Dual-view light-sheet imaging through tilted glass interface using a deformable mirror

Light-sheet microscopy has become one of the primary tools for imaging live developing organisms because of its high speed, low phototoxicity, and optical sectioning capabilities. Detection from multiple sides (multi-view imaging) additionally allows nearly isotropic resolution via computational merging of the views. However, conventional light-sheet microscopes require that the sample is suspended in a gel to allow optical access from two or more sides. At the same time, the use of microfluidic devices is highly desirable for many experiments, but geometric constrains and strong optical aberrations caused by the coverslip titled relative to objectives make the use of multi-view lightsheet challenging for microfluidics. In this paper we describe the use of adaptive optics (AO) to enable multi-view light-sheet microscopy in such microfluidic setup by correcting optical aberrations introduced by the tilted coverslip. The optimal shape of deformable mirror is computed by an iterative stochastic gradient-descent algorithm that optimizes PSF in two orthogonal planes simultaneously. Simultaneous AO correction in two optical arms is achieved via a knife-edge mirror that splits excitation path and combines the detection path. We characterize the performance of this novel microscope setup and, by dual-view light-sheet imaging of C. elegans inside a microfluidic channel, demonstrate a drastic improvement of image quality due to AO and dual-view reconstruction. Our microscope design allows multi-view light-sheet microscopy with microfluidic devices for precisely controlled experimental conditions and high-content screening

Dual-view light-sheet imaging through a tilted glass interface using a deformable mirror

Light-sheet microscopy has become indispensable for imaging developing organisms, and imaging from multiple directions (views) is essential to improve its spatial resolution. We combine multi-view light-sheet microscopy with microfluidics using adaptive optics (deformable mirror) which corrects aberrations introduced by the 45°-tilted glass coverslip. The optimal shape of the deformable mirror is computed by an iterative algorithm that optimizes the point-spread function in two orthogonal views. Simultaneous correction in two optical arms is achieved via a knife-edge mirror that splits the excitation path and combines the detection paths. Our design allows multi-view light-sheet microscopy with microfluidic devices for precisely controlled experiments and high-content screening

Cинтез та антимікробна активність продуктів аніонарилювання з сульфаніламідним фрагментом

Products containing the sulfonamide fragment have been synthesized by anionarylation reaction. 3-(4-Sulfonamidophenyl)2-thiocyanato(bromo)propanamides, 4-(2-thiocyanato(bromo, chloro)-2-phenylethyl)benzenesulfonamides, 2-(4-sulfonamidophenyl)fumaric and 2-bromo-3-(4-sulfonamidophenyl)butanedioic acids have been obtained by the copper-catalyst reaction of 4-sulfonamidophenyldiazonium tetrafluoroborate with acrylamide, styrene and fumaric acid with the yields of 36-82%. The anionarylation competing process is formation of 4-(iso)thiocyanato(chloro, bromo)benzenesulfonamides as Sandmeyer reaction products. In case of thiocyanatoarylation of fumaric acid 2-(4-sulfonamidophenyl)fumaric acid is selectively formed as an arylation product. The structure of the compounds synthesized has been confirmed by IR- and 1H NMR-spectra. The antimicrobial activity of these compounds in relation to the museum strains of staphylococcus, E.coli, aerobic bacillus and yeasts fungi has been studied. It has been found that sulfonamide derivatives are characterized by a high antibacterial and antifungal activity, which is the most pronounced for arylalkyl thiocyanates based on acrylamide. The research conducted has confirmed the positive impact of the sulfonamide fragment introduction in the structure of anionarylation products of unsaturated compounds to expand the range of the antimicrobial activity and decrease of the minimum inhibitory concentration.Реакцией анионарилирования синтезированы продукты, содержащие сульфаниламидный фрагмент. Взаимодействием тетрафторобората 4-сульфамидофенилдиазония с акриламидом, стиролом и фумаровой кислотой в каталитических условиях получены 3-(4-сульфамидофенил)-2-тиоцианато(бром) пропанамиды, 4-(2-тиоцианато(бром, хлор)-2-фенилэтил)бензолсульфонамиды, 2-(4-сульфамидофенил) фумаровая и 2-бром-3-(4-сульфамидофенил)бутандиовая кислоты с выходами 36-82%. Конкурирующим процессом реакции анионарилирования является образование продуктов реакции Зандмейера – 4 (изо) тиоцианато(хлор, бром)бензолсульфамидов. В случае тиоцианатоарилирования фумаровой кислоты селективно образуется продукт арилирования – 2-(4-сульфамидофенил)фумаровая кислота. Структура синтезированных соединений подтверждена данными ИК- и ЯМР 1Н-спектров. Исследовано противомикробное действие этих соединений относительно музейных штаммов стафилококков, кишечных палочек, аэробных бацилл и дрожжевых грибов. Установлено, что сульфамидные производные характеризуются противомикробной активностью, которая наиболее выражена для арилалкильних тиоцианатов на основе акриламида. Проведенные исследования подтвердили положительное влияние введения сульфаниламидного фрагмента в структуру продуктов анионарилирования непредельных соединений на расширение спектра их противомикробной активности и уменьшение значений минимальных ингибирующих концентраций.Реакцією аніонарилювання синтезовані продукти, що містять сульфаніламідний фрагмент. Взаємодією тетрафлуороборату 4-сульфамідофенілдіазонію із акриламідом, стиреном і фумаровою кислотою в умовах купрокаталізу одержані 3-(4-сульфамідофеніл)-2-тіоціанато(бромо)пропанаміди, 4-(2-тіоціанато(бромо,хлоро)2-фенілетил)бензенсульфонаміди, 2-(4-сульфамідофеніл)фумарова і 2-бромо-3-(4-сульфамідофеніл)бутандіова кислоти з виходами 36-82%. Конкуруючим процесом реакції аніонарилювання є утворення продуктів реакції Зандмейєра – 4-(ізо)тіоціанато(хлоро, бромо)бензенсульфонамідів. У випадку тіоціанатоарилювання фумарової кислоти селективно утворюється продукт арилювання – 2-(4-сульфамідофеніл)фумарова кислота. Структура синтезованих сполук підтверджена даними ІЧ- та ЯМР 1Н-спектрів. Досліджено антимікробну дію цих сполук відносно музейних штамів стафілококів, кишкових паличок, аеробних бацил, псевдомонад та дріжджових грибів. Встановлено, що сульфаніламідні похідні характеризуються антибактеріальною та протигрибковою активністю, яка найбільш виражена для арилалкільних тіоціанатів на основі акриламіду. Проведені дослідження підтвердили позитивний вплив введення сульфаніламідного фрагменту в структуру продуктів аніонарилювання ненасичених сполук на розширення спектра антимікробної активності і зменшення значень мінімальних інгібуючих концентрацій

Routes for breaching and protecting genetic privacy

We are entering the era of ubiquitous genetic information for research, clinical care, and personal curiosity. Sharing these datasets is vital for rapid progress in understanding the genetic basis of human diseases. However, one growing concern is the ability to protect the genetic privacy of the data originators. Here, we technically map threats to genetic privacy and discuss potential mitigation strategies for privacy-preserving dissemination of genetic data.Comment: Draft for comment

Crosstalk between Mitochondrial and Sarcoplasmic Reticulum Ca2+ Cycling Modulates Cardiac Pacemaker Cell Automaticity

Mitochondria dynamically buffer cytosolic Ca(2+) in cardiac ventricular cells and this affects the Ca(2+) load of the sarcoplasmic reticulum (SR). In sinoatrial-node cells (SANC) the SR generates periodic local, subsarcolemmal Ca(2+) releases (LCRs) that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na(+)-Ca(2+) exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP).To determine if mitochondrial Ca(2+) (Ca(2+) (m)), cytosolic Ca(2+) (Ca(2+) (c))-SR-Ca(2+) crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity.Inhibition of mitochondrial Ca(2+) influx into (Ru360) or Ca(2+) efflux from (CGP-37157) decreased [Ca(2+)](m) to 80 ± 8% control or increased [Ca(2+)](m) to 119 ± 7% control, respectively. Concurrent with inhibition of mitochondrial Ca(2+) influx or efflux, the SR Ca(2+) load, and LCR size, duration, amplitude and period (imaged via confocal linescan) significantly increased or decreased, respectively. Changes in total ensemble LCR Ca(2+) signal were highly correlated with the change in the SR Ca(2+) load (r(2) = 0.97). Changes in the spontaneous AP cycle length (Ru360, 111 ± 1% control; CGP-37157, 89 ± 2% control) in response to changes in [Ca(2+)](m) were predicted by concurrent changes in LCR period (r(2) = 0.84).A change in SANC Ca(2+) (m) flux translates into a change in the AP firing rate by effecting changes in Ca(2+) (c) and SR Ca(2+) loading, which affects the characteristics of spontaneous SR Ca(2+) release

Single-cell Hi-C reveals cell-to-cell variability in chromosome structure.

Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture (3C) assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single-cell Hi-C, combined with genome-wide statistical analysis and structural modelling of single-copy X chromosomes, to show that individual chromosomes maintain domain organization at the megabase scale, but show variable cell-to-cell chromosome structures at larger scales. Despite this structural stochasticity, localization of active gene domains to boundaries of chromosome territories is a hallmark of chromosomal conformation. Single-cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organization underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns

Anti-Neuroinflammatory effects of the extract of Achillea fragrantissima

<p>Abstract</p> <p>Background</p> <p>The neuroinflammatory process plays a central role in the initiation and progression of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, and involves the activation of brain microglial cells. During the neuroinflammatory process, microglial cells release proinflammatory mediators such as cytokines, matrix metalloproteinases (MMP), Reactive oxygen species (ROS) and nitric oxide (NO). In the present study, extracts from 66 different desert plants were tested for their effect on lipopolysaccharide (LPS) - induced production of NO by primary microglial cells. The extract of <it>Achillea fragrantissima </it>(<it>Af</it>)<it/>, which is a desert plant that has been used for many years in traditional medicine for the treatment of various diseases, was the most efficient extract, and was further studied for additional anti-neuroinflammatory effects in these cells.</p> <p>Methods</p> <p>In the present study, the ethanolic extract prepared from <it>Af </it>was tested for its anti-inflammatory effects on lipopolysaccharide (LPS)-activated primary cultures of brain microglial cells. The levels of the proinflammatory cytokines interleukin1β (IL-1β) and tumor necrosis factor-α (TNFα) secreted by the cells were determined by reverse transcriptase-PCR and Enzyme-linked immunosorbent assay (ELISA), respectively. NO levels secreted by the activate cells were measured using Griess reagent, ROS levels were measured by 2'7'-dichlorofluorescein diacetate (DCF-DA), MMP-9 activity was measured using gel zymography, and the protein levels of the proinflammatory enzymes cyclooxygenase-2 (COX-2) and induced nitric oxide synthase (iNOS) were measured by Western blot analysis. Cell viability was assessed using Lactate dehydrogenase (LDH) activity in the media conditioned by the cells or by the crystal violet cell staining.</p> <p>Results</p> <p>We have found that out of the 66 desert plants tested, the extract of <it>Af </it>was the most efficient extract and inhibited ~70% of the NO produced by the LPS-activated microglial cells, without affecting cell viability. In addition, this extract inhibited the LPS - elicited expression of the proinflammatory mediators IL-1β, TNFα, MMP-9, COX-2 and iNOS in these cells.</p> <p>Conclusions</p> <p>Thus, phytochemicals present in the <it>Af </it>extract could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology.</p

Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) overexpression in pancreatic ductal adenocarcinoma correlates with poor survival

<p>Abstract</p> <p>Background</p> <p>Pancreatic ductal adenocarcinoma is a lethal disease with a 5-year survival rate of 4% and typically presents in an advanced stage. In this setting, prognostic markers identifying the more agrressive tumors could aid in managment decisions. Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3, also known as IMP3 or KOC) is an oncofetal RNA-binding protein that regulates targets such as insulin-like growth factor-2 (IGF-2) and ACTB (beta-actin).</p> <p>Methods</p> <p>We evaluated the expression of IGF2BP3 by immunohistochemistry using a tissue microarray of 127 pancreatic ductal adenocarcinomas with tumor grade 1, 2 and 3 according to WHO criteria, and the prognostic value of IGF2BP3 expression.</p> <p>Results</p> <p>IGF2BP3 was found to be selectively overexpressed in pancreatic ductal adenocarcinoma tissues but not in benign pancreatic tissues. Nine (38%) patient samples of tumor grade 1 (n = 24) and 27 (44%) of tumor grade 2 (n = 61) showed expression of IGF2BP3. The highest rate of expression was seen in poorly differentiated specimen (grade 3, n = 42) with 26 (62%) positive samples. Overall survival was found to be significantly shorter in patients with IGF2BP3 expressing tumors (P = 0.024; RR 2.3, 95% CI 1.2-4.8).</p> <p>Conclusions</p> <p>Our data suggest that IGF2BP3 overexpression identifies a subset of pancreatic ductal adenocarcinomas with an extremely poor outcome and supports the rationale for developing therapies to target the IGF pathway in this cancer.</p