Placement of iliosacral screws using 3D image-guided (O-Arm) technology and Stealth Navigation: comparison with traditional fluoroscopy.

AIMS: We compared the accuracy, operating time and radiation exposure of the introduction of iliosacral screws using O-arm/Stealth Navigation and standard fluoroscopy. MATERIALS AND METHODS: Iliosacral screws were introduced percutaneously into the first sacral body (S1) of ten human cadavers, four men and six women. The mean age was 77 years (58 to 85). Screws were introduced using a standard technique into the left side of S1 using C-Arm fluoroscopy and then into the right side using O-Arm/Stealth Navigation. The radiation was measured on the surgeon by dosimeters placed under a lead thyroid shield and apron, on a finger, a hat and on the cadavers. RESULTS: There were no neuroforaminal breaches in either group. The set-up time for the O-Arm was significantly longer than for the C-Arm, while total time for placement of the screws was significantly shorter for the O-Arm than for the C-Arm (p = 0.001). The mean absorbed radiation dose during fluoroscopy was 1063 mRad (432.5 mRad to 4150 mRad). No radiation was detected on the surgeon during fluoroscopy, or when he left the room during the use of the O-Arm. The mean radiation detected on the cadavers was significantly higher in the O-Arm group (2710 mRem standard deviation (sd) 1922) than during fluoroscopy (11.9 mRem sd 14.8) (p < 0.01). CONCLUSION: O-Arm/Stealth Navigation allows for faster percutaneous placement of iliosacral screws in a radiation-free environment for surgeons, albeit with the same accuracy and significantly more radiation exposure to cadavers, when compared with standard fluoroscopy. TAKE HOME MESSAGE: Placement of iliosacral screws with O-Arm/Stealth Navigation can be performed safely and effectively. Cite this article: Bone Joint J 2016;98-B:696-702.

When do anterior external or internal fixators provide additional stability in an unstable (Tile C) pelvic fracture? A biomechanical study.

PURPOSE: This study aimed at evaluating the additional stability that is provided by anterior external and internal fixators in an unstable pelvic fracture model (OTA 61-C). METHODS: An unstable pelvic fracture (OTA 61-C) was created in 27 synthetic pelves by making a 5-mm gap through the sacral foramina (posterior injury) and an ipsilateral pubic rami fracture (anterior injury). The posterior injury was fixed with either a single iliosacral (IS) screw, a single trans-iliac, trans-sacral (TS) screw, or two iliosacral screws (S1S2). Two anterior fixation techniques were utilized: external fixation (Ex-Fix) and supra-acetabular external fixation and internal fixation (In-Fix); supra-acetabular pedicle screws connected with a single subcutaneous spinal rod. The specimens were tested using a nondestructive single-leg stance model. Peak-to-peak (P2P) displacement and rotation and conditioning displacement (CD) were calculated. RESULTS: The Ex-Fix group failed in 83.3 % of specimens with concomitant single-level posterior fixation (Total: 15/18-7 of 9 IS fixation, 8 of 9 TS fixation), and 0 % (0/9) of specimens with concomitant two-level (S1S2) posterior fixation. All specimens with the In-Fix survived testing except for two specimens treated with In-Fix combined with IS fixation. Trans-sacral fixation had higher pubic rotation and greater sacral and pubic displacement than S1S2 (p < 0.05). Rotation of the pubis and sacrum was not different between In-Fix constructs combined with single-level IS and TS fixation. CONCLUSION: In this model of an unstable pelvic fracture (OTA 61-C), anterior fixation with an In-Fix was biomechanically superior to an anterior Ex-Fix in the setting of single-level posterior fixation. There was no biomechanical difference between the In-Fix and Ex-Fix when each was combined with two levels of posterior sacral fixation.

IAA17/AXR3: biochemical insight into an auxin mutant phenotype.

The Aux/IAA genes are rapidly and specifically induced by the plant hormone auxin. The proteins encoded by this gene family are short-lived nuclear proteins that are capable of homodimerizing and heterodimerizing. Molecular, biochemical, and genetic data suggest that these proteins are involved in auxin signaling. The pleiotropic morphological phenotype and altered auxin responses of the semidominant axr3-1 mutant of Arabidopsis result from a single amino acid change in the conserved domain II of the Aux/IAA protein IAA17. Here, we show that the biochemical effect of this gain-of-function mutation is to increase the half-life of the iaa17/axr3-1 protein by sevenfold. Intragenic mutations that suppress the iaa17/axr3-1 phenotype have been described. The iaa17/axr3-1R3 revertant contains a second site mutation in domain I and the iaa17/axr3-1R2 revertant contains a second site mutation in domain III. Transient expression assays show that the mutant forms of IAA17/AXR3 retain the ability to accumulate in the nucleus. Using the yeast two hybrid system, we show that the iaa17/axr3-1 mutation does not affect homodimerization. However, the iaa17/axr3-1 revertants counteract the increased levels of iaa17/axr3-1 protein by decreasing the capacity of the mutant protein to homodimerize. Interestingly, heterodimerization of the revertant forms of IAA17/AXR3 with IAA3/SHY2, another Aux/IAA protein, and ARF1 or ARF5/MP proteins is affected only by changes in domain III. Collectively, the results provide biochemical evidence that the revertant mutations in the IAA17/AXR3 gene affect the capacity of the encoded protein to dimerize with itself, other members of the Aux/IAA protein family, and members of the ARF protein family. By extension, these findings may provide insight into the effects of analogous mutations in other members of the Aux/IAA gene family.

Goodbye to 'one by one' genetics.

The completion of the Arabidopsis thaliana (mustard weed) genome sequence constitutes a major breakthrough in plant biology. It will revolutionize how we answer questions about the biology and evolution of plants as well as how we confront and resolve world-wide agricultural problems.

Sequence and analysis of chromosome 1 of the plant Arabidopsis thaliana.

The genome of the flowering plant Arabidopsis thaliana has five chromosomes. Here we report the sequence of the largest, chromosome 1, in two contigs of around 14.2 and 14.6 megabases. The contigs extend from the telomeres to the centromeric borders, regions rich in transposons, retrotransposons and repetitive elements such as the 180-base-pair repeat. The chromosome represents 25% of the genome and contains about 6,850 open reading frames, 236 transfer RNAs (tRNAs) and 12 small nuclear RNAs. There are two clusters of tRNA genes at different places on the chromosome. One consists of 27 tRNA(Pro) genes and the other contains 27 tandem repeats of tRNA(Tyr)-tRNA(Tyr)-tRNA(Ser) genes. Chromosome 1 contains about 300 gene families with clustered duplications. There are also many repeat elements, representing 8% of the sequence.

Degradation of Aux/IAA proteins is essential for normal auxin signalling.

The growth substance auxin mediates many cellular processes, including division, elongation and differentiation. PSIAA6 is a member of the Aux/IAA family of short-lived putative transcriptional regulators that share four conserved domains and whose mRNAs are rapidly induced in the presence of auxin. Here PSIAA6 was shown to serve as a dominant transferable degradation signal when present as a translational fusion with firefly luciferase (LUC), with an in vivo half-life of 13.5 min in transgenic Arabidopsis seedlings. In a transient assay system in tobacco protoplasts using steady-state differences as an indirect measure of protein half-life, LUC fusions with full-length PSIAA6 and IAA1, an Aux/IAA protein from Arabidopsis, resulted in protein accumulations that were 3.5 and 1. 0%, respectively, of that with LUC alone. An N-terminal region spanning conserved domain II of PSIAA6 containing amino acids 18-73 was shown to contain the necessary cis-acting element to confer low protein accumulation onto LUC, while a fusion protein with PSIAA6 amino acids 71-179 had only a slight effect. Single amino acid substitutions of PSIAA6 in conserved domain II, equivalent to those found in two alleles of axr3, a gene that encodes Aux/IAA protein IAA17, resulted in a greater than 50-fold increase in protein accumulation. Thus, the same mutations resulting in an altered auxin response phenotype increase Aux/IAA protein accumulation, providing a direct link between these two processes. In support of this model, transgenic plants engineered to over-express IAA17 have an axr3-like phenotype. Together, these data suggest that rapid degradation of Aux/IAA proteins is necessary for a normal auxin response.

Genome-wide mapping with biallelic markers in Arabidopsis thaliana.

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

Biochemical characterization of recombinant polypeptides corresponding to the predicted betaalphaalpha fold in Aux/IAA proteins.

The plant hormone indoleacetic acid (IAA or auxin) transcriptionally activates a select set of early genes. The Aux/IAA class of early auxin-responsive genes encodes a large family of short-lived, nuclear proteins. Aux/IAA polypeptides homo- and heterodimerize, and interact with auxin-response transcription factors (ARFs) via C-terminal regions conserved in both protein families. This shared region contains a predicted betaalphaalpha motif similar to the prokaryotic beta-ribbon DNA binding domain, which mediates both protein dimerization and DNA recognition. Here, we show by circular dichroism spectroscopy and by chemical cross-linking experiments that recombinant peptides corresponding to the predicted betaalphaalpha region of three Aux/IAA proteins from Arabidopsis thaliana contain substantial alpha-helical secondary structure and undergo homo- and heterotypic interactions in vitro. Our results indicate a similar biochemical function of the plant betaalphaalpha domain and suggest that the betaalphaalpha fold plays an important role in mediating combinatorial interactions of Aux/IAA and ARF proteins to specifically regulate secondary gene expression in response to auxin.

Ethylene signalling: redundant receptors all have their say.

In Arabidopsis, the hormone ethylene is sensed by five related receptors, all of the 'two-component' variety. The receptors constitutively suppress a downstream signalling pathway, and are inactivated by ethylene, leading to the activation of genes necessary for the various ethylene-regulated biological responses.

age Mutants of Arabidopsis exhibit altered auxin-regulated gene expression.

An Arabidopsis transgenic line was constructed expressing beta-glucuronidase (GUS) via the auxin-responsive domains (AuxRDs) A and B (BA-GUS) of the PS-IAA4/5 gene in an indoleacetic acid (IAA)-dependent fashion. GUS expression was preferentially enhanced in the root elongation zone after treatment of young seedlings with 10(-7) M IAA. Expression of the BA-GUS gene in the axr1, axr4, and aux1 mutants required 10- to 100-fold higher auxin concentration than that in the wild-type background. GUS expression was nil in the axr 2 and axr 3 mutants. The transgene was used to isolate mutants exhibiting altered auxin-responsive gene expression (age). Two mutants, age1 and age2, were isolated and characterized. age1 showed enhanced sensitivity to IAA, with strong GUS expression localized in the root elongation zone in the presence of 10(-8) M IAA. In contrast, age2 exhibited ectopic GUS expression associated with the root vascular tissue, even in the absence of exogenous IAA. Morphological and molecular analyses indicated that the age1 and age2 alleles are involved in the regulation of gene expression in response to IAA.

Random mutagenesis of 1-aminocyclopropane-1-carboxylate synthase: a key enzyme in ethylene biosynthesis.

1-Aminocyclopropane-1-carboxylate synthase (ACC synthase, EC 4.4.1. 14) catalyzes the rate-limiting step in the ethylene biosynthetic pathway in plants. To determine the amino acid residues critical for the structure and function of this enzyme, the tomato Le-ACS2 isoenzyme has been subjected to both site-directed and PCR random mutagenesis. Mutant ACC synthases with reduced enzyme activity have been selected by using a genetic screen based on the functional complementation of an Escherichia coli Ile auxotroph that has been engineered to express ACC deaminase from Pseudomonas sp. The DNA sequence of almost 1,000 clones has been determined, and 334 single missense mutations have been selected for analysis. We have identified three classes of mutants based on their activity and expression in E. coli. Class I and II mutants have the same level of protein expression as the wild type, but their enzyme activity is reduced to 0-5% and 5-50%, respectively. Class III mutants have neither activity nor detectable protein expression. The inactive mutations are clustered in regions that are highly conserved among various ACC synthases. This library of mutants will facilitate the elucidation of structure-function relationships of this regulatory enzyme.

Complementation analysis of mutants of 1-aminocyclopropane- 1-carboxylate synthase reveals the enzyme is a dimer with shared active sites.

The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACS, EC 4.4.1.14) catalyzes the rate-limiting step in the ethylene biosynthetic pathway. ACS shares the conservation of 11 invariant residues with a family of aminotransferases that includes aspartate aminotransferase. Site-directed mutagenesis on two of these residues, Tyr-92 and Lys-278, in the tomato isoenzyme Le-ACS2 greatly reduces enzymatic activity, indicating their importance in catalysis. These mutants have been used in complementation experiments either in vivo in Escherichia coli or in an in vitro transcription/translation assay to study whether the enzyme functions as a dimer. When the Y92L mutant is coexpressed with the K278A mutant protein, there is partial restoration of enzyme activity, suggesting that the mutant proteins can dimerize and form active heterodimers. Coexpressing a double mutant with the wild-type protein reduces wild-type activity, indicating that inactive heterodimers are formed between the wild-type and the double mutant protein subunits. Furthermore, hybrid complementation shows that another tomato isoenzyme, Le-ACS4, can dimerize and that Le-ACS2 and Le-ACS4 have limited capacity for heterodimerization. The data suggest that ACS functions as a dimer with shared active sites.

Recessive and dominant mutations in the ethylene biosynthetic gene ACS5 of Arabidopsis confer cytokinin insensitivity and ethylene overproduction, respectively.

We identified a set of cytokinin-insensitive mutants by using a screen based on the ethylene-mediated triple response observed after treatment with low levels of cytokinins. One group of these mutants disrupts ACS5, a member of the Arabidopsis gene family that encodes 1-aminocyclopropane-1-carboxylate synthase, the first enzyme in ethylene biosynthesis. The ACS5 isoform is mainly responsible for the sustained rise in ethylene biosynthesis observed in response to low levels of cytokinin and appears to be regulated primarily by a posttranscriptional mechanism. Furthermore, the dominant ethylene-overproducing mutant eto2 was found to be the result of an alteration of the carboxy terminus of ACS5, suggesting that this domain acts as a negative regulator of ACS5 function.

Protein-protein interactions among the Aux/IAA proteins.

The plant hormone indoleacetic acid (IAA) transcriptionally activates early genes in plants. The Aux/IAA family of early genes encodes proteins that are short-lived and nuclear-localized. They also contain a putative prokaryotic betaalphaalpha DNA binding motif whose formation requires protein dimerization. Here, we show that the pea PS-IAA4 and Arabidopsis IAA1 and IAA2 proteins perform homo- and heterotypic interactions in yeast using the two-hybrid system. Gel-filtration chromatography and chemical cross-linking experiments demonstrate that the PS-IAA4 and IAA1 proteins interact to form homodimers in vitro. Deletion analysis of PS-IAA4 indicates that the betaalphaalpha containing acidic C terminus of the protein is necessary for homotypic interactions in the yeast two-hybrid system. Screening an Arabidopsis lambda-ACT cDNA library using IAA1 as a bait reveals heterotypic interactions of IAA1 with known and newly discovered members of the Arabidopsis Aux/IAA gene family. The new member IAA24 has similarity to ARF1, a transcription factor that binds to an auxin response element. Combinatorial interactions among the various members of the Aux/IAA gene family may regulate a variety of late genes as well as serve as autoregulators of early auxin-regulated gene expression. These interactions provide a molecular basis for the developmental and tissue-specific manner of auxin action.

Expression characteristics of OS-ACS1 and OS-ACS2, two members of the 1-aminocyclopropane-1-carboxylate synthase gene family in rice (Oryza sativa L. cv. Habiganj Aman II) during partial submergence.

Deepwater rice can grow in the regions of Southeast Asia that are flooded during the monsoon season because it has several adaptations allowing it to survive under flooded conditions. One such adaptation is the ability for rapid internode elongation upon partial submergence to maintain its foliage above the rising flood water levels. Ethylene is considered to be the trigger of this growth response because deepwater conditions not only trap ethylene in submerged organs, but also enhance the activity of 1-aminocyclopropane-1-carboxylate (ACC) synthase. Herein we have studied the expression characteristics of two members of the five-member multigene family encoding ACC synthase in rice OS-ACS1 and OS-ACS2 and show that partial submergence induces expression of OS-ACS1 and suppresses expression of OS-ACS2. The induction of OS-ACS1 occurs within 12 h of partial submergence and at low oxygen concentrations. The data also suggest that deepwater conditions posttranscriptionally regulate ACC synthase activity. OS-ACS1 gene expression may contribute to longer-term ethylene production, but not to the initial, growth-promoting increase in ethylene synthesis.

Li(+)-regulated 1-aminocyclopropane-1-carboxylate synthase gene expression in Arabidopsis thaliana.

In Arabidopsis thaliana, 1-aminocyclopropane-1-carboxylate synthase (ACS) is encoded by a multigene family consisting of at least five members whose expression is induced by hormones, developmental signals, and protein synthesis inhibition. Li+, known to interfere with the phosphoinositide (PI) second messenger system by inhibiting the activity of inositol-phosphate phosphatases, is one of the strongest inducers of ACC synthase activity in plants. Treatment of etiolated Arabidopsis seedlings with LiCl results in a rapid induction of the ACS5 gene. Also, LiCl represses the cycloheximide (CHX)-induced accumulation of the ACS2 mRNA. The effects of Li+ on the expression of ACS5 and ACS2 are specific, dose-dependent, and can be reversed by Ca2+ and mimicked by the protein kinase inhibitor K-252a. The results suggest that the regulation of some ACS genes by various inducers may involve protein kinase activity, which in turn may be controlled through an inositol 1,4,5-triphosphate (IP3)-mediated Ca2+ mobilization. Since plants contain no Li+, the cation appears to unmask pre-existing biochemical capacity that may be utilized by various unknown transducers during plant growth and development.

DNA elements responsive to auxin.

Genes induced by the plant hormone auxin are probably involved in the execution of vital cellular functions and developmental processes. Experimental approaches designed to elucidate the molecular mechanisms of auxin action have focused on auxin perception, genetic dissection of the signaling apparatus and specific gene activation. Auxin-responsive promoter elements of early genes provide molecular tools for probing auxin signaling in reverse. Functional analysis of several auxin-specific promoters of unrelated early genes suggests combinatorial utilization of both conserved and variable elements. These elements are arranged into autonomous domains and the combination of such modules generates uniquely composed promoters. Modular promoters allow for auxin-mediated transcriptional responses to be revealed in a tissue- and development-specific manner.

Differential activation of the primary auxin response genes, PS-IAA4/5 and PS-IAA6, during early plant development.

The plant growth hormone auxin typified by indoleacetic acid (IAA) transcriptionally activates early genes in pea, PS-IAA4/5 and PS-IAA6, that are members of a multigene family encoding short-lived nuclear proteins. To gain first insight into the biological role of PS-IAA4/5 and PS-IAA6, promoter-beta-glucuronidase (GUS) gene fusions were constructed and their expression during early development of transgenic tobacco seedlings was examined. The comparative analysis reveals spatial and temporal expression patterns of both genes that correlate with cells, tissues, and developmental processes known to be affected by auxin. GUS activity in seedlings of both transgenic lines is located in the root meristem, sites of lateral root initiation and in hypocotyls undergoing rapid elongation. In addition, mutually exclusive cell-specific expression is evident. For instance, PS-IAA4/5-GUS but not PS-IAA6-GUS is expressed in root vascular tissue and in guard cells, whereas only PS-IAA6-GUS activity is detectable in glandular trichomes and redistributes to the elongating side of the hypocotyl upon gravitropic stimulation. Expression of PS-IAA4/5 and PS-IAA6 in elongating, dividing, and differentiating cell types indicates multiple functions during development. The common and yet distinct activity patterns of both genes suggest a combinatorial code of spatio-temporal co-expression of the various PS-IAA4/5-like gene family members in plant development that may mediate cell-specific responses to auxin.