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OBJECTIVE: Coronavirus infectious disease, that emerged in 2019 (COVID-19) has been a major public health issue not only in Japan, but also worldwide, and the implementation of a proper vaccination strategy has been important. To promote vaccination, the present study compared impressions of COVID-19 vaccinations stratified by the number of vaccinations among healthcare professional university students in Okayama, Japan, and suggests better vaccination strategies. METHOD: A total of 212 Japanese healthcare professional university students were enrolled in this clinical qualitative study using the text mining method. A self-reported questionnaire, including questions such as "What do you think about COVID-19 vaccinations?" was performed. We also examined the number of vaccinations, sex, history of COVID-19 infection, and daily mask use. RESULTS: A total of 5,935 words were obtained and "Think" (169 times) was the most frequently used followed by "Inject" (108 times), "Inoculation" (97 times), "Vaccine" (83 times), "Corona" (66 times) and "Side effects" (49 times). Characteristic words were "Safety" in non-vaccinated subjects and "Side effects" and "Necessary" in vaccinated subjects. In addition, "Safety" in non-vaccinated men and "Frightening" in non-vaccinated women were characteristic and fundamental features. CONCLUSION: Impressions of COVID-19 vaccinations stratified by the number of vaccinations differed among healthcare professional university students. The provision of appropriate information on safety to non-vaccinated subjects and side effects to vaccinated subjects appears to be necessary. In addition, sex-specific information may be required for non-vaccinated subjects.
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Inborn errors of bile acid metabolism (IEBAM) cause cholestasis during the neonatal period, and 8 types of IEBAM have been reported to date. IEBAM accounts for approximately 2% of cases of cholestasis of unknown cause. As only 10 patients have been identified in Japan, IEBAM presents diagnostic challenges due to the similarity of clinical symptoms with biliary atresia, thus necessitating precise differentiation to avoid unnecessary invasive procedures. Laboratory tests in IEBAM are characterized by normal γ-glutamyltransferase (GGT) and serum total bile acid (STBA) levels despite the presence of cholestasis; therefore, measuring STBA and GGT is essential to distinguishing biliary atresia from IEBAM. With suspected IEBAM, liquid chromatography-mass spectrometry (LC/MS) analysis of urinary bile acids is needed to optimize diagnostic and therapeutic efficacy and avoid open cholangiography and initiate treatment for primary bile acids such as cholic acid or chenodeoxycholic acid. This prospective report aims to increase awareness of IEBAM by highlighting the characteristics of general blood test and bile acid profiles from LC/MS analyses of blood, urine, and stool samples.
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As a space project, in "Stem Cells" by the Japan Aerospace Exploration Agency (JAXA), frozen mouse ES cells were stored on the International Space Station (ISS) in the Minus Eighty Degree Laboratory Freezer for ISS (MELFI) for 1584 days. After taking these cells back to the ground, the cells were thawed and cultured, and their gene expressions were comprehensively analyzed using RNA sequencing in order to elucidate the early response of the cells to long-time exposure to space radiation consisting of various ionized particles. The comparisons of gene expression involved in double-stranded break (DSB) repair were examined. The expressions of most of the genes that were involved in homologous recombination (HR) and non-homologous end joining (NHEJ) were not significantly changed between the ISS-stocked cells and ground-stocked control cells. However, the transcription of Trp53inp1 (tumor protein 53 induced nuclear protein-1), Cdkn1a (p21), and Mdm2 genes increased in ISS-stocked cells as well as Fe ion-irradiated cells compared to control cells. This suggests that accumulated DNA damage caused by space radiation exposure would activate these genes, which are involved in cell cycle arrest for repair and apoptosis in a p53-dependent or -independent manner, in order to prevent cells with damaged genomes from proliferating and forming tumors.
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Quebras de DNA de Cadeia Dupla , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Reparo do DNA , Reparo do DNA por Junção de Extremidades , Análise de Sequência de RNA , Perfilação da Expressão GênicaRESUMO
Animals frequently eat less after a test-article treatment in nonclinical toxicological studies, and it can be difficult to distinguish test article-derived toxicities from secondary changes related to this reduced food intake. Therefore, in this study, we restricted the food intake of cynomolgus monkeys (Cambodian, male, n=2 or 3, 48 ± 3 months old) to 25% of the control for two weeks and evaluated the effects on toxicological parameters (general conditions, body weight, electrocardiography, urinalysis, hematology, blood chemistry, bone marrow analysis, pathological examination). After 2 weeks, the monkeys exhibited decreases in bone marrow erythropoiesis (e.g., decreases in reticulocytes and bone marrow erythrocytes), as well as glycogenesis induction (e.g., increase in aspartate aminotransferase (AST)) and malnutrition (e.g., decrease in triglyceride and systemic adipocytes atrophy). Additionally, histopathological analysis revealed granuloma and inflammatory cell infiltration in coronary fat, which had never been found in previous food restriction studies using other animal species. These findings will enable researchers to more accurately evaluate the toxicological risks of test articles that simultaneously induce food intake reduction.
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Ingestão de Alimentos , Alimentos , Masculino , Animais , Macaca fascicularis , Peso Corporal , EletrocardiografiaRESUMO
Therapeutic antibodies sometimes elicit anti-drug antibodies (ADAs) that can affect efficacy and safety. Engineered antibodies that contain artificial amino acid sequences are potentially highly immunogenic, but this is currently difficult to predict. Therefore, it is important to efficiently assess immunogenicity during the development of complex antibody-based formats. Here, we present an in vitro peripheral blood mononuclear cell-based assay that can be used to assess immunogenicity potential within 3 days. This method involves examining the frequency and function of interleukin (IL)-2-secreting CD4+ T cells induced by therapeutic antibodies. IL-2-secreting CD4+ T cells seem to be functionally relevant to the immunogenic potential due to their proliferative activity and the expression of several cytokines. The rates of the donors responding to low and high immunogenic proteins, mAb1, and keyhole limpet hemocyanin were 1.3% and 93.5%, respectively. Seven antibodies with known rates of immunogenicity (etanercept, emicizumab, abciximab, romosozumab, blosozumab, humanized anti-human A33 antibody, and bococizumab) induced responses in 1.9%, 3.8%, 6.4%, 10.0%, 29.2%, 43.8%, and 89.5% of donors, respectively. These data are comparable with ADA incidences in clinical settings. Our results show that this assay can contribute to the swift assessment and mechanistic understanding of the immunogenicity of therapeutic antibodies.
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Interleucina-2 , Linfócitos T , Interleucina-2/farmacologia , Leucócitos Mononucleares/metabolismo , Citocinas/metabolismo , Linfócitos T CD4-PositivosRESUMO
The aim of this study was to compare impressions of COVID-19 vaccination and influenza vaccination in Japan by analyzing social media (Twitter®) using a text-mining method. We obtained 10,000 tweets using the keywords "corona vaccine" and "influenza vaccine" on 15 December 2022 and 19 February 2023. We then counted the number of times the words were used and listed frequency of these words by a text-mining method called KH Coder. We also investigated concepts in the data using groups of words that often appeared together or groups of documents that contained the same words using multi-dimensional scaling (MDS). "Death" in relation to corona vaccine and "severe disease" for influenza vaccine were frequently used on 15 December 2022. The number of times the word "death" was used decreased, "after effect" was newly recognized for corona vaccine, and "severe disease" was not used in relation to influenza vaccine. Through this comprehensive analysis of social media data, we observed distinct variations in public perceptions of corona vaccination and influenza vaccination in Japan. These findings provide valuable insights for public health authorities and policymakers to better understand public sentiment and tailor their communication strategies accordingly.
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Extracellular vesicles, such as microvesicles (LEV) and exosomes (SEV), play an important role in intercellular signaling by encapsulating functional molecules and delivering them to specific cells. Recent studies showed that signal peptides (SPs), which are derived from sequences at the N-terminal of newly synthesized proteins, exhibited biological activity in the extracellular fluid. We previously reported that SPs were secreted into the extracellular fluid via SEV; however, it remains unclear whether the release of SPs occurs via LEV. In the present study, we demonstrated that SP fragments from human placental secreted alkaline phosphatase (SEAP) were present in LEV as well as SEV released from RAW-Blue cells, which stably express an NF-κB-inducible SEAP reporter. When RAW-Blue cells were treated with LPS at 0-10,000 ng/mL, SEAP SP fragments per particle were more abundant in LEV than in SEV, with fragments in LEV and SEV reaching a maximum at 1000 and 100 ng/mL, respectively. The content of SEAP SP fragments in LEV from IFNγ-stimulated RAW-Blue cells was higher than those from TNFα-stimulated cells, whereas that in SEV from TNFα-stimulated RAW-Blue cells was higher than those from IFNγ-stimulated cells. Moreover, the content of SEAP SP fragments in LEV and SEV decreased in the presence of W13, a calmodulin inhibitor. Collectively, these results indicate that the transportation of SP fragments to extracellular vesicles was changed by cellular activation, and calmodulin was involved in their transportation to LEV and SEV.
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Vesículas Extracelulares , Fator de Necrose Tumoral alfa , Feminino , Gravidez , Humanos , Sinais Direcionadores de Proteínas , Calmodulina , Placenta , MacrófagosRESUMO
Triple-FLAG (3 × FLAG)-tagged proteins can be affinity purified through binding to an anti-FLAG antibody and competitive elution with excess free 3 × FLAG peptide. To expand the availability of the 3 × FLAG purification system, we produced a recombinant His-tagged 3 × FLAG peptide in Brevibacillus choshinensis. The screening of connecting linkers between His-tag and the 3 × FLAG peptide, culture containers, and culture media showed that the His-tagged 3 × FLAG peptide with an LA linker was most expressed in 2SY medium using a baffled shake flask. The peptide was affinity-purified to give a yield of about 25 mg/L of culture. The peptide was effective for eluting 3 × FLAG-tagged α-amylase from anti-FLAG magnetic beads. Finally, the peptide remaining in the amylase fraction was removed by His-tag affinity purification. These results show that the recombinant His-tagged 3 × FLAG peptide can function as an easy-to-remove affinity peptide in the 3 × FLAG purification system.
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Brevibacillus , Proteínas Recombinantes/metabolismo , Brevibacillus/genética , Brevibacillus/metabolismo , Cromatografia de Afinidade/métodos , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Raspberry bushy dwarf virus (RBDV) is transmitted through seed in infected red raspberry plants after pollination with pollen grains from healthy red raspberry plants. Here, we show that RBDV is not transmitted through seeds in infected Nicotiana benthamiana (Nb) plants after pollination with virus-free Nb pollen grains. Chromogenic in situ hybridization revealed that the virus invades the shoot apical meristem and the ovule, including the embryo sac, of RBDV-infected Nb plants; however, in seeds that developed from infected embryo sacs after fertilization by virus-free sperm cells, RBDV was absent in the embryos and present in the endosperms. When we analyzed seed transmission of RBDV in Nb mutants with mutations in dicer-like enzyme 2 and 4 (NbDCL2&4) or RNA-dependent RNA polymerase 6 (NbRDR6), RBDV was not present in the offspring from seeds with embryos and endosperms that did not express NbDCL2&4 or NbRDR6. These results suggest that seed transmission of RBDV is prevented by evasion of infection by the embryo and that RNA silencing is not essential for preventing seed transmission of RBDV in Nb plants.
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Vírus de Plantas , Vírus de RNA , Rubus , Nicotiana , Sementes , Endosperma , Internalização do Vírus , Vírus de Plantas/genética , Doenças das Plantas , Vírus de RNA/genéticaRESUMO
We investigated the link between prolonged sedentary bouts and all-cause mortality in patients on chronic hemodialysis, using a prospective cohort. A total of 104 outpatients on chronic hemodialysis from 2013 to 2019, aged 71.4±11.4 years, were enrolled. Prolonged sedentary bouts (≥ 30 min and ≥60 min) (min and bouts) and relative prolonged sedentary bouts (≥ 30 min and ≥ 60 min) (%) on the patients' non-hemodialysis days were measured by a tri-accelerometer, and we also analyzed the patients' clinical parameters. The relationship between prolonged sedentary bouts and all-cause mortality was evaluated by a survival analysis and the Cox proportional hazard model. Thirty-five patients died during the follow-up period. A Kaplan-Meier analysis detected significant differences in the survival rate between two groups stratified by the median for all prolonged sedentary-bout parameters. After the adjustment for confounding factors, all of the prolonged sedentary-bout parameters were determinant factors for all-cause mortality. These results indicate that prolonged sedentary bouts on non-hemodialysis days were closely related to all-cause mortality in the patients on hemodialysis.
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Diálise Renal , Comportamento Sedentário , Humanos , Estudos Prospectivos , Modelos de Riscos Proporcionais , Pacientes AmbulatoriaisRESUMO
The present study investigated the use of complementary and alternative medicine (CAM) and its relationship with health-related quality of life (HRQOL) in patients with type 2 diabetes mellitus. A total of 421 patients with type 2 diabetes mellitus who completely met the inclusion criteria, aged 67.3 ± 12.8 years, among 622 outpatients were recruited in this cross-sectional study. We examined the use of CAM, such as supplements, Kampo, acupuncture, and yoga. HRQOL was assessed by EuroQOL. A total of 161 patients (38.2%) with type 2 diabetes mellitus used some type of CAM. The use of supplements and/or health foods was the highest among CAM users (112 subjects, 26.6%). HRQOL was significantly lower in patients who used some CAM (0.829 ± 0.221) than in those without any CAM use (0.881 ± 0.189), even after adjustments for confounding factors [F(1, 414) = 2.530, p = 0.014]. Proper information on CAM is needed for patients with type 2 diabetes mellitus.
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BACKGROUND: Problematic Internet use (PIU) remains a significant social concern in Japan and across the globe. Numerous studies have been conducted to examine the prevalence of PIU, and its association with lifestyle habits and mental health status. To the best of our knowledge, few studies have examined the association between parental attachment and PIU in Japan. Furthermore, preventive measures in Japan have primarily targeted adolescents via education. Therefore, in this study we explored the association between PIU and attachment styles among adolescents to seek a new approach for the development of appropriate, effective measures. METHODS: A self-administered survey was conducted at one private (in 2018) and two public junior high schools (in 2020) in Kagawa Prefecture, western Japan. We used the Internet Addiction Test (IAT), the Experiences in Close Relationships-Relationship Structures (ECR-RS) questionnaire, and the Birleson Depression Self-Rating Scale for Children (DSRS-C). Multiple regression analysis was performed to examine the association between IAT scores and attachment styles. RESULTS: Of 1,413 participants (grades 7-9; 700 boys and 713 girls), 39% of the students showed possible PIU and 3% showed PIU. Students with PIU tended to sleep for shorter durations, access the Internet for long hours, and have a poor mental health status (p < 0.01). PIU was significantly associated with insecure attachment (p < 0.0001). CONCLUSIONS: The findings of our study imply that improving attachment styles and developing secure attachment between the child and parent might help mitigate and/or prevent PIU.
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Comportamento Aditivo , Uso da Internet , Masculino , Feminino , Criança , Humanos , Adolescente , Japão/epidemiologia , Comportamento Aditivo/epidemiologia , Comportamento Aditivo/psicologia , Instituições Acadêmicas , Depressão/epidemiologia , InternetRESUMO
Oxygenic photoautotrophic bacteria, cyanobacteria, have the tricarboxylic acid (TCA) cycle, and metabolite production using the cyanobacterial TCA cycle has been spotlighted recently. The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (Synechocystis 6803) has been used in various studies on the cyanobacterial TCA cycle. Malate oxidation in the TCA cycle is generally catalyzed by malate dehydrogenase (MDH). However, Synechocystis 6803 MDH (SyMDH) is less active than MDHs from other organisms. Additionally, SyMDH uses only NAD+ as a coenzyme, unlike other TCA cycle enzymes from Synechocystis 6803 that use NADP+. These results suggest that MDH rarely catalyzes malate oxidation in the cyanobacterial TCA cycle. Another enzyme catalyzing malate oxidation is malic enzyme (ME). We clarified which enzyme oxidizes malate that originates from the cyanobacterial TCA cycle using analyses focusing on ME and MDH. In contrast to SyMDH, Synechocystis 6803 ME (SyME) showed high activity when NADP+ was used as a coenzyme. Unlike the Synechocystis 6803 mutant lacking SyMDH, the mutant lacking SyME accumulated malate in the cells. ME was more highly preserved in the cyanobacterial genomes than MDH. These results indicate that ME mainly oxidizes malate that originates from the cyanobacterial TCA cycle (named the ME-dependent TCA cycle). The ME-dependent TCA cycle generates NADPH, not NADH. This is consistent with previous reports that NADPH is an electron carrier in the cyanobacterial respiratory chain. Our finding suggests the diversity of enzymes involved in the TCA cycle in the organisms, and analyses such as those performed in this study are necessary to determine the enzymes. IMPORTANCE Oxygenic photoautotrophic bacteria, namely, cyanobacteria, have the tricarboxylic acid (TCA) cycle. Recently, metabolite production using the cyanobacterial TCA cycle has been well studied. To enhance the production volume of metabolites, understanding the biochemical properties of the cyanobacterial TCA cycle is required. Generally, malate dehydrogenase oxidizes malate in the TCA cycle. However, cyanobacterial malate dehydrogenase shows low activity and does not use NADP+ as a coenzyme, unlike other cyanobacterial TCA cycle enzymes. Our analyses revealed that another malate oxidation enzyme, the malic enzyme, mainly oxidizes malate that originates from the cyanobacterial TCA cycle. These findings provide better insights into metabolite production using the cyanobacterial TCA cycle. Furthermore, our findings suggest that the enzymes related to the TCA cycle vary from organism to organism and emphasize the importance of analyses to identify the enzymes such as those performed in this study.
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Ciclo do Ácido Cítrico , Synechocystis , NADP/metabolismo , Synechocystis/metabolismo , Oxirredução , Ácidos Tricarboxílicos/metabolismoRESUMO
Nowadays, ordinary people can travel in space, and the possibility of extended durations in an environment such as moon of the Earth and Mars with higher space radiation exposures compared to past missions, is increasing. Until now, the physical doses of space radiation have been measured, but measurement of direct biological effects has been hampered by its low dose and low dose-rate effect. To assess the biological effects of space radiation, we launched and kept frozen mouse embryonic stem (ES) cells in minus eighty degree Celsius freezer in ISS (MELFI) on the International Space Station (ISS) for a maximum of 1,584 days. The passive dosimeter for life science experiments in space (PADLES) was attached on the surface of the sample case of the ES cells. The physical dosimeter measured the absorbed dose in water. After return, the frozen cells were thawed and cultured and their chromosome aberrations were analyzed. Comparative experiments with proton and iron ion irradiation were performed at particle accelerators on Earth. The wild-type ES cells showed no differences in chromosomal aberrations between the ground control and ISS exposures. However, we detected an increase of chromosome aberrations in radio-sensitized histone H2AX heterozygous-deficient mouse ES cells and found that the rate of increase against the absorbed dose was 1.54-fold of proton irradiation at an accelerator. On the other hand, we estimated the quality factor of space radiation as 1.48 ± 0.2. using formulas of International Commission of Radiation Protection (ICRP) 60. The relative biological effectiveness (RBE) observed from our experiments (1.54-fold of proton) was almost equal (1.04-fold) to the physical estimation (1.48 ± 0.2). It should be important to clarify the relation between biological effect and physical estimates of space radiation. This comparative study paves a way to reveal the complex radiation environments to reduce the uncertainty for risk assessment of human stay in space.
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Background: Neonatal hyperbilirubinemia is a significant health problem in Myanmar. We introduced transcutaneous bilirubin (TcB) measurements in 2017 and developed an hour-specific TcB nomogram for early detection and treatment of hyperbilirubinemia in Myanmar neonates. This study aimed to evaluate whether our screening method for hyperbilirubinemia decreased the requirement of blood exchange therapy (ET). Methods: This retrospective cohort study was conducted at the Central Women's Hospital, Yangon. Two groups were included as follows: group 1 (control group; comprising infants born in 2016 and screened on the basis of Kramer's rule), and group 2 (intervention group; comprising infants born in 2019 and screened by TcB measurement using a nomogram). The number of ETs was analyzed based on causes of hyperbilirubinemia and number of days after birth. Results: Groups 1 and 2 comprised 12,968 and 10,090 infants, respectively. Forty-six and two infants in Groups 1 and 2, respectively, required an ET. The odds ratio for ET was 18.0 (Group 1 to Group 2; 95% confidence interval [CI]: 4.8-67.1; p = 0.000). Serum bilirubin values at the time ET was administered were significantly higher in Group 1 than those in Group 2 (median: 23.0 and 16.8, respectively). Conclusion: The management of hyperbilirubinemia using our screening method (TcB Nomogram) can effectively reduce the need for ET in neonates in Myanmar.
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The aim of this study was to investigate the pre-impressions of the third Coronavirus disease 2019 (COVID-19) vaccination among Japanese medical staff using quantitative analysis. Among 413 medical staff, 260 (60 men and 200 women) aged 40.4 ± 12.3 years were enrolled in this cross-sectional study. Pre-impressions of the third COVID-19 vaccination were analyzed using the text-mining analysis software, KH coder. Among 260 subjects, 242 (93.1%) agreed to the third vaccination, with the rate being lower among subjects in their 30s (87.3%) than those in the other age groups. The word "side effects" was characteristic of subjects in their 20s and nurses, and "pregnancy" of those in their 30s and administrative staff. Pre-impressions of the third COVID-19 vaccination varied among age groups and different professions. The results obtained provide useful information for promoting the third COVID-19 vaccination to Japanese adults.
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This study aimed to investigate the link between prolonged sedentary bouts and health-related quality of life (QOL) in patients on chronic hemodialysis (CHD). A total of 84 outpatients on CHD, aged 71.6±11.8 years, were enrolled in this cross-sectional study. Parameters for prolonged sedentary bouts [i.e., ⧠30 min (% and bout) and ⧠60 min (% and bout)] were measured using a triaxial accelerometer. Health-related QOL (HRQOL) was evaluated by the Euro-QOL (EQ-5D). Clinical parameters were obtained from medical records. Relatively prolonged sedentary bouts (%) were 44.0±18.2 (⧠30 min) and 29.8±16.5 (⧠60 min) for total days. Prolonged sedentary bouts (bouts) were 6.2±2.7 (⧠30 min) and 2.7±1.6 (⧠60 min) for total days. EQ-5D scores were 0.728±0.220. All prolonged sedentary bout parameters were negatively correlated with EQ-5D scores, except for prolonged sedentary bouts (⧠60 min) (min) and relatively prolonged sedentary bouts (%) on hemodialysis days. Multiple regression analysis showed that prolonged sedentary bout parameters were an important factor in EQ-5D scores even after adjusting for confounding factors for total and non-hemodialysis days. Our results suggested that prolonged sedentary bouts were closely associated with HRQOL in patients on CHD, especially on non-hemodialysis days.
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Doença Enxerto-Hospedeiro , Qualidade de Vida , Estudos Transversais , Humanos , Pacientes Ambulatoriais , Análise de Regressão , Diálise RenalRESUMO
Signal peptides (SPs) not only mediate targeting to the endoplasmic reticulum (ER) but also play important roles as biomarkers and substances with physiological activity in extracellular fluids including blood. SPs are thought to be degraded intracellularly, making it unclear how they are transported from the ER to the extracellular fluid. In a recent study, we showed that a C-terminal fragment of the SP of a type I membrane protein, amyloid precursor protein (APP), was secreted into the extracellular fluid via exosomes using transformed HEK293 cells expressing APP SP flanking a reporter protein. In the present study, we demonstrate that a N-terminal fragment of the SP from a type II membrane protein, human placental secreted alkaline phosphatase (SEAP), is contained in exosomes and secreted into the extracellular fluid using HEK-Blue hTLR3 cells, which express both a human toll-like receptor 3 gene and an inducible SEAP reporter gene. When HEK-Blue hTLR3 cells were stimulated with a TLR3 ligand, a N-terminal fragment of SEAP SP in exosomes was increased in parallel with SEAP secretion in a concentration-dependent manner. These results indicated that SP fragments are exosomal components. In addition, migrating SP fragments were determined by characteristics of the signal-anchor sequence of membrane proteins. Furthermore, we found that SP fragments could bind to calmodulin (CALM), which is a cytosolic protein and also a component of exosomes, suggesting its involvement in the transportation of SP fragments from the endoplasmic reticulum to exosomes.
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Exossomos , Sinais Direcionadores de Proteínas , Precursor de Proteína beta-Amiloide/metabolismo , Exossomos/metabolismo , Feminino , Células HEK293 , Humanos , Fragmentos de Peptídeos/metabolismo , Placenta/metabolismo , GravidezRESUMO
The gold standard test for identifying SARS-CoV-2, the causative agent of COVID-19, is polymerase chain reaction (PCR). Despite their limited sensitivity, SARS-CoV-2 antigen rapid diagnostic tests are vital tools in the fight against viral spread. Owing to its simplicity and low cost, the lateral flow assay (LFA) is the most extensively used point-of-care diagnostic test. Here, we report a newly designed LFA-NanoSuit method (LNSM) that works in conjunction with desktop scanning electron microscopy (SEM) to detect SARS-CoV-2. LNSM requires no standard SEM treatment, avoids cellulose and residual buffer deformation, and enables the capture of high-resolution images of antibody-labeled gold/platinum particles reacting with SARS-CoV-2 antigens. To assess its applicability, we compared clinical SARS-CoV-2 samples via visual detection of LFA, LSNM detection of LFA, and real-time reverse transcription-PCR (qRT-PCR). Compared to qRT-PCR, LNSM showed 86.7% sensitivity (26/30; 95% confidence interval (CI): 69.28-96.24%) and 93.3% specificity (14/15; 95% CI: 68.05-99.83%) for SARS-CoV-2. In samples with a relatively low SARS-CoV-2 RNA copy number (30 < Ct ≤ 40), the sensitivity of LNSM was greater (73.3%) than that of visual detection (0%). A simple, sensitive, and quantitative LNSM can be used to diagnose SARS-CoV-2.
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Signal peptides (SPs) and their fragments play important roles as biomarkers and substances with physiological functions in extracellular fluid. We previously reported that SP fragments were released into extracellular fluid via exosomes and bound to calmodulin (CaM), an exosomal component, in a cell-free system. However, it currently remains unclear whether CaM intracellularly interacts with SP fragments or is involved in the trafficking of these fragments to exosomes. Therefore, the present study examined the binding of CaM to SP fragments in T-REx AspALP cells, transformed HEK293 cells expressing amyloid precursor protein (APP) SP flanking a reporter protein, and their exosomes. APP SP fragments were detected in exosomes from T-REx AspALP cells in the absence of W13, a CaM inhibitor, but were present in lower amounts in exosomes from W13-treated cells. Cargo proteins, such as Alix, CD63, and CD81, were increased in W13-treated T-REx AspALP cells but were decreased in their exosomes. Furthermore, CaM interacted with heat shock protein 70 and CD81 in T-REx AspALP cells and this increased in the presence of W13. APP SP fragments were detected in intracellular CaM complexes in the absence of W13, but not in its presence. These results indicate that CaM functions as a key regulator of the transport of SP fragments into exosomes and plays novel roles in the sorting of contents during exosomal biogenesis.