A higher spectral range of beetle bioluminescence with infraluciferin.

Coleopteran bioluminescence is unique in that beetle luciferases emit colors ranging between green (ca.550 nm) and red (ca.600 nm), including intermediate colors such as yellow and orange, allowing up to 3 simultaneous parameters to be resolved in vitro with natural luciferin (D-LH2). Here, we report a more than doubling of the maximum bioluminescence wavelength range using a single synthetic substrate, infraluciferin (iLH2). We report that different luciferases can emit colors ranging from visible green to near-infrared (nIR) with iLH2, including in human cells. iLH2 was designed for dual color far-red to nIR bioluminescence imaging (BLI) in small animals and has been utilized in different mouse models of cancer (including a metastatic hepatic model showing detailed hepatic morphology) and for robust dual parameter imaging in vivo (including in systemic hematological models). Here, we report the properties of different enzymes with iLH2: Lampyrid wild-type (WT) Photinus pyralis (Ppy) firefly luciferase, Ppy-based derivatives previously engineered to be thermostable with D-LH2, and also color-shifted Elaterid-based enzymes: blue-shifted Pyrearinus termitilluminans derivative Eluc (reported D-LH2 λmax = 538 nm) and red-shifted Pyrophorus plagiopthalamus derivative click beetle red (CBR) luciferase (D-LH2 λmax = 618 nm). As purified enzyme, in bacteria or in human cells, Eluc emitted green light (λmax = 536 nm) with DL-iLH2 whereas Ppy Fluc (λmax = 689 nm), x2 Fluc (λmax = 704 nm), x5 Fluc (λmax = 694 nm), x11 Fluc (λmax = 694 nm) and CBR (λmax = 721 nm) produced far-red to nIR peak wavelengths. Therefore, with iLH2, enzyme λmaxes can be separated by ca.185nm, giving almost non-overlapping spectra. This is the first report of single-substrate bioluminescence color emission ranging from visible green to nIR in cells and may help shed light on the color tuning mechanism of beetle luciferases. We also report on the reason for the improvement in activity of x11 Fluc with iLH2 and engineer an improved infraluciferase (iluc) based on this mutant.

Activation of CYCD7;1 in the central cell and early endosperm overcomes cell-cycle arrest in the Arabidopsis female gametophyte, and promotes early endosperm and embryo development.

In angiosperms, double fertilization of the egg and central cell of the megagametophyte leads to the development of the embryo and endosperm, respectively. Control of cell cycle progression in the megagametophyte is essential for successful fertilization and development. Central cell-targeted expression of the D-type cyclin CYCD7;1 (end CYCD7;1) using the imprinted FWA promoter overcomes cycle arrest of the central cell in the Arabidopsis female gametophyte in the unfertilized ovule, leading to multinucleate central cells at high frequency. Unlike FERTILIZATION-INDEPENDENT SEED (fis) mutants, but similar to lethal RETINOBLASTOMA-RELATED (rbr) mutants, no seed coat development is triggered. Unlike the case with loss of rbr, post-fertilization end CYCD7;1 in the endosperm enhances the number of nuclei during syncytial endosperm development and induces the partial abortion of developing seeds, associated with the enhanced size of the surviving seeds. The frequency of lethality was less than the frequency of multinucleate central cells, indicating that these aspects are not causally linked. These larger seeds contain larger embryos composed of more cells of wild-type size, surrounded by a seed coat composed of more cells. Seedlings arising from these larger seeds displayed faster seedling establishment and early growth. Similarly, two different embryo-lethal mutants also conferred enlarged seed size in surviving siblings, consistent with seed size increase being a general response to sibling lethality, although the cellular mechanisms were found to be distinct. Our data suggest that tight control of CYCD activity in the central cell and in the developing endosperm is required for optimal seed formation.

Glutathione transport: a new role for PfCRT in chloroquine resistance.

AIMS: Chloroquine (CQ) kills Plasmodium falciparum by binding heme, preventing its detoxification to hemozoin in the digestive vacuole (DV) of the parasite. CQ resistance (CQR) is associated with mutations in the DV membrane protein P. falciparum chloroquine resistance transporter (PfCRT), mediating the leakage of CQ from the DV. However, additional factors are thought to contribute to the resistance phenotype. This study tested the hypothesis that there is a link between glutathione (GSH) and CQR. RESULTS: Using isogenic parasite lines carrying wild-type or mutant pfcrt, we reveal lower levels of GSH in the mutant lines and enhanced sensitivity to the GSH synthesis inhibitor l-buthionine sulfoximine, without any alteration in cytosolic de novo GSH synthesis. Incubation with N-acetylcysteine resulted in increased GSH levels in all parasites, but only reduced susceptibility to CQ in PfCRT mutant-expressing lines. In support of a heme destruction mechanism involving GSH in CQR parasites, we also found lower hemozoin levels and reduced CQ binding in the CQR PfCRT-mutant lines. We further demonstrate via expression in Xenopus laevis oocytes that the mutant alleles of Pfcrt in CQR parasites selectively transport GSH. INNOVATION: We propose a mechanism whereby mutant pfcrt allows enhanced transport of GSH into the parasite's DV. The elevated levels of GSH in the DV reduce the level of free heme available for CQ binding, which mediates the lower susceptibility to CQ in the PfCRT mutant parasites. CONCLUSION: PfCRT has a dual role in CQR, facilitating both efflux of harmful CQ from the DV and influx of beneficial GSH into the DV.

A bistable circuit involving SCARECROW-RETINOBLASTOMA integrates cues to inform asymmetric stem cell division.

In plants, where cells cannot migrate, asymmetric cell divisions (ACDs) must be confined to the appropriate spatial context. We investigate tissue-generating asymmetric divisions in a stem cell daughter within the Arabidopsis root. Spatial restriction of these divisions requires physical binding of the stem cell regulator SCARECROW (SCR) by the RETINOBLASTOMA-RELATED (RBR) protein. In the stem cell niche, SCR activity is counteracted by phosphorylation of RBR through a cyclinD6;1-CDK complex. This cyclin is itself under transcriptional control of SCR and its partner SHORT ROOT (SHR), creating a robust bistable circuit with either high or low SHR-SCR complex activity. Auxin biases this circuit by promoting CYCD6;1 transcription. Mathematical modeling shows that ACDs are only switched on after integration of radial and longitudinal information, determined by SHR and auxin distribution, respectively. Coupling of cell-cycle progression to protein degradation resets the circuit, resulting in a "flip flop" that constrains asymmetric cell division to the stem cell region.

Novel bioluminescent quantitative detection of nucleic acid amplification in real-time.

BACKGROUND: The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. PRINCIPAL FINDINGS: Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. CONCLUSIONS: The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings.

Plant homologs of the Plasmodium falciparum chloroquine-resistance transporter, PfCRT, are required for glutathione homeostasis and stress responses.

In Arabidopsis thaliana, biosynthesis of the essential thiol antioxidant, glutathione (GSH), is plastid-regulated, but many GSH functions, including heavy metal detoxification and plant defense activation, depend on cytosolic GSH. This finding suggests that plastid and cytosol thiol pools are closely integrated and we show that in Arabidopsis this integration requires a family of three plastid thiol transporters homologous to the Plasmodium falciparum chloroquine-resistance transporter, PfCRT. Arabidopsis mutants lacking these transporters are heavy metal-sensitive, GSH-deficient, and hypersensitive to Phytophthora infection, confirming a direct requirement for correct GSH homeostasis in defense responses. Compartment-specific measurements of the glutathione redox potential using redox-sensitive GFP showed that knockout of the entire transporter family resulted in a more oxidized glutathione redox potential in the cytosol, but not in the plastids, indicating the GSH-deficient phenotype is restricted to the cytosolic compartment. Expression of the transporters in Xenopus oocytes confirmed that each can mediate GSH uptake. We conclude that these transporters play a significant role in regulating GSH levels and the redox potential of the cytosol.

The AUXIN BINDING PROTEIN 1 is required for differential auxin responses mediating root growth.

BACKGROUND: In plants, the phytohormone auxin is a crucial regulator sustaining growth and development. At the cellular level, auxin is interpreted differentially in a tissue- and dose-dependent manner. Mechanisms of auxin signalling are partially unknown and the contribution of the AUXIN BINDING PROTEIN 1 (ABP1) as an auxin receptor is still a matter of debate. METHODOLOGY/PRINCIPAL FINDINGS: Here we took advantage of the present knowledge of the root biological system to demonstrate that ABP1 is required for auxin response. The use of conditional ABP1 defective plants reveals that the protein is essential for maintenance of the root meristem and acts at least on the D-type CYCLIN/RETINOBLASTOMA pathway to control entry into the cell cycle. ABP1 affects PLETHORA gradients and confers auxin sensitivity to root cells thus defining the competence of the cells to be maintained within the meristem or to elongate. ABP1 is also implicated in the regulation of gene expression in response to auxin. CONCLUSIONS/SIGNIFICANCE: Our data support that ABP1 is a key regulator for root growth and is required for auxin-mediated responses. Differential effects of ABP1 on various auxin responses support a model in which ABP1 is the major regulator for auxin action on the cell cycle and regulates auxin-mediated gene expression and cell elongation in addition to the already well known TIR1-mediated ubiquitination pathway.

Dissecting regulatory pathways of G1/S control in Arabidopsis: common and distinct targets of CYCD3;1, E2Fa and E2Fc.

Activation of E2F transcription factors at the G1-to-S phase boundary, with the resultant expression of genes needed for DNA synthesis and S-phase, is due to phosphorylation of the retinoblastoma-related (RBR) protein by cyclin D-dependent kinase (CYCD-CDK), particularly CYCD3-CDKA. Arabidopsis has three canonical E2F genes, of which E2Fa and E2Fb are proposed to encode transcriptional activators and E2Fc a repressor. Previous studies have identified genes regulated in response to high-level constitutive expression of E2Fa and of CYCD3;1, but such plants display significant phenotypic abnormalities. We have sought to identify targets that show responses to lower level induced changes in abundance of these cell cycle regulators. Expression of E2Fa, E2Fc or CYCD3;1 was induced using dexamethasone and the effects analysed using microarrays in a time course allowing short and longer term effects to be observed. Overlap between CYCD3;1 and E2Fa modulated genes substantiates their action in a common pathway with a key role in controlling the G1/S transition, with additional targets for CYCD3;1 in chromatin modification and for E2Fa in cell wall biogenesis and development. E2Fc induction led primarily to gene downregulation, but did not antagonise E2Fa action and hence E2Fc appears to function outside the CYCD3-RBR pathway, does not have a direct effect on cell cycle genes, and promoter analysis suggests a distinct binding site preference.

Enhanced transformation of tnt by tobacco plants expressing a bacterial nitroreductase.

The manufacture, disposal, and detonation of explosives have resulted in the pollution of large tracts of land and groundwater. Historically, 2,4,6-trinitrotoluene (TNT) is the most widely used military explosive and is toxic to biological systems and recalcitrant to degradation. To examine the feasibility of enhancing the ability of plants to detoxify the explosive TNT, we created transgenic tobacco (Nicotiana tabacum) constitutively expressing the nsfI nitroreductase gene from Enterobacter cloacae. The product of TNT reduction by the nitroreductase was found to be 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT). Characterization of the transgenic lines in sterile, aqueous conditions amended with TNT demonstrated that these plants were able to remove all of the TNT from the medium at an initial concentration of 0.5 mM (113 mg L(-1)) TNT. In contrast, growth was suppressed in wild-type plants at 0.1 mM (23 mg L(-1)). Following uptake, transgenic seedlings transformed TNT predominantly to 4-HADNT and its high levels appeared to correlate with enhanced tolerance and transformation of TNT. Transformation products of TNT were subsequently conjugated to plant macromolecules to a greater degree in transgenic tobacco, indicating enhanced detoxification compared to the wild type.

Synchronization, transformation, and cryopreservation of suspension-cultured cells.

We have recently described the selection of rapidly dividing Arabidopsis cell suspension cultures MM1 and MM2d that provide a powerful platform for plant cell-cycle research. Here we provide detailed protocols and procedures to achieve high levels of synchronization, either by starving the cell cultures of sucrose or by applying the toxin aphidicolin. Cell-cycle activity during cell-cycle reentry (starvation-induced synchrony) or further cell-cycle progression (aphidicolin-induced synchrony) can be conveniently followed by using various validation procedures, such as determination of labeling index and metaphase/anaphase index or flow cytometry. We also describe a procedure that allows clonal transformed cell-suspension lines to be produced using Agrobacterium-mediated transformation, and an optimized and straightforward method for the cryopreservation and recovery of both parental and transformed lines which is applicable both to Arabidopsis and the tobacco BY2 cell lines. Cell-cycle synchronization capacity of the parental lines is maintained after both transformation and recovery from cryopreservation. The techniques described here require no specialized equipment and are suitable for routine laboratory use, greatly facilitating the handling and maintenance of cell cultures. The ability to store easily large numbers of transformed lines opens the possibility of using Arabidopsis cell suspension cultures for future high-throughput cell-cycle analysis.

Cell cycle regulated D3-type cyclins form active complexes with plant-specific B-type cyclin-dependent kinase in vitro.

Tobacco (Nicotiana tabacum L.) cv Bright Yellow-2 (BY-2) cells are the most highly synchronizable plant cell culture, and previously we used them to analyze cell cycle regulation of cyclin-dependent kinases (CDKs) containing the cyclin binding motifs PSTAIRE (CDKA) and PPTA/TLRE (CDKB). Here we describe the analysis of tobacco CycD3 cyclins whose transcripts predominantly accumulate during G2 to M phase, which represents a unique feature of this type of cyclin D in plants. Although protein levels of CycD3s fluctuate with different patterns during the cell cycle, kinase assays revealed that the CycD3-associated kinases phosphorylate histone H1 and the tobacco retinoblastoma related protein (NtRBR1) with two peaks at the G1/S and G2/M boundaries. In vitro pull-down assays revealed that cell cycle-regulated CycD3s bind to CDKA, but more weakly than does CycD3;3, and that they also bind to CDKB and the CDK inhibitor NtKIS1a. Mutations in the cyclin box of the CycD3s showed that two amino acids are required for binding with CDKA and NtKIS1a, but no diminished interaction was observed with CDKB. A reconstituted kinase assay was adapted for use with bacterially produced GST-CycD3s, and kinase activity could be activated by incubation of extracts from exponentially growing BY-2 cells. Such activated complexes contained CDKA and CDKB, and the reconstituted GST-CycD3 mutants, retaining binding ability to CDKB, showed kinase activity, suggesting that these cell cycle-regulated CycD3s form active complexes with both A- and B-type CDKs in vitro.

Transcriptional activation of tobacco E2F is repressed by co-transfection with the retinoblastoma-related protein: cyclin D expression overcomes this repressor activity.

Evidence is emerging that the E2F family of transcription factors plays an important role in the regulation of gene expression at the G1/S transition in plants. Here, we show that in the tobacco proliferating cell nuclear antigen (PCNA), whose transcript is specifically expressed at G1/S phase, the two E2F binding sites are synergistically responsible for transcriptional activation at G1/S phase in synchronized tobacco BY-2 cells transformed with promoter constructs fused to a reporter gene. In addition, we have isolated the tobacco DP cDNA (NtDP) and showed that significant activation of the reporter gene was observed in transient expression assays by concomitantly transfecting with plasmids expressing NtE2F and NtDP. This transcriptional activation was repressed by co-transfection with a plasmid expressing NtRBR1; in vitro pull-down assays also revealed that NtRBR1 binds directly to NtE2F, thereby potentially blocking the transcriptional activation of NtE2F. Importantly, this repressor activity was cancelled when NtRBR1 was further co-transfected with a plasmid expressing cyclin D but not with cyclin A or cyclin B. These results are discussed with respect to the repression activity of NtRBR1 on the NtE2F/NtDP complex.

Abdominal approach to chronic diaphragmatic hernias: is it safe?

Standard teaching has been to approach chronic diaphragmatic hernias (CDH) via a thoracotomy. It has been our experience that CDH can be safely approached via an abdominal incision. The objective of this study was to evaluate the outcome of patients undergoing the transabdominal approach for repair of CDH and comparing the outcome with that of patients undergoing a transthoracic (TT) approach. This is a retrospective chart review and was performed of patients with CDH secondary to trauma. Patient demographics, presenting symptoms, operative approach, and complications were collected. Patients were stratified by the surgical approach, TA versus TT. The endpoints of analysis were need for second incision, intraoperative and postoperative complications (enterotomies, pneumonia), need for a chest tube, mechanical ventilation postoperatively, and ICU and hospital days. Twenty-eight patients with CDH repairs performed between Jan 1993 and Dec 2002 were identified. Nineteen patients were in the TA group, and nine were in the TT group. Patients in the TA group had a higher incidence of emergent surgery (68% vs 11%, P = 0.005) and had a lower incidence of postoperative pneumonia (0% vs 33%, P = 0.009). No case of enteric injury from lysis of adhesions in the chest was identified. The need for a second incision (11%), the mortality (11%), ICU stay, and hospital stay were the same between the two groups. It appears that repair of CDH can be performed safely through an abdominal approach.

Differential stability of Arabidopsis D-type cyclins: CYCD3;1 is a highly unstable protein degraded by a proteasome-dependent mechanism.

In Arabidopsis, the D-type cyclin CYCD3 is rate-limiting for transition of the G(1)/S boundary, and is transcriptionally upregulated at this point in cells re-entering the cell cycle in response to plant hormones and sucrose. However, little is known about the regulation of plant cell-cycle regulators at the protein level. We show here that CYCD3;1 is a phosphoprotein highly regulated at the level of protein abundance, whereas another D-type cyclin CYCD2;1 is not. The level of CYCD3;1 protein falls rapidly on sucrose depletion, correlated with the arrest of cells in G(1) phase, suggesting a rapid turnover of CYCD3;1. Treatment of exponentially growing cells with the protein synthesis inhibitor cycloheximide (CHX) confirms that CYCD3;1 is normally a highly unstable protein, with a half-life of approximately 7 min on CHX treatment. In both sucrose-starved and exponentially growing cells, CYCD3;1 protein abundance increases in response to treatment with MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), a reversible proteasome inhibitor, but not in response to the cysteine protease inhibitor E-64 or the calpain inhibitor ALLN (N-acetyl-leucyl-leucyl-norleucinal). The increase on MG132 treatment is because of de novo protein synthesis coupled with the blocking of CYCD3;1 degradation. Longer MG132 treatment leads to C-terminal cleavage of CYCD3;1, accumulation of a hyperphosphorylated form and its subsequent disappearance. We conclude that CYCD3;1 is a highly unstable protein whose proteolysis is mediated by a proteasome-dependent pathway, and whose levels are highly dependent on the rate of CYCD3;1 protein synthesis.

Cryopreservation of transformed and wild-type Arabidopsis and tobacco cell suspension cultures.

We have recently described Arabidopsis cell suspension cultures that can be effectively synchronised. Here, we describe procedures that allow clonal-transformed cell suspension lines to be produced using Agrobacterium-mediated transformation, and an optimised and straightforward procedure for the cryopreservation and recovery of both parental and transformed lines. Frozen cultures show 90% viability and rapid re-growth after recovery. We show that the cryopreservation procedure is equally applicable to the frequently used tobacco bright yellow (BY)2 cell suspension culture, and that cell cycle synchronisation capacity of parental lines is maintained after both transformation and recovery from cryopreservation. The techniques require no specialised equipment, and are suitable for routine laboratory use, greatly facilitating the handling and maintenance of cell cultures and providing security against both contamination and cumulative somaclonal variation. Finally, the ability to store easily large numbers of transformed lines opens the possibility of using Arabidopsis cell suspension cultures for high-throughput analysis.

The plant cell cycle.

Cell division in plants is controlled by the activity of cyclin-dependent kinase (CDK) complexes. Although this basic mechanism is conserved with all other eukaryotes, plants show novel features of cell-cycle control in the molecules involved and their regulation, including novel CDKs showing strong transcriptional regulation in mitosis. Plant development is characterized by indeterminate growth and reiteration of organogenesis and is therefore intimately associated with cell division. This may explain why plants have a large number of cell-cycle regulators that appear to have overlapping and distinct functions. Here we review the recent considerable progress in understanding how core cell-cycle regulators are involved in integrating and coordinating cell division at the molecular level.

Plant D-type cyclins and the control of G1 progression.

The basic pattern of controls that operate during the G1 phase of the plant cell cycle shows much closer similarity to animals than to the yeasts and other fungi. The activity of D-type cyclin (CycD) kinases is induced in response to stimulatory signals, and these phosphorylate the plant homologue of the retinoblastoma tumour susceptibility (Rb) protein. It is likely that Rb phosphorylation results in the activation of genes under the control of E2F transcription factors, including those required for S phase entry. As the initial triggers of the cascade, attention has focused on the CycDs, and a family of 10 genes is present in Arabidopsis, divided into three major and three minor groups. Analysis to date suggests that these groups are functionally distinct.

Handsewn versus stapled anastomosis in penetrating colon injuries requiring resection: a multicenter study.

BACKGROUND: Although the use of stapling devices in elective colon surgery has been shown to be as safe as handsewn techniques, there have been concerns about their safety in emergency trauma surgery. The purpose of this study was to compare stapled with handsewn colonic anastomosis following penetrating trauma. METHODS: This was a prospective multicenter study and included patients who underwent colon resection and anastomosis following penetrating trauma. Multivariate logistic regression analysis was used to identify independent risk factors for abdominal complications and compare outcomes between stapled and handsewn repairs. RESULTS: Two hundred seven patients underwent colon resection and primary anastomosis. In 128 patients (61.8%) the anastomosis was performed with handsewing and in the remaining 79 (38.2%) with stapling devices. There were no colon-related deaths and the overall incidence of colon-related abdominal complications was 22.7% (26.6% in the stapled group and 20.3% in the handsewn group, p = 0.30). The incidence of anastomotic leak was 6.3% in the stapled group and 7.8% in the handsewn group (p = 0.69). Multivariate analysis adjusting for blood transfusions, fecal contamination, and type of antibiotic prophylaxis showed that the adjusted odds ratio (OR) of complications in the stapled group was 0.83 (95% CI, 0.38-1.74, p = 0.63). In a second multivariate analysis adjusting for blood transfusions, hypotension, fecal contamination, Penetrating Abdominal Trauma Index, and preoperative delays the adjusted OR in the stapled group was 0.99 (95% CI, 0.46-2.11, p = 0.99). CONCLUSION: The results of this study suggest that the method of anastomosis following colon resection for penetrating trauma does not affect the incidence of abdominal complications and the choice should be surgeon's preference.

Influence of flail chest on outcome among patients with severe thoracic cage trauma.

Flail chest is associated with a higher morbidity compared with multiple rib fractures, and it requires early intubation. This was a prospective comparative uncontrolled study at an academic level 1 trauma center. Twenty-two patients with flail chest (FLAIL) were compared with 90 patients with more than two rib fractures but no flail chest (RIBS) to determine differences in outcomes such as mortality, significant respiratory complications (pneumonia and adult respiratory distress syndrome), need for mechanical ventilation, and length of hospital stay. Stepwise logistic regression identified independent risk factors of poor outcome. Despite similar age and rates of lung contusion and extrathoracic injury, FLAIL patients had a higher need for mechanical ventilation (86% versus 42%, P < 0.01), higher incidence of significant respiratory complications (64% versus 26%, P < 0.01), and longer hospital stay (28 +/- 21 versus 17 +/- 19 days, P = 0.04) compared with RIBS patients. Flail chest and extrathoracic injuries were independent risk factors of significant respiratory complications. Of 11 FLAIL patients who were not intubated on arrival, eight required intubation within the next 24 hours, often while receiving diagnostic studies in poorly monitored hospital areas; two of these patients suffered morbidity directly related to the delay in intubation. Three patients without associated injuries were managed successfully without intubation. Flail chest is an independent marker of poor outcome among patients with thoracic cage trauma. The majority of patients with flail chest need mechanical ventilatory support and develop significant respiratory complications. In the presence of associated injuries, intubation is unavoidable and should be done under controlled conditions early after arrival to avoid morbidity related to sudden respiratory decompensation.