Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4',6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detectedwith blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images.
COBISS.SI-ID: 30233561
Tandem autologous hematopoietic stem cell transplantation (ta-HSCT) is a standard treatment for multiple myeloma (MM). Patients receive a high-dose cyclophosphamide (CY), followed by two myeloablative cycles of melphalan (MEL). There are scarce data about long term cardiotoxicity. Patients and methods. We studied 12 patients (62.25 8.55 years) six years after the completion of MM treatment with ta-HCST. Late cardiotoxic effects were evaluated clinically and echocardiographically. Results. None of the patients developed clinical signs of heart failure, all were in sinus rhythm and NT-pro BNP concentration was elevated (778 902.76 pg/mL). The left ventricular (LV) size remained normal. The LV ejection fraction did not decrease (73.75 5.67%, 69.27 6.13%, p = NS). The LV diastolic function parameters (E, A, ratio E/A and A/a) did not change significantly. Conclusions. The treatment of MM with ta-HSCT, during which patients receive a high dose CY followed by two myeloablative cycles of MEL, causes mild, chronic, partially reversible and clinically silent cardiotoxic side-effects. However, ta-HSCT in patients with MM is a safe regarding cardiotoxic side effects, but, because of increasing life expectancy needs long term attention.
COBISS.SI-ID: 1577595
Amniotic membrane (AM) is the innermost, multilayered part of the placenta. When harvested, processed and stored properly, its properties, stemming from AM biological composition, make it a useful tissue for ophthalmic surgery. AM was shown to have several beneficial effects: it promotes epithelization, has antimicrobial effects, decreases inflammation, fibrosis and neovascularization. Many case reports and case series as well as practical experience (e.g. reconstruction of conjunctival and corneal defects, treatment of corneal ulcers) demonstrated the beneficial effect of AM for different ophthalmological indications. The combination of the above mentioned beneficial effects and reasonable mechanical properties are also the reason why AM is used as a substrate for ex vivo expansion of epithelial progenitor cells. Recently, amnion-derived cells, which also have stem cell characteristics, have been proposed as potential contributors to cell-based treatment of ocular surface disease. However, the use of AM remains one of the least standardized methods in ophthalmic surgery. In this review, the various properties of AM and its current clinical use in ophthalmology in Slovenia are discussed.
COBISS.SI-ID: 31054041
Toll-like receptor 4 (TLR4) in complex with its accessory protein MD-2 represents an emerging target for the treatment of severe sepsis and neuropathic pain. We performed structure-based and ligand-based virtual screening targeting the TLR4-MD-2 interface. Three in silico hit compounds showed promising TLR4 antagonistic activities with micromolar IC50 values. These compounds also suppressed cytokine secretion by human peripheral blood mononuclear cells. The specific affinity of the most potent hit was confirmed by surface plasmon resonance direct-binding experiments. The results of our study represent a very promising starting point for the development of potent small-molecule antagonists of TLR4.
COBISS.SI-ID: 3538289
Purpose: To evaluate the in vitro biomechanical characteristics of patellar tendon ligaments (BTB) when stored as fresh frozen or as glycerol cryopreserved allografts. Methods: Seventy patellar tendons were harvested from 35 cadaveric human donors and randomly assigned into seven groups. Grafts in group FRESH were mechanically tested within 2 h of harvesting. FROZ-3, FROZ-6, and FROZ-9 were deep-frozen to -80 degrees C for 3, 6, and 9 months, respectively. Grafts in groups CRYO-3, CRYO-6, and CRYO-9 were initially incubated with 10 % glycerol in a phosphate-buffered saline for 1 h and then stored in glycerol solution (10 % glycerol in PBS) at -80 degrees C for 3, 6, and 9 months, respectively. Grafts were mechanically tested with two cycling modes (50-250 degrees N and 150-500 degrees N) and then loaded to failure. Results: Cryopreserved grafts demonstrated more consistent results and expressed lower elongation rates after both cycling protocols. Moreover, cryopreserved grafts revealed similar ultimate elongation and uniformly higher ultimate stiffness and ultimate stress levels compared to fresh grafts. Conclusion: Cryopreservation with glycerol solution might be used to further improve the quality of preserved soft-tissue allografts.
COBISS.SI-ID: 30380505