The Search for New Screening Models of Pharmacoresistant Epilepsy: Is Induction of Acute Seizures in Epileptic Rodents a Suitable Approach?

Epilepsy, a prevalent neurological disease characterized by spontaneous recurrent seizures (SRS), is often refractory to treatment with anti-seizure drugs (ASDs), so that more effective ASDs are urgently needed. For this purpose, it would be important to develop, validate, and implement new animal models of pharmacoresistant epilepsy into drug discovery. Several chronic animal models with difficult-to-treat SRS do exist; however, most of these models are not suited for drug screening, because drug testing on SRS necessitates laborious video-EEG seizure monitoring. More recently, it was proposed that, instead of monitoring SRS, chemical or electrical induction of acute seizures in epileptic rodents may be used as a surrogate for testing the efficacy of novel ASDs against refractory SRS. Indeed, several ASDs were shown to lose their efficacy on acute seizures, when such seizures were induced by pentylenetetrazole (PTZ) in epileptic rather than nonepileptic rats, whereas this was not observed when using the maximal electroshock seizure test. Subsequent studies confirmed the loss of anti-seizure efficacy of valproate against PTZ-induced seizures in epileptic mice, but several other ASDs were more potent against PTZ in epileptic than nonepileptic mice. This was also observed when using the 6-Hz model of partial seizures in epileptic mice, in which the potency of levetiracetam, in particular, was markedly increased compared to nonepileptic animals. Overall, these observations suggest that performing acute seizure tests in epileptic rodents provides valuable information on the pharmacological profile of ASDs, in particular those with mechanisms inherent to disease-induced brain alterations. However, it appears that further work is needed to define optimal approaches for acute seizure induction and generation of epileptic/drug refractory animals that would permit reliable screening of new ASDs with improved potential to provide seizure control in patients with pharmacoresistant epilepsy.     

Evidence-based methodology for obtaining commercial insurance coverage of stereotactic radiosurgery for intractable epilepsy

ObjectivesThe coverage policies of many commercial insurers in the United States do not include coverage of stereotactic radiosurgery (SRS) for intractable epilepsy despite recent Level I evidence supporting its efficacy. We sought to assess the efficacy of an evidence-based methodology in obtaining coverage approval of SRS for intractable epilepsy.Patients and MethodsThe clinical policy guidelines from five of the largest United States commercial insurers were reviewed for their language regarding coverage of SRS for epilepsy. An evidence-based questionnaire was created for temporal lobe epilepsy and extratemporal lobe epilepsy based on recent evidence. Telephone interviewers of Insurers assessed the likelihood of SRS coverage for an epilepsy patient meeting the clinical inclusion criteria in the questionnaire. This likelihood was assessed numerically based on interviewee response (2 = yes, 1 = dependent on peer-to-peer, 0 = no).ResultsOf the five policy guidelines, none included literature more recent than 2017. For TLE, 3/5 insurance companies indicated likely SRS coverage; 2/5 indicated peer-to-peer discussion dependence for patients meeting questionnaire criteria for a score of 8/10. For extratemporal TLE, 2/5 companies indicated likely SRS coverage and 3/5 indicated peer-to-peer discussion dependence for a total score of 7/10.ConclusionCreation of an evidence-based methodology in approaching commercial insurers greatly increased the likelihood of SRS coverage for an indication (intractable epilepsy) widely perceived as investigational. These results should pave the way for epilepsy patients to receive coverage should they be appropriate SRS candidates.     

RASgrf1, a Potential Methylatic Mediator of Anti-epileptogenesis?

Epileptogenesis, induced by status epilepticus (SE), is a chronic process, and intervention in this progress may prevent chronic epilepsy. It has been proposed that DNA methylation might be related with epileptogenesis. RASgrf1 has a differentially methylated region at the promoter which can silence gene expression. We have previously observed the down-regulation of RASgrf1 in epilepsy patients and proved that hypermethylation of RASgrf1 reaches maximal level at the latent period in mice after kainate-induced SE (KA mice), with corresponding alteration of RASgrf1 expression. In the present study, N-phthalyl-L-tryptophan (RG108), a DNA methyltransferase inhibitor, was applied in KA mice at latent phase and the behavior, electroencephalogram and pathological changes were observed in chronic phase. Methylation and expression of RASgrf1 were determined by polymerase chain reaction (PCR), western blotting, and bisulfite sequencing PCR. The results showed that the incidence of spontaneous recurrent seizures (SRS) was significantly lower in the RG108 group than the normal saline (NS) group. Subgroup analysis showed significant hypermethylation and lower expression of RASgrf1 in the RG108–SRS subgroup and the NS–SRS subgroup but not in the RG108–NSRS (no SRS) subgroup and the NS–NSRS subgroup compared with the control group. No significant difference was found between the RG108–SRS and NS–SRS subgroups. Meanwhile, hippocampal neuronal loss was observed in RG108–SRS and NS–SRS subgroups. We thus demonstrated that RG108 could modify the progression of epileptogenesis after KA induced SE and prevent chronic epilepsy. Meanwhile, hypermethylation of RASgrf1 after KA induced SE could be reversed with corresponding changes of RASgrf1 expression. Additionally, we speculated that RASgrf1 might be a potential epigenetic mediator in epileptogenesis and chronic epilepsy.     

Does Pilocarpine-Induced Epilepsy in Adult Rats Require Status epilepticus?

Pilocarpine-induced seizures in rats provide a widely animal model of temporal lobe epilepsy. Some evidences reported in the literature suggest that at least 1 h of status epilepticus (SE) is required to produce subsequent chronic phase, due to the SE-related acute neuronal damage. However, recent data seems to indicate that neuro-inflammation plays a crucial role in epileptogenesis, modulating secondarily a neuronal insult. For this reason, we decided to test the following hypotheses: a) whether pilocarpine-injected rats that did not develop SE can exhibit long-term chronic spontaneous recurrent seizures (SRS) and b) whether acute neurodegeneration is mandatory to obtain chronic epilepsy. Therefore, we compared animals injected with the same dose of pilocarpine that developed or did not SE, and saline treated rats. We used telemetric acquisition of EEG as long-term monitoring system to evaluate the occurrence of seizures in non-SE pilocarpineinjected animals. Furthermore, histology and MRI analysis were applied in order to detect neuronal injury and neuropathological signs. Our observations indicate that non-SE rats exhibit SRS almost 8 (+/22) months after pilocarpine-injection, independently to the absence of initial acute neuronal injury. This is the first time reported that pilocarpine injected rats without developing SE, can experience SRS after a long latency period resembling human pathology. Thus, we strongly emphasize the important meaning of including these animals to model human epileptogenesis in pilocarpine induced epilepsy.     

The PPARγ agonist rosiglitazone prevents neuronal loss and attenuates development of spontaneous recurrent seizures through BDNF/TrkB signaling following pilocarpine-induced status epilepticus

Hippocampal neuronal loss plays an important role in epileptogenesis, and it is considered a trigger of repeated spontaneous recurrent seizures (SRS). The BDNF/TrkB signaling pathway regulates neuronal plasticity in the CNS, and promotes epileptogenesis. Previous studies have shown that Peroxisome proliferator-activated receptor gamma (PPARγ) agonists exert neuroprotective effects by inhibiting oxidative stress and inflammation in epilepsy. In the present study, the PPARγ agonist rosiglitazone inhibited increases in BDNF and TrkB after status epilepticus (SE), and also prevented hippocampal neuronal loss. More importantly, our study showed that rosiglitazone suppressed SRS. However, the effects of rosiglitazone were significantly reversed by cotreatment with K252a, an antagonist of TrkB. Additionally, rosiglitazone did not affect the development and severity of SE. Thus, our data provide evidence that rosiglitazone exerts neuroprotective and antiepileptic effects involve BDNF/TrkB signaling. Our study also offers new perspectives for the treatment of epilepsy.     

Degradation products of the mRNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean and transgenic petunia.

The degradation of a soybean ribulose-1,5-bisphosphate carboxylase small subunit RNA, SRS4, was investigated in soybean seedlings and in petunia plants transformed with an SRS4 gene construct. Polyacrylamide RNA gel blot, primer extension, and S1 nuclease analyses were used to identify and map fragments of the SRS4 mRNA generated in vivo. We showed that SRS4 mRNA is degraded to a characteristic set of fragments in soybean and transgenic petunia and that degradation is not dependent on position of insertion of the gene construct within the genome, on the expression level of the SRS4 mRNA, or on the rbcS promoter. Degradation products lacked poly(A) tails and fractionated with poly(A)-depleted RNA on oligo(dT)-sepharose columns. These products pelleted with polysomes and were released from polysomes prepared with EDTA. Sequences at the 5' end of the SRS4 mRNA were more stable than those at the 3' end of the mRNA. Three models for SRS4 mRNA degradation involving endonucleolytic and exonucleolytic degradation were presented to explain the origin of the 5' proximal fragments.     

Ecological Effects of Metals in Streams on a Defense Materials Processing Site in South Carolina,USA

The Savannah River Site (SRS) is a 780 km² U.S. Department of Energy facility near Aiken, South Carolina, established in 1950 to produce nuclear materials. SRS streams are “integrators” that potentially receive water transportable contaminants from all sources within their drainage basins, necessitating a watershed approach to organize contaminant distribution data and characterize the effects of multiple contaminants on aquatic organisms. This study used several lines-of-evidence to assess the ecological effects of metals in SRS streams, including contaminant exposure models for apex predators and bioassessments of fish and macroinvertebrate assemblages. Concentrations of metals in sediments, fish, and water were elevated in streams affected by SRS operations, but contaminant exposure models for the river otter Lontra Canadensis and belted kingfisher Ceryle alcyon indicated that toxicological reference values were exceeded only by Hg and Al. Macroinvertebrate assemblage structure was unrelated to sediment metal concentrations. Fish assemblage data were inconclusive. This study indicated that (1) modeling studies and field bioassessments provide a complementary basis for addressing the individual and cumulative effects of contaminants, (2) habitat effects must be controlled when assessing contaminant impacts, (3) sensitivity analyses of contaminant exposure models can help to apportion sampling effort, and (4) most individual metals in SRS streams are unlikely to have significant ecological effects.     

锂-匹罗卡品颞叶癫模型的建立及苔藓纤维出芽的动态变化

目的研究锂-匹罗卡品颞叶癫模型大鼠致后性发作的行为学特点及海马结构病理改变的动态变化。方法将所有Wistar大鼠随机分为对照组和实验组,实验组大鼠腹腔依次注射氯化锂、匹罗卡品诱发癫持续状态（SE）后,观察其自发性癫发作（SRS）,分别于SE后1周至10周5个不同时间点取材,Nissl染色和Timm染色分别观察海马神经元损伤及苔藓纤维出芽（MFS）的变化。结果注射匹罗卡品后84%的大鼠可诱发出SE,经过10～20d的缄默期后,可观察到Ⅰ～Ⅲ级的反复SRS,病理学检查可见海马神经元的损伤及齿状回内分子层MFS。结论锂-匹罗卡品颞叶癫模型与人类颞叶癫有类似发作特点及病理改变,是一种理想的颞叶癫动物模型。     

Common active site architecture and binding strategy of four phenylpropanoid P450s from Arabidopsis thaliana as revealed by molecular modeling

Despite extensive primary sequence diversity, crystal structures of several bacterial cytochrome P450 monooxygenases (P450s) and a single eukaryotic P450 indicate that these enzymes share a structural core of alpha-helices and beta-sheets and vary in the loop regions contacting individual substrates. To determine the extent to which individual structural features are conserved among divergent P450s existing in a single biosynthetic pathway, we have modeled the structures of four highly divergent P450s (CYP73A5, CYP84A1, CYP75B1, CYP98A3) in the Arabidopsis phenylpropanoid pathway synthesizing lignins, flavonoids and anthocyanins. Analysis of these models has indicated that, despite primary sequence identities as low as 13%, the structural cores and several loop regions of these P450s are highly conserved. Substrate docking indicated that all four enzymes employ a common strategy to identify their substrates in that their cinnamate-derived substrates align along helix I with their aromatic ring positioned towards the C-terminus of this helix and their aliphatic tails positioned towards the N-terminus. Further similarity was observed in the way the substrates contact the consensus P450 substrate recognition sites (SRS). Residues predicted to contact the aromatic ring region exist in SRS5, SRS6 and the C-terminal portion of SRS4 and residues contacting the distal end of each substrate exist in SRS1, SRS2 and the N-terminal portion of SRS4. Alignments of the regions contacting the aromatic ring region indicate that SRS4, SRS5 and SRS6 share higher degrees of sequence conservation than found in SRS1, SRS2 or the full-length protein.     

Antiepileptic and neuroprotective effects of human umbilical cord blood mononuclear cells in a pilocarpine-induced epilepsy model

Status epilepticus (SE) is a condition of persistent seizure that leads to brain damage and, frequently, to the establishment of chronic epilepsy. Cord blood is an important source of adult stem cells for the treatment of neurological disorders. The present study aimed to evaluate the effects of human umbilical cord blood mononuclear cells (HUCBC) transplanted into rats after induction of SE by the administration of lithium and pilocarpine chloride. Transplantation of HUCBC into epileptic rats protected against neuronal loss in the hippocampal subfields CA1, CA3 and in the hilus of the dentate gyrus, up to 300 days after SE induction. Moreover, transplanted rats had reduced frequency and duration of spontaneous recurrent seizures (SRS) 15, 120 and 300 days after the SE. Our study shows that HUCBC provide prominent antiepileptic and neuroprotective effects in the experimental model of epilepsy and reinforces that early interventions can protect the brain against the establishment of epilepsy.     

A Spatially Explicit Model of the Wild Hog for Ecological Risk Assessment Activities at the Department of Energy's Savannah River Site

In North America, wild hogs (Sus scrofa) are both sought after as prime game and despised due to their detrimental impacts to the environment from their digging and rooting behavior. They are also a potentially useful indicator species for environmental health for both ecological- and human-based risk assessments. An inductive approach was used to develop probabilistic resource selection models using logistic regression to quantify the likelihood of hogs being in any area of the Department of Energy's 805 km² Savannah River Site (SRS) in west-central South Carolina. These models were derived by using available SRS hog hunt data from 1993–2000 and a Geographic Information System database describing the habitat structure of the SRS. The model's significant parameters indicated that wild hogs preferred hardwoods and avoided pine and shrubby areas. Further, landscape metric analyses revealed that hogs preferred areas with large complex patch areas and low size variation. These resource selection models were then utilized to better estimate exposure of wild hogs to radionuclides and metals in a disturbed riparian ecosystem on the SRS using two different possible diets based on food availability. Contaminant exposure can be better estimated using these resource selection models than has been previously possible, because past practices did not consider home range and habitat utilization probability in heterogeneously contaminated habitats. Had these models not been used, risk calculations would assume that contaminated areas were utilized 100% of the time, thus overestimating exposure by a factor of up to 25.     

Bioclimatic modelling using Gaussian mixture distributions and multiscale segmentation

Aim To introduce Gaussian mixture distributions and sequential maximum a posteriori image segmentation (GM‐SMAP) as a model that predicts species ranges from mapped climatic variables, and to compare its predictive capacity with two commonly used bioclimatic models: regression tree analysis (RTA) and smoothed response surfaces (SRS). Location North‐west North America. Methods We compared models for their ability to predict the distributional range of western hemlock (Tsuga heterophylla). We calculated and projected nine climatic and water‐balance variables to a 2‐km grid up to 140 km from the T. heterophylla range. Models were trained using the five variables selected by RTA, as well as subsets of three variables. Goodness of fit was assessed using models trained and tested on the entire study area. Predictive capacity was assessed using 100 cross‐validation tests, each trained on a randomly sampled 1% of the study area and tested on the complement of the study area. Results Models using all five variables were significantly better than three‐variable models. Model fit was greatest for SRS. GM‐SMAP misclassified slightly more area and RTA misclassified almost twice the area compared to SRS. However, cross‐ validation showed that the predictive capacity was clearly greatest for GM‐SMAP and lowest for SRS, indicating that GM‐SMAP makes more accurate predictions from sparse data. Main conclusions GM distributions prevent overfitting using an information‐theoretic approach, and the SMAP algorithm minimizes the spatial extent of the largest misclassified area using a multiscale method. These properties, useful for image classification, also aid their strong predictive capacity as a bioclimatic model. SRS overfit the data, lowering its predictive capacity, and RTA failed to capture details of interactions among variables, yielding a poor fit. These results demonstrate the strong potential of GM‐SMAP as a bioclimatic model.     

分叉双歧杆菌对小鼠腹水瘤的抑制作用

本文观察了分叉双歧杆菌对小鼠腹腔移植的淋巴细胞腹水瘤(SRS)的抑制作用。结果发现,分叉双歧杆菌在SRS瘤细胞移植前或移植后应用,均能明显抑制瘤细胞的生长,特别在移植后应用,抗瘤效果更显著。主要表现为荷瘤小鼠存活时间延长、存活率提高。将该菌预先进行处理或不处理后加入体外培养的SRS瘤细胞中,发现该菌对离体的瘤细胞生长也有直接抑制作用。     

The SRS superfamily of Toxoplasma surface proteins

The surface of the protozoan parasite Toxoplasma gondii is coated with developmentally expressed, glycosylphosphatidylinositol-linked proteins structurally related to the highly immunogenic surface antigen SAG1. Collectively, these surface antigens are known as the SRS (SAG1-related sequences) superfamily of proteins. SRS proteins are thought to mediate attachment to host cells and activate host immunity to regulate the parasite's virulence. To better understand the number, evolution and developmental expression of SRS genes, this study has bioinformatically identified 161 unique SRS DNA sequences present in the T. gondii type II Me49 genome. The SRS superfamily of sequences phylogenetically bifurcates into two subfamilies, the prototypic members being SAG1 and SAG2A, respectively. Paralogous SRS sequences are 24-99% identical, are tandemly arrayed throughout the genome, and are present on most, if not all, chromosomes. All 11 SRS sequences on chromosomes Ia and Ib are clustered at sub-telomeric expression sites. Messenger RNA expression in the majority of SRS sequences for which multiple Expressed Sequence Tags exist is developmentally regulated. A consensus nucleotide sequence surrounding both the splice acceptor and donor sites was identified in those SRS sequences possessing an intron. Genotypic differences among SRS sequences are present at several loci (e.g. the absence of SAG5B, the truncation of SAG2D in Me49 compared with RH) indicating that different genotypes possess distinct sets of SRS sequences. Orthologous genes are restricted to tissue-dwelling coccidia (Neospora, Sarcocystis) with no related sequences present in other more distant apicomplexa such as Eimeria, Cryptosporidia, and Plasmodium spp.     

Outlook on sponge reproduction science in the last ten years: are we far from where we should be?

To comprehend the state of the art of sponge reproduction science (SRS), we quantified and analyzed the trends in SRS in the last decade, aiming to answer three questions: (i) Were there fewer SRS works presented during the last sponge conference? (ii) Did the number of SRS publications decline in the last decade? (iii) Does the number of abstracts at sponge conferences influence overall SRS publications? In addition, we checked whether the SRS community has answered Ereskovsky’s ‘five important questions’, enabling us to advance SRS enough to be considered as a fourth period of this scientific field. We found that SRS was less represented at the last sponge conference, despite an increase in the number of publications during the last decade. Moreover, the number of abstracts presented at sponge conferences contributed to a small portion (25%) of the published works in this area during the last decade. In addition, we found that two of the five Ereskovsky’s questions are still mostly not answered. Thus, we conclude that SRS is healthy and advancing steadily, especially in some subareas (e.g. developmental biology and life history). There are still much to advance, but this is still a strong field of biological science research.     

Growth in coculture stimulates metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2

Metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2 was significantly enhanced when the strain was grown in coculture with a soil bacterium (designated strain SRS1). Both members of this consortium were isolated from a highly enriched isoproturon-degrading culture derived from an agricultural soil previously treated regularly with the herbicide. Based on analysis of the 16S rRNA gene, strain SRS1 was assigned to the beta-subdivision of the proteobacteria and probably represents a new genus. Strain SRS1 was unable to degrade either isoproturon or its known metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, or 4-isopropyl-aniline. Pure culture studies indicate that Sphingomonas sp. SRS2 is auxotrophic and requires components supplied by association with other soil bacteria. A specific mixture of amino acids appeared to meet these requirements, and it was shown that methionine was essential for Sphingomonas sp. SRS2. This suggests that strain SRS1 supplies amino acids to Sphingomonas sp. SRS2, thereby leading to rapid metabolism of (14)C-labeled isoproturon to (14)CO(2) and corresponding growth of strain SRS2. Proliferation of strain SRS1 suggests that isoproturon metabolism by Sphingomonas sp. SRS2 provides unknown metabolites or cell debris that supports growth of strain SRS1. The role of strain SRS1 in the consortium was not ubiquitous among soil bacteria; however, the indigenous soil microflora and some strains from culture collections also stimulate isoproturon metabolism by Sphingomonas sp. strain SRS2 to a similar extent.     

Deciphering information encoded in birdsong: male songbirds with fertile mates respond most strongly to complex, low-amplitude songs used in courtship

Research on the function of acoustic signals has focused on high-amplitude long-range songs (LRS) and largely ignored low-amplitude songs produced by many species during close-proximity, conspecific interactions. Low-amplitude songs can be structurally identical to LRS (soft LRS), or they can be widely divergent, sharing few spectral and temporal attributes with LRS (short-range song [SRS]). SRS is often more complex than LRS and is frequently sung by males during courtship. To assess function, we performed two playback experiments on males of a socially monogamous songbird. We compared responses of males whose mates were fertile or nonfertile with differences in song structure (SRS vs. LRS and soft LRS), amplitude (SRS and soft LRS vs. LRS), and tempo (slow SRS vs. fast SRS). Males responded more strongly to SRS than to LRS or soft LRS, indicating that song structure had a greater effect on response than song amplitude. SRS tempo did not detectably affect male response. Importantly, males responded more strongly to SRS when their mates were fertile, presumably because hearing SRS can indicate that a male's mate is being courted by an intruding male and a strong response can deter extrapair competitors. We conclude that low-amplitude songs can function in both inter- and intrasexual communication and should receive greater attention in future studies of mate choice and male-male competition.     

Stage-specific expression of surface antigens by Toxoplasma gondii as a mechanism to facilitate parasite persistence

Toxoplasma persists in the face of a functional immune system. This success critically depends on the ability of parasites to activate a strong adaptive immune response during acute infection with tachyzoites that eliminates most of the parasites and to undergo stage conversion to bradyzoites that encyst and persist predominantly in the brain. A dramatic change in antigenic composition occurs during stage conversion, such that tachyzoites and bradyzoites express closely related but antigenically distinct sets of surface Ags belonging to the surface Ag 1 (SAG1)-related sequence (SRS) family. To test the contribution of this antigenic switch to parasite persistence, we engineered parasites to constitutively express the normally bradyzoite-specific SRS9 (SRS9(c)) mutants and tachyzoite-specific SAG1 (SAG1(c)) mutants. SRS9(c) but not wild-type parasites elicited a SRS9-specific immune response marked by IFN-gamma production, suggesting that stage-specificity of SRS Ags determines their immunogenicity in infection. The induction of a SRS9-specific immune response correlated with a continual decrease in the number of SRS9(c) cysts persisting in the brain. In contrast, SAG1(c) mutants produced reduced brain cyst loads early in chronic infection, but these substantially increased over time accompanying a hyperproduction of IFN-gamma, TNF-alpha, and IL-10, and severe encephalitis. We conclude that stage-specific expression of SRS Ags is among the key mechanisms by which optimal parasite persistency is established and maintained.     

The modeling of rat EEG signals in absence epilepsy in the analysis of brain connectivity

Simple models that describe some features of the electrical brain activity in rats with genetic absence epilepsy recorded before and after an epileptic seizure have been proposed in this study. These models can help to analyze the efficiency of the Granger causality analysis of the directional connectivity determination. The comparison of the results of the experimental and modeled signal analysis, on one hand, reveals a number of artifacts of this method, and on the other hand, proves its effectiveness in the research on absence epilepsy mechanisms.