ALBERT

Description: The Orthologous Matrix (OMA) project is a method and associated database inferring evolutionary relationships amongst currently 1706 complete proteomes (i.e. the protein sequence associated for every protein-coding gene in all genomes). In this update article, we present six major new developments in OMA: (i) a new web interface; (ii) Gene Ontology function predictions as part of the OMA pipeline; (iii) better support for plant genomes and in particular homeologs in the wheat genome; (iv) a new synteny viewer providing the genomic context of orthologs; (v) statically computed hierarchical orthologous groups subsets downloadable in OrthoXML format; and (vi) possibility to export parts of the all-against-all computations and to combine them with custom data for ‘client-side’ orthology prediction. OMA can be accessed through the OMA Browser and various programmatic interfaces at http://omabrowser.org .

Description: Phylogenetic inference can potentially result in a more accurate tree using data from multiple loci. However, if the loci are incongruent—due to events such as incomplete lineage sorting or horizontal gene transfer—it can be misleading to infer a single tree. To address this, many previous contributions have taken a mechanistic approach, by modeling specific processes. Alternatively, one can cluster loci without assuming how these incongruencies might arise. Such "process-agnostic" approaches typically infer a tree for each locus and cluster these. There are, however, many possible combinations of tree distance and clustering methods; their comparative performance in the context of tree incongruence is largely unknown. Furthermore, because standard model selection criteria such as AIC cannot be applied to problems with a variable number of topologies, the issue of inferring the optimal number of clusters is poorly understood. Here, we perform a large-scale simulation study of phylogenetic distances and clustering methods to infer loci of common evolutionary history. We observe that the best-performing combinations are distances accounting for branch lengths followed by spectral clustering or Ward’s method. We also introduce two statistical tests to infer the optimal number of clusters and show that they strongly outperform the silhouette criterion, a general-purpose heuristic. We illustrate the usefulness of the approach by 1) identifying errors in a previous phylogenetic analysis of yeast species and 2) identifying topological incongruence among newly sequenced loci of the globeflower fly genus Chiastocheta . We release treeCl, a new program to cluster genes of common evolutionary history ( http://git.io/treeCl ).

Description: The 2016 central Italy seismic sequence consists so far of a series of moderate-to-large earthquakes activating within a few months along a 60-km-long and Apenninic-trending normal-fault system. Regrettably, the high vulnerability of the local infrastructure and the shallowness of the largest events (depth around 8 km) resulted in 299 casualties and more than 20,000 homeless, with great difficulties in the disaster management. The sequence evolved around its largest event ( M w  6.5, 30 October) that occurred right in the middle of a fault system already activated two months before with a first M w  6.0 mainshock (on 24 August) located to the south near the town of Amatrice. Then, another M w  5.9 mainshock occurred just four days before the largest mainshock (26 October) at the northernmost extent of the sequence, near the town of Visso. We analyze the space–time evolution of the first four months of seismic activity through the relocation of ~26,000 earthquakes and the kinematic source models of the three mainshocks. All the main events nucleated at the base of a southwest-dipping normal-fault system segmented by the presence of crosscutting compressional structures. The presence of these inherited faults, separating diverse geological domains, appears to modulate evolution of the sequence interfering with coseismic slip distribution and fault segments interaction. Several secondary antithetic and synthetic faults are located at a shallow depth (〈4 km), both in the hanging wall and footwall. The whole normal fault system, confined within the first 8 km of the upper crust, is bounded below by a shallow east-dipping and 2–3-km-thick layer in which small events plus a series of large extensional aftershocks ( M w  4) occur, possibly decoupling the upper and lower crusts.

Description: Key Points Germline gain-of-function mutations in STAT3 lead to lymphoproliferation and autoimmunity with prominent cytopenias. Mutations in STAT3 cause altered regulatory T cells and cytokine signaling.

Description: Organ-specific endothelial cells (ECs) are both conduits for delivery of nutrients and also establish an instructive vascular niche. The vascular niche produces paracrine factors, (i.e., angiocrine factors), that balance self-renewal and differentiation of hematopoietic stem/progenitor cells (HSPCs) (1,2). Activation of Akt-mTOR pathway in sinusoidal ECs (SECs) stimulates physiological expression of angiocrine factors, including Kit-ligand, Notch-ligands, Wnts, FGFs, BMPs and TGFb, that expand long-term repopulating HSPCs. Activation of MAPkinase in ECs upregulates expression of GM-CSF, M-CSF, IL6, IL7, SDF-1 and G-CSF (..others) to accelerate HSPC multi-lineage differentiation. We developed an ex vivo vascular niche in which HSPC/EC co-cultures are maintained and expanded in serum-free conditions. This vascular niche platform produces physiologic levels of angiocrine factors that balance expansion/differentiation of human cord blood, mobilized peripheral blood, and steady state bone marrow HSPCs that maintain their ability to reconstitute hematopoiesis in vivo. In contrast to our vascular platform, co-culture with bone marrow-derived mesenchymal does not support long-term expansion of HSPCs. In collaboration with Drs. Kiem and Gori at Hutchinson Cancer Center, we have shown that ECs expand repopulating nonhuman primate marrow-derived HSPCs. Transplantation of the vascular-niche expanded gene-modified HSPCs reconstituted long-term multi-lineage hematopoiesis in autologous transplantation setting in nonhuman primates. Importantly, intravenous co-infusion of the vascular niche with HSPCs did not cause infusional toxicity. Vascular niche-expanded HSPCs supported robust hematopoietic recovery underscoring the essential function of vascular niche-signals in hematopoietic reconstitution without provoking fibrosis (3). The ECs also supplies key signals that induce emergence of HSPCs from hemogenic ECs. To prove this point, we transduced adult human or mouse ECs with Runx1/Spi1/Gfi1/FosB transcription factors along with vascular niche-induction allowing for conversion of these ECs into stable and long-term engraftable HSPCs, including functional immune cells (4). Importantly, transition through a pluripotent state results in poorly engraftable hematopoietic cells that are unstable and upon exposure to pathophysiological stressors differentiate aberrantly into other cell-types. Remarkably, signals from vascular niche support specification of repopulating multipotent-HSPCs from both human and nonhuman primate pluripotent stem cells (5). In summary, we developed and characterized a vascular niche platform that provides physiologically relevant levels of key angiocrine factors that stimulate safe clinical-scale expansion of authentic adult, cord blood, and primitive HSPCs under GMP-grade culture conditions. We are currently translating the vascular niche platform to the clinical setting, to evaluate the potential of co-transplantation of HSPCs with vascular niche cells to reconstruct injured EC niches thereby accelerating short- and long-term hematopoietic recovery. This first-in-man clinical application will set the stage for repopulation with true hematopoietic stem cells, thereby enabling use of a vascular niche for treatment of a wide range of acquired, inherited, and malignant hematopoietic diseases. 1. Butler JM …… Rafii S. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell, 3:251-64, 2010. 2. Nolan D........Rafii S. Molecular and cellular signatures of tissue-specific vascular heterogeneity in organ maintenance and regeneration. Developmental Cell, 26(2):204-19, 2013. 3. Ding BS …..Rafii S. Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis.Nature 505(7481):97-102, 2014. 4. Sandler VM, Lis R ...... Butler JM, Scandura JM, Rafii S. Reprogramming of Human Endothelium Into Engraftable Hematopoietic Progenitors by Vascular Niche Induction.Nature, 511(7509):312-8, 2014. 5. Gori J., Butler JM, .....Rafii S, Kiem HP. Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells. Journal of Clinical Investigation, 125(3): 1243-54, 2015. Disclosures Rafii: Angiocrine Bioscience: Consultancy, Equity Ownership.

Description: Tumor antigen directed T-cells, using chimeric antigen receptors (CAR), have shown remarkable clinical responses in B-cell malignancies, particularly acute lymphoblastic leukemia, diffuse large B-cell lymphoma and multiple myeloma. A number of challenges remain, however, in safe and effective cell therapy for durable responses broadly in hematologic malignancies and solid tumors. The durability of response is often reduced by antigen positive or antigen negative escape while the immunosuppressive tumor microenvironment can reduce the potency of the engineered T-cells. Cytokines can be effectively used to improve T-cell expansion and persistence to prevent antigen positive escape, enhance epitope spreading to prevent antigen negative escape, and to relieve the immunosuppressive tumor microenvironment. Indeed, anti-tumor responses with cytokine immunotherapy as well as cytokine-aided cell therapy have been observed. However, the utility of cytokines is often limited by systemic toxicity associated with their potent pharmacological effects. Locally restricted, on demand production of cytokines coupled with antigen directed T-cells can enable safe and effective cell therapy for the treatment of hematologic malignancies as well as solid tumors. Among the cytokines, Interleukin 12 (IL12) and Interleukin 15 (IL15) are of particular interest due to their role in remodeling the tumor microenvironment and improving T-cell persistence. To determine the effect of cytokine expression on CAR-T control of tumor growth, we generated bicistronic constructs expressing CD19-CAR and IL12 or CD19-CAR and membrane bound IL15 (mbIL15). The constructs were configured to provide a high level of CAR expression with high or low levels of cytokine expression for dose exploration. Even at low cytokine levels and low cell dose, expression of IL12 along with CD19-CAR in human T-cells durably regressed disseminated CD19+ Nalm6-luc tumors in Nod-scid IL2Rgnull (NSG mice), compared to CD19-CAR-expressing cells alone and improved survival at both low and high doses (n=8/group, p

Description: BACKGROUND: Peripheral blood (PB) hematopoietic progenitor cells (HPC) mobilized with G-CSF are the first-choice source for allogeneic stem cell transplantation. We carried out a prospective study on healthy donors (HDs), to identify donor characteristics that could influence the effectiveness of mobilization. STUDY DESIGN AND METHODS: PB-HPC allogeneic donations from sibling HDs were analyzed. We tested somatic variables (sex, age, weight, height, volemia) and blood counts (WBC, platelets, hemoglobin, CD34+ cell count). Two different determinations of CD34+ cells were done in each HD: baseline (before G-CSF administration) and in PB on the morning of the fifth day (after G-CSF administration). HDs received G-CSF subcutaneously at a dose of 10 µg/kg per day. RESULTS: 128 consecutive HDs (66 males) with a median age of 43 years were enrolled. The mean value of CD34+ on day 5 was 90.8 cells/µL, 84.2 cells/µL in females and 97.2 cells/µL in males. The median values of CD34+ on day 5 were 75.5 cells/µL for the overall sample, 74 cells/µL in females and 80 cells/µL in males. On univariate correlation analysis, donor weight r=0.19, P

Description: Transplantation of gene-modified autologous hematopoietic stem/progenitor cells (HSPCs) is an effective treatment for several hematologic diseases. However, a number of blood disorders may not be amenable to gene augmentation-based therapeutics. Targeted genome editing in human HSPCs could provide a therapeutic approach for these otherwise untreatable diseases. Here we demonstrate that CRISPR/Cas9 ribonucleoprotein (RNP) edits target genes in human HSPCs with high efficiency and precision. Human adult and umbilical cord blood (CB) CD34+ cells from 20 donors were electroporated with S. pyogenes or S. aureus Cas9 RNP targeting HBB, AAVS1, or CXCR4. Sequence analysis demonstrated up to 80% editing in CB CD34+ cells (mean±s.d: 61%±9%) and up to 57% in adult CD34+ cells (39%±13%). Delivery of Cas9 RNP and a single-stranded oligodeoxynucleotide donor (ssODN) led to up to 12% ssODN-mediated homology directed repair (HDR) and also led to a 20% increase in total gene editing (HDR+NHEJ)(RNP: 48%±15%; RNP+ssODN: 69%±8%). Both Cas9 RNP gene-edited CD34+ cells and donor-matched untreated control CD34+ cells reconstituted human hematopoiesis in primary and secondary recipient immunodeficient mice, with ~85% human CD45+ cell peripheral blood reconstitution 4 months after primary transplantation. Human T and B lymphoid, erythroid, and myeloid cells were detected in the spleen, thymus, and bone marrow with 20% CD34+ cell engraftment in the marrow of mice transplanted with RNP gene-edited or control CD34+ cells. The level of targeted gene editing in human erythroid, myeloid, and CD34+ cells that were recovered and enriched from the hematopoietic organs of primary recipients (~50%) was similar to the level of gene editing detected in the pre-infusion product (~60%). In summary, these results indicate that Cas9 gene-edited human HSPCs retain long-term engraftment potential and support multilineage blood reconstitution in vivo, thus supporting further investigation of CRISPR/Cas9 mediated gene-edited hematopoietic stem/progenitor cell therapies. Disclosures Heath: Editas Medicine: Employment. Chalishazar:Editas Medicine: Employment. Lee:Editas Medicine: Employment. Selleck:Editas Medicine: Employment. Cotta-Ramusino:Editas Medicine: Employment. Bumcrot:Editas Medicine: Employment. Gori:Editas Medicine: Employment.