Experience of an anatomic femoral stem in a UK orthopaedic centre beyond 20 years of follow-up.

INTRODUCTION: Increasing interest in the use of anatomical stems has developed as the prevalence of periprosthetic fractures (PPFs) continues to increase. The primary aim of this study was to determine the long-term survivorship and PPF rate of an anatomical femoral stem in a single UK centre. PATIENTS AND METHODS: Between 2000 and 2002, 94 consecutive THAs were performed using the 170 mm Lubinus SP II anatomical femoral stem in our institution. Patient demographics, operative details and clinical outcomes were collected prospectively in an arthroplasty database. Patient records and national radiographic archives were reviewed finally at a mean of 21.5 years (SD 0.7) following surgery to identify occurrence of subsequent revision surgery, dislocation or periprosthetic fracture. RESULTS: Mean patient age at surgery was 65.8 years (SD 12.5, 34-88 years). There were 48 women (51%). Osteoarthritis was the operative indication in 88 patients (94%). Analysis of all-cause THA failure demonstrated a survivorship of 98.5% (95% confidence interval [CI], 98.0-99.3%) at 10 years and 96.7% (94.5-98.9%) at 21 years. The 20-year stem survival for aseptic loosening was 100% with no cases of significant lysis found (lucent line > 2 mm) and no stems required revision. Patient demographics did not appear to influence risk of revision (p > 0.05). There were 2 revisions in total (2 for acetabular loosening with original stems retained). There were no PPFs identified at mean 21.5 year follow-up and 5 dislocations (5%). CONCLUSIONS: The Lubinus SP II 170 mm stem demonstrated excellent survivorship and negligible PPF rates over 20 years following primary THA.

Assigning forensic body fluids to donors in mixed body fluids by targeted RNA/DNA deep sequencing of coding region SNPs.

Biological traces found at crime scenes are analysed not only to genetically identify the donor(s) but also to determine the composition of the stain. For some cases, it is essential to associate a body fluid with a donor. Especially in mixed body fluid stains, but also in body fluid stains that appear to be single-source, this may be of importance. Linking a DNA profile (sub-source level) with evidence from a presumptive test or mRNA analysis (source level) is not straightforward. Our results support that associating donors and body fluids by means of comparing mixture ratios in RNA and DNA is not recommended. We introduce a set of 35 coding region SNPs (cSNPs) in body fluid-specific mRNA transcripts that represent a direct link between the body fluids and their donors. The discrimination power of the cSNPs was estimated based on allele frequencies calculated from a population sample (n = 188), and we investigated the practical application of the cSNPs in different scenarios. The results demonstrate that more cSNPs are needed to improve the discrimination power. However, the findings are promising as we were able to associate donors with body fluids in mixtures of different body fluids as well as in stains where both donors have contributed the same body fluid, e.g. a blood-blood mixture. In addition, the cSNP assay can be used for body fluid identification. The results of this proof-of-concept study support the use of cSNPs to assign body fluids to the respective donors.

Body fluid identification and assignment to donors using a targeted mRNA massively parallel sequencing approach - results of a second EUROFORGEN / EDNAP collaborative exercise.

In a previous EUROFORGEN/EDNAP collaborative exercise, we tested two assays for targeted mRNA massively parallel sequencing for the identification of body fluids/tissues, optimized for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms, respectively. The task of the second EUROFORGEN/EDNAP collaborative exercise was to analyze dried body fluid stains with two different multiplexes, the former Illumina 33plex mRNA panel for body fluid/tissue identification and a 35plex cSNP panel for assignment of body fluids/tissues to donors that was introduced in a proof-of-concept study recently. The coding region SNPs (cSNPs) are located within the body fluid specific mRNA transcripts and represent a direct link between the body fluid and the donor. We predicted the origin of the stains using a partial least squares discriminant analysis (PLS-DA) model, where most of the single source samples were correctly predicted. The mixed body fluid stains showed poorer results, however, at least one component was predicted correctly in most stains. The cSNP data demonstrated that coding region SNPs can give valuable information on linking body fluids/tissues with donors in mixed body fluid stains. However, due to the unfavorable performance of some cSNPs, the interpretation remains challenging. As a consequence, additional markers are needed to increase the discrimination power in each body fluid/tissue category.

Messenger RNA biomarker signatures for forensic body fluid identification revealed by targeted RNA sequencing.

The recovery of a DNA profile from the perpetrator or victim in criminal investigations can provide valuable 'source level' information for investigators. However, a DNA profile does not reveal the circumstances by which biological material was transferred. Some contextual information can be obtained by a determination of the tissue or fluid source of origin of the biological material as it is potentially indicative of some behavioral activity on behalf of the individual that resulted in its transfer from the body. Here, we sought to improve upon established RNA based methods for body fluid identification by developing a targeted multiplexed next generation mRNA sequencing assay comprising a panel of approximately equal sized gene amplicons. The multiplexed biomarker panel includes several highly specific gene targets with the necessary specificity to definitively identify most forensically relevant biological fluids and tissues (blood, semen, saliva, vaginal secretions, menstrual blood and skin). In developing the biomarker panel we evaluated 66 gene targets, with a progressive iteration of testing target combinations that exhibited optimal sensitivity and specificity using a training set of forensically relevant body fluid samples. The current assay comprises 33 targets: 6 blood, 6 semen, 6 saliva, 4 vaginal secretions, 5 menstrual blood and 6 skin markers. We demonstrate the sensitivity and specificity of the assay and the ability to identify body fluids in single source and admixed stains. A 16 sample blind test was carried out by one lab with samples provided by the other participating lab. The blinded lab correctly identified the body fluids present in 15 of the samples with the major component identified in the 16th. Various classification methods are being investigated to permit inference of the body fluid/tissue in dried physiological stains. These include the percentage of reads in a sample that are due to each of the 6 tissues/body fluids tested and inter-sample differential gene expression revealed by agglomerative hierarchical clustering.

Body fluid identification using a targeted mRNA massively parallel sequencing approach - results of a EUROFORGEN/EDNAP collaborative exercise.

In a previous study we presented an assay for targeted mRNA sequencing for the identification of human body fluids, optimised for the Illumina MiSeq/FGx MPS platform. This assay, together with an additional in-house designed assay for the Ion Torrent PGM/S5 platform, was the basis for a collaborative exercise within 17 EUROFORGEN and EDNAP laboratories, in order to test the efficacy of targeted mRNA sequencing to identify body fluids. The task was to analyse the supplied dried body fluid stains and, optionally, participants' own bona fide or mock casework samples of human origin, according to specified protocols. The provided primer pools for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms included 33 and 29 body fluid specific targets, respectively, to identify blood, saliva, semen, vaginal secretion, menstrual blood and skin. The results demonstrated moderate to high count values in the body fluid or tissue of interest with little to no counts in non-target body fluids. There was some inter-laboratory variability in read counts, but overall the results of the laboratories were comparable in that highly expressed markers showed high read counts and less expressed markers showed lower counts. We performed a partial least squares (PLS) analysis on the data, where blood, menstrual blood, saliva and semen markers and samples clustered well. The results of this collaborative mRNA massively parallel sequencing (MPS) exercise support targeted mRNA sequencing as a reliable body fluid identification method that could be added to the repertoire of forensic MPS panels.

Severe arthritis predicts greater improvements in function following total knee arthroplasty.

PURPOSE: Although excellent outcomes are routinely reported following total knee replacement, up to 20 % of patients remain dissatisfied. The aim of this study was to determine whether pre-operative radiographic classification was associated with functional outcomes following surgery. METHODS: A retrospective review of a prospective arthroplasty database identified 256 patients that fulfilled the inclusion criteria over an 18-month period. Baseline demographic data on all patients were collected prospectively. All pre-operative radiographs were assessed using the Kellgren and Lawrence (K&L) classification system. Patients were prospectively assessed using the American Knee Society Score pre-operatively and at 1, 3 and 5 years post-surgery. RESULTS: An association was found between the pre-operative radiographic severity of arthritis and the pre-operative American Knee Society Knee (AKSK) scores, with worsening radiographic grade corresponding to worsening AKSK scores (p = 0.020). There was an association between K&L classification and improvement in AKSK scores from pre-operative to 1 year (p = 0.003) and 3 years (p = 0.04), with K&L grades 3 and 4 demonstrating the most significant improvements. On multivariate regression analysis, K&L classification was the only significant predictor of improvement in AKSK at 1 year (p = 0.009). No correlation was found between K&L grade and the American Knee Society Functional Scores at any stage. CONCLUSIONS: The results of this study may help to improve satisfaction rates in total knee replacement by targeting treatment. Patients can be counselled that although radiographic severity of arthritic changes can predict knee-specific functional improvement, the extent of their global functional improvement cannot. LEVEL OF EVIDENCE: IV.

Enhanced recovery programmes after total hip arthroplasty can result in reduced length of hospital stay without compromising functional outcome.

AIMS: The primary aim of this study was to investigate the effect of an enhanced recovery program (ERP) on the short-term functional outcome after total hip arthroplasty (THA). Secondary outcomes included its effect on rates of dislocation and mortality. PATIENTS AND METHODS: Data were gathered on 1161 patients undergoing primary THA which included 611 patients treated with traditional rehabilitation and 550 treated with an ERP. RESULTS: The ERP was shown to be a significant independent factor which shortened length of stay (LOS) by a mean of 1.5 days (95% confidence interval (CI) 1.3 to 1.8, p < 0.001) after adjusting for confounding variables. The rates of dislocation (traditional 1.03% vs ERP 0.91%, p = 0.84) and mortality (1.5% vs 0.6%, p = 0.14) one year post-operatively were not significantly different. Both groups showed significant improvement in Harris Hip Score (42.8 vs 41.5) at 12 to 18 months post-operatively and there was no significant difference in the magnitude of improvement on univariate (p = 0.09) and multivariate analysis (p = 0.35). There was no significant difference in any of the eight domain scores of the Short-Form - 36 general health surveys post-operatively (p > 0.38). CONCLUSION: We conclude that an ERP after THA shortens LOS by a mean of 1.5 days and does not increase the rate of complications post-operatively. It gives equivalent functional outcomes to a traditional rehabilitation pathway. TAKE HOME MESSAGE: ERP reduces LOS after THA in comparison to traditional rehabilitation, without adversely affecting functional outcomes, dislocation rates or mortality.

RNA/DNA co-analysis from human skin and contact traces--results of a sixth collaborative EDNAP exercise.

The European DNA profiling group (EDNAP) organized a sixth collaborative exercise on RNA/DNA co-analysis for body fluid/tissue identification and STR profiling. The task was to identify skin samples/contact traces using specific RNA biomarkers and test three housekeeping genes for their suitability as reference genes. Eight stains, a skin RNA dilution series and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 22 participating laboratories using RNA extraction or RNA/DNA co-extraction methods. Two sets of previously described skin-specific markers were used: skin1 pentaplex (LCE1C, LCE1D, LCE2D, IL1F7 and CCL27) and skin2 triplex (LOR, KRT9 and CDSN) in conjunction with a housekeeping gene, HKG, triplex (B2M, UBC and UCE). The laboratories used different chemistries and instrumentation. All laboratories were able to successfully isolate and detect mRNA in contact traces (e.g., human skin, palm-, hand- and fingerprints, clothing, car interiors, computer accessories and electronic devices). The simultaneous extraction of RNA and DNA provides an opportunity for positive identification of the tissue source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling. The skin markers LCE1C and LOR and the housekeeping gene marker B2M were detected in the majority of contact traces. Detection of the other markers was inconsistent, possibly due to the low amounts and/or poor quality of the genetic material present in shed skin cells. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid/tissue identification method that can easily be combined with current STR typing technology.

A Y-short tandem repeat specific DNA enhancement strategy to aid the analysis of late reported (≥ 6 days) sexual assault cases.

The ability to obtain an autosomal short tandem repeat (STR) profile of the semen donor from the reproductive tract of a living victim rapidly diminishes as the post-coital interval increases. This is of concern where victims of sexual assault provide vaginal samples several days after the incident. In order to overcome the technological impediments inherent in autosomal DNA typing with extended interval samples, we previously employed the use of Y chromosome STR profiling which, by targeting only male DNA, can eliminate masking of the male profile (by the victim's alleles) or critical polymerase chain reaction reagent titration (due to excessive female DNA). Thus employing Y-STR profiling and additional enhancement strategies, we reported the ability to recover Y-STR profiles from samples collected 5 to 6 days after intercourse. However, the reproductive biology literature indicates that spermatozoa are found in the human cervix up to 7 to 10 days post coitus. Thus, even with improved extraction and profiling techniques, we failed to routinely recover profiles from samples collected ≥ 6 days after intercourse. The aim of the present work was to develop additional strategies to permit the recovery of male donor DNA profiles from ≥ 6 post-coital samples. Using nested polymerase chain reaction and DNA concentration procedures that together maximize the recovery and targeting of male DNA, we demonstrate the ability to obtain semen donor Y-STR profiles in extended interval post-coital samples collected 6 to 9 days after intercourse. This approaches the recognized time limits of sperm residence in the cervico-vaginal canal as described in the clinical literature.

RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: results of a fourth and fifth collaborative EDNAP exercise.

The European DNA Profiling Group (EDNAP) organized a fourth and fifth collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling. The task was to identify dried menstrual blood and vaginal secretion stains using specific RNA biomarkers, and additionally test 3 housekeeping genes for their suitability as reference genes. Six menstrual blood and six vaginal secretion stains, two dilution series (1/4-1/64 pieces of a menstrual blood/vaginal swab) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 24 participating laboratories, using RNA extraction or RNA/DNA co-extraction methods. Two novel menstrual blood mRNA multiplexes were used: MMP triplex (MMP7, MMP10, MMP11) and MB triplex (MSX1, LEFTY2, SFRP4) in conjunction with a housekeeping gene triplex (B2M, UBC, UCE). Two novel mRNA multiplexes and a HBD1 singleplex were used for the identification of vaginal secretion: Vag triplex (MYOZ1, CYP2B7P1 and MUC4) and a Lactobacillus-specific Lacto triplex (Ljen, Lcris, Lgas). The laboratories used different chemistries and instrumentation and all were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA allowed for positive identification of the tissue/fluid source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling, also from old and compromised casework samples. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid identification method that can easily be combined with current STR typing technology.

RNA/DNA co-analysis from human saliva and semen stains--results of a third collaborative EDNAP exercise.

A third collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Twenty saliva and semen stains, four dilution series (10-0.01 µl saliva, 5-0.01 µl semen) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 20 participating laboratories using an RNA extraction or RNA/DNA co-extraction method. Two novel mRNA multiplexes were used: a saliva triplex (HTN3, STATH and MUC7) and a semen pentaplex (PRM1, PRM2, PSA, SEMG1 and TGM4). The laboratories used different chemistries and instrumentation and a majority (16/20) were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA from individual stains not only permitted a confirmation of the presence of saliva/semen (i.e. tissue/fluid source of origin), but allowed an STR profile of the stain donor to be obtained as well. The method proved to be reproducible and sensitive, with as little as 0.05 µl saliva or semen, using different analysis strategies. Additionally, we demonstrated the ability to positively identify the presence of saliva and semen, as well as obtain high quality DNA profiles, from old and compromised casework samples. The results of this collaborative exercise involving an RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of saliva and semen in forensic casework that is compatible with current DNA analysis methodologies.

Specific and sensitive mRNA biomarkers for the identification of skin in 'touch DNA' evidence.

In forensic casework analysis it is often necessary to attempt to obtain DNA profiles from microscopic amounts of biological material left behind by perpetrators of crime. The ability to obtain profiles from trace biological evidence is routinely demonstrated with so-called 'touch DNA' evidence, which is generally perceived to be the result of DNA obtained from shed skin cells transferred from donor to an object or person during physical contact. Although a genetic profile from trace biological evidence is routinely obtained, the tissue source of the profile is rarely known. This merely perpetuates the 'mystery' of the nature of 'touch DNA' evidence allowing the significance or meaningfulness of genetic profiles obtained from these samples to be challenged. Numerous reports state that the tissue source of origin of 'touch DNA' evidence cannot be determined due to the small amount of biological material present, while others conclude that the DNA profiles are obtained from shed skin cells (as opposed to, say, buccal epithelial cells present in saliva traces) without any scientific basis for this assertion. Proper identification of the biological material present might be crucial to the investigation and prosecution of a criminal offense and a misrepresentation of the nature of the evidence can have undue influence on the perception of the circumstance of the crime. Thus far, research has failed to provide forensic scientists with feasible, definitive methods to identify the tissue origin of 'touch DNA'. In the present work, we sought to identify novel highly specific and sensitive messenger RNA (mRNA) biomarkers for the identification of skin. Gene candidates were identified using both literature searches and whole transcriptome deep sequencing (RNA-Seq). Utilizing this dual approach, we identified and evaluated over 100 gene candidates. Five mRNA markers were identified that demonstrated a high degree of specificity for skin. Using these markers, we have been able to successfully detect and identify skin using as little as 5-25 pg of input total RNA from skin and, significantly, in swabs of human skin and various touched objects. One of the markers, LCE1C, is particularly highly sensitive and was detected in the majority of skin samples tested including touched objects. We have been successful in incorporating the five skin biomarkers into two multiplex systems. Although further work is needed to optimize the assay for routine casework, the initial studies demonstrate that a molecular-based characterization of the biological material recovered from touch samples is possible.

RNA/DNA co-analysis from blood stains--results of a second collaborative EDNAP exercise.

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 µl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.

Does body mass index affect clinical outcome post-operatively and at five years after primary unilateral total hip replacement performed for osteoarthritis? A multivariate analysis of prospective data.

Studies describing the effect of body mass index (BMI) on the outcome of total hip replacement have been inconclusive and contradictory. We examined the effect of BMI on medium-term outcome in a cohort of 1617 patients who underwent a primary total hip replacement for osteoarthritis. These patients were followed prospectively for five years with the outcomes of dislocation, revision, duration of surgery and deep and superficial infection studied, as well as collecting Harris hip scores (HHS) and Short-Form 36 (SF-36) questionnaires pre-operatively and at review. A multivariate analysis was performed to see whether BMI is an independent predictor of poor outcome. We found that patients with a BMI of ? 35 kg/m(2) have a 4.42 times higher rate of dislocation than those with a BMI < 25 kg/m(2). Increasing BMI is also associated with superficial infection and poorer HHS and SF-36 scores at five years. These trends remain significant even when multivariate analysis adjusts for age, gender, prosthesis, operating consultant, pre-operative HHS and SF-36, and comorbidities including diabetes mellitus, cardiac disease and osteoporosis. Despite the increased risks, the five-year outcome scores indicate that obese patients have much to gain from total hip replacement. Thus total hip replacement should not be withheld from patients solely on the grounds of an elevated BMI. However, longer-term follow-up of this cohort is required to establish whether adverse outcomes become more evident with time.

Selection of highly specific and sensitive mRNA biomarkers for the identification of blood.

In the present work, we have evaluated eight reportedly blood-specific mRNA markers (HBB, HBA, ALAS2, CD3G, ANK1, PBGD, SPTB, AQP9) in an attempt to determine the most suitable ones for use in forensic applications based on their sensitivities, specificities and performance with casework samples. While varying levels of expression were observed, all markers were relatively sensitive requiring as little as 1ng of RNA input into the reverse transcription (RT) reaction. In singleplex reactions, seven of the eight analyzed blood markers (all except AQP9) demonstrated a high degree of specificity for blood. In multiplex reactions, non-reproducible cross-reactivity was observed for several of the mRNA markers, which was reduced and, in most cases, eliminated when less input total RNA was used. Additionally, some cross-reactivity was observed with tissue and animal samples. Despite differences in the observed sensitivity and specificity of the blood markers examined in this study, a number of the candidates appear to be suitable for inclusion in appropriately validated multiplex mRNA-based body fluid identification systems.

mRNA profiling for the identification of blood--results of a collaborative EDNAP exercise.

A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, using different kits, chemistries and instrumentation. The results demonstrate that HBB is expressed abundantly in blood, SPTB moderately and PBGD significantly less. All but one of the 16 participating laboratories were able to successfully isolate and detect RNA from the dried bloodstains even though a majority of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative to conventional serological tests.

Total hip replacement in morbidly obese patients with osteoarthritis: results of a prospectively matched study.

We compared 55 consecutive total hip replacements performed on 53 morbidly obese patients with osteoarthritis with a matched group of 55 total hip replacements in 53 non-obese patients. The groups were matched for age, gender, prosthesis type, laterality and preoperative Harris Hip Score. They were followed prospectively for five years and the outcomes were assessed using the Harris Hip Score, the Short-form 36 score and radiological findings. Survival at five years using revision surgery as an endpoint, was 90.9% (95% confidence interval 82.9 to 98.9) for the morbidly obese and 100% for the non-obese patients. The Harris Hip and the Short-form 36 scores were significantly better in the non-obese group (p < 0.001). The morbidly obese patients had a higher rate of complications (22% vs 5%, p = 0.012), which included dislocation and both superficial and deep infection. In light of these inferior results, morbidly obese patients should be advised to lose weight before undergoing a total hip replacement, and counselled regarding the complications. Despite these poorer results, however, the patients have improved function and quality of life.

Inhibition of glutamine synthetase during ammonia exposure in rainbow trout indicates a high reserve capacity to prevent brain ammonia toxicity.

Glutamine synthetase (GSase), the enzyme that catalyses the conversion of glutamate and ammonia to glutamine, is present at high levels in vertebrate brain tissue and is thought to protect the brain from elevated ammonia concentrations. We tested the hypothesis that high brain GSase activity is critical in preventing accumulation of brain ammonia and glutamate during ammonia loading in the ammonia-intolerant rainbow trout. Trout pre-injected with saline or the GSase inhibitor methionine sulfoximine (MSOX, 6 mg kg(-1)), were exposed to 0, 670 or 1000 micromol l(-1) NH(4)Cl in the water for 24 and 96 h. Brain ammonia levels were 3- to 6-fold higher in ammonia-exposed fish relative to control fish and MSOX treatment did not alter this. Brain GSase activity was unaffected by ammonia exposure, while MSOX inhibited GSase activity by approximately 75%. Brain glutamate levels were lower and glutamine levels were higher in fish exposed to ammonia relative to controls. While MSOX treatment had little impact on brain glutamate, glutamine levels were significantly reduced by 96 h. With ammonia treatment, significant changes in the concentration of multiple other brain amino acids occurred and these changes were mostly reversed or eliminated with MSOX. Overall the changes in amino acid levels suggest that multiple enzymatic pathways can supply glutamate for the production of glutamine via GSase during ammonia exposure and that alternative transaminase pathways can be recruited for ammonia detoxification. Plasma cortisol levels increased 7- to 15-fold at 24 h in response to ammonia and MSOX did not exacerbate this stress response. These findings indicate that rainbow trout possess a relatively large reserve capacity for ammonia detoxification and for preventing glutamate accumulation during hyperammonaemic conditions.

Cytochrome c oxidase is regulated by modulations in protein expression and mitochondrial membrane phospholipid composition in estivating African lungfish.

We examined some of the potential mechanisms lungfish (Protopterus dolloi) use to regulate cytochrome c oxidase (CCO), during metabolic depression. CCO activity was reduced by 67% in isolated liver mitochondria of estivating fish. This was likely accomplished, in part, by the 46% reduction in CCO subunit I protein expression in the liver. No change in the mRNA expression levels of CCO subunits I, II, III, and IV were found in the liver, suggesting CCO is under translational regulation; however, in the kidney, messenger limitation may be a factor as the expression of subunits I and II were depressed ( approximately 10-fold) during estivation, suggesting tissue-specific mechanisms of regulation. CCO is influenced by mitochondrial membrane phospholipids, particularly cardiolipin (CL). In P. dolloi, the phospholipid composition of the liver mitochondrial membrane changed during estivation, with a approximately 2.3-fold reduction in the amount of CL. Significant positive correlations were found between CCO activity and the amount of CL and phosphatidylethanolamine within the mitochondrial membrane. It appears CCO activity is regulated through multiple mechanisms in P. dolloi, and individual subunits of CCO are regulated independently, and in a tissue-specific manner. It is proposed that altering the amount of CL within the mitochondrial membrane may be a means of regulating CCO activity during metabolical depression in the African lungfish, P. dolloi.

RNA Profiling for the Identification of the Tissue Origin of Dried Stains in Forensic Biology.

Examination of crime scene items for biological evidence typically begins with a preliminary screening for the presence of biological fluids in order to identify possible sources of DNA. Conventional biochemical and immunological assays employed for this screening require multiple tests to be performed in a serial manner, can consume a significant amount of valuable evidentiary material, and can require a significant amount of time and labor for completion. Moreover, the presence of several biological fluids, such as saliva, vaginal secretions, and menstrual blood, cannot be conclusively identified using current methods. Due to the disadvantages of conventional body fluid testing, some operational crime laboratories have chosen to bypass the body fluid identification process and proceed directly to DNA analysis. However, while reducing the time spent on each case, this "shortcut" could result in a failure to provide important probative information regarding the nature of the crime as well as result in increased cost to crime laboratories if unnecessary DNA testing is performed. In the past several years, a number of forensic researchers have attempted to develop molecular-based approaches to body fluid identification that would provide operational crime laboratories with significantly improved specificity. This has resulted in an increased interest in the use of RNA profiling strategies for the identification of forensically relevant biological fluids. This review provides an overview of studies carried out on the use of both messenger RNA and small (micro) RNA profiling. The results of these studies are encouraging and presage the routine identification the tissue source(s) of forensic evidence using molecular-based approaches.