Adaptation of the Endolithic Biome in Antarctic Volcanic Rocks.

Endolithic microorganisms, ranging from microeukaryotes to bacteria and archaea, live within the cracks and crevices of rocks. Deception Island in Antarctica constitutes an extreme environment in which endoliths face environmental threats such as intense cold, lack of light in winter, high solar radiation in summer, and heat emitted as the result of volcanic eruptions. In addition, the endolithic biome is considered the harshest one on Earth, since it suffers added threats such as dryness or lack of nutrients. Even so, samples from this hostile environment, collected at various points throughout the island, hosted diverse and numerous microorganisms such as bacteria, fungi, diatoms, ciliates, flagellates and unicellular algae. These endoliths were first identified by Scanning Electron Microscopy (SEM). To understand the molecular mechanisms of adaptation of these endoliths to their environment, genomics techniques were used, and prokaryotic and eukaryotic microorganisms were identified by metabarcoding, sequencing the V3-V4 and V4-V5 regions of the 16S and 18S rRNA genes, respectively. Subsequently, the sequences were analyzed by bioinformatic methods that allow their metabolism to be deduced from the taxonomy. The results obtained concluded that some of these microorganisms have activated the biosynthesis routes of pigments such as prodigiosin or flavonoids. These adaptation studies also revealed that microorganisms defend themselves against environmental toxins by activating metabolic pathways for the degradation of compounds such as ethylbenzene, xylene and dioxins and for the biosynthesis of antioxidant molecules such as glutathione. Finally, these Antarctic endolithic microorganisms are of great interest in astrobiology since endolithic settings are environmentally analogous to the primitive Earth or the surfaces of extraterrestrial bodies.

Detection of Macromolecular Fractions in HCN Polymers Using Electrophoretic and Ultrafiltration Techniques.

Elucidating the origin of life involves synthetic as well as analytical challenges. Herein, for the first time, we describe the use of gel electrophoresis and ultrafiltration to fractionate HCN polymers. Since the first prebiotic synthesis of adenine by Oró, HCN polymers have gained much interest in studies on the origins of life due to the identification of biomonomers and related compounds within them. Here, we demonstrate that macromolecular fractions with electrophoretic mobility can also be detected within HCN polymers. The migration of polymers under the influence of an electric field depends not only on their sizes (one-dimensional electrophoresis) but also their different isoelectric points (two-dimensional electrophoresis, 2-DE). The same behaviour was observed for several macromolecular fractions detected in HCN polymers. Macromolecular fractions with apparent molecular weights as high as 250 kDa were detected by tricine-SDS gel electrophoresis. Cationic macromolecular fractions with apparent molecular weights as high as 140 kDa were also detected by 2-DE. The HCN polymers synthesized were fractionated by ultrafiltration. As a result, the molecular weight distributions of the macromolecular fractions detected in the HCN polymers directly depended on the synthetic conditions used to produce these polymers. The implications of these results for prebiotic chemistry will be discussed.

Proteomic analysis of the adaptation to warming in the Antarctic bacteria Shewanella frigidimarina.

Antarctica is subjected to extremely variable conditions, but the importance of the temperature increase in cold adapted bacteria is still unknown. To study the molecular adaptation to warming of Antarctic bacteria, cultures of Shewanella frigidimarina were incubated at temperatures ranging from 0°C to 30°C, emulating the most extreme conditions that this strain could tolerate. A proteomic approach was developed to identify the soluble proteins obtained from cells growing at 4°C, 20°C and 28°C. The most drastic effect when bacteria were grown at 28°C was the accumulation of heat shock proteins as well as other proteins related to stress, redox homeostasis or protein synthesis and degradation, and the decrease of enzymes and components of the cell envelope. Furthermore, two main responses in the adaptation to warm temperature were detected: the presence of diverse isoforms in some differentially expressed proteins, and the composition of chaperone interaction networks at the limits of growth temperature. The abundance changes of proteins suggest that warming induces a stress situation in S. frigidimarina forcing cells to reorganize their molecular networks as an adaptive response to these environmental conditions.

The hidden microbial ecosystem in the perennial ice from a Pyrenean ice cave.

Over the last years, perennial ice deposits located within caves have awakened interest as places to study microbial communities since they represent unique cryospheric archives of climate change. Since the beginning of the twentieth century, the temperature has gradually increased, and it is estimated that by the end of this century the increase in average temperature could be around 4.0°C. In this context of global warming the ice deposits of the Pyrenean caves are undergoing a significant regression. Among this type of caves, that on the Cotiella Massif in the Southern Pyrenees is one of the southernmost studied in Europe. These types of caves house microbial communities which have so far been barely explored, and therefore their study is necessary. In this work, the microbial communities of the Pyrenean ice cave A294 were identified using metabarcoding techniques. In addition, research work was carried out to analyze how the age and composition of the ice affect the composition of the bacterial and microeukaryotic populations. Finally, the in vivo effect of climate change on the cellular machinery that allow microorganisms to live with increasing temperatures has been studied using proteomic techniques.

Comparative Proteomic Analysis of Psychrophilic vs. Mesophilic Bacterial Species Reveals Different Strategies to Achieve Temperature Adaptation.

The old debate of nature (genes) vs. nurture (environmental variables) is once again topical concerning the effect of climate change on environmental microorganisms. Specifically, the Polar Regions are experiencing a drastic increase in temperature caused by the rise in greenhouse gas emissions. This study, in an attempt to mimic the molecular adaptation of polar microorganisms, combines proteomic approaches with a classical microbiological analysis in three bacterial species Shewanella oneidensis, Shewanella frigidimarina, and Psychrobacter frigidicola. Both shewanellas are members of the same genus but they live in different environments. On the other hand, Shewanella frigidimarina and Psychrobacter frigidicola share the same natural environment but belong to a different genus. The comparison of the strategies employed by each bacterial species estimates the contribution of genome vs. environmental variables in the adaptation to temperature. The results show a greater versatility of acclimatization for the genus Shewanella with respect to Psychrobacter. Besides, S. frigidimarina was the best-adapted species to thermal variations in the temperature range 4-30°C and displayed several adaptation mechanisms common with the other two species. Regarding the molecular machinery used by these bacteria to face the consequences of temperature changes, chaperones have a pivoting role. They form complexes with other proteins in the response to the environment, establishing cooperation with transmembrane proteins, elongation factors, and proteins for protection against oxidative damage.

Microbial Communities in Volcanic Glacier Ecosystems.

Glaciers constitute a polyextremophilic environment characterized by low temperatures, high solar radiation, a lack of nutrients, and low water availability. However, glaciers located in volcanic regions have special characteristics, since the volcanic foci provide them with heat and nutrients that allow the growth of microbial communities highly adapted to this environment. Most of the studies on these glacial ecosystems have been carried out in volcanic environments in the northern hemisphere, including Iceland and the Pacific Northwest. To better know, the microbial diversity of the underexplored glacial ecosystems and to check what their specific characteristics were, we studied the structure of bacterial communities living in volcanic glaciers in Deception Island, Antarctica, and in the Kamchatka peninsula. In addition to geographic coordinates, many other glacier environmental factors (like volcanic activity, altitude, temperature, pH, or ice chemical composition) that can influence the diversity and distribution of microbial communities were considered in this study. Finally, using their taxonomic assignments, an attempt was made to compare how different or similar are the biogeochemical cycles in which these microbiomes are involved.

Identification of Biomolecules Involved in the Adaptation to the Environment of Cold-Loving Microorganisms and Metabolic Pathways for Their Production.

Cold-loving microorganisms of all three domains of life have unique and special abilities that allow them to live in harsh environments. They have acquired structural and molecular mechanisms of adaptation to the cold that include the production of anti-freeze proteins, carbohydrate-based extracellular polymeric substances and lipids which serve as cryo- and osmoprotectants by maintaining the fluidity of their membranes. They also produce a wide diversity of pigmented molecules to obtain energy, carry out photosynthesis, increase their resistance to stress and provide them with ultraviolet light protection. Recently developed analytical techniques have been applied as high-throughoutput technologies for function discovery and for reconstructing functional networks in psychrophiles. Among them, omics deserve special mention, such as genomics, transcriptomics, proteomics, glycomics, lipidomics and metabolomics. These techniques have allowed the identification of microorganisms and the study of their biogeochemical activities. They have also made it possible to infer their metabolic capacities and identify the biomolecules that are parts of their structures or that they secrete into the environment, which can be useful in various fields of biotechnology. This Review summarizes current knowledge on psychrophiles as sources of biomolecules and the metabolic pathways for their production. New strategies and next-generation approaches are needed to increase the chances of discovering new biomolecules.

Glacial Ice Age Shapes Microbiome Composition in a Receding Southern European Glacier.

Glaciers and their microbiomes are exceptional witnesses of the environmental conditions from remote times. Climate change is threatening mountain glaciers, and especially those found in southern Europe, such as the Monte Perdido Glacier (northern Spain, Central Pyrenees). This study focuses on the reconstruction of the history of microbial communities over time. The microorganisms that inhabit the Monte Perdido Glacier were identified using high-throughput sequencing, and the microbial communities were compared along an altitudinal transect covering most of the preserved ice sequence in the glacier. The results showed that the glacial ice age gradient did shape the diversity of microbial populations, which presented large differences throughout the last 2000 years. Variations in microbial community diversity were influenced by glacial conditions over time (nutrient concentration, chemical composition, and ice age). Some groups were exclusively identified in the oldest samples as the bacterial phyla Fusobacteria and Calditrichaeota, or the eukaryotic class Rhodophyceae. Among groups only found in modern samples, the green sulfur bacteria (phylum Chlorobi) stood out, as well as the bacterial phylum Gemmatimonadetes and the eukaryotic class Tubulinea. A patent impact of human contamination was also observed on the glacier microbiome. The oldest samples, corresponding to the Roman Empire times, were influenced by the beginning of mining exploitation in the Pyrenean area, with the presence of metal-tolerant microorganisms. The most recent samples comprise 600-year-old ancient ice in which current communities are living.

Microbial Community Structure Driven by a Volcanic Gradient in Glaciers of the Antarctic Archipelago South Shetland.

It has been demonstrated that the englacial ecosystem in volcanic environments is inhabited by active bacteria. To know whether this result could be extrapolated to other Antarctic glaciers and to study the populations of microeukaryotes in addition to those of bacteria, a study was performed using ice samples from eight glaciers in the South Shetland archipelago. The identification of microbial communities of bacteria and microeukaryotes using 16S rRNA and 18S rRNA high throughput sequencing showed a great diversity when compared with microbiomes of other Antarctic glaciers or frozen deserts. Even the composition of the microbial communities identified in the glaciers from the same island was different, which may be due to the isolation of microbial clusters within the ice. A gradient in the abundance and diversity of the microbial communities from the volcano (west to the east) was observed. Additionally, a significant correlation was found between the chemical conditions of the ice samples and the composition of the prokaryotic populations inhabiting them along the volcanic gradient. The bacteria that participate in the sulfur cycle were those that best fit this trend. Furthermore, on the eastern island, a clear influence of human contamination was observed on the glacier microbiome.

Proteomic analysis of the response of an acidophilic strain of Chlamydomonas sp. (Chlorophyta) to natural metal-rich water.

A proteomic approach including 2-DE and MALDI-TOF analysis has been developed to identify the soluble proteins of the unicellular photosynthetic algae Chlamydomonas sp. isolated from an extreme acidic environment, Río Tinto (southwest Spain). We have analyzed the soluble proteome obtained from whole cells growing on metal-rich natural acidic water from the river in comparison with the same strain growing in artificial BG-11 media. The most drastic effect was the decrease in the abundance of the ribulose-1,5-biphosphate carboxylase as well as other enzymes related to photosynthesis. However, phytochrome B, phosphoribulokinase, and phosphoglycerate kinase were upregulated when cells were grown in metal-rich acidic water. Besides, increased accumulation of two Hsps, Hsp70 and Hsp90 as well as other stress-related enzymes were also found in the cells growing in natural acidic water. These results suggest that naturally occurring metal-rich water induces a stress response in acidophilic Chlamydomonas forcing algal cells to reorganize their metabolic pathways as an adaptive response to these environmental conditions.

Expression of heat shock protein 90 at the cell surface in human neuroblastoma cells.

In addition to the activity of heat shock protein 90 (Hsp90/HSPC) as a chaperone, some recent studies have reported expression of Hsp90 at the cell surface in certain types of cancer and nervous system cells. We study the expression of Hsp90 at the cell surface in human neuroblastoma (NB69) cells. Immunofluorescence experiments labeling with anti-Hsp90 antibodies on both nonpermeabilized cells and live cells detected Hsp90 at the cell surface. Hsp90 was also identified in a membrane fraction from subcellular fractionation. Cell-surface Hsp90 was significantly more expressed in undifferentiated proliferative spherical neuroblastoma cells than in differentiated flattened cells. In addition, spherical cells were significantly more sensitive to Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin compared to flattened cells. This paper describes the first evidence of cell-surface Hsp90 expression in a cancer cell line from nervous tissue and may indicate a novel target for anti-tumoral agents.

Taxonomic and functional characterization of a microbial community from a volcanic englacial ecosystem in Deception Island, Antarctica.

Glaciers are populated by a large number of microorganisms including bacteria, archaea and microeukaryotes. Several factors such as solar radiation, nutrient availability and water content greatly determine the diversity and abundance of these microbial populations, the type of metabolism and the biogeochemical cycles. Three ecosystems can be differentiated in glaciers: supraglacial, subglacial and englacial ecosystems. Firstly, the supraglacial ecosystem, sunlit and oxygenated, is predominantly populated by photoautotrophic microorganisms. Secondly, the subglacial ecosystem contains a majority of chemoautotrophs that are fed on the mineral salts of the rocks and basal soil. Lastly, the englacial ecosystem is the least studied and the one that contains the smallest number of microorganisms. However, these unknown englacial microorganisms establish a food web and appear to have an active metabolism. In order to study their metabolic potentials, samples of englacial ice were taken from an Antarctic glacier. Microorganisms were analyzed by a polyphasic approach that combines a set of -omic techniques: 16S rRNA sequencing, culturomics and metaproteomics. This combination provides key information about diversity and functions of microbial populations, especially in rare habitats. Several whole essential proteins and enzymes related to metabolism and energy production, recombination and translation were found that demonstrate the existence of cellular activity at subzero temperatures. In this way it is shown that the englacial microorganisms are not quiescent, but that they maintain an active metabolism and play an important role in the glacial microbial community.

Enzymatic and cell factory approaches to the production of human milk oligosaccharides.

Infant formula milk companies try to develop fortified formula milk that mimics human milk as closely as possible, since it is well-known that breast milk has considerable implications in the development of the infant in the first years of life. Human milk is unique in terms of complex oligosaccharides content, known as human milk oligosaccharides (HMOs). Their role in the development of intestinal flora blocking the attachment of pathogens and modulating the immune system of the infant are currently recognized. Due to these biological effects, there is a great interest to introduce the main HMOs in the infant formula milk. Therefore, efficient synthetic strategies for HMOs production are required. Here we present a complete review of HMO production using either (chemo)enzymatic syntheses or cell factory approaches, focusing on the strategies that produce HMOs at least at the milligram scale. 42 HMO structures have already been produced as free sugars. Whereas short HMOs are well obtained by cell factory approaches, complex and branched HMOs are better produced by chemoenzymatic strategies. Inspite of the current advances, production strategies of some biologically relevant HMOs are still missing.

Ion-sensitive field effect transistors using carbon nanotubes as the transducing layer.

We report a new type of ion-sensitive field effect transistor (ISFET). This type of ISFET incorporates a new architecture, containing a network of single-walled carbon nanotubes (SWCNTs) as the transduction layer, making an external reference electrode unnecessary. To show an example of its application, the SWCNT-based ISFET is able to detect at least 10(-8) M of potassium in water using an ion-selective membrane containing valinomycin.

Carbon nanotube field effect transistors for the fast and selective detection of human immunoglobulin G.

We report a field effect transistor (FET) based on a network of single-walled carbon nanotubes (SWCNTs) which can selectively detect human immunoglobulin G (HIgG). HIgG antibodies, which are strongly adsorbed onto the walls of the SWCNTs, are the basic elements of the recognition layer. The non-specific binding of proteins and the effects of other interferences are avoided by covering the non-adsorbed areas of the SWCNTs with Tween 20. The selectivity of the sensor has been tested against bovine serum albumin (BSA), the most abundant protein in plasma. HIgG in aqueous solution with concentrations from 1.25 mg L(-1) (8 nM) can be readily detected with response times of about 10 min. The SWCNT networks that form the basis of the sensor are easily grown by chemical vapour deposition. Silver screen-printed electrodes make the sensor quick to build. The sensitivity obtained with this sensor is similar to other FET devices based on SWCNTs built using much more complicated lithography processes. Moreover, the sensor is a reagentless device that does not need labels to detect HIgG.

Bacterial molecular machinery in the Martian cryosphere conditions.

The exploration of Mars is one of the main objectives of space missions since the red planet is considered to be, or was in the past, potentially habitable. Although the surface of Mars is now dry and arid, abundant research suggests that water covered Mars billions of years ago. Recently, the existence of liquid water in subglacial lakes has been postulated below the South pole of Mars. Until now, experiments have been carried out on the survival of microorganisms in Martian surface conditions, but it remains unknown how their adaptation mechanisms would be in the Martian cryosphere. In this work, two bacterial species (Bacillus subtilis and Curtobacterium flacumfaciens) were subjected to a simulated Martian environment during 24 h using a planetary chamber. Afterward, the molecular machinery of both species was studied to investigate how they had been modified. Proteomes, the entire set of proteins expressed by each bacterium under Earth (named standard) conditions and Martian conditions, were compared using proteomic techniques. To establish this evaluation, both the expression levels of each protein, and the variation in their distribution within the different functional categories were considered. The results showed that these bacterial species followed a different strategy. The Bacillus subtilis resistance approach consisted of improving its stress response, membrane bioenergetics, degradation of biomolecules; and to a lesser extent, increasing its mobility and the formation of biofilms or resistance endospores. On the contrary, enduring strategy of Curtobacterium flacumfaciens comprised of strengthening the cell envelope, trying to protect cells from the extracellular environment. These results are especially important due to their implications for planetary protection, missions to Mars and sample return since contamination by microorganisms would invalidate the results of these investigations.

Regulatory proteins of eukaryotic initiation factor 2-alpha subunit (eIF2 alpha) phosphatase, under ischemic reperfusion and tolerance.

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha), which is one of the substrates of protein phosphatase 1 (PP1), occurs rapidly during the first minutes of post-ischemic reperfusion after an episode of cerebral ischemia. In the present work, two experimental models of transient global ischemia and ischemic tolerance (IT) were used to study PP1 interacting/regulatory proteins following ischemic reperfusion. For that purpose we utilized PP1 purified by microcystin chromatography, as well as 2D DIGE of PP1alpha and PP1gamma immunoprecipitates. The highest levels of phosphorylated eIF2alpha found after 30 min reperfusion in rats without IT, correlated with increased levels in PP1 immunoprecipitates of the inhibitor DARPP32 as well as GRP78 and HSC70 proteins. After 4 h reperfusion, the levels of these proteins in PP1c complexes had returned to control values, in parallel to a significant decrease in eIF2alpha phosphorylated levels. IT that promoted a decrease in eIF2alpha phosphorylated levels after 30 min reperfusion induced the association of GADD34 with PP1c, while prevented that of DARPP32, GRP78, and HSC70. Different levels of HSC70 and DARPP32 associated with PP1alpha and PP1gamma isoforms, whereas GRP78 was only detected in PP1gamma immunoprecipitates. Here we suggest that PP1, through different signaling complexes with their interacting proteins, may modulate the eIF2alpha phosphorylation/dephosphorylation during reperfusion after a transient global ischemia in the rat brain. Of particular interest is the potential role of GADD34/PP1c complexes after tolerance acquisition.

Glaciers and Ice Sheets As Analog Environments of Potentially Habitable Icy Worlds.

Icy worlds in the solar system and beyond have attracted a remarkable attention as possible habitats for life. The current consideration about whether life exists beyond Earth is based on our knowledge of life in terrestrial cold environments. On Earth, glaciers and ice sheets have been considered uninhabited for a long time as they seemed too hostile to harbor life. However, these environments are unique biomes dominated by microbial communities which maintain active biochemical routes. Thanks to techniques such as microscopy and more recently DNA sequencing methods, a great biodiversity of prokaryote and eukaryote microorganisms have been discovered. These microorganisms are adapted to a harsh environment, in which the most extreme features are the lack of liquid water, extremely cold temperatures, high solar radiation and nutrient shortage. Here we compare the environmental characteristics of icy worlds, and the environmental characteristics of terrestrial glaciers and ice sheets in order to address some interesting questions: (i) which are the characteristics of habitability known for the frozen worlds, and which could be compatible with life, (ii) what are the environmental characteristics of terrestrial glaciers and ice sheets that can be life-limiting, (iii) What are the microbial communities of prokaryotic and eukaryotic microorganisms that can live in them, and (iv) taking into account these observations, could any of these planets or satellites meet the conditions of habitability? In this review, the icy worlds are considered from the point of view of astrobiological exploration. With the aim of determining whether icy worlds could be potentially habitable, they have been compared with the environmental features of glaciers and ice sheets on Earth. We also reviewed some field and laboratory investigations about microorganisms that live in analog environments of icy worlds, where they are not only viable but also metabolically active.

Antibodies reactive to heat shock protein 90 induce oligodendrocyte precursor cell death in culture. Implications for demyelination in multiple sclerosis.

Oligodendrocyte precursor cells (OPCs) are extremely efficient at remyelination. These cells persist in the adult human central nervous system and can proliferate. However, the failure to remyelinate is a pathological characteristic of the human demyelinating disease multiple sclerosis (MS), which suggests that these cells are ineffective in this disorder. This paper reports that IgG antibodies in the cerebrospinal fluid (CSF) of MS patients specifically recognize an antigen on OPCs in culture. Control patients were found not to possess these antibodies. The antigen was immunoprecipitated in cell extracts from cultures with purified IgG from MS CSF. Peptide mass fingerprinting identified it as the beta type of heat shock protein 90 (Hsp90). Two-dimensional electrophoresis and immunoblot showed that this antigen in fact corresponds to two specific isoforms of Hsp90beta. Several control assays using monoclonal and polyclonal anti-Hsp90 antibodies confirmed the specific expression of Hsp90 on OPCs. Labeling OPCs in vivo with MS CSF and anti-Hsp90 antibodies and subsequent immunofluorescence confocal microscopy located the antigen on the cell surface. The binding of CSF antibodies from MS patients to the OPC surface led to complement activation and significant extinction of the OPC population. These results suggest that OPCs may be a target of anti-Hsp90 antibodies in MS patients and that this could prevent remyelination.

Antifouling activity in some benthic Antarctic invertebrates by "in situ" experiments at Deception Island, Antarctica.

Competition for space is a remarkable ecological force, comparable to predation, producing a strong selective pressure on benthic invertebrates. Some invertebrates, thus, possess antimicrobial compounds to reduce surface bacterial growth. Antimicrobial inhibition is the first step in avoiding being overgrown by other organisms, which may have a negative impact in feeding, respiration, reproduction … The in situ inhibition of bacterial biofilm was used here as an indicator of antifouling activity by testing hydrophilic extracts of twelve Antarctic invertebrates. Using two different approaches (genetics and confocal techniques) different levels of activity were found in the tested organisms. In fact, differences within body parts of the studied organisms were determined, in agreement with the Optimal Defense Theory. Eight out of 15 extracts tested had negative effects on fouling after 28 days submerged in Antarctic waters. Thus, although chemical defenses may be quite species-specific in their ecological roles, these results suggest that different chemical strategies exist to deal with space competition.