Glioblastomas harbor diverse cell populations, including rare glioblastoma stem cells (GSCs) that drive tumorigenesis. To characterize functional diversity within this population, we performed ...single-cell RNA sequencing on >69,000 GSCs cultured from the tumors of 26 patients. We observed a high degree of inter- and intra-GSC transcriptional heterogeneity that could not be fully explained by DNA somatic alterations. Instead, we found that GSCs mapped along a transcriptional gradient spanning two cellular states reminiscent of normal neural development and inflammatory wound response. Genome-wide CRISPR-Cas9 dropout screens independently recapitulated this observation, with each state characterized by unique essential genes. Further single-cell RNA sequencing of >56,000 malignant cells from primary tumors found that the majority organize along an orthogonal astrocyte maturation gradient yet retain expression of founder GSC transcriptional programs. We propose that glioblastomas grow out of a fundamental GSC-based neural wound response transcriptional program, which is a promising target for new therapy development.
Brain-predicted age difference scores are calculated by subtracting chronological age from ‘brain’ age, which is estimated using neuroimaging data. Positive scores reflect accelerated ageing and are ...associated with increased mortality risk and poorer physical function. To date, however, the relationship between brain-predicted age difference scores and specific cognitive functions has not been systematically examined using appropriate statistical methods. First, applying machine learning to 1359 T1-weighted MRI scans, we predicted the relationship between chronological age and voxel-wise grey matter data. This model was then applied to MRI data from three independent datasets, significantly predicting chronological age in each dataset: Dokuz Eylül University (
n
= 175), the Cognitive Reserve/Reference Ability Neural Network study (
n
= 380), and The Irish Longitudinal Study on Ageing (
n
= 487). Each independent dataset had rich neuropsychological data. Brain-predicted age difference scores were significantly negatively correlated with performance on measures of general cognitive status (two datasets); processing speed, visual attention, and cognitive flexibility (three datasets); visual attention and cognitive flexibility (two datasets); and semantic verbal fluency (two datasets). As such, there is firm evidence of correlations between increased brain-predicted age differences and reduced cognitive function in some domains that are implicated in cognitive ageing.
One mechanism by which genetic factors influence complex traits and diseases is altering gene expression. Direct measurement of gene expression in relevant tissues is rarely tenable; however, ...genetically regulated gene expression (GReX) can be estimated using prediction models derived from large multi-omic datasets. These approaches have led to the discovery of many gene-trait associations, but whether models derived from predominantly European ancestry (EA) reference panels can map novel associations in ancestrally diverse populations remains unclear. We applied PrediXcan to impute GReX in 51,520 ancestrally diverse Population Architecture using Genomics and Epidemiology (PAGE) participants (35% African American, 45% Hispanic/Latino, 10% Asian, and 7% Hawaiian) across 25 key cardiometabolic traits and relevant tissues to identify 102 novel associations. We then compared associations in PAGE to those in a random subset of 50,000 White British participants from UK Biobank (UKBB50k) for height and body mass index (BMI). We identified 517 associations across 47 tissues in PAGE but not UKBB50k, demonstrating the importance of diverse samples in identifying trait-associated GReX. We observed that variants used in PrediXcan models were either more or less differentiated across continental-level populations than matched-control variants depending on the specific population reflecting sampling bias. Additionally, variants from identified genes specific to either PAGE or UKBB50k analyses were more ancestrally differentiated than those in genes detected in both analyses, underlining the value of population-specific discoveries. This suggests that while EA-derived transcriptome imputation models can identify new associations in non-EA populations, models derived from closely matched reference panels may yield further insights. Our findings call for more diversity in reference datasets of tissue-specific gene expression.
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Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in ...preventing sexual virus transmission. In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion-amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses.
Since genotype imputation was introduced, researchers have been relying on the estimated imputation quality from imputation software to perform post-imputation quality control (QC). However, this ...quality estimate (denoted as Rsq) performs less well for lower-frequency variants. We recently published MagicalRsq, a machine-learning-based imputation quality calibration, which leverages additional typed markers from the same cohort and outperforms Rsq as a QC metric. In this work, we extended the original MagicalRsq to allow cross-cohort model training and named the new model MagicalRsq-X. We removed the cohort-specific estimated minor allele frequency and included linkage disequilibrium scores and recombination rates as additional features. Leveraging whole-genome sequencing data from TOPMed, specifically participants in the BioMe, JHS, WHI, and MESA studies, we performed comprehensive cross-cohort evaluations for predominantly European and African ancestral individuals based on their inferred global ancestry with the 1000 Genomes and Human Genome Diversity Project data as reference. Our results suggest MagicalRsq-X outperforms Rsq in almost every setting, with 7.3%–14.4% improvement in squared Pearson correlation with true R2, corresponding to 85–218 K variant gains. We further developed a metric to quantify the genetic distances of a target cohort relative to a reference cohort and showed that such metric largely explained the performance of MagicalRsq-X models. Finally, we found MagicalRsq-X saved up to 53 known genome-wide significant variants in one of the largest blood cell trait GWASs that would be missed using the original Rsq for QC. In conclusion, MagicalRsq-X shows superiority for post-imputation QC and benefits genetic studies by distinguishing well and poorly imputed lower-frequency variants.
Ever-growing reference panels allow imputation of a huge number (∼108) of lower-frequency variants. However, the standard imputation quality metric poorly reflects the true quality of uncommon variants. We introduce MagicalRsq-X, an extension of MagicalRsq that allows model training across cohorts for which only the genotypes used for imputation are available.
There is an unmet need to develop imaging methods for the early and objective assessment of breast tumors to therapy. 3'-Deoxy-3'-18Ffluorothymidine (18FFLT)-positron emission tomography represents a ...new approach to imaging thymidine kinase activity, and hence, cellular proliferation. We compared graphical, spectral, and semiquantitative analytic methodologies for quantifying 18FFLT kinetics in tumor and normal tissue of patients with locally advanced and metastatic breast cancer. The resultant kinetic parameters were correlated with the Ki-67 labeling index from tumor biopsies. 18FFLT accumulation was detected in primary tumor, nodal disease, and lung metastasis. In large tumors, there was substantial heterogeneity in regional radiotracer uptake, reflecting heterogeneity in cellular proliferation; radiotracer uptake in primary tumors also differed from that of metastases. 18FFLT was metabolized in patients to a single metabolite 18FFLT-glucuronide. Unmetabolized 18FFLT accounted for 71.54 +/- 1.50% of plasma radioactivity by 90 minutes. The rate constant for the metabolite-corrected net irreversible uptake of 18FFLT (Ki) ranged from 0.6 to 10.4 x 10(-4) and from 0 to 0.6 x 10(-4) mL plasma cleared/s/mL tissue in tumor (29 regions, 15 patients) and normal tissues, respectively. Tumor Ki and fractional retention of radiotracer determined by spectral analysis correlated with Ki-67 labeling index (r = 0.92, P < 0.0001 and r = 0.92, P < 0.0001, respectively). These correlations were superior to those determined by semiquantitative methods. We conclude that 18FFLT-positron emission tomography is a promising clinical tool for imaging cellular proliferation in breast cancer, and is most predictive when analyzed by graphical and spectral methods.
Systemic low-grade inflammation is a feature of chronic disease. C-reactive protein (CRP) is a common biomarker of inflammation and used as an indicator of disease risk; however, the role of ...inflammation in disease is not completely understood. Methylation is an epigenetic modification in the DNA which plays a pivotal role in gene expression. In this study we evaluated differential DNA methylation patterns associated with blood CRP level to elucidate biological pathways and genetic regulatory mechanisms to improve the understanding of chronic inflammation. The racially and ethnically diverse participants in this study were included as 50% White, 41% Black or African American, 7% Hispanic or Latino/a, and 2% Native Hawaiian, Asian American, American Indian, or Alaska Native (total
= 13,433) individuals. We replicated 113 CpG sites from 87 unique loci, of which five were novel (
,
, and cg03282312), across a discovery set of 1,150 CpG sites associated with CRP level (
< 1.2E-7). The downstream pathways affected by DNA methylation included the identification of
and
CpG-gene transcript pairs which contributed to the innate immune response gene enrichment pathway along with
,
, and
. Gene enrichment analysis also identified the nuclear factor-kappaB transcription pathway. Using two-sample Mendelian randomization (MR) we inferred methylation at three CpG sites as causal for CRP levels using both White and Black or African American MR instrument variables. Overall, we identified novel CpG sites and gene transcripts that could be valuable in understanding the specific cellular processes and pathogenic mechanisms involved in inflammation.
This study sought to evaluate the reproducibility of (11)Ccholine-positron emission tomography and the effect of trastuzumab in breast cancer.
Twenty-one patients with newly diagnosed and recurrent ...breast cancer stage II-IV had a baseline dynamic (11)Ccholine-PET scan, 10 patients had a second (11)Ccholine-PET scan to examine reproducibility, and 6 patients had a second scan within a month after trastuzumab. Analysis of (11)Ccholine uptake was measured as the semiquantitative standardized uptake value at 30 and 60 minutes (SUV(30) and SUV(60)), and quantitatively as the net irreversible retention of the radiotracer at steady-state (Ki) and plasma to tissue exchange at 60 minutes (IRF60min).
Breast tumor lesions in all patients were visualized by (11)Ccholine PET. The difference in tumor versus normal tissue uptake was significant for SUV(30), SUV(60), Ki, and IRF60 minutes (Wilcoxon P < 0.0001). At 60 minutes postinjection, 15.1 +/- 2.16% of plasma radioactivity was due to unmetabolized (11)Ccholine radioactivity. (11)CCholine uptake was reproducible in breast tumor lesions (r(2) = 0.9 for SUV, 0.9 for Ki, and 0.8 for IRF60). Early responses to trastuzumab measured by (11)Ccholine-PET were significant in three lesions occurring in two patients who responded clinically.
(11)CCholine-PET uptake variables can be reproducibly assessed. Initial studies show that trastuzumab decreases (11)Ccholine uptake.
Purpose: We showed in preclinical models that thymidylate synthase (TS) inhibition leads to redistribution of the nucleoside transporter,
ENT1, to the cell membrane and hence increases tissue uptake ...of 18 Ffluorothymidine (FLT). In this study, we assessed for the first time the altered pharmacokinetics of FLT in patients following
administration of capecitabine, a drug whose mode of action has been reported to include TS inhibition.
Experimental Design: We analyzed 10 lesions from six patients with breast cancer by positron emission tomography before and after treatment with
capecitabine. Although drug treatment did not alter tumor delivery pharmacokinetic variables (K1 and permeability product
surface area) or blood flow, tumor FLT retention variables increased with drug treatment in all but one patient.
Results: The baseline average standardized uptake value at 60 minutes, rate constant for the net irreversible transfer of radiotracer
from plasma to tumor (Ki), and unit impulse response function at 60 minutes were 11.11 × 10 −5 m 2 /mL, 4.38 × 10 −2 mL plasma/min/mL tissue, and 4.93 × 10 −2 /min, respectively. One hour after capecitabine administration, the standardized uptake value was 13.55 × 10 −5 m 2 /mL ( P = 0.004), Ki 7.40 × 10 −2 mL plasma/min/mL tissue ( P = 0.004), and impulse response function was 7.40 × 10 −2 /min ( P = 0.002). FLT pharmacokinetics did not change in normal tissues, suggesting that the effect was largely restricted to tumors
( P = 0.55).
Conclusions: We have identified FLT positron emission tomography retention parameters that could be used in future early clinical studies
to measure the pharmacodynamics of TS inhibitors, as well as for identifying patients who are unlikely to benefit from TS
inhibition. (Clin Cancer Res 2009;15(21):6649–57)
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