. In healthy flies, Toll activated 4E-BP, which enabled fat catabolism, which resulted in sustaining of the bacteriome. The presence of gut bacteria kept Toll signalling activity thus ensuring the feedback loop of their own preservation. When Toll activity was absent, TOR-mediated suppression of 4E-BP made fat resources inaccessible and this correlated with loss of intestinal bacterial density. This could be overcome by genetic or pharmacological inhibition of TOR, which restored bacterial density. Our results give insights into how an animal integrates immune sensing and metabolism to maintain indigenous bacteria in a healthy gut.

A radiomics and genomics-derived model for predicting metastasis and prognosis in colorectal cancer.

Approximately 50% of colorectal cancer (CRC) patients would develop metastasis with poor prognosis, therefore, it is necessary to effectively predict metastasis in clinical treatment. In this study, we aimed to establish a machine-learning model for predicting metastasis in CRC patients by considering radiomics and transcriptomics simultaneously. Here, 1023 patients with CRC from three centers were collected and divided into five queues (Dazhou Central Hospital n = 517, Nanchong Central Hospital n = 120 and the Cancer Genome Atlas (TCGA) n = 386). A total of 854 radiomics features were extracted from tumor lesions on CT images, and 217 differentially expressed genes were obtained from non-metastasis and metastasis tumor tissues using RNA sequencing. Based on radiotranscriptomic (RT) analysis, a novel RT model was developed and verified through genetic algorithms (GA). Interleukin (IL)-26, a biomarker in RT model, was verified for its biological function in CRC metastasis. Furthermore, 15 radiomics variables were screened through stepwise regression, which was highly correlated with the IL26 expression level. Finally, a radiomics model (RA) was established by combining GA and stepwise regression analysis with radiomics features. The RA model exhibited favorable discriminatory ability and accuracy for metastasis prediction in two independent verification cohorts. We designed multicenter, multi-scale cohorts to construct and verify novel combined radiomics and genomics models for predicting metastasis in CRC. Overall, RT model and RA model might help clinicians in directing personalized diagnosis and therapeutic regimen selection for patients with CRC.

Irisin attenuates ventilator-induced diaphragmatic dysfunction by inhibiting endoplasmic reticulum stress through activation of AMPK.

Mechanical ventilation (MV) is an essential life-saving technique, but prolonged MV can cause significant diaphragmatic dysfunction due to atrophy and decreased contractility of the diaphragm fibres, called ventilator-induced diaphragmatic dysfunction (VIDD). It is not clear about the mechanism of occurrence and prevention measures of VIDD. Irisin is a newly discovered muscle factor that regulates energy metabolism. Studies have shown that irisin can exhibit protective effects by downregulating endoplasmic reticulum (ER) stress in a variety of diseases; whether irisin plays a protective role in VIDD has not been reported. Sprague-Dawley rats were mechanically ventilated to construct a VIDD model, and intervention was performed by intravenous administration of irisin. Diaphragm contractility, degree of atrophy, cross-sectional areas (CSAs), ER stress markers, AMPK protein expression, oxidative stress indicators and apoptotic cell levels were measured at the end of the experiment.Our findings showed that as the duration of ventilation increased, the more severe the VIDD was, the degree of ER stress increased, and the expression of irisin decreased.ER stress may be one of the causes of VIDD. Intervention with irisin ameliorated VIDD by reducing the degree of ER stress, attenuating oxidative stress, and decreasing the apoptotic index. MV decreases the expression of phosphorylated AMPK in the diaphragm, whereas the use of irisin increases the expression of phosphorylated AMPK. Irisin may exert its protective effect by activating the phosphorylated AMPK pathway.

Locational and functional characterization of PI4KB in the mouse embryo.

Phosphatidylinositol 4-kinase beta (PI4KB) is a member of the PI4K family, which is mainly enriched and functions in the Golgi apparatus. The kinase domain of PI4KB catalyzes the phosphorylation of phosphatidylinositol to form phosphatidylinositol 4-phosphate, a process that regulates various sub-cellular events, such as non-vesicular cholesterol and ceramide transport, protein glycosylation, and vesicle transport, as well as cytoplasmic division. In this study, a strain of PI4KB knockout mouse, immunofluorescence, reverse transcription polymerase chain reaction and microinjection were used to characterize the cytological location and biological function of PI4KB in the mouse embryos. we found that knocking down Pi4kb in mouse embryos resulted in embryonic lethality at around embryonic day (E) 7.5. Additionally, we observed dramatic fluctuations in PI4KB expression during the development of preimplantation embryos, with high expression in the 4-cell and morula stages. PI4KB colocalized with the Golgi marker protein TGN46 in the perinuclear and cytoplasmic regions in early blastomeres. Postimplantation, PI4KB was highly expressed in the epiblast of E7.5 embryos. Treatment of embryos with PI4KB inhibitors was found to inhibit the development of the morula into a blastocyst and the normal progression of cytoplasmic division during the formation of a 4-cell embryo. These findings suggest that PI4KB plays an important role in mouse embryogenesis by regulating various intracellular vital functions of embryonic cells.

Altered static and dynamic functional brain network in knee osteoarthritis: A resting-state functional magnetic resonance imaging study: Static and dynamic FNC in KOA.

This study aimed to investigate altered static and dynamic functional network connectivity (FNC) and its correlation with clinical symptoms in patients with knee osteoarthritis (KOA). One hundred and fifty-nine patients with KOA and 73 age- and gender-matched healthy subjects (HS) underwent resting-state functional magnetic resonance imaging (rs-fMRI) and clinical evaluations. Group independent component analysis (GICA) was applied, and seven resting-state networks were identified. Patients with KOA had decreased static FNC within the default mode network (DM), visual network (VS), and cerebellar network (CB) and increased static FNC between the subcortical network (SC) and VS (p < 0.05, FDR corrected). Four reoccurring FNC states were identified using k-means clustering analysis. Although abnormalities in dynamic FNCs of KOA patients have been found using the common window size (22 TR, 44 s), but the results of the clustering analysis were inconsistent when using different window sizes, suggesting dynamic FNCs might be an unstable method to compare brain function between KOA patients and HS. These recent findings illustrate that patients with KOA have a wide range of abnormalities in the static and dynamic FNCs, which provided a reference for the identification of potential central nervous therapeutic targets for KOA treatment and might shed light on the other musculoskeletal pain neuroimaging studies.

Zirconaaziridine-Mediated Ni-Catalyzed Diastereoselective C(sp2)-Glycosylation.

In the realm of organic synthesis, the catalytic and stereoselective formation of C-glycosidic bonds is a pivotal process, bridging carbohydrates with aglycones. However, the inherent chirality of the saccharide scaffold often has a substantial impact on the stereoinduction imposed by a chiral ligand. In this study, we have established an unprecedented zirconaaziridine-mediated asymmetric nickel catalysis, enabling the diastereoselective coupling of bench-stable glycosyl phosphates with a range of (hetero)aromatic and glycal iodides as feasible coupling electrophiles. Our developed method showcases a broad scope and a high tolerance for various functional groups. More importantly, precise stereocontrol toward both anomeric configurations of forming C(sp2)-glycosides can be realized by simply utilizing the popular chiral bioxazoline (biOx) ligands in this reductive Ni catalysis. Regarding the operating mechanism, both experimental and computational studies support the occurrence of a redox transmetalation process, leading to the formation of a transient, bimetallic Ni-Zr species that acts as a potent and efficient single-electron reductant in the catalytic process.

Efficient and Accurate pH Determination with pH Test Strips Based on Machine Learning.

The determination of pH values is crucial in various fields, such as analytical chemistry, medical diagnostics, and biochemical research. pH test strips, renowned for their convenience and cost-effectiveness, are commonly utilized for pH qualitative estimation. Recently, quantitative methods for determining pH values using pH test strips have been developed. However, these methods can be prone to errors due to environmental factors, such as lighting conditions, which affect the imaging quality of the pH test strips. To address these challenges, we developed an innovative approach that combines machine learning techniques with pH test strips for the quantitative determination of pH values. Our method involves extracting artificial features from the pH test strip images and combining them across multiple dimensions for comprehensive analysis. To ensure optimal feature selection, we developed a feature selection strategy based on SHAP importance. This strategy helps in identifying the most relevant features that contribute to accurate pH prediction. Furthermore, we integrated multiple machine learning algorithms, employing a robust stacking fusion strategy to establish a highly reliable pH value prediction model. Our proposed method automates the determination of pH values through pH test strips, effectively overcoming the limitations associated with environmental lighting interference. Experimental results demonstrate that this method is convenient, effective, and highly reliable for the determination of pH values.

Ligation-Based High-Performance Mimetic Enzyme Sensing Platform for Nucleic Acid Detection.

G-quadruplex (G4)/hemin DNAzyme is a promising candidate to substitute horseradish peroxidase in biosensing systems, especially for the detection of nucleic acids. However, the relatively suboptimal catalytic capacity limits its potential applications. This makes it imperative to develop an ideal signal for the construction of highly sensitive biosensing platforms. Herein, we integrated a novel chimeric peptide-DNAzyme (CPDzyme) with the ligase chain reaction (LCR) for the cost-efficient and highly sensitive detection of nucleic acids. By employing microRNA (miRNA) and single-nucleotide polymorphism detection as the model, we designed a G4-forming sequence on the LCR probe with a terminally labeled amino group. Subsequently, asymmetric hemin with carboxylic arms allowed assembly with the LCR products and peptide to form CPDzyme, followed by the magnetic separation of the extraneous components and chemiluminescence detection. Compared with the conventional G4/hemin signaling-based method, the LCR-CPDzyme system demonstrated 3 orders of magnitude improved sensitivity, with accurate quantification of as low as 25 aM miRNA and differentiation of 0.1% of mutant DNA from the pool containing a large amount of wild-type DNA. The proposed LCR-CPDzyme strategy is a potentially powerful method for in vitro diagnostics and serves as a reference for the development of other ligation- or hybridization-based nucleic acid amplification assays.

METTL16 deficiency attenuates apoptosis through translational control of extrinsic death receptor during nutrient deprivation.

METTL16 is a well-characterized m6A methyltransferase that has been reported to contribute to tumorigenesis in various types of cancer. However, the effect of METTL16 on tumor progression under restricted nutrient conditions, which commonly occur in tumor microenvironment, has yet to be elucidated. Herein, our study initially reported the inhibitory effect of METTL16 depletion on apoptosis under amino acid starvation conditions. Mechanistically, we determined that the METTL16 knockdown represses the expression of extrinsic death receptors at both transcription and translation levels. Depletion of METTL16 prevented protein synthesis of GCN2, resulting in diminished ATF4 expression in a GCN2-eIF2α-dependent manner. Reduction of ATF4 further declined the expression of apoptotic receptor protein DR5. Meanwhile, METTL16 deficiency directly hampered protein synthesis of FADD and DR5, thereby impairing apoptosis and promoting cancer cell survival. Taken together, our study provides novel evidence for the involvement of METTL16 in regulating cancer progression, suggesting that METTL16 as a potential therapeutic target for cancer treatment.