Ssc-miR-101-3p inhibits hypoxia-induced apoptosis and inflammatory response in alveolar type-II epithelial cells of Tibetan pigs via targeting FOXO3.

Tibetan pigs are a unique swine strain adapted to the hypoxic environment of the plateau regions in China. The unique mechanisms underlying the adaption by Tibetan pigs, however, are still elusive. Only few studies have investigated hypoxia-associated molecular regulation in the lung tissues of animals living in the plateau region of China. Our previous study reported that ssc-miR-101-3p expression significantly differed in the lung tissues of Tibetan pigs at different altitudes, suggesting that ssc-miR-101-3p plays an important role in the adaptation of Tibetan pigs to high altitude. To understand the underlying molecular mechanism, in this study, the target genes of ssc-miR-101-3p and their functions were analyzed via various methods including qRT-PCR and GO and KEGG pathway enrichment analyses. The action of ssc-miR-101-3p was investigated by culturing alveolar type-II epithelial cells (ATII) of Tibetan pigs under hypoxic conditions and transfecting ATII cells with vectors overexpressing or inhibiting ssc-miR-101-3p. Overexpression of ssc-miR-101-3p significantly increased the proliferation of ATII cells and decreased the expression of inflammatory and apoptotic factors. The target genes of ssc-miR-101-3p were significantly enriched in FOXO and PI3K-AKT signaling pathways required to mitigate lung injury. Further, FOXO3 was identified as a direct target of ssc-miR-101-3p. Interestingly, ssc-miR-101-3p overexpression reversed the damaging effect of FOXO3 in the ATII cells. In conclusion, ssc-miR-101-3p targeting FOXO3 could inhibit hypoxia-induced apoptosis and inflammatory response in ATII cells of Tibetan pigs. These results provided new insights into the molecular mechanisms elucidating the response of lung tissues of Tibetan pigs to hypoxic stress.

Tumor cell-intrinsic Piezo2 drives radioresistance by impairing CD8+ T cell stemness maintenance.

Changes in mechanosensitive ion channels following radiation have seldom been linked to therapeutic sensitivity or specific factors involved in antitumor immunity. Here, in this study, we found that the mechanical force sensor, Piezo2, was significantly upregulated in tumor cells after radiation, and Piezo2 knockout in tumor cells enhanced tumor growth suppression by radiotherapy. Specifically, loss of Piezo2 in tumor cells induced their IL-15 expression via unleashing JAK2/STAT1/IRF-1 axis after radiation. This increase in IL-15 activates IL-15Rα on tumor-infiltrating CD8+ T cells, thereby leading to their augmented effector and stem cell-like properties, along with reduced terminal exhausted feature. Importantly, Piezo2 expression was negatively correlated with CD8 infiltration, as well as with radiosensitivity of patients with rectum adenocarcinoma receiving radiotherapy treatment. Together, our findings reveal that tumor cell-intrinsic Piezo2 induces radioresistance by dampening the IRF-1/IL-15 axis, thus leading to impaired CD8+ T cell-dependent antitumor responses, providing insights into the further development of combination strategies to treat radioresistant cancers.

Creating large-scale genetic diversity in Arabidopsis via base editing-mediated deep artificial evolution.

BACKGROUND: Base editing is a powerful tool for artificial evolution to create allelic diversity and improve agronomic traits. However, the great evolutionary potential for every sgRNA target has been overlooked. And there is currently no high-throughput method for generating and characterizing as many changes in a single target as possible based on large mutant pools to permit rapid gene directed evolution in plants. RESULTS: In this study, we establish an efficient germline-specific evolution system to screen beneficial alleles in Arabidopsis which could be applied for crop improvement. This system is based on a strong egg cell-specific cytosine base editor and the large seed production of Arabidopsis, which enables each T1 plant with unedited wild type alleles to produce thousands of independent T2 mutant lines. It has the ability of creating a wide range of mutant lines, including those containing atypical base substitutions, and as well providing a space- and labor-saving way to store and screen the resulting mutant libraries. Using this system, we efficiently generate herbicide-resistant EPSPS, ALS, and HPPD variants that could be used in crop breeding. CONCLUSIONS: Here, we demonstrate the significant potential of base editing-mediated artificial evolution for each sgRNA target and devised an efficient system for conducting deep evolution to harness this potential.

Toxicity Evaluation and Transcriptome Analysis of Yellowstripe Goby (Mugilogobius chulae) in Response to 2,7-Dibromocarbazole Exposure during Early Development.

Polyhalogenated carbazoles (PHCZs) are a class of nitrogen-containing heterocyclic compounds that are widely distributed throughout the marine environment and sediment. These compounds share structural and toxicity similarities with dioxins. However, our understanding of the toxicological effects of PHCZs on marine organisms and their underlying molecular mechanisms remains limited. In this study, we employed the marine model organism Mugilogobius chulae as the experimental subject and selected 2,7-dibromocarbazole (2,7-DBCZ), a compound known for its high toxicity and detection frequency, to conduct both an acute toxicity test and transcriptome analysis on M. chulae embryos. Our findings revealed that the 96 h median lethal concentration (LC50) of 2,7-DBCZ for M. chulae embryos was 174 µg/L, with a median effective concentration (EC50) resulting in pericardial edema deformity of 88.82 µg/L. Transcriptome analysis revealed significant impacts on various systems in M. chulae embryos following exposure to 2,7-DBCZ, including the sensory, cardiovascular, immune, and endocrine systems. Furthermore, this compound perturbed signaling pathways such as phototransduction, protein folding and processing, amino acid metabolism, lipid transport, and exogenous compound metabolism. Notably, transcript abundance of the CYP1A gene associated with the activation of the AhR signaling pathway, similar to dioxin-like compounds, was 18.18 times higher than that in the control group. This observation suggests that M. chulae embryos mount a stress response when exposed to PHCZs. In summary, this study contributes to our understanding of the toxicological implications of PHCZ in marine fish and offers a theoretical foundation for risk assessment and regulatory frameworks for PHCZs in the marine environment.

Renin and angiotensin (1-7) offer predictive value in pediatric sepsis: findings from prospective observational cohorts.

BACKGROUND: Pediatric sepsis is a common and complex syndrome characterized by a dysregulated immune response to infection. Aberrations in the renin-angiotensin system (RAS) are factors in several infections of adults. However, the precise impact of RAS dysregulation in pediatric sepsis remains unclear. METHODS: Serum samples were collected from a derivation cohort (58 patients with sepsis, 14 critically ill control subjects, and 37 healthy controls) and validation cohort (50 patients with sepsis, 37 critically ill control subjects, and 46 healthy controls). Serum RAS levels on day of PICU admission were determined and compared with survival status and organ dysfunction. RESULTS: In the derivation cohort, the serum renin concentration was significantly higher in patients with sepsis (3678 ± 4746) than that in healthy controls(635.6 ± 199.8) (p < 0.0001). Meanwhile, the serum angiotensin (1-7) was significantly lower in patients with sepsis (89.7 ± 59.7) compared to that in healthy controls(131.4 ± 66.4) (p < 0.01). These trends were confirmed in a validation cohort. Non-survivors had higher levels of renin (8207 ± 7903) compared to survivors (2433 ± 3193) (p = 0.0001) and lower levels of angiotensin (1-7) (60.9 ± 51.1) compared to survivors (104.0 ± 85.1) (p < 0.05). A combination of renin, angiotensin (1-7) and procalcitonin achieved a model for diagnosis with an area under the receiver operating curve (AUROC) of 0.87 (95% CI: 0.81-0.92). CONCLUSION: Circulating renin and angiotensin (1-7) have predictive value in pediatric sepsis.

ClusterMatch aligns single-cell RNA-sequencing data at the multi-scale cluster level via stable matching.

MOTIVATION: Unsupervised clustering of single-cell RNA sequencing (scRNA-seq) data holds the promise of characterizing known and novel cell type in various biological and clinical contexts. However, intrinsic multi-scale clustering resolutions poses challenges to deal with multiple sources of variability in the high-dimensional and noisy data. RESULTS: We present ClusterMatch, a stable match optimization model to align scRNA-seq data at the cluster level. In one hand, ClusterMatch leverages the mutual correspondence by canonical correlation analysis and multi-scale Louvain clustering algorithms to identify cluster with optimized resolutions. In the other hand, it utilizes stable matching framework to align scRNA-seq data in the latent space while maintaining interpretability with overlapped marker gene set. Through extensive experiments, we demonstrate the efficacy of ClusterMatch in data integration, cell type annotation, and cross-species/timepoint alignment scenarios. Our results show ClusterMatch's ability to utilize both global and local information of scRNA-seq data, sets the appropriate resolution of multi-scale clustering, and offers interpretability by utilizing marker genes. AVAILABILITY AND IMPLEMENTATION: The code of ClusterMatch software is freely available at https://github.com/AMSSwanglab/ClusterMatch.

Potential SLA Hp-4.0 haplotype-restricted CTL epitopes identified from the membrane protein of PRRSV induce cell immune responses.

Swine leukocyte antigen (SLA) class I molecule-restricted T-cell epitopes, which induce cytotoxic T lymphocyte (CTL) responses, play a critical role in the clearance of porcine reproductive and respiratory syndrome virus (PRRSV) and the development of efficient protective vaccines. The SLA-1*04:01:01, SLA-2*04:01, and SLA-3*04:01 alleles, assigned the Hp-4.0 haplotype, are highly prevalent and usually present in all pig breeds. However, the SLA Hp-4.0 haplotype-restricted CTL epitopes in the structural membrane (M) protein of PRRSV are still unknown. In this study, we predicted 27 possible 9-mer epitope peptides in M protein with high binding scores for SLA-1*04:01:01 using CTL epitope prediction tools. In total, 45 SLA class I complexes, comprising the predicted peptide, extracellular region of the SLA-I molecules, and ß2-microglobulin, were constructed in vitro to detect the specific binding of these peptides to SLA-1*04:01:01 (27 complexes), SLA-2*04:01 (9 complexes), and SLA-3*04:01 (9 complexes), respectively. Our results showed that the M27 (T91WKFITSRC), M39 (N130HAFVVRRP), and M49 (G158RKAVKQGV) peptides bind specifically to SLA-1*04:01:01, SLA-2*04:01, and SLA-3*04:01, respectively. Subsequently, using peripheral blood mononuclear cells (PBMCs) isolated from the homozygous Hp-4.0 and Hp-26.0 haplotype piglets vaccinated with commercial PRRSV HuN4-F112 strain, we determined the capacities of these 27 potential peptides to stimulate their proliferation with a Cell Counting Kit-8 and their secretion and expression of interferon gamma (IFN-γ) with an ELISpot assay and real-time qPCR, respectively. The immunological activities of M27, M39, and M49 were therefore confirmed when they efficiently induced PBMC proliferation and IFN-γ secretion in PBMCs from piglets with the prevalent SLA Hp-4.0 haplotype. The amino acid sequence alignment revealed that M27, M39, and M49 are highly conserved among 248 genotype II PRRSV strains collected between 1998 and 2019. These findings contribute to the understanding of the mechanisms of cell-mediated immune responses to PRRSV. Our study also provides a novel strategy for identifying and confirming potential SLA haplotype-restricted CTL epitopes that could be used to develop novel peptide-based vaccines against swine diseases.

Unveiling the Influence of Copy Number Variations on Genetic Diversity and Adaptive Evolution in China's Native Pig Breeds via Whole-Genome Resequencing.

Copy number variations (CNVs) critically influence individual genetic diversity and phenotypic traits. In this study, we employed whole-genome resequencing technology to conduct an in-depth analysis of 50 pigs from five local swine populations [Rongchang pig (RC), Wuzhishan pig (WZS), Tibetan pig (T), Yorkshire (YL) and Landrace (LR)], aiming to assess their genetic potential and explore their prospects in the field of animal model applications. We identified a total of 96,466 CNVs, which were subsequently integrated into 7112 non-redundant CNVRs, encompassing 1.3% of the swine genome. Functional enrichment analysis of the genes within these CNVRs revealed significant associations with sensory perception, energy metabolism, and neural-related pathways. Further selective scan analyses of the local pig breeds RC, T, WZS, along with YL and LR, uncovered that for the RC variety, the genes PLA2G10 and ABCA8 were found to be closely related to fat metabolism and cardiovascular health. In the T breed, the genes NCF2 and CSGALNACT1 were associated with immune response and connective tissue characteristics. As for the WZS breed, the genes PLIN4 and CPB2 were primarily linked to fat storage and anti-inflammatory responses. In summary, this research underscores the pivotal role of CNVs in fostering the diversity and adaptive evolution of pig breeds while also offering valuable insights for further exploration of the advantageous genetic traits inherent to China's local pig breeds. This facilitates the creation of experimental animal models tailored to the specific characteristics of these breeds, contributing to the advancement of livestock and biomedical research.

Recommendations for the selection of nucleoside analogues as antihuman herpesvirus drugs: a real-world analysis of reported cases from the FDA adverse event reporting system.

OBJECTIVE: The aim of this study is to provide guidance for refining medication protocols, developing alternative strategies, and enhancing protection against herpesvirus infections in personalized clinical settings. METHODS: Adverse drug events (ADEs) data for anti-herpesvirus from the first quarter of 2004 to the fourth quarter of 2022 were collected from the FDA Adverse Event Reporting System (FAERS). Disproportionality analysis was performed using Reporting Odds Ratio (ROR), Proportional Reporting Ratio (PRR), and Bayesian Confidence Propagation Neural Network (BCPNN) methods for data mining. RESULTS: A total of 18,591, 24,206, 6,150, and 419 reports of ADEs associated with acyclovir (ACV), valacyclovir (VACV), ganciclovir (GCV), and famciclovir (FCV) were screened and extracted from the FAERS. In this study, the report summarized the high frequency and strong correlation of ADEs for the four drugs at the Preferred Term (PT) level. Additionally, the analysis also identified the relationship between ADEs and factors such as age, gender, and severity of outcome at the System Organ Class (SOC) level. CONCLUSION: The safety reports for the four-nucleoside analogue anti-herpesvirus drugs are diverse and interconnected. Dosing for patients with herpesvirus infections should be tailored to their specific conditions and the potential risk of disease.

Development of a triplex quantitative reverse transcription-polymerase chain reaction for the detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus A.

Porcine viral diarrhea is caused by many pathogens and can result in watery diarrhea, dehydration and death. Various detection methods, such as polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR), have been widely used for molecular diagnosis. We developed a triplex real-time quantitative reverse transcription PCR (qRT-PCR) for the simultaneous detection of three RNA viruses potentially associated with porcine viral diarrhea: porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus A (PoRVA). The triplex qRT-PCR had R2 values of 0.999 for the standard curves of PEDV, TGEV and PoRVA. Importantly, the limits of detection for PEDV, TGEV and PoRVA were 10 copies/µL. The specificity test showed that the triplex qRT-PCR detected these three pathogens specifically, without cross-reaction with other pathogens. In addition, the approach had good repeatability and reproducibility, with intra-and inter-assay coefficients of variation <1%. Finally, this approach was evaluated for its practicality in the field using 256 anal swab samples. The positive rates of PEDV, TGEV and PoRVA were 2.73% (7/256), 3.91% (10/256) and 19.14% (49/256), respectively. The co-infection rate of two or more pathogens was 2.73% (7/256). The new triplex qRT-PCR was compared with the triplex RT-PCR recommended by the Chinese national standard (GB/T 36871-2018) and showed 100% agreement for PEDV and TGEV and 95.70% for PoRVA. Therefore, the triplex qRT-PCR provided an accurate and sensitive method for identifying three potential RNA viruses for porcine viral diarrhea that could be applied to diagnosis, surveillance and epidemiological investigation.

Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity.

The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA1. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities2. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes. It remains uncertain whether members of this family can evolve additional mechanisms to counter viral invasions3,4. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel ß-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR-Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9-PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and 'growth evolutionary' trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs-such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.

Peptide REF1 is a local wound signal promoting plant regeneration.

Plants frequently encounter wounding and have evolved an extraordinary regenerative capacity to heal the wounds. However, the wound signal that triggers regenerative responses has not been identified. Here, through characterization of a tomato mutant defective in both wound-induced defense and regeneration, we demonstrate that in tomato, a plant elicitor peptide (Pep), REGENERATION FACTOR1 (REF1), acts as a systemin-independent local wound signal that primarily regulates local defense responses and regenerative responses in response to wounding. We further identified PEPR1/2 ORTHOLOG RECEPTOR-LIKE KINASE1 (PORK1) as the receptor perceiving REF1 signal for plant regeneration. REF1-PORK1-mediated signaling promotes regeneration via activating WOUND-INDUCED DEDIFFERENTIATION 1 (WIND1), a master regulator of wound-induced cellular reprogramming in plants. Thus, REF1-PORK1 signaling represents a conserved phytocytokine pathway to initiate, amplify, and stabilize a signaling cascade that orchestrates wound-triggered organ regeneration. Application of REF1 provides a simple method to boost the regeneration and transformation efficiency of recalcitrant crops.

Targeted genome-modification tools and their advanced applications in crop breeding.

Crop improvement by genome editing involves the targeted alteration of genes to improve plant traits, such as stress tolerance, disease resistance or nutritional content. Techniques for the targeted modification of genomes have evolved from generating random mutations to precise base substitutions, followed by insertions, substitutions and deletions of small DNA fragments, and are finally starting to achieve precision manipulation of large DNA segments. Recent developments in base editing, prime editing and other CRISPR-associated systems have laid a solid technological foundation to enable plant basic research and precise molecular breeding. In this Review, we systematically outline the technological principles underlying precise and targeted genome-modification methods. We also review methods for the delivery of genome-editing reagents in plants and outline emerging crop-breeding strategies based on targeted genome modification. Finally, we consider potential future developments in precise genome-editing technologies, delivery methods and crop-breeding approaches, as well as regulatory policies for genome-editing products.

Anlotinib enhanced CD8+ T cell infiltration via induction of CCL5 improves the efficacy of PD-1/PD-L1 blockade therapy in lung cancer.

Non-small cell lung cancer (NSCLC) is a leading cause of mortality worldwide and requires effective treatment strategies. Recently, the development of a novel multiple-target tyrosine kinase inhibitor, anlotinib, has drawn increasing attention, especially it shows advantages when combined with PD-1/PD-L1 blockade. However, the mechanism by which anlotinib improves immunotherapy and remodeling of the tumor microenvironment remains unclear. In this study, we found that anlotinib combined with PD-1 blockade significantly inhibited tumor growth and reduced tumor weight in a lung cancer xenograft model compared to any single treatment. Both immunofluorescence and flow cytometry analyses revealed that anlotinib induced a CD8+ T cell dominated tumor microenvironment, which might account for its improved role in immunotherapy. Further investigations showed that CCL5-mediated CD8+ T cell recruitment plays a critical role in anlotinib and PD-1 blockade strategies. The depletion of CD8+ T cells abrogated this process. In conclusion, our findings showed that the combination of anlotinib and PD-1 blockade produced promising effects in the treatment of lung cancer, and that the induction of CCL5-mediced CD8+ T cell recruitment by anlotinib provided a novel mechanism of action.

Hydrolytic endonucleolytic ribozyme (HYER) is programmable for sequence-specific DNA cleavage.

Ribozymes are catalytic RNAs with diverse functions including self-splicing and polymerization. This work aims to discover natural ribozymes that behave as hydrolytic and sequence-specific DNA endonucleases, which could be repurposed as DNA manipulation tools. Focused on bacterial group II-C introns, we found that many systems without intron-encoded protein propagate multiple copies in their resident genomes. These introns, named HYdrolytic Endonucleolytic Ribozymes (HYERs), cleaved RNA, single-stranded DNA, bubbled double-stranded DNA (dsDNA), and plasmids in vitro. HYER1 generated dsDNA breaks in the mammalian genome. Cryo-electron microscopy analysis revealed a homodimer structure for HYER1, where each monomer contains a Mg2+-dependent hydrolysis pocket and captures DNA complementary to the target recognition site (TRS). Rational designs including TRS extension, recruiting sequence insertion, and heterodimerization yielded engineered HYERs showing improved specificity and flexibility for DNA manipulation.

Hypoxia-Derived Exosomes Promote Lung Adenocarcinoma by Regulating HS3ST1-GPC4-Mediated Glycolysis.

OBJECTIVE: The diagnosis of lung adenocarcinoma (LUAD) is often delayed due to the typically asymptomatic nature of the early-stage disease, causing advanced-stage LUAD diagnosis in most patients. Hypoxia is widely recognized as a driving force in cancer progression. Exosomes originating from hypoxic tumor cells promote tumorigenesis by influencing glycolysis, migration, invasion, and immune infiltration. Given these insights, our study aimed to explore the role of hypoxia-derived exosomal long non-coding RNA (lncRNA) OIP5-AS1 in LUAD cell lines and mouse models. MATERIALS AND METHODS: Exosomes were meticulously isolated and authenticated based on their morphology and biomarkers. The interaction between heparan sulfate (glucosamine) 3-O-sulfotransferase 1 (HS3ST1) and Glypican 4 (GPC4) was examined using immunoprecipitation. The influence of the hypoxia-derived exosomal lncRNA OIP5-AS1 on glycolysis was assessed in LUAD cell lines. The effect of the hypoxia-derived exosomal lncRNA OIP5-AS1 on cell proliferation and metastasis was evaluated using colony formation, cell viability, cell cycle, and apoptosis analyses. Its effects on tumor size were confirmed in xenograft animal models. RESULTS: Our study revealed the mechanism of the hypoxia-derived exosomal lncRNA OIP5-AS1 in LUAD progression. We discovered that GPC4 promotes HS3ST1-mediated glycolysis and that the hypoxia-derived exosomal lncRNA OIP5-AS1 enhances glycolysis by regulating miR-200c-3p in LUAD cells. Notably, this lncRNA stimulates LUAD cell proliferation and metastasis and fosters LUAD tumor size via miR-200c-3p. Our findings underscore the potential role of the hypoxia-derived exosomal lncRNA OIP5-AS1 in LUAD progression. CONCLUSIONS: The hypoxia-derived exosomal lncRNA OIP5-AS1 promotes LUAD by regulating HS3ST1-GPC4-mediated glycolysis via miR-200c-3p.

Prime editing using CRISPR-Cas12a and circular RNAs in human cells.

Genome editing with prime editors based on CRISPR-Cas9 is limited by the large size of the system and the requirement for a G/C-rich protospacer-adjacent motif (PAM) sequence. Here, we use the smaller Cas12a protein to develop four circular RNA-mediated prime editor (CPE) systems: nickase-dependent CPE (niCPE), nuclease-dependent CPE (nuCPE), split nickase-dependent CPE (sniCPE) and split nuclease-dependent CPE (snuCPE). CPE systems preferentially recognize T-rich genomic regions and possess a potential multiplexing capacity in comparison to corresponding Cas9-based systems. The efficiencies of the nuclease-based systems are up to 10.42%, whereas niCPE and sniCPE reach editing frequencies of up to 24.89% and 40.75% without positive selection in human cells, respectively. A derivative system, called one-sniCPE, combines all three RNA editing components under a single promoter. By arraying CRISPR RNAs for different targets in one circular RNA, we also demonstrate low-efficiency editing of up to four genes simultaneously with the nickase prime editors niCPE and sniCPE.