Cross-tissue immune cell analysis reveals tissue-specific adaptations and clonal architecture in humans

Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. Here, we surveyed the immune compartment of 15 tissues of six deceased adult donors by single-cell RNA sequencing and paired VDJ sequencing. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of 45 finely phenotyped immune cell types and states, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. In summary, our multi-tissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis and antigen receptor sequencing. One Sentence Summary We provide an immune cell atlas, including antigen receptor repertoire profiling, across lymphoid and non-lymphoid human tissues

Evaluation of the current knowledge limitations in breast cancer research: a gap analysis

BACKGROUND A gap analysis was conducted to determine which areas of breast cancer research, if targeted by researchers and funding bodies, could produce the greatest impact on patients. METHODS Fifty-six Breast Cancer Campaign grant holders and prominent UK breast cancer researchers participated in a gap analysis of current breast cancer research. Before, during and following the meeting, groups in seven key research areas participated in cycles of presentation, literature review and discussion. Summary papers were prepared by each group and collated into this position paper highlighting the research gaps, with recommendations for action. RESULTS Gaps were identified in all seven themes. General barriers to progress were lack of financial and practical resources, and poor collaboration between disciplines. Critical gaps in each theme included: (1) genetics (knowledge of genetic changes, their effects and interactions); (2) initiation of breast cancer (how developmental signalling pathways cause ductal elongation and branching at the cellular level and influence stem cell dynamics, and how their disruption initiates tumour formation); (3) progression of breast cancer (deciphering the intracellular and extracellular regulators of early progression, tumour growth, angiogenesis and metastasis); (4) therapies and targets (understanding who develops advanced disease); (5) disease markers (incorporating intelligent trial design into all studies to ensure new treatments are tested in patient groups stratified using biomarkers); (6) prevention (strategies to prevent oestrogen-receptor negative tumours and the long-term effects of chemoprevention for oestrogen-receptor positive tumours); (7) psychosocial aspects of cancer (the use of appropriate psychosocial interventions, and the personal impact of all stages of the disease among patients from a range of ethnic and demographic backgrounds). CONCLUSION Through recommendations to address these gaps with future research, the long-term benefits to patients will include: better estimation of risk in families with breast cancer and strategies to reduce risk; better prediction of drug response and patient prognosis; improved tailoring of treatments to patient subgroups and development of new therapeutic approaches; earlier initiation of treatment; more effective use of resources for screening populations; and an enhanced experience for people with or at risk of breast cancer and their families. The challenge to funding bodies and researchers in all disciplines is to focus on these gaps and to drive advances in knowledge into improvements in patient care

Dual FGF-2 and Intergrin α5β1 Signaling Mediate GRAF-Induced RhoA Inactivation in a Model of Breast Cancer Dormancy

Interactions with the bone marrow stroma regulate dormancy and survival of breast cancer micrometastases. In an in vitro model of dormancy in the bone marrow, we previously demonstrated that estrogen-dependent breast cancer cells are partially re-differentiated by FGF-2, re-express integrin α5β1 lost with malignant transformation and acquire an activated PI3K/Akt pathway. Ligation of integrin α5β1 by fibronectin and activation of the PI3K pathway both contribute to survival of these dormant cells. Here, we investigated mechanisms responsible for the dormant phenotype. Experiments demonstrate that integrin α5β1 controls de novo cytoskeletal rearrangements, cell spreading, focal adhesion kinase rearrangement to the cell perimeter and recruitment of a RhoA GAP known as GRAF. This results in the inactivation of RhoA, an effect which is necessary for the stabilization of cortical actin. Experiments also demonstrate that activation of the PI3K pathway by FGF-2 is independent of integrin α5β1 and is also required for cortical actin reorganization, GRAF membrane relocalization and RhoA inactivation. These data suggest that GRAF-mediated RhoA inactivation and consequent phenotypic changes of dormancy depend on dual signaling by FGF-2-initiated PI3K activation and through ligation of integrin α5β1 by fibronectin

The Effect of Anandamide on Uterine Nitric Oxide Synthase Activity Depends on the Presence of the Blastocyst

Nitric oxide production, catalyzed by nitric oxide synthase (NOS), should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot−1 h−1) compared to days 4 (0.34±0.03) and 5 (0.35±0.02) of pregnancy and to day 6 implantation sites (0.33±0.01). This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA), an endocannabinoid, binds to cannabinoid receptors type 1 (CB1) and type 2 (CB2), and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA) and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04) and URB-597 (1.08±0.09 vs 0.83±0.06) inhibited NOS activity in the absence of a blastocyst (pseudopregnancy) through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05). While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02), a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01). Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These data establish cannabinoid receptors as an interesting target for the treatment of implantation deficiencies

The Open AUC Project

Progress in analytical ultracentrifugation (AUC) has been hindered by obstructions to hardware innovation and by software incompatibility. In this paper, we announce and outline the Open AUC Project. The goals of the Open AUC Project are to stimulate AUC innovation by improving instrumentation, detectors, acquisition and analysis software, and collaborative tools. These improvements are needed for the next generation of AUC-based research. The Open AUC Project combines on-going work from several different groups. A new base instrument is described, one that is designed from the ground up to be an analytical ultracentrifuge. This machine offers an open architecture, hardware standards, and application programming interfaces for detector developers. All software will use the GNU Public License to assure that intellectual property is available in open source format. The Open AUC strategy facilitates collaborations, encourages sharing, and eliminates the chronic impediments that have plagued AUC innovation for the last 20 years. This ultracentrifuge will be equipped with multiple and interchangeable optical tracks so that state-of-the-art electronics and improved detectors will be available for a variety of optical systems. The instrument will be complemented by a new rotor, enhanced data acquisition and analysis software, as well as collaboration software. Described here are the instrument, the modular software components, and a standardized database that will encourage and ease integration of data analysis and interpretation software

Mitochondrial Physiology and Gene Expression Analyses Reveal Metabolic and Translational Dysregulation in Oocyte-Induced Somatic Nuclear Reprogramming

While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT), the enucleated oocyte (ooplasm) must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene). We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism