Influence of Macrocyclic Chelators on the Targeting Properties of 68Ga-Labeled Synthetic Affibody Molecules: Comparison with 111In-Labeled Counterparts

Affibody molecules are a class of small (7 kDa) non-immunoglobulin scaffold-based affinity proteins, which have demonstrated substantial potential as probes for radionuclide molecular imaging. The use of positron emission tomography (PET) would further increase the resolution and quantification accuracy of Affibody-based imaging. The rapid in vivo kinetics of Affibody molecules permit the use of the generator-produced radionuclide ⁶⁸Ga (T_1/2 = 67.6 min). Earlier studies have demonstrated that the chemical nature of chelators has a substantial influence on the biodistribution properties of Affibody molecules. To determine an optimal labeling approach, the macrocyclic chelators 1,4,7,10-tetraazacylododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N,N-triacetic acid (NOTA) and 1-(1,3-carboxypropyl)-1,4,7- triazacyclononane-4,7-diacetic acid (NODAGA) were conjugated to the N-terminus of the synthetic Affibody molecule Z_HER2:S1 targeting HER2. Affibody molecules were labeled with ⁶⁸Ga, and their binding specificity and cellular processing were evaluated. The biodistribution of ⁶⁸Ga-DOTA-Z_HER2:S1, ⁶⁸Ga-NOTA-Z_HER2:S1 and ⁶⁸Ga-NODAGA-Z_HER2:S1, as well as that of their ¹¹¹In-labeled counterparts, was evaluated in BALB/C nu/nu mice bearing HER2-expressing SKOV3 xenografts. The tumor uptake for ⁶⁸Ga-DOTA-Z_HER2:S1 (17.9±0.7%IA/g) was significantly higher than for both ⁶⁸Ga-NODAGA-Z_HER2:S1 (16.13±0.67%IA/g) and ⁶⁸Ga-NOTA-Z_HER2:S1 (13±3%IA/g) at 2 h after injection. ⁶⁸Ga-NODAGA-Z_HER2:S1 had the highest tumor-to-blood ratio (60±10) in comparison with both ⁶⁸Ga-DOTA-Z_HER2:S1 (28±4) and ⁶⁸Ga-NOTA-Z_HER2:S1 (42±11). The tumor-to-liver ratio was also higher for ⁶⁸Ga-NODAGA-Z_HER2:S1 (7±2) than the DOTA and NOTA conjugates (5.5±0.6 vs.3.3±0.6). The influence of chelator on the biodistribution and targeting properties was less pronounced for ⁶⁸Ga than for ¹¹¹In. The results of this study demonstrate that macrocyclic chelators conjugated to the N-terminus have a substantial influence on the biodistribution of HER2-targeting Affibody molecules labeled with ⁶⁸Ga.This can be utilized to enhance the imaging contrast of PET imaging using Affibody molecules and improve the sensitivity of molecular imaging. The study demonstrated an appreciable difference of chelator influence for ⁶⁸Ga and ¹¹¹In.     

RASSF1A Promoter Methylation Levels Positively Correlate with Estrogen Receptor Expression in Breast Cancer Patients

The aim of this study was to investigate the relationship between the promoter methylation in five cancer-associated genes and clinicopathologic features for identification of molecular markers of tumor metastatic potential and hormone therapy response efficiency in breast cancer. The methylation levels in paraffin-embedded tumor tissues, plasma, and blood cells from 151 sporadic breast cancer patients and blood samples of 50 controls were evaluated by quantitative multiplex methylation-specific polymerase chain reaction. DNA methylation of RAS-association domain family member 1 (RASSF1A), estrogen receptor 1 (ESR1), cadherin 1, type 1, E-cadherin (CDH1), TIMP metallopeptidase inhibitor 3 (TIMP3) and spleen tyrosine kinase (SYK) genes was detected in the tumors of 124, 19, 15, 15, and 6 patients with mean levels of 48.45%, 3.81%, 2.36%, 27.55%, and 10.81%, respectively. Plasma samples exhibited methylation in the same genes in 25, 10, 15, 17, and 3 patients with levels of 22.54%, 17.20%, 22.87%, 31.93%, and 27.42%, respectively. Cumulative methylation results confirmed different spectra in tumor and plasma samples. Simultaneous methylation in tumors and plasma were shown in less than 17% of patients. RASSF1A methylation levels in tumor samples statistically differ according to tumor size (P = .029), estrogen receptor (ER) and progesterone receptor (PR) status (P = .000 and P = .004), and immunohistochemical subtype (P = .000). Moreover, the positive correlation was found between RASSF1A methylation levels and percentage of cancer cells expressing ER and PR. The direct relationship between RASSF1A promoter methylation and expression of ER could aid the prognosis of hormonal therapy response.     

Simultaneous RP‐HPLC‐DAD Separation,and Determination of Flavonoids and Phenolic Acids in Plantago L. Species

A rapid reversed‐phase (RP) high‐performance liquid chromatography method was developed and applied for simultaneous separation, and determination of flavonoids and phenolic acids in eight Plantago L. taxa (P. altissima L., P. argentea Chaix , P. coronopus L., P. holosteum Scop . ssp. depauperata Pilger , P. holosteum ssp. holosteum, P. holosteum ssp. scopulorum (Degen) Horvati? , P. lagopus L., and P. maritima L.) growing in Croatia. Chromatographic separation was carried out on Zorbax Eclipse XDB‐C18 using gradient elution with a H₂O (pH 2.5, adjusted with CF₃COOH) and MeCN mixture at 30°. The contents of analyzed phenolic compounds (% of the dry weight of the leaves, dw) varied among examined species: rutin (max. 0.024%, P. argentea), hyperoside (max. 0.020%, P. lagopus), quercitrin (max. 0.013%, P. holosteum ssp. holosteum), quercetin (max. 0.028%, P. holosteum ssp. scopulorum), chlorogenic acid (max. 0.115%, P. lagopus), and caffeic acid (max. 0.046%, P. coronopus). Isoquercitrin was detected only in P. argentea (0.020%), while isochlorogenic acid content was below limit of quantification in all investigated species. Multivariate analyses (UPGMA and PCA) showed significant differences in contents of investigated polyphenolic compounds between different Plantago taxa. Accordingly, investigated substances might be employed as chemotaxonomic markers in the study of the complex genus Plantago.     

Occurrence of Fatty Acid Short‐Chain‐Alkyl Esters in Fruits of Celastraceae Plants

Small amounts of a mixture of fatty acid short‐chain‐alkyl esters (FASCAEs) were obtained from the fruits of twelve plant species of Celastraceae family, and in five of them the FASCAEs were present not only in the arils but also in the seeds. These mixtures contained 32 individual FASCAE species, which formed four separate fractions, viz. FA methyl, ethyl, isopropyl, and butyl esters (FAMEs, FAEEs, FAIPEs, and FABEs, resp.). The FASCAE acyl components included the residues of 16 individual C₁₄–C₂₄ saturated, mono‐, di‐, and trienoic FAs. Linoleic, oleic, and palmitic acids, and, in some cases, also α‐linolenic acid predominated in FAMEs and FAEEs, while myristic acid was predominant in FAIPEs. It can be suggested that, in the fruit arils of some plant species, FAMEs and FAEEs were formed at the expense of a same FA pool characteristic of a given species and were strongly different from FAIPEs and FABEs esters regarding the mechanism of their biosynthesis. However, as a whole, the qualitative and quantitative composition of various FASCAE fractions, as well as their FA composition, varied considerably depending on various factors. Therefore, separate FASCAE fractions seem to be synthesized from different FA pools other than those used for triacylglycerol formation.     

Fluorescent (Au@SiO2)SiC Nanohybrids: Influence of Gold Nanoparticle Diameter and SiC Nanoparticle Surface Density

Gold@silica core–shell nanoparticles were prepared with various gold core diameters (ranging from 20 to 150 nm) and silica thicknesses (ranging from 10 to 30 nm). When the gold diameter is increased, the size dispersion became larger, leading to a broader plasmon band. Then, silicon carbide (SiC) nanoparticles were covalently immobilized onto silica to obtain hybrid (Au@SiO₂) SiC nanoparticles. The absorption properties of these hybrid nanoparticles showed that an excess of SiC nanoparticles in the dispersion can be identified by a strong absorption in the UV region. Compared to SiC reference samples, a blue shift of the fluorescence emission, from 582 to 523 nm, was observed, which was previously attributed to the strong surface modification of SiC when immobilized onto silica. Finally, the influence of several elaboration parameters (gold diameter, silica thickness, SiC concentration) on fluorescence enhancement was investigated. It showed that the highest enhancements were obtained with 10 nm silica thickness, low concentration of SiC nanoparticles, and surprisingly, with a 20-nm gold core diameter. This last result could be attributed to the broad plasmon band of big gold colloids. In this case, SiC emission strongly overlapped gold absorption, leading to possible quenching of SiC fluorescence by energy transfer.     

Paleo-Balkan and Slavic Contributions to the Genetic Pool of Moldavians: Insights from the Y Chromosome

Moldova has a rich historical and cultural heritage, which may be reflected in the current genetic makeup of its population. To date, no comprehensive studies exist about the population genetic structure of modern Moldavians. To bridge this gap with respect to paternal lineages, we analyzed 37 binary and 17 multiallelic (STRs) polymorphisms on the non-recombining portion of the Y chromosome in 125 Moldavian males. In addition, 53 Ukrainians from eastern Moldova and 54 Romanians from the neighboring eastern Romania were typed using the same set of markers. In Moldavians, 19 Y chromosome haplogroups were identified, the most common being I-M423 (20.8%), R-M17* (17.6%), R-M458 (12.8%), E-v13 (8.8%), R-M269* and R-M412* (both 7.2%). In Romanians, 14 haplogroups were found including I-M423 (40.7%), R-M17* (16.7%), R-M405 (7.4%), E-v13 and R-M412* (both 5.6%). In Ukrainians, 13 haplogroups were identified including R-M17 (34.0%), I-M423 (20.8%), R-M269* (9.4%), N-M178, R-M458 and R-M73 (each 5.7%). Our results show that a significant majority of the Moldavian paternal gene pool belongs to eastern/central European and Balkan/eastern Mediterranean Y lineages. Phylogenetic and AMOVA analyses based on Y-STR loci also revealed that Moldavians are close to both eastern/central European and Balkan-Carpathian populations. The data correlate well with historical accounts and geographical location of the region and thus allow to hypothesize that extant Moldavian paternal genetic lineages arose from extensive recent admixture between genetically autochthonous populations of the Balkan-Carpathian zone and neighboring Slavic groups.     

Ancient DNA Analysis Affirms the Canid from Altai as a Primitive Dog

The origin of domestic dogs remains controversial, with genetic data indicating a separation between modern dogs and wolves in the Late Pleistocene. However, only a few dog-like fossils are found prior to the Last Glacial Maximum, and it is widely accepted that the dog domestication predates the beginning of agriculture about 10,000 years ago. In order to evaluate the genetic relationship of one of the oldest dogs, we have isolated ancient DNA from the recently described putative 33,000-year old Pleistocene dog from Altai and analysed 413 nucleotides of the mitochondrial control region. Our analyses reveal that the unique haplotype of the Altai dog is more closely related to modern dogs and prehistoric New World canids than it is to contemporary wolves. Further genetic analyses of ancient canids may reveal a more exact date and centre of domestication.     

Identifying Emotions on the Basis of Neural Activation

We attempt to determine the discriminability and organization of neural activation corresponding to the experience of specific emotions. Method actors were asked to self-induce nine emotional states (anger, disgust, envy, fear, happiness, lust, pride, sadness, and shame) while in an fMRI scanner. Using a Gaussian Naïve Bayes pooled variance classifier, we demonstrate the ability to identify specific emotions experienced by an individual at well over chance accuracy on the basis of: 1) neural activation of the same individual in other trials, 2) neural activation of other individuals who experienced similar trials, and 3) neural activation of the same individual to a qualitatively different type of emotion induction. Factor analysis identified valence, arousal, sociality, and lust as dimensions underlying the activation patterns. These results suggest a structure for neural representations of emotion and inform theories of emotional processing.     

Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells

Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6′-carboxy isomers and not the 5′-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of ¹H NMR spectra to discriminate between 6′- and 5′-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae.