Description and validation of a new descriptive and multiparametric numeric rating scale to assess sedation in cats.

The objective of this study was to design and assess the validity and reliability of a new feline multiparametric sedation scale (FMSS). A total of 89 household cats were recruited, enabling a total of 534 sedation assessments. Every assessment was performed by 3 blinded observers with varying expertise levels (Level 1: Student; Level 2: RVT; Level 3: ACVAA diplomate or senior resident). For comparison purposes, a visual analogue scale (VAS) and a Simple Qualitative Scale (SQS) were also used concurrently, with the VAS considered the gold standard. The new scale had excellent inter-observer agreement among experience groups with weighted Kappa scores of 0.84 (Levels 1 versus 2), 0.82 (Levels 2 versus 3), and 0.84 (Levels 1 versus 3), with P < 0.0001 for all comparisons. There was a high degree of association between FMSS and VAS (r = 0.90, P < 0.0001) and between FMSS and SQS (r = 0.89, P < 0.0001). Final FMSS numerical values were paired with levels of sedation with None = 0 (0 to 5), Mild = 4 (1 to 7), Moderate = 6 (2 to 10), and Profound = 12 (7 to 12); furthermore, differences were detected between pre- and post-sedation evaluations (P = 0.001). This scale demonstrated internal consistency and sensitivity even when evaluating drugs or doses with minimal sedative effects and there was very strong interrater reliability, independent of experience level. Based on this clinical study, we concluded that the use of this sedation scale is appropriate when objective numerical sedation quantification is required, in either a clinical or research setting.

Description et validation d'une nouvelle échelle d'évaluation numérique descriptive et multiparamétrique pour évaluer la sédation chez le chat. L'objectif de cette étude était de concevoir et d'évaluer la validité et la fiabilité d'une nouvelle échelle de sédation multiparamétrique féline (FMSS). Un total de 89 chats domestiques a été recruté, permettant un total de 534 évaluations de sédation. Chaque évaluation a été effectuée par trois observateurs en aveugle avec différents niveaux d'expertise (Niveau 1 : étudiant; Niveau 2 : RVT; Niveau 3 : diplomate de l'ACVAA ou résident senior). À des fins de comparaison, une échelle visuelle analogique (VAS) et une échelle qualitative simple (SQS) ont également été utilisées simultanément, VAS étant considérée comme l'étalon. La nouvelle échelle présentait un excellent accord inter-observateurs parmi les groupes d'expérience avec des scores Kappa pondérés de 0,84 (niveaux 1 versus 2), 0,82 (niveaux 2 versus 3) et 0,84 (niveaux 1 versus 3), avec P < 0,0001 pour toutes les comparaisons. Il y avait un degré élevé d'association entre FMSS et VAS (r = 0,90, P < 0,0001) et entre FMSS et SQS (r = 0,89, P < 0,0001). Les valeurs numériques FMSS finales ont été appariées avec les niveaux de sédation avec Aucun = 0 (0 à 5), Léger = 4 (1 à 7), Modéré = 6 (2 à 10) et Profond = 12 (7 à 12); en outre, des différences ont été détectées entre les évaluations pré- et post-sédation (P = 0,001). Cette échelle a démontré une cohérence interne et une sensibilité même lors de l'évaluation de médicaments ou de doses avec des effets sédatifs minimes et il y avait une très forte fiabilité inter-évaluateur, indépendamment du niveau d'expérience. Sur la base de cette étude clinique, nous avons conclu que l'utilisation de cette échelle de sédation est appropriée lorsqu'une quantification numérique objective de la sédation est requise, dans un cadre clinique ou de recherche.(Traduit par Dr Serge Messier).

Biosafety Level 2 cabinet UV-C light exposure of sports antidoping human urine samples does not affect the stability of selected prohibited substances.

The current study examined the stability of several antidoping prohibited substances analytes in urine after 15-min exposure to UV-C light in a Biosafety Level 2 cabinet. The urine matrices were exposed within the original antidoping bottles with the aim to destroy DNA/RNA and possible SARS CoV-2. The analytes small molecules Phase I and Phase II metabolites and peptides, in total 444, endogenous, internal standards, and prohibited substances, pH, and specific gravity in urine were studied. The accredited analytical methods were used by Anti-Doping Laboratory Qatar for the comparison of data of the same urine samples analyzed with and without UV-C exposure. In the study conditions, no problems of stability were detected in the substances spiked in the urine samples exposed in the UV-C irradiation.

Dose and cardiopulmonary effects of propofol alone or with midazolam for induction of anesthesia in critically ill dogs.

OBJECTIVE: To determine the dose and cardiopulmonary effects of propofol alone or with midazolam for induction of anesthesia in American Society of Anesthesiologists status ≥III dogs requiring emergency abdominal surgery. STUDY DESIGN: Prospective, randomized, blinded, clinical trial. ANIMALS: A total of 19 client-owned dogs. METHODS: Dogs were sedated with fentanyl (2 µg kg-1) intravenously (IV) for instrumentation for measurement of heart rate, arterial blood pressure, cardiac index, systemic vascular resistance index, arterial blood gases, respiratory rate and rectal temperature. After additional IV fentanyl (3 µg kg-1), the quality of sedation was scored and cardiopulmonary variables recorded. Induction of anesthesia was with IV propofol (1 mg kg-1) and saline (0.06 mL kg-1; group PS; nine dogs) or midazolam (0.3 mg kg-1; group PM; 10 dogs), with additional propofol (0.25 mg kg-1) IV every 6 seconds until endotracheal intubation. Induction/intubation quality was scored, and anesthesia was maintained with isoflurane. Variables were recorded for 5 minutes with the dog in lateral recumbency, breathing spontaneously, and then in dorsal recumbency with mechanical ventilation for the next 15 minutes. A general linear mixed model was used with post hoc analysis for multiple comparisons between groups (p < 0.05). RESULTS: There were no differences in group demographics, temperature and cardiopulmonary variables between groups or within groups before or after induction. The propofol doses for induction of anesthesia were significantly different between groups, 1.9 ± 0.5 and 1.1 ± 0.5 mg kg-1 for groups PS and PM, respectively, and the induction/intubation score was significantly better for group PM. CONCLUSIONS AND CLINICAL RELEVANCE: Midazolam co-induction reduced the propofol induction dose and improved the quality of induction in critically ill dogs without an improvement in cardiopulmonary variables, when compared with a higher dose of propofol alone.

Trends of maxillofacial trauma: An update from the prospective register of a multicenter study in emergency services of Chile

Background: Determine the behavior of the maxillofacial trauma of adults treated in 3 tertiary care centers in the central zone of Chile. Material and Methods: descriptive, cross-sectional, multicenter study, based on the prospective records of maxillofacial trauma cases attended between May 2016 and April 2017 by dental and maxillofacial clinical teams of Adult Emergency Units of hospitals Dr. Sótero del Río (metropolitan region), Carlos Van Buren and Dr. Gustavo Fricke (region V). Age, sex, date of occurrence, type of trauma according to ICD-10, etiology, legal medical prognosis and associated injuries were recorded, stratifying by sex and age. Chi square and unpaired Wilcoxon tests were used to compare by groups. Results: 2.485 cases and 3.285 injuries were investigated. The male: female ratio was 1.7: 1 with age under 30 predominant, followed by older adults. Variability was observed in the yearly, weekly and daily presentation The highest frequencies were in January and September, weekends and at night. The main etiologies were violence (42.3%), falls (13.1%) and road traffic crashes (12.9%) with differences by age and sex (p <0.05). 31,9% of the injuries occurred in hard tissue, being fractures in nasal bones predominant (S02.2). Conclusions: the profile of the maxillofacial trauma in Chile seems to be mixed by age, affecting young people and the elderly. The male sex predominates; the main cause, which varies by age group, is violence. Their surveillance is possible from hospital emergency records

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Population reference ranges of urinary endogenous sulfate steroids concentrations and ratios as complement to the steroid profile in sports antidoping.

The population based Steroid Profile (SP) ratio of testosterone (T) and epitestosterone (E) has been considered as a biomarker approach to detect testosterone abuse in '80s. The contemporary Antidoping Laboratories apply the World Antidoping Agency (WADA) Technical Document (TD) for Endogenous Androgenic Anabolic Steroids (EAAS) in the analysis of SP during their screening. The SP Athlete Biological Passport (ABP) adaptive model uses the concentrations of the total of free and glucuronide conjugated forms of six EAASs concentrations and ratios measured by GC/MS. In the Antidoping Lab Qatar (ADLQ), the routine LC/MS screening method was used to quantitatively estimate the sulfate conjugated EAAS in the same analytical run as for the rest qualitative analytes. Seven sulfate EAAS were quantified for a number of routine antidoping male and female urine samples during screening. Concentrations, statistical parameters and selected ratios for the 6 EAAS, the 6 sulfate EAAS and 29 proposed ratios of concentrations from both EAAS and sulfate EAAS, which potentially used as SP ABP biomarkers, population reference limits and distributions have been estimated after the GC/MSMS analysis for EAAS and LC/Orbitrap/MS analysis for sulfate EAAS.

Comparison of 3 blind brachial plexus block techniques during maintenance of anesthesia and postoperative pain scores in dogs undergoing surgical procedures of the thoracic limb.

The degree of analgesia provided by blind techniques for brachial plexus blocks (BPBs) has not been compared in clinical cases undergoing surgery of the thoracic limb. The objective of this study was to evaluate the anesthetic conditions and postoperative analgesia resulting from 3 different BPB local anesthetic techniques in canine patients undergoing such surgery. Twenty-four client-owned dogs received a standardized premedication/induction protocol (hydromorphone and acepromazine/propofol), maintained with isoflurane in oxygen using mechanical ventilation, in a prospective, randomized, blinded clinical trial. Before surgery, dogs received 1 of 3 anatomical BPB techniques: traditional, perpendicular, or axillary, with 0.2 mL/kg body weight (BW) of bupivacaine 0.5%. Cardiorespiratory variables and isoflurane end-tidal concentrations were recorded throughout anesthesia. Scores for anesthetic maintenance (0-best to 4-responsive), recovery quality (0-not responsive to 3-responsive), and Glasgow pain scale were recorded for up to 24 h postoperatively. All dogs recovered uneventfully from anesthesia and no differences in the measured variables or scores were noted among groups, during and after anesthesia. When thoracic limb amputations in each of the 3 groups (n = 9; 4 in traditional, 3 in perpendicular, 2 in axillary) were compared to the other surgical procedures (n = 15); however, scores for anesthetic maintenance were higher [0 (0 to 1) versus 0 (0 to 0); median (interquartile range)], recovery [1 (0 to 2) versus 0 (0 to 0)], and pain [2.4 (2.4 to 3.0) versus 1.6 (1.4 to 2.2)] in the first 3 h post-extubation. Surgery times were also longer with amputations [115 min (100 to 138 min) versus 50 min (41 to 90 min)]. The 3 BPB techniques provided similar anesthesia and postoperative pain scores. Despite higher pain scores in thoracic limb amputations than in less invasive surgeries, the BPB appeared to provide significant comfort.

Le degré d'analgésie fournie par les blocs à l'aveugle du plexus brachial (BPBs) n'a pas été comparé lors de cas cliniques soumis à une chirurgie du membre thoracique. La présente étude visait à évaluer les conditions anesthésiques et l'analgésie post-opératoire résultant de trois techniques différentes d'anesthésie locale par BPB chez des patients canins soumis à une chirurgie. Vingt-quatre chiens appartenant à des propriétaires ont reçu une prémédication/induction standardisée (hydromorphone et acépromazine/propofol), avec maintien à l'isoflurane dans de l'oxygène en utilisant une ventilation mécanique, dans une étude clinique prospective, randomisée, et à l'aveugle. Avant la chirurgie, les chiens ont reçu un BPB par une des trois techniques anatomiques : traditionnelle, perpendiculaire, ou axillaire, avec 0,2 mL/kg de poids corporel de bupivacaïne 0,5 %. Les variables cardiorespiratoires et les concentrations d'isoflurane en fin d'expiration furent enregistrées tout au long de l'anesthésie. Les pointages pour le maintien de l'anesthésie (0-meilleur à 4-réactif), la qualité du rétablissement (0-non réactif à 3-réactif), et l'échelle de la douleur de Glasgow ont été notés jusqu'à 24 h post-opération. Tous les chiens ont récupéré sans problème de l'anesthésie et aucune différence mesurable dans les variables mesurées ou les pointages ne fut notée parmi les groupes, durant ou après l'anesthésie. Toutefois, lorsque les amputations du membre thoracique dans chacun des trois groupes (n = 9; 4 dans la traditionnelle, 3 dans la perpendiculaire, 2 dans l'axillaire) furent comparées aux autres procédures chirurgicales (n = 15), les pointages pour le maintien de l'anesthésie étaient plus élevés [0 (0 à 1) versus 0 (0 à 0); médiane (écart interquartile)], rétablissement [1 (0 à 2) versus 0 (0 à 0)], et douleur [2,4 (2,4 à 3,0) versus 1,6 (1,4 à 2,2)] dans les 3 h suivant l'extubation. La durée des chirurgies étaient également plus longues lors des amputations [115 min (100 à 138 min) versus 50 min (41 à 90 min)]. Les trois techniques de BPB produisaient des pointages similaires d'anesthésie et de douleur postopératoire. Malgré des pointages de douleur plus élevés lors d'amputations du membre thoracique que lors de chirurgies moins invasives, les BPB semblent fournir un confort significatif.(Traduit par Docteur Serge Messier).

High resolution full scan liquid chromatography mass spectrometry comprehensive screening in sports antidoping urine analysis.

The aim of this paper is to present the development and validation of a high-resolution full scan (HR-FS) electrospray ionization (ESI) liquid chromatography coupled to quadrupole Orbitrap mass spectrometer (LC/Q/Orbitrap MS) platform for the screening of prohibited substances in human urine according to World Antidoping Agency (WADA) requirements. The method was also validated for quantitative analysis of six endogenous steroids (epitestosterone, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone, androsterone and etiocholanolone) in their intact sulfates form. The sample preparation comprised a combination of a hydrolyzed urine liquid-liquid extraction and the dilute & shoot addition of original urine in the extracted aliquot. The HR-FS MS acquisition mode with Polarity Switching was applied in combination of the Quadrupole-Orbitrap mass filter. The HR-FS acquisition of analytical signal, for known and unknown small molecules, allows the inclusion of all analytes detectable with LC-MS for antidoping investigations to identify the use of known or novel prohibited substances and metabolites after electronic data files' reprocessing. The method has been validated to be fit-for-purpose for the antidoping analysis.

Evaluation of longitudinal steroid profiles from male football players in UEFA competitions between 2008 and 2013.

Testosterone and related compounds are the most recurrent doping substances. The steroid profile, consisting of the quantification of testosterone and its metabolites, has been described as the most significant biomarker to detect doping with pseudo-endogenous anabolic steroids. The steroidal module of the Athlete Biological Passport (ABP) was launched by the World Anti-Doping Agency (WADA) in 2014. To assess the value of introducing the module to its anti-doping programme, the Union of European Football Associations (UEFA) decided to analyze retrospectively the steroid profile data of 4195 urine samples, collected from 879 male football players and analyzed in 12 WADA-accredited laboratories between 2008 and mid-2013. This study focused on the evaluation of T/E ratios. The coefficient of variation (CV) and the adaptive model were the two statistical models used to study the longitudinal follow-up. A CV of 46% was determined to be the maximal natural intra-individual variation of the T/E when the sequence consisted of single data points analyzed in different laboratories. The adaptive model showed some profiles with an atypical T/E sequence and also enabled an estimate of the prevalence of external factors impacting the T/E sequences. Despite the limitations of this retrospective study, it clearly showed that the longitudinal and individual follow-up of the T/E biomarker of the players is a good tool for target testing in football. UEFA has therefore decided to implement the steroidal module of the ABP from the start of the next European football season in September 2015. Copyright © 2015 John Wiley & Sons, Ltd.

Involvement of spinal cord BDNF in the generation and maintenance of chronic neuropathic pain in rats.

Brain-derived neurotrophic factor (BDNF) is involved in neuronal survival and synaptic plasticity of the central and peripheral nervous system. In chronic pain, plastic changes in dorsal horn neurons contribute to a phenomenon of hypersensitivity to pain sensation that is maintained over time, known as central sensitization. This process is accompanied by BDNF overexpression, but the role of BDNF in the generation and maintenance of the hyperalgesic phenomenon is still unclear. The present study was aimed to investigate if exogenous BDNF administered to the rat spinal cord, in addition to trigger pain, participates in the maintenance of the central sensitization process (i.e., pain persistence) and to determine if the pain generated is comparable to that observed in a neuropathic pain model. Results showed that a single intrathecal injection of 0.003 ng of BDNF was able to decrease the nociceptive threshold (Randall-Selitto test) in normal rats, for at least a 42-day period. Furthermore, the hyperalgesia generated was comparable to that observed in rats with a 42-day history of mononeuropathy. Increasing the dose or administering additional doses of BDNF resulted neither in additional effectiveness in reducing the pain threshold nor in the prolongation of the hyperalgesic effect, thus showing that central sensitization induced by BDNF is a dose-independent, all-or-none process. It is concluded that BDNF alone is sufficient for generating a long-lasting neural excitability change in the spinal cord via tyrosine kinase B receptor signaling, similar to that observed in chronic pain models such as neuropathy.

The influence of diet on isotope ratio mass spectrometry values.

OBJECTIVES: Athletes have increasingly used testosterone (T) and other endogenous anabolic steroids that cannot be detected by conventional gas chromatography-mass spectrometry. This led to gas chromatography-combustion-isotope ratio mass spectrometry(GC/C/IRMS), which measures the relative amount of 13C in urinary steroids. Because exogenous testosterone is relatively low in 13C content, this study will determine if consuming a diet low in 13C plants, such as soy, can be confused with a GC/C/IRMS-positive test for exogenous testosterone. DESIGN: Cross-sectional study in which 22 vegetarians known to consume a diet depleted of 13C isotope were compared with a geographic control group of 14 subjects consuming a normal diet. SETTING: Two distinct subject populations with respect to diet. SUBJECTS: Subjects were recruited from a soy-based cooperative and control volunteers. Twenty-two of 24 research subjects completed the protocol compared with 14 of 22 control subjects. INTERVENTIONS: Independent variables were delta13C IRMS values,urinary steroid profile, and isoflavone analysis. MAIN OUTCOME MEASURES: Comparisons were made with respect to dietary analysis, isoflavones, and urinary steroid measurements using GC-C-IRMS. RESULTS: The delta13C values for 2 major metabolites of T (androsterone and etiocholanolone) were lower for the vegetarians than the controls (P = 0.005). The vegetarians excreted a median of 23 micromol/d of total isoflavones compared with 2.7 micromol/d for the control group (P =0.0002). CONCLUSIONS: The carbon isotope ratios of urinary testosterone metabolites of vegetarians consuming a diet that is markedly depleted of 13C content were lower than that of control subjects, but not low enough to result in World Anti-Doping Agency criteria for a positive IRMS analysis.

Drug testing data from the 2007 Pan American Games: delta13C values of urinary androsterone, etiocholanolone and androstanediols determined by GC/C/IRMS.

The main purpose of this article is to show the application of the CG/C/IRMS in real time during competition in the steroid confirmation analysis. For this reason, this paper summarizes the results obtained from the doping control analysis during the period of the 2007 Pan American Games held in Rio de Janeiro, Brazil. Approximately 5600 athletes from 42 different countries competed in the games. Testing was performed in accordance to World Anti-Doping Agency (WADA) technical note for prohibited substances. This paper reports data where abnormal urinary steroid profiles, have been found with the screening procedures. One 8 mL urine sample was used for the analysis of five steroid metabolites with two separate analyses by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Urine samples were submitted to GC/C/IRMS for confirmation analysis to determine the (13)C/(12)C ratio of selected steroids. Fifty-seven urine samples were analyzed by GC/C/IRMS and the delta(13)C values ( per thousand) of androsterone, etiocholanolone, 5beta-androstane-3alpha, 17beta-diol (5beta-diol), 5alpha-androstane-3alpha, 17beta-diol (5alpha-diol) and 5beta-pregnane-3alpha, 20alpha-diol (5beta-pdiol), the endogenous reference compound are presented. One urine sample with a testosterone/epitestosterone (T/E) ratio of 4.7 was confirmed to be positive of doping by GC/C/IRMS analysis. The delta values of 5beta-diol and 5alpha-diol were 3.8 and 10.8, respectively, compared to the endogenous reference compound 5beta-pdiol, which exceeded the WADA limit of 3 per thousand. The results obtained by CG/C/IRMS confirmation analyses, in suspicious samples, were conclusive in deciding whether or not a doping steroid violation had occurred.

A proteomic approach links decreased pyruvate kinase M2 expression to oxaliplatin resistance in patients with colorectal cancer and in human cell lines.

We aimed to gain further understanding of the molecular mechanisms involved in oxaliplatin resistance in colorectal cancer by using a proteomic approach. A 5-fold oxaliplatin-resistant cell line, HTOXAR3, was compared with its parental cell line, HT29, using two-dimensional PAGE. Mass spectrometry, Western blot, and real-time quantitative PCR confirmed the down-regulation of pyruvate kinase M2 (PK-M2) in HTOXAR3 cells. In a panel of eight colorectal cancer cell lines, we found a negative correlation between oxaliplatin resistance and PK-M2 mRNA levels (Spearman r=-0.846, P=0.008). Oxaliplatin exposure in both HT29 and HTOXAR3 led to PK-M2 mRNA up-regulation. PK-M2 mRNA levels were measured by real-time quantitative PCR in 41 tumors treated with oxaliplatin/5-fluorouracil. Tumors with the lowest PK-M2 levels attained the lowest response rates (20% versus 64.5%, P=0.026). High PK-M2 levels were associated with high p53 levels (P=0.032). In conclusion, the data provided clearly link PK-M2 expression and oxaliplatin resistance mechanisms and further implicate PK-M2 as a predictive marker of response in patients with oxaliplatin-treated colorectal cancer.

Association of MBP peptides with Hsp70 in normal appearing human white matter.

Multiple Sclerosis is an autoimmune disease directed against myelin proteins. The etiology of MS is poorly defined though, with no definitive causative agent yet identified. It has been hypothesized that MS may be a multifactorial disease resulting in the same end product: the destruction of myelin by the immune system. In this report we describe a potential role for heat shock proteins in the pathogenesis of MS. We isolated Hsp70 from the normal appearing white matter of both MS and normal human brain and found this was actively associated with, among other things, immunodominant MBP peptides. Hsp70-MBP peptide complexes prepared in vitro were shown to be highly immunogenic, with adjuvant-like effects stimulating MBP peptide-specific T cell lines to respond to normally sub-optimal concentrations of peptide. This demonstration of a specific interaction between Hsp70 and different MBP peptides, coupled with the adjuvanticity of this association is suggestive of a possible role for Hsp70 in the immunopathology associated with MS.

Identification of biofilm proteins in non-typeable Haemophilus Influenzae.

BACKGROUND: Non-typeable Haemophilus influenzae biofilm formation is implicated in a number of chronic infections including otitis media, sinusitis and bronchitis. Biofilm structure includes cells and secreted extracellular matrix that is "slimy" and believed to contribute to the antibiotic resistant properties of biofilm bacteria. Components of biofilm extracellular matrix are largely unknown. In order to identify such biofilm proteins an ex-vivo biofilm of a non-typeable Haemophilus influenzae isolate, originally from an otitis media patent, was produced by on-filter growth. Extracellular matrix fraction was subjected to proteomic analysis via LC-MS/MS to identify proteins. RESULTS: 265 proteins were identified in the extracellular matrix sample. The identified proteins were analyzed for COG grouping and predicted cellular location via the TMHMM and SignalP predictive algorithms. The most over-represented COG groups identified compared to their frequency in the Haemophilus influenzae genome were cell motility and secretion (group N) followed by ribosomal proteins of group J. A number of hypothetical or un-characterized proteins were observed, as well as proteins previously implicated in biofilm function. CONCLUSION: This study represents an initial approach to identifying and cataloguing numerous proteins associated with biofilm structure. The approach can be applied to biofilms of other bacteria to look for commonalities of expression and obtained information on biofilm protein expression can be used in multidisciplinary approaches to further understand biofilm structure and function.

Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione.

Aconitases are iron-sulfur cluster-containing proteins present both in mitochondria and cytosol of cells; the cubane iron-sulfur (Fe-S) cluster in the active site is essential for catalytic activity, but it also renders aconitase highly vulnerable to reactive oxygen and nitrogen species. This study examined the sites and mechanisms of aconitase inactivation by peroxynitrite (ONOO-), a strong oxidant and nitrating agent readily formed from superoxide anion and nitric oxide generated by mitochondria. ONOO- inactivated aconitase in a dose-dependent manner (half-maximal inhibition was observed with approximately 3 microM ONOO-). Low levels of ONOO- caused the conversion of the Fe-S cluster from the [4Fe-4S]2+ form to the inactive [3Fe-4S]1+ form with the loss of labile iron, as confirmed by low-temperature EPR analysis. In the presence of the substrate, citrate, 66-fold higher concentrations of ONOO- were required for half-maximal inhibition. The protective effects of citrate corresponded to its binding to the active site. The inactivation of aconitase in the presence of citrate was due to ONOO--mediated cysteine thiol loss and tyrosine nitration in the enzyme as shown by Western blot analyses. LC/MS/MS analyses revealed that ONOO- treatment to aconitase resulted in nitration of tyrosines 151 and 472 and oxidation to sulfonic acid of cysteines 126 and 385. The latter is one of the three cysteine residues in aconitase that binds to the Fe-S cluster. All other modified tyrosine and cysteine residues were adjacent to the binding site, thus suggesting that these modifications caused conformational changes leading to active-site disruption. Aconitase cysteine thiol modifications other than oxidation to sulfonic acid, such as S-glutathionylation, also decreased aconitase activity, thus indicating that glutathionylation may be an important means of modulating aconitase activity under oxidative and nitrative stress. Taken together, these results demonstrate that the Fe-S cluster in the active site, cysteine 385 bound to the Fe-S cluster, and tyrosine and cysteine residues in the vicinity of the active site are important targets of oxidative and/or nitrative attack, which is selectively controlled by the mitochondrial matrix citrate levels. The mechanisms inherent in aconitase inactivation by ONOO- are discussed in terms of the mitochondrial matrix metabolic and thiol redox state.

Purification and proteomic analysis of outer membrane vesicles from a clinical isolate of Leptospira interrogans serovar Copenhageni.

The severe pulmonary form of leptospirosis (SPFL) is an especially serious and rapid disease process characterized by alveolar hemorrhage and acute respiratory failure. The outer membrane of Leptospira facilitates direct interactions with the environs and likely contains important constituents involved during infection, transmission, survival, and adaptation to environmental conditions, including putative vaccinogen and diagnostic candidates. Outer membrane vesicles (OMVs) were purified by incubation in low-pH citrate buffer, treatment in a French press, and centrifugation over a continuous sucrose gradient. OMVs characterized by two-dimensional gel electrophoresis (2-DE) contained the previously described outer membrane proteins OmpL1, Qlp42, LipL32, LipL41, LipL36 and Loa22. In addition, unknown, hypothetical and putative outer membrane proteins were identified. High-performance liquid chromatography (HPLC) coupled with mass spectrometry and fraction collection (LC-MS+) measured the intact mass profile of the major outer membrane protein, LipL32, and the putative lipoprotein Qlp42. In contrast to a predicted molecularmass of 27,653.5 Da for LipL32 after cleavage of its signal peptide, intact mass proteomics measured the mass as ranging from 28,468 to 28,583 Da, consistent with lipidation of LipL32. In contrast to a predicted molecular mass of 39.8 kDa for Qlp42, the actual mass was measured as 24,811 and 26,461 Da consistent with a 30 kDa doublet observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and processing of the N-terminus of the mature protein. These studies indicate that purified OMVs are highly compatible with proteomics technologies including 2-DE and intact mass proteomics using LC-MS+ that facilitates definition of actual molecular masses of intact outer membrane proteins, and heterogeneity associated with them.

Temporal analysis of the antigenic composition of Borrelia burgdorferi during infection in rabbit skin.

The numbers of host-adapted Borrelia burgdorferi (HAB) organisms in rabbit skin were assessed by real-time PCR over the first 3 weeks of infection. Maximal numbers were found at day 11, while spirochete numbers decreased by more than 30-fold by day 21. The antigenic composition of HAB in skin biopsy samples was determined by use of a procedure termed hydrophobic antigen tissue Triton extraction. Immune sera from rabbits, sera from chronically infected mice, and monospecific antiserum to the antigenic variation protein, VlsE, were used to probe parallel two-dimensional immunoblots representing each time point. Individual proteins were identified using either specific antisera or by matching protein spots to mass spectrometry-identified protein spots from in vitro-cultivated Borrelia. There were significant changes in the relative expression of a variety of known and previously unrecognized HAB antigens during the 21-day period. OspC and the outer membrane proteins OspA and OspB were prominent at the earliest time point, day 5, when the antigenic variation protein VlsE was barely detected. OspA and OspB were not detected after day 5. OspC was not detected after day 9. VlsE was the most prominent antigen from day 7 through day 21. BmpA, ErpN, ErpP, LA7, OppA-2, DbpA, and an unidentified 15-kDa protein were also detected from day 7 through day 21. Immunoblot analysis using monospecific anti-VlsE revealed the presence of prominent distinct VlsE lower forms in HAB at days 9, 11, and 14; however, these lower forms were no longer detected at day 21. This marked diminution in VlsE lower forms paralleled the clearance of the spirochete from skin.

Subtle modification of isotope ratio proteomics; an integrated strategy for expression proteomics.

Use of minor modification of isotope ratio to code samples for expression proteomics is being investigated. Alteration of (13)C abundance to approximately 2% yields a measurable effect on peptide isotopic distribution and inferred isotope ratio. Elevation of (13)C abundance to 4% leads to extension of isotopic distribution and background peaks across every unit of the mass range. Assessment of isotope ratio measurement variability suggests substantial contributions from natural measurement variability. A better understanding of this variable will allow assessment of the contribution of sequence dependence. Both variables must be understood before meaningful mixing experiments for relative expression proteomics are performed. Subtle modification of isotope ratio ( approximately 1-2% increase in (13)C) had no effect upon either the ability of data-dependent acquisition software or database searching software to trigger tandem mass spectrometry or match MSMS data to peptide sequences. More severe modification of isotope ratio caused a significant drop in performance of both functionalities. Development of software for deconvolution of isotope ratio concomitant with protein identification using LC-MSMS, or any other proteomics strategy, is underway (Isosolv). The identified peptide sequence is then be used to provide elemental composition for accurate isotope ratio decoding and the potential to control for specific amino acid biases should these prove significant. It is suggested that subtle modification of isotope ratio proteomics (SMIRP) offers a convenient approach to in vivo isotope coding of plants and might ultimately be extended to mammals including humans.

Transit peptide cleavage sites of integral thylakoid membrane proteins.

A set of 58 nuclearly encoded thylakoid-integral membrane proteins from four plant species was identified, and their amino termini were assigned unequivocally based upon mass spectrometry of intact proteins and peptide fragments. The dataset was used to challenge the Web tools ChloroP, TargetP, SignalP, PSORT, Predotar, and MitoProt II for predicting organelle targeting and transit peptide proteolysis sites. ChloroP and TargetP reliably predicted chloroplast targeting but only reliably predicted transit peptide cleavage sites for soluble proteins targeted to the stroma. SignalP (eukaryote settings) accurately predicted the transit peptide cleavage site for soluble proteins targeted to the lumen. SignalP (Gram-negative bacteria settings) reliably predicted peptide cleavage of integral thylakoid proteins inserted into the membrane via the "spontaneous" pathway. The processing sites of more common thylakoid-integral proteins inserted by the signal recognition peptide-dependent pathway were not well predicted by any of the programs. The results suggest the presence of a second thylakoid processing protease that recognizes the transit peptide of integral proteins inserted via the spontaneous mechanism and that this mechanism may be related to the secretory mechanism of Gram-negative bacteria.

Full subunit coverage liquid chromatography electrospray ionization mass spectrometry (LCMS+) of an oligomeric membrane protein: cytochrome b(6)f complex from spinach and the cyanobacterium Mastigocladus laminosus.

Highly active cytochrome b(6)f complexes from spinach and the cyanobacterium Mastigocladus laminosus have been analyzed by liquid chromatography with electrospray ionization mass spectrometry (LCMS+). Both size-exclusion and reverse-phase separations were used to separate protein subunits allowing measurement of their molecular masses to an accuracy exceeding 0.01% (+/-3 Da at 30,000 Da). The products of petA, petB, petC, petD, petG, petL, petM, and petN were detected in complexes from both spinach and M. laminosus, while the spinach complex also contained ferredoxin-NADP(+) oxidoreductase (Zhang, H., Whitelegge, J. P., and Cramer, W. A. (2001) Flavonucleotide:ferredoxin reductase is a subunit of the plant cytochrome b(6)f complex. J. Biol. Chem. 276, 38159-38165). While the measured masses of PetC and PetD (18935.8 and 17311.8 Da, respectively) from spinach are consistent with the published primary structure, the measured masses of cytochrome f (31934.7 Da, PetA) and cytochrome b (24886.9 Da, PetB) modestly deviate from values calculated based upon genomic sequence and known post-translational modifications. The low molecular weight protein subunits have been sequenced using tandem mass spectrometry (MSMS) without prior cleavage. Sequences derived from the MSMS spectra of these intact membrane proteins in the range of 3.2-4.2 kDa were compared with translations of genomic DNA sequence where available. Products of the spinach chloroplast genome, PetG, PetL, and PetN, all retained their initiating formylmethionine, while the nuclear encoded PetM was cleaved after import from the cytoplasm. While the sequences of PetG and PetN revealed no discrepancy with translations of the spinach chloroplast genome, Phe was detected at position 2 of PetL. The spinach chloroplast genome reports a codon for Ser at position 2 implying the presence of a DNA sequencing error or a previously undiscovered RNA editing event. Clearly, complete annotation of genomic data requires detailed expression measurements of primary structure by mass spectrometry. Full subunit coverage of an oligomeric intrinsic membrane protein complex by LCMS+ presents a new facet to intact mass proteomics.