Akt-Induced Phosphorylation of N-CoR at Serine 1450 Contributes to Its Misfolded Conformational Dependent Loss (MCDL) in Acute Myeloid Leukemia of the M5 Subtype

The nuclear receptor co-repressor (N-CoR) is a key component of the generic co-repressor complex that plays an important role in the control of cellular growth and differentiation. As shown by us recently, the growth suppressive function of N-CoR largely relies on its capacity to repress Flt3, a key regulator of cellular gorwth during normal and malignant hematopoesis. We further demonstrated how de-repression of Flt3 due to the misfolded conformation dependent loss (MCDL) of N-CoR contributed to malignant growth in acute myeloid leukemia (AML). However, the molecular mechanism underlying the MCDL of N-CoR and its implication in AML pathogenesis is not fully understood. Here, we report that Akt-induced phosphorylation of N-CoR at the consensus Akt motif is crucial for its misfolding and subsequent loss in AML (AML-M5). N-CoR displayed significantly higher level of serine specific phosphorylation in almost all AML-M5 derived cells and was subjected to processing by AML-M5 specific aberrant protease activity. To identify the kinase linked to N-CoR phosphorylation, a library of activated kinases was screened with the extracts of AML cells; leading to the identification of Akt as the putative kinase linked to N-CoR phosphorylation. Consistent with this finding, a constitutively active Akt consistently phosphorylated N-CoR leading to its misfolding; while the therapeutic and genetic ablation of Akt largely abrogated the MCDL of N-CoR in AML-M5 cells. Site directed mutagenic analysis of N-CoR identified serine 1450 as the crucial residue whose phosphorylation by Akt was essential for the misfolding and loss of N-CoR protein. Moreover, Akt-induced phosphorylation of N-CoR contributed to the de-repression of Flt3, suggesting a cross talk between Akt signaling and N-CoR misfolding pathway in the pathogenesis of AML-M5. The N-CoR misfolding pathway could be the common downstream thread of pleiotropic Akt signaling activated by various oncogenic insults in some subtypes of leukemia and solid tumors.     

Plasmodium falciparum Erythrocytic Stage Parasites Require the Putative Autophagy Protein PfAtg7 for Normal Growth

Analysis of the Plasmodium falciparum genome reveals a limited number of putative autophagy genes, specifically the four genes involved in ATG8 lipidation, an essential step in formation of autophagosomes. In yeast, Atg8 lipidation requires the E1-type ligase Atg7, an E2-type ligase Atg3, and a cysteine protease Atg4. These four putative P. falciparum ATG (PfATG) genes are transcribed during the parasite’s erythrocytic stages. PfAtg7 has relatively low identity and similarity to yeast Atg7 (14.7% and 32.2%, respectively), due primarily to long insertions typical of P. falciparum. Excluding the insertions the identity and similarity are higher (38.0% and 70.8%, respectively). This and the fact that key residues are conserved, including the catalytic cysteine and ATP binding domain, we hypothesize that PfAtg7 is the activating enzyme of PfAtg8. To assess the role of PfAtg7 we have generated two transgenic parasite lines. In one, the PfATG7 locus was modified to introduce a C-terminal hemagglutinin tag. Western blotting reveals two distinct protein species, one migrating near the predicted 150 kDa and one at approximately 65 kDa. The second transgenic line introduces an inducible degradation domain into the PfATG7 locus, allowing us to rapidly attenuate PfAtg7 protein levels. Corresponding species are also observed in this parasite line at approximately 200 kDa and 100 kDa. Upon PfATG7 attenuation parasites exhibit a slow growth phenotype indicating the essentiality of this putative enzyme for normal growth.     

2-Hydroxy Arachidonic Acid: A New Non-Steroidal Anti-Inflammatory Drug

Background

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a family of COX1 and COX2 inhibitors used to reduce the synthesis of pro-inflammatory mediators. In addition, inflammation often leads to a harmful generation of nitric oxide. Efforts are being done in discovering safer NSAIDs molecules capable of inhibiting the synthesis of pro-inflammatory lipid mediators and nitric oxide to reduce the side effects associated with long term therapies.

Methodology/Principal Findings

The analogue of arachidonic acid (AA), 2-hydroxy-arachidonic acid (2OAA), was designed to inhibit the activities of COX1 and COX2 and it was predicted to have similar binding energies as AA for the catalytic sites of COX1 and COX2. The interaction of AA and 2OAA with COX1 and COX2 was investigated calculating the free energy of binding and the Fukui function. Toxicity was determined in mouse microglial BV-2 cells. COX1 and COX2 (PGH2 production) activities were measured in vitro. COX1 and COX2 expression in human macrophage-like U937 cells were carried out by Western blot, immunocytochemistry and RT-PCR analysis. NO production (Griess method) and iNOS (Western blot) were determined in mouse microglial BV-2 cells. The comparative efficacy of 2OAA, ibuprofen and cortisone in lowering TNF-α serum levels was determined in C57BL6/J mice challenged with LPS. We show that the presence of the –OH group reduces the likelihood of 2OAA being subjected to H* abstraction in COX, without altering significantly the free energy of binding. The 2OAA inhibited COX1 and COX2 activities and the expression of COX2 in human U937 derived macrophages challenged with LPS. In addition, 2OAA inhibited iNOS expression and the production of NO in BV-2 microglial cells. Finally, oral administration of 2OAA decreased the plasma TNF-α levels in vivo.

Conclusion/Significance

These findings demonstrate the potential of 2OAA as a NSAID.     

Model vs. playback experiments: The impact of sensory mode on predator-specific escape responses in saki monkeys

Although experimentally simulating predator presence helps improve sample sizes in studies of free-ranging animals, few studies have examined whether auditory playbacks and visual models produce similar results. Additionally, it is unclear if anti-predator strategies are specific to predator hunting styles in understudied Neotropical pitheciid primates, limiting what we can generalize about this phenomenon across this taxonomic order. We conducted predator simulation experiments to assess whether wild Rylands' bald-faced saki monkeys (Pithecia rylandsi) recognize predators based solely on acoustic cues, exhibit predator-specific responses to different predator types, and vary responses to presentations in different sensory modes. In our playback experiments, sakis had weak responses to non-predator control vocalizations compared to jaguar growls and harpy eagle shrieks. In most predator playbacks, subjects' first glance corresponded to the direction from which simulated predators would typically attack (above vs. below). However, although sakis exhibited appropriate movement responses to harpy playbacks (i.e., descending canopy), they exhibited no clear movement patterns when presented with jaguar playbacks. In contrast, jaguar model experiments consistently elicited fast approaches, mobbing-style responses, and long alarm calling bouts. Thus, if we had relied on playbacks alone, we might have concluded that sakis have only generalized responses to terrestrial ambush predators. In fact, in all variables measured (e.g., latency, number of calls, and response duration), models of both predator species elicited stronger reactions than playbacks. Results indicate that bald-faced sakis can identify predators based solely on vocalizations, but do not exhibit predator-specific escape responses to terrestrial predators based on acoustic cues alone. The differential response to playbacks and models calls into question the reliability of using acoustic-only stimuli to assess the specificity of anti-predator behavior to predator hunting styles in some primate species.     

Amyloid precursor protein is a restriction factor that protects against Zika virus infection in mammalian brains

Zika virus (ZIKV) is a neurotropic flavivirus that causes several diseases including birth defects such as microcephaly. Intrinsic immunity is known to be a frontline defense against viruses through host anti-viral restriction factors. Limited knowledge is available on intrinsic immunity against ZIKV in brains. Amyloid precursor protein (APP) is predominantly expressed in brains and implicated in the pathogenesis of Alzheimer''s diseases. We have found that ZIKV interacts with APP, and viral infection increases APP expression via enhancing protein stability. Moreover, we identified the viral peptide, HGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGL, which is capable of en-hancing APP expression. We observed that aging brain tissues with APP had protective effects on ZIKV infection by reducing the availability of the viruses. Also, knockdown of APP expression or blocking ZIKV-APP interactions enhanced ZIKV replication in human neural progenitor/stem cells. Finally, intracranial infection of ZIKV in APP-null neonatal mice resulted in higher mortality and viral yields. Taken together, these findings suggest that APP is a restriction factor that protects against ZIKV by serving as a decoy receptor, and plays a protective role in ZIKV-mediated brain injuries.     

Responses of ‘Conference’ Pear to Deficit Irrigation: Water Relations, Leaf Discrimination Against 13CO2, Tree Starch Content, Growth, and Recovery After Rewatering

Responses to deficit irrigation (DI) throughout the fruit-growing season were studied in ‘Conference’ pear grafted onto quince M-A rootstock and grown in large containers. The treatments were (1) full irrigation (FI), (2) DI during Stage I of fruit growth (DI-Stage I), and (3) DI during Stage II of fruit growth (DI-Stage II). Four whole trees were sampled before Stage I and from all treatments at the end of Stage I, end of Stage II (fruit harvest), and before leaf fall. There was less discrimination against ¹³CO₂ in DI leaves, indicative of reduced photosynthetic capacity. DI treated trees had lower starch content in branches and trunks but root starch concentration was the same between DI- and FI-treated trees. Compared to FI-treated trees, leaf, shoot, branch, and trunk dry biomass was reduced by 34, 50, 37, and 32 %, respectively, in DI-Stage I and by 45, 73, 37, and 22 % in DI-Stage II. Root growth was not affected by DI. Trees had limited capacity for storing starch in roots. Recovery of the aboveground starch concentration for DI treatments occurred within 1 month after rewatering but total starch content never recovered.     

Circadian system responses to nocturnal and diurnal hosts in the kissing bug,Triatoma infestans

ABSTRACT

Insects express diverse behavioral rhythms synchronized to environmental cycles. While circadian entrainment to light–dark cycles is ubiquitous in living organisms, synchronization to non-photic cycles may be critical for hematophagous bugs that depend on rhythmic hosts. The purpose was to determine whether Triatoma infestans are capable of synchronizing to the circadian rhythms of potential hosts with temporally distinct activity patterns; and, if so, if this synchronization occurs through masking or entrainment. Precise synchronization with the food source may be critical for the insects’ survival due to the specific predatory or defensive nature of each host. Kissing bugs were housed in a compartment in constant dark, air-flow-connected to another compartment with a nocturnal or a diurnal host; both hosts were synchronized to a light–dark cycle. The activity rhythms of kissing bugs were modulated by the daily activity rhythms of the vertebrates. Effects were a decrease in the endogenous circadian period, independent of the host being nocturnal or diurnal; in some cases relative coordination occurred and in others synchronization was clearly achieved. Moreover, splitting and bimodality arose, phenomena that were also affected by the host presence. The results indicate that T. infestans were able to detect the non-photic cycle of their potential hosts, an ability that surely facilitates feeding and hinders predation risk. Understanding triatomines behavior is of fundamental importance to the design of population control methods.     

New Insights into the Circadian Rhythm of Acute Myocardial Infarction in Subgroups

The aim of this study was to determine the existence of the circadian rhythm (CR) in the onset of acute myocardial infarction (AMI) in different patient subgroups. Information was collected about 41,244 infarctions from the database of the ARIAM (Analysis of Delay in AMI) Spanish multicenter study. CR in AMI were explored in subgroups of cases categorized by age, gender, previous ischemic heart disease (PIHD), outcome in coronary care unit, infarction electrocardiograph (ECG) characteristics (Q wave or non‐Q wave), and location of AMI. Cases were classified according to these variables in the different subgroups. To verify the presence of CR, a simple test of equality of time series based on the multiple‐sinusoid (24, 12, and 8 h periods) cosinor analysis was developed. For the groups as a whole, the time of pain onset as an indicator of the AMI occurrence showed a CR (p<0.0001), with a morning peak at 10:10 h. All the analyzed subgroups also showed CR. Comparison between subgroups showed significant differences in the PIHD (p<0.01) and infarction ECG characteristics (p<0.01) groups. The CR of the subgroup with Q‐wave infarction differed from that of non‐Q wave subgroup (p<0.01) when the patients had PIHD (23% in Q wave infarction vs. 39.2% in non‐Q wave). AMI onset followed a CR pattern, which is also observed in all analyzed subgroups. Differences in the CR according to the Q/non‐Q wave infarction characteristics could be determined by PIHD. The cosinor model fit with three components (24, 12, and 8 h periods) showed a higher sensitivity than the single 24 h period analysis.     

Genetic and phylogenetic evidence for horizontal gene transfer among ecologically disparate groups of marine Vibrio

Vibrio represents a diverse bacterial genus found in different niches of the marine environment, including numerous genera of marine sponges (phylum Porifera), inhabiting different depths and regions of benthic seas, that are potentially important in driving adaptive change among Vibrio spp. Using 16S rRNA gene sequencing, a previous study showed that sponge‐derived (SD) vibrios clustered with their mainstream counterparts present in shallow, coastal ecosystems, suggesting a genetic relatedness between these populations. Sequences from the topA, ftsZ, mreB, rpoD, rctB and toxR genes were used to investigate the degree of relatedness existing between these two separate populations by examining their phylogenetic and genetic disparity. Phylogenies were constructed from the concatenated sequences of the six housekeeping genes using maximum‐parsimony, maximum‐likelihood and neighbour‐joining algorithms. Genetic recombination was evaluated using the incongruence length difference test, Split decomposition and measuring overall compatibility of sites. This combined technical approach provided evidence that SD Vibrio strains are largely genetically homologous to their shallow‐water counterparts. Moreover, the analyses conducted support the existence of extensive horizontal gene transfer between these two groups, supporting the idea of a single panmictic population structure among vibrios from two seemingly distinct, marine environments.