Impact of reduced-representation sequencing protocols on detecting population structure in a threatened marsupial.

Reduced-representation sequencing methods have wide utility in conservation genetics of non-model species. Several methods are now available that reduce genome complexity to examine a wide range of markers in a large number of individuals. We produced two datasets collected using different laboratory techniques, comprising a common set of samples from the greater bilby (Macrotis lagotis). We examined the impact of differing data filtering thresholds on downstream population inferences. We found that choice of restriction enzyme and data filtering thresholds, especially the rate of allowable missing data, impacted our ability to detect population structure. Estimates of FST were robust to alterations in laboratory and bioinformatic protocols while principal coordinates and STRUCTURE analyses showed variation according to the number of loci and percent missing data. We advise researchers using reduced-representation sequencing in conservation projects to examine a range of data thresholds, and follow these through to downstream population inferences. Multiple measures of population differentiation should be used in order to fully understand how data filtering thresholds influence the final dataset, paying particular attention to the impact of allowable missing data. Our results indicate that failure to follow these checks could impact conclusions drawn, and conservation management decisions made.

Intracranial self-stimulation and the rapid decline of frustrative nonreward.

Animals deprived of reward for a task previously rewarded behave differently, depending on whether the reward is food or positive brain stimulation. Unlike the relatively stable frustration effects obtained with conventional reward, frustration produced by withholding brain stimulation dissipates rapidly with time.

DNA based method for determining source country of the short beaked echidna (Tachyglossus aculeatus) in the illegal wildlife trade.

The illegal trade in wild animals being sold as 'captive bred' is an emerging issue in the pet and zoo industry and has both animal welfare and conservation implications. DNA based methods can be a quick, inexpensive, and definitive way to determine the source of these animals, thereby assisting efforts to combat this trade. The short beaked echidna (Tachyglossus aculeatus) is currently one of the species suspected to be targeted in this trade. As this species is distributed throughout Australia and in New Guinea (currently comprising of five recognised sub-species), this project aimed to develop a DNA based method to definitively determine the source country of an echidna and explore the use of non-invasive sampling techniques. Here we use non-invasively sampled echidna quills and demonstrate the extraction of mitochondrial DNA and amplification of a region of the mitochondrial genome. Phylogenetically informative markers for analysis of a 322bp segment of the D-loop region were developed, and subsequently validated, using animals with known source locations allowing us to reliably distinguish between echidnas from New Guinea, and Australia. This research presents the first validated forensic protocols for short beaked echidnas and will be an integral tool in understanding the movement of animals in this emerging trade.

Depletion of myocardial creatine kinase, lactate dehydrogenase, myoglobin and K+ after coronary artery ligation in dogs.

In order to compare the time-course of disappearance of macromolecules and electrolytes from ischaemic myocardium, measurements of creatine kinase and lactate dehydrogenase activity and myoglobin, K+ and Na+ concentration were made on myocardial extracts from dogs which had left anterior descending coronary artery ligation for 3, 6, 12 and 24 h (4 groups of 6 dogs each). Intensity of ischaemia was assessed by myocardial blood flow measured with 15+/- 5 micrometers microspheres at 15 min after ligation. Creatine kinase and lactate dehydrogenase activities and K+/Na+ concentration ratios were at all times correlated with the magnitude of collateral blood flow in the ischaemic myocardium, while myoglobin concentration was correlated with blood flow only at 12 and 24h. Comparisons of the intensity of depletion at the various times after ligation showed that K+, K+/Na+ and creatine kinase had all reached a steady state at 12 h after ligation while lactate dehydrogenase and myoglobin had still to reach a steady state at 24 h. We conclude that these indices are mutually supportive markers of the intensity of ischaemia of 24 h duration, but K+ or K+/Na+ may be the most reliable indices for shorter periods of ischaemia of 3 to 6 h duration.

Severity of canine myocardial infarcts in relation to indices of oxygen demand: preservation of myocardial creatine kinase activity by vagal stimulation and propranolol.

The left anterior descending coronary artery was ligated in 58 open-chest anaesthetised dogs; 23 were controls, 15 were given intravenous propranolol 1 mg . kg-1 before and at 6 h intervals after coronary ligation, nine had bilateral cervical vagal nerve stimulation (VS) before and for 4 to 6 h after coronary ligation, and 11 had both VS and propranolol. None of the 20 dogs undergoing VS developed ventricular fibrillation within the first hour after coronary ligation compared to nine of the remaining 38 (P less than 0.05). Compared to controls, myocardial creatine kinase (CK) depletion in the epicardial layer of the infarct centre measured 24 h after coronary ligation was significantly less in the groups treated separately with vagal nerve stimulation and propranolol. Myocardial blood flow (MBF) measured at 15 min after coronary ligation was reduced to the normal myocardium by the interventions, but was unchanged at the infarct centre. Severely ischaemic myocardium (MBF less than or equal to 20% of normal) was better protected by the interventions than was moderately ischaemic myocardium. At 15 min after coronary ligation, the heart rate--blood pressure product (RPP) was reduced compared with controls by propranolol (18% reduction, P less than 0.05), reduced more by vagal stimulation (by 37%, P less than 0.001) and still more by vagal stimulation with propranolol (by 43%, P less than 0.001). Preservation of CK in myocardium with MBF less than or equal to 20% of normal was improved by VS and propranolol given separately roughly in proportion to reduction in RPP, but further reduction in RPP by VS and propranolol together did not improve CK levels further. We conclude that there may be an optimum level of indices of oxygen demand for preservation of very ischaemic myocardium in experimental infarction.

Effects of valproate and ethosuximide on thalamocortical excitability.

Sodium valproate and ethosuximide are anticonvulsants employed in the treatment of petit mal epilepsy; both drugs are considered to be thalamically active. Valproate and ethosuximide both decreased the average evoked response following the second of two stimuli delivered to the ventrolateral thalamus at stimulus frequencies in the region of 3 Hz. Ethosuximide, but not valproate, enhanced the average evoked response at high stimulus frequencies an action shared with several convulsant treatments having different modes of action. The clinical effects of valproate and ethosuximide can be related to this differential modulation of thalamocortical excitability.

Effects of antiepileptic drugs on thalamocortical excitability.

Comparative effects of anticonvulsant drugs on the thalamocortical system were analyzed quantitatively. Paired stimuli were delivered to the ventrolateral thalamus with evoked responses recorded from the ipsilateral sensorimotor cortex in the cat. Threshold and excitability profiles were developed with an on-line computer. Effects of phenytoin and diazepam were generally similar, with depression of excitability and slight elevation of thresholds. Ethosuximide produced a pronounced pair-interval dependent effect of unchanged or increased excitability and lowered threshold at shorter intervals, with depressed excitability and raised threshold at longer intervals. These data demonstrate a marked difference in effect of the petit mal and grand mal agents tested and suggest a basis for the effectiveness of ethosuximide in controlling 3-per-second repetitive activity.

Additional Plasmodium species from Anolis lizards of Hispaniola and Panama.

Saurian malaria parasites in the Caribbean were previously represented by only two species, Plasmodium azurophilum and P. floridense. An additional three species of Plasmodium occur on Hispaniola which appear, because of morphometric and qualitative similarities, to be related to South and Middle American species: a subspecies of Plasmodium tropiduri in Anolis cybotes; a population of Plasmodium minasense anolisi in A. cybotes and A. distichus; and another parasite in A. distichus which is designated as a subspecies of a previously undescribed Plasmodium species in Anolis limifrons. Taxonomic descriptions based upon morphology of mature schizonts and gametocytes are given, the previously unknown parasites are described as new species/subspecies, and comparison is made with P. floridense which also occurs in their respective host species. A Fallisia species was also present but is not described. The increased diversity of Plasmodium parasites in Hispaniolan anoles is attributed to colonization of the island by four separate evolutionary lines of Anolis lizards, in comparison to fewer invasions of Jamaica and Puerto Rico, where only P. floridense and P. azurophilum are found.

Intravitreal tissue plasminogen activator treatment of experimental vitreous hemorrhage.

Intraocular recombinant DNA human tissue-type plasminogen activator (tPA) can promote rapid resolution of intracular fibrin clots and hyphema. The efficacy of intravitreal tPA in promoting the short-term clearance of vitreous hemorrhage was investigated. Five treatment groups with experimentally induced vitreous hemorrhage were studied. The extent of the vitreous hemorrhage was graded based on the visibility of retinal details in each quadrant. Two weeks following intravitreal tPA injection (50,000 IU), the nonvitrectomized group and gas vitrectomy groups, each of which received a single tPA injection, showed a statistically significant reduction of the vitreous hemorrhage, although this was thought to represent only a modest clinical effect. Tractional retinal detachments were noted to develop more frequently in tPA-treated eyes as compared with controls.

Endolaser, cryopexy, and retinal reattachment in the air-filled eye. A clinicopathologic correlation.

We report the histopathology of intraocular argon blue-green laser photocoagulation lesions and transscleral cryopexy lesions in the air-filled human eye. Two days after treatment, the cryopexy lesions showed full-thickness retinal involvement, including disruption of the internal limiting lamina. The laser lesions showed full-thickness involvement as well. These observations emphasize the need for caution with thermal treatment modalities in the air-filled eye. Ophthalmoscopic examination 48 hours following vitrectomy, internal drainage of subretinal fluid, and gas fill of the vitreous cavity for diabetic macular detachment of several months' duration showed the retina to be attached. However, histopathologic examination revealed a thin layer of persistent subretinal fluid, demonstrating that it may take longer than is clinically apparent for true retinal reattachment to occur following gas tamponade of posterior retinal breaks. Prolonged gas tamponade may be necessary before retinal reattachment, with reestablishment of photoreceptor-pigment epithelial adherence, can be expected to help seal unrecognized or untreated posterior retinal breaks.

Histopathology of krypton red laser panretinal photocoagulation. A clinicopathologic correlation.

The histopathologic appearance of five-day-old and 14-day-old krypton red laser-created panretinal photocoagulation lesions in the diabetic human eye is described. The five-day-old lesions were examined near the equator. The 14-day-old lesions were near the temporal arcades and optic disc. Both groups of laser lesions showed a similar degree of damage and occlusion in the lamina choriocapillaris and deeper choroidal vessels. The 14-day-old posterior laser lesions showed outer retinal damage, whereas the five-day-old peripheral lesions showed full-thickness retinal involvement, probably because the peripheral retina is approximately half the thickness of the posterior retina. The five-day-old lesions showed no evidence of repair, whereas the 14-day-old lesions showed an adhesion formed with tight junctions binding Müller's fibers to retinal pigment epithelium.

Progressive enlargement of laser scars following grid laser photocoagulation for diffuse diabetic macular edema.

Laser photocoagulation in a grid pattern is effective in many eyes in resolving diffuse diabetic macular edema and stabilizing vision. We retrospectively studied 203 eyes of 203 patients, all of whom had diabetic macular edema treated with grid laser photocoagulation. During the follow-up period, 11 of these 203 eyes developed enlargement of the laser scars that progressed into the central fovea. Each of the 11 eyes experienced loss of vision, and in five, the visual loss was significant. Five of these eyes also developed a retinal pigment hyperplastic scar. After the laser treatment, but before the scars enlarged, the visual acuity of these eyes was unchanged from that before treatment. But after the atrophic scars enlarged into the central fovea, visual acuity in one eye decreased from 20/40 to 20/100, and the other 10 eyes had visual acuities of 20/200 or worse.

Fibrovascular tissue in bilateral juxtafoveal telangiectasis.

OBJECTIVE: To study the natural history and retinal findings associated with the intraretinal and subretinal fibrovascular tissues that develop in the late phases of bilateral juxtafoveal telangiectasis. METHODS: The records of 10 patients (11 eyes) with bilateral juxtafoveal telangiectasis who developed these fibrovascular tissues were examined. RESULTS: Throughout the follow-up period (average 44 months), only 2 eyes (18%) lost 2 or more lines of vision; the final visual acuities were similar for the eyes both with and without fibrovascular tissues. Sixty-four percent of fibrovascular tissues showed little to no growth. Eyes with fibrovascular tissue commonly had retinal pigment epithelial hyperplasia (72%), draining retinal venules (82%), and retinal vascular distortion (64%). CONCLUSIONS: Fibrovascular tissues of bilateral juxtafoveal telangiectasis have little proliferative potential and minimal effects on visual acuity. Nevertheless, these fibrovascular tissues do remodel over time, leading to retinal vascular distortion. Given these benign findings, the role of laser photocoagulation treatment of these tissues is questionable.

Transient hypopyon with marked anterior chamber fibrin following pars plana vitrectomy and silicone oil injection.

In a retrospective review of 30 cases of complex retinal detachment, which had been managed by pars plana vitrectomy techniques together with silicone oil injection, we found that two patients developed marked postoperative anterior chamber fibrin and hypopyon. In all 30 cases, the same silicone oil was used: purified and heat-sterilized trimethylsiloxy-terminated, polydimethylsiloxane of 1000-centistoke viscosity. Although the silicone oil itself remained clear and without emulsification, the differential diagnosis of this postoperative inflammation included microbial endophthalmitis. These two cases were treated with frequent topical steroids and antibiotics while we closely monitored the retraction of the anterior chamber fibrin. In both cases, clinical improvement and long-term retinal reattachment with satisfactory visual function were achieved. The use of intense scatter laser endophotocoagulation and the presence of impurities in the silicone oil may have contributed to the excessive postoperative inflammation in these cases.

Retinal toxicity of recombinant tissue plasminogen activator in the rabbit.

We studied the dose-dependent retinal toxicity of the available commercial preparation of human recombinant tissue plasminogen activator in the normal rabbit eye. Tissue plasminogen activator was injected into the midvitreous cavity of albino rabbits in doses (per 100 microL) of 25, 50, 75, and 100 micrograms. Control eyes received 100 microL of balanced salt solution or tissue plasminogen activator vehicle. No evidence of a retinal toxic reaction was seen in eyes receiving 25 micrograms of tissue plasminogen activator. One of four eyes injected with 50 micrograms showed loss of photoreceptor cells by light microscopy. Severe retinal damage was seen by ophthalmoscopy, electroretinography, and light microscopy in three of four eyes receiving 75 micrograms of tissue plasminogen activator and in all eyes treated with 100 micrograms of tissue plasminogen activator or equivalent vehicle. These results suggest that the commercial recombinant tissue plasminogen activator formulation has a narrow margin of safety in nonvitrectomized eyes and that a component of the vehicle is the toxic factor.

Crystalline retinopathy associated with chronic retinal detachment.

OBJECTIVE: To describe the presence of crystalline opacities located at the level of the inner retina in patients with chronic retinal detachment. METHODS: We reviewed the clinical records, fundus photographs, and fluorescein angiograms of patients with superficial retinal crystals in the presence of a chronic retinal detachment. RESULTS: Eleven eyes in 11 patients with chronic retinal detachment were found to have these peculiar crystalline opacities on the inner retinal surface. In 5 patients, the crystalline opacities were noted on initial assessment prior to surgery and persisted without change in appearance or number after surgical repair. In 6 eyes, the crystals were not appreciated until after surgical repair of the retinal detachment. The crystals appeared similar in all 11 eyes, were highly refractile, and were located in the posterior pole. Eight of the eyes had retinal detachment associated with retinal dialysis and 6 of these had a history of trauma. There was a definite history of vitreous hemorrhage in 2 eyes. The crystals did not seem to be associated with any visual deficit. CONCLUSIONS: Chronic retinal detachment can be associated with crystals on the inner retinal surface. The cause and composition of these crystals are unknown. They seem to be visually inconsequential and unchanging.

Echographic findings in benign reactive lymphoid hyperplasia of the choroid.

BACKGROUND: Benign reactive lymphoid hyperplasia is an uncommon uveal tumor. Misdiagnosis may lead to unnecessary enucleations. Clinical evaluation alone is usually not sufficient to diagnose this tumor. OBJECTIVE: To evaluate the utility of standardized echography in the recognition of this disease. METHODS: Retrospective review of the medical and echographic records of 6 patients with choroidal involvement of benign reactive lymphoid hyperplasia. RESULTS: Extraocular episcleral lymphoid nodules were present in 5 of the 6 patients and were usually seen as a late manifestation of the disease. Two patients had repeated echography after treatment of the tumor and showed marked regression of the episcleral nodules. CONCLUSIONS: A combination of clinical and echographic findings can assist in the accurate diagnosis of this rare uveal tumor. The presence of episcleral nodules on standardized echography supports the diagnosis.

Possible determination of the vector and reservoir of leishmaniasis in the Dominican Republic.

Two species of sand flies were collected by various methods from sites in the Dominican Republic. Lutzomyia cayennensis hispaniolae was the more common of the two. It was found in wooded habitats from sea level to an elevation of 442 m. This species was observed feeding on lizards (Anolis sp.) in the wild. In the laboratory, it fed only on lizards and only under lighted conditions. The other species, Lu. christophei was only found in the vicinity of seven leishmaniasis case sites. It readily fed on or probed rodents and humans. Although no naturally infected sand flies were collected, in the laboratory Lu. christophei was readily capable of transmitting the Dominican Leishmania parasite to uninfected BALB/c mice. We collected 167 specimens of three species of rodents and three Herpestes auropunctatus (mongoose) from the vicinity of two case sites. All four species are non-endemics introduced in post-Columbian times. Although we were unable to isolate parasites from any of these specimens, four of 44 Rattus rattus from one case site were seropositive for antibodies against Leishmania by indirect fluorescent antibody testing. This represents the first report of transmission of the Dominican Leishmania parasite by a sympatric species of sand fly and suggests that commensal rodents may play a role in the epidemiologic cycle.

Phlebotomine sand fly (Diptera: Psychodidae) seasonal distribution and infection rates in a defined focus of Leishmania tropica.

A two-year study was conducted of phlebotomine sand fly fauna in a defined focus of Leishmania tropica. A total of 17,947 sand flies representing 10 species were collected from the location. Phlebotomus guggisbergi, a vector of L. tropica in Kenya, was the most prevalent species through the entire period, representing about 80% of the total catch. There was marked seasonal fluctuation in the populations of the three most common species, with highest population levels reached in December and lowest levels reached in July and August. Leishmania-like infections were encountered in 489 P. guggisbergi. No flagellate infections were observed in any other species of sand fly. Although infected P. guggisbergi were collected during each month of the year, the percent parous infected flies was highest (27.5%) during the November through January time period. These data show that the greatest risk of transmission to humans at this focus occurs during December, when the vector is prevalent and infections are common.

Response of medullary neurons to step changes in carotid sinus pressure.

Moderate amplitude step changes in carotid sinus pressure were found, in an open-loop baroreflex preparation, to produce equivalent step changes in the firing rate of a majority of the medullary neurons tested. The average firing rate remained constant so long as the pressure was held constant (up to 24 sec). No rate sensitivity was observed in 32 out of 33 neurons; however, a single neuron showed mean firing rate changes only during pressure transitions. The variety in the activity patterns of cells which, on the average exhibited stereotyped responses to a simple repetitive stimulus, suggests that afferent information may undergo considerable integration and restructuring within the sensory nucleus, rather than being 'relayed' intact to other regions.