Aly and THO are required for assembly of the human TREX complex and association of TREX components with the spliced mRNA

The mRNA export complex TREX (TREX) is known to contain Aly, UAP56, Tex1 and the THO complex, among which UAP56 is required for TREX assembly. Here, we systematically investigated the role of each human TREX component in TREX assembly and its association with the mRNA. We found that Tex1 is essentially a subunit of the THO complex. Aly, THO and UAP56 are all required for assembly of TREX, in which Aly directly interacts with THO subunits Thoc2 and Thoc5. Both Aly and THO function in linking UAP56 to the cap-binding protein CBP80. Interestingly, association of UAP56 with the spliced mRNA, but not with the pre-mRNA, requires Aly and THO. Unexpectedly, we found that Aly and THO require each other to associate with the spliced mRNA. Consistent with these biochemical results, similar to Aly and UAP56, THO plays critical roles in mRNA export. Together, we propose that Aly, THO and UAP56 form a highly integrated unit to associate with the spliced mRNA and function in mRNA export.     

p53 and PI3K/AKT signalings are up-regulated in flies with defects in the THO complex

The THO complex (THO) is an evolutionary conserved protein required for the formation of export-competent mRNP. The growing evidence indicates that the metazoan THO plays important roles in cell differentiation and cellular stress response. But the underlying mechanisms are poorly understood. Herein we examined the relevance of THO to cellular signaling pathways involved in cell differentiation and cellular stress response. When we examined the endogenous p53 level in the testis, it was sustained much longer during spermatogenesis in the THO mutant compared to that of wild-type. In flies with impaired THO, overexpression of p53 by eye-specific GAL4 not only enhanced p53-mediated retinal degeneration, but p53 level was also elevated compared to the control flies. Since the body size of the THO mutant flies was significantly larger than control flies, we also examined whether the PI3K/AKT signaling is enhanced in the mutant flies. The results showed that the endogenous level of phosphorylated AKT, which is the active form, was highly elevated in the THO mutants. Taken together our results suggested that both p53 and PI3K/AKT signalings are up-regulated in the flies with impaired THO.     

Sumoylation of the THO complex regulates the biogenesis of a subset of mRNPs

Assembly of messenger ribonucleoparticles (mRNPs) is a pivotal step in gene expression, but only a few molecular mechanisms contributing to its regulation have been described. Here, through a comprehensive proteomic survey of mRNP assembly, we demonstrate that the SUMO pathway specifically controls the association of the THO complex with mRNPs. We further show that the THO complex, a key player in the interplay between gene expression, mRNA export and genetic stability, is sumoylated on its Hpr1 subunit and that this modification regulates its association with mRNPs. Altered recruitment of the THO complex onto mRNPs in sumoylation-defective mutants does not affect bulk mRNA export or genetic stability, but impairs the expression of acidic stress-induced genes and, consistently, compromises viability in acidic stress conditions. Importantly, inactivation of the nuclear exosome suppresses the phenotypes of the hpr1 non-sumoylatable mutant, showing that SUMO-dependent mRNP assembly is critical to allow a specific subset of mRNPs to escape degradation. This article thus provides the first example of a SUMO-dependent mRNP-assembly event allowing a refined tuning of gene expression, in particular under specific stress conditions.     

Saliva activated transmission (SAT) of Thogoto virus: relationship with vector potential of different haematophagous arthropods

Tick saliva (or salivary gland extract) potentiates the transmission of Thogoto (THO) virus to uninfected ticks feeding on a non-viraemic guinea-pig. This phenomenon has been named saliva activated transmission (SAT). To investigate the potential of different haematophagous arthropods to mediate SAT, guinea-pigs were infested with uninfected R.appendiculatus Neumann nymphs and inoculated with THO virus and salivary gland extract (SGE) derived from a range of ixodid (metastriate and prostriate) or argasid ticks, or mosquitoes; control guinea-pigs were inoculated with virus alone. Enhancement of THO virus transmission was observed only when SGE was derived from metastriate ticks. Comparison with the vector potential of these various arthropod species revealed that enhancement of THO virus transmission was specific for ticks which were competent vectors of the virus. The data indicate a correlation between vector competence and the ability of haematophagous arthropods to mediate SAT of THO virus.     

Viscous flow simulation in a stenosis model using discrete particle dynamics: a comparison between DPD and CFD

Flow and stresses induced by blood flow acting on the blood cellular constituents can be represented to a certain extent by a continuum mechanics approach down to the order of the?μm level. However, the molecular effects of, e.g., adhesion/aggregation bonds of blood clotting can be on the order of nm. The coupling of the disparate length and timescales between such molecular levels and macroscopic transport represents a major computational challenge. To address this challenge, a multiscale numerical approach based on discrete particle dynamics (DPD) methodology derived from molecular dynamics (MD) principles is proposed. The feasibility of the approach was firstly tested for its ability to simulate viscous flow conditions. Simulations were conducted in low Reynolds numbers flows (Re = 25–33) through constricted tubes representing blood vessels with various degrees of stenosis. Multiple discrete particles interacting with each other were simulated, with 1.24–1.36 million particles representing the flow domain and 0.4 million particles representing the vessel wall. The computation was carried out on the massive parallel supercomputer NY BlueGene/L employing NAMD-a parallel MD package for high performance computing (HPC). Typical recirculation zones were formed distal to the stenoses. The velocity profiles and recirculation zones were in excellent agreement with computational fluid dynamics (CFD) 3D Navier–Stokes viscous fluid flow simulations and with classic numerical and experimental results by YC Fung in constricted tubes. This feasibility analysis demonstrates the potential of a methodology that widely departs from a continuum approach to simulate multiscale phenomena such as flow induced blood clotting.     

Flow dependent changes in the effective surface area of microdialysis probes

The relative efficiencies of microdialysis probes were determined both in vitro and in vivo using tritiated water. Tritiated water (THO) freely distributes throughout the fluid spaces of an experimental animal and, at equilibrium, the brain extracellular concentration of THO is the same as the plasma concentration. Microdialysis probes were inserted into the right caudoputamen of anesthetized rats. The rats were injected with THO and after one hour microdialysis samples were collected at flow rates between 0.2 and 10.0 ul/min. The in vitro relative efficiency for THO was computed as the ratio of the THO concentration in the dialysate to that of the solution the probe was immersed in. The in vivo relative efficiency was computed as the ratio of the concentration of THO in the brain dialysate to that measured in the plasma of the rat. Both the in vitro and in vivo relative efficiencies for THO decrease with increasing flow rates, but they differ from each other except at very low flow rates (less than 0.25 ul/min). The in vitro relative efficiency at a given probe flow is the maximum efficiency that can be attained in vivo at that flow. The surface of effective exchange (Se) is the fraction of that maximum which is attained in vivo. This study also demonstrates how the effective surface area can be computed at any probe flow rate and how it can be used as a correction factor.     

Rhodococcus sp. strain TM1 plays a synergistic role in the degradation of piperidine by Mycobacterium sp. strain THO100

Mycobacterium sp. strain THO100 and Rhodococcus sp. strain TM1 were isolated from a morpholine-containing enrichment culture of activated sewage sludge. Strain THO100, but not strain TM1, was able to degrade alicyclic amines such as morpholine, piperidine, and pyrrolidine. The mixed strains THO100 and TM1 showed a better growth on piperidine as the substrate than the pure strain THO100 because strain TM1 was able to reduce the level of glutaraldehyde (GA) produced during piperidine degradation. GA was toxic to strain THO100 (IC₅₀ = 28.3 μM) but less toxic to strain TM1 (IC₅₀ = 215 μM). Strain THO100 possessed constitutive semialdehyde dehydrogenases, namely Sad1 and Sad2, whose activities toward succinic semialdehyde (SSA) were strongly inhibited by GA. The two isozymes were identified as catalase–peroxidase (KatG = Sad1) and semialdehyde dehydrogenase (Sad2) based on mass spectrometric analyses of tryptic peptides and database searches of the partial DNA sequences of their genes. In contrast, strain TM1 containing another constitutive enzyme Gad1 could oxidize both SSA and GA. This study suggested that strain TM1 possessing Gad1 played a synergistic role in reducing the toxic and inhibitory effects of GA produced in the degradation of piperidine by strain THO100.