Programmed cell death 4 blocks autophagy and promotes dopaminergic neuronal injury in Parkinson's disease.

Dysregulation of autophagy has previously been associated with the formation of toxic proteins, such as α-synuclein, in patients with Parkinson's disease (PD). In addition, it has been indicated that programmed cell death 4 (PDCD4) can inhibit autophagy in certain conditions, such as diabetic nephropathy, atherosclerosis and cardiac hypertrophy. Therefore, the hypothesis that PDCD4 can promote dopaminergic neuron damage through autophagy was proposed. To explore this hypothesis, the present study treated human neuroblastoma SK-N-SH cells with 1-methyl-4-phenylpyridinium (MPP+) to establish an in vitro model of PD. The potential effects of PDCD4 knockdown on lactate dehydrogenase (LDH) release, cell apoptosis, inflammatory response, oxidative stress and autophagy were then evaluated in this model of PD using an LDH assay kit, flow cytometry, western blotting, ELISA and immunofluorescence. The autophagy inhibitor 3-methyladenine (3-MA) was also applied to treat these cells, and its effects on these aforementioned parameters following PDCD4 knockdown were assessed. MPP+ was shown to increase the expression levels of PDCD4 in SK-N-SH cells. PDCD4 knockdown was revealed to suppress LDH release, cell apoptosis, secretion of inflammatory factors and oxidative stress. In addition, PDCD4 knockdown was demonstrated to enhance autophagy in cells treated with MPP+. By contrast, 3-MA treatment reversed the aforementioned effects of PDCD4 knockdown on cells, suggesting autophagy to be among the processes regulated by PDCD4 in SK-N-SH cells. The results of the present study suggested the existence of regulatory effects mediated by PDCD4 on autophagy in MPP+-induced SK-N-SH cells, offering potential future targets for PD therapy.

Global genomic diversity and conservation of SARS-CoV-2 since the COVID-19 outbreak.

IMPORTANCE: Our results indicate that most severe acute respiratory syndrome coronavirus 2 genomes sampled from patients had a mutation rate ≤1.07 ‰ and genome-tail proteins (including S protein) were the main sources of genetic polymorphism. The analysis of the virus-host interaction network of genome-tail proteins showed that they shared some antiviral signaling pathways, especially the intracellular protein transport pathway.

NEK7 inhibition attenuates Aß42-induced cognitive impairment by regulating TLR4/NF-κB and the NLRP3 inflammasome in mice.

NEK7 is a serine/threonine kinase that regulates cell mitosis and the activation of the nucleotide-binding oligomerization domain-like (NOD-like) receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, and is related to neuroinflammation and neuronal damage. The purpose of this study was to explore the role and mechanism of NEK7 in cognitive impairment in Alzheimer's disease (AD). BV2 cells, a microglia cell line, was treated with Aß42. NEK7 expression was measured with reverse transcription-quantitative polymerase chain reaction and Western blotting. An apoptosis kit was used to determine the apoptotic rate. APPswe/PS1dE9 (APP/PS1) transgenic mice were used as an in vivo AD model. The experimental mice were infected with sh-NEK7 lentivirus to downregulate NEK7. The Morris water maze was conducted to explore the effect of NEK7 downregulation on cognitive ability. The results showed that Aß42 significantly upregulated NEK7 in BV2 cells. Silencing NEK7 suppressed the decrease in BV2 viability and the increase in inflammation, oxidative stress and apoptosis induced by Aß42. NEK7 mediated it effects through the TLR4/NF-κB signalling pathway and the NLRP3 inflammasome. Finally, inhibition of NEK7 alleviated the cognitive impairment in APP/PS1 mice. In conclusion, Silencing NEK7 suppresses Aß42-induced cell apoptosis, inflammation and oxidative stress, and improves cognitive performance in AD mice. NEK7 may be a potential target for AD treatment.

A Bioinspired Skin UV Filter with Broadband UV Protection, Photostability, and Resistance to Oxidative Damage.

In recent years, sunscreens' adverse impacts on the environment and biology have gained wide attention. The improvement of sunscreen safety has become one of the major priorities in skin photoprotection research. It is an effective strategy to develop bionic photoprotective materials by simulating the photoprotective mechanism existing in nature. Inspired by the photoprotective mechanisms of skin and plant leaves, the bionic photoprotective material CS-SA-PDA nanosheet was developed using the free radical grafting method and Michael addition, with natural melanin analogue polydopamine (PDA) nanoparticles and plant sunscreen molecular sinapic acid (SA) as sun protection factors and natural polymer chitosan (CS) as the connecting arm. The results show that CS-SA-PDA can effectively shield UVB and UVA due to the possible synergistic effect between PDA and SA. The introduction of polymer CS significantly improved the photostability of SA and reduced the skin permeability of PDA nanoparticles. The CS-SA-PDA nanosheet can also effectively scavenge photoinduced free radicals. Furthermore, in vivo toxicity and anti-UV evaluations confirm that CS-SA-PDA has no skin irritation and is excellent against skin photodamage, which makes it an ideal skin photoprotective material.

Inhibition of circ_0004381 improves cognitive function via miR-647/PSEN1 axis in an Alzheimer disease mouse model.

Circ_0004381 promotes neuronal damage in Parkinson disease, but its role in Alzheimer disease (AD) is unreported. The goal of this study was to investigate the role and potential mechanisms of circ_0004381 effects in AD models. Primary hippocampal neurons were treated with amyloid-ß (Aß1-42) to construct AD cell models. We found that circ_0004381 was upregulated in Aß1-42-treated hippocampal neurons. Knockdown of circ_0004381 attenuated Aß1-42-induced apoptosis, oxidative stress, and mitochondrial dysfunction in hippocampal neurons. Next, we induced microglia activation with lipopolysaccharide (LPS). The results of flow cytometry experiments showed that knockdown of circ_0004381 promoted microglial M2-type polarization and knockdown of circ_0004381 inhibited the production of inflammatory factors by microglia. Furthermore, knockdown of circ_0004381 improved cognitive function of male APPswe/PS1dE9 transgenic mice. Mechanistically, circ_0004381 regulated presenilin-1 (PSEN1) expression by absorbing miR-647. MiR-647 inhibition attenuated the effects of circ_0004381 knockdown. In conclusion, knockdown of circ_0004381 attenuated hippocampal neuronal damage and promoted microglia M2-type polarization through the miR-647/PSEN1 axis, ultimately improving cognitive function in AD model mice.

Analysis of the miRNA-mRNA Regulatory Network Reveals the Biomarker Genes in the Progression of Myocardial Ischemic Reperfusion.

Objective: Cardiac injury induced by myocardial ischemic reperfusion (MI/R) is still an intractable question in clinical, and it has been confirmed as a major reason for the development of cardiovascular disease. Bioinformatics analysis has been widely used for revealing the pathogenic mechanism of diseases. This study attempted to identify the biomarkers and reveal the regulation mechanism of MI/R injury via bioinformatics analysis. Methods: The GSE67308 and GSE74951 were obtained from the GEO database. The datasets were analyzed with GEO2R tool, and the genes with |logFC| > 2 and p value <0.05 were identified as the differentially expressed genes (DEGs). The enrichment analysis of the DEGs was performed with the DAVID database and R language. Moreover, the protein-protein interaction (PPI) network of DEGs was performed with the STRING database and then visualized with Cytoscape. Result: The results showed that 195 downregulated mRNAs and 240 downregulated mRNAs were found in GSE67308, and 11 miRNAs were found in GSE7495. 152 common genes were screened in DEGs of GSE67308 and the targets of 11 miRNAs in GSE7495. Moreover, the enrichment analysis showed that the common genes were related with inflammatory response, immune response, PI3K/AKT, NF-κB, and TNF pathways. Besides, mmu-miR-92a-3p and mmu-miR-27b-3p were identified as the hubs miRNAs, and TNF, IL1B, and IFG1 were screened as the key nodes. Conclusion: This study established a miRNA-mRNA network for cardiac injury induced by MI/R and provided the evidence concerning the molecular mechanism of MI/R injury, which provided some reference for MI/R treatment.

Protective Effect of Isopulegol in Alleviating Neuroinflammation in Lipopolysaccharide-Induced BV-2 Cells and in Parkinson Disease Model Induced with MPTP.

BACKGROUND: Parkinson's disease (PD) is the most prevalent disease linked with age-associated neuronal degeneration. Phytotherapeutic compounds or agents have gained increased importance because of their increased specificity and minimal side effects. Isopulegol, a monoterpene, was utilized in the present study because of its wide range of therapeutic properties. Our aim was to examine the underlying mechanism of anti-neuroinflammatory action and neuroprotective efficacy of isopulegol in cell lines and in an experimental animal model of PD. METHODS: The MTT assay was performed in microglial BV-2 cells subjected to lipopolysaccharides (LPS). The release of NO and synthesis of ROS intracellularly in BV-2 cells were detected. C57BL/6 mice induced with MPTP were examined for motor function and coordination. Expression of proinflammatory mediators was also assessed both in vivo and in vitro. Histopathological sections of brain and expression of iNOS and COX-2 were also analyzed. RESULTS: BV-2 cells did not exhibit noticeable toxicity at selected concentrations and LPS-incubated cells showed marked elevation of NO levels and increased production of intracellular ROS. Increased expression of proinflammatory cytokines was also observed. Motor function and coordination deficits were observed in mice induced with MPTP. Histopathological abnormalities and increased iNOS and COX-2 expression were noted in MPTP-induced mice. Administration of isopulegol reversed the changes brought about by LPS and MPTP. CONCLUSION: The study indicated that isopulegol is a potential therapeutic drug against clinical complications of PD.

Functional composite hydrogels entrapping polydopamine hollow nanoparticles for highly efficient resistance of skin penetration and photoprotection.

Living organisms tend to evolve various naturally photoprotective mechanisms to avoid photodamage. Among them, polydopamine (PDA) is an effective sunscreen, a mimic of melanin, which is the main functional component of the photoprotective system of human skin. However, the concerns of its dark color, skin penetration and photoprotective efficiency remain yet to be solved. Herein, we have constructed melanin-inspired nanocomposite hydrogels (CS-PDAh-GP-HA) for photoprotection, in which PDA was prepared as hollow nanoparticles (PDAh NPs) and entrapped in a physically cross-linked hydrogel (CS-GP-HA) formed by chitosan (CS) and hyaluronic acid (HA) using ß-glycerophosphate (ß-GP) as a modulator. The CS-PDAh-GP-HA hydrogels exhibit a shear-thinning flow behavior with an elastic modulus of 300 Pa with the gel-sol transition temperature maintained at about 37 °C simply by adjusting the ß-GP content in the hydrogels. The CS-PDAh-GP-HA hydrogels also possess excellent resistance toward skin penetration. The photoprotective performances of CS-PDAh-GP-HA hydrogels were evaluated by the determination of sun protection factor (SPF) and in vitro UVA protection efficacy (UVAPE) along with UV-Vis spectroscopy. Compared with the TiO2 nanoparticles in CS-GP-HA hydrogel, the CS-PDAh-GP-HA hydrogels show stronger shielding ability in both UVA and UVB regions. When protected by the CS-PDAh-GP-HA hydrogels, the cell viability of NIH-3T3 fibroblasts increases to 96% while it was only 14% in the case of non-protecting group. These results suggest that the CS-PDAh-GP-HA hydrogels could efficiently shield the UV irradiation and protect the skin from photodamage. This work introduces PDA-based nanocomposite hydrogels with safe, biocompatible and photoprotective properties, and provides a melanin-mimicking photoprotection system for the application in sunscreens.

AITC induces MRP1 expression by protecting against CS/CSE-mediated DJ-1 protein degradation via activation of the DJ-1/Nrf2 axis.

The present study aimed to examine the effect of allyl isothiocyanate (AITC) on chronic obstructive pulmonary disease and to investigate whether upregulation of multidrug resistance-associated protein 1 (MRP1) associated with the activation of the PARK7 (DJ-1)/nuclear factor erythroid 2-related factor 2 (Nrf2) axis. Lung function indexes and histopathological changes in mice were assessed by lung function detection and H&E staining. The expression levels of Nrf2, MRP1, heme oxygenase-1 (HO-1), and DJ-1 were determined by immunohistochemistry, Western blotting and reverse transcription-quantitative polymerase chain reaction. Next, the expression of DJ-1 in human bronchial epithelial (16HBE) cells was silenced by siRNA, and the effect of DJ-1 expression level on cigarette smoke extract (CSE)-stimulated protein degradation and AITC-induced protein expression was examined. The expression of DJ-1, Nrf2, HO-1, and MRP1 was significantly decreased in the wild type model group, while the expression of each protein was significantly increased after administration of AITC. Silencing the expression of DJ-1 in 16HBE cells accelerated CSE-induced protein degradation, and significantly attenuated the AITC-induced mRNA and protein expression of Nrf2 and MRP1. The present study describes a novel mechanism by which AITC induces MRP1 expression by protecting against CS/CSEmediated DJ-1 protein degradation via activation of the DJ-1/Nrf2 axis.

TIPE regulates VEGFR2 expression and promotes angiogenesis in colorectal cancer.

Background: Metastasis is the leading cause of death in colorectal cancer (CRC) patients. It is regulated mainly by tumor cell angiogenesis, and angiogenesis is caused by the binding of vascular endothelial growth factor (VEGF) to vascular endothelial growth factor receptor 2 (VEGFR2). Tumor necrosis factor-α-induced protein 8 (TNFAIP8, hereto after TIPE) plays an important role in tumorigenesis, development, and prognosis. However, the relationship between TIPE and VEGFR2 in CRC angiogenesis and the mechanism of action remain unknown. Method: In this study, we used quantitative real-time PCR, Western blotting and immunohistochemistry to detect TIPE and VEGFR2 expression in 55 specimens from CRC patients. We also used HCT116 CRC cells and human umbilical vein endothelial cells (HUVECs) for in vitro experiments by stably transducing shTIPE and shRNA control lentivirus into HCT116 cells, detecting VEGFR2 expression after TIPE knockdown and repurposing the culture supernatant as conditioned medium to stimulate angiogenesis of HUVECs. In vivo experiments with chicken chorioallantoic membranes (CAMs) and a nude mouse matrix subcutaneous tumor model were performed to validate the effects of TIPE on angiogenesis. Additionally, we analyzed the expression and phosphorylation levels of PDK1 and blocked PDK1 expression using inhibitors to determine whether TIPE-induced changes in VEGFR2-mediated angiogenesis acted via the PI3K-Akt pathway. Results: We found that TIPE and VEGFR2 are highly expressed in CRC and act as oncogenes. TIPE knockdown also downregulated VEGFR2 expression, which resulted in simultaneous inhibition of cell proliferation, cell migration and angiogenesis. Then, in vivo experiments further demonstrated that TIPE promotes angiogenesis in CRC. Finally, we found that TIPE promotes VEGFR2-mediated angiogenesis by upregulating PDK1 expression and phosphorylation and that blocking PDK1 expression can inhibit this process. Conclusion: TIPE promotes angiogenesis in CRC by regulating the expression of VEGFR2, which may be a target for antiangiogenic cancer therapy.

Allyl isothiocyanate upregulates MRP1 expression through Notch1 signaling in human bronchial epithelial cells.

Multidrug resistance associated protein-1 (MRP1) and Notch signaling are closely related and both play a critical role in chronic obstructive pulmonary disease (COPD) establishment and progression. The aim of our work was to test whether Notch1 is involved in allyl isothiocyanate (AITC) induced MRP1 expression. We used cigarette smoke extract (CSE) to simulate the smoking microenvironment in vitro. The results demonstrated that CSE led to apoptosis as well as reduced the expression of Notch1, Hes1, and MRP1, while AITC significantly reversed this downregulation. Transfected with Notch1 siRNA downregulated MRP1 expression and activity, aggravated the suppression effect by CSE, and abolished the AITC-induced Notch1, Hes1, and MRP1 expression. Validation of the correlation between Notch1 and MRP1 was implemented by gel-shift assays (electrophoretic mobility shift assay). The result revealed an interaction between a specific promoter region of MRP1 and the intracellular domain of Notch1. In conclusion, Notch1 signaling positively regulated MRP1 in 16HBE cells and AITC induced MRP1 expression and function may be attributed to Notch1 signaling. These findings show that Notch1 and MRP1 might have a potential protective effect in the COPD process and become a new therapeutic target for COPD or other lung diseases. It also provides a theoretical basis for the therapeutic effects of AITC.

Preparation and in vitro/in vivo evaluation of azilsartan osmotic pump tablets based on the preformulation investigation.

The objective of this study was to design and evaluate azilsartan osmotic pump tablets. Preformulation properties of azilsartan were investigated for formulation design. Azilsartan osmotic pump tablets were prepared by incorporation of drug in the core and subsequent coating with cellulose acetate and polyethylene glycol 4000 as semi-permeable membrane, then drilled an orifice at the center of one side. The influence of different cores, compositions of semipermeable membrane and orifice diameter on azilsartan release were evaluated. The formulation of core tablet was optimized by orthogonal design and the release profiles of various formulations were evaluated by similarity factor (f2). The optimal formulation achieved to deliver azilsartan at an approximate zero-order up to 14 h. The pharmacokinetic study was performed in beagle dogs. The azilsartan osmotic pump tablets exhibited less fluctuation in blood concentration and higher bioavailability compared to immediate-release tablets. Moreover, there was a good correlation between the in vitro dissolution and in vivo absorption of the tablets. In summary, azilsartan osmotic pump tablets presented controlled release in vitro, high bioavailability in vivo and a good in vitro-in vivo correlation.

Pharmacokinetics, Tissue Distribution, Plasma Protein Binding Studies of 10-Dehydroxyl-12-Demethoxy-Conophylline, a Novel Anti-Tumor Candidate, in Rats.

10-Dehydroxyl-12-demethoxy-conophylline is a natural anticancer candidate. The motivation of this study was to explore the pharmacokinetic profiles, tissue distribution, and plasma protein binding of 10-dehydroxyl-12-demethoxy-conophylline in Sprague Dawley rats. A rapid, sensitive, and specific ultra-performance liquid chromatography (UPLC) system with a fluorescence (FLR) detection method was developed for the determination of 10-dehydroxyl-12-demethoxy-conophylline in different rat biological samples. After intravenous (i.v.) dosing of 10-dehydroxyl-12-demethoxy-conophylline at different levels (4, 8, and 12 mg/kg), the half-life t1/2α of intravenous administration was about 7 min and the t1/2ß was about 68 min. The AUC0→∞ increased in a dose-proportional manner from 68.478 µg/L·min for 4 mg/kg to 305.616 mg/L·min for 12 mg/kg. After intragastrical (i.g.) dosing of 20 mg/kg, plasma levels of 10-dehydroxyl-12-demethoxy-conophylline peaked at about 90 min. 10-dehydroxyl-12-demethoxy-conophyllinea absolute oral bioavailability was only 15.79%. The pharmacokinetics process of the drug was fit to a two-room model. Following a single i.v. dose (8 mg/kg), 10-dehydroxyl-12-demethoxy-conophylline was detected in all examined tissues with the highest in kidney, liver, and lung. Equilibrium dialysis was used to evaluate plasma protein binding of 10-dehydroxyl-12-demethoxy-conophylline at three concentrations (1.00, 2.50, and 5.00 µg/mL). Results indicated a very high protein binding degree (over 80%), reducing substantially the free fraction of the compound.

PEG-lipid-PLGA hybrid nanoparticles loaded with berberine-phospholipid complex to facilitate the oral delivery efficiency.

The natural product berberine (BBR), present in various plants, arouses great interests because of its numerous pharmacological effects. However, the further development and application of BBR had been hampered by its poor oral bioavailability. In this work, we report on polymer-lipid hybrid nanoparticles (PEG-lipid-PLGA NPs) loaded with BBR phospholipid complex using a solvent evaporation method for enhancing the oral BBR efficiency. The advantage of this new drug delivery system is that the BBR-soybean phosphatidylcholine complex (BBR-SPC) could be used to enhance the liposolubility of BBR and improve the affinity with the biodegradable polymer to increase the drug-loading capacity and controlled/sustained release. The entrapment efficiency of the PEG-lipid-PLGA NPs/BBR-SPC was observed to approach approximately 89% which is more than 2.4 times compared with that of the PEG-lipid-PLGA NPs/BBR. To the best of our knowledge, this is the first report on using polymer material for effective encapsulation of BBR to improve its oral bioavailability. The prepared BBR delivery systems demonstrated a uniform spherical shape, a well-dispersed core-shell structure and a small particle size (149.6 ± 5.1 nm). The crystallographic and thermal analysis has indicated that the BBR dispersed in the PEG-lipid-PLGA NPs matrix is in an amorphous form. More importantly, the enhancement in the oral relative bioavailability of the PEG-lipid-PLGA NPs/BBR-SPC was ∼343% compared with that of BBR. These positive results demonstrated that PEG-lipid-PLGA NPs/BBR-SPC may have the potential for facilitating the oral drug delivery of BBR.

Expression profiles of prostaglandin E2 receptor subtypes in aspirin tolerant adult Chinese with chronic rhinosinusitis.

BACKGROUND: Several studies have indicated that prostaglandin E2 and E-prostanoid (EP) receptors play a role in the pathogenesis of chronic rhinosinusitis (CRS) in white populations. However, until now there was no report about EP receptor expression and its role in the pathophysiology of CRS in Chinese patients. OBJECTIVE: To investigate the expression profiles of EP receptors, including EP1, EP2, EP3, and EP4 receptors in different Chinese patients with CRS with aspirin tolerance. METHODS: Nasal biopsy specimens were obtained from 12 controls, 12 patients with CRS without nasal polyps (CRSsNP), 12 with eosinophilic CRS with nasal polyps (CRSwNP), and 16 with noneosinophilic CRSwNP. Histopathologic characteristics were observed under a light microscope. Immunostaining was used to examine tissue localization of EP receptors. Messenger RNA and protein expression of EP receptors were examined by means of quantitative RT-polymerase chain reaction and Western blot, respectively. RESULTS: Different types of CRS presented different histopathologic hallmarks. EP receptors were expressed mainly on epithelium, glands, and infiltrating inflammatory cells in nasal tissue. In controls, patients with CRSsNP, and those with noneosinophilic CRSwNP, EP4 mRNA levels were higher than EP1, EP2, and EP3 receptors. EP2 was downexpressed, and EP1 was upexpressed in patients with eosinophilic CRSwNP. When comparing EP receptor expression among different groups, Messenger RNA and protein of EP1 receptor were significantly enhanced in eosinophilic CRSwNP, but EP2, EP3, and EP4 receptors did not show significant differences. CONCLUSION: EP receptor expressions present different features in healthy subjects and patients with CRS. The upregulated EP1 receptor in eosinophilic CRSwNP might be associated with excessive infiltrations of eosinophils and other inflammatory cells. The accurate role of the four EP receptors in the pathogenesis of different CRS remains to be further explored.

A multifunctional ß-CD-modified Fe3O4@ZnO:Er(3+),Yb(3+) nanocarrier for antitumor drug delivery and microwave-triggered drug release.

We constructed a novel core-shell structured Fe3O4@ZnO:Er(3+),Yb(3+)@(ß-CD) nanoparticles used as drug carrier to investigate the loading and controllable release properties of the chemotherapeutic drug etoposide (VP-16). The cavity of ß-cyclodextrin is chemically inert, it can store etoposide molecules by means of hydrophobic interactions. The Fe3O4 core and ZnO:Er(3+),Yb(3+) shell functioned successfully for magnetic targeting and up-conversion fluorescence imaging, respectively. In addition, the ZnO:Er(3+),Yb(3+) shell acts as a good microwave absorber with excellent microwave thermal response property for microwave triggered drug release (the VP-16 release of 18% under microwave irradiation for 15 min outclass the 2% within 6h without microwave irradiation release). The release profile could be controlled by the duration and number of cycles of microwave application. This material therefore promises to be a useful noninvasive, externally controlled drug-delivery system in cancer therapy.

Gratitude and suicidal ideation and suicide attempts among Chinese adolescents: direct, mediated, and moderated effects.

In a sample of 1252 Chinese adolescents (mean age = 15.00 years), this study examined the direct relations between gratitude and adolescents' suicidal ideation and suicide attempts. This study also examined indirect relations between gratitude and suicidal ideation and suicide attempts via two self-system beliefs--coping efficacy and self-esteem. Finally, this study examined the extent to which stressful life events moderated the direct and indirect relations between gratitude and suicidal ideation and suicide attempts. The odds of suicidal ideation and suicide attempts were lower among adolescents who scored higher on gratitude, after controlling for demographic variables. Self-esteem mediated the relations between gratitude and suicidal ideation and suicide attempts, while the mediating role of coping efficacy was not significant. Moreover, stressful life events moderated the mediated path through self-esteem. This indirect effect was stronger for adolescents low on stressful life events than that for those high on stressful life events. This study discusses the theoretical and practical implications of these findings.