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BACKGROUND: Current animal models of sensitive skin do not adequately reflect the objective symptoms or physiological manifestations observed in human sensitive skin. OBJECTIVE: To construct and validate a sensitive skin model in mice. METHODS: Tape stripping (TS) was used to induce partial mechanical disruption of the lipid film and stratum corneum. Subsequently, propylene glycol (PG) was applied to disrupt the lipid structure in the skin barrier, and capsaicin (CS) activate transient receptor potential vanilloid 1 (TRPV1) receptors of keratinocytes to simulate the formation of sensitive skin. Evident itching and tingling sensations, scaly skin, vasodilation, local congestion, increased transepidermal water loss (TEWL), elevated TRPV1 expression, and inflammatory symptoms were subsequently evaluated. RESULTS: TS combined with PG and CS application resulted in skin flakes; skin barrier disruption; vascular dilation; increased itching, stinging, and inflammation; TRPV1 upregulation in the epidermis; and a significant increase in lactic acid-induced itching and stinging. CONCLUSION: Using a combination of TS and PG, and CS application, a mouse model of sensitive skin was successfully established involving various skin phenotypes and physiological manifestations, including skin flakes, vasodilation, increased blood flow and TEWL, itching and stinging sensations, inflammation, and elevated TRPV1 expression.
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Inflammatory bowel disease (IBD) is an immunologically complex disorder involving genetic, microbial, and environmental risk factors. Its global burden has continued to rise since industrialization, with epidemiological studies suggesting that ambient particulate matter (PM) in air pollution could be a contributing factor. Prior animal studies have shown that oral PM10 exposure promotes intestinal inflammation in a genetic IBD model and that PM2.5 inhalation exposure can increase intestinal levels of pro-inflammatory cytokines. PM10 and PM2.5 include ultrafine particles (UFP), which have an aerodynamic diameter of <0.10 µm and biophysical and biochemical properties that promote toxicity. UFP inhalation, however, has not been previously studied in the context of murine models of IBD. Here, we demonstrated that ambient PM is toxic to cultured Caco-2 intestinal epithelial cells and examined whether UFP inhalation affected acute colitis induced by dextran sodium sulfate and 2,4,6-trinitrobenzenesulfonic acid. C57BL/6J mice were exposed to filtered air (FA) or various types of ambient PM reaerosolized in the ultrafine size range at ~300 µg/m3, 6 h/day, 3-5 days/week, starting 7-10 days before disease induction. No differences in weight change, clinical disease activity, or histology were observed between the PM and FA-exposed groups. In conclusion, UFP inhalation exposure did not exacerbate intestinal inflammation in acute, chemically-induced colitis models.
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Colitis , Sulfato de Dextran , Ratones Endogámicos C57BL , Material Particulado , Ácido Trinitrobencenosulfónico , Material Particulado/toxicidad , Animales , Colitis/inducido químicamente , Colitis/patología , Ratones , Humanos , Sulfato de Dextran/toxicidad , Células CACO-2 , Ácido Trinitrobencenosulfónico/toxicidad , Ácido Trinitrobencenosulfónico/efectos adversos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Mucosa Intestinal/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Células Epiteliales/metabolismo , Modelos Animales de Enfermedad , Masculino , Tamaño de la PartículaRESUMEN
Emu oil is the oil extracted from the body fat of the Australian bird emu. Although previous studies have reported that emu oil has anti-inflammatory effects, the effect and mechanism of emu oil on the treatment of atopic dermatitis have not been reported. Here, 2, 4-dinitrofluorobenzene was used to induce atopic dermatitis-like appearance on the back skin of C57BL/6 mice. And then, the effect of emu oil in the atopic dermatitis treatment was evaluated. We found that emu oil reduced the transdermal water loss in the atopic dermatitis model. Additionally, the epidermal thickness treated with emu oil was significantly thinner. The number of mast cells and inflammatory cells were significantly decreased. The thymic stromal lymphopoietin (TSLP), which is secreted by keratinocyte, was decreased significantly after treatment. Moreover, the serum levels of cytokines TSLP, interleukin-4, interleukin-13, and immunoglobulin (Ig) E were decreased after emu oil treatment. Surprisingly, we found that the high level of Cdc42 expression in the atopic dermatitis, which was decreased after emu oil treatment. To detect the role of Cdc42 in atopic dermatitis, we constructed atopic dermatitis model in mice with sustained activation of Cdc42 signaling. Furthermore, we have confirmed that emu oil demonstrates anti-inflammatory effects in atopic dermatitis by inhibiting the expression of Cdc42 signaling in keratinocytes. In conclusion, we discovered a new role of Cdc42 in the development of atopic dermatitis, which mediated the therapeutic effect of emu oil on atopic dermatitis.
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Antiinflamatorios , Citocinas , Dermatitis Atópica , Modelos Animales de Enfermedad , Queratinocitos , Ratones Endogámicos C57BL , Transducción de Señal , Proteína de Unión al GTP cdc42 , Animales , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/inducido químicamente , Dermatitis Atópica/inmunología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Citocinas/metabolismo , Transducción de Señal/efectos de los fármacos , Ratones , Proteína de Unión al GTP cdc42/metabolismo , Antiinflamatorios/uso terapéutico , Antiinflamatorios/farmacología , Linfopoyetina del Estroma Tímico , Aceites/farmacología , Aceites/uso terapéutico , Inmunoglobulina E/sangre , Dinitrofluorobenceno , Piel/efectos de los fármacos , Piel/patología , Piel/metabolismo , Humanos , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Mastocitos/metabolismo , MasculinoRESUMEN
Human pregnane X receptor (PXR) is critical for regulating the expression of key drug-metabolizing enzymes such as CYP3A and CYP2C. Our recent study revealed that treatment with rodent-specific PXR agonist pregnenolone-16α-carbonitrile (PCN) significantly induced hepatomegaly and promoted liver regeneration after two-thirds partial hepatectomy (PHx) in mice. However, it remains unclear whether PXR activation induces hepatomegaly and liver regeneration and simultaneously promotes metabolic function of the liver. Here, we investigated the metabolism activity of CYP1A2, CYP3A1/2 and CYP2C6/11 during PXR activation-induced liver enlargement and regeneration in rats after cocktail dosing of CYP probe drugs. For PCN-induced hepatomegaly, a notable increase in the metabolic activity of CYP3A1/2 and CYP2C6/11, as evidenced by the plasma exposure of probe substrates and the AUC ratios of the characteristic metabolites to its corresponding probe substrates. The metabolic activity of CYP1A2, CYP3A1/2 and CYP2C6/11 decreased significantly after PHx. However, PCN treatment obviously enhanced the metabolic activity of CYP2C6/11 and CYP3A1/2 in PHx rats. Furthermore, the protein expression levels of CYP3A1/2 and CYP2C6/11 in liver were up-regulated. Taken together, this study demonstrates that PXR activation not only induces hepatomegaly and liver regeneration in rats, but also promotes the protein expression and metabolic activity of the PXR downstream metabolizing enzymes such as CYP3A1/2 and CYP2C6/11 in the body.
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Citocromo P-450 CYP3A , Hepatomegalia , Regeneración Hepática , Hígado , Receptor X de Pregnano , Carbonitrilo de Pregnenolona , Animales , Receptor X de Pregnano/metabolismo , Receptor X de Pregnano/genética , Regeneración Hepática/efectos de los fármacos , Masculino , Citocromo P-450 CYP3A/metabolismo , Carbonitrilo de Pregnenolona/farmacología , Hígado/metabolismo , Hígado/enzimología , Hígado/efectos de los fármacos , Ratas , Hepatomegalia/metabolismo , Hepatomegalia/patología , Hidrocarburo de Aril Hidroxilasas/metabolismo , Hidrocarburo de Aril Hidroxilasas/genética , Familia 2 del Citocromo P450/metabolismo , Familia 2 del Citocromo P450/genética , Ratas Sprague-Dawley , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1A2/genética , Esteroide 16-alfa-Hidroxilasa/metabolismo , Esteroide 16-alfa-Hidroxilasa/genética , Esteroide 12-alfa-Hidroxilasa/metabolismo , Esteroide 12-alfa-Hidroxilasa/genética , HepatectomíaRESUMEN
Typical BCR::ABL1-negative myeloproliferative neoplasms (MPN) are mainly referred to as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofbrosis (PMF). Granulocytes in MPN patients are involved in their inflammation and form an important part of the pathophysiology of MPN patients. It has been shown that the immunophenotype of granulocytes in MPN patients is altered. We used flow cytometry to explore the immunophenotype of MPN patients and correlate it with clinical parameters. The results showed that PMF patients and PV patients had higher CD15+CD11b+ granulocytes than ET patients and normal controls. When grouped by gene mutation, changes in the granulocyte immunophenotype of MPN patients were independent of the JAK2V617F and CALR mutations. There was no significant heterogeneity in immunophenotype between ET patients and Pre-PMF, and between Overt-PMF and Pre-PMF patients. Granulocytes from some MPN patients showed an abnormal CD13/CD16 phenotype with a significant increase in mature granulocytes on molecular and cytomorphological grounds, and this abnormal pattern occurred significantly more frequently in PMF patients than in ET patients. CD15-CD11b- was negatively correlated with WBC and Hb and positively correlated with DIPSS score, whereas high CD10+ granulocytes were significantly and negatively associated with prognostic system IPSS and DIPSS scores in PMF patients. In conclusion, this study demonstrates the landscape of bone marrow granulocyte immunophenotypes in MPN patients. MPN patients, especially those with PMF, have a significant granulocyte developmental overmaturation phenotype. CD10+ granulocytes may be involved in the prognosis of PMF patients.
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Citometría de Flujo , Proteínas de Fusión bcr-abl , Granulocitos , Inmunofenotipificación , Trastornos Mieloproliferativos , Humanos , Masculino , Persona de Mediana Edad , Femenino , Granulocitos/patología , Adulto , Anciano , Proteínas de Fusión bcr-abl/genética , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/inmunología , Trastornos Mieloproliferativos/patología , Janus Quinasa 2/genética , Trombocitemia Esencial/genética , Trombocitemia Esencial/patología , Anciano de 80 o más Años , China , Adulto Joven , Calreticulina/genética , Antígeno CD11b/genética , Policitemia Vera/genética , Policitemia Vera/patología , Policitemia Vera/inmunología , Mutación , Pueblo Asiatico/genética , Pueblos del Este de AsiaRESUMEN
Exploration of variant allele frequency (VAF) of GATA2 mutations (GATA2mut) provides insights into acute myeloid leukemia (AML) prognosis. In this study, we analyzed GATA2mut and co-mutations in 166 Chinese patients with cytogenetically normal AML. This was done through targeted next-generation sequencing of 34 genes associated with myeloid leukemia. GATA2mut was identified in 17 (10%) patients being significantly correlated with co-mutations in CCAAT/enhancer-binding protein alpha (CEBPA) double mutation (P = 0.001). We observed that the N-terminal zinc finger domain (ZF1) was linked to CEBPA mutations, while the C-terminal zinc finger domain (ZF2) was associated with Wilms' tumor 1 (WT1) mutations. It was also noted that patients with GATA2mut had lower platelet counts at diagnosis (P = 0.032). In the entire cohort, GATA2mut had no significant prognostic impact on overall survival (OS) (P = 0.762) and relapse-free survival (RFS) (P = 0.369) compared to patients with GATA2wt. The OS (P = 0.737) and RFS (P = 0.894) of the ZF1 mutation were similar to those of the ZF2 mutation. Most patients with GATA2 mutations were classified in the ELN2022 favorable- and intermediate-risk groups. GATA2mut patients in the favorable-risk group were divided into GATA2High and GATA2Low groups using a median cutoff variant allele frequency (VAF) of 40.13%. GATA2High patients were associated with worse OS (P = 0.031) and RFS (P = 0.021) than GATA2Low patients. In the intermediate-risk group, the high median VAF of GATA2 (≥38.51%) had no significant effect in OS and RFS compared with the low median VAF (<38.51%). This study offers new insights on the prognosis of GATA2mut in the favorable-risk group, where VAF can be used as a guide.
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Factor de Transcripción GATA2 , Frecuencia de los Genes , Leucemia Mieloide Aguda , Mutación , Humanos , Factor de Transcripción GATA2/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Masculino , Femenino , Adulto , Persona de Mediana Edad , Pronóstico , Anciano , Pueblo Asiatico/genética , Adolescente , Adulto Joven , Proteínas Potenciadoras de Unión a CCAAT/genética , China/epidemiología , Anciano de 80 o más Años , Pueblos del Este de AsiaRESUMEN
OBJECTIVES: Dysbiosis of the sinonasal microbiome has been implicated in the pathogenesis of chronic rhinosinusitis (CRS). However, the mycobiome remains largely understudied, and microbial alterations associated with specific CRS subtypes have yet to be delineated. The objective of this study is to investigate the fungal and bacterial microbiome of sinus mucosa in CRS patients with and without nasal polyposis (CRSwNP and CRSsNP) versus healthy controls. METHODS: Sinus mucosa was obtained from 92 patients (31 CRSsNP, 31 CRSwNP, and 30 controls) undergoing endoscopic sinus/skull base surgery. Data regarding demographics, Lund-MacKay scores, and histopathology were collected. Fungal and bacterial microbiome analysis was performed utilizing internal transcribed spacer amplicon and 16S rRNA sequencing. RESULTS: Beta diversity of the sinonasal mycobiome differed significantly between CRS and controls (p = 0.001) and between CRSwNP and controls (p = 0.049), but not between CRSwNP and CRSsNP (p = 0.32) nor between CRSsNP and controls (p = 0.06). With respect to the bacterial microbiome, significantly lower alpha diversity was observed between CRS and controls (p < 0.001), CRSwNP versus controls (p < 0.001), and CRSsNP versus controls (p < 0.001). Beta diversity was also significantly different at the genus level between CRSwNP and CRSsNP (p = 0.019), CRSwNP and controls (p = 0.002)), and CRSsNP and controls (p < 0.001). However, alpha and beta diversity did not differ significantly between CRS patients with/without eosinophils or correlate with Lund-MacKay scores. CONCLUSIONS: Differences in mycobiota diversity in CRS patients in comparison with controls suggest that alterations in the mycobiome may contribute to disease pathogenesis. Our findings also confirmed that diminished diversity among bacterial communities is associated with CRS and that significant differences are present in microbial composition between CRSwNP and CRSsNP. LEVEL OF EVIDENCE: 3 Laryngoscope, 134:1054-1062, 2024.
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Microbiota , Pólipos Nasales , Rinitis , Rinosinusitis , Sinusitis , Humanos , Rinitis/cirugía , ARN Ribosómico 16S/genética , Enfermedad Crónica , Sinusitis/cirugía , Pólipos Nasales/complicaciones , Bacterias/genética , Membrana Mucosa/patologíaRESUMEN
BACKGROUND: Stress reactivity (SR) is associated with increased risk of flares in ulcerative colitis (UC) patients. Because both preclinical and clinical data support that stress can influence gut microbiome composition and function, we investigated whether microbiome profiles of SR exist in UC. METHODS: Ninety-one UC subjects in clinical and biochemical remission were classified into high and low SR groups by questionnaires. Baseline and longitudinal characterization of the intestinal microbiome was performed by 16S rRNA gene sequencing and fecal and plasma global untargeted metabolomics. Microbe, fecal metabolite, and plasma metabolite abundances were analyzed separately to create random forest classifiers for high SR and biomarker-derived SR scores. RESULTS: High SR reactivity was characterized by altered abundance of fecal microbes, primarily in the Ruminococcaceae and Lachnospiraceae families; fecal metabolites including reduced levels of monoacylglycerols (endocannabinoid-related) and bile acids; and plasma metabolites including increased 4-ethyl phenyl sulfate, 1-arachidonoylglycerol (endocannabinoid), and sphingomyelin. Classifiers generated from baseline microbe, fecal metabolite, and plasma metabolite abundance distinguished high vs low SR with area under the receiver operating characteristic curve of 0.81, 0.83, and 0.91, respectively. Stress reactivity scores derived from these classifiers were significantly associated with flare risk during 6 to 24 months of follow-up, with odds ratios of 3.8, 4.1, and 4.9. Clinical flare and intestinal inflammation did not alter fecal microbial abundances but attenuated fecal and plasma metabolite differences between high and low SR. CONCLUSIONS: High SR in UC is characterized by microbial signatures that predict clinical flare risk, suggesting that the microbiome may contribute to stress-induced UC flares.
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Colitis Ulcerosa , Humanos , Endocannabinoides , ARN Ribosómico 16S , Ácidos y Sales Biliares , ClostridialesRESUMEN
BACKGROUND: Ultrasonic for detecting and evaluating pleural effusion is an essential part of the Extended Focused Assessment with Sonography in Trauma (E-FAST) in emergencies. Our study aimed to develop an Artificial Intelligence (AI) diagnostic model that automatically identifies and segments pleural effusion areas on ultrasonography. METHODS: An Attention U-net and a U-net model were used to detect and segment pleural effusion on ultrasound images of 848 subjects through fully supervised learning. Sensitivity, specificity, precision, accuracy, F1 score, the receiver operating characteristic (ROC) curve, and the area under the curve (AUC) were used to assess the model's effectiveness in classifying the data. The dice coefficient was used to evaluate the segmentation performance of the model. RESULTS: In 10 random tests, the Attention U-net and U-net 's average sensitivity of 97% demonstrated that the pleural effusion was well detectable. The Attention U-net performed better at identifying negative images than the U-net, which had an average specificity of 91% compared to 86% for the U-net. Additionally, the Attention U-net was more accurate in predicting the pleural effusion region because its average dice coefficient was 0.86 as opposed to the U-net's average dice coefficient of 0.82. CONCLUSIONS: The Attention U-net showed excellent performance in detecting and segmenting pleural effusion on ultrasonic images, which is expected to enhance the operation and application of E-FAST in clinical work.
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Inteligencia Artificial , Derrame Pleural , Humanos , Derrame Pleural/diagnóstico por imagen , Ultrasonografía , Área Bajo la Curva , Curva ROCRESUMEN
This study aimed to design a novel mouse model of chronic photoaging. We used three different species of mice (C57BL/6J, ICR, and KM) to create a chronic photoaging model of the skin. The irradiation time was gradually increased for 40 consecutive days. The skins of the mice were removed on day 41 and subjected to staining to observe them for morphological changes. Immunohistochemistry was used to detect tumor necrosis factor-α (TNF-α) and p53 expression; superoxide dismutase (SOD) and malondialdehyde (MDA) were measured as well. Compared with C57BL/J mice, which showed hyperpigmentation, the irradiated skin of ICR and KM mice showed more obvious skin thickening and photoaging changes of the collagen and elastic fibers. KM mice had higher levels of inflammation, oxidative stress, and senescent cells. Compared with the 5-month-old KM mice, the photoaging changes of the 9-month-old KM mice were more pronounced, the SOD values were lower, and the MDA values were higher. In summary, KM mice have higher levels of abnormal elastic fibers, inflammation, cellular senescence, and oxidative stress than ICR mice, and are more suitable for studies related to chronic skin photoaging. C57BL/6J mice were found to be suitable for studies related to skin pigmentation due to photoaging.
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Envejecimiento de la Piel , Ratones , Animales , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Piel/metabolismo , Superóxido Dismutasa/metabolismo , Rayos Ultravioleta/efectos adversosRESUMEN
OBJECTIVE: To investigate the molecular characteristics and clinical prognosis of the neuroblastoma RAS viral oncogene (NRAS) in patients with primary cytogenetically normal acute myeloid leukemia (AML). METHODS: A total of 171 adult patients with cytogenetically normal primary AML were collected, and 34 gene mutations in these patients were detected by targeted next-generation sequencing. RESULTS: Among 171 patients with cytogenetically normal AML(CN-AML), 17 (9.9%) patients had found NRAS mutations. Among the 17 NRAS mutant patients, 16 cases were associated with the concomitant gene, and NRAS mutation (NRASmut) was significantly positively correlated with DNMT3A mutation (DNMT3Amut) (P=0.011) and KRAS mutation (P=0.008) compared with the NRAS wild-type (NRASwt) group. The frequency of NRASmutDNMT3Amut clone was significantly higher in CN-AML patients with NRAS mutation (8/17, 47%). The total NRASmut group showed no significant differences on clinical characteristics, CR rate after induction therapy, OS, and RFS as compared with NRASwt group. However, patients with NRASmutDNMT3Amut provided a shorter effect on OS (median:7 vs 15 months; P=0.036) and RFS (median: 3 vs 12 months; P=0.003) than those with NRASwt, though no statistic differences on demographics, lab parameters, treatment and CR rate of patients receiving induction therapy. Multivariate analysis showed that NRASmutDNMT3Amut subtype could independently affect the RFS of CN-AML patients (HR:3.210, 95%CI:1.078-9.557, P=0.036). CONCLUSION: NRASmutDNMT3Amut clones have a high frequency of occurrence and show a poor survival prognosis. Our findings highlight potentially novel aspects of the underlying biology of NRASmutDNMT3Amut commutation in adult de novo CN-AML.
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Leucemia Mieloide Aguda , Adulto , Humanos , Pronóstico , Leucemia Mieloide Aguda/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis Multivariante , Mutación/genética , Proteínas de la Membrana/genética , GTP Fosfohidrolasas/genéticaRESUMEN
Biotin is an essential vitamin and critical cofactor in several metabolic pathways, and its deficiency has been linked to several disorders including inflammatory bowel disease (IBD). We previously reported that biotin deficiency (BD) in mice, whether modeled through intestine-specific deletion of biotin transporter (SMVT-icKO) or through a biotin-deficient diet, resulted in intestinal inflammation consistent with an IBD-like phenotype. To assess whether the gut microbiome is associated with these BD-induced changes, we collected stool and intestinal samples from both of these mouse models and utilized them for 16S rRNA gene sequencing. We find that both diet-mediated and deletion-mediated BD result in the expansion of opportunistic microbes including Klebsiella, Enterobacter, and Helicobacter, at the expense of mucus-resident microbes including Akkermansia. Additionally, microbiome dysbiosis resulting from diet-mediated BD precedes the onset of the IBD-like phenotypic changes. Lastly, through the use of predictive metagenomics, we report that the resulting BD-linked microbiome perturbations exhibit increased biotin biosynthesis in addition to several other perturbed metabolic pathways. Altogether, these results demonstrate that biotin deficiency results in a specific microbiome composition, which may favor microbes capable of biotin synthesis and which may contribute to intestinal inflammation.
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Biotina , Enfermedades Inflamatorias del Intestino , Animales , Ratones , Disbiosis , ARN Ribosómico 16S/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Fenotipo , InflamaciónRESUMEN
Colorectal cancer (CRC) is associated with alterations of the fecal and tissue-associated microbiome. Preclinical models support a pathogenic role of the microbiome in CRC, including in promoting metastasis and modulating antitumor immune responses. To investigate whether the microbiome is associated with lymph node metastasis and T cell infiltration in human CRC, we performed 16S rRNA gene sequencing of feces, tumor core, tumor surface, and healthy adjacent tissue collected from 34 CRC patients undergoing surgery (28 fecal samples and 39 tissue samples). Tissue microbiome profiles-including increased Fusobacterium-were significantly associated with mesenteric lymph node (MLN) involvement. Fecal microbes were also associated with MLN involvement and accurately classified CRC patients into those with or without MLN involvement. Tumor T cell infiltration was assessed by immunohistochemical staining of CD3 and CD8 in tumor tissue sections. Tumor core microbiota, including members of the Blautia and Faecalibacterium genera, were significantly associated with tumor T cell infiltration. Abundance of specific fecal microbes including a member of the Roseburia genus predicted high vs. low total and cytotoxic T cell infiltration in random forests classifiers. These findings support a link between the microbiome and antitumor immune responses that may influence prognosis of locally advanced CRC.
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Neoplasias Colorrectales , Microbioma Gastrointestinal , Microbiota , Linfocitos T , Humanos , Neoplasias Colorrectales/patología , Heces/microbiología , Microbioma Gastrointestinal/fisiología , Ganglios Linfáticos , ARN Ribosómico 16S/genética , Linfocitos Infiltrantes de Tumor , Linfocitos T/inmunologíaRESUMEN
PURPOSE: The detection of abdominal free fluid or hemoperitoneum can provide critical information for clinical diagnosis and treatment, particularly in emergencies. This study investigates the use of deep learning (DL) for identifying peritoneal free fluid in ultrasonography (US) images of the abdominal cavity, which can help inexperienced physicians or non-professional people in diagnosis. It focuses specifically on first-response scenarios involving focused assessment with sonography for trauma (FAST) technique. METHODS: A total of 2985 US images were collected from ascites patients treated from 1 January 2016 to 31 December 2017 at the Shenzhen Second People's Hospital. The data were categorized as Ascites-1, Ascites-2, or Ascites-3, based on the surrounding anatomy. A uniform standard for regions of interest (ROIs) and the lack of obstruction from acoustic shadow was used to classify positive samples. These images were then divided into training (90%) and test (10%) datasets to evaluate the performance of a U-net model, utilizing an encoder-decoder architecture and contracting and expansive paths, developed as part of the study. RESULTS: Test results produced sensitivity and specificity values of 94.38% and 68.13%, respectively, in the diagnosis of Ascites-1 US images, with an average Dice coefficient of 0.65 (standard deviation [SD] = 0.21). Similarly, the sensitivity and specificity for Ascites-2 were 97.12% and 86.33%, respectively, with an average Dice coefficient of 0.79 (SD = 0.14). The accuracy and area under the curve (AUC) were 81.25% and 0.76 for Ascites-1 and 91.73% and 0.91 for Ascites-2. CONCLUSION: The results produced by the U-net demonstrate the viability of DL for automated ascites diagnosis. This suggests the proposed technique could be highly valuable for improving FAST-based preliminary diagnoses, particularly in emergency scenarios.
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Ascitis , Aprendizaje Profundo , Abdomen , Ascitis/diagnóstico por imagen , Humanos , Sensibilidad y Especificidad , UltrasonografíaRESUMEN
Based on our previous research, thirty new 5-amino-1H-1,2,4-triazoles possessing 3,4,5-trimethoxyphenyl moiety were synthesized, and evaluated for antiproliferative activities. Among them, compounds IIa, IIIh, and IIIm demonstrated significant antiproliferative activities against a panel of tumor cell lines, and the promising compound IIIm dose-dependently caused G2/M phase arrest in HeLa cells. Furthermore, analogue IIa exhibited the most potent tubulinpolymerization inhibitory activity with an IC50 value of 9.4 µM, and molecular modeling studies revealed that IIa formed stable interactions in the colchicine-binding site of tubulin, suggesting that 5-amino-1H-1,2,4-triazole scaffold has potential for further investigation to develop novel tubulin polymerization inhibitors with anticancer activity.
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Antineoplásicos/farmacología , Triazoles/farmacología , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Estructura Molecular , Polimerizacion/efectos de los fármacos , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/química , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/químicaRESUMEN
The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo(-)) and DNA polymerase ß (pol ß) using (19)F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol ß. The addition of a non-hydrolysable 2'-deoxycytosine-5'-[(α,ß)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo(-) complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol ß, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with (19)F NMR data. Surface plasmon resonance binding kinetics revealed that pol ß binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential.
Asunto(s)
Aductos de ADN/química , ADN Polimerasa I/química , ADN Polimerasa beta/química , Replicación del ADN , Desoxiguanosina/análogos & derivados , Fluorenos/química , Dominio Catalítico , Desoxiguanosina/química , Humanos , Cinética , Resonancia Magnética Nuclear Biomolecular , Especificidad por Sustrato , Resonancia por Plasmón de SuperficieRESUMEN
The environmental arylamine mutagens are implicated in the etiology of various sporadic human cancers. Arylamine-modified dG lesions were studied in two fully paired 11-mer duplexes with a -G*CN- sequence context, in which G* is a C8-substituted dG adduct derived from fluorinated analogs of 4-aminobiphenyl (FABP), 2-aminofluorene (FAF) or 2-acetylaminofluorene (FAAF), and N is either dA or dT. The FABP and FAF lesions exist in a simple mixture of 'stacked' (S) and 'B-type' (B) conformers, whereas the N-acetylated FAAF also samples a 'wedge' (W) conformer. FAAF is repaired three to four times more efficiently than FABP and FAF. A simple A- to -T polarity swap in the G*CA/G*CT transition produced a dramatic increase in syn-conformation and resulted in 2- to 3-fold lower nucleotide excision repair (NER) efficiencies in Escherichia coli. These results indicate that lesion-induced DNA bending/thermodynamic destabilization is an important DNA damage recognition factor, more so than the local S/B-conformational heterogeneity that was observed previously for FAF and FAAF in certain sequence contexts. This work represents a novel 3'-next flanking sequence effect as a unique NER factor for bulky arylamine lesions in E. coli.
Asunto(s)
2-Acetilaminofluoreno/química , Compuestos de Aminobifenilo/química , Aductos de ADN/química , Daño del ADN , Reparación del ADN , Desoxiguanosina/análogos & derivados , Fluorenos/química , Secuencia de Bases , Dicroismo Circular , Aductos de ADN/metabolismo , Desoxiguanosina/química , Ensayo de Cambio de Movilidad Electroforética , Endodesoxirribonucleasas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , TermodinámicaRESUMEN
We report a systematic spectroscopic investigation on the conformational evolution during primer extension of a bulky fluoroaminofluorene-modified dG adduct (FAF-dG) in chemically simulated translesion synthesis. FAF-dG was paired either with dC or dA (dC-match and dA-mismatch series, respectively). Dynamic (19)F NMR/CD results showed that the FAF-adduct exists in a syn/anti equilibrium and that its conformational characteristics are modulated by the identity of an inserted nucleotide at the lesion site and the extent of primer elongation. At the pre-insertion site, the adduct adopted preferentially a syn conformation where FAF stacked with preceding bases. Insertion of the correct nucleotide dC at the lesion site and subsequent elongation resulted in a gradual transition to the anti conformation. By contrast, the syn conformer was persistent along with primer extension in the dA-mismatch series. In the dC-match series, FAF-induced thermal (T(m)) and thermodynamic (-ΔG°(37 °C)) stabilities were significantly reduced relative to those of the controls. However, the corresponding T(m) and -ΔG°(37 °C) values were increased in the FAF-modified mismatched dA series. The lesion impact persisted up to three 5'-nucleotides from the lesion. Occupation of the minor groove of the W-conformer with the bulky carcinogenic fluorene moiety not only would limit the DNA mobility but also would impose a serious difficulty for the active site of a polymerase throughout the replication process. Our spectroscopic results are consistent with reported data on AF, which showed dramatic (~10(4)-fold) differences in the nucleotide insertion rates between the dC-match and dA-mismatch series. The results emphasize the importance of adduct-induced steric constraints for determining the replication fidelity of a polymerase.
Asunto(s)
Aductos de ADN/química , ADN/biosíntesis , Fluorenos/química , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Espectrofotometría Ultravioleta , Termodinámica , Temperatura de TransiciónRESUMEN
DNA damage by adduct formation is a critical step for the initiation of carcinogenesis. Aromatic amines are strong inducers of environmental carcinogenesis. Their DNA adducts are known to exist in an equilibrium between the major groove (B) and base-displaced stacked (S) conformations. However, the factors governing such heterogeneity remain unclear. Here we conducted extensive calorimetry/NMR/CD studies on the model DNA lesions caused by fluorinated 2-aminfluorene (FAF) and 4-aminobiphenyl (FABP) in order to gain thermodynamic and kinetic insights into the S/B conformational equilibrium. We demonstrate that there are large differences in enthalpy-entropy compensations for FABP and FAF. The small and flexible FABP exclusively adopts the less perturbed B conformer with small enthalpy (DeltaDeltaH-2.7 kcal/mol)/entropy (DeltaDeltaS-0.7 eu) change. In contrast, FAF stacks better and exists as a mixture of B and S conformers with large enthalpy (DeltaDeltaH-13.4 kcal/mol)/entropy (DeltaDeltaS-34.2 eu) compensation. van't Hoff analysis of dynamic (19)F NMR data indicated DeltaH(B<-->S) = 4.1 kcal/mol in favor of the B conformer and DeltaS(B<-->S) = 15.6 cal mol(-1) K(-1) in favor of the intercalated S conformer. These findings demonstrate that the favorable entropy of the S conformer over B conformer determines the S/B population ratios at physiological temperatures.
Asunto(s)
Carcinógenos/farmacología , ADN/química , Secuencia de Bases , Calorimetría , Rastreo Diferencial de Calorimetría/métodos , Dicroismo Circular , ADN/efectos de los fármacos , Aductos de ADN/síntesis química , Aductos de ADN/química , Aductos de ADN/efectos de los fármacos , Daño del ADN , Entropía , Espectroscopía de Resonancia Magnética/métodos , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Oligodesoxirribonucleótidos , TermodinámicaRESUMEN
We report synthesis and characterization of a complete set of alpha,beta-methylene-2'-dNTPs (alpha,beta-m-dNTP; N = A, C, T, G, 12-15) in which the alpha,beta-oxygen linkage of natural dNTP was replaced by a methylene group. These nucleotides were designed to be noncleavable substrates for DNA polymerases. Synthesis entails preparation of 2'-deoxynucleoside 5'-diphosphate precursors, followed by an enzymatic gamma-phosphorylation. All four synthesized alpha,beta-m-dNTPs were found to be potent inhibitors of polymerase beta, with K i values ranging 1-5 microM. During preparation of the dG and dT derivatives of alpha,beta-methylene diphosphate, we also isolated significant amounts of 3,5'-cyclo-dG (16) and 2,5'-cyclo-dT (17), respectively. These novel 2'-deoxycyclonucleosides were formed via a base-catalyzed intramolecular cyclization (N3 --> C5' and O2 --> C5', respectively). In acidic solution, both 16 and 17 underwent glycolysis, followed by complete depurination. When exposed to alkaline conditions, 16 underwent an oxidative deamination to produce 3,5'- cyclo-2'-deoxyxanthosine (19), whereas 17 was hydrolyzed exclusively to dT.