Improvement of Opal Multiplex Immunofluorescence Workflow for Human Tissue Sections

The Opal multiplex technique is an established methodology for the detection of multiple biomarkers in one section. The protocol encompasses iterative single stainings and heating-mediated removal of the primary and secondary antibodies after each staining round, leaving untouched the Opal fluorophores which are deposited onto the antigen of interest. According to our experience, repetitive heating of skin sections often results in tissue damage, indicating an urgent need for milder alternatives to strip immunoglobulins. In this study, we demonstrate that considerable heating-related damage was found not only in skin but also in tissues of different origin, mostly characterized by low cell density. Importantly, the morphology remained fully intact when sections were repetitively exposed to β-mercaptoethanol-containing stripping buffer instead of multiple heating cycles. However, target epitopes appeared sensitive at a differential degree to multiple treatments with stripping buffer, as shown by loss in staining intensity, but in all cases, the staining intensity could be restored by increment of the primary antibody concentrations. Application of β-mercaptoethanol-containing stripping buffer instead of heating for antibody removal markedly improved the quality of the Opal multiplex technique, as a substantial higher number of differently colored cells could be visualized within a well-conserved morphological context:     

Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

Alzheimer´s disease is the most prominent type of dementia and currently no causative treatment is available. According to recent studies, oligomeric species of the amyloid beta (Aβ) peptide appear to be the most toxic Aβ assemblies. Aβ monomers, however, may be not toxic per se and may even have a neuroprotective role. Here we describe a competitive mirror image phage display procedure that allowed us to identify preferentially Aβ_1–42 monomer binding and thereby stabilizing peptides, which destabilize and thereby eliminate toxic oligomer species. One of the peptides, called Mosd1 (monomer specific d-peptide 1), was characterized in more detail. Mosd1 abolished oligomers from a mixture of Aβ_1–42 species, reduced Aβ_1–42 toxicity in cell culture, and restored the physiological phenotype in neuronal cells stably transfected with the gene coding for human amyloid precursor protein.     

Leaf wax n‐alkane patterns of six tropical montane tree species show species‐specific environmental response

It remains poorly understood how the composition of leaf wax n‐alkanes reflects the local environment. This knowledge gap inhibits the interpretation of plant responses to the environment at the community level and, by extension, inhibits the applicability of n‐alkane patterns as a proxy for past environments. Here, we studied the n‐alkane patterns of five Miconia species and one Guarea species, in the Ecuadorian Andes (653–3,507 m a.s.l.). We tested for species‐specific responses in the average chain length (ACL), the C₃₁/(C₃₁ + C₂₉) ratio (ratio), and individual odd n‐alkane chain lengths across an altitudinally driven environmental gradient (mean annual temperature, mean annual relative air humidity, and mean annual precipitation). We found significant correlations between the environmental gradients and species‐specific ACL and ratio, but with varying magnitude and direction. We found that the n‐alkane patterns are species‐specific at the individual chain length level, which could explain the high variance in metrics like ACL and ratio. Although we find species‐specific sensitivity and responses in leaf n‐alkanes, we also find a general decrease in “shorter” (<C₂₉) and an increase in “longer” (>C₃₁) chain lengths with the environmental gradients, most strongly with temperature, suggesting n‐alkanes are useful for reconstructing past environments.     

Stability of interleukin 6, soluble interleukin 6 receptor,interleukin 10 and CC16 in human serum

The stability of interleukin 6 (IL-6), its soluble receptor (sIL-6R), IL-10 and CC16 or uteroglobin (an endogenous cytokine inhibitor) in human serum was examined using an accelerated stability testing protocol according to the Arrhenius equation. Further, the effect of time delay between blood sampling and sample processing, clotting temperature and repeated freeze-thaw cycles on serum levels of these proteins were determined. Paired serum samples were stored at 4 degrees C, 20 degrees C, 30 degrees C and 40 degrees C for 1 to 21 days. We found that IL-6 and CC16 concentrations did not change at 4 degrees C, 20 degrees C and 30 degrees C. Interleukin-6 concentrations significantly declined after 11 days at 40 degrees C. The concentrations of sIL-6R and IL-10 did not change at 4 degrees C but significantly decreased at 20 degrees C (after 21 and 14 days respectively), 30 degrees C and 40 degrees C (after 1 day at both temperatures for both cytokines). Arrhenius-plots indicated that sIL-6R and IL-10 are stable for at least several years at -20 degrees C and -70 degrees C, respectively. Since their relative stability, no Arrhenius-plot could be calculated for IL-6 and CC16. The concentrations of the proteins examined were not significantly altered by repeated freeze-thaw cycles, nor by extended clotting times at 4 degrees C or 20 degrees C. We conclude that serum samples for the determination of IL-6, sIL-6R and CC16 can be stored at -20 degrees C for several years, but for IL-10 determinations, storage at -70 degrees C is recommended.     

Conserved features of Y RNAs: a comparison of experimentally derived secondary structures

In this study, phylogenetically conserved structural features of the Ro RNP associated Y RNAs were investigated. The human, iguana, and frog Y3 and Y4 RNA sequences have been determined previously and the respective RNAs were subjected to enzymatic and chemical probing to obtain structural information. For all of the analyzed RNAs, the probing data were used to compose secondary structures, which partly deviate from previously predicted structures. Our results confirm the existence of two stem structures, which are also found at similar positions in hY1 and hY5 RNA. For the remaining parts of hY3 and hY4 RNA the secondary structures differ from those previously proposed based upon computer predictions. What might be more important is that certain parts of the RNAs appear to be flexible, i.e., to adopt several conformations. Another striking feature is that a characteristic pyrimidine-rich region, present in every Y RNA known, is single-stranded in all secondary structures. This may suggest that this region is readily available for base pairing interactions with other cellular nucleic acids, which might be important for the as yet unknown function of the RNAs.     

Involvement of distinct G-proteins, Gpa2 and Ras, in glucose- and intracellular acidification-induced cAMP signalling in the yeast Saccharomyces cerevisiae.

Adenylate cyclase activity in Saccharomyces cerevisiae is dependent on Ras proteins. Both addition of glucose to glucose-deprived (derepressed) cells and intracellular acidification trigger an increase in the cAMP level in vivo. We show that intracellular acidification, but not glucose, causes an increase in the GTP/GDP ratio on the Ras proteins independent of Cdc25 and Sdc25. Deletion of the GTPase-activating proteins Ira1 and Ira2, or expression of the RAS2(val19) allele, causes an enhanced GTP/GDP basal ratio and abolishes the intracellular acidification-induced increase. In the ira1Delta ira2Delta strain, intracellular acidification still triggers a cAMP increase. Glucose also did not cause an increase in the GTP/GDP ratio in a strain with reduced feedback inhibition of cAMP synthesis. Further investigation indicated that feedback inhibition by cAPK on cAMP synthesis acts independently of changes in the GTP/GDP ratio on Ras. Stimulation by glucose was dependent on the Galpha-protein Gpa2, whose deletion confers the typical phenotype associated with a reduced cAMP level: higher heat resistance, a higher level of trehalose and glycogen and elevated expression of STRE-controlled genes. However, the typical fluctuation in these characteristics during diauxic growth on glucose was still present. Overexpression of Ras2(val19) inhibited both the acidification- and glucose-induced cAMP increase even in a protein kinase A-attenuated strain. Our results suggest that intracellular acidification stimulates cAMP synthesis in vivo at least through activation of the Ras proteins, while glucose acts through the Gpa2 protein. Interaction of Ras2(val19) with adenylate cyclase apparently prevents its activation by both agonists.     

Quantitative proteomics suggests decrease in the secretogranin‐1 cerebrospinal fluid levels during the disease course of multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS with unknown cause. Proteins with different abundance in the cerebrospinal fluid (CSF) from relapsing‐remitting MS (RRMS) patients and neurological controls could give novel insight to the MS pathogenesis and be used to improve diagnosis, predict prognosis and disease course, and guide in therapy decisions. We combined iTRAQ labeling and Orbitrap mass spectrometry to discover proteins with different CSF abundance between six RRMS patients and 18 neurological disease controls. From 777 quantified proteins seven were selected as biomarker candidates, namely chitinase‐3‐like protein 1, secretogranin‐1 (Sg1), cerebellin‐1, neuroserpin, cell surface glycoprotein MUC18, testican‐2 and glutamate receptor 4. An independent sample set of 13 early‐MS patients, 13 RRMS patients and 13 neurological controls was used in a multiple reaction monitoring verification study. We found the intracellular calcium binding protein Sg1 to be increased in early‐MS patients compared to RRMS and neurological controls. Sg1 should be included in further studies to elucidate its role in the early phases of MS pathogenesis and its potential as a biomarker for this disease.