The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem

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 detected with 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. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.     

Diagnosis and phylogenetic analysis of orf virus in Aleppo and Saanen goats from an outbreak in Turkey

正Dear Editor,The Poxviridae family is divided into Chordopoxvirinae and Entomopoxvirinae genera.Parapoxvirus is one of 11genera in the Chordopoxvirinae genus,including orf virus(ORFV),bovine papular stomatitis virus(BPSV),pseudocowpox virus(PCPV)and parapoxvirus of red deer in New Zealand(PVNZ)(Skinner et al.,2012).This virus,also known as contagious ecthyma or contagious pustular dermatitis,is a viral skin disease causing edematous proliferative lesions,most often in hairless skin     

Low‐dose bisphenol A induces RIPK1‐mediated necroptosis in SH‐SY5Y cells: Effects on TNF‐α and acetylcholinesterase

Bisphenol A (BPA) is an endocrine disruptor chemical, which is commonly used in everyday products. Adverse effects of its exposure are reported even at picomolar doses. Effects of picomolar and nanomolar concentrations of BPA on cytotoxicity, nitric oxide (NO) levels, acetylcholinesterase (AChE) gene expression and activity, and tumor necrosis factor‐α (TNF‐α) and caspase‐8 levels were determined in SH‐SY5Y cells. The current study reveals that low‐dose BPA treatment induced cytotoxicity, NO, and caspase‐8 levels in SH‐SY5Y cells. We also evaluated the mechanism underlying BPA‐induced cell death. Ours is the first report that receptor‐interacting serine/threonine‐protein kinase 1–mediated necroptosis is induced by nanomolar BPA treatment in SH‐SY5Y cells. This effect is mediated by altered AChE and decreased TNF‐α levels, which result in an apoptosis‐necroptosis switch. Moreover, our study reveals that BPA is an activator of AChE.     

ABA treatment increases both the desiccation tolerance of photosynthesis,and nonphotochemical quenching in the moss Atrichum undulatum

Pulse amplitude modulation fluorescence was used to investigate whether abscisic acid (ABA) pretreatment increases the desiccation tolerance of photosynthesis in the moss Atrichum undulatum. In unstressed plants, ABA pretreatment decreased the F _V/F _m ratio, largely as a result of an increase in F _o. This indicated a reduction in energy transfer between LHCII and PSII, possibly hardening the moss to subsequent stress by reducing the production of the reactive oxygen species near PSII. During desiccation, F ₀, F _m, F _v/F _m, PSII, and NPQ and F ₀ quenching declined in ABA-treated and nontreated mosses. However, during rehydration, F ₀, F _m, F _v/F _m, and PSII recovered faster in ABA-treated plants, suggesting that ABA improved the tolerance of photosystem II to desiccation. NPQ increased upon rehydration in mosses from both treatments, but much more rapidly in ABA-treated plants; during the first hour of rehydration, NPQ was two-fold greater in plants treated with ABA. F ₀quenching followed a similar pattern, indicating that ABA treatment stimulated zeaxanthin-based quenching. The implications of these results for the mechanisms of ABA-induced desiccation tolerance in A. undulatum are discussed.     

Synthesis and biological activity of a new progestogen, 16-methylene-17alpha-hydroxy-18-methyl-19-norpregn-4-ene-3, 20-dione acetate

The progestational activity of second- and third-generation progestins in oral contraceptives were markedly increased by addition of an 18-methyl group. A new progestin, the 18-methyl analog of Nestorone, 16-methylene-17alpha-hydroxy-18-methyl-19-norpregn-4-ene-3,2 0-dione acetate (10), was synthesized. The relative binding affinity and biologic activity of 10 was compared with Nestorone, levonorgestrel, and progesterone using a binding assay for rat progesterone receptors, the Clauberg assay in the rabbit, and by assessing pregnancy maintenance in the rat. These studies, as summarized in Table 4, show that 10 is three to ten times more potent than Nestorone. The addition of the 18-methyl group to Nestorone markedly increased its potency as noted above, but is unlikely to change its rate of delivery from sustained release systems. 10 should be ideally suited for administration by implants or small skin patches.     

Comparison of ELISA with shell vial cell culture method for the detection of human rotavirus in fecal specimens

The aim of the study was to compare an enzyme immunoassay method with shell vial cell culture method for detection of rotavirus in fecal specimens. In addition, the correlation between laboratory results and clinical scores of patients with gastroenteritis was evaluated. A total of 219 fecal specimens from children (ages 3 weeks to 5 years) with acute gastroenteritis submitted to pediatric emergency room were evaluated by both ELISA and shell vial cell culture. A Vesikari score was used for assessing the severity of the illness. Among 219 stool samples tested, 107 (48.9%) were determined to be positive. Two specimens were positive by shell vial cell culture method while they were ELISA negative. According to these results the calculated sensitivity, specificity, PPV, and NPV of ELISA were 98.1%, 100%, 100%, and 98.2%, respectively. The mean severity score for the 107 episodes of rotavirus diarrhoea was 11.0 +/- 3.6 compared to 4.5 +/- 1.9 for the 112 episodes of non-rotavirus diarrhea in the same population. Our study indicates that ELISA, which is easier to perform, faster and cheaper than cell culture methods may be suitable for routine diagnosis of rotavirus infections. The severity of rotavirus positive gastroenteritis was significantly higher than that of rotavirus negative patients.     

Enantioseparation of Mandelic Acid Enantiomers With Magnetic Nano‐Sorbent Modified by a Chiral Selector

In this study, R(+)‐α‐methylbenzylamine‐modified magnetic chiral sorbent was synthesized and assessed as a new enantioselective solid phase sorbent for separation of mandelic acid enantiomers from aqueous solutions. The chemical structures and magnetic properties of the new sorbent were characterized by vibrating sample magnetometry, transmission electron microscopy, Fourier transform infrared spectroscopy, and dynamic light scattering. The effects of different variables such as the initial concentration of racemic mandelic acid, dosage of sorbent, and contact time upon sorption characteristics of mandelic acid enantiomers on magnetic chiral sorbent were investigated. The sorption of mandelic acid enantiomers followed a pseudo‐second‐order reaction and equilibrium experiments were well fitted to a Langmuir isotherm model. The maximum adsorption capacity of racemic mandelic acid on to the magnetic chiral sorbent was found to be 405 mg g^?1. The magnetic chiral sorbent has a greater affinity for (S)‐(+)‐mandelic acid compared to (R)‐(?)‐mandelic acid. The optimum resolution was achieved with 10 mL 30 mM of racemic mandelic acid and 110 mg of magnetic chiral sorbent. The best percent enantiomeric excess values (up to 64%) were obtained by use of a chiralpak AD‐H column. Chirality 27:835–842, 2015. © 2015 Wiley Periodicals, Inc.     

Ecophysiological responses of desiccation-tolerant cryptobiotic crusts

In our present studies, the recovery of photosynthetic activity after rehydration was demonstrated. We measured chlorophyll fluorescence, CO₂ gas exchange and the pigment composition in the previously long-term air-dried cryptogamic inselberg crusts collected from two tropical areas. The cryptobiotic crusts were collected from different localities on similar ecological and climatic conditions from extreme habitats of inselbergs (outcrops). These inselbergs are characterized by a dry microclimate and are covered by scarce soil. We found that the ecophysiological responses of both cryptogamic inselberg crusts showed an extremely high degree of desiccation-tolerance due to the fast and full recovery during rehydration. The photosynthetic activity of the cryptobiotic crusts were restored and regained within 15 and 40 min, respectively, after rehydration. Photosynthetic activity of the crusts was retained at all applied light intensities when enough water was available, however the degree of the recovery was different between the crusts. Photosynthetic pigment contents were strongly and positively correlated with water content. Our results indicated that tropical desiccation-tolerant cryptogamic crusts found on inselberg rock surfaces have CO₂ fixation ability in the range of cyanobacteria and lichens, suggesting that at a global scale they can assimilate CO₂ in a significant amount.