Improved and easy method for long-term in vitro culture of rumen ciliates Dasytricha ruminantium.

The rumen ciliates are a diverse group of protozoa residing in the rumen of ruminant animals. They are primarily found in the orders Entodiniomorphida and Vestibuliferida, playing crucial roles in the digestion and breakdown of feed within the host's rumen, closely intertwined with the host's nutrient absorption. In vitro monocultures of representatives of rumen ciliates are important to better study them. So far, Entodinium caudatum and Epidinium caudatum, representatives of the order Entodiniomorphida, have been successfully cultivated as a monoculture in vitro. However, for the order Vestibuliferida, no representative species has been established a stable monoculture in vitro up to date, which hampers to study their physiology and metabolism. Therefore, we have developed a simple method for the in vitro cultivation of Dasytricha ruminantium, a representative rumen ciliate in the order Vestibuliferida. Utilizing an optimized culture medium with easily obtainable components, and the cultivation process is simple. This will facilitate further research in metabolism and other studies requiring large pure live materials.1.Filtration and separation for enriching D. ruminantium.2.A culture medium (DRM) suitable for the growth of D. ruminantium, with easily obtainable components.3.Simple cultivation process, facilitating the obtainment of a large number of monocultured D. ruminantium.

Mitochondrial GRIM19 Loss Induces Liver Fibrosis through NLRP3/IL33 Activation via Reactive Oxygen Species/NF-кB Signaling.

Background and Aims: Hepatic fibrosis (HF) is a critical step in the progression of hepatocellular carcinoma (HCC). Gene associated with retinoid-IFN-induced mortality 19 (GRIM19), an essential component of mitochondrial respiratory chain complex I, is frequently attenuated in various human cancers, including HCC. Here, we aimed to investigate the potential relationship and underlying mechanism between GRIM19 loss and HF pathogenesis. Methods: GRIM19 expression was evaluated in normal liver tissues, hepatitis, hepatic cirrhosis, and HCC using human liver disease spectrum tissue microarrays. We studied hepatocyte-specific GRIM19 knockout mice and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) lentivirus-mediated GRIM19 gene-editing in murine hepatocyte AML12 cells in vitro and in vivo. We performed flow cytometry, immunofluorescence, immunohistochemistry, western blotting, and pharmacological intervention to uncover the potential mechanisms underlying GRIM19 loss-induced HF. Results: Mitochondrial GRIM19 was progressively downregulated in chronic liver disease tissues, including hepatitis, cirrhosis, and HCC tissues. Hepatocyte-specific GRIM19 heterozygous deletion induced spontaneous hepatitis and subsequent liver fibrogenesis in mice. In addition, GRIM19 loss caused chronic liver injury through reactive oxygen species (ROS)-mediated oxidative stress, resulting in aberrant NF-кB activation via an IKK/IкB partner in hepatocytes. Furthermore, GRIM19 loss activated NLRP3-mediated IL33 signaling via the ROS/NF-кB pathway in hepatocytes. Intraperitoneal administration of the NLRP3 inhibitor MCC950 dramatically alleviated GRIM19 loss-driven HF in vivo. Conclusions: The mitochondrial GRIM19 loss facilitates liver fibrosis through NLRP3/IL33 activation via ROS/NF-кB signaling, providing potential therapeutic approaches for earlier HF prevention.

Characterization of the complete mitochondrial genome of Ergasilus anchoratus Markevich, 1946 (Ergasilidae) and phylogeny of Copepoda.

The mitochondrial (mt) genome can provide data for phylogenetic analyses and evolutionary biology. Herein, we sequenced and annotated the complete mt genome of Ergasilus anchoratus. This mt genome was 13852 bp long and comprised 13 protein-coding genes (PCGs), 22 tRNAs and 2 rRNAs. All PCGs used the standard ATN start codons and complete TAA/TAG termination codons. A majority of tRNA genes exhibited standard cloverleaf secondary structures, with the exception of one tRNA that lacked the TψC arm (trnC), and three tRNAs that lacked the DHU arm (trnR, trnS1 and trnS2). Phylogenetic analyses conducted using Bayesian inference (BI) and maximum likelihood (ML) methods both supported Ergasilidae as a monophyletic family forming a sister group to Lernaea cyprinacea and Paracyclopina nana. It also supported the monophyly of orders Calanoida, Cyclopoida, and Siphonostomatoida; and the monophyly of families Harpacticidae, Ergasilidae, Diaptomidae, and Calanidae. The gene orders of E. anchoratus and Sinergasilus undulatus were identical, which represents the first instance of two identical gene orders in copepods. More mt genomes are needed to better understand the phylogenetic relationships within Copepoda in the future.

Comprehensive molecular characterisation of the complete mitogenome of Ergasilus tumidus and phylogenetic relationships of Copepoda inferred from mitogenomes.

Although parasitic copepods of the genus Ergasilus von Nordmann, 1832 are globally distributed parasites of fish, their phylogenetic relationships with other Copepoda are not clear, and the characteristics of their mitochondrial genomes (mitogenomes) are not thoroughly understood. The objective of this study was to address these knowledge gaps by sequencing the complete mitogenome of Ergasilus tumidus Markevich, 1940. The complete mitogenome (GenBank Acc. No. OQ596537) was 14,431 bp long and it comprised 13 protein-coding genes (PCGs), 22 tRNAs, two tRNAs, and two control regions (CRs). Phylogenetic analyses, conducted using concatenated nucleotide and amino acid sequences of 13 protein-coding genes, produced two partially incongruent topologies. While the order Calanoida was consistently resolved as the sister lineage to the other three orders, topological instability was observed in the relationships of the orders Cyclopoida, Siphonostomatoida and Harpacticoida. Siphonostomatoida clustered with Cyclopoida in the nucleotide-based phylogeny, but with Harpacticoida in the amino acid-based phylogeny. The latter topology conforms to the widely accepted relationships, but we speculate that the former topology is more likely to be the correct one. Our study provides a complete mitogenome sequence of E. tumidus, which helps us better understand the molecular evolution of the genus Ergasilus. Additionally, we suggest a different perspective on the controversial phylogenetic relationships among Siphonostomatoida, Cyclopoida and Harpacticoida, diverging from previously accepted views.

Evolutionary Rates, Divergence Rates, and Performance of Individual Mitochondrial Genes Based on Phylogenetic Analysis of Copepoda.

Copepoda is a large and diverse group of crustaceans, which is widely distributed worldwide. It encompasses roughly 9 orders, whose phylogeny remains unresolved. We sequenced the complete mitochondrial genome (mitogenome) of Sinergasilus major (Markevich, 1940) and used it to explore the phylogeny and mitogenomic evolution of Copepoda. The mitogenome of S. major (14,588 bp) encodes the standard 37 genes as well as a putative control region, and molecular features are highly conserved compared to other Copepoda mitogenomes. Comparative analyses indicated that the nad2 gene has relatively high nucleotide diversity and evolutionary rate, as well as the largest amount of phylogenetic information. These results indicate that nad2 may be a better marker to investigate phylogenetic relationships among closely related species in Copepoda than the commonly used cox1 gene. The sister-group relationship of Siphonostomatoida and Cyclopoida was recovered with strong support in our study. The only topological ambiguity was found within Cyclopoida, which might be caused by the rapid evolution and sparse taxon sampling of this lineage. More taxa and genes should be used to reconstruct the Copepoda phylogeny in the future.

Mitochondrial GRIM-19 loss in parietal cells promotes spasmolytic polypeptide-expressing metaplasia through NLR family pyrin domain-containing 3 (NLRP3)-mediated IL-33 activation via a reactive oxygen species (ROS) -NRF2- Heme oxygenase-1(HO-1)-NF-кB axis.

Spasmolytic polypeptide-expressing metaplasia (SPEM), as a pre-neoplastic precursor of intestinal metaplasia (IM), plays critical roles in the development of chronic atrophic gastritis (CAG) and gastric cancer (GC). However, the pathogenetic targets responsible for the SPEM pathogenesis remain poorly understood. Gene associated with retinoid-IFN-induced mortality 19 (GRIM-19), an essential subunit of the mitochondrial respiratory chain complex I, was progressively lost along with malignant transformation of human CAG, little is known about the potential link between GRIM-19 loss and CAG pathogenesis. Here, we show that lower GRIM-19 is associated with higher NF-кB RelA/p65 and NLR family pyrin domain-containing 3 (NLRP3) levels in CAG lesions. Functionally, GRIM-19 deficiency fails to drive direct differentiation of human GES-1 cells into IM or SPEM-like cell lineages in vitro, whereas parietal cells (PCs)-specific GRIM-19 knockout disturbs gastric glandular differentiation and promotes spontaneous gastritis and SPEM pathogenesis without intestinal characteristics in mice. Mechanistically, GRIM-19 loss causes chronic mucosal injury and aberrant NRF2 (Nuclear factor erythroid 2-related factor 2)- HO-1 (Heme oxygenase-1) activation via reactive oxygen species (ROS)-mediated oxidative stress, resulting in aberrant NF-кB activation by inducing p65 nuclear translocation via an IKK/IкB partner, while NRF2-HO-1 activation contributes to GRIM-19 loss-driven NF-кB activation via a positive feedback NRF2-HO-1 loop. Furthermore, GRIM-19 loss did not cause obvious PCs loss but triggers NLRP3 inflammasome activation in PCs via a ROS-NRF2-HO-1-NF-кB axis, leading to NLRP3-dependent IL-33 expression, a key mediator for SPEM formation. Moreover, intraperitoneal administration of NLRP3 inhibitor MCC950 drastically attenuates GRIM-19 loss-driven gastritis and SPEM in vivo. Our study suggests that mitochondrial GRIM-19 maybe a potential pathogenetic target for the SPEM pathogenesis, and its deficiency promotes SPEM through NLRP3/IL-33 pathway via a ROS-NRF2-HO-1-NF-кB axis. This finding not only provides a causal link between GRIM-19 loss and SPEM pathogenesis, but offers potential therapeutic strategies for the early prevention of intestinal GC.

Antidiabetic DPP-4 Inhibitors Reprogram Tumor Microenvironment That Facilitates Murine Breast Cancer Metastasis Through Interaction With Cancer Cells via a ROS-NF-кB-NLRP3 Axis.

Improvement of understanding of the safety profile and biological significance of antidiabetic agents in breast cancer (BC) progression may shed new light on minimizing the unexpected side effect of antidiabetic reagents in diabetic patients with BC. Our recent finding showed that Saxagliptin (Sax) and Sitagliptin (Sit), two common antidiabetic dipeptidyl peptidase-4 inhibitors (DPP-4i) compounds, promoted murine BC 4T1 metastasis via a ROS-NRF2-HO-1 axis in nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. However, the potential role of DPP-4i in BC progression under immune-competent status remains largely unknown. Herein, we extended our investigation and revealed that Sax and Sit also accelerated murine BC 4T1 metastasis in orthotopic, syngeneic, and immune-competent BALB/c mice. Mechanically, we found that DPP-4i not only activated ROS-NRF2-HO-1 axis but also triggered reactive oxygen species (ROS)-dependent nuclear factor kappa B (NF-κB) activation and its downstream metastasis-associated gene levels in vitro and in vivo, while NF-кB inhibition significantly abrogated DPP-4i-driven BC metastasis in vitro. Meanwhile, inhibition of NRF2-HO-1 activation attenuated DPP-4i-driven NF-кB activation, while NRF2 activator ALA enhanced NF-кB activation, indicating an essential role of ROS-NRF2-HO-1 axis in DPP-4i-driven NF-кB activation. Furthermore, we also found that DPP-4i increased tumor-infiltrating CD45, MPO, F4/80, CD4, and Foxp3-positive cells and myeloid-derived suppressor cells (MDSCs), and decreased CD8-positive lymphocytes in metastatic sites, but did not significantly alter cell viability, apoptosis, differentiation, and suppressive activation of 4T1-induced splenic MDSCs. Moreover, we revealed that DPP-4i triggered ROS-NF-κB-dependent NLRP3 inflammasome activation in BC cells, leading to increase in inflammation cytokines such as interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), intercellular cell adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), IL-1ß and IL-33, and MDSCs inductors granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and M-CSF, which play a crucial role in the remodeling of tumor immune-suppressive microenvironment. Thus, our findings suggest that antidiabetic DPP-4i reprograms tumor microenvironment that facilitates murine BC metastasis by interaction with BC cells via a ROS-NRF2-HO-1-NF-κB-NLRP3 axis. This finding not only provides a mechanistic insight into the oncogenic ROS-NRF2-HO-1 in DPP-4i-driven BC progression but also offers novel insights relevant for the improvement of tumor microenvironment to alleviate DPP-4i-induced BC metastasis.

Antidiabetic Agent DPP-4i Facilitates Murine Breast Cancer Metastasis by Oncogenic ROS-NRF2-HO-1 Axis via a Positive NRF2-HO-1 Feedback Loop.

Cancer has been as one of common comorbidities of diabetes. Long-term antidiabetic treatment may potentially exert uncertain impacts on diabetic patients with cancer including breast cancer (BC). Dipeptidyl peptidase-4 inhibitors (DPP-4i) are currently recommended by the AACE as first-line hypoglycemic drugs in type 2 diabetes mellitus (T2DM). Although the safety of DPP-4i has been widely evaluated, the potential side-effects of DPP-4i in cancer metastasis were also reported and remain controversial. Here, we revealed that Saxagliptin (Sax) and Sitagliptin (Sit), two common DPP-4i compounds, potentially promoted murine BC 4T1 metastasis in vitro and in vivo under immune-deficient status. Mechanically, we observed that DPP-4i treatment induced aberrant oxidative stress by triggering ROS overproduction, as well as ROS-dependent NRF2 and HO-1 activations in BC cells, while specific inhibition of ROS, NRF2 or HO-1 activations abrogated DPP-4i-driven BC metastasis and metastasis-associated gene expression in vitro. Furthermore, ALA, a NRF2 activator significantly promoted BC metastasis in vitro and in vivo, which can be abrogated by specific HO-1 inhibition in vitro. Moreover, specific HO-1 inhibition not only reversed DPP-4i-induced NRF2 activation but also abrogated ALA-induced NRF2 activation, resulting in a decrease of metastasis-associated genes, indicating a positive-feedback NRF2-HO-1 loop. Our findings suggest that DPP-4i accelerates murine BC metastasis through an oncogenic ROS-NRF2-HO-1 axis via a positive-feedback NRF2-HO-1 loop. Therefore, this study not only offers novel insights into an oncogenic role of DPP-4i in BC progression but also provides new strategies to alleviate the dark side of DPP-4i by targeting HO-1.

Complete mitochondrial genome of the copepod Sinergasilus undulates (Copepoda: Poecilostomatoida).

The total mitochondrial genome size of Sinergasilus undulatus is 14,239 bp in length, including 13 protein-coding genes (PCGs), two rRNA genes, 22 transfer RNA genes, and a non-coding control region (D-loop). The overall nucleotide composition of the mitochondrial DNA of S. undulatus is 34.9% A, 35.5% T, 15.7% C, 13.9% G, and 70.4% AT, respectively. Phylogenetic analysis suggests that the genus Sinergasilus is monophyletic, and S. undulatus is closely related to S. polycolpus. The complete mitochondrial genome of S. undulatus would be useful for species identification, epidemiology, and phylogenetics among Copepods.

Identification and evolutionary analysis of the nucleolar proteome of Giardia lamblia.

BACKGROUND: The nucleoli, including their proteomes, of higher eukaryotes have been extensively studied, while few studies about the nucleoli of the lower eukaryotes - protists were reported. Giardia lamblia, a protist with the controversy of whether it is an extreme primitive eukaryote or just a highly evolved parasite, might be an interesting object for carrying out the nucleolar proteome study of protists and for further examining the controversy. RESULTS: Using bioinformatics methods, we reconstructed G. lamblia nucleolar proteome (GiNuP) and the common nucleolar proteome of the three representative higher eukaryotes (human, Arabidopsis, yeast) (HEBNuP). Comparisons of the two proteomes revealed that: 1) GiNuP is much smaller than HEBNuP, but 78.4% of its proteins have orthologs in the latter; 2) More than 68% of the GiNuP proteins are involved in the "Ribosome related" function, and the others participate in the other functions, and these two groups of proteins are much larger and much smaller than those in HEBNuP, respectively; 3) Both GiNuP and HEBNuP have their own specific proteins, but HEBNuP has a much higher proportion of such proteins to participate in more categories of nucleolar functions. CONCLUSION: For the first time the nucleolar proteome of a protist - Giardia was reconstructed. The results of comparison of it with the common proteome of three representative higher eukaryotes -- HEBNuP indicated that the simplicity of GiNuP is most probably a reflection of primitiveness but not just parasitic reduction of Giardia, and simultaneously revealed some interesting evolutionary phenomena about the nucleolus and even the eukaryotic cell, compositionally and functionally.

Single-cell transcriptome sequencing of rumen ciliates provides insight into their molecular adaptations to the anaerobic and carbohydrate-rich rumen microenvironment.

Rumen ciliates are a specialized group of ciliates exclusively found in the anaerobic, carbohydrate-rich rumen microenvironment. However, the molecular and mechanistic basis of the physiological and behavioral adaptation of ciliates to the rumen microenvironment is undefined. We used single-cell transcriptome sequencing to explore the adaptive evolution of three rumen ciliates: two entodiniomorphids, Entodinium furca and Diplodinium dentatum; and one vestibuliferid, Isotricha intestinalis. We found that all three species are members of monophyletic orders within the class Litostomatea, with E. furca and D. dentatum in Entodiniomorphida and I. intestinalis in Vestibuliferida. The two entodiniomorphids might use H2-producing mitochondria and the vestibuliferid might use anaerobic mitochondria to survive under strictly anaerobic conditions. Moreover, carbohydrate-active enzyme (CAZyme) genes were identified in all three species, including cellulases, hemicellulases, and pectinases. The evidence that all three species have acquired prokaryote-derived genes by horizontal gene transfer (HGT) to digest plant biomass includes a significant enrichment of gene ontology categories such as cell wall macromolecule catabolic process and carbohydrate catabolic process and the identification of genes in common between CAZyme and HGT groups. These findings suggest that HGT might be an important mechanism in the adaptive evolution of ciliates to the rumen microenvironment.

Insights into the origin and evolution of Peritrichia (Oligohymenophorea, Ciliophora) based on analyses of morphology and phylogenomics.

Peritrichia is a large and distinctive assemblage of ciliated protists that was first observed by Antonie van Leeuwenhoek over 340 years ago. In the last two decades the evolutionary relationships of this subclass have been increasingly debated as morphological and molecular analyses have generated contrasting conclusions. In this study, we provide genomic-scale data from 12 typical representatives. We combine taxon- and gene-rich phylogenomic analyses, with up to 151 genes (43,956 amino acid residues) from 18 freshwater, brackish and marine isolates in order to assess the systematics and evolutionary history of the Peritrichia. The main findings were: (1) the subclass Peritrichia originates from the end of the Proterozoic to the Cambrian; (2) the monophyletic Peritrichia is sister to the Peniculia (represented by Paramecium) within the class Oligohymenophorea; (3) spasmin plays a significant role in peritrich evolution: we detected the spasmin gene in target ciliates and traced the molecular evolution of spasmin, a key spasmoneme component, together with phylogenetic relationships and morphology of the peritrichs. These findings provide evidence that spasmin is an important molecule to illustrate the phylogenetic position of Peritrichia within the class Oligohymenophorea, the monophyly of Peritrichia, and the diverse and rapid evolution of sessilid peritrichs.

Evolution and multiple origins of zona pellucida genes in vertebrates.

Animal egg coats are composed of different glycoproteins collectively named zona pellucida (ZP) proteins. The characterized vertebrate genes encoding ZP proteins have been classified into six subfamilies, and exhibit low similarity to the ZP genes characterized in certain invertebrates. The origin and evolution of the vertebrate ZP genes remain obscure. A search against 97 representative metazoan species revealed various numbers (ranging from three to 33) of different putative egg-coat ZP genes in all 47 vertebrates and several ZP genes in five invertebrate species, but no putative ZP gene was found in the other 45 species. Based on phylogenetic and synteny analyses, all vertebrate egg-coat ZP genes were classified into eight ZP gene subfamilies. Lineage- and species-specific gene duplications and gene losses occurred frequently and represented the main causes of the patchy distribution of the eight ZP gene subfamilies in vertebrates. Thorough phylogenetic analyses revealed that the vertebrate ZP genes could be traced to three independent origins but were not orthologues of the characterized invertebrate ZP genes. Our results suggested that vertebrate egg-coat ZP genes should be classified into eight subfamilies, and a putative evolutionary map is proposed. These findings would aid the functional and evolutionary analyses of these reproductive genes in vertebrates.

Phylogenomic analysis of Balantidium ctenopharyngodoni (Ciliophora, Litostomatea) based on single-cell transcriptome sequencing.

In this paper, we present transcriptome data for Balantidium ctenopharyngodoni Chen, 1955 collected from the hindgut of grass carp (Ctenopharyngodon idella). We evaluated sequence quality and de novo assembled a preliminary transcriptome, including 43.3 megabits and 119,141 transcripts. Then we obtained a final transcriptome, including 17.7 megabits and 35,560 transcripts, by removing contaminative and redundant sequences. Phylogenomic analysis based on a supermatrix with 132 genes comprising 53,873 amino acid residues and phylogenetic analysis based on SSU rDNA of 27 species were carried out herein to reveal the evolutionary relationships among six ciliate groups: Colpodea, Oligohymenophorea, Litostomatea, Spirotrichea, Heterotrichea and Protocruziida. The topologies of both phylogenomic and phylogenetic trees are discussed in this paper. In addition, our results suggest that single-cell sequencing is a sound method of obtaining sufficient omics data for phylogenomic analysis, which is a good choice for uncultivable ciliates. The transcriptome data for Balantidium ctenopharyngodoni are the first omics data within the subclass Trichostomatia, and provide a good basis for ciliate phylogenomic analysis, as well as related omics analysis.

Tracing the structural evolution of eukaryotic ATP binding cassette transporter superfamily.

The ATP binding cassette (ABC) transporters superfamily is one of the largest classes of membrane proteins. The core of the ABC transporter protein is composed of transmembrane domains (TMDs) and nucleotide binding domains (NBD). Eukaryotes ABC transporters are classified into seven main families (ABCA to ABCG) based on sequence similarity and domain organizations. With different domain number and domain organizations, eukaryote ABC transporters show diverse structures: the single structure (NBD or TMD), the ABC2 structure (NBD-NBD), the half structure (TMD-NBD or NBD-TMD) and the full structure (TMD-NBD-TMD-NBD or NBD-TMD-NBD-TMD). However, studies on how various ABC transporter gene structures evolved is still absent. Therefore, in this study, we comprehensively investigated the structural evolution of eukaryotic ABC transporters. The seven eukaryote ABC transporter families (A to G) fell into three groups: A&G group, B,C&D group and E&F group. There were at least four times the number of NBD and TMD fusion events in the origin of the half structure transporter. Two fusion modes were found in the full and ABC2 structure origination. Based on these findings, we present a putative structural evolutionary path of eukaryote ABC transporters that will increase our understanding on their origin, divergence and function.

Phylogenomic analyses reveal subclass Scuticociliatia as the sister group of subclass Hymenostomatia within class Oligohymenophorea.

Scuticociliates and hymenostomes are two groups of the ciliate class Oligohymenophorea, a diverse clade that includes two model genera, Tetrahymena and Paramecium, which have been intensively studied due to their ease of culture and their amenability to a wide range of biochemical and genetic investigations. However, phylogenetic relationships among the subclasses of the Oligohymenophorea, and especially between the Scuticociliatia and Hymenostomatia, are not clearly resolved. Here, we investigate the phylogenetic relationship between the subclasses Scuticociliatia and Hymenostomatia based on omics data. The transcriptomes of five species, comprising four oligohymenophoreans and one colpodean, were sequenced. A supermatrix was constructed for phylogenomic analyses based on 113 genes encoding 43,528 amino acid residues from 26 taxa, including ten representatives of the class Oligohymenophorea. Our phylogenomic analyses revealed that the monophyletic Scuticociliatia is sister to the monophyletic Hymenostomatia, which together form the terminal branch within the monophyletic class Oligohymenophorea. Competing hypotheses for this relationship were rejected by topological tests. Our results provide corroborative evidence for the close relationship between the subclasses Scuticociliatia and Hymenostomatia, justifying the possible use of the model hymenostome T. thermophila as an effective experimental system to study the molecular and cellular biology of the scuticociliates.

Characterization of a novel N-acetylneuraminic acid lyase favoring industrial N-acetylneuraminic acid synthesis.

N-Acetylneuraminic acid lyase (NAL, E.C. number 4.1.3.3) is a Class I aldolase that catalyzes the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) from pyruvate and N-acetyl-D-mannosamine (ManNAc). Due to the high Neu5Ac cleavage activity in most isozyme forms, the enzyme catalyzes the rate-limiting step of two biocatalytic reactions producing Neu5Ac in industry. We report the biochemical characterization of a novel NAL from a "GRAS" (General recognized as safe) strain C. glutamicum ATCC 13032 (CgNal). Compared to all previously reported NALs, CgNal exhibited the lowest kcat/Km value for Neu5Ac and highest kcat/Km values for ManNAc and pyruvate, which makes CgNal favor industrial Neu5Ac synthesis process in a non-equilibrium condition. The recombinant CgNal reached the highest expression level (480 mg/L culture), and the highest reported yield of Neu5Ac was achieved (194 g/L, 0.63 M). All these unique properties make CgNal a promising biocatalyst for industrial Neu5Ac biosynthesis. Additionally, although showing the best Neu5Ac synthesis activity among the NAL family, CgNal is more related to dihydrodipicolinate synthase (DHDPS) by phylogenetic analysis. The activities of CgNal towards both NAL's and DHDPS' substrates are fairly high, which indicates CgNal a bi-functional enzyme. The sequence analysis suggests that CgNal might have adopted a unique set of residues for substrates recognition.

The genome of the myxosporean Thelohanellus kitauei shows adaptations to nutrient acquisition within its fish host.

Members of Myxozoa, a parasitic metazoan taxon, have considerable detrimental effects on fish hosts and also have been associated with human food-borne illness. Little is known about their biology and metabolism. Analysis of the genome of Thelohanellus kitauei and comparative analysis with genomes of its two free-living cnidarian relatives revealed that T. kitauei has adapted to parasitism, as indicated by the streamlined metabolic repertoire and the tendency toward anabolism rather than catabolism. Thelohanellus kitauei mainly secretes proteases and protease inhibitors for nutrient digestion (parasite invasion), and depends on endocytosis (mainly low-density lipoprotein receptors-mediated type) and secondary carriers for nutrient absorption. Absence of both classic and complementary anaerobic pathways and gluconeogenesis, the lack of de novo synthesis and reduced activity in hydrolysis of fatty acids, amino acids, and nucleotides indicated that T. kitauei in this vertebrate host-parasite system has adapted to inhabit a physiological environment extremely rich in both oxygen and nutrients (especially glucose), which is consistent with its preferred parasitic site, that is, the host gut submucosa. Taking advantage of the genomic and transcriptomic information, 23 potential nutrition-related T. kitauei-specific chemotherapeutic targets were identified. This first genome sequence of a myxozoan will facilitate development of potential therapeutics for efficient control of myxozoan parasites and ultimately prevent myxozoan-induced fish-borne illnesses in humans.

New phylogenomic and comparative analyses provide corroborating evidence that Myxozoa is Cnidaria.

Myxozoa, a diverse group of morphologically simplified endoparasites, are well known fish parasites causing substantial economic losses in aquaculture. Despite active research, the phylogenetic position of Myxozoa remains ambiguous. After obtaining the genome and transcriptome data of the myxozoan Thelohanellus kitauei, we examined the phylogenetic position of Myxozoa from three different perspectives. First, phylogenomic analyses with the newly sequenced genomic data strongly supported the monophyly of Myxozoa and that Myxozoa is sister to Medusozoa within Cnidaria. Second, we detected two homologs to cnidarian-specific minicollagens in the T. kitauei genome with molecular characteristics similar to cnidarian-specific minicollagens, suggesting that the minicollagen homologs in T. kitauei may have functions similar to those in Cnidaria and that Myxozoa is Cnidaria. Additionally, phylogenetic analyses revealed that the minicollagens in myxozoans and medusozoans have a common ancestor. Third, we detected 11 of the 19 proto-mesodermalgenes in the T. kitauei genome, which were also present in the cnidarian Hydra magnipapillata, indicating Myxozoa is within Cnidaria. Thus, our results robustly support Myxozoa as a derived cnidarian taxon with an affinity to Medusozoa, helping to understand the diversity of the morphology, development and life cycle of Cnidaria and its evolution.