Development of a nomogram for identifying periodontitis cases in Denmark.

Although self-reported health outcomes are of importance, attempts to validate a clinical applicable instrument (e.g., nomogram) combining sociodemographic and self-reported information on periodontitis have yet to be performed to identify periodontitis cases. Clinical and self-reported periodontitis, along with sociodemographic data, were collected from 197 adults. Akaike information criterion models were developed to identify periodontitis, and nomograms developed based on its regression coefficients. The discriminatory capability was evaluated by receiver-operating characteristic curves. Decision curve analysis was performed. Smoking [OR 3.69 (95%CI 1.89, 7.21)], poor/fair self-rated oral health [OR 6.62 (95%CI 3.23, 13.56)], previous periodontal treatment [OR 9.47 (95%CI 4.02, 22.25)], and tooth loss [OR 4.96 (95%CI 2.47, 9.97)], determined higher probability of having "Moderate/Severe Periodontitis". Age [OR 1.08 (95%CI 1.05, 1.12)], low educational level [OR 1.65 (95%CI 1.34, 2.23)], poor/fair self-rated oral health [OR 3.57 (95%CI 1.82, 6.99)], and previous periodontal treatment [OR 6.66 (95%CI 2.83, 15.68)] determined higher probability for "Any Periodontitis". Both nomograms showed excellent discriminatory capability (AUC of 0.83 (95%CI 0.75, 0.91) and 0.81 (95% CI 0.74, 0.88), good calibration, and slight overestimation of high risk and underestimation of low risk. Hence, our nomograms could help identify periodontitis among adults in Denmark.

Biopsy-based optimization and calibration of a signal-intensity-ratio-based MRI method (1.5 Tesla) in a dextran-iron loaded mini-pig model, enabling estimation of very high liver iron concentrations.

OBJECTIVE: Magnetic resonance imaging (MRI)-based techniques for non-invasive assessing liver iron concentration (LIC) in patients with iron overload have a limited upper measuring range around 35 mg/g dry weight, caused by signal loss from accelerated T1-, T2-, T2* shortening with increasing LIC. Expansion of this range is necessary to allow evaluation of patients with very high LIC. AIM: To assess measuring range of a gradient-echo R2* method and a T1-weighted spin-echo (SE), signal intensity ratio (SIR)-based method (TE = 25 ms, TR = 560 ms), and to extend the upper measuring range of the SIR method by optimizing echo time (TE) and repetition time (TR) in iron-loaded minipigs. METHODS: Thirteen mini pigs were followed up during dextran-iron loading with repeated percutaneous liver biopsies for chemical LIC measurement and MRIs for parallel non-invasive estimation of LIC (81 examinations) using different TEs and TRs. RESULTS: SIR and R2* method had similar upper measuring range around 34 mg/g and similar method agreement. Using TE = 12 ms and TR = 1200 ms extended the upper measuring range to 115 mg/g and yielded good method of agreement. DISCUSSION: The wider measuring range is likely caused by lesser sensitivity of the SE sequence to iron, due to shorter TE, leading to later signal loss at high LIC, allowing evaluation of most severe hepatic iron overload. Validation in iron-loaded patients is necessary.

Multilocus Sequence Typing of Aggregatibacter actinomycetemcomitans Competently Depicts the Population Structure of the Species.

We developed a multilocus sequence typing scheme (MLST) for Aggregatibacter actinomycetemcomitans based on seven housekeeping genes, adk, atpG, frdB, mdh, pgi, recA, and zwf. A total of 188 strains of seven serotypes were separated into 57 sequence types. Whole-genome sequences were available for 140 strains, and in contrast to comparison of 16S rRNA genes, phylogenetic analysis of concatenated MLST gene fragments was in accordance with the population structure revealed by alignment of 785 core genes. MLST could not decisively identify the so-called JP2 clone associated with rapidly progressing periodontitis in adolescents, but noticeable clustering of JP2 genotype strains was revealed. The MLST scheme of A. actinomycetemcomitans can be assessed at www.pubmlst.org. IMPORTANCE Accurate diagnosis of infectious disease comprise identification, typing, and antimicrobial resistance of the infective agent. Bacteria are sometimes grouped within their species according to expression of specific toxins or particular antimicrobial resistance traits, but explicit typing for infection control and survey of pathogenesis necessitates genetic analysis such as multilocus sequence typing (MLST). Schemes for the most prevalent human pathogens have been available for more than 10 years, and time has come to extend the scrutiny to second-line infectious agents. One such pathogen is Aggregatibacter actinomycetemcomitans, which is commonly involved in periodontitis, and more rarely as the cause of infective endocarditis or spontaneous brain abscess. A MLST scheme for A. actinomycetemcomitans is now available at www.pubmlst.org. Whole-genome sequencing of a large number of isolates confirms that MLST competently depicts the population structure of the species.

Whole Genome Sequencing of Aggregatibacter actinomycetemcomitans Cultured from Blood Stream Infections Reveals Three Major Phylogenetic Groups Including a Novel Lineage Expressing Serotype a Membrane O Polysaccharide.

Twenty-nine strains of Aggregatibacter actinomycetemcomitans cultured from blood stream infections in Denmark were characterised. Serotyping was unremarkable, with almost equal proportions of the three major types plus a single serotype e strain. Whole genome sequencing positioned the serotype e strain outside the species boundary; moreover, one of the serotype a strains was unrelated to other strains of the major serotypes and to deposited sequences in the public databases. We identified five additional strains of this type in our collections. The particularity of the group was corroborated by phylogenetic analysis of concatenated core genes present in all strains of the species, and by uneven distribution of accessory genes only present in a subset of strains. Currently, the most accurate depiction of A. actinomycetemcomitans is a division into three lineages that differ in genomic content and competence for transformation. The clinical relevance of the different lineages is not known, and even strains excluded from the species sensu stricto can cause serious human infections. Serotyping is insufficient for characterisation, and serotypes a and e are not confined to specific lineages.

Comprehensive antimicrobial susceptibility testing of a large collection of clinical strains of Aggregatibacter actinomycetemcomitans does not identify resistance to amoxicillin.

AIM: The present study aims to determine the susceptibility of Aggregatibacter actinomycetemcomitans to amoxicillin by investigating a large collection of oral strains of diverse geographical origin. METHODS: Two hundred and fifty-seven A. actinomycetemcomitans strains were serotyped using a multiplex polymerase chain reaction, and minimal inhibitory concentration (MIC) values of amoxicillin were determined using the agar dilution method (range 0.25-8.0 mg/L). The plates were spot-wise inoculated with approximately 104 colony-forming units, incubated in 5% CO2 at 37 C°, and visually inspected after 24 and 48 hr. A MIC ≤ 2.00 mg/L was categorised as susceptible using EUCAST interpretative criteria for Haemophilus species. RESULTS: Amoxicillin MIC values varied from 0.25 mg/L to 2.00 mg/L, and all tested strains, including strains previously reported as resistant, were susceptible to amoxicillin. The MIC50 was 1.00 mg/L and the MIC90 was 2.00 mg/L. CONCLUSION: Meticulous investigation of strains including isolates previously reported as resistant could not confirm the emergence of resistance to ß-lactams in A. actinomycetemcomitans. Based on the present in vitro results, amoxicillin can be considered a key oral antimicrobial agent for treatment of A. actinomycetemcomitans.

MicroRNA-101 Regulates Multiple Developmental Programs to Constrain Excitation in Adult Neural Networks.

A critical feature of neural networks is that they balance excitation and inhibition to prevent pathological dysfunction. How this is achieved is largely unknown, although deficits in the balance contribute to many neurological disorders. We show here that a microRNA (miR-101) is a key orchestrator of this essential feature, shaping the developing network to constrain excitation in the adult. Transient early blockade of miR-101 induces long-lasting hyper-excitability and persistent memory deficits. Using target site blockers in vivo, we identify multiple developmental programs regulated in parallel by miR-101 to achieve balanced networks. Repression of one target, NKCC1, initiates the switch in γ-aminobutyric acid (GABA) signaling, limits early spontaneous activity, and constrains dendritic growth. Kif1a and Ank2 are targeted to prevent excessive synapse formation. Simultaneous de-repression of these three targets completely phenocopies major dysfunctions produced by miR-101 blockade. Our results provide new mechanistic insight into brain development and suggest novel candidates for therapeutic intervention.

Pharmacological characterisation of α6ß4⁎ nicotinic acetylcholine receptors assembled from three chimeric α6/α3 subunits in tsA201 cells.

The distribution and physiological functions of the α6 subunit-containing (α6⁎) nicotinic acetylcholine receptors in the central nervous system make them interesting putative therapeutic targets in several disorders. However, investigations into the receptors have been complicated by their inefficient functional expression in vitro. In the present study we have characterized and compared the pharmacological properties displayed by α6ß4 and α6ß4ß3 nicotinic acetylcholine receptors assembled in tsA201 cells from the classical α6/α3 chimera (C1) and two novel α6/α3 chimeras (C6F223L and C16F223L) identified in a recent study (Jensen et al., 2013). Whereas the Bmax values exhibited by [3H]epibatidine at wild-type α6ß4, C1ß4, C6F223Lß4 and C16F223Lß4 receptors differed substantially, the radioligand and seven orthosteric nicotinic agonists exhibited very similar KD and Ki values at the four receptors. In the FLIPR™ Membrane Potential Blue assay, the agonists exhibited the same rank order of potencies [(±)-epibatidine>sazetidine A>varenicline>(-)-cytisine~(S)-nicotine>acetylcholine>carbachol] at the C1ß4, C1ß4ß3, C6F223Lß4, C6F223Lß4ß3, C16F223Lß4 and C16F223Lß4ß3 receptors, albeit the absolute EC50 values differed somewhat between the receptors. Furthermore, four reference antagonists displayed the same rank order of inhibitory potencies at the six receptors [α-conotoxin PIA>2,2,6,6-tetramethylpiperidin-4-yl heptanoate>mecamylamine>dihydro-ß-erythroidine]. Although all interpretations based on these results should be made keeping the molecular modifications in the α6 surrogate subunits in mind, this study sheds light on the pharmacological properties of α6ß4⁎ nicotinic acetylcholine receptors and demonstrates the applicability of the C6F223L and C16F223L chimeras for studies of these receptors.

Elucidation of molecular impediments in the α6 subunit for in vitro expression of functional α6ß4* nicotinic acetylcholine receptors.

Explorations into the α6-containing nicotinic acetylcholine receptors (α6* nAChRs) as putative drug targets have been severely hampered by the inefficient functional expression of the receptors in heterologous expression systems. In this study, the molecular basis for the problem was investigated through the construction of chimeric α6/α3 and mutant α3 and α6 subunits and functional characterization of these co-expressed with ß4 or ß4ß3 subunits in tsA201 cells in a fluorescence-based assay and in Xenopus oocytes using two-electrode voltage clamp electrophysiology. Substitution of a small C-terminal segment in the second intracellular loop or the Phe(223) residue in transmembrane helix 1 of α6 with the corresponding α3 segment or residue was found to enhance α6ß4 functionality in tsA201 cells significantly, in part due to increased cell surface expression of the receptors. The gain-of-function effects of these substitutions appeared to be additive since incorporation of both α3 elements into α6 resulted in assembly of α6ß4* receptors exhibiting robust functional responses to acetylcholine. The pharmacological properties exhibited by α6ß4ß3 receptors comprising one of these novel α6/α3 chimeras in oocytes were found to be in good agreement with those from previous studies of α6* nAChRs formed from other surrogate α6 subunits or concatenated subunits and studies of other heteromeric nAChRs. In contrast, co-expression of this α6/α3 chimera with ß2 or ß2ß3 subunits in oocytes did not result in efficient formation of functional receptors, indicating that the identified molecular elements in α6 could be specific impediments for the expression of functional α6ß4* nAChRs.