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.

Quantification of tRNA m1A modification by templated-ligation qPCR.

N1-methyladenosine (m1A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. m1A is generally located in the T loop of cytosolic tRNA and between the acceptor and D stems of mitochondrial tRNAs; it is involved in the tertiary interaction that stabilizes tRNA. Human tRNA m1A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m1A, a PCR method to assess m1A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by a qPCR method (TL-qPCR) that measures m1A levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template, followed by qPCR using the ligation product as the template. m1A interferes with the ligation in specific ways, allowing for the quantitative assessment of m1A levels using subnanogram amounts of total RNA. We identify the features of specificity and quantitation for m1A-modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.

The multiple reference range of mean uterine artery pulsatility index for natural and in vitro fertilization singletons during 11-14 gestational weeks.

Background: Ultrasonography of the uterine artery (UtA) in the first and second trimesters of pregnancy can assess uterine-placental blood perfusion and guide early clinical prevention. Establishing normal ranges of the UtA pulsatility index (UtA-PI) at 11-14 weeks of pregnancy is helpful for the early identification of high-risk pregnant women and improving the prognosis. This study aimed to establish a reference range of UtA-PI based on crown-rump length (CRL) for spontaneous and in vitro fertilization (IVF) singleton pregnancy during 11-14 weeks, respectively. Methods: A prospective study was performed at Peking Union Medical College Hospital. Healthy, low-risk women with a singleton pregnancy at 11-14 gestational weeks were consecutively recruited for this study from December 2017 to December 2020. All participants underwent routine prenatal ultrasound examination. The CRL of the fetus and the UtA-PI were measured in both uterine arteries, and average values were calculated. The LMS method was used to fit the percentile (P)5, P10, P25, P50, P75, P90, and P95 curves of the UtA-PI value of spontaneous and IVF singleton pregnancy with CRL changes, respectively. Results: A total of 1,962 pregnant women with normal fetuses were included in this study, including 1,792 pregnancies conceived naturally and 170 IVF fetuses. The UtA-PI reference range in the spontaneous pregnancy group was consistently higher than that in the IVF group during 11-14 weeks, and showed a statistically significant difference in UtA-PI for spontaneous and IVF pregnancies (P<0.001). According to the LMS method, each percentile curve of UtA-PI decreased with the increase of CRL in both the natural pregnancy group and the IVF group. The P95 range of UtA-PI for pregnant women with naturally conceived and IVF pregnancy was 2.74 to 2.11 and 2.50 to 1.94, respectively. The overall change of UtA-PI differentials of the two groups showed a downward trend and decreased slightly with the increase of CRL. Conclusions: This study provided a single-center, large sample of data and constructed a CRL-based reference value of UtA-PI for spontaneous and IVF singleton pregnancy, which provides a reliable basis for early UtA evaluation and early clinical decision-making during 11-14 gestational weeks.

Validation of the diagnostic efficacy of O-RADS in adnexal masses.

The aim of this study was to validate the performance of the Ovarian-Adnexal Reporting and Data Systems (O-RADS) series models proposed by the American College of Radiology (ACR) in the preoperative diagnosis of adnexal masses (AMs). Two experienced sonologists examined 218 patients with AMs and gave the assessment results after the examination. Pathological findings were used as a reference standard. Of the 218 lesions, 166 were benign and 52 were malignant. Based on the receiver operating characteristic (ROC) curve, we defined a malignant lesion as O-RADS > 3 (i.e., lesions in O-RADS categories 4 and 5 were malignant). The area under the curve (AUC) of O-RADS (v2022) was 0.970 (95% CI 0.938-0.988), which wasn't statistically significantly different from the O-RADS (v1) combined Simple Rules Risk (SRR) assessment model with the largest AUC of 0.976 (95% CI 0.946-0.992) (p = 0.1534), but was significantly higher than the O-RADS (v1) (AUC = 0.959, p = 0.0133) and subjective assessment (AUC = 0.918, p = 0.0255). The O-RADS series models have good diagnostic performance for AMs. Where, O-RADS (v2022) has higher accuracy and specificity than O-RADS (v1). The accuracy and specificity of O-RADS (v1), however, can be further improved when combined with SRR assessment.

Docetaxel-loaded pH/ROS dual-responsive nanoparticles with self-supplied ROS for inhibiting metastasis and enhancing immunotherapy of breast cancer.

BACKGROUND: Although stimuli-responsive nanoplatforms were developed to deliver immunogenic cell death (ICD) inducers to enhance cancer immunotherapy, the complete release of ICD inducers into the tumor microenvironment (TME) was limited by the inadequate supplementation of endogenous stimulus (e.g., reactive oxygen species (ROS)). To address this issue, we synthesized a self-responsive nanomaterial with self-supplied ROS, which mainly consists of a ROS responsive moiety HPAP and cinnamaldehyde (CA) as the ROS-generating agent. The endogenous ROS can accelerate the degradation of HPAP in materials to release docetaxel (DTX, an ICD inducer). In intracellular acidic environment, the pH-sensitive acetal was cleaved to release CA. The released CA in turn induces the generation of more ROS through mitochondrial damage, resulting in amplified DTX release. Using this self-cycling and self-responsive nanomaterial as a carrier, DTX-loaded pH/ROS dual-responsive nanoparticles (DTX/FA-CA-Oxi-αCD NPs) were fabricated and evaluated in vitro and in vivo. RESULTS: In vitro experiments validated that the NPs could be effectively internalized by FA-overexpressed cells and completely release DTX in acidic and ROS microenvironments to induce ICD effect. These NPs significantly blocked 4T1 cell migration and decreased cell invasion. In vivo experiments demonstrated that the tumor-targeted NPs significantly inhibited tumor growth and blocked tumor metastasis. More importantly, these NPs significantly improved immunotherapy through triggering effector T-cell activation and relieving the immunosuppressive state of the TME. CONCLUSIONS: Our results demonstrated that DTX/FA-CA-Oxi-αCD NPs displayed great potential in preventing tumor metastasis, inhibiting tumor growth, and improving the efficacy of anti-PD-1antibody.

RBFOX2 recognizes N6-methyladenosine to suppress transcription and block myeloid leukaemia differentiation.

N6-methyladenosine (m6A) methylation can be deposited on chromatin-associated RNAs (caRNAs) by the RNA methyltransferase complex (MTC) to regulate chromatin state and transcription. However, the mechanism by which MTC is recruited to distinct genomic loci remains elusive. Here we identify RBFOX2, a well-studied RNA-binding protein, as a chromatin factor that preferentially recognizes m6A on caRNAs. RBFOX2 can recruit RBM15, an MTC component, to facilitate methylation of promoter-associated RNAs. RBM15 also physically interacts with YTHDC1 and recruits polycomb repressive complex 2 (PRC2) to the RBFOX2-bound loci for chromatin silencing and transcription suppression. Furthermore, we found that this RBFOX2/m6A/RBM15/YTHDC1/PRC2 axis plays a critical role in myeloid leukaemia. Downregulation of RBFOX2 notably inhibits survival/proliferation of acute myeloid leukaemia cells and promotes their myeloid differentiation. RBFOX2 is also required for self-renewal of leukaemia stem/initiation cells and acute myeloid leukaemia maintenance. Our study presents a pathway of m6A MTC recruitment and m6A deposition on caRNAs, resulting in locus-selective chromatin regulation, which has potential therapeutic implications in leukaemia.

Cyclic RGD-Functionalized pH/ROS Dual-Responsive Nanoparticle for Targeted Breast Cancer Therapy.

Breast cancer is the most common malignant tumor in women and is a big challenge to clinical treatment due to the high morbidity and mortality. The pH/ROS dual-responsive nanoplatforms may be an effective way to significantly improve the therapeutic efficacy of breast cancer. Herein, we report a docetaxel (DTX)-loaded pH/ROS-responsive NP that could achieve active targeting of cancer cells and selective and complete drug release for effective drug delivery. The pH/ROS-responsive NPs were fabricated using nanocarriers that consist of an ROS-responsive moiety (4-hydroxymethylphenylboronic acid pinacol ester, HPAP), cinnamaldehyde (CA, an aldehyde organic compound with anticancer activities) and cyclodextrin (α-CD). The NPs were loaded with DTX, modified with a tumor-penetration peptide (circular RGD, cRGD) and named DTX/RGD NPs. The cRGD could promote DTX/RGD NPs penetration into deep tumor tissue and specifically target cancer cells. After internalization by cancer cells through receptor-mediated endocytosis, the pH-responsive acetal was cleaved to release CA in the lysosomal acidic environment. Meanwhile, the high ROS in tumor cells induced the disassembly of NPs with complete release of DTX. In vitro cellular assays verified that DTX/RGD NPs could be effectively internalized by 4T1 cells, obviously inducing apoptosis, blocking the cell cycle of 4T1 cells and consequently, killing tumor cells. In vivo animal experiments demonstrated that the NPs could target to the tumor sites and significantly inhibit the tumor growth in 4T1 breast cancer mice. Both in vitro and in vivo investigations demonstrated that DTX/RGD NPs could significantly improve the antitumor effect compared to free DTX. Thus, the DTX/RGD NPs provide a promising strategy for enhancing drug delivery and cancer therapy.

Effect of differences in O-RADS lexicon interpretation between senior and junior sonologists on O-RADS classification and diagnostic performance.

PURPOSE: To assess the consistency of Ovarian-Adnexal Reporting and Data System (O-RADS) lexicon interpretation between senior and junior sonologists and to investigate its impact on O-RADS classification and diagnostic performance. METHODS: We prospectively studied 620 patients with adnexal lesions, all of whom underwent transvaginal or transrectal ultrasound performed by a senior sonologist (R1) who selected the O-RADS lexicon description and O-RADS category for the lesion after the examination. Meanwhile, the junior sonologist (R2) analyzed the images retained by R1 and divided the lesion in the same way. Pathological findings were used as a reference standard. kappa (к) statistics were used to assess the interobserver agreement. RESULTS: Of the 620 adnexal lesions, 532 were benign and 88 were malignant. When using the O-RADS lexicon, R1 and R2 had almost perfect agreement regarding lesion category, external contour of solid lesions, presence of papillary inside cystic lesions, and fluid echogenicity (к: 0.81-1.00). Substantial agreement in solid components, acoustic shadow, vascularity and O-RADS categories (к: 0.61-0.80). Consistency in classifying classic benign lesions in the O-RADS category was only moderate (к = 0.535). No significant difference in diagnostic performance between them using O-RADS (P = 0.1211). CONCLUSION: There was good agreement between senior and junior sonologists in the interpretation of the O-RADS lexicon and in the classification of O-RADS, except for a moderate agreement in the interpretation and classification of classic benign lesions. Differences in O-RADS category delineation between sonologists had no significant effect on the diagnostic performance of O-RADS.

A general and efficient approach to synthesize the phosphoramidites of 5'-18O labeled purine nucleosides.

5'-18O labeled RNA oligos are important probes to investigate the mechanism of 2'-O-transphosphorylation reactions. Here we describe a general and efficient synthetic approach to the phosphoramidite derivatives of 5'-18O labeled nucleosides starting from the corresponding commercially available 5'-O-DMT protected nucleosides. Using this method, we prepared 5'-18O-guanosine phosphoramidite in 8 steps (13.2% overall yield), 5'-18O-adenosine phosphoramidite in 9 steps (10.1% overall yield) and 5'-18O-2'-deoxyguanosine phosphoramidite in 6 steps (12.8% overall yield). These 5'-18O labeled phosphoramidites can be incorporated into RNA oligos by solid phase synthesis for determination of heavy atom isotope effects in RNA 2'-O-transphosphorylation reactions.

pH-Triggered Size-Transformable and Bioactivity-Switchable Self-Assembling Chimeric Peptide Nanoassemblies for Combating Drug-Resistant Bacteria and Biofilms.

Drug-resistant bacteria and biofilm-associated infections are prominent problems in the field of antibacterial medicine, seriously affecting human and animal health. Despite the great potential of nanomaterials in the antibacterial field, overcoming the paradox of size and charge, efficient penetration, and retention within biofilms remain a formidable challenge. Here, self-assembling chimeric peptide nanoassemblies composed of multiple functional fragments are designed for the treatment of drug-resistant bacteria and biofilm-associated infections. Notably, the chimeric peptide self-assembles into nanofibers at pH 7.4 and is transformable into nanoparticles in the acidic biofilm-infected microenvironment at pH 5.0, and thus achieves a size reduction and charge increase, improving the penetration into the bacterial biofilms and killing drug-resistant bacteria by a mechanism dominated by membrane cleavage. In vivo mouse and piglet infection models confirm the ability of chimeric peptide nanoassemblies to reduce bacterial load within biofilms. Collectively, this research on pathological-environment-driven nanostructural transformations may provide a theoretical basis for designing high-performance antibacterial nanomaterials and advance the application of peptide-based nanomaterials in medicine and animal husbandry.

Mid-infrared analogue polaritonic reversed Cherenkov radiation in natural anisotropic crystals.

Cherenkov radiation (CR) excited by fast charges can serve as on-chip light sources with a nanoscale footprint and broad frequency range. The reversed CR, which usually occurs in media with the negative refractive index or negative group-velocity dispersion, is highly desired because it can effectively separate the radiated light from fast charges thanks to the obtuse radiation angle. However, reversed CR at the mid-infrared remains challenging due to the significant loss of conventional artificial structures. Here we observe mid-infrared analogue polaritonic reversed CR in a natural van der Waals (vdW) material (i.e., α-MoO3), whose hyperbolic phonon polaritons exhibit negative group velocity. Further, the real-space image results of analogue polaritonic reversed CR indicate that the radiation distributions and angles are closely related to the in-plane isofrequency contours of α-MoO3, which can be further tuned in the heterostructures based on α-MoO3. This work demonstrates that natural vdW heterostructures can be used as a promising platform of reversed CR to design on-chip mid-infrared nano-light sources.

Serotonin acts through YAP to promote cell proliferation: mechanism and implication in colorectal cancer progression.

Serotonin, also known as 5-hydroxytryptamine (5-HT), is a key messenger that mediates several central and peripheral functions in the human body. Emerging evidence indicates that serotonin is critical in tumorigenesis, but its role in colorectal cancer remains elusive. Herein, we report that serotonin transporter (SERT) transports serotonin into colorectal cancer cells, enhancing Yes-associated protein (YAP) expression and promoting in vitro and in vivo colon cancer cell growth. Once within the cells, transglutaminase 2 (TG2) mediates RhoA serotonylated and activates RhoA-ROCK1/2 signalling to upregulate YAP expression in SW480 and SW1116 cells. Blocking SERT with citalopram reversed the serotonin-induced YAP expression and cell proliferation, inhibiting serotonin's effects on tumour formation in mice. Moreover, SERT expression was correlated with YAP in pathological human colorectal cancer samples and the levels of 5-HT were highly significant in the serum of patients with colorectal cancer. Together, our findings suggested that serotonin enters cells via SERT to activate RhoA/ROCK/YAP signalling to promote colon cancer carcinogenesis. Consequently, targeting serotonin-SERT-YAP axis may be a potential therapeutic strategy for colorectal cancer. Video abstract.

Correlation between urine vitamin D-binding protein and early-stage renal damage in Type 2 diabetes. / å°¿ç»´ç”Ÿç´ D结合蛋白与2åž‹ç³–å°¿ç— æ‚£è€ æ—©æœŸè‚¾æŸå®³çš„ç›¸å ³æ€§.

OBJECTIVES: The excretion of urinary vitamin D-binding protein (uVDBP) is related to the occurrence and development of early-stage renal damage in patients with Type 2 diabetes (T2DM). This study aims to explore the significance of detecting uVDBP in T2DM patients and its relationship with renal tubules, and to provide a new direction for the early diagnosis of T2DM renal damage. METHODS: A total of 105 patients with T2DM, who met the inclusion criteria, were included as a patient group, and recruited 30 individuals as a normal control group. The general information and blood and urine biochemical indicators of all subjects were collected; the levels of uVDBP, and a marker of tubular injury [urine kidney injury molecule 1 (uKIM-1), urine neutrophil gelatinase-associated lipocalin (uNGAL) and urine retinol-binding protein (uRBP)] were detected by enzyme-linked immunosorbent assay. The results were corrected by urinary creatinine (Cr) to uVDBP/Cr, uKIM-1/Cr, uNGAL/Cr and uRBP/Cr. The Pearson's and Spearman's correlation tests were used to analyze the correlation between uVDBP/Cr and urine albumin-to-creatinine ratio (UACR), estimated glomerular filtration rate (eGFR) and markers of tubular injury, and multivariate linear regression and receiver operating characteristic curve were used to analyze the correlation between uVDBP/Cr and UACR or eGFR. RESULTS: Compared with the normal control group, the uVDBP/Cr level in the patient group was increased (P<0.05), and which was positively correlated with UACR (r=0.774, P<0.01), and negatively correlated with eGFR (r=-0.397, P<0.01). There were differences in the levels of uKIM-1/Cr, uNGAL/Cr, and uRBP/Cr between the 2 groups (all P<0.01). The uVDBP/Cr was positively correlated with uKIM-1/Cr (r=0.752, P<0.01), uNGAL/Cr (r=0.644, P<0.01) and uRBP/Cr (r=0.812, P<0.01). The sensitivity was 90.0% and the specificity was 82.9% (UACR>30 mg/g) for evaluation of uVDBP/Cr on T2DM patients with early-stage renal damage, while the sensitivity was 75.0% and the specificity was 72.6% for evaluation of eGFR on T2DM patients with early-stage renal damage. CONCLUSIONS: The uVDBP/Cr can be used as a biomarker in early-stage renal damage in T2DM patients.

Simultaneous quantitative analysis and in vitro anti-arthritic effects of five polyphenols from Terminalia chebula.

Background: The fruit of Terminalia chebula has been widely used for a thousand years for treating diarrhea, ulcers, and arthritic diseases in Asian countries. However, the active components of this Traditional Chinese medicine and their mechanisms remain unclear, necessitating further investigation. Objectives: To perform simultaneous quantitative analysis of five polyphenols in T. chebula and evaluate their anti-arthritic effects including antioxidant and anti-inflammatory activity in vitro. Materials and methods: Water, 50% water-ethanol, and pure ethanol were used as extract solvents. Quantitative analysis of gallic acid, corilagin, chebulanin, chebulagic acid, and ellagic acid in the three extracts was performed using high-performance liquid chromatography (HPLC). Antioxidant activity was assessed by the 2,2-diphenylpicrylhydrazyl (DPPH) radical-scavenging assay, and anti-inflammatory activity was evaluated by detecting interleukin (IL)-6 and IL-8 expression in IL-1ß-stimulated MH7A cells. Results: The 50% water-ethanol solvent was the optimal solvent yielding the highest total polyphenol content, and the concentrations of chebulanin and chebulagic acid were much higher than those of gallic acid, corilagin, and ellagic acid in the extracts. The DPPH radical-scavenging assay showed that gallic acid and ellagic acid were the strongest antioxidative components, while the other three components showed comparable antioxidative activity. As for the anti-inflammatory effect, chebulanin and chebulagic acid significantly inhibited IL-6 and IL-8 expression at all three concentrations; corilagin and ellagic acid significantly inhibited IL-6 and IL-8 expression at high concentration; and gallic acid could not inhibit IL-8 expression and showed weak inhibition of IL-6 expression in IL-1ß-stimulated MH7A cells. Principal component analysis indicated that chebulanin and chebulagic acid were the main components responsible for the anti-arthritic effects of T. chebula. Conclusion: Our findings highlight the potential anti-arthritic role of chebulanin and chebulagic acid from T. chebula.

Plasmonic Cu Nanoparticles for the Low-temperature Photo-driven Water-gas Shift Reaction.

The activation of water molecules in thermal catalysis typically requires high temperatures, representing an obstacle to catalyst development for the low-temperature water-gas shift reaction (WGSR). Plasmonic photocatalysis allows activation of water at low temperatures through the generation of light-induced hot electrons. Herein, we report a layered double hydroxide-derived copper catalyst (LD-Cu) with outstanding performance for the low-temperature photo-driven WGSR. LD-Cu offered a lower activation energy for WGSR to H2 under UV/Vis irradiation (1.4 W cm-2 ) compared to under dark conditions. Detailed experimental studies revealed that highly dispersed Cu nanoparticles created an abundance of hot electrons during light absorption, which promoted *H2 O dissociation and *H combination via a carboxyl pathway, leading to the efficient production of H2 . Results demonstrate the benefits of exploiting plasmonic phenomena in the development of photo-driven low-temperature WGSR catalysts.

Dysregulation of the Epitranscriptomic Mark m1A in Ischemic Stroke.

Methylation of adenosine at N1 position yields N1-methyladenosine (m1A), which is an epitranscriptomic modification that regulates mRNA metabolism. Recent studies showed that altered m1A methylation promotes acute and chronic neurological diseases. We currently evaluated the effect of focal ischemia on cerebral m1A methylome and its machinery. Adult male C57BL/6J mice were subjected to transient middle cerebral artery occlusion, and the peri-infarct cortex was analyzed at 12 h and 24 h of reperfusion. The bulk abundance of m1A was measured by mass spectrometry and dot blot, and transcriptome-wide m1A alterations were profiled using antibody-independent m1A-quant-seq. Expression of the m1A writers and erasers was estimated by real-time PCR. Ischemia significantly decreased m1A levels and concomitantly upregulated m1A demethylase alkB homolog 3 at 24 h of reperfusion compared to sham. Transcriptome-wide profiling showed differential m1A methylation at 14 sites (8 were hypo- and 6 were hypermethylated). Many of those are located in the 3'-UTRs of unannotated transcripts proximal to the genes involved in regulating protein complex assembly, circadian rhythms, chromatin remodeling, and chromosome organization. Using several different approaches, we show for the first time that m1A epitranscriptomic modification in RNA is highly sensitive to cerebral ischemia.

Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution.

Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10-20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different 'writer' proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes.

Dominant-negative transforming growth factor-ß receptor-armoured mesothelin-targeted chimeric antigen receptor T cells slow tumour growth in a mouse model of ovarian cancer.

Ovarian cancer is a major cause of death among all gynaecological cancers. Although surgery, chemotherapy and targeted therapy have yielded successful outcomes, the 5-year survival rate remains < 30%. Adoptive immunotherapy, particularly chimeric antigen receptor (CAR) T-cell therapy, has demonstrated improved survival in acute lymphoblastic leukaemia with manageable toxicity. We explored CAR T-cell therapy in a preclinical mouse model of ovarian cancer. Second-generation CAR T cells were developed targeting mesothelin (MSLN), which is abundantly expressed in ovarian cancer. Cytotoxicity experiments were performed to verify the lethality of CAR T cells on target cells via flow cytometry. The in vivo antitumour activity of MSLN CAR T cells was also verified using a patient-derived xenograft (PDX) mouse model with human tumour-derived cells. We also evaluated the potency of CAR T cells directed to MSLN following co-expression of a dominant-negative transforming growth factor-ß receptor type II (dnTGFßRII). Our data demonstrate that anti-MSLN CAR T cells specifically eliminate MSLN-expressing target cells in an MSLN density-dependent manner. This preclinical research promises an effective treatment strategy to improve outcomes for ovarian cancer, with the potential for prolonging survival while minimizing risk of on-target off-tumour toxicity.

m7G-quant-seq: Quantitative Detection of RNA Internal N7-Methylguanosine.

Methods for the precise detection and quantification of RNA modifications are critical to uncover functional roles of diverse RNA modifications. The internal m7G modification in mammalian cytoplasmic tRNAs is known to affect tRNA function and impact embryonic stem cell self-renewal, tumorigenesis, cancer progression, and other cellular processes. Here, we introduce m7G-quant-seq, a quantitative method that accurately detects internal m7G sites in human cytoplasmic tRNAs at single-base resolution. The efficient chemical reduction and mild depurination can almost completely convert internal m7G sites into RNA abasic sites (AP sites). We demonstrate that RNA abasic sites induce a mixed variation pattern during reverse transcription, including G → A or C or T mutations as well as deletions. We calculated the total variation ratio to quantify the m7G modification fraction at each methylated site. The calibration curves of all relevant motif contexts allow us to more quantitatively determine the m7G methylation level. We detected internal m7G sites in 22 human cytoplasmic tRNAs from HeLa and HEK293T cells and successfully estimated the corresponding m7G methylation stoichiometry. m7G-quant-seq could be applied to monitor the tRNA m7G methylation level change in diverse biological processes.