AIMTo elucidate the genetic basis for the pronounced resistance that the oral pathogen, Porphyromonas gingivalis (P. gingivalis), exhibits towards the cationic antimicrobial peptide, polymyxin B.

METHODOLOGYA genetic screen of P. gingivalis clones generated by a Tn4400'-based random insertion mutagenesis strategy was performed to identify bacteria harboring novel genetic mutations that render P. gingivalis susceptible to killing by the cationic antimicrobial peptide, polymyxin B (PMB, 50 microg x mL(-1)).

RESULTSP. gingivalis (ATCC 33277) is unusually resistant to the cationic antimicrobial peptide, PMB at relatively high concentrations (200 microg x mL(-1)). Approximately 2,700 independent Tn4400'-derived mutants of P. gingivalis were examined for increased sensitivity to PMB killing at a relatively low dose (50 microg x mL(-1)). A single PMB-sensitive mutant was obtained in this phenotypic screen. We determined that the Tn4400' transposon was integrated into the gene encoding the lipid A 4'-phosphatase, PGN_0524, demonstrating that this insertion event was responsible for its increased susceptibility of this clone to PMB-dependent killing. The resulting mutant strain, designated 0524-Tn4400', was highly sensitive to PMB killing relative to wild-type P. gingivalis, and exhibited the same sensitivity as the previously characterized strain, 0524KO, which bears a genetically engineered deletion in the PGN_0524 locus. Positive ion mass spectrometric structural (MALDI-TOF MS) analyses revealed that lipid A isolates from 0524-Tn4400' and 0524KO strains displayed strikingly similar MALDI-TOF MS spectra that were substantially different from the wildtype P. gingivalis lipid A spectrum. Finally, intact 0524-Tn4400' and 0524KO mutant bacteria, as well as their corresponding LPS isolates, were significantly more potent in stimulating Toll-like receptor 4 (TLR4)-dependent E-selectin expression in human endothelial cells relative to intact wild-type P. gingivalis or its corresponding LPS isolate.

CONCLUSIONThe combined molecular evidence provided in this report suggests that PGN_0524, a lipid A 4'-phosphatase, is the sole genetic element conferring the ability of the periodontopathogen, P. gingivalis, to evade the killing activity of cationic antimicrobial peptides, such as PMB. These data strongly implicate PGN_0524 as a critical virulence factor for the ability of P. gingivalis to evade front-line host innate defenses that are dependent upon cationic antimicrobial peptide activity and TLR 4 sensing.

Anti-Bacterial Agents ; pharmacology ; Chromosome Mapping ; DNA Transposable Elements ; genetics ; Drug Resistance, Bacterial ; genetics ; E-Selectin ; analysis ; immunology ; Endothelial Cells ; immunology ; microbiology ; Gene Deletion ; Humans ; Lipid A ; analysis ; immunology ; Lipopolysaccharides ; analysis ; immunology ; Mutagenesis, Insertional ; genetics ; Open Reading Frames ; genetics ; Phosphoric Monoester Hydrolases ; genetics ; physiology ; Polymyxin B ; pharmacology ; Porphyromonas gingivalis ; enzymology ; genetics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Toll-Like Receptor 4 ; analysis ; immunology ; Virulence Factors ; physiology

Objectives: Calcaneal quantitative ultrasound measurement (QUS) has been considered an alternative to dual-energy X-ray absorptiometry (DXA) based bone mineral density (BMD) for assessing bone health. This study sought to examine the utility of QUS as an osteoporosis screening tool by evaluating the correlation between QUS and DXA. Methods: The study was a part of the Vietnam Osteoporosis Study that involved 1270 women and 773 men aged 18 years and older. BMD at the femoral neck, total hip and lumbar spine was measured using DXA. Osteoporosis was diagnosed based on the femoral neck T-score using World Health Organization criteria. Broadband ultrasound attenuation (BUA) at the calcaneus was measured by QUS. The concordance between BUA and BMD was analyzed by the linear regression model. Results: In all individuals, BUA modestly correlated with femoral neck BMD (r = 0.35; P < 0.0001) and lumbar spine BMD (r = 0.34; P < 0.0001) in both men and women. In individuals aged 50 years and older, approximately 16% (n = 92/575) of women and 3.2% (n = 10/314) of men were diagnosed to have osteoporosis. Only 0.9% (n = 5/575) women and 1.0% (n = 3/314) men were classified as “Low BUA”. The kappa coefficient of concordance between BMD and BUA classification was 0.09 (95% CI, 0.04 to 0.15) for women and 0.12 (95% CI, 0.03 to 0.22) for men. Conclusions In this population-based study, QUS BUA modestly correlated with DXA BMD, suggesting that BUA is not a reliable method for screening of osteoporosis.

Objectives: Calcaneal quantitative ultrasound measurement (QUS) has been considered an alternative to dual-energy X-ray absorptiometry (DXA) based bone mineral density (BMD) for assessing bone health. This study sought to examine the utility of QUS as an osteoporosis screening tool by evaluating the correlation between QUS and DXA. Methods: The study was a part of the Vietnam Osteoporosis Study that involved 1270 women and 773 men aged 18 years and older. BMD at the femoral neck, total hip and lumbar spine was measured using DXA. Osteoporosis was diagnosed based on the femoral neck T-score using World Health Organization criteria. Broadband ultrasound attenuation (BUA) at the calcaneus was measured by QUS. The concordance between BUA and BMD was analyzed by the linear regression model. Results: In all individuals, BUA modestly correlated with femoral neck BMD (r = 0.35; P < 0.0001) and lumbar spine BMD (r = 0.34; P < 0.0001) in both men and women. In individuals aged 50 years and older, approximately 16% (n = 92/575) of women and 3.2% (n = 10/314) of men were diagnosed to have osteoporosis. Only 0.9% (n = 5/575) women and 1.0% (n = 3/314) men were classified as “Low BUA”. The kappa coefficient of concordance between BMD and BUA classification was 0.09 (95% CI, 0.04 to 0.15) for women and 0.12 (95% CI, 0.03 to 0.22) for men. Conclusions In this population-based study, QUS BUA modestly correlated with DXA BMD, suggesting that BUA is not a reliable method for screening of osteoporosis.

BACKGROUND: Sedentary behaviors have recently become an important public health issue. We aimed to investigate the relationship between screen time and nutrient intake in children and adolescents. METHODS: The present study was conducted in 2013. Data were collected from children and adolescents aged between 6 and 15 years old in Shika town. Questionnaires were distributed to 1459 subjects, 1414 of whom participated in the study (96.9%). Sedentary behaviors were assessed based on participants' screen behaviors (television (TV) viewing, personal computer (PC) use, and mobile phone (MP) use). The main outcomes were the intake of nutrients from a validated food frequency questionnaire. Analysis of covariance (ANCOVA) was used to examine the significance of differences in nutrient intake estimates. Multivariate linear regression analyses, adjusting for age, BMI, and physical activity, were used to provide parameter estimates (β) and 95% CI for the relationship between screen time and nutrient intake. RESULTS: In boys, longer TV viewing times correlated or tended to correlate with a lower intake of protein, potassium, calcium, iron, vitamin K, vitamin B-2, and total dietary fiber. In girls, longer TV viewing times correlated with a lower intake of protein, sodium, calcium, vitamin D, and vitamin B-2. Longer TV viewing times correlated with a higher intake of n-6 fatty acids in girls. PC use was related or tended to be related to a lower intake of potassium, iron, vitamin K, and folic acid in boys, but not in girls. A relationship was observed between MP use and a lower intake of vitamin K in boys, and MP use and a higher intake of vitamin D in girls. CONCLUSIONS The present results revealed that longer TV viewing times are associated with less protein, minerals, vitamins, and total dietary fiber intake in children and adolescents. It was also revealed that boys with PC use have less minerals and vitamins. These results support the need to design intervention programs that focus on decreasing TV viewing time in both sexes and PC use in boys while encouraging adherence to dietary guidelines among children and adolescents.
Adolescent ; Body Mass Index ; Child ; Cross-Sectional Studies ; Diet ; Energy Intake ; Exercise ; Female ; Humans ; Japan ; Male ; Screen Time ; Sedentary Behavior ; Sex Factors ; Socioeconomic Factors