Understanding the cholesterol metabolism-perturbing effects of docosahexaenoic acid by gas chromatography-mass spectrometry targeted metabonomic profiling.

PURPOSE: Over the past few decades, docosahexaenoic acid (DHA) has gained special attention for management of cholesterol-associated metabolic disorders and neurodegenerative diseases such as Alzheimer's disease (AD) owing to its neuroprotective, anti-inflammatory and hypolipidemic properties. Several epidemiological studies have reported the effect of DHA in reducing the risk of developing AD by lowering cholesterol. Hypercholesterolemia is a pro-amyloidogenic factor influencing the enzymatic processing of amyloid-ß precursor protein (AßPP) to toxic ß-amyloid. However, the mechanism by which DHA modulates the cholesterol pathway has not been established. Thus, the objective of this study was to investigate the mechanism of regulation of cholesterol metabolism by DHA in an AßPP695 overexpressing AD cell model. METHODS: A gas chromatography/mass spectrometry method was developed and validated for the targeted profiling of 11 cholesterol metabolites in DHA-treated Chinese hamster ovary wild-type (CHO-wt) and AßPP695 overexpressing (CHO-AßPP695) cells. The differential metabolite profiles between DHA- and vehicle-treated groups were further analyzed using fold change values of the ratio of concentration of metabolites in CHO-AßPP695 to CHO-wt cells. Effect of DHA on key rate-limiting enzymatic activities within the cholesterol pathway was established using biochemical assays. RESULTS: Our results showed that DHA reduced the levels of key cholesterol anabolites and catabolites in CHO-AßPP695 cells as compared to CHO-wt cells. Further enzymatic studies revealed that the cholesterol-lowering effect of DHA was mediated by regulating HMG-CoA reductase and squalene epoxidase enzyme activities. CONCLUSION: We demonstrate for the first time the dual effects of DHA in inhibiting HMG-CoA reductase and squalene epoxidase and modulating the sterol biosynthesis axis of the cholesterol pathway in AßPP695 overexpressing AD. Our novel findings underscore the potential of DHA as a multi-target hypocholesterolemic agent for the prophylaxis of AD and other cholesterol-associated diseases.

A comparison of GOLD 2019 and 2023 recommendations to contemporaneous real-world inhaler treatment patterns for chronic obstructive pulmonary disease management in Singapore.

Background: In 2019 and 2023, the Global Initiative for Chronic Obstructive Lung Disease (GOLD) provided updated strategies for modifying the therapy of patients with chronic obstructive pulmonary disease (COPD) and high exacerbation risk. A key update since the 2019 guidelines recommends considering blood eosinophil count to guide decisions on inhaled corticosteroid (ICS) treatment. To evaluate the potential impact of these updated recommendations, this study aimed to assess how extensively future practice would diverge from contemporaneous prescribing practices at a single center in Singapore, assuming adherence to the 2019 and 2023 GOLD guidelines. Methods: Retrospective cohort analysis of the Changi General Hospital COPD data warehouse involving patients aged ≥40 years hospitalized for a COPD exacerbation (October 2018-April 2020) receiving long-acting muscarinic antagonist (LAMA), LAMA plus a long-acting beta2-agonist (LABA), or an ICS plus LABA at admission. The proportion of patients eligible for treatment escalations per GOLD 2019 and 2023 recommendations was calculated. Results: In total, 268 patients were included (mean age 73 years; 91% male). At admission, 19%, 59%, and 22% of patients were receiving LAMA, LAMA + LABA, and ICS + LABA, respectively. Overall, 226 patients would have been eligible for treatment escalation per GOLD 2019 or 2023 recommendations; 31 (13.7%) had treatment escalations consistent with GOLD 2019 guidelines and 34 (15%) received treatment escalations consistent with GOLD 2023 guidelines. A total of 205 patients (76.5%) remained on the same treatment regimen at hospital discharge as they were receiving at admission. Lower measured post-bronchodilator forced expiratory volume in 1 second was associated with treatment escalations that would have been GOLD-concordant (P=0.028), as was increased number of emergency department/hospital visits in the last year (P=0.048). Conclusions: Compared with real-world clinical practice, a significantly higher proportion of patients may be eligible for treatment escalation under the GOLD 2019 and 2023 eosinophil-directed algorithms.

Year-to-year trajectories of hospital utilisation rates among patients with COPD: a real-world, single-centre, retrospective cohort study.

OBJECTIVES: The long-term clinical trajectory of chronic obstructive pulmonary disease (COPD) in terms of year-to-year hospital utilisation rates can be highly variable and is not well studied. We investigated year-to-year trends of hospitalisation or emergency department (ED) visits among patients with COPD over 3 years, identified distinct trajectories and examined associated predictive factors. DESIGN: A retrospective cohort study. SETTING: Data were extracted from the Changi General Hospital, Singapore COPD data warehouse. PARTICIPANTS: Patients with COPD aged ≥40 years with 3 years of follow-up data. PRIMARY AND SECONDARY OUTCOME MEASURES: The yearly rates of hospitalisations or ED visits, stratified by COPD-related or all-cause, were described. Group-based trajectory modelling was used to identify clinically distinct trajectories year-by-year. Baseline predictive factors associated with different trajectories were examined. RESULTS: In total, 396 patients were analysed (median age 70 years; 87% male). Four trajectories were generated for year-to-year trends in COPD-related hospitalisations/ED visits (C1-C4: consistently frequent, consistently infrequent, improving and worsening); post-bronchodilator forced expiratory volume in 1 second (FEV1) was a significant predictor of trajectory, with worse lung function being the main factor associated with less favourable trajectories. For all-cause hospitalisations/ED visits, four trajectories were identified (A1-A4: infrequent and stable, frequent and stable, frequent and decreasing, frequent and increasing); significant differences in age (p=0.041), sex (p=0.016) and ethnicity (p=0.005) were found between trajectories. Higher overall comorbidity burden was a key determinant in less favourable trajectories of all-cause hospitalisations/ED visits. CONCLUSIONS: Distinct trajectories were demonstrated for hospitalisations/ED visits related to COPD or all causes, with predictive associations between FEV1 and COPD trajectory and between comorbidities and all-cause trajectory. Trajectories carry nuanced prognostic information and may be useful for clinical risk stratification to identify high-risk individuals for preventative treatments.

Characteristics of Patients with Chronic Obstructive Pulmonary Disease Treated with Long-Acting Bronchodilators in a Real-World Setting in Singapore: A Single-Center Observational Study.

Introduction: There is limited real-world evidence regarding clinical practice for chronic obstructive pulmonary disease (COPD) in Singapore. We compared baseline clinical characteristics and evaluated outcomes in patients with COPD who initiated treatment with either a long-acting muscarinic antagonist (LAMA) or a LAMA and a long-acting ß2-agonist (LAMA+LABA). Methods: This was a single-center observational study at Changi General Hospital, Singapore. Routine clinical data (hospital visits, case management, lung function, laboratory/imaging results, medication orders) were collected and compiled into a data warehouse. Eligible patients with COPD were ≥40 years old and newly prescribed LAMA or LAMA+LABA during the enrollment period. Patient characteristics in the baseline period (6 months) were compared between treatments. Clinical worsening was measured as a composite endpoint, defined as the first of a change in maintenance treatment class or a moderate-to-severe exacerbation during follow-up (12 months). Results: In total, 261 patients were included in the baseline period (LAMA: 73; LAMA+LABA: 188). In the baseline period, patients receiving LAMA+LABA versus LAMA had significantly lower body mass index, higher COPD Assessment Test score and worse lung function, and numerically higher exacerbation history. Prevalence of comorbidities was similar between treatment groups. In follow-up, high rates of clinical worsening were observed regardless of treatment regimen (LAMA: 38/73 [52%]; LAMA+LABA: 86/188 [46%]). Median time-to-clinical worsening was 340 days for the LAMA cohort and the raw median 154 days (interquartile range: 44-225) for the LAMA+LABA cohort. Median medication dispensation rate (0.86; interquartile range: 0.56-1.00) was similar between treatments. Conclusion: Patients initiating treatment with LAMA+LABA had more severe COPD than patients prescribed LAMA. The proportion of patients experiencing clinical worsening was similarly high in both cohorts, suggesting that early identification and treatment optimization are necessary.

Public health impact of pneumococcal conjugate vaccine infant immunization programs: assessment of invasive pneumococcal disease burden and serotype distribution.

INTRODUCTION: Pneumococcal conjugate vaccine (PCV) impact studies have reported substantial reductions in the incidence of invasive pneumococcal disease (IPD) after implementation of childhood PCV programs. Heterogeneity in surveillance systems, local epidemiology and PCV programs hampers comparisons between studies. We aimed to better understand the impact of childhood PCV programs on overall IPD and serotype distribution. AREAS COVERED: We analyzed the impact of PCV programs on the incidence of overall IPD, and the distribution of vaccine serotypes (VT) and non-vaccine serotypes (NVT) in children <5 years and adults ≥65 years old. We retrieved datasets from observational post-marketing studies and surveillance reports from countries with high-quality surveillance data available for at least 2 years before PCV program initiation and 3 years after higher-valent PCV implementation. We harmonized pre- and post-PCV analysis periods and assessment methods. EXPERT COMMENTARY: After introduction of pediatric PCV programs, the residual overall IPD burden in children was low and in a narrow range across countries with high vaccination coverage, irrespective of differences in PCV programs and pneumococcal epidemiology. Effects on overall IPD were more variable in the elderly. Whereas IPD was mainly due to VTs before PCV introduction, NVTs are the major contributor today in children and adults.

Wnt co-receptor LRP5/6 overexpression confers protection against hydrogen peroxide-induced neurotoxicity and reduces tau phosphorylation in SH-SY5Y cells.

Emerging studies have suggested the involvement of dysregulated Wnt/ß-catenin cascade in the etiology of Alzheimer's disease (AD). Recently, genetic variations in Wnt co-receptor low density lipoprotein receptor-related protein (LRP) 6 causing reduced Wnt signaling has been linked to late-onset AD. Here, we hypothesized that overexpression of Wnt co-receptors LRP5 and LRP6 would serve as an effective new approach in reducing neurotoxicity induced by oxidative stress and decreasing tau phosphorylation in SH-SY5Y human neuroblastoma cells. Our results showed that overexpression of LRP5 and LRP6 in SH-SY5Y cells activates Wnt signaling and downstream proliferation genes, whereas knockdown of the co-receptors represses Wnt signaling and the transcription of proliferative markers. We further demonstrated that overexpression of LRP5 and LRP6 protects SH-SY5Y from cell death caused by hydrogen peroxide-induced oxidative stress, inhibits GSK3ß activity and subsequently reduces tau phosphorylation. Together, our findings suggest that rescuing LRP5/6-mediated Wnt signaling improves neuronal cell survival and reduces tau phosphorylation, which support the hypothesis that Wnt signaling might be an attractive therapeutic strategy for managing AD.

Metabotyping of docosahexaenoic acid - treated Alzheimer's disease cell model.

BACKGROUND: Despite the significant amount of work being carried out to investigate the therapeutic potential of docosahexaenoic acid (DHA) in Alzheimer's disease (AD), the mechanism by which DHA affects amyloid-ß precursor protein (AßPP)-induced metabolic changes has not been studied. OBJECTIVE: To elucidate the metabolic phenotypes (metabotypes) associated with DHA therapy via metabonomic profiling of an AD cell model using gas chromatography time-of-flight mass spectrometry (GC/TOFMS). METHODS: The lysate and supernatant samples of CHO-wt and CHO-AßPP695 cells treated with DHA and vehicle control were collected and prepared for GC/TOFMS metabonomics profiling. The metabolic profiles were analyzed by multivariate data analysis techniques using SIMCA-P+ software. RESULTS: Both principal component analysis and subsequent partial least squares discriminant analysis revealed distinct metabolites associated with the DHA-treated and control groups. A list of statistically significant marker metabolites that characterized the metabotypes associated with DHA treatment was further identified. Increased levels of succinic acid, citric acid, malic acid and glycine and decreased levels of zymosterol, cholestadiene and arachidonic acid correlated with DHA treatment effect. DHA levels were also found to be increased upon treatment. CONCLUSION: Our study shows that DHA plays a role in mitigating AßPP-induced impairment in energy metabolism and inflammation by acting on tricarboxylic acid cycle, cholesterol biosynthesis pathway and fatty acid metabolism. The perturbations of these metabolic pathways by DHA in CHO-wt and CHO-AßPP695 cells shed further mechanistic insights on its neuroprotective actions.

Functionalized curcumin analogs as potent modulators of the Wnt/ß-catenin signaling pathway.

Osteosarcoma is a primary bone malignancy with aggressive metastatic potential and poor prognosis rates. In our earlier work we have investigated the therapeutic potential of curcumin as an anti-invasive agent in osteosarcoma by its ability to regulate the Wnt/ß-catenin signaling pathway. However, the clinical use of curcumin is limited owing to its low potency and poor pharmacokinetic profile. In this study, an attempt was made to achieve more potent Wnt inhibitory activity in osteosarcoma cells by carrying out synthetic chemical modifications of curcumin. We synthesized a total of five series consisting of 43 curcumin analogs and screened in HEK293T cells for inhibition of ß-catenin transcriptional activity. Six promising analogs, which were 6.5- to 60-fold more potent than curcumin in inhibiting Wnt activity, were further assessed for their anti-invasive activity and Wnt inhibitory mechanisms. Western blot analysis showed disruption of ß-catenin protein nuclear translocation following treatment with analogs 2f, 3c and 4f. Using transwell assays, we also found that these compounds were more potent than 1a (curcumin) in impeding the invasion of osteosarcoma cells, possibly through suppressing MMP-9 activity. Structure-activity-relationship studies revealed that Wnt inhibitory effects could be enhanced by shortening and restraining the flexibility of the 7-carbon linker moiety connecting the terminal aromatic rings of curcumin and substituting both rings with appropriate substituents. Our results demonstrate that the synthesized curcumin analogs are more potent Wnt inhibitors in osteosarcoma cell lines as compared to parental curcumin and are good lead compounds for further development. Future in vivo tests with these compounds will define their therapeutic potentials as promising drug candidates for clinical treatment of osteosarcoma.

Synthetic modifications of the immunomodulating peptide thymopentin to confer anti-mycobacterial activity.

Effective global control of tuberculosis (TB) is increasingly threatened by the convergence of multidrug-resistant TB and the human immunodeficiency virus (HIV) infection. TB/HIV coinfections exert a tremendous burden on the host's immune system, and this has prompted the clinical use of immunomodulators to enhance host defences as an alternative therapeutic strategy. In this study, we modified the clinically used synthetic immunomodulatory pentapeptide, thymopentin (TP-5, RKDVY), with six arginine residues (RR-6, RRRRRR) at the N- and C-termini to obtain the cationic peptides, RR-11 (RKDVYRRRRRR-NH2) and RY-11 (RRRRRRRKDVY-NH2), respectively. The arginine residues conferred anti-mycobacterial activity to TP-5 in the peptides as shown by effective minimum inhibitory concentrations of 125 mg/L and killing efficiencies of >99.99% against both rifampicin-susceptible and -resistant Mycobacterium smegmatis. The immunomodulatory action of the peptides remained unaffected as shown by their ability to stimulate TNF-α production in RAW 264.7 mouse macrophage cells. A distinct change in surface morphology after peptide treatment was observed in scanning electron micrographs, while confocal microscopy and dye leakage studies suggested bacterial membrane disruption by the modified peptides. The modified peptides were non-toxic and did not cause hemolysis of rat red blood cells up to a concentration of 2000 mg/L. Moreover, RY-11 showed synergism with rifampicin and reduced the effective concentration of rifampicin, while preventing the induction of rifampicin resistance. The synthetic peptides may have a potential application in both immunocompetent and immunocompromised TB patients.