Idiopathic central diabetes insipidus in a large cohort of patients: the hypopituitarism ENEA rare observational (HEROS) study.

Central Diabetes Insipidus (CDI) is mainly associated with structural pathologies of the hypothalamic-pituitary area. Etiologies underlying CDI are identified in most patients, however idiopathic CDI is reported in 13-17% of cases after excluding other etiologies. The Hypopituitarism ENEA Rare Observational Study (HEROS study) retrospectively collected data of patients with idiopathic CDI from 14 pituitary centers in 9 countries. The cohort included 92 patients (59 females 64%), mean age at diagnosis was 35.4 ± 20.7 years, and a mean follow up of 19.1 ± 13.5 years following CDI diagnosis. In 6 women, diagnosis was related to pregnancy. Of 83 patients with available data on pituitary imaging, 40(48%) had normal sellar imaging, and 43(52%) had pathology of the posterior pituitary or the stalk, including loss of the bright spot, posterior pituitary atrophy or stalk enlargement. Anterior pituitary hormone deficiencies at presentation included hypogonadism in 6 (6.5%) patients (5 females), and hypocortisolism in one; during follow-up new anterior pituitary deficiencies developed in 6 patients. Replacement treatment with desmopressin was given to all patients except one, usually with an oral preparation. During follow up, no underlying disease causing CDI was identified in any patient. Patients with idiopathic CDI following investigation at baseline are stable with no specific etiology depicted during long-term follow-up.

Peripapillary retinal nerve fibre layer thinning rate as a biomarker discriminating stable and progressing relapsing-remitting multiple sclerosis.

BACKGROUND AND PURPOSE: Peripapillary retinal nerve fibre layer (pRNFL) thickness is a strong candidate as a biomarker of axonal degeneration in multiple sclerosis (MS). The aim was to determine a cut-off value of pRNFL thinning rates in relapsing-remitting MS (RRMS) to discriminate between stable and progressing patients. METHODS: In this 3-year prospective longitudinal study on 141 RRMS patients, annual pRNFL thinning rates (aLpRNFL) were determined by individual linear regression models. The best possible cut-off value discriminating clinically progressing (physical progression or cognitive decline) and stable patients was defined by receiver operating characteristic analysis. Cut-off values were validated using a multivariate logistic regression model. RESULTS: Average aLpRNFL in progressing patients (2.4 µm, SD 2.1) was significantly higher compared to stable patients (0.5 µm, SD 1.2, P < 0.001). At a predefined specificity of 90%, aLpRNFL >1.5 µm was able to distinguish between stable and progressing RRMS with a sensitivity of 76.1%. aLpRNFL >1.5 µm was associated with a 15-fold increased risk of clinically progressing MS (P < 0.001). CONCLUSIONS: A cut-off of aLpRNFL discriminating clinically progressing and stable RRMS was identified. After validation in independent cohorts, this cut-off could be used as a biomarker of axonal degeneration supporting disease monitoring in daily clinical routine.

Long term persistence of introduced Amblyomma geoemydae tick population under indoor conditions in Austria.

An indoor terrarium population of Amblyomma geoemydae was established subsequent to the import of a single yellow-marginated box turtle Cuora flavomarginata. This indoor tick population revealed an unexpected resistance against de-ticking trials, with persistence between 2010 and 2015, when the ticks were successfully eliminated. Ticks were collected from the bodies and shells of turtles, as well as from terraria soil. Species diagnosis of ticks was carried out according to distinguishable morphological characters and supported by molecular analysis using DNA-barcoding. Introduced exotic ticks are potential vectors of pathogens and can have an impact on wildlife, domestic animals and the human population. This case emphasizes the need for sharp surveillance and control measures on imported reptiles.

Cerebrospinal fluid:serum glucose ratio in the ventricular and lumbar compartments: implications for clinical practice.

BACKGROUND AND PURPOSE: Calculation of the cerebrospinal fluid:serum glucose (CSF:SGlu ) ratio is part of the routine cerebrospinal fluid (CSF) work-up. Reference values have been defined for lumbar CSF, but are lacking for ventricular CSF. The objective of this study was to investigate whether the CSF:SGlu ratio is similar in lumbar and ventricular compartments, and to determine cut-off values for CSF:SGlu ratio in ventricular CSF. METHODS: We included CSF samples that were collected by either lumbar puncture or ventricular drainage, with a red blood cell count <500/µL, normal white blood cell count and age-related normal total protein content, with simultaneously withdrawn serum sample and time to laboratory processing of ≤2 h. This resulted in 1808 sample pairs. Glucose concentrations in CSF and serum were measured by enzymatic spectrophotometry. RESULTS: The CSF:SGlu ratio was similar in ventricular and lumbar compartments after controlling for age, sex, time between sample withdrawal and laboratory processing, CSF white blood cell and red blood cell count, CSF total protein and serum glucose concentration using a multiple linear regression model. Lower limits for CSF:SGlu ratio in the ventricular compartment, defined as 5th percentile, were 0.51 for patients with serum glucose concentration < 100 mg/dL, 0.45 for those with serum glucose concentration ≥ 100 mg/dL and <150 mg/dL, and 0.36 for those with serum glucose concentration ≥150 mg/dL. CONCLUSIONS: The CSF:SGlu ratio was similar in the ventricular and lumbar compartments, and depended mainly on time to laboratory processing and absolute serum glucose levels. Previously established lower limits for CSF:SGlu ratio in lumbar CSF can be also applied for ventricular CSF.

Quantitation of intrathecal immunoglobulin synthesis - a new empirical formula.

BACKGROUND AND PURPOSE: Intrathecal immunoglobulin (Ig) synthesis occurs in various chronic inflammatory neurological diseases. Different formulae have been developed for quantitative determination of Ig synthesis within the cerebrospinal fluid (CSF) compartment. The hyperbolic formula of Reiber is frequently used which, however, returns a considerable number of false positive results in empirical observations. METHODS: A computerized database of more than 19 000 paired CSF and serum samples was screened for patients presumed negative for local Ig synthesis and a new formula characterizing this collective was calculated. The validity of this formula was confirmed by several validation steps. RESULTS: A cohort of 1173 patients with normal CSF findings was used for quantile regression. The 97.5th quantile of the formula Qlim(IgX)=a×Qalbb was considered as the cut-off curve for intrathecal Ig synthesis using different constants a and b for IgG, IgA and IgM. Compared to the Reiber formula, a lower level of false positive results was produced especially for IgM and IgA which was confirmed in a separate clinically well defined validation cohort. In 77 patients with discrepant findings between Reiber and our formula no specific diagnoses were found confirming the low diagnostic value of borderline Ig synthesis. CONCLUSIONS: A new approximation formula was developed for determination of intrathecal Ig synthesis which produces fewer false positive results without reducing diagnostic sensitivity.

Supratentorial white matter blurring associated with voltage-gated potassium channel-complex limbic encephalitis.

INTRODUCTION: Limbic encephalitis (LE) associated with voltage-gated potassium channel-complex antibodies (VGKC-LE) is frequently non-paraneoplastic and associated with marked improvement following corticosteroid therapy. Mesial temporal lobe abnormalities are present in around 80 % of patients. If associated or preceded by faciobrachial dystonic seizures, basal ganglia signal changes may occur. In some patients, blurring of the supratentorial white matter on T2-weighted images (SWMB) may be seen. The purpose of this study was to evaluate the incidence of SWMB and whether it is specific for VGKC-LE. METHODS: Two experienced neuroradiologists independently evaluated signal abnormalities on FLAIR MRI in 79 patients with LE while unaware on the antibody type. RESULTS: SWMB was independently assessed as present in 10 of 36 (28 %) compared to 2 (5 %) of 43 non-VGKC patients (p = 0.009). It was not related to the presence of LGI1 or CASPR2 proteins of VGKC antibodies. MRI showed increased temporomesial FLAIR signal in 22 (61 %) VGKC compared to 14 (33 %) non-VGKC patients (p = 0.013), and extratemporomesial structures were affected in one VGKC (3 %) compared to 11 (26 %) non-VGKC patients (p = 0.005). CONCLUSION: SWMB is a newly described MRI sign rather specific for VGKC-LE.

Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification.

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification.

Evidence for direct electron transfer by a gram-positive bacterium isolated from a microbial fuel cell.

Despite their importance in iron redox cycles and bioenergy production, the underlying physiological, genetic, and biochemical mechanisms of extracellular electron transfer by Gram-positive bacteria remain insufficiently understood. In this work, we investigated respiration by Thermincola potens strain JR, a Gram-positive isolate obtained from the anode surface of a microbial fuel cell, using insoluble electron acceptors. We found no evidence that soluble redox-active components were secreted into the surrounding medium on the basis of physiological experiments and cyclic voltammetry measurements. Confocal microscopy revealed highly stratified biofilms in which cells contacting the electrode surface were disproportionately viable relative to the rest of the biofilm. Furthermore, there was no correlation between biofilm thickness and power production, suggesting that cells in contact with the electrode were primarily responsible for current generation. These data, along with cryo-electron microscopy experiments, support contact-dependent electron transfer by T. potens strain JR from the cell membrane across the 37-nm cell envelope to the cell surface. Furthermore, we present physiological and genomic evidence that c-type cytochromes play a role in charge transfer across the Gram-positive bacterial cell envelope during metal reduction.

Generalized schemes for high-throughput manipulation of the Desulfovibrio vulgaris genome.

The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA "parts" to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.

Engineering strategies to recapitulate epithelial morphogenesis within synthetic three-dimensional extracellular matrix with tunable mechanical properties.

The mechanical properties (e.g. stiffness) of the extracellular matrix (ECM) influence cell fate and tissue morphogenesis and contribute to disease progression. Nevertheless, our understanding of the mechanisms by which ECM rigidity modulates cell behavior and fate remains rudimentary. To address this issue, a number of two and three-dimensional (3D) hydrogel systems have been used to explore the effects of the mechanical properties of the ECM on cell behavior. Unfortunately, many of these systems have limited application because fiber architecture, adhesiveness and/or pore size often change in parallel when gel elasticity is varied. Here we describe the use of ECM-adsorbed, synthetic, self-assembling peptide (SAP) gels that are able to recapitulate normal epithelial acini morphogenesis and gene expression in a 3D context. By exploiting the range of viscoelasticity attainable with these SAP gels, and their ability to recreate native-like ECM fibril topology with minimal variability in ligand density and pore size, we were able to reconstitute normal and tumor-like phenotypes and gene expression patterns in nonmalignant mammary epithelial cells. Accordingly, this SAP hydrogel system presents the first tunable system capable of independently assessing the interplay between ECM stiffness and multi-cellular epithelial phenotype in a 3D context.

Oxytocin and cortisol concentrations in adults with and without autism spectrum disorder in response to physical exercise.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder, whose core symptoms consist of deficits in social interaction and communication as well as restricted and repetitive behavior. Brain oxytocin (OXT) has been associated with various prosocial behaviors, and might, therefore, be involved in the pathogenesis of disorders associated with socio-emotional dysfunctions such as ASD. However, significant associations between central and peripheral OXT levels may only be present in response to physiological or stressful stimuli but were not shown under baseline conditions. In this study, we, therefore, investigated salivary and plasma OXT in response to physical exercise in adults with ASD (n â€‹= â€‹33, mean age: 36.8 â€‹± â€‹10.7 years) without intellectual impairment (IQ â€‹> â€‹70) and neurotypical controls (n â€‹= â€‹31, mean age: 31.0 â€‹± â€‹11.7 years). To stimulate the OXT system, we used rapid cycling and measured cortisol (CORT) concentrations to monitor the physiological stress response. When controlling for age, neither salivary OXT (p â€‹= â€‹.469), plasma OXT (p â€‹= â€‹.297) nor CORT (p â€‹= â€‹.667) concentrations significantly differed between groups at baseline. In addition, neither OXT nor CORT concentrations significantly differed between groups after physical exercise. Social anxiety traits were negatively correlated with plasma, but not saliva OXT concentrations in neurotypicals at baseline, while empathetic traits were positively correlated with saliva, but not plasma concentrations in autistic patients at baseline. No significant correlations between salivary and plasma OXT concentrations were found at any time point. Future studies including adult participants should investigate the effect of age on CORT and OXT concentrations in response to stress.

Study of enzymatic digestion of cellulose by small angle neutron scattering.

Small angle neutron scattering (SANS) was used to study the structure of Avicel (FD100) microcrystalline cellulose during enzymatic digestion. Digestions were performed in either of two modes: a static, quiescent mode or a dynamic mode using a stirred suspension recycled through a flow cell. The scattering pattern for as-received Avicel in D(2)O buffer is comprised of a low Q power law region resulting from the surface fractal character of the microcrystalline fibers and a high Q roll-off due to scattering from water-filled nanopores with radii approximately 20 A. For digestions in the dynamic mode the high Q roll-off decreased in magnitude within approximately 1 h after addition of enzymes, whereas in the static digestions no change was observed in the high Q roll-off, even after 60 h. These results indicate that only with significant agitation does enzyme digestion affect the structure of the nanopores.

Biomechanical rupture risk assessment of abdominal aortic aneurysms: model complexity versus predictability of finite element simulations.

OBJECTIVE: Investigation of the predictability of finite element (FE) models regarding rupture risk assessment of abdominal aortic aneurysms (AAAs). MATERIALS AND MATERIALS: Peak wall stress (PWS) and peak wall rupture risk (PWRR) of ruptured (n = 20) and non-ruptured (n = 30) AAAs were predicted by four FE models of different complexities derived from computed tomography (CT) data. Two matching sub-groups of ruptured and non-ruptured aneurysms were used to investigate the usability of different FE models to discriminate amongst them. RESULTS: All FE models exhibited a strong positive correlation between PWS and PWRR with the maximum diameter. FE models, which excluded the intra-luminal thrombus (ILT) failed to discriminate between ruptured and non-ruptured aneurysms. The predictability of all applied FE models was strengthened by including wall strength data, that is, computing the PWRR. The most sophisticated FE model applied in this study predicted PWS and PWRR 1.17 (p = 0.021) and 1.43 (p = 0.016) times higher in ruptured than diameter-matched non-ruptured aneurysms, respectively. CONCLUSIONS: PWRR reinforces PWS as a biomechanical rupture risk index. The ILT has a major impact on AAA biomechanics and rupture risk, and hence, needs to be considered in meaningful FE simulations. The applied FE models, however, could not explain rupture in all analysed aneurysms.

Label-free in situ imaging of lignification in the cell wall of low lignin transgenic Populus trichocarpa.

Chemical imaging by confocal Raman microscopy has been used for the visualization of the cellulose and lignin distribution in wood cell walls. Lignin reduction in wood can be achieved by, for example, transgenic suppression of a monolignol biosynthesis gene encoding 4-coumarate-CoA ligase (4CL). Here, we use confocal Raman microscopy to compare lignification in wild type and lignin-reduced 4CL transgenic Populus trichocarpa stem wood with spatial resolution that is sub-microm. Analyzing the lignin Raman bands in the spectral region between 1,600 and 1,700 cm(-1), differences in lignin signal intensity and localization are mapped in situ. Transgenic reduction of lignin is particularly pronounced in the S2 wall layer of fibers, suggesting that such transgenic approach may help overcome cell wall recalcitrance to wood saccharification. Spatial heterogeneity in the lignin composition, in particular with regard to ethylenic residues, is observed in both samples.

Go east: phylogeographies of Mauremys caspica and M. rivulata- discordance of morphology, mitochondrial and nuclear genomic markers and rare hybridization.

In recent years many cases of hybridization and introgression became known for chelonians, requiring a better understanding of their speciation mechanisms. Phylogeographic investigations offer basic data for this challenge. We use the sister species Mauremys caspica and M. rivulata, the most abundant terrapins in the Near and Middle East and South-east Europe, as model. Their phylogeographies provide evidence that speciation of chelonians fits the allopatric speciation model, with both species being in the parapatric phase of speciation, and that intrinsic isolation mechanisms are developed during speciation. Hybridization between M. caspica and M. rivulata is very rare, suggesting that the increasing numbers of hybrids in other species are caused by human impact on environment (breakdown of ecological isolation). Genetic differentiation within M. caspica and M. rivulata resembles the paradigm of southern genetic richness and northern purity of European biota. However, in west Asia this pattern is likely to reflect dispersal and vicariance events older than the Holocene. For M. caspica three distinct Pleistocene refuges are postulated (Central Anatolia, south coast of Caspian Sea, Gulf of Persia). Morphologically defined subspecies within M. caspica are not supported by genetic data. This is one of the few studies available about the phylogeography of west and central Asian species.

Controlled microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography.

High-pressure freezing is the preferred method to prepare thick biological specimens for ultrastructural studies. However, the advantages obtained by this method often prove unattainable for samples that are difficult to handle during the freezing and substitution protocols. Delicate and sparse samples are difficult to manipulate and maintain intact throughout the sequence of freezing, infiltration, embedding and final orientation for sectioning and subsequent transmission electron microscopy. An established approach to surmount these difficulties is the use of cellulose microdialysis tubing to transport the sample. With an inner diameter of 200 microm, the tubing protects small and fragile samples within the thickness constraints of high-pressure freezing, and the tube ends can be sealed to avoid loss of sample. Importantly, the transparency of the tubing allows optical study of the specimen at different steps in the process. Here, we describe the use of a micromanipulator and microinjection apparatus to handle and position delicate specimens within the tubing. We report two biologically significant examples that benefit from this approach, 3D cultures of mammary epithelial cells and cochlear outer hair cells. We illustrate the potential for correlative light and electron microscopy as well as electron tomography.

Serum procalcitonin has the potential to identify Staphylococcus aureus endocarditis.

The role of procalcitonin (PCT) in the diagnosis of infective endocarditis (IE) remains unclear. The aim of our study was to test the accuracy of PCT in the early diagnosis of IE and analyse if the accuracy of PCT is dependent on the type of pathogen causing IE. We carried out a prospective analysis of hospitalised patients referred for transthoracic echocardiography to search for an IE. The plasma PCT value was measured at the time of echocardiography. The diagnosis of IE was made using the modified Duke criteria. A total of 77 patients were included. IE was confirmed in 15 patients. The mean PCT values were 6.9 (+/-21.6) ug/l in patients without IE and 6.4 (+/-11.7) ug/l in patients with confirmed IE (p=0.92). IE patients with Staphylococcus aureus bacteraemia (n=7) had significantly higher PCT values compared to IE patients with other types of bacteraemia (n=8) (13.1 vs. 0.435, p=0.0299). This study demonstrates that PCT levels markedly differ at the time when IE is diagnosed. While PCT values are very high in patients with S. aureus bacteraemia, they are surprisingly low in patients with Streptococcus viridans bacteraemia, which are common offenders of endocarditis. We conclude that serum PCT has the potential to be used in the early diagnosis of S. aureus endocarditis.

Comparison of H+-ATPase and Ca2+-ATPase suggests that a large conformational change initiates P-type ion pump reaction cycles.

BACKGROUND: Structures have recently been solved at 8 A resolution for both Ca2+-ATPase from rabbit sarcoplasmic reticulum and H+-ATPase from Neurospora crassa. These cation pumps are two distantly related members of the family of P-type ATPases, which are thought to use similar mechanisms to generate ATP-dependent ion gradients across a variety of cellular membranes. We have undertaken a detailed comparison of the two structures in order to describe their similarities and differences as they bear on their mechanism of active transport. RESULTS: Our first important finding was that the arrangement of 10 transmembrane helices was remarkably similar in the two molecules. This structural homology strongly supports the notion that these pumps use the same basic mechanism to transport their respective ions. Despite this similarity in the membrane-spanning region, the cytoplasmic regions of the two molecules were very different, both in their disposition relative to the membrane and in the juxtaposition of their various subdomains. CONCLUSIONS: On the basis of the crystallization conditions, we propose that these two crystal structures represent different intermediates in the transport cycle, distinguished by whether cations are bound to their transport sites. Furthermore, we propose that the corresponding conformational change (E2 to E1 ) has two components: the first is an inclination of the main cytoplasmic mass by 20 degrees relative to the membrane-spanning domain; the second is a rearrangement of the domains comprising the cytoplasmic part of the molecules. Accordingly, we present a rough model for this important conformational change, which relays the effects of cation binding within the membrane-spanning domain to the nucleotide-binding site, thus initiating the transport cycle.

State dependent dissociation of HERG channel inhibitors.

BACKGROUND AND PURPOSE: Inhibition of HERG channels prolongs the ventricular action potential and the QT interval with the risk of torsade de pointes arrhythmias and sudden cardiac death. Many drugs induce greater inhibition of HERG channels when the cell membrane is depolarized frequently. The dependence of inhibition on the pulsing rate may yield different IC(50) values at different frequencies and thus affect the quantification of HERG channel block. We systematically compared the kinetics of HERG channel inhibition and recovery from block by 8 blockers at different frequencies. EXPERIMENTAL APPROACH: HERG channels were expressed heterologously in Xenopus oocytes and currents were measured with the two-electrode voltage clamp technique. KEY RESULTS: Frequency-dependent block was observed for amiodarone, cisapride, droperidol and haloperidol (group 1) whereas bepridil, domperidone, E-4031 and terfenadine (group 2) induced similar pulse-dependent block at all frequencies. With the group 1 compounds, HERG channels recovered from block in the presence of drug (recovery being voltage-dependent). No substantial recovery from block was observed with the second group of compounds. Washing out of bepridil, domperidone, E-4031 and terfenadine was substantially augmented by frequent pulsing. Mutation D540K in the HERG channel (which exhibits reopening at negative voltages) facilitated recovery from block by these compounds at -140 mV. CONCLUSION AND IMPLICATIONS: Drug molecules dissociate at different rates from open and closed HERG channels ('use-dependent' dissociation). Our data suggest that apparently 'trapped' drugs (group 2) dissociated from the open channel state whereas group 1 compounds dissociated from open and resting states.

Structure of the P-type ATPases.

Electron cryocrystallography of precipitant-induced two-dimensional surface crystals of the neurospora plasma membrane H+ - ATPase and tubular crystals of the sarcoplasmic reticulum Ca(2+)-ATPase has recently yielded structure maps for these ion transporters at a resolution of about 8 A. The membrane-embedded regions of these closely related enzymes are similar, but the cytoplasmic regions appear to be significantly different.