Functional diversity of microbial eukaryotes in a meromictic lake: Coupling between metatranscriptomic and a trait-based approach.

The advent of high-throughput sequencing has led to the discovery of a considerable diversity of microbial eukaryotes in aquatic ecosystems, nevertheless, their function and contribution to the trophic food web functioning remain poorly characterized especially in freshwater ecosystems. Based on metabarcoding data obtained from a meromictic lake ecosystem (Pavin, France), we performed a morpho-physio-phenological traits-based approach to infer functional groups of microbial eukaryotes. Metatranscriptomic data were also analysed to assess the metabolic potential of these groups across the diel cycle, size fraction, sampling depth, and periods. Our analysis highlights a huge microbial eukaryotic diversity in the monimolimnion characterized by numerous saprotrophs expressing transcripts related to sulfur and nitrate metabolism as well as dissolved and particulate organic matter degradation. We also describe strong seasonal variations of microbial eukaryotes in the mixolimnion, especially for parasites and mixoplankton. It appears that the water mixing (occurring during spring and autumn) which benefits photosynthetic host communities also promotes parasitic fungi dissemination and over-expression of genes involved in the zoospore phototaxis and stage transition in the parasitic cycle. Mixoplanktonic haptophytes over-expressing photosynthesis-, endocytosis- and phagosome-linked genes under nutrient limitation also suggest that phagotrophy may provide them an advantage over non-phagotrophic phytoplankton.

A practical guide to separate and concentrate ALNs and femtoplankton entities.

The development of new technologies of microscopy, flow cytometry and genomics has allowed a profound reconsideration of the diversity and ecological role of femtoplankton entities (i.e., viruses, vesicles, aster like nanoparticles -ALNs-). Among these, the discovery of ALNs, raise serious questions about their exact nature and their biological and environmental roles. The elaboration of a practical guide for the concentration and separation of femtoplankton entities, including ALNs, is necessary for a better understanding of their diversity, ontogeny, and ecology. Here, we propose a step-by-step procedure for the enrichment and isolation of femtoplankton entities and prokaryotes. The established protocol couples tangential flow filtration to differential centrifugation, leading to differentiate enriched samples (with different target entity contents), usable as a matrix for sorting by flow cytometry. All entities were identified, characterized and counted by transmission electron microscopy and flow cytometry. The procedure allows an efficient detection, concentration and separation of femtoplankton entities (up to purity rate of 92, 67, 81 and 85% for virus like particles, vesicles, prokaryotes and ALNs, respectively), and different morphotypes of ALNs into different fractions (up to 51, 72, 52, 40 and 79% of total ALNs for 20-, 11-, budding 11-, 5-10- and 4-armed ALNs, respectively).

Variable impact of geochemical gradients on the functional potential of bacteria, archaea, and phages from the permanently stratified Lac Pavin.

BACKGROUND: Permanently stratified lakes contain diverse microbial communities that vary with depth and so serve as useful models for studying the relationships between microbial community structure and geochemistry. Recent work has shown that these lakes can also harbor numerous bacteria and archaea from novel lineages, including those from the Candidate Phyla Radiation (CPR). However, the extent to which geochemical stratification differentially impacts carbon metabolism and overall genetic potential in CPR bacteria compared to other organisms is not well defined. RESULTS: Here, we determine the distribution of microbial lineages along an oxygen gradient in Lac Pavin, a deep, stratified lake in central France, and examine the influence of this gradient on their metabolism. Genome-based analyses revealed an enrichment of distinct C1 and CO2 fixation pathways in the oxic lake interface and anoxic zone/sediments, suggesting that oxygen likely plays a role in structuring metabolic strategies in non-CPR bacteria and archaea. Notably, we find that the oxidation of methane and its byproducts is largely spatially separated from methane production, which is mediated by diverse communities of sediment methanogens that vary on the centimeter scale. In contrast, we detected evidence for RuBisCO throughout the water column and sediments, including form II/III and form III-related enzymes encoded by CPR bacteria in the water column and DPANN archaea in the sediments. On the whole, though, CPR bacteria and phages did not show strong signals of gene content differentiation by depth, despite the fact that distinct species groups populate different lake and sediment compartments. CONCLUSIONS: Overall, our analyses suggest that environmental gradients in Lac Pavin select for capacities of CPR bacteria and phages to a lesser extent than for other bacteria and archaea. This may be due to the fact that selection in the former groups is indirect and depends primarily on host characteristics. Video Abstract.

Occurrence and Seasonal Dynamics of ALNs in Freshwater Lakes Are Influenced by Their Biological Environment.

Aster-like nanoparticles (ALNs) are femtoentities, recently discovered in different aquatic environments, whose intrinsic nature and ecological features remain to be determined. In this study, we investigate the in situ temporal dynamics of ALNs during 1 year in 3 different lakes, in relation to the physico-chemical and biological environment. ALN abundances in investigated lakes showed a marked seasonal dynamic (from no detectable to 4.28 ± 0.75 × 106 ALNs mL-1), with characteristic peaks in spring. We recorded a correlation between ALNs and some prokaryotic phyla suggesting a broad and non-specific relationship. From their seasonal dynamics and potential link with prokaryotes, we conclude that ALNs represent an important ecological actor in the functioning of aquatic ecosystems.

Long-Term Incubation of Lake Water Enables Genomic Sampling of Consortia Involving Planctomycetes and Candidate Phyla Radiation Bacteria.

Microbial communities in lakes can profoundly impact biogeochemical processes through their individual activities and collective interactions. However, the complexity of these communities poses challenges, particularly for studying rare organisms such as Candidate Phyla Radiation bacteria (CPR) and enigmatic entities such as aster-like nanoparticles (ALNs). Here, a reactor was inoculated with water from Lake Fargette, France, and maintained under dark conditions at 4°C for 31 months and enriched for ALNs, diverse Planctomycetes, and CPR bacteria. We reconstructed draft genomes and predicted metabolic traits for 12 diverse Planctomycetes and 9 CPR bacteria, some of which are likely representatives of undescribed families or genera. One CPR genome representing the little-studied lineage "Candidatus Peribacter" was curated to completion (1.239 Mbp) and unexpectedly encodes the full gluconeogenesis pathway. Metatranscriptomic data indicate that some planctomycetes and CPR bacteria were active under the culture conditions, accounting for ∼30% and ∼1% of RNA reads mapping to the genome set, respectively. We also reconstructed genomes and obtained transmission electron microscope images for numerous viruses, including one with a >300-kbp genome and several predicted to infect Planctomycetes. Together, our analyses suggest that freshwater Planctomycetes are central players in a subsystem that includes ALNs, symbiotic CPR bacteria, and viruses. IMPORTANCE Laboratory incubations of natural microbial communities can aid in the study of member organisms and their networks of interaction. This is particularly important for understudied lineages for which key elements of basic biology are still emerging. Using genomics and microscopy, we found that members of the bacterial lineage Planctomycetes may be central players in a subset of a freshwater lake microbiome that includes other bacteria, archaea, viruses, and mysterious entities, called aster-like nanoparticles (ALNs), whose origin is unknown. Our results help constrain the possible origins of ALNs and provide insight into possible interactions within a complex lake ecosystem.

Femtoplankton: What's New?

Since the discovery of high abundances of virus-like particles in aquatic environment, emergence of new analytical methods in microscopy and molecular biology has allowed significant advances in the characterization of the femtoplankton, i.e., floating entities filterable on a 0.2 µm pore size filter. The successive evidences in the last decade (2010-2020) of high abundances of biomimetic mineral-organic particles, extracellular vesicles, CPR/DPANN (Candidate phyla radiation/Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota and Nanohaloarchaeota), and very recently of aster-like nanoparticles (ALNs), show that aquatic ecosystems form a huge reservoir of unidentified and overlooked femtoplankton entities. The purpose of this review is to highlight this unsuspected diversity. Herein, we focus on the origin, composition and the ecological potentials of organic femtoplankton entities. Particular emphasis is given to the most recently discovered ALNs. All the entities described are displayed in an evolutionary context along a continuum of complexity, from minerals to cell-like living entities.

Trophic Conditions Influence Widespread Distribution of Aster-Like Nanoparticles Within Aquatic Environments.

Aster-like nanoparticles (ALNs) are newly described femto-entities. Their ecology (e.g., geographic distribution, spatial dynamic, preferences, forcing factors) is still unknown. Here, we report that these entities, which have largely been ignored until now, can develop or maintain themselves in most aquatic environments in the Loire River catchment, France. We observed a significant influence of the trophic state on ALN ecological distributions. A positive relationship between prokaryotic abundance and ALN (r2 = 0.72, p < 0.01) has been identified, but its exact nature remains to be clarified. Combined with their ubiquitous distribution and high abundances (up to 7.9 × 106 ALNs mL-1) recorded in our samples, this probably makes ALNs an overlooked functional component in aquatic ecosystems.

Hormonal Therapy Resistance and Breast Cancer: Involvement of Adipocytes and Leptin.

Obesity, a recognized risk factor for breast cancer in postmenopausal women, is associated with higher mortality rates regardless of menopausal status, which could in part be explained by therapeutic escape. Indeed, adipose microenvironment has been described to influence the efficiency of chemo- and hormonal therapies. Residual cancer stem cells could also have a key role in this process. To understand the mechanisms involved in the reduced efficacy of hormonal therapy on breast cancer cells in the presence of adipose secretome, human adipose stem cells (hMAD cell line) differentiated into mature adipocytes were co-cultured with mammary breast cancer cells and treated with hormonal therapies (tamoxifen, fulvestrant). Proliferation and apoptosis were measured (fluorescence test, impedancemetry, cytometry) and the gene expression profile was evaluated. Cancer stem cells were isolated from mammospheres made from MCF-7. The impact of chemo- and hormonal therapies and leptin was evaluated in this population. hMAD-differentiated mature adipocytes and their secretions were able to increase mammary cancer cell proliferation and to suppress the antiproliferative effect of tamoxifen, confirming previous data and validating our model. Apoptosis and cell cycle did not seem to be involved in this process. The evaluation of gene expression profiles suggested that STAT3 could be a possible target. On the contrary, leptin did not seem to be involved. The study of isolated cancer stem cells revealed that their proliferation was stimulated in the presence of anticancer therapies (tamoxifen, fulvestrant, doxorubicine) and leptin. Our study confirmed the role of adipocytes and their secretome, but above all, the role of communication between adipose and cancer cells in interfering with the efficiency of hormonal therapy. Among the pathophysiological mechanisms involved, leptin does not seem to interfere with the estrogenic pathway but seems to promote the proliferation of cancer stem cells.

Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics.

This study reports the discovery of Aster-Like Nanoparticles (ALNs) in pelagic environments. ALNs are pleomorphic, with three dominant morphotypes which do not fit into any previously defined environmental entities [i.e., ultramicro-prokaryotes, controversed nanobes, and non-living particles (biomimetic mineralo-organic particles, natural nanoparticles or viruses)] of similar size. Elemental composition and selected-area electron diffraction patterns suggested that the organic nature of ALNs may prevail over the possibility of crystal structures. Likewise, recorded changes in ALN numbers in the absence of cells are at odds with an affiliation to until now described viral particles. ALN abundances showed marked seasonal dynamics in the lakewater, with maximal values (up to 9.0 ± 0.5 × 107 particles·mL-1) reaching eight times those obtained for prokaryotes, and representing up to about 40% of the abundances of virus-like particles. We conclude that (i) aquatic ecosystems are reservoirs of novel, abundant, and dynamic aster-like nanoparticles, (ii) not all virus-like particles observed in aquatic systems are necessarily viruses, and (iii) there may be several types of other ultra-small particles in natural waters that are currently unknown but potentially ecologically important.

Adipocyte/breast cancer cell crosstalk in obesity interferes with the anti-proliferative efficacy of tamoxifen.

BACKGROUND: Obesity is a well-known risk factor of breast cancer in post-menopausal women that also correlates with a diminished therapeutic response. The influence of adipocytes and their secretome, i.e. adipokines, on the efficacy of hormone therapy has yet to be elucidated. METHODS: We investigated, ex vivo, whether mature adipocytes, differentiated from adipose stem cells of normal-weight (MA20) or obese (MA30) women, and their secretions, were able to counteract the effects of tamoxifen (Tx) which is known to decrease neoplastic cell proliferation. RESULTS: In a tridimensional model and in a model of co-culture, the anti-proliferative effect of Tx on MCF-7 cancer cells was counteracted by MA30. These two models highlighted two different specific gene expression profiles for genes encoding cytokines or involved in angiogenesis based on the adipocyte microenvironment and the treatment. Thus it notably showed altered expression of genes such as TNFα that correlated with IL-6. In addition, leptin, IL-6 and TNFα, at concentrations reflecting plasma concentrations in obese patients, decreased the anti-proliferative efficacy of 4-hydroxytamoxifen (a major active metabolite of Tx). CONCLUSIONS: These findings bring insights on adipocytes and mammary cancer cell interactions in Tx therapy, particularly in overweight/obese people. Indeed, patient' adipokine status would give valuable information for developing individual strategies and avoid resistance to treatment.

Supernatants of Adipocytes From Obese Versus Normal Weight Women and Breast Cancer Cells: In Vitro Impact on Angiogenesis.

Breast cancer is correlated with a higher risk of metastasis in obese postmenopausal women. Adipokines, whose plasma concentrations are modulated in obese subjects and adipocytes surround mammary cells, suggesting that adipocyte secretome affect mammary tumorogenesis. We hypothesize that mature adipocyte secretions from obese women conditioned or not by breast neoplasic cells, increase changes on the angiogenesis stages. Supernatants of human mature adipocytes, differentiated from stem cells of either adipose tissue of normal weight (MA20) or obese (MA30) women or obtained from co-cultures between MA20 and MA30 and breast cancer cell line MCF-7, were collected. The impact of these supernatants was investigated on proliferation, migration, and tube formation by endothelial cells (HUVEC). MA20 and MA30 showed a preservation of their "metabolic memory" (increase of Leptin, ObR, VEGF, CYP19A1, and a decrease of Adiponectin expression in MA30 compared to MA20). Supernatants from obese-adipocytes increased HUVEC proliferation, migration, and sprouting like with supernatants obtained from co-cultures of MA/MCF-7 regardless the women's BMI. Additional analyses such as the use of neutralizing antibodies, analysis of supernatants (Milliplex®) and variations in gene expression (qRT-PCR), strongly suggest an implication of IL-6, or a synergistic action among adipokines, probably associated with that of VEGF or IL-6. As a conclusion, supernatants from co-cultures of MA30 and MCF-7 cells increase proliferation, migration, and sprouting of HUVEC cells. These results provide insights into the interaction between adipocytes and epithelial cancer cells, particularly in case of obesity. The identification of synergistic action of adipokines would therefore be a great interest in developing preventive strategies. J. Cell. Physiol. 232: 1808-1816, 2017. © 2016 Wiley Periodicals, Inc.

Leptin induces a proliferative response in breast cancer cells but not in normal breast cells.

Obesity is a risk factor for breast cancer in postmenopausal women. Leptin, a hormone excessively produced during obesity, is suggested to be involved in breast cancer. The aim of the study was to investigate procarcinogenic potential of leptin by evaluating influence of leptin on cell proliferation, cell cycle, apoptosis, and signaling on numerous breast cells lines, including 184B5 normal cells, MCF10A fibrocystic cells and MCF-7, MDA-MB-231, and T47D cancer cells. Expressions of leptin and Ob-R were analyzed using qRT-PCR and immunohistochemistry, proliferation using fluorimetric resazurin reduction test and xCELLigence system, apoptosis and cell cycle by flow cytometry, and effect of leptin on different signalling pathways using qRT-PCR and Western blot. Cells were exposed to increasing concentrations of leptin. All cell lines expressed mRNA and protein of leptin and Ob-R. Leptin stimulated proliferation of all cell lines except for 184B5 and MDA-MB-231 cells. Leptin inhibited apoptosis but didn't alter proportion of cells within cell cycle in MCF7 cells. Leptin induced overexpression of leptin, Ob-R, estrogen receptor, and aromatase mRNA in MCF-7 and T47D cells. Autoregulation induced by leptin, relationship with estrogen pathway, and proliferative and antiapoptic activity in breast cancer cells may explain that obesity-associated hyperleptinemia may be a breast cancer risk factor.

Assessing the dual activity of a chalcone-phthalocyanine conjugate: design, synthesis, and antivascular and photodynamic properties.

Photodynamic therapy (PDT) and vascular-disrupting agents (VDA) each have their advantages in the treatment of solid tumors, but also present drawbacks. In PDT, hypoxia at the center of the tumor limits conversion of molecular oxygen into singlet oxygen, while VDAs are deficient at affecting the rim of the tumor. A phthalocyanine-chalcone conjugate combining the VDA properties of chalcones with the PDT properties of phthalocyanines was designed to address these deficiencies. Its vascular targeting, photophysical, photochemical, photodynamic activities are reported herein.

Reciprocal interactions between breast tumor and its adipose microenvironment based on a 3D adipose equivalent model.

Breast cancer has become the most common cancer among women in industrialized countries. Obesity is well established as a risk factor, in particular owing to the attendant secretion of the entities called adipokines; there is growing evidence for a role of cells and factors present in the mammary tumor microenvironment such as fibroblasts, preadipocytes, adipocytes and their secretions. To study how the microenvironment influences breast cancer growth, we developed a novel tridimensional adipose model epithelialized with normal human keratinocytes or with breast cancer cell lines. These mimicked a breast tumor in contact with an adipose microenvironment and allowed monitoring of the interactions between the cells. Leptin and adiponectin, two major adipokines, and their respective receptors, ObRt and AdipoR1, were expressed in the model, but not the second adiponectin receptor, AdipoR2. The differentiation of preadipocytes into adipocytes was greater when they were in contact with the breast cancer cell lines. The contact of breast cancer cell lines with the microenvironment completely modified their transcriptional programs by increasing the expression of genes involved in cell proliferation (cyclinD1, MAPK), angiogenesis (MMP9, VEGF) and hormonal pathways (ESR1, IL6). This tridimensional adipose model provides new insights into the interactions between breast cancer cells and their adipose microenvironment, and provides a tool to develop new drugs for the treatment of both cancer and obesity.

Breast cancer and obesity: in vitro interferences between adipokines and proangiogenic features and/or antitumor therapies?

Obesity is now considered as a risk factor for breast cancer in postmenopausal women. Adipokine levels are modulated in obesity, and may play a role in carcinogenesis. Moreover, obesity increases risk of cancer mortality. Here, we hypothesized that this increase could be due to a modification in angiogenesis, capital event in the development of metastases, and/or in effectiveness of cancer treatments. To test these assumptions, following a same experimental design and simultaneously the effects of leptin and adiponectin on angiogenesis were investigated, and the impact of hyperleptinemia on anticancer drug effectiveness was measured in physiological and obesity situations. Focusing on angiogenesis, the proliferation of endothelial cells (HUVEC), which expressed leptin and adiponectin receptors, was stimulated by leptin and inhibited by adiponectin. Both adipokines globally reduced apoptosis and caspase activity. Leptin increased migration whereas adiponectin decreased migration, and leptin enhanced the area of the tubes formed by HUVEC cells while adiponectin inhibited their formation. MCF7 and MDA-MB-231 cells treated with leptin secreted more VEGF than untreated cells, whereas adiponectin treatment inhibited VEGF secretion. Finally, MCF7 cells pre-treated with leptin were more invasive than untreated cells. This effect was not reproduced in MDA-MB-231 cells. In the MCF7 breast cancer cell line, leptin could induce cell proliferation and reduced the efficacy of all breast cancer therapies (tamoxifen, 5-fluorouracil, taxol and vinblastin). These results suggest that, in obesity situation, leptin- in contrast to adiponectin - may promote tumor invasion and angiogenesis, leading to metastases 'apparition, and reduce treatment efficacy, which could explain the increased risk of cancer mortality in cases of overweight. The evidence suggests adipokines influence breast cancer issue and could play a significant role, especially in obese patients for which hyperleptinemia, hypoadiponectinemia and increased metastatic potential are described.

Zinc-α2-glycoprotein: a proliferative factor for breast cancer? In vitro study and molecular mechanisms.

Zinc-α2-glycoprotein (ZAG) is a new adipokine whose gene expression is downregulated in obese patients. We recently reported ZAG expression in breast tumor or healthy breast tissue and detected this expression at high levels in ductal carcinoma and in normal epithelial adjacent tissue but not in normal tissue of healthy women. In the present study, we used two human breast tumor cell lines (MCF-7 and MDA-MB­231) and one fibrocystic breast cell line (MCF­10a) to examine whether recombinant ZAG has an effect on proliferative/apoptotic response in breast cancer cell lines. ZAG seemed to exert a proliferative effect on breast cancer cell proliferation [+11 to 27% in MCF-7 with (ZAG) = 5-20 µg/ml; +13% in MDA-MB-231 with (ZAG) = 5 µg/ml] and, on the contrary, an anti-proliferative effect in the fibrocystic breast cell line [-5 to -8% in MCF-10a with (ZAG) = 5-10 µg/ml]. ZAG was able to modulate gene and protein expression involved in the apoptotic response. However, further studies are required to fully elucidate the effects of ZAG on the proliferation of mammary cells.

Human langerhans cells are more efficient than CD14(-)CD1c(+) dermal dendritic cells at priming naive CD4(+) T cells.

Few data are available regarding the role of human skin dendritic cells (DCs) in driving T-cell responses. In this study we analyzed the relative capacity of Langerhans cells (LCs) and dermal CD14(-)CD1c(+) DCs (DDCs) to trigger naive CD4(+) T-cell proliferation and differentiation. DC subsets were purified after a 2-day migration from epidermis and dermis of the same skin sample. Migratory LCs showed far more activated phenotype than CD1c(+)DDCs and distinct expression of new molecules of the B7 family; when compared with LCs, CD1c(+)DDCs showed higher PD-L1 and lower inducible co-stimulator ligand (ICOS-L) expression. As expected, CD1c(+)DDCs showed lower allostimulatory property than LCs, a process that was partly reversed by anti-PD-L1 mAb. LCs were significantly more efficient than CD1c(+)DDCs at inducing allogeneic naive CD4(+) T cells to secrete both T helper cell 1 (Th1; IFN-gamma and tumor necrosis factor-alpha ) and Th2 (IL-4 and IL-5) cytokines. Moreover, anti-PD-L1 mAb increased the production of IFN-gamma by both LC- and CD1c(+)DDC-stimulated T cells. Globally, these results argue for a preponderant role of human LCs in inducing naive CD4(+) T-cell priming. Low expression of co-stimulatory molecules together with high expression of PD-L1 might limit the efficiency of CD1c(+)DDCs at inducing naive CD4(+) T-cell proliferation and secretion of cytokines.

Poly(I:C)-Treated human langerhans cells promote the differentiation of CD4+ T cells producing IFN-gamma and IL-10.

Epidermal Langerhans cells (LCs) are the first dendritic cells to encounter skin pathogens. However, their function has recently been challenged, especially in the initiation of T-cell responses to viral antigens. We have previously reported that fresh immature human LCs express mRNA encoding TLR3. Here we analyze the response of highly purified human LCs to poly(I:C), a synthetic mimetic of viral dsRNA recognized by TLR3. We show that LCs exposed for 2 days to poly(I:C) under serum-free conditions up-regulated co-stimulatory molecules, a process associated with increased allostimulatory capacity. Furthermore, poly(I:C) significantly enhanced LC survival and induced them to produce CXCL10, IL-6, and IL-12 p40. Bioactive IL-12 p70, IL-1beta, IL-15, IL-18, and IL-23 were never detected, even after CD40 ligation. LC incubation in the presence of bafilomycin completely reversed the effect of poly(I:C) on LC phenotypic activation and survival, indicating that endosomal TLR3 is involved in this process. Most interestingly, we report here that poly(I:C)-treated LCs favored alloreactive CD4(+) T-cell differentiation toward a Th1 profile and concomitant differentiation of IL-10-producing CD4(+) T cells that might limit, at another time, the inflammatory response and subsequent tissue damage.