Complete genome sequence of Peptostreptococcus porci isolated from porcine endocarditis in Japan.

Peptostreptococcus porci is a recently described bacterium belonging to the Peptostreptococcaceae family, which was isolated in 2016 from pig intestine. Herein, we report the complete genome sequence of a clinical isolate of P. porci (GAI11004) obtained from porcine endocarditis in Japan. The genome contains a 2.4-Mb circular chromosome.

Identification of cfxA gene variants and susceptibility patterns in ß-lactamase-producing Prevotella strains.

OBJECTIVES: Antimicrobial-resistant isolates of Prevotella species, especially those resistant to ß-lactams, have become increasingly common. Here, we aimed to elucidate the underlying mechanisms contributing to the emergence and spread of antimicrobial resistance in Prevotella species. METHODS: Prevotella species were isolated from a variety of clinical specimens. ß-lactamase production was determined using nitrocefin discs, and the determination of minimum inhibitory concentration (MIC) to ten antimicrobials was done by the agar dilution method. Four resistance genes (cfxA, tetQ, ermF, and nim) and cfxA-flanking regions were detected using polymerase chain reaction. cfxA and the flanking regions were sequenced, and a phylogenetic tree was constructed based on CfxA amino acid sequences using the UPGMA method. RESULTS: Among the 45 Prevotella isolates identified, 35 (77.8%) produced ß-lactamases and had the cfxA genes. The tetQ, ermF, and nim genes were detected in 53.3%, 17.8%, and 0% of the 45 isolates, respectively. Among the 33 sequenced cfxA alleles, cfxA2 (45.5%) was the most frequent, followed by cfxA3 (42.4%) and a novel variant (cfxA7, 12.1%). The novel CfxA7 ß-lactamase had a novel L155F substitution not previously reported in CfxA variants. The MICs of all ß-lactam agents tested, excluding cefmetazole and meropenem, were lower among cfxA7-positive isolates than in cfxA2-and cfxA3-positive isolates. CONCLUSIONS: Differences in MICs of penicillins and cephalosporins may be due to amino acid substitutions in the CfxA variants, CfxA2, CfxA3, and CfxA7, among Prevotella isolates. Possession of cfxA-mobA, tetQ, and ermF may increase the risks of the emergence and spread of multidrug-resistant Prevotella species.

Identification of Key Genes and Pathways Associated with Preeclampsia by a WGCNA and an Evolutionary Approach.

Preeclampsia (PE) is the serious obstetric-related disease characterized by newly onset hypertension and causes damage to the kidneys, brain, liver, and more. To investigate genes with key roles in PE's pathogenesis and their contributions, we used a microarray dataset of normotensive and PE patients and conducted a weighted gene co-expression network analysis (WGCNA). Cyan and magenta modules that are highly enriched with differentially expressed genes (DEGs) were revealed. By using the molecular complex detection (MCODE) algorithm, we identified five significant clusters in the cyan module protein-protein interaction (PPI) network and nine significant clusters in the magenta module PPI network. Our analyses indicated that (i) human accelerated region (HAR) genes are enriched in the magenta-associated C6 cluster, and (ii) positive selection (PS) genes are enriched in the cyan-associated C3 and C5 clusters. We propose these enriched HAR and PS genes, i.e., EIF4E, EIF5, EIF3M, DDX17, SRSF11, PSPC1, SUMO1, CAPZA1, PSMD14, and MNAT1, including highly connected hub genes, HNRNPA1, RBMX, PRKDC, and RANBP2, as candidate key genes for PE's pathogenesis. A further clarification of the functions of these PPI clusters and key enriched genes will contribute to the discovery of diagnostic biomarkers for PE and therapeutic intervention targets.

Immunohistological analysis of pathogenic infiltrates in the epidermis and liver of a patient with toxic epidermal necrolysis accompanied by vanishing bile duct syndrome.

Toxic epidermal necrolysis (TEN) is a severe cutaneous adverse drug reaction characterized by extensive epidermal detachment, which is reportedly mediated by drug-specific cytotoxic CD8+ T cells, inflammatory monocytes, and neutrophils. Besides the skin, TEN often damages other organs, and it remains unknown whether they are mediated by similar pathogenic cells that cause epidermal damage. We experienced a case who developed TEN complicated with vanishing bile duct syndrome. Immunohistological analysis revealed the infiltration of CD8+ T cells, inflammatory monocytes, and neutrophil extracellular trap-forming neutrophils in the lesions of both the skin and liver with different degree of infiltration of these cells. These data suggest a difference of dominant pathogenic cells between skin and liver of patients with TEN.

Complete Genome Sequences of Three Phocaeicola vulgatus Strains Isolated from a Healthy Japanese Individual.

Phocaeicola vulgatus (formerly Bacteroides vulgatus) is a pathogenic anaerobic bacterium frequently involved in human infections. We present the complete genome sequences of three Phocaeicola vulgatus strains isolated from the same healthy person, determined by hybrid assembly using Nanopore long-read sequencing and DNBseq short-read sequencing.

Neutrophil Extracellular Traps in Skin Diseases.

Neutrophils are the primary innate immune cells, and serve as sentinels for invading pathogens. To this end, neutrophils exert their effector functions via phagocytosis, degranulation, reactive oxygen species generation, and neutrophil extracellular trap (NET) release. Pathogens and pathogen-derived components trigger NET formation, leading to the clearance of pathogens. However, NET formation is also induced by non-related pathogen proteins, such as cytokines and immune complexes. In this regard, NET formation can be induced under both non-sterile and sterile conditions. NETs are enriched by components with potent cytotoxic and inflammatory properties, thereby occasionally damaging tissues and cells and dysregulating immune homeostasis. Research has uncovered the involvement of NETs in the pathogenesis of several connective tissue diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and ANCA-associated vasculitis. In dermatology, several skin diseases clinically develop local or systemic sterile pustules and abscesses. The involvement of neutrophils and subsequent NET formation has recently been elucidated in these skin diseases. Therefore, this review highlights the NETs in these neutrophil-associated diseases.

Neuropsychiatric Adverse Events of Montelukast: An Analysis of Real-World Datasets and drug-gene Interaction Network.

Montelukast is a selective leukotriene receptor antagonist that is widely used to treat bronchial asthma and nasal allergy. To clarify the association between montelukast and neuropsychiatric adverse events (AEs), we evaluated case reports recorded between January 2004 and December 2018 in the Food and Drug Administration Adverse Event Reporting System (FAERS). Furthermore, we elucidated the potential toxicological mechanisms of montelukast-associated neuropsychiatric AEs through functional enrichment analysis of human genes interacting with montelukast. The reporting odds ratios of suicidal ideation and depression in the system organ class of psychiatric disorders were 21.5 (95% confidence interval (CI): 20.3-22.9) and 8.2 (95% CI: 7.8-8.7), respectively. We explored 1,144 human genes that directly or indirectly interact with montelukast. The molecular complex detection (MCODE) plug-in of Cytoscape detected 14 clusters. Functional analysis indicated that several genes were significantly enriched in the biological processes of "neuroactive ligand-receptor interaction." "Mood disorders" and "major depressive disorder" were significant disease terms related to montelukast. Our retrospective analysis based on the FAERS demonstrated a significant association between montelukast and neuropsychiatric AEs. Functional enrichment analysis of montelukast-associated genes related to neuropsychiatric symptoms warrant further research on the underlying pharmacological mechanisms.

Network and Evolutionary Analysis of Human Epigenetic Regulators to Unravel Disease Associations.

We carried out a system-level analysis of epigenetic regulators (ERs) and detailed the protein-protein interaction (PPI) network characteristics of disease-associated ERs. We found that most diseases associated with ERs can be clustered into two large groups, cancer diseases and developmental diseases. ER genes formed a highly interconnected PPI subnetwork, indicating a high tendency to interact and agglomerate with one another. We used the disease module detection (DIAMOnD) algorithm to expand the PPI subnetworks into a comprehensive cancer disease ER network (CDEN) and developmental disease ER network (DDEN). Using the transcriptome from early mouse developmental stages, we identified the gene co-expression modules significantly enriched for the CDEN and DDEN gene sets, which indicated the stage-dependent roles of ER-related disease genes during early embryonic development. The evolutionary rate and phylogenetic age distribution analysis indicated that the evolution of CDEN and DDEN genes was mostly constrained, and these genes exhibited older evolutionary age. Our analysis of human polymorphism data revealed that genes belonging to DDEN and Seed-DDEN were more likely to show signs of recent positive selection in human history. This finding suggests a potential association between positive selection of ERs and risk of developmental diseases through the mechanism of antagonistic pleiotropy.

Systems Level Analysis and Identification of Pathways and Key Genes Associated with Delirium.

Delirium is a complex pathophysiological process, and multiple contributing mechanisms have been identified. However, it is largely unclear how the genes associated with delirium contribute and which of them play key roles. In this study, the genes associated with delirium were retrieved from the Comparative Toxicogenomics Database (CTD) and integrated through a protein-protein interaction (PPI) network. Delirium-associated genes formed a highly interconnected PPI subnetwork, indicating a high tendency to interact and agglomerate. Using the Molecular Complex Detection (MCODE) algorithm, we identified the top two delirium-relevant network modules, M1 and M5, that have the most significant enrichments for the delirium-related gene sets. Functional enrichment analysis showed that genes related to neurotransmitter receptor activity were enriched in both modules. Moreover, analyses with genes located in human accelerated regions (HARs) provided evidence that HAR-Brain genes were overrepresented in the delirium-relevant network modules. We found that four of the HAR-Brain genes, namely APP, PLCB1, NPY, and HTR2A, in the M1 module were highly connected and appeared to exhibit hub properties, which might play vital roles in delirium development. Further understanding of the function of the identified modules and member genes could help to identify therapeutic intervention targets and diagnostic biomarkers for delirium.

Genome-wide analyses reveal genes subject to positive selection in Toxoplasma gondii.

Toxoplasma gondii is an important protozoan pathogen that infects many wild and domestic animals and causes infections in immunocompromised humans. However, there has been little investigation of the molecular evolutionary trajectories of this pathogenic protozoa using comparative genomics data. Here, we employed a comparative evolutionary genomics approach to identify genes that are under site- and lineage-specific positive selection in nine strains of T. gondii, including two closely related species, Neospora caninum and Hammondia hammondi. Based on the analyses of five coccidian core genomes, 4.5% of the 5788 core genome genes showed strong signals for positive selection in the site model. In addition, the branch-site model analyses in the nine T. gondii core genomes indicated that 2 to 20 genes underwent significant positive selection along each lineage leading to T. gondii strains. Many of the protein products encoded by the positively selected genes are secretory or surface proteins that have previously been implicated in host pathogenesis. The adaptive changes in these positively selected genes might be related to dynamic interactions between the host immune systems and might play a crucial role in the infection and pathogenic processes of T. gondii.

Heterotrimeric G protein Gαs subunit attenuates PLEKHG2, a Rho family-specific guanine nucleotide exchange factor, by direct interaction.

PLEKHG2 is a Gßγ-dependent guanine nucleotide exchange factor (GEF) for the small GTPases Rac and Cdc42, and has been shown to mediate signalling pathways such as actin cytoskeleton reorganization and serum response element (SRE)-dependent gene transcription. Here we show that the constitutively active mutant of the Gαs subunit significantly attenuated PLEKHG2-induced SRE-mediated gene transcription. Strikingly, we observed that the constitutive activation of endogenous Gαs by treatment with CTx caused a similar inhibitory effect on PLEKHG2-induced activation of SRE. However, both the enforced expression of the catalytic subunit ß of protein kinase A and the treatment with dibutyl-cyclic AMP failed to mimic the inhibitory effect of Gαs on PLEKHG2. Furthermore, the dominant negative mutant of protein kinase A had no effect on PLEKHG2-mediated SRE activation. Performing immunoprecipitation and an in vitro pulldown assay, we found that PLEKHG2 directly interacted with the active form of the Gαs subunit in cells. The interaction between PLEKHG2 and Gαs required the N-terminal region of PLEKHG2, which includes the DH domain, a functional domain of GEF, suggesting that Gαs directly masks the DH domain of PLEKHG2. In a previous study, we reported that Gßγ accelerates PLEKHG2-mediated SRE-dependent gene transcription. Interestingly, Gαs also inhibited the hyperactivation of SRE induced by the co-expression of Gßγ and PLEKHG2; however, Gαs and Gßγ bind to different regions of PLEKHG2. This is the first report to show that PLEKHG2 is a novel effector of Gαs, and is negatively regulated by the Gαs subunit through direct interaction.

Molecular Evolution of the TET Gene Family in Mammals.

Ten-eleven translocation (TET) proteins, a family of Fe(2+)- and 2-oxoglutarate-dependent dioxygenases, are involved in DNA demethylation. They also help regulate various cellular functions. Three TET paralogs have been identified (TET1, TET2, and TET3) in humans. This study focuses on the evolution of mammalian TET genes. Distinct patterns in TET1 and TET2 vs. TET3 were revealed by codon-based tests of positive selection. Results indicate that TET1 and TET2 genes have experienced positive selection more frequently than TET3 gene, and that the majority of codon sites evolved under strong negative selection. These findings imply that the selective pressure on TET3 may have been relaxed in several lineages during the course of evolution. Our analysis of convergent amino acid substitutions also supports the different evolutionary dynamics among TET gene subfamily members. All of the five amino acid sites that are inferred to have evolved under positive selection in the catalytic domain of TET2 are localized at the protein's outer surface. The adaptive changes of these positively selected amino acid sites could be associated with dynamic interactions between other TET-interacting proteins, and positive selection thus appears to shift the regulatory scheme of TET enzyme function.

Adaptive evolution of formyl peptide receptors in mammals.

The formyl peptide receptors (FPRs) are a family of chemoattractant receptors with important roles in host defense and the regulation of inflammatory reactions. In humans, three FPR paralogs have been identified (FPR1, FPR2, and FPR3) and may have functionally diversified by gene duplication and adaptive evolution. However, the evolutionary mechanisms operating in the diversification of FPR family genes and the changes in selection pressures have not been characterized to date. Here, we have made a comprehensive evolutionary analysis of FPR genes from mammalian species. Phylogenetic analysis showed that an early duplication was responsible for FPR1 and FPR2/FPR3 splitting, and FPR3 originated from the latest duplication event near the origin of primates. Codon-based tests of positive selection reveal interesting patterns in FPR1 and FPR2 versus FPR3, with the first two genes showing clear evidence of positive selection at some sites while the majority of them evolve under strong negative selection. In contrast, our results suggest that the selective pressure may be relaxed in the FPR3 lineage. Of the six amino acid sites inferred to evolve under positive selection in FPR1 and FPR2, four sites were located in extracellular loops of the protein. The electrostatic potential of the extracellular surface of FPR might be affected more frequently with amino acid substitutions in positively selected sites. Thus, positive selection of FPRs among mammals may reflect a link between changes in the sequence and surface structure of the proteins and is likely to be important in the host's defense against invading pathogens.

Genome-wide evidence of positive selection in Bacteroides fragilis.

We used an evolutionary genomics approach to identify genes that are under lineage-specific positive selection in six species of the genus Bacteroides, including three strains of pathogenic Bacteroides fragilis. Using OrthoMCL, we identified 1275 orthologous gene clusters present in all eight Bacteroides genomes. A total of 52 genes were identified as under positive selection in the branch leading to the B. fragilis lineage, including a number of genes encoding cell surface proteins such as TonB-dependent receptor. Three-dimensional structural mapping of positively selected sites indicated that many residues under positive selection occur in the extracellular loops of the proteins. The adaptive changes in these positively selected genes might be related to dynamic interactions between the host immune systems and the surrounding intestinal environment.

Increased bioavailability of tricin-amino acid derivatives via a prodrug approach.

Tricin (4',5,7-trihydroxy-3',5'-dimethoxyflavone) has demonstrated diverse biological activities. This compound has a high anti-human cytomegalovirus (HCMV) activity; however, its oral availability is low. To improve its bioavailability, we synthesized tricin-amino acid derivatives as prodrugs and investigated their cell permeability, stability in vitro, and oral availability in vivo. The results demonstrated that the tricin-alanine-glutamic acid conjugate exhibited enhanced permeability, stability in MDCK cells, and excellent bioavailability after oral administration in Crl:CD (SD) male rats. Tricin-alanine-glutamic acid conjugate is a potential new anti-HCMV drug.

Evolutionary features and intracellular behavior of the PRTB protein.

Human PRTB encodes a proline-rich protein of 168 amino acids (PRTB). We analyzed the evolutionary patterns of PRTB from various vertebrate species. Maximum likelihood analyses indicated that while mammalian PRTB has been very well conserved and underwent a significantly slower rate of evolution, only the branch leading to fish PRTB has undergone adaptive evolution. We generated several mutant PRTBs fused to the GFP variant, Venus, and found that the degradation of PRTB was enhanced by the transfection of an E2, UbcH5. Since mutation of the K153 site in PRTB was refractory to its degradation, proteolysis was suggested to be mediated by ubiquitination of K153. The subcellular localization of PRTB was also investigated, which showed that mutation of the K4 site completely prevented the nuclear localization of this protein. Together, these results suggest that Lys residues might play important roles in regulating the intracellular dynamics of the PRTB protein.

An evolutionarily conserved leucine-rich repeat protein CLERC is a centrosomal protein required for spindle pole integrity.

The centrosome functions as the microtubule-organizing center (MTOC) and plays a vital role in organizing spindle poles during mitosis. The pair of centrioles, which are the core components of the centrosome, duplicate once per cell cycle, and this feature is essential for the establishment of spindle bipolarity. Here we describe the molecular characterization of a novel protein called CLERC (Centrosomal leucine-rich repeat and coiled-coil containing protein) which is a human ortholog of Chlamydomonas Vfl1 protein. CLERC is a protein of 1032 amino acids with a calculated molecular mass of 120 kDa and possesses leucine-rich repeat and coiled-coil domains. Database searches revealed that CLERC has homologs in a wide variety of eukaryotes and is evolutionarily conserved. Endogenous CLERC protein associated with the centrosomes throughout the cell cycle and accumulated during mitosis. RNAi-mediated depletion of CLERC blocked formation of normal mitotic spindles and led to multipolar spindles. Moreover, many of the spindle poles in CLERC depleted cells contained only one centriole, indicating that centrosomes split into fractions containing a single centriole. These data indicate that the major function of CLERC during mitosis is to maintain the structural integrity of centrosomes, thereby contributing to spindle bipolarity.

Phosphorylated retinoid X receptor alpha loses its heterodimeric activity with retinoic acid receptor beta.

A malfunction in retinoid X receptor (RXR) alpha due to phosphorylation is associated with the development of hepatocellular carcinoma. However, the precise mechanisms by which phosphorylated RXRalpha loses its physiological function remain unclear. In the present study we examined whether phosphorylation of RXRalpha affects its dimeric activity. Fluorescence resonance energy transfer studies and immunoprecipitation assays showed that the physical interaction between RXRalpha and retinoic acid receptor beta was impaired when 293T cells were transfected with phosphomimic mutant RXRalpha (T82D/S260D), whereas this interaction was activated at a level similar to wild-type RXRalpha-transfected cells when the cells were transfected with an unphosphorylated mutant RXRalpha (T82A/S260A). Treating the T82A/S260A-transfected cells with retinoid resulted in a significant increase in the transcriptional activities of the retinoic acid receptor responsive element and RXR responsive element promoters, whereas these transcriptional activities did not increase in the T82D/S260D-transfected cells. Transfection with T82A/S260A enhanced both the inhibition of cell growth and the induction of apoptosis caused by retinoid, although the T82D/S260D-transfected cells lost their responsiveness to retinoid. Moreover, transfection with T82A/S260A caused an inhibition of cell growth and a reduction of colony-forming ability in soft agar in HuH7 human hepatocellular carcinoma cells. These findings suggest that phosphorylation of RXRalpha abolishes its ability to form homodimers and heterodimers with RXR and retinoic acid receptor beta, thus resulting in the loss of cell growth control and the acceleration of cancer development. In conclusion, the inhibition of RXRalpha phosphorylation and the restoration of its original function as a master regulator of nuclear receptors might therefore be an effective strategy for controlling cancer cell growth.

Neuronal protein NP25 interacts with F-actin.

Neuronal protein NP25 is a neuron-specific protein present in highly differentiated neural cells, but its functional properties have not been well characterized. NP25 shows high amino acid sequence homology with the smooth muscle cell cytoskeleton-associated proteins, SM22, mp20, and calponin. To gain an insight into the biological functions of NP25, we first examined its subcellular localization in the human neuroblastoma cell line, SK-N-SH. NP25 diffusely distributed in the cytoplasm and fiber-like staining was also observed. It showed that NP25 co-localized with F-actin on stress fibers. A co-sedimentation assay demonstrated that NP25 bound to filamentous actin. Further investigations using fluorescence resonance energy transfer (FRET) technique revealed intracellular binding of NP25 and actin. The significance of the interaction between NP25 and F-actin is discussed.