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[This corrects the article DOI: 10.3389/fimmu.2024.1420351.].
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BACKGROUND: We recently reported that the dopamine (DA) analogue CA140 modulates neuroinflammatory responses in lipopolysaccharide-injected wild-type (WT) mice and in 3-month-old 5xFAD mice, a model of Alzheimer's disease (AD). However, the effects of CA140 on Aß/tau pathology and synaptic/cognitive function and its molecular mechanisms of action are unknown. METHODS: To investigate the effects of CA140 on cognitive and synaptic function and AD pathology, 3-month-old WT mice or 8-month-old (aged) 5xFAD mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 10, 14, or 17 days. Behavioral tests, ELISA, electrophysiology, RNA sequencing, real-time PCR, Golgi staining, immunofluorescence staining, and western blotting were conducted. RESULTS: In aged 5xFAD mice, a model of AD pathology, CA140 treatment significantly reduced Aß/tau fibrillation, Aß plaque number, tau hyperphosphorylation, and neuroinflammation by inhibiting NLRP3 activation. In addition, CA140 treatment downregulated the expression of cxcl10, a marker of AD-associated reactive astrocytes (RAs), and c1qa, a marker of the interaction of RAs with disease-associated microglia (DAMs) in 5xFAD mice. CA140 treatment also suppressed the mRNA levels of s100ß and cxcl10, markers of AD-associated RAs, in primary astrocytes from 5xFAD mice. In primary microglial cells from 5xFAD mice, CA140 treatment increased the mRNA levels of markers of homeostatic microglia (cx3cr1 and p2ry12) and decreased the mRNA levels of a marker of proliferative region-associated microglia (gpnmb) and a marker of lipid-droplet-accumulating microglia (cln3). Importantly, CA140 treatment rescued scopolamine (SCO)-mediated deficits in long-term memory, dendritic spine number, and LTP impairment. In aged 5xFAD mice, these effects of CA140 treatment on cognitive/synaptic function and AD pathology were regulated by dopamine D1 receptor (DRD1)/Elk1 signaling. In primary hippocampal neurons and WT mice, CA140 treatment promoted long-term memory and dendritic spine formation via effects on DRD1/CaMKIIα and/or ERK signaling. CONCLUSIONS: Our results indicate that CA140 improves neuronal/synaptic/cognitive function and ameliorates Aß/tau pathology and neuroinflammation by modulating DRD1 signaling in primary hippocampal neurons, primary astrocytes/microglia, WT mice, and aged 5xFAD mice.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Ratones Transgénicos , Enfermedades Neuroinflamatorias , Receptores de Dopamina D1 , Transducción de Señal , Animales , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Ratones , Péptidos beta-Amiloides/metabolismo , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Receptores de Dopamina D1/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Sinapsis/patología , Cognición/efectos de los fármacos , Dopamina/metabolismo , Ratones Endogámicos C57BL , Masculino , HumanosRESUMEN
Many types of neurons exhibit a daily rhythm of intrinsic excitability. Here, we present a protocol for assessing circadian regulation of dentate granule cell excitability using a mouse model for conditional knockout of the molecular clock protein BMAL1. We describe steps for obtaining healthy oblique horizontal slices that contain the hippocampus and measuring intrinsic excitability and synaptic potentials by combining whole-cell patch-clamp recordings and perforant-path electric stimulation. We then detail procedures for validating single-cell genetic deletion of Bmal1 by immunohistochemistry. For complete details on the use and execution of this protocol, please refer to Gonzalez et al.1.
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Background: Pre-transplant donor-specific anti-human leukocyte antigen antibody (HLA-DSA) is a recognized risk factor for acute antibody-mediated rejection (ABMR) and allograft failure. However, the clinical relevance of pre-transplant crossmatch (XM)-negative HLA-DSA remains unclear. Methods: We investigated the effect of XM-negative HLA-DSA on post-transplant clinical outcomes using data from the Korean Organ Transplantation Registry (KOTRY). This study included 2019 living donor kidney transplant recipients from 40 transplant centers in South Korea: 237 with HLA-DSA and 1782 without HLA-DSA. Results: ABMR developed more frequently in patients with HLA-DSA than in those without (5.5% vs. 1.5%, p<0.0001). Multivariable analysis identified HLA-DSA as a significant risk factor for ABMR (odds ratio = 3.912, 95% confidence interval = 1.831-8.360; p<0.0001). Furthermore, the presence of multiple HLA-DSAs, carrying both class I and II HLA-DSAs, or having strong HLA-DSA were associated with an increased incidence of ABMR. However, HLA-DSA did not affect long-term clinical outcomes, such as allograft function and allograft survival, patient survival, and infection-free survival. Conclusion: Pre-transplant XM-negative HLA-DSA increased the risk of ABMR but did not affect long-term allograft outcomes. HLA-incompatible kidney transplantation in the context of XM-negative HLA-DSA appears to be feasible with careful monitoring and ensuring appropriate management of any occurrence of ABMR. Furthermore, considering the characteristics of pre-transplant XM-negative HLA-DSA, the development of a more detailed and standardized desensitization protocol is warranted.
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Rechazo de Injerto , Antígenos HLA , Prueba de Histocompatibilidad , Isoanticuerpos , Trasplante de Riñón , Sistema de Registros , Humanos , Trasplante de Riñón/efectos adversos , Rechazo de Injerto/inmunología , Masculino , Femenino , Antígenos HLA/inmunología , República de Corea , Persona de Mediana Edad , Isoanticuerpos/sangre , Isoanticuerpos/inmunología , Adulto , Supervivencia de Injerto/inmunología , Factores de Riesgo , Resultado del Tratamiento , Donantes de TejidosRESUMEN
Distant metastasis, the leading cause of cancer death, is efficiently kept in check by immune surveillance. Studies have uncovered peripheral natural killer (NK) cells as key antimetastatic effectors and their dysregulation during metastasis. However, the molecular mechanism governing NK cell dysfunction links to metastasis remains elusive. Herein, MAP4K1 encoding HPK1 is aberrantly overexpressed in dysfunctional NK cells in the periphery and the metastatic site. Conditional HPK1 overexpression in NK cells suffices to exacerbate melanoma lung metastasis but not primary tumor growth. Conversely, MAP4K1-deficient mice are resistant to metastasis and further protected by combined immune-checkpoint inhibitors. Mechanistically, HPK1 restrains NK cell cytotoxicity and expansion via activating receptors. Likewise, HPK1 limits human NK cell activation and associates with melanoma NK cell dysfunction couples to TGF-ß1 and patient response to immune checkpoint therapy. Thus, HPK1 is an intracellular checkpoint controlling NK-target cell responses, which is dysregulated and hijacked by tumors during metastatic progression.
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Progresión de la Enfermedad , Células Asesinas Naturales , Melanoma , Células Asesinas Naturales/inmunología , Animales , Ratones , Melanoma/genética , Melanoma/patología , Melanoma/inmunología , Humanos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Modelos Animales de Enfermedad , Metástasis de la Neoplasia , Línea Celular TumoralRESUMEN
BACKGROUND: The skin microbiome is essential in guarding against harmful pathogens and responding to environmental changes by generating substances useful in the cosmetic and pharmaceutical industries. Among these microorganisms, Streptococcus is a bacterial species identified in various isolation sources. In 2021, a strain of Streptococcus infantis, CX-4, was identified from facial skin and found to be linked to skin structure and elasticity. As the skin-derived strain differs from other S. infantis strains, which are usually of oral origin, it emphasizes the significance of bacterial variation by the environment. OBJECTIVE: This study aims to explore the unique characteristics of the CX-4 compared to seven oral-derived Streptococcus strains based on the Whole-Genome Sequencing data, focusing on its potential role in skin health and its possible application in cosmetic strategies. METHODS: The genome of the CX-4 strain was constructed using PacBio Sequencing, with the assembly performed using the SMRT protocol. Comparative whole-genome analysis was then performed with seven closely related strains, utilizing web-based tools like PATRIC, OrthoVenn3, and EggNOG-mapper, for various analyses, including protein association analysis using STRING. RESULTS: Our analysis unveiled a substantial number of Clusters of Orthologous Groups in diverse functional categories in CX-4, among which sphingosine kinase (SphK) emerged as a unique product, exclusively present in the CX-4 strain. SphK is a critical enzyme in the sphingolipid metabolic pathway, generating sphingosine-1-phosphate. The study also brought potential associations with isoprene formation and retinoic acid synthesis, the latter being a metabolite of vitamin A, renowned for its crucial function in promoting skin cell growth, differentiation, and maintaining of skin barrier integrity. These findings collectively suggest the potential of the CX-4 strain in enhancing of skin barrier functionality. CONCLUSION: Our research underscores the potential of the skin-derived S. infantis CX-4 strain by revealing unique bacterial compounds and their potential roles on human skin.
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Genoma Bacteriano , Streptococcus , Humanos , Filogenia , Streptococcus/genética , Secuenciación Completa del GenomaRESUMEN
Sex and gender disparities in biomedical research have been emphasized to improve scientific knowledge applied for the health of both men and women. Despite sex differences in cancer incidence, prognosis, and responses to therapeutic agents, mechanistic explanations at molecular levels are far from enough. Recent studies suggested that cell sex is an important biological variable due to differences in sex chromosome gene expression and differences in events associated with developmental biology. The objective of this study was to analyze the reporting of sex of cells used in cancer research using articles published in Cancer Cell, Molecular Cancer, Journal of Hematology & Oncology, Journal for ImmunoTherapy of Cancer, and Cancer Research in 2020, and to examine whether there exists any sex bias. We found that the percentage of cells with sex notation in the article was 36.5%. Primary cells exhibited higher sex notation compared to cell lines. A higher percentage of female cells were used in cell cultures with sex notation. Also, sex-common cells omitted sex description more often compared to sex-specific cells. None of the cells isolated from embryo and esophagus reported the cell sex in the article. Our results indicate cell sex report in cancer research is limited to a small proportion of cells used in the study. These results call for acknowledging the sex of cells to increase the applicability of biomedical research discoveries.
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Investigación Biomédica , Células Cultivadas , Neoplasias , Femenino , Humanos , Masculino , Publicaciones , Factores Sexuales , SexismoRESUMEN
In consideration of the limited number of FDA-approved drugs for autism spectrum disorder (ASD), significant efforts have been devoted to identifying novel drug candidates. Among these, 5-HT7R modulators have garnered considerable attention due to their potential in alleviating autism-like behaviors in ASD animal models. In this study, we designed and synthesized biphenyl-3-ylmethylpyrrolidines 3 and biphenyl-3-yl-dihydroimidazoles 4 as 5-HT7R modulators. Through extensive biological tests of 3 and 4 in G protein and ß-arrestin signaling pathways of 5-HT7R, it was determined that 2-(2'-methoxy-[1,1'-biphenyl]-3-yl)-4,5-dihydro-1H-imidazole 4h acted as a 5-HT7R antagonist in both signaling pathways. In in vivo study with Shank3-/- transgenic (TG) mice, the self-grooming behavior test was performed with 4h, resulting in a significant reduction in the duration of self-grooming. In addition, an immunohistochemical experiment with 4h restored reduced neurogenesis in Shank3-/- TG mice, which is confirmed by the quantification of doublecortin (DCX) positive neurons, suggesting the promising therapeutic potential of 4h.
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Trastorno del Espectro Autista , Compuestos de Bifenilo , Animales , Ratones , Serotonina , beta-Arrestinas , Transducción de Señal , Ratones Transgénicos , Proteínas de Unión al GTP , Modelos Animales de Enfermedad , Proteínas de Microfilamentos , Proteínas del Tejido NerviosoRESUMEN
Adult-born granule cells (abGCs) exhibit a transient period of elevated synaptic plasticity that plays an important role in hippocampal function. Various mechanisms have been implicated in this critical period for enhanced plasticity, including minimal GABAergic inhibition and high intrinsic excitability conferred by T-type Ca2+ channels. Here we assess the contribution of synaptic inhibition and intrinsic excitability to long-term potentiation (LTP) in abGCs of adult male and female mice using perforated patch recordings. We show that the timing of critical period plasticity is unaffected by intact GABAergic inhibition such that 4-6-week-old abGCs exhibit LTP that is absent by 8â weeks. Blocking GABAA receptors, or partial blockade of GABA release from PV and nNos-expressing interneurons by a µ-opioid receptor agonist, strongly enhances LTP in 4-week-old GCs, suggesting that minimal inhibition does not underlie critical period plasticity. Instead, the closure of the critical period coincides with a reduction in the contribution of T-type Ca2+ channels to intrinsic excitability, and a selective T-type Ca2+ channel antagonist prevents LTP in 4-week-old but not mature GCs. Interestingly, whole-cell recordings that facilitate T-type Ca2+ channel activity in mature GCs unmasks LTP (with inhibition intact) that is also sensitive to a T-type Ca2+ channel antagonist, suggesting T-type channel activity in mature GCs is suppressed by native intracellular signaling. Together these results show that abGCs use T-type Ca2+ channels to overcome inhibition, providing new insight into how high intrinsic excitability provides young abGCs a competitive advantage for experience-dependent synaptic plasticity.
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Potenciación a Largo Plazo , Neuronas , Ratones , Animales , Masculino , Femenino , Neuronas/fisiología , Potenciación a Largo Plazo/fisiología , Plasticidad Neuronal/fisiología , Hipocampo/fisiología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
OBJECTIVES: This study investigated the association between exacerbated economic hardship during the coronavirus disease 2019 (COVID-19) pandemic and changes in the health behaviors of Korean adolescents. METHODS: We analyzed data from the 2021 Korea Youth Risk Behavior Survey and included 44 908 students (22 823 boys and 22 085 girls) as study subjects. The dependent variables included changes in health behaviors (breakfast habits, physical activity, and alcohol use) that occurred during the COVID-19 pandemic. The aggravation of economic hardship by COVID-19 and the subjective economic status of the family were used as exposure variables. Multiple logistic regression analysis was utilized to calculate the prevalence odds ratios (PORs). RESULTS: Severe exacerbation of a family's economic hardship due to COVID-19 was negatively associated with the health behaviors of adolescents, including increased breakfast skipping (POR, 1.85; 95% confidence interval [CI], 1.55 to 2.21 for boys and POR, 1.56; 95% CI, 1.27 to 1.92 for girls) and decreased physical activity (POR, 1.37; 95% CI, 1.19 to 1.57 for boys and POR, 1.38; 95% CI, 1.19 to 1.60 for girls). These negative changes in health behaviors were further amplified when combined with a low subjective family economic status. CONCLUSIONS: The experience of worsening household hardship can lead to negative changes in health behavior among adolescents. It is crucial to implement measures that address the economic challenges that arise from stressful events such as COVID-19 and to strive to improve the lifestyles of adolescents under such circumstances.
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Conducta del Adolescente , COVID-19 , Masculino , Femenino , Humanos , Adolescente , Estudios Transversales , Pandemias , Estrés Financiero , Salud del Adolescente , COVID-19/epidemiología , Conductas Relacionadas con la Salud , Asunción de Riesgos , República de Corea/epidemiologíaRESUMEN
Dry eye disease (DED) is caused by a loss of homeostasis of the tear film, which results in visual disturbance, ocular surface inflammation and damage, and neurosensory abnormalities. Although it is prevalent in 5-50% of the global population, there are limited clinical options for its treatment. This study explored the potential use of human intravenous immunoglobulin (IVIg) and its enriched fractions of sialylation, sialylated IVIg (sIVIg), as a treatment for DED. Fifteen female New Zealand white rabbits were topically instilled with 0.2% benzalkonium chloride (BAC) twice daily for five consecutive days to induce experimental dry eye. Saline, 0.4% IVIg, or 0.04% sIVIg eye drops were instilled twice daily for 20 consecutive days. Clinical evaluations, such as non-invasive tear break-up time (NIBUT) and corneal fluorescein staining (CFS), were conducted. mRNA levels of mucin 4, mucin 16, TNF-α, IL-1ß, MMP9, IL-10, TGF-ß, and CD209 in rabbit conjunctival tissues were examined using reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR). The relationships between CD209 family members in rabbits and various mammalian species were analyzed using a phylogenetic tree. IVIg or sIVIg treatment resulted in clinical improvements in the rabbit DED model. The inflammatory cytokines, TNF-α and IL-1ß, were increased and mucin 4 and mucin 16, cell surface-associated mucins, were decreased in BAC-induced dry eye. Following IVIg or sIVIg treatment, inflammatory cytokines decreased, whereas the anti-inflammatory cytokine, IL-10, increased substantially. Moreover, a 10-fold lower sIVIg treatment dose resulted in prolonged IL-10 production, representing a significantly improved DED compared to IVIg. Furthermore, the expression of rabbit CD209 mRNA in the rabbit conjunctiva and its close relationship with primate homologs suggest that it may interact with IVIg or sIVIg to promote IL-10 expression, as previously described in humans. At a lower dosage, sIVIg showed a more efficient improvement in DED, making it a promising new candidate medication for DED.
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Citocinas , Síndromes de Ojo Seco , Conejos , Humanos , Animales , Citocinas/genética , Citocinas/metabolismo , Inmunoglobulinas Intravenosas/uso terapéutico , Inmunoglobulinas Intravenosas/metabolismo , Interleucina-10/efectos adversos , Interleucina-10/metabolismo , Mucina 4/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Antígeno Ca-125 , Filogenia , Síndromes de Ojo Seco/metabolismo , Lágrimas/metabolismo , Compuestos de Benzalconio , ARN Mensajero/genética , ARN Mensajero/metabolismo , MamíferosRESUMEN
Men living in refugee settings are often exposed to violence, poverty, and social instability, which impacts physical and mental health and increases the risk of perpetrating sexual and gender-based violence. Healthy Men Healthy Communities was developed as a male-led health promotion program to address men's physical and mental health and their role in creating healthy relationships and families. Three community leaders from the settlements were trained to facilitate the program, which was implemented among six groups consisting of twelve men in each group. Pre/post surveys and feedback were collected among the facilitators and participants. Facilitators suggested culturally appropriate ways to present physical activities as a stress reduction technique and the importance of spacing out births. The small group setting facilitated open conversations on topics such as birth spacing and healthy partner communication. Participants experienced an increase in knowledge and confidence in practicing the program content, such as stress-reduction techniques and healthy communication strategies. Participants recommended additional topics such as fertility and sexually transmitted infections. The Healthy Men Healthy Communities program has the potential for wider implementation among male South Sudanese refugees to promote their health as well as the health of their families.
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OBJECTIVE: Liberia experiences an unmet need for cesarean section with about 5% population coverage, lower than 9%-19% coverage associated with improved maternal and newborn outcomes. Delays in the referral process for comprehensive emergency obstetric and newborn care (CEmONC) services due to ineffective communication between a rural health facility (RHF) and a district hospital contribute to the low CS rate. This study examined the association between mobile obstetric emergency system (MORES) implementation and referral time for obstetric emergencies as well as maternal/newborn outcomes. METHODS: A pre-post descriptive analysis was conducted on data collected from 20 rural health facilities (RHFs) and two hospitals in Bong County. Women with referral data from both RHFs and hospitals were matched and information including transfer time, reasons for referral, and maternal and newborn outcomes were extracted. Descriptive analysis and logistic regression models examined the relationship between the intervention's implementation and mode of delivery, maternal outcome, newborn outcome, and transfer time from RHF to district hospital. Ethics approval was obtained from two study centers. RESULTS: Women had higher odds of undergoing a CS at endline (OR: 1.86 95% CI: 0.99-3.46) compared to baseline. Additionally, newborns had lower odds of showing non-vigorous symptoms (OR: 0.31; 95% CI: 0.14-0.68), defined as a newborn with poor respiratory effort, muscle tone, or heart rate. There was no significant association between the intervention's implementation and transfer time. CONCLUSION: The MORES intervention is a promising means to increase timely care seeking along the referral pathway which may enhance access to cesarean section as well as improved newborn outcome in low- and middle-income countries.
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Cesárea , Accesibilidad a los Servicios de Salud , Embarazo , Femenino , Recién Nacido , Humanos , Liberia , Derivación y ConsultaRESUMEN
BACKGROUND: The skin microbiome, a diverse community of microorganisms, plays a crucial role in maintaining skin health. Among these microorganisms, the gram-positive bacterium Micrococcus luteus exhibits potential for promoting skin health. This study focuses on postbiotics derived from M. luteus YM-4, a strain isolated from human skin. OBJECTIVE: Our objective is to explore the beneficial effects of YM-4 culture filtrate on dermatological health, including enhancing barrier function, modulating immune response, and aiding recovery from environmental damage. METHODS: The effects of the YM-4 culture filtrate were tested on human keratinocytes and fibroblasts under various conditions using real-time PCR for gene expression analysis and fibroblast migration assays. A dehydration-simulated model was employed to prepare RNA-Seq samples from HaCaT cells treated with the YM-4 culture filtrate. Differentially expressed genes were identified and functionally classified through k-means clustering, gene ontology terms enrichment analyses, and protein-protein interactions mapping. RESULTS: The YM-4 culture filtrate enhanced the expression of genes involved in skin hydration, hyaluronic acid synthesis, barrier function, and cell proliferation. It also reduced inflammation markers in keratinocytes and fibroblasts under stress conditions. It mitigated UVB-induced collagen degradation while promoted collagen synthesis, suggesting anti-aging properties, and accelerated wound healing processes by promoting cell proliferation and migration. RNA sequencing analysis revealed that the YM-4 culture filtrate could reverse dehydration-induced transcriptional changes towards a state similar to untreated cells. CONCLUSION: M. luteus YM-4 culture filtrate exhibits significant therapeutic potential for dermatological applications.
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Deshidratación , Epirrubicina/análogos & derivados , Micrococcus luteus , Humanos , Deshidratación/metabolismo , Piel/metabolismo , Colágeno/metabolismoRESUMEN
BACKGROUND: Existing models have performed poorly when predicting mortality for patients undergoing venovenous extracorporeal membrane oxygenation (VV-ECMO). This study aimed to develop and validate a machine learning (ML)-based prediction model to predict 90-day mortality in patients undergoing VV-ECMO. METHODS: This study included 368 patients with acute respiratory failure undergoing VV-ECMO from 16 tertiary hospitals across South Korea between 2012 and 2015. The primary outcome was the 90-day mortality after ECMO initiation. The inputs included all available features (n=51) and those from the electronic health record (EHR) systems without preprocessing (n=40). The discriminatory strengths of ML models were evaluated in both internal and external validation sets. The models were compared with conventional models, such as respiratory ECMO survival prediction (RESP) and predicting death for severe acute respiratory distress syndrome on VV-ECMO (PRESERVE). RESULTS: Extreme gradient boosting (XGB) (areas under the receiver operating characteristic curve, AUROC 0.82, 95% CI (0.73 to 0.89)) and light gradient boosting (AUROC 0.81 (95% CI 0.71 to 0.88)) models achieved the highest performance using EHR's and all other available features. The developed models had higher AUROCs (95% CI 0.76 to 0.82) than those of RESP (AUROC 0.66 (95% CI 0.56 to 0.76)) and PRESERVE (AUROC 0.71 (95% CI 0.61 to 0.81)). Additionally, we achieved an AUROC (0.75) for 90-day mortality in external validation in the case of the XGB model, which was higher than that of RESP (0.70) and PRESERVE (0.67) in the same validation dataset. CONCLUSIONS: ML prediction models outperformed previous mortality risk models. This model may be used to identify patients who are unlikely to benefit from VV-ECMO therapy during patient selection.
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Oxigenación por Membrana Extracorpórea , Síndrome de Dificultad Respiratoria , Humanos , Estudios Retrospectivos , Mortalidad Hospitalaria , Síndrome de Dificultad Respiratoria/terapia , Aprendizaje Automático SupervisadoRESUMEN
PCR has been a reliable and inexpensive method for nucleic acid detection in the past several decades. In particular, multiplex PCR is a powerful tool to analyze many biomarkers in the same reaction, thus maximizing detection sensitivity and reducing sample usage. However, balancing the amplification kinetics between amplicons and distinguishing them can be challenging, diminishing the broad adoption of high order multiplex PCR panels. Here, we present a new paradigm in PCR amplification and multiplexed detection using UltraPCR. UltraPCR utilizes a simple centrifugation workflow to split a PCR reaction into â¼34 million partitions, forming an optically clear pellet of spatially separated reaction compartments in a PCR tube. After in situ thermocycling, light sheet scanning is used to produce a 3D reconstruction of the fluorescent positive compartments within the pellet. At typical sample DNA concentrations, the magnitude of partitions offered by UltraPCR dictate that the vast majority of target molecules occupy a compartment uniquely. This single molecule realm allows for isolated amplification events, thereby eliminating competition between different targets and generating unambiguous optical signals for detection. Using a 4-color optical setup, we demonstrate that we can incorporate 10 different fluorescent dyes in the same UltraPCR reaction. We further push multiplexing to an unprecedented level by combinatorial labeling with fluorescent dyes - referred to as "comboplex" technology. Using the same 4-color optical setup, we developed a 22-target comboplex panel that can detect all targets simultaneously at high precision. Collectively, UltraPCR has the potential to push PCR applications beyond what is currently available, enabling a new class of precision genomics assays.
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In this study, the effects of organic acid-soaking (malic, citric, tartaric, and succinic acid) and sonication on the formation of flavor and α-dicarbonyl compounds in Robusta (C. canephora syn. Coffea robusta) green beans were investigated. A total of 20 volatile compounds were identified in Robusta coffee. Furfural and 5-methyl furfural, two dominant volatile compounds in Arabica coffee, increased after organic acid pretreatment. In Robusta coffee processed from 3% malic acid-soaked coffee beans, furfural and 5-methyl furfural increased by 90.99% and 24.92%, respectively, compared to the control. In Robusta coffee processed from 3% malic acid-sonicated (280 W, 1 h) coffee beans, furfural and 5-methyl furfural increased by 236.03% and 114.77%, respectively. α-Dicarbonyls (glyoxal, methylglyoxal, and diacetyl) were significantly (p < 0.05) decreased in all Robusta coffees after organic acid pretreatment. In Robusta coffee processed from coffee beans soaked and sonicated in tartaric acid solution, the α-dicarbonyls decreased by up to 44% and 58%, respectively, compared to the control. This study suggested the pretreatment methods to enhance the flavor substances and reduce the α-DCs in Robusta coffee.
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Coffea , Sonicación , Furaldehído , Malatos , Compuestos OrgánicosRESUMEN
A non-motile, novel actinobacterial strain, Kera-3T, which is a gram-positive, aerobic, rod-shaped bacterium, was isolated from human keratinocytes on 1/10 diluted R2A agar. Whole-cell hydrolysis of amino acids revealed the presence of meso-DAP, alanine, and glutamic acid. The predominant menaquinone was MK-9 (H8), whereas the primary fatty acids were C16:0 and C18:1 ω9c. The major phospholipids included diphosphatidylglycerol and aminophospholipids, along with an unidentified phosphoglycolipid and an aminophosphoglycolipid. The G+C content of the genomic DNA was 73.2%, based on the complete genome sequence. Phylogenetic analyses of the 16S rRNA gene sequence and phylogenomic analysis of 91 core genes showed that strain Kera-3T formed a new lineage in the family Iamiaceae, with the closest neighbour Rhabdothermincola sediminis SYSU G02662T having 91.19% 16S rRNA gene sequence identity. A comparative genomic study of the predicted general metabolism and carbohydrate-active enzymes supported the phylogenetic and phylogenomic data. Based on the analysis of physiological, biochemical, and genomic characteristics, strain Kera-3T can be distinguished from known genera in the family Iamiaceae and represents a novel genus and species. Therefore, the name Dermatobacter hominis gen. nov., sp. nov. was proposed, with the type strain Kera-3T (= KACC 22415T = LMG 32493T).