time-resolved experiments
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The present work provides a proof-of-concept that the singlet oxygen-sensitized delayed fluorescence (SOSDF) can be detected from individual living mammalian cells in a time-resolved microscopy experiment. To this end, 3T3 mouse fibroblasts incubated with 100 μM TPPS4 or TMPyP were used and the microsecond kinetics of the delayed fluorescence (DF) were recorded. The analysis revealed that SOSDF is the major component of the overall DF signal. The microscopy approach enables precise control of experimental conditions - the DF kinetics are clearly influenced by the presence of the (1)O2 quencher (sodium azide), H2O/D2O exchange, and the oxygen concentration. Analysis of SOSDF kinetics, which was reconstructed as a difference DF kinetics between the unquenched and the NaN3-quenched samples, provides a cellular (1)O2 lifetime of τΔ = 1-2 μs and a TPPS4 triplet lifetime of τT = 22 ± 5 μs in agreement with previously published values. The short SOSDF acquisition times, typically in the range of tens of seconds, enable us to study the dynamic cellular processes. It is shown that SOSDF lifetimes increase during PDT-like treatment, which may provide valuable information about changes of the intracellular microenvironment. SOSDF is proposed and evaluated as an alternative tool for (1)O2 detection in biological systems.
- MeSH
- analýza jednotlivých buněk přístrojové vybavení metody MeSH
- azid sodný chemie MeSH
- buňky 3T3 MeSH
- design vybavení MeSH
- fibroblasty chemie MeSH
- fluorescence * MeSH
- kinetika MeSH
- kyslík chemie MeSH
- mikroskopie přístrojové vybavení metody MeSH
- myši MeSH
- oxid deuteria chemie MeSH
- singletový kyslík chemie MeSH
- voda chemie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
INTRODUCTION: Competitive binding assays can be used to decipher not only the binding kinetics of studied ligands but also the binding site preference. Such assays are an essential step in the characterization of radioligands. However, the currently used competition assays require high concentrations of usually expensive ligands and still provide only binding site preference. By employing the time-resolved competition assay presented in this paper, binding characteristics including binding site preference can be obtained using less ligand. METHODS: To demonstrate the appropriateness of the time-resolved competition assay, we developed an assay in which the ligand binding was interrupted with a competitor. Experiments were performed on human carcinoma cell lines expressing epidermal growth factor receptor (EGFR). The targeting of the receptor was performed with radio-iodinated epidermal growth factor (EGF). The employed competitors involved either natural ligand transforming growth factor alpha (TGF-α) or anti-EGFR antibodies cetuximab and panitumumab targeting the same EGFR domain. RESULTS: Radio-iodinated EGF bound to EGFR was displaced with either low concentrations of cetuximab or high concentrations of panitumumab. In the case of TGF-α, we observed no competitive displacement of bound EGF at either high or low concentrations. When comparing the time-resolved competition assay with a manual competition assay, the resulting data of measured inhibition constants were in agreement. DISCUSSION: The results summarised in this study confirm the appropriateness of the time-resolved competition assay for assessing ligand binding properties. The assay has the potential to complement or replace conventional competition assays for determining binding site preference in the future.
- MeSH
- časové faktory MeSH
- epidermální růstový faktor chemie metabolismus MeSH
- erbB receptory antagonisté a inhibitory chemie metabolismus MeSH
- humanizované monoklonální protilátky chemie farmakologie MeSH
- kompetitivní vazba účinky léků MeSH
- lidé MeSH
- ligandy MeSH
- monoklonální protilátky chemie farmakologie MeSH
- nádorové buňky kultivované MeSH
- substrátová specifita MeSH
- transformující růstový faktor alfa chemie metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chloroplast thylakoid membranes contain virtually all components of the energy-converting photosynthetic machinery. Their energized state, driving ATP synthesis, is enabled by the bilayer organization of the membrane. However, their most abundant lipid species is a non-bilayer-forming lipid, monogalactosyl-diacylglycerol; the role of lipid polymorphism in these membranes is poorly understood. Earlier 31P-NMR experiments revealed the coexistence of a bilayer and a non-bilayer, isotropic lipid phase in spinach thylakoids. Packing of lipid molecules, tested by fluorescence spectroscopy of the lipophilic dye, merocyanine-540 (MC540), also displayed heterogeneity. Now, our 31P-NMR experiments on spinach thylakoids uncover the presence of a bilayer and three non-bilayer lipid phases; time-resolved fluorescence spectroscopy of MC540 also reveals the presence of multiple lipidic environments. It is also shown by 31P-NMR that: (i) some lipid phases are sensitive to the osmolarity and ionic strength of the medium, (ii) a lipid phase can be modulated by catalytic hydrogenation of fatty acids and (iii) a marked increase of one of the non-bilayer phases upon lowering the pH of the medium is observed. These data provide additional experimental evidence for the polymorphism of lipid phases in thylakoids and suggest that non-bilayer phases play an active role in the structural dynamics of thylakoid membranes.
Experimental and theoretical foundations for femtosecond time-resolved circular dichroism (TRCD) spectroscopy of excitonic systems are presented. In this method, the system is pumped with linearly polarized light and the signal is defined as the difference between the transient absorption spectrum probed with left and with right circularly polarized light. We present a new experimental setup with a polarization grating as key element to generate circularly polarized pulses. Herein the positive (negative) first order of the diffracted light is left-(right-)circularly polarized and serves as a probe pulse in a TRCD experiment. The grating is capable of transferring ultrashort broadband pulses ranging from 470 nm to 720 nm into two separate beams with opposite ellipticity. By applying a specific chopping scheme we can switch between left and right circular polarizations and detect transient absorption (TA) and TRCD spectra on a shot-to-shot basis simultaneously. We perform experiments on a squaraine polymer, investigating excitonic dynamics, and we develop a general theory for TRCD experiments of excitonically coupled systems that we then apply to describe the experimental data in this particular example. At a magic angle of 54.7° between the pump-pulse polarization and the propagation direction of the probe pulse, the TRCD and TA signals become particularly simple to analyze, since the orientational average over random orientations of complexes factorizes into that of the interaction with the pump and the probe pulse, and the intrinsic electric quadrupole contributions to the TRCD signal average to zero for isotropic samples. Application of exciton theory to linear absorption and to linear circular dichroism spectra of squaraine polymers reveals the presence of two fractions of polymer conformations, a dominant helical conformation with close interpigment distances that are suggested to lead to short-range contributions to site energy shifts and excitonic couplings of the squaraine molecules, and a fraction of unfolded random coils. Theory demonstrates that TRCD spectra of selectively excited helices can resolve state populations that are practically invisible in TA spectroscopy due to the small dipole strength of these states. A qualitative interpretation of TRCD and TA spectra in the spectral window investigated experimentally is offered. The 1 ps time component found in these spectra is related to the slow part of exciton relaxation obtained between states of the helix in the low-energy half of the exciton manifold. The dominant 140 ps time constant reflects the decay of excited states to the electronic ground state.
- Publikační typ
- časopisecké články MeSH
Fluorescence solvent relaxation experiments are based on the characterization of time-dependent shifts in the fluorescence emission of a chromophore, yielding polarity and viscosity information about the chromophore's immediate environment. A chromophore applied to a phospholipid bilayer at a well-defined location (with respect to the z-axis of the bilayer) allows monitoring of the hydration and mobility of the probed segment of the lipid molecules. Specifically, time-resolved fluorescence experiments, fluorescence quenching data and molecular dynamic (MD) simulations show that 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) probes the hydration and mobility of the sn-1 acyl groups in a phosphatidylcholine bilayer. The time-dependent fluorescence shift (TDFS) of Laurdan provides information on headgroup compression and expansion induced by the addition of different amounts of cationic lipids to phosphatidylcholine bilayers. Those changes were predicted by previous MD simulations. Addition of truncated oxidized phospholipids leads to increased mobility and hydration at the sn-1 acyl level. This experimental finding can be explained by MD simulations, which indicate that the truncated chains of the oxidized lipid molecules are looping back into aqueous phase, hence creating voids below the glycerol level. Fluorescence solvent relaxation experiments are also useful in understanding salt effects on the structure and dynamics of lipid bilayers. For example, such experiments demonstrate that large anions increase hydration and mobility at the sn-1 acyl level of phosphatidylcholine bilayers, an observation which could not be explained by standard MD simulations. If polarizability is introduced into the applied force field, however, MD simulations show that big soft polarizable anions are able to interact with the hydrophilic/hydrophobic interface of the lipid bilayer, penetrating to the level probed by Laurdan, and that they expand and destabilize the bilayer making it more hydrated and mobile.
Time-resolved X-ray crystallography experiments were first performed in the 1980s, yet they remained a niche technique for decades. With the recent advent of X-ray free electron laser (XFEL) sources and serial crystallographic techniques, time-resolved crystallography has received renewed interest and has become more accessible to a wider user base. Despite this, time-resolved structures represent < 1 % of models deposited in the world-wide Protein Data Bank, indicating that the tools and techniques currently available require further development before such experiments can become truly routine. In this chapter, we demonstrate how applying data multiplexing to time-resolved crystallography can enhance the achievable time resolution at moderately intense monochromatic X-ray sources, ranging from synchrotrons to bench-top sources. We discuss the principles of multiplexing, where this technique may be advantageous, potential pitfalls, and experimental design considerations.
Electron and x-ray magnetic microscopies allow for high-resolution magnetic imaging down to tens of nanometers. However, the samples need to be prepared on transparent membranes which are very fragile and difficult to manipulate. We present processes for the fabrication of samples with magnetic micro- and nanostructures with spin configurations forming magnetic vortices suitable for Lorentz transmission electron microscopy and magnetic transmission x-ray microscopy studies. The samples are prepared on silicon nitride membranes and the fabrication consists of a spin coating, UV and electron-beam lithography, the chemical development of the resist, and the evaporation of the magnetic material followed by a lift-off process forming the final magnetic structures. The samples for the Lorentz transmission electron microscopy consist of magnetic nanodiscs prepared in a single lithography step. The samples for the magnetic x-ray transmission microscopy are used for time-resolved magnetization dynamic experiments, and magnetic nanodiscs are placed on a waveguide which is used for the generation of repeatable magnetic field pulses by passing an electric current through the waveguide. The waveguide is created in an extra lithography step.
V období 1989 - 1996 zvolili autoři po radikální cystoprostatektomii pro karcinom u 43 mužů kontinentní ortotopickou derivaci moč ortotopickou derivaci moče. K vytvoření neoveziky použili detubulizované kličky terminálního ilea, tvarované v různých modifikacích. Vytvořenou neovezikou anastomózovali s uretrou. S časovým odstupem 6 - 12 měsíců zjišťovali anamnestický stav kontinence a provedli kompletní urodynamickou studii s uroflowmetrií u 25 nemocných. V krátkém časovém odstupu do 6 měsíců od operace pozorovali významnou nestabihtu neoveziky. Po 9 12 měsících šlo u všech nemocných o vysokokapacitní nízkotlaký pouch. Se stavem kontinence bylo spokojeno 90 % nemocných. Vyšší procento plně kontinentních bylo přes den (80 %) než v noci (46 %). Inkontinence v různém stupni (8 -10 %) byla uspokojivě řešena modenrími pomůckami pro inkontinenci.
During 1989 -1996 the authors selected after radical cystoprostatectomy on account of carcinoma in 43 men continent orthotopic derivation of urine. To create a neovesica the authors used a detubulized loop of the terminal ileum shaped in different modifications. The neovesica was connected with the urethra. After a time interval of ileum shaped in different modifications. The neovesica was connected with the urethra. After a time interval ot uroflowmetry in 25 patients. After a 6-month time interval following operation they observed a significant instability of the neovesica. After 9 -12 months all patients had a high-capacity low-pressure pouch. 90 % of the patients were satisfied with the state of continence. The percentage of fully continent patients was higher in daytime (80 %) than during the night (46 %). Incontinence of varying extent (8 -10 %) was resolved satisfactorily by modem aids for incontinence.
Cíl: Laparoskopická uretero-pyeloanastomóza (UPA) představuje alternativu v léčbě zdvojených anomálií ledvin se zachovanou funkcí horní moiety nebo tam, kde je heminefrektomie obtížná či riskantní. Cílem je zhodnotit naše dlouhodobé zkušenosti s touto technikou. Soubor: Retrospektivní hodnocení souboru 35 pacientů, u kterých byla v letech 2011–2023 provedena laparoskopická UPA. Ve třech případech šlo o inkompletní zdvojení ledviny s hydronefrózou dolního segmentu, v ostatních případech (32) o kompletní zdvojení s megaureterem horního segmentu zakončeným ureterokélou v devíti a ektopií močovodu ve 23 případech. Laparoskopickou operaci jsme indikovali u dětí starších jednoho roku, s minimální hmotností 10 kg, s významnou dilatací zdvojeného systému horních močových cest, bez vezikoureterálního refluxu do dolního a zachovalou funkcí horního segmentu. Hodnotili jsme věk pacientů v době operace, délku výkonu, úspěšnost, délku sledování a komplikace. Výsledky: Střední věk pacientů byl 29 (14–184) měsíců, střední délka výkonu 210 (110–320) minut. U všech pacientů došlo k významnému zmenšení či vymizení dilatace horních močových cest. Střední délka sledování byla 18 (5–120) měsíců, žádný z pacientů nevyžadoval reoperaci. Nejčastější komplikací byl časný únik moči z anastomózy u sedmi (20 %) dětí. Příčinou byla v pěti případech porucha drenáže (2× malfunkce nefrostomie, 3× malfunkce ureterální cévky), ve dvou případech pacient operovaný bez použití derivace. Komplikaci jsme vyřešili zavedením ureterálního katétru (3×) a ureterálního stentu (4×). Závěr: Laparoskopická UPA představuje alter- nativní způsob řešení některých forem zdvojených anomálií ledvin u dětí. Má vysokou úspěšnost a nízký výskyt komplikací. Nejčastější komplikací je únik moči z anastomózy, proto je nezbytné zvolit spolehlivý typ derivace moči.
Aim: Laparoscopic ureteropyelostomy (UPS) represents alternative method in treatment of duplex kidney anomalies with preserved function of upper pole moiety, where the heminephrectomy is risky or impossible. We present long-time experiences with this technique. Materials and methods: Retrospective evaluation of 35 patients after UPS done between 2011 and 2023. In 3 cases we performed surgery because of hydronephrosis of lower moiety in patients with incomplete duplication. Remaining cases (32) were operated because of complete duplication. With ectopic megaureter in 23 cases, with ectopic ureterocele in 9. Laparoscopic operation was recommended in children older than one year, minimally 10 kg, significant kidney dilatation, no reflux and preserved upper segment function. We evaluated age of patients, operating time, success rate, follow up and complications. Results: Median age was 29 (14-184) months, median operating time 210 (110-320) minutes. In all patients decreased or vanished upper tract dilatation. Median follow up was 18 (5-120) months. No reoperations were recorded. The most frequent complication was urinary leak from anastomosis in 7 (20%) cases. The reason was failure of urine diversion in 5 children (2 nephrostomy failure, ureteral catheter dysfunction in 3), remaining 2 patients were operated without urine diversion. Complications were resolved by ureteral catheter insertion in 3 and ureteral stent insertion in 4 cases. Conclusion: Laparoscopic UPS represents alternative mini-invasive method in duplex kidney anomalies treatment. It has high success rate and low rate of complications. The most frequent problem is urine leak from anastomosis. Therefore, the choice of reliable urine diversion is critical.
- Klíčová slova
- Zdvojení ledviny, uretero-pyeloanastomóza,
- MeSH
- anastomóza chirurgická * metody MeSH
- dítě MeSH
- diverze moči metody MeSH
- hydronefróza chirurgie etiologie MeSH
- laparoskopie * metody MeSH
- ledviny abnormality chirurgie embryologie MeSH
- lidé MeSH
- retrospektivní studie MeSH
- stenty MeSH
- urogenitální abnormality * chirurgie embryologie MeSH
- urologické chirurgické výkony metody MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Cíl: Výhřez meziobratlové ploténky v bederní oblasti patří k nejčastějším indikacím operace páteře. Zlatým standardem chirurgické léčby výhřezu meziobratlové ploténky je otevřená diskektomie z transligamentózního přístupu. V poslední dekádě se čím dál více prosazuje endoskopická technika, která má srovnatelný efekt z hlediska výsledku léčby a navíc přináší řadu výhod pro operatéra i pacienta díky minimální invazivnosti endoskopické techniky. Neurochirurgická klinika FN Brno je prvním pracovištěm v ČR, které začalo získávat zkušenosti s tímto typem operací. V předkládaném článku přinášíme první zkušenosti a výsledky získané od roku 2017, kdy u nás byla endoskopická metoda léčby výhřezu meziobratlové ploténky zavedena. Metody: Do souboru jsou zařazování pacienti ve věkovém rozmezí 20– 70 let, u kterých je indikováno operační řešení výhřezu meziobratlové ploténky v etáži L4/ L5 nebo L5/ S1 a kteří dříve nepodstoupili žádnou operaci v inkriminovaných etážích. Dosud bylo do souboru zařazeno 15 pacientů, u kterých byla hodnocena míra intenzity bolesti charakteru dorzalgií a radikulární symptomatologie pomocí Visual Analogue Score (VAS) a dále míra omezení v běžných činnostech pomocí Oswestry Disability Index (ODI). Údaje byly hodnoceny předoperačně a dále s odstupem 6 týdnů a 6 měsíců po operaci. Výsledky: Předoperační hodnoty VAS skóre byly u celého souboru signifikantně vyšší než hodnoty 6 týdnů a 6 měsíců po operaci, a to jak v případě dorzalgie, tak v případě radikulárních bolestí. Obdobné výsledky byly v podskupinách mužů a žen, kdy došlo u obou typů iritací ke značnému zlepšení 6 týdnů i 6 měsíců po operaci. U souboru jako celku byly signifikantně vyšší předoperační hodnoty ODI v porovnání s hodnotami 6 týdnů a 6 měsíců po operaci. V podskupině mužů nebyl prokázán signifikantní rozdíl mezi předoperačními hodnotami ODI a hodnotami 6 týdnů po operaci, zatímco 6 měsíců po operaci byly hodnoty výrazně nižší. V podskupině žen byly předoperační hodnoty ODI signifikantně vyšší než hodnoty 6 týdnů i 6 měsíců po operaci. Pooperačně nastalo u zkoumaného souboru značné zmírnění subjektivních obtíží, které ve většině případů odpovídalo normalizaci stavu. K výraznému zlepšení až k ústupu problémů došlo ve všech sledovaných parametrech, kterými byly dorzalgie, radikulární symptomatologie a omezení běžných denních aktivit v důsledku bolestí dolní části zad. Recidiva výhřezu disku se ve sledovaném období vyskytla v jednom případě. Závěr: Naše dosavadní zkušenosti s použitím endoskopické techniky k operaci výhřezu meziobratlové ploténky hodnotíme maximálně pozitivně a vzhledem k těmto zkušenostem i s odkazem na literární zdroje z jiných pracovišť plánujeme další rozvoj této operační metody.
Aim: Lumbar disc herniation is the most frequent indication for spinal surgery. Open discectomy is considered as a standard surgical procedure; however, the endoscopic technique has evolved recently as an alternative method of treatment. Compared to open discectomy, the endoscopic technique has a similar effect in terms of outcome and additionally, it is beneficial for both surgeon and patient, because the endoscopic technique is a minimally invasive procedure. Department of Neurosurgery in The University Hospital Brno is the first department in the Czech Republic in which endoscopic discectomy has been implemented. The aim of this article is to present the first results and experiences with this technique, which has been used in our department since 2017. Methods: So far, 15 patients (20– 70 years old) underwent endoscopic surgery for L4– 5 or L5– S1 herniation. Evaluation parameters were pain intensity (dorsalgia and radiculopathy) as ses sed by Visual Analogue Score (VAS) and limitations of com mon activities as ses sed by Oswestry Disability Index (ODI). The parameters were evaluated before surgery and after the surgery at the 6-week and 6-month time- -points. Results: In all study groups the VAS score (for both dorsalgia and radiculopathy) was signifi cantly higher before surgery compared to the 6-week and 6-month time-points. Similar results were found in male and female subgroups, and significant improvement was observed at both the 6-week and 6-month time-points. The ODI before surgery was significantly higher in all patients before surgery compared to the 6-week and 6-month time-points. In the male subgroup, there was no significant difference between ODI before surgery and the 6-week time-point, while ODI at the 6-month time-point was signifi cantly lower. In the female subgroup, ODI at both the 6-week and 6-month timepoints was signifi cantly lower than before surgery. Recur rent herniation had occurred in one case and was resolved by reoperation. Conclusion: In conclusion, endoscopic lumbar discectomy is a safe and effective option for lumbar disc herniation surgery.
- MeSH
- dospělí MeSH
- endoskopie * metody MeSH
- klinická studie jako téma MeSH
- lidé středního věku MeSH
- lidé MeSH
- posuzování pracovní neschopnosti MeSH
- senioři MeSH
- vizuální analogová stupnice MeSH
- výhřez meziobratlové ploténky * chirurgie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
