Mutational analysis of the C-terminal FATC domain of Saccharomyces cerevisiae Tra1

Tra1 is a component of the Saccharomyces cerevisiae SAGA and NuA4 complexes and a member of the PIKK family, which contain a C-terminal phosphatidylinositol 3-kinase-like (PI3K) domain followed by a 35-residue FATC domain. Single residue changes of L3733A and F3744A, within the FATC domain, resulted in transcriptional changes and phenotypes that were similar but not identical to those caused by mutations in the PI3K domain or deletions of other SAGA or NuA4 components. The distinct nature of the FATC mutations was also apparent from the additive effect of tra1-L3733A with SAGA, NuA4, and tra1 PI3K domain mutations. Tra1-L3733A associates with SAGA and NuA4 components and with the Gal4 activation domain, to the same extent as wild-type Tra1; however, steady-state levels of Tra1-L3733A were reduced. We suggest that decreased stability of Tra1-L3733A accounts for the phenotypes since intragenic suppressors of tra1-L3733A restored Tra1 levels, and reducing wild-type Tra1 led to comparable growth defects. Also supporting a key role for the FATC domain in the structure/function of Tra1, addition of a C-terminal glycine residue resulted in decreased association with Spt7 and Esa1, and loss of cellular viability. These findings demonstrate the regulatory potential of mechanisms targeting the FATC domains of PIKK proteins

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Spatial and temporal uplift history of South America from calibrated drainage analysis

A multidisciplinary approach is used to analyze the Cenozoic uplift history of South America. Residual depth anomalies of oceanic crust abutting this continent help to determine the pattern of present-day dynamic topography. Admittance analysis and crustal thickness measurements indicate that the elastic thickness of the Borborema and Altiplano regions is ≤₁₀ km with evidence for sub-plate support at longer wavelengths. A drainage inventory of 1827 river profiles is assembled and used to investigate landscape development. Linear inverse modeling enables river profiles to be fitted as a function of the spatial and temporal history of regional uplift. Erosional parameters are calibrated using observations from the Borborema Plateau and tested against continent-wide stratigraphic and thermochronologic constraints. Our results predict that two phases of regional uplift of the Altiplano plateau occurred in Neogene times. Regional uplift of the southern Patagonian Andes also appears to have occurred in Early Miocene times. The consistency between observed and predicted histories for the Borborema, Altiplano, and Patagonian plateaux implies that drainage networks record coherent signals that are amenable to simple modeling strategies. Finally, the predicted pattern of incision across the Amazon catchment constrains solid sedimentary flux at the Foz do Amazonas. Observed and calculated flux estimates match, suggesting that erosion and deposition were triggered by regional Andean uplift during Miocene times

Sustained alterations in biodistribution of stem/progenitor cells in Tie2Cre+α4(f/f) mice are hematopoietic cell autonomous

We have generated Tie2Cre+α4(f/f) mice with documented α4-integrin ablation in hematopoietic and endothelial cells. A prominent feature in this model is a sustained, significant increase in circulating progenitors at levels higher than the levels seen with Tie2Cre+VCAM-1(f/f) mice. To test whether phenotypic differences are due to contributions by ligands other than VCAM-1 in bone marrow, or to α4-deficient endothelial cells or pericytes, we carried out transplantation experiments using these mice as donors or as recipients. Changes in progenitor biodistribution after transplantation were seen only with α4-deficient donor cells, suggesting that these cells were necessary and sufficient to reproduce the phenotype with no discernible contribution by α4-deficient nonhematopoietic cells. Because several similarities are seen after transplantation between our results and those with CXCR4(−/−) donor cells, the data suggest that VLA4/VCAM-1 and CXCR4/CXCL12 pathways contribute to a nonredundant, ongoing signaling required for bone marrow retention of progenitor cells during homeostasis

Deletion of α4 Integrins from Adult Hematopoietic Cells Reveals Roles in Homeostasis, Regeneration, and Homing

We have explored the functional implications of inducible α4 integrin deletion during adult hematopoiesis by generating a conditional-knockout mouse model, and we show that α4 integrin-deficient hematopoietic progenitor cells accumulate in the peripheral blood soon after interferon-induced gene deletion. Although their numbers gradually stabilize at a lower level, progenitor cell influx into the circulation continues at above-normal levels for more than 50 weeks. Concomitantly, a progressive accumulation of progenitors occurs within the spleen. In addition, the regeneration of erythroid and myeloid progenitor cells is delayed during stress hematopoiesis induced by phenylhydrazine or by 5-fluorouracil, suggesting impairment in early progenitor expansion in the absence of α4 integrin. Moreover, in transplantation studies, homing of α4(−/−) cells to the bone marrow, but not to the spleen, is selectively impaired, and short-term engraftment is critically delayed in the early weeks after transplantation. Thus, conditional deletion of α4 integrin in adult mice is accompanied by a novel hematopoietic phenotype during both homeostasis and recovery from stress, a phenotype that is distinct from the ones previously described in α4 integrin-null chimeras and β1 integrin-conditional knockouts

Hierarchy of molecular-pathway usage in bone marrow homing and its shift by cytokines

Efficient bone marrow (BM) homing is a prerequisite for successful engraftment of transplanted hematopoietic cells (HPCs). Contradictory conclusions about the contribution of SDF-1/CXCR4 have clouded our understanding of its role within the molecular pathway cooperation needed for BM homing, particularly with the well-defined hierarchic network of adhesion molecules. In the present study we sought to unravel cooperative and compensatory molecular pathways guiding BM homing. Fresh BM-HPCs, rendered either SDF-1 unresponsive or Gi-signaling refractory, homed quite efficiently, because of compensation by α4-integrin interacting with VCAM-1. The contribution of SDF-1/CXCR4- or Gi-protein-mediated signals to BM homing became apparent after their blockade was combined with deletion of α4-integrin, leading to dramatic reduction in BM homing. Similar conclusions were revealed when VCAM-1-deficient hosts were used. Cytokine incubation changed the functional properties of BM-HPCs and hierarchy of molecular pathway usage in homing, by shifting the dominance among the homing mediators: loss of CXCR4 or Gi-signaling now significantly reduced BM homing, with only partial compensation through α4/VCAM-1 and endothelial selectins. These studies depict a flexible hierarchy of cooperating homing pathways, in which dominant players are repositioned with changing cytokine milieu, and possibly source of HPCs

Lack of α4 integrin expression in stem cells restricts competitive function and self-renewal activity

Alpha4 integrin or VLA4 (CD49d/CD29) is a multitask molecule with wide expression within and outside the hematopoietic system. Because targeted ablation of α4 integrin leads to embryonic lethality, to study its effects on adult hematopoiesis, we used animals with conditional excision of α4 integrin (α4Δ/Δ) in hematopoietic cells. In such animals, we previously documented weakened bone marrow retention of progenitor cells during homeostasis and impaired homing and short-term engraftment after transplantation. In the present study we show that long-term repopulating cells lacking α4 integrins display a competitive disadvantage in hematopoietic reconstitution compared to normal competitors. Although initial dominance of α4+ competitors is due to their better homing and proliferative expansion early after transplantation, a progressive decline in contribution of α4Δ/Δ hematopoiesis is compatible with neither normal homing nor normal function of α4Δ/Δ hematopoietic stem cells (HSCs) in post-homing hematopoiesis. In the absence of α4+ competitor cells, α4Δ/Δ HSCs can establish long-term hematopoiesis in primary recipients, however, some resurgence of host hematopoiesis is evident, and it becomes dominant in secondary transplants, so that no survivors with exclusively α4Δ/Δ cells are seen in tertiary transplants. Collectively, our data provide compelling evidence that under regenerative stress α4 integrin assumes a greater importance than for maintenance of steady-state hematopoiesis

Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice: involvement in mobilization of stem/progenitor cells.

International audienceIt was previously reported that treatment with the sulfated polysaccharide fucoidan or the structurally similar dextran sulfate increased circulating mature white blood cells and hematopoietic progenitor/stem cells (HPCs) in mice and nonhuman primates; however, the mechanism mediating these effects was unclear. It is reported here that plasma concentrations of the highly potent chemoattractant stromal-derived factor 1 (SDF-1) increase rapidly and dramatically after treatment with fucoidan in monkeys and in mice, coinciding with decreased levels in bone marrow. In vitro and in vivo data suggest that the SDF-1 increase is due to its competitive displacement from heparan sulfate proteoglycans that sequester the chemokine on endothelial cell surfaces or extracellular matrix in bone marrow and other tissues. Although moderately increased levels of interleukin-8, MCP1, or MMP9 were also present after fucoidan treatment, studies in gene-ablated mice (GCSFR(-/-), MCP1(-/-), or MMP9(-/-)) and the use of metalloprotease inhibitors do not support their involvement in the concurrent mobilization. Instead, SDF-1 increases, uniquely associated with sulfated glycan-mobilizing treatments and not with several other mobilizing agents tested, are likely responsible. To the authors' knowledge, this is the first published report of disrupting the SDF-1 gradient between bone marrow and peripheral blood through a physiologically relevant mechanism, resulting in mobilization with kinetics similar to other mobilizing CXC chemokines. The study further underscores the importance of the biological roles of carbohydrates