Sulthiame use in children with pharmacoresistant epilepsies: A retrospective study.

OBJECTIVE: This retrospective study aimed to assess the efficacy and tolerability of sulthiame as an add-on treatment in children with pharmacoresistant epilepsies. METHODS: All patients with epilepsy who received sulthiame at Montreal Children's Hospital over an 11-year period were included. Medical charts were reviewed, and extracted data included patient age and sex, seizure types, epilepsy syndrome, electroencephalography (EEG) reports, brain imaging reports, antiseizure treatments trialed, starting and final dose of sulthiame, duration of sulthiame treatment, adverse events attributed to sulthiame, and seizure frequency before and after sulthiame treatment. EEG studies were also analyzed and spike-wave index (SWI) in the first 10 min of sleep was calculated. RESULTS: Sixteen patients were included, all of whom had pharmacoresistant epilepsies (mean of 9.9 trials of other antiseizure treatments). Six had genetic diagnoses, four had in utero/perinatal acquired brain injury, one had a suspected focal cortical dysplasia, and five were idiopathic. Ten patients had developmental and epileptic encephalopathy with spike-wave activation in sleep, three had Lennox-Gastaut syndrome, and one each had sleep-related hyperkinetic epilepsy, self-limited epilepsy with centrotemporal spikes, and mixed generalized and multifocal epilepsy. Of the 12 patients with uncontrolled seizures at the time of sulthiame initiation, 4 had improvement in seizure frequency, including 2 who became seizure free. Eight patients had EEG data available that allowed calculation of sleep SWI; from this group, SWI decreased from 81.1% +/- 17.6% to 45.1% +/- 36.5% (p = .007). The most common adverse events reported were somnolence/drowsiness, aggression, and increased seizure frequency. Of the patients with genetic etiologies, significant positive responses were seen in patients with pathogenic variants in NDUFS1 and SATB1. SIGNIFICANCE: These data demonstrate the therapeutic potential of sulthiame, even in patients with highly pharmacoresistant epilepsy. Improvements may be seen in both seizure frequency and sleep SWI.

Defining the Genetic Landscape of Congenital Mirror Movements in 80 Affected Individuals.

BACKGROUND: Congenital mirror movements (CMM) is a rare neurodevelopmental disorder characterized by involuntary movements from one side of the body that mirror voluntary movements on the opposite side. To date, five genes have been associated with CMM, namely DCC, RAD51, NTN1, ARHGEF7, and DNAL4. OBJECTIVE: The aim of this study is to characterize the genetic landscape of CMM in a large group of 80 affected individuals. METHODS: We screened 80 individuals with CMM from 43 families for pathogenic variants in CMM genes. In large CMM families, we tested for presence of pathogenic variants in multiple affected and unaffected individuals. In addition, we evaluated the impact of three missense DCC variants on binding between DCC and Netrin-1 in vitro. RESULTS: Causal pathogenic/likely pathogenic variants were found in 35% of probands overall, and 70% with familial CMM. The most common causal gene was DCC, responsible for 28% of CMM probands and 80% of solved cases. RAD51, NTN1, and ARHGEF7 were rare causes of CMM, responsible for 2% each. Penetrance of CMM in DCC pathogenic variant carriers was 68% and higher in males than females (74% vs. 54%). The three tested missense variants (p.Ile164Thr; p.Asn176Ser; and p.Arg1343His) bind Netrin-1 similarly to wild type DCC. CONCLUSIONS: A genetic etiology can be identified in one third of CMM individuals, with DCC being the most common gene involved. Two thirds of CMM individuals were unsolved, highlighting that CMM is genetically heterogeneous and other CMM genes are yet to be discovered. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Utility of genetic testing in the pre-surgical evaluation of children with drug-resistant epilepsy.

We evaluated the utility of genetic testing in the pre-surgical evaluation of pediatric patients with drug-resistant focal epilepsy. This single-center retrospective study reviewed the charts of all pediatric patients referred for epilepsy surgery evaluation over a 5-year period. We extracted and analyzed results of genetic testing as well as clinical, EEG, and neuroimaging data. Of 125 patients referred for epilepsy surgical evaluation, 86 (69%) had some form of genetic testing. Of these, 18 (21%) had a pathogenic or likely pathogenic variant identified. Genes affected included NPRL3 (3 patients, all related), TSC2 (3 patients), KCNH1, CHRNA4, SPTAN1, DEPDC5, SCN2A, ARX, SCN1A, DLG4, and ST5. One patient had ring chromosome 20, one a 7.17p12 duplication, and one a 15q13 deletion. In six patients, suspected epileptogenic lesions were identified on brain MRI that were thought to be unrelated to the genetic finding. A specific medical therapy choice was allowed due to genetic diagnosis in three patients who did not undergo surgery. Obtaining a molecular diagnosis may dramatically alter management in pediatric patients with drug-resistant focal epilepsy. Genetic testing should be incorporated as part of standard investigations in the pre-surgical work-up of pediatric patients with drug-resistant focal epilepsy.

Epilepsy in Legius syndrome: Coincidence or causation?

Legius syndrome is a rare genetic disorder, caused by heterozygous SPRED1 pathogenic variants, which shares phenotypic features with neurofibromatosis type 1 (NF1). Both conditions typically involve café-au-lait macules, axillary freckling, and macrocephaly; however, patients with NF1 are also at risk for tumors, such as optic nerve gliomas and neurofibromas. Seizure risk is known to be elevated in NF1, but there has been little study of this aspect of Legius syndrome. The reported epilepsy incidence is 3.3%-5%, well above the general population incidence of ~0.5%-1%, but the few reports in the literature have very little data regarding epilepsy phenotype. We identified two unrelated individuals, both with Legius syndrome and epilepsy, and performed thorough phenotyping. One individual's mother also had Legius syndrome and now-resolved childhood epilepsy, as well as reports of more distant relatives who also had multiple café-au-lait macules and seizures. Both probands had experienced childhood-onset focal seizures, with normal brain MRI. In one patient, EEG later showed apparently generalized epileptiform abnormalities. Based on the data from this small case series and literature review, seizure risk is increased in people with Legius syndrome, but the epilepsy prognosis appears to be generally good, with patients having either self-limited or pharmacoresponsive courses.

mTOR Pathway Somatic Pathogenic Variants in Focal Malformations of Cortical Development: Novel Variants, Topographic Mapping, and Clinical Outcomes.

Background and Objectives: Somatic and germline pathogenic variants in genes of the mammalian target of rapamycin (mTOR) signaling pathway are a common mechanism underlying a subset of focal malformations of cortical development (FMCDs) referred to as mTORopathies, which include focal cortical dysplasia (FCD) type II, subtypes of polymicrogyria, and hemimegalencephaly. Our objective is to screen resected FMCD specimens with mTORopathy features on histology for causal somatic variants in mTOR pathway genes, describe novel pathogenic variants, and examine the variant distribution in relation to neuroimaging, histopathologic classification, and clinical outcomes. Methods: We performed ultra-deep sequencing using a custom HaloPlexHS Target Enrichment kit in DNA from 21 resected fresh-frozen histologically confirmed FCD type II, tuberous sclerosis complex, or hemimegalencephaly specimens. We mapped the variant alternative allele frequency (AAF) across the resected brain using targeted ultra-deep sequencing in multiple formalin-fixed paraffin-embedded tissue blocks. We also functionally validated 2 candidate somatic MTOR variants and performed targeted RNA sequencing to validate a splicing defect associated with a novel DEPDC5 variant. Results: We identified causal mTOR pathway gene variants in 66.7% (14/21) of patients, of which 13 were somatic with AAF ranging between 0.6% and 12.0%. Moreover, the AAF did not predict balloon cell presence. Favorable seizure outcomes were associated with genetically clear resection borders. Individuals in whom a causal somatic variant was undetected had excellent postsurgical outcomes. In addition, we demonstrate pathogenicity of the novel c.4373_4375dupATG and candidate c.7499T>A MTOR variants in vitro. We also identified a novel germline aberrant splice site variant in DEPDC5 (c.2802-1G>C). Discussion: The AAF of somatic pathogenic variants correlated with the topographic distribution, histopathology, and postsurgical outcomes. Moreover, cortical regions with absent histologic FCD features had negligible or undetectable pathogenic variant loads. By contrast, specimens with frank histologic abnormalities had detectable pathogenic variant loads, which raises important questions as to whether there is a tolerable variant threshold and whether surgical margins should be clean, as performed in tumor resections. In addition, we describe 2 novel pathogenic variants, expanding the mTORopathy genetic spectrum. Although most pathogenic somatic variants are located at mutation hotspots, screening the full-coding gene sequence remains necessary in a subset of patients.

SCN1A as a therapeutic target for Dravet syndrome.

INTRODUCTION: Dravet syndrome is a severe early infancy-onset developmental and epileptic encephalopathy. Patients have drug-resistant seizures, as well as significant co-morbidities, including developmental impairment, crouch gait, sleep disturbance, and early mortality. The underlying cause is mutations in SCN1A, encoding the sodium channel subunit NaV1.1, in >90% of patients. At present, approved Dravet syndrome treatments are symptomatic, primarily aimed at reducing seizure frequency, but having little to no effect on co-morbidities. AREAS COVERED: We discuss the potential to treat Dravet syndrome by targeting NaV1.1 directly. Anti-seizure medications that act as sodium channel inhibitors are generally minimally effective and can actually exacerbate seizures. However, other interventions are currently under investigation, including gene therapies that increase the amount of functional NaV1.1. Some of these interventions have encouraging pre-clinical data from in vitro and animal models. EXPERT OPINION: Increasing functional NaV1.1 via antisense oligonucleotides or virus-borne vectors is the most promising avenue for meaningful improvement in Dravet syndrome treatment, with the potential to not only reduce seizures but also address the multiple co-morbidities associated with this disease. However, human clinical trial data are necessary to determine safety and to clarify if, and to what extent, these interventions modify the natural history of Dravet syndrome.

Awake Craniotomy Language Mapping in Children With Drug-Resistant Epilepsy due to Focal Cortical Dysplasia.

BACKGROUND: Language mapping during awake craniotomy can allow for precise resection of epileptogenic lesions, while reducing the risk of damage to eloquent cortex. There are few reports in the literature of language mapping during awake craniotomy in children with epilepsy. Some centers may avoid awake craniotomy in the pediatric age group due to concerns that children are unable to cooperate with such procedures. METHODS: We reviewed pediatric patients from our center with drug-resistant focal epilepsy who underwent language mapping during awake craniotomy and subsequent resection of the epileptogenic lesion. RESULTS: Two patients were identified, both female, aged 17 years and 11 years at the time of surgery. Both patients had frequent and disabling focal seizures despite trials of multiple antiseizure medications. Both patients had resection of their epileptogenic lesions with the aid of intraoperative language mapping; in both cases pathology was consistent with focal cortical dysplasia. Both patients had transient language difficulties in the immediate postoperative period but no deficits at six-month follow-up. Both patients are now seizure-free. CONCLUSIONS: Awake craniotomy should be considered in pediatric patients with drug-resistant epilepsy in whom the suspected epileptogenic lesion is in close proximity to cortical language areas.

WWOX developmental and epileptic encephalopathy: Understanding the epileptology and the mortality risk.

OBJECTIVE: WWOX is an autosomal recessive cause of early infantile developmental and epileptic encephalopathy (WWOX-DEE), also known as WOREE (WWOX-related epileptic encephalopathy). We analyzed the epileptology and imaging features of WWOX-DEE, and investigated genotype-phenotype correlations, particularly with regard to survival. METHODS: We studied 13 patients from 12 families with WWOX-DEE. Information regarding seizure semiology, comorbidities, facial dysmorphisms, and disease outcome were collected. Electroencephalographic (EEG) and brain magnetic resonance imaging (MRI) data were analyzed. Pathogenic WWOX variants from our cohort and the literature were coded as either null or missense, allowing individuals to be classified into one of three genotype classes: (1) null/null, (2) null/missense, (3) missense/missense. Differences in survival outcome were estimated using the Kaplan-Meier method. RESULTS: All patients experienced multiple seizure types (median onset = 5 weeks, range = 1 day-10 months), the most frequent being focal (85%), epileptic spasms (77%), and tonic seizures (69%). Ictal EEG recordings in six of 13 patients showed tonic (n = 5), myoclonic (n = 2), epileptic spasms (n = 2), focal (n = 1), and migrating focal (n = 1) seizures. Interictal EEGs demonstrated slow background activity with multifocal discharges, predominantly over frontal or temporo-occipital regions. Eleven of 13 patients had a movement disorder, most frequently dystonia. Brain MRIs revealed severe frontotemporal, hippocampal, and optic atrophy, thin corpus callosum, and white matter signal abnormalities. Pathogenic variants were located throughout WWOX and comprised both missense and null changes including five copy number variants (four deletions, one duplication). Survival analyses showed that patients with two null variants are at higher mortality risk (p-value = .0085, log-rank test). SIGNIFICANCE: Biallelic WWOX pathogenic variants cause an early infantile developmental and epileptic encephalopathy syndrome. The most common seizure types are focal seizures and epileptic spasms. Mortality risk is associated with mutation type; patients with biallelic null WWOX pathogenic variants have significantly lower survival probability compared to those carrying at least one presumed hypomorphic missense pathogenic variant.

Surgically Remediable Secondary Network Epileptic Encephalopathies With Continuous Spike Wave in Sleep: Lesions May Not Be Visible on Brain Magnetic Resonance Imaging (MRI).

BACKGROUND: Continuous spike wave in sleep (CSWS) is an electroencephalogram (EEG) pattern associated with developmental and epileptic encephalopathy with spike-and-wave activation in sleep (DEE-SWAS). This etiologically heterogeneous syndrome may occur because of genetic factors and congenital or acquired brain lesions. We studied the pattern of clinical presentation and underlying etiologies in patients with DEE-SWAS that respond to resective surgery. METHODS: We reviewed our clinical and research databases for patients who had resolution of CSWS following surgical resection of a focal lesion. RESULTS: We identified 5 patients meeting inclusion criteria. In 3 of 5, an epileptogenic structural abnormality was not apparent on brain magnetic resonance imaging (MRI). In all 3 patients, focal cortical dysplasia was identified through intracranial EEG monitoring. SIGNIFICANCE: DEE-SWAS may be a secondary bilateral network epilepsy syndrome, which can be treated with resection of the inciting focal lesion. In patients with drug-resistant CSWS, clinicians should consider a complete epilepsy presurgical workup, including intracranial EEG monitoring.

De Novo GLI3 Pathogenic Variants May Cause Hypotonia and a Range of Brain Malformations Without Skeletal Abnormalities.

BACKGROUND: GLI3 encodes a zinc finger transcription factor that plays a role in the sonic hedgehog pathway. Germline pathogenic GLI3 variants are associated with Greig cephalopolysyndactyly and Pallister-Hall syndromes, two syndromes involving brain malformation and polydactyly. METHODS: We identified patients with pathogenic GLI3 variants and brain malformations in the absence of polydactyly or other skeletal malformation. RESULTS: Two patients were identified. Patient #1 is a 4-year-old boy with hypotonia and global developmental delay. Brain MRI showed a focal cortical dysplasia, but he had no history of seizures. Genetic testing identified a de novo likely pathogenic GLI3 variant: c.4453A>T, p.Asn1485Tyr. Patient #2 is a 4-year-old boy with hypotonia, macrocephaly, and global developmental delay. His brain MRI showed partial agenesis of the corpus callosum, dilatation of the right lateral ventricle, and absent hippocampal commissure. Genetic testing identified a de novo pathogenic GLI3 variant: c.4236_4237del, p.Gln1414AspfsTer21. Neither patient had polydactyly or any apparent skeletal abnormality. CONCLUSIONS: These patients widen the spectrum of clinical features that may be associated with GLI3 pathogenic variants to include hypotonia, focal cortical dysplasia, and other brain malformations, in the absence of apparent skeletal malformation. Further study is needed to determine if GLI3 pathogenic variants are a more common cause of focal cortical dysplasia or corpus callosum agenesis than presently recognized.

Septins guide noncentrosomal microtubules to promote focal adhesion disassembly in migrating cells.

Endothelial cell migration is critical for vascular angiogenesis and is compromised to facilitate tumor metastasis. The migratory process requires the coordinated assembly and disassembly of focal adhesions (FA), actin, and microtubules (MT). MT dynamics at FAs deliver vesicular cargoes and enhance actomyosin contractility to promote FA turnover and facilitate cell advance. Noncentrosomal (NC) MTs regulate FA dynamics and are sufficient to drive cell polarity, but how NC MTs target FAs to control FA turnover is not understood. Here, we show that Rac1 induces the assembly of FA-proximal septin filaments that promote NC MT growth into FAs and inhibit mitotic centromere-associated kinesin (MCAK)-associated MT disassembly, thereby maintaining intact MT plus ends proximal to FAs. Septin-associated MT rescue is coupled with accumulation of Aurora-A kinase and cytoplasmic linker-associated protein (CLASP) localization to the MT between septin and FAs. In this way, NC MTs are strategically positioned to undergo MCAK- and CLASP-regulated bouts of assembly and disassembly into FAs, thereby regulating FA turnover and cell migration.

New interinstitutional, multimodal presurgical evaluation protocol associated with improved seizure freedom for poorly defined cases of focal epilepsy in children.

OBJECTIVE: In an attempt to improve postsurgical seizure outcomes for poorly defined cases (PDCs) of pediatric focal epilepsy (i.e., those that are not visible or well defined on 3T MRI), the authors modified their presurgical evaluation strategy. Instead of relying on concordance between video-electroencephalography and 3T MRI and using functional imaging and intracranial recording in select cases, the authors systematically used a multimodal, 3-tiered investigation protocol that also involved new collaborations between their hospital, the Montreal Children's Hospital, and the Montreal Neurological Institute. In this study, the authors examined how their new strategy has impacted postsurgical outcomes. They hypothesized that it would improve postsurgical seizure outcomes, with the added benefit of identifying a subset of tests contributing the most. METHODS: Chart review was performed for children with PDCs who underwent resection following the new strategy (i.e., new protocol [NP]), and for the same number who underwent treatment previously (i.e., preprotocol [PP]); ≥ 1-year follow-up was required for inclusion. Well-defined, multifocal, and diffuse hemispheric cases were excluded. Preoperative demographics and clinical characteristics, resection volumes, and pathology, as well as seizure outcomes (Engel class Ia vs > Ia) at 1 year postsurgery and last follow-up were reviewed. RESULTS: Twenty-two consecutive NP patients were compared with 22 PP patients. There was no difference between the two groups for resection volumes, pathology, or preoperative characteristics, except that the NP group underwent more presurgical evaluation tests (p < 0.001). At 1 year postsurgery, 20 of 22 NP patients and 10 of 22 PP patients were seizure free (OR 11.81, 95% CI 2.00-69.68; p = 0.006). Magnetoencephalography and PET/MRI were associated with improved postsurgical seizure outcomes, but both were highly correlated with the protocol group (i.e., independent test effects could not be demonstrated). CONCLUSIONS: A new presurgical evaluation strategy for children with PDCs of focal epilepsy led to improved postsurgical seizure freedom. No individual presurgical evaluation test was independently associated with improved outcome, suggesting that it may be the combined systematic protocol and new interinstitutional collaborations that makes the difference rather than any individual test.

Heart Rate Variability in Insulo-Opercular Epilepsy.

BACKGROUND: We aimed to evaluate heart rate variability (HRV) changes in insulo-opercular epilepsy (IOE) and after insulo-opercular surgery. METHODS: We analyzed 5-min resting HRV of IOE patients before and after surgery. Patients' SUDEP-7 risk inventory scores were also calculated. Results were compared with age- and sex-matched patients with temporal lobe epilepsy (TLE) and healthy individuals. RESULTS: There were no differences in HRV measurements between IOE, TLE, and healthy control groups (and within each IOE group and TLE group) in preoperative and postoperative periods. In IOE patients, the SUDEP-7 score was positively correlated with pNN50 (percentage of successive RR intervals that differ by more than 50 ms) (p = 0.008) and RMSSD (root mean square of successive RR interval differences) (p = 0.019). We stratified IOE patients into those whose preoperative RMSSD values were below (Group 1a = 7) versus above (Group 1b = 9) a cut-off threshold of 31 ms (median value of a healthy population from a previous study). In group 1a, all HRV values significantly increased after surgery. In group 1b, time-domain parameters significantly decreased postoperatively. CONCLUSIONS: Our results suggest that in IOE, HRV may be either decreased in parasympathetic tone or increased globally in both sympathetic and parasympathetic tones. We found no evidence that insulo-opercular surgeries lead to major autonomic dysfunction when a good seizure outcome is reached. The increase in parasympathetic tone observed preoperatively may be of clinical concern, as it was positively correlated with the SUDEP-7 score.

Transcriptome analysis of a ring chromosome 20 patient cohort.

Ring chromosomes occur when the ends of normally rod-shaped chromosomes fuse. In ring chromosome 20 (ring 20), intellectual disability and epilepsy are usually present, even if there is no deleted coding material; the mechanism by which individuals with complete ring chromosomes develop seizures and other phenotypic abnormalities is not understood. We investigated altered gene transcription as a contributing factor by performing RNA-sequencing (RNA-seq) analysis on blood from seven patients with ring 20, and 11 first-degree relatives (all parents). Geographic analysis did not identify altered expression in peritelomeric or other specific chromosome 20 regions. RNA-seq analysis revealed 97 genes potentially differentially expressed in ring 20 patients. These included one epilepsy gene, NPRL3, but this finding was not confirmed on reverse transcription Droplet Digital polymerase chain reaction analysis. Molecular studies of structural chromosomal anomalies such as ring chromosome are challenging and often difficult to interpret because many patients are mosaic, and there may be genome-wide chromosomal instability affecting gene expression. Our findings nevertheless suggest that peritelomeric altered transcription is not the likely pathogenic mechanism in ring 20. Underlying genetic mechanisms are likely complex and may involve differential expression of many genes, the majority of which may not be located on chromosome 20.

A proposed guideline for vagus nerve stimulator handling in palliative care and after death.

Vagus nerve stimulation (VNS) is often used for patients with drug-resistant epilepsy. Although this intervention may improve seizure control and mood, a number of factors must be considered when patients with VNS near end of life. We reviewed relevant literature to create a proposed guideline for management of patients with VNS in palliative care and after death. VNS has multiple possible side effects, including cough and swallowing difficulties. For patients with neurologic disease in palliative care, such adverse effects can severely affect quality of life and increase the risk for complications such as aspiration pneumonia. Patients with VNS should be screened regularly for such side effects, and VNS parameters should be adjusted if they are identified. If a patient requires urgent cardiac resuscitation involving external defibrillation, the VNS should be interrogated immediately afterwards to evaluate its function. During defibrillation, paddles should be placed perpendicular to the VNS, and as far as possible away from it. The VNS can be acutely turned off by taping the magnet to the patient's chest, thereby preventing any possible interference with restoration of a normal heart rhythm. After death, any staff involved with handling the body should be notified that a VNS is in place. The device must be removed prior to cremation, as it can explode with high heat. If the cause of death is unclear, a full postmortem examination should be undertaken, per sudden unexpected death in epilepsy guidelines. If there is concern about device malfunction, the device should be returned to the manufacturer for evaluation.

X-Linked Familial Focal Epilepsy Associated With Xp22.31 Deletion.

BACKGROUND: The genetic basis for familial focal epilepsy is poorly understood, with most of the known genetic causes occurring via autosomal dominant inheritance. X-linked familial focal epilepsy has not been previously reported. METHODS: We reviewed our research database for cases of X-linked focal epilepsy. RESULTS: We identified three boys with X-linked ichthyosis and focal epilepsy, including two maternal cousins. Age of seizure onset ranged from seven to 10 years, and all three patients had seizures that were relatively easily controlled. The epilepsy phenotype in all boys was consistent with self-limited focal epilepsy of childhood, most closely resembling childhood epilepsy with centrotemporal spikes. Brain magnetic resonance imaging was normal in two of the boys, with a third found to have a suspected focal cortical dysplasia. All three boys carried maternally inherited hemizygous Xp22.31 deletions (estimated size 0.9 to 1.66 Mb), affecting four to six genes. Of the affected genes, only STS has clear clinical relevance; deletions, and pathogenic variants in STS cause X-linked ichthyosis, although all patients described had only minor skin findings. CONCLUSIONS: The findings in these patients illustrate that X-linked familial focal epilepsy can occur, although it is a rare entity. Although STS pathogenic variants are likely better categorized as an epilepsy risk factor, variants in this gene may partially explain the male predominance observed in specific epilepsy phenotypes, namely childhood epilepsy with centrotemporal spikes.

Ferrochelatase Deficiency Abrogated the Enhancement of Aminolevulinic Acid-mediated Protoporphyrin IX by Iron Chelator Deferoxamine.

Aminolevulinic acid (ALA) is a prodrug that is metabolized in the heme biosynthesis pathway to produce protoporphyrin IX (PpIX) for tumor fluorescence detection and photodynamic therapy (PDT). The iron chelator deferoxamine (DFO) has been widely used to enhance PpIX accumulation by inhibiting the iron-dependent bioconversion of PpIX to heme, a reaction catalyzed by ferrochelatase (FECH). Tumor response to DFO treatment is known to be highly variable, and some tumors even show no response. Given the fact that tumors often exhibit reduced FECH expression/enzymatic activity, we examined how reducing FECH level affected the DFO enhancement effect. Our results showed that reducing FECH level by silencing FECH in SkBr3 breast cancer cells completely abrogated the enhancement effect of DFO. Although DFO enhanced ALA-PpIX fluorescence and PDT response in SkBr3 vector control cells, it caused a similar increase in MCF10A breast epithelial cells, resulting in no net gain in the selectivity toward tumor cells. We also found that DFO treatment induced less increase in ALA-PpIX fluorescence in tumor cells with lower FECH activity (MDA-MB-231, Hs 578T) than in tumor cells with higher FECH activity (MDA-MB-453). Our study demonstrates that FECH activity is an important determinant of tumor response to DFO treatment.

LANA oligomeric architecture is essential for KSHV nuclear body formation and viral genome maintenance during latency.

The molecular basis for the formation of functional, higher-ordered macro-molecular domains is not completely known. The Kaposi's Sarcoma-Associated Herpesvirus (KSHV) genome forms a super-molecular domain structure during latent infection that is strictly dependent on the DNA binding of the viral nuclear antigen LANA to the viral terminal repeats (TR). LANA is known to form oligomeric structures that have been implicated in viral episome maintenance. In this study, we show that the LANA oligomerization interface is required for the formation of higher-order nuclear bodies that partially colocalize with DAXX, EZH2, H3K27me3, and ORC2 but not with PML. These nuclear bodies assemble at the periphery of condensed cellular chromosomes during mitotic cell division. We demonstrate that the LANA oligomerization interface contributes to the cooperative DNA binding at the viral TR and the recruitment of ORC to the viral episome. Oligomerization mutants failed to auto-regulate LANA/ORF73 transcription, and this correlated with the loss of a chromosome conformational DNA-loop between the TR and LANA promoter. Viral genomes with LANA oligomerization mutants were subject to genome rearrangements including the loss of subgenomic DNA. Our data suggests that LANA oligomerization drives stable binding to the TR and formation of an epigenetically stable chromatin architecture resulting in higher-order LANA nuclear bodies important for viral genome integrity and long-term episome persistence.

Disruption of microtubule function in cultured human cells by a cytotoxic ruthenium(ii) polypyridyl complex.

Treatment of malignant and non-malignant cultured human cell lines with a cytotoxic IC50 dose of ∼2 µM tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(ii) chloride (RPC2) retards or arrests microtubule motion as tracked by visualizing fluorescently-tagged microtubule plus end-tracking proteins. Immunofluorescent microscopic images of the microtubules in fixed cells show substantial changes to cellular microtubule network and to overall cell morphology upon treatment with RPC2. Flow cytometry with MCF7 and H358 cells reveals only minor elevations of the number of cells in G2/M phase, suggesting that the observed cytotoxicity is not tied to mitotic arrest. In vitro studies with purified tubulin reveal that RPC2 acts to promote tubulin polymerization and when imaged by electron microscopy, these microtubules look normal in appearance. Isothermal titration calorimetry measurements show an associative binding constant of 4.8 × 106 M-1 for RPC2 to preformed microtubules and support a 1 : 1 RPC2 to tubulin dimer stoichiometry. Competition experiments show RPC2 does not compete for the taxane binding site. Consistent with this tight binding, over 80% of the ruthenium in treated cells is co-localized with the cytoskeletal proteins. These data support RPC2 acting as an in vivo microtubule stabilizing agent and sharing many similarities with cells treated with paclitaxel.