Here we assess the active role of ice sheets in the global carbon cycle and potential ramifications of enhanced melt and ice discharge in a warming world.The cycling of carbon on Earth exerts a fundamental influence upon the greenhouse gas content of the atmosphere, and hence global climate over millennia. Until recently, ice sheets were viewed as inert components of this cycle and largely disregarded in global models. Research in the past decade has transformed this view, demonstrating the existence of uniquely adapted microbial communities, high rates of biogeochemical/physical weathering in ice sheets and storage and cycling of organic carbon (>10 Ice sheets have long been overlooked as regulators of the global carbon cycle. In this Review, Wadham and colleagues show how the growth and retreat of ice sheets support the productivity of the oceans and variably store or release organic carbon–in effect, these frozen landscapes must be considered in future assessments of climate impacts on biogeochemical cycling. More recent work has highlighted the capacity of glacier surfaces to act as sinks for carbon-containing aerosols from anthropogenic or natural sources3. However, both these mechanisms represent passive processes of carbon storage and release by glaciers. Only in the last 15 years have glacial systems started to be considered as active cyclers of carbon, arising from the discovery that they include a range of aquatic environments4 which host abundant and diverse populations of microorganisms5 and are hot spots for biogeochemical weathering6. These processes create the potential for ice sheets to directly or indirectly impact the global carbon cycle  during the microbial respiration of organic matter (OM) stored within ice sheets. Examples of indirect impacts include the fertilisation of downstream ecosystems, promoted by either the release of nutrient-rich glacial meltwaters9 or by subglacial meltwater-induced upwelling of nutrient replete marine water at tidewater glacier margins14. Ocean fertilisation by glaciers may be accompanied by significant CO2 drawdown by phytoplankton, intensifying the biological pump16.For a component of the Earth’s system to impact the global carbon cycle, and potentially influence atmospheric concentrations of carbon dioxide or methane, it must either directly sequester and/or release carbon, or indirectly influence carbon uptake/release in other parts of the Earth system. The notion that ice sheets contain significant carbon stores has its roots in the early 2000s–the great ice sheets were hypothesised to advance over soil and vegetation carbon during glacial periods, with this fossil carbon being released back to the atmosphere once exposed by retreating ice during deglaciation18 and magnitude of nutrient supply to surface ocean waters by meltwaters and icebergs19 make it crucial to evaluate the role of ice sheets as open systems in regulating the global carbon cycle via the direct and indirect mechanisms identified in Fig. The size of the carbon stores entombed in ice sheets20 driving a major shift in our world-view of glaciers and ice sheets as abiotic systems to extensive icy biomes5. Microbial communities have since been found ubiquitously in glacial systems5, ranging from sub-micron diameter liquid ice veins between ice crystals21 to large subglacial water bodies, such as Subglacial Lake Whillans situated 800 m beneath the West Antarctic Ice Sheet22. The survival of these seemingly diverse communities is due to their adaptation to a unique combination of physical and geochemical conditions that are not encountered anywhere else on Earth.In 1999, the first communities of microorganisms were discovered beneath a valley glacier in the Swiss Alps23. These drainage systems evolve seasonally from slow-inefficient distributed drainage to fast-efficient drainage , with residence times shifting from months to <1 day23. In contrast, surface melt is largely absent in Antarctica, apart from in marginal locations25. Nonetheless, the bed of the Antarctic Ice Sheet is hydrologically active, with subglacial lakes, swamps, channels and groundwater aquifers fed by the melting of basal ice layers4. Water flowing in such long residence time drainage systems here may take years to decades to emerge at the ice margin26.First, liquid water is abundant in ice sheets, generated by the melting of surface ice and snow and by geothermal/frictional heating and pressure melting of ice at the glacier bed. Surface melt is often widespread on the Greenland Ice Sheet and supplies melt to well-developed drainage systems at the ice sheet bed (where ice is at the pressure melting point)27, as well as reduced elemental species such as Fe(II) in iron sulphide minerals), micro-nutrients and macro-nutrients, liberated by chemical dissolution. In the deep, dark and cold ecosystems beneath ice sheets, these rock-sourced redox pairs and nutrients promote the metabolism of a diverse mix of chemolithotrophic and chemoorganotrophic microorganisms, able to fix and supply autochthonous carbon to the wider subglacial ecosystem. In Antarctic Subglacial Lake Whillans, a large proportion of phylotypes in sediments were related to chemolithoautotrophic species that use reduced forms of sulphur, nitrogen and iron as energy sources22.Second, the sliding of glaciers over their bedrock generates very fine, highly reactive rock flour by glacial crushing and grinding. This is the source of a potent mixture of electron acceptors ), electron donors . The release of fresh, buoyant subglacial meltwaters often some 100 s of m below the ocean surface at the margins of marine-terminating outlet glaciers also stimulates the upwelling of marine water and transport of associated nutrients to the ocean surface14. It is this combination of in situ microbial activity, dynamic processes of meltwater export and glacial advance/retreat cycles that creates an active connection between ice sheets, the ocean and the atmosphere 178. Bare ice surfaces are dominated by ice algae179, which are commonly green algae on the Greenland Ice Sheet181. In cryoconite holes, the dominant active taxa are cyanobacteria and alpha-proteobacteria and/or beta-proteobacteria182. The activity of these microorganisms has recently drawn attention via the hypothesis that the biologically driven accumulation of OM accelerates ice melting, via its effect upon ice surface darkening183. Net bioaccumulation depends upon the balance of CO2 fixed from the atmosphere by phototrophs and CO2 returned to the atmosphere by respiration, and is influenced by a wide range of physical and biogeochemical processes. Recent work on the Greenland Ice Sheet suggests that carbon fixation exceeds carbon consumption, making the ice sheet surface net autotrophic  and enabling the accumulation of OM and pigmented algae179. Autochthonous OM is supplemented by wind-blown material, which becomes biologically altered on the ice sheet surface by the release of exopolymeric substances which bind particles185. The direct impact of the bioalbedo effect has yet to be fully quantified. However, initial investigations indicate that algal abundance on Greenland ice surfaces explains 70% of the variation ice reflectance, suggesting a significant impact of ice algae on ice surface albedo28. The resultant early exposure of underlying dark  glacial ice following snowmelt has the potential to further enhance glacier melting186.The bioalbedo effect refers to the darkening effect of biological impurities on snow and ice4+, NO3−, NO2−) and P (PO43−), organic forms of N and P, Si and micronutrients such as Fe , promote the chemical dissolution and alteration of nutrient-rich minerals such as aluminosilicates and P-bearing apatite. Thus, meltwaters and icebergs exported from ice sheets are also often enriched in bioavailable forms of dissolved and particulate phosphorous and silicon44. For example, yields of soluble reactive P generated in large ice sheet catchments are among the highest in the literature (17–27 kg P m−2 a−1)42, and an order of magnitude higher than from small valley glaciers46. Total P fluxes are an order of magnitude higher than those for other world rivers, largely sourced from minerals such as fluorapatite42. This suggests that high physical and chemical erosion rates in ice sheets47 are important in enhancing total P fluxes. Traditional notions of depressed silicate mineral weathering beneath small valley glaciers, inferred from low concentrations of dissolved Si in glacial runoff48, may also not hold for large ice sheets43. Recent work has shown significant concentrations of amorphous silicon (ASi) associated with suspended particulate matter in Greenland Ice Sheet runoff (~1% by mass). When this particulate component is included in total Greenland Ice Sheet Si fluxes via bulk runoff, the total flux rises by an order of magnitude to 50% of the total Arctic riverine Si flux43. Potential formation mechanisms include either production of ASi during rock grinding, a chemically leached surface layer or a precipitated weathering crust arising from dissolution/re-precipitation on freshly ground and leached particles43. Inclusion of ASi in total glacial Si fluxes has the potential to increase Si fluxes by an order of magnitude43. Glacially sourced ASi is highly soluble in seawater and its export, together with DSi, has the potential to change the oceanic Si isotopic signature and inventory on glacial-interglacial timescales49 and stimulate the productivity of diatoms and other siliceous organisms50.By far the largest source of nutrients released directly via ice sheet meltwaters is the subglacial environmentty41 Box . Long fl52. A suite of microbially -mediated processes including silicate dissolution, sulphide oxidation53 and dissimilatory iron reduction34 generate ice sheet runoff enriched in filterable  and highly reactive particulate forms of iron57 that contain bioavailable Fe(II)59. Icebergs are particularly important Fe exporters, containing enhanced concentrations of bioavailable (ascorbic acid extractable) iron (oxy)hydroxides  in iceberg rafted debris (IRD)19. There are likely important interactions between iron and other chemical species which are critical to consider within the context of wider ice sheet nutrient cycles, for example, the sorption of soluble reactive P (SRP) onto highly reactive Fe (oxy)hydroxide nanoparticles or colloids42.Perhaps one of the most well studied nutrients released from glaciers and ice sheets is iron (Fe). The liberation of bioavailable Fe from bedrock via biogeochemical weathering see Box  is thoug44 compared to deep ocean water 42. In comparison, the dissolved organic components of N and P in ice sheet meltwaters can be significant, acquired via the activity of micro-organisms on the glacier surface and at the bed. For example, dissolved organic N (DON) comprised 50% of the total dissolved N load of Greenland Ice Sheet runoff31. Dissolved organic matter (DOM) may also act as an important stabiliser for key redox-sensitive or phase-sensitive trace elements such as iron. A direct relationship between dissolved organic carbon (DOC) and dissolved iron observed in sub-Antarctic meltwaters was attributed to the stabilisation of filterable Fe (<0.45 μm) by complexation with DOM56, although further investigation is needed to determine if DOM-Fe linkage is important in glacial meltwaters.Organic phases of key nutrients are important in some contexts within ice sheets, resulting from the cycling of allochthonous OM and production of autochthonous OM. The organic phases of particulate P and N are measurable in ice sheet runoff, but comprise a much smaller proportion of the total N and P fluxes compared with those from Arctic rivers61. High contemporary fluxes of bioavailable Fe and Si from ice sheets are observed (1.8 Tg a−1 Fe and 9.4 Tg a−1 Si), where >60% of these estimated Si and Fe fluxes are sourced from the Greenland Ice Sheet  for these nutrient species62  lower than those for Fe, Si and organic carbon . However, these estimates exclude the more refractory mineral forms of P associated with particles , which display fluxes orders of magnitude higher than the traditionally ascribed bioavailable P flux 42. Fluxes of bioavailable P from ice sheets at the present day are of a similar order of magnitude (25 Gg a−1) to other riverine sources such as Arctic rivers (70 Gg a−1 bioavailable P)42. Present day N fluxes from ice sheets are moderate  and exceed those from a major Arctic river (20 Gg a−1 for the Mackenzie River63) , P fluxes via upwelling are 2–9 Gg N a−1 and those of N are 12–40 Gg P a−1, compared with estimates of  15 Gg P a−1 and  30 Gg N a−1 via meltwater and ice discharge from the entire Greenland Ice Sheet 2). The peet Fig. . Present−1 and 21 Tg a−1, respectively). This flux of Si is almost equivalent to the total global river discharge of Si during the same time interval, which is notable given that the primary source of Si in the oceans is riverine inputs43. The impact of these boosted nutrient inputs on primary productivity in oceans bordering ice sheets during the last glaciation and glacial termination has been hinted at refs. 65, but is poorly constrained and requires a comprehensive modelling study in its own right. What is known about the fate of glacially exported nutrients at the present day is examined in the following section.Figure Box 2 Sources and cycling of nutrientsAtmospheric-derived nutrients187. Liberation of this ice-entombed nutrient is controlled by ice sheet hydrology and differs between ice sheets. On the Greenland Ice Sheet, atmospheric nutrients in snow and ice are released to surface meltwater streams in summer melt in the ablation zone187. In zones of ice sheets where surface air temperatures are well below 0 °C , zero surface melting means that this atmospheric nutrient release does not occur until the ice is advected to melting zones of the ice sheet bed. An exception is found in marginal regions, such as the McMurdo Dry Valleys, where solar radiation-driven melting of debris entombed in surface glacier ice causes localised melting and meltwater release to streams24.The annual deposition of snow and subsequent transformation to ice locks up atmospheric-derived nutrients, such as nitrateIce sheet surfaces190. These microbial processes further enhance the nutrient content of meltwaters prior to their discharge either to the ice sheet bed via moulins or at the margin. While nitrate concentrations can be several μM, surface meltwaters generally have much lower concentrations of crustally derived species such as P, Si and Fe8.A wide range of ecosystems, such as cryoconite holes (see photo) on melting ice sheet surfaces, harbour productive microbiota with active nutrient cycling, including nitrogen fixation and nitrificationIce sheet beds191. Such flour is highly geochemically reactive, due to its large surface area: volume ratio and the exposure of reactive minerals such as iron sulphides and carbonates48. Traditionally, chemical weathering beneath ice sheets was assumed to be low to negligible192, but contemporary field studies in glacierised catchments indicate chemical weathering rates that exceed or are similar to the global mean48. Marine-based work using paleo-proxies also indicates that sub-ice sheet chemical dissolution of rock material is enhanced during deglaciation (21–7 kyr B.P) when large expanses of glacial sediments become exposed as the ice retreats194. The chemical weathering of glacial sediments is an important process driving the release of crustal nutrients, such as Si, P, Fe and other micronutrients in dissolved or bioavailable particulate forms. The first reaction to occur upon wetting of glacial flour is carbonate hydrolysis  production of fresh mineral surfaces by glacial grinding; and (2) OM cycling, including that of ancient overridden OM. The immense capacity of glaciers to erode their underlying bedrocks via a combination of plucking and abrasion, generates an abundant supply of very fine rock flour. This gives glacial meltwater rivers a distinctive milky appearance, visible as turbid plumes when they enter downstream fjord and coastal waters producti2 in some cases195), by protons released during the  oxidation of sulphide minerals .Further chemical dissolution, including that of silicate minerals, is fuelled by carbonation reactions 53 and dissimilatory iron reduction33 , advection by ocean currents66, phytoplankton utilisation pathways69 and spatial patterns of phytoplankton nutrient limitation51. Observations of heightened biological activity in marine waters surrounding glaciers has been noted as early as 1938, when brown zones (representing turbid melt plumes) in front of tidewater glaciers were noted to be particularly productive regions for biota71, with more recent research reinforcing this connection72.The fate of glacially exported or cycled nutrients and their impact on marine ecosystems depends upon a complex interplay of physical and biological processes, including proglacial/lake/fjord/estuarine filter effects  which limit phytoplankton in these waters and turbid melt plumes supress productivity in the summer months10. Here, the most important influence of the ice sheet is heralded to be the pumping of N-replete and P-replete deep ocean water to the surface during the upwelling of buoyant subglacial meltwater at marine-terminating glacier margins44. This indirect supply of nutrients has been linked to spring and summer phytoplankton blooms, and supports fisheries in the coastal zone10. An exception may be the direct export of silicon (both dissolved and amorphous particulate phases) in glacial meltwaters83, which has been demonstrated to support diatom blooms in Greenlandic fjords11, corroborating previous studies suggesting high silica concentrations and high diatom abundance in glacially fed fjords and coastal waters84.In contrast, runoff from northern hemisphere ice sheets often enters fjords and complex estuarine environments, modulating total fluxes to the ocean via flocculation and scavenging86. Radium isotopes measured on surface ocean waters off the coast of Greenland strongly suggest the rapid offshore transport of glacial particles into shelf waters and into the open ocean83. Taking these inferences a step further, remote sensing and numerical modelling have showed a strong correlation between the timing and spatial extent of summer phytoplankton blooms in the Labrador Sea and the arrival of Greenland meltwater and subsequent patterns of advection via ocean currents16. The inferred driver for these summer  blooms is the advection of ice sheet derived Fe, which must be transported some 500 km offshore16. These summer/autumn blooms account for 40% of the annual net primary production for the region, indicating a process of regional significance16.While the impacts of glacially exported nutrients upon fjord and coastal productivity around Greenland may be limited, there is mounting evidence that there is a net export of glacial nutrients from land to the open ocean. For example, the concentrations of filterable and particulate iron in the surface waters at the mouths of Greenland fjords and in near coastal regions downstream of glacial meltwater inputs can be orders of magnitude higher than that in the open ocean87 in the 21st century are predicted to be accompanied by rising nutrient and organic carbon export from ice sheets9. However, this increase in glacially sourced nutrient fluxes may not be a simple linear function of the freshwater flux. For example, seasonal records of nutrient fluxes from a large Greenland outlet glacier in extreme melt years demonstrated that a doubling of glacial runoff was accompanied by a similar increase in dissolved nutrient loading8, but sediment-bound fluxes declined due to the intricate (supply-limited) relationship of particulate loading and bulk meltwater discharge8. Decreased glacier size, and hence erosion rates, may in fact also reduce sediment-bound nutrient export to the ocean, as seen for P export from two Greenland catchments of contrasting size42. In addition, expansion of the proglacial zone in land-terminating systems may drive fundamental shifts in microbial community size and composition and additional nutrient cycling steps may occur in proglacial aquatic ecosystems  prior to nutrient export to the ocean89. In regions where marine ice retreats to land, changes in the mechanisms of freshwater (and nutrient) input to ocean waters may occur. Thus, there may be a shift from sub-surface to surface (turbid) meltwater flows10 at the land-ocean margin, and cessation of the entrainment of nutrient-replete ocean water by rising buoyant melt plumes from depth at marine margins. These changes are likely to lead to light limitation and reduced macronutrient (N and P) supply, respectively, in surface waters90. They may explain the high biological productivity and fish catches found in the vicinity of fjords headed by marine terminating glaciers in Greenland compared with fjords dominated by land terminating glacier inputs10.Increasing ice sheet freshwater discharge and glacier retreat22 or imported to the glacier system via wind-blown material 3 or from biomes buried by ice during periods of glacial advance18. The cycling of this OM within glaciers and ice sheets creates the potential for a series of more direct impacts on the global carbon cycle 18 . Such processes of active carbon cycling create a direct link between ice sheets and carbon cycles of the atmosphere and oceans  from the ice sheet, storage in subglacial basins and frozen bed zones, and active respiration via microbial activity to COans Fig.  and are −1 have been plausibly modelled for Antarctic ice stream tributaries101 and rates of up to 5 mm a−1 are inferred at the margins of the Greenland Ice Sheet47. However, erosion rates in inland regions of ice sheets must be much smaller to be compatible with the preservation of sedimentary basins, such as those found in Antarctica even after 30 Ma of glaciation102. Consistent with this are calculated erosion rates in East Antarctica of 0.04 mm a−1 (Slessor Glacier102) and 0.001 mm a−1 (Lambert Basin103). The erosion and export of organic carbon from the polar ice sheets has recently become pertinent to the global carbon cycle, because of the potential for this material to become buried in long term geological carbon sinks104, such as the dark, oxygen-starved bottom waters of fjords105. The global rate of carbon burial in fjords is estimated to be 18 Tg a−1105,, but at many sites the proportions of old vs. newly produced OM are unknown. It is notable that the percentage organic carbon contents reported in Greenland fjord sediments (1.6%)105 are orders of magnitude higher than those recorded in suspended particulate material exported in runoff from large Greenland outlet glaciers (<0.1%)106.Glacial erosion may lead to some OM removal or re-distribution over time beneath ice sheets. Indeed, where ice is warm at the bed and exhibits high rates of flow , erosion rates could be considerable. For example, erosion rates of up to 0.6 mm a−1, including ice-rafted debris (IRD)) compared to the total reserve held in ice or sediments . This is consistent with the observation that the 14C radiocarbon ages of bulk POC associated with suspended particulate material in Greenland runoff are old (5–9 kyr) and become progressively older through a melt season as the snowline retreats inland99. We estimate that the amount of OC liberated via Antarctic subglacial meltwater discharge, is 0.17 Tg C a−1 as DOC and 0.33 Tg C a−1 as POC , which is comparable to previous estimates9  organic carbon on glacier surfaces and in glacial runoff also suggests that glacially exported DOC is also paradoxically ancient108. The source of this ancient DOC has been debated, as has the reason for its high bioavailability in comparison with non-glacial sources of DOC. Ice core data demonstrate a significant contribution of anthropogenic-derived carbon in glacier ice, concurrent with the onset of the industrial era110. The molecular composition of OM within Alaskan and Tibetan glaciers indicates high relative contributions of condensed aromatics, which is consistent with the presence of combustion derived OM 93. Other studies have suggested an alternative explanation for aged DOC in glacial runoff (but not the glacier surface), arguing that it reflects cycling of overridden subglacial material 108 or glacial erosion and associated reactivation of ancient OM in bedrock111. The high bioavailability of DOC emanating from glacial surfaces (and hence runoff) has been hypothesised to reflect OM sourced from the incomplete combustion of fossil fuels enriched in high abundances of nitrogen-rich aliphatic compounds3. However, more recent work also shows the potential role of microorganisms in cycling organic carbon to more bioavailable forms, giving rise to bioavailable DOC both on the glacier surface113 and at the glacier bed114. Future projections indicate that the total magnitude of DOC exported in glacier runoff between now and 2050 will be approximately 48 Tg C as annual glacier mass loss increases, particularly from mountain glaciers9. This increasing flux of glacier DOC provides a subsidy of bioavailable carbon to receiving streams, rivers, estuaries and coastal systems and will be disproportionally impactful in heavily glaciated coastal systems  that are home to commercially important fisheries.Although fluxes of DOC and POC are relatively small on a global scale , a growing number of studies have observed that glacier derived DOC and POC are highly bioavailable4, under the anaerobic conditions that are inferred from a small number of data points recovered from ice sheet beds117. This may be supplemented by CH4 generated during erosion of underlying bedrock or from deep thermogenic sources in geothermically active zones95, such as West Antarctica118. Supersaturated CH4 concentrations of microbial origin have been reported recently in subglacial runoff draining from a small and much larger Greenlandic catchment117, Antarctic Subglacial Lake Whillans116 and in low temperature (1–10 °C) anaerobic incubation experiments of subglacial sediment sampled from a range of glaciers95. Concentrations of CH4  > 15 times greater than atmospheric values have also been recorded in air expelled with meltwater via subglacial channels at the margins of a large glacier in SW Greenland119. However, there is high uncertainty regarding the magnitude of sinks for the CH4 produced, and how much CH4 is oxidised to CO2 before being released to the subglacial drainage system. This would reduce the positive radiative forcing associated with CH4 emissions from ice sheets by approximately a factor of 20. In very long residence time  subglacial hydrological systems, such as Subglacial Lake Whillans, results suggest that most CH4 generated in lake sediments is oxidised to CO2 before reaching the lake water116. However, observations from the Greenland Ice Sheet indicate that if subglacial meltwaters/sediments are transported rapidly via efficient channels to the margin during summer melt, this oxidative sink is much reduced and lateral fluxes of CH4 at the ice margin are high119 and rival those from other world rivers117. This suggests that subglacial hydrology is key in determining how much subglacial CH4 is released to the atmosphere. This uncertainty regarding CH4 export calls for wider study of CH4 production and export from large glacier catchments draining ice sheets.Sedimentary OM beneath ice sheets which is not eroded and exported from the glacier system is available as a substrate for subglacial microbial metabolism. An important fate for sedimentary OM buried beneath the ice is microbial respiration to greenhouse gases, and specifically CH4 in ice sheet runoff at the present day are poorly quantified. Conservative estimates via this manuscript suggest potential fluxes of <1 Tg a−1, employing modelled meltwater discharge from both ice sheets121 and concentrations of CH4 measured in runoff from two end member estimates for subglacial meltwaters (min concentrations = 0.024 μM116 and maximum concentrations of 83 μM115). However, high pressure/low temperature conditions that typify the beds of thick ice sheets should favour methane hydrate formation122, once saturation with respect to CH4 is attained in sediment porewaters95  showed extremely high concentrations of CH4 in sediment cores 128. Such high concentrations were not found in cores further offshore. It is probable that these processes also prevail beneath the ice sheet, as indicated by the supersaturated CH4 concentrations in Subglacial Lake Whillans, West Antarctica116 (0.024 μM in lake waters to up to 300 µM in sediments).Fluxes of CH4 hydrate reserves because of the difficulties of accessing sediments in subglacial sedimentary basins. Global subglacial methane hydrate stocks at the present day are likely to be dominated by those in Antarctic sedimentary basins (estimated at up to 300 Pg C as methane hydrate and free gas95). At the LGM, the global sub-ice sheet hydrate reserve could have been much larger , with hydrate also present beneath former northern hemisphere ice sheets122  that reduces the potential for methane loss via Anaerobic Oxidation of Methane (AOM), frozen conditions creating a seal over the ice sheet bed, and finally glaciation periods of 105–106 years which promotes CH4 accumulation in sediment porewaters.Methane hydrate (see inset image) is one of the three phases in which CH129. The fastest present day relative net mass losses are apparent on mountain glaciers, but non-linear changes and high gross losses are expected for the Greenland and Antarctic ice sheets due to the collapse of floating ice tongues and marine ice sheet instability129. The potential impacts of ice sheet melting upon global sea level are well studied129, but the impacts of thinning ice and enhanced freshwater fluxes upon local, regional and global carbon cycles  impacts of ice sheets on the global carbon cycle during past phases of ice sheet growth and retreat during the Quaternary . To introduce this idea and to provide some testable hypotheses for future work, we turn our attention to the Fe-limited51 Southern Ocean over the last glacial-interglacial transition. Here, the productivity of phytoplankton is limited primarily by the availability of Fe in surface ocean waters, and variations in the Fe supply over glacial-interglacial cycles has been argued as a plausible driver for CO2 drawdown132, via its influence upon the strength of the biological pump which controls carbon export to the deep ocean133. Specifically, enhanced Fe supply to the Sub-Antarctic zone of the Southern Ocean, and associated changes in export production, has been linked to the step decreases in atmospheric CO2 recorded in ice cores during the last glacial period, particularly during Marine Isotope Stages (MIS) 4/5 transition (c. 70 kyr B.P.) and around the LGM137.Climate warming over the last glacial-interglacial transition was accompanied by the disappearance of ice sheets over much of northern Europe and America132, coastal sediments141, hydrothermal inputs142 and more recently, iceberg rafted debris (IRD)143 and meltwater from the Antarctic Ice Sheet144. Inferring the precise mechanism(s) driving Southern Ocean fertilisation at key periods of Earth’s history is challenging, in part due to the overlapping geographical impacts of different Fe sources. For example, inputs of Fe from dust, coastal sediments or icebergs are often concentrated in the Atlantic Sector of the Southern Ocean, via transport off Patagonia  and via ocean currents along the so called iceberg alley adjacent to the Antarctic Peninsula. In general, discerning the importance of each of these potential Fe sources involves correlating past CO2 variations to reconstructions of iron/nutrient supply via elevated Fe inputs (the iron hypothesis132) and associated proxies for export production 147.There are a number of potential Fe sources in the Southern Ocean fertilisation game, all with their own spatial and temporal complexities. These include aeolian dust148. Supporting the dust hypothesis for Southern Ocean fertilisation is the existence of excellent temporal records of past lithogenic fluxes (inferred to reflect dust inputs) to both the ice sheet149 and to marine sediments152, which lend themselves well to correlation with palaeo records of export production and atmospheric CO2149 , and therefore, of potential Fe fertilisation of the Southern Ocean149. The fertilisation impacts of this Fe-bearing dust have been inferred in the SE Pacific and Atlantic (sub-Antarctic) sectors of the Southern Ocean at the LGM and have been linked to elevated rates of opal accumulation and overall export production154  Fe fluxes associated with Antarctic IRD at the present day   inputs are likely limited to the coastal zone15.A new player on the field of Southern Ocean Fe fertilisation is the Antarctic Ice Sheet. Early work on ice sheet contributions focussed upon iceberg rafted debris, which contains bioavailable Fe (oxy)hydroxide nanoparticlesday Fig.  are c. 6160 . However, research on the grain size distribution of debris of glacial sediments, including those entombed in icebergs, often have an important fine  fraction163. Thus, while the presence of larger particles  in marine cores might point towards IRD rather than dust inputs, the presence of silt/clays could reflect inputs from either IRD or dust. Geochemical provenance studies may help elucidate the precise origin of the lithogenic fraction in marine cores150, as may the use of biomarkers. The presence of n-alkanes derived from leaf waxes in the lithogenic material in marine cores is often interpreted as indicative of a dusty source156. While it seems unlikely that leaf waxes are present in sediments beneath the Antarctic Ice Sheet, the presence of these biomarkers in Antarctic IRD has not been evaluated. Finally, biogeochemical models have strong potential to reveal the magnitude of fertilisation impacts that could be possible in a glacial ocean via various Fe inputs, including dust and IRD. Model simulation of dust and ice sheet-sourced Fe impacts on Southern Ocean fertilisation and thus, productivity, have been attempted for a modern ocean and suggest strong influences by ice sheet Fe inputs76. However, these simulations have not been conducted for an LGM ocean. Part of the challenge of conducting such model studies is the grave uncertainty regarding potential Fe fluxes from the ice sheet to the ocean, which need to be better constrained. In summary, determining the role of iceberg-borne Fe in fertilising the Southern Ocean is no simple task, but has the potential to reveal powerful insights regarding the relationship between Fe export from the ice sheet (via melt and icebergs) and export production in the Southern Ocean, which may become more pertinent in a future warming world.It is difficult with the current data available to conclusively evaluate the role of iceberg-associated Fe in fertilising the Southern Ocean during the last glacial period alongside the more widely acclaimed dust. Certainly, the magnitude of present day IRD–Fe fluxes, when combined with the similarity in temporal and spatial patterns of IRD export and those for dust Fig. , suggest4 release. Ice sheet thinning or retreat has the potential to dramatically alter in situ temperature and pressure conditions in sub-ice sheet sediments, triggering methane hydrate destabilisation and release of the resultant CH4 gas to the atmosphere  methane hydrate dissociation at depth, and the latter arise from AOM-driven saturation of sediment porewaters with respect to calcite. These features are contended to reflect CH4 release as the ice overburden was removed during the last deglaciation164 and provide compelling evidence that hydrate forms beneath ice sheets and is destabilised as the ice thins and retreats, in this case over a period of ~10 kyr164. Similar evidence has yet to be uncovered from the Antarctic Ice Sheet, where hydrate reservoirs are predicted at the present day95. Ice thickness reductions in marginal areas of the Antarctic Ice Sheet during the last deglaciation were significant  and sufficient to destabilise potential subglacial hydrate reserves in these areas95. The potential impacts of such release events on atmospheric CH4 concentrations are unclear, and are complicated by the uncertainty regarding the fate of CH4 in marine waters . For example, recent work off the coast of Svalbard has indicated that high CH4 gas fluxes from the seafloor instead stimulate enhanced marine productivity, likely due to indirect transportation of nutrient-rich water and CO2 (from dissociated CH4) from depth. In this case, the negative radiative forcing exceeded positive radiative forcing associated with CH4 release166. Resolving the influence of these opposing influences is important in determining the net impact of subglacial CH4 hydrate destabilisation on atmospheric CO2. If the CH4 is released to the atmosphere during non-linear ice retreat, it could impact the effectiveness of the UN Framework Convention on Climate Change emissions-based agreements to keep climate warming to a minimum of 2 °C by 2100.Turning our attention to potential direct effects of ice sheets upon the global carbon cycle, we examine the geological record for past periods of methane hydrate destabilisation beneath ice sheets, and associated CHere Fig. . Non-lin−1) but have potential regional importance for marine and lacustrine food webs due to the high lability of this material. The important indirect effect of meltwater and iceberg nutrient export upon the carbon cycle, via impacts on marine productivity, is illustrated by modelled 100 s Tg C a−1 of export production in the Southern Ocean associated with iron export from the ice sheet which reduces outgassing from the Southern Ocean by 30%76.The balance of evidence presented in the previous sections supports our opening hypothesis that ice sheets have an important impact on local, regional and global carbon cycles via a suite of direct and indirect effects. The estimated magnitude of these effects is summarised in Fig. 2 recorded in the EPICA Dome C ice core . On the other side of the world, the observation of pock marks aligned to the position of former ice sheets are consistent with the release of CH4 gas flares as the northern hemisphere deglaciated122. Despite these clues from the past, the impact of future climate warming in the Polar regions on the feedbacks between ice sheets and the global carbon cycle is highly uncertain. Predictions suggest that there may be increased fluxes of bioavailable nutrients to the ocean with rising freshwater discharge8 and destabilisation of hydrate reserves beneath ice sheets as ice thins95. These two processes have opposing potential impacts upon atmospheric CO2 concentrations, the former (nutrient fluxes to the ocean) acting as a negative feedback on warming and the latter (hydrate destabilisation) as a positive feedback. Discerning the relative importance of these impacts is hampered by a currently poor representation of ice sheet biogeochemical processes in global biogeochemical models due to gaps in data and understanding.Scrutiny of the geological record also hints that these impacts were greater during periods of rapid ice sheet change over the last glacial-interglacial cycle. For example, a reduction in iceberg fluxes  to the sub-Antarctic sector of the Southern Ocean from 20 to 11 kyr B.P. correlates in timing with the increase in atmospheric CO167. Second, a shift in focus towards the downstream impact of ice sheets will help drive new understanding. This should encompass research spanning the full land-to-ocean continuum, together with paleo-environmental studies that seek to detect past changes in the ocean and/or atmosphere systems in response to biogeochemical perturbations related to ice sheets. Large-scale uncertainty, however, still centres upon the fate of nutrients in fjords, beneath ice shelves and in nearshore coastal environments and in constraining changes to nutrient fluxes from glacial runoff, icebergs and via meltwater induced  upwelling as the freshwater flux increases. Last, coupled biogeochemical models, including feedbacks between the glacial cryosphere, atmosphere and oceans are required to test the sensitivity of carbon sinks or sources to changes in the terrestrial cryosphere. From a field where life was thought absent until two decades ago, the possibility for new discovery is immense, but demands creativity, tenacity and technological investment in order to narrow current uncertainties and to reveal the true role of ice sheets in the global carbon cycle.Thus, high uncertainty surrounds the estimates in Fig. Supplementary Information


The cube of an ice grain’s radius (r3) increased proportionately with time (t), and its slope gave the recrystallization rate (k). There was a significant difference in the IRI efficiency between the samples, and the fungal AFP possessing the activity with the smallest amount (0.27 μM) exhibited an affinity to multiple ice planes. These results suggest that the IRI efficiency is maximized when AFPs bind to a whole set of ice planes.Ice recrystallization is a phenomenon observed as the increase in ice crystal size within an already frozen material. Antifreeze proteins (AFPs), a class of proteins capable of arresting ice crystal growth, are known to inhibit this phenomenon even at sub milli-molar concentrations. A tremendous range in the possible applications of AFPs is hence expected in both medical and industrial fields, while a key determinant of the ice recrystallization inhibition (IRI) is hardly understood. Here, IRI efficiency and ice plane affinity were examined for the wild-type AFPI–III, a defective AFPIII isoform, and a fungal AFP isoform. To simplify the IRI analysis using the formal representation of Ostwald-ripening ( Large ice crystals grow at the expense of smaller ones through the process called “Ice Recrystallization”. The recrystallization rate is evaluated using Ostwald-ripening principle, represented formally by r3 = r03 + kt, where r0 is the radius of an averaged ice grain size at t = 0, and k is the recrystallization rate (μm3·min−1)2. The resultant expansion in size of ice grains physically destroys the texture of any frozen materials from both inside and outside, especially during the freeze-thaw cycle conducted at temperatures around zero4. Antifreeze protein (AFP) and its mimics are known to possess the ability of ice recrystallization inhibition (IRI)7. The mechanism of inhibition and what factors determine a strong IRI efficiency however, are not understood, although many tools and software have been developed to appreciate thousands of ice grains in photomicroscope snapshots12. Here, an alternative method, which monitors just 13–15 ice grains that exhibit only uniform growth, was examined in a video file. Consistency between the IRI efficiency parameter obtained from this method and from previous knowledge is discussed in light of the ice-binding properties of each AFP species.A general ice block is made of an infinite number of single ice crystals, whose size and shape are not uniform14. Examples include AFP I from barfin plaice Liposetta pinnifasciata, an alanine-rich amphipathic α-helical polypeptide (Mw = 3.5 kDa), AFP II from longsnout poacher Brachyopsis rostratus, a disulfide-bond-rich globular protein exhibiting high structural similarity with a carbohydrate-recognition domain of C-type lectin (Mw = 14 kDa), and AFP III from notched-fin eelpout Zoarces elongatus Kner, another globular protein composed of twisted loops folded into triple-strand β-sheets (Mw = 6.5 kDa)17. Fungal AFP was also identified from a mid-latitude snow-mold Typhula ishikariensis, whose 8th isoform (Tis8) exhibited a strong ability of ice binding, and constructs a 7-ladder β-helical structure with a triangular cross-section (Mw = 22 kDa)19. In contrast, a defective isoform of AFPIII, whose A20 is replaced with Leu (A20L), was suggested to bind only weakly to ice crystal surface20. Figure AFPs initially isolated from the polar sea area have also been discovered in various organisms living in mid-latitude waters, whose amino acid sequences are partially differenta1–a3 and c-axes, in which an ice crystal plane is represented by Miller–Bravais indices1. For example, the indices (0001), 21, which leads to the creation of a curved ice front on that plane between the bound AFPs, through the Gibbs–Thomson effect22. Such an ice front is energetically unfavorable for further adsorption of water, and its crystal growth is terminated23. This mechanism lowers the nonequilibrium freezing temperature (Tf) and slightly elevates the equilibrium melting temperature (Tm) of the AFP solution24. The resultant difference between Tf and Tm is termed thermal hysteresis (TH)25.AFPs are commonly capable of binding to a single ice crystal composed water forming a hexagonal unit defined by the 26. By submerging a single ice crystal hemisphere of 2–3-cm in diameter into a vessel containing a solution of fluorescence-labeled AFPs, their adsorptions show specific patterns on the ice hemisphere under UV light. If six equally distant ellipses are illuminated on the equator, they show that the AFP binds to six equivalent prism planes of a single ice crystal. Similarly, the illumination of six ellipses on the mid-latitude implies that the AFP is binding to six equivalent pyramidal planes. Entire illumination of the hemisphere implies that the AFP sample binds to multiple ice planes28. The present study examined the IRI efficiency of the five AFP samples, for which the time-dependent change of specific ice grains was analyzed to elucidate ice recrystallization kinetics during subzero temperature annealing. The relationship with their TH value and the FIPA pattern will be considered.A technique named fluorescence-based ice plane affinity (FIPA) analysis is useful in determining the ice planes to which AFPs bindt) in different manners including; (i) uniform growth with keeping a relatively round shape (indicated by arrows), (ii) shrinkage by melting and disappearing, or (iii) merging to become larger ice grains 30. The (i) and (ii) are known as migratory recrystallization6. These processes progressed consecutively in a variety of combinations to generate a range of diverse single and overlapping crystals. The time-lapse images of AFPI-III, A20L, and Tis8 whose concentration was adjusted to 1.5 μM, are also shown in Fig. 31 originally deduced the Ostwald-ripening formula to describe the kinetic process, whereby diffusion effects cause the precipitation of grains of a second phase in a supersaturated solid solution31. We hence marked specific grains that increase in size by diffusion, while discarding the others that showed signs of the other two processes, (ii) disappearance and (iii) accretive recrystallization. A total of 13–15 ice grains were chosen from the last 20 min of the video files, whose ice volume fraction per all ice crystals are constant with time (t)  was employed for this analysis, which automatically extracts the area (A) and perimeter (P) of the ice grains to calculate their circularity (R = 4 πAP−2), where R = 1 implies perfect roundness. The radius (r) was calculated as the half-length of the longest distance of the ice grains, or the maximum Feret dimeter  of ice grains examined in 1.5, 3.0, 6.0, 8.0, and 10.0 μM solutions of AFPI. The solvent data (40% sucrose) without AFP was also examined. The r3 values increased linearly with time (t), and their slopes represent the ice recrystallization rates (k) (102 μm3·min−1), which became less steep with increasing AFP concentration (μM). A similar linear dependence was also obtained for the AFPII, AFPIII, Tis8, and A20L solutions. In addition to the IRI endpoint, ice recrystallization kinetics has been examined by fitting of a sigmoidal curve of the formula33 to a set of the k values determined at different AFP concentrations37. The semi-log plot version clarifies the lower concentration data, so that an inflection point in this plot has been used for determination of a unique concentration of AFP (Ci) at which it is assumed to occur a turnover from diffusion-limited growth to liquid-to-ice-transfer-limited growth induced by ice-binding of AFP33. The Ci value hence represents the IRI efficiency of an AFP species. Here we observed that all of our data can be fitted by the S-shaped curves in the semi-log plot , 0.60 μM (AFPII), 3.00 μM (AFPIII), 4.69 μM (AFPI), and 7.69 μM (A20L), respectively. Olijve et al.37 evaluated the Ci values of 5.8 and 5.9 μM for recombinant proteins of AFPI and III, respectively, which are comparable to our data37. We also estimated 0.06 μM of the Ci value for native AFGP sample, for which we assumed an averaged molecular weight of 15 kDa although not clarified  or Tris-buffered saline (150 mM NaCl and 10 mM Tris-HCl) (pH 7.5), leading to a lower IRI endpoint at just 98 nM and 780 nM, respectively38. They also reported an extremely small value (0.16 μM) of the IRI endpoint for Sea raven AFPII with this the Tris-buffered saline, which also agree with the high IRI activity obtained for AFPII in the present study. Dissolved ions were assumed to recruit water molecules around themselves and reduce the amount of freezable waters that can join the ice crystal38, which may reduce the AFP consumption to halt the ice growth.The known methods evaluated an averaged ice grain size from time-lapse snapshots from photomicroscope images, and determined the minimum AFP concentration necessary to halt ice recrystallizationlot Fig. , implyinTf and Tm of the solution, for which two types of techniques have been developed. One way is to observe a single ice crystal with a photomicroscope, where the stage temperature is computer-controlled to allow direct measurement of the growth-initiation temperature (Tf) and the melting temperature (Tm) of a single ice crystal. The other method is to detect the latent heat emission at the moments when the water freezes or melts, without observing the ice crystrals. Examples of the former method are to use a nanoliter osmometer or the previously reported photomicroscope system40, and the latter method includes differential scanning calorimetry and a sonocrystallization method42. In the latter method, heat emisson originates from an uncontrolled number of single ice crystals born in a solution at the moment of freezing. The number of surface-bound-AFPs per ice crystal will be hence be fewer, leading to smaller TH values compared with those from a photomicroscope-based method37. Here, we evaluated TH values of AFPI–III, A20L, and Tis8 from Tf and Tm determined by observation of a single ice crystal  were adjusted throughout the experiments. The highest TH value (2.2 °C) was obtained for Tis8, a hyperactive species of AFP19, which is 2–3 fold greater than that of AFPII (1.1 °C), AFPIII (0.9 °C) and AFPI (0.7 °C). These values are in good agreement with the TH values reported previously25. For A20L, its growth inhibition ability was not enough to arrest ice growth, no TH value was evaluated. Tis8 exhibited the strongest IRI  and constructs an α-helical structure13. On the basis of this property, AFPI protrudes four OH-groups on one side of this molecule at intervals of 16.5 Å, indicated by the yellow box in Fig. 14. Two IBSs bound to the  for each AFP sample (Supplementary Table 2) > AFPII (1149 Å2) > AFPIII (820 Å2) > A20L (696 Å2) > AFPI (564 Å2)43. The ice-binding mechanism has recently been attributed to a semi-clathrate water network on the protein, but not the amino acid residues constructing the IBS; it merges with and freezes to a disordered water layer constructing the ice crystal surface46. Such water network may determine the ice-binding specificity of the present AFPs characterized based on the ice crystal morphology and the FIPA pattern.The mechanism of ice-binding has been thought to be based on the tertiary structure of the AFPs Fig. . For exa47. A single ice crystal, which is composed of hexagonally arranged waters, is rounded to form an ice disk without AFP  is much lower than that concentration. These results suggest that (1) AFP can shape ice crystals into hexagonal plates even in the presence of 40% sucrose, and (2) IRI activity is aroused by extremely small amounts of AFP that can modify the shape of ice crystals from disks to hexgonoal plates  of AFPIII mixed with known cryoprotectants improves the viability of cells and tissues after frozen storage52. The excess amount of AFPs are generally harmful, which might be due to the sharpened edges of the ice crystals formed at higher AFP concentrations  or a3  are observed. For secondary prism planes . These homogenates were used as the source materials for our protein purification. The recombinant isoforms of fish AFPIII whose A20 is replaced with Leu (A20L) as well as a fungal AFP isoform from snow-mold Typhura ishikariensis (Tis8) were prepared using standard overlap extension PCR methodologies19. Briefly, Escherichia coli BL21 (DE3) containing each expression vector was cultured with Luria–Bertani medium, and the products were purified via cation-exchange chromatography  with a linear NaCl gradient (0–0.5 M) using 10 mM acidic buffer (pH 3.0). The five AFP samples  were lyophilized after dialysis against Milli-Q water for overnight, whose purity was checked with SDS-PAGE and silver staining. Each sample was dissolved in degassed Milli-Q water containing 40% sucrose for the IRI measurements.The samples of AFP I–III were purified from the barfin plaice, longsnout poacher, and notched-fin eelpout, respectivelyet al. 40. This system is composed of a Leica DMLB 100 photomicroscope  equipped with a cooling stage, whose temperature was manipulated between −196 °C and +25 °C with an accuracy of ±0.2 °C using a Linkam THMS600 temperature controller . The ice crystal images were captured using a color-video 3CCD camera , and the temperature status was simultaneously viewed on a display to be saved as a video file. A 1 μl droplet of each AFP solution containing 40% (W/V) sucrose was sandwiched by two cover slips (ϕ = 13 mm) and set onto the stage. This sample was then frozen entirely by decreasing the temperature to −40 °C at a rate of −20 °C/min, and heating to −6 °C at a rate of 10 °C/min. Following exactly the same temperature manipulation, a video was captured to monitor the IRI process of the ice crystals at the temperature of −6 °C.The ability of IRI was examined with a photomicroscope system described by Takamichi Tf and Tm values, respectively. A difference between the two temperatures was estimated as the TH value 40.For evaluation of the TH value, the entirely frozen sample on the photomicroscope stage was heated until one single ice crystal was separately observed in the 1 μl AFP solution. The single ice crystal was then carefully cooled down or warmed up to observe its growth initiation or melting processes, whose critical temperatures were used as the nonequilibrium 26. Briefly, a macroscopic single ice crystal of 2–3 cm diameter was initially prepared in a cylindrical mold. Following determination of its c-axis using a polarizer, a half-cut of this cylindrical crystal with a known orientation was mounted on a hollow copper tube (ϕ = 15 mm), in which −0.8 °C coolant was circulated by a refrigerant pump, Hitachi AMS-007 . A method called “ice pitting” generated a six-sided star mark on the polar region of the single ice crystal hemisphere, which indicates its a1–a3 directions. The hemisphere with known orientation is then mounted onto a chilled probe, so as to face the desired ice plane down. Following 1–2 hours incubation with a 0.1 mg/ml solution of a fluorescence-labeled AFP sample, the FIPA pattern illuminated on the ice crystal hemisphere was observed under UV light.The fluorescence-based ice plane affinity was also examined for the studied AFP samples according to the procedures described previouslySupplementary Information


Understanding how transcription factors (TFs) regulate mammalian gene expression in space and time is a central topic in biology. To activate a gene, a TF has first to diffuse in the available space of the nucleus until it reaches a target DNA sequence or protein (target site). This eventually results in the recruitment of the whole transcriptional machinery. All these processes take place in the mammalian nucleoplasm, a highly organized and dynamic environment, in which some complexes transiently assemble and break apart, whereas others appear more stable. This diversity of dynamic behaviors arises from the number of biomolecules that make up the nucleoplasm and their pairwise interactions. Indeed, interactions energies that span several orders of magnitude, from covalent bounds to transient and dynamic interactions, can shape nuclear landscapes. Thus, the nuclear environment determines how frequently and how fast a TF contacts its target site, and it indirectly regulates gene expression. How exactly transient interactions are involved in the regulation of TF diffusion is unclear, but are reflected by live cell imaging techniques, including single-particle tracking (SPT). Overall, the macroscopic result of these microscopic interactions is almost always anomalous diffusion, a phenomenon widely studied and modeled. Here, we review the connections between the anomalous diffusion of a TF observed by SPT and the microscopic organization of the nucleus, including recently described topologically associated domains and dynamic phase-separated compartments. We propose that anomalous diffusion found in SPT data result from weak and transient interactions with dynamic nuclear substructures, and that SPT data analysis would benefit from a better description of such structures. Mammalian gene expression and its regulation take place in the nucleus, a highly complex and subcompartmented organelle. Interactions strengths between nuclear constituents span several orders of magnitude, from covalent bounds to ‘strong’ noncovalent interactions. These interactions lead to the formation of macromolecular structures, either stable , right.cis-regulatory sequences such as enhancers  affect the kinetic constants uchowski  that theSince  on DNA, –8) or , lamina-associated domains, nucleoli, noncoding RNAs, transcription factories, phase-separated domains, etc. They constitute substructures with a high valency amenable to weak interactions that can qualitatively influence diffusion and target search.Here, we first review anomalous diffusion models applied to protein motion and relate them to a potential physical generative model. Then, we emphasize recent advances in the characterization of regions of reduced dimensionality in mammalian nuclei, both aspecific through volume exclusion and specific through transient, weak-but-specific interactions. Finally, we propose that these weak interactions shape TF dynamics, and that single-particle tracking (SPT) analysis would greatly benefit from a better understanding of the pairwise interaction map between nuclear proteins.The technique of choice to investigate protein motion in the nucleus of live cells is light microscopy of fluorescently tagged proteins. Different imaging and modeling modalities have provided significant insights, including fluorescence recovery after photobleaching (FRAP), fluorescence correlation microscopy (FCS) or SPT. In this review, we focus on SPT, because it directly provides access to the dynamics of single diffusing molecules. In a SPT experiment, a small subset of the proteins of interest are imaged and tracked over short times at the resolution of a few tens of nanometers, allowing to resolve isolated single molecules even within clusters of high densities.η) through the Stokes–Einstein relationship In solution, the diffusion coefficient lar mass ,14, it iThus, the diffusion of TFs cannot be described by simple friction/viscosity relationships, and their behavior, perhaps unsurprisingly, has to be seen from the angle of transient interactions with a dense matrix of interactants. In the context of this review, we define transient (or ‘weak’) interactions as interactions that are usually too short-lived to be captured by traditional biochemistry techniques that typically involve one or several wash steps, during which proteins interacting specifically but transiently get diluted and washed out.α is called the anomalous diffusion exponent. This anomalous diffusion exponent does not fully characterizes the type of diffusion and additional metrics, such as the distribution of translocation angles ∼tn angles  20,21] ,21 (t)∼tn angles  22–24].–24.(t)∼treduced dimensionality.From a physical perspective, proteins can adsorb and diffuse on nuclear substructures. When this happens, the exploration properties of the protein are universally given by two parameters: first, the dimension of the random walk fractals . For insDepending on ncompact –27. In aStructures of reduced dimensionality, including fractals, emerge naturally from various processes, including diffusion-limited aggregation and hierarchical assembly of macromolecular scaffolds, such as the multi-scale organization of chromatin. The goal of the next sections is to highlight a few structures of reduced dimensionality in the nucleus and how they influence kinetics of TFs.Far from constituting a homogeneous medium, the nucleus is a highly organized and subcompartmentalized organelle. The main organizing structure, chromatin, constitutes ∼10–30% of the nuclear volume ,29 and pOver the past years, organizing principles of chromatin have emerged: at large scale, the genome is segregated in chromosome territories and regions of heterochromatin/euchromatin, lamina-associated domains at the periphery and nucleoli lying more at the center. At higher magnification, chromatin is organized in areas of preferential interactions such as A/B compartments and TADs that reflect the functional organization of chromatin .Overall, although highly heterogeneous, chromatin in the mammalian nucleus is well described by a self-similar, fractal structure that occupies a nonzero volume. This was initially postulated , and latAs a consequence, factors diffusing in the available volume are constrained by this structure , possiblα) is mild for proteins of weight <150 kDa . Indeed, FRAP experiments performed with protein or nonprotein tracers of increasing molecular mass suggest that low molecular mass tracers diffuse almost freely in the nucleus, allowing to infer a viscosity close to the one of water [Several lines of argument point to the fact that although steric hindrance induces a significant decrease in the diffusion coefficient  win vitro or in vivo is quickly growing, supporting the vision that the emergence of structures of reduced dimensionality is closer to a general organizing principle than an anecdotal biophysical phenomenon, and some of them were linked with transcriptional regulation [Unlike inert tracers whose diffusion is only determined by volume exclusion, proteins have both a relevant shape and charge pattern that determine their interaction landscape and thus their diffusive properties. These noncovalent interactions are obviously crucial to form biochemically stable complexes such as the transcription preinitiation complex or the spliceosome , left, bgulation ,54–57.multivalent partner that can nucleate the aggregation. As such, many abundant constituents of the mammalian nucleus have been shown to nucleate a structure of reduced dimensionality. These structures include the formation of nucleoli in which diffusion is highly constrained, phase-separation of heterochromatin protein 1 domains  and  and multns  they usually exist as an extremely dynamic equilibrium rather than a stable structure ,64,73, aThe characterization of structures of reduced dimensionality emerging from weak interactions is still in its infancy, but appears more and more strongly as a clear organizing principle of mammalian nuclei. These structures create the matrix upon which fast-diffusing factors can specifically and transiently bind, diffuse and unbind, thus dynamically shaping the ‘diffusion landscape’ of the whole transcriptional machinery.Even though live imaging approaches specifically characterize the behavior of one single factor, they are blind to all these substructures. Indeed, SPT reflects the dynamics of proteins transiently interacting with those structures of reduced dimensionality and one TF potentially visits several of them in the span of a few tens of milliseconds . Such coIn a complex mammalian nucleus, the diffusion of a TF is ruled by transient interactions with underlying structures of reduced dimensionality. From a more general perspective, the question arises of how gene expression regulation processes relate to the multiplicity of structures of reduced dimensionality?Proteins often harbor several domains , holdingFurthermore, structures of reduced dimensionality have been proved to be functionally relevant. For example, the dynamic and regulated switching of a TF between structures of reduced dimensionality determines its dynamics  and funcAlthough theoretical and experimental support for the importance of weak interactions as an architectural principle of the nucleus and gene expression regulation is being actively investigated, several questions remain unaddressed:First, how many distinct types of structures of reduced dimensionality exist? Since the numbers of types of low-complexity domains are likely to be limited, one can expect that a limited number of such structures actually coexist at a given time in a nucleus . This imSecond, can we determine the pairwise interaction matrix between low-complexity protein domains, which would allow us to derive predictive dynamics of a given TF modification? Ideally, such matrix will encompass all known low-complexity domains, but also abundant multivalent RNAs and DNA sequences, and each element of this matrix will reflect the affinity between two domains under physiological conditions .Third, how much detail is required to describe these structures of reduced dimensionality? Is the pairwise interaction between protein domains a good approximation of the properties of the nucleus? Conversely, one can imagine substructures of reduced dimensionality arising from interactions more complex than simple pairwise-interactions. Indeed, it is widely known that cooperativity plays a role in the assembly of many more or less stable macromolecular structures , includiFourth, how do key biological transitions such as differentiation intertwine with these structures of reduced dimensionality? In a similar way, as pluripotency or cell-type specific TF networks have been identified, can pluripotency or cell-type specific structures of reduced dimensionality be evidenced, integrating the expression levels of TFs and providing a framework to better understand such key processes?To answer those questions, our understanding of nuclear processes need to be drastically expanded. Hitherto, a dynamic picture of spatially segregated factors, together with their interaction matrix, is currently missing. Promising tools to access those parameters include quantitative FRET , in cellAlthough the so far identified key players in gene expression regulation are biochemically stable complexes that can be purified using traditional methods, increasing evidence suggest that higher-order, weaker-interaction structures, acting as structures of reduced dimensionality, play a central role in transcriptional regulation. They do so by providing a remarkably versatile way of specifically and timely regulating TF target search dynamics and thus gene expression. All in all, it appears that the functional properties of the nucleus are shaped by not only stable macromolecular complexes but also transient structures (arising from a continuum of weak interactions that might seem spurious). In this context, the saying from Heraclitus makes probably more sense than ever: ‘The fairest order in the world is alike a heap of random sweepings’.


Flavonoids are natural compounds that have been extensively studied due to their positive effects on human health. There are over 4000 flavonoids found in higher plants and their beneficial effects have been shown in vitro as well as in vivo. However, data on their pharmacokinetics and influence on metabolic enzymes is scarce. The aim of this study was to focus on possible interactions between the 30 most commonly encountered flavonoid aglycones on the metabolic activity of CYP3A4 enzyme. 6β-hydroxylation of testosterone was used as marker reaction of CYP3A4 activity. Generated product was determined by HPLC coupled with diode array detector. Metabolism and time dependence, as well as direct inhibition, were tested to determine if inhibition was reversible and/or irreversible. Out of the 30 flavonoids tested, 7 significantly inhibited CYP3A4, most prominent being acacetin that inhibited 95% of enzyme activity at 1 µM concentration. Apigenin showed reversible inhibition, acacetin, and chrysin showed combined irreversible and reversible inhibition while chrysin dimethylether, isorhamnetin, pinocembrin, and tangeretin showed pure irreversible inhibition. These results alert on possible flavonoid–drug interactions on the level of CYP3A4. Flavonoids are natural compounds abundantly present in nature, widely distributed in seeds, leaves, and flowers of plants. There are over 4000 flavonoids identified to date, which makes them one of the largest groups of natural products known. They present benzo-γ-pyrone derivatives consisting of phenolic and pyrane rings . Based oO-methylation can influence metabolic faith and their in vitro activity [It is well-known that the polyphenolic compounds, in particular flavonoids, show various beneficial effects on the human health, that have been demonstrated by various studies. Amongst the many promising health benefits, flavonoids have important biological activities—such as antiallergic, anti-inflammatory, antioxidative, antimicrobial, antitumorigenic, and antimutagenic—thus preventing cancer, heart disease, bone loss, and a number of diseases ,5,6,7,8.activity .As many xenobiotics, including flavonoids, are metabolized in vivo, they can interfere with the activity of the metabolic enzymes in the human liver and cause potential risks of drug–herbal drug interactions ,17,18,19The aim of the present study was to evaluate the inhibitory effect of thirty flavonoid aglycones, most commonly found in herbal drugs and propolis, on the most important drug metabolizing enzyme CYP3A4. Although flavonoids are found mainly as glycosides in natural sources, they are susceptible to hydrolysis in gut . Thus, tFor enzyme activity assessment testosterone 6β-hydroxylation was used as it represents a marker reaction of CYP3A4 activity. Generated product/metabolite was monitored by high performance liquid chromatography coupled with diode array detector (HPLC-DAD). If the inhibitory effect has been observed, the type of inhibition was determined as this can have repercussion on the possible drug–flavonoid interactions.p < 0.001).To test the CYP3A4 enzyme inhibition, residual activity was assessed based on the product of the marker reaction generated by monitoring incubations with addition of flavonoid, as a potential inhibitor, vs. incubations without inhibitor. p < 0.05.Screening of all flavonoids was conducted against metabolism dependent inhibition experimental setup, as it includes preincubation required for time dependent inhibition, as well as incubation of flavonoids and marker substrate at the same time, consequently detecting direct inhibition. Seven flavonoids have been shown to inhibit CYP3A4 with statistical significance Flavanones acacetin and pinocembrin decreased enzyme activity by 95% and 50% respectively, acacetin being the most prominent inhibitor . AcacetiPinocembrin has been extensively studied due to its positive effects on cardiovascular system. This flavanone protects endothelial cells from oxidized LDL-induced injury, and has neuroprotective effects on ischemia/reperfusion-induced brain injury by inhibiting autophagy ,37. AlthResidual activity of CYP3A4 after incubations with flavones apigenin, chrysin, chrysin dimethyl ether, and tangerine was 24%, 17%, 61%, and 42% . ApigeniChrysin dimethylether has been reported to have antitrypanosomal and antileishmanial properties while chrysin inhibits diabetic renal tubulointerstitial fibrosis, xanthine oxidase activity, and foam cell formation having potentially positive effects in diabetes, gout, and atherosclerosis ,47,48,49Tangeretin, a component of orange juice, has been reported to have positive effects on human health that include antioxidant, antiviral, and anticancer properties ,52,53. QIsorhamnetin was the only flavonol to show inhibitory effect decreasing residual enzyme activity to 73% . IsorhamInterestingly, stimulation of enzyme activity of CYP3A4 was observed with tamarixetin; observed residual activity was 195% . TamarixLooking at the structural features responsible for the inhibitory effect , it can 2+ form occurred, giving sufficient time for the potential inhibitor to interact with the enzyme.To distinguish if the observed metabolism dependent inhibition (of the seven confirmed inhibitors of CYP3A4 in this study) was a result of time dependent inhibition, in which additional time is needed for the inhibitor to interact with enzyme to cause the inhibition, preincubations were conducted without presence of NADPH coenzyme. In this way, no reduction of CYP3A4 heme iron to Fep = 0.047), 34 ± 10% (p = 0.005), and 45 ± 3% (p = 0.019), respectively , 35 ± 18% (p = 0.049), and 54 ± 23% (p = 0.044), respectively , and chrysin dimethylether, isorhamnetin, pinocembrin, and tangeretin (pure irreversible inhibitors). Irreversible inhibition cannot be overcome by discontinuation and substitution of a medicinal drug, as the enzyme is inactivated and time is needed for a new enzyme to be re-expressed. As the half-life of cytochromes P450 is between 24 and 72 h, regeneration of enzyme activity can take up to two weeks, preventing successful therapy. This is especially of importance in cases where medicinal drug is exclusively metabolized by inhibited enzyme or inhibited enzyme is susceptible to genetic polymorphism, as well as concomitant use of drugs and flavonoid rich foods metabolizing through same enzyme due to possible drug–flavonoid interactions .A significant number of papers on metabolism of flavonoids and their inhibition of metabolic enzymes has been published ,61,64,65Thirty flavonoids used in this study  were acquired commercially from Sigma-Aldrich , TransMIT GmbH , Extrasynthèse . For the evaluation of activity of CYP3A4 testosterone was used as marker substrate (Sigma-Aldrich). 6β-hydroxytestosterone was purchased from Cayman Europe . Positive controls used to test direct inhibition (ketoconazole) and irreversible inhibition (troleandomycin) were obtained from Sigma-Aldrich .5, was used as source of enzyme. Baculosomes were commercially obtained from Thermo Fisher Scientific , and all other chemicals from Sigma-Aldrich if not otherwise indicated. A 1.0 M solution phosphate buffer of pH 7.4 was prepared in house. NADPH generating system was prepared of 0.1 M glucose-6-phosphate, 10 mg mL−1 NADP+, and 1000 IU mL−1 glucose-6-phosphate dehydrogenase in ratio 100:50:2 (v/v/v) [The recombinant baculosomes with hyperexpressed CYP3A4 and coexpressed NADPH cytochrome P450 reductase and cytochrome b (v/v/v) .g for 10 min. The organic layer was transferred, and the solvent was evaporated under a stream of nitrogen. The dried sample was dissolved in methanol for HPLC analysis [6β-Hydroxylation of testosterone was used as marker reaction to monitor activity of the CYP3A4 enzyme . Incubatanalysis .Three types of experiments were conducted to determine metabolism dependent inhibition, time dependent inhibition, and direct inhibition . To deteIf metabolism-dependent inhibition was determined, time dependent inhibition was assessed by preincubating flavonoids and enzymes, after which residual activity was determined by adding NADPH generating system along with substrate. In this instance, the direct inhibition was also tested without preincubation, i.e., NADPH generating system, was added to incubation mixture containing flavonoid and substrate .−1 Chromatograms were recorded at 240 nm, and the amount of generated product (6β-hydroxytestosterone) was determined as the area under the curve based on the calibration curve of the standard [HPLC analysis was performed on Agilent 1100 instrument  coupled with diode array detector on Agilent Zorbax SB C18 column . The mobile phase used consisted of 64% methanol and 36% water; analysis was isocratic at a flow rate of 1.0 mL minstandard .t-test in the program R . Mann–Whitney U-test was used to test normality (p = 0.508) and Levene’s test homogeneity (p = 0.882) of the inhibitory data for apigenin justifying the use of the selected statistical analysis.All incubations were conducted in triplicate. The results are expressed as residual activity of the enzyme, i.e., percentage of product generated in incubation with the addition of flavonoid in ratio to the control without flavonoid. Statistical significance was tested with Student’s Out of 30 flavonoids screened for potential CYP3A4 enzyme inhibition, 7 showed statistically significant inhibition. Apigenin showed reversible inhibition; acacetin and chrysin showed combined irreversible and reversible inhibition; while chrysin dimethylether, isorhamnetin, pinocembrin, and tangeretin showed pure irreversible inhibition. These results bring attention to possible flavonoid–drug interactions on the level of CYP3A4. As CYP3A4 is the most significant enzyme for the metabolism of xenobiotics including drugs, further in vivo studies are needed to assess clinical significance of the observed inhibitions, and possible drug–flavonoid interactions.


Permafrost contains one of the least known soil microbiomes, where microbial populations reside in an ice-locked environment. Here, 56 prokaryotic metagenome-assembled genome (MAG) sequences from 13 phyla are reported. These MAGs will provide information on metabolic pathways that could mediate biogeochemical cycles in Svalbard permafrost. Permafrost contains one of the least known soil microbiomes, where microbial populations reside in an ice-locked environment. Here, 56 prokaryotic metagenome-assembled genome (MAG) sequences from 13 phyla are reported. These MAGs will provide information on metabolic pathways that could mediate biogeochemical cycles in Svalbard permafrost. Permafrost covers over 25% of the exposed land surface of the Northern Hemisphere and hosts a diversity of microbes proposed to be unique to cold habitats . These f1–Soil samples were obtained from an ice-wedge polygon site in the Adventdalen Valley in Svalbard, Norway . The site soil geochemistry was described previously , 1; candidate phylum Levybacteria, 1; Firmicutes, 1; Nitrospirae, 1; and Thaumarchaeota, 1  database under the study number


Habranthus jamesonii, Phycella herbertiana, Rhodophiala mendocina and Zephyranthes filifolia collected in the Argentinian Andean region all contained galanthamine, and showed a strong AChE inhibitory activity (IC50 between 1.2 and 2 µg/mL). To our knowledge, no previous reports on alkaloid profiles and AChEIs activity of wild Argentinian Amarillydaceae species have been publisihed. The demand for renewable sources of industrial products like galanthamine and the need to protect plant biodiversity creates an opportunity for Argentinian farmers to produce such crops.The Amaryllidaceae family is well known for its pharmacologically active alkaloids. An important approach to treat Alzheimer’s disease involves the inhibition of the enzyme acetylcholinesterase (AChE). Galanthamine, an Amaryllidaceae alkaloid, is an effective, selective, reversible, and competitive AChE inhibitor. This work was aimed at studying the alkaloid composition of four wild Argentinian Amarillydaceae species for the first time, as well as analyzing their inhibitory activity on acetylcholinesterase. Alkaloid content was characterized by means of GC-MS analysis. Chloroform basic extracts from  Taxus brevifolia), vinblastine from the Madagascar periwinkle (Cataranthus roseus), and digoxin from the foxglove  [Many species of medicinal and aromatic plants are cultivated for such industrial uses, but most are still collected in the wild. The demand for renewable sources of industrial products and the need to protect plant biodiversity create an opportunity for farmers to produce such plants as crops. More than 25% of the pharmaceutical drugs used in the World today are derived from plant natural products . In the  lanata) .®), was originally isolated from Galanthus woronowii. While several total syntheses of the alkaloid galanthamine are available [Leucojum aestivum from Turkey , or from small plantations of this species in Bulgaria, which are insufficient to meet current pharmaceutical company demand. The worldwide production of galanthamine is about 250 kg per year. Around 61 species of the Amaryllidaceae family grow in Argentina, covering a wide variety of genera  [The alkaloids of the Amaryllidaceae family are extensively studied for their biological activities in several pharmaceutical areas, for example, Alzheimer’s disease (AD), a neurodegenerative problem of enormous economic and social impact . The treatment is based on drugs that increase levels of acetylcholine. Galanthamine is a long-acting, selective, reversible and competitive inhibitor of acetylcholinesterase (AChE) and an allosteric modulator of the neuronal nicotinic receptor for acetylcholine. AChE is responsible for the degradation of acetylcholine at the neuromuscular junction, in peripheral and central cholinergic synapses. Galanthamine has the ability to cross the blood-brain barrier and to act within the central nervous system ,4. Accorvailable ,7,8,9,10ranthes) . To our Habranthus jamesonii, Phycella herbertiana, Rhodophiala mendocina, and Zephyranthes filifolia (Amaryllidaceae species that grow in Argentine) to find new sources of production of galanthamine, and other potential alkaloids for treating AD. AChE inhibitory activity was determined by the spectrophotometric method by Ellman et al. [Our search for plant raw materials for medicinal products is now aimed at investigating the acetylcholinesterase inhibitory activity (AChE) of basic chloroform extracts (BCE) obtained from n et al. . AlkaloiHabranthus jamesonii, Phycella herbertiana, Rodophiala mendocina and Zephyranthes filifolia species, collected from the Andean region of San Juan (SJ), Mendoza (MDZ), and Neuquén (NQN) provinces (Argentine), were tested according to the methodology developed by Ellman et al. [50 values (µg/mL) are shown in Table 1. BCE showed the highest acetylcholinesterase inhibitory activity, with IC50 values ranging from 1 to 2 µg/mL . The BCE-Z. filifolia MZA and BCE-H. jamesonii SJ displayed the highest inhibition towards AChE with similar values (IC50 1 ± 0.01 and 1 ± 0.08 µg/mL respectively) only three times higher than that of galanthamine. BCE-P. herbertiana SJ was found to have the second highest inhibition on AChE . Acetylcholinesterase inhibition was similar to that of specie R. mendocina regardless of collection site . BCE-H. jamesonii SJ showed a similar AChE inhibitory activity . The yield percentages of the basic chloroform extract (BCE) (g/100 g dry bulbs) are reported in Zephyranthes filifolia SJ had the lowest percentage at 0.21%, whereas BCE-Rhodophiala mendocina SJ gave the highest one at 0.38%.The AChE inhibitory activity of the BCE from n et al.  with somn et al. . GalanthH. jamesonii MZA, BCE-H. jamesonii SJ, BCE-P. herbertiana SJ, BCE-R. mendocina NQN, BCE-R. mendocina SJ and BCE-Z. filifolia SJ are listed in The alkaloids detected by GC-MS in the BCE-4) was found in all the species and it ranged from 0.6 to 17.8% of total ion current (TIC). Zephyranthes filifolia presented the highest galanthamine (4) content . The highest AChE inhibitory activity of these species (IC50 1.0 ± 0.08 µg/mL) belonged to BCE-Z. filifolia SJ a fact that could be related to the high content of galanthamine (4).Galanthamine (4) content was collection site dependent: the BCE-H. jamesonii SJ sample presented 1.4% galanthamine TIC, while the BCE-H. jamesonii MZA with 4.3% TIC was four times higher. The differences in alkaloid content, depending on geographical distribution of H. jamesonii populations, coincides with a previous report on the European species. Berkov et al. [Galanthus elwesii and G. nivalis populations collected in different locations in Bulgaria. They presented galanthamine TIC between 0 and 46%. The main alkaloid types (chemotypes) showed a wide variation in the number of compounds comprising their alkaloid mixture. Genetic and environmental factors and their interaction play a role in determining alkaloid profiles.Galanthamine , identified in BCE-H. jamesonii MZA (0.5% TIC), has a hydroxyl group at C9 instead of a methoxyl group, and is around 10 times more active than galanthamine (4). Although the differential content could indicate that some environmental parameters might be influencing galanthamine (4) production, this specie could be considered for the sustainable production of galanthamine. A similar galanthamine (4) content (4.2% TIC) has been found in BCE-P. herbertiana SJ. BCE-R. mendocina SJ and BCE-R. mendocina NQN, showed a similar galanthamine content (<1% TIC). Narwedine (8), another AChE inhibitor [H. jamesonii and R. mendocina collected in San Juan province), but this compound comprised no more than 1% TIC. Other main alkaloids characterized by GC-MS in the BCE-P. herbertiana SJ were lycorine (21) (33%), lycoramine (5) (27%) and tazettine (16) (5.4%).Additionally, sanguinine (nhibitor  was foun1), galanthamine (4), lycoramine (5), vittatine (7), anhydrolycorine (9), montanine (14), haemanthamine/crinamine (15), tazettine (16), 11-hydroxyvittatine (19), galanthine (20), lycorine (21) and tazettamide (29) are reported for the first time in Habranthus jamesonii from Argentina. According to the literature, haemanthamine (15) and galanthine (20), have been reported previously in Habranthus brachyandrus, a specie of the genus [R. mendocina SJ, whereas five of them montanine (14), vittatine (7), haemanthamine (15), tazettine (16) and lycorine (21) have been previously reported as constituents of Rhodophiala bifida [The occurrence of trisphaeridine  and tazettine (16) as percentage of the total ion current (TIC) is consistent with that observed in TLC alkaloid profiles of chloroform basic extract  has shown significant AChE inhibitory activity [21) and haemanthamine (15) [5) and tazettine (16) have no AChE inhibitory activity [20), 11-hydroxyvittatine, 2-O-acetyllycorine (27). The AChE inhibitory activity of these species can be explained by the presence of other AChE inhibitors in the alkaloid mixtures. Montanine  . The othactivity  while, t15) is a potent inducer of apoptosis [7) has shown cytotoxic activity [7) and 11-hydroxyvittatine (19) [21) exhibits citotoxic, apoptotic, antiviral, antifungal, anti-protozoan [20) [16) [10, 11, 12, 13, 17, 26, 28, 30, 32, 33, 34, and 35) were detected in minor quantities, showing mass spectral patterns that also suggest structures related to the Amaryllidaceae alkaloids. Isolation studies are currently being developed.The alkaloids found in the Argentinian species studied possess other interesting biological properties besides their AChE inhibitory activity. Haemanthamine  . Lycorinrotozoan , and antrotozoan . It is arotozoan .Analgesioan [20) . Moderat20) [16)  which is20) [16) . In addiHabranthus jamesonii (BAK) Rav, Phycella herbertiana LINDL, Rhodophiala mendocina (PHIL.) Rav., and Zephyranthes filifolia (HERB.)ex Kraenzlin (Amaryllidaceae) were collected in the Andean regions of San Juan (SJ), Mendoza (MZA), and Neuquén (NQN) provinces (Argentina), during the flowering period between October and March 2009-2010 and then transferred to flowerpots and kept under greenhouse conditions. The species collected and identified, and voucher numbers are shown in Wild plants of the species 2SO4 (2% v/v) and neutral material was removed with CHCl3 (200 mL). Then, the aqueous solutions were basified with 25% NaOH up to pH 10-12 and the alkaloids were extracted with CHCl3 (3 ´ 500 mL) to obtain the basic chloroform extract (BCE). After evaporation of the organic solvent, the dry alkaloid fractions were dissolved in MeOH for GC/MS analysis. The BCE were named as BCE-H. jamesonii MZA, BCE-H. jamesonii SJ, BCE-P. herbertiana SJ, BCE-R. mendocina NQN, BCE-R. mendocina SJ and BCE-Z. filifolia SJ after their origin.Dried bulbs (100 g per each plant) were extracted under reflux three times with MeOH (300 mL) for 1 h each. The solvent was evaporated under reduced pressure to give the methanolic crude extracts (MCEs). MCEs were dissolved in H−1, 1 min hold at 180 °C, 180–300 °C at 5 °C min−1, and 1 min hold at 300 °C. Injector temperature was 280 °C. The flow rate of carrier gas (He) was 0.8 mL min−1. The split ratio was 1:20. The results obtained were analyzed using AMDIS 2.64 software (NIST). Compounds were identified through the comparison of their mass spectral patterns and retention indexes, with the data recorded in literature.GC-MS analyses were performed on a Hewlett Packard 6890/MSD 5975 instrument  operating in EI mode at 70 eV. A DB-5 MS column (30 m ´ 0.25 mm ´ 0.25 μm) was used. The temperature program was: 100–180 °C at 15 °C minet al. [2HPO4, 2.3 mM NaH2PO4, 0.15 M NaCl, 0.05% Tween 20, pH 7.6) and 50 μL of the sample dissolved in the same buffer were added to the wells. The plates were incubated for 30 minutes at room temperature before the addition of 100 μL of the substrate solution . The absorbance was read in a Labsystems microplate reader  at 405 nm after three minutes. Enzyme activity was calculated as a percentage compared to an assay using a buffer without any inhibitor. The AChE inhibitory data were analyzed with the software package Prism . IC50 values are means ± SD of three individual determinations each performed in triplicate.AChE activity was assayed as described by Ellman et al.  with somet al. . Fifty μTLC was carried out on Merck Silica gel 60 F254 plates, using chloroform-methanol-ammonia (99:9:1) mixtures as mobile phase. TLC plates were sprayed with Dragendorff’s reagent; main alkaloids gave orange spots.H. jamesonii MDZ and Z. filifolia SJ. The demand for renewable sources of galanthamine and the need to protect plant biodiversity create an opportunity for Argentinian farmers to produce such crops. Studies of domestication of some of these species are currently in progress in order to determine which crops can be cultivated outdoors in the particular climate and soil, and which can be grown in greenhouses. Production cost and galanthamine levels in traditional cultivars are also being analyzed.The findings of the present study demonstrate the potential of wild Argentinian Amaryllidaceae species collected in the central Andean region, as a new renewable source of galanthamine. The most promising species seen to be