Vertebrate retinas are generally composed of rod (dim-light) and cone (bright-light) photoreceptors with distinct morphologies that evolved as adaptations to nocturnal/crepuscular and diurnal light environments. Over 70 years ago, the "transmutation" theory was proposed to explain some of the rare exceptions in which a photoreceptor type is missing, suggesting that photoreceptors could evolutionarily transition between cell types. Although studies have shown support for this theory in nocturnal geckos, the origins of allcone retinas, such as those found in diurnal colubrid snakes, remain a mystery. Here we investigate the evolutionary fate of the rods in a diurnal garter snake and test two competing hypotheses: (i) that the rods, and their corresponding molecular machinery, were lost or (ii) that the rods were evolutionarily modified to resemble, and function, as cones. Using multiple approaches, we find evidence for a functional and unusually blue-shifted rhodopsin that is expressed in small single "cones." Moreover, these cones express rod transducin and have rod ultrastructural features, providing strong support for the hypothesis that they are not true cones, as previously thought, but rather are modified rods. Several intriguing features of garter snake rhodopsin are suggestive of a more cone-like function. We propose that these cone-like rods may have evolved to regain spectral sensitivity and chromatic discrimination as a result of ancestral losses of middle-wavelength cone opsins in early snake evolution. This study illustrates how sensory evolution can be shaped not only by environmental constraints but also by historical contingency in forming new cell types with convergent functionality. rhodopsin evolution | visual evolution | reptile vision | snake photoreceptors | visual pigment H ow complex structures can arise has long fascinated evolutionary biologists, and the evolution of the eye, as noted by Charles Darwin (1), is perhaps the most famous example. Within the vertebrate eye, the light-sensing photoreceptors are complex, highly specialized cellular structures that can be divided into two general types based on their distinct morphologies and functions: cones, which are active during the day and contain cone opsin pigments, and rods, which mediate dim-light vision and contain rhodopsin (RH1) (2-4). The visual pigments contained in cone photoreceptors are classified into four different subtypes that mediate vision across the visible spectrum from the UV to the red (2). Although most vertebrate retinas are duplex, containing both cones and rods, squamate reptiles (lizards and snakes) are unusual, not only in having highly variable photoreceptor morphologies, but also for several instances of the absence of an entire class of photoreceptors, resulting in simplex retinas composed of only cones or rods (4).In a seminal book published in 1942, Walls (4) hypothesized that, during evolution, vertebrate photoreceptors could transform from one type to another, a process that he termed photoreceptor "transm...

RUNX3/AML2 is a Runt domain transcription factor like RUNX1/AML1 and RUNX2/ AML3. Regulated by 2 promoters P1 and P2, RUNX3 is frequently inactivated by P2 methylation in solid tumors. Growing evidence has suggested a role of this transcription factor in hematopoiesis. However, genetic alterations have not been reported in blood cancers. In this study on 73 acute myeloid leukemia (AML) patients (44 children and 29 adults), we first showed that high RUNX3 expression among childhood AML was associated with a shortened event-free survival, and RUNX3 was significantly underexpressed in the prognostically favorable subgroup of AML with the t(8;21) and inv(16) translocations. We further demonstrated that this RUNX3 repression was mediated not by P2 methylation, but RUNX1-ETO and CBF␤-MYH11, the fusion products of t(8; 21) and inv(16), via a novel transcriptional mechanism that acts directly or indirectly in collaboration with RUNX1, on 2 conserved RUNX binding sites in the P1 promoter. In in vitro studies, ectopically expressed RUNX1-ETO and CBF␤-MYH11 also inhibited endogenous RUNX3 expression. Taken together, RUNX3 was the first transcriptional target found to be commonly repressed by the t(8;21) and inv (16) IntroductionThe Runt domain transcription factor family includes RUNX1/ AML1, RUNX2/AML3, and RUNX3/AML2. These transcription factors share a conserved Runt domain for binding to a 6-base pair (bp) DNA sequence (TGT/cGGT) and heterodimerization with core-binding factor ␤ (CBF␤). CBF␤ does not bind DNA directly but increases the ability of RUNX proteins to bind DNA and regulate transcription. 1 RUNX1 and RUNX2 are essential for hematopoiesis and osteogenesis, respectively. 2,3 Moreover, RUNX1 regulates neuron and muscle function. 4,5 On the other hand, RUNX3 is involved in neurogenesis and thymopoiesis and acts as a tumor suppressor in gastric cancer. [6][7][8] In addition, RUNX3 is inactivated frequently by promoter methylation and less frequently by gene deletion, point mutations, and protein mislocalization in various solid tumors. 9 RUNX3 and RUNX1 show prominent expression in hematopoietic cells and different subsets of neurons, 4,10 while RUNX2 is expressed mainly in bone and also other tissues including hematopoietic stem cells. 11 The expression of RUNX3 and RUNX1 can be induced by retinoic acid, suggesting that these transcription factors may jointly regulate retinoic acid-mediated hematopoietic differentiation. 10 The functional overlap was supported by the observations that hematopoietic defects due to RUNX1 deficiency could be rescued by RUNX3. 12,13 Further evidence suggesting a role of RUNX3 in hematopoiesis was reduction of mature blood cell formation in zebrafish by RUNX3 depletion. 14 The role of RUNX3 in tumorigenesis and its potential involvement in hematopoiesis suggest a role of this transcription factor in hematological malignancies. However, genetic alterations of RUNX3 have not been reported in acute myeloid leukemia (AML). 15 t(8;21)(q22;q22) and inv(16)(p13;q22) are the 2 most common t...

© F e r r a t a S t o r t i F o u n d a t i o nH929 cells using increasing doses over a period of 96 h. Results of WST-1 assays ( Figure 1A) and trypan blue exclusion ( Figure 1B) showed that PF4 markedly inhibited the growth of these cell lines in time-and dose-dependent manners. A significant decrease in cell number was observed for OPM2, NCI-H929 and U266 after 24, 72 and 96 h of incubation with PF4. The inhibitory concentration at 50% (IC 50 ) for these three cell lines were approximately 2, 4 and 4 μM, respectively. Next, we investigated whether the observed inhibitory effects of PF4 on cell growth were due to cell cycle arrest, apoptosis, or both. The effect of PF4 on the cellular DNA content was determined using flow cytometric analysis in U266 and NCI-H929 cell lines. While changes in G0/G1, S, and G2/M phases were not distinctively different, we observed a population of cells in the sub-G1 phase indicative of increased apoptosis after PF4 treatment (data not shown). To further confirm that apoptosis was induced by PF4, we treated U266, OPM2 and NCI-H929 cells with increasing doses of PF4 and determined the percentage of apoptotic cells by flow cytometric analysis of annexin V and 7-amino-actinomycin D (7-AAD). Results showed that PF4 led to an increase in apoptotic cells (annexin V+ and/or 7AAD+) in all three of these MM cell lines ( Figure 1C). Pretreatment of cells with cycloheximide, a protein synthesis inhibitor, inhibited PF4-induced apoptosis of MM cells (P=0.001) (Online Supplementary Figure S1), indicating that the induction of apoptosis by PF4 is likely dependent on upregulation of pro-apoptotic proteins. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays in U266 and OPM2 cells further confirmed that PF4 induced apoptosis in MM cells, as evidenced by the observed increase in staining of nuclear DNA fragments (Online Supplementary Figure S2). In addition, treatment of OPM2 and U266 cells with PF4 triggered a marked increase in proteolytic cleavage of PARP, a signature event during apoptosis ( Figure 1D). Similarly, PF4 increased caspase-3 activity, an upstream activator of PARP, by 2.6-fold in U266 cells and by 3.2-fold in OPM2 cells ( Figure 1E).We also examined the effect of PF4 on purified cells from patients with MM. CD138 + plasma cells were isolated from 26 patients diagnosed with MM as described in Online Supplementary Table S2. Cells were treated with PF4 for 48 h and the levels of apoptosis were measured by annexin V-7AAD staining. To compare the cytotoxicity of PF4 in MM and normal cells, normal plasma cells from the bone marrow of healthy donors and normal mononuclear cells from the peripheral blood of healthy donors were obtained. We found minimal changes and a significant increase of mean percentages of apoptotic cells in PF4-treated normal (bone marrow plasma cells and peripheral blood mononuclear cells) and patients' MM cells, respectively (0.01±2.78%, 0.06±1.36%, and 15.16±2.52%) ( Figure 1F). Taken together, our findings suggest that PF4 inhib...