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.

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.

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.

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.

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.

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.

Snf2 Proteins Are Required to Generate Gamete Pronuclei in Tetrahymena thermophila.

During sexual reproduction/conjugation of the ciliate Tetrahymena thermophila, the germinal micronucleus undergoes meiosis resulting in four haploid micronuclei (hMICs). All hMICs undergo post-meiotic DNA double-strand break (PM-DSB) formation, cleaving their genome. DNA lesions are subsequently repaired in only one 'selected' hMIC, which eventually produces gametic pronuclei. DNA repair in the selected hMIC involves chromatin remodeling by switching from the heterochromatic to the euchromatic state of its genome. Here, we demonstrate that, among the 15 Tetrahymena Snf2 family proteins, a core of the ATP-dependent chromatin remodeling complex in Tetrahymena, the germline nucleus specific Iswi in Tetrahymena IswiGTt and Rad5Tt is crucial for the generation of gametic pronuclei. In either gene knockout, the selected hMIC which shows euchromatin markers such as lysine-acetylated histone H3 does not appear, but all hMICs in which markers for DNA lesions persist are degraded, indicating that both IswiGTt and Rad5Tt have important roles in repairing PM-DSB DNA lesions and remodeling chromatin for the euchromatic state in the selected hMIC.

Identification of potent anti-Cryptosporidium new drug leads by screening traditional Chinese medicines.

Cryptosporidium spp. are gastrointestinal opportunistic protozoan parasites that infect humans, domestic animals, and wild animals all over the world. Cryptosporidiosis is the second leading infectious diarrheal disease in infants less than 5 years old. Cryptosporidiosis is a common zoonotic disease associated with diarrhea in infants and immunocompromised individuals. Consequently, cryptosporidiosis is considered a serious economic, veterinary, and medical concern. The treatment options for cryptosporidiosis are limited. To address this problem, we screened a natural product library containing 87 compounds of Traditional Chinese Medicines for anti-Cryptosporidium compounds that could serve as novel drug leads and therapeutic targets against C. parvum. To examine the anti-Cryptosporidium activity and half-maximal inhibitory doses (EC50) of these compounds, we performed in vitro assays (Cryptosporidium growth inhibition assay and host cell viability assay) and in vivo experiments in mice. In these assays, the C. parvum HNJ-1 strain was used. Four of the 87 compounds (alisol-A, alisol-B, atropine sulfate, and bufotalin) showed strong anti-Cryptosporidium activity in vitro (EC50 values = 122.9±6.7, 79.58±13.8, 253.5±30.3, and 63.43±18.7 nM, respectively), and minimum host cell cytotoxicity (cell survival > 95%). Furthermore, atropine sulfate (200 mg/kg) and bufotalin (0.1 mg/kg) also showed in vivo inhibitory effects. Our findings demonstrate that atropine sulfate and bufotalin are effective against C. parvum infection both in vitro and in vivo. These compounds may, therefore, represent promising novel anti-Cryptosporidium drug leads for future medications against cryptosporidiosis.

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.

Shifts in xylanases and the microbial community associated with xylan biodegradation during treatment with rumen fluid.

Treatment with rumen fluid improves methane production from non-degradable lignocellulosic biomass during subsequent methane fermentation; however, the kinetics of xylanases during treatment with rumen fluid remain unclear. This study aimed to identify key xylanases contributing to xylan degradation and their individual activities during xylan treatment with bovine rumen microorganisms. Xylan was treated with bovine rumen fluid at 37°C for 48 h under anaerobic conditions. Total solids were degraded into volatile fatty acids and gases during the first 24 h. Zymography showed that xylanases of 24, 34, 85, 180, and 200 kDa were highly active during the first 24 h. Therefore, these xylanases are considered to be crucial for xylan degradation during treatment with rumen fluid. Metagenomic analysis revealed that the rumen microbial community's structure and metabolic function temporally shifted during xylan biodegradation. Although statistical analyses did not reveal significantly positive correlations between xylanase activities and known xylanolytic bacterial genera, they positively correlated with protozoal (e.g., Entodinium, Diploplastron, and Eudiplodinium) and fungal (e.g., Neocallimastix, Orpinomyces, and Olpidium) genera and unclassified bacteria. Our findings suggest that rumen protozoa, fungi, and unclassified bacteria are associated with key xylanase activities, accelerating xylan biodegradation into volatile fatty acids and gases, during treatment of lignocellulosic biomass with rumen fluid.

Peptoid-based reprogrammable template for cell-permeable inhibitors of protein-protein interactions.

The development of inhibitors of intracellular protein-protein interactions (PPIs) is of great significance for drug discovery, but the generation of a cell-permeable molecule with high affinity to protein is challenging. Oligo(N-substituted glycines) (oligo-NSGs), referred to as peptoids, are attractive as potential intracellular PPI inhibitors owing to their high membrane permeability. However, their intrinsically flexible backbones make the rational design of inhibitors difficult. Here, we propose a peptoid-based rational approach to develop cell-permeable PPI inhibitors using oligo(N-substituted alanines) (oligo-NSAs). The rigid structures of oligo-NSAs enable independent optimization of each N-substituent to improve binding affinity and membrane permeability, while preserving the backbone shape. A molecule with optimized N-substituents inhibited a target PPI in cells, which demonstrated the utility of oligo-NSA as a reprogrammable template to develop intracellular PPI inhibitors.

Characteristics of various fibrolytic isozyme activities in the rumen microbial communities of Japanese Black and Holstein Friesian cattle under different conditions.

Rumen microorganisms produce various fibrolytic enzymes and degrade lignocellulosic materials into nutrient sources for ruminants; therefore, the characterization of fibrolytic enzymes contributing to the polysaccharide degradation in the rumen microbiota is important for efficient animal production. This study characterized the fibrolytic isozyme activities of a rumen microbiota from four groups of housed cattle (1, breeding Japanese Black; 2, feedlot Japanese Black; 3, lactating Holstein Friesian; 4, dry Holstein Friesian). Rumen fluids in all cattle groups showed similar concentrations of total volatile fatty acids and reducing sugars, whereas acetic acid contents and pH were different among them. Predominant genera were commonly detected in all cattle, although the bacterial compositions were different among cattle groups. Zymograms of whole proteins in rumen fluids showed endoglucanase activities at 55 and 57 kDa and xylanase activity at 44 kDa in all cattle. Meanwhile, several fibrolytic isozyme activities differed among cattle groups and individuals. Treponema, Succinivibrio, Anaeroplasma, Succiniclasticum, Ruminococcus, and Butyrivibrio showed positive correlations with fibrolytic isozyme activities. Further, endoglucanase activity at 68 kDa was positively correlated with pH. This study suggests the characteristics of fibrolytic isozyme activities and their correlations with the rumen microbiota.

Exploration of microbial communities contributing to effective methane production from scum under anaerobic digestion.

Scum is formed by the adsorption of long-chain fatty acids (LCFAs) onto biomass surface in anaerobic digestion of oily substrates. Since scum is a recalcitrant substrate to be digested, it is disposed via landfilling or incineration, which results in biomass washout and a decrease in methane yield. The microbes contributing to scum degradation are unclear. This study aimed to investigate the cardinal microorganisms in anaerobic scum digestion. We pre-incubated a sludge with scum to enrich scum-degrading microbes. Using this sludge, a 1.3-times higher methane conversion rate (73%) and a faster LCFA degradation compared with control sludge were attained. Then, we analyzed the cardinal scum-degrading microbes in this pre-incubated sludge by changing the initial scum-loading rates. Increased 16S rRNA copy numbers for the syntrophic fatty-acid degrader Syntrophomonas and hydrogenotrophic methanogens were observed in scum high-loaded samples. 16S rRNA amplicon sequencing indicated that Syntrophomonas was the most abundant genus in all the samples. The amino-acid degrader Aminobacterium and hydrolytic genera such as Defluviitoga and Sporanaerobacter became more dominant as the scum-loading rate increased. Moreover, phylogenic analysis on Syntrophomonas revealed that Syntrophomonas palmitatica, which is capable of degrading LCFAs, related species became more dominant as the scum-loading rate increased. These results indicate that a variety of microorganisms that degrade LCFAs, proteins, and sugars are involved in effective scum degradation.

Depletion of Intracellular Glutamine Pools Triggers Toxoplasma gondii Stage Conversion in Human Glutamatergic Neurons.

Toxoplasma gondii chronically infects the brain as latent cysts containing bradyzoites and causes various effects in the host. Recently, the molecular mechanisms of cyst formation in the mouse brain have been elucidated, but those in the human brain remain largely unknown. Here, we show that abnormal glutamine metabolism caused by both interferon-γ (IFN-γ) stimulation and T. gondii infection induce cyst formation in human neuroblastoma cells regardless of the anti-T. gondii host factor nitric oxide (NO) level or Indoleamine 2,3-dioxygenase-1 (IDO1) expression. IFN-γ stimulation promoted intracellular glutamine degradation in human neuronal cells. Additionally, T. gondii infection inhibited the mRNA expression of the host glutamine transporters SLC38A1 and SLC38A2. These dual effects led to glutamine starvation and triggered T. gondii stage conversion in human neuronal cells. Furthermore, these mechanisms are conserved in human iPSC-derived glutamatergic neurons. Taken together, our data suggest that glutamine starvation in host cells is an important trigger of T. gondii stage conversion in human neurons.

Diversity of Sulfur-oxidizing Bacteria at the Surface of Cattle Manure Composting Assessed by an Analysis of the Sulfur Oxidation Gene soxB.

Sulfur-oxidizing bacterial diversity at the surface of cattle manure was characterized throughout the composting process using a sulfur oxidation gene (soxB) clone library approach. In the mesophilic phase, clones related to the genera Hydrogenophaga and Hydrogenophilus were characteristically detected. In the thermophilic phase, clones related to the genera Hydrogenophaga and Thiohalobacter were predominant. In the cooling phase, the predominant soxB sequences were related to the genus Pseudaminobacter and a new sulfur-oxidizing bacterium belonging to the class Alphaproteobacteria. The present study showed changes in the community composition of sulfur-oxidizing bacteria at the surface of compost throughout the composting process.

Distribution of Cryptosporidium species isolated from diarrhoeic calves in Japan.

Cryptosporidium spp. are enteric protozoan parasites that infect a wide range of hosts including humans, and domestic and wild animals. The aim of this study was to molecularly characterize the Cryptosporidium spp. found in calf faeces in Japan. A total of 80 pre-weaned beef and dairy calves' diarrhoeic faecal specimens were collected from nine different prefectures in Japan. A nested polymerase chain reaction targeting the small subunit 18S rRNA and GP60 genes were used to detect the Cryptosporidium genotypes and subtypes. 83.8% (67 out of 80) of the specimens were positive for Cryptosporidium spp.; Cryptosporidium was found in both beef and dairy calves. Cryptosporidium parvum was the predominant species, detected in 77.5% (31/40) of beef calves and 80% (32/40) of dairy calves. Cryptosporidium bovis was also detected, 5.0% (2/40) of dairy calves, and C. ryanae was also found 2.5% (1/40) of dairy calves. One mixed-species infection, 2.5% (1/40) was detected in a beef calf having C. parvum, and C. ryanae. We detected the most common subtype of C. parvum (i.e., IIaA15G2R1), as well as other subtypes (i.e., IIaA14G3R1, IIaA14G2R1, and IIaA13G1R1) that have not previously been detected in calves in Japan. Our results demonstrate the widespread diversity of Cryptosporidium infection in calves in Japan.