Gut Microbiota-mediated Alleviation of Dextran Sulfate Sodium-induced Colitis in Mice.

Background and Aims: Gut dysbiosis characterized by an imbalanced microbiota is closely involved in the pathogenesis of a widespread gastrointestinal inflammatory disorder, inflammatory bowel disease. However, it is unclear how the complex intestinal microbiota affects development or resistant of mucosal inflammation. Our aim was to investigate the impact of the gut microbiota on susceptibility in a mouse model of ulcerative colitis. Methods: We compared the susceptibility to dextran sulfate sodium (DSS)-induced colitis of inbred BALB/c mice obtained from the 3 main distributors of laboratory animals in Japan. Clinical symptoms of the colitis and the faecal microbiota were assessed. Cohousing approach was used to identify whether the gut microbiota is a primary factor determining disease susceptibility. Results: Here, we showed differences in the susceptibility of BALB/c mice from the vendors to DSS colitis. Analysis of the gut microbiota using 16S ribosomal RNA sequencing revealed clear separation of the gut microbial composition among mice from the vendors. Notably, the abundance of the phylum Actinobacteriota was strongly associated with disease activity. We also observed the expansion of butyrate-producing Roseburia species in mice with decreased susceptibility of the disease. Further cohousing experiments showed that variation in clinical outcomes was more correlated with the gut microbiota than genetic variants among substrains from different suppliers. Conclusion: A BALB/c substrain that was resistant to DSS-induced colitis was observed, and the severity of DSS-induced colitis was mainly influenced by the gut microbiota. Targeting butyrate-producing bacteria could have therapeutic potential for ulcerative colitis.

Time-course study of genetic changes in periodontal ligament regeneration after tooth replantation in a mouse model.

This research focused on analyzing gene expression changes in the periodontal ligament (PDL) after tooth re-plantation to identify key genes and pathways involved in healing and regeneration. Utilizing a mouse model, mRNA was extracted from the PDL at various intervals post-replantation for RNA sequencing analysis, spanning from 3 to 56 days. The results revealed significant shifts in gene expression, particularly notable on day 28, supported by hierarchical clustering and principal component analysis. Gene ontology (GO) enrichment analysis highlighted an upregulation in olfactory receptor and G protein-coupled receptor signaling pathways at this time point. These findings were validated through reverse transcription-quantitative PCR (RT-qPCR), with immunochemical staining localizing olfactory receptor gene expression to the PDL and surrounding tissues. Moreover, a scratch assay indicated that olfactory receptor genes might facilitate wound healing in human PDL fibroblasts. These results underscore the importance of the 28-day post-transplant phase as a potential "tipping point" in PDL healing and regeneration. In conclusion, this research sheds light on the potential role of olfactory receptor genes in PDL regeneration, providing a foundation for developing new therapeutic approaches in tooth replantation and transplantation, with broader implications for regenerative medicine in oral health.

Hybrid sequence-based analysis reveals the distribution of bacterial species and genes in the oral microbiome at a high resolution.

Bacteria in the oral microbiome are poorly identified owing to the lack of established culture methods for them. Thus, this study aimed to use culture-free analysis techniques, including bacterial single-cell genome sequencing, to identify bacterial species and investigate gene distribution in saliva. Saliva samples from the same individual were classified as inactivated or viable and then analyzed using 16S rRNA sequencing, metagenomic shotgun sequencing, and bacterial single-cell sequencing. The results of 16S rRNA sequencing revealed similar microbiota structures in both samples, with Streptococcus being the predominant genus. Metagenomic shotgun sequencing showed that approximately 80 % of the DNA in the samples was of non-bacterial origin, whereas single-cell sequencing showed an average contamination rate of 10.4 % per genome. Single-cell sequencing also yielded genome sequences for 43 out of 48 wells for the inactivated samples and 45 out of 48 wells for the viable samples. With respect to resistance genes, four out of 88 isolates carried cfxA, which encodes a ß-lactamase, and four isolates carried erythromycin resistance genes. Tetracycline resistance genes were found in nine bacteria. Metagenomic shotgun sequencing provided complete sequences of cfxA, ermF, and ermX, whereas other resistance genes, such as tetQ and tetM, were detected as fragments. In addition, virulence factors from Streptococcus pneumoniae were the most common, with 13 genes detected. Our average nucleotide identity analysis also suggested five single-cell-isolated bacteria as potential novel species. These data would contribute to expanding the oral microbiome data resource.

Characteristic craniofacial defects associated with a novel USP9X truncation mutation.

Germline loss-of-function mutations in USP9X have been reported to cause a wide spectrum of congenital anomalies. Here, we report a Japanese girl with a novel heterozygous nonsense mutation in USP9X who exhibited intellectual disability with characteristic craniofacial abnormalities, including hypotelorism, brachycephaly, hypodontia, micrognathia, severe dental crowding, and an isolated submucous cleft palate. Our findings provide further evidence that disruptions in USP9X contribute to a broad range of congenital craniofacial abnormalities.

Comparison of teaching methods for the emergence and maintenance of untaught relations in foreign language vocabulary acquisition: A systematic replication.

In a replication of Daly and K. Dounavi (2020), the researchers evaluated the effect of foreign tact and bidirectional intraverbal teaching on the emergence of untaught relations. Three university students learned three stimulus sets through three types of teaching: native-foreign intraverbal teaching (vocalizing Spanish words that refer to a Japanese textual stimulus), foreign-native intraverbal teaching (reversed relation of native-foreign condition), and foreign-tact teaching (tacting a picture in Spanish). The researchers used an adapted alternating-treatments design to assess the differential effect of each teaching condition on the emergence of untaught relations in a foreign language and collected data on response maintenance. The results replicated previous findings that native-foreign intraverbal and foreign-tact teachings were more effective than foreign-native intraverbal teaching despite previous reporting that the maintenance outcomes may be a result of carryover effects.

Type I interferon regulation by USP18 is a key vulnerability in cancer.

Precise regulation of Type I interferon signaling is crucial for combating infection and cancer while avoiding autoimmunity. Type I interferon signaling is negatively regulated by USP18. USP18 cleaves ISG15, an interferon-induced ubiquitin-like modification, via its canonical catalytic function, and inhibits Type I interferon receptor activity through its scaffold role. USP18 loss-of-function dramatically impacts immune regulation, pathogen susceptibility, and tumor growth. However, prior studies have reached conflicting conclusions regarding the relative importance of catalytic versus scaffold function. Here, we develop biochemical and cellular methods to systematically define the physiological role of USP18. By comparing a patient-derived mutation impairing scaffold function (I60N) to a mutation disrupting catalytic activity (C64S), we demonstrate that scaffold function is critical for cancer cell vulnerability to Type I interferon. Surprisingly, we discovered that human USP18 exhibits minimal catalytic activity, in stark contrast to mouse USP18. These findings resolve human USP18's mechanism-of-action and enable USP18-targeted therapeutics.

Analysis of FctB3 crystal structure and insight into its structural stabilization and pilin linkage mechanisms.

Streptococcus pyogenes harboring an FCT type 3 genomic region display pili composed of three types of pilins. In this study, the structure of the base pilin FctB from a serotype M3 strain (FctB3) was determined at 2.8 Å resolution. In accordance with the previously reported structure of FctB from a serotype T9 strain (FctB9), FctB3 was found to consist of an immunoglobulin-like domain and proline-rich tail region. Data obtained from structure comparison revealed main differences in the omega (Ω) loop structure and the proline-rich tail direction. In the Ω loop structure, a differential hydrogen bond network was observed, while the lysine residue responsible for linkage to growing pili was located at the same position in both structures, which indicated that switching of the hydrogen bond network in the Ω loop without changing the lysine position is advantageous for linkage to the backbone pilin FctA. The difference in direction of the proline-rich tail is potentially caused by a single residue located at the root of the proline-rich tail. Also, the FctB3 structure was found to be stabilized by intramolecular large hydrophobic interactions instead of an isopeptide bond. Comparisons of the FctB3 and FctA structures indicated that the FctA structure is more favorable for linkage to FctA. In addition, the heterodimer formation of FctB with Cpa or FctA was shown to be mediated by the putative chaperone SipA. Together, these findings provide an alternative FctB structure as well as insight into the interactions between pilin proteins.

Involvement of ribonuclease Y in pilus production by M49 Streptococcus pyogenes strain via modulation of messenger RNA level of transcriptional regulator.

Streptococcus pyogenes displays a wide variety of pili, which is largely dependent on serotype. A distinct subset of S. pyogenes strains that possess the Nra transcriptional regulator demonstrates thermoregulated pilus production. Findings obtained in the present study of an Nra-positive serotype M49 strain revealed involvement of conserved virulence factor A (CvfA), also referred to as ribonuclease Y (RNase Y), in virulence factor expression and pilus production, while a cvfA deletion strain showed reduced pilus production and adherence to human keratinocytes as compared with wild-type and revertant strains. Furthermore, transcript levels of pilus subunits and srtC2 genes were decreased by cvfA deletion, which was remarkable at 25°C. Likewise, both messenger RNA (mRNA) and protein levels of Nra were remarkably decreased by cvfA deletion. Whether the expression of other pilus-related regulators, including fasX and CovR, was subject to thermoregulation was also examined. While the mRNA level of fasX, which inhibits cpa and fctA translation, was decreased by cvfA deletion at both 37°C and 25°C, CovR mRNA and protein levels, as well as its phosphorylation level were not significantly changed, suggesting that neither fasX nor CovR is necessarily involved in thermosensitive pilus production. Phenotypic analysis of the mutant strains revealed that culture temperature and cvfA deletion had varied effects on streptolysin S and SpeB activities. Furthermore, bactericidal assay data showed that cvfA deletion decreased the rate of survival in human blood. Together, the present findings indicate that CvfA is involved in regulation of pilus production and virulence-related phenotypes of the serotype M49 strain of S. pyogenes.

Pneumococcal BgaA Promotes Host Organ Bleeding and Coagulation in a Mouse Sepsis Model.

Streptococcus pneumoniae is a major cause of invasive diseases such as pneumonia, meningitis, and sepsis, with high associated mortality. Our previous molecular evolutionary analysis revealed that the S. pneumoniae gene bgaA, encoding the enzyme ß-galactosidase (BgaA), had a high proportion of codons under negative selection among the examined pneumococcal genes and that deletion of bgaA significantly reduced host mortality in a mouse intravenous infection assay. BgaA is a multifunctional protein that plays a role in cleaving terminal galactose in N-linked glycans, resistance to human neutrophil-mediated opsonophagocytic killing, and bacterial adherence to human epithelial cells. In this study, we performed in vitro and in vivo assays to evaluate the precise role of bgaA as a virulence factor in sepsis. Our in vitro assays showed that the deletion of bgaA significantly reduced the bacterial association with human lung epithelial and vascular endothelial cells. The deletion of bgaA also reduced pneumococcal survival in human blood by promoting neutrophil-mediated killing, but did not affect pneumococcal survival in mouse blood. In a mouse sepsis model, mice infected with an S. pneumoniae bgaA-deleted mutant strain exhibited upregulated host innate immunity pathways, suppressed tissue damage, and blood coagulation compared with mice infected with the wild-type strain. These results suggest that BgaA functions as a multifunctional virulence factor whereby it induces host tissue damage and blood coagulation. Taken together, our results suggest that BgaA could be an attractive target for drug design and vaccine development to control pneumococcal infection.

Unique virulence role of post-translocational chaperone PrsA in shaping Streptococcus pyogenes secretome.

Streptococcus pyogenes (group A Streptococcus, GAS) is a strict human pathogen causing a broad spectrum of diseases and a variety of autoimmune sequelae. The pathogenesis of GAS infection mostly relies on the production of an extensive network of cell wall-associated and secreted virulence proteins, such as adhesins, toxins, and exoenzymes. PrsA, the only extracellular parvulin-type peptidyl-prolyl isomerase expressed ubiquitously in Gram-positive bacteria, has been suggested to assist the folding and maturation of newly exported proteins to acquire their native conformation and activity. Two PrsA proteins, PrsA1 and PrsA2, have been identified in GAS, but the respective contribution of each PrsA in GAS pathogenesis remains largely unknown. By combining comparative proteomic and phenotypic analysis approaches, we demonstrate that both PrsA isoforms are required to maintain GAS proteome homeostasis and virulence-associated traits in a unique and overlapping manner. The inactivation of both PrsA in GAS caused remarkable impairment in biofilm formation, host adherence, infection-induced cytotoxicity, and in vivo virulence in a murine soft tissue infection model. The concordance of proteomic and phenotypic data clearly features the essential role of PrsA in GAS full virulence.

GP96 Drives Exacerbation of Secondary Bacterial Pneumonia following Influenza A Virus Infection.

Influenza A virus (IAV) infection predisposes the host to secondary bacterial pneumonia, known as a major cause of morbidity and mortality during influenza virus epidemics. Analysis of interactions between IAV-infected human epithelial cells and Streptococcus pneumoniae revealed that infected cells ectopically exhibited the endoplasmic reticulum chaperone glycoprotein 96 (GP96) on the surface. Importantly, efficient pneumococcal adherence to epithelial cells was imparted by interactions with extracellular GP96 and integrin αV, with the surface expression mediated by GP96 chaperone activity. Furthermore, abrogation of adherence was gained by chemical inhibition or genetic knockout of GP96 as well as addition of RGD peptide, an inhibitor of integrin-ligand interactions. Direct binding of extracellular GP96 and pneumococci was shown to be mediated by pneumococcal oligopeptide permease components. Additionally, IAV infection induced activation of calpains and Snail1, which are responsible for degradation and transcriptional repression of junctional proteins in the host, respectively, indicating increased bacterial translocation across the epithelial barrier. Notably, treatment of IAV-infected mice with the GP96 inhibitor enhanced pneumococcal clearance from lung tissues and ameliorated lung pathology. Taken together, the present findings indicate a viral-bacterial synergy in relation to disease progression and suggest a paradigm for developing novel therapeutic strategies tailored to inhibit pneumococcal colonization in an IAV-infected respiratory tract. IMPORTANCE Secondary bacterial pneumonia following an influenza A virus (IAV) infection is a major cause of morbidity and mortality. Although it is generally accepted that preceding IAV infection leads to increased susceptibility to secondary bacterial infection, details regarding the pathogenic mechanism during the early stage of superinfection remain elusive. Here, we focused on the interaction of IAV-infected cells and Streptococcus pneumoniae, which revealed that human epithelial cells infected with IAV exhibit a cell surface display of GP96, an endoplasmic reticulum chaperon. Notably, extracellular GP96 was shown to impart efficient adherence for secondary infection by S. pneumoniae, and GP96 inhibition ameliorated lung pathology of superinfected mice, indicating it to be a useful target for development of therapeutic strategies for patients with superinfection.

Perspectives on the development of first-in-class protein degraders.

Targeted protein degradation is a broad and expanding field aimed at the modulation of protein homeostasis. A focus of this field has been directed toward molecules that hijack the ubiquitin proteasome system with heterobifunctional ligands that recruit a target protein to an E3 ligase to facilitate polyubiquitination and subsequent degradation by the 26S proteasome. Despite the success of these chimeras toward a number of clinically relevant targets, the ultimate breadth and scope of this approach remains uncertain. Here we highlight recent advances in assays and tools available to evaluate targeted protein degradation, including and beyond the study of E3-targeted chimeric ligands. We note several challenges associated with degrader development and discuss various approaches to expanding the protein homeostasis toolbox.

Streptococcus pyogenes upregulates arginine catabolism to exert its pathogenesis on the skin surface.

The arginine deiminase (ADI) pathway has been found in many kinds of bacteria and functions to supplement energy production and provide protection against acid stress. The Streptococcus pyogenes ADI pathway is upregulated upon exposure to various environmental stresses, including glucose starvation. However, there are several unclear points about the advantages to the organism for upregulating arginine catabolism. We show that the ADI pathway contributes to bacterial viability and pathogenesis under low-glucose conditions. S. pyogenes changes global gene expression, including upregulation of virulence genes, by catabolizing arginine. In a murine model of epicutaneous infection, S. pyogenes uses the ADI pathway to augment its pathogenicity by increasing the expression of virulence genes, including those encoding the exotoxins. We also find that arginine from stratum-corneum-derived filaggrin is a key substrate for the ADI pathway. In summary, arginine is a nutrient source that promotes the pathogenicity of S. pyogenes on the skin.

Epidemiological analysis of pneumococcal strains isolated at Yangon Children's Hospital in Myanmar via whole-genome sequencing-based methods.

Streptococcus pneumoniae causes over one million deaths from lower respiratory infections per annum worldwide. Although mortality is very high in Southeast Asian countries, molecular epidemiological information remains unavailable for some countries. In this study, we report, for the first time, the whole-genome sequences and genetic profiles of pneumococcal strains isolated in Myanmar. We isolated 60 streptococcal strains from 300 children with acute respiratory infection at Yangon Children's Hospital in Myanmar. We obtained whole-genome sequences and identified the species, serotypes, sequence types, antimicrobial resistance (AMR) profiles, virulence factor profiles and pangenome structure using sequencing-based analysis. Average nucleotide identity analysis indicated that 58 strains were S. pneumoniae and the other 2 strains were Streptococcus mitis. The major serotype was 19F (11 strains), followed by 6E (6B genetic variant; 7 strains) and 15 other serotypes; 5 untypable strains were also detected. Multilocus sequence typing analysis revealed 39 different sequence types, including 11 novel ones. In addition, genetic profiling indicated that AMR genes and mutations spread among pneumococcal strains in Myanmar. A minimum inhibitory concentration assay indicated that several pneumococcal strains had acquired azithromycin and tetracycline resistance, whereas no strains were found to be resistant against levofloxacin and high-dose penicillin G. Phylogenetic and pangenome analysis showed various pneumococcal lineages and that the pneumococcal strains contain a rich and mobile gene pool, providing them with the ability to adapt to selective pressures. This molecular epidemiological information can help in tracking global infection and supporting AMR control in addition to public health interventions in Myanmar.

[Investigation of pneumococcal virulence factors in the infection process].

This review summarizes current knowledge regarding the pathological mechanism of Streptococcus pneumoniae, a major cause of pneumonia, sepsis, and meningitis, with focus on our previously presented studies.To identify pneumococcal adhesins or invasins on cell surfaces, we investigated several proteins with an LPXTG anchoring motif and identified one showing interaction with human fibronectin, which was designated PfbA. Next, the mechanism of pneumococcal evasion form host immunity system in blood was examined and pneumococcal α-Enolase was found to function as a neutrophil extracellular trap induction factor. Although S. pneumoniae organisms are partially killed by iron ion-induced free radicals, they have an ability to invade red blood cells and then evade antibiotics, neutrophil phagocytosis, and H2O2 killing. In addition, our findings have indicated that zinc metalloprotease ZmpC suppresses pneumococcal virulence by inhibiting bacterial invasion of the central nervous system. Since evolutionarily conserved virulence factors are potential candidate therapeutic targets, we performed molecular evolutionary analyses, which revealed that cbpJ had the highest rate of codons under negative selection to total number of codons among genes encoding choline-binding proteins. Our experimental analysis results indicated that CbpJ functions as a virulence factor in pneumococcal pneumonia by contributing to evasion of neutrophil killing.Use of a molecular biological approach based on bacterial genome sequences, clinical disease states, and molecular evolutionary analysis is an effective strategy for revealing virulence factors and important therapeutic targets.

Role of BgaA as a Pneumococcal Virulence Factor Elucidated by Molecular Evolutionary Analysis.

Streptococcus pneumoniae is a major cause of pneumonia, sepsis, and meningitis. Previously, we identified a novel virulence factor by investigating evolutionary selective pressure exerted on pneumococcal choline-binding cell surface proteins. Herein, we focus on another pneumococcal cell surface protein. Cell wall-anchoring proteins containing the LPXTG motif are conserved in Gram-positive bacteria. Our evolutionary analysis showed that among the examined genes, nanA and bgaA had high proportions of codon that were under significant negative selection. Both nanA and bgaA encode a multi-functional glycosidase that aids nutrient acquisition in a glucose-poor environment, pneumococcal adherence to host cells, and evasion from host immunity. However, several studies have shown that the role of BgaA is limited in a mouse pneumonia model, and it remains unclear if BgaA affects pneumococcal pathogenesis in a mouse sepsis model. To evaluate the distribution and pathogenicity of bgaA, we performed phylogenetic analysis and intravenous infection assay. In both Bayesian and maximum likelihood phylogenetic trees, the genetic distances between pneumococcal bgaA was small, and the cluster of pneumococcal bgaA did not contain other bacterial orthologs except for a Streptococcus gwangjuense gene. Evolutionary analysis and BgaA structure indicated BgaA active site was not allowed to change. The mouse infection assay showed that the deletion of bgaA significantly reduced host mortality. These results indicated that both nanA and bgaA encode evolutionally conserved pneumococcal virulence factors and that molecular evolutionary analysis could be a useful alternative strategy for identification of virulence factors.

Evaluation of decontamination methods of oral biofilms formed on screw-shaped, rough and machined surface implants: an ex vivo study.

BACKGROUND: To evaluate the effect of several representative decontamination methods of oral biofilms on different implant surfaces. MATERIAL AND METHODS: Eleven participants wore a hard resin splint carrying 6 rough (GC Aadva® implant; 3.3-mm diameter, 8-mm length) or machined (not commercially available) surface implants for 4 days to accumulate dental plaque naturally on the titanium surfaces of the implants. Apart from surface roughness, the morphology of all implants was identical. After detaching the implants from the splints, the ability of the following decontamination methods-gauze soaked in saline (G), ultrasonic scaler (US), air abrasive (Air), rotary stainless steel instrument (Rot), and Er:YAG laser (Las)-to cleanse the contaminated implant surface for 1 min extra-orally was tested. The control (Cont) group did not receive any decontamination. Scanning electron microscopic (SEM) investigation of one participant's samples was employed to examine the post-instrumented implant surface for qualitative analysis, and bacterial culture of the remaining 10 participants' samples was performed to count the number of colony-forming units (CFU) for quantitative analysis. The experimental sequence was initially performed for the rough surface implants and then similarly repeated for the machined surface implants. Bacterial CFU counts among the six groups were analyzed using the Steel-Dwass test, and differences between rough and machined surface implants were determined using the Mann-Whitney U test. RESULTS: G and Rot eliminated most biofilms on machined surface implants according to SEM analysis. G, Air, and Rot removed significantly more of the biofilms on rough and machined surface implants compared with US according to CFU counts. Moreover, G significantly reduced more biofilms than Las on machined surface implants. The analysis between rough and machined surface implants showed that Cont, G, and US were better able to cleanse biofilms on machined surface implants compared with rough surface implants. CONCLUSIONS: Gauze soaked in saline and rotary stainless steel instruments may be advantageous for cleansing contaminated implant surfaces based on the qualitative and quantitative analyses. In contrast, air abrasives were not shown to be preferable in the qualitative analyses. Additionally, apart from the Er:YAG laser, the reduction of biofilms assessed in both qualitative and quantitative analyses demonstrated that all decontamination methods were better at cleansing machined surface implants compared with rough surface implants.

Paradoxical mitotic exit induced by a small molecule inhibitor of APC/CCdc20.

The anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase that initiates anaphase and mitotic exit. APC/C is activated by Cdc20 and inhibited by the mitotic checkpoint complex (MCC), which delays mitotic exit when the spindle assembly checkpoint (SAC) is activated. We previously identified apcin as a small molecule ligand of Cdc20 that inhibits APC/CCdc20 and prolongs mitosis. Here we find that apcin paradoxically shortens mitosis when SAC activity is high. These opposing effects of apcin arise from targeting of a common binding site in Cdc20 required for both substrate ubiquitination and MCC-dependent APC/C inhibition. Furthermore, we found that apcin cooperates with p31comet to relieve MCC-dependent inhibition of APC/C. Apcin therefore causes either net APC/C inhibition, prolonging mitosis when SAC activity is low, or net APC/C activation, shortening mitosis when SAC activity is high, demonstrating that a small molecule can produce opposing biological effects depending on regulatory context.