Body fluid identification using a targeted mRNA massively parallel sequencing approach - results of a EUROFORGEN/EDNAP collaborative exercise.

In a previous study we presented an assay for targeted mRNA sequencing for the identification of human body fluids, optimised for the Illumina MiSeq/FGx MPS platform. This assay, together with an additional in-house designed assay for the Ion Torrent PGM/S5 platform, was the basis for a collaborative exercise within 17 EUROFORGEN and EDNAP laboratories, in order to test the efficacy of targeted mRNA sequencing to identify body fluids. The task was to analyse the supplied dried body fluid stains and, optionally, participants' own bona fide or mock casework samples of human origin, according to specified protocols. The provided primer pools for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms included 33 and 29 body fluid specific targets, respectively, to identify blood, saliva, semen, vaginal secretion, menstrual blood and skin. The results demonstrated moderate to high count values in the body fluid or tissue of interest with little to no counts in non-target body fluids. There was some inter-laboratory variability in read counts, but overall the results of the laboratories were comparable in that highly expressed markers showed high read counts and less expressed markers showed lower counts. We performed a partial least squares (PLS) analysis on the data, where blood, menstrual blood, saliva and semen markers and samples clustered well. The results of this collaborative mRNA massively parallel sequencing (MPS) exercise support targeted mRNA sequencing as a reliable body fluid identification method that could be added to the repertoire of forensic MPS panels.

Quantitative phase measurement in coherent diffraction imaging.

We demonstrate high spatial resolution phase retrieval of a non-periodic gold nano-structure using the method of Fresnel coherent diffractive imaging. The result is quantitative to better than 10% and does not rely on any a priori knowledge of the sample.

A 3D view of the host cell compartment in P. falciparum-infected erythrocytes.

The most deadly of the human malaria parasites, Plasmodium falciparum, invades the erythrocytes of its host and initiates a remarkable series of morphological rearrangements within the host cell cytoplasm. The mature erythrocyte is effectively a floating sack of haemoglobin with no endogenous protein synthesis or protein trafficking machinery. In order to colonise and remodel its extracellular space, the parasite generates a series of novel structures that are involved in the export of virulence factors to the surface of the host cell. These include extensions of the parasite's vacuolar membrane, known as the tubulovesicular network, and structures referred to as Maurer's clefts. Maurer's clefts are convoluted collections of distorted discs that are tethered to the red blood cell membrane by structures with stalk-like profiles. Recently electron tomography has enabled visualisation--in three dimensions and at unprecedented resolution--the complexity of the membrane systems within the infected RBC cytoplasm.

Coeliac disease, unilateral occipital calcifications, and drug-resistant epilepsy: successful lesionectomy.

PURPOSE: To draw attention to the triad of coeliac disease (CD), occipital calcifications, and drug-resistant epilepsy, with focus on the outcome of epilepsy surgery. METHODS: We describe a male patient who despite a diagnosis of CD from the age of 9 did not comply with the gluten-free diet. At the age of 11 he developed simple and complex partial seizures with visual symptoms, anxiety, and ambulatory automatisms. His epilepsy appeared to be drug resistant, and after having tried nine antiepileptic drugs (AEDs), alone or in combinations, he underwent a presurgical evaluation at the age of 30. Interictal standard electroencephalograms (EEGs) disclosed frequent biparieto-occipital epileptiform discharges. Computed tomography showed cortical-subcortical punctate calcifications in the right parieto-occipital region, where his seizures seemed to start, according to ictal EEG registrations from intracranial strip electrodes. RESULTS: At the age of 31 he underwent epilepsy surgery. A 5 x 6 cm large area of the right parieto-occipital region was resected, including the area with calcifications. Except for a few short-lasting episodes of anxiety (simple partial seizures?) he has now been seizure-free for 12 years. AEDs were withdrawn 5 years ago. Postoperatively he was left with an upper left-sided quadrant anopsia, which is not bothering him. CONCLUSIONS: In patients with CD, unilateral occipital calcifications, and drug-resistant epilepsy, epilepsy surgery should be considered, as a lesionectomy might be very successful.

Synthesis and structural organization of zonular fibers during development and aging.

Zonular fibers are a specific form of extracellular matrix composed mainly of fibrillins. The purpose of this study was to determine which cells secrete fibrillin-1 during development and aging. A specific guinea pig fibrillin-1 mRNA probe was designed and cloned in order to identify fibrillin-secreting cells in guinea pig eye, using in situ hybridization. Immunofluorescence, with a specific guinea pig monoclonal antibody, was used to compare protein levels at different stages from birth to 35 months of age. Electron microscopy and immunolabeling were used to investigate the organization of zonular microfibril bundles. We identified the cells of non-pigmented epithelium of the ciliary body as the main source of fibrillin secreted into the zonule. Moreover, while mRNA expression decreased during aging, there was no decrease in fibrillin immunoreactivity, as previously described in human aorta. These data indicate a very slow turnover of the zonular microfibrils which can be correlated with the appearance during aging of a new periodic fibrillar structure. This new structure may reflect an increased cross-linking in the long-lived zonular microfibrillar bundles.

Surgical treatment of anterior circulation aneurysms.

The purpose of this paper is to present the results, assessed by an independent observer, of surgical treatment of 428 consecutive patients harbouring aneurysms of the anterior circulation, together with a review of relevant anatomy and operative strategy. At follow-up (mean 5.6 years) 89.3% lived at home and were independent, 5.1% lived at home but needed some kind of assistance, 2.0% lived in institution, whereas information was unavailable in 3.6% of living patients. Two hundred and fifty-three patients (64.5%) had unchanged employment status, 0.3% worked in sheltered environment, whereas 30.9% went out of work due to their subarachnoid hemorrhage (SAH). Information about employment status was unavailable in 4.3%. For aneurysms of the internal carotid, anterior communicating and middle cerebral artery, respectively, mortality was 3.2, 3.9 and 5.6%, whereas 92.0, 88.1 and 89.0% of surviving patients lived at home and were independent and 67.0, 63.6 and 63.0% had unchanged employment status. Three-months mortality of all causes was 4.2%. In the postoperative period 53 (12.4%) patients developed clinical signs of vasospasms, 6 (1.4%) had cardiac infarction, 4 (0.9%) lung oedema, 4 (0.9%) deep vein thrombosis, and 7 patients (1.6%) infection. During the follow-up period shunt-dependent hydrocephalus developed in 4.2% and 0.2% had a subsequent SAH from the same aneurysm. Forty-three patients were on anticonvulsive therapy.

Atomic force microscopy and modeling of natural elastic fibrillin polymers.

A central issue in the understanding of Marfan syndrome deals with the functional architecture of fibrillin-containing microfibrils. Fibrillin-rich microfibrils are long extracellular matrix fibrillar components exhibiting a 50 nm periodic beaded-structure with a width of around 20-25 nm after rotary shadowing and a 10-12 nm diameter when observed in ultra-thin sections. They are composed of fibrillin monomers more or less associated with many other components which are, for the most part, poorly characterized up to date. They are known to be elastic but few data have been accumulated to understand their properties. Atomic force microscopy (AFM) allowed us to morphologically differentiate fibrillin-rich microfibrils from other fibrillar components and to investigate the thin structure of these beaded filaments in their native state. They showed, in AFM, a periodic beaded structure ranging from 50 to 60 nm and a width of about 40 nm. The different sizes of fibrillin-containing microfibrils previously observed after rotary shadowing and in ultra-thin sections was resolved with our technique and is revealed to be 10 nm in diameter. Each beaded microfibril appears to be composed of heterogeneous beads connected by 2-3 arms. An orientation of the microfibrils has been shown, and allows us to propose a complementary model of microfibrillar monomer association.

Fibrillin-rich microfibrils: structural modifications during ageing in normal human zonule.

Ageing is marked by ultrastructural and functional changes in most tissues. In part, these changes are caused by a loss of elasticity in the elastic fibers of the extracellular matrix. These fibers are composed of the protein elastin associated with microfibrils of 8 to 12 nm in diameter. Microfibrils contain fibrillins as major constituents. Mutations in fibrillin genes are considered as primary causes of Marfan syndrome, a genetic disorder with pathological manifestations in the cardiovascular and skeletal systems, in addition to dysfunctions in the eye. Fibrillin is also the major protein of the ciliary zonule fibers. During ageing, these fibers become more fragile, and concomitantly, an increased risk for ocular pathologies is observed. We have investigated structural modifications in fibrillin-rich microfibrils during ageing of human ciliary zonule. Observations using light microscopy and transmission electron microscopy after rotary shadowing allowed us to describe the organization of the zonule fibers and their insertion into the ciliary body. Our results emphasize qualitative differences between young and old zonules, which are likely due to modifications in the structure of microfibrils.

Purification of fibrillin-containing microfibrils and collagen VI microfibrils by density gradient centrifugation.

A method is described for the purification of collagen VI microfibrils and fibrillin-containing microfibrils, respectively. High M(r) microfibril-rich preparations isolated from nuchal ligament by bacterial collagenase digestion and size fractionation were purified by CsCl density gradient centrifugation. Localization of collagen VI and fibrillin within the gradient was achieved by SDS-PAGE/Western blotting. Large collagen VI microfibrillar aggregates were present at the top of the gradient. Hyaluronidase pretreatment dissociated these aggregates and enabled purification of collagen VI microfibrils at a density of 1.33 g/ml. Fibrillin-containing microfibrils separated at 1.37 g/ml and copurified with MAGP1, but not LTBP1, LTBP2, or fibronectin. Confirmation of the intact status of the purified microfibrils was obtained by rotary shadowing. The ability to separate and purify these complex macromolecules provides a powerful means of addressing their molecular composition, organization, and structure:function relationships.

Amino-acid release from human cerebral cortex during simulated ischaemia in vitro.

The aim of the present study was to investigate the release of amino-acids in human cerebral cortex during membrane depolarization and simulated ischaemia (energy deprivation). Superfluous tissue from temporal Iobe resections for epilepsy was cut into 500 microns thick slices and incubated in vitro. Membrane depolarization with 50 mM K+ caused a release of glutamate, aspartate, GABA and glycine, but not glutamine or leucine. The release of glutamate and GABA was Ca(++)-dependent. Slices were exposed to simulated ischaemia (energy deprivation; ED) by combined glucose/oxygen deprivation. This caused a Ca(++)-independent release of glutamate, aspartate, GABA, glycine, and taurine which started after 8 min, peaked at the end or shortly after the 27 min period of ED, and returned to control levels within 11 min following termination of ED. Preloaded D-[3H]aspartate was released both during K(+)-stimulation and ED. Release of D-[3H]aspartate during ED was delayed compared to glutamate supporting an initial phase of synaptic glutamate release. Uptake of L-[3H]glutamate was increased during the period of glutamate release, suggesting passive diffusion across the cell membrane or enhanced transport efficacy in cellular elements with functioning uptake mechanisms.

Surgical treatment for partial epilepsy among Norwegian adults.

We conducted a retrospective longitudinal self-controlled study of 124 adult patients treated with resective surgery for medically uncontrolled partial epilepsy from 1949 to 1988. Approximately 65% of the patients experienced > 95% reduction in seizure frequency, and 75% had worthwhile improvement of at least 75% seizure reduction. Significant reductions were noted in all major seizure types treatable with resective surgery; complex partial (CPS), simple partial (SPS), and secondarily generalized tonic-clonic seizures (GTC) (all p < 0.05). Tissue pathology and region of resection did not provide significant information with respect to seizure outcome. EEG in the first postoperative year was an important predictor of long-term seizure outcome (p = 0.03). One third of the temporal lobe resected patients had neurologic deficits as a consequence of the resection as compared with 14% of patients with frontal resections (p = 0.03). One third of the deficits among the temporal lobe resected patients were considerable, with possible social implications. Half of the patients with preoperative focal spike activity had a normal EEG postoperatively. One fifth of patients maintained their preoperative epileptic focus after the operation, and about one fifth displayed new foci. Approximately one fourth of the patients were free of medication for a median of 16 years postoperatively, and 60% of patients who were seizure-free were still receiving medication. There was no operative mortality, but the late mortality, as expected, was higher than that of the general population. Two male patients (1.6%) committed suicide.

Surgical treatment for partial epilepsy among Norwegian children and adolescents.

We conducted a retrospective longitudinal self-controlled study of 64 patients aged 4-19 years treated with resective surgery for partial epilepsy from 1952 to 1988. Approximately 60% of patients experienced > 95% reduction in seizure frequency, and 70% had worthwhile improvement of at least 75% reduction. Seizure relief was more frequent among patients who underwent operation after 1978, and significant differences by time period of operation were noted for those with temporal lobe excisions and patients with normal tissue histology. The region of resection and the age at treatment did not provide significant information with respect to seizure outcome. Postresection electrocorticography (ECoG) and EEG of the first postoperative year predicted later seizure outcome. Small neurologic deficits were more common among patients resected in the temporal lobe than in patients resected in the frontal lobe. Half of the patients with preoperative unilateral focal activity and a third of those with bilateral focal activity had normal EEG postoperatively. One fourth had discontinued antiepileptic drug (AED) therapy. As expected, long-term mortality was significantly higher than the mortality of the general population. Seven patients died during follow-up. Two male patients committed suicide.

Predictive factors for success in surgical treatment for partial epilepsy: a multivariate analysis.

We conducted a longitudinal self-controlled study of 131 patients aged 4-60 treated with resective surgery for medically uncontrolled partial epilepsy from 1949 to 1988. Using multivariate logistic regression, we showed that pre- and perioperative variables can be used to predict "success" or "failure" of surgical resective treatment in approximately 79% of cases. If the predicted probability is > 0.75 or < 0.25, the model predicts a correct result in 87% of cases. Eight predictive factors emerged with a backward multivariate logistic regression model with the likelihood-ratio (LR) test to exclude variables from the equation: (a) the influence of the surgical team and surgical procedure, (b) the presence of paresis preoperatively, (c) duration of disease, (d) age at treatment, (e) positive neuroradiologic findings in preoperative investigations, (f) preoperative complex partial seizures (CPS), (g) nonepileptic EEG abnormalities, and (h) generalized spike activity in EEG preoperatively. Sex, age at first seizure, area of resection, presence of simple or generalized seizures preoperatively, preoperative seizure frequency, tissue pathology, use of computed tomography/nuclear magnetic resonance (CT/NMR) in preoperative investigations, degree of preoperative neurologic deficit, perioperative electrocorticographic results, and bilateral EEG spikes did not have predictive value in the model.

Calcium-dependent release of glutamate from human cerebral cortex.

The release of the excitatory amino acids glutamate and aspartate from human neocortex was investigated in vitro by utilizing brain tissue removed during anterior temporal lobectomies for tumor or epilepsy. Depolarization (50 mM K+) increased the glutamate release to 291% of control (809 pmol/mg/min) during blocked synaptic transmission and to 669% (1859 pmol/mg/min) when synaptic transmission was not blocked. Aspartate release increased to 141% (326 pmol/mg/min) and 178% (412 pmol/mg/min) respectively. The difference between release with and without blocked synaptic transmission was statistically significant only for glutamate (P less than 0.01). These data provides evidence for a Ca(2+)-dependent release of glutamate, supporting a possible role of this amino acid as a neurotransmitter in human neocortex.

The prognostic value of intra-operative observations during thalamotomy for parkinsonian tremor.

Data from 27 thalamotomies were analyzed with respect to possible correlations between certain intra-operative observations and the long-term effect on parkinsonian tremor. Tremor reduction caused by mechanical impact of the electrodes in the target area was not correlated with the long-term effect on the tremor. The same was true for the threshold intensities during the intra-operative electrical stimulation of the target area, stimulation that facilitates and/or inhibits the tremor. In a minority of the patients, all with good long-term results, a combination of a pronounced tremor inhibition from the electrode insertion and a low threshold intensity was observed. Variations in other lesion parameters were not correlated with the outcome. The results are discussed within the framework of a "tremor" vs. a "tonus" mechanism underlying the thalamotomy effect.

Surgical versus medical treatment for epilepsy. II. Outcome related to social areas.

We conducted a retrospective parallel cohort study comparing surgical and medical treatment for epilepsy. The surgical group contained all 201 patients treated with resective surgery for epilepsy in Norway since the first operation in 1949 until January 1988. The 185 patients in the control group, medically treated only, were closely matched for year of treatment, age at treatment, sex, seizure type, and neurologic deficit before treatment. Between 75 and 95% of the survivors (median 17 years after treatment) completed two questionnaires on their social situation. Although surgical treatment improved the seizure situation (about one-fourth had some neurologic deficit), a considerably smaller long-range influence on different social aspects was observed. There were no significant differences between the two groups in educational status, social pensions, social status, marital status, fertility, dependency in residential situation, the need for aid in daily activities of living (ADL), or the need for being looked after, when we controlled for pretreatment status. In all, 25.3% of the surgically treated patients and 8.5% of the controls were not receiving anti-epileptic drugs (AEDs) at the time of investigation (Mann-Whitney U test, two-tailed p = 0.0011). A considerably higher proportion of the surgically treated (53.2%) than control patients (24.2%) claimed that the treatment had improved their "working ability" (Mann-Whitney U test, two-tailed p less than 0.0001), but this resulted in significant improvements in the actual working situation only for those in regular education or work before treatment (chi 2 = 6.514, p = 0.038).

Surgical versus medical treatment for epilepsy. I. Outcome related to survival, seizures, and neurologic deficit.

We conducted a retrospective parallel longitudinal cohort study comparing surgical and medical treatment for epilepsy. The surgical group contained 201 patients treated with resective surgery for epilepsy in Norway since the first operation in 1949 until January 1988. The 185 control group patients treated medically only were closely matched for year of treatment, age at treatment, sex, seizure type, and neurologic deficit before treatment. There was no significant difference in survival between the two groups. The total monthly seizure frequency in the first and second year after operation and last year of registration (median 9 years) was significantly lower in the surgical group than in the control group (Mann-Whitney U test, two-tailed p less than 0.0001). The patterns were similar, with significant differences for subgroups with similar pretreatment status, such as seizure frequency, age, etiology and EEG-focality. Twenty-three and four-tenths percent (n = 40) of the surgically treated, and 2.9% of the controls had contracted neurologic deficits within 2 years after treatment. The difference was significant (chi square = 32.89, p less than 0.0001). Psychosis or permanent psychotic symptoms were reported in 6.7% (n = 11) of the surgically treated patients, and we suspect a higher proportion of psychotic development in the surgical group than in the control group. We conclude that surgical treatment for partial epilepsy is more successful than medical treatment in producing seizure reduction, provided the indications for operation exist. Surgical treatment produces more neurologic deficits than medical treatment (and possibly more psychiatric morbidity), and this factor must be weighed against the reduction in seizure frequency. The two treatments are equal for longterm survival.

Stereotaxic thalamotomy--experiences from the levodopa era.

After the introduction of levodopa drugs in the late 1960s, the number of thalamotomies fell dramatically world wide. However, as the Parkinsonian tremor proved rather resistant to levodopa treatment, the interest in this operation has been reviewed. During 1978-86, 51 stereotaxic thalamotomies were performed in 48 patient in our department. Thirty three of these patients had Parkinsonism, nine multiple sclerosis (MS) and the remaining six had various other involuntary movement disorders. The operation was most useful in the Parkinsonian group. Nearly 80% of these patients gained a substantial benefit in their daily lives. Patients with MS were all in advanced stages of the disease, and the operation was tried as a last resort. They had less benefit and more complications from operation than the other patients.

A randomized study of SMS 201-995 versus bromocriptine treatment in acromegaly: clinical and biochemical effects.

Twenty-six acromegalic patients were randomized to treatment with either SMS 201-995 or bromocriptine in increasing doses and were investigated before treatment, after 2, 4, and 8 weeks of treatment, and 2 weeks after discontinuation of treatment. There were two dropouts from the bromocriptine group and one from the SMS 201-995 group. Amelioration of clinical signs and symptoms was seen in both groups during treatment. After 8 weeks mean 12-h GH concentrations had declined from 13.8 +/- 5.2 to 2.9 +/- 4.4 (mean +/- SEM) in SMS 201-995-treated and from 18.8 +/- 7.5 to 5.4 +/- 1.2 micrograms/L in bromocriptine-treated patients. Somatomedin-C concentrations fell from 3.04 +/- 0.36 to 1.43 +/- 0.36 in SMS 201-995-treated and from 2.93 +/- 0.40 to 2.13 +/- 0.27 U/mL in bromocriptine-treated patients. Size reduction of the pituitary tumor was seen in one patient receiving bromocriptine. Gastrointestinal glucose absorption was delayed, and insulin secretion suppressed during treatment with SMS 201-995. Hemoglobin-A1 concentrations remained unchanged in SMS 201-995-treated patients, but declined in the bromocriptine group. Side-effects were common, but usually tolerable, with both treatments. It is concluded that both drugs are of benefit in the treatment of acromegaly.

[Surgical treatment of epilepsy. Needs, examination, resource demands and results]. / Kirurgisk behandling av epilepsi. Behov, utredning, krav til ressurser og resultater.

This review gives the current status of epilepsy surgery, the diagnostic procedures involved and the resources needed. Relatively few of the epilepsy patients are possible candidates for surgery. However, only a small fraction of these possible candidates are actually offered this treatment, in Norway as well as world-wide. The need for increased activity within this field is emphasized, as is the necessity of multi-disciplinary, regionalized epilepsy centers.