Microglia-neuron signalling in the spinal cord is a key mediator of mechanical allodynia caused by peripheral nerve injury. We recently reported sex differences in microglia in pain signalling in ...mice: spinal mechanisms underlying nerve injury-induced allodynia are microglial dependent in male but not female mice. Whether this sex difference in pain hypersensitivity mechanisms is conserved in other species is unknown. Here, we show that in rats, the spinal mechanisms of nerve injury-induced hypersensitivity in males differ from those in females, with microglial P2X4 receptors (P2X4Rs) being a key point of divergence. In rats, nerve injury produced comparable allodynia and reactive microgliosis in both sexes. However, inhibiting microglia in the spinal cord reversed allodynia in male rats but not female rats. In addition, pharmacological blockade of P2X4Rs, by an intrathecally administered antagonist, attenuated pain hypersensitivity in male rats only. Consistent with the behavioural findings, nerve injury increased cell surface expression and function of P2X4Rs in acutely isolated spinal microglia from male rats but not from female rats. Moreover, in microglia cultured from male rats, but not in those from female rats, stimulating P2X4Rs drove intracellular signalling through p38 mitogen-activated protein kinase. Furthermore, chromatin immunoprecipitation-qPCR revealed that the transcription factor IRF5 differentially binds to the P2rx4 promoter region in female rats vs male rats. Finally, mechanical allodynia was produced in otherwise naive rats by intrathecally administering P2X4R-stimulated microglia from male rats but not those from female rats. Together, our findings demonstrate the existence of sexually dimorphic pain signalling in rats, suggesting that this sex difference is evolutionarily conserved, at least across rodent species.
Despite compelling antitumour activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in lung cancer, resistance to these therapies has ...increasingly been observed. In this study, to elucidate mechanisms of adaptive resistance, we analyse the tumour immune microenvironment in the context of anti-PD-1 therapy in two fully immunocompetent mouse models of lung adenocarcinoma. In tumours progressing following response to anti-PD-1 therapy, we observe upregulation of alternative immune checkpoints, notably T-cell immunoglobulin mucin-3 (TIM-3), in PD-1 antibody bound T cells and demonstrate a survival advantage with addition of a TIM-3 blocking antibody following failure of PD-1 blockade. Two patients who developed adaptive resistance to anti-PD-1 treatment also show a similar TIM-3 upregulation in blocking antibody-bound T cells at treatment failure. These data suggest that upregulation of TIM-3 and other immune checkpoints may be targetable biomarkers associated with adaptive resistance to PD-1 blockade.
Transcriptional elongation by RNA polymerase II (Pol II) is regulated by positive transcription elongation factor b (P-TEFb) in association with bromodomain-containing protein 4 (BRD4). We used ...genome-wide chromatin immunoprecipitation sequencing in primary human CD4+ T cells to reveal that BRD4 co-localizes with Ser-2-phosphorylated Pol II (Pol II Ser-2) at both enhancers and promoters of active genes. Disruption of bromodomain-histone acetylation interactions by JQ1, a small-molecule bromodomain inhibitor, resulted in decreased BRD4 binding, reduced Pol II Ser-2, and reduced expression of lineage-specific genes in primary human CD4+ T cells. A large number of JQ1-disrupted BRD4 binding regions exhibited diacetylated H4 (lysine 5 and -8) and H3K27 acetylation (H3K27ac), which correlated with the presence of histone acetyltransferases and deacetylases. Genes associated with BRD4/H3K27ac co-occupancy exhibited significantly higher activity than those associated with H3K27ac or BRD4 binding alone. Comparison of BRD4 binding in T cells and in human embryonic stem cells revealed that enhancer BRD4 binding sites were predominantly lineage-specific. Our findings suggest that BRD4-driven Pol II phosphorylation at serine 2 plays an important role in regulating lineage-specific gene transcription in human CD4+ T cells.
Background: BRD4 interacts with P-TEFb, which regulates Pol II elongation.
Results: Disruption of BRD4 binding by JQ1 resulted in reduced Pol II Ser-2 in CD4+ T cells.
Conclusion: BRD4 affects Pol II Ser-2 phosphorylation at a subset of lineage-specific active genes in primary human CD4+ T cells.
Significance: BRD4 binding may represent a means of identifying active promoters and lineage-specific enhancer elements.
Data from the 1000 Genomes project is quite often used as a reference for human genomic analysis. However, its accuracy needs to be assessed to understand the quality of predictions made using this ...reference. We present here an assessment of the genotyping, phasing, and imputation accuracy data in the 1000 Genomes project. We compare the phased haplotype calls from the 1000 Genomes project to experimentally phased haplotypes for 28 of the same individuals sequenced using the 10X Genomics platform.
We observe that phasing and imputation for rare variants are unreliable, which likely reflects the limited sample size of the 1000 Genomes project data. Further, it appears that using a population specific reference panel does not improve the accuracy of imputation over using the entire 1000 Genomes data set as a reference panel. We also note that the error rates and trends depend on the choice of definition of error, and hence any error reporting needs to take these definitions into account.
The quality of the 1000 Genomes data needs to be considered while using this database for further studies. This work presents an analysis that can be used for these assessments.
Squamous cell carcinomas (SCCs) are one of the most frequent forms of human malignancy, but, other than TP53 mutations, few causative somatic aberrations have been identified. We identified NOTCH1 or ...NOTCH2 mutations in ∼75% of cutaneous SCCs and in a lesser fraction of lung SCCs, defining a spectrum for the most prevalent tumor suppressor specific to these epithelial malignancies. Notch receptors normally transduce signals in response to ligands on neighboring cells, regulating metazoan lineage selection and developmental patterning. Our findings therefore illustrate a central role for disruption of microenvironmental communication in cancer progression. NOTCH aberrations include frameshift and nonsense mutations leading to receptor truncations as well as point substitutions in key functional domains that abrogate signaling in cell-based assays. Oncogenic gain-of-function mutations in NOTCH1 commonly occur in human T-cell lymphoblastic leukemia/lymphoma and B-cell chronic lymphocytic leukemia. The bifunctional role of Notch in human cancer thus emphasizes the context dependency of signaling outcomes and suggests that targeted inhibition of the Notch pathway may induce squamous epithelial malignancies.
Reduced knee weight-bearing from prescription or sedentary lifestyles are associated with cartilage degradation; effects on the meniscus are unclear. Rodents exposed to spaceflight or hind limb ...unloading (HLU) represent unique opportunities to evaluate this question. This study evaluated arthritic changes in the medial knee compartment that bears the highest loads across the knee after actual and simulated spaceflight, and recovery with subsequent full weight-bearing. Cartilage and meniscal degradation in mice were measured via microCT, histology, and proteomics and/or biochemically after: (1) ~ 35 days on the International Space Station (ISS); (2) 13-days aboard the Space Shuttle Atlantis; or (3) 30 days of HLU, followed by a 49-day weight-bearing readaptation with/without exercise. Cartilage degradation post-ISS and HLU occurred at similar spatial locations, the tibial-femoral cartilage-cartilage contact point, with meniscal volume decline. Cartilage and meniscal glycosaminoglycan content were decreased in unloaded mice, with elevated catabolic enzymes (e.g., matrix metalloproteinases), and elevated oxidative stress and catabolic molecular pathway responses in menisci. After the 13-day Shuttle flight, meniscal degradation was observed. During readaptation, recovery of cartilage volume and thickness occurred with exercise. Reduced weight-bearing from either spaceflight or HLU induced an arthritic phenotype in cartilage and menisci, and exercise promoted recovery.
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