Characterization of the NMD mechanism in Arabidopsis thaliana – identification of new factors, analysis of new interaction and functional role of NMD mechanism

Prawidłowy metabolizm RNA wymaga obecności zróżnicowanych mechanizmów kontroli w celu monitorowania i sprawdzania ilości oraz jakości powstających cząsteczek RNA. Mechanizmy te zachodzą podczas transkrypcji i wielu procesów post-transkrypcyjnych, zapewniając równowagę puli prawidłowych RNA poprzez degradację nieprawidłowo zsyntetyzowanych lub niefunkcjonalnych oraz zbędnych transkryptów. Jedną z najistotniejszych ścieżek kontroli jakości RNA jest mechanizm NMD (ang. Nonsense Mediated Decay), który prowadzi do rozpoznania nieprawidłowych transkryptów zawierających przedwczesny kodon terminacji translacji PTC. Cząsteczki takie powstają najczęściej w wyniku alternatywnego splicingu, mutacji lub błędów podczas transkrypcji. Główną funkcją NMD jest ochrona przed akumulacją potencjalnie szkodliwych białek. Ponadto NMD bierze udział w regulacji ekspresji informacji genetycznej prawidłowych transkryptów oraz pełni rolę w odpowiedzi na stres. NMD jest nie tylko mechanizmem kontroli jakości RNA ale ma również znaczenie dla homeostazy komórkowej. W pracy doktorskiej przedstawiono wyniki badań roślinnego mechanizmu NMD u modelowej rośliny Arabidopsis thaliana dotyczące określenia interaktorów białka UPF1, które jest głównym efektorem NMD. Wśród białek oddziałujących z UPF1 zidentyfikowano zarówno znane czynniki NMD jak również nowe składniki potencjalnie zaangażowane w ten proces. Wykazano udział roślinnych helikaz DEAD-box w przebiegu NMD, w szczególności homologów ludzkiej helikazy DDX3 (RH11, RH37, RH52) i helikazy DDX6 (białka RH6, RH8 i RH12) jako, odpowiednio, pozytywnych i negatywnych regulatorów tego procesu. Potwierdzono także ko-lokalizację tych helikaz z głównymi czynnikami NMD, co dodatkowo wskazuje na ich rolę w mechanizmie NMD. Ponadto wyniki wstępnych badań potwierdziły związek mechanizmu NMD z translacją i wskazały na fizjologiczną rolę NMD w reakcji na stres cieplny. Co więcej, eksperymenty pilotażowe zasugerowały możliwość, że modyfikacje m6A w cząsteczkach mRNA mogą przyczynić się do regulacji wydajności NMD.Metabolism of all RNA molecules requires various precise quality control mechanisms to monitor their quality and quantity. They occur during transcriptional and post-transcriptional processes, providing balance between RNA synthesis and decay by degrading incorrectly synthesized or non-functional and unnecessary transcripts. One of the most important cytoplasmic RNA quality control pathway is Nonsense-Mediated mRNA Decay mechanism (NMD), which recognizes aberrant mRNAs carrying premature termination codons (PTC). Aberrant PTC-containing transcripts most often result from alternative splicing, mutations or transcription errors. The major function of NMD is to protect against accumulation of potentially harmful proteins, but it also regulates the expression of genetic information of normal transcripts and plays a role in response to stress. Therefore, NMD is not only an RNA quality mechanism, but also contributes to cellular homeostasis. The dissertation presents the results of analysis of the plant NMD mechanism in the model organism Arabidopsis thaliana, mainly related to establishing interactors of UPF1 protein, which is a major NMD factor. The identified UPF1-interacting proteins included not only known NMD factors, but also novel potential components of the NMD machinery. The most interesting proteins were a group of helicases from the DEAD-box family, homologues of human DDX3 (RH11, RH37, RH52) and DDX6 (RH6, RH8 and RH12). Functional studies of these helicases confirmed their involvement in NMD efficiency as positive and negative regulators, respectively. Also their subcellular localization and co-localization with major NMD factors corroborated their role in this mechanism. In addition, results of preliminary studies supported the association of the NMD mechanism with translation and indicated a physiological role of NMD in response to heat stress. Moreover, pilot experiments suggested a possibility that m6A modifications in mRNA molecules may contribute to the regulation of NMD efficiency

Risk of transmission of blood-derived pathogens by transfusion in Poland

Blood transfusion in Poland is the safest in history. High virological level of safety has been achieved mainly by improving not only the qualification of donors and methods used for donor screening, but also applying leukoreduction, pathogen reduction technology and grace period for serum.In this article, we discuss the improvement of the epidemic situation among blood donors for hepatitis B virus (HBV) and hepatitis C virus (HCV) and the increasing trend for HIV. Preliminary results of residual risk calculation for these pathogens are presented.Hepatitis E virus (HEV) and Babesia microti were considered as new factors potentially relevant for the safety of blood transfusion in our country. Due to evidence of West Nile virus (WNV) circulation in the ecosystem in Poland, it is also necessary to monitor the infections with this pathogen.In this article, it was emphasized that the reporting of all possible complications associated with transfusion and meticulous implementation of the look-back procedure play a key role for monitoring the risk of transmission of infectious agents by blood. It is especially important in view of the increasing epidemiological problems associated with emerging infectious agents

Diagnostyka molekularna SARS-CoV-2

Zakażenia nowym koronawirusem — SARS-CoV-2 — stały się w ostatnich miesiącachgłównym problemem epidemiologicznym oraz klinicznym na świecie, w tym Polsce w zakresiechorób zakaźnych. Niniejszy artykuł powstał na podstawie wystąpienia zaprezentowanegow trakcie webinaru zatytułowanego „Hematologia i transfuzjologia a COVID-19”, który odbyłsię w maju 2020 roku. Praca ma na celu przybliżenie procedury diagnostyki zakażenia nowymkoronawirusem. Zwrócono w niej uwagę na krytyczne dla jakości wykonywanych badańaspekty fazy przedanalitycznej oraz analitycznej, na które ma wpływ zarówno osoba zlecająca,jak i personel wykonujący badanie. Na podstawie aktualnego piśmiennictwa przedstawionotakże dalsze kierunki doskonalenia diagnostyki molekularnej SARS-CoV-2

Analysis of the role of education and sport in the heavy burden of obesity

Obesity has become the global pandemic of the world. The epidemiological rise has only happened in recent decades. Unfortunately, the disease affects people of all ages. The most prominent characteristics of the disorder are excessive fat accumulation, body mass index exceeding the number of 30, and general lowering of the quality of life. The consequences are observed in the whole organism. The disruptions affect one’s overall homeostasis. Certain diseases are specifically associated with excessive fat mass. When not properly managed, obesity leads to chronic illnesses and severe comorbidities, the most morbid one being death. Thus, effective therapeutic approaches are needed. Focus on education and physical activity are the best way to tackle this problem. The aim of this study is to check the factors and changes necessary to lose weight in obese patients in Poland. The main question regarded the role of sport and proper education in maintaining or losing weight in the Polish society. In order to analyse the issue, an Internet survey was prepared and disseminated in March and April 2023. Over half of the respondents have ever used a diet. Over 8 out of ten of them achieved the success in the set task. When asked about physical activity, the most respondents chose option 30-60 minutes per day. The preferred type of physical activity was most commonly walking. When asked if sport enabled the respondents to lose or maintain weight – 63,5% confirmed. 72% of respondents experienced education regarding healthy lifestyle, most commonly at home. Over 60% of respondents believed they were well informed about recommended behaviours. Over 50% of them confirmed their education is sufficient to lose weight. Maintaining healthy regimes is key to a healthy life. However, not all people are properly educated how to do that. In fact, there is a tendency of overestimation of one’s education in those topics. All in all, proper experience and routines are necessary

Charakterystyka testu cobas® DPX przeznaczonego do badania dawców krwi na obecność DNA parwowirusa B19 (B19V) i RNA HAV przy użyciu analizatora cobas® 6800 oraz aparatu cobas p 680 firmy Roche

Introduction The cobas DPX test for use on the cobas 6800 system, is real-time PCR test for the identification and quantitation of human parvovirus B19 (B19V) DNA and the detection of hepatitis A virus (HAV) RNA in human plasma. Optional component to the cobas 6800 system is the cobas p 680 instrument that creates pools from individual samples. Aim To evaluate the performance of cobas® DPX test and to demonstrate its ability to detect HAV and polymorphic forms of B19V during plasma testing. Sensitivity, correct identification of reactive donations and risk of false positive results was estimated. Material and method The analytical sensitivity of cobas® DPX test was assessed by testing dilutions of the WHO B19V Genotype 1-3 Reference Panels (09/110) and IS WHO RNA HAV (00/562) dilution panel. Six dilutions of each B19 panel were tested ranging from 31.6 IU/mL to 0.1 IU /mL of genotype 1, 2, 3 and six dilutions of RNA HAV panel ranging from 10-0,03 IU/ml (24 replicates/dilution). Detection and identification of reactive donations tested in minipools were evaluated by testing 767 negative and 1 high DNA B19V reactive donations in 8 pools. Results The 95% limits of detection [LOD] (95% confidence range) of the cobas® DPX test for DNA B19V were respectively: genotype 1 - 8.70 IU/mL (4.92-21.69), genotype 2 - 37.80 IU/ml (22.24 – 93.65), genotype 3 - 29.84 IU/ml (19.04-65.60) and for RNA HAV were: 0.82 IU/ml (0.49 -1.96). Results of the cobas® DPX test from samples analyzed in minipools were 100% concordant with the expected results. No false reactive or invalid results was observed during donation screening. Conclusion The cobas® DPX test on the cobas 6800 system allows to identify donations infected with HAV and genotypes 1-3 of B19V that could result contamination of plasma production pools with HAV and B19V beyond acceptable level (>104 IU/mL, according to recommendations). There was no significant risk of false results during evaluation procedure.Wstęp. Test cobas® DPX typu multiplex jest przeznaczony do badań dawców krwi w analizatorze cobas® 6800 fi rmy Roche. Wykorzystuje metodę real-time PCR i służy do ilościowego badania kwasu deoksyrybonukleinowego (DNA) parwowirusa B19 (B19V) oraz wykrywania kwasu rybonukleinowego RNA wirusa zapalenia wątroby typu A (HAV) w ludzkim osoczu. Opcjonalnym elementem systemu jest cobas p 680 — w pełni zautomatyzowane urządzenie pipetujące przeznaczone do pulowania pojedynczych próbek osocza. Cel. Celem badań była ocena przydatności testów cobas® DPX do prowadzenia badań w krwiodawstwie. Przeanalizowano między innymi czułość wykrywania markerów, poprawność identyfi kacji donacji zakażonych oraz ryzyko wystąpienia wyników fałszywych. Materiał i metody. Czułość analityczną określano, badając w 24 powtórzeniach każdą próbkę z panelu rozcieńczeń materiału referencyjnego Światowej Organizacji Zdrowia (WHO) genotypów B19V (09/110) — sześć rozcieńczeń genotypów 1–3 (31,6–0,1 IU/ml) oraz IS WHO RNA HAV (00/562) zawierających 10–0,03 IU/ml. Poprawność identyfi kacji zakażeń testem DPX oceniono, badając w ośmiu pulach 767 donacji, w tym jedną z wysoką wiremią B19V. Wyniki. Czułość analityczna testu cobas® DPX na poziomie 95% wykrywalności (LOD) (95-proc. przedział ufności) wyniosła 8,70 (4,92–21,69) IU/ml dla genotypu 1; 37,80 (22,24–93,65) IU/ml dla genotypu 2 i 29,84 (19,04–65,60) IU/ml dla genotypu 3 DNA B19V oraz 0,82 (0,49–1,96) IU/ml dla RNA HAV. Wyniki badań imitujących badania przeglądowe były zgodne z wynikami spodziewanymi. W trakcie oceny nie obserwowano wyników fałszywych. Wnioski. Badanie testem DPX wykonane na cobas® 6800 i p 680 pozwala prawidłowo identyfikować DNA-emię B19V, która może spowodować przekroczenie dopuszczalnego poziomu 104 IU B19V/ml w puli produkcyjnej składników osoczopochodnych. Równolegle prowadzone jest badanie RNA HAV. W badaniach nie stwierdzono istotnego ryzyka występowania wyników fałszywych

SARS-CoV-2 molecular diagnostics

The new coronavirus — SARS-CoV-2 infections have recently become the main epidemiological and clinical challenge worldwide (Poland included). This article is based on the presentation entitled “Hematology and transfusion medicine and COVID-19” given/conferred during the webinar in May 2020. The aim was to introduce the procedure for diagnosing the new coronavirus infection. The procedure highlights the aspects of the pre-analytical and analytical phase that are critical for the quality of the performed tests and affected by both the commissioning and test-performing personnel. On the basis of current literature, further recommendations for the improvement of molecular diagnostics of SARS-CoV-2 are also presented

RNA Helicases from the DEA(D/H)-Box Family Contribute to Plant NMD Efficiency

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic RNA surveillance mechanism that degrades aberrantm RNAs comprising a premature translation termination codon. The adenosine triphosphate (ATP)-dependent RNA helicase up-frameshift 1 (UPF1) is a major NMD factor in all studied organisms; however, the complexity of this mechanism has not been fully characterized in plants. To identify plant NMD factors, we analyzed UPF1-interacting proteins using tandem affinity purification coupled to mass spectrometry.Canonical members of the NMD pathway were found along with numerous NMD candidate factors, including conserved DEA(D/H)-box RNA helicase homologs of human DDX3, DDX5 and DDX6, translation initiation factors, ribosomal proteins and transport factors. Our functional studies revealed that depletion of DDX3 helicases enhances the accumulation of NMD target reporterm RNAs but does not result in increased protein levels. In contrast, silencing of DDX6 group leads to decreased accumulation of the NMD substrate. The inhibitory effect of DDX6-like helicases on NMD was confirmed by transient over-expression of RH12 helicase. These results indicate that DDX3 and DDX6 helicases in plants have a direct and opposing contribution to NMD and act as functional NMD factors

mRNA Decapping and 5′-3′ Decay Contribute to the Regulation of ABA Signaling in Arabidopsis thaliana

Defects in RNA processing and degradation pathways often lead to developmental abnormalities, impaired hormonal signaling and altered resistance to abiotic and biotic stress. Here we report that components of the 5′-3′ mRNA decay pathway, DCP5, LSM1-7 and XRN4, contribute to a proper response to a key plant hormone abscisc acid (ABA), albeit in a different manner. Plants lacking DCP5 are more sensitive to ABA during germination, whereas lsm1a lsm1b and xrn4-5 mutants are affected at the early stages of vegetative growth. In addition, we show that DCP5 and LSM1 regulate mRNA stability and act in translational repression of the main components of the early ABA signaling, PYR/PYL ABA receptors and SnRK2s protein kinases. mRNA decapping DCP and LSM1-7 complexes also appear to modulate ABA-dependent expression of stress related transcription factors from the AP2/ERF/DREB family that in turn affect the level of genes regulated by the PYL/PYR/RCAR-PP2C-SnRK2 pathway. These observations suggest that ABA signaling through PYL/PYR/RCAR receptors and SnRK2s kinases is regulated directly and indirectly by the cytoplasmic mRNA decay pathway

PyLasso: a PyMOL plugin to identify lassos

Entanglement in macromolecules is an important phenomenon and a subject of multidisciplinary research. As recently discovered, around 4% of proteins form new entangled motifs, called lassos. Here we present the PyLasso—a PyMOL plugin to identify and analyse properties of lassos in proteins and other (bio)polymers, as well as in other biological, physical and mathematical systems. The PyLasso is a useful tool for all researchers working on modeling of macromolecules, structure prediction, properties of polymers, entanglement in fluids and fields, etc