Live-animal imaging of native haematopoietic stem and progenitor cells.

The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow3-5. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches.

EVI1 Interferes with Myeloid Maturation via Transcriptional Repression of Cebpa, via Binding to Two Far Downstream Regulatory Elements.

One mechanism by which oncoproteins work is through perturbation of cellular maturation; understanding the mechanisms by which this occurs can lead to the development of targeted therapies. EVI1 is a zinc finger oncoprotein involved in the development of acute myeloid leukemia; previous work has shown it to interfere with the maturation of granulocytes from immature precursors. Here we investigate the mechanism by which that occurs, using an immortalized hematopoietic progenitor cell line, EML-C1, as a model system. We document that overexpression of EVI1 abrogates retinoic acid-induced maturation of EML cells into committed myeloid cells, a process that can be documented by the down-regulation of stem cell antigen-1 and acquisition of responsiveness to granulocyte-macrophage colony-stimulating factor. We show that this requires DNA binding capacity of EVI1, suggesting that downstream target genes are involved. We identify the myeloid regulator Cebpa as a target gene and identify two EVI1 binding regions within evolutionarily conserved enhancer elements at +35 and +37 kb relative to the gene. EVI1 can strongly suppress Cebpa transcription, and add-back of Cebpa into EVI1-expressing EML cells partially corrects the block in maturation. We identify the DNA sequences to which EVI1 binds at +35 and +37 kb and show that mutation of one of these releases Cebpa from EVI1-induced suppression. We observe a more complex picture in primary bone marrow cells, where EVI1 suppresses Cebpa in stem cells but not in more committed progenitors. Our data thus identify a regulatory node by which EVI1 contributes to leukemia, and this represents a possible therapeutic target for treatment of EVI1-expressing leukemia.

Fossil palm beetles refine upland winter temperatures in the Early Eocene Climatic Optimum.

Eocene climate and associated biotic patterns provide an analog system to understand their modern interactions. The relationship between mean annual temperatures and winter temperatures-temperature seasonality-may be an important factor in this dynamic. Fossils of frost-intolerant palms imply low Eocene temperature seasonality into high latitudes, constraining average winter temperatures there to >8 °C. However, their presence in a paleocommunity may be obscured by taphonomic and identification factors for macrofossils and pollen. We circumvented these problems by establishing the presence of obligate palm-feeding beetles (Chrysomelidae: Pachymerina) at three localities (a fourth, tentatively) in microthermal to lower mesothermal Early Eocene upland communities in Washington and British Columbia. This provides support for warmer winter Eocene climates extending northward into cooler Canadian uplands.

Essential role of PR-domain protein MDS1-EVI1 in MLL-AF9 leukemia.

A subgroup of leukemogenic mixed-lineage leukemia (MLL) fusion proteins (MFPs) including MLL-AF9 activates the Mecom locus and exhibits extremely poor clinical prognosis. Mecom encodes EVI1 and MDS1-EVI1 (ME) proteins via alternative transcription start sites; these differ by the presence of a PRDI-BF1-RIZ1 (PR) domain with histone methyltransferase activity in the ME isoform. Using an ME-deficient mouse, we show that ME is required for MLL-AF9-induced transformation both in vitro and in vivo. And, although Nup98-HOXA9, MEIS1-HOXA9, and E2A-Hlf could transform ME-deficient cells, both MLL-AF9 and MLL-ENL were ineffective, indicating that the ME requirement is specific to MLL fusion leukemia. Further, we show that the PR domain is essential for MFP-induced transformation. These studies clearly indicate an essential role of PR-domain protein ME in MFP leukemia, suggesting that ME may be a novel target for therapeutic intervention for this group of leukemias.

The role of EVI1 in myeloid malignancies.

The EVI1 oncogene at human chr 3q26 is rearranged and/or overexpressed in a subset of acute myeloid leukemias and myelodysplasias. The EVI1 protein is a 135 kDa transcriptional regulator with DNA-binding zinc finger domains. Here we provide a critical review of the current state of research into the molecular mechanisms by which this gene plays a role in myeloid malignancies. The major pertinent cellular effects are blocking myeloid differentiation and preventing cellular apoptosis, and several potential mechanisms for these phenomena have been identified. Evidence supports a role for EVI1 in inducing cellular quiescence, and this may contribute to the resistance to chemotherapy seen in patients with neoplasms that overexpress EVI1. Another isoform, MDS1-EVI1 (or PRDM3), encoded by the same locus as EVI1, harbors an N-terminal histone methyltransferase(HMT) domain; experimental findings indicate that this protein and its HMT activity are critical for the progression of a subset of AMLs, and this provides a potential target for therapeutic intervention.

Brain morphometric correlates of metabolic variables in HIV: the CHARTER study.

Obesity and other metabolic variables are associated with abnormal brain structural volumes and cognitive dysfunction in HIV-uninfected populations. Since individuals with HIV infection on combined antiretroviral therapy (CART) often have systemic metabolic abnormalities and changes in brain morphology and function, we examined associations among brain volumes and metabolic factors in the multisite CNS HIV AntiRetroviral Therapy Effects Research (CHARTER) cohort, cross-sectional study of 222 HIV-infected individuals. Metabolic variables included body mass index (BMI), total blood cholesterol (C), low- and high-density lipoprotein C (LDL-C and HDL-C), blood pressure, random blood glucose, and diabetes. MRI measured volumes of cerebral white matter, abnormal white matter, cortical and subcortical gray matter, and ventricular and sulcal CSF. Multiple linear regression models allowed us to examine metabolic variables separately and in combination to predict each regional volume. Greater BMI was associated with smaller cortical gray and larger white matter volumes. Higher total cholesterol (C) levels were associated with smaller cortex volumes; higher LDL-C was associated with larger cerebral white matter volumes, while higher HDL-C levels were associated with larger sulci. Higher blood glucose levels and diabetes were associated with more abnormal white matter. Multiple atherogenic metabolic factors contribute to regional brain volumes in HIV-infected, CART-treated patients, reflecting associations similar to those found in HIV-uninfected individuals. These risk factors may accelerate cerebral atherosclerosis and consequent brain alterations and cognitive dysfunction.

Master regulatory GATA transcription factors: mechanistic principles and emerging links to hematologic malignancies.

Numerous examples exist of how disrupting the actions of physiological regulators of blood cell development yields hematologic malignancies. The master regulator of hematopoietic stem/progenitor cells GATA-2 was cloned almost 20 years ago, and elegant genetic analyses demonstrated its essential function to promote hematopoiesis. While certain GATA-2 target genes are implicated in leukemogenesis, only recently have definitive insights emerged linking GATA-2 to human hematologic pathophysiologies. These pathophysiologies include myelodysplastic syndrome, acute myeloid leukemia and an immunodeficiency syndrome with complex phenotypes including leukemia. As GATA-2 has a pivotal role in the etiology of human cancer, it is instructive to consider mechanisms underlying normal GATA factor function/regulation and how dissecting such mechanisms may reveal unique opportunities for thwarting GATA-2-dependent processes in a therapeutic context. This article highlights GATA factor mechanistic principles, with a heavy emphasis on GATA-1 and GATA-2 functions in the hematopoietic system, and new links between GATA-2 dysregulation and human pathophysiologies.

A revision of the late Eocene snakeflies (Raphidioptera) of the Florissant Formation, Colorado, with special reference to the wing venation of the Raphidiomorpha.

The snakeflies (Raphidioptera) of the late Eocene Florissant Formation (Colorado, USA) are revised. Seven species of Raphidiidae are assigned to three genera, i.e., Megaraphidia Cockerell, 1907, Archiraphidia Handlirsch, 1910, and Florissantoraphidia gen. nov. Dictyoraphidia Handlirsch, 1910 is assigned to Baissopteridae, a first Cenozoic record of the family. Archiraphidia tumulata (Scudder, 1890), A. tranquilla (Scudder, 1890) and A.? somnolenta (Scudder, 1890), stat. res. are treated as distinct species, and A. eventa (Scudder, 1890) as a new synonym of A. tranquilla. The lectotype of A. eventa is designated. 'Raphidia' exhumata Cockerell, 1909 is transferred to Megaraphidia. 'Raphidia' mortua Rohwer, 1909 and 'R.' funerata Engel, 2003 constitute Florissantoraphidia gen. nov. Our findings support the treatment of the single Florissant species of Inocelliidae as preliminary assigned to Fibla Navás, 1915. We examine venational synapomorphies of Raphidiomorpha and provide a new diagnosis for it based on these characters. We evaluate putative derived venational character states of 'Neoraphidioptera' (Inocelliidae and Raphidiidae), finding no clear synapomorphy supporting its validity; these families may nest separately within a paraphyletic Mesoraphidiidae (s.l.). We provide diagnoses for the families occurring at Florissant (Baissopteridae, Inocelliidae and Raphidiidae) based on wing venation.

Disentangling the effect of growth from development in size-related trait scaling relationships.

In plant ecology, the terms growth and development are often used interchangeably. Yet these constitute two distinct processes. Plant architectural traits (e.g. number of successive forks) can estimate development stages. Here, we show the importance of including the effect of development stages to better understand size-related trait scaling relationships (i.e. between height and stem diameter). We focused on one common savanna woody species (Senegalia nigrescens) from the Greater Kruger Area, South Africa. We sampled 406 individuals that experience different exposure to herbivory, from which we collected four traits: plant height, basal stem diameter, number of successive forks (proxy for development stage), and resprouting. We analysed trait relationships (using standardized major axis regression) between height and stem diameter, accounting for the effect of ontogeny, exposure to herbivory, and resprouting. The number of successive forks affects the scaling relationship between height and stem diameter, with the slope and strength of the relationship declining in more developed individuals. Herbivory exposure and resprouting do not affect the overall height-diameter relationship. However, when height and stem diameter were regressed separately against number of successive forks, herbivory exposure and resprouting had an effect. For example, resprouting individuals allocate more biomass to both primary and secondary growth than non-resprouting plants in more disturbed conditions. We stress the need to include traits related to ontogeny so as to disentangle the effect of biomass allocation to primary and secondary growth from that of development in plant functional relationships.

Stolleagrion foghnielseni (Odonata, Cephalozygoptera, Dysagrionidae) gen. et sp. nov.: a new odonatan from the PETM recovery phase of the earliest Ypresian Fur Formation, Denmark.

We describe the new genus and species Stolleagrion foghnielseni n. gen. et sp. from the Fur Formation in northwestern Denmark based on a single fossil wing. This is the first odonatan described from the earliest part of the PETM recovery phase of the early Eocene. A combination of nine wing character states are considered to be diagnostic of the Dysagrionidae Cockrell only together with the cephalozygopteran head; however, the combination of these nine plus the presence of Ax0 is also diagnostic without the head. By this, we assign Stolleagrion foghnielseni to the Dysagrionidae and reassess the position of other odonates previously treated as cf. Dysagrionidae.

Overexpression of Evi-1 oncoprotein represses TGF-ß signaling in colorectal cancer.

Human colorectal cancer (CRC) cells are resistant to the anti-proliferative effect of transforming growth factor-ß (TGF-ß), suggesting that disruption of TGF-ß signaling plays an important role in colorectal carcinogenesis. Ecotropic virus integration site-1 (Evi-1) oncoprotein represses TGF-ß signaling by interacting with Smads, but its role in CRC has not been established. The purpose of this study is to determine whether Evi-1 plays role(s) in CRCs and to characterize Evi-1 transcript(s) in CRCs. Evi-1 was overexpressed in 53% of human CRC samples, 100% of colon adenoma samples, and 100% of human colon cancer cell lines tested. Using 5' RACE, we cloned a novel Evi-1 transcript (Evi-1e) from a human CRC tissue and found that this novel transcript was expressed at a higher level in CRC tissues than in normal tissues and was the major Evi-1 transcript in CRCs. Transient Evi-1 transfection inhibited TGF-ß-induced transcriptional activity and reversed the growth inhibitory effect of TGF-ß in MC-26 mouse colon cancer cells. In conclusion, we have identified overexpression of Evi-1 oncoprotein as a novel mechanism by which a subset of human CRCs may escape TGF-ß regulation. We have also identified a novel Evi-1 transcript, Evi-1e, as the major Evi-1 transcript expressed in human CRCs.

PR-domain-containing Mds1-Evi1 is critical for long-term hematopoietic stem cell function.

The Mds1 and Evi1 complex locus (Mecom) gives rise to several alternative transcripts implicated in leukemogenesis. However, the contribution that Mecom-derived gene products make to normal hematopoiesis remains largely unexplored. To investigate the role of the upstream transcription start site of Mecom in adult hematopoiesis, we created a mouse model with a lacZ knock-in at this site, termed ME(m1), which eliminates Mds1-Evi1 (ME), the longer, PR-domain-containing isoform produced by the gene (also known as PRDM3). ß-galactosidase-marking studies revealed that, within hematopoietic cells, ME is exclusively expressed in the stem cell compartment. ME deficiency leads to a reduction in the number of HSCs and a complete loss of long-term repopulation capacity, whereas the stem cell compartment is shifted from quiescence to active cycling. Genetic exploration of the relative roles of endogenous ME and EVI1 isoforms revealed that ME preferentially rescues long-term HSC defects. RNA-seq analysis in Lin(-)Sca-1(+)c-Kit(+) cells (LSKs) of ME(m1) documents near complete silencing of Cdkn1c, encoding negative cell-cycle regulator p57-Kip2. Reintroduction of ME into ME(m1) LSKs leads to normalization of both p57-Kip2 expression and growth control. Our results clearly demonstrate a critical role of PR-domain-containing ME in linking p57-kip2 regulation to long-term HSC function.

New species of Nymphites Haase (Neuroptera: Nymphidae) from the Middle Jurassic of China, with a redescription of the type species of the genus.

Nymphites priscus (Weyenbergh, 1869) from the Late Jurassic of Solnhofen (Germany), type species of the genus Nymphites Haase, 1890, is redescribed. The genus is assigned to the Nymphidae. The taxon Nymphitidae is not valid; it is an artificial aggregation of fossil genera. Two species new to science, one named, of Nymphites from the Middle Jurassic locality of Daohugou (Inner Mongolia, China) are described.

Cimbicidae (Hymenoptera, 'Symphyta') in the Paleogene: revision, the new subfamily Cenocimbicinae, and new taxa from the Eocene Okanagan Highlands.

We erect the Cenocimbicinae, a new subfamily of Cimbicidae (Hymenoptera, Symphyta), restricted to the Selandian Menat Formation of France, the oldest occurrence of the family, and the Ypresian Okanagan Highlands of far-western North America. We describe new taxa from the Okanagan Highlands: Allenbycimbex morrisae gen. et sp. nov. and Leptostigma n. gen. with seven new species: L. alaemacula n. sp., L. brevilatum n. sp., L. fasciatum n. sp., L. longiclava n. sp., L. longipallidum n. sp., L. longitenebricum n. sp., and L. proxivena n. sp. We revise the Cimbicidae from the Ypresian Green River Formation and the Priabonian Florissant Formation, both in Colorado, USA. The oldest fossil of a modern cimbicid subfamily appears with a single pachylostictine specimen in the Green River Formation, and all Cimbicidae are in modern subfamilies after the Ypresian (we did not examine one larva known from Priabonian Baltic amber). Pseudocimbex clavatus Rohwer 1908 from the Florissant Formation is not a cimbicid; we treat it as Tenthredinoidea incertae sedis. We transfer Cimbex vetusculus Cockerell to Floricimbex n. gen.

The damselfly genus Furagrion Petrulevicius et al. (Odonata, Zygoptera) from the early Eocene Fur Formation of Denmark and the dysagrionoid grade.

The earliest Eocene odonate genus Furagrion Petrulevicius et al. from the Danish Fur Formation is revised based on eighteen specimens, two of which apparently have been lost since their publication. The holotype of Phenacolestes jutlandicus Henriksen, type species of Furagrion, is incomplete and lacks the characters currently used to differentiate species, genera and higher taxa in Odonata. We, therefore, propose that the holotype is set aside and a recently discovered nearly complete Fur Formation fossil is designated as neotype. Furagrion possesses all of the nine wing character states currently used along with head shape for diagnosing the Dysagrionidae; however, Furagrion has a characteristically zygopteran head, not the distinctive head shape of the suborder Cephalozygoptera. We, therefore, treat it as a zygopteran unassigned to family. These nine wing character states appear in different combinations not only in various Zygoptera and Cephalozygoptera, but also in the Frenguelliidae, an Eocene family of Argentina that may represent an unnamed suborder. We recognise these taxa as constituting a dysagrionoid grade, in which these character states appear either convergently or as symplesiomorphies. Furagrion morsi Zessin is synonymized with Phenacolestes jutlandicus Henriksen, syn. nov. and Morsagrion Zessin with Furagrion Petrulevicius, Wappler, Wedmann, Rust, and Nel, syn. nov.

Highly efficient Runx1 enhancer eR1-mediated genetic engineering for fetal, child and adult hematopoietic stem cells.

A cis-regulatory genetic element which targets gene expression to stem cells, termed stem cell enhancer, serves as a molecular handle for stem cell-specific genetic engineering. Here we show the generation and characterization of a tamoxifen-inducible CreERT2 transgenic (Tg) mouse employing previously identified hematopoietic stem cell (HSC) enhancer for Runx1, eR1 (+24 m). Kinetic analysis of labeled cells after tamoxifen injection and transplantation assays revealed that eR1-driven CreERT2 activity marks dormant adult HSCs which slowly but steadily contribute to unperturbed hematopoiesis. Fetal and child HSCs that are uniformly or intermediately active were also efficiently targeted. Notably, a gene ablation at distinct developmental stages, enabled by this system, resulted in different phenotypes. Similarly, an oncogenic Kras induction at distinct ages caused different spectrums of malignant diseases. These results demonstrate that the eR1-CreERT2 Tg mouse serves as a powerful resource for the analyses of both normal and malignant HSCs at all developmental stages.

Neurocognitive deficits in HIV-infected women and victims of childhood trauma.

The study investigated the behavioral and brain effects of childhood trauma and human immunodeficiency virus (HIV) infection, both separately and in combination, and assessed potential interactions in women who were dually affected. Eighty-three HIV-positive and 47 matched HIV-negative South African women underwent neuromedical, neuropsychiatric, and neurocognitive assessments. Univariate tests of significance assessed if either HIV infection or childhood trauma, or the combination, had a significant effect on neurocognitive performance. The majority of women were Black (96%) and had an average age of 30 years. An analysis of covariance revealed significant HIV effects for the Hopkins Verbal Learning Test (HVLT) learning and delay trials (p < 0.01) and the Halstead Category Test (HCT) (p < 0.05). A significant trauma effect was seen on the HVLT delay trial (p < 0.05). The results provide evidence for neurocognitive dysfunction in memory and executive functions in HIV-infected women and memory disturbances in trauma exposed women.

The early Eocene Eourocerus anguliterreus gen. et sp. nov (Hymenoptera, Siricidae) from Republic, Washington.

A new fossil horntail wood wasp (Hymenoptera, Symphyta, Siricidae), Eourocerus anguliterreus gen. et sp. nov. from an early Eocene Okanagan Highlands locality at Republic, Washington, USA is described. Its forewing is most like that of the extant and fossil genus Urocerus Geoffroy, 1785. It is the third fossil siricid described from North America. We treat Xeris dorbnikensis Manukyan Smirnova, 2021 and Xeris sp. of Manukyan Smirnova (2021) as Urocerus dorbnikensis (Manukyan Smirnova, 2021), comb. nov. and Urocerus sp., both tentatively belonging to the genus, Urocerus klebsi Brues, 1926 as Xeris klebsi (Brues, 1926) comb. nov., and Afrotremex or Eriotremex sp. of Wedmann (1998) as a species of Eriotremex Benson, 1943. A key to extant and extinct genera of Siricinae by forewing characters is given.

Kishenehna prima, a new genus and species of darner dragonfly (Odonata, Aeshnidae, Gomphaeschninae) from the early middle Eocene Kishenehn Formation of Montana, USA.

We describe the darner dragonfly Kishenehna prima n. gen. and sp. (Odonata, Aeshnidae, Gomphaeschninae) based on a well-preserved, nearly complete female hind wing from the Lutetian Coal Creek Member of the Kishenehn Formation, northwestern Montana, USA. Kishenehna is morphologically close to the late Paleocene genus Alloaeschna Wighton Wilson of Alberta, Canada. This is the first dragonfly (Anisoptera) described from the Kishenehn Formation and the first from the Lutetian of the Western Hemisphere.