Characterization of the Structural Requirements for the NADase Activity of Bacterial Toll/IL-1R domains in a Course-based Undergraduate Research Experience.

TLRs initiate innate immune signaling pathways via Toll/IL-1R (TIR) domains on their cytoplasmic tails. Various bacterial species also express TIR domain-containing proteins that contribute to bacterial evasion of the innate immune system. Bacterial TIR domains, along with the mammalian sterile α and TIR motif-containing protein 1 and TIRs from plants, also have been found to exhibit NADase activity. Initial X-ray crystallographic studies of the bacterial TIR from Acinetobacter baumannii provided insight into bacterial TIR structure but were unsuccessful in cocrystallization with the NAD+ ligand, leading to further questions about the TIR NAD binding site. In this study, we designed a Course-Based Undergraduate Research Experience (CURE) involving 16-20 students per year to identify amino acids crucial for NADase activity of A. baumannii TIR domain protein and the TIR from Escherichia coli (TIR domain-containing protein C). Students used structural data to identify amino acids that they hypothesized would play a role in TIR NADase activity, and created plasmids to express mutated TIRs through site-directed mutagenesis. Mutant TIRs were expressed, purified, and tested for NADase activity. The results from these studies provide evidence for a conformational change upon NAD binding, as was predicted by recent cryogenic electron microscopy and hydrogen-deuterium exchange mass spectrometry studies. Along with corroborating recent characterization of TIR NADases that could contribute to drug development for diseases associated with dysregulated TIR activity, this work also highlights the value of CURE-based projects for inclusion of a diverse group of students in authentic research experiences.

The structure of NAD+ consuming protein Acinetobacter baumannii TIR domain shows unique kinetics and conformations.

Toll-like and interleukin-1/18 receptor/resistance (TIR) domain-containing proteins function as important signaling and immune regulatory molecules. TIR domain-containing proteins identified in eukaryotic and prokaryotic species also exhibit NAD+ hydrolase activity in select bacteria, plants, and mammalian cells. We report the crystal structure of the Acinetobacter baumannii TIR domain protein (AbTir-TIR) with confirmed NAD+ hydrolysis and map the conformational effects of its interaction with NAD+ using hydrogen-deuterium exchange-mass spectrometry. NAD+ results in mild decreases in deuterium uptake at the dimeric interface. In addition, AbTir-TIR exhibits EX1 kinetics indicative of large cooperative conformational changes, which are slowed down upon substrate binding. Additionally, we have developed label-free imaging using the minimally invasive spectroscopic method 2-photon excitation with fluorescence lifetime imaging, which shows differences in bacteria expressing native and mutant NAD+ hydrolase-inactivated AbTir-TIRE208A protein. Our observations are consistent with substrate-induced conformational changes reported in other TIR model systems with NAD+ hydrolase activity. These studies provide further insight into bacterial TIR protein mechanisms and their varying roles in biology.

Cobbling Together the Myddosome.

An organism's ability to recognize and respond quickly and appropriately to pathogenic stimuli is a fundamental aspect of innate immunity. Harnessing the dynamic nature of fluorescent microscopy and the resolution of cryo-electron microscopy, Moncrieffe et al. (2020) characterize MyD88-only filaments and provide insight into the mechanisms underlying innate immune signaling.

Prevalence of metabolic syndrome among Hispanics/Latinos of diverse background: the Hispanic Community Health Study/Study of Latinos.

OBJECTIVE: Approximately one-third of the adult U.S. population has the metabolic syndrome. Its prevalence is the highest among Hispanic adults, but variation by Hispanic/Latino background is unknown. Our objective was to quantify the prevalence of the metabolic syndrome among men and women 18-74 years of age of diverse Hispanic/Latino background. RESEARCH DESIGN AND METHODS: Two-stage area probability sample of households in four U.S. locales, yielding 16,319 adults (52% women) who self-identified as Cuban, Dominican, Mexican, Puerto Rican, Central American, or South American. The metabolic syndrome was defined according to the American Heart Association/National Heart, Lung, and Blood Institute 2009 Joint Scientific Statement. The main outcome measures were age-standardized prevalence of the metabolic syndrome per the harmonized American Heart Association/National Heart, Lung, and Blood Institute definition and its component abnormalities. RESULTS: The metabolic syndrome was present in 36% of women and 34% of men. Differences in the age-standardized prevalence were seen by age, sex, and Hispanic/Latino background. The prevalence of the metabolic syndrome among those 18-44, 45-64, and 65-74 years of age was 23%, 50%, and 62%, respectively, among women; and 25%, 43%, and 55%, respectively, among men. Among women, the metabolic syndrome prevalence ranged from 27% in South Americans to 41% in Puerto Ricans. Among men, prevalences ranged from 27% in South Americans to 35% in Cubans. In those with the metabolic syndrome, abdominal obesity was present in 96% of the women compared with 73% of the men; more men (73%) than women (62%) had hyperglycemia. CONCLUSIONS: The burden of cardiometabolic abnormalities is high in Hispanic/Latinos but varies by age, sex, and Hispanic/Latino background. Hispanics/Latinos are thus at increased, but modifiable, predicted lifetime risk of diabetes and its cardiovascular sequelae.

Short-term repeatability of electrocardiographic Tpeak-Tend and QT intervals.

BACKGROUND: The electrocardiographic (ECG) Tpeak-Tend interval (TpTe) is associated with arrhythmias and sudden cardiac death. TpTe offers a supplementary measure for the QT interval (QT), yet its repeatability has not been established. PURPOSE: Evaluate short-term repeatability of TpTe and QT. METHODS: Four ECGs were obtained on sixty participants. The sources of variation, intra-class correlation coefficient (ICC) - an index of reproducibility - and minimal detectable change (MDC) were estimated for TpTe and QT. The impact of repeated measurements on repeatability was estimated for a hypothetical clinical trial designed to detect drug-induced prolongation of TpTe and QT. RESULTS: We used heart rate-adjusted QT [(QT)a] but TpTe in the study group was rate-invariant. The ICC [95% confidence interval (CI)] was 0.77 (0.69, 0.85) for TpTe, 0.75 (0.65, 0.85) for QT and 0.60 (0.47, 0.73) for (QT)a. The MDC (ms) was 21, 32 and 26 for TpTe, QT and (QT)a respectively. CONCLUSION: TpTe has excellent repeatability supporting its use as a supplement to QT in observational and clinical studies.

Redistribution of heart failure as the cause of death: the Atherosclerosis Risk in Communities Study.

BACKGROUND: Heart failure is sometimes incorrectly listed as the underlying cause of death (UCD) on death certificates, thus compromising the accuracy and comparability of mortality statistics. Statistical redistribution of the UCD has been used to examine the effect of misclassification of the UCD attributed to heart failure, but sex- and race-specific redistribution of deaths on coronary heart disease (CHD) mortality in the United States has not been examined. METHODS: We used coarsened exact matching to infer the UCD of vital records with heart failure as the UCD from 1999 to 2010 for decedents 55 years old and older from states encompassing regions under surveillance by the Atherosclerosis Risk in Communities (ARIC) Study (Maryland, Minnesota, Mississippi, and North Carolina). Records with heart failure as the UCD were matched on decedent characteristics (five-year age groups, sex, race, education, year of death, and state) to records with heart failure listed among the multiple causes of death. Each heart failure death was then redistributed to plausible UCDs proportional to the frequency among matched records. RESULTS: After redistribution the proportion of deaths increased for CHD, chronic obstructive pulmonary disease, diabetes, hypertensive heart disease, and cardiomyopathy, P < 0.001. The percent increase in CHD mortality after redistribution was the highest in Mississippi (12%) and lowest in Maryland (1.6%), with variations by year, race, and sex. Redistribution proportions for CHD were similar to CHD death classification by a panel of expert reviewers in the ARIC study. CONCLUSIONS: Redistribution of ill-defined UCD would improve the accuracy and comparability of mortality statistics used to allocate public health resources and monitor mortality trends.

Complement protein C3 and coronary artery calcium in middle-aged women with polycystic ovary syndrome and controls.

Circulating complement protein C3 (C3) levels have been associated with coronary artery calcification (CAC) in women with systemic lupus erythematosus, but have yet to be evaluated in women with polycystic ovary syndrome (PCOS). We aimed to determine whether C3 levels were elevated in women with PCOS compared to controls and to quantify the association of C3 with cardiovascular disease (CVD) risk factors and CAC and if PCOS modified this association. This cross-sectional analysis included 132 women with PCOS and 155 controls, 35-62 years old, from the third visit of a case-control study. CAC was measured during the study visit, and circulating C3 was measured in stored sera. The presence of CAC and CAC categories (Agatston score 0, 1-9.9 and ≥ 10) were used for logistic and ordinal regression analysis, respectively. C3 levels were not significantly different between women with PCOS and controls. Among all women, C3 was associated with the presence of CAC and increasing CAC groups after adjusting for age, PCOS status and insulin or body mass index (BMI), all p<0.05. In addition, C3 was associated with the presence of CAC after adjusting for age, PCOS status, BMI, insulin and African American race, p=0.049. PCOS status did not modify these associations. In conclusion, circulating C3 levels may prove beneficial in identifying women at risk of CVD in women with PCOS and the general population.

Short-term repeatability of electrocardiographic P wave indices and PR interval.

BACKGROUND: P wave indices and PR interval from 12-lead electrocardiograms (ECGs) are predictors of cardiovascular morbidity and mortality, but their repeatability has not been examined. OBJECTIVES: Determine the short-term repeatability of P wave indices (P axis, maximum P area and duration, P dispersion and P terminal force in V1) and PR interval. METHODS: Participants (n=63) underwent two standard ECGs at each of two visits, two weeks apart. We calculated the intra-class correlation coefficient (ICC), weighted kappa, and minimal detectable change and difference. RESULTS: ICCs were 0.93 for PR interval, 0.78 for P axis, 0.77 for maximum P area, and 0.58 for maximum P duration. Within- and between-visit Kappa were 0.30 and 0.11 for P dispersion, and 0.68 and 0.46 for P terminal force. CONCLUSION: Repeatability of PR duration was excellent, that of P wave axis and maximum area was fair, and maximum P wave duration and terminal force was poor. Repeatability of P wave dispersion was fair within visit, yet poor between visits. These results illustrate potential biases when measurement error of some P wave indices is ignored in clinical and epidemiologic studies.

Bacterial discrimination: Dictyostelium's discerning taste.

New research indicates that the social amoeba Dictyostelium discoideum recognizes distinctions between Gram(-) and Gram(+) bacterial prey and responds discriminately to these two groups of bacteria. These findings may lend insight to the origins of microbial pattern recognition in innate immunity.

Lipopolysaccharide induction of autophagy is associated with enhanced bactericidal activity in Dictyostelium discoideum.

Innate immune cells respond to microbial invaders using pattern recognition receptors that detect conserved microbial patterns. Among the cellular processes stimulated downstream of pattern recognition machinery is the initiation of autophagy, which plays protective roles against intracellular microbes. We have shown recently that Dictyostelium discoideum, which takes up bacteria for nutritive purposes, may employ pattern recognition machinery to respond to bacterial prey, as D. discoideum cells upregulate bactericidal activity upon stimulation by lipopolysaccharide (LPS). Here we extend these findings, showing that LPS treatment leads to induction of autophagosomal maturation in cells responding to the bacteria Staphylococcus aureus. Cells treated with the autophagy-inducing drug rapamycin clear internalized bacteria at an accelerated rate, while LPS-enhanced clearance of bacteria is reduced in cells deficient for the autophagy-related genes atg1 and atg9. These findings link microbial pattern recognition with autophagy in the social amoeba D. discoideum.

Lipopolysaccharide enhances bactericidal activity in Dictyostelium discoideum cells.

Innate immune cells respond to invading microbes upon detection of pathogen-associated molecular patterns (PAMPS). PAMP-recognition machinery is evolutionarily conserved, allowing for characterization in model organisms. The model organism Dictyostelium discoideum can exist as single-celled amoebae, which phagocytize bacteria for nutrients. Although D. discoideum is used extensively to study phagocytosis, it has not been determined if D. discoideum detects bacterial PAMPs using pattern-recognition machinery. Here we show that D. discoideum mounts responses against the bacterial cell wall PAMP, lipopolysaccharide (LPS). Upon treatment with LPS or its active component Lipid A, D. discoideum cells more efficiently clear phagocytized bacteria. LPS-enhanced bactericidal activity appears dependent both on MAPK signaling pathways as well as on the D. discoideum toll/interleukin-1 receptor domain-containing protein, TirA. These findings indicate that pattern-recognition machinery required to detect and respond to bacterial PAMPs may be conserved in D. discoideum.