Exploring the spatial dimension of estrogen and progesterone signaling: detection of nuclear labeling in lobular epithelial cells in normal mammary glands adjacent to breast cancer

Background

Comprehensive spatial assessment of hormone receptor immunohistochemistry staining in digital whole slide images of breast cancer requires accurate detection of positive nuclei within biologically relevant regions of interest. Herein, we propose a combination of automated region labeling at low resolution and subsequent detailed tissue evaluation of subcellular structures in lobular structures adjacent to breast cancer, as a proof of concept for the approach to analyze estrogen and progesterone receptor expression in the spatial context of surrounding tissue.

Methods

Routinely processed paraffin sections of hormone receptor-negative ductal invasive breast cancer were stained for estrogen and progesterone receptor by immunohistochemistry. Digital whole slides were analyzed using commercially available image analysis software for advanced object-based analysis, applying textural, relational, and geometrical features. Mammary gland lobules were targeted as regions of interest for analysis at subcellular level in relation to their distance from coherent tumor as neighboring relevant tissue compartment. Lobule detection quality was evaluated visually by a pathologist.

Results

After rule set optimization in an estrogen receptor-stained training set, independent test sets (progesterone and estrogen receptor) showed acceptable detection quality in 33% of cases. Presence of disrupted lobular structures, either by brisk inflammatory infiltrate, or diffuse tumor infiltration, was common in cases with lower detection accuracy. Hormone receptor detection tended towards higher percentage of positively stained nuclei in lobules distant from the tumor border as compared to areas adjacent to the tumor. After adaptations of image analysis, corresponding evaluations were also feasible in hormone receptor positive breast cancer, with some limitations of automated separation of mammary epithelial cells from hormone receptor-positive tumor cells.

Conclusions

As a proof of concept for object-oriented detection of steroid hormone receptors in their spatial context, we show that lobular structures can be classified based on texture-based image features, unless brisk inflammatory infiltration disrupts the normal morphological structure of the tubular gland epithelium. We consider this approach as prototypic for detection and spatial analysis of nuclear markers in defined regions of interest. We conclude that advanced image analysis at this level of complexity requires adaptation to the individual tumor phenotypes and morphological characteristics of the tumor environment.

     

Effects of inactivation methods on the analysis of Bacillus atrophaeus endospores using real‐time PCR and MALDI‐TOF‐MS

A wide range of analytical methods are available for the detection and identification of biological warfare agents. These technologies are often hampered in their performance when the inactivated samples are analyzed. To work with pathogens outside of biosafety level 3 laboratories, a complete inactivation is mandatory when appropriate protection equipment is unavailable. When methods of inactivation are used, the detection of bacteria becomes more difficult. In contrast to measuring viable organisms, inactivation steps can have a massive impact on the intrinsic cellular information. This study examined the effects of autoclaving and chemical inactivation methods on Bacillus spores using biological warfare detection setups like real‐time PCR and MALDI‐TOF‐MS. Here, the inactivation of Bacillus atrophaeus spores with formaldehyde, which is a suggested model for biological warfare spore agents, was compared with other inactivation reagents like Wofasteril^®E400, a commercially available decontaminant based on peroxyacetic acid. With Wofasteril^®E400 the critical factor of inactivation time was reduced to about 15 min and a limit of detection of 8500 spores by PCR was still measurable using five‐times‐washed spores. It has also been shown that MALDI‐TOF‐MS peak information can be hampered by inactivation methods.     

Environmental impacts of bioenergy wood production from poplar short‐rotation coppice grown at a marginal agricultural site in Germany

For avoiding competition with food production, marginal land is economically and environmentally highly attractive for biomass production with short‐rotation coppices (SRCs) of fast‐growing tree species such as poplars. Herein, we evaluated the environmental impacts of technological, agronomic, and environmental aspects of bioenergy production from hybrid poplar SRC cultivation on marginal land in southern Germany. For this purpose, different management regimes were considered within a 21‐year lifetime (combining measurements and modeling approaches) by means of a holistic Life Cycle Assessment (LCA). We analyzed two coppicing rotation lengths (7 × 3 and 3 × 7 years) and seven nitrogen fertilization rates and included all processes starting from site preparation, planting and coppicing, wood chipping, and heat production up to final stump removal. The 7‐year rotation cycles clearly resulted in higher biomass yields and reduced environmental impacts such as nitrate (NO₃) leaching and soil nitrous oxide (N₂O) emissions. Fertilization rates were positively related to enhanced biomass accumulation, but these benefits did not counterbalance the negative impacts on the environment due to increased nitrate leaching and N₂O emissions. Greenhouse gas (GHG) emissions associated with the heat production from poplar SRC on marginal land ranged between 8 and 46 kg CO₂‐eq. GJ^?1 (or 11–57 Mg CO₂‐eq. ha^?1). However, if the produced wood chips substitute oil heating, up to 123 Mg CO₂‐eq. ha^?1 can be saved, if produced in a 7‐year rotation without fertilization. Dissecting the entire bioenergy production chain, our study shows that environmental impacts occurred mainly during combustion and storage of wood chips, while technological aspects of establishment, harvesting, and transportation played a negligible role.     

Microbial inactivation for safe and rapid diagnostics of infectious samples

The high risk associated with biological threat agents dictates that any suspicious sample be handled under strict surety and safety controls and processed under high-level containment in specialized laboratories. This study attempted to find a rapid, reliable, and simple method for the complete inactivation of a wide range of pathogens, including spores, vegetative bacteria, and viruses, while preserving microbial nucleic acid fragments suitable for PCRs and proteinaceous epitopes for detection by immunoassays. Formaldehyde, hydrogen peroxide, and guanidium thiocyanate did not completely inactivate high titers of bacterial spores or viruses after 30 min at 21°C. Glutaraldehyde and sodium hypochlorite showed high microbicidal activity but obliterated the PCR or enzyme-linked immunosorbent assay (ELISA) detection of bacterial spores or viruses. High-level inactivation (more than 6 log(10)) of bacterial spores (Bacillus atrophaeus), vegetative bacteria (Pseudomonas aeruginosa), an RNA virus (the alphavirus Pixuna virus), or a DNA virus (the orthopoxvirus vaccinia virus) was attained within 30 min at 21°C by treatment with either peracetic acid or cupric ascorbate with minimal hindrance of subsequent PCR tests and immunoassays. The data described here should provide the basis for quickly rendering field samples noninfectious for further analysis under lower-level containment and considerably lower cost.     

Quantitative analysis of trifluoroacetic acid in body fluids of patients treated with halothane

A simple procedure for the quantitative analysis of trifluoroscetic acid (TFA) in urine and serum from patients narcotized with halothane is described. This involves addition of sodium hydroxide to the body fluid, evaporation of the aqueous phase and esterification of TFA in concentrated sulphuric acid with 2,2,2-trichloroethanol. The gaseous phases above the reaction mixture were then analyzed by gas chromatography with a nickel-63 electron-capture detector. The detection limit was 1 μg of TFA per mililitre of body fluid (200 μg of body fluid are analysed) and the relative standard deviation was ±6%. Patients treated with ethrane, another commercial ansesthetic, did not produce any detectable TFA.     

Nachschrift zu: V?gel der Ukerewe-Insel

Ohne Zusammenfassung     

Chemoenzymatic synthesis of 3'-O-(carboxyalkyl)fluorescein labels.

A general and versatile method is described for the synthesis of fluorescent labels. Coupling of the 3'-phenol of fluorescein methyl ester with hydroxyalkyl benzyl esters, followed by benzyl ester hydrolysis, provided a series of fluorescein carboxyalkyl ethers. Use of the Mitsunobu reaction allowed for the introduction of linkers of different lengths onto the 3'-phenol of fluorescein. Chemoenzymatic benzyl ester hydrolysis was achieved with LPL-80 lipase, providing pH-independent labels useful for the preparation of fluorescent conjugates.