Development of the yeast and lactic acid bacteria co-culture agent for atmospheric ammonia removing: Genomic features and on-site applications.

Odorous gas (e.g. atmospheric ammonia) in low ventilation public places, such as public toilets and waste transfer stations, causes severe health problems. Many technologies are developed to purify the atmospheric ammonia, among which the microbial agents are supposed to be a green and economical approach. In this study, we developed a yeast, Pichia sp. J1, and a lactic acid bacterium (LAB), Lactobacillus paracasei B1, co-culture agent for atmospheric ammonia removing. The on-site application results indicated the yeast and LAB mixed fermented agent had a maximum ammonia removing efficiency of 98.78%, which is significantly higher than the pure cultures (78.93% for B1 and 75.00% for J1), indicating the co-culture agent is an excellent biological product for ammonia removal. The excellent performance of the agent is closely related to the synergy behaviors between the yeast and LAB. In the co-culture agents, some of the LAB cells adhered closely to the yeast, and the growth and lactic acid producing ability of LAB were significantly promoted by yeast. Genomic analysis indicated the complementary of nutrients, i.e. carbon and nitrogen resources, signal transduction, and adhesion proteins (regulates adhesion behavior) played roles in regulating the synergy effects. Our study offers a novel biological solution of odorous gas purification.

Peptide Vaccine Formulation Controls the Duration of Antigen Presentation and Magnitude of Tumor-Specific CD8+ T Cell Response.

Despite remarkable progresses in vaccinology, therapeutic cancer vaccines have not achieved their full potential. We previously showed that an excessively long duration of Ag presentation critically reduced the quantity and quality of vaccination-induced T cell responses and subsequent antitumor efficacy. In this study, using a murine model and tumor cell lines, we studied l-tyrosine amino acid-based microparticles as a peptide vaccine adjuvant with a short-term Ag depot function for the induction of tumor-specific T cells. l-Tyrosine microparticles did not induce dendritic cell maturation, and their adjuvant activity was not mediated by inflammasome activation. Instead, prolonged Ag presentation in vivo translated into increased numbers and antitumor activity of vaccination-induced CD8+ T cells. Indeed, prolonging Ag presentation by repeated injection of peptide in saline resulted in an increase in T cell numbers similar to that observed after vaccination with peptide/l-tyrosine microparticles. Our results show that the duration of Ag presentation is critical for optimal induction of antitumor T cells, and can be manipulated through vaccine formulation.

Effective innate and adaptive antimelanoma immunity through localized TLR7/8 activation.

Intratumoral immune activation can induce local and systemic antitumor immunity. Imiquimod is a cream-formulated, TLR7 agonist that is Food and Drug Administration approved for the treatment of nonmelanoma skin cancers, but it has limited activity against melanoma. We studied the antitumor activity and mechanism of action of a novel, injectable, tissue-retained TLR7/8 agonist, 3M-052, which avoids systemic distribution. Intratumoral administration of 3M-052 generated systemic antitumor immunity and suppressed both injected and distant, uninjected wild-type B16.F10 melanomas. Treated tumors showed that an increased level of CCL2 chemokines and infiltration of M1 phenotype-shifted macrophages, which could kill tumor cells directly through production of NO and CCL2, were essential for the antitumor activity of 3M-052. CD8(+) T cells, B cells, type I IFN, IFN-γ, and plasmacytoid dendritic cells were contributed to efficient tumor suppression, whereas perforin, NK cells, and CD4 T cells were not required. Finally, 3M-052 therapy potentiated checkpoint blockade therapy with anti-CTLA-4 and anti-programmed death ligand 1 Abs, even when checkpoint blockade alone was ineffective. Our findings suggest that intratumoral treatment with 3M-052 is a promising approach for the treatment of cancer and establish a rational strategy and mechanistic understanding for combination therapy with intratumoral, tissue-retained TLR7/8 agonist and checkpoint blockade in metastatic cancer.

Compensating the electron beam energy spread by the natural transverse gradient of laser undulator in all-optical x-ray light sources.

All-optical ideas provide a potential to dramatically cut off the size and cost of x-ray light sources to the university-laboratory scale, with the combination of the laser-plasma accelerator and the laser undulator. However, the large longitudinal energy spread of the electron beam from laser-plasma accelerator may hinder the way to high brightness of these all-optical light sources. In this paper, the beam energy spread effect is proposed to be significantly compensated by the natural transverse gradient of a laser undulator when properly transverse-dispersing the electron beam. Theoretical analysis and numerical simulations on conventional laser-Compton scattering sources and high-gain all-optical x-ray free-electron lasers with the electron beams from laser-plasma accelerators are presented.

RubisCO gene clusters found in a metagenome microarray from acid mine drainage.

The enzyme responsible for carbon dioxide fixation in the Calvin cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), is always detected as a phylogenetic marker to analyze the distribution and activity of autotrophic bacteria. However, such an approach provides no indication as to the significance of genomic content and organization. Horizontal transfers of RubisCO genes occurring in eubacteria and plastids may seriously affect the credibility of this approach. Here, we presented a new method to analyze the diversity and genomic content of RubisCO genes in acid mine drainage (AMD). A metagenome microarray containing 7,776 large-insertion fosmids was constructed to quickly screen genome fragments containing RubisCO form I large-subunit genes (cbbL). Forty-six cbbL-containing fosmids were detected, and six fosmids were fully sequenced. To evaluate the reliability of the metagenome microarray and understand the microbial community in AMD, the diversities of cbbL and the 16S rRNA gene were analyzed. Fosmid sequences revealed that the form I RubisCO gene cluster could be subdivided into form IA and IB RubisCO gene clusters in AMD, because of significant divergences in molecular phylogenetics and conservative genomic organization. Interestingly, the form I RubisCO gene cluster coexisted with the form II RubisCO gene cluster in one fosmid genomic fragment. Phylogenetic analyses revealed that horizontal transfers of RubisCO genes may occur widely in AMD, which makes the evolutionary history of RubisCO difficult to reconcile with organismal phylogeny.

Proposal for an x-ray free electron laser oscillator with intermediate energy electron beam.

Harmonic lasing of low-gain free electron laser oscillators has been experimentally demonstrated in the terahertz and infrared regions. Recently, the low-gain oscillator has been reconsidered as a promising candidate for hard x-ray free electron lasers, through the use of high reflectivity, high-resolution x-ray crystals. In this Letter, it is proposed to utilize a crystal-based cavity resonant at a higher harmonic of the undulator radiation, together with phase shifting, to enable harmonic lasing of the x-ray free electron laser oscillator, and hence allow the generation of hard x-ray radiation at a reduced electron beam energy. Results show that fully coherent free electron laser radiation with megawatt peak power, in the spectral region of 10-25 keV, can be generated with a 3.5 GeV electron beam.

The co-culture of Acidithiobacillus ferrooxidans and Acidiphilium acidophilum enhances the growth, iron oxidation, and CO2 fixation.

Although the synergetic interactions between chemolithoautotroph Acidithiobacillus ferrooxidans and heterotroph Acidiphilium acidophilum have drawn a share of attention, the influence of Aph. acidophilum on growth and metabolic functions of At. ferrooxidans is still unknown on transcriptional level. To assess this influence, a co-culture composed by At. ferrooxidans and Aph. acidophilum was successfully acclimated in this study. Depending on the growth dynamics, At. ferrooxidans in co-culture had 2 days longer exponential phase and 5 times more cell number than that in pure culture. The ferrous iron concentration in culture medium and the expression of iron oxidation-related genes revealed that the energy acquisition of At. ferrooxidans in co-culture was more efficient than that in pure culture. Besides, the analysis of CO2 fixation-related genes in At. ferrooxidans indicated that the second copy of RuBisCO-encoding genes cbbLS-2 and the positive regulator-encoding gene cbbR were up-regulated in co-culture system. All of these results verified that Aph. acidophilum could heterotrophically grow with At. ferrooxidans and promote the growth of it. By means of activating iron oxidation-related genes and the second set of cbbLS genes in At. ferrooxidans, the Aph. acidophilum facilitated the iron oxidation and CO2 fixation by At. ferrooxidans.

Inequalities for the fractional convolution operator on differential forms.

The purpose of this paper is to derive some Coifman type inequalities for the fractional convolution operator applied to differential forms. The Lipschitz norm and BMO norm estimates for this integral type operator acting on differential forms are also obtained.

Cancer vaccine formulation dictates synergy with CTLA-4 and PD-L1 checkpoint blockade therapy.

Anticancer vaccination is a promising approach to increase the efficacy of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death ligand 1 (PD-L1) checkpoint blockade therapies. However, the landmark FDA registration trial for anti-CTLA-4 therapy (ipilimumab) revealed a complete lack of benefit of adding vaccination with gp100 peptide formulated in incomplete Freund's adjuvant (IFA). Here, using a mouse model of melanoma, we found that gp100 vaccination induced gp100-specific effector T cells (Teffs), which dominantly forced trafficking of anti-CTLA-4-induced, non-gp100-specific Teffs away from the tumor, reducing tumor control. The inflamed vaccination site subsequently also sequestered and destroyed anti-CTLA-4-induced Teffs with specificities for tumor antigens other than gp100, reducing the antitumor efficacy of anti-CTLA-4 therapy. Mechanistically, Teffs at the vaccination site recruited inflammatory monocytes, which in turn attracted additional Teffs in a vicious cycle mediated by IFN-γ, CXCR3, ICAM-1, and CCL2, dependent on IFA formulation. In contrast, nonpersistent vaccine formulations based on dendritic cells, viral vectors, or water-soluble peptides potently synergized with checkpoint blockade of both CTLA-4 and PD-L1 and induced complete tumor regression, including in settings of primary resistance to dual checkpoint blockade. We conclude that cancer vaccine formulation can dominantly determine synergy, or lack thereof, with CTLA-4 and PD-L1 checkpoint blockade therapy for cancer.

Development and evaluation of 50-mer oligonucleotide arrays for detecting microbial populations in Acid Mine Drainages and bioleaching systems.

To effectively monitor microbial populations in acidic environments and bioleaching systems, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the known genes associated with the acidophiles. This array contained 1,072 probes in which there were 571 related to 16S rRNA and 501 related to functional genes. The functional genes in the microarray were involved in carbon metabolism (158), nitrogen metabolism (72), sulfur metabolism (39), iron metabolism (68), DNA replication and repair (97), metal-resistance (27), membrane-relate gene (16), transposon (13) and IST sequence (11). Based on the results of microarray hybridizations, specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. The detection limit was 5 ng of genomic DNA in the absence of background DNA. Strong linear relationships between the signal intensity and the target DNA were observed (r(2) approximately 0.98). Application of this type of the microarray to analyze the acidic environments and bioleaching systems demonstrated that the developed microarray appeared to be useful for profiling differences in microbial community structures of acidic environments and bioleaching systems. Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of genes related with acidophilic microorganism and the microbial community in acidic environments and bioleaching systems, although more work is needed to improve.

Intratumoral CD40 activation and checkpoint blockade induces T cell-mediated eradication of melanoma in the brain.

CD40 agonists bind the CD40 molecule on antigen-presenting cells and activate them to prime tumor-specific CD8+ T cell responses. Here, we study the antitumor activity and mechanism of action of a nonreplicating adenovirus encoding a chimeric, membrane-bound CD40 ligand (ISF35). Intratumoral administration of ISF35 in subcutaneous B16 melanomas generates tumor-specific, CD8+ T cells that express PD-1 and suppress tumor growth. Combination therapy of ISF35 with systemic anti-PD-1 generates greater antitumor activity than each respective monotherapy. Triple combination of ISF35, anti-PD-1, and anti-CTLA-4 results in complete eradication of injected and noninjected subcutaneous tumors, as well as melanoma tumors in the brain. Therapeutic efficacy is associated with increases in the systemic level of tumor-specific CD8+ T cells, and an increased ratio of intratumoral CD8+ T cells to CD4+ Tregs. These results provide a proof of concept of systemic antitumor activity after intratumoral CD40 triggering with ISF35 in combination with checkpoint blockade for multifocal cancer, including the brain.

IL2 Variant Circumvents ICOS+ Regulatory T-cell Expansion and Promotes NK Cell Activation.

Clinical responses to high-dose IL2 therapy are limited due to selective expansion of CD4+CD25+Foxp3+ T-regulatory cells (Treg), especially ICOS+ Tregs, rather than natural killer (NK) cells and effector T cells. These ICOS+ Tregs are highly suppressive and constitutively express high levels of IL2Rα (CD25) and CD39. Here, we characterized the effect of a mutant form of IL2 (F42K), which preferentially binds to the lower affinity IL2Rßγ with reduced binding to CD25, on Tregs, effector NK cells, and T-cell subsets. Unlike wild-type (WT) IL2, F42K did not efficiently induce the expansion of highly suppressive ICOS+ Tregs in peripheral blood mononuclear cells (PBMC) from healthy controls and melanoma patients. Instead, it promoted the expansion of CD16+CD56+ NK cells and CD56hiCD16- NK cell subsets in both short- and long-term cultures, with enhanced Bcl-2 expression. Stimulation of PBMCs with F42K induced expression of more NK cell activation molecules, such as NKp30, NKp44, DNAM-1, NKG2D, 4-1BB/CD137, and Tim-3, than WT IL2. F42K induced greater upregulation of TRAIL, and NK-mediated cytolytic activity was increased against both autologous and HLA-mismatched melanoma cells compared with WT IL2. Gene expression analysis revealed distinct gene expression profiles stimulated by F42K, WT IL2, and IL15. F42K therapy in vivo also induced a dramatic reduction in the expansion of ICOS+ Tregs, promoted NK cell expansion, and inhibited melanoma tumor growth more efficiently than WT IL2 and more effectively than anti-CTLA-4. Our findings suggest that F42K could be a potential substitute for WT IL2 as a cytokine therapy for cancer. Cancer Immunol Res; 4(11); 983-94. ©2016 AACR.

Identification of nitrogen-fixing genes and gene clusters from metagenomic library of acid mine drainage.

Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

Persistent antigen at vaccination sites induces tumor-specific CD8⁺ T cell sequestration, dysfunction and deletion.

To understand why cancer vaccine-induced T cells often do not eradicate tumors, we studied immune responses in mice vaccinated with gp100 melanoma peptide in incomplete Freund's adjuvant (peptide/IFA), which is commonly used in clinical cancer vaccine trials. Peptide/IFA vaccination primed tumor-specific CD8(+) T cells, which accumulated not in tumors but rather at the persisting, antigen-rich vaccination site. Once there, primed T cells became dysfunctional and underwent antigen-driven, interferon-γ (IFN-γ)- and Fas ligand (FasL)-mediated apoptosis, resulting in hyporesponsiveness to subsequent vaccination. Provision of CD40-specific antibody, Toll-like receptor 7 (TLR7) agonist and interleukin-2 (IL-2) reduced T cell apoptosis but did not prevent vaccination-site sequestration. A nonpersisting vaccine formulation shifted T cell localization toward tumors, inducing superior antitumor activity while reducing systemic T cell dysfunction and promoting memory formation. These data show that persisting vaccine depots can induce specific T cell sequestration, dysfunction and deletion at vaccination sites; short-lived formulations may overcome these limitations and result in greater therapeutic efficacy of peptide-based cancer vaccines.

Insights into the pH up-shift responsive mechanism of Acidithiobacillus ferrooxidans by microarray transcriptome profiling.

To define the molecular response of Acidithiobacillus ferrooxidans under pH up-shift, temporal gene expression profiles were examined by using whole-genome DNA microarrays for A. ferrooxidans. Approximately 30% of the 3,132 genes represented on the microarray were significantly upregulated over a 160-min period, while about 14% were significantly downregulated. Our results revealed that A. ferrooxidans showed potential self-protection and self-regulation performance in response to pH up-shift stress. Many genes involved in regulation of membrane components were differentially expressed under the pH up-shift stress. Likewise, most of genes involved in phosphate metabolism, sulfur assimilation, and CO(2) fixation were obviously induced. Conversely, the transcription of a polyphosphate kinase gene (AFE1210) associated with phosphate storage was significantly repressed, which probably stemmed from the depletion of polyphosphate. Besides, most of the genes involved in hydrogen uptake were significantly induced, whereas many genes involved in nitrogen fixation were obviously repressed, which suggested that hydrogen uptake and nitrogen fixation could contribute to cytoplasmic pH homeostasis.

Molecular diversity of 16S rRNA and gyrB genes in copper mines.

The molecular diversities of the microbial communities from four sites impacted by acid mine drainage (AMD) at Dexing Copper Mine in Jiangxi province of China were studied using 16S rRNA sequences and gyrB sequences. Of the four sampled sites, each habitat exhibited distinct geochemical characteristics and the sites were linked geographically allowing us to correlate microbial community structure to geochemical characteristics. In the present study, we examined the molecular diversity of 16S rRNA and gyrB genes from water at these sites using a PCR-based cloning approach. We found that the microbial community appears to be composed primarily of Proteobacteria, Acidobacteria, Actinobacteria, Nitrospira, Firmicutes, Chlorella and unknown phylotypes. Of clones affiliated with Nitrospira, Leptospirillum ferrooxidans, Leptospirillum ferriphilum and Leptospirillum group III were all detected. Principal-component analysis (PCA) revealed that the distribution of the microbial communities was influenced greatly by geochemical characteristics. The overall PCA profiles showed that the sites with similar geochemical characteristics had more similar microbial community structures. Moreover, our results also indicated that gyrB sequence analysis may be very useful for differentiating very closely related species in the study of microbial communities.

A new inorganic-organic hybrid copper iodate with potentially large void volume.

A new one-dimensional hybrid copper iodate, (2,2'-bipy)(2)Cu(2)(IO(3))(3)(+), with potentially large void volume and a high amount of accessible volume, was synthesized by hydrothermal reactions and characterized by single crystal X-ray diffraction.

Low-dimensional hybrid copper halides with novel D6R Cu(6)I(6) cores.

Two low-dimensional hybrid copper halides, (2,2'-bipy)Cu(3)I(3) (1) and (2,2'-bipy)Cu(4)I(4) (2) (2,2'-bipy = 2,2'-bipyridine), with novel D6R (double six-membered rings) Cu(6)I(6) cores were synthesized by hydro(solvo)thermal reactions and characterized by single-crystal X-ray diffraction.

Hydrothermal syntheses and crystal structures of complex-linked three-Dimensional coordination vanadium selenites: M(4,4'-bipy)(H(2)O)V(2)Se(2)O(10) (M = Co, Ni).

Two inorganic-organic hybrid compounds with the formula M(4,4'-bipy)(H(2)O)V(2)Se(2)O(10) (M = Co, Ni) were hydrothermally synthesized and characterized by single-crystal X-ray diffraction. Compounds Co(4,4'-bipy)(H(2)O)V(2)Se(2)O(10) (1) and Ni(4,4'-bipy)(H(2)O)V(2)Se(2)O(10) (2), which are structural analogues, crystallize in the triclinic space group Ponemacr; with crystal data a = 7.9665(3) A, b = 8.1974(3) A, c = 13.8096(4) A, alpha = 85.704(2) degrees, beta = 73.5180(10) degrees, gamma = 75.645(2) degrees, V = 837.76(5) A(3), and Z = 2 and a = 7.9489(19) A, b = 8.128(2) A, c = 13.709 A, alpha = 85.838(6) degrees, beta = 73.736(8) degrees, gamma = 75.594(9) degrees, V = 823.5(4) A(3), and Z = 2, respectively. [M(4,4'-bipy)(H(2)O)V(2)Se(2)O(10)] (M = Co, Ni) have a three-dimensional structure and consist of two subunits, [(VO(2))(SeO(3))](-) infinite chains and [M(4,4'-bipy)(H(2)O)](2+) fragments. The [(VO(2))(SeO(3))](-) chains are composed of [V(2)Se(4)O(14)](4)(-) clusters linked by VO(4)N triangular bipyramids. The 4,4'-bipy molecule as a bifunctional organic ligand is directly linked to Co or Ni and V atoms, affording the three-dimensionality. The compounds were characterized by infrared spectroscopy and differential thermal and thermogravimetric analyses.

Hydrothermal syntheses, crystal structures, and magnetic properties of inorganic-organic hybrid vanadium selenites with zero- to three-dimensional structures: (1,10-phenanthroline)(2)V(2)SeO(7), (2,2'-bipyridine)VSeO(4), (4,4'-bipyridine)V(2)Se(2)O(8), and (4,4'-bipyridine)(2)V(4)Se(3)O(15).H(2)O.

A family of inorganic-organic hybrid vanadium selenites with zero-, one-, two-, and three-dimensional structures, (1,10-phen)(2)V(2)SeO(7), (2,2'-bipy)VSeO(4), (4,4'-bipy)V(2)Se(2)O(8), and (4,4'-bipy)(2)V(4)Se(3)O(15).H(2)O (where phen = phenanthroline and bipy = bipyridine), were hydrothermally synthesized and characterized by single-crystal X-ray diffraction. Different bidentate organodiamine ligands and reactant concentrations were used in the four reaction systems, which are responsible for the variety of structural dimensions of the compounds. (1,10-phen)(2)V(2)SeO(7) crystallizes in a monoclinic system with space group P2(1)/n and cell parameters a = 8.6509(3) A,( )b = 7.8379(2) A, c = 34.0998(13) A, beta = 91.503(2) degrees, and Z = 4. (2,2'-bipy)VSeO(4) crystallizes in a monoclinic system with space group C2/c and cell parameters a = 17.0895(12) A, b = 14.7707(10) A, c = 11.7657(8) A, beta = 131.354(3) degrees, and Z = 8. (4,4'-bipy)V(2)Se(2)O(8) crystallizes in a triclinic system with space group Ponemacr; and cell parameters a = 7.1810(10) A, b = 10.8937(13) A, c = 11.1811(15) A, alpha = 115.455(3) degrees, beta = 107.582(3) degrees, gamma = 91.957(4) degrees, and Z = 2. (4,4'-bipy)(2)V(4)Se(3)O(15).H(2)O crystallizes in a monoclinic system with space group Pc and cell parameters a = 7.9889(9) A, b = 7.8448 A, c = 23.048(3) A, beta = 99.389(4) degrees, and Z = 2. (1,10-phen)(2)V(2)SeO(7) has an isolated structure, (2,2'-bipy)VSeO(4) has a chain structure, (4,4'-bipy)V(2)Se(2)O(8) has a layered structure, and (4,4'-bipy)(2)V(4)Se(3)O(15).H(2)O has a framework structure. The chains are constructed from VO(4)N(2) octahedra and SeO(3) pyramids, laced by organic ligands (2,2'-bipy). The layers consist of vanadium selenite chains [(VO)(2)(SeO(3))(2)]( infinity ), linked by 4,4'-bipy molecules. The framework is composed of vanadium selenite sheets [V(4)Se(3)O(16)]( infinity ), pillared by 4,4'-bipy molecules. All of the compounds are thermally stable to 300 degrees C, and the magnetic susceptibilities confirm the existence of tetravalent V atoms in the antiferromagnetic (4,4'-bipy)V(2)Se(2)O(8) complex and mixed tetravalent and pentavalent V atoms in the paramagnetic complex (4,4'-bipy)(2)V(4)Se(3)O(15).H(2)O.