Engineering a precise adenine base editor with minimal bystander editing.

Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise safety concerns. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1-2 nucleotides with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited the A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision over ABE8e) and was further confirmed through a library of guide RNA-target sequence pairs. Owing to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic single-nucleotide variants when fused to Cas9 variants with expanded protospacer adjacent motif compatibility. bpNLS, bipartite nuclear localization signals.

Identification of Cell Types and Transcriptome Landscapes of Porcine Epidemic Diarrhea Virus-Infected Porcine Small Intestine Using Single-Cell RNA Sequencing.

Swine coronavirus-porcine epidemic diarrhea virus (PEDV) with specific susceptibility to pigs has existed for decades, and recurrent epidemics caused by mutant strains have swept the world again since 2010. In this study, single-cell RNA sequencing was used to perform for the first time, to our knowledge, a systematic analysis of pig jejunum infected with PEDV. Pig intestinal cell types were identified by representative markers and identified a new tuft cell marker, DNAH11. Excepting enterocyte cells, the goblet and tuft cells confirmed susceptibility to PEDV. Enrichment analyses showed that PEDV infection resulted in upregulation of cell apoptosis, junctions, and the MAPK signaling pathway and downregulation of oxidative phosphorylation in intestinal epithelial cell types. The T cell differentiation and IgA production were decreased in T and B cells, respectively. Cytokine gene analyses revealed that PEDV infection downregulated CXCL8, CXCL16, and IL34 in tuft cells and upregulated IL22 in Th17 cells. Further studies found that infection of goblet cells with PEDV decreased the expression of MUC2, as well as other mucin components. Moreover, the antimicrobial peptide REG3G was obviously upregulated through the IL33-STAT3 signaling pathway in enterocyte cells in the PEDV-infected group, and REG3G inhibited the PEDV replication. Finally, enterocyte cells expressed almost all coronavirus entry factors, and PEDV infection caused significant upregulation of the coronavirus receptor ACE2 in enterocyte cells. In summary, this study systematically investigated the responses of different cell types in the jejunum of piglets after PEDV infection, which deepened the understanding of viral pathogenesis.

Structure of the human cation-chloride cotransport KCC1 in an outward-open state.

Cation-chloride cotransporters (CCCs) catalyze electroneutral symport of Cl- with Na+ and/or K+ across membranes. CCCs are fundamental in cell volume homeostasis, transepithelia ion movement, maintenance of intracellular Cl- concentration, and neuronal excitability. Here, we present a cryoelectron microscopy structure of human K+-Cl- cotransporter (KCC)1 bound with the VU0463271 inhibitor in an outward-open state. In contrast to many other amino acid-polyamine-organocation transporter cousins, our first outward-open CCC structure reveals that opening the KCC1 extracellular ion permeation path does not involve hinge-bending motions of the transmembrane (TM) 1 and TM6 half-helices. Instead, rocking of TM3 and TM8, together with displacements of TM4, TM9, and a conserved intracellular loop 1 helix, underlie alternate opening and closing of extracellular and cytoplasmic vestibules. We show that KCC1 intriguingly exists in one of two distinct dimeric states via different intersubunit interfaces. Our studies provide a blueprint for understanding the mechanisms of CCCs and their inhibition by small molecule compounds.

UHMK1 promotes gastric cancer progression through reprogramming nucleotide metabolism.

UHMK1 is a nuclear serine/threonine kinase recently implicated in carcinogenesis. However, the functions and action mechanisms of UHMK1 in the pathogenesis of human gastric cancer (GC) are unclear. Here, we observed that UHMK1 was markedly upregulated in GC. UHMK1 silencing strongly inhibited GC aggressiveness. Interestingly, UHMK1-induced GC progression was mediated primarily via enhancing de novo purine synthesis because inhibiting purine synthesis reversed the effects of UHMK1 overexpression. Mechanistically, UHMK1 activated ATF4, an important transcription factor in nucleotide synthesis, by phosphorylating NCOA3 at Ser (S) 1062 and Thr (T) 1067. This event significantly enhanced the binding of NCOA3 to ATF4 and the expression of purine metabolism-associated target genes. Conversely, deficient phosphorylation of NCOA3 at S1062/T1067 significantly abrogated the function of UHMK1 in GC development. Clinically, Helicobacter pylori and GC-associated UHMK1 mutation induced NCOA3-S1062/T1067 phosphorylation and enhanced the activity of ATF4 and UHMK1. Importantly, the level of UHMK1 was significantly correlated with the level of phospho-NCOA3 (S1062/T1067) in human GC specimens. Collectively, these results show that the UHMK1-activated de novo purine synthesis pathway significantly promotes GC development.

Genetic signatures associated with the virulence of porcine epidemic diarrhea virus AH2012/12.

IMPORTANCE: Porcine epidemic diarrhea (PED) caused by PED virus (PEDV) remains a big threat to the swine industry worldwide. Vaccination with live attenuated vaccine is a promising method to prevent and control PED, because it can elicit a more protective immunity than the killed vaccine, subunit vaccine, and so on. In this study, we found two obvious deletions in the genome of a high passage of AH2012/12. We further confirmed the second deletion which contains seven amino acids at the carboxy-terminus of the S2 gene and the start codon of ORF3 can reduce its pathogenicity in vivo. Animal experiments indicated that the recombinant PEDV with deleted carboxy-terminus of S gene showed higher IgG, IgA, neutralization antibodies, and protection effects against virus challenge than the killed vaccine. These data reveal that the engineering of the carboxy-terminus of the S2 gene may be a promising method to develop live attenuated vaccine candidates of PEDV.

Polymer Microspheres and Their Application in Cancer Diagnosis and Treatment.

Cancer is a significant global public health issue with increasing morbidity and mortality rates. To address this challenge, novel drug carriers such as nano-materials, liposomes, hydrogels, fibers, and microspheres have been extensively researched and utilized in oncology. Among them, polymer microspheres are gaining popularity due to their ease of preparation, excellent performance, biocompatibility, and drug-release capabilities. This paper categorizes commonly used materials for polymer microsphere preparation, summarizes various preparation methods (emulsification, phase separation, spray drying, electrospray, microfluidics, and membrane emulsification), and reviews the applications of polymer microspheres in cancer diagnosis, therapy, and postoperative care. The current status and future development directions of polymer microspheres in cancer treatment are analyzed, highlighting their importance and potential for improving patient outcomes.

Nonstructural Protein 1 of Variant PEDV Plays a Key Role in Escaping Replication Restriction by Complement C3.

Zoonotic coronaviruses represent an ongoing threat to public health. The classical porcine epidemic diarrhea virus (PEDV) first appeared in the early 1970s. Since 2010, outbreaks of highly virulent PEDV variants have caused great economic losses to the swine industry worldwide. However, the strategies by which PEDV variants escape host immune responses are not fully understood. Complement component 3 (C3) is considered a central component of the three complement activation pathways and plays a crucial role in preventing viral infection. In this study, we found that C3 significantly inhibited PEDV replication in vitro, and both variant and classical PEDV strains induced high levels of interleukin-1ß (IL-1ß) in Huh7 cells. However, the PEDV variant strain reduces C3 transcript and protein levels induced by IL-1ß compared with the PEDV classical strain. Examination of key molecules of the C3 transcriptional signaling pathway revealed that variant PEDV reduced C3 by inhibiting CCAAT/enhancer-binding protein ß (C/EBP-ß) phosphorylation. Mechanistically, PEDV nonstructural protein 1 (NSP1) inhibited C/EBP-ß phosphorylation via amino acid residue 50. Finally, we constructed recombinant PEDVs to verify the critical role of amino acid 50 of NSP1 in the regulation of C3 expression. In summary, we identified a novel antiviral role of C3 in inhibiting PEDV replication and the viral immune evasion strategies of PEDV variants. Our study reveals new information on PEDV-host interactions and furthers our understanding of the pathogenic mechanism of this virus. IMPORTANCE The complement system acts as a vital link between the innate and the adaptive immunity and has the ability to recognize and neutralize various pathogens. Activation of the complement system acts as a double-edged sword, as appropriate levels of activation protect against pathogenic infections, but excessive responses can provoke a dramatic inflammatory response and cause tissue damage, leading to pathological processes, which often appear in COVID-19 patients. However, how PEDV, as the most severe coronavirus causing diarrhea in piglets, regulates the complement system has not been previously reported. In this study, for the first time, we identified a novel mechanism of a PEDV variant in the suppression of C3 expression, showing that different coronaviruses and even different subtype strains differ in regulation of C3 expression. In addition, this study provides a deeper understanding of the mechanism of the PEDV variant in immune escape and enhanced virulence.

High C-reactive protein to lymphocyte ratio predicts mortality outcomes of patients with severe fever with thrombocytopenia syndrome: A multicenter study in China.

Severe fever with thrombocytopenia syndrome (SFTS) is a life-threatening infectious disease caused by the SFTS virus (SFTSV). This study aimed to evaluate the predictive power of C-reactive protein to lymphocyte ratio (CLR) and establish an early-warning model for SFTS mortality. We retrospectively analyzed hospitalized SFTS patients in six clinical centers from May 2011 to 2022. The efficacy of CLR prediction was evaluated by the receiver operating characteristic (ROC) analysis. A nomogram was established and validated. Eight hundred and eighty-two SFTS patients (median age 64 years, 48.5% male) were enrolled in this study, with a mortality rate of 17.8%. The area under the ROC curve (AUC) of CLR was 0.878 (95% confidence interval [CI]: 0.850-0.903, p < 0.001), which demonstrates high predictive strength. The least absolute shrinkage and selection operator regression selected seven potential predictors. Multivariate logistic regression analysis determined three independent risk factors, including CLR, to construct the nomogram. The performance of the nomogram displayed excellent discrimination and calibration, with significant net benefits in clinical uses. CLR is a brand-new predictor for SFTS mortality. The nomogram based on CLR can serve as a convenient tool for physicians to identify critical SFTS cases in clinical practice.

Procapra Przewalskii Tracking Autonomous Unmanned Aerial Vehicle Based on Improved Long and Short-Term Memory Kalman Filters.

This paper presents an autonomous unmanned-aerial-vehicle (UAV) tracking system based on an improved long and short-term memory (LSTM) Kalman filter (KF) model. The system can estimate the three-dimensional (3D) attitude and precisely track the target object without manual intervention. Specifically, the YOLOX algorithm is employed to track and recognize the target object, which is then combined with the improved KF model for precise tracking and recognition. In the LSTM-KF model, three different LSTM networks (f, Q, and R) are adopted to model a nonlinear transfer function to enable the model to learn rich and dynamic Kalman components from the data. The experimental results disclose that the improved LSTM-KF model exhibits higher recognition accuracy than the standard LSTM and the independent KF model. It verifies the robustness, effectiveness, and reliability of the autonomous UAV tracking system based on the improved LSTM-KF model in object recognition and tracking and 3D attitude estimation.

Molecular Mechanisms of Mercury-Sensitive Aquaporins.

Aquaporins are transmembrane channels that allow for the passive permeation of water and other small molecules across biological membranes. Their channel activities are sensitive to mercury ions. Intriguingly, while most aquaporins are inhibited by mercury ions, several aquaporins are activated by mercury ions. The molecular basis of the opposing aquaporin regulation by mercury remains poorly understood. Herein, we investigated AqpZ inhibition and AQP6 activation upon binding of mercury ions using solid-state NMR (ssNMR) and molecular dynamics (MD) simulations. Based on the structure of the Hg-AqpZ complex constructed by MD simulations and ssNMR, we identified that the pore closure was caused by mercury-induced conformational changes of the key residue R189 in the selectivity filter region, while pore opening was caused by conformational changes of residues H181 and R196 in the selectivity filter region in AQP6. Both conformational changes were caused by the disruption of the H-bond network of R189/R196 by mercury. The molecular details provided a structural basis for mercury-mediated functional changes in aquaporins.

Annotating unknown species of urban microorganisms on a global scale unveils novel functional diversity and local environment association.

In urban ecosystems, microbes play a key role in maintaining major ecological functions that directly support human health and city life. However, the knowledge about the species composition and functions involved in urban environments is still limited, which is largely due to the lack of reference genomes in metagenomic studies comprises more than half of unclassified reads. Here we uncovered 732 novel bacterial species from 4728 samples collected from various common surface with the matching materials in the mass transit system across 60 cities by the MetaSUB Consortium. The number of novel species is significantly and positively correlated with the city population, and more novel species can be identified in the skin-associated samples. The in-depth analysis of the new gene catalog showed that the functional terms have a significant geographical distinguishability. Moreover, we revealed that more biosynthetic gene clusters (BGCs) can be found in novel species. The co-occurrence relationship between BGCs and genera and the geographical specificity of BGCs can also provide us more information for the synthesis pathways of natural products. Expanded the known urban microbiome diversity and suggested additional mechanisms for taxonomic and functional characterization of the urban microbiome. Considering the great impact of urban microbiomes on human life, our study can also facilitate the microbial interaction analysis between human and urban environment.

A comprehensive rat transcriptome built from large scale RNA-seq-based annotation.

The rat is an important model organism in biomedical research for studying human disease mechanisms and treatments, but its annotated transcriptome is far from complete. We constructed a Rat Transcriptome Re-annotation named RTR using RNA-seq data from 320 samples in 11 different organs generated by the SEQC consortium. Totally, there are 52 807 genes and 114 152 transcripts in RTR. Transcribed regions and exons in RTR account for ∼42% and ∼6.5% of the genome, respectively. Of all 73 074 newly annotated transcripts in RTR, 34 213 were annotated as high confident coding transcripts and 24 728 as high confident long noncoding transcripts. Different tissues rather than different stages have a significant influence on the expression patterns of transcripts. We also found that 11 715 genes and 15 852 transcripts were expressed in all 11 tissues and that 849 house-keeping genes expressed different isoforms among tissues. This comprehensive transcriptome is freely available at http://www.unimd.org/rtr/. Our new rat transcriptome provides essential reference for genetics and gene expression studies in rat disease and toxicity models.

A case report of renal calyceal diverticulum with hypertension in children and review of literature.

BACKGROUND: Renal calyx diverticulum refers to a cystic lesion covered with the transitional epithelium in the renal parenchyma. Although there is no clear evidence that calyx diverticulum can cause hypertension, there exists a close association between the two, and there are few related reports. Herein, we reported the case of a child with renal calyx diverticulum complicated with hypertension and summarized the diagnosis and treatment. CASE PRESENTATION: Physical examination of the patient, an 11-year-old child, revealed a left renal cyst with hypertension (155/116 mmHg). There were no related symptoms. Routine urine and blood biochemical examinations showed no abnormalities. Imaging revealed left renal cyst compression causing the hypertension. She underwent renal cyst fluid aspiration and injection of a sclerosing agent into the capsule, but her blood pressure increased again 3 days postoperatively. Color Doppler ultrasonography showed that the size of the left renal cyst was the same as that preoperatively. To further confirm the diagnosis, cystoscopic retrograde ureteropyelography was performed to confirm the diagnosis of renal calyx diverticulum. Subsequently, renal calyceal diverticulum resection and calyx neck enlargement were performed. The operation went smoothly and the blood pressure returned to normal postoperatively. No abnormalities were noted at the 7-month postoperative follow-up. CONCLUSION: There exists an association between renal calyx diverticulum and hypertension. Therefore, hypertension can be considered a surgical indication for renal calyx diverticulum. Moreover, renal calyceal diverticulum in children can be easily misdiagnosed as a renal cyst. Therefore, it is important to be vigilant to prevent a series of complications, such as postoperative urine leakage, in such cases.

Core Role of Hydrophobic Core of Polymeric Nanomicelle in Endosomal Escape of siRNA.

Efficient endosomal escape is the most essential but challenging issue for siRNA drug development. Herein, a series of quaternary ammonium-based amphiphilic triblock polymers harnessing an elaborately tailored pH-sensitive hydrophobic core were synthesized and screened. Upon incubating in an endosomal pH environment (pH 6.5-6.8), mPEG45-P(DPA50-co-DMAEMA56)-PT53 (PDDT, the optimized polymer) nanomicelles (PDDT-Ms) and PDDT-Ms/siRNA polyplexes rapidly disassembled, leading to promoted cytosolic release of internalized siRNA and enhanced silencing activity evident from comprehensive analysis of the colocalization and gene silencing using a lysosomotropic agent (chloroquine) and an endosomal trafficking inhibitor (bafilomycin A1). In addition, PDDT-Ms/siPLK1 dramatically repressed tumor growth in both HepG2-xenograft and highly malignant patient-derived xenograft models. PDDT-Ms-armed siPD-L1 efficiently blocked the interaction of PD-L1 and PD-1 and restored immunological surveillance in CT-26-xenograft murine model. PDDT-Ms/siRNA exhibited ideal safety profiles in these assays. This study provides guidelines for rational design and optimization of block polymers for efficient endosomal escape of internalized siRNA and cancer therapy.

Spectral editing of alanine, serine, and threonine in uniformly labeled proteins based on frequency-selective homonuclear recoupling in solid-state NMR.

Spectral editing is crucial to simplify the crowded solid-state NMR spectra of proteins. New techniques are introduced to edit 13C-13C correlations of uniformly labeled proteins under moderate magic-angle spinning (MAS), based on our recent frequency-selective homonuclear recoupling sequences [Zhang et al., J. Phys. Chem. Lett. 2020, 11, 8077-8083]. The signals of alanine, serine, or threonine residues are selected out by selective 13Cα-13Cß double-quantum filtering (DQF). The 13Cα-13Cß correlations of alanine residues are selectively established with efficiency up to ~ 1.8 times that by dipolar-assisted rotational resonance (DARR). The techniques are shown in 2D/3D NCCX experiments and applied to the uniformly 13C, 15N labeled Aquaporin Z (AqpZ) membrane protein, demonstrating their potential to simplify spectral analyses in biological solid-state NMR.

Extensively sparse 13C labeling to simplify solid-state NMR 13C spectra of membrane proteins.

Solid-state Nuclear Magnetic Resonance (ssNMR) is an emerging technique to investigate the structures and dynamics of membrane proteins in an artificial or native membrane environment. However, the structural studies of proteins by ssNMR are usually prolonged or impeded by signal assignments, especially the assignments of signals for collection of distance restraints, because of serious overlapping of signals in 2D 13C-13C spectra. Sparse labeling of 13C spins is an effective approach to simplify the 13C spectra and facilitate the extractions of distance restraints. Here, we propose a new reverse labeling combination of six types of amino acid residues (Ile, Leu, Phe, Trp, Tyr and Lys), and show a clean reverse labeling effect on a model membrane protein E. coli aquaporin Z (AqpZ). We further combine this reverse labeling combination and alternate 13C-12C labeling, and demonstrate an enhanced dilution effect in 13C-13C spectra. In addition, the influences of reverse labeling on the labeling of the other types of residues are quantitatively analyzed in the two strategies (1, reverse labeling and 2, reverse labeling combining alternate 13C-12C labeling). The signal intensities of some other types of residues in 2D 13C-13C spectra are observed to be 20-50% weaker because of the unwanted reverse labeling. The extensively sparse 13C labeling proposed in this study is expected to be useful in the collection of distance restraints using 2D 13C-13C spectra of membrane proteins.

Antigen-Presenting Hybrid Colloidal Crystal Clusters for Promoting T cells Expansion.

T cell based-immunotherapy has been a powerful strategy to eradicate tumor cells in clinical trials. Effectively expanding the therapeutic T cells for clinical demand is still a challenge. Here, artificial antigen-presenting scaffolds are created for T cell ex vivo expansion. The antigen-presenting hybrid colloidal crystal clusters (HCCCs) with multiple stimuli are generated by internal encapsulation with prosurvival cytokines and surface decoration with activating antibodies to CD3ε and CD28, respectively. With the large loading capacity endowed by their abundant nanoporous structures, the antigen-presenting HCCCs can constantly release prosurvival cytokine IL-2. It is found that following the direct and multiple stimulations, the antigen-presenting HCCCs can effectively promote the expansion of T cells, which exhibits robust antitumor activity in vitro. Thus, the antigen-presenting HCCCs provide a novel expansion platform for clinical manufacturing of T cells.

Current strategies of virotherapy in clinical trials for cancer treatment.

As a novel immune-active agent for cancer treatment, viruses have the ability of infecting and replicating in tumor cells. The safety and efficacy of viruses has been tested and confirmed in preclinical and clinical trials. In the last decade, virotherapy has been adopted as a monotherapy or combined therapy with immunotherapy, chemotherapy, or radiotherapy, showing promising outcomes against cancer. In this review, the current strategies of viruses used in clinical trials are classified and described. Besides this, the challenge and future prospects of virotherapy in the management for cancer patients are discussed in this review.

Development and application of reverse genetic technology for the influenza virus.

Influenza virus is a common virus in people's daily lives, and it has certain infectivity in humans and animals. Influenza viruses have the characteristics of a high mutation rate and wide distribution. Reverse genetic technology is primarily used to modify viruses at the DNA level through targeted modification of the virus cDNA. Genetically modified influenza viruses have a unique advantage when researching the transmission and pathogenicity of influenza. With the continuous development of oncolytic viruses in recent years, studies have found that influenza viruses also have certain oncolytic activity. Influenza viruses can specifically recognize tumor cells; activate cytotoxic T cells, NK cells, dendritic cells, etc.; and stimulate the body to produce an immune response, thereby killing tumor cells. This article will review the development and application of influenza virus reverse genetic technology.