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BACKGROUND: Per- and polyfluoroalkyl Substances (PFAS) are synthetic chemicals widely detected in humans and the environment. Exposure to perfluorooctanesulfonic acid (PFOS) or perfluorohexanesulfonic acid (PFHxS) was previously shown to cause dark-phase hyperactivity in larval zebrafish. OBJECTIVES: The objective of this study was to elucidate the mechanism by which PFOS or PFHxS exposure caused hyperactivity in larval zebrafish. METHODS: Swimming behavior was assessed in 5-d postfertilization (dpf) larvae following developmental (1-4 dpf) or acute (5 dpf) exposure to 0.43-7.86 [micro]M PFOS, 7.87-120 [micro]M PFHxS, or 0.4% dimethyl sulfoxide (DMSO). After developmental exposure and chemical washout at 4 dpf, behavior was also assessed at 5-8 dpf. RNA sequencing was used to identify differences in global gene expression to perform transcriptomic benchmark concentration--response ([BMC.sub.T]) modeling, and predict upstream regulators in PFOS- or PFHxS-exposed larvae. CRISPR/Cas9-based gene editing was used to knockdown peroxisome proliferator-activated receptors (ppars) pparaa/ab, pparda/db, or pparg at day 0. Knockdown crispants were exposed to 7.86 [micro]M PFOS or 0.4% DMSO from 1-4 dpf and behavior was assessed at 5 dpf. Coexposure with the ppard antagonist GSK3787 and PFOS was also performed. RESULTS: Transient dark-phase hyperactivity occurred following developmental or acute exposure to PFOS or PFHxS, relative to the DMSO control. In contrast, visual startle response (VSR) hyperactivity only occurred following developmental exposure and was irreversible up to 8 dpf. Similar global transcriptomic profiles, [BMC.sub.T] estimates, and enriched functions were observed in PFOS- and PFHxS-exposed larvae, and ppars were identified as putative upstream regulators. Knockdown of pparda/db, but not pparaa/ab or pparg, blunted PFOS-dependent VSR hyperactivity to control levels. This finding was confirmed via antagonism of ppard in PFOS-exposed larvae. DISCUSSION: This work identifies a novel adverse outcome pathway for VSR hyperactivity in larval zebrafish. We demonstrate that developmental, but not acute, exposure to PFOS triggered persistent VSR hyperactivity that required ppard function. https://doi.org/10.1289/EHP13667, Introduction Per- and polyfluoroalkyl substances (PFAS) are a diverse class of synthetic chemicals containing >14,000 unique structures used in industrial manufacturing (US EPA PFASSTRUCTv3, last checked December 2023 (1)). PFAS [...]

Newborn mammalian cardiomyocytes quickly transition from a fetal to an adult phenotype that utilizes mitochondrial oxidative phosphorylation but loses mitotic capacity. We tested whether forced reversal of adult cardiomyocytes back to a fetal glycolytic phenotype would restore proliferative capacity. We deleted Uqcrfsl (mitochondrial Rieske iron- sulfur protein, RISP) in hearts of adult mice. As RISP protein decreased, heart mitochondrial function declined, and glucose utilization increased. Simultaneously, the hearts underwent hyperplastic remodeling during which cardiomyocyte number doubled without cellular hypertrophy. Cellular energy supply was preserved, AMPK activation was absent, and mTOR activation was evident. In ischemic hearts with RISP deletion, new cardiomyocytes migrated into the infarcted region, suggesting the potential for therapeutic cardiac regeneration. RNA sequencing revealed upregulation of genes associated with cardiac development and proliferation. Metabolomic analysis revealed a decrease in a-ketoglutarate (required for TET-mediated demethylation) and an increase in 5-adenosylmethionine (required for methyltransferase activity). Analysis revealed an increase in methylated CpGs near gene transcriptional start sites. Genes that were both differentially expressed and differentially methylated were linked to upregulated cardiac developmental pathways. We conclude that decreased mitochondrial function and increased glucose utilization can restore mitotic capacity in adult cardiomyocytes, resulting in the generation of new heart cells, potentially through the modification of substrates that regulate epigenetic modification of genes required for proliferation., Introduction Mitochondria generate ATP through oxidative phosphorylation (OXPHOS), but they also participate in diverse biological functions that include redox signaling (1), metabolite signaling (2), calcium signaling (3), and the generation [...]

Trisomy 21 (T21), a recurrent aneuploidy occurring in 1:800 births, predisposes to congenital heart disease (CHD) and multiple extracardiac phenotypes. Despite a definitive genetic etiology, the mechanisms by which T21 perturbs development and homeostasis remain poorly understood. We compared the transcriptome of CHD tissues from 49 patients with T21 and 226 with euploid CHD (eCHD). We resolved cell lineages that misexpressed T21 transcripts by cardiac single-nucleus RNA sequencing and RNA in situ hybridization. Compared with eCHD samples, T21 samples had increased chr21 gene expression; 11-fold-greater levels (P = 1.2 x [10.sup.8]) of SOST (chr17), encoding the Wnt inhibitor sclerostin; and 1.4-fold-higher levels (P = 8.7 x [10.sup.-8]) of the SOST transcriptional activator ZNF467 (chr7). Euploid and T21 cardiac endothelial cells coexpressed SOST and ZNF467; however, T21 endothelial cells expressed 6.9-fold more SOST than euploid endothelial cells (P = 2.7 x [10.sup.-27]). Wnt pathway genes were downregulated in T21 endothelial cells. Expression of DSCAM, residing within the chr21 CHD critical region, correlated with SOST(P = 1.9 x [10.sup.-5]) and ZNF467 (P = 2.9 x [10.sup.-4]). Deletion of DSCAM from T21 endothelial cells derived from human induced pluripotent stem cells diminished sclerostin secretion. As Wnt signaling is critical for atrioventricular canal formation, bone health, and pulmonary vascular homeostasis, we concluded that T21-mediated increased sclerostin levels would inappropriately inhibit Wnt activities and promote Down syndrome phenotypes. These findings imply therapeutic potential for anti-sclerostin antibodies in T21., Introduction Trisomy 21 (T21), the most common genetic cause of syndromic congenital heart disease (CHD), is diagnosed in approximately 5,000 newborns in the US annually. Almost half of affected infants [...]

We previously demonstrated that a subset of acute myeloid leukemia (AML) patients with concurrent RAS pathway and TP53 mutations have an extremely poor prognosis and that most of these TP53 mutations are missense mutations. Here, we report that, in contrast to the mixed AML and T cell malignancy that developed in [Nras.sup.G12D/+] [p53.sup.-/-] ([NP.sup.-/- ]) mice, [Nras.sup.G120/+] [p53.sup.R172H/+] ([NP.sup.mut]) mice rapidly developed inflammation-associated AML. Under the inflammatory conditions, [NP.sup.mut] hematopoietic stem and progenitor cells (HSPCs) displayed imbalanced myelopoiesis and lymphopoiesis and mostly normal cell proliferation despite MEK/ERK hyperactivation. RNA-Seq analysis revealed that oncogenic NRAS signaling and mutant p53 synergized to establish an [NP.sup.mut]-AML transcriptome distinct from that of [NP.sup.-/-] cells. The [NP.sup.mut]-AML transcriptome showed GATA2 downregulation and elevated the expression of inflammatory genes, including those linked to NF-[kappa]B signaling. NF- [kappa]B was also upregulated in human NRAS TP53 AML. Exogenous expression of GATA2 in human [NP.sup.mut] KY821 AML cells downregulated inflammatory gene expression. Mouse and human [NP.sup.mut] AML cells were sensitive to MEK and NF- [kappa]B inhibition in vitro. The proteasome inhibitor bortezomib stabilized the NF-KB-inhibitory protein iKBa, reduced inflammatory gene expression, and potentiated the survival benefit of a MEK inhibitor in [NP.sup.mut] mice. Our study demonstrates that a p53 structural mutant synergized with oncogenic NRAS to promote AML through mechanisms distinct from p53 loss., Introduction Acute myeloid leukemia (AML) is an aggressive and devastating hematologic malignancy characterized by the accumulation of partially differentiated myeloid blast cells ([greater than or equal to]20%) in bone marrow [...]

Tissue-resident lymphocytes provide organ-adapted protection against invading pathogens. Whereas their biology has been examined in great detail in various infection models, their generation and functionality in response to vaccination have not been comprehensively analyzed in humans. We therefore studied SARS-CoV-2 mRNA vaccine-specific T cells in surgery specimens of kidney, liver, lung, bone marrow, and spleen compared with paired blood samples from largely virus-naive individuals. As opposed to lymphoid tissues, nonlymphoid organs harbored significantly elevated frequencies of spike-specific [CD4.sup.+] T cells compared with blood showing hallmarks of tissue residency and an expanded memory pool. Organ-derived [CD4.sup.+] T cells further exhibited increased polyfunctionality over those detected in blood. Single- cell RNA-Seq together with T cell receptor repertoire analysis indicated that the clonotype rather than organ origin is a major determinant of transcriptomic state in vaccine-specific [CD4.sup.+] T cells. In summary, our data demonstrate that SARS-CoV-2 vaccination entails acquisition of tissue memory and residency features in organs distant from the inoculation site, thereby contributing to our understanding of how local tissue protection might be accomplished., Introduction Vaccination against SARS-CoV-2 has been proven in large cohort studies (1, 2) to be a powerful strategy to protect from severe coronavirus disease 2019 (COVID-19). Since the availability of [...]

The progression of proteinuric kidney diseases is associated with podocyte loss, but the mechanisms underlying this process remain unclear. Podocytes reenter the cell cycle to repair double-stranded DNA breaks. However, unsuccessful repair can result in podocytes crossing the [G.sub.1]/S checkpoint and undergoing abortive cytokinesis. In this study, we identified Pfn1 as indispensable in maintaining glomerular integrity--its tissue-specific loss in mouse podocytes resulted in severe proteinuria and kidney failure. Our results suggest that this phenotype is due to podocyte mitotic catastrophe (MC), characterized histologically and ultrastructurally by abundant multinucleated cells, irregular nuclei, and mitotic spindles. Podocyte cell cycle reentry was identified using FUCCI2aR mice, and we observed altered expression of cellcycle associated proteins, such as p21, p53, cyclin B1, and cyclin D1. Podocyte-specific translating ribosome affinity purification and RNA-Seq revealed the downregulation of ribosomal RNA-processing 8 (Rrp8). Overexpression of Rrp8 in Pfn1-KO podocytes partially rescued the phenotype in vitro. Clinical and ultrastructural tomographic analysis of patients with diverse proteinuric kidney diseases further validated the presence of MC podocytes and reduction in podocyte PFN1 expression within kidney tissues. These results suggest that profilinl is essential in regulating the podocyte cell cycle and its disruption leads to MC and subsequent podocyte loss., Introduction Podocytes are specialized terminally differentiated epithelial cells that line the kidney filtration barrier. Unlike typical epithelial cells that are compressed against their basement membrane, podocytes are situated on the [...]

* Context.--Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are common methods to detect ALK status in inflammatory myofibroblastic tumors (IMTs). However, equivocal ALK FISH signals and inconsistency between FISH and IHC are occasionally observed. Objective.--To study the inconsistency between FISH and IHC, and clarify ALK status in IMT by targeted RNA sequencing (RNAseq). Design.--In this study, 12 consultation cases preliminarily diagnosed as uncommon IMTs with ALK IHC positivity but FISH negativity, plus 3 ALK-positive and 3 ALK-negative IMTs, were re-analyzed by IHC, FISH, and RNAseq. Results.--As a result, 1 case with FUS-TFCP2 fusion was detected by RNAseq, which was previously misdiagnosed as IMT. In the other 11 uncommon IMTs, 90.9% (10 of 11) showed equivocal ALK FISH signals, and all were confirmed to harbor ALK fusion by RNAseq, except for 1 failure, suggesting that a low threshold for ALK FISH might be proposed in IMT. Furthermore, RNAseq also identified IGFBP5-ALK in 1 case with ALK IHC positivity but typical FISH negativity, suggesting the possibility of false negatives for ALK FISH. For the typical IMTs, ALK fusion was identified by RNAseq in all 3 ALK-positive IMTs as expected, and additionally FN1-ROS1 fusions were identified in 2 of 3 ALK-negative IMTs. Conclusions.--These findings indicated that RNAseq can simultaneously detect multiple gene fusions and provide fusion forms and breakpoints, which is of great value for differential diagnosis, especially for those uncommon IMTs with equivocal FISH findings or inconsistency between IHC and FISH. doi: 10.5858/arpa.2021-0230-OA, Inflammatory myofibroblastic tumor (IMT) is a rare, mesenchymal neoplasm with a tendency for local recurrence. While it can occur at any age and in multiple anatomic locations, it arises primarily [...]

Background Polymyalgia rheumatica (PMR) is a common inflammatory disease in elderly persons whose mechanism of pathogenesis has not been elucidated. Glucocorticoids are the main first-line treatments but result in numerous side effects. Therefore, there is a need to explore pathogenetic factors and identify possible glucocorticoid-sparing agents. We aimed to study the pathogenetic features of the disease and assess the efficacy and safety of Janus tyrosine kinase (JAK)-inhibitor tofacitinib in patients with PMR. Methods and findings We recruited treatment-naïve PMR patients from the First Affiliated Hospital, Zhejiang University School of Medicine, between September 2020 and September 2022. In the first cohort, we found that the gene expression patterns of peripheral blood mononuclear cells (PBMCs) in 11 patients (10 female, 1 male, age 68.0 ± 8.3) with newly diagnosed PMR were significantly different from 20 healthy controls (17 female, 3 male, age 63.7 ± 9.8) by RNA sequencing. Inflammatory response and cytokine-cytokine receptor interaction were the most notable pathways affected. We observed marked increases in expression of IL6R, IL1B, IL1R1, JAK2, TLR2, TLR4, TLR8, CCR1, CR1, S100A8, S100A12, and IL17RA, which could trigger JAK signaling. Furthermore, tofacitinib suppressed the IL-6R and JAK2 expression of CD4.sup.+ T cells from patients with PMR in vitro. In the second cohort, patients with PMR were randomized and treated with tofacitinib or glucocorticoids (1/1) for 24 weeks. All PMR patients underwent clinical and laboratory examinations at 0, 4, 8, 12, 16, 20, and 24 weeks, and PMR activity disease scores (PMR-AS) were calculated. The primary endpoint was the proportion of patients with PMR-AS [less than or equal to]10 at weeks 12 and 24. Secondary endpoints: PMR-AS score, c-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) at weeks 12 and 24. Thirty-nine patients with newly diagnosed PMR received tofacitinib, and 37 patients received glucocorticoid. Thirty-five patients (29 female, 6 male, age 64.4 ± 8.4) and 32 patients (23 female, 9 male, age 65.3 ± 8.7) patients completed the 24-week intervention, respectively. There were no statistically significant differences in primary or secondary outcomes. At weeks 12 and 24, all patients in both groups had PMR-AS Conclusions We found that JAK signaling was involved in the pathogenesis of PMR. Tofacitinib effectively treated patients with PMR as glucocorticoid does in this randomized, monocenter, open-label, controlled trial (ChiCTR2000038253). Trial registration This investigator-initiated clinical trial (IIT) had been registered on the website (http://www.chictr.org.cn/, ChiCTR2000038253)., Author(s): Xinlei Ma 1, Fan Yang 2, Jinzhi Wu 1, Bei Xu 1, Mengdi Jiang 1, Yiduo Sun 1, Chuanying Sun 1, Ye Yu 1, Danyi Xu 1, Lanlan Xiao [...]

Characterization of the dynamic change in the immunological landscape during malignant transformation from precancerous lesions to cancerous lesions in squamous cell carcinoma (SCC) is critical for the application of immunotherapy. Here, we performed single-cell RNA-Seq (scRNA-Seq) of 131,702 cells from 13 cancerous tissues of oral squamous cell carcinoma (OSCC), 3 samples of precancerous oral leukoplakia, and 8 adjacent normal samples. We found that tumor-infiltrating [CD4.sup.+] and [CD8.sup.+] T cells were functionally inhibited by immunosuppressive ligands expressed on various types of myeloid cells or neutrophils in the process of oral carcinogenesis. Notably, we identified a subset of myofibroblasts that exclusively expressed tryptophan 2,3-dioxygenase (TDO2). These [TDO2.sup.+] myofibroblasts were located distally from tumor nests, and both [CD4.sup.+] and [CD8.sup.+] T cells were enriched around them. Functional experiments revealed that [TDO2.sup.+] myofibroblasts were more likely to possess the ability for chemotaxis toward T cells but induced the transformation of [CD4.sup.+] T cells into Tregs and caused [CD8.sup.+] T cell dysfunction. We further showed that use of the TDO2 inhibitor LM10 attenuated the inhibitory states of T cells, restored the T cell antitumor response, and prevented the progression of OSCC malignant transformation in murine models. Our study reveals a multistep transcriptomic landscape of OSCC and demonstrates that [TDO2.sup.+] myofibroblasts are potential targets for immunotherapy., Introduction Oral squamous cell carcinoma (OSCC), a malignancy that occurs in the epithelium of the oral cavity, is the predominant type of head and neck squamous cell carcinoma (HNSCC). It [...]

Immune checkpoint blockade (ICB) has revolutionized cancer therapeutics. Desmoplastic malignancies, such as cholangiocarcinoma (CCA), have an abundant tumor immune microenvironment (TIME). However, to date, ICB monotherapy in such malignancies has been ineffective. Herein, we identify tumor-associated macrophages (TAMs) as the primary source of programmed death-ligand 1 (PD-L1) in human and murine CCA. In a murine model of CCA, recruited [PD-L1.sup.+] TAMs facilitated CCA progression. However, TAM blockade failed to decrease tumor progression due to a compensatory emergence of granulocytic myeloid-derived suppressor cells (G-MDSCs) that mediated immune escape by impairing T cell response. Singlecell RNA sequencing (scRNA-Seq) of murine tumor G-MDSCs highlighted a unique ApoE G-MDSC subset enriched with TAM blockade; further analysis of a human scRNA-Seq data set demonstrated the presence of a similar G-MDSC subset in human CCA. Finally, dual inhibition of TAMs and G-MDSCs potentiated ICB. In summary, our findings highlight the therapeutic potential of coupling ICB with immunotherapies targeting immunosuppressive myeloid cells in CCA., Introduction Cholangiocarcinoma (CCA) is the most common biliary malignancy and the second most common primary hepatic malignancy (1). CCAs are highly lethal neoplasms with limited therapeutic options and a dismal [...]

Intrahepatic neutrophil infiltration has been implicated in severe alcoholic hepatitis (SAH) pathogenesis; however, the mechanism underlying neutrophil-induced injury in SAH remains obscure. This translational study aims to describe the patterns of intrahepatic neutrophil infiltration and its involvement in SAH pathogenesis. Immunohistochemistry analyses of explanted livers identified two SAH phenotypes despite a similar clinical presentation, one with high intrahepatic neutrophils ([Neu.sup.hi]), but low levels of CD8* T cells, and vice versa. RNA-Seq analyses demonstrated that neutrophil cytosolic factor 1 (NCF1), a key factor in controlling neutrophilic ROS production, was upregulated and correlated with hepatic inflammation and disease progression. To study specifically the mechanisms related to [Neu.sup.hi] in AH patients and liver injury, we used the mouse model of chronic-plus-binge ethanol feeding and found that myeloid-specific deletion of the Ncf1 gene abolished ethanol-induced hepatic inflammation and steatosis. RNA-Seq analysis and the data from experimental models revealed that neutrophilic NCFI-dependent ROS promoted alcoholic hepatitis (AH) by inhibiting AMP-activated protein kinase (a key regulator of lipid metabolism) and microRNA-223 (a key antiinflammatory and antifibrotic microRNA). In conclusion, two distinct histopathological phenotypes based on liver immune phenotyping are observed in SAH patients, suggesting a separate mechanism driving liver injury and/or failure in these patients., Introduction Alcohol use disorder is a major contributor to several medical morbidities, including alcohol-associated liver disease (ALD) (1). There has been an exponential increase in the incidence of ALD worldwide, [...]

Mechanisms driving tumor progression from less aggressive subtypes to more aggressive states represent key targets for therapy. We identified a subset of luminal A primary breast tumors that give rise to HER2-enriched (HER2E) subtype metastases, but remain clinically HER2 negative (cHER2). By testing the unique genetic and transcriptomic features of these cases, we developed the hypothesis that FGFR4 likely participates in this subtype switching. To evaluate this, we developed 2 FGFR4 genomic signatures using a patient-derived xenograft (PDX) model treated with an FGFR4 inhibitor, which inhibited PDX growth in vivo. Bulk tumor gene expression analysis and single-cell RNA sequencing demonstrated that the inhibition of FGFR4 signaling caused molecular switching. In the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohort, FGFR4-induced and FGFR4-repressed signatures each predicted overall survival. Additionally, the FGFR4-induced signature was an independent prognostic factor beyond subtype and stage. Supervised analysis of 77 primary tumors with paired metastases revealed that the FGFR4-induced signature was significantly higher in luminal/[ER.sup.+] tumor metastases compared with their primaries. Finally, multivariate analysis demonstrated that the FGFR4-induced signature also predicted site-specific metastasis for lung, liver, and brain, but not for bone or lymph nodes. These data identify a link between FGFR4-regulated genes and metastasis, suggesting treatment options for FGFR4-positive patients, whose high expression is not caused by mutation or amplification., Introduction Treating metastatic breast cancer patients has become increasingly complex, in part due to the large number of therapeutic options in the second- and third-line settings. Tumor heterogeneity imparts another [...]

[beta] Cell apoptosis and dedifferentiation are 2 hotly debated mechanisms underlying [beta] cell loss in type 2 diabetes; however, the molecular drivers underlying such events remain largely unclear. Here, we performed a side-by-side comparison of mice carrying [beta] cell-specific deletion of ER-associated degradation (ERAD) and autophagy. We reported that, while autophagy was necessary for [beta] cell survival, the highly conserved Sel1L-Hrd1 ERAD protein complex was required for the maintenance of [beta] cell maturation and identity. Using single-cell RNA-Seq, we demonstrated that Sel1L deficiency was not associated with [beta] cell loss, but rather loss of [beta] cell identity. Sel1L-Hrd1 ERAD controlled [beta] cell identity via TGF-[beta] signaling, in part by mediating the degradation of TGF-[beta] receptor 1. Inhibition of TGF-[beta] signaling in Sel1L-deficient [beta] cells augmented the expression of [beta] cell maturation markers and increased the total insulin content. Our data revealed distinct pathogenic effects of 2 major proteolytic pathways in [beta] cells, providing a framework for therapies targeting distinct mechanisms of protein quality control., Introduction Type 2 diabetes (T2D) is a heterogeneous multicomponent disease, in which disease initiation and progression are triggered and propelled by pancreatic [beta] cell dysfunction and death. Recent studies have [...]

Highly structured RNA molecules usually interact with each other, and associate with various RNA-binding proteins, to regulate critical biological processes. However, RNA structures and interactions in intact cells remain largely unknown. Here, by coupling proximity ligation mediated by RNA-binding proteins with deep sequencing, we report an RNA in situ conformation sequencing (RIC-seq) technology for the global profiling of intra- and intermolecular RNA-RNA interactions. This technique not only recapitulates known RNA secondary structures and tertiary interactions, but also facilitates the generation of three-dimensional (3D) interaction maps of RNA in human cells. Using these maps, we identify noncoding RNA targets globally, and discern RNA topological domains and trans-interacting hubs. We reveal that the functional connectivity of enhancers and promoters can be assigned using their pairwise-interacting RNAs. Furthermore, we show that CCAT1-5L--a super-enhancer hub RNA--interacts with the RNA-binding protein hnRNPK, as well as RNA derived from the MYC promoter and enhancer, to boost MYC transcription by modulating chromatin looping. Our study demonstrates the power and applicability of RIC-seq in discovering the 3D structures, interactions and regulatory roles of RNA. RNA in situ conformation sequencing (RIC-seq) enables the generation of three-dimensional interaction maps of RNA in cells, which sheds light on the interactions and regulatory functions of RNA., Author(s): Zhaokui Cai [sup.1] [sup.2] , Changchang Cao [sup.1] , Lei Ji [sup.1] , Rong Ye [sup.1] [sup.2] , Di Wang [sup.1] [sup.2] , Cong Xia [sup.3] , Sui Wang [...]

Unequal utilization of synonymous codons is a well-known phenomenon among living organisms. This phenomenon plays a major role in the enhancement of the accuracy and efficiency of translation. Gymnosperms are rarely paid attention in this aspect. Understanding the degree of and determining the forces influencing codon usage bias (CUB) in Taxus contorta, an endangered Himalayan gymnosperm, will prove useful in interpreting the evolutionary characteristics of this species. Using RNAseq data, 93 790 assembled transcripts were clustered into 32 701 unigenes. Around 13 061 full-length sequences were utilized for the analysis of CUB. Compositional properties showed that GC-content ranged from 28.76% to 65.22%, with an average value of 44.28%, suggesting an AT-rich genome. The mean effective number of codons (ENC) value revealed that CUB is not strong in T. contorta. The preferred codons tended to be A/U ending, whereas the avoided codons tended to be G/C ending. A P2 index of 0.54 and a Mutation Responsive Index (MRI) value of -0.02 in addition to the results revealed by the neutrality, ENC, and parity plots showed that natural selection is a predominating factor governing CUB. Mutational pressure, gene length, hydropathiciy, aromaticity, and nucleotide composition influence CUB weakly. Key words: Taxus contorta, codon usage bias, selection, mutation, RNAseq. Resume : Un usage inegal de codons synonymes est un phenomene bien connu chez les organismes vivants. Ce phenomene joue un role important dans l'accroissement de la justesse et de l'efficience de la traduction. Cet aspect a cependant peu ete etudie chez les gymnospermes.La comprehension de l'etendue de ce phenomene et des forces qui influencent le biais des codons (CUB pour << codon usage bias >>) chez le Taxus contorta, une gymnosperme himalayenne menacee, sera utile pour interpreter les caracteristiques evolutives de cette espece. A l'aide de donnees RNA-seq, 93 790 transcrits assembles ont ete groupes en 32 701 unigenes. Environ 13 061 sequences completes ont ete employees pour effectuer l'analyse du CUB. Une analyse de la composition a revele que le contenu en GC variait entre 28,76 et 65,22 %, pour une moyenne de 44,28 %, ce qui suggere qu'il s'agit d'un genome riche en AT. En moyenne, la valeur ENC (<< effective number of codons >>) a revele que le CUB n'est pas fort chez le T. contorta. Les codons favorises tendaient a se terminer en A/U, tandis que les codons evites se terminaient le plus souvent en G/C. Un indice P2 de 0,54 et une valeur MRI (<< mutation responsive index >>) de -0,02, en plus des resultats reveles par la neutralite, l'ENC et les graphiques de parite, ont montre que la selection naturelle serait le facteur predominant determinent le CUB. La pression mutationnelle, la longueur des genes, l'hydropathie, l'aromaticite et la composition nucleotidique influencent faiblement le CUB. [Traduit par la Redaction] Mots-cles : Taxus contorta, biais dans l'usage des codons, selection, mutation, RNA-seq., Introduction Codon degeneracy ensures that certain amino acids are coded by more than one synonymous codon (Crick et al. 1961). The process of unequal usage of synonymous codons for specifying [...]

Despite advancements in targeting the immune checkpoints program cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) for cancer immunotherapy, a large number of patients and cancer types remain unresponsive. Current immunotherapies focus on modulating an antitumor immune response by directly or indirectly expanding antitumor CD8 T cells. A complementary strategy might involve inhibition of Tregs that otherwise suppress antitumor immune responses. Here, we sought to identify functional immune molecules preferentially expressed on tumor-infiltrating Tregs. Using genome-wide RNA-Seq analysis of purified Tregs sorted from multiple human cancer types, we identified a conserved Treg immune checkpoint signature. Using immunocompetent murine tumor models, we found that antibody-mediated depletion of 4-1BB-expressing cells (4-1BB is also known as TNFRSF9 or CD137) decreased tumor growth without negatively affecting CD8 T cell function. Furthermore, we found that the immune checkpoint 4-1BB had a high selectivity for human tumor Tregs and was associated with worse survival outcomes in patients with multiple tumor types. Thus, antibody-mediated depletion of 4-1BB-expressing Tregs represents a strategy with potential activity across cancer types., Introduction Immune checkpoint molecules are a group of surface proteins expressed on various immune cell subsets that can function as either stimulatory or inhibitory mediators, depending upon cell-specific and contextual [...]

Acute graft-versus-host disease (GVHD) can affect the central nervous system (CNS). The role of microglia in CNS-GVHD remains undefined. In agreement with microglia activation, we found that profound morphological changes and MHC-II and CD80 upregulation occurred upon GVHD induction. RNA sequencing-based analysis of purified microglia obtained from mice with CNS-GVHD revealed TNF upregulation. Selective TNF gene deletion in microglia of [Cx3cr1.sup.creER] [Tnf.sup.fl-] mice reduced MHC-II expression and decreased CNS T cell infiltrates and [VCAM-1.sup.+] endothelial cells. GVHD increased microglia TGF-[beta]-activated kinase-1 (TAK1) activation and NF-[kappa]B/p38 MAPK signaling. Selective Tak1 deletion in microglia using [Cx3cr1.sup.creER] [Tak1.sup.fl/fl] mice resulted in reduced TNF production and microglial MHC-II and improved neurocognitive activity. Pharmacological TAK1 inhibition reduced TNF production and MHC-II expression by microglia, Th1 and Th17 T cell infiltrates, and [VCAM-1.sup.+] endothelial cells and improved neurocognitive activity, without blocking graft- versus-leukemia effects. Consistent with these findings in mice, we observed increased activation and TNF production of microglia in the CNS of GVHD patients. In summary, we prove a role for microglia in CNS-GVHD, identify the TAK1/TNF/MHC-II axis as a mediator of CNS-GVHD, and provide a TAK1 inhibitor-based approach against GVHD-induced neurotoxicity., Introduction Acute graft-versus-host disease (GVHD) is a life-threatening complication after allogeneic hematopoietic cell transplantation (allo-HCT). About 50% of patients with severe acute GVHD fail to respond to corticosteroids, and steroid-refractory [...]

Pre-harvest soybean seeds in the field are susceptible to high temperature and humidity (HTH) stress, leading to pre-harvest seed deterioration, which will result in a reduction in grain quality, yield, and seed vigor. To understand the gene expression involved in seed deterioration response under HTH stress, in this study, we conducted an RNA-Seq analysis using two previously screened soybean cultivars with contrasting seed deterioration resistance. HTH stress induced 1081 and 357 differentially expressed genes (DEGs) in the sensitive cultivar Ningzhen No. 1 and resistant cultivar Xiangdou No. 3, respectively. The majority of DEGs in the resistant cultivar were up-regulated, while down-regulated DEGs were predominant in the sensitive cultivar. KEGG pathway analysis revealed that metabolic pathways, biosynthesis of secondary metabolites, and protein processing in endoplasmic reticulum were the predominant pathways in both cultivars during seed deterioration under HTH stress. The genes involved in photosynthesis, carbohydrate metabolism, lipid metabolism, and heat shock proteins pathways might contribute to the different response to seed deterioration under HTH treatment in the two soybean cultivars. Our study extends the knowledge of gene expression in soybean seed under HTH stress and further provides insight into the molecular mechanism of seed deterioration as well as new strategies for breeding soybean with improved seed deterioration resistance. Key words: soybean, high temperature and humidity, RNA-Seq, pre-harvest seed deterioration. Avant la recolte, les graines de soja au champ sont sensibles a un stress cause par une temperature et une humidite elevees (HTH), ce qui peut entrainer une deterioration des grains en pre-recolte, laquelle diminue la qualite des grains, le rendement et la vigueur des graines. Pour comprendre les profils d'expression genique associes a cette deterioration en reponse au stress HTH, les auteurs ont realise une etude RNA-Seq sur deux cultivars prealablement identifies comme presentant une resistance contrastee a cette deterioration. Le stress HTH a permis d'identifier, respectivement, 1081 et 357 genes a expression differentielle (DEG) chez le cultivar sensible Ningzhen No. 1 et le cultivar resistant Xiangdou No. 3. La majorite des DEG chez le cultivar resistant etaient exprimes a la hausse, tandis que les genes moins fortement exprimes etaient predominants chez le cultivar sensible. Une analyse des sentiers KEGG a revele que les 'sentiers metaboliques', la 'biosynthese des metabolites secondaires' et la 'maturation des proteines dans le reticulum endoplasmique' etaient les sentiers predominants touches lors de la deterioration des graines soumis a un stress HTH. Les genes impliques dans la photosynthese, le metabolisme des hydrates de carbone, le metabolisme des lipides et les proteines de choc thermique pourraient contribuer a la reponse differente observee entre les deux cultivars soumis a un traitement de stress HTH. Cette etude accroit les connaissances sur l'expression genique chez les graines de soja en reponse au stress HTH et fournit un eclairage sur le mecanisme moleculaire de la deterioration des graines ainsi que sur de nouvelles strategies pour selectionner des sojas dotes d'une meilleure resistance a la deterioration des graines. [Traduit par la Redaction] Mots-cles : soja, temperature et humidite elevees, RNA-Seq, deterioration des graines en pre-recolte., Introduction Soybean seeds provide major sources of oil and protein globally for human consumption and animal feed (Saha and Sultana 2008). Seed quality is determined by its composition of protein, [...]