SRS-22r Self-Image After Surgery for Adolescent Idiopathic Scoliosis at 10-year Follow-up.

STUDY DESIGN: Retrospective cohort. OBJECTIVE: To examine SRS-Self Image scores at up to 10 years after surgery for adolescent idiopathic scoliosis (AIS). SUMMARY OF BACKGROUND DATA: Self-image is complex with implications for surgical and patient-reported outcomes after AIS surgery. Surgically modifiable factors that impact self-image are inconsistently reported in the literature with few longer-term reports. We examined the rate and durability of self-image improvement. MATERIALS AND METHODS: An AIS registry was queried for patients with up to 10 years of follow-up after AIS surgery. A mixed effects model estimated change in SRS-22 Self Image from baseline to 6 weeks, 1 year, 2 years, 5 years, and 10 years. All enrolled patients contributed data to the mixed effects models. A sub-analysis of patients with 1-year and 10-year follow-up evaluated worsening/static/improved SRS-22 Self Image scores examined stability of scores over that timeline. Baseline demographic data and 1-year deformity magnitude data were compared between groups using parametric and nonparametric tests as appropriate. RESULTS: Data from 4608 patients contributed data to the longitudinal model; 162 had 1-year and 10-year data. Mean SRS-Self Image improvement at 10-year follow-up was 1.0 (95% CI: 0.9-1.1) point. No significant changes in Self-Image domain scores were estimated from 1-year to 10-year (all P >0.05) postoperative. Forty (25%) patients had SRS-Self Image worsening from 1 year to 10 years, 36 (22%) improved, and 86 (53%) were unchanged. Patients who worsened over 10 years had lower SRS-Self Image at baseline than those unchanged at enrollment (3.3 vs. 3.7, P =0.007). Neither radiographic parameters nor SRS-Mental Health were different at baseline for the enrolled patients. CONCLUSION: Ten years after surgery, 75% of patients reported similar or better SRS-Self Image scores than one year after surgery. Nearly 25% of patients reported worsening self-image at 10 years. Patients who worsened had lower baseline SRS-Self Image scores, without radiographic or mental health differences at baseline or follow-up.

Late spinal infections following posterior spinal fusion in pediatric deformities: treatment using single-stage titanium implant exchange.

PURPOSE: Late infection following posterior spinal fusion (PSF) for deformity is a leading cause of revision. The purpose of this study is to evaluate clinical and radiographic outcomes following a single-stage debridement and exchange of spinal implants with titanium in adolescent patients with late-onset infections following PSF METHODS: A retrospective review of prospectively collected data of adolescent patients with spinal deformity, who were surgically treated with PSF was collected. Patients were included for the study if they developed late arising infection (> 1 year after index posterior fusion for the deformity) from 2006-2019. Treatment consisted of irrigation, debridement, implant exchange with titanium screws and rods, and antibiotics. Parameters evaluated include radiographic Cobb angles, operative data, and clinical data, all at minimum 2-year follow-up. RESULTS: 31 patients (29 with AIS and 2 with Scheuermann's kyphosis) developed late spinal infections. Mean age was 11.4 ± 2.3 years, 84% female, mean time from index surgery was 52.5 months. 25 had all stainless steel implants and 6 had cobalt chrome during the index procedure. Positive cultures were obtained in 5 patients (2 Staphylococcus Aureus, 1 Staphylococcus epidermidis, 1 Peptostreptococcus, 1 Pseudomonas aeruginosa) with cultures followed till 7 days post-operatively. At 2-years following the exchange, there was no change in coronal and sagittal alignment. Three (9%) patients developed subsequent infection necessitating implant removal. CONCLUSION: A single-stage procedure consisting of implant removal, irrigation, and debridement, and replacement with all titanium implants is an effective treatment strategy in patients developing late wound infection following PSF with regards to maintenance of curve correction and minimizing recurrent infections.

Percutaneous and open iliac screw safety and accuracy using a tactile technique with adjunctive anteroposterior fluoroscopy.

BACKGROUND CONTEXT: All currently described percutaneous iliac screw placement methods are entirely dependent on fluoroscopy. PURPOSE: The purpose of this study was to determine the safety and the accuracy of percutaneous and open iliac screw placement using a primarily tactile technique with adjunctive anteroposterior (AP) fluoroscopy. STUDY DESIGN/CONTEXT: All patients who underwent open and percutaneous iliac screw placement over a 5-year period were identified. Charts were reviewed to assess for any instances of neurologic or vascular injury associated with iliac screw placement. Screw accuracy was judged with postoperative computed tomography (CT) scans. PATIENT SAMPLE: A total of 133 patients were identified who underwent open or percutaneous iliac screw placement. Computed tomography scans were available for 57 patients, and all of these patients were included in the study, with a total of 115 iliac screws. OUTCOME MEASURES: Radiographic measurements were performed, consisting of the distance of the iliac screw to the sciatic notch on postoperative radiographs and CT scans. Computed tomography scans were used to determine iliac screw accuracy. METHODS: Charts were reviewed to assess for any neurologic or vascular injuries related to screw placement. The distance of the iliac screw to the sciatic notch was measured and compared on AP radiography and CT scans. Computed tomography scans were assessed for any screw violation of the iliac cortex or the sciatic notch. The accuracy of open iliac screw placement was compared with minimally invasive percutaneous placement. RESULTS: There were no neurologic or vascular injuries related to screw placement in the 133 patients. Computed tomography scans were available for 115 iliac screws, with 3 cortical breaches, all by less than 2 mm. All 112 other screws were accurately intraosseous. There was a strong correlation between the iliac screw to the sciatic notch distance when measured by CT scan compared with AP radiography (r=0.9), thus validating the accuracy of AP fluoroscopy in guiding iliac screw placement with respect to the sciatic notch. Iliac screw accuracy was equal with the open and percutaneous insertion techniques. CONCLUSIONS: The described surgical technique represents a safe and reliable surgical option for iliac screw placement. Intraoperative AP fluoroscopy accurately reflects the distance of the iliac screw to the sciatic notch. Percutaneous iliac screws placed with this technique are as accurate as open iliac screws.

Molecular responses of European flounder (Platichthys flesus) chronically exposed to contaminated estuarine sediments.

Molecular responses to acute toxicant exposure can be effective biomarkers, however responses to chronic exposure are less well characterised. The aim of this study was to determine chronic molecular responses to environmental mixtures in a controlled laboratory setting, free from the additional variability encountered with environmental sampling of wild organisms. Flounder fish were exposed in mesocosms for seven months to a contaminated estuarine sediment made by mixing material from the Forth (high organics) and Tyne (high metals and tributyltin) estuaries (FT) or a reference sediment from the Ythan estuary (Y). Chemical analyses demonstrated that FT sediment contained significantly higher concentrations of key environmental pollutants (including polycyclic aromatic hydrocarbons (PAHs), chlorinated biphenyls and heavy metals) than Y sediment, but that chronically exposed flounder showed a lack of differential accumulation of contaminants, including heavy metals. Biliary 1-hydroxypyrene concentration and erythrocyte DNA damage increased in FT-exposed fish. Transcriptomic and (1)H NMR metabolomic analyses of liver tissues detected small but statistically significant alterations between fish exposed to different sediments. These highlighted perturbance of immune response and apoptotic pathways, but there was a lack of response from traditional biomarker genes. Gene-chemical association annotation enrichment analyses suggested that polycyclic aromatic hydrocarbons were a major class of toxicants affecting the molecular responses of the exposed fish. This demonstrated that molecular responses of sentinel organisms can be detected after chronic mixed toxicant exposure and that these can be informative of key components of the mixture.

Transcriptomic responses of European flounder (Platichthys flesus) liver to a brominated flame retardant mixture.

Male European flounder (Platichthys flesus) were exposed to a technical mixture of brominated diphenyl ethers (PDBEs, DE-71, Pentamix) that had been purified to remove contaminating dioxins. Controls were exposed to carrier solvent alone. Fish were exposed to decadally increasing concentrations of Pentamix via both sediment and spiked food. The GENIPOL P. flesus cDNA microarray, differentially expressed gene profiling (DEG) and quantitative PCR were employed to detect hepatic transcriptional differences between exposed fish and controls. Gene transcriptional changes were more sensitive to Pentamix exposure than biomarkers measured previously. Pentamix exposure induced transcripts coding for enzymes of xenobiotic metabolism (CYP1A, aldo-keto reductases) and elicited endocrine disruption (vitellogenin and thyroid hormone receptor alpha), with effects on CYP1A and VTG occurring at the highest exposure. Ontology analysis clearly showed dose-responsive changes indicative of oxidative stress, induction of mitochondrial dysfunction, and apoptosis. We conclude that exposure to PBDEs in both sediment and food has a significant adverse effect on a broad range of crucial biochemical processes in the livers of this widely distributed estuarine fish species, the flounder.

Towards a system level understanding of non-model organisms sampled from the environment: a network biology approach.

The acquisition and analysis of datasets including multi-level omics and physiology from non-model species, sampled from field populations, is a formidable challenge, which so far has prevented the application of systems biology approaches. If successful, these could contribute enormously to improving our understanding of how populations of living organisms adapt to environmental stressors relating to, for example, pollution and climate. Here we describe the first application of a network inference approach integrating transcriptional, metabolic and phenotypic information representative of wild populations of the European flounder fish, sampled at seven estuarine locations in northern Europe with different degrees and profiles of chemical contaminants. We identified network modules, whose activity was predictive of environmental exposure and represented a link between molecular and morphometric indices. These sub-networks represented both known and candidate novel adverse outcome pathways representative of several aspects of human liver pathophysiology such as liver hyperplasia, fibrosis, and hepatocellular carcinoma. At the molecular level these pathways were linked to TNF alpha, TGF beta, PDGF, AGT and VEGF signalling. More generally, this pioneering study has important implications as it can be applied to model molecular mechanisms of compensatory adaptation to a wide range of scenarios in wild populations.

Copper induces Cu-ATPase ATP7A mRNA in a fish cell line, SAF1.

Copper transporting ATPase, ATP7A, is an ATP dependent copper pump present in all vertebrates, critical for the maintenance of intracellular and whole body copper homeostasis. Effects of copper treatment on ATP7A gene expression in fibroblast cells (SAF1) of the sea bream (Sparus aurata) were investigated by qRT-PCR and by a medium density microarray from a closely related species, striped sea bream (Lithognathus mormyrus). To discriminate between the effects of Cu and other metals, SAF1 cells were exposed to sub-toxic levels of Cu, Zn and Cd. Expression of Cu homeostasis genes copper transporter 1 (CTR1), Cu ATPase (ATP7A), Cu chaperone (ATOX1) and metallothionein (MT) together with the oxidative stress markers glutathione reductase (GR) and Cu/Zn superoxide dismutase (CuZn/SOD) were measured 0, 4 and 24 hours post-exposure by qRT-PCR. Microarray was conducted on samples from 4 hours post Cu exposure. Cu, Zn and Cd increased MT and GR mRNA levels, while only Cu increased ATP7A mRNA levels. Microarray results confirmed the effects of Cu on ATP7A and MT and in addition showed changes in the expression of genes involved in protein transport and secretion. Results suggest that ATP7A may be regulated at the transcriptional level directly by Cu and by a mechanism that is different from that exerteted by metals on MT genes.

Integrating omic technologies into aquatic ecological risk assessment and environmental monitoring: hurdles, achievements, and future outlook.

BACKGROUND: In this commentary we present the findings from an international consortium on fish toxicogenomics sponsored by the U.K. Natural Environment Research Council (Fish Toxicogenomics-Moving into Regulation and Monitoring, held 21-23 April 2008 at the Pacific Environmental Science Centre, Vancouver, BC, Canada). OBJECTIVES: The consortium from government agencies, academia, and industry addressed three topics: progress in ecotoxicogenomics, regulatory perspectives on roadblocks for practical implementation of toxicogenomics into risk assessment, and dealing with variability in data sets. DISCUSSION: Participants noted that examples of successful application of omic technologies have been identified, but critical studies are needed to relate molecular changes to ecological adverse outcome. Participants made recommendations for the management of technical and biological variation. They also stressed the need for enhanced interdisciplinary training and communication as well as considerable investment into the generation and curation of appropriate reference omic data. CONCLUSIONS: The participants concluded that, although there are hurdles to pass on the road to regulatory acceptance, omics technologies are already useful for elucidating modes of action of toxicants and can contribute to the risk assessment process as part of a weight-of-evidence approach.

Multiple Cu-ATPase genes are differentially expressed and transcriptionally regulated by Cu exposure in sea bream, Sparus aurata.

Copper (Cu) is an essential metal, although in excess is highly toxic due to its redox properties and, therefore intracellular Cu homeostasis is a highly regulated process. Cu-ATPases are pivotal regulatory, proteins of intracellular and bodily Cu homeostasis. Two Cu-ATPases, ATP7A and ATP7B with distinct, functions are found in mammals and herein we report the structure and expression under Cu stress of, homologues of ATP7A and ATP7B in gilthead sea bream (Sparus aurata), the first such report for any, fish. The deduced protein sequences of S. aurata ATP7A (saATP7A) and ATP7B (saATP7B), displayed 63% and 75% identity respectively to their human homologues. All characteristic structural, features of Cu-ATPases were conserved between fish and mammals, although the number of Cu-binding, domains was less in fish ATP7B than in mammalian ATP7B. The tissue expression of sea bream, Cu-ATPases was similar to that observed in mammals, saATP7A being ubiquitously expressed, although low in liver, whilst saATP7B was mainly expressed in the intestine and liver. By analysis of the sequenced genomes of other species we have confirmed the presence of ATP7A and ATP7B genes in fish and propose that the presence of two Cu-ATPase genes in vertebrates represents a retention and neo-functionalization of a duplicated ancestral gene coincident with the development of a closed circulatory system and discrete hepato-biliary system. Expression of Cu-ATPase mRNA was changed after exposure to excess Cu in a manner dependent on exposure route and tissue type. Excess dietary Cu (130mgkg(-1) Cu dry diet) reduced saATP7A mRNA levels in intestine, gill, kidney and liver, and increased hepatic saATP7B mRNA consistent with increased biliary excretion. Whilst after waterborne Cu exposure (0.3mgL(-1) Cu), expression of ATP7A mRNA was increased in intestine and liver and toxic responses were observed in gill and liver. Our results indicate that Cu-ATPases in both fish and mammals have similar functions in maintenance of Cu homeostasis and are consistent with previous physiological evidence from various fish species for the involvement of multiple Cu-ATPases in Cu transport. Furthermore, our evidence suggests that fish can detoxify excess dietary Cu relatively efficiently but are unable to cope with excess dissolved Cu in the water, demonstrating that the exposure route is critical to toxicity.

Hepatic gene expression in flounder chronically exposed to multiply polluted estuarine sediment: Absence of classical exposure 'biomarker' signals and induction of inflammatory, innate immune and apoptotic pathways.

The effects of chronic long-term exposure to multiply polluted environments on fish are not well understood, but environmental surveys suggest that such exposure may cause a variety of pathologies, including cancers. Transcriptomic profiling has recently been used to assess gene expression in European flounder (Platichthys flesus) living in several polluted and clean estuaries. However, the gene expression changes detected were not unequivocally elicited by pollution, most likely due to the confounding effects of natural estuarine ecosystem variables. In this study flounder from an uncontaminated estuary were held on clean or polluted sediments in mesocosms, allowing control of variables such as salinity, temperature, and diet. After 7 months flounder were removed from each mesocosm and hepatocytes prepared from fish exposed to clean or polluted sediments. The hepatocytes were treated with benzo(a)pyrene (BAP), estradiol (E2), copper, a mixture of these three, or with the vehicle DMSO. A flounder cDNA microarray was then used to measure hepatocyte transcript abundance after each treatment. The results show that long-term chronic exposure to a multiply polluted sediment causes increases in the expression of mRNAs coding for proteins of the endogenous apoptotic programme, of innate immunity and inflammation. Contrary to expectation, the expression of mRNAs which are commonly used as biomarkers of environmental exposure to particular contaminants were not changed, or were changed contrary to expectation. However, acute treatment of hepatocytes from flounder from both clean and polluted sediments with BAP or E2 caused the expected changes in the expression of these biomarkers. Thus transcriptomic analysis of flounder exposed long-term to chronic pollution causes a different pattern of gene expression than in fish acutely treated with single chemicals, and reveals novel potential biomarkers of environmental contaminant exposure. These novel biomarkers include Diablo, a gene involved in apoptotic pathways and highly differentially regulated by both chronic and acute exposure to multiple pollutants.

Transcriptomic responses of European flounder (Platichthys flesus) to model toxicants.

The temporal transcriptomic responses in liver of Platichthys flesus to model environmental pollutants were studied over a 16-day time span after intraperitoneal injection with cadmium chloride (50 microg/kg in saline), 3-methylcholanthrene (25 mg/kg in olive oil), Aroclor 1254 (50 mg/kg in olive oil), tert-butyl-hydroperoxide (5 mg/kg in saline), Lindane (25mg/kg in olive oil), perfluoro-octanoic acid (100 mg/kg in olive oil) and their vehicles, olive oil (1 ml/kg) or saline (0.9%). Statistical, gene ontology and supervised analysis clearly demonstrated the progression from acute effects, biological responses to and recovery from the treatments. Key biological processes disturbed by the individual treatments were characterised by gene ontology analyses and individual toxicant-responsive genes and pathways were identified by supervised analyses. Responses to the polyaromatic and chlorinated aromatic compounds showed a degree of commonality but were distinguishable and they were clearly segregated from the responses to the pro-oxidants cadmium and the organic hydroperoxide, as well as from the peroxisomal proliferator, perfluoro-octanoic acid. This study demonstrated the utility of the microarray technique in the identification of toxicant-responsive genes and in discrimination between modes of toxicant action.

Adaptive differences in gene expression in European flounder (Platichthys flesus).

Population structure was previously believed to be very limited or absent in classical marine fishes, but recently, evidence of weakly differentiated local populations has been accumulating using noncoding microsatellite markers. However, the evolutionary significance of such minute genetic differences remains unknown. Therefore, in order to elucidate the relationship between genetic markers and adaptive divergence among populations of marine fishes, we combined cDNA microarray and microsatellite analysis in European flounders (Platichthys flesus). We demonstrate that despite extremely low levels of neutral genetic divergence, a high number of genes were significantly differentially expressed between North Sea and Baltic Sea flounders maintained in a long-term reciprocal transplantation experiment mimicking natural salinities. Several of the differentially regulated genes could be directly linked to fitness traits. These findings demonstrate that flounders, despite little neutral genetic divergence between populations, are differently adapted to local environmental conditions and imply that adaptation in gene expression could be common in other marine organisms with similar low levels of population subdivision.

Piscine UDP-glucuronosyltransferase 1B.

Glucuronidation is an important detoxification pathway for organic pollutants in fish. We report here the isolation and characterisation of UDP-glucuronosyltransferases (UGT) genes from the closely related marine flatfish, plaice (Pleuronectes platessa) and flounder (Platichthys flesus). The deduced amino acid sequences share greater similarity with mammalian UGT1 family genes than UGT2 genes (44-47% and 39-40% amino acid identity, respectively) and have been designated UGT1B. Both plaice and flounder UGT1B mRNAs are expressed in all tissues and are most highly expressed in liver, with high levels in intestine, gill, kidney and adipose tissue and much lower levels in muscle, heart and brain. Plaice UGT1B mRNA is undetectable in gametes or fertilised eggs and there is a large increase in expression between gastrulation and myotome formation after which levels decline some 5-10-fold. Flounder UGT1B mRNA was increased in liver after intraperitoneal injection of Arochlor 1254 or lindane (gamma-hexachlorocyclohexane), but not after perflourooctanoic acid or 3-methylcholanthrene treatment. In isolated flounder hepatocytes UGT1B mRNA was increased after exposure to benzo(a)pyrene but not by 17alpha-ethynylestradiol. Expression of a cDNA for plaice UGT1B in cos7 cells resulted in higher 1-naphthol conjugation in cell homogenates compared to steroid conjugation, whilst bilirubin and bile acid conjugation were undetectable. This indicates that the plaice gene codes for the phenol-conjugating UGT previously purified in our laboratory from this species and that it is likely to play a major role in the detoxification of polyaromatic hydrocarbons in flatfish. Its role in development is unknown. UGT1B genes are also present in pufferfish (Tetraodon nigroviridis) and zebrafish (Danio rerio) genomes, but they differ in their genic organisation. Pufferfish possess multiple (repeated) complete UGT1 genes and Southern blots indicate that the homologous plaice UGT1B gene may also be organised in this way. In contrast, zebrafish appear to have two UGT1 loci whose sequences and intron/exon structures are closely related to that of plaice, however, the organisation of these genes is similar to the mammalian UGT1 family since each has multiple repeated exon 1's which are alternatively spliced to a common set of exons encoding the aglycone binding domain. Taken together with evidence from phylogenetic comparison of fish sequences with UGT1 and UGT2 families in mammals, we suggest these homologous fish UGTs should all be included within the vertebrate UGT1 family and designated as UGT1B.

Estimating the efficiency of fish cross-species cDNA microarray hybridization.

Using an available cross-species cDNA microarray is advantageous for examining multigene expression patterns in non-model organisms, saving the need for construction of species-specific arrays. The aim of the present study was to estimate relative efficiency of cross-species hybridizations across bony fishes, using bioinformatics tools. The methodology may serve also as a model for similar evaluations in other taxa. The theoretical evaluation was done by substituting comparative whole-transcriptome sequence similarity information into the thermodynamic hybridization equation. Complementary DNA sequence assemblages of nine fish species belonging to common families or suborders and distributed across the bony fish taxonomic branch were selected for transcriptome-wise comparisons. Actual cross-species hybridizations among fish of different taxonomic distances were used to validate and eventually to calibrate the theoretically computed relative efficiencies.

Gene expression responses of European flounder (Platichthys flesus) to 17-beta estradiol.

Male European flounder (Platichthys flesus) were intraperitoneally injected with 10mg/kg 17-beta estradiol and tissues taken from individuals over a timecourse of 16 days. The GENIPOL P. flesus cDNA microarray was employed to detect hepatic gene expression differences between fish treated with estradiol and saline controls. Known biomarkers of estrogen exposure, choriogenin L and vitellogenins, showed sustained induction over the time-course. Among 175 identified clones showing sustained statistically significant induction or repression, those associated with the Gene Ontology terms mitochondria, amino acid synthesis, ubiquitination and apoptosis were included amongst those induced while those associated with immune function, electron transport, cell signalling and protein phosphorylation were repressed. Thus, we show the gene expression response of an environmentally relevant fish species to a high dose of an estrogenic endocrine disruptor and also report the sequencing of a further 2121 flounder ESTs.

Development of the GENIPOL European flounder (Platichthys flesus) microarray and determination of temporal transcriptional responses to cadmium at low dose.

We have constructed a high density, 13 270-clone cDNA array for the sentinel fish species European flounder (Platichthys flesus), combining clones from suppressive subtractive hybridization and a liver cDNA library; DNA sequences of 5211 clones were determined. Fish were treated by single intraperitoneal injection with 50 micrograms cadmium chloride per kilogram body weight, a dose relevant to environmental exposures, and hepatic gene expression changes were determined at 1, 2, 4, 8, and 16 days postinjection in comparison to saline-treated controls. Gene expression responses were confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR). Blast2GO gene ontology analysis highlighted a general induction of the unfolded protein response, response to oxidative stress, protein synthesis, transport, and degradation pathways, while apoptosis, cell cycle, cytoskeleton, and cytokine genes were also affected. Transcript levels of cytochrome P450 1A (CYP1A) were repressed and vitellogenin altered, real-time PCR showed induction of metallothionein. We thus describe the establishment of a useful resource for ecotoxicogenomics and the determination of the temporal molecular responses to cadmium, a prototypical heavy metal pollutant.

Molecular evidence for multiple UDP-glucuronosyltransferase gene familes in fish.

The presence of multiple distinct UGT genes in fish was derived by analysis of DNA sequence data derived the zebrafish EST project, confirming indications from previous protein purification studies in another fish species, the plaice, for a diversity of isoforms in lower vertebrates. At least 10 different UGTs can be identified from nucleotide sequence data in zebrafish. Phylogenetic analysis of exon 1 sequences of the zebrafish, plaice and human UGTs indicates that six of these genes are related to the 1A, 1B and 2 families and that a further four genes were of more ancient lineage. Importantly data for the 3' sequences of the zebrafish clones, both from the database and our own sequences of the publicly available clones did not provide any evidence for elaboration of family 1A genes by alternative splicing in this lower vertebrate.