MiR-26 down-regulates TNF-α/NF-κB signalling and IL-6 expression by silencing HMGA1 and MALT1.

MiR-26 has emerged as a key tumour suppressor in various cancers. Accumulating evidence supports that miR-26 regulates inflammation and tumourigenicity largely through down-regulating IL-6 production, but the underlying mechanism remains obscure. Here, combining a transcriptome-wide approach with manipulation of cellular miR-26 levels, we showed that instead of directly targeting IL-6 mRNA for gene silencing, miR-26 diminishes IL-6 transcription activated by TNF-α through silencing NF-κB signalling related factors HMGA1 and MALT1. We demonstrated that miR-26 extensively dampens the induction of many inflammation-related cytokine, chemokine and tissue-remodelling genes that are activated via NF-κB signalling pathway. Knocking down both HMGA1 and MALT1 by RNAi had a silencing effect on NF-κB-responsive genes similar to that caused by miR-26. Moreover, we discovered that poor patient prognosis in human lung adenocarcinoma is associated with low miR-26 and high HMGA1 or MALT1 levels and not with levels of any of them individually. These new findings not only unravel a novel mechanism by which miR-26 dampens IL-6 production transcriptionally but also demonstrate a direct role of miR-26 in down-regulating NF-κB signalling pathway, thereby revealing a more critical and broader role of miR-26 in inflammation and cancer than previously realized.

Evidence for Hydroxamate Siderophores and Other N-Containing Organic Compounds Controlling (239,240)Pu Immobilization and Remobilization in a Wetland Sediment.

Pu concentrations in wetland surface sediments collected downstream of a former nuclear processing facility in F-Area of the Savannah River Site (SRS), USA, were ∼2.5 times greater than those measured in the associated upland aquifer sediments; similarly, the Pu concentration solid/water ratios were orders of magnitude greater in the wetland than in the low-organic matter content aquifer soils. Sediment Pu concentrations were correlated to total organic carbon and total nitrogen contents and even more strongly to hydroxamate siderophore (HS) concentrations. The HS were detected in the particulate or colloidal phases of the sediments but not in the low molecular weight fractions (<1000 Da). Macromolecules which scavenged the majority of the potentially mobile Pu were further separated from the bulk mobile organic matter fraction ("water extract") via an isoelectric focusing experiment (IEF). An electrospray ionization Fourier-transform ion cyclotron resonance ultrahigh resolution mass spectrometry (ESI FTICR-MS) spectral comparison of the IEF extract and a siderophore standard (desferrioxamine; DFO) suggested the presence of HS functionalities in the IEF extract. This study suggests that while HS are a very minor component in the sediment particulate/colloidal fractions, their concentrations greatly exceed those of ambient Pu, and HS may play an especially important role in Pu immobilization/remobilization in wetland sediments.

Effects of a novel encapsulating technique on the temperature tolerance and anti-colitis activity of the probiotic bacterium Lactobacillus kefiranofaciens M1.

Lactobacillus kefiranofaciens M1 (M1) has been shown to possess many different beneficial health effects including anti-colitis activity. The purpose of this study was to develop a novel and easily scaled-up encapsulating technique that would improve the temperature tolerance of the bacterium and reduce the sensitivity of the organism to gastrointestinal fluid. A mixture of sodium alginate, gellan gum and skim milk powder was used as a coating material to entrap M1. The M1 gel was then directly freeze dried in order to dehydrate the covering and form microcapsules. The viable cell numbers of M1 present only dropped ten folds after the freeze-drying encapsulation process. The viable cell counts remained constant at 5 × 10(7) CFU/g after heating from 25 °C to 75 °C and holding at 75 °C for 1 min. The viable cell counts were reduced to 10(6) CFU/g and 10(5) CFU/g after 8-week storage at 4 °C and subsequent heat treatment with simulated gastrointestinal fluid test (SGFT) and bile salts, respectively. The effect of encapsulated M1 on the organism's anti-colitis activity was evaluated using the dextran sodium sulfate (DSS) induced colitis mouse model. An in vivo study indicated that administration of heat treated encapsulated M1 was able to ameliorate DSS-induced colitis producing a significant reduction in the bleeding score and an attenuation of inflammatory score. These findings clearly demonstrate that encapsulation of M1 using this novel technique is able to provide good protection from temperature changes and SGFT treatment and also does not affect the organism's anti-colitis activity.

Superoxide production by a manganese-oxidizing bacterium facilitates iodide oxidation.

The release of radioactive iodine (i.e., iodine-129 and iodine-131) from nuclear reprocessing facilities is a potential threat to human health. The fate and transport of iodine are determined primarily by its redox status, but processes that affect iodine oxidation states in the environment are poorly characterized. Given the difficulty in removing electrons from iodide (I(-)), naturally occurring iodide oxidation processes require strong oxidants, such as Mn oxides or microbial enzymes. In this study, we examine iodide oxidation by a marine bacterium, Roseobacter sp. AzwK-3b, which promotes Mn(II) oxidation by catalyzing the production of extracellular superoxide (O2(-)). In the absence of Mn(2+), Roseobacter sp. AzwK-3b cultures oxidized ∼90% of the provided iodide (10 µM) within 6 days, whereas in the presence of Mn(II), iodide oxidation occurred only after Mn(IV) formation ceased. Iodide oxidation was not observed during incubations in spent medium or with whole cells under anaerobic conditions or following heat treatment (boiling). Furthermore, iodide oxidation was significantly inhibited in the presence of superoxide dismutase and diphenylene iodonium (a general inhibitor of NADH oxidoreductases). In contrast, the addition of exogenous NADH enhanced iodide oxidation. Taken together, the results indicate that iodide oxidation was mediated primarily by extracellular superoxide generated by Roseobacter sp. AzwK-3b and not by the Mn oxides formed by this organism. Considering that extracellular superoxide formation is a widespread phenomenon among marine and terrestrial bacteria, this could represent an important pathway for iodide oxidation in some environments.

Temporal variation of iodine concentration and speciation (127I and 129I) in wetland groundwater from the Savannah River Site, USA.

(129)I derived from a former radionuclide disposal basin located on the Savannah River Site (SRS) has concentrated in a wetland 600 m downstream. To evaluate temporal environmental influences on iodine speciation and mobility in this subtropical wetland environment, groundwater was collected over a three-year period (2010-2012) from a single location. Total (127)I and (129)I showed significant temporal variations, ranging from 68-196 nM for (127)I and <5-133 pCi/L for (129)I. These iodine isotopes were significantly correlated with groundwater acidity and nitrate, two parameters elevated within the contaminant plume. Additionally, (129)I levels were significantly correlated with those of (127)I, suggesting that biogeochemical controls on (127)I and (129)I are similar within the SRS aquifer/wetland system. Iodine speciation demonstrates temporal variations as well, reflecting effects from surface recharges followed by acidification of groundwater and subsequent formation of anaerobic conditions. Our results reveal a complex system where few single ancillary parameters changed in a systematic manner with iodine speciation. Instead, changes in groundwater chemistry and microbial activity, driven by surface hydrological events, interact to control iodine speciation and mobility. Future radiological risk models should consider the flux of (129)I in response to temporal changes in wetland hydrologic and chemical conditions.

Plutonium immobilization and remobilization by soil mineral and organic matter in the far-field of the Savannah River Site, U.S.

To study the effects of natural organic matter (NOM) on Pu sorption, Pu(IV) and (V) were amended at environmentally relevant concentrations (10(-14) M) to two soils of contrasting particulate NOM concentrations collected from the F-Area of the Savannah River Site. More Pu(IV) than (V) was bound to soil colloidal organic matter (COM). A de-ashed humic acid (i.e., metals being removed) scavenged more Pu(IV,V) into its colloidal fraction than the original HA incorporated into its colloidal fraction, and an inverse trend was thus observed for the particulate-fraction-bound Pu for these two types of HAs. However, the overall Pu binding capacity of HA (particulate + colloidal-Pu) decreased after de-ashing. The presence of NOM in the F-Area soil did not enhance Pu fixation to the organic-rich soil when compared to the organic-poor soil or the mineral phase from the same soil source, due to the formation of COM-bound Pu. Most importantly, Pu uptake by organic-rich soil decreased with increasing pH because more NOM in the colloidal size desorbed from the particulate fraction in the elevated pH systems, resulting in greater amounts of Pu associated with the COM fraction. This is in contrast to previous observations with low-NOM sediments or minerals, which showed increased Pu uptake with increasing pH levels. This demonstrates that despite Pu immobilization by NOM, COM can convert Pu into a more mobile form.

Iodine-129 and iodine-127 speciation in groundwater at the Hanford site, US: iodate incorporation into calcite.

The geochemical transport and fate of radioiodine depends largely on its chemical speciation that is greatly affected by environmental factors. This study reports, for the first time, the speciation of stable and radioactive iodine in the groundwater from the Hanford Site. Iodate was the dominant species and accounted for up to 84% of the total iodine present. The alkaline pH (pH ∼ 8) and predominantly oxidizing environment may have prevented reduction of the iodate. In addition, groundwater samples were found to have large amounts of calcite precipitate which were likely formed as a result of CO2 degassing during removal from the deep subsurface (>70m depth). Further analyses indicated that between 7 and 40% of the dissolved (127)I and (129)I that was originally in the groundwater had coprecipitated in the calcite. Iodate was the main species incorporated into calcite and this incorporation process could be impeded by elevating the pH and decreasing ionic strength in groundwater. This study provides critical information for predicting the long-term fate and transport of (129)I. Furthermore, the common sampling artifact resulting in the precipitation of calcite by degassing CO2, had the unintended consequence of providing insight into a potential solution for the in situ remediation of groundwater (129)I.

Bacterial production of organic acids enhances H2O2-dependent iodide oxidation.

To develop an understanding of the role that microorganisms play in the transport of (129)I in soil-water systems, bacteria isolated from subsurface sediments were assessed for iodide oxidizing activity. Spent liquid medium from 27/84 bacterial cultures enhanced iodide oxidation 2-10 fold in the presence of H(2)O(2). Organic acids secreted by the bacteria were found to enhance iodide oxidation by (1) lowering the pH of the spent medium, and (2) reacting with H(2)O(2) to form peroxy carboxylic acids, which are extremely strong oxidizing agents. H(2)O(2)-dependent iodide oxidation increased exponentially from 8.4 to 825.9 µM with decreasing pH from 9 to 4. Organic acids with ≥2 carboxy groups enhanced H(2)O(2)-dependent iodide oxidation (1.5-15-fold) as a function of increasing pH above pH 6.0, but had no effect at pH ≤ 5.0. The results indicate that as pH decreases (≤5.0), increasing H(2)O(2) hydrolysis is the driving force behind iodide oxidation. However, at pH ≥ 6.0, spontaneous decomposition of peroxy carboxylic acids, generated from H(2)O(2) and organic acids, contributes significantly to iodide oxidation. The results reveal an indirect microbial mechanism, organic acid secretion coupled to H(2)O(2) production, that could enhance iodide oxidation and organo-iodine formation in soils and sediments.

Artificial-epitope mapping for CK-MB assay.

A quantitative method using artificial antibody to detect creatine kinases was developed. Linear epitope sequences were selected based on an artificial-epitope mapping strategy. Nine different MIPs corresponding to the selected peptides were then fabricated on QCM chips. The subtle conformational changes were also recognized by these chips.

Sequestration and remobilization of radioiodine (129I) by soil organic matter and possible consequences of the remedial action at Savannah River Site.

In order to investigate the distributions and speciation of (129)I (and (127)I) in a contaminated F-Area groundwater plume of the Savannah River Site that cannot be explained by simple transport models, soil resuspension experiments simulating surface runoff or stormflow and erosion events were conducted. Results showed that 72-77% of the newly introduced I(-) or IO(3)(-) were irreversibly sequestered into the organic-rich riparian soil, while the rest was transformed by the soil into colloidal and truly dissolved organo-iodine, resulting in (129)I remobilization from the soil greatly exceeding the 1 pCi/L drinking water permit. This contradicts the conventional view that only considers I(-) or IO(3)(-) as the mobile forms. Laboratory iodination experiments indicate that iodine likely covalently binds to aromatic structures of the soil organic matter (SOM). Under very acidic conditions, abiotic iodination of SOM was predominant, whereas under less acidic conditions (pH ≥5), microbial enzymatically assisted iodination of SOM was predominant. The organic-rich soil in the vadose zone of F-Area thus acts primarily as a "sink," but may also behave as a potentially important vector for mobile radioiodine in an on-off carrying mechanism. Generally the riparian zone provides as a natural attenuation zone that greatly reduces radioiodine release.

Role of natural organic matter on iodine and (239)(,240)Pu distribution and mobility in environmental samples from the northwestern Fukushima Prefecture, Japan.

In order to assess how environmental factors are affecting the distribution and migration of radioiodine and plutonium that were emitted from the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident, we quantified iodine and (239,240)Pu concentration changes in soil samples with different land uses (urban, paddy, deciduous forest and coniferous forest), as well as iodine speciation in surface water and rainwater. Sampling locations were 53-63 km northwest of the FDNPP within a 75-km radius, in close proximity of each other. A ranking of the land uses by their surface soil (<4 cm) stable (127)I concentrations was coniferous forest > deciduous forest > urban > paddy, and (239,240)Pu concentrations ranked as deciduous forest > coniferous forest > paddy ≥ urban. Both were quite distinct from that of (134)Cs and (137)Cs: urban > coniferous forest > deciduous forest > paddy, indicating differences in their sources, deposition phases, and biogeochemical behavior in these soil systems. Although stable (127)I might not have fully equilibrated with Fukushima-derived (129)I, it likely still works as a proxy for the long-term fate of (129)I. Surficial soil (127)I content was well correlated to soil organic matter (SOM) content, regardless of land use type, suggesting that SOM might be an important factor affecting iodine biogeochemistry. Other soil chemical properties, such as Eh and pH, had strong correlations to soil (127)I content, but only within a given land use (e.g., within urban soils). Organic carbon (OC) concentrations and Eh were positively, and pH was negatively correlated to (127)I concentrations in surface water and rain samples. It is also noticeable that (127)I in the wet deposition was concentrated in both the deciduous and coniferous forest throughfall and stemfall water, respectively, comparing to the bulk rainwater. Further, both forest throughfall and stemflow water consisted exclusively of organo-iodine, suggesting all inorganic iodine in the original bulk deposition (∼ 28.6% of total iodine) have been completely converted to organo-iodine. Fukushima-derived (239,240)Pu was detectable at a distance ∼ 61 km away, NW of FDNPP. However, it is confined to the litter layer, even three years after the FDNPP accident-derived emissions. Plutonium-239,240 activities were significantly correlated with soil OC and nitrogen contents, indicating Pu may be associated with nitrogen-containing SOM, similar to what has been observed at other locations in the United States. Together, these finding suggest that natural organic matter (NOM) plays a key role in affecting the fate and transport of I and Pu and may warrant greater consideration for predicting long-term stewardship of contaminated areas and evaluating various remediation options in Japan.

Radioiodine sorption/desorption and speciation transformation by subsurface sediments from the Hanford Site.

During the last few decades, considerable research efforts have been extended to identify more effective remediation treatment technologies to lower the (129)I concentrations to below federal drinking water standards at the Hanford Site (Richland, USA). Few studies have taken iodate into consideration, though recently iodate, instead of iodide, was identified as the major species in the groundwater of 200-West Area within the Hanford Site. The objective of this study was thus to quantify and understand aqueous radioiodine species transformations and uptake by three sediments collected from the semi-arid, carbonate-rich environment of the Hanford subsurface. All three sediments reduced iodate (IO3(-)) to iodide (I(-)), but the loamy-sand sediment reduced more IO3(-) (100% reduced within 7 days) than the two sand-textured sediments (∼20% reduced after 28 days). No dissolved organo-iodine species were observed in any of these studies. Iodate uptake Kd values ([Isolid]/[Iaq]; 0.8-7.6 L/kg) were consistently and appreciably greater than iodide Kd values (0-5.6 L/kg). Furthermore, desorption Kd values (11.9-29.8 L/kg) for both iodate and iodide were consistently and appreciably greater than uptake Kd values (0-7.6 L/kg). Major fractions of iodine associated with the sediments were unexpectedly strongly bound, such that only 0.4-6.6 % of the total sedimentary iodine could be exchanged from the surface with KCl solution, and 0-1.2% was associated with Fe or Mn oxides (weak NH2HCl/HNO3 extractable fraction). Iodine incorporated into calcite accounted for 2.9-39.4% of the total sedimentary iodine, whereas organic carbon (OC) is likely responsible for the residual iodine (57.1-90.6%) in sediments. The OC, even at low concentrations, appeared to be controlling iodine binding to the sediments, as it was found that the greater the OC concentrations in the sediments, the greater the values of uptake Kd, desorption Kd, and the greater residual iodine concentrations (non-exchangeable, non-calcite-incorporated and non-Mn, Fe-oxide associated). This finding is of particular interest because it suggests that even very low OC concentrations, <0.2%, may have an impact on iodine geochemistry. The findings that these sediments can readily reduce IO3(-), and that IO3(-) sorbs to a greater extent than I(-), sheds light into earlier unexplained Hanford field data that demonstrated increases in groundwater (127)I(-)/(127)IO3(-) ratios and a decrease groundwater (129)IO3(-) concentrations along a transect away from the point sources, where iodine was primarily introduced as IO3(-). While a majority of the radioiodine does not bind to these alkaline sediments, there is likely a second smaller iodine fraction in the Hanford subsurface that is strongly bound, presumably to the sediment OC (and carbonate) phases. This second fraction may have an impact on establishing remediation goals and performance assessment calculations.

Radioiodine concentrated in a wetland.

Most subsurface environmental radioactivity contamination is expected to eventually resurface in riparian zones, or wetlands. There are a number of extremely sharp biogeochemical interfaces in wetlands that could alter radionuclide speciation and promote accumulation. The objective of this study was to determine if a wetland concentrated (129)I emanating from a former waste disposal basin located on the Savannah River Site (SRS) in South Carolina, USA. Additionally, studies were conducted to evaluate the role of sediment organic matter in immobilizing the radioiodine. Groundwater samples were collected along a 0.7-km transect away from the seepage basin and in the downstream wetlands. The samples were analyzed for (129)I speciation (iodide (I(-)), iodate (IO3(-)), and organo-I). Groundwater (129)I concentrations in many locations in the wetlands (as high as 59.9 Bq L(-1)(129)I) were greatly elevated with respect to the source term (5.9 Bq L(-1)(129)I). (129)I concentration profiles in sediment cores were closely correlated to organic matter concentrations (r(2) = 0.992; n = 5). While the sediment organic matter promoted the uptake of (129)I to the wetland sediment, it also promoted the formation of a soluble organic fraction: 74% of the wetland groundwater (129)I could pass through a 1 kDa (<1 nm) membrane and only 26% of the (129)I was colloidal. Of that fraction that could pass through a 1 kDa membrane, 39% of the (129)I was organo-I. Therefore, while wetlands may be highly effective at immobilizing aqueous (129)I, they may also promote the formation of a low-molecular-weight organic species that does not partition to sediments. This study provides a rare example of radioactivity concentrations increasing rather than decreasing as it migrates from a point source and brings into question assumptions in risk models regarding continuous dilution of released contaminants.

Speciation of iodine isotopes inside and outside of a contaminant plume at the Savannah River Site.

A primary obstacle in understanding the fate and transport of the toxic radionuclide (129)I (a thyroid seeker) is an accurate method to distinguish it from the stable isotope, (127)I, and to quantify the various species at environmentally relevant concentrations (~10(-8) M). A pH-dependent solvent extraction and combustion method was paired with accelerator mass spectrometry (AMS) to measure ambient levels of (129)I/(127)I isotope ratios and iodine speciation (iodide (I(-)), iodate (IO3(-)), and organo-I (OI)) in aquatic systems. The method exhibited an overall uncertainty of 10% or less for I(-) and IO3(-), and less than 30% for OI species concentrations and enabled (129)I measurements as low as 0.001 Bq/L (1 Bq/L=10(-13) M). The method was used to analyze groundwater from the Savannah River Site (SRS), South Carolina, USA, along a pH, redox potential (Eh), and organic carbon gradient (8-60 µM DOC). The data confirmed that the (129)I/(127)I ratios and species distribution were strongly pH dependent and varied in a systematic manner from the strongly acidic source. While (129)I speciation in plume samples containing total I concentrations >1.7 Bq/L was similar whether measured by AMS or GC-MS ([I(-)]≫[IO3(-)]=[OI]), AMS enabled (129)I speciation measurements at much lower concentrations than what was possible with GC-MS. AMS analyses demonstrated that groundwater samples minimally impacted by the plume were still orders of magnitude higher than ambient (129)I concentrations typically found elsewhere in the USA groundwaters and rivers. This is likely due to past atmospheric releases of volatile (129)I species by SRS nuclear reprocessing facilities near the study site. Furthermore, the results confirmed the existence of (129)I not only as I(-), but also as OI and IO3(-) species.

Novel molecular-level evidence of iodine binding to natural organic matter from Fourier transform ion cyclotron resonance mass spectrometry.

Major fractions of radioiodine ((129)I) are associated with natural organic matter (NOM) in the groundwater and surface soils of the Savannah River Site (SRS). Electrospray ionization coupled to Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) was applied to elucidate the interactions between inorganic iodine species (iodide and iodate) and a fulvic acid (FA) extracted from a SRS surface soil. Iodate is likely reduced to reactive iodine species by the lignin- and tannin-like compounds or the carboxylic-rich alicyclic molecules (CRAM), during which condensed aromatics and lignin-like compounds were generated. Iodide is catalytically oxidized into reactive iodine species by peroxides, while FA is oxidized by peroxides into more aliphatic and less aromatic compounds. Only 9% of the total identified organo-iodine compounds derived from molecules originally present in the FA, whereas most were iodine binding to newly-produced compounds. The resulting iodinated molecules were distributed in three regions in the van Krevelen diagrams, denoting unsaturated hydrocarbons, lignin and protein. Moreover, characteristics of these organo-iodine compounds, such as their relatively low O/C ratios (<0.2 or <0.4) and yet some degree of un-saturation close to that of lignin, have multiple important environmental implications concerning possibly less sterically-hindered aromatic ring system for iodine to get access to and a lower hydrophilicity of the molecules thus to retard their migration in the natural aquatic systems. Lastly, ~69% of the identified organo-iodine species contains nitrogen, which is presumably present as NH2 or HNCOR groups and a ring-activating functionality to favor the electrophilic substitution. The ESI-FTICR-MS technique provides novel evidence to better understand the reactivity and scavenging properties of NOM towards radioiodine and possible influence of NOM on (129)I migration.

Factors controlling mobility of 127I and 129I species in an acidic groundwater plume at the Savannah River Site.

In order to quantify changes in iodine speciation and to assess factors controlling the distribution and mobility of iodine at an iodine-129 ((129)I) contaminated site located at the U.S. Department of Energy's Savannah River Site (SRS), spatial distributions and transformation of (129)I and stable iodine ((127)I) species in groundwater were investigated along a gradient in redox potential (654 to 360 mV), organic carbon concentration (5 to 60 µmol L(-1)), and pH (pH 3.2 to 6.8). Total (129)I concentration in groundwater was 8.6±2.8 Bq L(-1) immediately downstream of a former waste seepage basin (well FSB-95DR), and decreased with distance from the seepage basin. (127)I concentration decreased similarly to that of (129)I. Elevated concentrations of (127)I or (129)I were not detected in groundwater collected from wells located outside of the mixed waste plume of this area. At FSB-95DR, the majority (55-86%) of iodine existed as iodide for both (127)I and (129)I. Then, as the iodide move down gradient, some of it transformed into iodate and organo-iodine. Considering that iodate has a higher K(d) value than iodide, we hypothesize that the production of iodate in groundwater resulted in the removal of iodine from the groundwater and consequently decreased concentrations of (127)I and (129)I in downstream areas. Significant amounts of organo-iodine species (30-82% of the total iodine) were also observed at upstream wells, including those outside the mixed waste plume. Concentrations of groundwater iodide decreased at a faster rate than organo-iodine along the transect from the seepage basin. We concluded that removal of iodine from the groundwater through the formation of high molecular weight organo-iodine species is complicated by the release of other more mobile organo-iodine species in the groundwater.

Structure of the alkalohyperthermophilic Archaeoglobus fulgidus lipase contains a unique C-terminal domain essential for long-chain substrate binding.

Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 A resolution. This enzyme has optimal activity in the temperature range of 70-90 degrees C and pH 10-11. AFL consists of an N-terminal alpha/beta-hydrolase fold domain, a small lid domain, and a C-terminal beta-barrel domain. The N-terminal catalytic domain consists of a 6-stranded beta-sheet flanked by seven alpha-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a beta-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL-substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. AFL has a higher preference toward long-chain substrates whose binding site is provided by a hydrophobic tunnel in the C-terminal domain. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications.