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Invasive lionfish (Pterois volitans/miles) pose a serious threat to marine ecosystems throughout the western Atlantic Ocean and Caribbean Sea. The development of a fishery for lionfish has been proposed as a strategy for controlling populations; however, there is concern about consumption of this species by humans due to its high trophic position and potential for bioaccumulation of mercury. We analyzed total mercury (THg) in tissues of lionfish from two locations on the east coast of Florida. THg in lionfish increased with size and differed by location and sex. THg was highest in muscle tissue and was strongly positively correlated among tissues. THg in lionfish was lower than other commonly consumed marine fishes, and falls into Florida’s least restrictive advisory level. Consumption of lionfish poses a low risk and concerns over mercury bioaccumulation should not present a significant barrier to lionfish harvest.

We reviewed the results of testing in patients from the West of Scotland attending adult epilepsy clinics with MRI-negative drug-resistant epilepsies or epilepsies with co-morbid intellectual disability,Methods45 eligible (>18y) cases were referred for testing. Clinicians completed a structured electronic clinical referral proforma to aid variant interpretation. Cases were tested on a custom-designed 104-gene panel focusing on disorders for which MRI would not provide a diagnosis. Variants were classified using UK Association of Clinical Genetic Science Guidelines. Variants of uncertain significance (VUS), likely pathogenic, and definitely pathogenic variants were discussed by a multidisciplinary team. Diagnostic variants were confirmed by Sanger sequencing.ResultsAmong 45 cases, likely pathogenic and pathogenic (diagnostic) variants were identified in 8 (18%), with VUS in 10 (22%). Pathogenic variants were identified in the following genes; GAMT, CHRNB2, SCN2A, PCDH19, PRRT2, SLC2A1, SCARB2, and SBTX1B.All cases with abnormal results or VUS were offered referral to a Genetic Epilepsy Clinic for counselling. Genetic findings altered therapy in several cases: some with VUS became seizure-free with targeted changes in treatment. Determination of clinical significance was sometimes helped by further familial testing.ConclusionGenetic testing is an essential tool in the management of adult patients with drug-resistant epilepsy, particularly those with childhood onset and/or accompanying learning difficulties. Abnormalities can be expected in approximately 40% using these criteria, which can aid both management and family counselling.

Achievement of effective, safe and long-term immunosuppression represents one of the challenges in experimental allogeneic and xenogeneic cell and organ transplantation. The goal of the present study was to develop a reliable, long-term immunosuppression protocol in Sprague-Dawley (SD) rats by: 1) comparing the pharmacokinetics of four different subcutaneously delivered/implanted tacrolimus (TAC) formulations, including: i) caster oil/saline solution, ii) unilamellar or multilamellar liposomes, iii) biodegradable microspheres, and iv) biodegradable 3-month lasting pellets; and 2) defining the survival and immune response in animals receiving spinal injections of human neural precursors at 6 weeks to 3 months after cell grafting. In animals implanted with TAC pellets (3.4 mg/kg/day), a stable 3-month lasting plasma concentration of TAC averaging 19.1 +/- 4.9 ng/ml was measured. Analysis of grafted cell survival in SOD + or spinal trauma-injured SD rats immunosuppressed with 3-month lasting TAC pellets (3.4-5.1 mg/kg/day) showed the consistent presence of implanted human neurons with minimal or no local T-cell infiltration. These data demonstrate that the use of TAC pellets can represent an effective, long-lasting immunosuppressive drug delivery system that is safe, simple to implement and is associated with a long-term human neural precursor survival after grafting into the spinal cord of SOD+ or spinal trauma-injured SD rats. Published by Elsevier Inc.

In previous studies, we have demonstrated that spinal grafting of human or rat fetal spinal neural precursors leads to amelioration of spasticity and improvement in ambulatory function in rats with spinal ischemic injury. In the current study, we characterize the survival and maturation of three different human embryonic stem (ES) cell line-derived neural precursors (hNPCs) once grafted into ischemia-injured lumbar spinal cord in rats or in naive immunosuppressed minipigs. Proliferating HUES-2, HUES-7, or HUES-9 colonies were induced to form embryoid bodies. During the nestin-positive stage, the rosettes were removed and CD184+/CD271-/CD44-/CD24+population of ES-hNPCs FAC-sorted and expanded. Male Sprague–Dawley rats with spinal ischemic injury or naive immunosuppressed Gottingen–Minnesota minipigs received 10 bilateral injections of ES-NPCs into the L2–L5 gray matter. After cell grafting, animals survived for 2 weeks to 4.5 months, and the presence of grafted cells was confirmed after staining spinal cord sections with a combination of human-specific (hNUMA, HO14, hNSE, hSYN) or nonspecific (DCX, MAP2, CHAT, GFAP, APC) antibodies. In the majority of grafted animals, hNUMA-positive grafted cells were identified. At 2–4 weeks after grafting, double-labeled hNUMA/ DCX-immunoreactive neurons were seen with extensive DCX+processes. At survival intervals of 4–8 weeks, hNSE+neurons and expression of hSYN was identified. Some hSYN-positive terminals formed putative synapses with the host neurons. Quantitative analysis of hNUMA+cells at 2 months after grafting showed comparable cell survival for all three cell lines. In the presence of low-level immunosuppression, no grafted cell survival was seen at 4.5 months after grafting. Spinal grafting of proliferating pluripotent HUES-7 cells led to consistent teratoma formation at 2–6 weeks after cell transplantation. These data show that ES-derived, FAC-sorted NPCs can represent an effective source of human NPCs to be used in CNS cell replacement therapies.

Background A proportion of patients with seemingly “uncontrolled” epilepsy could still control their epilepsy with further pharmacological manipulations. It is possible that their epilepsy might not be truly “drug-resistant”. We audited the patients with “uncontrolled epilepsy” using the recent ILAE definition of drug-resistant epilepsy. Methods Patients with newly diagnosed epilepsy at Glasgow and patients with chronic epilepsy treated in Hong Kong were independently assessed at their last clinic visit. If the patient was not seizure-free, the epilepsy was considered “uncontrolled”. In this latter situation, if the patient had adequate trials of two or more tolerated, appropriately chosen, and appropriately used AED schedules, the epilepsy was classified as “drug-resistant” in accordance with the ILAE definition. If not, the outcome was classified as “undefined”, and the reason(s) for this was documented. Results In the newly diagnosed cohort with uncontrolled epilepsy (n = 311), outcome was “undefined” in 175 (56%). The most common reasons were trying just one AED usually at the patient's behest (n = 68; 39%); intermittent compliance (60; 34%); adverse effects at low dosage (51; 29%); inadequate dosing (49; 28%); social issues such as imprisonment, alcohol, and recreational drug use (34; 19%); psychiatric problems affecting documentation, attendance, etc. (32; 18%); patient choice accepting less than optimal control (14; 8%); and seizure freedom of less than 12 months (12.7%). In the chronic cohort of 194 patients with uncontrolled epilepsy, drug responsiveness was “undefined” in just 79 (41%). The most common reasons were inadequate use of the AED(s) (35; 44%), followed by a lack of information on treatment response in the medical records (18; 23%) and failure of only one adequately used AED (11; 14%). Conclusion Uncontrolled epilepsy is not necessarily the same as drug-resistant epilepsy. Efforts should be made to understand why a patient is not seizure-free so that appropriate adjustment in AED regimen can be taken to enable the patient to attain long-term seizure freedom.

Introduction Intraspinal grafting of human neural stem cells represents a promising approach to promote recovery of function after spinal trauma. Such a treatment may serve to: I) provide trophic support to improve survival of host neurons; II) improve the structural integrity of the spinal parenchyma by reducing syringomyelia and scarring in trauma-injured regions; and III) provide neuronal populations to potentially form relays with host axons, segmental interneurons, and/or α-motoneurons. Here we characterized the effect of intraspinal grafting of clinical grade human fetal spinal cord-derived neural stem cells (HSSC) on the recovery of neurological function in a rat model of acute lumbar (L3) compression injury. Methods Three-month-old female Sprague–Dawley rats received L3 spinal compression injury. Three days post-injury, animals were randomized and received intraspinal injections of either HSSC, media-only, or no injections. All animals were immunosuppressed with tacrolimus, mycophenolate mofetil, and methylprednisolone acetate from the day of cell grafting and survived for eight weeks. Motor and sensory dysfunction were periodically assessed using open field locomotion scoring, thermal/tactile pain/escape thresholds and myogenic motor evoked potentials. The presence of spasticity was measured by gastrocnemius muscle resistance and electromyography response during computer-controlled ankle rotation. At the end-point, gait (CatWalk), ladder climbing, and single frame analyses were also assessed. Syrinx size, spinal cord dimensions, and extent of scarring were measured by magnetic resonance imaging. Differentiation and integration of grafted cells in the host tissue were validated with immunofluorescence staining using human-specific antibodies. Results Intraspinal grafting of HSSC led to a progressive and significant improvement in lower extremity paw placement, amelioration of spasticity, and normalization in thermal and tactile pain/escape thresholds at eight weeks post-grafting. No significant differences were detected in other CatWalk parameters, motor evoked potentials, open field locomotor (Basso, Beattie, and Bresnahan locomotion score (BBB)) score or ladder climbing test. Magnetic resonance imaging volume reconstruction and immunofluorescence analysis of grafted cell survival showed near complete injury-cavity-filling by grafted cells and development of putative GABA-ergic synapses between grafted and host neurons. Conclusions Peri-acute intraspinal grafting of HSSC can represent an effective therapy which ameliorates motor and sensory deficits after traumatic spinal cord injury.

Background Due to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions. Methodology/Principal Findings HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions. Conclusions/Significance The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types, identification and isolation of stem cell subpopulations, and generation of single cell clones. Finally, these results demonstrate an additional application of ROCK inhibition to hESC research.