Association of Immune Cell Subsets with Incident Hip Fracture: The Cardiovascular Health Study.

In this study, we aimed to evaluate the association of innate and adaptive immune cell subsets in peripheral blood mononuclear cells (PBMCs) with hip fracture. To conduct this study, we used data from the Cardiovascular Health Study (CHS), a U.S. multicenter observational cohort of community-dwelling men and women aged ≥ 65 years. Twenty-five immune cell phenotypes were measured by flow cytometry from cryopreserved PBMCs of CHS participants collected in 1998-1999. The natural killer (NK), γδ T, T helper 17 (Th17), and differentiated/senescent CD4+CD28- T cell subsets were pre-specified as primary subsets of interest. Hip fracture incidence was assessed prospectively by review of hospitalization records. Multivariable Cox hazard models evaluated associations of immune cell phenotypes with incident hip fracture in sex-stratified and combined analyses. Among 1928 persons, 259 hip fractures occurred over a median 9.7 years of follow-up. In women, NK cells were inversely associated with hip fracture [hazard ratio (HR) 0.77, 95% confidence interval (CI) 0.60-0.99 per one standard deviation higher value] and Th17 cells were positively associated with hip fracture [HR 1.18, 95% CI 1.01-1.39]. In men, γδ T cells were inversely associated with hip fracture [HR 0.60, 95% CI 0.37-0.98]. None of the measured immune cell phenotypes were significantly associated with hip fracture incidence in combined analyses. In this large prospective cohort of older adults, potentially important sex differences in the associations of immune cell phenotypes and hip fracture were identified. However, immune cell phenotypes had no association with hip fracture in analyses combining men and women.

Dietary inflammatory index and cardiovascular disease risk in Hispanic women from the Women's Health Initiative.

BACKGROUND: To evaluate the association between the dietary inflammatory index (DII®) and incident cardiovascular disease (CVD) in Hispanic women from the Women's Health Initiative (WHI), and to determine if body mass index (BMI) interacted with the DII scores. METHODS: Secondary analysis of baseline dietary data and long-term CVD outcomes among 3,469 postmenopausal women who self-identified as Hispanic enrolled in WHI. DII scores were calculated from self-administered food frequency questionnaires. The CVD outcomes included coronary heart disease (CHD) and stroke. Stratified Cox regression models were used to assess the relationship between DII scores and CVD in women with and without obesity. Models were adjusted for age, lifestyle risk factors, known risk factors, and neighborhood socioeconomic status. RESULTS: The incidence of CHD was 3.4 and 2.8% for stroke after a median follow-up of 12.9 years. None of the DIIs were associated with CVD risk in this sample of Hispanic women. BMI interacted with the DII (p < 0.20) and stratified models showed that the associations between the DII and CVD were only significant in women with overweight (p < 0.05). In this group, higher DII scores were associated with a higher risk of CHD (HR 1.27; 95% CI: 1.08, 1.51) and a higher risk of stroke (HR 1.32; 95% CI: 1.07, 1.64). CONCLUSION: Among postmenopausal Hispanic women with overweight, greater adherence to pro-inflammatory diets was associated with higher risk of CVD. Additional research is needed to understand how to promote long-term heart-healthy dietary habits to reduce inflammation and prevent CVD in at-risk Hispanic women.

Age-Related Factors Associated With The Risk of Hip Fracture.

OBJECTIVE: Advancing age is a powerful risk factor for hip fractures. The biological mechanisms through which aging impacts the risk of hip fractures have not been well studied. METHODS: Biological factors associated with "advancing age" that help to explain how aging is associated with the risk of hip fractures are reviewed. The findings are based on analyses of the Cardiovascular Health Study, an ongoing observational study of adults aged ≥65 years with 25 years of follow-up. RESULTS: The following 5 age-related factors were found to be significantly associated with the risk of hip fractures: (1) microvascular disease of the kidneys (albuminuria and/or elevated urine-albumin-to-creatinine ratio) and brain (abnormal white matter disease on brain magnetic resonance imaging); (2) increased serum levels of carboxymethyl-lysine, an advanced glycation end product that reflects glycation and oxidative stress; (3) reduced parasympathetic tone, as derived from 24-hour Holter monitoring; (4) carotid artery atherosclerosis in the absence of clinical cardiovascular disease; and (5) increased transfatty acid levels in the blood. Each of these factors was associated with a 10% to 25% increased risk of fractures. These associations were independent of traditional risk factors for hip fractures. CONCLUSION: Several factors associated with older age help to explain how "aging" may be associated with the risk of hip fractures. These same factors may also explain the high risk of mortality following hip fractures.

Life-Course Genome-wide Association Study Meta-analysis of Total Body BMD and Assessment of Age-Specific Effects.

Bone mineral density (BMD) assessed by DXA is used to evaluate bone health. In children, total body (TB) measurements are commonly used; in older individuals, BMD at the lumbar spine (LS) and femoral neck (FN) is used to diagnose osteoporosis. To date, genetic variants in more than 60 loci have been identified as associated with BMD. To investigate the genetic determinants of TB-BMD variation along the life course and test for age-specific effects, we performed a meta-analysis of 30 genome-wide association studies (GWASs) of TB-BMD including 66,628 individuals overall and divided across five age strata, each spanning 15 years. We identified variants associated with TB-BMD at 80 loci, of which 36 have not been previously identified; overall, they explain approximately 10% of the TB-BMD variance when combining all age groups and influence the risk of fracture. Pathway and enrichment analysis of the association signals showed clustering within gene sets implicated in the regulation of cell growth and SMAD proteins, overexpressed in the musculoskeletal system, and enriched in enhancer and promoter regions. These findings reveal TB-BMD as a relevant trait for genetic studies of osteoporosis, enabling the identification of variants and pathways influencing different bone compartments. Only variants in ESR1 and close proximity to RANKL showed a clear effect dependency on age. This most likely indicates that the majority of genetic variants identified influence BMD early in life and that their effect can be captured throughout the life course.

Insights into Thiotemplated Pyrrole Biosynthesis Gained from the Crystal Structure of Flavin-Dependent Oxidase in Complex with Carrier Protein.

Sequential enzymatic reactions on substrates tethered to carrier proteins (CPs) generate thiotemplated building blocks that are then delivered to nonribosomal peptide synthetases (NRPSs) to generate peptidic natural products. The underlying diversity of these thiotemplated building blocks is the principal driver of the chemical diversity of NRPS-derived natural products. Structural insights into recognition of CPs by tailoring enzymes that generate these building blocks are sparse. Here we present the crystal structure of a flavin-dependent prolyl oxidase that furnishes thiotemplated pyrrole as the product, in complex with its cognate CP in the holo and product-bound states. The thiotemplated pyrrole is an intermediate that is well-represented in natural product biosynthetic pathways. Our results delineate the interactions between the CP and the oxidase while also providing insights into the stereospecificity of the enzymatic oxidation of the prolyl heterocycle to the aromatic pyrrole. Biochemical validation of the interaction between the CP and the oxidase demonstrates that NRPSs recognize and bind to their CPs using interactions quite different from those of fatty acid and polyketide biosynthetic enzymes. Our results posit that structural diversity in natural product biosynthesis can be, and is, derived from subtle modifications of primary metabolic enzymes.

The Association of Aromatic Amino Acids with Incident Hip Fracture, aBMD, and Body Composition from the Cardiovascular Health Study.

In 5187 persons from the Cardiovascular Health Study, there was no significant association of dietary intakes of aromatic amino acids (AAA) with areal BMD of the hip or body composition. However, those who had the lowest dietary intakes of AAA were at increased risk for incident hip fractures. Prior studies of the association of protein intake with osteoporosis are conflicting and have not directly examined the relationship of aromatic amino acids (AAA) with fractures, areal bone mineral density (aBMD), and body composition. We sought to determine the relationship of dietary intakes of AAA with osteoporosis parameters in elderly men and women. 5187 men and women aged ≥ 65 years from the Cardiovascular Health Study (CHS) with dietary intakes of AAA (tryptophan, phenylalanine, tyrosine) estimated by food frequency questionnaire (FFQ) were included. We examined the relationship between a one-time estimate of daily dietary AAA intake with risk of incident hip fractures over a median of 13.2 years of fracture follow-up. A subset (n = 1336) who had dual energy X-ray absorptiometry (DXA) performed were included in a cross-sectional analysis of the association of dietary AAA intake with aBMD of the total hip and measurements of body composition. In multivariable models adjusted for demographic and clinical variables, medication use, and diet, higher dietary AAA intake was not significantly associated with incident hip fractures. All hazard ratios (HR) were less than one (tryptophan, HR 0.14, 95% CI 0.01 to 1.89; phenylalanine, HR 0.60, 95% CI 0.23 to 1.55; tyrosine, HR 0.59, 95% CI 0.27 to 1.32), but confidence intervals were wide and included no difference. However, in post hoc analyses, the lowest quartile of intake for each AAA was associated with an increased risk for hip fracture compared to higher quartiles (p ≤ 0.047 for all). Dietary AAA intakes were not significantly associated with total hip aBMD or any measurements of body composition. Overall, there was no significant association of dietary AAA intake with hip fractures, aBMD of the hip, or body composition. However, there may be a subset of elderly individuals with low dietary intakes of AAA who are at increased for hip fractures.

Formate Oxidase (FOx) from Aspergillus oryzae: One Catalyst Enables Diverse H2 O2 -Dependent Biocatalytic Oxidation Reactions.

An increasing number of biocatalytic oxidation reactions rely on H2 O2 as a clean oxidant. The poor robustness of most enzymes towards H2 O2 , however, necessitates more efficient systems for in situ H2 O2 generation. In analogy to the well-known formate dehydrogenase to promote NADH-dependent reactions, we here propose employing formate oxidase (FOx) to promote H2 O2 -dependent enzymatic oxidation reactions. Even under non-optimised conditions, high turnover numbers for coupled FOx/peroxygenase catalysis were achieved.

Photoirradiation Generates an Ultrastable 8-Formyl FAD Semiquinone Radical with Unusual Properties in Formate Oxidase.

Formate oxidase (FOX) was previously shown to contain a noncovalently bound 8-formyl FAD (8-fFAD) cofactor. However, both the absorption spectra and the kinetic parameters previously reported for FOX are inconsistent with more recent reports. The ultraviolet-visible (UV-vis) absorption spectrum reported in early studies closely resembles the spectra observed for protein-bound 8-formyl flavin semiquinone species, thus suggesting FOX may be photosensitive. Therefore, the properties of dark and light-exposed FOX were investigated using steady-state kinetics and site-directed mutagenesis analysis along with inductively coupled plasma optical emission spectroscopy, UV-vis absorption spectroscopy, circular dichroism spectroscopy, liquid chromatography and mass spectrometry, and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, these experimental results demonstrate that FOX is deactivated in the presence of light through generation of an oxygen stable, anionic (red) 8-fFAD semiquinone radical capable of persisting either in an aerobic environment for multiple weeks or in the presence of a strong reducing agent like sodium dithionite. Herein, we study the photoinduced formation of the 8-fFAD semiquinone radical in FOX and report the first EPR spectrum of this radical species. The stability of the 8-fFAD semiquinone radical suggests FOX to be a model enzyme for probing the structural and mechanistic features involved in stabilizing flavin semiquinone radicals. It is likely that the photoinduced formation of a stable 8-fFAD semiquinone radical is a defining characteristic of 8-formyl flavin-dependent enzymes. Additionally, a better understanding of the radical stabilization process may yield a FOX enzyme with more robust activity and broader industrial usefulness.

Targeted sequencing of genome wide significant loci associated with bone mineral density (BMD) reveals significant novel and rare variants: the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) targeted sequencing study.

Background: Bone mineral density (BMD) is a heritable phenotype that predicts fracture risk. We performed fine-mapping by targeted sequencing at WLS, MEF2C, ARHGAP1/F2 and JAG1 loci prioritized by eQTL and bioinformatic approaches among 56 BMD loci from our previous GWAS meta-analysis. Targeted sequencing was conducted in 1,291 Caucasians from the Framingham Heart Study ( n = 925) and Cardiovascular Health Study ( n = 366), including 206 women and men with extreme low femoral neck (FN) BMD. A total of 4,964 sequence variants (SNVs) were observed and 80% were rare with MAF <1%. The associations between previously identified SNPs in these loci and BMD, while nominally significant in sequenced participants, were no longer significant after multiple testing corrections. Conditional analyses did not find protein-coding variants that may be responsible for GWAS signals. On the other hand, in the sequenced subjects, we identified novel associations in WLS , ARHGAP1 , and 5' of MEF2C ( P- values < 8x10 - 5 ; false discovery rate (FDR) q-values < 0.01) that were much more strongly associated with BMD compared to the GWAS SNPs. These associated SNVs are less-common; independent from previous GWAS signals in the same loci; and located in gene regulatory elements. Our findings suggest that protein-coding variants in selected GWAS loci did not contribute to GWAS signals. By performing targeted sequencing in GWAS loci, we identified less-common and rare non-coding SNVs associated with BMD independently from GWAS common SNPs, suggesting both common and less-common variants may associate with disease risks and phenotypes in the same loci.

Mechanistic studies of formate oxidase from Aspergillus oryzae: A novel member of the glucose-Methanol-choline oxidoreductase enzyme superfamily that oxidizes carbon acids.

Formate oxidase (FOX) from Aspergillus oryzae is the only GMC member that oxidizes a carbon acid rather than alcohols; thus, its catalytic mechanism may be different from that of other GMC members. We have used pH, solvent viscosity, and deuterium kinetic isotope effects, to investigate the catalytic mechanism of FOX. The enzyme followed a Bi-Bi sequential steady-state kinetic mechanism. The kcat value was pH-independent between pH 2.8 and 6.8, suggesting a lack of ionizable groups in kinetic step(s) that limit the overall turnover of the enzyme. The kcat/Kformate value decreased from a value of 10,000 M-1s-1 at low pH with a pKa value of 4.4, consistent with the requirement of a protonated group for substrate binding. An inverse viscosity dependence on the kcat/Kformate value indicated an isomerization of the Michaelis complex. The kcat/Koxygen value was 340,000 M-1s-1 and pH independent up to pH 6.0. The Dkcat and D(kcat/Kformate) values were 2.5 and 1.9, respectively, indicating that substrate CH bond cleavage is rate-limiting for FOX catalysis. Analytical ultracentrifugation indicated a concentration dependence of the oligomeric state of FOX. The appkred,H value was ∼75% that of kcat,H, indicating that the anaerobic reduction of FOX was dependent on the oligomeric state of FOX.

A high-throughput pH-based colorimetric assay: application focus on alpha/beta hydrolases.

Research involving α/ß hydrolases, including α-amino acid ester hydrolase and cocaine esterase, has been limited by the lack of an online high throughput screening assay. The development of a high throughput screening assay capable of detecting α/ß hydrolase activity toward specific substrates and/or chemical reactions (e.g., hydrolysis in lieu of amidase activity and/or synthesis instead of thioesterase activity) is of interest in a broad set of scientific questions and applications. Here we present a general framework for pH-based colorimetric assays, as well as the mathematical considerations necessary to estimate de novo the experimental response required to assign a 'hit' or a 'miss,' in the absence of experimental standard curves. This combination is valuable for screening the hydrolysis and synthesis activity of α/ß hydrolases on a variety of substrates, and produces data comparable to the current standard technique involving High Performance Liquid Chromatography (HPLC). In contrast to HPLC, this assay enables screening experiments to be performed with greater efficiency.

Strategies for imputing missing covariates in accelerated failure time models.

Missing covariates often occur in biomedical studies with survival outcomes. Multiple imputation via chained equations (MICE) is a semi-parametric and flexible approach that imputes multivariate data by a series of conditional models, one for each incomplete variable. When applying MICE, practitioners tend to specify the conditional models in a simple fashion largely dictated by the software, which could lead to suboptimal results. Practical guidelines for specifying appropriate conditional models in MICE are lacking. Motivated by a study of time to hip fractures in the Women's Health Initiative Observational Study using accelerated failure time models, we propose and experiment with some rationales leading to appropriate MICE specifications. This strategy starts with specifying a joint model for the variables involved. We first derive the conditional distribution of each variable under the joint model, then approximate these conditional distributions to the extent which can be characterized by commonly used regression models. We propose to fit separate models to impute incomplete variables by the failure status, which is key to generating appropriate MICE specifications for survival outcomes. The proposed strategy can be conveniently implemented with all available imputation software that uses fully conditional specifications. Our simulation results show that some commonly used simple MICE specifications can produce suboptimal results, while those based on the proposed strategy appear to perform well and be robust toward model misspecifications. Hence, we warn against a mechanical use of MICE and suggest careful modeling of the conditional distributions of variables to ensure proper performance.

Enzyme-Mediated Conversion of Flavin Adenine Dinucleotide (FAD) to 8-Formyl FAD in Formate Oxidase Results in a Modified Cofactor with Enhanced Catalytic Properties.

Flavins, including flavin adenine dinucleotide (FAD), are fundamental catalytic cofactors that are responsible for the redox functionality of a diverse set of proteins. Alternatively, modified flavin analogues are rarely found in nature as their incorporation typically results in inactivation of flavoproteins, thus leading to the disruption of important cellular pathways. Here, we report that the fungal flavoenzyme formate oxidase (FOX) catalyzes the slow conversion of noncovalently bound FAD to 8-formyl FAD and that this conversion results in a nearly 10-fold increase in formate oxidase activity. Although the presence of an enzyme-bound 8-formyl FMN has been reported previously as a result of site-directed mutagenesis studies of lactate oxidase, FOX is the first reported case of 8-formyl FAD in a wild-type enzyme. Therefore, the formation of the 8-formyl FAD cofactor in formate oxidase was investigated using steady-state kinetics, site-directed mutagenesis, ultraviolet-visible, circular dichroism, and fluorescence spectroscopy, liquid chromatography with mass spectrometry, and computational analysis. Surprisingly, the results from these studies indicate not only that 8-formyl FAD forms spontaneously and results in the active form of FOX but also that its autocatalytic formation is dependent on a nearby arginine residue, R87. Thus, this work describes a new enzyme cofactor and provides insight into the little-understood mechanism of enzyme-mediated 8α-flavin modifications.

Incidence and Predictors of Repeat Bone Mineral Densitometry: A Longitudinal Cohort Study.

BACKGROUND: Existing guidelines for repeat screening and treatment monitoring intervals regarding the use of dual-energy x-ray absorptiometry (DXA) scans are conflicting or lacking. The Choosing Wisely campaign recommends against repeating DXA scans within 2 years of initial screening. It is unclear how frequently physicians order repeat scans and what clinical factors contribute to their use. OBJECTIVE: To estimate cumulative incidence and predictors of repeat DXA for screening or treatment monitoring in a regional health system. DESIGN: Retrospective longitudinal cohort study PARTICIPANTS: A total of 5992 women aged 40-84 years who received initial DXA screening from 2006 to 2011 within a regional health system in Sacramento, CA. MAIN MEASURES: Two- and five-year cumulative incidence and hazard rations (HR) of repeat DXA by initial screening result (classified into three groups: low or high risk of progression to osteoporosis, or osteoporosis) and whether women were prescribed osteoporosis drugs after initial DXA. KEY RESULTS: Among women not treated after initial DXA, 2-year cumulative incidence for low-risk, high-risk, and osteoporotic women was 8.0%, 13.8%, and 19.6%, respectively, increasing to 42.9%, 60.4%, and 57.4% by 5 years after initial screening. For treated women, median time to repeat DXA was over 3 years for all groups. Relative to women with low-risk initial DXA, high-risk initial DXA significantly predicted repeat screening for untreated women [adjusted HR 1.67 (95% CI 1.40-2.00)] but not within the treated group [HR 1.09 (95% CI 0.91-1.30)]. CONCLUSIONS: Repeat DXA screening was common in women both at low and high risk of progression to osteoporosis, with a substantial proportion of women receiving repeat scans within 2 years of initial screening. Conversely, only 60% of those at high-risk of progression to osteoporosis were re-screened within 5 years. Interventions are needed to help clinicians make higher-value decisions regarding repeat use of DXA scans.

Association of Plasma SDF-1 with Bone Mineral Density, Body Composition, and Hip Fractures in Older Adults: The Cardiovascular Health Study.

Aging is associated with an increase in circulating inflammatory factors. One, the cytokine stromal cell-derived factor 1 (SDF-1 or CXCL12), is critical to stem cell mobilization, migration, and homing as well as to bone marrow stem cell (BMSC), osteoblast, and osteoclast function. SDF-1 has pleiotropic roles in bone formation and BMSC differentiation into osteoblasts/osteocytes, and in osteoprogenitor cell survival. The objective of this study was to examine the association of plasma SDF-1 in participants in the cardiovascular health study (CHS) with bone mineral density (BMD), body composition, and incident hip fractures. In 1536 CHS participants, SDF-1 plasma levels were significantly associated with increasing age (p < 0.01) and male gender (p = 0.04), but not with race (p = 0.63). In multivariable-adjusted models, higher SDF-1 levels were associated with lower total hip BMD (p = 0.02). However, there was no significant association of SDF-1 with hip fractures (p = 0.53). In summary, circulating plasma levels of SDF-1 are associated with increasing age and independently associated with lower total hip BMD in both men and women. These findings suggest that SDF-1 levels are linked to bone homeostasis.

Sleep-disordered breathing is associated with higher carboxymethyllysine level in elderly women but not elderly men in the cardiovascular health study.

CONTEXT: Carboxymethyl-lysine (CML) results from oxidative stress and has been linked to cardiovascular disease. OBJECTIVE: The objective of this study is to investigate the association between sleep-disordered breathing (SDB) - a source of oxidative stress - and CML. MATERIALS AND METHODS: About 1002 participants in the Cardiovascular Health Study (CHS) were studied. RESULTS: Women with SDB had significantly higher CML concentration compared with those without SDB (OR = 1.63, 95%CI = 1.03-2.58, p = 0.04). The association was not significant among men. DISCUSSION: SDB was associated with CML concentration among elderly women but not men in the Cardiovascular Health Study. CONCLUSION: Accumulation of CML may be an adverse health consequence of SDB.

Toward a new vaccine for pertussis.

To overcome the limitations of the current pertussis vaccines, those of limited duration of action and failure to induce direct killing of Bordetella pertussis, a synthetic scheme was devised for preparing a conjugate vaccine composed of the Bordetella bronchiseptica core oligosaccharide with one terminal trisaccharide to aminooxylated BSA via their terminal ketodeoxyoctanate residues. Conjugate-induced antibodies, by a fraction of an estimated human dose injected into young outbred mice as a saline solution, were bactericidal against B. pertussis, and their titers correlated with their ELISA values. The carrier protein is planned to be genetically altered pertussis toxoid. Such conjugates are easy to prepare, stable, and should add both to the level and duration of immunity induced by current vaccine-induced pertussis antibodies and reduce the circulation of B. pertussis.

Underuse and Overuse of Osteoporosis Screening in a Regional Health System: a Retrospective Cohort Study.

BACKGROUND: The United States Preventive Services Task Force (USPSTF) recommends screening for osteoporosis with dual-energy x-ray absorptiometry (DXA) for women aged ≥ 65 years and younger women with increased risk. "Choosing Wisely" initiatives advise avoiding DXA screening in women younger than 65 years without osteoporosis risk factors. OBJECTIVE: We aimed to determine the extent to which DXA screening is used in accordance with USPSTF recommendations within a regional health system. DESIGN: This was a retrospective longitudinal cohort study within 13 primary care clinics in the Sacramento, CA region. PATIENTS: The study included 50,995 women aged 40-85 years without prior osteoporosis screening, diagnosis, or treatment attending primary care visits from 2006 to 2012, observed for a mean of 4.4 years. MAIN MEASURES: We examined incidence of DXA screening. Covariates included age, race/ethnicity, and osteoporosis risk factors (body mass index < 20, glucocorticoid use, secondary osteoporosis, prior high-risk facture, rheumatoid arthritis, alcohol abuse, and current smoking). KEY RESULTS: Among previously unscreened women for whom the USPSTF recommends screening, 7-year cumulative incidence of DXA screening was 58.8 % among women aged 60-64 years with ≥ 1 risk factor (95 % CI: 51.9-65.8 %), 57.8 % for women aged 65-74 years (95 % CI: 55.6-60.0 %), and 42.7 % for women aged ≥ 75 years (95 % CI: 38.7-46.7 %). Among women for whom the USPSTF does not recommend screening, 7-year cumulative incidence was 45.5 % among women aged 50-59 years (95 % CI 44.1-46.9 %) and 58.6 % among women aged 60-64 years without risk factors (95 % CI 55.9-61.4 %). CONCLUSIONS: DXA screening was underused in women at increased fracture risk, including women aged ≥ 65 years. Meanwhile, DXA screening was common among women at low fracture risk, such as younger women without osteoporosis risk factors. Interventions may be needed to augment the value of population screening for osteoporosis.

Steady-state kinetic isotope effects support a complex role of Arg226 in the proposed desulfonation mechanism of alkanesulfonate monooxygenase.

The alkanesulfonate monooxygenase system catalyzes the desulfonation of alkanesulfonates through proposed acid-base mechanistic steps that involves the abstraction of a proton from the alkane peroxyflavin intermediate and protonation of the FMN-O(-) intermediate. Both solvent and kinetic isotope studies were performed to define the proton transfer steps involved in the SsuD reaction. Substitution of the protium at the C1 position of octanesulfonate with deuterium resulted in an observed primary isotope effect of 3.0 ± 0.2 on the kcat parameter, supporting abstraction of the α-proton from the alkane peroxyflavin as the rate-limiting step in catalysis. Previous studies implicated Arg226 as the acid involved in the reprotonation of the hydroxyflavin intermediate. Solvent isotope kinetic studies gave an inverse isotope effect on (D2O)kcat of 0.75 ± 0.04 with no observable effect on (D2O)kcat/Km. This resulted in equivalent solvent isotope effects on (D2O)kcat and (D2O)(kcat)D, suggesting a solvent equilibrium isotope effect on a step occurring after the first irreversible step through product release. Data from proton inventory studies on kcat were best fit to a dome-shaped curve consistent with a conformational change to an open conformation during product release. The solvent isotope effect data coupled with the corresponding proton inventory results support and extend our previous observations that Arg226 donates a proton to the FMN-O(-) intermediate, triggering a conformational change that opens the enzyme to solvation and promotes product release.

Exemestane for breast-cancer prevention in postmenopausal women.

BACKGROUND: Tamoxifen and raloxifene have limited patient acceptance for primary prevention of breast cancer. Aromatase inhibitors prevent more contralateral breast cancers and cause fewer side effects than tamoxifen in patients with early-stage breast cancer. METHODS: In a randomized, placebo-controlled, double-blind trial of exemestane designed to detect a 65% relative reduction in invasive breast cancer, eligible postmenopausal women 35 years of age or older had at least one of the following risk factors: 60 years of age or older; Gail 5-year risk score greater than 1.66% (chances in 100 of invasive breast cancer developing within 5 years); prior atypical ductal or lobular hyperplasia or lobular carcinoma in situ; or ductal carcinoma in situ with mastectomy. Toxic effects and health-related and menopause-specific qualities of life were measured. RESULTS: A total of 4560 women for whom the median age was 62.5 years and the median Gail risk score was 2.3% were randomly assigned to either exemestane or placebo. At a median follow-up of 35 months, 11 invasive breast cancers were detected in those given exemestane and in 32 of those given placebo, with a 65% relative reduction in the annual incidence of invasive breast cancer (0.19% vs. 0.55%; hazard ratio, 0.35; 95% confidence interval [CI], 0.18 to 0.70; P=0.002). The annual incidence of invasive plus noninvasive (ductal carcinoma in situ) breast cancers was 0.35% on exemestane and 0.77% on placebo (hazard ratio, 0.47; 95% CI, 0.27 to 0.79; P=0.004). Adverse events occurred in 88% of the exemestane group and 85% of the placebo group (P=0.003), with no significant differences between the two groups in terms of skeletal fractures, cardiovascular events, other cancers, or treatment-related deaths. Minimal quality-of-life differences were observed. CONCLUSIONS: Exemestane significantly reduced invasive breast cancers in postmenopausal women who were at moderately increased risk for breast cancer. During a median follow-up period of 3 years, exemestane was associated with no serious toxic effects and only minimal changes in health-related quality of life. (Funded by Pfizer and others; NCIC CTG MAP.3 ClinicalTrials.gov number, NCT00083174.).