Development of standard fuel models in boreal forests of Northeast China through calibration and validation.

Understanding the fire prediction capabilities of fuel models is vital to forest fire management. Various fuel models have been developed in the Great Xing'an Mountains in Northeast China. However, the performances of these fuel models have not been tested for historical occurrences of wildfires. Consequently, the applicability of these models requires further investigation. Thus, this paper aims to develop standard fuel models. Seven vegetation types were combined into three fuel models according to potential fire behaviors which were clustered using Euclidean distance algorithms. Fuel model parameter sensitivity was analyzed by the Morris screening method. Results showed that the fuel model parameters 1-hour time-lag loading, dead heat content, live heat content, 1-hour time-lag SAV(Surface Area-to-Volume), live shrub SAV, and fuel bed depth have high sensitivity. Two main sensitive fuel parameters: 1-hour time-lag loading and fuel bed depth, were determined as adjustment parameters because of their high spatio-temporal variability. The FARSITE model was then used to test the fire prediction capabilities of the combined fuel models (uncalibrated fuel models). FARSITE was shown to yield an unrealistic prediction of the historical fire. However, the calibrated fuel models significantly improved the capabilities of the fuel models to predict the actual fire with an accuracy of 89%. Validation results also showed that the model can estimate the actual fires with an accuracy exceeding 56% by using the calibrated fuel models. Therefore, these fuel models can be efficiently used to calculate fire behaviors, which can be helpful in forest fire management.

Deletion of CXCR4 in cardiomyocytes exacerbates cardiac dysfunction following isoproterenol administration.

Altered alpha- and beta-adrenergic receptor signaling is associated with cardiac hypertrophy and failure. Stromal cell-derived factor-1α (SDF-1α) and its cognate receptor CXCR4 have been reported to mediate cardioprotection after injury through the mobilization of stem cells into injured tissue. However, little is known regarding whether SDF-1/CXCR4 induces acute protection following pathological hypertrophy and if so, by what molecular mechanism. We have previously reported that CXCR4 physically interacts with the beta-2 adrenergic receptor and modulates its downstream signaling. Here we have shown that CXCR4 expression prevents beta-adrenergic receptor-induced hypertrophy. Cardiac beta-adrenergic receptors were stimulated with the implantation of a subcutaneous osmotic pump administrating isoproterenol and CXCR4 expression was selectively abrogated in cardiomyocytes using Cre-loxP-mediated gene recombination. CXCR4 knockout mice showed worsened fractional shortening and ejection fraction. CXCR4 ablation increased susceptibility to isoproterenol-induced heart failure, by upregulating apoptotic markers and reducing mitochondrial function; cardiac function decreases whereas fibrosis increases. In addition, CXCR4 expression was rescued with the use of cardiotropic adeno-associated viral-9 vectors. CXCR4 gene transfer reduced cardiac apoptotic signaling, improved mitochondrial function and resulted in a recovered cardiac function. Our results represent the first evidence that SDF-1/CXCR4 signaling mediates acute cardioprotection through modulating beta-adrenergic receptor signaling in vivo.

[Pulmonary mucormycosis: difficulties in diagnosis and treatment]. / Mucormycoses pulmonaires: difficultés diagnostiques et thérapeutiques.

INTRODUCTION: Mucormycosis is a rare opportunistic fungal infection due to filamentous fungi of the order Mucorales in the class Zygomycetes. Rhino-cerebral and pulmonary manifestations predominate on account of the airborn spread of the spores. Gastro-intestinal, cutaneous and disseminated disease is less common. The principal risk factors are immuno-suppression and diabetic keto-acidosis. CASE REPORTS: One case of fatal pulmonary mucormycosis and two cases of colonisation illustrate both the extreme severity of this disease and the diagnostic difficulties facing the physician. The ubiquitous nature of the organism leads to frequent colonisation and, moreover, the symptomatology readily mimics that of invasive pulmonary aspergillosis. The diagnosis of mucormycosis can only be confirmed by pathological and mycological examination of biopsy specimens. These requirements conflict with the need for urgent treatment with surgical debridement, amphotericin B and control of the underlying pathology. Sadly the mortality remains very high, between 50 and 80% in published series. CONCLUSION: Currently there is hope of new therapeutic approaches with posaconozole but the ineffectiveness of voriconozole and the echinocandines, used more and more against aspergillus, raises the possibility of an increase in mucormycosis by selection.

Effect of combined cytostatic cyclosporin A and cytolytic suicide gene therapy on the prevention of experimental graft-versus-host disease.

The immunosuppressive drug cyclosporin A (CsA) represents the standard preventive treatment of graft-versus-host disease (GVHD), the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). However, its efficacy is only partial and many patients develop lethal GVHD despite CsA. A strategy of genetic immunosuppression based on conditional elimination of donor T cells expressing the Herpes simplex type 1 thymidine kinase (TK) suicide gene was recently developed. In this system, ganciclovir (GCV) selectively kills dividing but not quiescent TK T cells. Since CsA is known to have a cytostatic effect on T cells, it could negatively interfere with the division-dependent TK gene therapy. We thus tested whether administration of CsA would antagonize elimination of alloreactive donor TK T cells mediated by GCV in a murine model of GVHD. In vivo experiments revealed that, contrary to GCV, CsA only transiently controlled alloactivation-induced T cell proliferation, and likewise could not prevent lethal GVHD. When T cells resumed proliferation under CsA, they were however still sensitive to GCV. Survival, as well as immune reconstitution, was excellent in mice treated with GCV alone or in combination with CsA. These observations should help to design improved suicide gene therapy trials in the field of allogeneic HSCT.

Role of conserved nucleotides in building the 16 S rRNA binding site for ribosomal protein S15.

Ribosomal protein S15 recognizes a highly conserved target on 16 S rRNA, which consists of two distinct binding regions. Here, we used extensive site-directed mutagenesis on a Escherichia coli 16 S rRNA fragment containing the S15 binding site, to investigate the role of conserved nucleotides in protein recognition and to evaluate the relative contribution of the two sites. The effect of mutations on S15 recognition was studied by measuring the relative binding affinity, RNA probing and footprinting. The crystallographic structure of the Thermus thermophilus complex allowed molecular modelling of the E. coli complex and facilitated interpretation of biochemical data. Binding is essentially driven by site 1, which includes a three-way junction constrained by a conserved base triple and cross-strand stacking. Recognition is based mainly on shape complementarity, and the role of conserved nucleotides is to maintain a unique backbone geometry. The wild-type base triple is absolutely required for protein interaction, while changes in the conserved surrounding nucleotides are partially tolerated. Site 2, which provides functional groups in a conserved G-U/G-C motif, contributes only modestly to the stability of the interaction. Binding to this motif is dependent on binding at site 1 and is allowed only if the two sites are in the correct relative orientation. Non-conserved bulged nucleotides as well as a conserved purine interior loop, although not directly involved in recognition, are used to provide an appropriate flexibility between the two sites. In addition, correct binding at the two sites triggers conformational adjustments in the purine interior loop and in a distal region, which are known to be involved for subsequent binding of proteins S6 and S18. Thus, the role of site 1 is to anchor S15 to the rRNA, while binding at site 2 is aimed to induce a cascade of events required for subunit assembly.

The ski7 antiviral protein is an EF1-alpha homolog that blocks expression of non-Poly(A) mRNA in Saccharomyces cerevisiae.

We mapped and cloned SKI7, a gene that negatively controls the copy number of L-A and M double-stranded RNA viruses in Saccharomyces cerevisiae. We found that it encodes a nonessential 747-residue protein with similarities to two translation factors, Hbs1p and EF1-alpha. The ski7 mutant was hypersensitive to hygromycin B, a result also suggesting a role in translation. The SKI7 product repressed the expression of nonpolyadenylated [non-poly(A)] mRNAs, whether capped or uncapped, thus explaining why Ski7p inhibits the propagation of the yeast viruses, whose mRNAs lack poly(A). The dependence of the Ski7p effect on 3' RNA structures motivated a study of the expression of capped non-poly(A) luciferase mRNAs containing 3' untranslated regions (3'UTRs) differing in length. In a wild-type strain, increasing the length of the 3'UTR increased luciferase expression due to both increased rates and duration of translation. Overexpression of Ski7p efficiently cured the satellite virus M2 due to a twofold-increased repression of non-poly(A) mRNA expression. Our experiments showed that Ski7p is part of the Ski2p-Ski3p-Ski8p antiviral system because a single ski7 mutation derepresses the expression of non-poly(A) mRNA as much as a quadruple ski2 ski3 ski7 ski8 mutation, and the effect of the overexpression of Ski7p is not obtained unless other SKI genes are functional. ski1/xrn1Delta ski2Delta and ski1/xrn1Delta ski7Delta mutants were viable but temperature sensitive for growth.

Ski6p is a homolog of RNA-processing enzymes that affects translation of non-poly(A) mRNAs and 60S ribosomal subunit biogenesis.

We mapped and cloned SKI6 of Saccharomyces cerevisiae, a gene that represses the copy number of the L-A double-stranded RNA virus, and found that it encodes an essential 246-residue protein with homology to a tRNA-processing enzyme, RNase PH. The ski6-2 mutant expressed electroporated non-poly(A) luciferase mRNAs 8- to 10-fold better than did the isogenic wild type. No effect of ski6-2 on expression of uncapped or normal mRNAs was found. Kinetics of luciferase synthesis and direct measurement of radiolabeled electroporated mRNA indicate that the primary effect of Ski6p was on efficiency of translation rather than on mRNA stability. Both ski6 and ski2 mutants show hypersensitivity to hygromycin, suggesting functional alteration of the translation apparatus. The ski6-2 mutant has normal amounts of 40S and 60S ribosomal subunits but accumulates a 38S particle containing 5'-truncated 25S rRNA but no 5.8S rRNA, apparently an incomplete or degraded 60S subunit. This suggests an abnormality in 60S subunit assembly. The ski6-2 mutation suppresses the poor expression of the poly(A)- viral mRNA in a strain deficient in the 60S ribosomal protein L4. Thus, a ski6 mutation bypasses the requirement of the poly(A) tail for translation, allowing better translation of non-poly(A) mRNA, including the L-A virus mRNA which lacks poly(A). We speculate that the derepressed translation of non-poly(A) mRNAs is due to abnormal (but full-size) 60S subunits.

Identification in a pseudoknot of a U.G motif essential for the regulation of the expression of ribosomal protein S15.

The ribosomal protein S15 from Escherichia coli binds to a pseudoknot in its own messenger. This interaction is an essential step in the mechanism of S15 translational autoregulation. In a previous study, a recognition determinant for S15 autoregulation, involving a U.G wobble pair, was located in the center of stem I of the pseudoknot. In this study, an extensive mutagenesis analysis has been conducted in and around this U.G pair by comparing the effects of these mutations on the expression level of S15. The results show that the U.G wobble pair cannot be substituted by A.G, C.A, A.C, G.U, or C.G without loss of the autocontrol. In addition, the base pair C.G, adjacent to the 5' side of U, cannot be flipped or changed to another complementary base pair without also inducing derepression of translation. A unique motif, made of only two adjacent base pairs, U.G/C.G, is essential for S15 autoregulation and is presumably involved in direct recognition by the S15 protein.

Pseudoknot and translational control in the expression of the S15 ribosomal protein.

Translational autocontrol of the expression of the ribosomal protein S15 proceeds through the transitory formation of a pseudoknot. A synopsis of the known data is used to propose a molecular model of the mechanism involved and for the role of the pseudoknot. This latter structure is able to recruit 30S ribosomal subunits to initiate translation, but also to bind S15 and to stop translation by trapping the ribosome on its loading site. Information on the S15 protein recognition of the messenger RNA site was deduced from mutational analyses and chemical probing. A comparison of this messenger site with the S15 ribosomal binding site was conducted by analysing hydroxyl radical footprintings of these two sites. The existence of two subsites in 16S RNA suggests that the ribosomal protein S15 might present either two different binding sites or at least one common subsite. Clues for the presence of a common site between the messenger and 16S RNA are given which cannot rule out that recognition specificity is linked to a few other determinants. Whether these determinants are different or not remains an open question.

A pseudoknot is required for efficient translational initiation and regulation of the Escherichia coli rpsO gene coding for ribosomal protein S15.

Escherichia coli ribosomal protein S15 down regulates its own synthesis by binding to its mRNA in a region overlapping the ribosome binding site, called the translational operator. This binding stabilizes a pseudoknot structure that exists in equilibrium with two stem-loop structures. When synthesized in excess over 16S rRNA, S15 binds to its translational operator and traps the ribosome on its loading site in a transient state, preventing the formation of the active ternary (30S-mRNA-rRNA(f)Met) complex. This inhibition can be suppressed by 16S rRNA, which displaces S15 from the mRNA. An extensive mutational analysis showed that the pseudoknot is the structural element required for S15 recognition and in vivo translational control. Specific sequence determinants are located in limited regions of the structure formed by the pseudoknot. An unexpected result is that the pseudoknot can exist in a variety of topologically equivalent structures recognizable and shapable by S15. Based on footprinting experiments and computer graphic modelling, S15 shields the two stems of the pseudoknot, sitting in the major groove of the coaxial stack.

Molecular dissection of the pseudoknot governing the translational regulation of Escherichia coli ribosomal protein S15.

The ribosomal protein S15 controls its own translation by binding to a mRNA region overlapping the ribosome binding site. That region of the mRNA can fold in two mutually exclusive conformations that are in dynamic equilibrium: a structure with two hairpins and a pseudoknot. A mutational analysis provided evidence for the existence and requirement of the pseudoknot for translational control in vivo and S15 recognition in vitro. In this study, we used chemical probing to analyze the structural consequences of mutations and their effect on the stem-loop/pseudoknot equilibrium. Interactions between S15 and the pseudoknot structure were further investigated by footprinting experiments. These data, combined with computer modelling and the previously published data on S15 binding and in vivo control, provide important clues on pseudoknot formation and S15 recognition. An unexpected result is that the relevant control element, here the pseudoknot form, can exist in a variety of topologically equivalent structures recognizable and shapable by S15. S15 sits on the deep groove of the co-axial stack and makes contacts with both stems, shielding the bridging adenine. The only specific sequence determinants are found in the helix common to the pseudoknot and the hairpin structures.

Mutational analysis of the pseudoknot structure of the S15 translational operator from Escherichia coli.

Expression of rpsO, the gene encoding the small ribosomal protein S15, is autoregulated at the translational level by S15, which binds to its mRNA in a region overlapping the ribosome-binding site. By measuring the effect of mutations on the expression of a translational rpsO-lacZ fusion and the S15 binding affinity for the translational operator, the formation of a pseudoknot in the operator site in vivo is fully demonstrated and appears to be a prerequisite for S15 binding. The mutational analysis suggests also that specific determinants for S15 binding are located in very limited regions of the structure formed by the pseudoknot. It is deduced that a specific pseudoknot conformation is a key element for autoregulation.

Structural elements of rps0 mRNA involved in the modulation of translational initiation and regulation of E. coli ribosomal protein S15.

Previous experiments showed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site. This binding was postulated to stabilize a pseudoknot structure that exists in equilibrium with two stem-loops and to trap the ribosome on its mRNA loading site in a transitory state. In this study, we investigated the effect of mutations in the translational operator on: the binding of protein S15, the formation of the 30S/mRNA/tRNA(fMet) ternary initiation complex, the ability of S15 to inhibit the formation of this ternary complex. The results were compared to in vivo expression and repression rates. The results show that (1) the pseudoknot is required for S15 recognition and translational control; (2) mRNA and 16S rRNA efficiently compete for S15 binding and 16S rRNA suppresses the ability of S15 to inhibit the formation of the active ternary complex; (3) the ribosome binds more efficiently to the pseudoknot than to the stem-loop; (4) sequences located between nucleotides 12 to 47 of the S15 coding phase enhances the efficiency of ribosome binding in vitro; this is correlated with enhanced in vivo expression and regulation rates.

Ribosomal protein S15 from Escherichia coli modulates its own translation by trapping the ribosome on the mRNA initiation loading site.

From genetic and biochemical evidence, we previously proposed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site. This binding was postulated to stabilize a pseudoknot structure that exists in equilibrium with two stem-loops. Here, we use "toeprint" experiments with Moloney murine leukemia virus reverse transcriptase to analyze the effect of S15 on the formation of the ternary mRNA-30S-tRNA(fMet) complex. We show that the binding of the 30S subunit on the mRNA stops reverse transcriptase near position +10, corresponding to the 3' terminus of the pseudoknot, most likely by stabilizing the pseudoknot conformation. Furthermore, S15 is found to stabilize the binary 30S-mRNA complex. When the ternary 30S-mRNA-tRNA(fMet) complex is formed, a toeprint is observed at position +17. This toeprint progressively disappears when the ternary complex is formed in the presence of increasing concentrations of S15, while a shift from position +17 to position +10 is observed. Beside, RNase T1 footprinting experiments reveal the simultaneous binding of S15 and 30S subunit on the mRNA. Otherwise, we show by filter binding assays that initiator tRNA remains bound to the 30S subunit even in the presence of S15. Our results indicate that S15 prevents the formation of a functional ternary 30S-mRNA-tRNA(fMet) complex, the ribosome being trapped in a preternary 30S-mRNA-tRNA(fMet) complex.