FLT3-ITDs Instruct a Myeloid Differentiation and Transformation Bias in Lymphomyeloid Multipotent Progenitors

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Influence of physiological concentrations of androgens on the developmental competence and sex ratio of in vitro produced bovine embryos

This study was designed to determine if the addition of androgens at ovarian follicular fluid (FF) concentrations to oocyte maturation media would alter the development and sex ratio of bovine embryos. To maximize hormone bioavailability, oil was removed and glass culture dishes were used during in vitro maturation (IVM) phase; this modified system was then used in the present experiment along with the standard IVM system utilizing plastic containers and incubation under oil. Ethanol (0.2%) was the vector for steroid hormone delivery. Oocytes were incubated for 22 h in the presence of two doses (“low” and “high”) of androstenedione (A₄) or testosterone (T); the doses were based on the concentrations of both androgens in preovulatory bovine follicles (A₄: 337.5 and 562.5 ng/ml; T: 22.2 and 42.6 ng/ml). The results of hormone assays indicated that bioavailability of steroid hormones remained relatively constant, regardless of the IVM system used. The plasticware with the addition of T resulted in significantly higher cleavage rates (80.0 ± 2.1%) than any other combination of treatments (plasticware × A₄: 71.5 ± 2.6%; glassware × T: 71.2 ± 1.9%; and glassware × A₄: 71.4 ± 2.4%). The blastocyst formation rate for the plasticware × T treatment (39.7 ± 2.5%) was significantly greater than for all other combinations (glassware × T: 28.7 ± 2.2%; glassware × A₄: 24.0 ± 2.8%; and plasticware × A₄: 19.8 ± 3.0%) and the low dose of T (37.1 ± 2.5%) resulted in higher (p < 0.05) blastocyst formation rates than all other treatments (T high dose: 29.2 ± 2.5%; A₄ high dose: 27.1 ± 2.9%; and A₄ low dose: 20.2 ± 3.0%). The proportion of male embryos was greater (p < 0.05) in plastic than glass dishes in the low-dose A₄ group (59.1 ± 8.7% vs. 38.2 ± 5.5%, plasticware vs. glassware, respectively) and it tended to be greater (p < 0.08) in the control groups and high-dose A₄ group, but not in the T groups. There was a moderate positive correlation between blastocyst formation rates across all treatment and control groups, and the percentage of male bovine embryos (r = 0.38, p < 0.05). In summary, specific combinations of androgen and glassware/plasticware treatments did alter early bovine embryo development and sex ratio. The addition of T to IVM media increased the cleavage and blastocyst formation rates in plasticware and may be employed to improve the efficiency of the standard in vitro embryo production systems. Androstenedione appeared to enhance whereas testosterone nullified the deviation in sex ratio (pro-femaleness) associated with the use of glass IVM dishes.     

Reversible senescence in human CD4+CD45RA+CD27- memory T cells

Persistent viral infections and inflammatory syndromes induce the accumulation of T cells with characteristics of terminal differentiation or senescence. However, the mechanism that regulates the end-stage differentiation of these cells is unclear. Human CD4(+) effector memory (EM) T cells (CD27(-)CD45RA(-)) and also EM T cells that re-express CD45RA (CD27(-)CD45RA(+); EMRA) have many characteristics of end-stage differentiation. These include the expression of surface KLRG1 and CD57, reduced replicative capacity, decreased survival, and high expression of nuclear γH2AX after TCR activation. A paradoxical observation was that although CD4(+) EMRA T cells exhibit defective telomerase activity after activation, they have significantly longer telomeres than central memory (CM)-like (CD27(+)CD45RA(-)) and EM (CD27(-)CD45RA(-)) CD4(+) T cells. This suggested that telomerase activity was actively inhibited in this population. Because proinflammatory cytokines such as TNF-α inhibited telomerase activity in T cells via a p38 MAPK pathway, we investigated the involvement of p38 signaling in CD4(+) EMRA T cells. We found that the expression of both total and phosphorylated p38 was highest in the EM and EMRA compared with that of other CD4(+) T cell subsets. Furthermore, the inhibition of p38 signaling, especially in CD4(+) EMRA T cells, significantly enhanced their telomerase activity and survival after TCR activation. Thus, activation of the p38 MAPK pathway is directly involved in certain senescence characteristics of highly differentiated CD4(+) T cells. In particular, CD4(+) EMRA T cells have features of telomere-independent senescence that are regulated by active cell signaling pathways that are reversible.     

Structure of the Pseudorabies Virus Capsid: Comparison with Herpes Simplex Virus Type 1 and Differential Binding of Essential Minor Proteins

The structure of pseudorabies virus (PRV) capsids isolated from the nucleus of infected cells and from PRV virions was determined by cryo-electron microscopy (cryo-EM) and compared to herpes simplex virus type 1 (HSV-1) capsids. PRV capsid structures closely resemble those of HSV-1, including distribution of the capsid vertex specific component (CVSC) of HSV-1, which is a heterodimer of the pUL17 and pUL25 proteins. Occupancy of CVSC on all PRV capsids is near 100%, compared to ~ 50% reported for HSV-1 C-capsids and 25% or less that we measure for HSV-1 A- and B-capsids. A PRV mutant lacking pUL25 does not produce C-capsids and lacks visible CVSC density in the cryo-EM-based reconstruction. A reconstruction of PRV capsids in which green fluorescent protein was fused within the N-terminus of pUL25 confirmed previous studies with a similar HSV-1 capsid mutant localizing pUL25 to the CVSC density region that is distal to the penton. However, comparison of the CVSC density in a 9-Å-resolution PRV C-capsid map with the available crystal structure of HSV-1 pUL25 failed to find a satisfactory fit, suggesting either a different fold for PRV pUL25 or a capsid-bound conformation for pUL25 that does not match the X-ray model determined from protein crystallized in solution. The PRV capsid imaged within virions closely resembles C-capsids with the addition of weak but significant density shrouding the pentons that we attribute to tegument proteins. Our results demonstrate significant structure conservation between the PRV and HSV capsids.     

Downsizing a giant: re-evaluating Dreadnoughtus body mass

Estimates of body mass often represent the founding assumption on which biomechanical and macroevolutionary hypotheses are based. Recently, a scaling equation was applied to a newly discovered titanosaurian sauropod dinosaur (Dreadnoughtus), yielding a 59 300 kg body mass estimate for this animal. Herein, we use a modelling approach to examine the plausibility of this mass estimate for Dreadnoughtus. We find that 59 300 kg for Dreadnoughtus is highly implausible and demonstrate that masses above 40 000 kg require high body densities and expansions of soft tissue volume outside the skeleton several times greater than found in living quadrupedal mammals. Similar results from a small sample of other archosaurs suggests that lower-end mass estimates derived from scaling equations are most plausible for Dreadnoughtus, based on existing volumetric and density data from extant animals. Although volumetric models appear to more tightly constrain dinosaur body mass, there remains a clear need to further support these models with more exhaustive data from living animals. The relative and absolute discrepancies in mass predictions between volumetric models and scaling equations also indicate a need to systematically compare predictions across a wide size and taxonomic range to better inform studies of dinosaur body size.     

Serpin Treatment Suppresses Inflammatory Vascular Lesions in Temporal Artery Implants (TAI) from Patients with Giant Cell Arteritis

Giant cell arteritis (GCA) and Takayasu’s disease are inflammatory vasculitic syndromes (IVS) causing sudden blindness and widespread arterial obstruction and aneurysm formation. Glucocorticoids and aspirin are mainstays of treatment, predominantly targeting T cells. Serp-1, a Myxomavirus-derived serpin, blocks macrophage and T cells in a wide range of animal models. Serp-1 also reduced markers of myocardial injury in a Phase IIa clinical trial for unstable coronary disease. In recent work, we detected improved survival and decreased arterial inflammation in a mouse Herpesvirus model of IVS. Here we examine Serp-1 treatment of human temporal artery (TA) biopsies from patients with suspected TA GCA arteritis after implant (TAI) into the aorta of immunodeficient SCID (severe combined immunodeficiency) mice. TAI positive for arteritis (GCA^pos) had significantly increased inflammation and plaque when compared to negative TAI (GCA^neg). Serp-1 significantly reduced intimal inflammation and CD11b⁺ cell infiltrates in TAI, with reduced splenocyte Th1, Th17, and Treg. Splenocytes from mice with GCA^pos grafts had increased gene expression for interleukin-1beta (IL-1β), IL-17, and CD25 and decreased Factor II. Serp-1 decreased IL-1β expression. In conclusion, GCA^pos TAI xenografts in mice provide a viable disease model and have increased intimal inflammation as expected and Serp-1 significantly reduces vascular inflammatory lesions with reduced IL-1β.     

Effect of Pasteurization on Survival of Certain Psychrophilic Bacteria

The effect of pasteurization on survival in milk of four strains of psychrophilic pseudomonads and of one strain of Alcaligenes was determined. Cell numbers were very low immediately after pasteurization, but increased after storage. The survival of psychrophilic bacteria in pasteurized milk appeared to depend on the initial number of organisms, and detection depended partly on the length of time of storage at 3 to 5 C.     

Correcting for Purifying Selection: An Improved Human Mitochondrial Molecular Clock

There is currently no calibration available for the whole human mtDNA genome, incorporating both coding and control regions. Furthermore, as several authors have pointed out recently, linear molecular clocks that incorporate selectable characters are in any case problematic. We here confirm a modest effect of purifying selection on the mtDNA coding region and propose an improved molecular clock for dating human mtDNA, based on a worldwide phylogeny of > 2000 complete mtDNA genomes and calibrating against recent evidence for the divergence time of humans and chimpanzees. We focus on a time-dependent mutation rate based on the entire mtDNA genome and supported by a neutral clock based on synonymous mutations alone. We show that the corrected rate is further corroborated by archaeological dating for the settlement of the Canary Islands and Remote Oceania and also, given certain phylogeographic assumptions, by the timing of the first modern human settlement of Europe and resettlement after the Last Glacial Maximum. The corrected rate yields an age of modern human expansion in the Americas at ∼15 kya that—unlike the uncorrected clock—matches the archaeological evidence, but continues to indicate an out-of-Africa dispersal at around 55–70 kya, 5–20 ky before any clear archaeological record, suggesting the need for archaeological research efforts focusing on this time window. We also present improved rates for the mtDNA control region, and the first comprehensive estimates of positional mutation rates for human mtDNA, which are essential for defining mutation models in phylogenetic analyses.