Identification of rootstock-specific miRNAs in apple tree

Wydział BiologiiCząsteczki mikroRNA (miRNA) odgrywają niezwykle istotną rolę w regulacji ekspresji genów u roślin i zwierząt poprzez wyciszanie ekspresji transkryptów docelowych. Dla jabłoni (Malus domestica) jest obecnie znanych i dostępnych w bazie „miRBase” 206 cząsteczek miRNA. Identyfikacja nowych cząsteczek, jak również poznawanie funkcji już istniejących może przyczynić się do ich praktycznego wykorzystania w celu lepszej ochrony roślin przed patogenami. Celem niniejszej pracy było zidentyfikowanie cząsteczek miRNA jabłoni odmiany „Gala” i określenie ich zróżnicowanej ekspresji w zależności od podkładki. Zwrócono szczególną uwagę na miRNA zaangażowane w kształtowanie odporności na zarazę ogniową, chorobę wywoływaną przez bakterię Erwinia amylovora. W badaniach opierano się głównie na metodach głębokiego sekwencjonowania i analizach bioinformatycznych, których wyniki potwierdzano przy użyciu metod eksperymentalnych. Materiałem badawczym były drzewa odmiany „Gala” rosnące na podkładkach o różnej odporności na zarazę ogniową (M.27, B.9, M.111 i G.30). W pierwszym etapie pracy, poprzez porównanie ekspresji miRNA zidentyfikowanych w zdrowych drzewach, skoncentrowano się na wytypowaniu cząsteczek charakterystycznych dla poszczególnych podkładek. Następnie zidentyfikowano transkrypty docelowe dla miRNA oraz przeanalizowano potencjalny związek pomiędzy profilem ekspresji miRNA a odpornością jabłoni na zarazę ogniową. Spośród dwustu pięćdziesięciu dwóch zidentyfikowanych w tej części pracy cząsteczek, biorąc pod uwagę wszystkie późniejsze wyniki, wytypowano cztery cząsteczki potencjalnie zaangażowane w kształtowanie oporności na zarazę ogniową: mdm-miR535b,c, mdm-miR169a, mdm-miR167b-g oraz mdm-miR168a,b. Celem drugiego etapu pracy było zidentyfikowanie miRNA zaangażowanych w odpowiedź jabłoni na zakażenie bakteryjne poprzez porównanie poziomów ekspresji miRNA pochodzących z drzew zakażonych i niezakażonych bakteriami E amylovora. W tej części pracy wytypowano trzy cząsteczki wykazujące zróżnicowaną ekspresję w odpowiedzi na zakażenie bakteryjne: mdm-miR168a,b, mdm-miR194C i mdm-miR1392C. Dodatkowo, analiza transkryptów docelowych wymienionych cząsteczek potwierdziła ich potencjalną rolę w odpowiedzi na stres biotyczny. Przeprowadzone badania znacznie poszerzyły katalog znanych dla jabłoni cząsteczek miRNA oraz pozwoliły wysnuć hipotezę o udziale cząsteczki mdm-miR535b,c i noduliny w reakcjach obronnych jabłoni na zakażenie zarazą ogniową. Ponadto przyczyniły się do lepszego poznania funkcji innych cząsteczek miRNA, zwłaszcza w odniesieniu do choroby jaką jest zaraza ogniowa.Micro RNAs (miRNAs) play crucial role in gene expression regulation in plants and animals through the silencing mechanism of target genes. Currently, there are 206 miRNA records for apple tree (Malus domestica) available in the miRBase database. The identification of novel miRNAs, as well as discovery of functions of already known molecules may help in developing practical applications of miRNAs in order to protect plants against pathogenes. The main goal of this research was to identify miRNAs from “Gala” apple and to characterize rootstock-specific miRNA expression. Particularly, miRNAs involved in fire blight resistance, disease caused by Erwinia amylovora, were analyzed and described. The project methodology was based on Next Generation Sequencing and bioinformatics analyses followed by the experimental validation. The plant material was “Gala” apple scions grafted on four rootstocks with various fire blight resistance (M.27, B.9, M.111 and G.30). In the first part of the project, through the comparison of miRNA expression profiles in non-inoculated scions, rootstock-dependent miRNAs were indicated. Then, miRNA targets were identified and the relation between miRNA expression and fire blight resistance was analyzed. Out of 252 newly identified miRNAs, taking into consideration also following results, four miRNAs were indicated as potentially involved in fire blight resistance of apple: mdm-miR535b,c, mdm-miR169a, mdm-miR167b-g and mdm-miR168a,b. The goal of the second part of the project was the identification of miRNAs involved in apple response to bacterial infection through the comparison of miRNA expression levels from E. amylovora infected and non-infected trees. Three miRNAs showing differential expression in response to bacterial infection were selected: mdm-miR168a,b, mdm-miR194C i mdm-miR1392C. Additional analysis of their targets confirmed their role in response to biotic stress. Performed analyses significantly extended apple miRNA repertoire and allowed to create the hypothesis that mdm-miR535b,c and nodulins are involved in apple defense mechanisms in response to fire blight infection. Additionally, the results shed light on other miRNA functions, especially in relation to fire blight disease

Comparing Mfd- and UvrD-dependent models of transcription coupled DNA repair in live Escherichia coli using single-molecule tracking

During transcription-coupled DNA repair (TCR) the detection of DNA damage and initiation of nucleotide excision repair (NER) is performed by translocating RNA polymerases (RNAP), which are arrested upon encountering bulky DNA lesions. Two opposing models of the subsequent steps of TCR in bacteria exist. In the first model, stalled RNAPs are removed from the damage site by recruitment of Mfd which dislodges RNAP by pushing it forwards before recruitment of UvrA and UvrB. In the second model, UvrD helicase backtracks RNAP from the lesion site. Recent studies have proposed that both UvrD and UvrA continuously associate with RNAP before damage occurs, which forms the primary damage sensor for NER. To test these two models of TCR in living E. coli, we applied super-resolution microscopy (PALM) combined with single particle tracking to directly measure the mobility and recruitment of Mfd, UvrD, UvrA, and UvrB to DNA during ultraviolet-induced DNA damage. The intracellular mobilities of NER proteins in the absence of DNA damage showed that most UvrA molecules could in principle be complexed with RNAP, however, this was not the case for UvrD. Upon DNA damage, Mfd recruitment to DNA was independent of the presence of UvrA, in agreement with its role upstream of this protein in the TCR pathway. In contrast, UvrD recruitment to DNA was strongly dependent on the presence of UvrA. Inhibiting transcription with rifampicin abolished Mfd DNA-recruitment following DNA damage, whereas significant UvrD, UvrA, and UvrB recruitment remained, consistent with a UvrD and UvrA performing their NER functions independently of transcribing RNAP. Together, although we find that up to ∼8 UvrD-RNAP-UvrA complexes per cell could potentially form in the absence of DNA damage, our live-cell data is not consistent with this complex being the primary DNA damage sensor for NER

COVID-19: Specific and Non-Specific Clinical Manifestations and Symptoms: The Current State of Knowledge

Coronavirus disease 2019 (COVID-19), due to the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has become an epidemiological threat and a worldwide concern. SARS-CoV-2 has spread to 210 countries worldwide and more than 6,500,000 confirmed cases and 384,643 deaths have been reported, while the number of both confirmed and fatal cases is continually increasing. COVID-19 is a viral disease that can affect every age group-from infants to the elderly-resulting in a wide spectrum of various clinical manifestations. COVID-19 might present different degrees of severity-from mild or even asymptomatic carriers, even to fatal cases. The most common complications include pneumonia and acute respiratory distress syndrome. Fever, dry cough, muscle weakness, and chest pain are the most prevalent and typical symptoms of COVID-19. However, patients might also present atypical symptoms that can occur alone, which might indicate the possible SARS-CoV-2 infection. The aim of this paper is to review and summarize all of the findings regarding clinical manifestations of COVID-19 patients, which include respiratory, neurological, olfactory and gustatory, gastrointestinal, ophthalmic, dermatological, cardiac, and rheumatologic manifestations, as well as specific symptoms in pediatric patients

The eye wipes with polyhexanide (HexaClean) in preoperative prophylaxis of cataract surgery

Background: Progressively increasing number of eye surgeries forces the development of simple-to-use, effective methods to reduce the risk of postoperative endophthalmitis. We wondered whether the eyelid margin wipes containing 0.1% polyhexamethylene biguanide (PHMB) (HexaClean, VERCO, Poland) influence the reduction of bacterial flora localized in the conjunctival sac and whether it can be used for prophylaxis before the cataract surgery. Material and methods: 95 patients before the cataract surgery were included in the study. The conjunctival swab was collected from patients twice — before using eye wipes and after 5 days of eye wipes usage. The swabs were plated on microbiological enriched media and incubated under aerobic and microaerophilic conditions for 24–48 hours at 35°C ± 2°C. Then the identification of microorganisms was carried out using classic microbiological methods and tests. Results: Bacterial strains were isolated from the conjunctival sac in 84% of patients before using the eye wipes. The largest group of isolated pathogens was Gram-positive cocci, and these were mainly methicillin-sensitive and methicillin-resistant coagulase-negative staphylococci, which accounted for 72% of isolated strains. When the eye wipes were used, the bacterial flora was eliminated from the conjunctival sac in 54% of patients. A reduction in isolated strains and decreased variety of bacteria was observed in another 22% of patients. Conclusion: These results indicate that the application of eyelid wipes with polyhexanide reduces a significant amount of the conjunctival sac microbiota, which may prevent inflammation after cataract surgery

The evaluation of the inflammatory status and systemic antioxidant-oxidant balance of women with breast cancer during adjuvant chemotherapy

Background: Chemotherapy may cause systemic inflammation. Therefore, reliable markers monitoring inflammation during cancer treatment are intensively investigated. In our study, we analyzed the concentration of high-sensitivity C-reactive protein (hs-CRP) and selected oxidative stress markers, such as malondialdehyde (MDA), glutathione peroxidase activity (GPx), and total antioxidant capacity (TAC), in breast cancer women before and during adjuvant chemotherapy. Materials and methods: The study included 90 women with breast cancer stratified according to clinicopathological and anthropometric features. Blood samples were taken before and after two cycles of adjuvant chemotherapy. Results: During adjuvant chemotherapy, a significant increase in hs-CRP concentration was noticed in the entire group of patients with breast cancer. After division into appropriate groups, a twofold increase in hs-CRP concentration was particularly observed in patients not expressing steroid hormone receptors and those without metastases in regional lymph nodes. A significant rise in hs-CRP was observed in patients with smaller tumor sizes (2 cm ≤) and with a lower stage of disease [I–IIA according to the tumor–node–metastasis (TNM) classification]. Adjuvant chemotherapy resulted in a significant decrease in GPx activity, especially in patients diagnosed with larger (> 2 cm) and more advanced tumors (IIB–IIIC according to the TNM classification), without metastasis in regional lymph nodes, and without HER-2 expression. A significant decrease in glutathione peroxidase (GPx) activity during adjuvant chemotherapy was also observed in patients with abnormal body mass index (BMI) and body fat content. TAC and MDA values remained unchanged in the entire group of patients and individual subgroups during adjuvant chemotherapy. Conclusion: Our study showed that adjuvant chemotherapy causes systemic inflammation, manifested by increased hs-CRP and altered markers of oxidative stress in the blood of breast cancer patients. The severity of inflammatory processes during adjuvant chemotherapy may depend on specific characteristics of breast cancer and body composition

Calmodulin as Ca2+-Dependent Interactor of FTO Dioxygenase

FTO is an N(6)-methyladenosine demethylase removing methyl groups from nucleic acids. Several studies indicate the creation of FTO complexes with other proteins. Here, we looked for regulatory proteins recognizing parts of the FTO dioxygenase region. In the Calmodulin (CaM) Target Database, we found the FTO C-domain potentially binding CaM, and we proved this finding experimentally. The interaction was Ca(2+)-dependent but independent on FTO phosphorylation. We found that FTO–CaM interaction essentially influences calcium-binding loops in CaM, indicating the presence of two peptide populations—exchanging as CaM alone and differently, suggesting that only one part of CaM interacts with FTO, and the other one reminds free. The modeling of FTO–CaM interaction showed its stable structure when the half of the CaM molecule saturated with Ca(2+) interacts with the FTO C-domain, whereas the other part is disconnected. The presented data indicate calmodulin as a new FTO interactor and support engagement of the FTO protein in calcium signaling pathways

A global effort to dissect the human genetic basis of resistance to SARS-CoV-2 infection

SARS-CoV-2 infections display tremendous interindividual variability, ranging from asymptomatic infections to life-threatening disease. Inborn errors of, and autoantibodies directed against, type I interferons (IFNs) account for about 20% of critical COVID-19 cases among SARS-CoV-2-infected individuals. By contrast, the genetic and immunological determinants of resistance to infection per se remain unknown. Following the discovery that autosomal recessive deficiency in the DARC chemokine receptor confers resistance to Plasmodium vivax, autosomal recessive deficiencies of chemokine receptor 5 (CCR5) and the enzyme FUT2 were shown to underlie resistance to HIV-1 and noroviruses, respectively. Along the same lines, we propose a strategy for identifying, recruiting, and genetically analyzing individuals who are naturally resistant to SARS-CoV-2 infection.The Laboratory of Human Genetics of Infectious Diseases is supported by the National Institutes of Health (NIH) (R01AI088364), the National Center for Advancing Translational Sciences (NCATS), NIH Clinical and Translational Science Award (CTSA) program (UL1TR001866), a Fast Grant from Emergent Ventures, Mercatus Center at George Mason University, the Yale Center for Mendelian Genomics and the GSP Coordinating Center funded by the National Human Genome Research Institute (NHGRI) (UM1HG006504 and U24HG008956), the Fisher Center for Alzheimer’s Research Foundation, the Meyer Foundation, the French National Research Agency (ANR) under the Investments for the Future program (ANR-10-IAHU-01), the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID), the French Foundation for Medical Research (FRM) (EQU201903007798), the FRM and ANR GENCOVID project (ANR-20-COVI-0003), ANRS-COV05, the Fondation du Souffle, the Square Foundation, Grandir - Fonds de solidarité pour l’enfance, the SCOR Corporate Foundation for Science, the Howard Hughes Medical Institute, the Rockefeller University, the St. Giles Foundation, Institut National de la Santé et de la Recherche Médicale (INSERM), and the University of Paris. E.A. is supported by research grants from the European Commission’s Horizon 2020 research and innovation program (IMMUNAID, grant no. 779295, CURE, grant no. 767015 and TO_AITION grant no. 848146) and the Hellenic Foundation for Research and Innovation (INTERFLU, no. 1574). C.O.F. is supported in part by the Science Foundation Ireland COVID-19 Program. G.N. is supported by a grant awarded to Regione Lazio (Research Group Projects 2020) no. A0375-2020-36663, GecoBiomark. A.P. is supported in part by the Horizon 2020 program under grant no. 824110 (EasiGenomics grant no. COVID-19/PID12342) and the CERCA Program/Generalitat de Catalunya. H.S. is supported in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. A.S. is supported in part by the European Union’s Horizon 2020 research and innovation program (Marie Sklodowska-Curie grant no. 789645)

Exome-wide association study to identify rare variants influencing COVID-19 outcomes : Results from the Host Genetics Initiative

Publisher Copyright: Copyright: © 2022 Butler-Laporte et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Host genetics is a key determinant of COVID-19 outcomes. Previously, the COVID-19 Host Genetics Initiative genome-wide association study used common variants to identify multiple loci associated with COVID-19 outcomes. However, variants with the largest impact on COVID-19 outcomes are expected to be rare in the population. Hence, studying rare variants may provide additional insights into disease susceptibility and pathogenesis, thereby informing therapeutics development. Here, we combined whole-exome and whole-genome sequencing from 21 cohorts across 12 countries and performed rare variant exome-wide burden analyses for COVID-19 outcomes. In an analysis of 5,085 severe disease cases and 571,737 controls, we observed that carrying a rare deleterious variant in the SARS-CoV-2 sensor toll-like receptor TLR7 (on chromosome X) was associated with a 5.3-fold increase in severe disease (95% CI: 2.75–10.05, p = 5.41x10-7). This association was consistent across sexes. These results further support TLR7 as a genetic determinant of severe disease and suggest that larger studies on rare variants influencing COVID-19 outcomes could provide additional insights.Peer reviewe