Cryo-focused ion beam sample preparation for imaging vitreous cells by cryo-electron tomography

Cryo-electron tomography (CET) is a well-established technique for imaging cellular and molecular structures at sub-nanometer resolution. As the method is limited to samples that are thinner than 500 nm, suitable sample preparation is required to attain CET data from larger cell volumes. Recently, cryo-focused ion beam (cryo-FIB) milling of plunge-frozen biological material has been shown to reproducibly yield large, homogeneously thin, distortion-free vitreous cross-sections for state-of-the-art CET. All eukaryotic and prokaryotic cells that can be plunge-frozen can be thinned with the cryo-FIB technique. Together with advances in low-dose microscopy, this has shifted the frontiers of in situ structural biology. In this protocol we describe the typical steps of the cryo-FIB technique, starting with fully grown cell cultures. Three recently investigated biological samples are given as examples

Trans synaptic assemblies link synaptic vesicles and neuroreceptors

Synaptic transmission is characterized by fast, tightly coupled processes and complex signaling pathways that require a precise protein organization, such as the previously reported nanodomain colocalization of pre and postsynaptic proteins. Here, we used cryo electron tomography to visualize synaptic complexes together with their native environment comprising interacting proteins and lipids on a 2 to 4 nm scale. Using template free detection and classification, we showed that tripartite trans synaptic assemblies subcolumns link synaptic vesicles to postsynaptic receptors and established that a particular displacement between directly interacting complexes characterizes subcolumns. Furthermore, we obtained de novo average structures of ionotropic glutamate receptors in their physiological composition, embedded in plasma membrane. These data support the hypothesis that synaptic function is carried by precisely organized trans synaptic units. It provides a framework for further exploration of synaptic and other large molecular assemblies that link different cells or cellular regions and may require weak or transient interactions to exert their functio

Synucleins Have Multiple Effects on Presynaptic Architecture

Synucleins ( a , b , g -synuclein) are abundant presynaptic proteins, with a -synuclein linked to the pathogenesis of Parkinson’s disease. Vargas et al. investigate the effects of deleting synucleins and overexpressing mutated a -synuclein on synapse architecture using electron microscopy. They find that synucleins regulate presynaptic terminal size and synaptic vesicle distribution

Evaluation of composition and antioxidant activity of the functional teas extracts

Vaistinės arbatos mišiniai vertingi dėl jose esančių komponentų, kaip fenoliniai junginiai (atsakingi už antimikrobinį veikimą) ir flavonoidai (natūralūs antioksidantai, surišantys laisvuosius radikalus). Flavonoidų cheminės savybės apžvelgtos darbe. Šio darbo tikslas - atlikti flavonoidų ir fenolinių junginių kiekybinę ir kokybinę analizę bei nustatyti bendrą antioksidacinį aktyvumą vaistinių arbatų mišinių ekstraktuose. Paruošti metanoliniai vaistinių arbatų mišinių ekstraktai po kietafazės ekstrakcijos buvo analizuojami spektrofotometru, kapiliarine elektroforeze ir efektyviąja skysčių chromatografija su reakcijos detektoriumi. Spektrofotometru išmatuotas bendras fenolinių junginių, flavonoidų kiekis vaistinėse arbatose. Įvertintos metanolinių ekstraktų laikymo sąlygos šaldytuve bei sausos žaliavos laikymo sąlygos sausoje, vėsioje vietoje. Efektyviąja skysčių chromatografija įvertintas fenolinių junginių kiekis ir jų antioksidacinis aktyvumas su 2,2 - difenil – 1 - pikrilhidrazilo (DPPH ) laisvuoju radikalu. Blukinimo reakcija matuota UV detektoriumi ties 517 nm. Efektyviaja skysčių chromatografija išskirstyti fenoliniai junginiai, taikant gradientą. Atliktas kapiliarinės elektroforezės metodo optimizavimas: įvertintas buferio pH, organinio tirpiklio priedo, įtampos įtaka skirstymo efektyvumui, skiriamajai gebai. Atlikti įteisinimo žingsniai: įvertintos aptikimo ir nustatymo ribos, matavimų glaudumas, stabilumas ir atrankumas. Priedo metodu buvo identifikuoti flavonoidų standartai vaistinių arbatų mišiniuose. Gauti rezultatai kapiliarine elektroforeze, efektyviąja skysčių chromatografija buvo lyginami su spektrofotometru gautais rezultatais. Įvertintos sausos žaliavos ir metanolinių ekstraktų vaistinių arbatų mišinių laikymo sąlygos. Didžiausias fenolinių junginių kiekis nustatytas „Prakaitavimą skatinančioje“ arbatoje 3,53mg/g. Flavonoidų didžiausias kiekis nustatytas „Tulžį varančioje“ arbatos mišinyje 2,08mg/g. Nustatytas suminis flavonoidų kiekis ne visiškai atitinka fenolinių junginių kiekio pasiskirstymą, tačiau fenolinių junginių kiekiai koreliuoja su antioksidaciniu aktyvumu.Previous studies refer mostly to the HPLC analysis of the flavonoids, for oxidative product deterioration and radicals scavenging action. Methanolic extracts after solid phase extraction of 6 different functional teas were analyzed by means of capillary zone electrophoresis (CZE) and high performence liquid chromatography (HPLC) with reaction detector. A rapid on-line method for screening components was developed using methanolic solution 2,2 – diphenyl – picrylhydrazyl (DPPH) as stable free radical. Bleaching reaction is detected as a negative peak by an absorbance detector at 517 nm. HPLC gradient runs with mobile phase composition ranging from 33 to 90% organic solvent (methanol) in water with 0.1% TFA. The activity of compounds in functional teas (complex mixtures) was determined. Development and validation of the CZE method was carried out.. Limits of detection and quantification were determined. The results obtained using CZE are comparable to the spectrophotometric and HPLC analysis results. The influence of the extraction conditions (with solid phase extraction (SPE) and without SPE extraction), amount of flavonoids and phenolic compounds in the various functional teas was studied. The highest content of flavonoids was obtained in Choleretic herbs mixture 2,08 mg/g and the highest content of phenolic compounds was determined in Diaphoretic functional tea 3,53mg/g in raw dry medicinal functional tea herbs. The total amount of flavonoids did not directly correlate with the antioxidant activity, however amount of phenolic compounds in functional teas has shown a correlation to the antioxidant activity.Vytauto Didžiojo universiteta

Ekologinės ir įprastinės žemės ūkio gamybos augalinės produkcijos kokybės vertinimas

Vytauto Didžiojo universitetasŽemės ūkio akademij

MAN1 Restricts BMP Signaling During Synaptic Growth in Drosophila

Bone morphogenic protein (BMP) signaling is crucial for coordinated synaptic growth and plasticity. Here, we show that the nuclear LEM-domain protein MAN1 is a negative regulator of synaptic growth at Drosophila larval and adult neuromuscular junctions (NMJs). Loss of MAN1 is associated with synaptic structural defects, including floating T-bars, membrane attachment defects, and accumulation of vesicles between perisynaptic membranes and membranes of the subsynaptic reticulum. In addition, MAN1 mutants accumulate more heterogeneously sized vesicles and multivesicular bodies in larval and adult synapses, the latter indicating that MAN1 may function in synaptic vesicle recycling and endosome-to-lysosome trafficking. Synaptic overgrowth in MAN1 is sensitive to BMP signaling levels, and loss of key BMP components attenuate BMP-induced synaptic overgrowth. Based on these observations, we propose that MAN1 negatively regulates accumulation and distribution of BMP signaling components to ensure proper synaptic growth and integrity at larval and adult NMJs

Hierarchical detection and analysis of macromolecular complexes in cryo-electron tomograms using Pyto software

Molecular complexes, arguably the basic units carrying cellular function, can be visualized directly in their native environment by cryo-electron tomography. Here we describe a procedure for the detection of small, pleomorphic membrane-bound molecular complexes in cryo-tomograms by a hierarchical connectivity segmentation. Validation on phantom and real data showed above 90% true positive rates. This segmentation procedure is implemented in the Pyto software package, together with methods for quantitative characterization and classification of complexes detected by our segmentation procedure and for statistical analysis between experimental conditions. Therefore, the methods presented provide a means for the detection and quantitative interpretation of structures captured in cryo-electron tomograms, as well as for the elucidation of their cellular function. (C) 2016 Published by Elsevier Inc

The influence of the sample preparation of carrots (Daucus carota L. Neptun) on the antioxidant activity and phenolic compounds

Skirtingomis ekstrakcijos sąlygomis – derinant sausos masės kiekį ir tirpiklio procentinį santykį, atliekant reakcijas su laisvuoju radikalu DPPH bei matuojant spektrofotometriškai ties 515 nm bangos ilgiu – siekta nustatyti antioksidacinį ‘Neptun’ morkų veislės aktyvumą. Šioje morkų veislėje rasti fenolinių junginių kiekiai svyravo tarp 0,07 ir 0,31 mg/ml metanolio ekstrakto ir tarp 1,51 ir 7,1 mg/g sausos morkų masės pagal galo rūgšties ekvivalentus (Folin- Ciocalteu modifikuotas metodas). Didžiausias antioksidacinis aktyvumas – 82 % nustatytas sukapotų ir vaisių džiovyklėje išdžiovintų morkų, kai 0,5 g sausos morkų žaliavos ekstrahuota 75 % metanoliu. Mažiausias aktyvumas – apie 8 % gautas ekstrahuojant 0,25 g sausos morkų žaliavos 100 % metanolio tirpikliu. Išmatavus antioksidacinio aktyvumo kaitą laiko atžvilgiu kas 15min., reikšmingi skirtumai gauti iki 45min. taikant 100 % metanolio ekstrakciją 0,5 g kapotai sausai medžiagai (p > 0,05). Su 75 % metanolio ekstrakcija gauti skirtumai nereikšmingi laiko atžvilgiu. 0,5 g kapotose morkose, džiovintose vaisių džiovyklėje, pasiekus 80 % aktyvumą, reakcija pasiekdavo pusiausvyrą. Džiovinimo poveikis antioksidaciniam aktyvumui buvo nedidelis (p > 0,05), bet reikšmingas bendram fenolinių junginių kiekiui (p Vytauto Didžiojo universitetasŽemės ūkio akademij

Aging Drosophila melanogaster Display Altered Pre- and Postsynaptic Ultrastructure at Adult Neuromuscular Junctions

Although age-related changes in synaptic plasticity are an important focus within neuroscience, little is known about ultrastructural changes of synaptic morphology during aging. Here we report how aging affects synaptic ultrastructure by using fluorescence and electron microscopy at the adult Drosophila neuromuscular junction (NMJ) of ventral abdominal muscles. Mainly four striking morphological changes of aging NMJs were revealed. 1) Bouton size increases with proportionally rising number of active zones (AZs). 2) Synaptic vesicle density at AZs is increased in old flies. 3) Late endosomes, cisternae, and multivesicular bodies accumulate in the presynaptic terminal, and vesicles accumulate between membranes of the terminal bouton and the subsynaptic reticulum. 4) The electron-dense pre- and postsynaptic apposition is expanded in aging NMJs, which is accompanied by an expansion of the postsynaptic glutamate receptor fields. These findings suggest that aging is possibly accompanied by impaired synaptic vesicle release and recycling and a potentially compensatory expansion of AZs and postsynaptic densities. (C) 2015 Wiley Periodicals, Inc