Fuzheng Huayu recipe inhibits bleomycin-induced pulmonary fibrosis in rats by inhibiting M2 polarization of macrophages via the oxidative phosphorylation pathway.

Fuzheng Huayu recipe (FZHYR) is a Chinese patent medicine for the treatment of fibrosis. The effects of FZHYR on pulmonary fibrosis and macrophage polarization were investigated in vitro. FZHYR inhibited pulmonary inflammation and fibrosis and M2 polarization of macrophages in bleomycin-induced pulmonary fibrosis (BPF) of rat model. Differentially expressed genes were screened by high-throughput mRNA sequencing and GSEA showed that oxidative phosphorylation (OXPHOS) was correlated with BPF. FZHYR inhibited expressions of Ndufa2 and Ndufa6 in lung tissues of BPF rats. These findings suggest that OXPHOS pathway serves as a possible target for pulmonary fibrosis therapy by FZHYR.

A tensor decomposition reveals ageing-induced differences in muscle and grip-load force couplings during object lifting.

Do motor patterns of object lifting movements change as a result of ageing? Here we propose a methodology for the characterization of these motor patterns across individuals of different age groups. Specifically, we employ a bimanual grasp-lift-replace protocol with younger and older adults and combine measurements of muscle activity with grip and load forces to provide a window into the motor strategies supporting effective object lifts. We introduce a tensor decomposition to identify patterns of muscle activity and grip-load force ratios while also characterizing their temporal profiles and relative activation across object weights and participants of different age groups. We then probe age-induced changes in these components. A classification analysis reveals three motor components that are differentially recruited between the two age groups. Linear regression analyses further show that advanced age and poorer manual dexterity can be predicted by the coupled activation of forearm and hand muscles which is associated with high levels of grip force. Our findings suggest that ageing may induce stronger muscle couplings in distal aspects of the upper limbs, and a less economic grasping strategy to overcome age-related decline in manual dexterity.

Quantitative profiling of m6A at single base resolution across the life cycle of rice and Arabidopsis.

N6-methyladenosine (m6A) plays critical roles in regulating mRNA metabolism. However, comprehensive m6A methylomes in different plant tissues with single-base precision have yet to be reported. Here, we present transcriptome-wide m6A maps at single-base resolution in different tissues of rice and Arabidopsis using m6A-SAC-seq. Our analysis uncovers a total of 205,691 m6A sites distributed across 22,574 genes in rice, and 188,282 m6A sites across 19,984 genes in Arabidopsis. The evolutionarily conserved m6A sites in rice and Arabidopsis ortholog gene pairs are involved in controlling tissue development, photosynthesis and stress response. We observe an overall mRNA stabilization effect by 3' UTR m6A sites in certain plant tissues. Like in mammals, a positive correlation between the m6A level and the length of internal exons is also observed in plant mRNA, except for the last exon. Our data suggest an active m6A deposition process occurring near the stop codon in plant mRNA. In addition, the MTA-installed plant mRNA m6A sites correlate with both translation promotion and translation suppression, depicting a more complicated regulatory picture. Our results therefore provide in-depth resources for relating single-base resolution m6A sites with functions in plants and uncover a suppression-activation model controlling m6A biogenesis across species.

Small-molecule inhibition of the METTL3/METTL14 complex suppresses neuroblastoma tumor growth and promotes differentiation.

The N6-methyladenosine (m6A) RNA modification is an important regulator of gene expression. m6A is deposited by a methyltransferase complex that includes methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14). High levels of METTL3/METTL14 drive the growth of many types of adult cancer, and METTL3/METTL14 inhibitors are emerging as new anticancer agents. However, little is known about the m6A epitranscriptome or the role of the METTL3/METTL14 complex in neuroblastoma, a common pediatric cancer. Here, we show that METTL3 knockdown or pharmacologic inhibition with the small molecule STM2457 leads to reduced neuroblastoma cell proliferation and increased differentiation. These changes in neuroblastoma phenotype are associated with decreased m6A deposition on transcripts involved in nervous system development and neuronal differentiation, with increased stability of target mRNAs. In preclinical studies, STM2457 treatment suppresses the growth of neuroblastoma tumors in vivo. Together, these results support the potential of METTL3/METTL14 complex inhibition as a therapeutic strategy against neuroblastoma.

Chemical manipulation of m1A mediates its detection in human tRNA.

N 1-methyl adenosine (m1A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m1A modification sites in tRNAs are evolutionarily conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m1A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m1A can notably modulate RNA structure. It also blocks Watson-Crick-Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m1A. In this study, we introduce a reduction-based m1A sequencing (red-m1A-seq). We report that NaBH4 reduction of m1A can improve the mutation and readthrough rates using commercially available RT enzymes to give a better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m1A to m6A to provide good controls, allowing the detection of m1A with higher sensitivity and accuracy. We applied red-m1A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m1A sites, but also new m1A sites in mt-tRNAAsn-GTT and 5.8S rRNA.

Postpartum weight retention and the early evolution of cardiovascular risk over the first 5 years after pregnancy.

BACKGROUND: The cumulative effect of postpartum weight retention from each pregnancy in a woman's life may contribute to her risk of ultimately developing type 2 diabetes and cardiovascular disease. However, there is limited direct evidence supporting this hypothesis. Thus, we sought to characterize the impact of postpartum weight retention on the trajectories of cardiovascular risk factors over the first 5-years after pregnancy. METHODS: In this prospective observational cohort study, 330 women (mean age 35.7 ± 4.3 years, mean pre-pregnancy body mass index 25.2 ± 4.8 kg/m2, 50.9% primiparous) underwent serial cardiometabolic characterization (anthropometry, blood pressure, lipids, oral glucose tolerance test, insulin sensitivity/resistance (Matsuda index, HOMA-IR), C-reactive protein (CRP), adiponectin) at 1-year, 3-years, and 5-years postpartum. Based on the magnitude of weight change between pre-pregnancy and 5-years postpartum, they were stratified into the following 3 groups: weight loss (n = 100), weight gain 0-6% (n = 110), and weight gain ≥ 6% (n = 120). RESULTS: At 1-year postpartum, cardiovascular risk factors did not differ between the groups. However, an adverse risk factor profile progressively emerged in the weight retention groups at 3- and 5-years. Indeed, after covariate adjustment, there was stepwise worsening (from the weight loss group to weight gain 0-6% to weight gain ≥ 6% group) of the following cardiovascular risk factors at 5-years: triglycerides (p = 0.001), HDL (p = 0.02), LDL (p = 0.01), apolipoprotein-B (p = 0.003), Matsuda index (p < 0.0001), HOMA-IR (p < 0.0001), fasting glucose (p = 0.07), and CRP (p = 0.01). Moreover, on logistic regression analyses, weight gain ≥ 6% emerged as an independent predictor of pre-diabetes/diabetes at 5-years (adjusted OR = 3.40, 95%CI: 1.63-7.09). CONCLUSION: Postpartum weight retention predicts trajectories of worsening cardiovascular risk factors and glucose intolerance over the first 5-years after delivery, consistent with its postulated contribution to future vascular disease in women.

Ultrafast bisulfite sequencing detection of 5-methylcytosine in DNA and RNA.

Bisulfite sequencing (BS-seq) to detect 5-methylcytosine (5mC) is limited by lengthy reaction times, severe DNA damage, overestimation of 5mC level and incomplete C-to-U conversion of certain DNA sequences. We present ultrafast BS-seq (UBS-seq), which uses highly concentrated bisulfite reagents and high reaction temperatures to accelerate the bisulfite reaction by ~13-fold, resulting in reduced DNA damage and lower background noise. UBS-seq allows library construction from small amounts of purified genomic DNA, such as from cell-free DNA or directly from 1 to 100 mouse embryonic stem cells, with less overestimation of 5mC level and higher genome coverage than conventional BS-seq. Additionally, UBS-seq quantitatively maps RNA 5-methylcytosine (m5C) from low inputs of mRNA and allows the detection of m5C stoichiometry in highly structured RNA sequences. Our UBS-seq results identify NSUN2 as the major 'writer' protein responsible for the deposition of ~90% of m5C sites in HeLa mRNA and reveal enriched m5C sites in 5'-regions of mammalian mRNA, which may have functional roles in mRNA translation regulation.

Establishment of a novel minigenome system for the identification of drugs targeting Nipah virus replication.

Nipah virus (NiV) is a deadly zoonotic pathogen with high potential to cause another pandemic. Owing to biosafety concerns, studies on living NiV must be performed in biosafety level 4 (BSL-4) laboratories, which greatly hinders the development of anti-NiV drugs. To overcome this issue, minigenome systems have been developed to study viral replication and screen for antiviral drugs. This study aimed to develop two minigenome systems (transient and stable expression) based on a helper cell line expressing the NiV P, N and L proteins required to initiate NiV RNA replication. Stable minigenome cells were resistant to ribavirin, remdesivir and favipiravir but sensitive to interferons. Cells of the transient replication system were sensitive to ribavirin and favipiravir and suitable for drug screening. Our study demonstrates a feasible and effective platform for studying NiV replication and shows great potential for high-throughput drug screening in a BSL-2 laboratory environment.

Sequencing of N6-methyl-deoxyadenosine at single-base resolution across the mammalian genome.

Although DNA N6-methyl-deoxyadenosine (6mA) is abundant in bacteria and protists, its presence and function in mammalian genomes have been less clear. We present Direct-Read 6mA sequencing (DR-6mA-seq), an antibody-independent method, to measure 6mA at base resolution. DR-6mA-seq employs a unique mutation-based strategy to reveal 6mA sites as misincorporation signatures without any chemical or enzymatic modulation of 6mA. We validated DR-6mA-seq through the successful mapping of the well-characterized G(6mA)TC motif in the E. coli DNA. As expected, when applying DR-6mA-seq to mammalian systems, we found that genomic DNA (gDNA) 6mA abundance is generally low in most mammalian tissues and cells; however, we did observe distinct gDNA 6mA sites in mouse testis and glioblastoma cells. DR-6mA-seq provides an enabling tool to detect 6mA at single-base resolution for a comprehensive understanding of DNA 6mA in eukaryotes.

Profiling of RNA-binding protein binding sites by in situ reverse transcription-based sequencing.

RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA targets are still lacking, especially when the interaction is dynamic or samples are limited. Here we present an assay of reverse transcription-based RBP binding site sequencing (ARTR-seq), which relies on in situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites. ARTR-seq avoids ultraviolet crosslinking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section. Taking advantage of rapid formaldehyde fixation, ARTR-seq enables capturing the dynamic RNA binding by RBPs over a short period of time, as demonstrated by the profiling of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 minutes.

BID-seq for transcriptome-wide quantitative sequencing of mRNA pseudouridine at base resolution.

Pseudouridine (Ψ) is an abundant RNA modification that is present in and affects the functions of diverse non-coding RNA species, including rRNA, tRNA and small nuclear RNA. Ψ also exists in mammalian mRNA and probably exhibits functional roles; however, functional investigations of mRNA Ψ modifications in mammals have been hampered by the lack of a quantitative method that detects Ψ at base precision. We have recently developed bisulfite-induced deletion sequencing (BID-seq), which provides the community with a quantitative method to map RNA Ψ distribution transcriptome-wide at single-base resolution. Here, we describe an optimized BID-seq protocol for mapping Ψ distribution across cellular mRNAs, which includes fast steps in both library preparation and data analysis. This protocol generates highly reproducible results by inducing high deletion ratios at Ψ modification within diverse sequence contexts, and meanwhile displayed almost zero background deletions at unmodified uridines. When used for transcriptome-wide Ψ profiling in mouse embryonic stem cells, the current protocol uncovered 8,407 Ψ sites from as little as 10 ng of polyA+ RNA input. This optimized BID-seq workflow takes 5 days to complete and includes four main sections: RNA preparation, library construction, next-generation sequencing (NGS) and data analysis. Library construction can be completed by researchers who have basic knowledge and skills in molecular biology and genetics. In addition to the experimental protocol, we provide BID-pipe ( https://github.com/y9c/pseudoU-BIDseq ), a user-friendly data analysis pipeline for Ψ site detection and modification stoichiometry quantification, requiring only basic bioinformatic and computational skills to uncover Ψ signatures from BID-seq data.

Early diagnosis and prognostic potential of RAC3 in bladder tumor.

BACKGROUND AND PURPOSE: Bladder tumors are among the most prevalent malignancies in the urinary system, and RAC3 has been linked to various types of cancer. This article seeks to explore the potential of RAC3 as both an early diagnostic marker for bladder tumors and a novel therapeutic target. METHODS/PATIENTS: The expression of RAC3 in bladder tissue was detected using immunohistochemical staining. Additionally, the protein expression of RAC3 was measured and quantified through enzyme-linked immunosorbent assay (ELISA). Subsequently, the correlation between the expression level of RAC3 and bladder tumors was investigated through multifactorial analysis and survival analysis. RESULTS: Our findings revealed that RAC3 expression was upregulated in bladder tumor tissues. Moreover, we observed higher levels of RAC3 expression in the serum and urine of patients with bladder tumors compared to those with non-bladder tumors. Additionally, we identified a significant positive correlation between RAC3 expression levels and the stage, degree of differentiation, and infiltration of bladder tumors. Importantly, high RAC3 expression emerged as an influential factor in the poor prognosis of bladder tumors, as patients with high RAC3 expression exhibited a lower overall survival rate than those with low RAC3 expression. CONCLUSION: Based on our results, RAC3 shows promise as both a marker for early diagnosis of bladder tumors and a potential therapeutic target.

Enhanced performance of enzymes confined in biocatalytic hydrogen-bonded organic frameworks for sensing of glutamate in the central nervous system.

Glutamate (Glu) is a key excitatory neurotransmitter associated with various neurological disorders in the central nervous system, so its measurement is vital to both basic research and biomedical application. In this work, we propose the first example of using biocatalytic hydrogen-bonded organic frameworks (HOFs) as the hosting matrix to encapsulate glutamate oxidase (GLOD) via a de novo approach, fabricating a cascaded-enzyme nanoreactor for Glu biosensing. In this design, the ferriporphyrin ligands can assemble to form Fe-HOFs with high catalase-like activity, while offering a scaffold for the in-situ immobilization of GLOD. Moreover, the formed GLOD@Fe-HOFs are favorable for the efficient diffusion of Glu into the active sites of GLOD via the porous channels, accelerating the cascade reaction with neighboring Fe-HOFs. Consequently, the constructed nanoreactor can offer superior activity and operational stability in the catalytic cascade for Glu biosensing. More importantly, rapid and selective detection can be achieved in the cerebrospinal fluid (CSF) collected from mice in a low sample consumption. Therefore, the successful fabrication of enzyme@HOFs may offer promise to develop high-performance biosensor for further biomedical applications.

Impact of the diagnosis of gestational diabetes on maternal physical activity after pregnancy.

AIM: The diagnosis of gestational diabetes (GDM) identifies women who are at future risk of developing type 2 diabetes. However, it is unclear if diagnosing GDM thus motivates women to increase physical activity after pregnancy or if this medicalization has the opposite effect of decreasing activity, possibly reflecting assumption of a sick role. We thus sought to evaluate the impact of diagnosing GDM on changes in maternal physical activity after pregnancy. METHODS: In this prospective cohort study, physical activity patterns were assessed by the Baecke questionnaire for the year before pregnancy and the first year postpartum in 405 white women comprising the following three gestational glucose tolerance groups: (a) those who did not have GDM (non-GDM; n = 247), (b) women with undiagnosed GDM (n = 46) and (c) those diagnosed with GDM (n = 112). RESULTS: In the year before pregnancy, mean adjusted total physical activity progressively decreased from non-GDM to undiagnosed GDM to diagnosed GDM (p = .067). Conversely, at 1 year postpartum, total physical activity was highest in those who had been diagnosed with GDM (p = .02). Compared with non-GDM, diagnosed GDM predicted an increase in total physical activity from pre-pregnancy to 1 year postpartum (t = 2.3, p = .02) whereas undiagnosed GDM predicted a concurrent decrease in leisure-time activity (t = -2.74, p = .006). Accordingly, the mean adjusted increase in body mass index from pre-pregnancy to 1 year postpartum was lowest in those with diagnosed GDM (0.26 ± 0.25 kg/m2 ), highest in undiagnosed GDM (1.23 ± 0.38 kg/m2 ) and intermediate in non-GDM (0.89 ± 0.22 kg/m2 ) (overall p = .04). CONCLUSION: Diagnosis of GDM leads to increased physical activity after pregnancy that may partially attenuate postpartum weight retention.

Light-induced LLPS of the CRY2/SPA1/FIO1 complex regulating mRNA methylation and chlorophyll homeostasis in Arabidopsis.

Light regulates chlorophyll homeostasis and photosynthesis via various molecular mechanisms in plants. The light regulation of transcription and protein stability of nuclear-encoded chloroplast proteins have been extensively studied, but how light regulation of mRNA metabolism affects abundance of nuclear-encoded chloroplast proteins and chlorophyll homeostasis remains poorly understood. Here we show that the blue light receptor cryptochrome 2 (CRY2) and the METTL16-type m6A writer FIONA1 (FIO1) regulate chlorophyll homeostasis in response to blue light. In contrast to the CRY2-mediated photo-condensation of the mRNA adenosine methylase (MTA), photoexcited CRY2 co-condenses FIO1 only in the presence of the CRY2-signalling protein SUPPRESSOR of PHYTOCHROME A (SPA1). CRY2 and SPA1 synergistically or additively activate the RNA methyltransferase activity of FIO1 in vitro, whereas CRY2 and FIO1, but not MTA, are required for the light-induced methylation and translation of the mRNAs encoding multiple chlorophyll homeostasis regulators in vivo. Our study demonstrates that the light-induced liquid-liquid phase separation of the photoreceptor/writer complexes is commonly involved in the regulation of photoresponsive changes of mRNA methylation, whereas the different photo-condensation mechanisms of the CRY/FIO1 and CRY/MTA complexes explain, at least partially, the writer-specific functions in plant photomorphogenesis.

Cryo-EM structure of TMEM63C suggests it functions as a monomer.

The TMEM63 family proteins (A, B, and C), calcium-permeable channels in animals that are preferentially activated by hypo-osmolality, have been implicated in various physiological functions. Deficiency of these channels would cause many diseases including hearing loss. However, their structures and physiological roles are not yet well understood. In this study, we determine the cryo-electron microscopy (cryo-EM) structure of the mouse TMEM63C at 3.56 Å, and revealed structural differences compared to TMEM63A, TMEM63B, and the plant orthologues OSCAs. Further structural guided mutagenesis and calcium imaging demonstrated the important roles of the coupling of TM0 and TM6 in channel activity. Additionally, we confirm that TMEM63C exists primarily as a monomer under physiological conditions, in contrast, TMEM63B is a mix of monomer and dimer in cells, suggesting that oligomerization is a regulatory mechanism for TMEM63 proteins.

Chemical Proteomic Discovery of Isotype-Selective Covalent Inhibitors of the RNA Methyltransferase NSUN2.

5-Methylcytosine (m5 C) is an RNA modification prevalent on tRNAs, where it can protect tRNAs from endonucleolytic cleavage to maintain protein synthesis. The NSUN family (NSUN1-7 in humans) of RNA methyltransferases are capable of installing the methyl group onto the C5 position of cytosines in RNA. NSUNs are implicated in a wide range of (patho)physiological processes, but selective and cell-active inhibitors of these enzymes are lacking. Here, we use cysteine-directed activity-based protein profiling (ABPP) to discover azetidine acrylamides that act as stereoselective covalent inhibitors of human NSUN2. Despite targeting a conserved catalytic cysteine in the NSUN family, the NSUN2 inhibitors show negligible cross-reactivity with other human NSUNs and exhibit good proteome-wide selectivity. We verify that the azetidine acrylamides inhibit the catalytic activity of recombinant NSUN2, but not NSUN6, and demonstrate that these compounds stereoselectively disrupt NSUN2-tRNA interactions in cancer cells, leading to a global reduction in tRNA m5 C content. Our findings thus highlight the potential to create isotype-selective and cell-active inhibitors of NSUN2 with covalent chemistry targeting a conserved catalytic cysteine.

Analysis of genome-wide 5-hydroxymethylation of blood samples stored in different anticoagulants: opportunities for the expansion of clinical resources for epigenetic research.

BACKGROUND: Elucidating epigenetic mechanisms could provide new biomarkers for disease diagnosis and prognosis. Technological advances allow genome-wide profiling of 5-hydroxymethylcytosines (5hmC) in liquid biopsies. 5hmC-Seal followed by NGS is a highly sensitive technique for 5hmC biomarker discovery in cfDNA. Currently, 5hmC Seal is optimized for EDTA blood collection. We asked whether heparin was compatible with 5hmC Seal as many clinical and biobanked samples are stored in heparin. METHODS: We obtained 60 samples in EDTA matched to 60 samples in heparin from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. Samples were comprised of 30 controls and 30 individuals who were later diagnosed with colon cancer. We profiled genome-wide 5hmC in cfDNA using 5hmC-Seal assay followed by NGS. The 5hmC profiling data from samples collected in EDTA were systematically compared to those in heparin across various genomic features. RESULTS: cfDNA isolation and library construction appeared comparable in heparin vs. EDTA. Typical genomic distribution patterns of 5hmC, including gene bodies and enhancer markers, were comparable in heparin vs. EDTA. 5hmC analysis of cases and controls yielded highly correlated differential features suggesting that both anticoagulants were compatible with 5hmC Seal assay. CONCLUSIONS: While not currently recommended for the 5hmC-Seal protocol, blood samples stored in heparin were successfully used to generate analysable and biologically relevant genome-wide 5hmC profiling. Our findings are the first to support opportunities to expand the biospecimen resource to heparin samples for 5hmC Seal and perhaps other PCR-based technologies in epigenetic research.