3D reconstruction of a gastrulating human embryo.

Progress in understanding early human development has been impeded by the scarcity of reference datasets from natural embryos, particularly those with spatial information during crucial stages like gastrulation. We conducted high-resolution spatial transcriptomics profiling on 38,562 spots from 62 transverse sections of an intact Carnegie stage (CS) 8 human embryo. From this spatial transcriptomic dataset, we constructed a 3D model of the CS8 embryo, in which a range of cell subtypes are identified, based on gene expression patterns and positional register, along the anterior-posterior, medial-lateral, and dorsal-ventral axis in the embryo. We further characterized the lineage trajectories of embryonic and extra-embryonic tissues and associated regulons and the regionalization of signaling centers and signaling activities that underpin lineage progression and tissue patterning during gastrulation. Collectively, the findings of this study provide insights into gastrulation and post-gastrulation development of the human embryo.

MARK2 variants cause autism spectrum disorder via the downregulation of WNT/ß-catenin signaling pathway.

Microtubule affinity-regulating kinase 2 (MARK2) contributes to establishing neuronal polarity and developing dendritic spines. Although large-scale sequencing studies have associated MARK2 variants with autism spectrum disorder (ASD), the clinical features and variant spectrum in affected individuals with MARK2 variants, early developmental phenotypes in mutant human neurons, and the pathogenic mechanism underlying effects on neuronal development have remained unclear. Here, we report 31 individuals with MARK2 variants and presenting with ASD, other neurodevelopmental disorders, and distinctive facial features. Loss-of-function (LoF) variants predominate (81%) in affected individuals, while computational analysis and in vitro expression assay of missense variants supported the effect of MARK2 loss. Using proband-derived and CRISPR-engineered isogenic induced pluripotent stem cells (iPSCs), we show that MARK2 loss leads to early neuronal developmental and functional deficits, including anomalous polarity and dis-organization in neural rosettes, as well as imbalanced proliferation and differentiation in neural progenitor cells (NPCs). Mark2+/- mice showed abnormal cortical formation and partition and ASD-like behavior. Through the use of RNA sequencing (RNA-seq) and lithium treatment, we link MARK2 loss to downregulation of the WNT/ß-catenin signaling pathway and identify lithium as a potential drug for treating MARK2-associated ASD.

A mobile robotic chemist.

Technologies such as batteries, biomaterials and heterogeneous catalysts have functions that are defined by mixtures of molecular and mesoscale components. As yet, this multi-length-scale complexity cannot be fully captured by atomistic simulations, and the design of such materials from first principles is still rare1-5. Likewise, experimental complexity scales exponentially with the number of variables, restricting most searches to narrow areas of materials space. Robots can assist in experimental searches6-14 but their widespread adoption in materials research is challenging because of the diversity of sample types, operations, instruments and measurements required. Here we use a mobile robot to search for improved photocatalysts for hydrogen production from water15. The robot operated autonomously over eight days, performing 688 experiments within a ten-variable experimental space, driven by a batched Bayesian search algorithm16-18. This autonomous search identified photocatalyst mixtures that were six times more active than the initial formulations, selecting beneficial components and deselecting negative ones. Our strategy uses a dexterous19,20 free-roaming robot21-24, automating the researcher rather than the instruments. This modular approach could be deployed in conventional laboratories for a range of research problems beyond photocatalysis.

Ribosomal frameshifting at normal codon repeats recodes functional chimeric proteins in human.

Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats, which may expand the coding capacity of mRNA and diversify the regulation in human.

Post-translational modification localizes MYC to the nuclear pore basket to regulate a subset of target genes involved in cellular responses to environmental signals.

The transcription factor MYC (also c-Myc) induces histone modification, chromatin remodeling, and the release of paused RNA polymerase to broadly regulate transcription. MYC is subject to a series of post-translational modifications that affect its stability and oncogenic activity, but how these control MYC's function on the genome is largely unknown. Recent work demonstrates an intimate connection between nuclear compartmentalization and gene regulation. Here, we report that Ser62 phosphorylation and PIN1-mediated isomerization of MYC dynamically regulate the spatial distribution of MYC in the nucleus, promoting its association with the inner basket of the nuclear pore in response to proliferative signals, where it recruits the histone acetyltransferase GCN5 to bind and regulate local gene acetylation and expression. We demonstrate that PIN1-mediated localization of MYC to the nuclear pore regulates MYC target genes responsive to mitogen stimulation that are involved in proliferation and migration pathways. These changes are also present at the chromatin level, with an increase in open regulatory elements in response to stimulation that is PIN1-dependent and associated with MYC chromatin binding. Taken together, our study indicates that post-translational modification of MYC controls its spatial activity to optimally regulate gene expression in response to extrinsic signals in normal and diseased states.

TIPE2 inhibits the migration and invasion of epithelial ovarian cancer cells by targeting Smad2 to reverse TGF-ß1-induced EMT.

Epithelial ovarian cancer is the deadliest gynecologic malignancy, characterized by high metastasis. Transforming growth factor-ß1 (TGF-ß1) drives epithelial- mesenchymal transformation (EMT), a key process in tumor metastasis. Tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like 2 (TIPE2) acts as a negative regulator of innate and adaptive immunity and involves in various cancers. However, its relationship with TGF-ß1 in ovarian cancer and its role in reversing TGF-ß1-induced EMT remain unclear. This study examined TIPE2 mRNA and protein expression using quantitative RT-PCR (qRT-PCR), western blot and immunohistochemistry. The effects of TIPE2 overexpression and knockdown on the proliferation, migration and invasion of epithelial ovarian cancer cells were assessed through 5-ethynyl-2-deoxyuridine, colony-forming, transwell migration and invasion assays. The relationship between TIPE2 and TGF-ß1 was investigated using qRT-PCR and enzyme-linked immunosorbent assay, while the interaction between TIPE2 and Smad2 was identified via co-immunoprecipitation. The results revealed that TIPE2 protein was significantly down-regulated in epithelial ovarian cancer tissues and correlated with the pathological type of tumor, patients' age, tumor differentiation degree and FIGO stage. TIPE2 and TGF-ß1 appeared to play an opposite role to each other during the progression of human ovarian cancer cells. Furthermore, TIPE2 inhibited the metastasis and EMT of ovarian cancer cells by combining with Smad2 in vitro or in an intraperitoneal metastasis model. Consequently, these findings suggest that TIPE2 plays a crucial inhibitory role in ovarian cancer metastasis by modulating the TGF-ß1/Smad2/EMT signaling pathway and may serve as a potential target for ovarian cancer, providing important direction for future diagnostic and therapeutic strategies.

Candesartan upregulates angiotensin-converting enzyme 2 in kidneys of male animals by decreased ubiquitination.

Candesartan is a common angiotensin-II receptor-1 blocker used for patients with cardiovascular and renal diseases. Angiotensin-converting enzyme 2 (ACE2) is a negative regulator of blood pressure (BP), and also a major receptor for coronaviruses. To determine whether and how candesartan upregulates ACE2, we examined BP and ACE2 in multi-organs from male and female C57BL/6J mice treated with candesartan (1 mg/kg, i.p.) for 7 days. Relative to the vehicle, candesartan lowered BP more in males than females; ACE2 protein abundances were increased in kidneys, not lungs, hearts, aorta, liver, spleen, brain, or serum, only from males. Ace2-mRNA was similar in kidneys. Candesartan also decreased BP in normal, hypertensive, and nephrotic male rats. The renal ACE2 was increased by the drug in normal and nephrotic male rats but not spontaneously hypertensive ones. In male mouse kidneys, ACE2 was distributed at sodium-hydrogen-exchanger-3 positive proximal-convoluted-tubules; ACE2-ubiquitination was decreased by candesartan, accompanied with increased ubiquitin-specific-protease-48 (USP48). In candesartan-treated mouse renal proximal-convoluted-tubule cells, ACE2 abundances and activities were increased while ACE2-ubiquitination and colocalization with lysosomal and proteosomal markers were decreased. The silence of USP48 by siRNA caused a reduction of ACE2 in the cells. Thus, the sex-differential ACE2 upregulation by candesartan in kidney from males may be due to the decreased ACE2-ubiquitination, associated with USP48, and consequent degradation in lysosomes and proteosomes. This is a novel mechanism and may shed light on candesartan-like-drug choice in men and women prone to coronavirus infections.

Passer, a highly active transposon from a fish genome, as a potential new robust genetic manipulation tool.

The discovery of new, active DNA transposons can expand the range of genetic tools and provide more options for genomic manipulation. In this study, a bioinformatics analysis suggested that Passer (PS) transposons, which are members of the pogo superfamily, show signs of recent and current activity in animals and may be active in some species. Cell-based transposition assays revealed that the native PS transposases from Gasterosteus aculeatus and Danio rerio displayed very high activity in human cells relative to the Sleeping Beauty transposon. A typical overproduction inhibition phenomenon was observed for PS, and transposition capacity was decreased by ∼12% with each kilobase increase in the insertion size. Furthermore, PS exhibited a pronounced integration preference for genes and their transcriptional regulatory regions. We further show that two domesticated human proteins derived from PS transposases have lost their transposition activity. Overall, PS may represent an alternative with a potentially efficient genetic manipulation tool for transgenesis and mutagenesis applications.

Kirigami-based metastructures with programmable multistability.

SignificanceDifferent from most existing multistable structures whose multiple stable states are achieved through the combinational effect of bistable units, we invent a generic tristable kirigami cuboid. The three stable states have fundamentally distinct geometric configurations and chirality, and the transformation among them can be realized by tension/compression or clockwise/counterclockwise twist. Tessellating the units in series, a family of multistable metamaterials can be constructed, the mechanical behaviors of which are programmable by the unit geometry, the material of the elastic joints, the number of units, and the loading conditions. As a demonstration of the potential applications, a frequency reconfigurable antenna for 5G triple-band communication is developed based on a tristable unit, and the frequency tunability is verified by experiments.

Uridylation and the SKI complex orchestrate the Calvin cycle of photosynthesis through RNA surveillance of TKL1 in Arabidopsis.

RNA uridylation, catalyzed by terminal uridylyl transferases (TUTases), represents a conserved and widespread posttranscriptional RNA modification in eukaryotes that affects RNA metabolism. In plants, several TUTases, including HEN1 SUPPRESSOR 1 (HESO1) and UTP: RNA URIDYLYLTRANSFERASE (URT1), have been characterized through genetic and biochemical approaches. However, little is known about their physiological significance during plant development. Here, we show that HESO1 and URT1 act cooperatively with the cytoplasmic 3'-5' exoribonucleolytic machinery component SUPERKILLER 2 (SKI2) to regulate photosynthesis through RNA surveillance of the Calvin cycle gene TRANSKETOLASE 1 (TKL1) in Arabidopsis. Simultaneous dysfunction of HESO1, URT1, and SKI2 resulted in leaf etiolation and reduced photosynthetic efficiency. In addition, we detected massive illegitimate short interfering RNAs (siRNAs) from the TKL1 locus in heso1 urt1 ski2, accompanied by reduced TKL1/2 expression and attenuated TKL activities. Consequently, the metabolic analysis revealed that the abundance of many Calvin cycle intermediates is dramatically disturbed in heso1 urt1 ski2. Importantly, all these molecular and physiological defects were largely rescued by the loss-of-function mutation in RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), demonstrating illegitimate siRNA-mediated TKL silencing. Taken together, our results suggest that HESO1- and URT1-mediated RNA uridylation connects to the cytoplasmic RNA degradation pathway for RNA surveillance, which is crucial for TKL expression and photosynthesis in Arabidopsis.

Neutrophil-Associated Proteins as Novel Biomarkers Elevated in Cerebrospinal Fluid of Patients With Neurosyphilis.

BACKGROUND: The immunopathological mechanisms underlying neurosyphilis remain incompletely elucidated, and the diagnosis of neurosyphilis presents challenges. METHODS: We used an antibody microarray to detect 640 proteins in cerebrospinal fluid (CSF) samples collected from 6 patients with non-neurosyphilis and 10 with neurosyphilis. The levels of CSF CXCL1, CXCL8, G-CSF, LCN2, MMP8, and MMP9 in 46 patients with non-neurosyphilis, 51 with untreated neurosyphilis, and 31 posttreatment for neurosyphilis were quantified using enzyme-linked immunosorbent assay. The associations between the levels of these proteins and clinical parameters in neurosyphilis were evaluated using Spearman analysis, and the diagnostic performance of these proteins in neurosyphilis was assessed using receiver operating characteristic curve. RESULTS: A total of 102 differentially expressed proteins between neurosyphilis and non-neurosyphilis were identified. The levels of significantly elevated neutrophil-associated proteins (CXCL1, CXCL8, G-CSF, LCN2, MMP8, and MMP9) in neurosyphilis positively correlated with white blood cell counts, rapid plasma regain (RPR) titer, and protein concentration in CSF. The combination of CSF CXCL8, MMP9, and LCN2 yielded an area under the curve of 0.92 for diagnosing neurosyphilis, surpassing that of CSF RPR. CONCLUSIONS: CXCL1, CXCL8, G-CSF, LCN2, MMP8, and MMP9 could be associated with central nervous system damage of neurosyphilis. The combination of CSF CXCL8, MMP9, and LCN2 is a promising biomarker for diagnosing neurosyphilis.

Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice.

Hem1 (hematopoietic protein 1), a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins, is essential for lymphopoiesis and innate immunity as well as for the transition of hematopoiesis from the fetal liver to the bone marrow. However, the role of Hem1 in bone cell differentiation and bone remodeling is unknown. Here, we show that deletion of Hem1 resulted in a markedly increase in bone mass because of defective bone resorption in mice of both sexes. Hem1-deficient osteoclast progenitors were able to differentiate into osteoclasts, but the osteoclasts exhibited impaired osteoclast fusion and decreased bone-resorption activity, potentially because of decreased mitogen-activated protein kinase and tyrosine kinase c-Abl activity. Transplantation of bone marrow hematopoietic stem and progenitor cells from wildtype into Hem1 knockout mice increased bone resorption and normalized bone mass. These findings indicate that Hem1 plays a pivotal role in the maintenance of normal bone mass.

Indobufen or Aspirin on Top of Clopidogrel After Coronary Drug-Eluting Stent Implantation (OPTION): A Randomized, Open-Label, End Point-Blinded, Noninferiority Trial.

BACKGROUND: Dual antiplatelet therapy (DAPT) with aspirin as a background therapy has become the standard care after percutaneous coronary intervention. However, some adverse noncardiac effects limited the use of aspirin in clinical practice. Thus, evaluation of pharmacological alternatives to aspirin is attractive. Previous data indicated that indobufen could lessen the unwanted side effects of aspirin while retaining the antithrombotic efficacy, but its combination with a P2Y12 inhibitor still lacks randomized clinical trial evidence. METHODS: In this randomized, open-label, noninferiority trial, patients with negative cardiac troponin undergoing coronary drug-eluting stent implantation were randomly assigned in a 1:1 ratio to receive either indobufen-based DAPT (indobufen 100 mg twice a day plus clopidogrel 75 mg/d for 12 months) or conventional DAPT (aspirin 100 mg/d plus clopidogrel 75 mg/d for 12 months). The primary end point was a 1-year composite of cardiovascular death, nonfatal myocardial infarction, ischemic stroke, definite or probable stent thrombosis, or Bleeding Academic Research Consortium criteria type 2, 3, or 5 bleeding. The end points were adjudicated by an independent Clinical Event Committee. RESULTS: Between January 11, 2018, and October 12, 2020, 4551 patients were randomized in 103 cardiovascular centers: 2258 patients to the indobufen-based DAPT group and 2293 to the conventional DAPT group. The primary end point occurred in 101 patients (4.47%) in the indobufen-based DAPT group and 140 patients (6.11%) in the conventional DAPT group (absolute difference, -1.63%; Pnoninferiority<0.001; hazard ratio, 0.73 [95% CI, 0.56-0.94]; P=0.015). Cardiovascular death, nonfatal myocardial infarction, ischemic stroke, and stent thrombosis were observed in 0.13%, 0.40%, 0.80%, and 0.22% of patients in the indobufen-based DAPT group and 0.17%, 0.44%, 0.83%, and 0.17% of patients in the conventional DAPT group (all P>0.05). The occurrence of Bleeding Academic Research Consortium criteria type 2, 3, or 5 bleeding events was lower in the indobufen-based DAPT group compared with the conventional DAPT group (2.97% versus 4.71%; hazard ratio, 0.63 [95% CI, 0.46-0.85]; P=0.002), with the main decrease in type 2 bleeding (1.68% versus 3.49%; hazard ratio, 0.48 [95% CI, 0.33-0.70]; P<0.001). CONCLUSIONS: In Chinese patients with negative cardiac troponin undergoing drug-eluting stent implantation, indobufen plus clopidogrel DAPT compared with aspirin plus clopidogrel DAPT significantly reduced the risk of 1-year net clinical outcomes, which was driven mainly by a reduction in bleeding events without an increase in ischemic events. REGISTRATION: URL: https://www.chictr.org.cn; Unique identifier: ChiCTR-IIR-17013505.

Omicron breakthrough infections after triple-dose inactivated COVID-19 vaccination: A comprehensive analysis of antibody and T-cell responses.

This study longitudinally evaluated the immune response in individuals over a year after receiving three doses of an inactivated SARS-CoV-2 vaccine, focusing on reactions to Omicron breakthrough infections. From 63 blood samples of 37 subjects, results showed that the third booster enhanced the antibody response against Alpha, Beta, and Delta VOCs but was less effective against Omicron. Although antibody titres decreased post-vaccination, SARS-CoV-2-specific T-cell responses, both CD4+ and CD8+, remained stable. Omicron breakthrough infections significantly improved neutralization against various VOCs, including Omicron. However, the boost in antibodies against WT, Alpha, Beta, and Delta variants was more pronounced. Regarding T cells, breakthrough infection predominantly boosted the CD8+ T-cell response, and the intensity of the spike protein-specific T-cell response was roughly comparable between WT and Omicron BA.5.

R-loops' m6A modification and its roles in cancers.

R-loops are three-stranded nucleic acid structures composed of an RNA-DNA hybrid and a displaced DNA strand. They are widespread and play crucial roles in regulating gene expression, DNA replication, and DNA and histone modifications. However, their regulatory mechanisms remain unclear. As R-loop detection technology advances, changes in R-loop levels have been observed in cancer models, often associated with transcription-replication conflicts and genomic instability. N6-methyladenosine (m6A) is an RNA epigenetic modification that regulates gene expression by affecting RNA localization, splicing, translation, and degradation. Upon reviewing the literature, we found that R-loops with m6A modifications are implicated in tumor development and progression. This article summarizes the molecular mechanisms and detection methods of R-loops and m6A modifications in gene regulation, and reviews recent research on m6A-modified R-loops in oncology. Our goal is to provide new insights into the origins of genomic instability in cancer and potential strategies for targeted therapy.

T cells in testicular germ cell tumors: new evidence of fundamental contributions by rare subsets.

BACKGROUND: Immune cell infiltration is heterogeneous but common in testicular germ cell tumors (TGCT) and pre-invasive germ cell neoplasia in situ (GCNIS). Tumor-infiltrating T cells including regulatory T (Treg) and follicular helper T (Tfh) cells are found in other cancer entities, but their contributions to TGCT are unknown. METHODS: Human testis specimens from independent patient cohorts were analyzed using immunohistochemistry, flow cytometry and single-cell RNA sequencing (scRNA-seq) with special emphasis on delineating T cell subtypes. RESULTS: Profound changes in immune cell composition within TGCT, shifting from macrophages in normal testes to T cells plus B and dendritic cells in TGCT, were documented. In most samples (96%), the CD4+ T cell frequency exceeded that of CD8+ cells, with decreasing numbers from central to peripheral tumor areas, and to tumor-free, contralateral testes. T cells including Treg and Tfh were most abundant in seminoma compared to mixed tumors and embryonal carcinoma. CONCLUSION: Despite considerable heterogeneity between patients, T cell subtypes form a key part of the TGCT microenvironment. The novel finding of rare Treg and Tfh cells in human testis suggests their involvement in TGCT pathobiology, with implications for understanding tumor progression, to assess patients' prognosis, and as putative targets for personalized immunotherapy.

A Nanofluorescent Probe for Evaluating the Fluctuation of Aminopeptidase N in Nonalcoholic Fatty Liver Disease and Hepatic Fibrosis.

Aminopeptidase N (APN/CD13) is a widely expressed transmembrane ectoenzyme that is crucial for maintaining normal physiological activities. It exhibits abnormal activity closely associated with hepatic fibrosis and nonalcoholic fatty liver disease (NAFLD). Therefore, there is a high demand for noninvasive detection of aminopeptidase N (APN) in the diagnosis and research of related diseases. Here, we developed a small molecule fluorescent probe, Hcy-APN, which is a fluorescent probe with high sensitivity and selectivity for the detection of APN. Furthermore, we synthesized the fluorescent nanoprobe Hcy-APN@MSN by self-assembling Hcy-APN and mesoporous silica nanoparticles in solution using a combination of molecular probe design and nanofunctionalization strategies. The detection limit of this probe was 1.5 ng/mL. Hcy-APN@MSN exhibits more stable spectral characteristics compared to Hcy-APN and is suitable for detecting APN activity in live cells and mice. Hcy-APN@MSN was utilized for in vivo and intracellular imaging of NAFLD and hepatic fibrosis at different stages, as well as for a systematic assessment of APN levels in the liver. The results confirm an elevation in the expression levels of APN in NAFLD and hepatic fibrosis models. Furthermore, we investigated the inhibitory effect of the APN inhibitor bestatin in nonalcoholic fatty liver and hepatic fibrosis disease models, confirming its regulatory effect on APN levels in cells and in vivo in both disease models. Therefore, this study may offer diagnostic possibilities for detecting NAFLD and hepatic fibrosis.

Confinement Effect Enhanced Bipolar Electrochemistry: Structural Color Coding Coupled with Wireless Electrochemiluminescence Imaging Technology.

In this work, SiO2/CNTs photonic crystal beads were constructed by doping CNTs into SiO2 photonic crystals, which have an angle-independent responsive structural color and can be used as bipolar electrodes due to their good electrical conductivity. In addition, the bipolar electrode-electrochemiluminescence (BPE-ECL) experiments and finite element simulation prove that the low driving voltage can trigger the bipolar electrode electrochemical reactions by confinement effect. Inspired by this, it is the first to combine the SiO2/CNTs structural color coding scheme with low-drive voltage induced wireless BPE-ECL imaging based on the confinement effect of microchannels to achieve simultaneous immune detection of ovarian cancer biomarkers (CA125, CEA, AFP). The detection limits of successfully constructed high-throughput BPE-ECL biosensor for AFP, CEA, and CA125 are 0.72 ng/mL, 0.95 ng/mL, and 1.03 U/mL, respectively, and have good stability and specificity, which expands the application of electrochemiluminescence and lays a foundation for the development of electrochemiluminescence coding technology.

Relevance of pyroptosis-associated genes in nasopharyngeal carcinoma diagnosis and subtype classification.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a highly aggressive and metastatic malignancy originating in the nasopharyngeal tissue. Pyroptosis is a relatively newly discovered, regulated form of necrotic cell death induced by inflammatory caspases that is associated with a variety of diseases. However, the role and mechanism of pyroptosis in NPC are not fully understood. METHODS: We analyzed the differential expression of pyroptosis-related genes (PRGs) between patients with and without NPC from the GSE53819 and GSE64634 datasets of the Gene Expression Omnibus (GEO) database. We mapped receptor operating characteristic profiles for these key PRGs to assess the accuracy of the genes for disease diagnosis and prediction of patient prognosis. In addition, we constructed a nomogram based on these key PRGs and carried out a decision curve analysis. The NPC patients were classified into different pyroptosis gene clusters by the consensus clustering method based on key PRGs, whereas the expression profiles of the key PRGs were analyzed by applying principal component analysis. We also analyzed the differences in key PRGs, immune cell infiltration and NPC-related genes between the clusters. Finally, we performed differential expression analysis for pyroptosis clusters and obtained differentially expressed genes (DEGs) and performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. RESULTS: We obtained 14 differentially expressed PRGs from GEO database. Based on these 14 differentially expressed PRGs, we applied least absolute shrinkage and selection operator analysis and the random forest algorithm to obtain four key PRGs (CHMP7, IL1A, TP63 and GSDMB). We completely distinguished the NPC patients into two pyroptosis gene clusters (pyroptosis clusters A and B) based on four key PRGs. Furthermore, we determined the immune cell abundance of each NPC sample, estimated the association between the four PRGs and immune cells, and determined the difference in immune cell infiltration between the two pyroptosis gene clusters. Finally, we obtained and functional enrichment analyses 259 DEGs by differential expression analysis for both pyroptosis clusters. CONCLUSIONS: PRGs are critical in the development of NPC, and our research on the pyroptosis gene cluster may help direct future NPC therapeutic approaches.

Novel underlying regulatory mechanism of the MsDAD2-mediated salt stress response in alfalfa.

Alfalfa (Medicago sativa L.), a crucial and widely grown forage legume, faces yield and quality challenges due to salinity stress. The defender against apoptotic death (DAD) gene, recognized initially as an apoptosis suppressor in mammals, plays a pivotal role in catalyzing N-glycosylation, acting as a positive regulator for protein folding and endoplasmic reticulum (ER) export. Here, we found that the MsDAD2 gene was specially induced in the salt-tolerant alfalfa cultivar (DL) under salinity stress, but not in the salt-sensitive cultivar (SD). Overexpression of MsDAD2 enhanced the salinity resistance of transgenic alfalfa by promoting NAD(P)H-quinone oxidoreductase (NQO1) and cytochrome b6f complex subunit (Cyt b6/f) expression, thereby mitigating reactive oxygen species (ROS) production. ChIP-qPCR analysis suggested that the differential expression of MsDAD2 in DL and SD under salinity stress may be linked to dynamic histone modifications in its promoter. Therefore, our findings elucidate a novel regulatory mechanism of MsDAD2 in alfalfa's response to salinity stress, underscoring its significance as a target for alfalfa breeding to enhance salt tolerance.