Comprehensive Application of XFEL Microcrystallography for Challenging Targets in Various Organic Compounds.

There is a growing demand for structure determination from small crystals, and the three-dimensional electron diffraction (3D ED) technique can be employed for this purpose. However, 3D ED has certain limitations related to the crystal thickness and data quality. We here present the application of serial X-ray crystallography (SX) with X-ray free electron lasers (XFELs) to small (a few µm or less) and thin (a few hundred nm or less) crystals of novel compounds dispersed on a substrate. For XFEL exposures, two-dimensional (2D) scanning of the substrate coupled with rotation enables highly efficient data collection. The recorded patterns can be successfully indexed using lattice parameters obtained through 3D ED. This approach is especially effective for challenging targets, including pharmaceuticals and organic materials that form preferentially oriented flat crystals in low-symmetry space groups. Some of these crystals have been difficult to solve or have yielded incomplete solutions using 3D ED. Our extensive analyses confirmed the superior quality of the SX data regardless of crystal orientations. Additionally, 2D scanning with XFEL pulses gives an overall distribution of the samples on the substrate, which can be useful for evaluating the properties of crystal grains and the quality of layered crystals. Therefore, this study demonstrates that XFEL crystallography has become a powerful tool for conducting structure studies of small crystals of organic compounds.

Oligo(N-methylalanine) as a Peptide-Based Molecular Scaffold with a Minimal Structure and High Density of Functionalizable Sites.

Functionalizable synthetic molecules with nanometer sizes and defined shapes in water are useful as molecular scaffolds to mimic the functions of biomacromolecules and develop chemical tools for manipulating biomacromolecules. Herein, we propose oligo(N-methylalanine) (oligo-NMA) as a peptide-based molecular scaffold with a minimal structure and a high density of functionalizable sites. Oligo-NMA forms a defined shape in water without hydrogen-bonding networks or ring constraints, which enables the molecule to act as a scaffold with minimal atomic composition. Furthermore, functional groups can be readily introduced on the nitrogens and α-carbons of oligo-NMA. Computational and NMR spectroscopic analysis suggested that the backbone structure of oligo-NMA is not largely affected by functionalization. Moreover, the usefulness of oligo-NMA was demonstrated by the design of protein ligands. The ease of synthesis, minimal structure, and high functionalization flexibility makes oligo-NMA a useful scaffold for chemical and biological applications.

Functional Group Distribution of the Carrier Surface Influences Adhesion of Methanothermobacter thermautotrophicus.

Various support carriers are used for high-density retention of methanogenic archaea in anaerobic wastewater treatment systems. Although the physicochemical properties of carrier materials and microorganisms influence the adhesion of methanogenic archaea, details about the underlying mechanism remain poorly characterized. We applied seven types of chemical surface modifications to carbon felts to clarify the adhesion properties of Methanothermobacter thermautotrophicus, a representative thermophilic hydrogenotrophic methanogen. The relationship between carrier surface properties and methanogen adhesion was evaluated. M. thermautotrophicus adhesion was significantly increased up to 2.6 times in comparison with control on carbon felts treated with NaOH, HCl, H2SO4, or Na2HPO4. Treated carbon felts showed a lower water contact angle, but no correlation between the carrier surface contact angle and methanogen adhesion was observed. On the other hand, at the surface of the carrier that showed improved adhesion of methanogens, the ratio of -COOH : -OH was 1 : 0.65. Such a ratio was not observed with treated carriers for which methanogen adhesion was not improved. Therefore, in the adhesion of M. thermautotrophicus, the functional group abundance was important as well as physical surface properties such as the hydrophobicity. Hydrogenotrophic methanogens are involved in active methanation during the startup of anaerobic digestion. Additionally, these methanogenic archaea function as methanogenic cathode catalysts. Therefore, anaerobic digestion performance will greatly improve by controlling the adhesion of hydrogenotrophic methanogens such as M. thermautotrophicus.

A Peptoid with Extended Shape in Water.

The term "peptoids" was introduced decades ago to describe peptide analogues that exhibit better physicochemical and pharmacokinetic properties than peptides. Oligo(N-substituted glycine) (oligo-NSG) was previously proposed as a peptoid due to its high proteolytic resistance and membrane permeability. However, oligo-NSG is conformationally flexible, and ensuring a defined shape in water is difficult. This conformational flexibility severely limits the biological application of oligo-NSG. Here, we propose oligo(N-substituted alanine) (oligo-NSA) as a peptoid that forms a defined shape in water. The synthetic method established in this study enabled the first isolation and conformational study of optically pure oligo-NSA. Computational simulations, crystallographic studies, and spectroscopic analysis demonstrated the well-defined extended shape of oligo-NSA realized by backbone steric effects. This new class of peptoid achieves the constrained conformation without any assistance of N-substituents and serves as a scaffold for displaying functional groups in well-defined three-dimensional space in water, which leads to effective biomolecular recognition.

Compositionally distinct nuclear pore complexes of functionally distinct dimorphic nuclei in the ciliate Tetrahymena.

The nuclear pore complex (NPC), a gateway for nucleocytoplasmic trafficking, is composed of ∼30 different proteins called nucleoporins. It remains unknown whether the NPCs within a species are homogeneous or vary depending on the cell type or physiological condition. Here, we present evidence for compositionally distinct NPCs that form within a single cell in a binucleated ciliate. In Tetrahymena thermophila, each cell contains both a transcriptionally active macronucleus (MAC) and a germline micronucleus (MIC). By combining in silico analysis, mass spectrometry analysis for immuno-isolated proteins and subcellular localization analysis of GFP-fused proteins, we identified numerous novel components of MAC and MIC NPCs. Core members of the Nup107-Nup160 scaffold complex were enriched in MIC NPCs. Strikingly, two paralogs of Nup214 and of Nup153 localized exclusively to either the MAC or MIC NPCs. Furthermore, the transmembrane components Pom121 and Pom82 localize exclusively to MAC and MIC NPCs, respectively. Our results argue that functional nuclear dimorphism in ciliates is likely to depend on the compositional and structural specificity of NPCs.

Prevalence and first genotyping of Giardia duodenalis in beef calves in Vietnam.

Little information is available on the epidemiology of Giardia duodenalis in beef cattle from Vietnam. This study was performed to determine the prevalence and genotypes/assemblages of G. duodenalis in native beef calves younger than 6 months in the region. A total of 412 calf fecal samples, randomly selected from 99 small-scale farms located in DacLac and KhanhHoa provinces, central Vietnam, were screened for the presence of G. duodenalis cysts using the zinc-sulfate flotation method followed by iodine staining. The overall prevalence on the sample and herd levels were 13.8% (57/412) and 42.4% (42/99), respectively. Molecular analysis in the ß-giardin and triosephosphate isomerase genes demonstrated the presence of only G. duodenalis assemblage E in the animals. Since assemblage E has been rarely reported in humans, the zoonotic risk in beef calves in the region appears to be minimal.

Role of the Cytosolic Heat Shock Protein 70 Ssa5 in the Ciliate Protozoan Tetrahymena thermophila.

Heat shock protein 70 (Hsp70) is a member of a family of conserved chaperone proteins whose function is well investigated in many model organisms. Here we focus on an Hsp70 called Ssa5 in the ciliate protozoan Tetrahymena thermophila, and reveal that its translation is heat inducible as for general Hsps. Moreover, the protein is abundantly expressed in the cytoplasm during sexual reproduction (conjugation) as well as in response to heat-stress. Knocking out of SSA5 (ΔSSA5) does not affect the survival of the cell under heat-stress, likely due to other Hsp70 paralogs compensating for the defect. During conjugation, ΔSSA5 leads to a fertilization defect in which the two pronuclei are in close proximity but never fuse. The unfertilized pronuclei differentiate, resulting in a heterokaryon with developed haploid germline and somatic nuclei. In addition, degeneration of the parental somatic nucleus is not affected. These results suggest a specific involvement of Ssa5 in pronuclear fusion and fertilization.

Circular metagenome-assembled genome of Candidatus Cloacimonadota recovered from anaerobic digestion sludge.

This study reports a circular metagenome-assembled genome (cMAG) of Candidatus Cloacimonadota recovered from a mesophilic full-scale food waste treatment plant. The cMAG spans 2,298,113 bp, with 980× coverage and 1 contig.

Circular metagenome-assembled genome of Candidatus Patescibacteria recovered from anaerobic digestion sludge.

A single-contig, circular metagenome-assembled genome (cMAG) of Candidatus (Ca.) Patescibacteria was reconstructed from a mesophilic full-scale food waste treatment plant in Japan. The genome is of small size and lacks fundamental biosynthetic pathways. Taxonomic analysis using the Genome Taxonomy Database revealed that this cMAG belonged to the genus JAEZRQ01 (Ca. Parcubacteria).

Goethite and Hematite Nanoparticles Show Promising Anti-Toxoplasma Properties.

Toxoplasma gondii is an intracellular parasitic protozoan with a high infection rate in mammals, including humans, and birds. There is no effective vaccine, and treatment relies on antiparasitic drugs. However, existing antiprotozoal drugs have strong side effects and other problems; therefore, new treatment approaches are needed. Metal nanoparticles have attracted increased interest in the biomedical community in recent years because of their extremely high surface area to volume ratio and their unique reactivity that could be exploited for medicinal purposes. Previously, we confirmed the anti-Toxoplasma effects of gold, silver, and platinum nanoparticles, in a growth inhibition test. Here, we asked whether the anti-Toxoplasma effect could be confirmed with less expensive metal nanoparticles, specifically iron oxide nanoparticles (goethite and hematite). To improve the selective action of the nanoparticles, we modified the surface with l-tryptophan as our previous findings showed that the bio-modification of nanoparticles enhances their selectivity against T. gondii. Fourier-Transform Infrared Spectroscopy (FTIR) analysis confirmed the successful coating of the iron oxide nanoparticles with l-tryptophan. Subsequently, cytotoxicity and growth inhibition assays were performed. L-tryptophan-modified nanoparticles showed superior anti-Toxoplasma action compared to their naked nanoparticle counterparts. L-tryptophan enhanced the selective toxicity of the iron oxide nanoparticles toward T. gondii. The bio-modified nanoparticles did not exhibit detectable host cell toxicity in the effective anti-Toxoplasma doses. To elucidate whether reactive oxygen species contribute to the anti-Toxoplasma action of the bio-modified nanoparticles, we added Trolox antioxidant to the assay medium and found that Trolox appreciably reduced the nanoparticle-induced growth inhibition.

Toxoplasma gondii chitinase-like protein TgCLP1 regulates the parasite cyst burden.

Toxoplasma, an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Although Toxoplasma secretory proteins during acute infection (tachyzoite, which divides rapidly and causes inflammation) have been extensively characterized, those involved in chronic infection (bradyzoite, which divides slowly and is surrounded by a cyst wall) remain uncertain. Regulation of the cyst wall is essential to the parasite life cycle, and polysaccharides, such as chitin, in the cyst wall are necessary to sustain latent infection. Toxoplasma secretory proteins during the bradyzoite stage may have important roles in regulating the cyst wall via polysaccharides. Here, we focused on characterizing the hypothetical T. gondii chitinase, chitinase-like protein 1 (TgCLP1). We found that the chitinase-like domain containing TgCLP1 is partially present in the bradyzoite microneme and confirmed, albeit partially, its previous identification in the tachyzoite microneme. Furthermore, although parasites lacking TgCLP1 could convert from tachyzoites to bradyzoites and make an intact cyst wall, they failed to convert from bradyzoites to tachyzoites, indicating that TgCLP1 is necessary for bradyzoite reactivation. Taken together, our findings deepen our understanding of the molecular basis of recrudescence and could contribute to the development of novel strategies for the control of toxoplasmosis.

A high-resolution structural characterization and physicochemical study of how a peptoid binds to an oncoprotein MDM2.

Peptoids are a promising drug modality targeting disease-related proteins, but how a peptoid engages in protein binding is poorly understood. This is primarily due to a lack of high-resolution peptoid-protein complex structures and systematic physicochemical studies. Here, we present the first crystal structure of a peptoid bound to a protein, providing high-resolution structural information about how a peptoid binds to a protein. We previously reported a rigid peptoid, oligo(N-substituted alanine) (oligo-NSA), and developed an oligo-NSA-type peptoid that binds to MDM2. X-ray crystallographic analysis of the peptoid bound to MDM2 showed that the peptoid recognizes the MDM2 surface predominantly through the interaction of the N-substituents, while the main chain acts as a scaffold. Additionally, conformational, thermodynamic, and kinetic analysis of the peptoid and its derivatives with a less rigid main chain revealed that rigidification of the peptoid main chain contributes to improving the protein binding affinity. This improvement is thermodynamically attributed to an increased magnitude of the binding enthalpy change, and kinetically to an increased association rate and decreased dissociation rate. This study provides invaluable insights into the design of protein-targeting peptoids.

Prevalence and molecular characterization of Cryptosporidium in ostriches (Struthio camelus) on a farm in central Vietnam.

This study was performed to determine the prevalence and molecular characterization of Cryptosporidium in ostriches on a farm in Khanh Hoa province, central Vietnam. A total of 464 ostrich fecal samples were examined Cryptosporidium oocysts using the modified Ziehl-Neelsen staining method, and 110 (overall prevalence 23.7%) were identified as positive by microscopy. Prevalence of Cryptosporidium in animals of <45 days, 45-60 days, 61-90 days, 91 days-12 months and >12 months was 23.5% (16/68), 33.3% (22/66), 35.2% (68/193), 0 and 5.8% (4/69), respectively (p<0.05). The majority of positive samples scored as the 3+ level of intensity of infection were from 61 to 90 days ostriches. Molecular analysis in the 18S ribosomal RNA, 70 kDa heat shock protein and actin genes demonstrated the presence of only Cryptosporidium avian genotype II in ostriches in central Vietnam.

Molecular characterization of Cryptosporidium in pigs in central Vietnam.

Little information is available on the epidemiology of Cryptosporidium in pigs in central Vietnam. The aims of this study were to investigate the prevalence and to characterize the genotype distribution of Cryptosporidium isolates in pigs in this region. A total of 193 pig fecal samples were screened for the presence of Cryptosporidium oocysts using the modified Ziehl-Neelsen staining method, and 28 (overall prevalence 14.5 %) were identified as positive by microscopic observation. Positive samples were further analyzed by polymerase chain reaction amplification and sequencing. Genetic identification based on the 18S ribosomal RNA and 70 kDa heat shock protein genes revealed that pigs in Vietnam are infected with two species/genotypes (Cryptosporidium suis and Cryptosporidium pig genotype II). This study is the first molecular characterization of Cryptosporidium in pigs in Vietnam. The presence of these host-adapted species/genotypes suggests that pigs may not pose a significant public health risk in this area. More extensive studies are necessary to ascertain the zoonotic potential of Cryptosporidium in porcine hosts in Vietnam.

Species-specific Primer and Probe Sets for Detection of Syntrophic Long-chain Fatty Acid-degrading Bacteria in Anaerobic Digestion Using Quantitative PCR.

Lipid-rich wastes are energy-dense substrates for anaerobic digestion. However, long-chain fatty acids (LCFAs), key intermediates in lipid degradation, inhibit methanogenic activity. In this study, TaqMan-based qPCR assays targeting the 16S rRNA gene of the cardinal LCFA-degrading bacterial species Syntrophomonas palmitatica and S. zehnderi were developed and validated. A trial experiment showed the advantage of species-specific quantification versus genus-specific quantification in assessing bacterial capacity for lipidic waste degradation. These qPCR assays will serve as monitoring tools for estimating the LCFA-degrading capacity of anaerobic digester communities and developing an effective strategy to enrich LCFA-degrading bacteria.

Computational capability of ecological dynamics.

Ecological dynamics is driven by complex ecological networks. Computational capabilities of artificial networks have been exploited for machine learning purposes, yet whether an ecological network possesses a computational capability and whether/how we can use it remain unclear. Here, we developed two new computational/empirical frameworks based on reservoir computing and show that ecological dynamics can be used as a computational resource. In silico ecological reservoir computing (ERC) reconstructs ecological dynamics from empirical time series and uses simulated system responses for information processing, which can predict near future of chaotic dynamics and emulate nonlinear dynamics. The real-time ERC uses real population dynamics of a unicellular organism, Tetrahymena thermophila. The temperature of the medium is an input signal and population dynamics is used as a computational resource. Intriguingly, the real-time ecological reservoir has necessary conditions for computing (e.g. synchronized dynamics in response to the same input sequences) and can make near-future predictions of empirical time series, showing the first empirical evidence that population-level phenomenon is capable of real-time computations. Our finding that ecological dynamics possess computational capability poses new research questions for computational science and ecology: how can we efficiently use it and how is it actually used, evolved and maintained in an ecosystem?

The role of atypical MAP kinase 4 in the host interaction with Cryptosporidium parvum.

Cryptosporidium parvum is an apicomplexan parasite that causes severe zoonotic diarrhea in humans and calves. Since there are no effective treatments or vaccines for infants or immunocompromised patients, it is important to understand the molecular mechanisms of the parasite-host interaction for novel drug discovery. Mitogen-activated protein kinase (MAP kinase) is a key host factor in interactions between host and various pathogens, including parasites. Although the function of conventional MAP kinases against parasite infection has been investigated, that of atypical MAP kinases remains largely unknown. Therefore, we focused on one of the atypical MAP kinases, MAPK4, and its effect on C. parvum infection in human intestinal cells. Here, we report that MAPK4-deficient intestinal cells showed a significant reduction in C. parvum infection. We also show that host MAPK4 has a role in host cell survival from C. parvum infection. In addition, we show that C. parvum requires host MAPK4 for its successful invasion and asexual reproduction. Taken together, our data suggest that MAPK4 is an important host factor contributing to C. parvum infection in human intestinal cells.

Antiplasmodial and interferon-gamma-modulating activities of the aqueous extract of stone breaker (Phyllanthus niruri Linn.) in malaria infection.

Plasmodium falciparum parasites are the primary cause of malaria across Africa. The problem of drug resistance to malaria is ever growing and novel therapeutic strategies need to be developed, particularly those targeting the parasite and also the host or host-pathogen interaction. Previous studies have shown that the development of cerebral malaria (CM) is related to dysregulation of the immune system in a murine malaria model of experimental cerebral malaria. It involves a complex interaction of events and interferon-gamma seems to be the unifying factor. Therefore, the antiplasmodial activity targeting the parasite and immunomodulatory strategies that reduce overall host inflammation, with IFN-γ in focus, could delay CM onset and prove beneficial in malaria infection therapy. Phyllanthus niruri is used to treat fever and other symptoms of malaria in Nigeria. Its modes of action as an anti-malarial remedy have not been exhaustively investigated. This study therefore examined the aqueous extract of P. niruri (PE) for its antiplasmodial activity in vitro using the Plasmodium falciparum HB3 strain. Furthermore, in vivo murine malaria model using the Plasmodium berghei ANKA strain was used to investigate its anti-malarial effects. We showed that PE has multiple anti-malarial effects, including anti-parasitic and host immunomodulatory activities. Co-culture of P. falciparum with PE and some of its phytoconstituents drastically reduced parasite number. PE also decreased parasitemia, and increased the survival of infected mice. We also observed that the integrity of the blood-brain barrier was maintained in the PE-treated mice. The results confirmed that PE showed moderate antiplasmodial activity. In vivo murine malaria model using P. berghei ANKA for experimental cerebral malaria revealed that PE suppressed parasite growth, and modulate the production of interferon-gamma. The findings demonstrate that PE affects malaria progression, targeting parasites and host cells.

Relationship Between Rumen Microbial Composition and Fibrolytic Isozyme Activity During the Biodegradation of Rice Straw Powder Using Rumen Fluid.

Rumen fibrolytic microorganisms have been used to increase the rate of lignocellulosic biomass biodegradation; however, the microbial and isozymatic characteristics of biodegradation remain unclear. Therefore, the present study investigated the relationship between rumen microorganisms and fibrolytic isozymes associated with lignocellulosic biomass hydrolysis. Rice straw, a widely available agricultural byproduct, was ground and used as a substrate. The biodegradation of rice straw powder was performed anaerobically in rumen fluid for 48 h. The results obtained revealed that 31.6 and 23.3% of cellulose and hemicellulose, respectively, were degraded. The total concentration of volatile fatty acids showed a 1.8-fold increase (from 85.4 to 151.6| |mM) in 48 h, and 1,230.1| |mL L-1 of CO2 and 523.5| |mL L-1 of CH4 were produced. The major isozymes identified by zymograms during the first 12| |h were 51- and 140-kDa carboxymethyl cellulases (CMCases) and 23- and 57-kDa xylanases. The band densities of 37-, 53-, and 58-kDa CMCases and 38-, 44-, and 130-kDa xylanases increased from 24 to 36 h. A microbial ana-lysis indicated that the relative abundances of Prevotella, Fibrobacter, and Bacteroidales RF16 bacteria, Neocallimastix and Cyllamyces fungi, and Dasytricha and Polyplastron protozoa were related to fibrolytic isozyme activity. The present results provide novel insights into the relationships between fibrolytic isozymes and rumen microorganisms during lignocellulose biodegradation.

Molecular characterization of Cryptosporidium in native beef calves in central Vietnam.

The aims of this study were to investigate the prevalence of Cryptosporidium and to characterize the genotype distribution of Cryptosporidium isolates in native beef calves 2-6 months old in Dac Lac province, central Vietnam. The presence of Cryptosporidium oocysts was determined using the modified Ziehl-Neelsen staining method. The overall prevalence on the sample and herd levels were 18.9% (44/232) and 50% (20/40), respectively. Genotyping based on PCR and sequence analysis of the 18 S rRNA gene revealed occurrence of the two nonzoonotic species Cryptosporidium ryanae and Cryptosporidium bovis, with the former as a dominant species in the animals. The absence of the zoonotic species Cryptosporidium parvum in calves examined suggests that the native beef calves 2-6 months old in the study area are unlikely to contribute to human cryptosporidiosis transmission.