Structural evidence for protein-protein interaction between the non-canonical methyl-CpG-binding domain of SETDB proteins and C11orf46.

SETDB1 and SETDB2 mediate trimethylation of histone H3 lysine 9 (H3K9), an epigenetic hallmark of repressive chromatin. They contain a non-canonical methyl-CpG-binding domain (MBD) and bifurcated SET domain, implying interplay between H3K9 trimethylation and DNA methylation in SETDB functions. Here, we report the crystal structure of human SETDB2 MBD bound to the cysteine-rich domain of a zinc-binding protein, C11orf46. SETDB2 MBD comprises the conserved MBD core and a unique N-terminal extension. Although the MBD core has the conserved basic concave surface for DNA binding, it utilizes it for recognition of the cysteine-rich domain of C11orf46. This interaction involves the conserved arginine finger motif and the unique N-terminal extension of SETDB2 MBD, with a contribution from intermolecular ß-sheet formation. Thus, the non-canonical MBD of SETDB1/2 seems to have lost methylated DNA-binding ability but gained a protein-protein interaction surface. Our findings provide insight into the molecular assembly of SETDB-associated repression complexes.

Biotypic and genotypic diversity in Pasteurella canis isolated from host animals and humans: differences in trehalose fermentation and nucleotide sequences encoding trehalose-6-phosphate hydrolase (treC).

The biotypic and genotypic features of Pasteurella canis isolated from dogs, cats, and humans were clarified by repetitive sequence-based fingerprinting and nucleotide sequences encoding trehalose-6-phosphate hydrolase (treC). Thirty P. canis and 48 P. multocida isolates were collected from dogs, cats, and humans to perform biotyping. The genotyping of P. canis by fingerprinting was followed by dendrogram construction. The whole-genome sequences (WGSs) were searched for the enzyme-coding nucleotide sequences around the main and adjacent loci constituting the operon. Full-length nucleotide sequences encoding the enzyme were determined using polymerase chain reaction and direct sequencing. Biotypic results were compared to the dendrogram and nucleotide sequence data. We observed a difference in trehalose fermentation with a positivity rate of 46.7%. Two (A-1/A-2) and three (B-1/B-2/B-3) clades were located on the dendrograms generated based on two repetitive sequence-based fingerprinting techniques, showing no association between trehalose fermentation and the clades. Based on the WGSs, two variants of the gene, namely, a 1,641 bp gene treC and a pseudogene (1,335 bp) of treC with its first 306 nucleotides deleted, were observed. Trehalose-positive isolates harbored treC, whereas trehalose-negative isolates lacked treC with or without the pseudogene. Our observations suggest biotypic and genotypic diversity among the P. canis isolates from animal and human hosts, with respect to trehalose fermentation and treC nucleotide sequences. This is the first report on the diversity of treC nucleotide sequences among these isolates.

Virulence-associated Genome Sequences of Pasteurella canis and Unique Toxin Gene Prevalence of P. canis and Pasteurella multocida Isolated from Humans and Companion Animals.

Background: Comparative analysis of virulence factors (VFs) between Pasteurella canis and Pasteurella multocida are lacking, although both cause zoonotic infections. We determined the virulence-associated genome sequence characteristics of P. canis and assessed the toxin gene prevalence unique to P. canis among clinical isolates of P. canis and P. multocida. Methods: We selected 10 P. canis and 16 P. multocida whole-genome sequences (WGSs) from the National Center for Biotechnology database. The VFanalyzer tool was used to estimate P. canis-characteristic VFs. Amino acid sequences of VFs were compared with multiple-aligned sequences. The genome structure containing P. canis-characteristic and adjacent loci was compared to the corresponding P. multocida genome structure. After designing primer sequences and assessing their accuracy, we examined the gene prevalence of the P. canis-characteristic VFs using PCR among clinical isolates of P. multocida and P. canis. Results: Using VFanalyzer, we found virulence-associated cytolethal distending toxin (cdt)A-cdtB-cdtC loci common to all P. canis WGSs that were not found in P. multocida WGSs. Similarities in the multiple alignments of CdtA-CdtB-CdtC amino acid sequences were found among the 10 P. canis WGSs. Shared or similar loci around cdtA-cdtB-cdtC were identified between the P. canis and P. multocida genome structures. The PCR-based cdtA-cdtB-cdtC prevalence differed for P. canis and P. multocida clinical isolates. Conclusions: P. canis-specific cdtA-cdtB-cdtC prevalence was identified among clinical isolates. These three loci may be unique toxin genes and promising targets for the rapid identification of P. canis in clinical settings.

Genotypic and Phenotypic Features of Eye-Origin Streptococcus canis Isolates from Dogs in 2021: Relatedness with Clonal Complex 46 and Antimicrobial Resistance.

The eye (including the cornea) and ear canal are the major sources of Streptococcus canis in companion animal practice. In this study, we aimed to clarify the genotypic and phenotypic features of eye-origin isolates collected in 2021 compared to ear-origin isolates collected in 2021 and eye-origin isolates collected in 2017. Of the 102 isolates in 2021, 9 eye-origin isolates were enrolled. Twenty ear-origin isolates in 2021 and 13 eye-origin isolates in 2017 were included as controls. Genotypic analyses included profiling of virulence-associated genes (VAGs; inl, sagA, slo, scp, lbp, fbp, gbp, ap1, fp1, and brp), S. canis M-like protein (SCM) allele typing, multilocus sequence typing, and antimicrobial resistance (AMR) genotyping and phenotyping analyses including hemolytic activity (HA) measurement and AMR phenotyping. One 2017-eye-origin isolate displayed high-level HA; the others displayed low-level HA. No association was evident between the 2021-eye-origin population and the detection rate of each VAG. There was no association between the 2021-eye-origin population and the main SCM allele 2. A significant association was evident between the 2021-eye-origin population and the main clonal complex (CC) 46 containing sequence type (ST) 46/ST2. A significant association was also detected between the 2021-eye-origin population and AMR phenotypes/genotypes. Our observations suggest unique microbiological features (CC46 with AMR phenotypes/genotypes) among the 2021-eye-origin population.

Human inactive X chromosome is compacted through a PRC2-independent SMCHD1-HBiX1 pathway.

Human inactive X chromosome (Xi) forms a compact structure called the Barr body, which is enriched in repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3) and Lys27 (H3K27me3). These two histone marks are distributed in distinct domains, and X-inactive specific transcript (XIST) preferentially colocalizes with H3K27me3 domains. Here we show that Xi compaction requires HBiX1, a heterochromatin protein 1 (HP1)-binding protein, and structural maintenance of chromosomes hinge domain-containing protein 1 (SMCHD1), both of which are enriched throughout the Xi chromosome. HBiX1 localization to H3K9me3 and XIST-associated H3K27me3 (XIST-H3K27me3) domains was mediated through interactions with HP1 and SMCHD1, respectively. Furthermore, HBiX1 was required for SMCHD1 localization to H3K9me3 domains. Depletion of HBiX1 or SMCHD1, but not Polycomb repressive complex 2 (PRC2), resulted in Xi decompaction, similarly to XIST depletion. Thus, the molecular network involving HBiX1 and SMCHD1 links the H3K9me3 and XIST-H3K27me3 domains to organize the compact Xi structure.

Unexpected instability of family of repeats (FR), the critical cis-acting sequence required for EBV latent infection, in EBV-BAC systems.

A group of repetitive sequences, known as the Family of Repeats (FR), is a critical cis-acting sequence required for EBV latent infection. The FR sequences are heterogeneous among EBV strains, and they are sometimes subject to partial deletion when subcloned in E. coli-based cloning vectors. However, the FR stability in EBV-BAC (bacterial artificial chromosome) system has never been investigated. We found that the full length FR of the Akata strain EBV was not stably maintained in a BAC vector. By contrast, newly obtained BAC clones of the B95-8 strain of EBV stably maintained the full length FR during recombinant virus production and B-cell transformation. Investigation of primary DNA sequences of Akata-derived EBV-BAC clones indicates that the FR instability is most likely due to a putative secondary structure of the FR region. We conclude that the FR instability in EBV-BAC clones can be a pitfall in E. coli-mediated EBV genetics.

Raftlin is involved in the nucleocapture complex to induce poly(I:C)-mediated TLR3 activation.

The double-stranded RNA analog, poly(I:C), extracellularly activates both the endosomal Toll-like receptor (TLR) 3 and the cytoplasmic RNA helicase, melanoma differentiation-associated gene 5, leading to the production of type I interferons (IFNs) and inflammatory cytokines. The mechanism by which extracellular poly(I:C) is delivered to TLR3-positive organelles and the cytoplasm remains to be elucidated. Here, we show that the cytoplasmic lipid raft protein, Raftlin, is essential for poly(I:C) cellular uptake in human myeloid dendritic cells and epithelial cells. When Raftlin was silenced, poly(I:C) failed to enter cells and induction of IFN-ß production was inhibited. In addition, cellular uptake of B-type oligodeoxynucleotide that shares its uptake receptor with poly(I:C) was suppressed in Raftlin knockdown cells. Upon poly(I:C) stimulation, Raftlin was translocated from the cytoplasm to the plasma membrane where it colocalized with poly(I:C), and thereafter moved to TLR3-positive endosomes. Thus, Raftlin cooperates with the uptake receptor to mediate cell entry of poly(I:C), which is critical for activation of TLR3.

Distinctive effects of the Epstein-Barr virus family of repeats on viral latent gene promoter activity and B-lymphocyte transformation.

The Epstein-Barr virus (EBV), a human B-lymphotropic gamma herpesvirus, contains multiple repetitive sequences within its genome. A group of repetitive sequences, known as the family of repeats (FR), contains multiple binding sites for the viral trans-acting protein EBNA-1. The FR sequences are important for viral genome maintenance and for the regulation of the promoter involved in viral latent gene expression. It has been reported that a palindromic sequence with a putative secondary structure exists at the 3' end of the FR in the genome of the EBV B95-8 strain and that this palindromic sequence has been deleted from the FR of the commonly used EBV miniplasmids. For the first time, we cloned an EBV B95-8 DNA fragment containing the full-length FR, which enabled us to examine the functional difference between full-length and deleted FRs. The full-length FR, like the deleted FR, functioned as a transcriptional enhancer of the viral latent gene promoter, but that transactivation was significantly attenuated in the case of the full-length FR. No significant enhancement of replication was observed when the deleted FR was replaced with the full-length FR in an EBV miniplasmid. By contrast, when the same set of FR sequences were tested in the context of the complete EBV genome, the full-length FR resulted in more-efficient B-cell transformation than the deleted FR. We propose that the presence of the full-length FR contributes to the precise regulation of the viral latent promoter and increases the efficiency of B-cell transformation.

Transcervical needle biopsy for the differential diagnosis between uterine sarcoma and leiomyoma.

BACKGROUND: The clinical differential diagnosis between uterine sarcoma and benign leiomyoma is difficult even with magnetic resonance imaging (MRI). Therefore, a considerable number of patients have undergone hysterectomies due to an indication of "suspected malignancy" based on tumor size alone. However, approximately 80% of these hysterectomies have been judged to have been recommended inappropriately. In such situations, reliable preoperative diagnostic tests are required. The authors have evaluated the accuracy of needle biopsy for uterine myoma-like tumors, a procedure that to the authors' knowledge has been performed infrequently. METHODS: Transcervical needle biopsy was performed in 435 patients with uterine myoma-like tumors. The biopsy specimens were scored for degree of malignancy according to the histopathologic criteria proposed by Bell et al. Histopathologic evaluation of surgical specimens and clinical outcome after 2 years of follow-up were used as the reference standards. RESULTS: Of 435 patients, 7 had uterine sarcomas, 4 of which were scored as > or = 4 points and were diagnosed as "sarcoma" by needle biopsy alone. No sarcoma cases were included in the group of patients with a score of 0. The cutoff score combining the highest sensitivity and specificity with respect to distinguishing uterine leiomyosarcoma from uterine leiomyoma was 2; sensitivity, specificity, and positive and negative predictive values were 100%, 98.6%, 58%, and 100.0%, respectively. CONCLUSIONS: Transcervical needle biopsy using histopathologic scoring is a reliable diagnostic test for the differential diagnosis between uterine sarcoma and leiomyoma. This diagnostic method, combined with MRI screening, could reduce the number of patients currently undergoing unnecessary surgery.