Compatibility of Distinct Label-Free Proteomic Workflows in Absolute Quantification of Proteins Linked to the Oocyte Quality in Human Follicular Fluid

We present two separate label-free quantitative workflows based on different high-resolution mass spectrometers and LC setups, which are termed after the utilized instrument: Quad-Orbitrap (nano-LC) and Triple Quad-TOF (micro-LC) and their directed adaptation toward the analysis of human follicular fluid proteome. We identified about 1000 proteins in each distinct workflow using various sample preparation methods. With assistance of the Total Protein Approach, we were able to obtain absolute protein concentrations for each workflow. In a pilot study of twenty samples linked to diverse oocyte quality status from four donors, 455 and 215 proteins were quantified by the Quad-Orbitrap and Triple Quad-TOF workflows, respectively. The concentration values obtained from both workflows correlated to a significant degree. We found reasonable agreement of both workflows in protein fold changes between tested groups, resulting in unified lists of 20 and 22 proteins linked to oocyte maturity and blastocyst development, respectively. The Quad-Orbitrap workflow was best suited for an in-depth analysis without the need of extensive fractionation, especially of low abundant proteome, whereas the Triple Quad-TOF workflow allowed a more robust approach with a greater potential to increase in effectiveness with the growing number of analyzed samples after the initial effort of building a comprehensive spectral library

The identification of discontinuous epitope in the human cystatin C – Monoclonal antibody HCC3 complex

Human cystatin C (hCC) is a cysteine proteinase inhibitor involved in pathophysiological processes of dimerization and amyloid formation. These processes are directly associated with a number of neurodegenerative disorders such as Alzheimer disease or hereditary cystatin C amyloid angiopathy (HCCAA). One of the ideas on how to prevent amyloid formation is to use immunotherapy. HCC3 is one of a group of antibodies binding to hCC and reducing the in vitro formation of cystatin C dimers. Therefore, identification of the binding sites in the hCC-HCC3 complex may facilitate a search of effective drugs against HCCAA as well as understanding the mechanisms of neurodegenerative disorders. In this work we present epitope identification of the hCC-HCC3 complex using methods such as affinity chromatography, epitope excision and extraction MS approach, enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry (HDX MS). Comprehensive analysis of the obtained results allowed us to identify the epitope sequence with the key fragment covering hCC L1 loop and two potential epitopic fragments – α-helical part, hCC (17–28) and β4 strand in C-terminal part of hCC. The presence of the L1 loop in the epitope sequence accounts for the significant reduction of hCC dimer formation in the presence of HCC3 antibody. Significance of the study: Deciphering the mechanism of the cystatin C aggregation process and detailed analysis of the interactions between hCC, or its pathogenic variant, and monoclonal antibodies, potentially constituting aggregation inhibitors, might be of great value as there still is a complete lack of any kind of efficient therapy for young people with the pathogenic mutation of hCC

The phonological skills of bilingual preschool children speaking Polish and Ukrainian in Poland

Zagadnienie jakościowych i ilościowych różnic w rozwoju fonologicznym dwujęzycznych oraz ich jednojęzycznych równieśników jest problemem nierozstrzygniętym, zwłaszcza w kontekście języków zbliżonych ze sobą typologicznie. Niniejsza publikacja prezentuje badanie, służące analizie rozwoju fonologicznego dwujęzycznych dzieci przedszkolnych, mówiących po polsku i ukraińsku, czyli niezbadanych pod tym kątem języków słowiańskich. W badaniu wzięło udział 57 dzieci w wieku od 4;0-6;0 - 18 z nich to dzieci dwujęzyczne z Polski, 19 to dzieci jednojęzyczne z Polski i 20 to dzieci jednojęzyczne z Ukrainy. W badaniu użyto kwestionariuszy obrazkowych za pomocą których zebrano realizacje dzieci. Wyniki badania pokazały dwujęzyczny rozwój fonologiczny w większości z użyciem typowych wzorców, silnymi wpływami międzyjęzykowymi i rzadkimi procesami, które były trudne do interpretacji. Autorzy wysunęli hipotezę, iż ogólny rozwój fonologiczny dzieci dwujęzycznych może opierać się na typowych wzorcach, zwłaszcza między językami podobnymi do siebie. Badanie potwierdziło również konieczność dwujęzycznego badania dzieci. Błędy, które nie są zinterpretowane jako typowe dla rozwoju albo wynikające z kontaktu językowych mogą być objawem zaburzenia.The extent of quantitative and qualitative differences in phonological development between bilingual children and their monolingual counterparts remains unresolved, especially with regard to typologically related languages. The current study used a comparative research design to examine the phonological skills of preschool children speaking Polish and Ukrainian, a pair of Slavic languages hitherto unexplored in this context. It involved 57 typically-developing children aged 4;0-6;0, i.e. 18 bilingual children speaking Polish and Ukrainian in Poland, 19 monolingual Polish children in Poland and 20 monolingual Ukrainian children in Ukraine. The qualitative and quantitative analyses of data collected through single word elicitation tests involved between-group comparisons of bilingual and monolingual children’s speech and within-group comparisons of the bilingual children’s phonological systems. Results paint a complex picture of bilingual phonological development with mostly typical patterns, a strong crosslinguistic influence, and rare processes which are difficult to interpret. Authors hypothesise that such general patterns of phonological process use might be typical of bilingual development involving typologically-related and geographically close languages. Findings underscore the need to assess bilingual children in all their languages and dialects. Errors which cannot be interpreted through either rules of normal development or crosslinguistic interaction could be a sign of disorder