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Accelerated reactions in microdroplets have been reported for a wide range of reactions with some microdroplet reactions occurring over a million times faster than the same reaction in bulk solution. Unique chemistry at the air-water interface has been implicated as a primary factor for accelerated reaction rates, but the role of analyte concentration in evaporating droplets has not been as well studied. Here, theta-glass electrospray emitters and mass spectrometry are used to rapidly mix two solutions on the low to sub-microsecond time scale and produce aqueous nanodrops with different sizes and lifetimes. We demonstrate that for a simple bimolecular reaction where surface chemistry does not appear to play a role, reaction rate acceleration factors are between 102 and 107 for different initial solution concentrations, and these values do not depend on nanodrop size. A rate acceleration factor of 107 is among the highest reported and can be attributed to concentration of analyte molecules, initially far apart in dilute solution, but brought into close proximity in the nanodrop through evaporation of solvent from the nanodrops prior to ion formation. These data indicate that analyte concentration phenomenon is a significant factor in reaction acceleration where droplet volume throughout the experiment is not carefully controlled.

Periodontitis is a public issue and imaging periodontal pocket is important to evaluate periodontitis. Regular linear transducers have limitations in imaging the posterior teeth due to their geometry restrictions. Here we characterized a transducer that can image the posterior teeth including assessment of periodontal pockets via a combination of photoacoustic and ultrasound imaging. Unlike conventional transducer design, this device has a toothbrush-shaped form factor with a side-view transducer to image molars (total size: 1 ×1.9 cm). A laser diode was integrated as the light source to reduce the cost and size and facilitates clinical transition. The in vivo imaging of a molar of a periodontal patient demonstrated that the transducer could image in the posterior area of gum in vivo; the value determined by imaging was within 7 % of the value measured clinically.

Porphyromonas gingivalis is a keystone pathogen in periodontal disease. We herein report a dual-modal fluorescent and photoacoustic imaging probe for the detection of gingipain proteases secreted by P. gingivalis. Upon proteolytic cleavage by Arg-specific gingipain (RgpB), five-fold photoacoustic enhancement and >100-fold fluorescence activation was measured with detection limits of 1.1 nM RgpB and 5.0E4 CFU mL-1 bacteria in vitro. RgpB activity was imaged in porcine jaws with low-nanomolar sensitivity. Diagnostic efficacy was evaluated in gingival crevicular fluid samples from subjects with and without periodontal disease, wherein activation was correlated to qPCR-based detection of P. gingivalis (Pearson's r=0.71). Finally, photoacoustic imaging of RgpB-cleaved probe was achieved in murine brains ex vivo, with relevance and potential utility for disease models of general infection by P. gingivalis, motivated by the recent biological link between gingipain and Alzheimer's disease.

This study examined the microbial diversity and community assembly of oral microbiota in periodontal health and disease and after nonsurgical periodontal treatment. The V4 region of 16S rRNA gene from DNA of 238 saliva and subgingival samples of 21 healthy and 48 diseased subjects was amplified and sequenced. Among 1979 OTUs identified, 28 were overabundant in diseased plaque. Six of these taxa were also overabundant in diseased saliva. Twelve OTUs were overabundant in healthy plaque. There was a trend for disease-associated taxa to decrease and health-associated taxa to increase after treatment with notable variations among individual sites. Network analysis revealed modularity of the microbial communities and identified several health- and disease-specific modules. Ecological drift was a major factor that governed community turnovers in both plaque and saliva. Dispersal limitation and homogeneous selection affected the community assembly in plaque, with the additional contribution of homogenizing dispersal for plaque within individuals. Homogeneous selection and dispersal limitation played important roles, respectively, in healthy saliva and diseased pre-treatment saliva between individuals. Our results revealed distinctions in both taxa and assembly processes of oral microbiota between periodontal health and disease. Furthermore, the community assembly analysis has identified potentially effective approaches for managing periodontitis.

Spontaneous ionization/breakup of water at the surface of aqueous droplets has been reported with evidence ranging from formation of hydrogen peroxide and hydroxyl radicals, indicated by ions at m/z 36 attributed to OH⋅‐H3O+ or (H2O‐OH2)+⋅ as well as oxidation products of radical scavengers in mass spectra of water droplets formed by pneumatic nebulization. Here, aqueous droplets are formed both by nanoelectrospray, which produces highly charged nanodrops with initial diameters ~100 nm, and a vibrating mesh nebulizer, which produces 2–20 μm droplets that are initially less highly charged. The lifetimes of these droplets range from 10s of μs to 560 ms and the surface‐to‐volume ratios span ~100‐fold range. No ions at m/z 36 are detected with pure water, nor are significant oxidation products for the two radical scavengers that were previously reported to be formed in high abundance. These and other results indicate that prior conclusions about spontaneous hydroxyl radical formation in unactivated water droplets are not supported by the evidence and that water appears to be stable at droplet surfaces over a wide range of droplet size, charge and lifetime. [ABSTRACT FROM AUTHOR]

Microbes frequently experience nutrient deprivations in the natural environment and may enter dormancy. Aggregatibacter actinomycetemcomitans is known to establish long-term infections in humans. This study examined the dormancy-like phenotype of an A. actinomycetemcomitans strain D7S-1 and its isogenic smooth-colony mutant D7SS. A tissue culture medium RPMI-1640 was nutrient-deficient (ND) and unable to support A. actinomycetemcomitans growth. RPMI-1640 amended with bases was nutrient-limited (NL) and supported limited growth of A. actinomycetemcomitans less than the nutrient-enriched (NE) laboratory medium did. Strain D7S-1, after an initial 2-log reduction in viability, maintained viability from day 4 to day 15 in the NL medium. Strain D7SS, after 1-log reduction in viability, maintained viability from day 3 to day 5. In contrast, bacteria in the NE medium were either non-recoverable (D7S-1; >6-log reduction) or continued to lose viability (D7SS; 3-log reduction) on day 5 and beyond. Scanning and transmission electron microscopy showed that A. actinomycetemcomitans in the NL medium formed robust biofilms similar to those in the NE medium but with evidence of stress. A. actinomycetemcomitans in the ND medium revealed scant biofilms and extensive cellular damage. We concluded that A. actinomycetemcomitans grown in the NL medium exhibited a dormancy-like phenotype characterized by minimum growth, prolonged viability, and distinct cellular morphology. [ABSTRACT FROM AUTHOR]

Objective: To develop a novel technique for localizing and reconstructing the greater palatine artery (GPA) using three-dimensional (3D) technology. Methods: A miniaturized intraoral ultrasound transducer was used to imaging landmarks including the GPA, gingival margin (GM), and palatal masticatory mucosa (PMM). A 5-mm-thick solid hydrogel couplant was integrated to replace traditional ultrasound gel and avoid bubbles when moving the transducer. Results: A panorama image provided the relative localization of landmarks including the GPA, PMM, and hard palate. Short- and long-axis imaging of GPA was performed in five subjects including 3D mapping of GPA branches and surrounding tissues in a volume of 10 mm ×8 mm × 10mm. Full-mouth Doppler imaging was also demonstrated on both the dorsal and ventral tongue as well as buccal mucosa and sublingual region on two subjects. Conclusions: This study can measure the vertical distance from the GM to the GPA and depth from PMM to GPA and visualize the GPA localization in a 3D manner, which is critical to evaluate the available volume of palatal donor tissues and avoid sectioning of GPA during surgical harvesting of the tissues. Finally, the transducer’s small size facilitates full-mouth Doppler imaging with the potential to improve the assessment, diagnosis, and management of oral mucosa. [ABSTRACT FROM AUTHOR]

Aggregatibacter actinomycetemcomitans, a Gram‐negative oral pathobiont causing aggressive periodontitis and systemic infections, demonstrates serum resistance. We have identified a dsDNA‐tailed bacteriophage, S1249, which was found to convert from this microorganism inducible by human serum into a lytic state to kill the bacterium. This phage demonstrated active transcripts when exposed to human serum: 20% of genes were upregulated more than 10‐fold, and 45% of them were upregulated 5–10‐fold when the bacterium was grown in the presence of human serum compared to without the presence of human serum. Transcriptional activation when grown in equine serum was less pronounced. This phage demonstrated a tail with inner rigid tubes and an outer contractile sheath, features of Myoviridae spp. Further characterization revealed that the lysogenized integration of the phage in the chromosome of A. actinomycetemcomitans occurred between the genes encoding cold‐shock DNA‐binding domain‐containing protein (csp) and glutamyl‐tRNA synthetase (gltX). Both phage DNA integrated lysogeny and nonintegrated pseudolysogeny were identified in the infected bacterium. A newly generated, lysogenized strain using this phage displayed similar attributes, including 63% growth inhibition compared to its isogenic phage‐free strain when in the presence of human serum. Our data suggest that bacteriophage S1249 can be induced in the presence of human serum and enters the lytic cycle, which reduces the viability of infected bacteria in vivo. [ABSTRACT FROM AUTHOR]

Actinobacillus actinomycetemcomitans is naturally competent for transformation, with a transformation system similar to that of Haemophilus influenzae that preferentially takes up DNA bearing uptake signal sequences (USS) with the same 9-base USS core. This study examined the function of the extended 29-base USS, which comprises a highly conserved 1st region (containing the 9-base core) and 2nd and 3rd semi-conserved AT-rich regions, in transformation of A. actinomycetemcomitans. Transformation frequency was not affected by either location (in middle or at 5' end) or quantity (one or two) of USS in donor DNA. Relative transformation efficiencies (in comparison to the positive control) were 28-67% for linear DNA with single-base mutations in the USS 1st region, and 47% and 73%, respectively, for linear DNA with USS that contained either a non-consensus 2nd or a non-consensus 3rd region. Plasmids with a stand-alone 1st or a stand-alone 2nd-3rd region exhibited 21% and 6% relative transformation efficiencies, respectively. It was also noted that A. actinomyceterncomitans and H. influenzae were similar in the frequencies and distribution patterns of USS in their genomes. In conclusion, all three regions of the extended 29-base USS are required for optimum transformation in A. actinomyceterncomitans.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolves rapidly under the pressure of host immunity, as evidenced by waves of emerging variants despite effective vaccinations, highlighting the need for complementing antivirals. We report that targeting a pyrimidine synthesis enzyme restores inflammatory response and depletes the nucleotide pool to impede SARS-CoV-2 infection. SARS-CoV-2 deploys Nsp9 to activate carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase (CAD) that catalyzes the rate-limiting steps of the de novo pyrimidine synthesis. Activated CAD not only fuels de novo nucleotide synthesis but also deamidates RelA. While RelA deamidation shuts down NF-γB activation and subsequent inflammatory response, it up-regulates key glycolytic enzymes to promote aerobic glycolysis that provides metabolites for de novo nucleotide synthesis. A newly synthesized smallmolecule inhibitor of CAD restores antiviral inflammatory response and depletes the pyrimidine pool, thus effectively impeding SARS-CoV-2 replication. Targeting an essential cellular metabolic enzyme thus offers an antiviral strategy that would be more refractory to SARS-CoV-2 genetic changes. [ABSTRACT FROM AUTHOR]

Research has been conducted on Actinobacillus actinomycetemcomitans. The identification of A. actinomycetemcomitans clinical isolate has been carried out and its transformation properties and uptake signal sequence have been investigated and discussed.

It appears that two bacteria types responsible for gum disease can be transmitted between close family members through saliva. However, the transmission patterns may be different. Children of parents infected with Actinobacillus actinomycetemcomitans are significantly more likely to harbor identical bacterial strains. However, children of parents infected with Porphyromonas (P.) gingivalis are not more likely to test positive for this bacteria. Spouses with gum disease appear more receptive to infections with P. gingivalis. Gum disease status does not affect spousal transmission of A. actinomycetemcomitans.

Abstract Background Bacterial phenotype may be profoundly affected by the physical arrangement of their genes in the genome. The Gram-negative species Aggregatibacter actinomycetemcomitans is a major etiologic agent of human periodontitis. Individual clonal types of A. actinomycetemcomitans may exhibit variable virulence and different patterns of disease association. This study examined the genome arrangement of A. actinomycetemcomitans using the genome sequences of serotypes a-c strains. The genome alignment and rearrangement were analyzed by the MAUVE and the GRIMM algorithms. The distribution patterns of genes along the leading/lagging strands were investigated. The occurrence and the location of repeat sequences relative to the genome rearrangement breakpoints were also determined. Results The genome arrangement of the serotype a strain D7S-1 is markedly different from the serotype b strain HK1651 or the serotype c strain D11S-1. Specific genome arrangements appear to be conserved among strains of the same serotypes. The reversal distance between D7S-1 and HK1651 by GRIMM analysis is also higher than the within-species comparisons of 7 randomly selected bacterial species. The locations of the orthologous genes are largely preserved between HK1651 and D11S-1 but not between D7S-1 and HK1651 (or D11S-1), irrespective of whether the genes are categorized as essential/nonessential or highly/nonhighly expressed. However, genome rearrangement did not disrupt the operons of the A. actinomycetemcomitans strains. A higher proportion of the genome in strain D7S-1 is occupied by repeat sequences than in strains HK1651 or D11S-1. Conclusion The results suggest a significant evolutionary divergence between serotype a strains and serotypes b/c strains of A. actinomycetemcomitans. The distinct patterns of genome arrangement may suggest phenotypic differences between serotype a and serotypes b/c strains.

Abstract Background Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressive forms of periodontitis. Increasing evidence points to a link between periodontitis and cardiovascular diseases, however, the underlying mechanisms are poorly understood. This study investigated the pathogenic potential of free-soluble surface material, released from live planktonic and biofilm A. actinomycetemcomitans cells. Results By employing an ex vivo insert model (filter pore size 20 nm) we demonstrated that the A. actinomycetemcomitans strain D7S and its derivatives, in both planktonic and in biofilm life-form, released free-soluble surface material independent of outer membrane vesicles. This material clearly enhanced the production of several proinflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8, MIP-1β) in human whole blood, as evidenced by using a cytokine antibody array and dissociation-enhanced-lanthanide-fluorescent-immunoassay. In agreement with this, quantitative real-time PCR indicated a concomitant increase in transcription of each of these cytokine genes. Experiments in which the LPS activity was blocked with polymyxin B showed that the stimulatory effect was only partly LPS-dependent, suggesting the involvement of additional free-soluble factors. Consistent with this, MALDI-TOF-MS and immunoblotting revealed release of GroEL-like protein in free-soluble form. Conversely, the immunomodulatory toxins, cytolethal distending toxin and leukotoxin, and peptidoglycan-associated lipoprotein, appeared to be less important, as evidenced by studying strain D7S cdt/ltx double, and pal single mutants. In addition to A. actinomycetemcomitans a non-oral species, Escherichia coli strain IHE3034, tested in the same ex vivo model also released free-soluble surface material with proinflammatory activity. Conclusion A. actinomycetemcomitans, grown in biofilm and planktonic form, releases free-soluble surface material independent of outer membrane vesicles, which induces proinflammatory responses in human whole blood. Our findings therefore suggest that release of surface components from live bacterial cells could constitute a mechanism for systemic stimulation and be of particular importance in chronic localized infections, such as periodontitis.

Abstract Background Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL) among A. actinomycetemcomitans strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles. Results The pal locus and its gene product were confirmed in clinical A. actinomycetemcomitans strains by PCR-restriction fragment length polymorphism and immunoblotting. Culturing under different growth conditions revealed no apparent requirement for the AaPAL expression. Inactivation of pal in a wild-type strain (D7S) and in its spontaneous laboratory variant (D7SS) resulted in pleiotropic cellular effects. In a cell culture insert model (filter pore size 0.02 μm), AaPAL was detected from filtrates when strains D7S and D7SS were incubated in serum or broth in the inserts. Electron microscopy showed that A. actinomycetemcomitans vesicles (0.05–0.2 μm) were larger than the filter pores and that there were no vesicles in the filtrates. The filtrates were immunoblot negative for a cytoplasmic marker, cyclic AMP (cAMP) receptor protein. An ex vivo model indicated cytokine production from human whole blood stimulated by AaPAL. Conclusion Free-soluble AaPAL can be extracellularly released in a process independent of vesicles.