Growth patterns in Onychophora (velvet worms): lack of a localised posterior proliferation zone

<p>Abstract</p> <p>Background</p> <p>During embryonic development of segmented animals, body segments are thought to arise from the so-called "posterior growth zone" and the occurrence of this "zone" has been used to support the homology of segmentation between arthropods, annelids, and vertebrates. However, the term "posterior growth zone" is used ambiguously in the literature, mostly referring to a region of increased proliferation at the posterior end of the embryo. To determine whether such a localised posterior proliferation zone is an ancestral feature of Panarthropoda (Onychophora + Tardigrada + Arthropoda), we examined cell division patterns in embryos of Onychophora.</p> <p>Results</p> <p>Using in vivo incorporation of the DNA replication marker BrdU (5-bromo-2'-deoxyuridine) and anti-phospho-histone H3 immunolabelling, we found that a localised posterior region of proliferating cells does not occur at any developmental stage in onychophoran embryos. This contrasts with a localised pattern of cell divisions at the posterior end of annelid embryos, which we used as a positive control. Based on our data, we present a mathematical model, which challenges the paradigm that a localised posterior proliferation zone is necessary for segment patterning in short germ developing arthropods.</p> <p>Conclusions</p> <p>Our findings suggest that a posterior proliferation zone was absent in the last common ancestor of Onychophora and Arthropoda. By comparing our data from Onychophora with those from annelids, arthropods, and chordates, we suggest that the occurrence of a "posterior growth zone" currently cannot be used to support the homology of segmentation between these three animal groups.</p

A kinome RNAi screen in Drosophila identifies novel genes interacting with Lgl, aPKC and Crb cell polarity genes in epithelial tissues

In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila ‘cell polarity’ eye and wing phenotypes. Initially we identified kinase or phosphatase genes whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, JNK, and Notch signalling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways, not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras and lipid/Phospho-inositol-3-kinase (PI3K) signalling pathways. Additionally, we demonstrated that the ‘nutrient sensing’ kinases, Salt Inducible Kinase 2 and 3 (SIK2 and 3) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, our screen has revealed novel cell-polarity interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development.This work was funded by a Contributing to Australian Scholarship and Science Foundation Science and Medicine Grant (SM-14-5398) awarded to L.M.P. and National Health and Medical Research Council (NHMRC) Project grant 628401 awarded to H.E.R. and N.G. H.E.R. was supported by a NHMRC fellowship (1020056), and funds from La Trobe University and the La Trobe Institute of Molecular Science

Cell cycle and growth stimuli regulate different steps of RNA polymerase I transcription

Transcription of the ribosomal RNA genes (rDNA) by RNA polymerase I (Pol I) is a major control step for ribosome synthesis and is tightly linked to cellular growth. However, the question of whether this process is modulated primarily at the level of transcription initiation or elongation is controversial. Studies in markedly different cell types have identified either initiation or elongation as the major control point. In this study, we have re-examined this question in NIH3T3 fibroblasts using a combination of metabolic labeling of the 47S rRNA, chromatin immunoprecipitation analysis of Pol I and overexpression of the transcription initiation factor Rrn3. Acute manipulation of growth factor levels altered rRNA synthesis rates over 8-fold without changing Pol I loading onto the rDNA. In fact, robust changes in Pol I loading were only observed under conditions where inhibition of rDNA transcription was associated with chronic serum starvation or cell cycle arrest. Overexpression of the transcription initiation factor Rrn3 increased loading of Pol I on the rDNA but failed to enhance rRNA synthesis in either serum starved, serum treated or G0/G1 arrested cells. Together these data suggest that transcription elongation is rate limiting for rRNA synthesis. We propose that transcription initiation is required for rDNA transcription in response to cell cycle cues, whereas elongation controls the dynamic range of rRNA synthesis output in response to acute growth factor modulation

Drosophila Ribosomal Protein Mutants Control Tissue Growth Non-Autonomously via Effects on the Prothoracic Gland and Ecdysone

The ribosome is critical for all aspects of cell growth due to its essential role in protein synthesis. Paradoxically, many Ribosomal proteins (Rps) act as tumour suppressors in Drosophila and vertebrates. To examine how reductions in Rps could lead to tissue overgrowth, we took advantage of the observation that an RpS6 mutant dominantly suppresses the small rough eye phenotype in a cyclin E hypomorphic mutant (cycEJP). We demonstrated that the suppression of cycEJP by the RpS6 mutant is not a consequence of restoring CycE protein levels or activity in the eye imaginal tissue. Rather, the use of UAS-RpS6 RNAi transgenics revealed that the suppression of cycEJP is exerted via a mechanism extrinsic to the eye, whereby reduced Rp levels in the prothoracic gland decreases the activity of ecdysone, the steroid hormone, delaying developmental timing and hence allowing time for tissue and organ overgrowth. These data provide for the first time a rationale to explain the counter-intuitive organ overgrowth phenotypes observed for certain members of the Minute class of Drosophila Rp mutants. They also demonstrate how Rp mutants can affect growth and development cell non-autonomously

International genome-wide meta-analysis identifies new primary biliary cirrhosis risk loci and targetable pathogenic pathways.

Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5 × 10(-8)) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine-cytokine pathways, for which relevant therapies exist

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste

Report on computational assessment of Tumor Infiltrating Lymphocytes from the International Immuno-Oncology Biomarker Working Group

Funder: U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)Funder: National Center for Research Resources under award number 1 C06 RR12463-01, VA Merit Review Award IBX004121A from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service, the DOD Prostate Cancer Idea Development Award (W81XWH-15-1-0558), the DOD Lung Cancer Investigator-Initiated Translational Research Award (W81XWH-18-1-0440), the DOD Peer Reviewed Cancer Research Program (W81XWH-16-1-0329), the Ohio Third Frontier Technology Validation Fund, the Wallace H. Coulter Foundation Program in the Department of Biomedical Engineering and the Clinical and Translational Science Award Program (CTSA) at Case Western Reserve University.Funder: Susan G Komen Foundation (CCR CCR18547966) and a Young Investigator Grant from the Breast Cancer Alliance.Funder: The Canadian Cancer SocietyFunder: Breast Cancer Research Foundation (BCRF), Grant No. 17-194Abstract: Assessment of tumor-infiltrating lymphocytes (TILs) is increasingly recognized as an integral part of the prognostic workflow in triple-negative (TNBC) and HER2-positive breast cancer, as well as many other solid tumors. This recognition has come about thanks to standardized visual reporting guidelines, which helped to reduce inter-reader variability. Now, there are ripe opportunities to employ computational methods that extract spatio-morphologic predictive features, enabling computer-aided diagnostics. We detail the benefits of computational TILs assessment, the readiness of TILs scoring for computational assessment, and outline considerations for overcoming key barriers to clinical translation in this arena. Specifically, we discuss: 1. ensuring computational workflows closely capture visual guidelines and standards; 2. challenges and thoughts standards for assessment of algorithms including training, preanalytical, analytical, and clinical validation; 3. perspectives on how to realize the potential of machine learning models and to overcome the perceptual and practical limits of visual scoring

Pitfalls in assessing stromal tumor infiltrating lymphocytes (sTILs) in breast cancer

Abstract: Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls

Application of a risk-management framework for integration of stromal tumor-infiltrating lymphocytes in clinical trials

Funder: Breast Cancer Research Foundation (BCRF); doi: https://doi.org/10.13039/100001006Abstract: Stromal tumor-infiltrating lymphocytes (sTILs) are a potential predictive biomarker for immunotherapy response in metastatic triple-negative breast cancer (TNBC). To incorporate sTILs into clinical trials and diagnostics, reliable assessment is essential. In this review, we propose a new concept, namely the implementation of a risk-management framework that enables the use of sTILs as a stratification factor in clinical trials. We present the design of a biomarker risk-mitigation workflow that can be applied to any biomarker incorporation in clinical trials. We demonstrate the implementation of this concept using sTILs as an integral biomarker in a single-center phase II immunotherapy trial for metastatic TNBC (TONIC trial, NCT02499367), using this workflow to mitigate risks of suboptimal inclusion of sTILs in this specific trial. In this review, we demonstrate that a web-based scoring platform can mitigate potential risk factors when including sTILs in clinical trials, and we argue that this framework can be applied for any future biomarker-driven clinical trial setting