Sex dependent alterations in mitochondrial electron transport chain proteins following neonatal rat cerebral hypoxic-ischemia.

Males are more susceptible to brain mitochondrial bioenergetic dysfunction following neonatal cerebral hypoxic-ischemia (HI) than females. Mitochondrial biogenesis has been implicated in the cellular response to HI injury, but sex differences in biogenesis following HI have not been described. We tested the hypothesis that mitochondrial biogenesis or the expression of mitochondrial electron transport chain (ETC) proteins are differentially stimulated in the brains of 8 day old male and female rats one day following HI, and promoted by treatment with acetyl-L-carnitine (ALCAR). There were no sex differences in mitochondrial mass, as reflected by the ratio of mitochondrial to nuclear DNA (mtDNA/nDNA) and citrate synthase enzyme activity present one day following HI or sham surgery. There was an increase in mtDNA/nDNA, however, in the hypoxic and ischemic (ipsilateral) hemisphere after HI in both male and female brains at one day post-injury, which was suppressed by ALCAR. Citrate synthase activity was increased in the ipsilateral hemisphere of ALCAR treated male and female brain. Most importantly, the levels of representative mitochondrial proteins present in ETC complexes I, II and IV increased substantially one day following HI in female, but not male brain. This sex difference is consistent with the increase in the mitochondrial biogenesis-associated transcription factor NRF-2/GABPα following HI in females, in contrast to the decrease observed with males. In conclusion, the female sex-selective increase in ETC proteins following HI may at least partially explain the relative female resilience to mitochondrial respiratory impairment and neuronal death that occur after HI.

Organizational role for pubertal androgens on adult hypothalamic-pituitary-adrenal sensitivity to testosterone in the male rat.

The inhibitory effect of androgens on the hypothalamic-pituitary-adrenal (HPA) axis in basal and stress conditions in adult male rats is well documented. Major sex-related neuroendocrine changes take place during puberty. There is a robust rise in production and secretion of gonadal steroids, which is thought to underlie numerous neural and behavioural changes brought on after puberty. The present study investigated the effect of the pubertal rise in gonadal steroid levels on the subsequent adult corticosterone profile, particularly the sensitivity of the adult HPA axis to testosterone. Animals were castrated either prepubertally (28 days) or in adulthood (11 weeks) and adult animals were subsequently treated with subcutaneous implants containing either testosterone or cholesterol. Using an automated blood sampling system, blood was collected from each freely moving, conscious rat every 10 min (i) over a 24 h period; (ii) in response to 10 min of noise stress, and (iii) following an immunological challenge with lipopolysaccharide (LPS). Analysis revealed that testosterone treatment did not significantly affect overall corticosterone release over the 24 h period in adult animals castrated before puberty in contrast to animals castrated in adulthood in which testosterone significantly suppressed corticosterone secretion. Following either a noise stress or LPS injection, testosterone treatment did not affect the hypothalamic or adrenal stress response in animals castrated prepubertally. Testosterone significantly suppressed the corticotrophin-releasing hormone and arginine vasopressin mRNA as well as the corticosterone response to LPS in castrated animals that had had their testes intact over puberty. These data provide evidence that puberty is a critical organizational period during which rising levels of gonadal steroids programme the sensitivity of the adult HPA axis to gonadal steroids in adulthood.

Sex-dependent mitophagy and neuronal death following rat neonatal hypoxia-ischemia.

Males are more susceptible than females to long-term cognitive deficits following neonatal hypoxic-ischemic encephalopathy (HIE). Mitochondrial dysfunction is implicated in the pathophysiology of cerebral hypoxia-ischemia (HI), but the influence of sex on mitochondrial quality control (MQC) after HI is unknown. Therefore, we tested the hypothesis that mitophagy is sexually dimorphic and neuroprotective 20-24h following the Rice-Vannucci model of rat neonatal HI at postnatal day 7 (PN7). Mitochondrial and lysosomal morphology and degree of co-localization were determined by immunofluorescence in the cerebral cortex. No difference in mitochondrial abundance was detected in the cortex after HI. However, net mitochondrial fission increased in both hemispheres of female brain, but was most extensive in the ipsilateral hemisphere of male brain following HI. Basal autophagy, assessed by immunoblot for the autophagosome marker LC3BI/II, was greater in males suggesting less intrinsic reserve capacity for autophagy following HI. Autophagosome formation, lysosome size, and TOM20/LAMP2 co-localization were increased in the contralateral hemisphere following HI in female, but not male brain. An accumulation of ubiquitinated mitochondrial protein was observed in male, but not female brain following HI. Moreover, neuronal cell death with NeuN/TUNEL co-staining occurred in both hemispheres of male brain, but only in the ipsilateral hemisphere of female brain after HI. In summary, mitophagy induction and neuronal cell death are sex dependent following HI. The deficit in elimination of damaged/dysfunctional mitochondria in the male brain following HI may contribute to male vulnerability to neuronal death and long-term neurobehavioral deficits following HIE.

Implementing Algorithm-Guided Warfarin Dosing in an Ethnically Diverse Patient Population Using Electronic Health Records and Preemptive CYP2C9 and VKORC1 Genetic Testing.

Implementation of pharmacogenetic-guided warfarin dosing has been hindered by inconsistent results from reported clinical trials and a lack of available algorithms that include alleles prevalent in non-white populations. However, current evidence indicates that algorithm-guided dosing is more accurate than empirical dosing. To facilitate multiethnic algorithm-guided warfarin dosing using preemptive genetic testing, we developed a strategy that accounts for the complexity of race and leverages electronic health records for algorithm variables and deploying point-of-care dose recommendations.

The clinical analgesic efficacy of oral nefopam hydrochloride.

The analgesic efficacy of 60 and 120 mg nefopam hydrochloride was compared to 650 mg aspirin and placebo in a double-blind single-dose study. Oral doses were administered to 120 patients suffering from acute postsurgical or fracture pain. All active medications demonstrated analgesic activity in comparison to placebo. Patients on 120 mg nefopam obtained the greatest degree of analgesia. Side effects were minor and did not interfere with the course of therapy. The incidence of side effects (sweating, nausea, and lightheadedness) was greater on 120 mg nefopam than on 650 mg aspirin).

Crossover and parallel study of oral analgesics.

Ten years ago, analgesics were studied using crossover designs. In recent years, analgesics have been studied only in parallel designs primarily because biostatisticians do not like crossover studies. The advantages of crossover studies are numerous: (1) patients serve as their own control; (2) there is less variability of responses among patients; and (3) a smaller number of patients is needed to provide statistically significant data. As long as crossover of treatment medications does not occur within 4 to 6 hours, the problem of carryover effect of the previous medication is insignificant or negligible. Two studies will be presented. One is a crossover study of Percodan with and without naloxone to placebo. The other is a parallel study comparing the effects of propoxyphene with naloxone to those of propoxyphene alone. The results of these studies reaffirm the value of the crossover method of evaluating analgesics.

The study of analgesics following single and repeated doses.

The efficacy and safety of 0.3 mg buprenorphine on single and repeated intramuscular administration (every 4 to 8 hours as needed) were compared to those of 10 mg intramuscular morphine. Fifty adult patients experiencing moderate to severe postoperative pain were evaluated up to three days following surgery. Results showed that 0.3 mg buprenorphine was as effective as 10 mg morphine, whether given as a single dose or on a repeat-dose schedule. The patterns of analgesia were similar and without indication of increasing dosage requirements with time. Minor side effects encountered were brief and minimal, including such conditions as drowsiness, dizziness, diaphoresis, flushing, and nausea.

The oral analgesic efficacy of bicifadine hydrochloride in postoperative pain.

The analgesic efficacy of 75 and 150 mg bicifadine hydrochloride was compared to 650 mg aspirin and placebo in a double-blind, single-dose study. Oral doses were administered to 100 patients suffering from moderate to severe postoperative pain. Significant analgesic activity was demonstrated with 650 mg aspirin and 150 mg bicifadine as compared to 75 mg bicifadine or placebo. No significant treatment difference was found between 75 mg bicifadine and placebo. Side effects were minor and did not interfere with the course of therapy.

Effect of HEPES buffer systems upon the pH, growth and survival of Mycoplasma mycoides subsp. mycoides small colony (MmmSC) vaccine cultures.

The use of a buffer system based on N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), in conjunction with standard Gourlay's culture medium was investigated for the growth and maintenance of Mycoplasma mycoides subsp. mycoides SC vaccine strain T(1)44. When the initial pH of the culture medium was adjusted to 8.0, 0.075 M HEPES-NaOH was found to be sufficient to prevent the pH falling below 7.1 at any stage during the growth cycle, even in the presence of 0.5% glucose. Compared to growth in standard unbuffered Gourlay's medium, the final culture titre was found to be one log(10) higher, at 10(11) colour changing units (CCU) per ml, and considerably extended culture survival was observed at 37 degrees C. The titre remained above 10(10) CCU ml(-1) for 4 days, and above 10(8) CCU ml(-1) in excess of 1 month. After 4 month's storage at 37 degrees C the titre had fallen to 5x10(4) CCU ml(-1). In contrast, no viable bacteria could be detected in standard unbuffered medium 3 days after the onset of stationary phase, at which point the pH had dropped to 5.4. No significant difference in growth rate between the two media was observed. Adoption of a HEPES-NaOH buffer system by African vaccine manufacturers should require minimal changes to current formulations and procedures, and should enhance both the final titre and thermostability of freeze-dried and liquid broth vaccines against contagious bovine pleuropneumonia (CBPP).

Prehospital cardiac arrest--a critical analysis of factors affecting survival.

During a 10-year period, 5631 cardiac arrests were treated in our paramedic system. In all, 4216 resuscitations were attempted, of which 533 (12.6%) resulted in saves, defined as hospital discharges. Patients presenting with an initial rhythm of coarse ventricular fibrillation or ventricular tachycardia were found to have significantly increased save rates in comparison to those presenting with an initial arrest rhythm of asystole/fine ventricular fibrillation or electromechanical dissociation (P less than or equal to 0.01). When controlling for witnessed arrest, 303 of 1905 (15.9%) of all witnessed arrests were saves vs. 230 of 2311 (10%) of unwitnessed arrests (P less than or equal to 0.01). Witnessed bystander/first responder external cardiac compression- cardiopulmonary resuscitation (ECC-CPR) was found not to influence save rate. One hundred eighty-one of 1248 bystander/first responder witnessed arrests (14.5%) who had external ECC-CPR initiated before paramedic advanced life support arrival were saves, compared with 38 of 252 (15.1%) who had no ECC-CPR initiated until paramedic arrival; this was not statistically significant. Advanced life support response times in saved patients with witnessed cardiac arrests were analyzed. Ninety-five percent of all saves had a response time of less than 10 min. We conclude that, when evaluating the effectiveness of CPR, the variables of witnessing of arrest, presenting arrest rhythm, and respective response times must be controlled or analyzed.

Electromechanical dissociation: six years prehospital experience.

Electromechanical dissociation (EMD) is the presenting rhythm in approximately 17% of all prehospital cardiorespiratory arrests. Yet, we know comparatively little about the demographic profile of these patients. The purpose of this study was to review historical and resuscitative parameters to help create a demographic profile. For a 6-year period of time from January 1st, 1980 to December 31st, 1985, 503 adult patients presented to a prehospital system in non-traumatic, nonpoisoned, cardiorespiratory arrest with an initial rhythm of electromechanical dissociation. The overall average response time was 6.1 +/- 3.2 min. Sixty percent of the patients were witnessed arrests and 65% had bystander initiated CPR. Forty-six percent of the patients had a cardiac history: myocardial infarction 13%, CHF 11% and other 21%. Other pertinent past medical history included diabetes 15%, COPD 10% and seizures 3%. The average age was 69.8 +/- 13.7 years. Fifty-seven percent were male. Forty-three percent were on cardiac medication including: digoxin, 24%; nitroglycerin, 12%; potassium supplements, 9%; propranolol, 8%; isordil, 6%; quinidine, 3%; nitropaste, 3%; and other cardiac medications, 15%. One hundred forty-eight (29%) patients developed a pulse at some time during resuscitative efforts, of these 17 (3.4%) patients responded with a pulse immediately after intubation. The mean time of resuscitation to sustaining pulse was 20 +/- 11 min and the mean resuscitation time to sustaining pressure was 22 +/- 11 min. Nineteen percent were successfully resuscitated, defined as a conveyance of a patient with a pulse and a rhythm to an emergency department. Four point four percent were saved, defined as a patient discharged alive from the hospital. Approximately 53% of the successfully resuscitated patients and 45% of the save patients were determined to have a probable respiratory event as the primary etiology of their arrest. This study attempts to provide some insight into the demographic profile of the patients in EMD.

The effect of bystander CPR on neurologic outcome in survivors of prehospital cardiac arrests.

The efficacy of CPR has been questioned. A major criticism is that neurologic outcomes have not been adequately studied. For a 26-month period, 138 patients from six major receiving hospitals were discharged alive following prehospital cardiac arrests. For 65/138 (47.1%) patients, either the patient or a direct family member was contacted for information concerning neurologic outcome. For 63/138 (45.7%) patients, contact with patient or family was unsuccessful, consequently neurologic outcome at time of discharge was obtained from the medical record. For 10/138 (7.2%) patients, no data on neurologic outcome was obtainable. Neurologic outcome was rated by a 5-point Cerebral Performance Categories Scale (CPC); (1) Minimal Disability; (2) Moderate; (3) Severe; (4) Vegetative; and (5) Brain Dead. The bystander/first responder CPR group had 55.1% CPC-1; 24.4% CPC-2; 16.7% CPC-3; and 3.8% CPC-4 outcomes. The bystander/first responder NO CPR group had 58.0% CPC-1; 18.0% CPC-2; 16.0% CPC-3; and 8.0% CPC-4 outcomes. There was no significant difference at any CPC level (P not significant). Furthermore, there was no statistical difference between either group when compared for age, response time, resuscitation time, witnessing of arrest or distribution of presenting rhythms. In conclusion, no significant effect in neurologic outcome among saved cardiac arrest victims was found between bystander/first responder CPR and bystander/first responder NO CPR groups in the paramedic program studied.

A review of the methodological aspects of aspartic acid racemization analysis for use in forensic science.

Accurate age determination of adult cadavers and human remains is a key requirement in forensic practice. The current morphological methods lack accuracy and precision, require specialist training and are costly. The use of aspartic acid racemization (AAR) in human dentine provides a simple, cost-effective solution and the method can achieve accuracies of +/- 3 years at best. Currently, there are differences in AAR methodology between laboratories which produce different results on the rate of racemization in teeth. These inconsistencies must be resolved if the technique is to be successfully applied to age determinations in forensic cases. This paper reviews the differences in protocol which have been used, discusses how each method will affect the results obtained from AAR analysis and gives recommendations for optimization of the methological protocol as a first step towards international standardization.

Capsular polysaccharide conjugate vaccines against contagious bovine pleuropneumonia: Immune responses and protection in mice.

The immunogenicity of Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) vaccines was investigated in BALB/c mice. Groups of mice were vaccinated with either (1) unconjugated capsular polysaccharide (CPS), (2) CPS covalently conjugated to ovalbumin via a carbodiimide reaction, (3) CPS non-covalently bound to latex microspheres, (4) CPS non-covalently complexed with rabbit anti-CPS IgG, and (5) whole inactivated, ultrasonically disrupted (WID) MmmSC. Only mice immunized with the CPS-ovalbumin conjugate exhibited a significant (P<0 small middle dot001) antibody response against CPS. Mice immunized with WID vaccine exhibited a high ELISA antibody titre against non-CPS (protein) antigens only. Mice given WID vaccine were immune against challenge with live MmmSC, and exhibited a significantly reduced degree of mycoplasmaemia (both in incidence and duration) as compared with non-vaccinated controls (P<0 small middle dot001). Mice immunized with the CPS-ovalbumin conjugate did not exhibit a reduction in mycoplasmaemia. The bactericidal activity of rabbit MmmSC-antiserum in an in-vitro growth inhibition test was related to the CPS antibody titre. This was not observed with antisera from the vaccinated mice. None of the mouse antisera exhibited growth inhibiting activity, irrespective of a high CPS or protein antibody titre (CPS-ovalbumin or WID vaccine groups, respectively). Thus, it would seem that protection against an MmmSC-induced mycoplasmaemia in the mouse is based upon cell-mediated rather than humoral immunity. The results suggest that conjugation to ovalbumin significantly increases the antibody response to CPS in the mouse; the lack of bactericidal activity of mouse anti-CPS as compared with rabbit anti-CPS in vitro suggests either that the titre of growth inhibiting antibodies is lower in the mouse or that the mechanism of growth inhibition differs between antibodies of the two species.

A glucocorticoid sensitive biphasic rhythm of testosterone secretion.

Studies of the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-testicular (HPT) axis have revealed a reciprocal relationship between these two endocrine pathways. In rats, for example, disruption of the HPT axis alters the circadian secretion of corticosterone. Stress, on the other hand, can have varying effects on testosterone secretion in both rats and humans. Furthermore, in contrast to humans, where several pulses of testosterone secretion can be detected across the 24-h period with the largest in the morning, rats appear to exhibit a diurnal rhythm of testosterone secretion. In the present study, we used an automated blood sampling system to investigate the true circadian pattern of testosterone secretion under basal conditions and investigated how this responds to changes in levels of circulating corticosteroids. Analysis of plasma testosterone revealed the expected bimodal pattern of basal testosterone secretion. The two secretory episodes were 12.59 h +/- 41 min apart and 4.04 h +/- 16 min long, with one in the light phase and the other in the dark phase of the cycle. Interestingly, when both testosterone and corticosterone diurnal profile were compared, we found that the circadian rise in plasma corticosterone levels falls neatly between the two testosterone secretory episodes. Treatment of rats with the synthetic glucocorticoid methylprednisolone in their drinking water abolished the normal bimodal profile of testosterone secretion. These rats show transient pulses of testosterone throughout the 24 h, but no circadian pattern. By contrast, adrenalectomised rats maintain their bimodal circadian pattern, suggesting that an intact HPA axis is not necessary for generation of the endogenous HPT rhythm. Thus, although the circadian rhythm of testosterone does not depend on normal HPA function, increased levels of glucocorticoids can abolish normal HPT rhythmicity.

Predicting protein decomposition: the case of aspartic-acid racemization kinetics.

The increase in proportion of the non-biological (D-) isomer of aspartic acid (Asp) relative to the L-isomer has been widely used in archaeology and geochemistry as a tool for dating. the method has proved controversial, particularly when used for bones. The non-linear kinetics of Asp racemization have prompted a number of suggestions as to the underlying mechanism(s) and have led to the use of mathematical transformations which linearize the increase in D-Asp with respect to time. Using one example, a suggestion that the initial rapid phase of Asp racemization is due to a contribution from asparagine (Asn), we demonstrate how a simple model of the degradation and racemization of Asn can be used to predict the observed kinetics. A more complex model of peptide bound Asx (Asn + Asp) racemization, which occurs via the formation of a cyclic succinimide (Asu), can be used to correctly predict Asx racemization kinetics in proteins at high temperatures (95-140 degrees C). The model fails to predict racemization kinetics in dentine collagen at 37 degrees C. The reason for this is that Asu formation is highly conformation dependent and is predicted to occur extremely slowly in triple helical collagen. As conformation strongly influences the rate of Asu formation and hence Asx racemization, the use of extrapolation from high temperatures to estimate racemization kinetics of Asx in proteins below their denaturation temperature is called into question. In the case of archaeological bone, we argue that the D:L ratio of Asx reflects the proportion of non-helical to helical collagen, overlain by the effects of leaching of more soluble (and conformationally unconstrained) peptides. Thus, racemization kinetics in bone are potentially unpredictable, and the proposed use of Asx racemization to estimate the extent of DNA depurination in archaeological bones is challenged.

Hydroxyproline interference during the gas chromatographic analysis of D/L aspartic acid in human dentine.

The proportion of D- to L-enantiomers of aspartic acid in metabolically isolated proteins has been used by forensic scientists to estimate age at death. We have demonstrated the interference of a derivative of hydroxyproline (N-TFA isopropyl Hyp ester) with the N-TFA isopropyl L-Aspartic (Asp) acid ester during gas chromatography of amino acids. This has serious implications for the accurate quantification of the D- to L-Asp ratio extracted from collagenous proteins. Having demonstrated the potential for this co-elution in amino acid standards, acid-soluble dentine proteins and non-mineralised collagen, we argue that this problem can be overcome either by high resolution separation or by analysis of the (Hyp-poor) non-collagenous protein fraction.

Quality assurance in age estimation based on aspartic acid racemisation.

Estimates of the age of living and dead individuals, obtained in order to answer legal or social questions, require minimum quality standards in order to guarantee data quality. We present an outline strategy (with recommendations) for the attainment of quality assurance in age estimation based on aspartic acid racemisation. The strategy is based on a definition of minimum standards for laboratories, including documentation of procedures, methodology and levels of expertise, and the formulation of guidelines for intralaboratory and interlaboratory quality control.

Age estimation: the state of the art in relation to the specific demands of forensic practise.

Age estimation in cadavers, human remains and living individuals may clarify issues with significant legal and social ramifications for individuals as well as for the community. In such cases methods for estimating age should fulfil the following specific demands: (1) they must have been presented to the scientific community, as a rule by publication in peer-reviewed journals, (2) clear information concerning accuracy of age estimation by the method should be available, (3) the methods need to be sufficiently accurate and (4) in cases of age estimation in living individuals principles of medical ethics and legal regulations have to be considered. We have identified and summarized the methods that essentially fulfil these specific demands. In childhood and adolescence morphological methods based on the radiological examination of dental and skeletal development are to be recommended. In adulthood, the accuracy of most morphological methods is much reduced. Here a biochemical method based on aspartic acid racemization in dentine provides the most accurate estimates of age, followed by special morphological dental and skeletal methods. The choice of method has to take account of the individual circumstances of each case. Most methods require either the consultation of specialised and trained scientists or an adequate calibration by the "user". Very few attempts have been made to find common standardisation, calibration and evaluation procedures or to develop means of quality assurance for methods of age estimation. Efforts in these directions are necessary to guarantee quality standards and adequate answers to the important legal and social issue of age estimation in forensic medicine.