Proteomic Investigation of Novel Nuclear JAK2 Pathways

Myeloproliferative neoplasms represent different cancer entities, which all originate from clonal deregulation of hematopoietic stem cells (HSCs). Most disease entities share the same activating mutation of the tyrosine kinase JAK2. Constitutively activated JAK2-V617F mutated HSCs are thought to promote disease progression by stimulating uncontrolled gene expression through the activation of signal transducers and activators of transcription (STAT). Whereas this canonical JAK2/STAT pathway has been thoroughly investigated, research within the nuclear compartment revealed previously unknown functions of JAK2. The kinase was shown to be involved with other transcription factors like NF1-C2 or RUSH. Moreover, through phosphorylation of histone H3 and subsequent release of HP1a, JAK2 is also responsible for epigenetic regulation. Discovery of these pathways gives rise to the question: which other pathways are influenced by nuclear JAK2? Striving to uncover further mechanisms of nuclear JAK2, I initially utilized mass spectrometric pYome and interactome analyses which led me to the recognition of proteins involved in mRNA splicing as thus far unknown targets: hnRNPA1-Y341&Y347, hnRNPA2B1-Y336&Y347, Raver1-Y303 and SF3b155-Y84. Introducing a mutated hnRNPA1 sequence and applying a CRISPR-Cas9 based approach to knock-out the endogenous one, ensured that phosphorylation of the mentioned tyrosine-residues was inhibited. Through a subsequent RNA-sequencing analysis, the differences in phosphorylated vs. non-phosphorylatable hnRNPA1 transcriptomes were examined. With this, I was able to show that nuclear JAK2 mediated phosphorylation of hnRNPA1-Y341&Y347 aids in activating the NFkB pathway by fostering expression of the easily degradable IkBb-1 isoform. In conclusion, I showed that inhibition of hnRNPA1 phosphorylation led to reduced expression of NFkB target genes

A rapid and concise setup for the fast screening of FRET pairs using bioorthogonalized fluorescent dyes

One of the most popular means to follow interactions between bio(macro)molecules is Forster resonance energy transfer (FRET). There is large interest in widening the selection of fluorescent FRET pairs especially in the region of the red/far red range, where minimal autofluorescence is encountered. A set of bioorthogonally applicable fluorescent dyes, synthesized recently in our lab, were paired (Cy3T/Cy5T; Cy1A/Cy3T and Cy1A/CBRD1A) based on their spectral characteristics in order to test their potential in FRET applications. For fast elaboration of the selected pairs we have created a bioorthogonalized platform based on complementary 17-mer DNA oligomers. The cyclooctynylated strands were modified nearly quantitatively with the fluorophores via bioorthogonal chemistry steps, using azide- (Cy1; CBRD1) or tetrazine-modified (Cy3; Cy5) dyes. Reactions were followed by capillary electrophoresis using a method specifically developed for this project. FRET efficiencies of the fluorescent dye pairs were compared both in close proximity (5' and 3' matched) and at larger distance (5' and 5' matched). The specificity of FRET signals was further elaborated by denaturation and competition studies. Cy1A/Cy3T and Cy1A/CBRD1A introduced here as novel FRET pairs are highly recommended for FRET applications based on the significant changes in fluorescence intensities of the donor and acceptor peaks. Application of one of the FRET pairs was demonstrated in live cells, transfected with labeled oligos. Furthermore, the concise installation of the dyes allows for efficient fluorescence modification of any selected DNA strands as was demonstrated in the construction of Cy3T labeled oligomers, which were used in the FISH-based detection of Helicobacter pylori

Are Co-operatives a Way to Integrate Small Farmers in Supply Chain Networks? Preliminary Thoughts on Hungary

Vertical coordination in agri-food chains is a significant and increasing phenomenon in Central and Eastern European Countries (CEEC). It has been observed that this development prefers large scale production. However, the agricultural sector is still a mixture of small scale and large scale farming in these countries. Hence, for small scale farmers, horizontal collaboration can be assumed to be a prerequisite for remaining in the market. Therefore, the goal of this paper is to investigate whether co-ops are appropriate means for integrating small farmers into modern supply systems. Further, we want to analyze how cooperatives can cope with the quality demands they face in modern distribution channels

Latvian co-operatives: Agents of vertical coordination?

Vertical coordination has become increasingly important in the agri-food business in countries such as Latvia, making the use of contractual arrangements such as marketing and production contracts more common. In this work, we analyse the use of these contracts in the Latvian agri-food sector. Because of the Latvian dualistic production structure, which consists of more small farms and fewer large farms, we pay particular attention to co-operatives in this region and evaluate whether co-operatives can act as agents of change by linking small farms to vertically coordinated chains

Genetic identification of members of the prominent Báthory aristocratic family

The Báthory family was one of the most powerful noble families in the medieval Hungarian Kingdom. Their influence peaked during the Ottoman occupation of Hungary, when the only partially autonomous region of the country was Transylvania, under Turkish protectorate. Several members of the family became Princes of Transylvania, and one of them, István Báthory, was also the elected King of Poland. We hereby present the first genetic data about this extinct family. Archaeological excavations in Pericei, a settlement now part of Romania, revealed the former family chapel of the Báthory family. Through this work, two Báthory family members were successfully identified among the 13 skeletons found at the site. The presence of Y chromosome haplogroup R-S498 fits the historical account describing the family’s German (Swabian) origins. Their genomic composition also indicates a family of Germanic origin that intermixed with medieval Hungarians

Peripheral re-localization of constitutive heterochromatin advances its replication timing and impairs maintenance of silencing marks.

The replication of the genome is a highly organized process, both spatially and temporally. Although a lot is known on the composition of the basic replication machinery, how its activity is regulated is mostly unknown. Several chromatin properties have been proposed as regulators, but a potential role of the nuclear DNA position remains unclear. We made use of the prominent structure and well-defined heterochromatic landscape of mouse pericentric chromosome domains as a well-studied example of late replicating constitutive heterochromatin. We established a method to manipulate its nuclear position and evaluated the effect on replication timing, DNA compaction and epigenetic composition. Using time-lapse microscopy, we observed that constitutive heterochromatin, known to replicate during late S-phase, was replicated in mid S-phase when repositioned to the nuclear periphery. Out-of-schedule replication resulted in deficient post-replicative maintenance of chromatin modifications, namely silencing marks. We propose that repositioned constitutive heterochromatin was activated in trans according to the domino model of origin firing by nearby (mid S) firing origins. In summary, our data provide, on the one hand, a novel approach to manipulate nuclear DNA position and, on the other hand, establish nuclear DNA position as a novel mechanism regulating DNA replication timing and epigenetic maintenance

Genetic identification of members of the prominent Báthory aristocratic family

Summary: The Báthory family was one of the most powerful noble families in the medieval Hungarian Kingdom. Their influence peaked during the Ottoman occupation of Hungary, when the only partially autonomous region of the country was Transylvania, under Turkish protectorate. Several members of the family became Princes of Transylvania, and one of them, István Báthory, was also the elected King of Poland. We hereby present the first genetic data about this extinct family. Archaeological excavations in Pericei, a settlement now part of Romania, revealed the former family chapel of the Báthory family. Through this work, two Báthory family members were successfully identified among the 13 skeletons found at the site. The presence of Y chromosome haplogroup R-S498 fits the historical account describing the family’s German (Swabian) origins. Their genomic composition also indicates a family of Germanic origin that intermixed with medieval Hungarians

Az intestinalis gél bevezetésekor rögzített jellemzők tízéves változásai előrehaladott Parkinson-kóros betegekben

Bevezetés: A Parkinson-kór olyan neurodegeneratív kórkép, melynek tüneti kezelése hatékonyan megoldható, bár a terá- piás ajánlások gyakran szorulnak finomításokra a gyarapodó tapasztalatok birtokában. Célkitűzés: Azt kívántuk elemezni, hogy előrehaladott Parkinson-kóros betegeinknél az időközben megjelent szakértői ajánlások hogyan tükröződtek az intestinalis gél bevezetését megelőző időszakban. Módszer: Retrospektíven vizsgáltuk azokat az azonos szempontok alapján nyert adatokat, amelyek levodopa-karbidopa intestinalis gél kezelésben részesülő betegekre vonatkoztak. A 2011 és 2021 közötti periódust két ötévesre osztottuk fel, mivel az első öt év után fogadták el a klinikai döntéshozatalban az „5-2-1-es szabályt”. Eredmények: A vizsgált időszakban 150 betegnél kezdtük el a levodopa-karbidopa intestinalis gél kezelést. A második periódusban a betegek átlagéletkora alacsonyabb, a diagnózis óta eltelt idő rövidebb volt. Csúcsdózis dyskinesisei (p = 0,02), bifázisos dyskinesisei (p<0,001), hajnali akinesisei (p = 0,02) szignifikánsan kevesebb betegnek voltak a második öt évben. Szintén az utóbbi öt évben kevesebb beteget érintett a megkésett „on” (p = 0,03), a „no on” (p = 0,02) és a „freezing” jelenség (p = 0,01). A Hoehn–Yahr-skála átlagos pontszáma is kisebb volt a második periódusban, míg az MMSE átlagos pontszáma nagyobb volt (p<0,001). A levodopa napi adagjai nagyobbak (p<0,01) voltak a második csoportban, az adagolási frekvencia nem változott jelentősen. Következtetés: Tízéves időszakot vizsgálva azt tapasztaltuk, hogy a második öt évben szignifikánsan jobb fizikális és kognitív állapotban került sor az előrehaladott Parkinson-kóros betegeknél a levodopa-karbidopa intestinalis gél bevezetésére. A szakértői ajánlásokhoz viszonyítva még mindig súlyosabb klinikai képet tapasztalunk az eszközös kezelés elkezdésekor, de ennek az invazív módszernek már javult az elfogadása mind a betegek, mind az őket alapszinten ellátó családorvosok és területi neurológusok részéről