Integrative Approach for the Reliable Detection and Specific Identification of the Microsporidium Loma morhua in Atlantic Cod (Gadus morhua).

Microsporidia are fungal parasites that infect diverse invertebrate and vertebrate hosts. Finfish aquaculture supports epizootics due to high host density and the high biotic potential of these parasites. Reliable methods for parasite detection and identification are a necessary precursor to empirical assessment of strategies to mitigate the effects of these pathogens during aquaculture. We developed an integrative approach to detect and identify Loma morhua infecting Atlantic cod. We show that the spleen is more reliable than the commonly presumed gills as best organ for parasite detection in spite of substantial morphological plasticity in xenoma complexes. We developed rDNA primers with 100% sensitivity in detecting L. morhua and with utility in distinguishing some congeneric Loma species. ITS sequencing is necessary to distinguish L. morhua from other congeneric microsporidia due to intraspecific nucleotide variation. 64% of L. morhua ITS variants from Atlantic cod have a 9-nucleotide motif that distinguishes it from Loma spp. infecting non-Gadus hosts. The remaining 36% of ITS variants from Atlantic cod are distinguished from currently represented Loma spp., particularly those infecting Gadus hosts, based on a 14-nucleotide motif. This research approach is amenable to developing templates in support of reliable detection and identification of other microsporidian parasites in fishes.

AZD9272 and AZD2066: selective and highly central nervous system penetrant mGluR5 antagonists characterized by their discriminative effects.

The metabotropic glutamate receptor 5 (mGluR5) antagonists fenobam, MPEP (2-methyl-6-(phenylethynyl)pyridine), and MTEP (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine) were previously shown to not cause N-methyl-D-aspartate antagonist-like psychoactive effects in phencyclidine (PCP) drug discrimination studies, but to cause MTEP-like discrimination in rats, suggesting that the psychoactive and psychotomimetic effects reported with fenobam in humans were likely mediated by mGluR5 antagonist mechanisms. The present study was designed to characterize AZD9272 (3-fluoro-5-(3-(5-fluoropyridin-2-yl)-1,2,4-oxadiazol5-yl)benzonitrile) and AZD2066 [4-(5-{(1R)-1-[5-(3-chlorophenyl)isoxazol-3-yl]ethoxy}-4-methyl-4H-1,2,4-triazol-3-yl)pyridine], two mGluR5 antagonists taken to clinical development for analgesia. AZD9272 was evaluated in several groups of rats trained to discriminate cocaine, PCP, chlordiazepoxide, (-)-Δ(9)-tetrahydrocannabinol [(-)-Δ(9)-THC], or MTEP from no drug. AZD9272 shared discriminative properties with MTEP only. The discriminative half-life was 3.23 hours for MTEP and 21.93 hours for AZD9272 in rats trained to discriminate MTEP from no drug. Other rats were successfully trained to discriminate AZD9272 from no drug. Due to the long duration of action of AZD9272, discrimination training was conducted every other day. AZD9272 caused a dose-dependent increase in AZD9272-appropriate responding. PCP did not cause AZD9272-appropriate responding, whereas MTEP, fenobam, and the mGluR5 antagonist AZD2066 did. The discriminative half-life of AZD9272 was 24.3 hours in rats trained to discriminate AZD9272 from no drug. It is concluded that the discriminative effects of AZD9272 and AZD2066 are similar to those of previously investigated mGluR5 antagonists and dissimilar to those of cocaine, PCP, chlordiazepoxide, and (-)-Δ(9)-THC. The discriminative half-life of AZD9272 is approximately 7-fold longer than for MTEP. These data support and extend previous findings suggesting that mGluR5 antagonism causes psychoactive effects selectively mediated by mGluR5 mechanisms.

mGluR5 antagonist-induced psychoactive properties: MTEP drug discrimination, a pharmacologically selective non-NMDA effect with apparent lack of reinforcing properties.

Fenobam [N-(3-chlorophenyl)-N'-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea], a potent metabotropic glutamate mGluR5 receptor antagonist, reported to have analgesic effects in animals and anxiolytic effects in humans, also caused adverse events, including psychostimulant-type effects and "derealization phenomena." Recent electrophysiologic, pharmacologic, and anatomic data show that the mGluR5 antagonists 2-methyl-6-(phenylethynyl)pyridine (MPEP) and (E)-2-methyl-6-styryl-pyridine (SIB-1893) can inhibit NMDA receptor-mediated activity and that mGluR5 receptors are highly expressed in limbic and forebrain regions. The present studies first evaluated the potential of mGluR5 receptor antagonists to cause PCP-like psychoactive effects in a rat drug discrimination procedure and, second, explored and characterized the selective mGluR5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) as a discriminative stimulus and compared MTEP with other drugs known to be psychoactive in humans. Additionally, the reinforcing potential of MPEP and MTEP was compared with phencyclidine (PCP) in a rat intravenous self-administration procedure. Dizocilpine [(+)-MK-801] and ketamine caused full PCP-appropriate responding. Memantine and the mGluR5 antagonists caused no or weak partial PCP-appropriate responding. In MTEP-trained rats, MTEP, MPEP, and fenobam caused full and equipotent MTEP-appropriate responding. (+)-MK-801 and memantine caused MTEP-appropriate responding below 70%, whereas PCP, chlordiazepoxide and LSD caused MTEP-appropriate responding below 50%. Δ(9)-Tetrahydrocannabinol, yohimbine, arecoline, and pentylenetetrazole all caused MTEP-appropriate responding below 20%. Rats self-administered PCP but not MPEP or MTEP, indicating a lack of reinforcing effects of the mGluR5 antagonists. These data suggest that the mGluR5 antagonists appear not to have reinforcing properties, that the discriminative effects of mGluR5 antagonists and PCP are dissimilar, and that mGluR5 antagonists may produce psychoactive effects different from NMDA-antagonists and other drugs with known psychotomimetic properties.

Prediction and modeling of effects on the QTc interval for clinical safety margin assessment, based on single-ascending-dose study data with AZD3839.

Corrected QT interval (QTc) prolongation in humans is usually predictable based on results from preclinical findings. This study confirms the signal from preclinical cardiac repolarization models (human ether-a-go-go-related gene, guinea pig monophasic action potential, and dog telemetry) on the clinical effects on the QTc interval. A thorough QT/QTc study is generally required for bioavailable pharmaceutical compounds to determine whether or not a drug shows a QTc effect above a threshold of regulatory interest. However, as demonstrated in this AZD3839 [(S)-1-(2-(difluoromethyl)pyridin-4-yl)-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3-amine hemifumarate] single-ascending-dose (SAD) study, high-resolution digital electrocardiogram data, in combination with adequate efficacy biomarker and pharmacokinetic data and nonlinear mixed effects modeling, can provide the basis to safely explore the margins to allow for robust modeling of clinical effect versus the electrophysiological risk marker. We also conclude that a carefully conducted SAD study may provide reliable data for effective early strategic decision making ahead of the thorough QT/QTc study.

A proactive nonclinical drug abuse and dependence liability assessment strategy: a sponsor perspective.

This paper outlines a strategy and process for proactive nonclinical assessment of drug abuse and dependence liability of new compounds intended for clinical use. Documentation of the potential for causing abuse and dependence liability is required for registration of a new drug; hence, proactive timing and planning of these studies allows for appropriate documentation of nonclinical as well as clinical data in time for registration. In cases for which an abuse and dependence liability label may not be acceptable, a proactive approach to abuse and dependence liability assessment allows for replacement of selected compounds at an early stage of development, thereby saving time and resources and avoiding late attrition.

Effects of grazing management in brachiaria grass-forage peanut pastures on canopy structure and forage intake1.

Maintenance of mixed grass-legume pastures for stand longevity and improved animal utilization is a challenge in warm-season climates. The goal of this study was to assess grazing management on stand persistence, forage intake, and N balance of beef heifers grazing mixed pastures of Brachiaria brizantha and Arachis pintoi. A 2-yr experiment was carried out in Brazil, where four grazing management were assessed: rest period interrupted at 90%, 95%, and 100% of light interception (LI) and a fixed rest period of 42 d (90LI, 95LI, 100LI, and 42D, respectively). The LI were taken at 50 points at ground level and at 5 points above the canopy for each paddock using a canopy analyzer. For all treatments, the postgrazing stubble height was 15 cm. Botanical composition and canopy structure characteristics such as canopy height, forage mass, and vertical distribution of the morphological composition were evaluated pre- and post-grazing. Forage chemical composition, intake, and microbial synthesis were also determined. A randomized complete block design was used, considering the season of the year as a repeated measure over time. Grazing management and season were considered fixed, while block and year were considered random effects. In the summer, legume mass accounted for 19% of the canopy at 100LI, which was less than other treatments (a mean of 30%). The 100LI treatment had a greater grass stem mass compared with other treatments. In terms of vertical distribution for 100LI, 38.6% of the stem mass was above the stubble height, greater than the 5.7% for other treatments. The canopy structure limited NDF intake (P = 0.007) at 100LI (1.02% of BW/d), whereas 42D, 90LI, and 95LI treatments had NDF intake close to 1.2% of BW/d. The intake of digestible OM (P = 0.007) and the ratio of CP/digestible OM (P < 0.001) were less at 100LI in relation to the other treatments. The production of microbial N (P < 0.001) and efficiency of microbial synthesis (P = 0.023) were greater at 95LI and 90LI, followed by 42D and less at 100LI. Overall, the range from 90% to 95% of LI is the recommendation to interrupt the rest period, since this strategy enhanced community stability, forage intake, and nutritional value of the diet. Under on-farm conditions, brachiaria grass and forage peanut pastures should be managed at a range height of 24 to 30 cm.

Toward the total synthesis of spirastrellolide A. Part 3: intelligence gathering and preparation of a ring-expanded analogue.

Different methods for the formation of the C.25-C.26 bond of spirastrellolide A () are evaluated that might qualify for the end game of the projected total synthesis, with emphasis on metathetic ways to forge the macrocyclic frame.

The myxozoan fauna of Fundulus diaphanus (Cyprinodontidae) from freshwater localities in eastern North America: prevalence, community structure, and geographic distribution.

Membership and richness of infracommunities and component communities of myxozoan fauna of the banded killifish (Fundulus diaphanus) from freshwater localities in Ontario, Quebec, New York State, New Brunswick, Nova Scotia, and Maryland were studied. Five species of parasites were found: Myxobolus diaphanus (Fantham, Porter, and Richardson, 1940) (connective tissue throughout the body and head), Myxobolus funduli (Kudo, 1918) (interlamellar), Myxobolus neurophilus (Guilford, 1963) (optic tectum of the brain), Myxobolus sp. (connective tissue, typically adjacent to vertebrae), and Sphaerospora sp. (kidney tubules). The most abundant species locally and regionally was M. diaphanus, occurring at prevalences of 14.2 to 93.3% at 6 of 9 localities. Myxobolus funduli and Myxobolus sp. were at 3 and 2 localities respectively, while M. neurophilus and Sphaerospora each occurred at single localities. Four of the 5 myxozoans appear to be specific to fundulids, the exception being M. neurophilus, which is typically a parasite of Perca flavescens. Mean infracommunity richness was 0-1.2. Component community richness was 0-3 species. The fauna is similar in composition to that described from the spottail shiner (Notropis hudsonius) in the Great Lakes in being dominated by histozoic myxobolids and in having maximum prevalence at any single locality correlate positively with geographical distribution. Moreover, mean infracommunity richness was correlated with percentage of hosts infected with any species at a locality, and maximum infracommunity richness was correlated with component community richness. Probably because fewer species of myxozoans of fundulids occur in the regional pool, myxozoan communities encountered in the present study are generally less rich than those described from N. hudsonius. It appears that dispersal of relatively resilient myxospores through such a mechanism as piscivory effectively distributes these parasites over the landscape, while the more delicate actinospores serve to ensure colonization by amplifying species' prevalence at a specific locality and thereby contributing to initial establishment. As such, these types of myxozoans, though they are autogenic, having their entire life cycle normally completed within the aquatic environment, behave more like allogenic parasites that rely on birds and mammals as definitive hosts.

Drug discrimination: A versatile tool for characterization of CNS safety pharmacology and potential for drug abuse.

Drug discrimination studies for assessment of psychoactive properties of drugs in safety pharmacology and drug abuse and drug dependence potential evaluation have traditionally been focused on testing novel compounds against standard drugs for which drug abuse has been documented, e.g. opioids, CNS stimulants, cannabinoids etc. (e.g. Swedberg & Giarola, 2015), and results are interpreted such that the extent to which the test drug causes discriminative effects similar to those of the standard training drug, the test drug would be further characterized as a potential drug of abuse. Regulatory guidance for preclinical assessment of abuse liability by the European Medicines Agency (EMA, 2006), the U.S. Food and Drug Administration (FDA, 2010), the International Conference of Harmonization (ICH, 2009), and the Japanese Ministry of Health Education and Welfare (MHLW, 1994) detail that compounds with central nervous system (CNS) activity, whether by design or not, need abuse and dependence liability assessment. Therefore, drugs with peripheral targets and a potential to enter the CNS, as parent or metabolite, are also within scope (see Swedberg, 2013, for a recent review and strategy). Compounds with novel mechanisms of action present a special challenge due to unknown abuse potential, and should be carefully assessed against defined risk criteria. Apart from compounds sharing mechanisms of action with known drugs of abuse, compounds intended for indications currently treated with drugs with potential for abuse and or dependence are also within scope, regardless of mechanism of action. Examples of such compounds are analgesics, anxiolytics, cognition enhancers, appetite control drugs, sleep control drugs and drugs for psychiatric indications. Recent results (Swedberg et al., 2014; Swedberg & Raboisson, 2014; Swedberg, 2015) on the metabotropic glutamate receptor type 5 (mGluR5) antagonists demonstrate that compounds causing hallucinatory effects in humans did not exhibit clear discriminative effects when tested against classical drugs of abuse in drug discrimination studies, and were not self-administered by rats. However, these compounds did cause salient discriminative effects of their own in animals trained to discriminate them from no drug. Therefore, from a safety pharmacology perspective, novel compounds that do not cause discriminative effects similar to classical drugs of abuse, may still cause psychoactive effects in humans and carry the potential to maintain drug abuse, suggesting that proactive investigation of drug abuse potential is warranted (Swedberg, 2013). These and other findings will be discussed, and the application of drug discrimination procedures beyond the typical standard application of testing novel compounds against known and well characterized reference drugs will be addressed.

Anticonvulsant prophylaxis and timing of seizures after aneurysmal subarachnoid hemorrhage.

OBJECTIVE: There is no evidence that seizure prophylaxis is indicated after aneurysmal subarachnoid hemorrhage (SAH). This study examines prophylactic antiepileptic drug (AED) prescription and the occurrence of seizures within a single university-affiliated institution. METHODS: The authors reviewed 95 SAH patient charts using standardized forms. Variables included prophylaxis duration, seizure incidence and timing, CT findings, AED adverse events, and 1-year patient follow-up. RESULTS: Prehospital seizures occurred in 17.9% (17/95) of patients; another 7.4% (7/95) had a questionable prehospital seizure. In-hospital seizures occurred in 4.1% (4/95) of patients, a mean of 14.5 +/- 13.7 days from ictus; three of these four patients were receiving an AED at the time of seizure. Inpatient AED were prescribed to 99% of the cohort for a median of 12 (range 1 to 68) days. Approximately 8% of the cohort had posthospital discharge seizures; this included the patients who had prehospital or in-hospital seizures, 50% of whom were receiving AED therapy at the time of the seizure. Adverse effects occurred in 4. 1%; none were serious. The thickness of cisternal clot was associated with having a seizure; no other clinical predictors were identified. Having a seizure at any time did not adversely affect outcome. CONCLUSIONS: In this SAH population, the majority of seizures happened before medical presentation. In-hospital seizures were rare and occurred more than 7 days postictus for patients receiving AED prophylaxis. The vast majority of putative clinical predictors did not help predict the occurrence of seizures; only the thickness of the cisternal clot was of value in predicting seizures. Patient selection for and the efficacy and timing of AED prophylaxis after SAH deserve prospective evaluation.

Coincidence of head and cervical spine injury.

The association of head and cervical spine injury has long been recognized. Reports of the coincidence of these injuries in the literature range from 1.2 to 19%. This study was undertaken to determine the coincidence and examine the mechanisms of head and cervical spine injuries at a major trauma hospital. All cases of cervical spine injury (CSI) and head injury (HI) admitted to The Detroit Receiving Hospital during 1987 were identified using the hospital computer data bank and spine unit log book. There were 359 admissions for CSI including concussion, skull fracture, and intracranial hemorrhage. There were 92 admissions for CSI including complete or incomplete neurologic syndromes with fractures. There were 22 patients with both HI and CSI. Thus the coincidence of "primary" CSI with HI (both/CSI) is 24% while the coincidence of primary HI with CSI (both/HI) is 6%. The variety of injuries is demonstrated with appropriate imaging studies. Our study supports the view that all seriously head-injured patients should be treated as if a concomitant cervical spine injury is present until proven otherwise. It also shows that nearly one-quarter of patients with cervical spine injury have also experienced head injury. This coincidence may be an important consideration in the rehabilitation of such injured patients.

Patterns of heat-shock protein 70 biosynthesis following human traumatic brain injury.

Heat-shock protein 70 (hsp70) is activated upon cellular stress/injury and participates in the folding and intracellular transport of damaged proteins. The expression of hsp70 following CNS trauma has been speculated to be part of a cellular response which is involved in the repair of damaged proteins. In this study, we measured hsp70 mRNA and protein levels within human cerebral cortex subjected to traumatic brain injury. Specimens were obtained during routine neurosurgery for trauma and processed for Northern mRNA and Western protein analysis. The largest increase in hsp70 mRNA levels was detected in trauma tissue obtained 4-6 h following injury. By 24 h, hsp70 mRNA levels were similar to nontrauma comparison tissues. hsp70 protein expression exhibited its greatest increases at 12-20 h post-injury. Immunocytological techniques revealed hsp70 protein expression in cells with neuronal-like morphology at 12 h after injury. These results suggest a role for hsp70 in human cortex following TBI. Moreover, since the temporal induction pattern of hsp70 biosynthesis is similar to that reported in the rodent, our observations validate the importance of rodent brain injury models in providing useful information directly applicable to human brain injury.

Heat-shock protein 72 expression in excitotoxic versus penetrating injuries of the rodent cerebral cortex.

The induction of heat shock protein 72 (hsp72) has been described in various experimental models of brain injury. The present study examined hsp72 expression patterns within the rodent cerebral cortex in experimental paradigms designed to mimic two mechanisms of damage produced by penetration of the cerebral cortex: (1) tissue tearing from the missile track and (2) diffuse excitotoxicity during temporary cavitation and shock wave formation. Adult male Spaque-Dawley rats received controlled penetration (stab) or injection of the NMDA receptor excitotoxin, quinolinic acid (QA), into the frontal cortex and were killed 1-24 h later. Tissue from the lesioned, sham-operated, or contralateral uninjected cortex was processed for Western and immunocytochemical analyses of hsp72 protein expression. By 12 h, both controlled penetration and excitotoxic brain injuries produced significant increases in hsp72 immunoreactivity, which decreased toward control levels at 24 h. However, the severity and regional distribution of hsp72 expression varied between the two models. Specifically, the controlled penetration injury produced many hsp72-expressing cells near the needle track, while immunoreactive cells within the QA-injected cortex were found in the periphery of the lesion site. Morphological assessment of brain sections subjected to dual-labeling procedures demonstrated that cells expressing hsp72 were primarily neuronal in both models of injury. These results suggest that although controlled penetration and diffuse excitotoxicity may induce similar temporal and cellular patterns of hsp72 expression, the spatial location of hsp72-immunoreactive cells may differ between the two models.

A formal construction of fasicularin.

[reaction: see text] Our synthetic approach toward fasicularin is presented. Key steps in this construction are a siloxy-epoxide semipinacol rearrangement, a B-alkyl Suzuki reaction and an intramolecular S(N)2 reaction.

Innervation of the pituitary gland by supraependymal neurons.

Retrograde transport of horseradish peroxidase (HRP) was used to identify supraependymal neurons projecting to the pituitary gland in the hamster. Supraependymal neurons overlying the median eminence were labeled by HRP injections into the neural and intermediate lobes of the pituitary gland; no neurons were labeled following HRP injections confined to the anterior lobe. Supraependymal neurons innervating the pituitary gland may provide a means whereby cerebrospinal fluid-borne substances modulate neuro-intermediate lobe function.

Measurement of free fatty acids in cerebrospinal fluid from patients with hemorrhagic and ischemic stroke.

Free fatty acid (FFA) concentrations in cerebrospinal fluid (CSF) from patients with ischemic and hemorrhagic stroke (n=25) and in contemporary controls (n=73) were examined using HPLC. Concentrations of CSF FFAs from ischemic and hemorrhagic stroke patients obtained within 48 h of the insult were significantly greater than in control patients. Higher concentrations of polyunsaturated fatty acids (PUFAs) in CSF obtained within 48 h of insult were associated with significantly lower (P<0.05) admission Glasgow Coma Scale scores and worse outcome at the time of hospital discharge, using the Glasgow Outcome Scale (P<0.01).

Adult rhombencephalosynapsis. Case report.

Rhombencephalosynapsis (RS) is a relatively rare developmental disorder of the cerebellum in which the cerebellar hemispheres are fused across the midline without being separated by a cleft or the vermis. The condition may be associated with hydrocephalus and other intracranial and extracranial abnormalities. The authors report on the case of a symptomatic adult who was successfully treated with suboccipital decompression and duraplasty. A 39-year-old woman presented with intractable pain radiating from the thoracolumbar column to the occiput. A general examination yielded normal findings and a neurological examination revealed only subtle ataxia of tandem gait. The patient underwent magnetic resonance (MR) imaging, the results of which revealed an absent cerebellar vermis with fusion of the cerebellum and mild hydrocephalus. A cine-MR image obtained to evaluate her cerebrospinal fluid flow (CSF) revealed attenuated flow in the posterior fossa and cerebral aqueduct. Preoperative intracranial pressure (ICP) monitoring demonstrated no elevation of ICP (mean 4.3 mm Hg). The patient consented to undergo suboccipital craniectomy and duraplasty. Despite an increase in postoperative ICP (mean 10.77 mm Hg; difference from preoperative level according to a t-test, p = 0.002), the patient experienced symptomatic relief, which has persisted for 3 years. One year postoperatively, a cine-MR image was obtained, which revealed improvement in the patient's CSF dynamics. The authors conclude that, although RS may cause altered flow in the adult, their patient has experienced symptomatic relief, suggesting that her pain was related to local pressure in the posterior fossa.

Gene expression in neurotrauma.

It has been established that following injury to the central nervous system two types of damage take place, the initial insult and the secondary response to injury. This review will focus on the secondary molecular aspects of neurotrauma. These responses may be either deleterious or have protective effects upon the injured cell population. Molecular responses include the regulation of genes which change cellular architecture, up-regulate of growth factors, induce reparative stress responses, influence apoptosis and regulate the transcriptional process. The purpose of this study is to provide the reader with a brief overview of some of the molecular mechanisms which are activated following a neurological insult.

Post-traumatic hydrocephalus.

The syndrome of post-traumatic hydrocephalus (PTH) has been recognized since Dandy's report in 1914. The incidence of symptomatic PTH ranges from 0.7%-29%. If CT criteria of ventriculomegaly are used the incidence has been reported to be from 30%-86%. Differences in diagnostic criteria and classification have contributed to the variation in reported incidence. The diagnosis of PTH is established using a combination of clinical, imaging and physiologic data. Symptomatic PTH is to be distinguished from post-traumatic ventriculomegaly resulting from atrophy. Symptomatic PTH patients are likely to improve when treated by shunting. Ventriculomegaly secondary to atrophy is less likely to respond to shunting. A series of traumatic brain injury patients at Wayne State University has been followed since 1989. The overall incidence of shunt placement in this group is 3.65%. Future studies of PTH should be aimed at refining diagnostic classification and criteria. Analysis of a large PTH population may then identify alterable risk factors in the early post-traumatic brain injury period. Minimizing these factors will help prevent subsequent PTH and obviate the need for shunting.

An unusual subdural empyema: case report.

Subdural empyema in a 38-year-old patient with congenital hemangioma, suppurative parotitisis, soft tissue phlegmonia and osteomyelitis is reported. The clinical, radiological and surgical features are outlined. A review of the literature reveals the uniqueness of this case.