A family of lanthanide-containing molecular open frameworks with high porosity: [Ln(abdc)(Habdc), nH2O]∞ with Ln = La-Eu and 8 ? n ? 11

Reactions in water between the di-sodium salt of amino terepthalic acid (C₈H₃NO₄Na₂) and a lanthanide chloride lead to a family of 3D-coordination polymers with general chemical formula [Ln(C₈H₃NO₄)(C₈H₄NO₄), O]_∞ where Ln = La-Eu (except Pm) and 8 ? n ? 11. All these compounds are isostructural. High quality single crystals of [Ln(C₈H₃NO₄)(C₈H₄NO₄), nH₂O]_∞ with Ln = La-Sm (except Pm) and 8 ? n ? 11 have been obtained by slow diffusion in agar-agar gels. The crystal structure has been solved for the Nd-containing compound. This compound crystallizes in the cubic system, space group Ia-3 (no. 206) with a = 26.8056(5) Å. The crystal structure can be described as the juxtaposition of large channels with square cross-section.The channels are filled by highly disordered crystallization water molecules. The dehydration of the compounds by freeze-drying is possible and most of the crystallization water molecules can be removed without destruction of the molecular skeleton. The partially dehydrated compounds have general chemical formula [Ln(abdc)(Habdc), 2H₂O]_∞ with Ln = La-Eu except Pm. The porosity of the Nd-containing compound has been estimated by computational methods to 2170 m² g⁻¹. This dehydrated compound reversibly binds water when exposed to wet atmosphere restoring the initial hydrated phase.     

Body temperatures during three long-distance polar swims in water of 0–3 °C

1.

We report body temperature responses in a single individual to 3 swims of 1000 m or longer in ice-cold water (0–3 °C) during which he swam the normal crawl stroke with his face in the water whilst wearing only a swimming costume, swimming cap and goggles.     

Key aromatic residues at subsites + 2 and + 3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates

Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest k_cat/K_m for maltotriose, while sugar beet α-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased k_cat/K_m values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the k_cat/K_m values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest k_cat/K_m value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at + 2 and + 3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AGs.     

Preparation of (1 → 3)-β-d-glucan oligosaccharides by hydrolysis of curdlan with commercial α-amylase

Water soluble (1 → 3)-β-d-glucan oligosaccharides were prepared by hydrolyzing curdlan with α-amylase. The hydrolysis process was monitored by the DE values of the hydrolysates. Under the optimized conditions (pH, 5.98; temperature, 55.92 °C; α-amylase amount, 31.94 mg α-amylase/500 mL of reaction mixture containing 5 g curdlan; reaction time, 30 min), maximum DE value (15.62%) was obtained. The resulting products were composed of (1 → 3)-β-d-glucan oligosaccharides of DP 2-9. The hydrolysates were filtered, concentrated to ∼20% (w/v), and precipitated with 5 volumes of ethanol, which were then freeze dried to yield a water soluble powder. The (1 → 3)-β-d-glucan oligosaccharides content of the product and the yield were 97.7% and 97.6% (w/w), respectively.     

Interleukin-1α − 899 (+ 4845) C→T polymorphism increases the risk of chronic periodontitis: Evidence from a meta-analysis of 23 case–control studies

Many epidemiological studies have indicated that interleukin-1α (IL-1α) − 899 (+ 4845) C→T polymorphism increases the risk of chronic periodontitis (CP), whereas some studies have reported opposite results. Accordingly, the aim of this meta-analysis is to investigate the association of the IL-1α − 899 (+ 4845) C→T polymorphism with CP. We searched the PubMed database up to May 1, 2013 and finally obtained 23 case–control studies. After data extraction, we performed meta-analysis using Comprehensive Meta-Analysis v2.2 software. The overall result based on the fixed-effect model showed that IL-1α − 899 (+ 4845) C→T polymorphism was significantly associated with increased risk of CP: [odds ratio (OR) = 1.29, 95% confidence interval (CI) = 1.15–1.44, p < 0.001] for T vs. C; (OR = 1.59, 95%CI = 1.22–2.07, p = 0.0005) for TT vs. CC; (OR = 1.30, 95% CI = 1.12–1.51, p = 0.0004) for CT vs. CC; and (OR = 1.40, 95% CI = 1.21–1.61, p < 0.001) for (CT+TT) vs. CC; (OR = 1.47, 95% CI = 1.16–1.87, p = 0.002) for TT vs. (CT+CC). Stratified analyses revealed that there was a significantly increased risk for Caucasians and Asians. In conclusion, current evidence showed that IL-1α − 899 (+ 4845) C→T polymorphism probably increased the risk of CP.     

Cyclooxygenase-2 169C > G and 8473T > C gene polymorphisms and prostaglandin E2 level in breast cancer: A case–control study

Aims

Cyclooxygenase 2 (COX-2) with the resulting prostaglandin E2 (PGE2) is linked to increased risk of human breast cancer (BC). The aim of this study was to determine COX-2 169C > G and 8473T > C gene polymorphisms and PGE2 level at various stages of BC clarifying the role of COX-2 gene polymorphism and PGE2 in relation to BC.

Methods

The study population comprised 160 women at different stages of BC and 150 gender- and age-matched healthy control subjects. Plasma PGE2 was measured by ELISA, the COX-2 gene polymorphisms were determined using PCR-RFLP.

Results

The variant alleles COX-2 169G and 8473C were significantly associated with BC susceptibility [OR = 3.1, 95% CI (2.2–4.4), P < 0.001 for 169C>G and OR = 1.74, 95%CI (1.3–2.4), P = 0.005 for 8473C]. However, both COX-2 gene polymorphisms were not associated with breast cancer stage. Plasma PGE2 levels were significantly increased in patients compared to the controls. In early and late stages of BC, there was a significant increase in the plasma PGE2 levels towards the presence of homozygous GG compared with homozygous CC (P < 0.001) for 169 C>G, also towards the presence of CC than TT (P < 0.001) for 8473T>C SNP.

Conclusion

The 169C>G and 8473T>C polymorphisms of the COX-2 gene were associated with the BC in Egyptian women. Furthermore, individuals with COX-2 169GG and 8473CC genotypes showed significant increase in plasma PGE2 levels. PGE2 levels may serve as a predictor of poor prognosis in patients with BC.     

Synthesis, crystal structures, and spectroscopic characterization of the neutral monomeric tetrahedral [M(Diap)2(OAc)2] · H2O complexes (M = Zn,Cd; Diap = 1,3-diazepane-2-thione; OAc = acetate) with N-H?O and O-H?O intra- and intermolecular hydrogen bonding interactions

The title complexes, [M(Diap)₂(OAc)₂] · H₂O (M = Zn,Cd; Diap = 1,3-diazepane-2-thione; OAc = acetate) with an MO₂S₂ configuration, have been characterized by X-ray crystallography as well as FT-IR, ¹H and ¹³C NMR spectroscopy. In these complexes, the metal atoms lie in a pseudo-tetrahedral environment and are coordinated by the thione sulfur atoms of two neutral 1,3-diazepane-2-thione ligands and one oxygen atom from each of two monodentate acetate anions. In both complexes, there are two intramolecular N-H?O hydrogen bonds, each being between one NH group of a Diap ligand and the uncoordinated O atom of an OAc ligand. The water molecule is also involved in hydrogen bonds, as an acceptor and as a donor twice, linking together three symmetry-related complexes. The Cd complex undergoes a structural phase transition from a monoclinic form at 150 K with Z′ = 2 to a smaller monoclinic cell at room temperature with Z′ = 1 without loss of crystallinity. The Zn complex does not exhibit an equivalent phase transition, and at 150 K is isostructural with the room-temperature form of the Cd complex. All three crystallographically independent molecules found for the Cd complex (two at low temperature and one at room temperature) have essentially the same structure except for small changes in the conformations of the ligands. Tetrahedral coordination with monodentate carboxylate ligands is common for Zn complexes of this kind, but is unusual for Cd complexes, and is the result of the bulky Diap ligands.     

Biomass and primary production of a 8–11 m depth meadow versus <3 m depth meadows of the seagrass Cymodocea nodosa (Ucria) Ascherson

Current knowledge about the abundance, growth, and primary production of the seagrass Cymodocea nodosa (Ucria) Ascherson is biased towards shallow (depth <3 m) meadows although this species also forms extensive meadows at larger depths along the coastlines. The biomass and primary production of a C. nodosa meadow located at a depth of 8–11 m was estimated at the time of maximum annual vegetative development (summer) using reconstruction techniques, and compared with those available from shallow meadows of this species. A depth-referenced data base of values at the time of maximum annual development was compiled to that end. The vegetative development of C. nodosa at 8–11 m depth was not different from that achieved by shallow (depth <3 m) meadows of this species. Only shoot density, which decreased from 1637 to 605 shoots m⁻², and the annual rate of elongation of the horizontal rhizome, which increased from 23 to 71 cm apex⁻¹ year⁻¹, were different as depth increased from <3 to 8–11 m. Depth was a poor predictor of the vegetative development and primary production of C. nodosa. The biomass of rhizomes and roots decreased with depth (g DW m⁻² = 480 (±53, S.E.) − 32 (±15, S.E.) depth (in m); R² = 0.12, F = 4.65, d.f. = 35, P = 0.0381) which made total biomass of the meadow to show a trend of decrease with depth but the variance of biomass data explained by depth was low. The annual rate of elongation of the horizontal rhizome showed a significant positive relationship with depth (cm apex⁻¹ year⁻¹ = 18 (±5.1, S.E.) + 5.0 (±1.33, S.E.) depth (in m); R² = 0.50, F = 14.07, d.f. = 14, P = 0.0021). As shoot size and growth did not change significantly with depth, the reduction of shoot density should drive any changes of biomass and productivity of C. nodosa as depth increases. The processes by which this reduction of C. nodosa abundance with depth occur remain to be elucidated.     

Functional properties of the newly observed γ-chain fetal hemoglobin variant Hb F-Monserrato-Sassari (HBG2:c.280T > C) or [γ93 (F9) Cys → Arg]

Background

HbF-Monserrato-Sassari is a newly discovered abnormal fetal hemoglobin observed in an apparently normal newborn baby during a hemoglobinopathies survey at birth in North Sardinian population.

Methods

Electrophoretic analysis of the cord blood lysate evidenced for an abnormal tetramer due to a mutated fetal globin chain. Electrospray ionisation-mass spectrometry and gene sequencing were used to identify the mutation. Oxygen binding ability of the variant Hb was determined.

Results

Sequencing of the γ globin genes revealed the TGT → CGT transition at codon 93 in one of the two ^Gγ genes, which leads to the Arg for Cys amino acid replacement at position 9 of the F α-helix. The amino acid substitution was confirmed by mass spectrometric analysis of the globin chains. Since modifications or substitutions at position β93 are known to affect the arrangement of a salt bridge at the α1β2 sliding contacts that are crucial for subunit cooperativity, the functional properties of the variant were studied to evaluate the effect of the replacement at the same position in the γ globin chain. With respect to normal HbF, the variant showed a significant increase in oxygen affinity and a slight decrease of both Bohr effect and cooperativity.

General significance

Result indicates a key role of the Cys γ93 residue for subunit cooperativity in the T → R transition of the HbF tetramer. Substitutions at the F9 position of the ^Gγ globin may result in stabilization of the high affinity R-state of the Hb tetramer. Because of the loss of Cys γ93 residue, this variant is considered to be potentially compromised in nitric oxide transport.     

ER stress-mediated regulation of immune function under glucose-deprived condition in glial cells: Up- and down-regulation of PGE2 + IFNγ-induced IL-6 and iNOS expressions

Glucose metabolism plays central role in maintaining brain function. Under ischemic condition, where glucose levels were reduced, glial cells induce pro-inflammatory cytokine production. In the present study, we found prostaglandin (PG) E₂ + interferon (IFN) γ-induced interleukin (IL)-6 production was enhanced under glucose-deprived condition in the primary cultured glial cells. On the other hand, to our surprise, we found that PGE₂ + IFNγ-induced iNOS expression was attenuated under glucose-deprived condition. These dual effects would be mediated through endoplasmic reticulum (ER) stress, because we observed (1) up-regulation of GRP78 and CHOP under glucose-deprived condition, which was inhibited by chemical chaperon TMAO, and (2) treatment with TMAO inhibited IL-6 production under glucose-deprived condition. By activating theses responses glial cells may protect neurons because we observed increased neuronal cell viability in the immune-activated glial cell conditioned medium. Overall, our results suggest a link between ER stress and immune reactions under glucose-deprived condition in the glial cells.     

DNA aptamers bind specifically and selectively to (1 → 3)-β-d-glucans

(1 → 3)-β-d-Glucans are structural cell wall components of fungi, plants, and some bacteria and have been linked with human respiratory symptoms following aerosol exposure. A clear interpretation of the health impact of (1 → 3)-β-d-glucans is limited by the high cost and uncertainties associated with current glucan quantitation methods. The objective of this research is to develop DNA aptamers for the measurement of (1 → 3)-β-d-glucans. Aptamers are synthetic DNA functional binding molecules that fold into unique conformations, allowing them to bind specifically to their target. Through the in vitro selection process SELEX, we have produced aptamers that are able to bind with sub-micromolar affinity to curdlan, a linear unbranched form of (1 → 3)-β-d-glucans. These aptamers display high selectivity to curdlan and do not bind to non-(1 → 3)-β-d-polysaccharides, suggesting specificity for the β-(1 → 3)-glycosidic linkage. The aptamers produced here will enable the production of more cost-effective, less ambiguous assays for the environmental measurement of (1 → 3)-β-d-glucans.     

Molecular modeling of the rabbit colonic (HKα2a) H<Superscript>+</Superscript>, K<Superscript>+</Superscript> ATPase

A model of the HK2a subunit of the rabbit colonic H⁺, K⁺ ATPase has been generated using the crystal structure of the Ca⁺² ATPase as a template. A pairwise sequence alignment of the deduced primary sequences of the two proteins demonstrated that they share 29% amino acid sequence identity and 47% similarity. Using O (version 7) the model of HK2a was constructed by interactively mutating, deleting, and inserting the amino acids that differed between the pairwise sequence alignment of the Ca⁺² ATPase and HK2a. Insertions and deletions in the HK2a sequence occur in apparent extra-membraneous loop regions. The HK2a model was energy minimized and globally refined to a level comparable to that of the Ca⁺² ATPase structure using CNS. The charge distribution over the surface of HK2a was evaluated in GRASP and possible secondary structure elements of HK2a were visualized in BOBSCRIPT. Conservation and placement of residues that may be involved in ouabain binding by the H⁺, K⁺ ATPase were considered and a putative location for the subunit was postulated within the structure.Figure Possible architecture of the HK2a subunit. The residue in green is the lysine (position 517, Fig. 1) that lies in the nucleotide binding pocket and the residue in red is the aspartic acid at the phosphorylation site (position 385). Based on an alignment with the Ca⁺² ATPase, ten transmembrane helices were modeled into HK2a. The ten transmembrane helices are drawn as rods and shown in different colors for clarity. From left to right, the transmembrane helix designations are M10 (blue), M7 (gray), M8 (purple), M9 (orange), M5 (pink), M6 (green), M3 (brown), M4 (cyan), M2 (teal), and M1 (almond).     

Establishment and characterization of a unique 1 μm diameter liver-derived progenitor cell line

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 μm in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers.     

Bidimensional self-assembling of [Hg(RPhNNNPhR′)2] (R = acetyl, R′ = F) through metal-η-arene π-interactions and non classical C-H?O bonding: Synthesis and X-ray characterization of a bis diaryl asymmetric-substituted triazenide complex polymer of Hg(II)

Deprotonated 3-(2-fluorophenyl)-1-(4-acetylphenyl)triazene reacts with Hg(CH₃COO)₂ in tetrahydrofuran to give light yellow crystals of [Hg^II(RPhNNNPhR′)₂]_n (R = acetyl, R′ = F). The new polymeric triazenide complex of Hg(II) belongs to the monoclinic space group C2/c. The lattice of [Hg^II(RPhNNNPhR′)₂]_n can be viewed as a bidimensional assembly of planar tectons [Hg^II(RPhNNNPhR′)₂], occurring through metallocene alike Hg(II)-η²,η²-arene π-interactions along the crystallographic axis b and non classical C-H?O bonding along the axis a.     

Variation of the electron population by four units in the cluster series [(η-Cp′)3Mo3S4Co(L)] (L = I, CO, PPh3, NO; n = 0, 1)

The versatility of cuboidal Mo₃S₄Co clusters for the preparation of complexes with different numbers of valence shell electrons (VSE) in the cluster is described. The reaction of the geometrically incomplete cuboidal cluster salt [(η⁵-Cp′)₃Mo₃S₄][pts] (pts = p-toluenesulfonate) with one molar equivalent of [Co₂(CO)₈] afforded almost quantitatively the electroneutral 60 VSE cluster [(η⁵-Cp′)₃Mo₃S₄Co(CO)] (1), which previously has been prepared in low yield by Curtis et al. in autoclave syntheses [M.D. Curtis, U. Riaz, O.J. Curnow, J.W. Kampf, Organometallics 14 (1995) 5337]. Cluster 1 was also obtained in high yield by reaction of [(η⁵-Cp′)₃Mo₃S₄][pts] with [(η⁵-Cp^∗)Co(CO)₂]. Reaction of [(η⁵-Cp′)₃Mo₃S₄][pts] with two molar equivalents of [Co(I)(CO)₃(PPh₃)] led to a complex mixture of products, of which the electron deficient 58 VSE cluster salt [(η⁵-Cp′)₃Mo₃S₄Co(I)][Co(I)₃(thf)] ([2][Co(I)₃(thf)]) was isolated as single crystals. In the crystal structures of 1 and [2][Co(I)₃(thf)], the Co-Mo bond lengths are almost identical, indicating a delocalization of the electron deficiency in [2]⁺. The reduced form of [2]⁺, [(η⁵-Cp′)₃Mo₃S₄Co(I)] (2), was prepared by oxidative substitution of the carbonyl ligand in 1 by I₂. Further reactions of 1 with PPh₃ and NO leading to the 60 and 61 VSE cluster complexes [(η⁵-Cp′)₃Mo₃S₄Co(PPh₃)] (3) and [(η⁵-Cp′)₃Mo₃S₄Co(NO)] (4), respectively, enabled the preparation of Mo₃S₄Co clusters in altogether four different oxidation states.     

Theoretical studies of the spectroscopic properties of (L)Pt [(1,2-η)-Ph-(CC)n-Ph] (L = dppp or (PPh3)2, n = 1 or 2)

Electronic structures and spectroscopic properties of (L)Pt[(1,2-η²)-Ph-(CC)_n-Ph] (n = 1, L = (PPh₃)₂ (1), n = 1, L = dppp (2), and n = 2, L = (PPh₃)₂ (3)) are studied by the ab initio and DFT methods, respectively. The ground- and excited-state structures are optimized by the B3LYP and CIS methods, respectively. The calculated bond lengths and bond angles in the ground-state agree well with the corresponding experimental values and the structures in the ground and excited-states have only slightly change. At the TD-DFT level with the PCM model, the absorption and emission spectra in solution are obtained. The lowest-energy absorptions of 1-3 are attributed to the mixing MLCT/ILCT transitions and phosphorescent emissions are attributed to coming from the combination of ³MLCT/³ILCT transitions. Furthermore, the lowest-energy absorptions and emissions of 1-3 are red-shifted in the order 1 < 2 < 3. It is shown that with the increase of the π-conjugated effect of alkyne or electron-donating ability of the phosphane atom, lowest-energy emission energy for 1-3 is correspondingly decreased.     

Coupling of Na<Superscript>+</Superscript>/α-ketoglutarate symport and PAH/α-ketoglutarate antiport in epithelial cells. Estimation of probability of the anion exchanger spontaneous re-orientation

In this work, dynamics was studied of uptake of p-aminohippurate by basolateral membrane vesicles isolated from rat kidney proximal tubules. The uphill PAH transport into the basolateral membrane vesicles was shown to occur in the presence of α-ketoglutarate and Na⁺-gradient. Based on mathematical model of symport and antiport cooperation, the mechanism of energy coupling of PAH transport via exchanger with Na⁺-dicarboxylate symport is discussed. Based on comparison of our own and literature data, the data analysis shows adequacy of the proposed mathematical model to describe the symport and antiport cooperation. This model has been shown to enable estimation of re-orientation probability of the empty anion exchanger (without substrate) from one membrane side to the other.     

Mixed-valent and radical states of complexes [(bpy)2M(μ-abpy)M′(bpy)2], M,M′ = Ru or Os, abpy = 2,2′-azobispyridine: Electron transfer vs. hole transfer mechanism in azo ligand-bridged complexes

The title complexes were obtained as M^IIM′^II species [(bpy)₂M(μ-abpy)M′(bpy)₂](PF₆)₄, M,M′ = Ru or Os, using the new mononuclear precursor [(bpy)₂Os(abpy)](PF₆)₂ for the osmium-containing dinuclear complexes. One-electron reduction produces radical complexes [(bpy)₂M(μ-abpy)M′(bpy)₂]³⁺ and [(bpy)₂M(abpy)]⁺ with significant contributions from the metals, as evident from the EPR effects on successive replacement of ruthenium by osmium with its much higher spin-orbit coupling constant. The diruthenium and diosmium radical complexes were also studied by EPR at high-frequency (285 GHz), the latter shows an unusually large g anisotropy g₁ − g₃ = 0.25 in frozen solution. Further reduction was monitored by UV/Vis spectroelectrochemistry. Oxidation produced Os^III EPR signals for [(bpy)₂Os(abpy)]³⁺ and [(bpy)₂Os(μ-abpy)Ru(bpy)₂]⁵⁺, indicating a Ru^IIOs^III species for the latter. The diosmium(III,II) and diruthenium(III,II) mixed-valent species remained EPR silent at 4 K, however, they exhibit weak inter-valence charge transfer (IVCT) bands at about 1460 nm. Whereas the cyclic voltammetric response towards reduction is only marginally different for the three dinuclear complexes, successive replacement of ruthenium by osmium causes the first oxidation potential to decrease. The much higher comproportionation constant K_c for the mixed valent diosmium(III,II) state (K_c > 10¹⁵) in comparison to the diruthenium(III,II) analogue with K_c = 10¹⁰ confirms the electron transfer alternative for the valence exchange mechanism, in contrast to the hole transfer established for analogous dinuclear complexes with the formally related diacylhydrazido(2−) bridging ligands.     

Spin crossover behavior of a one-dimensional polymeric-chain compound {[Fe(abpt)2(μ-Ni(CN)4)]·xH2O}n (x = 0.5 → 0): Synthesis, spectral, thermal and magnetic properties

A one-dimensional polymeric-chain iron(II)-nickel(II) cyanido-bridged complex of the composition {[Fe(abpt)₂(μ-Ni(CN)₄)]·0.5H₂O}_n (1·0.5H₂O), where abpt = 4-amino-3,5-di-2-pyridyl-4H-1,2,4-triazole, was prepared and characterized by elemental and thermal analyses, FTIR and ⁵⁷Fe Mössbauer spectroscopies, and magnetic measurements. The incomplete spin crossover phenomenon was observed with approximately 12% of the frozen high-spin fraction at low temperatures and with the spin transition critical temperature above room temperature.