Metagenomics-driven predictions in Archaea from hydrocarbon-rich Arctic hydrothermal systems: Phylogenetic and metabolic analyses of methane and short-chain alkane-degrading lineages

Postponed access: the file will be accessible after 2023-10-14Metan og hydrokarboner er potente klimagasser som produseres og nedbrytes hovedsakelig biotisk. I havet akkumuleres metan og hydrokarboner i sedimenter og hydrotermiske systemer. Nylige metagenomstudier har utvidet mangfoldet av slektslinjer av arker involvert i metan- og hydrokarbonsyklus. De har vist at metabolske moduler for omsetningsreaksjoner i hydrokarbonsyklus er vanlige i arker og kan forekomme i heterotrofe slektslinjer som bestemmer en mixotrofisk livsstil. Ytterligere metagenomstudier kan bidra til å øke forståelsen av den miljømessige rollen mikroorganismer involvert i omsetning av hydrokarboner har. I løpet av det siste tiåret har hydrokarbon-anrikede hydrotermiske systemer blitt oppdaget langs de arktiske midthavsryggene. I dette studiet har fokuset vært å beskrive det fylogenetiske og metabolske mangfoldet av anaerobe hydrokarbonnedbrytende slektslinjer i disse systemene, hovedsakelig ved å analysere genomer rekonstruert fra metagenomdata. Flere nye slektslinjer av anaerobe metanotrofe arker av typen ANME-1, ble identifisert, inkludert en ny familie. To slektslinjer som kunne oksidere kortkjedede hydrokarboner, henholdsvis etan og propan/butan ble også identifisert. Samtlige av slektslinjene benyttet etablerte metabolismeveier for syntrof anaerob oksidasjon av metan og hydrokarboner. Tidligere ubeskrevne funksjonelle forskjeller ble imidlertid identifisert mellom ulike ANME-1. Basert på tidligere funn i terrestriske hydrotermiske systemer, ble potensialet for metanoksidasjon også evaluert i rekonstruerte genomer av Korarchaeia. Korarchaeia fra marine hydrotermiske system, ble funnet å mangle gen for anaerob oksidasjon av metan. De ble i stedet identifisert som fermenterende mikroorganismer med evne til å benytte sukker og aminosyrer. En komplett Wood-Ljungdahl metabolismevei ble identifisert i dypforgrenede slektslinjer av Korarchaeia og gir sannsynligvis grunnlag for homoacetogenese. Totalt sett har denne studien bekreftet at hydrokarbonrike hydrotermiske systemer ved de arktiske midthavsryggene er tilholdssted for slektslinjer med potensial for hydrokarbonnedbrytning og bidrar til å utvide det fylogenetiske og funksjonelle mangfoldet av slektslinjer som bryter ned hydrokarboner i marine hydrotermiske systemer.Methane and short chain alkanes are potent greenhouse gases generated and degraded mainly biotically. In the ocean, methane and hydrocarbons accumulate in sediments and hydrothermal vents. Recent metagenomic studies have dramatically expanded the diversity of archaeal lineages involved in methane and hydrocarbon cycling. They also have revealed that metabolic modules at the basis of hydrocarbon cycling are relatively conserved and common in Archaea and can occur in heterotrophic lineages determining a mixotrophic lifestyle. Further metagenomic studies can contribute to expand such diversity and describe the environmental role of microorganisms involved in cycling of hydrocarbons. In the last decade, hydrocarbon-enriched hydrothermal vents have been discovered along the Arctic Mid Ocean Ridges (AMOR). This project aimed at identifying lineages of anaerobic hydrocarbon-degraders in these vents and describe their phylogenetic and metabolic diversity, mainly by reconstructing and analyzing metagenome-assembled genomes (MAGs) from various anoxic and actively venting hydrothermal locations. Potential for methane oxidation was also evaluated in MAGs of Korarchaeia since they have been previously proposed as methane oxidizers in terrestrial environments. Overall, several new lineages of anaerobic methanotrophic archaea ANME-1 were identified, including one new family. Two lineages of short-chain alkane oxidizers were found, one an ethane oxidizer and the other a butane/propane oxidizer. All encoded canonical routes for syntrophic anaerobic oxidation of methane and short-chain alkanes. Previously undescribed functional differences were found between ANME-1 lineages. Marine hydrothermal Korarchaeia did not encode genes for anaerobic oxidation of methane. They were instead identified as sugars and amino acids fermenters. Deep-branching lineages of Korarchaeia encoded a complete Wood-Ljungdahl pathway that is likely used reductively as electron sink during fermentation resulting in a homoacetogenic metabolism. Overall, this study confirms that hydrocarbon-rich hydrothermal vents at AMOR host microbial lineages with the potential for degradation of hydrocarbons and contributes to expanding the known phylogenetic and functional diversity of hydrocarbon-degrading lineages in marine hydrothermal systems.Doktorgradsavhandlin

HDAC inhibition is associated to valproic acid induction of early megakaryocytic markers

Valproic acid (VPA), a histone deacetylase inhibitor, causes differentiation in different cell lines and in a cell-specific manner; yet, its effect on megakaryocytic (MK) differentiation has not been studied. We evaluated whether VPA induces MK differentiation in a UT-7 cell line through histone acetylation in the GpIIIa gene region and activation of the ERK pathway. UT-7 cells, derived from megakaryoblastic leukemia, were treated with VPA at various concentrations, and the expression of differentiation markers as well as the gene expression profile was assessed. Flow cytometry, immunoblot analysis, and RT-PCR demonstrated that VPA induced the expression of the early MK markers GpIIIa (CD61) and GpIIb/IIIa (CD41) in a dose-dependent manner. The VPA-treated cells showed hyperacetylation of the histones H3 and H4; in particular, histone acetylation was found to have been associated with CD61 expression, in that the GpIIIa promoter showed H4 hyperacetylation, as demonstrated by the chromatin immunoprecipitation assay. Furthermore, activation of the ERK pathway was involved in VPA-mediated CD61/CD41 expression and in cell adhesion, as demonstrated by using the MEK/ERK inhibitor U0126. In conclusion, the capacity of VPA to commit UT-7 cells to MK differentiation is mediated by its inhibitory action on HDAC and the long-lived activation of ERK1/2

Potential for homoacetogenesis via the Wood–Ljungdahl pathway in Korarchaeia lineages from marine hydrothermal vents

The Wood–Ljungdahl pathway (WLP) is a key metabolic component of acetogenic bacteria where it acts as an electron sink. In Archaea, despite traditionally being linked to methanogenesis, the pathway has been found in several Thermoproteota and Asgardarchaeota lineages. In Bathyarchaeia and Lokiarchaeia, its presence has been linked to a homoacetogenic metabolism. Genomic evidence from marine hydrothermal genomes suggests that lineages of Korarchaeia could also encode the WLP. In this study, we reconstructed 50 Korarchaeia genomes from marine hydrothermal vents along the Arctic Mid-Ocean Ridge, substantially expanding the Korarchaeia class with several taxonomically novel genomes. We identified a complete WLP in several deep-branching lineages, showing that the presence of the WLP is conserved at the root of the Korarchaeia. No methyl-CoM reductases were encoded by genomes with the WLP, indicating that the WLP is not linked to methanogenesis. By assessing the distribution of hydrogenases and membrane complexes for energy conservation, we show that the WLP is likely used as an electron sink in a fermentative homoacetogenic metabolism. Our study confirms previous hypotheses that the WLP has evolved independently from the methanogenic metabolism in Archaea, perhaps due to its propensity to be combined with heterotrophic fermentative metabolisms.publishedVersio

Key role of MEK/ERK pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem-like cell population

The identification of signaling pathways that affect the cancer stem-like phenotype may provide insights into therapeutic targets for combating embryonal rhabdomyosarcoma. The aim of this study was to investigate the role of the MEK/ERK pathway in controlling the cancer stem-like phenotype using a model of rhabdospheres derived from the embryonal rhabdomyosarcoma cell line (RD)

Radioresistance in rhabdomyosarcomas: Much more than a question of dose

Management of rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, frequently accounting the genitourinary tract is complex and requires a multimodal therapy. In particular, as a consequence of the advancement in dose conformity technology, radiation therapy (RT) has now become the standard therapeutic option for patients with RMS. In the clinical practice, dose and timing of RT are adjusted on the basis of patients' risk stratification to reduce late toxicity and side effects on normal tissues. However, despite the substantial improvement in cure rates, local failure and recurrence frequently occur. In this review, we summarize the general principles of the treatment of RMS, focusing on RT, and the main molecular pathways and specific proteins involved into radioresistance in RMS tumors. Specifically, we focused on DNA damage/repair, reactive oxygen species, cancer stem cells, and epigenetic modifications that have been reported in the context of RMS neoplasia in both in vitro and in vivo studies. The precise elucidation of the radioresistance-related molecular mechanisms is of pivotal importance to set up new more effective and tolerable combined therapeutic approaches that can radiosensitize cancer cells to finally ameliorate the overall survival of patients with RMS, especially for the most aggressive subtypes

MS-275 (Entinostat) Promotes Radio-sensitivity in PAX3-FOXO1 Rhabdomyosarcoma cells

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. About 25% of RMS expresses fusion oncoproteins such as PAX3/PAX7‐FOXO1 (fusion‐positive, FP) while fusion‐negative (FN)‐RMS harbors RAS mutations. Radiotherapy (RT) plays a crucial role in local control but metastatic RMS is often radio‐resistant. HDAC inhibitors (HDACi) radio‐sensitize different cancer cells types. Thus, we evaluated MS−275 (Entinostat), a Class I and IV HDACi, in combination with RT on RMS cells in vitro and in vivo. MS−275 reversibly hampered cell survival in vitro in FN‐RMS RD (RASmut) and irreversibly in FP‐RMS RH30 cell lines down‐regulating cyclin A, B, and D1, up‐regulating p21 and p27 and reducing ERKs activity, and c‐Myc expression in RD and PI3K/Akt/mTOR activity and N‐Myc expression in RH30 cells. Further, MS−275 and RT combination reduced colony formation ability of RH30 cells. In both cell lines, co‐treatment increased DNA damage repair inhibition and reactive oxygen species formation, down‐regulated NRF2, SOD, CAT and GPx4 anti‐oxidant genes and improved RT ability to induce G2 growth arrest. MS−275 inhibited in vivo growth of RH30 cells and completely prevented the growth of RT‐unresponsive RH30 xenografts when combined with radiation. Thus, MS−275 could be considered as a radio‐sensitizing agent for the treatment of intrinsically radio‐resistant PAX3‐FOXO1 RMS

Tailoring Hydrothermal Vent Biodiversity Toward Improved Biodiscovery Using a Novel in situ Enrichment Strategy

Deep-sea hydrothermal vents are amongst the most extreme environments on Earth and represent interesting targets for marine bioprospecting and biodiscovery. The microbial communities in hydrothermal vents are often dominated by chemolithoautotrophs utilizing simple chemical compounds, though the full extent of their heterotrophic abilities is still being explored. In the bioprocessing industry, where degradation of complex organic materials often is a major challenge, new microbial solutions are heavily needed. To meet these needs, we have developed novel in situ incubators and tested if deployment of recalcitrant materials from fish farming and wood-pulping industries introduced changes in the microbial community structure in hot marine hydrothermal sediments. The incubation chambers were deployed in sediments at the Bruse vent site located within the Jan Mayen vent field for 1 year, after which the microbial populations in the chambers were profiled by 16S rRNA Ion Torrent amplicon sequencing. A total of 921 operational taxonomic units (OTUs) were assigned into 74 different phyla where differences in community structure were observed depending on the incubated material, chamber depth below the sea floor and/or temperature. A high fraction of putative heterotrophic microbial lineages related to cultivated members within the Thermotogales were observed. However, considerable fractions of previously uncultivated and novel Thermotogales and Bacteroidetes were also identified. Moreover, several novel lineages (e.g., members within the DPANN superphylum, unidentified archaeal lineages, unclassified Thermoplasmatales and Candidatus division BRC-1 bacterium) of as-yet uncultivated thermophilic archaea and bacteria were identified. Overall, our data illustrate that amendment of hydrothermal vent communities by in situ incubation of biomass induces shifts in community structure toward increased fractions of heterotrophic microorganisms. The technologies utilized here could aid in subsequent metagenomics-based enzyme discovery for diverse industries.publishedVersio

The botanical drug PBI-05204, a supercritical CO2 extract of Nerium oleander, sensitizes alveolar and embryonal rhabdomyosarcoma to radiotherapy in vitro and in vivo

: Treatment of rhabdomyosarcoma (RMS), the most common a soft tissue sarcoma in childhood, provides intensive multimodal therapy, with radiotherapy (RT) playing a critical role for local tumor control. However, since RMS efficiently activates mechanisms of resistance to therapies, despite improvements, the prognosis remains still largely unsatisfactory, mainly in RMS expressing chimeric oncoproteins PAX3/PAX7-FOXO1, and fusion-positive (FP)-RMS. Cardiac glycosides (CGs), plant-derived steroid-like compounds with a selective inhibitory activity of the Na+/K+-ATPase pump (NKA), have shown antitumor and radio-sensitizing properties. Herein, the therapeutic properties of PBI-05204, an extract from Nerium oleander containing the CG oleandrin already studied in phase I and II clinical trials for cancer patients, were investigated, in vitro and in vivo, against FN- and FP-RMS cancer models. PBI-05204 induced growth arrest in a concentration dependent manner, with FP-RMS being more sensitive than FN-RMS, by differently regulating cell cycle regulators and commonly upregulating cell cycle inhibitors p21Waf1/Cip1 and p27Cip1/Kip1. Furthermore, PBI-05204 concomitantly induced cell death on both RMS types and senescence in FN-RMS. Notably, PBI-05204 counteracted in vitro migration and invasion abilities and suppressed the formation of spheroids enriched in CD133+ cancer stem cells (CSCs). PBI-05204 sensitized both cell types to RT by improving the ability of RT to induce G2 growth arrest and counteracting the RT-induced activation of both Non-Homologous End-Joining and homologous recombination DSBs repair pathways. Finally, the antitumor and radio-sensitizing proprieties of PBI-05204 were confirmed in vivo. Notably, both in vitro and in vivo evidence confirmed the higher sensitivity to PBI-05204 of FP-RMS. Thus, PBI-05204 represents a valid radio-sensitizing agent for the treatment of RMS, including the intrinsically radio-resistant FP-RMS

RV Kronprins Håkon (cruise no. 2019708) Longyearbyen – Longyearbyen 19.09. – 16.10.2019

The HACON cruise is a major component of the FRINATEK HACON project, which aims at investigating the role of the Gakkel Ridge and Arctic Ocean in biological connectivity amongst ocean basins and global biogeography of chemosynthetic ecosystems. The HACON study area is centered in the Aurora seamount and Aurora vent field

Hot Vents Beneath an Icy Ocean: The Aurora Vent Field, Gakkel Ridge, Revealed

Evidence of hydrothermal venting on the ultra-slow spreading Gakkel Ridge in the Central Arctic Ocean has been available since 2001, with first visual evidence of black smokers on the Aurora Vent Field obtained in 2014. But it was not until 2021 that the first ever remotely operated vehicle (ROV) dives to hydrothermal vents under permanent ice cover in the Arctic were conducted, enabling the collection of vent fluids, rocks, microbes, and fauna. In this paper, we present the methods employed for deep-sea ROV operations under drifting ice. We also provide the first description of the Aurora Vent Field, which includes three actively venting black smokers and diffuse flow on the Aurora mound at ~3,888 m depth on the southern part of the Gakkel Ridge (82.5°N). The biological communities are dominated by a new species of cocculinid limpet, two small gastropods, and a melitid amphipod. The ongoing analyses of Aurora Vent Field samples will contribute to positioning the Gakkel Ridge hydrothermal vents in the global biogeographic puzzle of hydrothermal vents