Allotetraploidization in Brachypodium May Have Led to the Dominance of One Parent’s Metabolome in Germinating Seeds

Seed germination is a complex process during which a mature seed resumes metabolic activity to prepare for seedling growth. In this study, we performed a comparative metabolomic analysis of the embryo and endosperm using the community standard lines of three annual Brachypodium species, i.e., B. distachyon (Bd) and B. stacei (Bs) and their natural allotetraploid B. hybridum (BdBs) that has wider ecological range than the other two species. We explored how far the metabolomic impact of allotetraploidization would be observable as over-lapping changes at 4, 12, and 24 h after imbibition (HAI) with water when germination was initiated. Metabolic changes during germination were more prominent in Brachypodium embryos than in the endosperm. The embryo and endosperm metabolomes of Bs and BdBs were similar, and those of Bd were distinctive. The Bs and BdBs embryos showed increased levels of sugars and the tricarboxylic acid cycle compared to Bd, which could have been indicative of better nutrient mobilization from the endosperm. Bs and BdBs also showed higher oxalate levels that could aid nutrient transfer through altered cellular events. In Brachypodium endosperm, the thick cell wall, in addition to starch, has been suggested to be a source of nutrients to the embryo. Metabolites indicative of sugar metabolism in the endosperm of all three species were not prominent, suggesting that mobilization mostly occurred prior to 4 HAI. Hydroxycinnamic and monolignol changes in Bs and BdBs were consistent with cell wall remodeling that arose following the release of nutrients to the respective embryos. Amino acid changes in both the embryo and endosperm were broadly consistent across the species. Taking our data together, the formation of BdBs may have maintained much of the Bs metabolome in both the embryo and endosperm during the early stages of germination. In the embryo, this conserved Bs metabolome appeared to include an elevated sugar metabolism that played a vital role in germination. If these observations are confirmed in the future with more Brachypodium accessions, it would substantiate the dominance of the Bs metabolome in BdBs allotetraploidization and the use of metabolomics to suggest important adaptive changes

Chromatin signatures at Notch-regulated enhancers reveal large-scale changes in H3K56ac upon activation.

The conserved Notch pathway functions in diverse developmental and disease-related processes, requiring mechanisms to ensure appropriate target selection and gene activation in each context. To investigate the influence of chromatin organisation and dynamics on the response to Notch signalling, we partitioned Drosophila chromatin using histone modifications and established the preferred chromatin conditions for binding of Su(H), the Notch pathway transcription factor. By manipulating activity of a co-operating factor, Lozenge/Runx, we showed that it can help facilitate these conditions. While many histone modifications were unchanged by Su(H) binding or Notch activation, we detected rapid changes in acetylation of H3K56 at Notch-regulated enhancers. This modification extended over large regions, required the histone acetyl-transferase CBP and was independent of transcription. Such rapid changes in H3K56 acetylation appear to be a conserved indicator of enhancer activation as they also occurred at the mammalian Notch-regulated Hey1 gene and at Drosophila ecdysone-regulated genes. This intriguing example of a core histone modification increasing over short timescales may therefore underpin changes in chromatin accessibility needed to promote transcription following signalling activation.This work was supported by a BBSRC project grant [BB/J008842/1] to SJB, BA and SR and by a MRC programme grant [G0800034] to SJB. JL is the recipient of a scholarship from the China Scholarship Council Cambridge.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.15252/embj.20148992

Defining the Cell Wall, Cell Cycle and Chromatin Landmarks in the Responses of Brachypodium distachyon to Salinity

Excess salinity is a major stress that limits crop yields. Here, we used the model grass Brachypodium distachyon (Brachypodium) reference line Bd21 in order to define the key molecular events in the responses to salt during germination. Salt was applied either throughout the germination period (“salt stress”) or only after root emergence (“salt shock”). Germination was affected at 100 mM and root elongation at 75 mM NaCl. The expression of arabinogalactan proteins (AGPs), FLA1, FLA10, FLA11, AGP20 and AGP26, which regulate cell wall expansion (especially FLA11), were mostly induced by the “salt stress” but to a lesser extent by “salt shock”. Cytological assessment using two AGP epitopes, JIM8 and JIM13 indicated that “salt stress” increases the fluorescence signals in rhizodermal and exodermal cell wall. Cell division was suppressed at >75 mM NaCl. The cell cycle genes (CDKB1, CDKB2, CYCA3, CYCB1, WEE1) were induced by “salt stress” in a concentration-dependent manner but not CDKA, CYCA and CYCLIN-D4-1-RELATED. Under “salt shock”, the cell cycle genes were optimally expressed at 100mMNaCl. These changes were consistent with the cell cycle arrest, possibly at the G1 phase. The salt-induced genomic damage was linked with the oxidative events via an increased glutathione accumulation. Histone acetylation and methylation and DNA methylation were visualized by immunofluorescence. Histone H4 acetylation at lysine 5 increased strongly whereas DNA methylation decreased with the application of salt. Taken together, we suggest that salt-induced oxidative stress causes genomic damage but that it also has epigenetic effects, which might modulate the cell cycle and AGP expression gene. Based on these landmarks, we aim to encourage functional genomics studies on the responses of Brachypodium to salt

Migration without interbreeding: Evolutionary history of a highly selfing Mediterranean grass inferred from whole genomes

Wild plant populations show extensive genetic subdivision and are far from the ideal of panmixia which permeates population genetic theory. Understanding the spatial and temporal scale of population structure is therefore fundamental for empirical population genetics –and of interest in itself, as it yields insights into the history and biology of a species. In this study we extend the genomic resources for the wild Mediterranean grass Brachypodium distachyon to investigate the scale of population structure and its underlying history at whole-genome resolution. A total of 86 accessions were sampled at local and regional scales in Italy and France, which closes a conspicuous gap in the collection for this model organism. The analysis of 196 accessions, spanning the Mediterranean from Spain to Iraq, suggests that the interplay of high selfing and seed dispersal rates has shaped genetic structure in B. distachyon. At the continental scale, the evolution in B. distachyon is characterized by the independent expansion of three lineages during the Upper Pleistocene. Today, these lineages may occur on the same meadow yet do not interbreed. At the regional scale, dispersal and selfing interact and maintain high genotypic diversity, thus challenging the textbook notion that selfing in finite populations implies reduced diversity. Our study extends the population genomic resources for B. distachyon and suggests that an important use of this wild plant model is to investigate how selfing and dispersal, two processes typically studied separately, interact in colonizing plant species

Role of co-repressor genomic landscapes in shaping the Notch response.

Repressors are frequently deployed to limit the transcriptional response to signalling pathways. For example, several co-repressors interact directly with the DNA-binding protein CSL and are proposed to keep target genes silenced in the absence of Notch activity. However, the scope of their contributions remains unclear. To investigate co-repressor activity in the context of this well defined signalling pathway, we have analysed the genome-wide binding profile of the best-characterized CSL co-repressor in Drosophila, Hairless, and of a second CSL interacting repressor, SMRTER. As predicted there was significant overlap between Hairless and its CSL DNA-binding partner, both in Kc cells and in wing discs, where they were predominantly found in chromatin with active enhancer marks. However, while the Hairless complex was widely present at some Notch regulated enhancers in the wing disc, no binding was detected at others, indicating that it is not essential for silencing per se. Further analysis of target enhancers confirmed differential requirements for Hairless. SMRTER binding significantly overlapped with Hairless, rather than complementing it, and many enhancers were apparently co-bound by both factors. Our analysis indicates that the actions of Hairless and SMRTER gate enhancers to Notch activity and to Ecdysone signalling respectively, to ensure that the appropriate levels and timing of target gene expression are achieved

Distribution of plantar pressure in healthy controls and patients with type 1 and 2 diabetes

WSTĘP. Celem pracy jest ocena rozkładu podeszwowych nacisków w grupie osób zdrowych i chorych na cukrzycę typu 1 i 2 przy obecności lub braku neuropatii ruchowo-czuciowej. Opisane badania stanowią wstęp do opracowania pierwszego polskiego obuwia profilaktycznego, uwzględniającego odciążenie miejsc wysokiego ryzyka owrzodzenia na stopie. MATERIAŁ I METODY. Przebadano grupę 215 zdrowych osób, 56 osób chorych na cukrzycę typu 1, 61 chorych na cukrzycę typu 2. Badano zaawansowanie przewlekłych powikłań cukrzycy, szczególnie neuropatii, którą oceniano na podstawie skali NDS, NSS i przewodnictwa nerwowego. Pomiar nacisku [N/cm2] wykonano za pomocą systemu Emed-SF V2.1. WYNIKI BADAŃ. Wśród osób zdrowych stwierdzono największe naciski pod 2 (38,8 N/cm2) i 3 głową (33,4 N/cm2) kości śródstopia. Podobne wyniki uzyskano w populacji osób chorych na cukrzycę typu 1. U chorych na cukrzycę typu 2 ciśnienie pod 2 (45,5 N/cm2), 3 (39,6 N/cm2), 4 (31,8 N/cm2) głową kości śródstopia było statystycznie istotnie wyższe w porównaniu z populacją zdrowych osób. Podobnie wysokie ciśnienie stwierdzono pod 3 i 4 głową kości śródstopia w cukrzycy typu 2 powikłanej neuropatią. WNIOSKI. 1. Miejscami największego nacisku u osób zdrowych są: paluch, pięta, 2 i 3 głowa kości śródstopia. 2. U chorych na cukrzycę typu 2 naciski na 2, 3, 4 i 5 głowie kości śródstopia są istotnie statystycznie większe niż u osób zdrowych i chorych na cukrzycę typu 1. 3. U chorych na cukrzycę typu 2 powikłaną neuropatią ruchową i czuciową najwyższe naciski występują na 3 i 4 głowie kości śródstopia i różnią się one istotnie statystycznie od grupy osób zdrowych. 4. Szczególnych zabiegów prewencyjnych w postaci odciążenia główek kości śródstopia wymagają chorzy na cukrzycę typu 2, zwłaszcza powikłaną neuropatią ruchowo-czuciową.OBJECTIVE. To investigate the distribution of plantar pressures in healthy subjects and in patients with type 1 and 2 diabetes with or without sensorimotor neuropathy (SMN). The paper opens a series of studies aiming at the construction of the first Polish prophylactic footwear, which would offload the sites at high risk of plantar foot ulceration. MATERIAL AND METHODS. We studied 215 healthy subjects, 56 patients with type 1 diabetes, and 61 patients with type 2 diabetes. Chronic complications of diabetes were evaluated, especially neuropathy based upon NDS score, NSS score and neural conduction. We used the Emed-SF V2.1 system to measure plantar pressures [N/cm2]. RESULTS. Among the healthy individuals the highest pressures were observed below the second and the third metatarsal head (38,8 N/cm2 and 33,4 N/cm2, respectively). Similar results were found in the group of type 1 diabetes patients. However, the patients with type 2 diabetes mellitus had statistically significant higher pressures below the second, the third, and the fourth metatarsal head when compared with non-diabetic controls (45,5 N/cm2, 39,6 N/cm2, 31,8 N/cm2, respectively). Similar results below the third and fourth metatarsal head were observed in the group of type 2 diabetes patients complicated by diabetic neuropathy. CONCLUSIONS. 1. The highest pressures in healthy subjects were identified under great toe, the second and third metatarsal head. 2. Patients with type 2 diabetes have significantly higher pressures under the second through fifth metatarsal heads as compared with healthy subjects and type 1 diabetics. 3. In patients with type 2 diabetes complicated by SMN the highest pressures are found under the third and fourth metatarsal head, being significantly different from healthy subjects. 4. Special preventive procedures i.e. offloading metatarsal heads are necessary in patients with type 2 diabetes, especially those with concomitant SMN

Retrospektywna analiza skuteczności i bezpieczeństwa leczenia kabazytakselem chorych z rozsianym rakiem gruczołu krokowego opornym na kastrację po niepowodzeniu leczenia docetakselem

Wstęp. Kabazytaksel został zarejestrowany przez FDA i EMEA do leczenia chorych na przerzutowego raka gruczołu krokowego opornego na kastrację (mCRPC) po niepowodzeniu chemioterapii opartej na docetakselu. W okresie od czerwca 2011 do listopada 2013 roku kabazytaksel był dostępny dla polskich pacjentów z mCRPC w ramach programu refundacji chemioterapii niestandardowej. Celem pracy jest retrospektywna analiza danych dotyczących skuteczności oraz bezpieczeństwa terapii kabazytakselem prowadzonej w tym okresie. Materiał i metody. Zebrano retrospektywnie dane 48 chorych na mCRPC, którzy otrzymali kabazytaksel jako chemioterapię drugiej lub trzeciej linii, po niepowodzeniu leczenia docetakselem. Dane dotyczyły: wyjściowej charakterystyki chorych, historii choroby nowotworowej oraz przebiegu leczenia kabazytakselem w zakresie skutków i bezpieczeństwa. Przeżycie wolne od (radiologicznej/klinicznej/biochemicznej) progresji (PFS) i czas całkowitego przeżycia (OS) oceniono przy pomocy metody Kaplana–Meiera; oceniono również odsetki obiektywnych odpowiedzi i korzyści klinicznych. Wyniki. W badanej grupie 48 chorych mediana PFS wyniosła 4,2 (95% CI 3,4–5,1) miesiąca, a OS — 15,1 (95% CI 12,7–17,4) miesiąca. Wartość OS liczona od momentu rozpoczęcia leczenia docetakselem u chorych, którzy otrzymali kabazytaksel w ramach drugiej linii leczenia, wyniósł 28,7 (95% CI 25,3–32,1) miesiąca. Odsetki przeżyć 1-, 2- i 3-letnich wyniosły odpowiednio 65%, 25% i 15%. Ogółem podano 289 cykli kabazytakselu (średnio 6 na chorego). U 41 chorych dokonano oceny odpowiedzi biochemicznej. Redukcję PSA > 50% w porównaniu z wartościami wyjściowymi uzyskano u 19/41 (46%) pacjentów, w tym u 3/41 zaobserwowano obniżenie stężenia PSA > 50% po początkowym wzroście. Zdarzenia niepożądane dotyczyły najczęściej układu krwiotwórczego (26 chorych) i pokarmowego (14 chorych). Zgłoszono 10 poważnych zdarzeń niepożądanych, w tym jeden zgon z powodu ostrej niewydolności nerek. Wniosek. Leczenie kabazytakselem chorych na uogólnionego raka gruczołu krokowego opornego na kastrację po niepowodzeniu leczenia docetakselem stanowi wartościową opcję terapeutyczną, o akceptowalnej toksyczności, w aspekcie stabilizacji stanu klinicznego i możliwości wydłużenia czasu przeżycia

Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors