Influence of phospholipid head and tail molecular structures on cell membrane mechanical response under tension.

Biological cell membranes are primarily comprised of a diverse lipid bilayer with multiple phospholipid (lipid) types, each of which is comprised of a hydrophilic headgroup and two hydrophobic hydrocarbon tails. The lipid type determines the molecular structure of head and tail groups, which can affect membrane mechanics at nanoscale and subsequently cell viability under mechanical loading. Hence, using molecular dynamics simulations, the current study investigated seven membrane phospholipids and the effect of their structural differences on physical deformation, mechanoporation damage, and mechanical failure of the membranes under tension. The inspected phospholipids showed similar yield stresses and strains, as well as pore evolution and damage, but significantly different failure strains. In general, failure occurred at a lower strain for lipids with a larger equilibrium area per lipid. The obtained results suggest that larger headgroup structure, greater degree of unsaturation, and tail-length asymmetry influenced the phospholipids' ability to pack against each other, increased the fluidity and equilibrium area per lipid of the membrane, and resulted in lower failure strain. Overall, this study provides insights on how different phospholipid structures affect membrane physical responses at the molecular level and serves as a reference for future studies of more complex membrane systems with intricate biophysical properties.

Anterior temporal encephaloceles: Elusive, important, and rewarding to treat.

OBJECTIVE: To investigate the etiology and longitudinal clinical, neuropsychological, psychosocial, and surgical outcome profile of patients with medication refractory epilepsy and temporal encephaloceles with a view to highlight diagnostic clues and management strategies. METHODS: The comprehensive epilepsy program databases at two surgical epilepsy centers from January 2000 to October 2018 were reviewed for this observational study, to identify patients with encephaloceles causing temporal lobe epilepsy (TLE) and treated with surgical resection. Their clinical, radiological, neuropsychological, psychiatric, and surgical data were obtained. Body mass index (BMI) data were also reviewed due to possible correlation between idiopathic intracranial hypertension and encephaloceles. RESULTS: Thirteen patients (eight female) were identified; only three were recognized on initial magnetic resonance imaging (MRI) report. Temporal encephaloceles were identified on the left in eight patients, on the right in three patients, and bilaterally in two patients. One patient had a strong family history of encephaloceles. The median BMI for patients with seizure onset ≤20 years of age was 22.4, whereas for patients with onset >20 years median BMI was 32.6 (P = .06). Five patients underwent a focal lesionectomy, three patients had limited temporal lobectomy, and five patients had standard anterior temporal lobectomy. Median postoperative follow-up was 5.5 years. All but one patient were free of disabling seizures. Nine of ten neuropsychologically tested patients had no discernable cognitive decline postoperatively. Postoperative psychosocial adjustment features were present in four patients. SIGNIFICANCE: Genetic factors and a possible association with idiopathic intracranial hypertension (given female predominance and elevated BMI) may contribute to the causation of temporal lobe encephaloceles. It is notable that a targeted surgical approach in the management of patients with TLE associated with encephaloceles has an excellent long-term clinical and neuropsychological outcome. Subtle encephaloceles should be actively searched for in patients with drug-resistant TLE because they significantly change surgical strategy and prognostication.

Interictal and ictal source localization for epilepsy surgery using high-density EEG with MEG: a prospective long-term study.

Drug-resistant focal epilepsy is a major clinical problem and surgery is under-used. Better non-invasive techniques for epileptogenic zone localization are needed when MRI shows no lesion or an extensive lesion. The problem is interictal and ictal localization before propagation from the epileptogenic zone. High-density EEG (HDEEG) and magnetoencephalography (MEG) offer millisecond-order temporal resolution to address this but co-acquisition is challenging, ictal MEG studies are rare, long-term prospective studies are lacking, and fundamental questions remain. Should HDEEG-MEG discharges be assessed independently [electroencephalographic source localization (ESL), magnetoencephalographic source localization (MSL)] or combined (EMSL) for source localization? Which phase of the discharge best characterizes the epileptogenic zone (defined by intracranial EEG and surgical resection relative to outcome)? Does this differ for interictal and ictal discharges? Does MEG detect mesial temporal lobe discharges? Thirteen patients (10 non-lesional, three extensive-lesional) underwent synchronized HDEEG-MEG (72-94 channel EEG, 306-sensor MEG). Source localization (standardized low-resolution tomographic analysis with MRI patient-individualized boundary-element method) was applied to averaged interictal epileptiform discharges (IED) and ictal discharges at three phases: 'early-phase' (first latency 90% explained variance), 'mid-phase' (first of 50% rising-phase, 50% mean global field power), 'late-phase' (negative peak). 'Earliest-solution' was the first of the three early-phase solutions (ESL, MSL, EMSL). Prospective follow-up was 3-21 (median 12) months before surgery, 14-39 (median 21) months after surgery. IEDs (n = 1474) were recorded, seen in: HDEEG only, 626 (42%); MEG only, 232 (16%); and both 616 (42%). Thirty-three seizures were captured, seen in: HDEEG only, seven (21%); MEG only, one (3%); and both 25 (76%). Intracranial EEG was done in nine patients. Engel scores were I (9/13, 69%), II (2/13,15%), and III (2/13). MEG detected baso-mesial temporal lobe epileptogenic zone sources. Epileptogenic zone OR [odds ratio(s)] were significantly higher for earliest-solution versus early-phase IED-surgical resection and earliest-solution versus all mid-phase and late-phase solutions. ESL outperformed EMSL for ictal-surgical resection [OR 3.54, 95% confidence interval (CI) 1.09-11.55, P = 0.036]. MSL outperformed EMSL for IED-intracranial EEG (OR 4.67, 95% CI 1.19-18.34, P = 0.027). ESL outperformed MSL for ictal-surgical resection (OR 3.73, 95% CI 1.16-12.03, P = 0.028) but was outperformed by MSL for IED-intracranial EEG (OR 0.18, 95% CI 0.05-0.73, P = 0.017). Thus, (i) HDEEG and MEG source solutions more accurately localize the epileptogenic zone at the earliest resolvable phase of interictal and ictal discharges, not mid-phase (as is common practice) or late peak-phase (when signal-to-noise ratios are maximal); (ii) from empirical observation of the differential timing of HDEEG and MEG discharges and based on the superiority of ESL plus MSL over either modality alone and over EMSL, concurrent HDEEG-MEG signals should be assessed independently, not combined; (iii) baso-mesial temporal lobe sources are detectable by MEG; and (iv) MEG is not 'more accurate' than HDEEG-emphasis is best placed on the earliest signal (whether HDEEG or MEG) amenable to source localization. Our findings challenge current practice and our reliance on invasive monitoring in these patients. 10.1093/brain/awz015_video1 awz015media1 6018582479001.

COX2 induction: a mechanism of endocrine breast cancer resistance?

PURPOSE: Urine prostaglandin E2 (PGE2) levels have shown to be a risk factor of breast cancer, and the use of nonsteroidal anti-inflammatory drugs (NSAIDs) is known to be beneficial in preventing breast cancer risk and/or recurrence with or without aromatase inhibitors. We hypothesized that the use of an aromatase inhibitor triggers the activation of the inflammatory pathway via release of PGE2. METHODS: A single oral 25 mg dose of an aromatase inhibitor (exemestane) was given to 14 healthy postmenopausal female volunteers. Blood and urine samples were collected between 0 and 72 h post-dosing for pharmacokinetic and pharmacodynamic analysis. RESULTS: Our findings showed that urine PGE2 levels were markedly increased 72 h after exemestane administration (average pre-dosing PGE2 levels, 4061.1 pg/mL vs. post-dosing average PGE2 levels, 10732.5 pg/mL, P = 0.001, Wilcoxon Rank Test). Out of 14 subjects enrolled in the study, one subject showed no change in PGE2; another showed a 23-fold decreased in PGE2; and the remaining 12 showed an average of 8.4-fold increase in PGE2 levels (range 1.3-30.5, standard deviation 9.2) after exemestane administration. We found no statistically significant correlations between fold increase in urine PGE2 levels and the pharmacokinetics of either exemestane or 17-hydroexemestane (major in vivo metabolite of exemestane). CONCLUSION: Our results indicate that one of the pharmacological effects to aromatase inhibitors (e.g., exemestane) involves the activation of the inflammatory pathway via release of PGE2. Further in vitro mechanistic and in vivo translational studies designed to elucidate the role of this newly discovered effect are now warranted.

Health care professionals' perspectives of living and dying with primary malignant glioma: Implications for a unique cancer trajectory.

OBJECTIVES: Health care professionals (HCPs) caring for people with primary malignant glioma (PMG) and their carers see many of the profound challenges facing this group, yet their perspectives are not documented. This study aimed to understand and document the unique perspective of HCPs in relation to the supportive and palliative care needs of patients with PMG and their carers, with a view to developing a model of care. METHODS: Qualitative study involving semi-structured focus groups and interviews with 35 medical, nursing and allied health staff actively engaged in providing care for this patient group. Purposive and theoretical sampling from two major metropolitan hospitals and one community palliative care service in Australia was utilised to seek perspectives from a variety of disciplines and health care settings. Thematic analysis was conducted by three independent researchers, using a constant comparative method influenced by grounded theory. RESULTS: Key themes relating to the needs of people with PMG which were apparent from the HCPs included: The difference in the illness course of glioma compared to other cancers; Limitations of current medical care; Challenges in balancing hope with reality of the illness; and Recommendations to improve care, including recognising the role of family and moving from a model where services are offered in response to demonstrated needs. Significance of the results: Current models of care based upon the classic cancer trajectory are unresponsive to the needs of people with PMG. Care may be enhanced by moving towards a proactive approach, extending the goals of care beyond medical needs and broadening the focus of care to include family needs.

Mapping the patterns of care, the receipt of palliative care and the site of death for patients with malignant glioma.

High-grade malignant glioma patients face a poor prognosis, preceded by rapid functional and neurobehavioural changes, making multidisciplinary care incorporating supportive and palliative care important. This study aimed to quantify the association between symptoms,receipt of supportive and palliative care and site of death. We undertook a retrospective cohort study between 2003 and 2009 of incident malignant glioma cases who survived for at least 120 days between their first hospitalisation and their death (n = 678) in Victoria, Australia, using linked hospital, emergency department and death data. The median age of patients was 62 years, 40% were female, and the median survival was 11 months. Twenty-six percent of patients died outside of hospital, 49% in a palliative care bed/hospice setting and 25% in an acute hospital bed. Patients having 1 or more symptoms were more than five times as likely to receive palliative care. Patients who receive palliative care are 1.7 times more likely to die outside of hospital. In conclusion malignant glioma patients with a high burden of symptoms are more likely to receive palliative care and, in turn, patients who receive palliative care are more likely to die at home.

Clinical presentation and patterns of care for short-term survivors of malignant glioma.

Palliative care provision for patients with high-grade malignant glioma is often under-utilised. Difficulties in prognostication and inter-patient variability in survival may limit timely referral. This study sought to (1) describe the clinical presentation of short-term survivors of malignant glioma (survival time <120 days); (2) map their hospital utilisation, including palliative and supportive care service use, and place of death; (3) identify factors which may be important to serve as a prompt for palliative care referral. A retrospective cohort study of incident malignant glioma cases between 2003-2009 surviving <120 days in Victoria, Australia was undertaken (n = 482). Cases were stratified according to the patient's survival status (dead vs. alive) at the end of the diagnosis admission, and at 120 days from diagnosis. Palliative care was received by 78 % of patients who died during the diagnosis admission. Only 12 % of patients who survived the admission and then deteriorated rapidly dying in the following 120 days were referred to palliative care in their hospital admission, suggesting an important clinical subgroup that may miss out on being linked into palliative care services. The strongest predictor of death during the diagnosis admission was the presence of cognitive or behavioural symptoms, which may be an important prompt for early palliative care referral.

"I'm just waiting ": an exploration of the experience of living and dying with primary malignant glioma.

PURPOSE: Referral to supportive and palliative care services for people with high-grade primary malignant glioma (PMG) often occurs late in the illness course, despite significant care needs and overall poor prognosis. This study aimed to understand patient experience at the end of life and document supportive and palliative care needs. METHODS: A qualitative study was conducted involving ten PMG patients who were at different stages in the illness course including the end of life and had varying levels of physical and cognitive function. Consecutive, eligible patients attending neurosurgery, oncology, and palliative care services of two metropolitan hospitals were recruited. In-depth interviews explored supportive and palliative care needs across the disease trajectory. Interviews were analysed independently by three investigators consistent with a grounded theory approach, and emerging ideas were compared and refined to define key patient experiences. RESULTS: Despite the medical treatment and supportive care available, there remains a gap in services addressing complex existential and psychosocial needs that were markedly valued by patients. Patient experience was characterised by a pervasive loss of all that encompassed their former sense of self and a focus on immediate needs. CONCLUSIONS: Patients in this study had substantial needs, which were often not shared and not addressed by the current medical system of care. An improved multidisciplinary care model is indicated, which proactively (1) engages care coordination and advocacy; (2) minimises patients' sense of waiting and uncertainty through mapping out a plan, including involvement of palliative care in a timely fashion; and (3) actively invites discussion around goals and preferences for care to promote patients' sense of self.

Tolerability, safety, and side effects of levetiracetam versus phenytoin in intravenous and total prophylactic regimen among craniotomy patients: a prospective randomized study.

PURPOSE: Practical choice in parenteral antiepileptic drugs (AEDs) remains limited despite formulation of newer intravenous agents and requirements of special patient groups. This study aims to compare the tolerability, safety, and side effect profiles of levetiracetam (LEV) against the standard agent phenytoin (PHT) when given intravenously and in total regimen for seizure prophylaxis in a neurosurgical setting. METHODS: This prospective, randomized, single-center study with appropriate blinding comprised evaluation pertaining to intravenous use 3 days following craniotomy and at discharge, and to total intravenous-plus-oral AED regimen at 90 days. Primary tolerability end points were discontinuation because of side effect and first side effect. Safety combined end point was major side effect or seizure. Seizure occurrence and side effect profiles were compared as secondary outcomes. KEY FINDINGS: Of 81 patients randomized, 74 (36 LEV, 38 PHT) received parenteral AEDs. No significant difference attributable to intravenous use was found between LEV and PHT in discontinuation because of side effect (LEV 1/36, PHT 2/38, p = 1.00) or number of patients with side effect (LEV 1/36, PHT 4/38, p = 0.36). No significant difference was found between LEV and PHT total intravenous-plus-oral regimen in discontinuation because of side effect (hazard ratio [HR] 0.78, 95% confidence interval [CI] 0.21-2.92, p = 0.72) or number of patients with side effect (HR 1.51, 95% CI 0.77-2.98, p = 0.22). More patients assigned PHT reached the undesirable clinical end point for safety of major side effect or seizure (HR 0.09, 95% CI 0.01-0.70, p = 0.002). Seizures occurred only in patients assigned PHT (n = 6, p = 0.01). Although not significant, trends were observed for major side effect in more patients assigned PHT (p = 0.08) and mild side effect in more assigned LEV (p = 0.09). SIGNIFICANCE: Both LEV and PHT are well-tolerated perioperatively in parenteral preparation, and in total intravenous-plus-oral prophylactic regimen. Comparative safety and differing side effect profile of intravenous LEV supports use as an alternative to intravenous PHT.

Effect of Force Field Resolution on Membrane Mechanical Response and Mechanoporation Damage under Deformation Simulations.

Damage induced by transient disruption and mechanoporation in an intact cell membrane is a vital nanoscale biomechanical mechanism that critically affects cell viability. To complement experimental studies of mechanical membrane damage and disruption, molecular dynamics (MD) simulations have been performed at different force field resolutions, each of which follows different parameterization strategies and thus may influence the properties and dynamics of membrane systems. Therefore, the current study performed tensile deformation MD simulations of bilayer membranes using all-atom (AA), united-atom (UA), and coarse-grained Martini (CG-M) models to investigate how the damage biomechanics differs across atomistic and coarse-grained (CG) simulations. The mechanical response and mechanoporation damage were qualitatively similar but quantitatively different in the three models, including some progressive changes based on the coarse-graining level. The membranes yielded and reached ultimate strength at similar strains; however, the coarser systems exhibited lower average yield stresses and failure strains. The average failure strain in the UA model was approximately 7% lower than the AA, and the CG-M was 20% lower than UA and 27% lower than AA. The CG systems also nucleated a higher number of pores and larger pores, which resulted in higher damage during the deformation process. Overall, the study provides insight on the impact of force field-a critical factor in modeling biomolecular systems and their interactions-in inspecting membrane mechanosensitive responses and serves as a reference for justifying the appropriate force field for future studies of more complex membranes and more diverse biomolecular assemblies.

In vitro and in vivo characterization of microRNA-targeted alphavirus replicon and helper RNAs.

Alphavirus-based replicon vector systems (family Togaviridae) have been developed as expression vectors with demonstrated potential in vaccine development against both infectious diseases and cancer. The single-cycle nature of virus-like replicon particles (VRP), generated by supplying the structural proteins from separate replicable helper RNAs, is an attractive safety component of these systems. MicroRNAs (miRNAs) have emerged as important cellular RNA regulation elements. Recently, miRNAs have been employed as a mechanism to attenuate or restrict cellular tropism of replication-competent viruses, such as oncolytic adenoviruses, vesicular stomatitis virus, and picornaviruses as well as nonreplicating lentiviral and adenoviral vectors. Here, we describe the incorporation of miRNA-specific target sequences into replicable alphavirus helper RNAs that are used in trans to provide the structural proteins required for VRP production. VRP were found to be efficiently produced using miRNA-targeted helper RNAs if miRNA-specific inhibitors were introduced into cells during VRP production. In the absence of such inhibitors, cellular miRNAs were capable of downregulating helper RNA replication in vitro. When miRNA targets were incorporated into a replicon RNA, cellular miRNAs were capable of downregulating replicon RNA replication upon delivery of VRP into animals, demonstrating activity in vivo. These data provide the first example of miRNA-specific repression of alphavirus replicon and helper RNA replication and demonstrate the feasibility of miRNA targeting of expression vector helper functions that are provided in trans.

Effect of Aromatase Inhibition (Exemestane) on Urine Concentration of Osteoprotegerin in Healthy Postmenopausal Women.

This pilot study examined how exemestane (an aromatase inhibitor [AI]) affected osteoprotegerin (OPG) urine concentrations in postmenopausal women. Exemestane (25 mg, single dose) was given to 14 disease-free women past menopause in this nonrandomized, open-label study. Before dosing, urine specimens were gathered. Three days later, these women returned to provide urine specimens for pharmacokinetic (measurement of major parent drug and enzymatic product) and pharmacodynamic (profiling of OPG) analysis. Urine concentrations of the major parent drug (exemestane) and enzymatic product (17-hydroexemestane) were quantified using liquid chromatography-tandem mass spectrometry. An analyst software package was used for data processing. Following the manufacturer's guidelines, OPG urine concentrations were quantified using a human osteoprotegerin TNFRSF11b ELISA kit from Sigma-Aldrich. A microplate reader helped to carry out OPG data analysis and processing. Our results highlight that OPG urine concentrations were decreased 3 days after drug dosage (mean predosage OPG concentration, 61.4 ± 24.1 pg/mL; vs mean postdosage OPG concentration, 45.7 ± 22.1 pg/mL; P = .02, Wilcoxon rank test). Among the 14 volunteers enrolled in the study, 4 subjects had an increase of less than 1-fold, and the rest showed an average of a 2-fold decrease in OPG concentration (range, 1.1-5.4; standard deviation, 1.3) after exemestane administration. There was no association between fold decrease in OPG urine concentration and the pharmacokinetics of the major parent drug (exemestane) and its enzymatic product (17-hydroexemestane). We concluded that one of the off-target pharmacological effects of AIs (eg ,exemestane) may result in the reduction of osteoprotegerin.

Comparative proteomic analysis highlights metabolic dysfunction in α-synucleinopathy.

The synaptic protein α-synuclein is linked through genetics and neuropathology to the pathogenesis of Parkinson's disease and related disorders. However, the mechanisms by which α-synuclein influences disease onset and progression are incompletely understood. To identify pathogenic pathways and therapeutic targets we performed proteomic analysis in a highly penetrant new Drosophila model of α-synucleinopathy. We identified 476 significantly upregulated and 563 significantly downregulated proteins in heads from α-synucleinopathy model flies compared to controls. We then used multiple complementary analyses to identify and prioritize genes and pathways within the large set of differentially expressed proteins for functional studies. We performed Gene Ontology enrichment analysis, integrated our proteomic changes with human Parkinson's disease genetic studies, and compared the α-synucleinopathy proteome with that of tauopathy model flies, which are relevant to Alzheimer's disease and related disorders. These approaches identified GTP cyclohydrolase (GCH1) and folate metabolism as candidate mediators of α-synuclein neurotoxicity. In functional validation studies, we found that the knockdown of Drosophila Gch1 enhanced locomotor deficits in α-synuclein transgenic flies, while folate supplementation protected from α-synuclein toxicity. Our integrative analysis suggested that mitochondrial dysfunction was a common downstream mediator of neurodegeneration. Accordingly, Gch1 knockdown enhanced metabolic dysfunction in α-synuclein transgenic fly brains while folate supplementation partially normalized brain bioenergetics. Here we outline and implement an integrative approach to identify and validate potential therapeutic pathways using comparative proteomics and genetics and capitalizing on the facile genetic and pharmacological tools available in Drosophila.

Predicting human health from biofluid-based metabolomics using machine learning.

Biofluid-based metabolomics has the potential to provide highly accurate, minimally invasive diagnostics. Metabolomics studies using mass spectrometry typically reduce the high-dimensional data to only a small number of statistically significant features, that are often chemically identified-where each feature corresponds to a mass-to-charge ratio, retention time, and intensity. This practice may remove a substantial amount of predictive signal. To test the utility of the complete feature set, we train machine learning models for health state-prediction in 35 human metabolomics studies, representing 148 individual data sets. Models trained with all features outperform those using only significant features and frequently provide high predictive performance across nine health state categories, despite disparate experimental and disease contexts. Using only non-significant features it is still often possible to train models and achieve high predictive performance, suggesting useful predictive signal. This work highlights the potential for health state diagnostics using all metabolomics features with data-driven analysis.

Compressive Mechanical Properties of Porcine Brain: Experimentation and Modeling of the Tissue Hydration Effects.

Designing protective systems for the human head-and, hence, the brain-requires understanding the brain's microstructural response to mechanical insults. We present the behavior of wet and dry porcine brain undergoing quasi-static and high strain rate mechanical deformations to unravel the effect of hydration on the brain's biomechanics. Here, native 'wet' brain samples contained ~80% (mass/mass) water content and 'dry' brain samples contained ~0% (mass/mass) water content. First, the wet brain incurred a large initial peak stress that was not exhibited by the dry brain. Second, stress levels for the dry brain were greater than the wet brain. Third, the dry brain stress-strain behavior was characteristic of ductile materials with a yield point and work hardening; however, the wet brain showed a typical concave inflection that is often manifested by polymers. Finally, finite element analysis (FEA) of the brain's high strain rate response for samples with various proportions of water and dry brain showed that water played a major role in the initial hardening trend. Therefore, hydration level plays a key role in brain tissue micromechanics, and the incorporation of this hydration effect on the brain's mechanical response in simulated injury scenarios or virtual human-centric protective headgear design is essential.

Spontaneous third ventriculostomy.

Spontaneous ventriculostomy (SV) is a rare condition where there is a spontaneous communication between the ventricle and the subarachnoid space as a result of hydrocephalus. Only four cases of SV through the floor of the third ventricle have been previously reported. Two further cases are presented and the literature reviewed.

Impact of UGT2B17 Gene Deletion on the Pharmacokinetics of 17-Hydroexemestane in Healthy Volunteers.

Exemestane is an aromatase inhibitor drug used for the treatment of hormone-dependent breast cancer. 17-Hydroexemestane, the major and biologically active metabolite of exemestane in humans, is eliminated via glucuronidation by the polymorphic UGT2B17 phase II drug-metabolizing enzyme. Previous microsomal studies have shown that UGT2B17 gene deletion affects the intrinsic hepatic clearances of 17-hydroexemestane in vitro. In this open-label study we set out to assess the effect of UGT2B17 gene deletion on the pharmacokinetics of 17-hydroexemestane in healthy female volunteers with and without UGT2B17. To achieve this goal, 14 healthy postmenopausal women (8 carriers of the homozygous UGT2B17 wild-type allele and 6 carriers of the homozygous UGT2B17 gene-deletion allele) were enrolled and invited to receive a single 25-mg oral dose of exemestane. Pharmacokinetics was assessed over 72 hours postdosing. Our results showed that there were statistically significant differences in plasma 17-hydroexemestane AUC0-∞ (P = .0007) and urine 17-hydroexemestane C24h (P = .001) between UGT2B17 genotype groups. Our data suggest that UGT2B17 gene deletion influences 17-hydroexemestane pharmacokinetics in humans.

A brightness-area-product-based protocol for the quantitative assessment of antigen abundance in fluorescent immunohistochemistry.

A problem frequently facing researchers examining abundance of expression of a given antigen is measurement. When the antigen is confined to the nucleus, absolute numbers of nuclei or a percentage of nuclei expressing the antigen in a given region can be estimated. When the antigen is localized to cytoplasm, cytoplasmic organelles or processes or membranes, the assessment becomes more difficult. In these settings, an observer/experimenter may assign a density score but intra- and inter-observer agreement using a three-tiered system, and finer resolution than this, is unlikely to be reproducible. Digital image analysis provides an opportunity to minimize observer bias in quantification of immunohistochemical staining. Previously, reported digital methods have mostly employed chromogen-staining methods and often report mean image brightness. We report a method for quantitatively assessing and expressing abundance of expression of an antigen in neural tissue stained with immunofluorescent methods by determining the brightness-area-product (BAP). The described protocol utilizes simple to use commercially available software and calculates BAP rather than mean brightness as a measure more representative of antigen abundance and visual interpretation. Accordingly, we propose this protocol as a useful adjunct to observer interpretation of fluorescent immunohistochemistry and its application to assessment of antigen abundance for varying patterns of antigen localization.

Multimodality image-guided surgery for the treatment of medically refractory epilepsy.

OBJECT: The aim of this study was to review seizure outcome, imaging modalities used, and complications following surgery in patients with epilepsy who had undergone multimodality image-guided surgery at our institution. METHODS: Data from patients with epilepsy who had undergone surgery between April 1999 and October 2001 were reviewed. During this time period, 116 operations were performed in 109 patients with medically refractory epilepsy. Among these patients, 22 were selected to undergo multimodality image-guided surgery primarily on the basis of whether they had no lesion visible on conventional magnetic resonance (MR) imaging sequences, multiple lesions, or one very large lesion that could not be completely resected without the risk of significant postoperative morbidity. A fourth group of patients in whom there was a single lesion in the eloquent cortex, a location associated with a significant risk of postoperative morbidity, was also included in the analysis. This latter group was assessed with the aid of intracranial grid electrodes that were coregistered to the MR image and were used intraoperatively to minimize electrode position error. Other imaging modalities used included positron emission tomography (PET), fluid-attenuated inversion recovery (FLAIR) MR imaging, and subtracted ictal-interictal single-photon positron emission computerized tomography (SPECT) coregistered with MR imaging (SISCOM). After coregistration, images were then downloaded onto an image-guided surgical system and the epileptogenic area was then resected. The mean patient age was 33 years (range 17-46 years), and there was a mean follow up of 27 months (range 14-41 months). Multimodality coregistrations used were as follows: nine PET scans, seven subdural electrode grids, four SISCOM studies, one FLAIR MR image, and one combined PET/subdural grid. Seizure outcome was excellent in 17 patients (77%) and not excellent in five (23%), or favorable in 19 (86%) and unfavorable in three (14%). Six patients (27%) had a transient neurological deficit, one patient (5%) a permanent major deficit, and three patients (15%) a permanent minor deficit. Five patients (24%) had a transient psychiatric problem postoperatively. CONCLUSIONS: Multimodality image-guided surgery offers a new perspective in surgery for epilepsy. Functional imaging modalities previously lateralized and often localized a seizure focus, but did not provide enough anatomical information to resect the epileptogenic zone confidently and safely. The coregistration of these modalities to a volumetric MR image and their incorporation into an image-guided system has allowed surgeons to offer surgery to patients who may not previously have been considered eligible, with outcomes comparable to those in patients with more straightforward lesional epilepsy.

Single oral dose study of two isosorbide-based aspirin prodrugs in the dog.

The objective of this study was to compare two aspirin prodrugs, isosorbide diaspirinate (ISDA) and a nitroaspirin (ISMNA), with aspirin in terms of effects on dog platelet function after administration of a single oral dose. Groups of six dogs were administered ISDA (2mg kg(-1)), ISMNA (4 mg kg(-1)) or aspirin (2mg kg(-1)). Blood was sampled at 1, 2, 4, 8, 12 and 24 h post-dosing and evaluated for capacity to generate post-clotting thromboxane (TX)B2. The aggregation response to arachidonic acid (AA) (100 microM), ADP (30 microM) or collagen (10 microg mL(-1)) was estimated at each time-point using the whole blood impedance method. Plasma ISMN following oral administration of ISMNA was also measured and compared with plasma ISMN following administration of a physical mixture of ISMN and aspirin. ISDA administration (2 mg kg(-1)) was associated with a significant reduction (P < 0.05) in serum TXB2 at 12 and 24 h (>90%) post-dosing and persistent inhibition of AA-induced platelet aggregation. ISDA administration caused a more marked depression of post-clotting TXB2 levels than aspirin in this study, although its ability to inhibit platelet aggregation was less consistent than that of aspirin. The nitroaspirin ISMNA was least effective at inhibiting platelet aggregation response or TXB2 production. The ISMN AUC(0-24 h) for the ISMNA-treated dogs was 77% of that for the physical mix-treated dogs and the tmax was delayed. This study indicates that the two aspirin esters cause aspirin-like effects on platelet function, probably through aspirin release, when administered orally to dogs.