Role of BH4 deficiency as a mediator of oxidative stress-related endothelial dysfunction in menopausal women.

Endothelial function (brachial artery flow-mediated dilation [FMD]) is reduced in estrogen-deficient postmenopausal women, mediated, in part, by reduced nitric oxide (NO) bioavailability, secondary to tetrahydrobiopterin (BH4) deficiency and oxidative stress. FMD is increased, but not fully restored, in postmenopausal women after acute intravenous vitamin C (VITC; superoxide scavenger) or oral BH4 supplementation. In vitro studies demonstrate that coadministration of VITC with BH4 prevents endothelial nitric oxide synthase (eNOS) uncoupling and reductions in NO by peroxynitrite. To investigate mechanisms of endothelial dysfunction in women, we assessed the separate and combined effects of VITC and BH4 to determine whether coadministration of VITC + BH4 improves FMD in healthy postmenopausal women (n = 19, 58 ± 5 yr) to premenopausal (n = 14, 36 ± 9 yr) levels, with exploratory testing in perimenopausal women (n = 8, 51 ± 3 yr). FMD was measured during acute intravenous infusions of saline (control) and VITC (∼2-3 g) ∼3 h after a single dose of oral BH4 (KUVAN, 10 mg/kg body wt) or placebo (randomized crossover, separated by ∼1 mo). Under the placebo condition, FMD was reduced in postmenopausal compared with premenopausal women during the saline infusion (5.6 ± 0.7 vs. 11.6 ± 0.9%, P < 0.001) and increased in postmenopausal women during VITC (+3.5 [1.4, 5.6]%, P = 0.001) and acute BH4 (+1.8 [0.37, 3.2]%, P = 0.01) alone. Coadministration of VITC + BH4 increased FMD in postmenopausal women (+3.0 [1.7, 4.3]%, P < 0.001), but FMD remained reduced compared with premenopausal women (P = 0.02). Exploratory analyses revealed that VITC + BH4 did not restore FMD in perimenopausal women to premenopausal levels (P = 0.045). Coadministration of VITC + BH4 does not restore FMD in menopausal women, suggesting that additional mechanisms may be involved.NEW & NOTEWORTHY Endothelial function is reduced across the menopausal stages related to increased oxidative stress associated with estrogen deficiency. In vitro studies demonstrate that coadministration of VITC with BH4 prevents endothelial nitric oxide synthase (eNOS) uncoupling and reductions in NO by peroxynitrite; however, this remains untested in humans. We demonstrate that the coadministration of BH4 + VITC does not restore endothelial function in perimenopausal and postmenopausal women to the level of premenopausal women, suggesting that other mechanisms contribute.

Characterizing mucociliary clearance in young children with cystic fibrosis.

BACKGROUND: This research characterized mucociliary clearance (MCC) in young children with cystic fibrosis (CF). METHODS: Fourteen children (5-7 years old) with CF underwent: two baseline MCC measurements (Visits 1 and 2); one MCC measurement approximately 1 year later (Visit 3); and measurements of lung clearance index (LCI), a measure of ventilation inhomogeneity. RESULTS: Median (range) percent MCC through 60 min (MCC60) was similar on Visits 1 and 2 with 11.0 (0.9-33.7) and 12.8 (2.7-26.8), respectively (p = 0.95), and reproducible (Spearman Rho = 0.69; p = 0.007). Mucociliary clearance did not change significantly over 1 year with median percent MCC60 on Visit 3 [12.8 (3.7-17.6)] similar to Visit 2 (p = 0.58). Lower percent MCC60 on Visit 3 was significantly associated with higher LCI scores on Visit 3 (N = 14; Spearman Rho = -0.56; p = 0.04). CONCLUSIONS: Tests of MCC were reproducible and reliable over a 2-week period and stable over a 1-year period in 5-7-year-old children with CF. Lower MCC values were associated with increased ventilation inhomogeneity. These results suggest that measurements of MCC could be used in short-term clinical trials of interventions designed to modulate MCC and as a new, non-invasive test to evaluate early lung pathology in children with CF. IMPACT: This is the first study to characterize mucociliary clearance (MCC) in children with cystic fibrosis (CF) who were 5-7 years old. Measurements of mucociliary clearance were reproducible and reliable over a 2-week period and stable over a 1-year period. Variability in MCC between children was associated with differences in ventilation homogeneity, such that children with lower MCC values had increased ventilation inhomogeneity. These results suggest that measurements of MCC could be used in short-term clinical trials of interventions designed to modulate MCC and as a new, non-invasive test to evaluate early lung pathology in children with CF.

Potential unrealized mortality benefit of glucagon-like peptide-1 receptor agonists and sodium-glucose co-transport-2 inhibitors: A report from the Veterans Health Administration Clinical Assessment, Reporting and Tracking program.

AIM: To assess the unrealized potential of glucagon-like peptide-1 receptor agonist (GLP-1RA) or sodium-glucose co-transport-2 inhibitor (SGLT2i) use to reduce mortality in veterans with type 2 diabetes (T2D), coronary artery disease (CAD), and other characteristics congruent with clinical trial cohorts that established the efficacy of these agents. METHODS: Veterans with T2D and CAD on angiography in 2014 who were untreated with either a GLP-1RA or a SGLT2i were assessed for key eligibility criteria of the LEADER (GLP-1RA) and EMPA-REG OUTCOME (SGLT2i) trials. Trial hazard ratios and 95% confidence intervals for all-cause death were applied to deaths observed in veterans through 2018 to estimate the potential benefit of GLP-1RA or SGLT2i use. RESULTS: Median observation was 4.3 years. Of 15 987 veterans with T2D and CAD, 1186 (7.4%) were excluded for GLP-1RA or SGLT2i treatment, and 1386 lacked glycated haemoglobin measurement. Of the remaining 13 415 patients, 4103 (30.1%) and 5313 (39.6%) fulfilled the key criteria for the LEADER and EMPA-REG OUTCOME trials, respectively. Death occurred in 1009 (24.6%) of LEADER-eligible patients and 1335 (25.1%) of EMPA-REG OUTCOME-eligible patients. Under treatment with liraglutide in LEADER-eligible veterans, a 3.5% (0.7%-6.2%) potential absolute mortality reduction, corresponding to 144 (28-253) fewer deaths (0.88 [0.17-1.56] per 100 person-years), might have been expected. Similarly, under treatment with empagliflozin in EMPA-REG OUTCOME-eligible veterans, a 7.9% (4.5%-10.8%) potential absolute mortality reduction, corresponding to 418 (230-573) fewer deaths (1.98 [1.14-2.72] per 100 person-years), might have been expected. CONCLUSIONS: This analysis indicates unrealized opportunities to reduce mortality in selected veterans with T2D and CAD via increased GLP-1RA and SGLT2i use.

A model-specific role of microRNA-223 as a mediator of kidney injury during experimental sepsis.

Sepsis outcomes are heavily dependent on the development of septic organ injury, but no interventions exist to interrupt or reverse this process. microRNA-223 (miR-223) is known to be involved in both inflammatory gene regulation and host-pathogen interactions key to the pathogenesis of sepsis. The goal of this study was to determine the role of miR-223 as a mediator of septic kidney injury. Using miR-223 knockout mice and multiple models of experimental sepsis, we found that miR-223 differentially influences acute kidney injury (AKI) based on the model used. In the absence of miR-223, mice demonstrated exaggerated AKI in sterile models of sepsis (LPS injection) and attenuated AKI in a live-infection model of sepsis (cecal ligation and puncture). We demonstrated that miR-223 expression is induced in kidney homogenate after cecal ligation and puncture, but not after LPS or fecal slurry injection. We investigated additional potential mechanistic explanations including differences in peritoneal bacterial clearance and host stool virulence. Our findings highlight the complex role of miR-223 in the pathogenesis of septic kidney injury, as well as the importance of differences in experimental sepsis models and their consequent translational applicability.

Biomechanical Comparison of Impaction Techniques and Cross-Sectional Femoral Stem Shapes for Cementless Total Hip Arthroplasty.

Introduction: Traditional mallet broaching and stem seating in cementless total hip arthroplasty (THA) can result in femoral stem misalignment, potentially reducing implant longevity. This study aimed to compare the pullout strength of cementless THA femoral stems with different cross-sectional designs achieved through the powered impactor method versus the traditional mallet method. Methods: The authors utilized 24 polyurethane foam femurs and two femoral bone preservation stems with different proximal cross-sectional shapes (double taper: ACTIS®, size 5; flat taper: TRI-LOCK®, size 5). A single orthopedic surgeon broached each femur from size 0 to size 5 using either the powered impactor or mallet impaction methods. Broaching time and component implantation times were recorded. A load-to-failure pullout strength test was conducted, and the ultimate pullout load was recorded. Results: The broaching time for the TRI-LOCK® stem showed a statistically significant difference between the two impaction methods (powered: 37±7 seconds, mallet: 75±29 seconds, F[3, 20] = 4.56, p = 0.002), but no statistically significant difference was detected for the ACTIS® stem between the two impaction methods (powered: 47±22 seconds, mallet: 59±9 seconds, F[3, 20] = 4.56, p = 0.304). There was a statistically significant difference in pullout strength between the two impaction groups, and this strength was influenced by the implant cross-sectional shape (ACTIS®: 774±75N versus 679±22N, F(3,20) = 16.38, p = 0.018; TRI-LOCK®: 616±57N versus 859±85N, F(3, 20) = 16.38, p <0.001). Conclusions: The technique used for femoral bone preparation (powered impactor versus mallet) and the cross-sectional design of the cementless femoral stem are crucial factors that affect initial stem stability and operation time.

General improvements versus interruptive or non-interruptive alerts in the blood order set: study protocol for a randomized control trial to improve packed red blood cell utilization.

BACKGROUND: Blood transfusions can serve as a life-saving treatment, but inappropriate blood product transfusions can result in patient harm and excess costs for health systems. Despite published evidence supporting restricted packed red blood cell (pRBC) usage, many providers transfuse outside of guidelines. Here, we report a novel prospective, randomized control trial to increase guideline-concordant pRBC transfusions comparing three variations of clinical decision support (CDS) in the electronic health record (EHR). METHODS: All inpatient providers at University of Colorado Hospital (UCH) who order blood transfusions were randomized in a 1:1:1 fashion to the three arms of the study: (1) general order set improvements, (2) general order set improvements plus non-interruptive in-line help text alert, and (3) general order set improvements plus interruptive alert. Transfusing providers received the same randomized order set changes for 18 months. The primary outcome of this study is the guideline-concordant rate of pRBC transfusions. The primary objective of this study is to compare the group using the new interface (arm 1) versus the two groups using the new interface with interruptive or non-interruptive alerts (arms 2 and 3, combined). The secondary objectives compare guideline-concordant transfusion rates between arm 2 and arm 3 as well as comparing all of arms of the study in aggregate to historical controls. This trial concluded after 12 months on April 5, 2022. DISCUSSION: CDS tools can increase guideline-concordant behavior. This trial will examine three different CDS tools to determine which type is most effective at increasing guideline-concordant blood transfusions. TRIAL REGISTRATION: Registered on ClinicalTrials.gov 3/20/21, NCT04823273 . Approved by University of Colorado Institutional Review Board (19-0918), protocol version 1 4/19/2019, approved 4/30/2019.

Preliminary data demonstrate the Geriatric Surgery Verification program reduces postoperative length of stay.

OBJECTIVES/BACKGROUND: The Geriatric Surgery Verification (GSV) Program promotes clinical standards aimed to optimize the quality of surgical care delivered to older adults. The purpose of this study was to determine if preliminary implementation of the GSV Program standards improves surgical outcomes. DESIGN: Prospective study with cohort matching. SETTING: Data from a single institution compared with a national data set cohort. PARTICIPANTS: All patients aged ≥75 years undergoing inpatient operations between January 2018 and December 2019 were included. Cohort matching by age and procedure code was performed using a national data set. MEASUREMENTS: Baseline pre- and intraoperative characteristics prospectively recorded using Veterans Affairs Surgical Quality Improvement Program (VASQIP) variable definitions. Postoperative outcomes were recorded including complications as defined by VASQIP, 30-day mortality, and length of stay. RESULTS: A total of 162 patients participated in the GSV program, and 308 patients comprised the matched comparison group. There was no difference in postoperative occurrence of one or more complications (p = 0.81) or 30-day mortality (p = 0.61). Patients cared for by the GSV Program had a reduced postoperative length of stay (median 4 days [range 1,31] vs. 5 days [range 1,86]; p < 0.01; and mean 5.4 ± 4.8 vs. 8.8 ± 11.8 days; p < 0.01) compared with the matched cohort. In a multivariable regression model, the GSV Program's reduced length of stay was independent of other associated covariates including age, operative time, and comorbidities (p < 0.01). CONCLUSION: Preliminary implementation of the GSV Program standards reduces length of stay in older adults undergoing inpatient operations. This finding demonstrates both the clinical and financial value of the GSV Program.

Disulfide disruption reverses mucus dysfunction in allergic airway disease.

Airway mucus is essential for lung defense, but excessive mucus in asthma obstructs airflow, leading to severe and potentially fatal outcomes. Current asthma treatments have minimal effects on mucus, and the lack of therapeutic options stems from a poor understanding of mucus function and dysfunction at a molecular level and in vivo. Biophysical properties of mucus are controlled by mucin glycoproteins that polymerize covalently via disulfide bonds. Once secreted, mucin glycopolymers can aggregate, form plugs, and block airflow. Here we show that reducing mucin disulfide bonds disrupts mucus in human asthmatics and reverses pathological effects of mucus hypersecretion in a mouse allergic asthma model. In mice, inhaled mucolytic treatment loosens mucus mesh, enhances mucociliary clearance, and abolishes airway hyperreactivity (AHR) to the bronchoprovocative agent methacholine. AHR reversal is directly related to reduced mucus plugging. These findings establish grounds for developing treatments to inhibit effects of mucus hypersecretion in asthma.

Changes in mucociliary clearance over time in children with cystic fibrosis.

OBJECTIVES: (a) To quantify changes in mucociliary clearance (MCC) over time in children with cystic fibrosis (CF) and the relationship between MCC and rate of infection with Pseudomonas aeruginosa (PA); (b) to determine the impact of MCC on the evolution of CF lung disease; and (c) to explore the role of mucus composition as a determinant of MCC. METHODS: Children with CF, who had previously undergone an MCC measurement (visit 1), underwent the following tests 3 to 10 years later: (a) second MCC measurement (visit 2); (b) multiple breath washout to assess ventilation inhomogeneity, expressed as lung clearance index (LCI); (c) high resolution computed tomography lung scan (HRCT); and (d) induced sputum test. Number of PA + cultures/year between visits was documented and mucus dry weight was quantified in the children and adult controls. RESULTS: Nineteen children completed both visits. Median time between visits was 4.6 years. Clearance declined 30% between visits. Lower MCC on visit 2 was associated with more PA+ cultures/year between visits. Lower MCC values on visit 1 were associated with higher LCI values and higher HRCT scores on visit 2. Mucus dry weight was significantly higher in children with CF compared with controls. Higher dry weights were associated with lower MCC. CONCLUSIONS: Mucociliary clearance declines significantly over time in children with CF. The decline is associated with PA infection rate and is affected by mucus composition. Children with early slowing of MCC appear to be at risk for developing ventilation inhomogeneity and parenchymal lung damage when they are older.

Requirement for MUC5AC in KRAS-dependent lung carcinogenesis.

With more than 150,000 deaths per year in the US alone, lung cancer has the highest number of deaths for any cancer. These poor outcomes reflect a lack of treatment for the most common form of lung cancer, non-small cell lung carcinoma (NSCLC). Lung adenocarcinoma (ADC) is the most prevalent subtype of NSCLC, with the main oncogenic drivers being KRAS and epidermal growth factor receptor (EGFR). Whereas EGFR blockade has led to some success in lung ADC, effective KRAS inhibition is lacking. KRAS-mutant ADCs are characterized by high levels of gel-forming mucin expression, with the highest mucin levels corresponding to worse prognoses. Despite these well-recognized associations, little is known about roles for individual gel-forming mucins in ADC development causatively. We hypothesized that MUC5AC/Muc5ac, a mucin gene known to be commonly expressed in NSCLC, is crucial in KRAS/Kras-driven lung ADC. We found that MUC5AC was a significant determinant of poor prognosis, especially in patients with KRAS-mutant tumors. In addition, by using mice with lung ADC induced chemically with urethane or transgenically by mutant-Kras expression, we observed significantly reduced tumor development in animals lacking Muc5ac compared with controls. Collectively, these results provide strong support for MUC5AC as a potential therapeutic target for lung ADC, a disease with few effective treatments.

Ten pearls every dermatologist should know about the appropriate use of relaxers.

Chemical relaxers are products marketed to straighten the hair and have been principally used by African Americans since the early twentieth century. They contain alkaline agents that break and reform hydrogen and disulfide bonds, leaving hair permanently straightened. Relaxers cause loss of tensile strength and increased fragility of the hair shaft that lead to increased risk of hair breakage and thinning. When used improperly, they can also cause local irritant contact dermatitis and chemical burns to the scalp and hairline that can potentially lead to a scarring alopecia. After interviewing several cosmetology educators in the field and reviewing the literature, we generated a list of ten pearls that all dermatologists should know about relaxers to provide professional guidance to their patients.

Temporal Decay in Timber Species Composition and Value in Amazonian Logging Concessions.

Throughout human history, slow-renewal biological resource populations have been predictably overexploited, often to the point of economic extinction. We assess whether and how this has occurred with timber resources in the Brazilian Amazon. The asynchronous advance of industrial-scale logging frontiers has left regional-scale forest landscapes with varying histories of logging. Initial harvests in unlogged forests can be highly selective, targeting slow-growing, high-grade, shade-tolerant hardwood species, while later harvests tend to focus on fast-growing, light-wooded, long-lived pioneer trees. Brazil accounts for 85% of all native neotropical forest roundlog production, and the State of Pará for almost half of all timber production in Brazilian Amazonia, the largest old-growth tropical timber reserve controlled by any country. Yet the degree to which timber harvests beyond the first-cut can be financially profitable or demographically sustainable remains poorly understood. Here, we use data on legally planned logging of ~17.3 million cubic meters of timber across 314 species extracted from 824 authorized harvest areas in private and community-owned forests, 446 of which reported volumetric composition data by timber species. We document patterns of timber extraction by volume, species composition, and monetary value along aging eastern Amazonian logging frontiers, which are then explained on the basis of historical and environmental variables. Generalized linear models indicate that relatively recent logging operations farthest from heavy-traffic roads are the most selective, concentrating gross revenues on few high-value species. We find no evidence that the post-logging timber species composition and total value of forest stands recovers beyond the first-cut, suggesting that the commercially most valuable timber species become predictably rare or economically extinct in old logging frontiers. In avoiding even more destructive land-use patterns, managing yields of selectively-logged forests is crucial for the long-term integrity of forest biodiversity and financial viability of local industries. The logging history of eastern Amazonian old-growth forests likely mirrors unsustainable patterns of timber depletion over time in Brazil and other tropical countries.

Patterns of disease and treatment of cold sores.

BACKGROUND: Cold sores are a common condition that can cause significant morbidity and mortality. Antivirals are the typical treatment for cold sores, but the ways in which these medications are used to treat cold sores are not well studied. PURPOSE: To determine the main treatments prescribed for cold sores and trends in their management over time. METHODS: A retrospective analysis of the National Ambulatory Medical Care Survey database was used to analyze outpatient visits for cold sores from 1993 to 2009. Patients were included in the data analysis if they had one of the following three diagnoses reported for their reason-for-visit codes: cold sores (CS), herpes simplex (HS) or herpes simplex with cold sores (HS/CS). RESULTS: There was a decreasing trend in the number of annual patient visits for cold sores. The majority of patients were mainly young to middle adulthood, white women. The top two most commonly prescribed medications were acyclovir followed by valacyclovir. Valacyclovir use increased in all three populations, while acyclovir use decreased. CONCLUSIONS: The trends observed may indicate that physicians are evolving their treatment strategies to implement newer antiviral medications. This may prove more efficacious for the treatment of cold sores.

Replacing the wild type loxP site in BACs from the public domain with lox66 using a lox66 transposon.

BACKGROUND: Chromatin adjoining the site of integration of a transgene affects expression and renders comparisons of closely related transgenes, such as those derived from a BAC deletion series retrofitted with enhancer-traps, unreliable. Gene targeting to a pre-determined site on the chromosome is likely to alleviate the problem. FINDINGS: A general procedure to replace the loxP site located at one end of genomic DNA inserts in BACs with lox66 is described. Truncating insert DNA from the loxP end with a Tn10 transposon carrying a lox66 site simultaneously substitutes the loxP with a lox66 sequence. The replacement occurs with high stringency, and the procedure should be applicable to all BACs in the public domain. Cre recombination of loxP with lox66 or lox71 was found to be as efficient as another loxP site during phage P1 transduction of small plasmids containing those sites. However the end-deletion of insert DNA in BACs using a lox66 transposon occurred at no more than 20% the efficiency observed with a loxP transposon. Differences in the ability of Cre protein available at different stages of the P1 life cycle to recombine identical versus non-identical lox-sites is likely responsible for this discrepancy. A possible mechanism to explain these findings is discussed. CONCLUSIONS: The loxP/lox66 replacement procedure should allow targeting BACs to a pre-positioned lox71 site in zebrafish chromosomes; a system where homologous recombination-mediated "knock-in" technology is unavailable.

Coiled-coil regions play a role in the function of the Shigella flexneri O-antigen chain length regulator WzzpHS2.

Regulation of the length of the O-antigen (Oag) chain attached to LPS in Shigella flexneri is important for virulence and is dependent on the inner-membrane protein Wzz. A lack of high-resolution structural data for Wzz has hampered efforts so far to correlate mutations affecting function of Wzz with structure and describe a mechanism for chain length regulation. Here we have used secondary structure prediction to show that the periplasmic domain of the Wzz(pHS2) protein has three regions of significant coiled-coil (CC) potential, two of which lie within an extended helical region. We describe here the first site-directed mutagenesis study to investigate the role of individual predicted CC regions (CCRs) in Wzz function and oligomerization. We found that CCRs 2 and 3 are necessary for wild-type Oag chain length regulation by Wzz(pHS2). The in vivo cross-linking profile of mutants affected in the three CCRs was not altered, indicating that individually each CCR is not required for oligomerization. Interestingly, the CCR3 mutation resulted in a temperature-sensitive phenotype and an inhibitory effect on Oag polymerization. Analysis of Wzz(pHS2) and the mutant constructs in a S. flexneri degP mutant showed that DegP did not affect the function of wild-type Wzz(pHS2) but its presence influenced the phenotype of the Wzz(pHS2) CCR3 mutant. Additionally, the phenotype of the Wzz(pHS2) CCR3 mutant was suppressed by a cis mutation near the putative cytoplasmic C-terminus of Wzz(pHS2).