Aberrant splice variants provide a potentially rich source of novel pathogenic drivers and biomarkers. Using the example of clear cell renal cell carcinoma, we describe a robust and widely applicable ...pipeline to identify disease-specific aberrant splice variants and explore associations with underlying disease biology and clinical outcomes.
Alternative mRNA splicing can be dysregulated in cancer, resulting in the generation of aberrant splice variants (SVs). Given the paucity of actionable genomic mutations in clear cell renal cell carcinoma (ccRCC), aberrant SVs may be an avenue to novel mechanisms of pathogenesis.
To identify and characterize aberrant SVs enriched in ccRCC.
Using RNA-seq data from the Cancer Cell Line Encyclopedia, we identified neojunctions uniquely expressed in ccRCC. Candidate SVs were then checked for expression across normal tissue in the Genotype-Tissue Expression Project and primary tumor tissue from The Cancer Genome Atlas (TCGA), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and our institutional Total Cancer Care database.
Clinicopathologic, genomic, and survival data were available for all cohorts. Epigenetic data were available for the TCGA and CPTAC cohorts. Proteomic data were available for the CPTAC cohort. The association of aberrant SV expression with these variables was examined using the Kruskal-Wallis test, pairwise t test, Spearman correlation test, and Cox regression analysis.
Our pipeline identified 16 ccRCC-enriched SVs. EGFR, HPCAL1-SV and RNASET2-SV expression was negatively correlated with gene-specific CpG methylation. We derived a survival risk score based primarily on the expression of five SVs (RNASET2, FGD1, PDZD2, COBLL1, and PTPN14), which was consistent and applicable across multiple cohorts on multivariate analysis. The splicing factor RBM4, which modulates splicing of HIF-1α, exhibited significantly lower expression at the protein level in the high-risk group, as defined by our SV-based score.
We describe 16 aberrant SVs enriched in ccRCC, many of which are associated with disease biology and/or clinical outcomes. This study provides a novel strategy for identifying and characterizing disease-specific aberrant SVs.
We describe a method to identify disease targets and biomarkers using transcriptomic analysis beyond somatic mutations or gene expression. Kidney tumors express unique splice variants that may provide additional prognostic information following surgery.
To describe a step-by-step methodology to establish a reproducible staining protocol for the evaluation of human corneal endothelial cells.
Four procedures were performed to determine the best ...protocol. (1) To determine the optimal trypan blue staining method, goat corneas were stained with 4 dilutions of trypan blue (0.4%, 0.2%, 0.1%, and 0.05%) and 1% alizarin red. (2) To determine the optimal alizarin red staining method, goat corneas were stained with 2 dilutions of alizarin red (1% and 0.5%) and 0.2% trypan blue. (3) To ensure that trypan blue truly stains damaged cells, goat corneas were exposed to either 3% hydrogen peroxide or to balanced salt solution, and then stained with 0.2% trypan blue and 0.5% alizarin red. (4) Finally, fresh human corneal buttons were examined; 1 group was stained with 0.2% trypan blue and another group with 0.4% trypan blue.
For the 4 procedures performed, the results are as follows: (1) trypan blue staining was not observed in any of the normal corneal samples; (2) 0.5% alizarin red demonstrated sharper cell borders than 1% alizarin red; (3) positive trypan blue staining was observed in the hydrogen peroxide exposed tissue in damaged areas; (4) 0.4% trypan blue showed more distinct positive staining than 0.2% trypan blue.
We were able to determine the optimal vital dye staining conditions for human corneal endothelial cells using 0.4% trypan blue and 0.5% alizarin red.
Profiling gene expression in human ocular tissues provides invaluable information for understanding ocular biology and investigating numerous ocular diseases. Accurate measurement of gene expression ...requires high-quality RNA, which often is a challenge with postmortem ocular tissues.
We examined the effect of various death to preservation (DP) times on the RNA quality of ten different ocular tissues. We used 16 eyes from eight different human donors. The eyes were preserved immediately in RNAlater or preserved after initial storage at 4 °C to create a range of DP times from 2 to 48 h. Ten ocular tissues were dissected from each eye. After total RNA was extracted from each dissected ocular tissue, the RNA integrity number (RIN) was determined using an Agilent Bioanalyzer.
The RIN values from corneal and trabecular meshwork tissues were significantly (p<0.05) higher than those from the ciliary body at an earlier DP time (<6 h), but were not different among all tissues after 8 h. Interestingly, the RIN values from non-vascularized tissues were significantly (p=0.0002) higher than those from vascularized ocular tissues at early DP times (<6 h). The RIN value from the cornea was significantly (p<0.05) higher at short DP times compared to longer DP times. The RIN values from corneal tissues were significantly correlated to DP time according to regression analysis (p<0.05).
In this study, we determined RNA quality from postmortem ocular tissues with various DP times. Our results emphasize the need for rapid preservation and processing of postmortem human donor eye tissues, especially for vascularized ocular tissues.
Background
Aged individuals are highly susceptible to adverse outcomes following traumatic brain injury (TBI). Despite worsened correlates related to functional outcomes following injury, as well as ...increased likelihood to acquire a TBI, our understanding of the cellular mechanisms governing the poorer outcomes associated with aged individuals post‐TBI are not well‐characterized.
Method
In the current study, we examined microglial heterogeneity following TBI, in our mouse model of focal TBI across both acute (3d) and chronic (28d) intervals in both young (4m) and aged (18m) male C57B6J mice. At the appropriate interval, brains were collected and processed for single cell RNAseq (scRNAseq), spatial transcriptomics, and histology. A follow up study examining the interaction of TBI and aging upon T cell receptor (TCR) sequences was utilized to examine clonal expansion and lymphocyte heterogeneity in young and aged mice chronically after TBI.
Result
Our findings demonstrate that TBI drives diverse transcriptional heterogeneity of microglia in both the young and aged condition. Acutely at 3 days post injury an inflammatory response is seen both in the young and aged mice. However, chronically at 28 days post injury, the microglia from young animals more closely resemble their pre‐injury (i.e. Sham) phenotypes, in comparison to the aged brain where there is a persistence of Apoe‐linked ‘DAM‐like’ microglial response. Inferential analyses point toward these aging‐related chronic populations of microglia as having distinct transcription factor utilization and metabolic pathway enrichment, including Hif1α upregulation and interferon gamma (INFγ) signaling. Furthermore, linkage with chronically accumulated CD8+ T cells and interferon signaling may underlie these phenotypes. Using single cell TCR sequencing, we demonstrate stark contrasts in the responses due to aging and TBI in the clonal expansion of infiltrated CD8+ T cells chronically following TBI.
Conclusion
Our studies demonstrate that aging is a centrally associated with the persistence of chronically reactive microglia in the brain and that these responses are linked, in part, with the protracted accumulation of CD8+ T cells following TBI.
The 5 th -Generation Intel Xeon Scalable Processors (codenamed Emerald Rapids) support up to 64 cores, greater than 300MB shared L3 cache, 8 DDR5 channels at 5600MT/s with 1DPC, 32GT/s PCIe/CXL ...lanes, 20GT/s UPI lanes composed of 2 die (Fig. 2.3.1) in a multichip package. This generation delivers an 18% performance improvement for general integer compute workloads and a 24% improvement for floating-point workloads at iso power vs. the 4 th -Gen Xeon processors 1. This is achieved through an improved core, enhanced process technology, increased core count, significantly larger cache, higher DDR memory speeds, die reductions, and power efficiency improvements at idle conditions. Manufacturing is on the Intel 7 process technology optimized for server usage with improved transistor speed and a focus on leakage and dynamic capacitance reduction enabling +3% frequency/W enhancements over the prior generation process revision. The key power-efficiency improvements at idle were achieved through improvements to the fully integrated voltage regulators (FIVR) 2, 3 to decrease the regulator losses at lower utilization, enhanced active idle detection and power savings, and package C6 power reduction.
An Itanium® processor implemented in 32 nm CMOS with nine layers of Cu contains 3.1 billion transistors. The die measures 18.2 mm by 29.9 mm. The processor has eight multi-threaded cores, a ring ...based system interface and combined cache on the die is 50 MB. High-speed links allow for peak processor-to-processor bandwidth of up to 128 GB/s and memory bandwidth of up to 45 GB/s.
Traumatic brain injury (TBI) is the most common cause of death and disability in young adults, yet the molecular mechanisms that follow TBI are poorly understood. We previously reported a ...perturbation in iron (Fe) levels following TBI. Here we report that the distribution of cobalt (Co) is modulated in post-mortem human brain following injury. We also investigated how the distribution of other biologically relevant elements changes in TBI. Cobalt is increased due to TBI while copper (Cu), magnesium (Mg), manganese (Mn), phosphorus (P), potassium (K), rubidium (Rb), selenium (Se) and zinc (Zn) remain unchanged. The elevated Co has important implications for positron emission tomography neuroimaging. This is the first demonstration of the accumulation of Co in injured tissue explaining the previous utility of (55)Co-PET imaging in TBI.
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