In animals a discrete class of small RNAs, the piwi-interacting RNAs (piRNAs), guard germ cell genomes against the activity of mobile genetic elements. piRNAs are generated, via an unknown mechanism, ...from apparently single-stranded precursors that arise from discrete genomic loci, termed piRNA clusters. Presently, little is known about the signals that distinguish a locus as a source of piRNAs. It is also unknown how individual piRNAs are selected from long precursor transcripts. To address these questions, we inserted new artificial sequence information into piRNA clusters and introduced these marked clusters as transgenes into heterologous genomic positions in mice and flies. Profiling of piRNA from transgenic animals demonstrated that artificial sequences were incorporated into the piRNA repertoire. Transgenic piRNA clusters are functional in non-native genomic contexts in both mice and flies, indicating that the signals that define piRNA generative loci must lie within the clusters themselves rather than being implicit in their genomic position. Comparison of transgenic animals that carry insertions of the same artificial sequence into different ectopic piRNA-generating loci showed that both local and long-range sequence environments inform the generation of individual piRNAs from precursor transcripts.
Genomic alterations shape cell phenotypes and the structure of tumor ecosystems in poorly defined ways. To investigate these relationships, we used imaging mass cytometry to quantify the expression ...of 37 proteins with subcellular spatial resolution in 483 tumors from the METABRIC cohort. Single-cell analysis revealed cell phenotypes spanning epithelial, stromal and immune types. Distinct combinations of cell phenotypes and cell-cell interactions were associated with genomic subtypes of breast cancer. Epithelial luminal cell phenotypes separated into those predominantly impacted by mutations and those affected by copy number aberrations. Several features of tumor ecosystems, including cellular neighborhoods, were linked to prognosis, illustrating their clinical relevance. In summary, systematic analysis of single-cell phenotypic and spatial correlates of genomic alterations in cancer revealed how genomes shape both the composition and architecture of breast tumor ecosystems and will enable greater understanding of the phenotypic impact of genomic alterations.
To evaluate single-agent liposomal daunorubicin chemotherapy in the management of early HIV-related Kaposi's sarcoma (KS).
Randomized cross-over comparison of liposomal daunorubicin versus ...observation.
Study conducted at single site in tertiary referral HIV unit.
Twenty-nine HIV-seropositive men with < 20 cutaneous KS, no visceral involvement and CD4 cell counts < 400 x 10(6)/I were randomized. Adequate haematological, hepatic and renal function was required for entry. A left ventricular ejection fraction of > 45% was necessary for eligibility.
Patients were randomized to 12 weeks observation or 12 weeks of liposomal daunorubicin 40 mg/m2 every 2 weeks. After 12 weeks, or at disease progression, patients were crossed over to receive the alternative arm.
Disease evaluation was according to AIDS Clinical Trials Group criteria for response assessment and toxicity was recorded using the World Health Organization standardized grading.
Response rate to initial liposomal daunorubicin was six out of 15 (40%) and none experienced a spontaneous response during the observation arm. Six patients (40%) randomized to the initial chemotherapy arm progressed during chemotherapy, while 10(72%) in the observation arm progressed. Neutropenia was the main toxicity associated with liposomal daunorubicin and was documented following 20 out of 139(14%) treatment cycles.
Liposomal daunorubicin is a well tolerated and efficacious treatment for early KS; however, the duration of response is brief.
A holistic understanding of tissue and organ structure and function requires the detection of molecular constituents in their original three-dimensional (3D) context. Imaging mass cytometry (IMC) ...enables simultaneous detection of up to 40 antigens and transcripts using metal-tagged antibodies but has so far been restricted to two-dimensional imaging. Here we report the development of 3D IMC for multiplexed 3D tissue analysis at single-cell resolution and demonstrate the utility of the technology by analysis of human breast cancer samples. The resulting 3D models reveal cellular and microenvironmental heterogeneity and cell-level tissue organization not detectable in two dimensions. 3D IMC will prove powerful in the study of phenomena occurring in 3D space such as tumor cell invasion and is expected to provide invaluable insights into cellular microenvironments and tissue architecture.
RNA interference (RNAi) regulates gene expression by the cleavage of messenger RNA, by mRNA degradation and by preventing protein synthesis. These effects are mediated by a ribonucleoprotein complex ...known as RISC (RNA-induced silencing complex). We have previously identified four Drosophila components (short interfering RNAs, Argonaute 2 (ref. 2), VIG and FXR) of a RISC enzyme that degrades specific mRNAs in response to a double-stranded-RNA trigger. Here we show that Tudor-SN (tudor staphylococcal nuclease)-a protein containing five staphylococcal/micrococcal nuclease domains and a tudor domain-is a component of the RISC enzyme in Caenorhabditis elegans, Drosophila and mammals. Although Tudor-SN contains non-canonical active-site sequences, we show that purified Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases. Notably, both purified Tudor-SN and RISC are inhibited by a specific competitive inhibitor of micrococcal nuclease. Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi.
Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion ...microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale.
The heterogeneity of breast cancer plays a major role in drug response and resistance and has been extensively characterized at the genomic level. Here, a single-cell breast cancer mass cytometry ...(BCMC) panel is optimized to identify cell phenotypes and their oncogenic signalling states in a biobank of patient-derived tumour xenograft (PDTX) models representing the diversity of human breast cancer. The BCMC panel identifies 13 cellular phenotypes (11 human and 2 murine), associated with both breast cancer subtypes and specific genomic features. Pre-treatment cellular phenotypic composition is a determinant of response to anticancer therapies. Single-cell profiling also reveals drug-induced cellular phenotypic dynamics, unravelling previously unnoticed intra-tumour response diversity. The comprehensive view of the landscapes of cellular phenotypic heterogeneity in PDTXs uncovered by the BCMC panel, which is mirrored in primary human tumours, has profound implications for understanding and predicting therapy response and resistance.
This study aimed to test the hypothesis that the total daily energy expenditure (TDEE) of male academy soccer players is greater than players not enrolled on a formalised academy programme. English ...Premier League academy (ACAD: n = 8, 13 years, 50 ± 6 kg, 88 ± 3% predicted adult stature, PAS) and non-academy players (NON-ACAD: n = 6, 13 years, 53 ± 12 kg, 89 ± 3% PAS) were assessed for TDEE (via doubly labelled water) during a 14-day in-season period. External loading was evaluated during training (ACAD: 8 sessions, NON-ACAD: 2 sessions) and games (2 games for both ACAD and NON-ACAD) via GPS, and daily physical activity was evaluated using triaxial accelerometry. Accumulative duration of soccer activity (ACAD: 975 ± 23 min, NON-ACAD: 397 ± 2 min; p < 0.01), distance covered (ACAD: 54.2 ± 8.3 km, NON-ACAD: 21.6 ± 4.7 km; p < 0.05) and time engaged in daily moderate-to-vigorous (ACAD: 124 ± 17 min, NON-ACAD: 79 ± 18 min; p < 0.01) activity was greater in academy players. Academy players displayed greater absolute (ACAD: 3380 ± 517 kcal · d
−1
, NON-ACAD: 2641 ± 308 kcal · d
−1
; p < 0.05) and relative TDEE (ACAD: 66 ± 6 kcal · kg · d
−1
, NON-ACAD: 52 ± 10 kcal · kg · d
−1
; p < 0.05) versus non-academy players. Given the injury risk associated with high training volumes during growth and maturation, data demonstrate the requirement for academy players to consume sufficient energy (and carbohydrate) intake to support the enhanced energy cost of academy programmes.
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