Confounding amplifies the effect of environmental factors on COVID-19.

The global COVID-19 pandemic has severely impacted human health and socioeconomic development, posing an enormous public health challenge. Extensive research has been conducted into the relationship between environmental factors and the transmission of COVID-19. However, numerous factors influence the development of pandemic outbreaks, and the presence of confounding effects on the mechanism of action complicates the assessment of the role of environmental factors in the spread of COVID-19. Direct estimation of the role of environmental factors without removing the confounding effects will be biased. To overcome this critical problem, we developed a Double Machine Learning (DML) causal model to estimate the debiased causal effects of the influencing factors in the COVID-19 outbreaks in Chinese cities. Comparative experiments revealed that the traditional multiple linear regression model overestimated the impact of environmental factors. Environmental factors are not the dominant cause of widespread outbreaks in China in 2022. In addition, by further analyzing the causal effects of environmental factors, it was verified that there is significant heterogeneity in the role of environmental factors. The causal effect of environmental factors on COVID-19 changes with the regional environment. It is therefore recommended that when exploring the mechanisms by which environmental factors influence the spread of epidemics, confounding factors must be handled carefully in order to obtain clean quantitative results. This study offers a more precise representation of the impact of environmental factors on the spread of the COVID-19 pandemic, as well as a framework for more accurately quantifying the factors influencing the outbreak.

Microneedle Array Electrodes Fabricated With 3D Printing Technology for High-Quality Electrophysiological Acquisition.

Electrophysiological recordings are vital in assessing biological functions, diagnosing diseases, and facilitating biofeedback and rehabilitation. The applications of conventional wet (gel) electrodes often come with some limitations. Microneedle array electrodes (MAEs) present a possible solution for high-quality electrophysiological acquisition, while the prior technologies for MAE fabrication have been either complex, expensive, or incapable of producing microneedles with uniform dimensions. This work employed a projection stereolithography (P µ SL) 3D printing technology to fabricate MAEs with micrometer-level precision. The MAEs were compared with gel and flat electrodes on electrode-skin interface impedance (EII) and performances of EMG and ECG acquisition. The experimental results indicate that the P µ SL 3D printing technology contributed to an easy-to-perform and low-cost fabrication approach for MAEs. The developed MAEs exhibited promising EII and enabled a stable EMG and ECG acquisition in different conditions without inducing skin allergies, inflammation, or injuries. This research lies in the development of a type of customizable MAE with considerable biomedical application potentials for ultra-minimally invasive or non-invasive electrophysiological acquisition.

Mediating role of depression in linking rheumatoid arthritis to all-cause and cardiovascular-related mortality: A prospective cohort study.

OBJECTIVE: This study aimed to analyze the associations between rheumatoid arthritis (RA) and all-cause mortality and cardiovascular disease (CVD)-related mortality using data from the National Health and Nutrition Examination Survey (NHANES) and examine the potential mediating role of depression in these correlations. METHODS: 19,165 participants across five NHANES cycles from 2007 to 2016 participated in this study. Multifactorial Cox regression models between RA, depression and two mortality outcomes and multifactorial regression models between RA and depression were constructed to examine their associations. The mediating role of depression has also been investigated. RESULTS: The prevalence of RA in this study was 6.57 %, the all-cause mortality of RA patients was 20.57 %, and the CVD-related mortality was 6.12 %. In the fully adjusted model, RA was associated with all-cause mortality [hazard ratio (HR) = 1.28, 95 % confidence interval (CI) = 1.12 to 1.48] and CVD-related mortality (HR = 1.33, 95 % CI = 1.03 to 1.72), without detectable interaction among subgroups (P for interaction >0.05). RA also had a positive correlation with depression. Depression score demonstrated pronounced mediating effects in the connections between RA and two types of mortality, with mediation ratios of 18.2 % and 18.9 %. LIMITATIONS: The diagnosis of RA is self-reported and may be subject to recall bias. CONCLUSIONS: RA was positively correlated with the risk of all-cause mortality and CVD-related mortality. Depression partially mediates these associations. Close attention to and active improvement of mental health in RA patients will be critical to decrease all-cause mortality and CVD-related mortality.

Development of the second version of Global Prediction System for Epidemiological Pandemic.

Coronavirus disease 2019 (COVID-19) is a severe global public health emergency that has caused a major crisis in the safety of human life, health, global economy, and social order. Moreover, COVID-19 poses significant challenges to healthcare systems worldwide. The prediction and early warning of infectious diseases on a global scale are the premise and basis for countries to jointly fight epidemics. However, because of the complexity of epidemics, predicting infectious diseases on a global scale faces significant challenges. In this study, we developed the second version of Global Prediction System for Epidemiological Pandemic (GPEP-2), which combines statistical methods with a modified epidemiological model. The GPEP-2 introduces various parameterization schemes for both impacts of natural factors (seasonal variations in weather and environmental impacts) and human social behaviors (government control and isolation, personnel gathered, indoor propagation, virus mutation, and vaccination). The GPEP-2 successfully predicted the COVID-19 pandemic in over 180 countries with an average accuracy rate of 82.7%. It also provided prediction and decision-making bases for several regional-scale COVID-19 pandemic outbreaks in China, with an average accuracy rate of 89.3%. Results showed that both anthropogenic and natural factors can affect virus spread and control measures in the early stages of an epidemic can effectively control the spread. The predicted results could serve as a reference for public health planning and policymaking.

An overview for monitoring and prediction of pathogenic microorganisms in the atmosphere.

Corona virus disease 2019 (COVID-19) has exerted a profound adverse impact on human health. Studies have demonstrated that aerosol transmission is one of the major transmission routes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Pathogenic microorganisms such as SARS-CoV-2 can survive in the air and cause widespread infection among people. Early monitoring of pathogenic microorganism transmission in the atmosphere and accurate epidemic prediction are the frontier guarantee for preventing large-scale epidemic outbreaks. Monitoring of pathogenic microorganisms in the air, especially in densely populated areas, may raise the possibility to detect viruses before people are widely infected and contain the epidemic at an earlier stage. The multi-scale coupled accurate epidemic prediction system can provide support for governments to analyze the epidemic situation, allocate health resources, and formulate epidemic response policies. This review first elaborates on the effects of the atmospheric environment on pathogenic microorganism transmission, which lays a theoretical foundation for the monitoring and prediction of epidemic development. Secondly, the monitoring technique development and the necessity of monitoring pathogenic microorganisms in the atmosphere are summarized and emphasized. Subsequently, this review introduces the major epidemic prediction methods and highlights the significance to realize a multi-scale coupled epidemic prediction system by strengthening the multidisciplinary cooperation of epidemiology, atmospheric sciences, environmental sciences, sociology, demography, etc. By summarizing the achievements and challenges in monitoring and prediction of pathogenic microorganism transmission in the atmosphere, this review proposes suggestions for epidemic response, namely, the establishment of an integrated monitoring and prediction platform for pathogenic microorganism transmission in the atmosphere.

KR158 Spheres Harboring Slow-Cycling Cells Recapitulate High-Grade Glioma Features in an Immunocompetent System.

Glioblastoma (GBM) poses a significant challenge in clinical oncology due to its aggressive nature, heterogeneity, and resistance to therapies. Cancer stem cells (CSCs) play a critical role in GBM, particularly in treatment resistance and tumor relapse, emphasizing the need to comprehend the mechanisms regulating these cells. Also, their multifaceted contributions to the tumor microenvironment (TME) underline their significance, driven by their unique properties. This study aimed to characterize glioblastoma stem cells (GSCs), specifically slow-cycling cells (SCCs), in an immunocompetent murine GBM model to explore their similarities with their human counterparts. Using the KR158 mouse model, we confirmed that SCCs isolated from this model exhibited key traits and functional properties akin to human SCCs. KR158 murine SCCs, expanded in the gliomasphere assay, demonstrated sphere forming ability, self-renewing capacity, positive tumorigenicity, enhanced stemness and resistance to chemotherapy. Together, our findings validate the KR158 murine model as a framework to investigate GSCs and SCCs in GBM pathology, and explore specifically the SCC-immune system communications, understand their role in disease progression, and evaluate the effect of therapeutic strategies targeting these specific connections.

Product Enhanced Reverse Transcriptase for assessing replication competent virus in vectors retroviral vectors pseudotyped with GALV and VSV-G envelopes.

We evaluated the use of the Product Enhanced Reverse Transcriptase (PERT) assay as a means of detecting virus in retroviral vectors products pseudotyped with Gibbon Ape Leukemia Virus (GALV) and Vesicular Stomatitis Virus G (VSVG) envelopes. PERT provides greater standardization than the S+/L- assay which has been used extensively in virus detection. A challenge is that PERT will also detect residual retroviral vectors as vector particles contain reverse transcriptase. Vector products were cultured for 3 weeks on HEK293 cells to amplify any potential virus. In addition, vector supernatant and end-of-production cells were spiked with GALV to evaluate for inhibition by the test article. Results of PERT and the S+/L- assay were compared. PERT and S+/L- assays were both effective in detecting virus. Vector supernatants were negative at the end of 3 weeks of culture by PERT for both GAVL and VSVG pseudotyped vector. In contrast, end-of-production cells were positive by PERT due to persistent vector producing cells. A one-week culture of cell-free media obtained at the 3 weeks timepoint allowed distinction of virus-free test articles from those with virus. The PERT assay is suitable for detecting replication competent retrovirus in vector products pseudotyped with GALV and VSVG envelopes.

Asymmetric magma plumbing system beneath Axial Seamount based on full waveform inversion of seismic data.

The architecture of magma plumbing systems plays a fundamental role in volcano eruption and evolution. However, the precise configuration of crustal magma reservoirs and conduits responsible for supplying eruptions are difficult to explore across most active volcanic systems. Consequently, our understanding of their correlation with eruption dynamics is limited. Axial Seamount is an active submarine volcano located along the Juan de Fuca Ridge, with known eruptions in 1998, 2011, and 2015. Here we present high-resolution images of P-wave velocity, attenuation, and estimates of temperature and partial melt beneath the summit of Axial Seamount, derived from multi-parameter full waveform inversion of a 2D multi-channel seismic line. Multiple magma reservoirs, including a newly discovered western magma reservoir, are identified in the upper crust, with the maximum melt fraction of ~15-32% in the upper main magma reservoir (MMR) and lower fractions of 10% to 26% in other satellite reservoirs. In addition, a feeding conduit below the MMR with a melt fraction of ~4-11% and a low-velocity throat beneath the eastern caldera wall connecting the MMR roof with eruptive fissures are imaged. These findings delineate an asymmetric shallow plumbing system beneath Axial Seamount, providing insights into the magma pathways that fed recent eruptions.

Is refractive error a factor affecting scoliosis?

BACKGROUND: Scoliosis is one of the most common surgical disorders of the pediatric spine. Refractive errors are commonly associated with vision impairment worldwide. However, it is currently unclear whether refractive error correlates directly with the development of scoliosis. METHODS: A cross-sectional study was performed in 2023, and a stratified cluster sampling technique was employed among school-aged students in Nantong City, China. Univariate and multivariate logistic regression analyses were used to investigate specific correlations between scoliosis and related parameters; various types of refractive errors were also included in the study. RESULTS: The prevalence of scoliosis among school-aged students was 2.2% in Nantong city. Multiple logistic regression analyses showed that myopia, hyperopia, astigmatism, and anisometropia were not correlated with the development of scoliosis (all, p≥0.05). Lower body mass index (BMI) [adjusted odds ratio (aOR) = 0.92; 95% confidence interval (CI): 0.88-0.95; p<0.001], living in rural areas (aOR = 1.40; 95% CI: 1.05-1.86; p = 0.020), and older age (aOR = 1.32; 95% CI: 1.25-1.38; p<0.001) had significantly higher risks of scoliosis. CONCLUSIONS: Refractive errors did not correlate with the development of scoliosis. However, BMI, living in rural areas and older age did correlate with the development of scoliosis.

RNA aggregates harness the danger response for potent cancer immunotherapy.

Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create "onion-like" multi-lamellar RNA lipid particle aggregates (LPAs) to substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became "hot" within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy.

Reconciling East Asia's mid-Holocene temperature discrepancy through vegetation-climate feedback.

The term "Holocene temperature conundrum" refers to the inconsistencies between proxy-based reconstructions and transient model simulations, and it challenges our understanding of global temperature evolution during the Holocene. Climate reconstructions indicate a cooling trend following the Holocene Thermal Maximum, while model simulations indicate a consistent warming trend due to ice-sheet retreat and rising greenhouse gas concentrations. Various factors, such as seasonal biases and overlooked feedback processes, have been proposed as potential causes for this discrepancy. In this study, we examined the impact of vegetation-climate feedback on the temperature anomaly patterns in East Asia during the mid-Holocene (∼6 ka). By utilizing the fully coupled Earth system model EC-Earth and performing simulations with and without coupled dynamic vegetation, our objective was to isolate the influence of vegetation changes on regional temperature patterns. Our findings reveal that vegetation-climate feedback contributed to warming across most of East Asia, resulting in spatially diverse temperature changes during the mid-Holocene and significantly improved model-data agreement. These results highlight the crucial role of vegetation-climate feedback in addressing the Holocene temperature conundrum and emphasize its importance for simulating accurate climate scenarios.

Genomic and transcriptomic significance of multiple primary lung cancers detected by next-generation sequencing in clinical settings.

Effective diagnosis and understanding of the mechanism of intrapulmonary metastasis (IM) from multiple primary lung cancers (MPLC) aid clinical management. However, the actual detection panels used in the clinic are variable. Current research on tumor microenvironment (TME) of MPLC and IM is insufficient. Therefore, additional investigation into the differential diagnosis and discrepancies in TME between two conditions is crucial. Two hundred and fourteen non-small cell lung cancer patients with multiple tumors were enrolled and 507 samples were subjected to DNA sequencing (NGS 10). Then, DNA and RNA sequencing (master panel) were performed on the specimens from 32 patients, the TME profiles between tumors within each patient and across patients and the differentially expressed genes were compared. Four patients were regrouped with NGS 10 results. Master panel resolved the classifications of six undetermined patients. The TME in MPLC exhibited a high degree of infiltration by natural killer (NK) cells, CD56dim NK cells, endothelial cells, etc., P < 0.05. Conversely, B cells, activated B cells, regulatory cells, immature dendritic cells, etc., P < 0.001, were heavily infiltrated in the IM. NECTIN4 and LILRB4 mRNA were downregulated in the MPLC (P < 0.0001). Additionally, NECTIN4 (P < 0.05) and LILRB4 were linked to improved disease-free survival in the MPLC. In conclusion, IM is screened from MPLC by pathology joint NGS 10 detections, followed by a large NGS panel for indistinguishable patients. A superior prognosis of MPLC may be associated with an immune-activating TME and the downregulation of NECTIN4 and LILRB4 considered as potential drug therapeutic targets.

Smart osteoclasts targeted nanomedicine based on amorphous CaCO3 for effective osteoporosis reversal.

BACKGROUND: Osteoporosis is characterized by an imbalance in bone homeostasis, resulting in the excessive dissolution of bone minerals due to the acidified microenvironment mediated by overactive osteoclasts. Oroxylin A (ORO), a natural flavonoid, has shown potential in reversing osteoporosis by inhibiting osteoclast-mediated bone resorption. The limited water solubility and lack of targeting specificity hinder the effective accumulation of Oroxylin A within the pathological environment of osteoporosis. RESULTS: Osteoclasts' microenvironment-responsive nanoparticles are prepared by incorporating Oroxylin A with amorphous calcium carbonate (ACC) and coated with glutamic acid hexapeptide-modified phospholipids, aiming at reinforcing the drug delivery efficiency as well as therapeutic effect. The obtained smart nanoparticles, coined as OAPLG, could instantly neutralize acid and release Oroxylin A in the extracellular microenvironment of osteoclasts. The combination of Oroxylin A and ACC synergistically inhibits osteoclast formation and activity, leading to a significant reversal of systemic bone loss in the ovariectomized mice model. CONCLUSION: The work highlights an intelligent nanoplatform based on ACC for spatiotemporally controlled release of lipophilic drugs, and illustrates prominent therapeutic promise against osteoporosis.

Advancements in chimeric antigen receptor-expressing T-cell therapy for glioblastoma multiforme: Literature review and future directions.

Glioblastoma multiforme (GBM) is an aggressive cancer that has been difficult to treat and often requires multimodal therapy consisting of surgery, radiotherapy, and chemotherapy. Chimeric antigen receptor-expressing (CAR-T) cells have been efficacious in treating hematological malignancies, resulting in several FDA-approved therapies. CAR-T cells have been more recently studied for the treatment of GBM, with some promising preclinical and clinical results. The purpose of this literature review is to highlight the commonly targeted antigens, results of clinical trials, novel modifications, and potential solutions for challenges that exist for CAR-T cells to become more widely implemented and effective in eradicating GBM.

Modified patient-specific surgical-guide-assisted precise treatment of unilateral comminuted zygomaticomaxillary complex fractures: A 5-year retrospective study.

Our study aimed to evaluate modified patient-specific surgical-guide-assisted precise treatment of unilateral comminuted zygomaticomaxillary complex (ZMC) fractures. The retrospective non-randomized study was conducted in a single hospital in China. All patients diagnosed with unilateral comminuted ZMC fractures between January 1, 2018 and December 31, 2022 were retrospectively reviewed. All patients underwent preoperative spiral computed tomography (CT). CT data were processed using software to DICOM format and transferred to Proplan CMF3.0 for preoperative virtual surgical planning and postoperative evaluation. All data were extracted from standardized electronic medical records. All statistical analyses were performed using SPSS version 20.0. The chi-square test and t-test were used for statistical analyses. The 54 included patients were divided into two comparable, equal cohorts of 27 patients, and followed up for at least 6 months. Fracture reduction was assisted using the modified patient-specific surgical guides in the guide group (23 males, four females; mean age 37.74 ± 12.07 years) and without the modified patient-specific surgical guides in the control group (20 males, seven females; mean age 37.44 ± 13.58 years). In the guide group, the mean eminence deviation between the affected and unaffected sides was 1.01 ± 0.92 mm, and the mean width deviation between the affected and unaffected sides was 1.29 ± 1.32 mm. In the control group, the mean eminence deviation between the affected and unaffected sides was 1.99 ± 1.69 mm, and the mean width deviation between the affected and unaffected sides was 2.68 ± 2.01 mm. The differences in facial protrusion (p = 0.001) and width (p = 0.003) symmetry between the affected and healthy sides of the two groups were statistically significant (p < 0.05). In conclusion, applying the modified patient-specific surgical guides to unilateral comminuted zygomaticomaxillary complex fracture reduction has the advantages of greater predictability and effectiveness, and improved bilateral ZMC symmetry. It should be noted that this approach would be especially beneficial for less-experienced surgeons.

KR158 spheres harboring slow-cycling cells recapitulate GBM features in an immunocompetent system.

Glioblastoma (GBM) poses a significant challenge in clinical oncology due to its aggressive nature, heterogeneity, and resistance to therapies. Cancer stem cells (CSCs) play a critical role in GBM, particularly in treatment-resistance and tumor relapse, emphasizing the need to comprehend the mechanisms regulating these cells. Also, their multifaceted contributions to the tumor-microenvironment (TME) underline their significance, driven by their unique properties. This study aimed to characterize glioblastoma stem cells (GSCs), specifically slow-cycling cells (SCCs), in an immunocompetent murine GBM model to explore their similarities with their human counterparts. Using the KR158 mouse model, we confirmed that SCCs isolated from this model exhibited key traits and functional properties akin to human SCCs. KR158 murine SCCs, expanded in the gliomasphere assay, demonstrated sphere forming ability, self-renewing capacity, positive tumorigenicity, enhanced stemness and resistance to chemotherapy. Together, our findings validate the KR158 murine model as a framework to investigate GSCs and SCCs in GBM-pathology, and explore specifically the SCC-immune system communications, understand their role in disease progression, and evaluate the effect of therapeutic strategies targeting these specific connections.

The role of mountains in shaping the global meridional overturning circulation.

The meridional overturning circulation (MOC) in the ocean is a key player in the global climate system, while continental topography provides an essential backdrop to the system. In this study, we design a series of coupled model sensitivity experiments to investigate the influence of various mountain ranges on the global thermohaline circulation. The results highlight the influence of the Tibetan Plateau (TP) on the global thermohaline circulation. It emerges as a requisite for establishing the Atlantic MOC (AMOC) and a determining factor for the cessation of the Pacific MOC (PMOC). Additionally, the Antarctic continent plays a vital role in facilitating the TP to form the AMOC. While the formation of the AMOC cannot be attributed to any single mountain range, the TP alone can inhibit the PMOC's development. By modifying the global hydrological cycle, the TP is likely to have been crucial in molding the global thermohaline circulation.

Identification and characterization of camptothecin tailoring enzymes in Nothapodytes tomentosa.

Camptothecin is a complex monoterpenoid indole alkaloid with remarkable antitumor activity. Given that two C-10 modified camptothecin derivatives, topotecan and irinotecan, have been approved as potent anticancer agents, there is a critical need for methods to access other aromatic ring-functionalized congeners (e.g., C-9, C-10, etc.). However, contemporary methods for chemical oxidation are generally harsh and low-yielding when applied to the camptothecin scaffold, thereby limiting the development of modified derivatives. Reported herein, we have identified four tailoring enzymes responsible for C-9 modifications of camptothecin from Nothapodytes tomentosa, via metabolomic and transcriptomic analysis. These consist of a cytochrome P450 (NtCPT9H) which catalyzes the regioselective oxidation of camptothecin to 9-hydroxycamptothecin, as well as two methyltransferases (NtOMT1/2, converting 9-hydroxycamptothecin to 9-methoxycamptothecin), and a uridine diphosphate-glycosyltransferase (NtUGT5, decorating 9-hydroxycamptothecin to 9-ß-D-glucosyloxycamptothecin). Importantly, the critical residues that contribute to the specific catalytic activity of NtCPT9H have been elucidated through molecular docking and mutagenesis experiments. This work provides a genetic basis for producing camptothecin derivatives through metabolic engineering. This will hasten the discovery of novel C-9 modified camptothecin derivatives, with profound implications for pharmaceutical manufacture.

Can the aerosol pollution extreme events be revealed by global reanalysis products?

Extreme aerosol pollution poses significant risks to the climate, environment, and human health. To investigate the formation and impacts of aerosol pollution extreme events (APEE), the reanalysis product presents meticulous spatiotemporal information on the three-dimensional distribution of aerosols. However, there is a lack of comprehensive evaluation and information regarding the data quality of reanalysis products employed in APEE research, as well as limited understanding of their spatial and temporal distribution, variation, and long-term trends. To address this scientific gap, we conducted a global study for distribution and variation patterns of APEE using two widely-used reanalysis products, MERRA-2 (Modern-Era Retrospective Analysis for Research-2) and CAMS (Copernicus Atmospheric Monitoring Service). The APEE was defined here as a day when the daily aerosol optical depth (AOD) exceeding its 90th percentile for a given station and month. Eleven distinct land regions worldwide were selected for evaluation by comparing both reanalysis products with MODIS satellite products and ground-based observations in terms of frequency, intensity, and temporal trends of APEE. The analysis indicates that MERRA-2 and CAMS exhibit high matching rates (70 % and 80 %, respectively) in terms of occurrence timeline for APEE at monthly and seasonal scales, while also exhibiting strong monthly correlation coefficients (>0.65) with ground-based observations over selected regions. The total AOD (-0.002 âˆ¼ -0.123 decade-1), APEE AOD (-0.004 âˆ¼ -0.293 decade-1), and APEE frequency (-0.264 âˆ¼ -1.769 day month-1 decade-1) of both observations and reanalysis products in most regions showed a decreasing trend with various magnitude, except for some regions such as South Asia where the trend is increasing. Based on the aforementioned evaluation, it is evident that reanalysis products are effective and useful in identifying the temporal trends associated with APEE.