Seafloor incubation experiments at deep-sea hydrothermal vents reveal distinct biogeographic signatures of autotrophic communities.

The discharge of hydrothermal vents on the seafloor provides energy sources for dynamic and productive ecosystems, which are supported by chemosynthetic microbial populations. These populations use the energy gained by oxidizing the reduced chemicals contained within the vent fluids to fix carbon and support multiple trophic levels. Hydrothermal discharge is ephemeral and chemical composition of such fluids varies over space and time, which can result in geographically distinct microbial communities. To investigate the foundational members of the community, microbial growth chambers were placed within the hydrothermal discharge at Axial Seamount (Juan de Fuca Ridge), Magic Mountain Seamount (Explorer Ridge), and Kama'ehuakanaloa Seamount (Hawai'i hotspot). Campylobacteria were identified within the nascent communities, but different amplicon sequence variants were present at Axial and Kama'ehuakanaloa Seamounts, indicating that geography in addition to the composition of the vent effluent influences microbial community development. Across these vent locations, dissolved iron concentration was the strongest driver of community structure. These results provide insights into nascent microbial community structure and shed light on the development of diverse lithotrophic communities at hydrothermal vents.

A robust regression analysis method to determine the significance of trends in concentrations of heavy metals in UK ambient air and improve network design and emission inventories.

A novel application of the Theil-Sen robust regression method for determining the temporal trends in the concentration of heavy metals in UK ambient air over the period 2005-2020 is presented and compared to other regression methods. We have demonstrated improvements over non-robust methods of regression, proving the ability to tease out trends that are small with respect to the variability of the concentration measurement. The method is used to identify, in general, large and significant trends in the concentrations of Ni, As, Pb and V over the period 2005-2020, either across the UK as a whole or at groupings of site classifications in the UK. These trends have been compared to trends in emission data determined in the same manner. Although the results for most metals provide confidence that the UK metal network of monitoring sites is successful in appropriately capturing changes in emissions, a key finding of this work is the disagreement between trends in measured concentrations and emissions for Cu, Mn and Ni, for which we suggest improvements in future network design. The results also indicate that UK emission data for V should be reviewed, as we propose that the rate of reduction of V emissions is likely to have been overestimated.

Air-quality networks collect environmental DNA with the potential to measure biodiversity at continental scales.

One of the biggest planetary challenges is the accelerating loss of biodiversity threatening ecosystem functioning on a global scale. The WWF Living Planet Report (https://livingplanet.panda.org/) estimates a 69% decline in populations since 1970. The Convention on Biological Diversity and related international treaties ask countries to monitor shifts in community composition and assess rates of species decline to quantify extant biodiversity relative to global targets1. However, quantifying biodiversity is a challenge, and monitoring continual change is impossible at almost any scale due to a lack of standardized data and indicators2,3. A common problem is that the required infrastructure for such global monitoring does not exist. Here, we challenge this notion by analysing environmental DNA (eDNA) captured along with particulate matter by routine ambient air quality monitoring stations in the UK. In our samples, we identified eDNA from >180 vertebrate, arthropod, plant and fungal taxa representative of local biodiversity. We contend that air monitoring networks are in fact gathering eDNA data reflecting local biodiversity on a continental scale, as a result of their routine function. In some regions, air quality samples are stored for decades, presenting the potential for high resolution biodiversity time series. With minimal modification of current protocols, this material provides the best opportunity to date for detailed monitoring of terrestrial biodiversity using an existing, replicated transnational design and it is already in operation.

Globally-distributed microbial eukaryotes exhibit endemism at deep-sea hydrothermal vents.

Single-celled microbial eukaryotes inhabit deep-sea hydrothermal vent environments and play critical ecological roles in the vent-associated microbial food web. 18S rRNA amplicon sequencing of diffuse venting fluids from four geographically- and geochemically-distinct hydrothermal vent fields was applied to investigate community diversity patterns among protistan assemblages. The four vent fields include Axial Seamount at the Juan de Fuca Ridge, Sea Cliff and Apollo at the Gorda Ridge, all in the NE Pacific Ocean, and Piccard and Von Damm at the Mid-Cayman Rise in the Caribbean Sea. We describe species diversity patterns with respect to hydrothermal vent field and sample type, identify putative vent endemic microbial eukaryotes, and test how vent fluid geochemistry may influence microbial community diversity. At a semi-global scale, microbial eukaryotic communities at deep-sea vents were composed of similar proportions of dinoflagellates, ciliates, Rhizaria, and stramenopiles. Individual vent fields supported distinct and highly diverse assemblages of protists that included potentially endemic or novel vent-associated strains. These findings represent a census of deep-sea hydrothermal vent protistan communities. Protistan diversity, which is shaped by the hydrothermal vent environment at a local scale, ultimately influences the vent-associated microbial food web and the broader deep-sea carbon cycle.

Falling nickel concentrations in ambient air in South Wales - 50 years of progress.

Measurement of the composition of ambient air has become increasingly widespread over the last 50 years as the detrimental health effects of some air pollutants have become clearer and requirements for these measurements has been embedded in national and international legislation. The aim of this has been not only to assess exposure of the general population to air pollutants but also to assess the effectiveness of abatement strategies to reduce emissions of these pollutants at source. With a rich industrial heritage, the Swansea Valley (South Wales, UK) has long been associated with the refining and production of metal products, especially nickel. Despite a decline in output during the latter part of the twentieth century there is still sufficient activity to prompt a requirement for targeted air monitoring in the area. This is most important for nickel where there is a local history of measured concentrations exceeding legislative target values. This work demonstrates the effectiveness of nickel emissions abatement strategies over the last 50 years by tracking the falling air concentration of nickel over this period. It also demonstrates how the monitoring network in the Swansea Valley has expanded over this time and become significantly more sensitive to nickel emissions. The data presented represents a significant public health achievement - it is likely that the exposure to nickel in air of the population in the Swansea Valley has decreased more than 100-fold over the last 50 years - and reflects the progress in regulation, industrial efficiency, emissions abatement technology and air quality monitoring science achieved during this period.

Metabolic Strategies Shared by Basement Residents of the Lost City Hydrothermal Field.

Alkaline fluids venting from chimneys of the Lost City hydrothermal field flow from a potentially vast microbial habitat within the seafloor where energy and organic molecules are released by chemical reactions within rocks uplifted from Earth's mantle. In this study, we investigated hydrothermal fluids venting from Lost City chimneys as windows into subseafloor environments where the products of geochemical reactions, such as molecular hydrogen (H2), formate, and methane, may be the only available sources of energy for biological activity. Our deep sequencing of metagenomes and metatranscriptomes from these hydrothermal fluids revealed a few key species of archaea and bacteria that are likely to play critical roles in the subseafloor microbial ecosystem. We identified a population of Thermodesulfovibrionales (belonging to phylum Nitrospirota) as a prevalent sulfate-reducing bacterium that may be responsible for much of the consumption of H2 and sulfate in Lost City fluids. Metagenome-assembled genomes (MAGs) classified as Methanosarcinaceae and Candidatus Bipolaricaulota were also recovered from venting fluids and represent potential methanogenic and acetogenic members of the subseafloor ecosystem. These genomes share novel hydrogenases and formate dehydrogenase-like sequences that may be unique to hydrothermal environments where H2 and formate are much more abundant than carbon dioxide. The results of this study include multiple examples of metabolic strategies that appear to be advantageous in hydrothermal and subsurface alkaline environments where energy and carbon are provided by geochemical reactions. IMPORTANCE The Lost City hydrothermal field is an iconic example of a microbial ecosystem fueled by energy and carbon from Earth's mantle. Uplift of mantle rocks into the seafloor can trigger a process known as serpentinization that releases molecular hydrogen (H2) and creates unusual environmental conditions where simple organic carbon molecules are more stable than dissolved inorganic carbon. This study provides an initial glimpse into the kinds of microbes that live deep within the seafloor where serpentinization takes place, by sampling hydrothermal fluids exiting from the Lost City chimneys. The metabolic strategies that these microbes appear to be using are also shared by microbes that inhabit other sites of serpentinization, including continental subsurface environments and natural springs. Therefore, the results of this study contribute to a broader, interdisciplinary effort to understand the general principles and mechanisms by which serpentinization-associated processes can support life on Earth and perhaps other worlds.

Metabolic Features of Brain Function with Relevance to Clinical Features of Alzheimer and Parkinson Diseases.

Brain metabolism is comprised in Alzheimer's disease (AD) and Parkinson's disease (PD). Since the brain primarily relies on metabolism of glucose, ketone bodies, and amino acids, aspects of these metabolic processes in these disorders-and particularly how these altered metabolic processes are related to oxidative and/or nitrosative stress and the resulting damaged targets-are reviewed in this paper. Greater understanding of the decreased functions in brain metabolism in AD and PD is posited to lead to potentially important therapeutic strategies to address both of these disorders, which cause relatively long-lasting decreased quality of life in patients.

Challenges and policy implications of long-term changes in mass absorption cross-section derived from equivalent black carbon and elemental carbon measurements in London and south-east England in 2014-2019.

Determining the concentration of carbonaceous particles in ambient air is important for climate modelling, source attribution and air quality management. This study presents the difficulties associated with the interpretation of apparent long-term changes in the mass absorption cross section (MAC) of carbonaceous particles in London and south-east England based on equivalent black carbon (eBC) and elemental carbon (EC) measurements between 2014 and 2019. Although these two measurement techniques were used to determine the concentration of carbonaceous aerosols, the concentrations of eBC and EC changed at different rates at all sites, and exhibited different long-term trends. eBC measurements obtained using aethalometer instruments for traffic and urban background sites demonstrated consistent trends, showing decreases in concentrations of up to -12.5% y-1. The EC concentrations showed no change at the urban background location, a similar change to eBC at the traffic site and a significant upward trend of +10% y-1 was observed at the rural site. Despite these differences, the trends in the MAC values decreased at all sites in a similar way, with rates of change from -5.5% y-1 to -10.1% y-1. The different trends and magnitudes of change for the eBC and EC concentrations could lead to uncertainty in quantifying the efficacy of intervention measures and to different conclusions for policy making. This paper provides possible explanations of the observed decrease in MAC values over time.

Seafloor Incubation Experiment with Deep-Sea Hydrothermal Vent Fluid Reveals Effect of Pressure and Lag Time on Autotrophic Microbial Communities.

Depressurization and sample processing delays may impact the outcome of shipboard microbial incubations of samples collected from the deep sea. To address this knowledge gap, we developed a remotely operated vehicle (ROV)-powered incubator instrument to carry out and compare results from in situ and shipboard RNA stable isotope probing (RNA-SIP) experiments to identify the key chemolithoautotrophic microbes and metabolisms in diffuse, low-temperature venting fluids from Axial Seamount. All the incubations showed microbial uptake of labeled bicarbonate primarily by thermophilic autotrophic Epsilonbacteraeota that oxidized hydrogen coupled with nitrate reduction. However, the in situ seafloor incubations showed higher abundances of transcripts annotated for aerobic processes, suggesting that oxygen was lost from the hydrothermal fluid samples prior to shipboard analysis. Furthermore, transcripts for thermal stress proteins such as heat shock chaperones and proteases were significantly more abundant in the shipboard incubations, suggesting that depressurization induced thermal stress in the metabolically active microbes in these incubations. Together, the results indicate that while the autotrophic microbial communities in the shipboard and seafloor experiments behaved similarly, there were distinct differences that provide new insight into the activities of natural microbial assemblages under nearly native conditions in the ocean.IMPORTANCE Diverse microbial communities drive biogeochemical cycles in Earth's ocean, yet studying these organisms and processes is often limited by technological capabilities, especially in the deep ocean. In this study, we used a novel marine microbial incubator instrument capable of in situ experimentation to investigate microbial primary producers at deep-sea hydrothermal vents. We carried out identical stable isotope probing experiments coupled to RNA sequencing both on the seafloor and on the ship to examine thermophilic, microbial autotrophs in venting fluids from an active submarine volcano. Our results indicate that microbial communities were significantly impacted by the effects of depressurization and sample processing delays, with shipboard microbial communities being more stressed than seafloor incubations. Differences in metabolism were also apparent and are likely linked to the chemistry of the fluid at the beginning of the experiment. Microbial experimentation in the natural habitat provides new insights into understanding microbial activities in the ocean.

Safe Sampling Volume determinations of 12 volatile organic compounds on Carboxen 1003, Carbopack-X & Tenax-TA.

Pumped sorbent tube sampling is a well established method for the sampling of volatile organic compounds (VOCs) and semi volatile organic compounds (SVOCs) in ambient, indoor and workplace atmospheres1. Safe sampling volumes and breakthrough volumes have been published for commonly found VOCs on widely used sorbents such as Tenax, however for newer sorbents and less commonly found VOCs there is less robust data. The Safe Sampling Volumes (SSVs) were determined from 15 tests of Retention Volume on 12 VOCs across the 3 sorbents. VOCs tested were: Aldehydes (C5, C6, C8, C9), Ketones (C4, C6), Alcohols (C3, C4), Furan, Limonene, Isoprene and Ethyl Acetate. 12 VOC / sorbent combinations gave SSVs large enough for practical sampling of indoor atmospheres, while SSVs for Furan on Carbopack-X, Isovaleraldehyde on Tenax TA and Methyl Ethyl Ketone on Tenax TA gave SSVs that were too small to be of practical use. This work identifies suitable sorbents and sampling volumes for the complete range of species tested.

Validating the Cyc2 Neutrophilic Iron Oxidation Pathway Using Meta-omics of Zetaproteobacteria Iron Mats at Marine Hydrothermal Vents.

Zetaproteobacteria create extensive iron (Fe) oxide mats at marine hydrothermal vents, making them an ideal model for microbial Fe oxidation at circumneutral pH. Comparison of neutrophilic Fe oxidizer isolate genomes has revealed a hypothetical Fe oxidation pathway, featuring a homolog of the Fe oxidase Cyc2 from Acidithiobacillus ferrooxidans However, Cyc2 function is not well verified in neutrophilic Fe oxidizers, particularly in Fe-oxidizing environments. Toward this, we analyzed genomes and metatranscriptomes of Zetaproteobacteria, using 53 new high-quality metagenome-assembled genomes reconstructed from Fe mats at Mid-Atlantic Ridge, Mariana Backarc, and Loihi Seamount (Hawaii) hydrothermal vents. Phylogenetic analysis demonstrated conservation of Cyc2 sequences among most neutrophilic Fe oxidizers, suggesting a common function. We confirmed the widespread distribution of cyc2 and other model Fe oxidation pathway genes across all represented Zetaproteobacteria lineages. High expression of these genes was observed in diverse Zetaproteobacteria under multiple environmental conditions and in incubations. The putative Fe oxidase gene cyc2 was highly expressed in situ, often as the top expressed gene. The cyc2 gene showed increased expression in Fe(II)-amended incubations, with corresponding increases in carbon fixation and central metabolism gene expression. These results substantiate the Cyc2-based Fe oxidation pathway in neutrophiles and demonstrate its significance in marine Fe-mineralizing environments.IMPORTANCE Iron oxides are important components of our soil, water supplies, and ecosystems, as they sequester nutrients, carbon, and metals. Microorganisms can form iron oxides, but it is unclear whether this is a significant mechanism in the environment. Unlike other major microbial energy metabolisms, there is no marker gene for iron oxidation, hindering our ability to track these microbes. Here, we investigate a promising possible iron oxidation gene, cyc2, in iron-rich hydrothermal vents, where iron-oxidizing microbes dominate. We pieced together diverse Zetaproteobacteria genomes, compared these genomes, and analyzed expression of cyc2 and other hypothetical iron oxidation genes. We show that cyc2 is widespread among iron oxidizers and is highly expressed and potentially regulated, making it a good marker for the capacity for iron oxidation and potentially a marker for activity. These findings will help us understand and potentially quantify the impacts of neutrophilic iron oxidizers in a wide variety of marine and terrestrial environments.

Unique Solid Phase Microextraction Sampler Reveals Distinctive Biogeochemical Profiles among Various Deep-Sea Hydrothermal Vents.

Current methods for biochemical and biogeochemical analysis of the deep-sea hydrothermal vent ecosystems rely on water sample recovery, or in situ analysis using underwater instruments with limited range of analyte detection and limited sensitivity. Even in cases where large quantities of sample are recovered, labile dissolved organic compounds may not be detected due to time delays between sampling and preservation. Here, we present a novel approach for in situ extraction of organic compounds from hydrothermal vent fluids through a unique solid phase microextraction (SPME) sampler. These samplers were deployed to sample effluent of vents on sulphide chimneys, located on Axial Seamount in the North-East Pacific, in the Urashima field on the southern Mariana back-arc, and at the Hafa Adai site in the central Mariana back-arc. Among the compounds that were extracted, a wide range of unique organic compounds, including labile dissolved organic sulfur compounds, were detected through high-resolution LC-MS/MS, among which were biomarkers of anammox bacteria, fungi, and lower animals. This report is the first to show that SPME can contribute to a broader understanding of deep sea ecology and biogeochemical cycles in hydrothermal vent ecosystems.

Concentration trends of metals in ambient air in the UK: a review.

The monitoring of metals in ambient air has been undertaken for over 40 years on a national basis in the UK. During this period, the UK pollution landscape has continued to evolve in terms of emission sources, and the measurement framework for metals in ambient air, the UK Heavy Metals Monitoring Network, has also been subject to significant configuration changes. Therefore, this work provides a timely review of more recent concentration trends in the context of current emission profiles. Overall, throughout this time period, there has been a significant downward trend in the emissions and consequently, the measured concentrations of most metals in UK ambient air. Ambient concentrations were generally found to be well correlated with emission estimates. Analysis of the sensitivity of measured concentrations to emissions suggests that concentrations have fallen faster than the reduction in emission estimates would have predicted at typical median urban background sites.

Active subseafloor microbial communities from Mariana back-arc venting fluids share metabolic strategies across different thermal niches and taxa.

There are many unknowns regarding the distribution, activity, community composition, and metabolic repertoire of microbial communities in the subseafloor of deep-sea hydrothermal vents. Here we provide the first characterization of subseafloor microbial communities from venting fluids along the central Mariana back-arc basin (15.5-18°N), where the slow-spreading rate, depth, and variable geochemistry along the back-arc distinguish it from other spreading centers. Results indicated that diverse Epsilonbacteraeota were abundant across all sites, with a population of high temperature Aquificae restricted to the northern segment. This suggests that differences in subseafloor populations along the back-arc are associated with local geologic setting and resultant geochemistry. Metatranscriptomics coupled to stable isotope probing revealed bacterial carbon fixation linked to hydrogen oxidation, denitrification, and sulfide or thiosulfate oxidation at all sites, regardless of community composition. NanoSIMS (nanoscale secondary ion mass spectrometry) incubations at 80 °C show only a small portion of the microbial community took up bicarbonate, but those autotrophs had the highest overall rates of activity detected across all experiments. By comparison, acetate was more universally utilized to sustain growth, but within a smaller range of activity. Together, results indicate that microbial communities in venting fluids from the Mariana back-arc contain active subseafloor communities reflective of their local conditions with metabolisms commonly shared across geologically disparate spreading centers throughout the ocean.

Fluid geochemistry, local hydrology, and metabolic activity define methanogen community size and composition in deep-sea hydrothermal vents.

The size and biogeochemical impact of the subseafloor biosphere in oceanic crust remain largely unknown due to sampling limitations. We used reactive transport modeling to estimate the size of the subseafloor methanogen population, volume of crust occupied, fluid residence time, and nature of the subsurface mixing zone for two low-temperature hydrothermal vents at Axial Seamount. Monod CH4 production kinetics based on chemostat H2 availability and batch-culture Arrhenius growth kinetics for the hyperthermophile Methanocaldococcus jannaschii and thermophile Methanothermococcus thermolithotrophicus were used to develop and parameterize a reactive transport model, which was constrained by field measurements of H2, CH4, and metagenome methanogen concentration estimates in 20-40 °C hydrothermal fluids. Model results showed that hyperthermophilic methanogens dominate in systems where a narrow flow path geometry is maintained, while thermophilic methanogens dominate in systems where the flow geometry expands. At Axial Seamount, the residence time of fluid below the surface was 29-33 h. Only 1011 methanogenic cells occupying 1.8-18 m3 of ocean crust per m2 of vent seafloor area were needed to produce the observed CH4 anomalies. We show that variations in local geology at diffuse vents can create fluid flow paths that are stable over space and time, harboring persistent and distinct microbial communities.

Spatially distinct, temporally stable microbial populations mediate biogeochemical cycling at and below the seafloor in hydrothermal vent fluids.

At deep-sea hydrothermal vents, microbial communities thrive across geochemical gradients above, at, and below the seafloor. In this study, we determined the gene content and transcription patterns of microbial communities and specific populations to understand the taxonomy and metabolism both spatially and temporally across geochemically different diffuse fluid hydrothermal vents. Vent fluids were examined via metagenomic, metatranscriptomic, genomic binning, and geochemical analyses from Axial Seamount, an active submarine volcano on the Juan de Fuca Ridge in the NE Pacific Ocean, from 2013 to 2015 at three different vents: Anemone, Marker 33, and Marker 113. Results showed that individual vent sites maintained microbial communities and specific populations over time, but with spatially distinct taxonomic, metabolic potential, and gene transcription profiles. The geochemistry and physical structure of each vent both played important roles in shaping the dominant organisms and metabolisms present at each site. Genomic binning identified key populations of SUP05, Aquificales and methanogenic archaea carrying out important transformations of carbon, sulfur, hydrogen, and nitrogen, with groups that appear unique to individual sites. This work highlights the connection between microbial metabolic processes, fluid chemistry, and microbial population dynamics at and below the seafloor and increases understanding of the role of hydrothermal vent microbial communities in deep ocean biogeochemical cycles.

Community Structure of Lithotrophically-Driven Hydrothermal Microbial Mats from the Mariana Arc and Back-Arc.

The Mariana region exhibits a rich array of hydrothermal venting conditions in a complex geological setting, which provides a natural laboratory to study the influence of local environmental conditions on microbial community structure as well as large-scale patterns in microbial biogeography. We used high-throughput amplicon sequencing of the bacterial small subunit (SSU) rRNA gene from 22 microbial mats collected from four hydrothermally active locations along the Mariana Arc and back-arc to explore the structure of lithotrophically-based microbial mat communities. The vent effluent was classified as iron- or sulfur-rich corresponding with two distinct community types, dominated by either Zetaproteobacteria or Epsilonproteobacteria, respectively. The Zetaproteobacterial-based communities had the highest richness and diversity, which supports the hypothesis that Zetaproteobacteria function as ecosystem engineers creating a physical habitat within a chemical environment promoting enhanced microbial diversity. Gammaproteobacteria were also high in abundance within the iron-dominated mats and some likely contribute to primary production. In addition, we also compare sampling scale, showing that bulk sampling of microbial mats yields higher diversity than micro-scale sampling. We present a comprehensive analysis and offer new insights into the community structure and diversity of lithotrophically-driven microbial mats from a hydrothermal region associated with high microbial biodiversity. Our study indicates an important functional role of for the Zetaproteobacteria altering the mat habitat and enhancing community interactions and complexity.

Standardisation of a European measurement method for organic carbon and elemental carbon in ambient air: results of the field trial campaign and the determination of a measurement uncertainty and working range.

The European Committee for Standardisation (CEN) Technical Committee 264 'Air Quality' has recently produced a standard method for the measurements of organic carbon and elemental carbon in PM2.5 within its working group 35 in response to the requirements of European Directive 2008/50/EC. It is expected that this method will be used in future by all Member States making measurements of the carbonaceous content of PM2.5. This paper details the results of a laboratory and field measurement campaign and the statistical analysis performed to validate the standard method, assess its uncertainty and define its working range to provide clarity and confidence in the underpinning science for future users of the method. The statistical analysis showed that the expanded combined uncertainty for transmittance protocol measurements of OC, EC and TC is expected to be below 25%, at the 95% level of confidence, above filter loadings of 2 µg cm-2. An estimation of the detection limit of the method for total carbon was 2 µg cm-2. As a result of the laboratory and field measurement campaign the EUSAAR2 transmittance measurement protocol was chosen as the basis of the standard method EN 16909:2017.

Evidence of the immunomodulatory role of dual PI3K/mTOR inhibitors in transplantation: an experimental study in mice.

The PI3K/mTOR signaling cascade is fundamental in T-cell activation and fate decisions. We showed the distinct regulation of PI3K/mTOR in regulatory and effector T-cells and proposed the potential therapeutic benefit of targeting this pathway to control the balance between effector and regulatory T-cell activities. Substantial adverse effects in long-term clinical usage of rapamycin suggest the use of alternative treatments in restraining effector T-cell function in transplant patients. We hypothesize that dual PI3K/mTOR inhibitors may represent an immunosuppressant alternative. Here we show that dual PI3K/mTOR PI-103 and PKI-587 inhibitors interfered IL-2-dependent responses in T-cells. However, in contrast to the inhibitory effects in non-Treg T-cell proliferation and effector functions, dual inhibitors increased the differentiation, preferential expansion, and suppressor activity of iTregs. Rapamycin, PI-103, and PKI-587 targeted different signaling events and induced different metabolic patterns in primary T-cells. Similar to rapamycin, in vivo administration of PI-103 and PKI-587 controlled effectively the immunological response against allogeneic skin graft. These results characterize specific regulatory mechanisms of dual PI3K/mTOR inhibitors in T-cells and support their potential as a novel therapeutic option in transplantation.

Sorafenib and FH535 in combination act synergistically on hepatocellular carcinoma by targeting cell bioenergetics and mitochondrial function.

Treatment of advanced hepatocellular carcinoma (HCC) remains a challenge due to the high tumor heterogeneity. In the present study, we aim to evaluate the impact of the ß-catenin inhibitor, FH535, alone or in combination with the Ras/Raf/MAPK inhibitor Sorafenib, on the bioenergetics profiles of the HCC cell lines Huh7 and PLC/PRF/5. Single low-dose treatments with FH535 or Sorafenib promoted different effects on mitochondrial respiration and glycolysis in a cell type specific manner. However, the combination of these drugs significantly reduced both mitochondrial respiration and glycolytic rates regardless of the HCC cells. The significant changes in mitochondrial respiration observed in cells treated with the Sorafenib-FH535 combination may correspond to differential targeting of ETC complexes and changes in substrate utilization mediated by each drug. Moreover, the bioenergetics changes and the loss of mitochondrial membrane potential that were evidenced by treatment of HCC cells with the combination of FH535 and Sorafenib, preceded the induction of cell apoptosis. Overall, our results demonstrated that Sorafenib-FH535 drug combination induce the disruption of the bioenergetics of HCC by the simultaneous targeting of mitochondrial respiration and glycolytic flux that leads the synergistic effect on inhibition of cell proliferation. These findings support the therapeutic potential of combinatory FH535-Sorafenib treatment of the HCC heterogeneity by the simultaneous targeting of different molecular pathways.