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Introduction: Nicotine degradation is a new strategy to block nicotine-induced pathology. The potential of human microbiota to degrade nicotine has not been explored. Aims: This study aimed to uncover the genomic potentials of human microbiota to degrade nicotine. Methods: To address this issue, we performed a systematic annotation of Nicotine-Degrading Enzymes (NDEs) from genomes and metagenomes of human microbiota. A total of 26,295 genomes and 1,596 metagenomes for human microbiota were downloaded from public databases and five types of NDEs were annotated with a custom pipeline. We found 959 NdhB, 785 NdhL, 987 NicX, three NicA1, and three NicA2 homologs. Results: Genomic classification revealed that six phylum-level taxa, including Proteobacteria, Firmicutes, Firmicutes_A, Bacteroidota, Actinobacteriota, and Chloroflexota, can produce NDEs, with Proteobacteria encoding all five types of NDEs studied. Analysis of NicX prevalence revealed differences among body sites. NicX homologs were found in gut and oral samples with a high prevalence but not found in lung samples. NicX was found in samples from both smokers and non-smokers, though the prevalence might be different. Conclusion: This study represents the first systematic investigation of NDEs from the human microbiota, providing new insights into the physiology and ecological functions of human microbiota and shedding new light on the development of nicotine-degrading probiotics for the treatment of smoking-related diseases.
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Graphene oxide (GO) is a two-dimensional metastable nanomaterial. Interestingly, GO formed oxygen clusterings in addition to oxidized and graphitic phases during the low-temperature thermal annealing process, which could be further used for biomolecule bonding. By harnessing this property of GO, we created a bio-interface with patterned structures with a common laboratory hot plate that could tune cellular behavior by physical contact. Due to the regional distribution of oxygen clustering at the interface, we refer to it as patterned annealed graphene oxide (paGO). In addition, since the paGO was a heterogeneous interface and bonded biomolecules to varying degrees, arginine-glycine-aspartic acid (RGD) was modified on it and successfully regulated cellular-directed growth and migration. Finally, we investigated the FRET phenomenon of this heterogeneous interface and found that it has potential as a biosensor. The paGO interface has the advantages of easy regulation and fabrication, and the one-step thermal reduction method is suitable for biological applications. We believe that this low-temperature thermal annealing method would make GO interfaces more accessible, especially for the development of nano-interfacial modifications for biological applications, revealing its potential for biomedical applications.
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Background: Hypoxic conditions and Pseudomonas aeruginosa (P. aeruginosa) infection are significant factors influencing the prognosis and treatment of patients with bronchiectasis. This study aimed to explore the potential for breath analysis to detect hypoxic conditions and P. aeruginosa infection in bronchiectasis patients by analyzing of volatile organic compounds (VOCs) in exhaled breath condensate (EBC). Methods: EBC samples were collected from stable bronchiectasis patients and analyzed using solid phase microextraction-gas chromatography-mass spectrometry (SPME-GCMS). The association of VOCs with bronchiectasis patients' phenotypes including hypoxic conditions and P. aeruginosa isolation was analyzed, which may relate to the severity of bronchiectasis disease. Results: Levels of 10-heptadecenoic acid, heptadecanoic acid, longifolene, and decanol in the hypoxia group were higher compared to the normoxia group. Additionally, the levels of 13-octadecenoic acid, octadecenoic acid, phenol, pentadecanoic acid, and myristic acid were increased in P. aeruginosa (+) group compared to the P. aeruginosa (-) group. Subgroup analysis based on the bronchiectasis severity index (BSI)reveled that the levels of 10-heptadecenoic acid, heptadecanoic acid, decanol, 13-octadecenoic acid, myristic acid, and pentadecanoic acid were higher in the severe group compared to the moderate group. Multivariate linear regression showed that 10-heptadecenoic acid and age were independent prognostic factors for bronchiectasis patients with hypoxia. Furthermore, octadecenoic acid, phenol and gender were identified as independent prognostic factors for bronchiectasis patients with P. aeruginosa isolation. Conclusion: The study provides evidence that specific VOCs in EBC are correlated with the severity of bronchiectasis, and 10-heptadecenoic acid is shown to be a predictive marker for hypoxia condition in bronchiectasis patients.
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Patient-derived organoids (PDOs) developed ex vivo and in vitro are increasingly used for therapeutic screening. They provide a more physiologically relevant model for drug discovery and development compared to traditional cell lines. However, several challenges remain to be addressed to fully realize the potential of PDOs in therapeutic screening. This paper summarizes recent advancements in PDO development and the enhancement of PDO culture models. This is achieved by leveraging materials engineering and microfabrication technologies, including organs-on-a-chip and droplet microfluidics. Additionally, this work discusses the application of PDOs in therapy screening to meet diverse requirements and overcome bottlenecks in cancer treatment. Furthermore, this work introduces tools for data processing and analysis of organoids, along with their microenvironment. These tools aim to achieve enhanced readouts. Finally, this work explores the challenges and future perspectives of using PDOs in drug development and personalized screening for cancer patients.
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Aerobic granular sludge is one of the most promising biological wastewater treatment technologies, yet maintaining its stability is still a challenge for its application, and predicting the state of the granules is essential in addressing this issue. This study explored the potential of dynamic texture entropy, derived from settling images, as a predictive tool for the state of granular sludge. Three processes, traditional thickening, often overlooked clarification, and innovative particle sorting, were used to capture the complexity and diversity of granules. It was found that rapid sorting during settling indicates stable granules, which helps to identify the state of granules. Furthermore, a relationship between sorting time and granule heterogeneity was identified, helping to adjust selection pressure. Features of the dynamic texture entropy well correlated with the respirogram, i.e., R2 were 0.86 and 0.91 for the specific endogenous respiration rate (SOURe) and the specific quasi-endogenous respiration rate (SOURq), respectively, providing a biologically based approach for monitoring the state of granules. The classification accuracy of models using features of dynamic texture entropy as an input was greater than 0.90, significantly higher than the input of conventional features, demonstrating the significant advantage of this approach. These findings contributed to developing robust monitoring tools that facilitate the maintenance of stable granular sludge operations.
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Rationale: The relationship between symptoms, measured using a validated disease-specific questionnaire, and longitudinal exacerbation risk has not been demonstrated in bronchiectasis. Objectives: The aim of this study is to investigate whether baseline symptoms, assessed using the Quality-of-Life Bronchiectasis Respiratory Symptom Scale (QoL-B-RSS) and its individual component scores, could predict future exacerbation risk in patients with bronchiectasis. Methods: The study included 436 adults with bronchiectasis from three tertiary hospitals. Symptoms were measured using the QoL-B-RSS, with scores ranging from 0 to 100, where lower scores indicated more severe symptoms. We examined whether symptoms as continuous measures were associated with the risk of exacerbation over 12 months. The analysis was also repeated for individual components of the QoL-B-RSS score. Results: The baseline QoL-B-RSS score was associated with an increased risk of exacerbations (rate ratio, 1.25 for each 10-point decrease; 95% confidence interval [CI], 1.15-1.35; P < 0.001), hospitalizations (rate ratio, 1.24; 95% CI, 1.05-1.43; P = 0.02), and reduced time to the first exacerbation (hazard ratio, 1.12; 95% CI, 1.03-1.21; P = 0.01) over 12 months, even after adjusting for relevant confounders, including exacerbation history. The QoL-B-RSS score was comparable to exacerbation history in its association with future frequent exacerbations (defined as three or more exacerbations per year) and hospitalization (area under the curve, 0.86 vs. 0.84; P = 0.46; and area under the curve, 0.81 vs. 0.83; P = 0.41, respectively). Moreover, patients with more severe symptoms in the majority of individual components of the QoL-B-RSS were more likely to experience exacerbations. Conclusions: Symptoms can serve as useful indicators for identifying patients at increased risk of exacerbation in bronchiectasis. Beyond relying solely on exacerbation history, a comprehensive assessment of symptoms could facilitate timely and cost-effective implementation of interventions for exacerbation prevention.
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Bronquiectasia , Qualidade de Vida , Adulto , Humanos , Estudos Prospectivos , Bronquiectasia/complicações , Hospitalização , Centros de Atenção TerciáriaRESUMO
The efficient operation of activated sludge systems is frequently hindered by low temperatures, and extensive research has been conducted to overcome this difficulty. However, the effect of varying temperatures on heat generation during substrate degradation remains unclear. In this study, results from laboratory-scale reactors show that sludge generated 5.36 ± 0.58 J/mg COD, 4.45 ± 0.24 J/mg COD, and 4.22 ± 0.26 J/mg COD at 10 °C, 20 °C, and 30 °C under aerobic conditions, respectively. Similarly, the sludge generated 4.05 ± 0.31 J/mg COD, 2.37 ± 0.15 J/mg COD, and 2.89 ± 0.18 J/mg COD under anoxic conditions. Despite the decreased respiration rates and hence reduced pollutant removal efficiency, sludge exhibited effective heat generation at low temperatures. Results from the full-scale plant also show a negative correlation between the heat generation capacity of microorganisms and the temperatures. 14.2 °C is considered the critical wastewater temperature for microorganisms' heat generation to offset the investigated plant's heat dissipation. This observation verified that thermal compensation for low temperatures was also significant in the full-scale plant. The mechanism of low-temperature compensation is attributed to non-growth processes being less dependent on temperature than growth processes, resulting in slow microbial growth but high heat generation at low temperatures. These findings provide valuable insights into the design and sustainable operation of wastewater treatment plants.
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Temperatura Alta , Esgotos , Esgotos/química , Temperatura , Eliminação de Resíduos Líquidos/métodos , Reatores BiológicosRESUMO
Increased prevalence of cardiovascular disease and potentially life-threatening complications of myocardial infarction (MI) has led to emerging therapeutic approaches focusing on myocardial regeneration and restoration of physiologic function following infarction. Extracellular vesicle (EV) technology has gained attention owing to the biological potential to modulate cellular immune responses and promote the repair of damaged tissue. Also, EVs are involved in local and distant cellular communication following damage and play an important role in initiating the repair process. Vesicles derived from stem cells and cardiomyocytes (CM) are of particular interest due to their ability to promote cell growth, proliferation, and angiogenesis following MI. Although a promising candidate for myocardial repair, EV technology is limited by the short retention time of vesicles and rapid elimination by the body. There have been several successful attempts to address this shortcoming, which includes hydrogel technology for the sustained bioavailability of EVs. This review discusses and summarizes current understanding regarding EV technology in the context of myocardial repair.
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Graphene oxide (GO) has been widely used in biological sensing studies because of its excellent physical and chemical properties. In particular, the rich functional groups on the surface of GO can effectively enhance the bonding of biomolecules and serve as an efficient sensing substrate. However, when biomolecules are labeled with fluorescence, the GO interface affects the biomolecules by reducing the fluorescence properties and limiting their applications in biosensing. Here, we establish an annealed GO (aGO) substrate through the annealing process, which can effectively increase the bonding amount of a DNA probe because of the accumulation of oxygen atoms on the surface without significantly damaging the nanosheet structure. Furthermore, we prove that the aGO substrate can effectively maintain its fluorescence performance and stability by exposing more graphic domains. Overall, this study successfully verifies that GO's interface annealing modification can be used as an alternative innovative interface application in biosensing.
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Grafite , Óxidos , Óxidos/química , FluorescênciaRESUMO
BACKGROUND: Emerging experimental and epidemiological evidence highlights a crucial cross-talk between the intestinal flora and the lungs, termed the "gut-lung axis". However, the function of the gut microbiota in bronchiectasis remains undefined. In this study, we aimed to perform a multi-omics-based approach to identify the gut microbiome and metabolic profiles in patients with bronchiectasis. METHODS: Fecal samples collected from non-CF bronchiectasis patients (BE group, n = 61) and healthy volunteers (HC group, n = 37) were analyzed by 16 S ribosomal RNA (rRNA) sequencing. The BE group was divided into two groups based on their clinical status: acute exacerbation (AE group, n = 31) and stable phase (SP group, n = 30). Further, metabolome (lipid chromatography-mass spectrometry, LC-MS) analyses were conducted in randomly selected patients (n = 29) and healthy volunteers (n = 31). RESULTS: Decreased fecal microbial diversity and differential microbial and metabolic compositions were observed in bronchiectasis patients. Correlation analyses indicated associations between the differential genera and clinical parameters such as bronchiectasis severity index (BSI). Disease-associated gut microbiota was screened out, with eight genera exhibited high accuracy in distinguishing SP patients from HCs in the discovery cohort and validation cohort using a random forest model. Further correlation networks were applied to illustrate the relations connecting disease-associated genera and metabolites. CONCLUSION: The study uncovered the relationships among the decreased fecal microbial diversity, differential microbial and metabolic compositions in bronchiectasis patients by performing a multi-omics-based approach. It is the first study to characterize the gut microbiome and metabolome in bronchiectasis, and to uncover the gut microbiota's potentiality as biomarkers for bronchiectasis. TRIAL REGISTRATION: This study is registered with ClinicalTrials.gov, number NCT04490447.
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Bronquiectasia , Microbiota , Adulto , Humanos , Bronquiectasia/diagnóstico , Fibrose , Metaboloma , Microbiota/genética , RNA Ribossômico 16S/genéticaRESUMO
Based on the dataset derived from January to March between 2015 and 2021 in Beijing, the PM2.5 pollution characteristics and its potential source regions during the historical period of the Beijing 2022 Olympic Winter Games and Paralympic Winter Games were investigated. From 2015 to 2018, both the number of severely polluted days (daily average ρ(PM2.5)>75 µg·m-3) and the average PM2.5 concentrations during severe pollution episodes decreased significantly in the period of January to March. While, neither variable has changed obviously since 2018. On average, severely polluted days occurred 23 times in each year between 2018 and 2021 during the period of January to March, and the average of ρ(PM2.5) was approximately 120.0 µg·m-3 during such polluted days. From January to March in 2015-2021, the severely polluted event with more than 5 consecutive polluted days occurred 2-3 times in each year, and the severest one lasted 8 d. During the historical period of the Beijing 2022 Olympic Winter Games, severely polluted days took place 2-9 d every year. The large quantities of fireworks during the Spring Festival maybe one of important primary sources of the PM2.5. The number of severely polluted days during the historical period of the Paralympic Winter Games ranged from 1 to 5 d, except for 2021 with 9 d owing to the frequent stagnant weather condition. The PM2.5 chemical composition was dominated by secondary species on severely polluted days during the historical period of the Beijing 2022 Olympic Winter Games and Paralympic Winter Games. Nitrate accounted for 46% of the measurable chemical components of PM2.5 during severe pollution events in 2020, which was remarkably higher than that during clean days in the same year (11%). The mass fraction of SO42- ranged from 12% to 19% in 2018-2020, indicating that the contribution of sulfate was much less, but cannot be ignored. The main potential source regions of PM2.5 in Beijing during the period concerned in this study were central and western Inner Mongolia, Hebei Province, Tianjin City, Shanxi Province, Shaanxi Province, central and western Shandong Province, and northern Henan Province.
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Poluentes Atmosféricos , Poluição do Ar , Poluentes Atmosféricos/análise , Poluição do Ar/análise , Pequim , China , Monitoramento Ambiental , Material Particulado/análise , Estações do AnoRESUMO
Retinal prostheses offer viable vision restoration therapy for patients with blindness. However, a critical requirement for maintaining the stable performance of electrical stimulation and signal transmission is the biocompatibility of the electrode interface. Here, we demonstrated a functionalized electrode-neuron biointerface composed of an annealed graphene oxide-collagen (aGO-COL) composite and neuronal cells. The aGO-COL exhibited an electroactive 3D crumpled surface structure and enhanced the differentiation efficiency of PC-12 âcells. It is integrated into a photovoltaic self-powered retinal chip to develop a biohybrid retinal implant that facilitates biocompatibility and tissue regeneration. Moreover, aGO-COL micropatterns fabricated via 3D bioprinting can be used to create neuronal cell microarrays, which supports the possibility of retaining the high spatial resolution achieved through electrical stimulation of the retinal chip. This study paves the way for the next generation of biohybrid retinal implants based on biointerfaces.
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With the continuing progress in space exploration, a new and perplexing condition related to spaceflight ocular syndrome has emerged in the past four decades. National Aeronautics and Space Administration (NASA) has named this condition "spaceflight-associated neuro-ocular syndrome" (SANS). This article gives an overview of the current research about SANS and traditional Chinese medicine (TCM) by analyzing the existing publications on PubMed and CNKI and reports from NASA about SANS, summarizing the potential pathogenesis of SANS and physical interventions for treating SANS, and discussing the feasibility of treating SANS with TCM. Due to the unique characteristics of the space environment, it is infeasible to conduct large-scale human studies of SANS. SANS may be the result of the interaction of multiple factors, including inflammation and fluid displacement in the optic nerve sheath and cerebrospinal fluid. We should pay attention to SANS. Visual function is not only related to the health of astronauts but also closely related to space operations. TCM has antioxidative stress and antiapoptotic effects and is widely used for optic nerve diseases. TCM has great potential to prevent SANS.
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Pulmonary sequelae following COVID-19 pneumonia have been emerging as a challenge; however, suitable cell sources for studying COVID-19 mechanisms and therapeutics are currently lacking. In this paper, we present a standardized primary alveolar cell culture method for establishing a human alveolar epithelium model that can recapitulate viral infection and cellular plasticity. The alveolar model is infected with a SARS-CoV-2 pseudovirus, and the clinically relevant features of the viral entry into the alveolar type-I/II cells, cytokine production activation, and pulmonary surfactant destruction are reproduced. For this damaged alveolar model, we find that the inhibition of Wnt signaling via XAV939 substantially improves alveolar repair function and prevents subsequent pulmonary fibrosis. Thus, the proposed alveolar cell culture strategy exhibits potential for the identification of pathogenesis and therapeutics in basic and translational research.
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Células Epiteliais Alveolares/virologia , COVID-19/fisiopatologia , Plasticidade Celular , Cultura Primária de Células/métodos , SARS-CoV-2/fisiologia , Humanos , Internalização do VírusRESUMO
Chronic obstructive pulmonary disease (COPD) is one of the most lethal chronic disease worldwide; however, the establishment of reliable in vitro models for exploring the biological mechanisms of COPD remains challenging. Here, we determined the differences in the expression and characteristics of the autophagic protein LC3B in normal and COPD human small airway epithelial cells and found that the nucleus of COPD cells obviously accumulated LC3B. We next established 3D human small airway tissues with distinct disease characteristics by regulating the biological microenvironment, extracellular matrix, and air-liquid interface culture methods. Using this biomimetic model, we found that LC3B affects the differentiation of COPD cells into basal, secretory, mucous, and ciliated cells. Moreover, although chloroquine and ivermectin effectively inhibited the expression of LC3B in the nucleus, chloroquine specifically maintained the performance of LC3B in cytoplasm, thereby contributing to the differentiation of ciliated cells and subsequent improvement in the beating functions of the cilia, whereas ivermectin only facilitated differentiation of goblet cells. We demonstrated that the autophagic mechanism of LC3B in the nucleus is one factor regulating the ciliary differentiation and function of COPD cells. Our innovative model can be used to further analyze the physiological mechanisms in the in vitro airway environment.
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BACKGROUND: As of 14 October 2021, coronavirus disease 2019 (COVID-19) has affected more than 246 million individuals and caused more than 4.9 million deaths worldwide. COVID-19 has caused significant damage to the health, economy and lives of people worldwide. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not as lethal as SARS coronavirus or Middle East respiratory syndrome coronavirus, its high transmissibility has had disastrous consequences for public health and health-care systems worldwide given the lack of effective treatment at present. OBJECTIVES: To clarify the mechanisms by which SARS-CoV-2 caused lung inflammation and injury, from the molecular mechanism to lung damage and tissue repair, from research to clinical practice, and then presented clinical requirements. SOURCES: References for this review were identified through searches '(COVID-19 [Title]) OR (SARS-CoV-2 [Title])' on PubMed, and focused on the pathological damage and clinical practice of COVID-19. CONTENT: We comprehensively reviewed the process of lung inflammation and injury during SARS-CoV-2 infection, including pyroptosis of alveolar epithelial cells, cytokine storm and thrombotic inflammatory mechanisms. IMPLICATIONS: This review describes SARS-CoV-2 in comparison to SARS and explores why most people have mild inflammatory responses, even asymptomatic infections, and only a few develop severe disease. It suggests that future therapeutic strategies may be targeted antiviral therapy, the pathogenic pathways in the lung inflammatory response, and enhancing repair and regeneration in lung injury.
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COVID-19 , Coronavírus da Síndrome Respiratória do Oriente Médio , COVID-19/complicações , Síndrome da Liberação de Citocina , Humanos , Pulmão/patologia , SARS-CoV-2Assuntos
Criptococose/diagnóstico , Criptococose/patologia , Pneumopatias Fúngicas/diagnóstico , Pneumopatias Fúngicas/patologia , Nódulos Pulmonares Múltiplos/diagnóstico , Nódulos Pulmonares Múltiplos/patologia , Adulto , Idoso , Estudos de Casos e Controles , China , Criptococose/diagnóstico por imagem , Diagnóstico Diferencial , Feminino , Humanos , Pulmão/diagnóstico por imagem , Pneumopatias Fúngicas/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Nódulos Pulmonares Múltiplos/diagnóstico por imagem , Reprodutibilidade dos Testes , Tomografia Computadorizada por Raios X/métodosRESUMO
As one of the most conservative proteins in evolution, Y-box-binding protein 1 (YB-1) has long been considered as a potential cancer target. YB-1 is usually poorly expressed in normal cells and exerts cellular physiological functions such as DNA repair, pre-mRNA splicing and mRNA stabilizing. In cancer cells, the expression of YB-1 is up-regulated and undergoes nuclear translocation and contributes to tumorigenesis, angiogenesis, tumor proliferation, invasion, migration and chemotherapy drug resistance. During the past decades, a variety of pharmacological tools such as siRNA, shRNA, microRNA, circular RNA, lncRNA and various compounds have been developed to target YB-1 for cancer therapy. In this review, we describe the physiological characteristics of YB-1 in detail, highlight the role of YB-1 in tumors and summarize the current therapeutic methods for targeting YB-1 in cancer.
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Neoplasias , Proteína 1 de Ligação a Y-Box , Linhagem Celular Tumoral , Reparo do DNA , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , RNA Interferente Pequeno , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/metabolismoRESUMO
Background: Corticosteroid usage in acute respiratory distress syndrome (ARDS) remains controversial. We aim to explore the correlation between the different doses of corticosteroid administration and the prognosis of ARDS. Methods: All patients were diagnosed with ARDS on initial hospital admission and received systemic corticosteroid treatment for ARDS. The main outcomes were the effects of corticosteroid treatment on clinical parameters and the mortality of ARDS patients. Secondary outcomes were factors associated with the mortality of ARDS patients. Results: 105 ARDS patients were included in this study. Corticosteroid treatment markedly decreased serum interleukin-18 (IL-18) level (424.0 ± 32.19 vs. 290.2 ± 17.14; p = 0.0003) and improved arterial partial pressure of oxygen/fraction of inspired oxygen (PaO2/FiO2) (174.10 ± 65.28 vs. 255.42 ± 92.49; p < 0.0001). The acute physiology and chronic health evaluation (APACHE II) score (16.15 ± 4.41 vs. 14.88 ± 4.57, p = 0.042) decreased significantly on the seventh day after systemic corticosteroid treatment. Interestingly, the serum IL-18 decreased significantly (304.52 ± 286.00 vs. 85.85 ± 97.22, p < 0.0001), whereas the improvement of PaO2/FiO2 (24.78 ± 35.03 vs. 97.17 ± 44.82, p < 0.001) was inconspicuous after systemic corticosteroid treatment for non-survival patients, compared with survival patients. Furthermore, the receiver operating characteristic (ROC) model revealed, when equivalent methylprednisolone usage was 146.5 mg/d, it had the best sensitivity and specificity to predict the death of ARDS. Survival analysis by Kaplan-Meier curves presented the higher 45-day mortality in high-dose corticosteroid treatment group (logrank test p < 0.0001). Multivariate Cox regression analyses demonstrated that serum IL-18 level, APACHE II score, D-dimer, and high-dose corticosteroid treatment were associated with the death of ARDS. Conclusion: Appropriate dose of corticosteroids may be beneficial for ARDS patients through improving the oxygenation and moderately inhibiting inflammatory response. The benefits and risks should be carefully weighed when using high-dose corticosteroid for ARDS. Trial registration: This work was registered in ClinicalTrials.gov. Name of the registry: Corticosteroid Treatment for Acute Respiratory Distress Syndrome. Trial registration number: NCT02819453. URL of trial registry record: https://register.clinicaltrials.gov.