Amending reduced fish-meal feeds with marine lecithin, but not soy lecithin, improves the growth of juvenile cobia and may attenuate heightened responses to stress challenge.

Sparing of marine resources in aquafeeds can be environmentally and economically advantageous; however, fish meal (FM) replacement can affect the production performance and physiological competence. Phospholipids are increasingly understood to be involved in maintaining growth and vigour in fish and may be deficient in reduced FM formulations. Accordingly, we evaluated the growth and stress tolerance of juvenile cobia fed typical (50% FM) or reduced FM feeds (12% FM) with or without phospholipid amendment [1% marine lecithin (12% FM + Marine PL) or soy lecithin (12% FM + Soy PL)] for 6 weeks in triplicate tanks (N = 3) in a recirculation aquaculture system. The 50% FM feed yielded significantly superior growth and growth efficiency in comparison with the 12% FM and 12% FM+ Soy PL feeds, but the 12% FM+ Marine PL feed yielded comparable results to 50% FM feed. A low-water stress challenge induced elevated plasma glucose, cortisol and lactate levels in all treatments. However, a significant interaction (diet × stress) effect suggested a lesser cortisol response among fish fed the 12% FM+ Marine PL and 50% FM diets. These findings demonstrate that growth performance and, perhaps, resilience of cobia raised on reduced FM feeds may be improved by the addition of marine-origin phospholipid to the diet.

Novel automatic electrochemical-mechanical polishing (ECMP) of metals for scanning electron microscopy.

A low-stress automated polishing device was developed for preparing titanium and nickel alloys for scanning electron microscopy imaging. The system used pulsed electrochemical reactions within an alkaline electrolyte to generate a thin passivation layer on the surface of the sample, which was removed by the mechanical vibration of the system. The passivation layer development and removal were documented for Ti-6Al-4V and IN718 samples subjected to varying electrical potential cycles and polishing times. Results indicated that the applied cyclic potentials removed material faster than typical removal techniques. In addition, electron back scatter diffraction data showed a decrease in subsurface damage using the developed electrochemical-mechanical process compared to standard mechanical polishing techniques.

Optimizing Storage and Handling of DNA Extracts.

Nucleic acid sample storage is of paramount importance in forensic science as well as in epidemiological, clinical, and genetic laboratories. Millions of biological samples, including cells, viruses, and DNA/RNA, are stored every year for diagnostics, research, and forensic science. PCR has permitted the analysis of minute sample quantities. Samples such as bone, teeth, touch samples, and some sexual assault evidence may yield only low-quality and low-quantity DNA/RNA. Efficient storage of the extracted DNA/RNA is needed to ensure the stability of the sample over time for retesting of the CODIS STRs, mtDNA, YSTRs, mRNA, and other future marker-typing systems. Amplification of some or all of these markers may fail because the biological material has been highly degraded, contains inhibitors, is too low in quantity, or is contaminated with contemporary DNA. Reduction in recovery has been observed with refrigerated liquid DNA extracts and also those exposed to multiple freeze-thaw cycles. Therefore, the development of optimal storage and amplification methods is critical for successful recovery of profiles from these types of samples since, in many cases, retesting is necessary. This review is divided into three sections. The Introduction and Background covers forensic DNA storage, factors that influence DNA stability, and a brief review of molecular strategies to type non-optimal DNA. Section I covers the importance of DNA extract storage in forensic and non-forensic DNA databanks and the mechanisms responsible for loss during storage. Finally, Section II covers strategies and technologies being utilized to store DNA.

Quantification of sarin and cyclosarin metabolites isopropyl methylphosphonic acid and cyclohexyl methylphosphonic acid in minipig plasma using isotope-dilution and liquid chromatography- time-of-flight mass spectrometry.

An analysis method for determining isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA), the metabolic hydrolysis products of toxic organophosphorus nerve agents isopropyl methylphosphonofluoridate (sarin, GB) and cyclohexyl methylphosphonofluoridate (cyclosarin, GF), respectively, has been developed and validated using high-performance liquid chromatography-mass spectrometry with negative ion electrospray ionization with time-of-flight detection (LC-ESI-MS-TOF). The linear range of quantitation was 5 to 125 ng/mL in plasma with a method detection limit of 2 ng/mL for each compound. This method was developed to determine the amount of metabolic hydrolysis that was formed during and after nerve agent exposure in minipigs to account for a major pathway of GB and GF elimination that had not been previously characterized in the bloodstream, particularly during low-level whole-body inhalation experiments. Metabolic hydrolysis accounted for 70% to 90% of the recoverable agent in the bloodstream during exposure, when compared to both unbound and cholinesterase bound agent recovered by fluoride ion reactivation analysis for the same samples. The estimated half-life of IMPA and CMPA in plasma was determined to be 44 and 61 min, respectively. The method utilizes the mass selectivity of LC-ESI-MS-TOF using a bench-top instrument to achieve a detection limit that is consistent with reported LC-MS-MS methods analyzing blood samples.

Effects of whole-body VX vapor exposure on lethality in rats.

Male and female rats were whole-body exposed to VX vapor in a 1000-L single-pass exposure chamber. Estimated exposure dosages producing lethal (LCT50) effects in 50% of exposed male and female rats were established for 10, 60, and 240 min exposure durations. A potency comparison with GB and GF shows that VX becomes increasingly more potent than these G agents with increasing exposure duration. VX is approximately 4-30 times more potent than GB and 5-15 times more potent than GF. Gender differences in the estimated median dosages were not significant at the 10, 60, and 240 min exposure durations. An empirical toxic load model was developed and the toxic load exponent for lethality (n) in the equation Cn x T = k was determined to be n = 0.92. The VX-G regeneration assay was successfully used as a biomarker for the presence of VX in the blood plasma and RBC fractions of the blood 24 h postexposure.

Quantitation of fluoride ion released sarin in red blood cell samples by gas chromatography-chemical ionization mass spectrometry using isotope dilution and large-volume injection.

A new method for measuring fluoride ion released isopropyl methylphosphonofluoridate (sarin, GB) in the red blood cell fraction was developed that utilizes an autoinjector, a large-volume injector port (LVI), positive ion ammonia chemical ionization detection in the SIM mode, and a deuterated stable isotope internal standard. This method was applied to red blood cell (RBC) and plasma ethyl acetate extracts from spiked human and animal whole blood samples and from whole blood of minipigs, guinea pigs, and rats exposed by whole-body sarin inhalation. Evidence of nerve agent exposure was detected in plasma and red blood cells at low levels of exposure. The linear method range of quantitation was 10-1000 pg on-column with a detection limit of approximately 2-pg on-column. In the course of method development, several conditions were optimized for the LVI, including type of injector insert, injection volume, initial temperature, pressure, and flow rate. RBC fractions had advantages over the plasma with respect to assessing nerve agent exposure using the fluoride ion method especially in samples with low serum butyrylcholinesterase activity.

Inhalation toxicity of Cyclosarin (GF) vapor in rats as a function of exposure concentration and duration: potency comparison to sarin (GB).

The inhalation toxicity of cyclohexyl methylphosphonofluoridate (GF) was examined in male and female Sprague-Dawley rats exposed by whole body in a dynamic 750-L chamber. The objectives of this study were to (1) generate GF vapor in a dynamic inhalation chamber system, starting in the lethal to near-lethal concentration range, (2) examine dose-response effects of inhaled GF vapor and analyze the relationship between concentration (C) and exposure duration (T) in determining probability of lethality, and (3) establish a lethal potency ratio between GF and the more volatile agent Sarin (GB). Using a syringe pump, GF vapor concentrations were generated for exposure times of 10, 60, and 240 min. Dose-response curves with associated slopes were determined for each exposure duration by the Bliss probit method. GF vapor exposures were associated with sublethal clinical signs such as tremors, convulsions, salivation, and miosis. Concentration-exposure time values for lethality in 50% of the exposed population (LCT(50)) were calculated for 24-h and 14-day postexposure periods for 10-, 60-, and 240-min exposures. In general, LCT(50) values were lower in female rats than males and increased with exposure duration; that is, CT was not constant over time. The GF LCT(50) values for female rats were 253 mg min/m(3) at 10 min, 334 mg min/m(3) at 60 min, and 533 mg min/m(3) at 240 min, while the values for males were 371, 396, and 585 mg min/m(3), respectively. The GB LCT(50) values for female rats were 235 mg min/m(3) at 10 min, 355 mg min/m(3) at 60 min, and 840 mg min/m(3) at 240 min, while the values for males were 316, 433, and 1296 mg min/m(3), respectively. At longer exposure durations, the LCT(50) for GF was less than that found for GB but at shorter exposure durations, the LCT(50) for GF was more than that found for GB. Empirical models, consisting of the toxic load model plus higher order terms, were developed and successfully fit to the data.

Statistical analyses of 14 short tandem repeat loci in Brazilian populations from Rio de Janeiro and Mato Grosso do Sul states for forensic and identity testing purposes.

This work describes the statistical features of a database for two Brazilian populations (one from the Rio de Janeiro State (southeast region), and one from the Mato Grosso do Sul State (central western region) using fourteen short tandem repeat loci (STR).

Child abuse and the otolaryngologist: part I.

Nearly 1 million infants and children are neglected and abused yearly in the United States, with a greater than 1% resulting mortality rate. One half of these children are seen by physicians for abuse-related injuries, and nearly 75% have injuries of the head and neck. Physicians, however, account for reporting only 11% of all cases. As experts trained in diseases and injuries of the head and neck, otolaryngologists are particularly well positioned to recognize abuse in the clinic and in the emergency room and during other consultations. We present an overview of child abuse definitions, risk factors, and legal obligations of the physician. We also review the manifestations of child abuse within the head and neck, with particular attention to the role of the otolaryngologist. We briefly discuss some conditions that may be mistaken for abuse and suggest a practical protocol for management of suspected cases in the clinic.

Implementation of forensic DNA analysis on casework evidence at the Palm Beach County Sheriff's Office Crime Laboratory: historical perspective.

Palm Beach County is the largest of the 64 counties in the state of Florida, USA, with most of the area uninhabited and the population concentrated near the coastal region. The Serology/DNA Section of the Palm Beach County Sheriff's Office (PBSO) Crime Laboratory serves a community of approximately one million residents, and an additional million tourists visit Palm Beach County every year. In addition to the unincorporated county regions, there are thirty-four city police agencies, the Florida State Highway Patrol, several university security agencies, the local Federal Bureau of Investigation, and the county Medical Examiners Office that all use the PBSO Serology/DNA Laboratory for the analysis of casework evidence. The purpose of this manuscript is to provide laboratories that are in the process of initiating DNA analysis on casework with practical information regarding the decision-making processes that occurred during the development of the DNA testing program at PBSO. Many of the concerns addressed in the early 1990's are still a guide to the development of a quality forensic DNA analysis program in the year 2001. Issues, such as personnel, laboratory space, internal standard operating procedures, implementation of DNA analysis on casework evidence, and building a relationship with law enforcement personnel are discussed.

Paradoxical effects of ketamine on the memory of fetuses of different ages.

Brain N-methyl-D-aspartate (NMDA) glutamate receptors have been implicated as important mediators of both learning and neuronal development. The current study investigated how ketamine (a well-known NMDA-receptor blocking drug) influences taste-mediated conditioned motor responses (CMRs) in perinatal rats. Dams pregnant with either embryonic day 18 (E18) or E19 rat fetuses were injected with 0 or 100 mg/kg ketamine HCl (i.p.). One-half hour later, a reversible spinal block was performed on the dam and fetuses received oral lavage with 10 microl, 0.3% saccharin (SAC) or water (control) in utero. After the oral injection, fetuses received either a saline (control) or lithium chloride (LiCl) injection (81 mg/kg, i.p.). The uterus was replaced and, 2 days later (E20 or E21), some rats received oral lavage with SAC. Other litters were born via normal vaginal delivery or Cesarean section and orally exposed to SAC on post-natal day 3 (P3). Motor responses were observed immediately after the oral lavage of SAC. If SAC had been paired with LiCl in utero, pups generally exhibited conditioned suppression of orofacial movements (as compared to controls). Ketamine significantly attenuated this taste-mediated CMR of animals conditioned on E19. However, the same treatments did not disrupt CMRs of rats treated with ketamine before CS-US pairing on E18. Our findings indicate an age-dependent role for NMDA receptors in the formation of CMRs in perinatal rats.

Detection of novelty by perinatal rats.

This study investigated the development of fetal/neonatal rats' ability to distinguish between a novel and familiar taste. Here, we report that neonatal rats alter their orofacial movements (e.g., mouth movements and licks) upon tasting saccharin (SAC) if it was experienced previously. We also sought to determine the origins and duration of this response. Fetuses of embryonic ages E17, E18, or E19 received an oral injection of 10 microL 0.3% SAC while in utero. These animals were then reexposed to SAC on postnatal day 3, (P3) and observations of orofacial motor responses were recorded. Only neonates that first experienced SAC on E19 exhibited a SAC-induced stimulation of mouthing and licking on P3. These data suggested that a taste-recognition memory (TRM) is maintained for up to 5 days (i.e., E19 to P3). However, in this paradigm, the youngest fetuses also have the longest retention interval. Could these data also reflect the limitations of the E17 and E18 fetuses in retaining the TRM? In a second study, we shortened the taste exposure-reexposure interval to 2 days in an attempt to detect the TRM in younger fetuses. As expected, E19 rats exhibited a TRM when tested on E21. However, neither the E17 nor E18 fetuses showed SAC-induced increases in mouthing and licking when tested 2 days after their initial exposure (E19 or E20). Finally, in order to determine whether a TRM could be detected in fetuses as well as neonates (see above), we conducted an additional study wherein E21 fetuses were tested before parturition. Like E21 neonates, E21 rat fetuses that had received SAC on E19 showed a differential response to SAC depending on whether it was novel or familiar. Thus, although E21 fetal orofacial movements were less frequent than those of the E21 neonate, the fetal-testing procedures were not sufficient to obscure the detection of a TRM. In summary, the data indicate that E19 rat fetuses can acquire a TRM and retain it for at least 2-5 days, whereas E17 and E18 fetuses cannot.

Ketamine blocks a taste-mediated conditioned motor response in perinatal rats.

Brain N-methyl-D-aspartate (NMDA) glutamate receptors have been implicated as important mediators of both learning and neuronal development. The current study investigated how ketamine HCl (a well-known NMDA-receptor blocking drug) would influence taste-mediated conditioned motor responses in perinatal rats. Dams pregnant with E19 rat fetuses were injected with 0, 50, or 100 mg/kg ketamine HCl (IP). One-half hour later, a reversible spinal block was performed on the dam, and fetuses received an oral injection of 10 microl 0.3% Saccharin (SAC) or water while in utero. After the oral injection, fetuses received either saline or LiCl (81 mg/kg, IP). The uterus was replaced and, 2 days later (E21), rats received oral lavage with SAC. Rats in other litters were born via a normal vaginal delivery and were exposed to SAC on postnatal day 3 (P3). Observations of motor responses were recorded immediately after the oral lavage of SAC. If SAC had been paired with LiCl in utero, both E21 and P3 pups exhibited a conditioned suppression of orofacial movements (compared to controls). Both doses of ketamine significantly attenuated this taste-mediated conditioned motor response. These data reinforce the current conception of the fetus and neonate as sophisticated sensors and responders to the uterine and extrauterine environment. Further, our findings indicate a role for NMDA receptors in the formation of a conditioned motor response in fetal rats.

Ketamine blocks a taste recognition memory in fetal rats.

Decisions about novelty/familiarity are critical in determining whether or not information should be attended to, and possibly encoded, for long-term storage. We have reported that fetal and neonatal rats exhibit an increase in orofacial movements (e.g., perseverative mouthing and mouth movements, and licks) upon tasting saccharin (SAC), if it was experienced previously. E19 rat fetuses can acquire this taste recognition memory and retain it for at least 5 days (P3). In the current study, we sought to evaluate the role of N-methyl-D-aspartate (NMDA) receptors in establishing a taste recognition memory. Pregnant Sprague-Dawley rats received ketamine (NMDA receptor antagonist) (doses: 0, 50, or 100 mg/kg, i.p.). One-half hour later, we performed a reversible spinal block on each pregnant dam, and E19 fetuses received an oral injection of 10 microl, 0.3% SAC or water (control) while in utero. The uterus was replaced and the pups were later born via a normal vaginal delivery. On P3, all pups experienced oral lavage of 10 microl, 0.3% SAC, and motor responses were recorded. As expected, non-drugged control neonates tasting familiar SAC exhibited significantly more perseverative mouth movements, as well as total mouth movements and licks, than did pups tasting novel SAC. However, this taste recognition memory response was not observed in rats exposed to ketamine in utero. The data suggest that early non-associative taste memories may be disrupted by NMDA receptor blockade.

TWGDAM validation of a nine-locus and a four-locus fluorescent STR multiplex system.

The Gene Print PowerPlex 1.1/Amelogenin and FFFL Fluorescent STR Systems have been validated following the recommendations presented by the Technical Working Group on DNA Analysis Methods (TWGDAM). The PowerPlex 1.1/Amelogenin System supports simultaneous amplification of eight short tandem repeat loci and the Amelogenin gender identification marker. The loci D16S539, D7S820, D13S317, and D5S818 are labeled with fluorescein (FL) while the loci CSF1PO, TP0X, TH01, vWA and Amelogenin are labeled with carboxy-tetramethylrhodamine (TMR). The FFFL Multiplex System is composed of the loci F13A01, FESFPS, F13B, and LPL, each labeled with fluorescein. We have observed no overlap of alleles across loci labeled with an individual fluorescent dye. Samples of each system were amplified and labeled in a single reaction, separated by electrophoresis through a denaturing polyacrylamide gel, and amplified alleles detected using a Hitachi FMBIO Fluorescent Scanner. Alterations from the standard amplification protocols in cycle number and annealing temperature generally produced excellent results. In experiments testing sensitivity as little as 0.2 ng of DNA template could be detected. As expected, different body fluids from the same individuals generated identical DNA profile results. Template DNA derived from blood-strains deposited on a variety of matrix supports displayed robust amplification except for material derived from deposits on wood and Japanese orchid leaves. Mixtures of DNA templates could be interpreted with the minor component present in as little as ten percent of the total sample. Monoplex and multiplex amplifications produced identical amplified allele patterns, indicating that STR multiplex systems save template and increase efficiency in the amplification procedure without loss of quality. Analyses of genotype frequencies in African-American, Caucasian-American and Hispanic-American populations using all twelve loci were used to determine matching probabilities smaller than 1 in 1.14 x 10(8) and 1 in 2658 for the PowerPlex 1.1 and the FFFL Multiplex Systems, respectively. The matching probability achieved with the two systems combined is smaller than 1 in 3.03 x 10(11). The independence of alleles within loci was generally demonstrated by applying the exact test to demonstrate Hardy-Weinberg Equilibrium. All of the studies performed indicate that the PowerPlex 1.1/Amelogenin and FFFL Multiplex Systems are powerful, robust, and reliable investigative tools that can be used in the analysis of forensic samples.

Analysis and interpretation of short tandem repeat microvariants and three-banded allele patterns using multiple allele detection systems.

The Palm Beach County Sheriffs Office (PBSO) Crime Laboratory and the Alabama Department of Forensic Sciences (ADFS) have validated and implemented analysis of short tandem repeat (STR) sequences on casework using silver staining kit and SYBR Green I detection systems and are presently validating fluorescently tagged STR alleles using the Hitachi FMBIO 100 instrument. Concurrently, the Broward County Sheriff's Office (BSO) Crime Laboratory is validating the ABI Prism310 Genetic Analyzer capillary electrophoresis STR detection system (ABI CE310) from Perkin Elmer Applied BioSystems. During the course of analyzing over 10,000 individuals for the STR loci CSF1PO, TPOX and THO1 (CTT) using silver staining for allele detection, 42 samples demonstrated alleles that were "off ladder," contained three-banded patterns at a single locus, or exhibited an apparent THO1 "9.3,10" allele pattern. PBSO, ADFS and BSO Crime Laboratories have collaborated on the verification of the allele patterns observed in these 42 samples using the following allele detection systems: (1) manual silver staining, (2) SYBR Green I staining, and/or (3) fluorescently tagged amplified products separated by polyacrylamide gel electrophoresis or capillary electrophoresis followed by laser detection. Regardless of the CTT allele detection system utilized, concordant results were obtained for 41 of the 42 samples. The only exception was a sample in which a wide band within the THO1 locus was identified as a THO1 "9.3, 10" genotype by silver staining kit and SYBR Green I staining but was verified to be a THO1 "9.3" homozygote by all other allele detection systems. Manual allele detection could readily identify microvariants, as a visual assessment of stained gels clearly shows that alleles do not migrate coincident with well-characterized allele size standards. As would be predicted, however, the manual detection systems did not provide adequate resolution to approximate the basepair size for off-ladder variants. All fluorescent software program systems were consistent in designating alleles "not in range" or "off ladder," thereby indicating true microvariants. All single-locus three-banded patterns were detected using all of the STR multiplex systems. In addition, individual locus-specific primers verified multiplexed amplified products were specific for the locus in question.

Chemical and toxicological evaluation of pyrotechnically disseminated terephthalic acid smoke.

The terephthalic acid (TPA) smoke obscurants (M-83 grenade and M-8 smoke pot) were developed by the U.S. Army for training purposes to replace the more toxic hexachloroethane (HC) smoke. Inhalation toxicity testing and chemical characterization of pyrotechnically generated TPA was conducted to assess the health hazard potential of TPA and its combustion products. Fisher 344 rats were subjected to acute and repeated exposures to TPA smoke generated from the M-83 grenade. Acute exposure levels ranged from 150-1,900 mg/m3 for 30 minutes and repeated dose exposures ranged from 128-1,965 mg/m3 for 30 min/day for 5 days. Exposed and control rats were evaluated for toxic signs, and histopathologic changes. During exposure, the rats exhibited slight to moderate lacrimation, rhinorrhea, lethargy and dyspnea, which reversed within 1-hr post-exposure. No deaths occurred, even at the highest smoke concentrations. Histopathological changes were confined to exposure related nasal necrosis and inflammation in both the acute and repeated dose exposures at levels above 900 mg/m3. Chemical characterization of the M-83 grenade and the M-8 smoke pot showed that formaldehyde, benzene and carbon monoxide were the major organic vapor by-products formed. These by-products were above their respective ACGIH threshold limit values at various concentrations, but should not pose a hazard if the smoke is deployed in an open area. Overall, TPA is a safer training smoke to replace the HC smoke.