Unveiling winter survival strategies: physiological and metabolic responses to cold stress of Monochamus saltuarius larvae during overwintering.

BACKGROUND: Monochamus saltuarius is a destructive trunk-borer of pine forest and an effective dispersal vector for pinewood nematode (PWN), a causative agent of pine wilt disease (PWD), which leads to major ecological disasters. Cold winter temperatures determine insect survival and distribution. However, little is known about the cold tolerance and potential physiological mechanisms of M. saltuarius. RESULTS: We demonstrated that dead Pinus koraiensis trunks do not provide larvae with insulation. The M. saltuarius larvae are freeze-tolerant species. Unlike most other freeze-tolerant insects, they can actively freeze extracellular fluid at higher subzero temperatures by increasing their supercooling points. The main energy sources for larvae overwintering are glycogen and the mid-late switch to lipid. The water balance showed a decrease in free and an increase in bound water of small magnitude. Cold stress promoted lipid peroxidation, thus activating the antioxidant system to prevent cold-induced oxidative damage. We found eight main pathways linked to cold stress and 39 important metabolites, ten of which are cryoprotectants, including maltose, UDP-glucose, d-fructose 6P, galactinol, dulcitol, inositol, sorbitol, l-methionine, sarcosine, and d-proline. The M. saltuarius larvae engage in a dual respiration process involving both anaerobic and aerobic pathways when their bodily fluids freeze. Cysteine and methionine metabolism, as well as alanine, aspartate, and glutamate metabolism, are the most important pathways linked to antioxidation and energy production. CONCLUSIONS: The implications of our findings may help strengthen and supplement the management strategies for monitoring, quarantine, and control of this pest, thereby contributing to controlling the further spread of PWD. © 2024 Society of Chemical Industry.

Determining the mechanical and decomposition properties of high energetic materials (α-RDX, ß-HMX, and ε-CL-20) using a neural network potential.

Molecular simulations of high energetic materials (HEMs) are limited by efficiency and accuracy. Recently, neural network potential (NNP) models have achieved molecular simulations of millions of atoms while maintaining the accuracy of density functional theory (DFT) levels. Herein, an NNP model covering typical HEMs containing C, H, N, and O elements is developed. The mechanical and decomposition properties of 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), hexahydro-1,3,5-trinitro-1,3,5-triazine (HMX), and 2,4,6,8,10,12-hexanitrohexaazaisowurtzitane (CL-20) are determined by employing the molecular dynamics (MD) simulations based on the NNP model. The calculated results show that the mechanical properties of α-RDX, ß-HMX, and ε-CL-20 agree with previous experiments and theoretical results, including cell parameters, equations of state, and elastic constants. In the thermal decomposition simulations, it is also found that the initial decomposition reactions of the three crystals are N-NO2 homolysis, corresponding radical intermediates formation, and NO2-induced reactions. This decomposition trajectory is mainly divided into two stages separating from the peak of NO2: pyrolysis and oxidation. Overall, the NNP model for C/H/N/O elements in this work is an alternative reactive force field for RDX, HMX, and CL-20 HEMs, and it opens up new potential for future kinetic study of nitramine explosives.

The thermal decomposition mechanism of RDX/AP composites: ab initio neural network MD simulations.

A neural network potential (NNP) is developed to investigate the decomposition mechanism of RDX, AP, and their composites. Utilizing an ab initio dataset, the NNP is evaluated in terms of atomic energy and forces, demonstrating strong agreement with ab initio calculations. Numerical stability tests across a range of timesteps reveal excellent stability compared to the state-of-the-art ReaxFF models. Then the thermal decomposition of pure RDX, AP, and RDX/AP composites is performed using NNP to explore the coupling effect between RDX and AP. The results highlight a dual interaction between RDX and AP, i.e., AP accelerates RDX decomposition, particularly at low temperatures, and RDX promotes AP decomposition. Analyzing RDX trajectories at the RDX/AP interface unveils a three-part decomposition mechanism involving N-N bond cleavage, H transfer with AP to form Cl-containing acid, and chain-breaking reactions generating small molecules such as N2, CO, and CO2. The presence of AP enhances H transfer reactions, contributing to its role in promoting RDX decomposition. This work studies the reaction kinetics of RDX/AP composites from the atomic point of view, and can be widely used in the establishment of reaction kinetics models of composite systems with energetic materials.

Genome-Wide Identification of the Odorant Receptor Gene Family and Revealing Key Genes Involved in Sexual Communication in Anoplophora glabripennis.

Insects use a powerful and complex olfactory recognition system to sense odor molecules in the external environment to guide behavior. A large family of odorant receptors (ORs) mediates the detection of pheromone compounds. Anoplophora glabripennis is a destructive pest that harms broad-leaved tree species. Although olfactory sensation is an important factor affecting the information exchange of A. glabripennis, little is known about the key ORs involved. Here, we identified ninety-eight AglaORs in the Agla2.0 genome and found that the AglaOR gene family had expanded with structural and functional diversity. RT-qPCR was used to analyze the expression of AglaORs in sex tissues and in adults at different developmental stages. Twenty-three AglaORs with antennal-biased expression were identified. Among these, eleven were male-biased and two were female-biased and were more significantly expressed in the sexual maturation stage than in the post-mating stage, suggesting that these genes play a role in sexual communication. Relatively, two female-biased AglaORs were overexpressed in females seeking spawning grounds after mating, indicating that these genes might be involved in the recognition of host plant volatiles that may regulate the selection of spawning grounds. Our study provides a theoretical basis for further studies into the molecular mechanism of A. glabripennis olfaction.

Comparative mitochondrial genomic analysis provides new insights into the evolution of the subfamily Lamiinae (Coleoptera: Cerambycidae).

The genus Monochamus within the subfamily Lamiinae is the main vector of Bursaphelenchus xylophilus, which causes pine wilt disease and induces substantial economic and ecological losses. Only three complete mitochondrial genomes of the genus Monochamus have been sequenced to date, and no comparative mitochondrial genomic studies of Lamiinae have been conducted. Here, the mitochondrial genomes of two Monochamus species, M. saltuarius and M. urussovi, were newly sequenced and annotated. The composition and order of genes in the mitochondrial genomes of Monochamus species are conserved. All transfer RNAs exhibit the typical clover-leaf secondary structure, with the exception of trnS1. Similar to other longhorn beetles, Lamiinae mitochondrial genomes have an A + T bias. All 13 protein-coding genes have experienced purifying selection, and tandem repeat sequences are abundant in the A + T-rich region. Phylogenetic analyses revealed congruent topologies among trees inferred from the five datasets, with the monophyly of Acanthocinini, Agapanthiini, Batocerini, Dorcaschematini, Pteropliini, and Saperdini receiving high support. The findings of this study enhance our understanding of mitochondrial genome evolution and will provide a basis for future studies of population genetics and phylogenetic investigations in this group.

Cloning and Molecular Characterization of Hsp Genes from Anoplophora glabripennis and Their Responses to Cold Stress.

Anoplophora glabripennis (Agla) is an important global quarantine pest due to its highly destructive impacts on forests. It is widely distributed in many countries in Asia, Europe, and North America. The survival of A. glabripennis larvae has been facilitated by its high adaptability to low temperature. When insects are subjected to temperature stress, heat shock proteins (Hsps) limit cell damage and improve cell tolerance via their protein folding, localization, and degradation activities. However, the temperature adaptation mechanisms of A. glabripennis Hsps remain unclear. In this study, four A. glabripennis Hsp genes, AglaHsp20.43, AglaHsp71.18, AglaHsp82.09, and AglaHsp89.76, were cloned. Sequence analysis showed that all four Hsps had specific conserved domains. Phylogenetic analysis revealed that Hsps from different subfamilies were evolutionarily conserved, and that AglaHsps were highly similar to those of Coleoptera species. Protein expression vectors (pET30a-AglaHsps) were constructed and used to express AglaHsps in E. coli, where all four proteins were expressed in inclusion bodies. Western blot analysis showed that AglaHsps were expressed at a range of temperatures, from -10 °C to 25 °C. AglaHsp82.09 and AglaHsp89.76 showed high expressions with treatment at 0 °C. Our results will facilitate clarification of the molecular mechanisms underlying A. glabripennis responses to environmental stress.

Thermal decomposition of nano Al-based energetic composites with fluorinated energetic polyurethane binders: experimental and theoretical understandings for enhanced combustion and energetic performance.

Energetic composites composed of polymeric binders and metallic fuels are widely used in industrial and military fields, and their performance is largely dependent on the combustion process. Fluorinated energetic polymeric binders can facilitate the combustion of metallic fuels such as aluminum particles and enhance the energetic level of the energetic composites. In this report, fluorinated energetic polyurethanes (FPUs) were applied as binders for energetic composites with aluminum nanoparticles (AlNPs). The fluorinated components in the energetic binder could be a uniform dispersion inside the composites, endowing the composites with decent mechanical properties and high combustion rate. Most significantly, compared with the composites without fluorine, FPU/AlNP energetic composites not only showed a remarkably improved combustion efficiency, but also, surprisingly, a dramatic enhancement in the heat of explosion by 91.2%, despite the low content of fluorine. By analyzing the combustion products together with kinetic simulations derived from chemical reaction neural network (CRNN) modelling, a detailed mechanistic understanding of the combustion process was provided, suggesting the importance of synergistic effects brought by the fluorinated and energetic components.

Optical Properties of Polycyclic Aromatic Hydrocarbon Clusters with Oxygenated Substitutions: A Theoretical Analysis.

The introduction of functional groups at high coverage levels can have significant impacts on the band structures of polycyclic aromatic hydrocarbon (PAH) clusters. The HOMO-LUMO gaps are highly sensitive to the type and distribution of functional groups. An in-house method is proposed to build PAH (naphthalene, pyrene, coronene, and ovalene) clusters with surface functionalization of -OH, -COOH and -CHO groups using the DFT method. The -CHO groups are found to reduce the gap value the most, but exceptions exist due to the spatial distribution of functional groups. Considering the impact of -CHO groups only, we can approximate that the impact of functional groups lies in the range of 0.14-0.89 eV. Applying further analysis on the possible energy number of energy transitions of substituted PAH clusters, it is shown that PAH clusters with oxygenated functions still behave like an indirect band gap material. The coupling effect of PAH stacking and PAH size is also addressed. A simple expression is proposed to estimate the bandgap of a mixed system using the HOMO and LUMO energy of the two components. Further attempts are made to interpret recent experiments from the impact of PAH stacking, PAH size, and functional groups.

Genome-wide identification and expression analysis of the Hsp gene superfamily in Asian long-horned beetle (Anoplophora glabripennis).

The environmental adaptability of insects has been a key focus of ecological research. As molecular chaperones, Heat shock proteins (Hsps) play an important role in insect responses to environmental stress. Anoplophora glabripennis is a destructive pest of broad-leaved trees such as poplars. The ability to adapt to low temperature is an important factor for successful colonization of A. glabripennis in new diffusion area. However, the roles of Hsp in the stress responses in A. glabripennis have not been established. In this study, we identified 47 Hsp genes, including 3 Hsp90, 14 Hsp70, 9 Hsp60, and 21 sHsp genes. The Hsp gene family expanded substantially in A. glabripennis. The differences in expression patterns may be related to the type and intensity of stress. Larval overwintering transcriptomes showed that 13 Hsp genes were not induced during overwintering and 21 Hsp genes were involved in the regulation of life activities under non-stress conditions. In a quantitative RT-PCR analysis, AglaHsp90-2 responded more quickly under gradient cooling treatments; AglaHsp90-2 and AglaHsp90-3 were sensitive to treatment at 0 °C for 6 h under instantaneous cooling. Our results provide a theoretical basis for clarifying the molecular mechanism of Hsp genes in A. glabripennis in responsing to environmental stresses.

Effects of Initial Turbulence on the Explosion Limit and Flame Propagation Behaviors of Premixed Syngas-Air Mixtures.

Syngas with important industrial applications has explosive hazards because of its flammability. It is necessary and valuable to study the combustion and explosion characteristics of syngas under actual working conditions. To explore the effects of initial turbulence on the explosion limits and the flame propagation behavior of the syngas-air mixtures, the explosion limits were tested by the explosive limit instrument, and the flame propagation process in the spherical pressure vessel was recorded by a high-speed camera. By adjusting the rotating speed of the stirrer to obtain turbulence of different intensities, the explosion limit and flame propagation behavior of syngas under different turbulent conditions were analyzed. The explosion limit of syngas in the macro-static state was 9.5-76.1%, and its flame front was relatively smooth. However, with the increase in turbulence intensity, both the upper and lower explosion limits of syngas decreased. The disturbance of turbulence made the flame shape change. The flame front was wrinkled, and the flame boundary was blurred, which became more and more obvious with the increase in turbulence intensity. The maximum velocity and duration of flame propagation increased with the increase in turbulence intensity. Under the same turbulence intensity, the flame propagation velocity generally augmented first and then lessened.

Transcriptomic and Metabolomic Data Reveal the Key Metabolic Pathways Affecting Streltzoviella insularis (Staudinger) (Lepidoptera: Cossidae) Larvae During Overwintering.

Streltzoviella insularis (Staudinger) (Lepidoptera: Cossidae) is a woodboring insect feeding on Fraxinus pennsylvanica, Sophora japonica, and Ginkgo biloba, as well as many other species used for urban greening and plain afforestation in northern China, including the temperate north. There is also a risk that S. insularis could spread through the transportation of seedlings, thereby increasing urban greening costs. However, how S. insularis increases the cold tolerance then reduces it to survive winter temperature below 0°C remains unclear. In the transcriptomic of S. insularis, we identified three profiles (profile 25, 27, and 13) whose trends related to the cold tolerance. We detected 1,783 differentially expressed genes (in profile 25) and identified 522 genes enriched in the AMPK signaling pathway. The metabolome analysis identified 122 differential metabolites. We identified four co-pathways, among which "Glycerophospholipid metabolism" was the pathway most enriched in differentially expressed genes and differential metabolites. The AMPK signaling and glycerophospholipid metabolism pathways play key roles in the natural overwintering physiological process of S. insularis larvae.

Expression analysis of genes related to cold tolerance in Dendroctonus valens.

Pine beetles are well known in North America for their widespread devastation of pine forests. However, Dendroctonus valens LeConte is an important invasive forest pest in China also. Adults and larvae of this bark beetle mainly winter at the trunks and roots of Pinus tabuliformis and Pinus sylvestris; larvae, in particular, result in pine weakness or even death. Since the species was introduced from the United States to Shanxi in 1998, its distribution has spread northward. In 2017, it invaded a large area at the junction of Liaoning, Inner Mongolia and Hebei provinces, showing strong cold tolerance. To identify genes relevant to cold tolerance and the process of overwintering, we sequenced the transcriptomes of wintering and non-wintering adult and larval D. valens using the Illumina HiSeq platform. Differential expression analysis methods for other non-model organisms were used to compare transcript abundances in adults and larvae at two time periods, followed by the identification of functions and metabolic pathways related to genes associated with cold tolerance. We detected 4,387 and 6,091 differentially expressed genes (DEGs) between sampling dates in larvae and adults, respectively, and 1,140 common DEGs, including genes encoding protein phosphatase, very long-chain fatty acids protein, cytochrome P450, and putative leucine-rich repeat-containing proteins. In a Gene Ontology (GO) enrichment analysis, 1,140 genes were assigned to 44 terms, with significant enrichment for cellulase activity, hydrolase activity, and carbohydrate metabolism. Kyoto Encyclopedia of Genes and Genomes (KEGG) classification and enrichment analyses showed that the lysosomal and purine metabolism pathways involved the most DEGs, the highly enriched terms included autophagy-animal, pentose and glucuronate interconversions and lysosomal processes. We identified 140 candidate genes associated with cold tolerance, including genes with established roles in this trait (e.g., genes encoding trehalose transporter, fructose-1,6-bisphosphatase, and trehalase). Our comparative transcriptome analysis of adult and larval D. valens in different conditions provides basic data for the discovery of key genes and molecular mechanisms underlying cold tolerance.

Identification of key genes associated with overwintering in Anoplophora glabripennis larva using gene co-expression network analysis.

BACKGROUND: Anoplophora glabripennis (Coleoptera: Cerambycidae) is a major quarantine pest in forestry. It is widely distributed throughout many regions such as Asia, Europe, and North America, and has enormous destructive potential for forests. The larvae of A. glabripennis overwinter in a dormant state with strong cold tolerance, and whether the larvae survive winter determines the population density in the following year. However, the molecular mechanisms of this process are not clear. RESULTS: RNA sequencing (RNA-Seq) analysis of A. glabripennis larvae at five overwintering stages identified 6876 differentially expressed genes (DEGs). Among these, 46 functional genes that might respond to low temperature were identified. Weighted gene co-expression network analysis revealed that the MEturquoise module was correlated with the overwintering process. The STPK, PP2A, DGAT, and HSF genes were identified as hub genes using visualization of gene network. In addition, four genes related to sugar transport, gluconeogenesis and glycosylation were screened, which may be involved in the metabolic regulation of overwintering larvae. The protein-protein interaction network indicated that ribosomal protein and ATP synthase may play an important role in connecting with other proteins. The expression levels of fifteen hub genes were further validated by quantitative RT-PCR, and the results were consistent with RNA-Seq. CONCLUSION: This study demonstrates key genes that may reveal the molecular mechanism of overwintering in A. glabripennis larvae. The genes may be the potential targets to prevent larvae from surviving the cold winter by developing new biological agents using genetic engineering.

Reference Gene Selection for Expression Analyses by qRT-PCR in Dendroctonus valens.

Dendroctonus valens is the main pest of the genus Pinus. To facilitate gene expression analyses, suitable reference genes for adults and mature larvae of D. valens under different temperature conditions were determined. In particular, we obtained the sequences of candidate reference genes, ACT, TUB, SHDA, PRS18, 18S rRNA, and CYP4G55, from transcriptome data. Real-time quantitative PCR was used to analyze gene expression, and geNorm, NormFinder, and BestKeeper were used to evaluate expression stability. Under different temperature conditions, the expression levels of 18S rRNA, PRS18, and TUB were stable in adults, in which 18S rRNA > PRS18 > TUB. In mature larvae, the expression levels of TUB, 18S rRNA, and SDHA were stable, in which TUB > 18S rRNA > SDHA. The combination of 18S rRNA and PRS18 is recommended for studies of gene expression in adults and the combination of 18S rRNA and TUB is effective for studies of gene expression in mature larvae of D. valens under different temperature conditions.

Neuregulin-1/ErbB signaling and chronic heart failure.

Neuregulin-1 (NRG-1), a cardioactive growth factor released from endothelial cells, is indispensable for cardiac development, structural maintenance, and functional integrity of the heart. In recent years, a growing number of studies have focused on NRG-1 and members of the ErbB family that serve as receptors for NRG-1 in order to better understand the role of this signaling pathway in physiology and pathophysiology of the heart. An essential role for NRG-1 and ErbB in heart development and functionality has been suggested by studies in conditional NRG-1/ErbB-deficient mice and by the cardiac-related side effects of anti-ErbB2 antibody therapies used for treatment of breast cancer. In vitro and in vivo studies using recombinant human neuregulin-1 (rhNRG-1), which contains the epidermal growth factor (EGF)-like domain (necessary for ErbB2/ErbB4 activation), have further supported the hypothesis that NRG-1 plays an important role in heart function. Consistent with other studies, expression of rhNRG-1 not only restored normal cardiomyocytic structure altered by nutritional deficiency in cell cultures, but also improved the pumping function of the heart in several animal models of chronic heart failure (CHF). As a result of these findings, proteins involved in the NRG-1/ErbB-signaling pathway have been explored as potential drug targets for treatment of heart failure. Clinical trials to evaluate the safety and efficacy of rhNRG-1 have been conducted in both China and Australia. As predicted, rhNRG-1 treatment improved both cardiac function and reversed remodeling of the heart. Therefore, rhNRG-1 may represent a new drug for treatment of CHF with a novel therapeutic mechanism.

Cardiac functional improvement in rats with myocardial infarction by up-regulating cardiac myosin light chain kinase with neuregulin.

AIMS: Recombinant human neuregulin-1 (rhNRG-1) improves cardiac function in experimental heart failure models, but the underlying mechanism remains largely unknown. In this study, we evaluated whether rhNRG-1 could improve cardiac function via the cardiac myosin light chain kinase/myosin light chain 2 ventricular (cMLCK/MLC-2v) pathway in rats with myocardial infarction (MI). METHODS AND RESULTS: Rats with MI were intravenously infused with rhNRG-1 (5 µg/kg/h) for 7 days through osmotic pumps. The mechanism of action of rhNRG-1 was investigated by assaying the non-infarcted myocardium with gene chips. The cMLCK expression, phosphorylated MLC-2v and cardiac function were significantly up-regulated, as assessed by real-time PCR, Western blot and echocardiography, in those animals treated with rhNRG-1. Moreover, the restoration of rhNRG-1-induced sarcomeric organization in serum-free cultured neonatal rat cardiomyocytes with rhNRG-1 was inhibited by cMLCK RNA interference or ML-7, an inhibitor of MLCKs. Adenovirus containing the rat cMLCK coding region was injected into non-infarcted myocardium, and cardiac function was monitored using echocardiography and a haemodynamic machine. The dP/dt and fractional shortening decreasing significantly after MI, and improved by 15.7 and 32.1%, respectively, following local cMLCK application (all P < 0.05). CONCLUSION: Our results suggest that cMLCK is a downstream effector of rhNRG-1 involved in rhNRG-1-induced cardiac function improvement, and that myocardial cMLCK up-regulation can improve cardiac function in rats with MI.

A Phase II, randomized, double-blind, multicenter, based on standard therapy, placebo-controlled study of the efficacy and safety of recombinant human neuregulin-1 in patients with chronic heart failure.

OBJECTIVES: The purpose of this study was to assess the safety and efficacy of recombinant human neuregulin-1 (rhNRG-1) in chronic heart failure (CHF) patients. BACKGROUND: Neuregulin-1 plays important roles in maintaining cardiomyocyte structure and cardiac pumping functionality and physiology. Previously, rhNRG-1 was proven to be effective in treating heart failure in animals by reducing end-diastolic volume (EDV) and end-systolic volume (ESV) and increasing left ventricular ejection fraction (LVEF%). METHODS: A total of 44 CHF patients designated as New York Heart Association functional class II or III were enrolled in a double-blind, randomized manner and treated with a placebo or rhNRG-1 (0.3, 0.6, or 1.2 microg/kg/day) for 10 days, in addition to standard therapies. The follow-up period was 90 days; left ventricular function and structure measured by magnetic resonance imaging were the primary end points. RESULTS: Although not statistically different from placebo, the LVEF% was significantly increased by 27.11 +/- 31.12% (p = 0.009) at day 30 after rhNRG-1 treatment in the 0.6-microg/kg group, whereas it was only increased 5.83 +/- 25.75% in the placebo group (p = 0.49). In addition, there were decreases in ESV (-11.58 +/- 12.74%, p = 0.002) and EDV (-5.64 +/- 10.03%, p = 0.05) in the 0.6-microg/kg/day group at day 30; more importantly, both ESV and EDV levels continued to decrease at day 90 (-20.79 +/- 17.03% and -14.03 +/- 13.17%, respectively), accompanied by a sustained increase in LVEF%. This suggests that short-term treatment with rhNRG-1 results in a long-term reversal of remodeling. The effective dose was proven to be tolerable and safe for CHF patients. CONCLUSIONS: rhNRG-1 improved the cardiac function of CHF patients by increasing the LVEF% and showed the capability of antiremodeling by decreasing ESV and EDV compared with pre-treatment. (A Randomized, Double-Blind, Multi-Center, Placebo Parallel controlled, Standard Therapy Based Phase II Clinical Trial to Evaluate the Efficacy and Safety of Recombinant Human Neuregulin-1 for Injection in Patients with Chronic Heart Failure; ChiCTR-TRC-00000414).

Neuregulin-1/ErbB signaling: a druggable target for treating heart failure.

Neuregulin-1s are widely expressed signaling molecules that are involved in cell differentiation, proliferation, growth, survival, and apoptosis. They transmit their signals by interacting with cell membrane receptors of the ErbB family, resulting in the activation of intracellular signaling cascades, which participate in various physiological and etiological processes. Besides their essential function in the development of the heart and the physiology of cardiac pumping, there is emerging evidence for the involvement of neuregulin-1/ErbB signals in human disease, including heart failure. These reasons prompt the development of new therapeutic agents based on neuregulin-1.

Leukemogenic AML1-ETO fusion protein upregulates expression of connexin 43: the role in AML 1-ETO-induced growth arrest in leukemic cells.

AML1-ETO, a fusion protein generated by the chromosomal translocation t(8;21), is frequently associated with acute myeloid leukemia (AML). In addition to blocking differentiation, AML1-ETO is also shown to induce growth arrest in AML cells, which is unfavorable for leukemogenesis harboring the t(8;21) translocation. However, its precise mechanism is still unclear. Here we provide the first demonstration that the conditional expression of AML1-ETO by the ecdysone-inducible system dramatically increases the expression of connexin 43 (CX43), together with growth arrest at G1 phase in leukemic U937 cells. We also show that the CX43 induction inhibits the proliferation of U937 cells at G1 phase, while the suppression of CX43 expression by small interfering RNA (siRNA) effectively overcomes the growth-inhibitory effect of AML1 -ETO in leukemic cells. Furthermore, either AML1-ETO or CX43 induction elevates cell-cycle negative regulator P27(kip1) protein by inhibiting its degradation, which is antagonized by siRNA against CX43. Taken together, our data indicate that CX43 plays a role in AML1-ETO-induced growth arrest possibly through the accumulation of P27(kip1) protein. The potential mutation or/and epigenetic alterations of CX43 and its related gene(s) deserve to be explored in AML1-ETO-positive AML patients.

Induction of tumor arrest and differentiation with prolonged survival by intermittent hypoxia in a mouse model of acute myeloid leukemia.

We showed previously that mild real hypoxia and hypoxia-mimetic agents induced in vitro cell differentiation of acute myeloid leukemia (AML). We here investigate the in vivo effects of intermittent hypoxia on syngenic grafts of leukemic blasts in a PML-RARalpha transgenic mouse model of AML. For intermittent hypoxia, leukemic mice were housed in a hypoxia chamber equivalent to an altitude of 6000 m for 18 hours every consecutive day. The results show that intermittent hypoxia significantly prolongs the survival of the leukemic mice that received transplants, although it fails to cure the disease. By histologic and cytologic analyses, intermittent hypoxia is shown to inhibit the infiltration of leukemic blasts in peripheral blood, bone marrow, spleen, and liver without apoptosis induction. More intriguingly, intermittent hypoxia also induces leukemic cells to undergo differentiation with progressive increase of hypoxia-inducible factor-1alpha protein, as evidenced by morphologic criteria of maturating myeloid cells and increased expression of mouse myeloid cell differentiation-related antigens Gr-1 and Mac-1. Taken together, this study represents the first attempt to characterize the in vivo effects of hypoxia on an AML mouse model. Additional investigations may uncover ways to mimic the differentiative effects of hypoxia in a manner that will benefit human patients with AML.