Integrating EM and Patch-seq data: Synaptic connectivity and target specificity of predicted Sst transcriptomic types.

Neural circuit function is shaped both by the cell types that comprise the circuit and the connections between those cell types 1 . Neural cell types have previously been defined by morphology 2, 3 , electrophysiology 4, 5 , transcriptomic expression 6-8 , connectivity 9-13 , or even a combination of such modalities 14-16 . More recently, the Patch-seq technique has enabled the characterization of morphology (M), electrophysiology (E), and transcriptomic (T) properties from individual cells 17-20 . Using this technique, these properties were integrated to define 28, inhibitory multimodal, MET-types in mouse primary visual cortex 21 . It is unknown how these MET-types connect within the broader cortical circuitry however. Here we show that we can predict the MET-type identity of inhibitory cells within a large-scale electron microscopy (EM) dataset and these MET-types have distinct ultrastructural features and synapse connectivity patterns. We found that EM Martinotti cells, a well defined morphological cell type 22, 23 known to be Somatostatin positive (Sst+) 24, 25 , were successfully predicted to belong to Sst+ MET-types. Each identified MET-type had distinct axon myelination patterns and synapsed onto specific excitatory targets. Our results demonstrate that morphological features can be used to link cell type identities across imaging modalities, which enables further comparison of connectivity in relation to transcriptomic or electrophysiological properties. Furthermore, our results show that MET-types have distinct connectivity patterns, supporting the use of MET-types and connectivity to meaningfully define cell types.

Peptide-bicelle interaction: Following variations in size and morphology by a combined NMR-SAXS approach.

Changes in membrane properties occurring upon protein interaction are key questions in understanding membrane protein function. To report on the occurring size and shape variation we present here a combined NMR-SAXS method performed under physiological conditions using the same samples, enabling determination of a global parameter, the hydration radius (rH) and estimating the bicelle shape. We use zwitterionic (DMPC/DHPC) and negatively charged (DMPC/DHPC/DMPG) bicelles and investigate the interaction with model transmembrane and surface active peptides (KALP23 and melittin). 1H NMR measurements based mostly on the translational diffusion coefficient D determination are used to characterize cmc values of DHPC micelles under the investigated conditions, to describe DHPC distribution with exact determination of the q (long chain/short chain) lipid ratio, to estimate aggregation numbers and effective rH values. The scattering curve is used to fit a lenticular core-shell model enabling us to describe the bicelle shape in terms of ellipsoidal axis length parameters. For all studied systems formation of oblate ellipsoids is found. Even though the rG/rH ratio would be an elegant way to characterize shape variations, we show that changes occurring upon peptide-bicelle interaction in the "effective" size and in the measure on the anisometry - morphology - of the objects can be described by using rH and the simplistic ellipsoidal core-shell model. While the influence of the transmembrane KALP peptide is significant, effects upon addition of surface active melittin peptide seem negligible. This synergy of techniques under controlled conditions can provide information about bicellar shape modulation occurring during peptide-bicelle interactions.

Fast and accurate mutation detection in whole genome sequences of multiple isogenic samples with IsoMut.

BACKGROUND: Detection of somatic mutations is one of the main goals of next generation DNA sequencing. A wide range of experimental systems are available for the study of spontaneous or environmentally induced mutagenic processes. However, most of the routinely used mutation calling algorithms are not optimised for the simultaneous analysis of multiple samples, or for non-human experimental model systems with no reliable databases of common genetic variations. Most standard tools either require numerous in-house post filtering steps with scarce documentation or take an unpractically long time to run. To overcome these problems, we designed the streamlined IsoMut tool which can be readily adapted to experimental scenarios where the goal is the identification of experimentally induced mutations in multiple isogenic samples. METHODS: Using 30 isogenic samples, reliable cohorts of validated mutations were created for testing purposes. Optimal values of the filtering parameters of IsoMut were determined in a thorough and strict optimization procedure based on these test sets. RESULTS: We show that IsoMut, when tuned correctly, decreases the false positive rate compared to conventional tools in a 30 sample experimental setup; and detects not only single nucleotide variations, but short insertions and deletions as well. IsoMut can also be run more than a hundred times faster than the most precise state of art tool, due its straightforward and easily understandable filtering algorithm. CONCLUSIONS: IsoMut has already been successfully applied in multiple recent studies to find unique, treatment induced mutations in sets of isogenic samples with very low false positive rates. These types of studies provide an important contribution to determining the mutagenic effect of environmental agents or genetic defects, and IsoMut turned out to be an invaluable tool in the analysis of such data.

A homozygous genetic variant of mitochondrial uncoupling protein 4 affects the occurrence of leukoaraiosis.

OBJECTIVES: We hypothesized that an appropriate balance of the mitochondrial energy production is essential in the maintenance of the glia cells in the brain. The aim of this study was to examine the roles of the rs10807344 and rs2270450 genetic variants of mitochondrial uncoupling protein 4 in the development of vascular demyelinization of the white matter of the brain, referred to as leukoaraiosis (LA). The mUCPs are presumed to be of great importance in the regulation of the mitochondrial membrane potential (MMP) and the cellular energy metabolism. MATERIALS AND METHODS: An analysis was performed on the clinical and genetic data on 401 LA patients without infarction and on 451 neuroimaging alteration-free subjects. After univariate statistical approaches, logistic regression models were also used to adjust differences in significant clinical factors between the patients and controls. RESULTS: The rs10807344 CC genotype proved to exert a protective effect on the occurrence of LA (neuroimaging alteration-free controls: 57.7%, LA group: 44.9%, P < 0.0002; adjusted OR: 0.41, 95% CI: 0.2-0.68, P < 0.005). CONCLUSION: The present findings indirectly raise the possibility that a shift or imbalance in the finely regulated MMP may play a role in the development of LA.

Endothelial nitric oxide synthase gene interactions and the risk of ischaemic stroke.

OBJECTIVE: Endothelial nitric oxide synthase (eNOS), which produces NO, plays an important role in the endothelial function under a wide range of physiological conditions. eNOS exon 7 polymorphism (Glu298Asp, G894T) has been considered to influence the risk of coronary artery disease. Alone, however, it has not been shown to be a genetic risk factor for ischaemic stroke. With the assumption of additive interactions, we examined whether the eNOS G894T or eNOS 894TT genotypes in combination with the methylenetetrahydrofolate reductase 677TT (MTHFR 677TT) or angiotensin-converting enzyme (ACE) D/D genotype could contribute to acute ischaemic stroke. MATERIAL AND METHODS: The data on 407 consecutive patients with acute ischaemic stroke who had never suffered a previous stroke event were analysed. As a control group, 295 stroke and neuroimaging alteration-free Caucasian subjects were examined. With the use of the PCR technique, the eNOS G894T, eNOS 894TT, MTHFR 677TT and ACE D/D mutations, as unfavourable common genotypes were determined in the participants. Logistic regression models were used to evaluate the roles of the genotypes and their combinations in the development of ischaemic stroke. RESULTS: The MTHFR C677TT genotype combined with the eNOS G894T or eNOS 894TT genotypes occurred significantly more frequently in the subjects with ischaemic stroke (7.1%; P < 0.025) than in the control group (3.1%). The co-occurrence of the ACE D/D genotype and eNOS G894T or eNOS 894TT was calculated to be more frequent in the ischaemic stroke group (20.9%, P < 0.0001) than in the control group (5.4%). CONCLUSION: The eNOS G894T or eNOS 894TT genotypes in combination with the MTHFR 677TT or ACE D/D genotype increases the risk of ischaemic stroke.

Dietary exposures to mineral hydrocarbons from food-use applications in the United States.

Food-use applications of mineral hydrocarbons (MHC) derived from petroleum sources result in dietary exposure to these compounds by consumers. Food applications of MHC, including white mineral oils, paraffin waxes, microcrystalline waxes and petrolatum, include both direct-additive uses in which the MHC is intentionally applied to the food and indirect-additive uses in which the MHC become components of the food due to migration from food-contact surfaces. A key consideration in evaluating the safety of these uses of MHC is the level of exposure that results. We estimated exposures to MHC in the US from food applications based primarily on a food-consumption approach, in which MHC concentrations in foods were multiplied by the amount of these foods consumed. This was a conservative estimate, because it assumes that all foods that might contain MHC in fact do so at maximum possible concentrations. A "poundage approach", in which the amount of MHC used in food applications was divided by the US population to determine maximum potential per capita exposures, was used to validate the consumption-based estimates. Exposures to MHC from food-packaging applications were estimated using the FDA's food-factor approach, which takes into account the volume and kinds of food packaged with specific types of materials. A conservative estimate of mean exposure to all MHC types combined is 0.875 mg/kg BW/day. Half of this, 0.427 mg/kg BW/day, is white mineral oils used as pan-release lubricants in baking, for de-dusting of stored grain, in confectioneries, and in coatings for fruits and vegetables. Nearly all of the remainder, 0.404 mg/kg BW/day, is petrolatum, primarily from its use as trough grease in bakery applications. Exposure to paraffin and microcrystalline waxes combined is only 0.044 mg/kg BW/day.

[Introduction of liposomes: examination methods and their importance in research and quality assurance]. / Liposzómák vizsgálati módszereinek bemutatása, jelentóségük a kutatásban, és a minóségbiztosításban.

After a short introduction, the most important and most commonly used liposome examination methods are described in two parts. At first, we would like to review some of the methods used to examine physical parameters (electronmicroscope, high pressure liquid chromatography, light scattering measurement etc.). In the second part, the methods are reviewed with changes during phase transition of lipids can be described (differential scanning calorimetry, fluorescence).

19F NMR study of the equilibria and dynamics of the Al3+/F- system.

A careful reinvestigation by high-field 19F NMR (470 MHz) spectroscopy has been made of the Al3+/F- system in aqueous solution under carefully controlled conditions of pH, concentration, ionic strength (I), and temperature. The 19F NMR spectra show five distinct signals at 278 K and I = 0.6 M (TMACl) which have been attributed to the complexes AlFi(3-i)+(aq) with i < or = 5. There was no need to invoke AlFi(OH)j(3-i-j)+ mixed complexes in the model under our experimental conditions (pH < or = 6.5), nor was any evidence obtained for the formation of AlF6(3-)(aq) at very high ratios of F-/Al3+. The stepwise equilibrium constants obtained for the complexes by integration of the 19F signals are in good agreement with literature data given the differences in medium and temperature. In I = 0.6 M TMACl at 278 K and in I = 3 M KCl at 298 K the log Ki values are 6.42, 5.41, 3.99, 2.50, and 0.84 (for species i = 1-5) and 6.35, 5.25, and 4.11 (for species i = 1-3), respectively. Disappearance of the 19F NMR signals under certain conditions was shown to be due to precipitation. Certain 19F NMR signals exhibit temperature- and concentration-dependent exchange broadening. Detailed line shape analysis of the spectra and magnetization transfer measurements indicate that the kinetics are dominated by F- exchange rather than complex formation. The detected reactions and their rate constants are AlF2(2+) + *F- reversible AlF*F2+ + F- (k02 = (1.8 +/- 0.3) x 10(6) M-1 s-1), AlF3(0) + *F- reversible AlF2*F0 + F- (k03 = (3.9 +/- 0.9) x 10(6) M-1 s-1), and AlF3(0) + H*F reversible AlF2*F0 + HF (kH03 = (6.6 +/- 0.5) x 10(4) M-1 s-1). The rates of these exchange reactions increase markedly with increasing F- substitution. Thus, the reactions of AlF2+(aq) were too inert to be detected even on the T1 NMR time scale, while some of the reactions of AlF3(0)(aq) were fast, causing large line broadening. The ligand exchange appears to follow an associative interchange mechanism. The cis-trans isomerization of AlF2+(aq), consistent with octahedral geometry for that complex, is slowed sufficiently to be observed at temperatures around 270 K. Difference between the Al3+/F- system and the much studied Al3+/OH- system are briefly commented on.

Study on steroidal oximinoethers: synthesis and stereostructure by NMR spectroscopy.

The condensation of O-substituted hydroxylamines with conjugated 3-oxo-4-en steroids results in a mixture of syn-anti configurations. Syn-anti ratios are influenced by sterical hindrance between the oximino substituents (e.g. O-benzyl) and the steroidal skeleton. Stereostructures and isomeric ratios were proved by detailed 1H and 13C NMR studies and also by using 2D-COSY, 2D-HSC, DEPT, 2D-COLOC and DNOE measurements.

Voluntary consumption of ethanol in WSP, WSC and WSR selectively bred mouse lines.

The genetic correlation between voluntary consumption of ethanol solutions and severity of withdrawal seizures after chronic ethanol exposure was assessed using the selectively bred Withdrawal Seizure Prone (WSP) and Resistant (WSR) mouse lines. WSP mice have at least ten-fold more severe withdrawal than WSR mice after equal chronic ethanol exposure, and withdrawal in a nonselected control line (WSC) is intermediate to withdrawal in the WSP and WSR lines. In the first experiment, mice from the WSP, WSC and WSR lines were offered a choice between 2.2, 4.6 and 10.0% ethanol solutions and water in three consecutive eight-day sessions. WSR mice consumed more ethanol than WSP mice, and WSC mice were intermediate. In a second experiment, WSP and WSR mice were offered ethanol solutions in concentrations that were adjusted up or down every two days depending upon the amount of ethanol consumed. WSP and WSR mice displayed very different patterns of drinking, with WSP mice drinking more ethanol in early stages of the experiment, and WSR mice drinking more ethanol later. Results of these experiments suggest that some genes influencing severity of withdrawal from ethanol also influence voluntary ethanol drinking.

Refractory supraventricular reentry tachycardia treated by radiofrequency atrial pacemaker.

The authors report on a case of drug-resistant supraventricular tachycardia with frequent and long-lasting paroxysmal attacks. Electrophysiologic study revealed that the supraventricular tachycardia was based on a reentry circuit. The attack was regularly induced by premature delay 340-400 ms atrial stimulation and it could always be terminated by rapid atrial stimulation within 1-2 seconds. Atrioventricular 2:1 block occurred with very rapid atrial stimulation of greater than 190/min. The patient received a radiofrequency atrial pacemaker, and at the first sign of a tachyarrhythmia he switched on the instrument for 1-2 sec, and the attack was promptly terminated.