Targeted pseudouridylation: An approach for suppressing nonsense mutations in disease genes.

Nonsense mutations create premature termination codons (PTCs), activating the nonsense-mediated mRNA decay (NMD) pathway to degrade most PTC-containing mRNAs. The undegraded mRNA is translated, but translation terminates at the PTC, leading to no production of the full-length protein. This work presents targeted PTC pseudouridylation, an approach for nonsense suppression in human cells. Specifically, an artificial box H/ACA guide RNA designed to target the mRNA PTC can suppress both NMD and premature translation termination in various sequence contexts. Targeted pseudouridylation exhibits a level of suppression comparable with that of aminoglycoside antibiotic treatments. When targeted pseudouridylation is combined with antibiotic treatment, a much higher level of suppression is observed. Transfection of a disease model cell line (carrying a chromosomal PTC) with a designer guide RNA gene targeting the PTC also leads to nonsense suppression. Thus, targeted pseudouridylation is an RNA-directed gene-specific approach that suppresses NMD and concurrently promotes PTC readthrough.

Pericyte signaling via soluble guanylate cyclase shapes the vascular niche and microenvironment of tumors.

Pericytes and endothelial cells (ECs) constitute the fundamental components of blood vessels. While the role of ECs in tumor angiogenesis and the tumor microenvironment is well appreciated, pericyte function in tumors remains underexplored. In this study, we used pericyte-specific deletion of the nitric oxide (NO) receptor, soluble guanylate cyclase (sGC), to investigate via single-cell RNA sequencing how pericytes influence the vascular niche and the tumor microenvironment. Our findings demonstrate that pericyte sGC deletion disrupts EC-pericyte interactions, impairing Notch-mediated intercellular communication and triggering extensive transcriptomic reprogramming in both pericytes and ECs. These changes further extended their influence to neighboring cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) through paracrine signaling, collectively suppressing tumor growth. Inhibition of pericyte sGC has minimal impact on quiescent vessels but significantly increases the vulnerability of angiogenic tumor vessels to conventional anti-angiogenic therapy. In conclusion, our findings elucidate the role of pericytes in shaping the tumor vascular niche and tumor microenvironment and support pericyte sGC targeting as a promising strategy for improving anti-angiogenic therapy for cancer treatment.

A UHM-ULM interface with unusual structural features contributes to U2AF2 and SF3B1 association for pre-mRNA splicing.

During spliceosome assembly, the 3' splice site is recognized by sequential U2AF2 complexes, first with Splicing Factor 1 (SF1) and second by the SF3B1 subunit of the U2 small nuclear ribonuclear protein particle. The U2AF2-SF1 interface is well characterized, comprising a U2AF homology motif (UHM) of U2AF2 bound to a U2AF ligand motif (ULM) of SF1. However, the structure of the U2AF2-SF3B1 interface and its importance for pre-mRNA splicing are unknown. To address this knowledge gap, we determined the crystal structure of the U2AF2 UHM bound to a SF3B1 ULM site at 1.8-Å resolution. We discovered a distinctive trajectory of the SF3B1 ULM across the U2AF2 UHM surface, which differs from prior UHM/ULM structures and is expected to modulate the orientations of the full-length proteins. We established that the binding affinity of the U2AF2 UHM for the cocrystallized SF3B1 ULM rivals that of a nearly full-length U2AF2 protein for an N-terminal SF3B1 region. An additional SF3B6 subunit had no detectable effect on the U2AF2-SF3B1 binding affinities. We further showed that key residues at the U2AF2 UHM-SF3B1 ULM interface contribute to coimmunoprecipitation of the splicing factors. Moreover, disrupting the U2AF2-SF3B1 interface changed splicing of representative human transcripts. From analysis of genome-wide data, we found that many of the splice sites coregulated by U2AF2 and SF3B1 differ from those coregulated by U2AF2 and SF1. Taken together, these findings support distinct structural and functional roles for the U2AF2-SF1 and U2AF2-SF3B1 complexes during the pre-mRNA splicing process.

Single-domain antibodies as promising experimental tools in imaging and isolation of porcine epidemic diarrhea virus.

Single-domain antibody (sdAb) or nanobody possesses specific features non-accessible for conventional antibodies that make them suitable for research and biotechnological applications. Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in piglets, resulting in great economic losses all over the world. To detect and isolate PEDV rapidly and accurately is important for the control and further research of the clinical PEDV strains. In this study, four sdAb fragments (sdAb-Mc19/29/30/37) targeting the membrane (M) protein of PEDV were selected from sdAb library that was constructed through M protein-immunized Camelus bactrianus. The selected sdAb-Mcs were solubly expressed in Escherichia coli. The functional characteristics analysis revealed that the recombinant sdAb-Mcs have excellent binding activity and specificity to M protein but have no neutralizing activity to PEDV. For further application, sdAb-Mc37 was conjugated with quantum dots to synthesize a nanoprobe for imaging PEDV in vero cells. The observed fluorescence in vero cells clearly reflects that PEDV virions can be reliably recognized and labeled by the nanoprobe. Furthermore, the sdAb-Mc29 was conjugated with superparamagnetic nanobeads to construct immunomagnetic nanobeads (IMNBs) used to isolate PEDV. One PEDV strain was successfully isolated from clinical fecal sample, suggesting IMNBs as a novel and efficient tool suitable for PEDV isolation from clinical samples. This study provided a novel application and substantiated the suitability of sdAb as a specific binder for the isolation of viruses.

Updated Pseudo-seq Protocol for Transcriptome-Wide Detection of Pseudouridines.

Pseudouridine (Ψ), the most prevalent modified base in cellular RNAs, has been mapped to numerous sites not only in rRNAs, tRNAs, and snRNAs but also mRNAs. Although there have been multiple techniques to identify Ψs, due to the recent development of sequencing technologies some reagents are not compatible with the current sequencer. Here, we show the updated Pseudo-seq, a technique enabling the genome-wide identification of pseudouridylation sites with single-nucleotide precision. We provide a comprehensive description of Pseudo-seq, covering protocols for RNA isolation from human cells, library preparation, and detailed data analysis procedures. The methodology presented is easily adaptable to any cell or tissue type with high-quality mRNA isolation. It can be used for discovering novel pseudouridylation sites, thus constituting a crucial initial step toward understanding the regulation and function of this modification. Key features • Identification of Ψ sites on mRNAs. • Updated Pseudo-seq provides precise positional and quantitative information of Ψ. • Uses a more efficient library preparation with the latest, currently available materials.

Risk factors and a prediction model for unruptured intracranial aneurysms in patients with ischemic stroke using carotid intima-media thickness and systemic atherosclerosis.

Background: Systemic atherosclerosis and carotid intima-media thickness (IMT) have been widely used in clinical practice for ischemic stroke; however, little is known about the risk factors for unruptured intracranial aneurysms (UIAs) in patients with ischemic stroke (IS). Therefore, we performed this study to identify the risk factors and construct a prediction model for UIA in patients with IS. Methods: Data were retrospectively collected from patients with IS from 2015 to 2022 at the First Hospital of Quanzhou City, Quanzhou, Fujian, China. Risk factors for UIA in patients with IS were identified using a multivariate logistic regression model, and a receiver operating characteristic (ROC) curve was applied to construct the prediction model. Results: Out of the 122 patients with IS, 52 who presented with UIA (ISUIA) were categorized into the study group and the remaining 70 IS patients without UIA into the control group. Patients in the ISUIA group had lower carotid IMT and carotid artery plaque scores than those in the IS group (P < 0.05). Multivariate analyses found that aspirin use (OR: 12.987; P = 0.031), elevated C-reactive protein (CRP) level (OR: 1.019; P = 0.004), and carotid IMT > 0.09 mm (OR: 0.218; P < 0.001) were significantly associated with the risk of UIA in patients with IS. However, UIA in patients with IS was unaffected by the carotid artery plaque score (P = 0.114). The constricted prediction model based on the abovementioned factors for UIA in IS patients was 0.79 (95% CI: 0.71-0.87). Conclusion: The findings revealed that the risk factors for UIA in patients with IS included aspirin use, elevated CRP level, and smaller carotid IMT, and the predictive value of the prediction model was relatively better.

Modeling human ectopic pregnancies with trophoblast and vascular organoids.

Ruptured ectopic pregnancy (REP), a pregnancy complication caused by aberrant implantation, deep invasion, and overgrowth of embryos in fallopian tubes, could lead to rupture of fallopian tubes and accounts for 4%-10% of pregnancy-related deaths. The lack of ectopic pregnancy phenotypes in rodents hampers our understanding of its pathological mechanisms. Here, we employed cell culture and organoid models to investigate the crosstalk between human trophoblast development and intravillous vascularization in the REP condition. Compared with abortive ectopic pregnancy (AEP), the size of REP placental villi and the depth of trophoblast invasion are correlated with the extent of intravillous vascularization. We identified a key pro-angiogenic factor secreted by trophoblasts, WNT2B, that promotes villous vasculogenesis, angiogenesis, and vascular network expansion in the REP condition. Our results reveal the important role of WNT-mediated angiogenesis and an organoid co-culture model for investigating intricate communications between trophoblasts and endothelial/endothelial progenitor cells.

Activating NO-sGC crosstalk in the mouse vascular niche promotes vascular integrity and mitigates acute lung injury.

Disruption of endothelial cell (ECs) and pericytes interactions results in vascular leakage in acute lung injury (ALI). However, molecular signals mediating EC-pericyte crosstalk have not been systemically investigated, and whether targeting such crosstalk could be adopted to combat ALI remains elusive. Using comparative genome-wide EC-pericyte crosstalk analysis of healthy and LPS-challenged lungs, we discovered that crosstalk between endothelial nitric oxide and pericyte soluble guanylate cyclase (NO-sGC) is impaired in ALI. Indeed, stimulating the NO-sGC pathway promotes vascular integrity and reduces lung edema and inflammation-induced lung injury, while pericyte-specific sGC knockout abolishes this protective effect. Mechanistically, sGC activation suppresses cytoskeleton rearrangement in pericytes through inhibiting VASP-dependent F-actin formation and MRTFA/SRF-dependent de novo synthesis of genes associated with cytoskeleton rearrangement, thereby leading to the stabilization of EC-pericyte interactions. Collectively, our data demonstrate that impaired NO-sGC crosstalk in the vascular niche results in elevated vascular permeability, and pharmacological activation of this crosstalk represents a promising translational therapy for ALI.

Transgenic nude mouse with green fluorescent protein expression-based human glioblastoma multiforme animal model with EGFR expression and invasiveness.

Previously, we developed an orthotopic xenograft model of human glioblastoma multiforme (GBM) with high EGFR expression and invasiveness in Balb/c nu/nu nude mice. Now we also developed the same orthotopic xenograft model in transgenic nude mice with green fluorescent protein (GFP) expression. The present orthotopic xenografts labeled by phycoerythrin fluorescing red showed high EGFR expression profile, and invasive behavior under a bright green-red dual-color fluorescence background. A striking advantage in the present human GBM model is that the change of tumor growth can be observed visually instead of sacrificing animals in our further antitumor therapy studies.

Mucosal-Associated Invariant T Cells Expressing the TRAV1-TRAJ33 Chain Are Present in Pigs.

Mucosal-associated invariant T (MAIT) cells are a subpopulation of evolutionarily conserved innate-like T lymphocytes bearing invariant or semi-invariant TCRα chains paired with a biased usage of TCRß chains and restricted by highly conserved monomorphic MHC class I-like molecule, MR1. Consistent with their phylogenetically conserved characteristics, MAIT cells have been implicated in host immune responses to microbial infections and non-infectious diseases, such as tuberculosis, typhoid fever, and multiple sclerosis. To date, MAIT cells have been identified in humans, mice, cows, sheep, and several non-human primates, but not in pigs. Here, we cloned porcine MAIT (pMAIT) TCRα sequences from PBMC cDNA, and then analyzed the TCRß usage of pMAIT cells expressing the TRAV1-TRAJ33 chain, finding that pMAIT cells use a limited array of TCRß chains (predominantly TRBV20S and TRBV29S). We estimated the frequency of TRAV1-TRAJ33 transcripts in peripheral blood and tissues, demonstrating that TRAV1-TRAJ33 transcripts are expressed in all tested tissues. Analysis of the expression of TRAV1-TRAJ33 transcripts in three T-cell subpopulations from peripheral blood and tissues showed that TRAV1-TRAJ33 transcripts can be expressed by CD4+CD8-, CD8+CD4-, and CD4-CD8- T cells. Using a single-cell PCR assay, we demonstrated that pMAIT cells with the TRAV1-TRAJ33 chain express cell surface markers IL-18Rα, IL-7Rα, CCR9, CCR5, and/or CXCR6, and transcription factors PLZF, and T-bet and/or RORγt. In conclusion, pMAIT cells expressing the TRAV1-TRAJ33 chain have characteristics similar to human and mouse MAIT cells, further supporting the idea that the pig is an animal model for investigating MAIT cell functions in human disease.

Treatment of Blood Blister-like Aneurysms with Stent-Assisted Coiling: A Retrospective Multicenter Study.

OBJECTIVE: To clarify safety and efficacy of stent-assisted coiling for treatment of blood blister-like aneurysm (BBA) in a multicenter experience. METHODS: A total of 212 consecutive cases (213 BBAs) treated with stent-assisted coiling were retrospectively reviewed and included in the final analysis. Outcomes including complete occlusion, recurrence, perioperative morbidity and mortality, and overall good neurologic outcome during follow-up were analyzed. RESULTS: Of 212 patients, 102 (48.1%) were treated with a single stent, 77 (36.3%) were treated with 2 stents, and 33 (15.6%) were treated with ≥3 stents. Angiographic follow-up data were available for 96 BBAs; 64.6% of BBAs showed complete obliteration, and 22.9% showed recurrence. Complete obliteration rates of patients treated with single, 2, and ≥3 stents were 42.9%, 78.4%, and 88.2%. Recurrence rates of patients treated with single, 2, and ≥3 stents were 38.1%, 13.5%, and 5.9%. Using ≥2 stents seemed to result in higher obliteration rates (P < 0.001) and lower recurrence rates (P = 0.002). Clinical follow-up data available for 180 patients showed 91.1% of patients had a good clinical outcome. No difference was found between the 3 stent treatments. There were 14 (6.6%) perioperative ischemic complications and 12 (5.7%) hemorrhagic complications, with 10 (4.7%) cases of perioperative deaths. Using ≥2 stents seemed to result in a lower perioperative hemorrhagic complication rate compared with single stent (P = 0.037). CONCLUSIONS: Our data support the use of overlapping (≥2) stents combined with coiling as a safe and effective therapeutic modality for BBA. Overlapping (≥2) stents may provide higher obliteration rate, lower recurrence rate, and lower perioperative hemorrhagic risk in treatment of BBA.