Estudio comparativo entre un test de antígeno y la prueba de la reacción en cadena de la polimerasa en tiempo real (RT-PCR) para el diagnóstico de infección por COVID-19 en pediatría / Comparative study between an antigen test and the reverse transcription-polymerase chain reaction (RT-PCR) test for the diagnosis of COVID-19 in pediatrics

Introducción. La pandemia por COVID-19 ha puesto de manifiesto la necesidad de pruebas diagnósticas rápidas. La prueba de referencia es la reacción en cadena de la polimerasa en tiempo real (RT-PCR). Requiere un equipo y personal capacitado, y su resultado puede llevar un tiempo de espera prolongado. El sistema BD Veritor® es el método rápido cromatográfico utilizado para la detección del antígeno del coronavirus de tipo 2 del síndrome respiratorio agudo grave, en individuos sintomáticos. El objetivo primario del siguiente trabajo es evaluar sensibilidad y especificidad del test de antígeno (TA) comparadas con la RT-PCR en población pediátrica. Población y métodos. Estudio prospectivo, de prueba diagnóstica. Se incluyó a todo menor de 17 años en los primeros 5 días de inicio de síntomas, que consultó desde julio de 2021 hasta febrero de 2022. Se calculó un mínimo de 300 muestras para lograr una precisión de ± 8,76 % y de ± 3,68 % para sensibilidad y especificidad respectivamente. Se analizaron en paralelo las muestras por ambas metodologías. Resultados. De 316 muestras pareadas, 33 fueron positivas por ambos métodos; 6 fueron positivas solo por RT-PCR. La especificidad del TA fue del 100 %; la sensibilidad, del 84,6 %, con un valor predictivo positivo y negativo del 100 % y del 98 % respectivamente. Conclusiones. El TA demostró ser útil en el diagnóstico de pacientes pediátricos con COVID-19 en los primeros 5 días de inicio de síntomas, aunque aquellos con TA negativo y alta sospecha clínica deberían confirmar su resultado con la RT-PCR.

Introduction. The COVID-19 pandemic has brought to light the need for rapid diagnostic tests. The gold standard test is reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR requires equipment and trained personnel, and results may take a long waiting time. The BD Veritor® System is a rapid chromatographic method used for the detection of severe acute respiratory syndrome coronavirus 2 antigen in symptomatic individuals. The primary objective of this study is to assess the sensitivity and specificity of the antigen test (AT) compared to the RT-PCR in the pediatric population. Population and methods. Prospective study with a diagnostic test. All children younger than 17 years in the first 5 days of symptom onset, who consulted between July 2021 and February 2022, were included. A minimum of 300 specimens was estimated to achieve an accuracy of ±8.76% and ±3.68% for sensitivity and specificity, respectively. Specimens were analyzed in parallel using both methodologies. Results. Of 316 paired samples, 33 were positive by both methods; 6 were positive only by RT-PCR. The specificity of the AT was 100%; sensitivity was 84.6%, with a positive and negative predictive value of 100% and 98%, respectively. Conclusions. The AT proved to be useful in the diagnosis of pediatric patients with COVID-19 in the first 5 days of symptom onset, although those with a negative AT and high clinical suspicion should confirm their result with a RT-PCR.

Real-time reverse transcription-polymerase chain reaction assay panel for the detection of severe acute respiratory syndrome coronavirus 2 and its variants / 中华医学杂志(英文版)

BACKGROUND@#With the ongoing worldwide coronavirus disease 2019 (COVID-19) pandemic, an increasing number of viral variants are being identified, which poses a challenge for nucleic acid-based diagnostic tests. Rapid tests, such as real-time reverse transcription-polymerase chain reaction (rRT-PCR), play an important role in monitoring COVID-19 infection and controlling its spread. However, the changes in the genotypes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants may result in decreased sensitivity of the rRT-PCR assay and it is necessary to monitor the mutations in primers and probes of SARS-CoV-2 detection over time.@*METHODS@#We developed two rRT-PCR assays to detect the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes of SARS-CoV-2. We evaluated these assays together with our previously published assays targeting the ORF1ab and N genes for the detection and confirmation of SARS-CoV-2 and its variants of concern (VOCs). In addition, we also developed two rRT-PCR assays (S484K and S501Y) targeting the spike gene, which when combined with the open reading frames (ORF)1ab assay, respectively, to form duplex rRT-PCR assays, were able to detect SARS-CoV-2 VOCs (lineages B.1.351 and B.1.1.7).@*RESULTS@#Using a SARS-CoV-2 stock with predetermined genomic copies as a standard, the detection limit of both assays targeting RdRp and N was five copies/reaction. Furthermore, no cross-reactions with six others human CoVs (229E, OC43, NL63, HKU1, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus) were observed using these assays. In addition, the S484K and S501Y assays were combined with the ORF1ab assay, respectively.@*CONCLUSIONS@#Four rRT-PCR assays (RdRp, N, S484K, and S501Y) were used to detect SARS-CoV-2 variants, and these assays were shown to be effective in screening for multiple virus strains.

Validation of snort-spit saliva in detecting COVID-19 using RT-PCR and Rapid Antigen Detection Test

@#Objective. To determine the diagnostic accuracy of self-collected snorted and spit saliva in detecting COVID-19 using RT-PCR (ssRT-PCR) and lateral flow antigen test (ssLFA) versus nasopharyngeal swab RT-PCR (npRT-PCR). Methods. One hundred ninety-seven symptomatic subjects for COVID-19 testing in a tertiary hospital underwent snort-spit saliva self-collection for RT-PCR and antigen testing and nasopharyngeal swab for RT-PCR as reference. Positivity rates, agreement, sensitivity, specificity, and likelihood ratios were estimated. Results. Estimated prevalence of COVID-19 using npRT-PCR was 9% (exact 95% CI of 5.5% - 14.1%). A higher positivity rate of 13% in the ssRT-PCR assay suggested possible higher viral RNA in the snort-spit samples. There was 92.9% agreement between ssRT-PCR and npRT-PCR (exact 95% CI of 88.4% to 96.1%; Cohen’s Kappa of 0.6435). If npRT-PCR will be assumed as reference standard, the estimated Sensitivity was 83.3% (exact 95% CI of 60.8% to 94.2%), Specificity 93.9% (exact 95% CI of 89.3% to 96.5%), Positive predictive value of 57.7% (exact 95% CI of 38.9% to 74.5%), Negative predictive value of 98.2% (exact 95% CI of 95% to 99.4%), positive likelihood ratio of 3.65 (95% CI of 7.37 to 24.9), negative likelihood ratio of 0.178 (95% CI of 0.063 to 0.499). There was 84.84% agreement (95% exact CI of 79.1% to 89.5%; Cohen’s Kappa of 0.2356) between ssLFAvs npRT-PCR, sensitivity of 38.9% (exact 95% CI of 20.3% to 61.4%), specificity of 89.4% (exact 95% CI of 84.1% to 93.1%), PPV of 26.9% (95% CI of 13.7% to 46.1%), NPV of 93.6% (exact 95% CI of 88.8% to 96.4%), LR+ of 3.67 (95% CI of 1.79 - 7.51), LR – of 0.68 (95% CI of 0.47 - 0.99). Conclusion. Our data showed that snort-spit saliva RT-PCR testing had acceptable diagnostic performance characteristics and can potentially be used as an alternative to the standard nasopharyngeal/oropharyngeal swab RT-PCR test for COVID-19 in certain situations. However, our data also showed that snort-spit saliva antigen testing using lateral flow assay did not offer acceptable performance.

Detection of coronavirus-2 by real-time reverse transcription polymerase chain reaction in conjunctival swabs from patients with severe form of Coronavirus disease 2019 in São Paulo, Brazil

OBJECTIVES: To test conjunctival swabs from patients with laboratory-confirmed severe forms of coronavirus disease 2019 (COVID-19) for the presence of SARS-CoV-2 on real-time reverse-transcription polymerase chain reaction (rRT-PCR). METHODS: Fifty conjunctival swabs were collected from 50 in-patients with laboratory-confirmed severe forms of COVID-19 at the largest teaching hospital and referral center in Brazil (HCFMUSP, São Paulo, SP). The samples were tested for SARS-CoV-2 on rRT-PCR with the primers and probes described in the CDC protocol which amplify the region of the nucleocapsid N gene (2019_nCoV_N1 and 2019_nCoV_N2) of SARS-CoV-2 RNA and compared with naso/oropharyngeal swabs collected within 24 hours of the conjunctival swabs. RESULTS: Five conjunctival samples (10%) tested positive (amplification of the N1 and N2 primer/probe sets) while two conjunctival samples (4%) yielded inconclusive results (amplification of the N1 primer/probe set only). The naso/oropharyngeal swabs were positive for SARS-CoV-2 on rRT-PCR in 34 patients (68%), negative in 14 (28%) and inconclusive in 2 (4%). The 5 patients with positive conjunctival swabs had positive (n=2), negative (n=2) or inconclusive (n=1) naso/oropharyngeal swabs on rRT-PCR. Patients with negative or inconclusive naso/oropharyngeal swabs had the diagnosis of COVID-19 confirmed by previous positive rRT-PCR results or by serology. CONCLUSION: This is the first study to present conjunctival swab rRT-PCR results for SARS-CoV-2 in a Brazilian population. In our sample of 50 patients with severe forms of COVID-19, 10% had positive conjunctival swabs, most of which were correlated with positive naso/oropharyngeal rRT-PCR results.

Serum expression of microRNA-16 in a cohort of Egyptian patients with ulcerative colitis and its correlation with disease extent and severity / Expressão sérica do microRNA-16 em uma coorte de pacientes egípcios com colite ulcerosa e sua correlação com a extensão e gravidade da doença

Abstract Ulcerative colitis is one of the IBDs. Its etiology and pathogenesis remain undefined with an interaction between environmental, genetic and immunological factors is the most accepted explanation. Several recent studies have examined microRNA expression in the peripheral blood and tissues from IBD patients. The study aims at assessing the expression of serum miR-16 in ulcerative colitis patients and its correlation with disease extent, activity and severity. It included 30 treatment naïve ulcerative colitis patients of different presentations. Serum miR-16 expression was assessed using reverse transcriptase quantitative real time PCR (RT-qPCR), and then correlated with that of a group of 20 healthy subjects to assess its role in diagnosis of ulcerative colitis. Also, it was correlated with disease extent (proctitis, left sided colitis, extensive colitis) and disease activity and severity indices (Truelove and Witts criteria, fecal calprotectin and UCEIS). Thirty ulcerative colitis patients were enrolled, 53% had mild, 37% had moderate, while 10% had severe disease. Concerning endoscopic extent, 8 had proctitis, 14 had left sided colitis and 8 had extensive colitis. Serum expression of miR-16 in the 30 patients were compared to that of the healthy control subjects. The patients' group showed median serum miR-16 expression of 1.91, 1.13 for the control group with a significant difference between both groups. Correlation between serum miR-16 expression with disease extent, activity and severity showed no significant relation. From the current study we can conclude that increased serum expression of miR-16 is associated with ulcerative colitis despite no significant relation to disease activity extent or severity.

Resumo A colite ulcerativa é uma das DII. Sua etiologia e patogênese permanecem indefinidas; a interação entre fatores ambientais, genéticos e imunológicos é a explicação mais aceita. Vários estudos recentes avaliaram a expressão de microRNA no sangue e tecidos periféricos em pacientes com DII. O presente estudo teve como objetivo avaliar a expressão do miR-16 sérico em pacientes com colite ulcerativa e sua correlação com a extensão, atividade e gravidade da doença. Foram incluídos 30 pacientes de colite ulcerativa, com diferentes apresentações, que ainda não haviam sido submetidos a nenhum tipo de tratamento. A expressão sérica de miR-16 foi avaliada usando transcrição reversa seguida de reação em cadeia da polimerase quantitativa (RT-qPCR) e, em seguida, correlacionada com a de um grupo de 20 indivíduos saudáveis para avaliar seu papel no diagnóstico de colite ulcerativa. Além disso, foi feita uma correlação com a extensão da doença (proctite, colite do lado esquerdo, colite extensa) e com os índices de atividade e gravidade da doença (critérios de Truelove e Witts, calprotectina fecal e UCEIS). Trinta pacientes com colite ulcerativa foram incluídos no estudo, classificada como leve em 53%, moderada em 37% e grave em 10%. Quanto à extensão endoscópica, oito apresentavam proctite, 14 apresentavam colite do lado esquerdo e oito apresentavam colite extensa. A expressão sérica de miR-16 nos 30 pacientes foi comparada à dos indivíduos controle saudáveis. No, grupo de pacientes, a expressão sérica de miR-16 foi de 1,91 (grupo controle: 1,13), uma diferença estatisticamente significativa entre os dois grupos. Não foi observada relação significativa entre a expressão sérica de miR-16 e a extensão, atividade e gravidade da doença. A partir do presente estudo, pode-se concluir que o aumento da expressão sérica do miR-16 está associado à colite ulcerativa, apesar de não haver relação significativa com a extensão ou gravidade da atividade da doença.

Frecuencia de aberraciones moleculares en pacientes cubanos con leucemia mieloide aguda / Frequency of molecular disorders in Cuban patients with acute myeloid leukemia

Introducción: En el Instituto de Hematología e Inmunología se realiza el estudio molecular de las leucemias mieloides agudas (LMA). Para las leucemias mieloides agudas no promielocíticas (LPM) se determinan cuatro biomarcadores: los genes de fusión RUNX1-RUNX1T1 y CBF(-MYH11, la duplicación interna en tándem del gen FLT3 (DIT FLT3) y la mutación A del gen NPM1 (NPM1-A). Objetivo: Determinar la frecuencia de estos cuatro biomarcadores, en pacientes cubanos con leucemias mieloides agudas primaria no promielocíticas. Métodos: Se incluyeron 91 pacientes entre niños y adultos, estudiados en el Instituto durante tres años desde el debut. A partir de ARN de sangre medular se obtuvo ADN complementario por transcripción inversa; se amplificaron los fragmentos correspondientes mediante la reacción en cadena de la polimerasa y el producto se analizó por electroforesis capilar. Resultados: El RUNX1-RUNX1T1 apareció en el 24,2 por ciento, fue más frecuente en los pacientes pediátricos y disminuyó significativamente con la edad. El CBFβ-MYH11 solo se encontró en adultos (4,8 por ciento). La NPM1-A con 41 por ciento fue mayoritaria entre los adultos. La DIT FLT3 se observó en el 21,6 por ciento y no mostró relación con la edad. NPM1-A y DIT FLT3 fueron las aberraciones con mayor presencia simultánea. Conclusiones: Por primera vez se describe la frecuencia de los cuatro biomarcadores moleculares en los pacientes cubanos con leucemias mieloides agudas primaria no promielocíticas; su comportamiento fue similar a lo descrito por otros autores, aunque se encontraron algunas particularidades(AU)

Introduction: At the Institute of Hematology and Immunology, the molecular study of acute myeloid leukemias (AML) is carried out. For nonpromyelocytic acute myeloid leukemias, four biomarkers are determined: the RUNX1-RUNX1T1 and CBF(-MYH11 fusion genes, the internal tandem duplication of the FLT3 gene (DIT FLT3), and the A mutation of the NPM1 gene (NPM1-A). Objective: To determine the frequency of these four biomarkers in Cuban patients with nonpromyelocytic primary acute myeloid leukemias. Methods: 91 patients were included, children and adults, who were studied at the Institute for three years from their disease debut. Complementary DNA was obtained from medullary blood RNA by reverse transcription. The corresponding fragments were amplified by polymerase chain reaction and the product was analyzed by capillary electrophoresis. Results: RUNX1-RUNX1T1 appeared in 24.2 percent; it was more frequent in pediatric patients and decreased significantly with age. CBFβ-MYH11 was found only in adults (4.8 percent). NPM1-A, accounting for 41 percent, represented the majority among adults. FLT3 DIT was observed in 21.6 por ciento and was not related to age. NPM1-A and DIT FLT3 were the disorders with the greatest concurrence. Conclusions: For the first time, the frequency of the four molecular biomarkers is described in Cuban patients with primary non-promyelocytic acute myeloid leukemias. Its characterization was similar to that described by other authors, although some peculiarities were found(AU)

Upregulated Long Noncoding RNA LINC01234 Predicts Unfavorable Prognosis for Colorectal Cancer and Negatively Correlates With KLF6 Expression

BACKGROUND: LINC01234, a long noncoding RNA (lncRNA), is overexpressed in several cancers, including colorectal cancer (CRC). We investigated the role of LINC01234 in CRC development and confirmed its correlation with Krüppel-like factor 6 (KLF6), a tumor suppressor gene that is dysregulated in CRC. METHODS: We tested mRNA levels using quantitative reverse transcription PCR (qRT-PCR). Tissue samples from patients with CRC, inflammatory bowel disease (IBD), hyperplastic polyp, and adenoma were included. Correlations between clinicopathological parameters, overall survival (OS) rate, and LINC01234 were analyzed using Kruskal-Wallis H test. Additionally, cell proliferation, apoptosis, and tumor formation in nude mice were tested to investigate the mechanism of LINC01234. Western blotting was used to determine protein levels. RESULTS: LINC01234 expression was significantly upregulated in CRC tissues and CRC cell lines than in non-tumor tissues and normal epithelial cells, respectively. LINC01234 was associated with high tumor stage, larger tumor size, and metastasis. Patients with higher LINC01234 expression showed reduced OS. Cell proliferation was inhibited by LINC01234 knockdown, whereas apoptosis was enhanced. Mice injected with SW480 cells with LINC01234 knockdown displayed decreased tumor volume, weight, and Ki-67 levels compared with those injected with control cells. KLF6 was negatively regulated by LINC01234. Overexpression of KLF6 showed effects similar to those observed following LINC01234 knockdown on cell proliferation and apoptosis. CONCLUSIONS: LINC01234 could be a prognostic biomarker in CRC patients. Upregulation of LINC01234 in CRC promotes tumor development through negative regulation of KLF6.

Role of Efflux Pump Gene adeIJK to Multidrug Resistance in Acinetobacter baumannii Clinical Isolates / 대한임상미생물학회지

BACKGROUND: The emergence of multidrug-resistant Acinetobacter baumannii as a nosocomial pathogen is one of the major public health problems. The aim of this study was to evaluate the role of an efflux pump gene adeJ for the multidrug resistance of A. baumannii clinical isolates.METHODS: Two groups (MDRAB and SAB) of A. baumannii clinical isolates were studied. The SAB group consisted of strains that did not meet the criteria of MDRAB and were susceptible to more categories of antibiotics than MDRAB. Antimicrobial susceptibility results obtained by VITEKII system were used in data analysis and bacterial group allocation. We performed real-time reverse transcription PCR to determine relative expression of adeJ. We compared relative expression of adeJ in comparison groups by considering two viewpoints: i) MDRAB and SAB groups and ii) susceptible and non-susceptible groups for each antibiotic used in this study.RESULTS: The mean value of relative expression of adeJ of MDRAB and SAB groups was 1.4 and 0.92, respectively, and showed significant difference (P=0.002). The mean values of relative expression of adeJ of susceptible and non-susceptible groups to the antibiotics cefepime, ceftazidime, ciprofloxacin, imipenem, meropenem, tigecycline, piperacillin/tazobactam, ticarcillin/clavulanic acid, trimethoprim/sulfamethoxazole, piperacillin, and gentamicin showed statistically significant differences.CONCLUSION: The overexpression of adeIJK might contribute to the multi-drug resistance in A. baumannii clinical isolates. Further, the overexpression of adeIJK might be one of the factors contributing to the resistance to numerous antibiotics.

Monitorización de la capacidad de degradación de las nuevas bacterias haloalcalifílicas resistentes a cloruro de tributiltina (TBTCl) en una ubicación contaminada por butiltina / Monitoring the degradation capability of novel haloalkaliphilic tributyltin chloride (TBTCl) resistant bacteria from butyltin-polluted site

Tributyltin (TBT) is recognized as a major environmental problem at a global scale. Haloalkaliphilic tributyltin (TBT)-degrading bacteria may be a key factor in the remediation of TBT polluted sites. In this work, three haloalkaliphilic bacteria strains were isolated from a TBT-contaminated site in the Mediterranean Sea. After analysis of the 16S rRNA gene sequences the isolates were identified as Sphingobium sp. HS1, Stenotrophomonas chelatiphaga HS2 and Rhizobium borbori HS5. The optimal growth conditions for biodegradation of TBT by the three strains were pH 9 and 7% (w/v) salt concentration. S. chelatiphaga HS2 was the most effective TBT degrader and has the ability to transform most TBT into dibutyltin and monobutyltin (DBT and MBT). A gene was amplified from strain HS2 and identified as TBTB-permease-like, that encodes an ArsB-permease. A reverse transcription polymerase chain reaction analysis in the HS2 strain confirmed that the TBTB-permease-like gene contributes to TBT resistance. The three novel haloalkaliphilic TBT degraders have never been reported previously.

Se considera a la tributiltina (TBT) como un problema medioambiental serio a escala global. Las bacterias haloalcalifílicas degradadoras de TBT pueden constituir un factor clave para remediar áreas contaminadas con dicho xenobiótico. En este estudio se aislaron 3 cepas de bacterias haloalcalifílicas procedentes de un sitio contaminado con TBT en el mar Mediterráneo. Tras analizar las secuencias del gen de 16S del ARNr, se identificaron los aislados como Sphingo-bium sp. HS1, Stenotrophomonas chelatiphaga HS2 y Rhizobium borbori HS5. Las condiciones de crecimiento óptimas para la biodegradación de TBT por parte de las 3 cepas fueron pH 9 y 7% (p/v) de concentración de sal. S. chelatiphaga HS2 fue el degradador de TBT más efectivo, con capacidad de transformar la mayor parte de ese compuesto en dibutiltina y monobutiltina (DBT y MBT). Se amplificó un gen de la cepa HS2, que fue identificado como tipo TBTB-permeasa, que codifica para una ArsB permeasa. Un análisis de la cepa HS2 por reacción en cadena de la polimerasa con transcriptasa inversa (RT PCR) confirmó que el gen TBTB-permeasa contribuye a la resistencia al TBT. Estos 3 nuevos degradadores haloalcalifílicos de TBT no habían sido reportados previamente.

Myricetin Protects Against High Glucose-Induced β-Cell Apoptosis by Attenuating Endoplasmic Reticulum Stress via Inactivation of Cyclin-Dependent Kinase 5

BACKGROUND: Chronic hyperglycemia has deleterious effects on pancreatic β-cell function and turnover. Recent studies support the view that cyclin-dependent kinase 5 (CDK5) plays a role in β-cell failure under hyperglycemic conditions. However, little is known about how CDK5 impair β-cell function. Myricetin, a natural flavonoid, has therapeutic potential for the treatment of type 2 diabetes mellitus. In this study, we examined the effect of myricetin on high glucose (HG)-induced β-cell apoptosis and explored the relationship between myricetin and CDK5. METHODS: To address this question, we subjected INS-1 cells and isolated rat islets to HG conditions (30 mM) in the presence or absence of myricetin. Docking studies were conducted to validate the interaction between myricetin and CDK5. Gene expression and protein levels of endoplasmic reticulum (ER) stress markers were measured by real-time reverse transcription polymerase chain reaction and Western blot analysis. RESULTS: Activation of CDK5 in response to HG coupled with the induction of ER stress via the down regulation of sarcoendoplasmic reticulum calcium ATPase 2b (SERCA2b) gene expression and reduced the nuclear accumulation of pancreatic duodenal homeobox 1 (PDX1) leads to β-cell apoptosis. Docking study predicts that myricetin inhibit CDK5 activation by direct binding in the ATP-binding pocket. Myricetin counteracted the decrease in the levels of PDX1 and SERCA2b by HG. Moreover, myricetin attenuated HG-induced apoptosis in INS-1 cells and rat islets and reduce the mitochondrial dysfunction by decreasing reactive oxygen species production and mitochondrial membrane potential (Δψm) loss. CONCLUSION: Myricetin protects the β-cells against HG-induced apoptosis by inhibiting ER stress, possibly through inactivation of CDK5 and consequent upregulation of PDX1 and SERCA2b.

Seasonal patterns and etiologies of croup in children during the period 2010–2015: A multicenter retrospective study

PURPOSE: Croup is known to have epidemics in seasonal and biennial trends, and to be strongly associated with epidemics of parainfluenza virus. However, seasonal and annual epidemics of croup have not been clearly reported in Korea. This study aimed to examine the seasonal/annual patterns and etiologies of childhood croup in Korea during a consecutive 6-year period. METHODS: Pediatric croup data were collected from 23 centers in Korea from 1 January 2010 to 31 December 2015. Electronic medical records, including multiplex reverse transcription polymerase chain reaction (RT-PCR) results, demographics and clinical information were cross-sectionally reviewed and analyzed. RESULTS: Overall, 2,598 childhood croup patients requiring hospitalization were identified during the study period. Among them, a total of 927 who underwent RT-PCR were included in the analysis. Males (61.5%) predominated, and most (63.0%) of them were younger than 2 years of age (median, 19 months; interquartile range, 11–31 months). Peak hospitalization occurred in 2010 and 2012 in even-numbered years, and parainfluenza virus (PIV, 39.7%) was the most common cause of childhood croup requiring hospitalization, followed by respiratory syncytial virus (14.9%), human rhinovirus (12.5%), Mycoplasma pneumonaie (10.6%), and human coronavirus (7.3%). CONCLUSION: It is concluded that croup hospitalization has a biennial pattern in even-numbered years. PIV may be the most common cause of childhood croup; however, croup epidemics could be attributed to other viruses.

Combined Detection of Serum MiR-221-3p and MiR-122-5p Expression in Diagnosis and Prognosis of Gastric Cancer

PURPOSE: To investigate the clinical value of serum miR-221-3p and miR-122-5p expression levels in the diagnosis and prognosis of gastric cancer. MATERIALS AND METHODS: Serum samples from 141 gastric cancer cases (gastric cancer group), 110 gastric polyps (gastric polyp group), and 75 healthy people (healthy control) were used to detect miR-221-3p and miR-122-5p expression using real-time reverse transcription polymerase chain reaction. RESULTS: Serum miR-221-3p expression was significantly higher in the gastric cancer group than in the gastric polyp group, and it was significantly lower than that before operation. The miR-221-3p expression was significantly higher in the death group than in the survival group. The proliferation and migration ability significantly increased and the apoptosis rate significantly decreased by miR-221-3p transfection in gastric cancer cells. In contrast, the function of miR-122-5p in gastric cancer cells was opposite of miR-221-3p. Serum miR-221-3p expression was negatively correlated with that of miR-122-5p in gastric cancer. Serum miR-221-3p and miR-122-5p expressions were significantly correlated with the degree of differentiation, tumor, node, metastasis stage, lymph node metastasis, and invasion depth. miR-221-3p and miR-122-5p expression levels were independent prognostic factors for postoperative gastric cancer. In the diagnosis and predicting prognosis of gastric cancer, receiver operating characteristic analysis revealed that the area under curve of combined detection of serum miR-221-3p and miR-122-5p expression had a greater diagnostic effect than either single maker. CONCLUSIONS: The miR-221-3p and miR-122-5p are involved in the development of gastric cancer, and they have important clinical values in gastric cancer diagnosis and prognosis.

Inflammatory Effect of Light-Emitting Diodes Curing Light Irradiation on Raw264.7 Macrophage / 치위생과학회지

BACKGROUND: The light-emitting diode (LED) curing light used is presumed to be safe. However, the scientific basis for this is unclear, and the safety of LED curing light is still controversial. The purpose of this study was to investigate the effect of LED curing light irradiation according to the conditions applied for the polymerization of composite resins in dental clinic on the cell viability and inflammatory response in Raw264.7 macrophages and to confirm the stability of LED curing light. METHODS: Cell viability and cell morphology of Raw264.7 macrophages treated with 100 ng/ml of lipopolysaccharide (LPS) or/and LED curing light with a wavelength of 440~490 nm for 20 seconds were confirmed by methylthiazolydiphenyl-tetrazolium bromide assay and microscopic observation. The production of nitric oxide (NO) and prostaglandin E2 (PGE2) was confirmed by NO assay and PGE2 enzyme-linked immunosorbent assay kit. Expression of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in total RNA and protein was confirmed by reverse transcription polymerase chain reaction and Western blot analysis. RESULTS: The LED curing light did not affect the viability and morphology of normal Raw264.7 cells but affected the cell viability and induced cytotoxicity in the inflammation-induced Raw264.7 cells by LPS. The irradiation of the LED curing light did not progress to the inflammatory state in the inflammation-induced Raw264.7 macrophage. However, LED curing light irradiation in normal Raw264.7 cells induced an increase in NO and PGE2 production and mRNA and protein expression of IL-1β and TNF-α, indicating that it is possible to induce the inflammatory state. CONCLUSION: The irradiation of LED curing light in RAW264.7 macrophage may induce an excessive inflammatory reaction and damage oral tissues. Therefore, it is necessary to limit the long-term irradiation which is inappropriate when applying LED curing light in a dental clinic.

Anticancer Activity of Tubulosine through Suppression of Interleukin-6-Induced Janus Kinase 2/Signal Transducer and Activation of Transcription 3 Signaling / 한국유방암학회지

PURPOSE: The chemical structure of tubulosine has been known since the mid-1960s. However, little is known about its biological and pharmacological functions. The aim of this study was to investigate the novel functions of tubulosine in cancer treatment, specifically in breast cancer. METHODS: An Unpaired (Upd)-induced Drosophila cell line and interleukin (IL)-6-stimulated human breast cancer cell lines were used to investigate the biological and pharmacological activities of tubulosine in vitro. To investigate the activities of tubulosine, we performed molecular and cellular experiments such as Western blot and reverse transcription polymerase chain reaction analyses, immunoprecipitation and terminal deoxynucleotidyl transferase dUTP nick end labeling assays, and immunofluorescence staining using breast cancer cell lines. RESULTS: Tubulosine exhibited anticancer activity in IL-6-stimulated human breast cancer cells. Moreover, tubulosine reduced the tyrosine phosphorylation level and transcriptional activity of signal transducer and activator of transcription (STAT) protein at 92E in Upd-induced Drosophila cells. Additionally, tubulosine suppressed IL-6-induced Janus kinase 2 (JAK2)/STAT3 signaling, resulting in decreased viability and induction of apoptotic cell death in breast cancer cells. Interestingly, inhibition of IL-6-induced JAK2/STAT3 signaling by tubulosine was associated with the blocking of IL-6 receptor (IL-6R) and glycoprotein 130 (gp130) binding. CONCLUSION: Tubulosine exhibits anticancer activity through functional inhibition of IL-6-induced JAK2/STAT3 signaling by targeting IL-6Rα/gp130 binding in breast cancer cells. These findings suggest that tubulosine may hold promise for the treatment of inflammation-associated cancers, including breast cancer.

Effects of Oxytocin on Cell Proliferation in a Corticotroph Adenoma Cell Line

BACKGROUND: Oxytocin (OXT) has been reported to act as a growth regulator in various tumor cells. However, there is a paucity of data on the influence of OXT on cell proliferation of corticotroph adenomas. This study aimed to examine whether OXT affects cell growth in pituitary tumor cell lines (AtT20 and GH3 cells) with a focus on corticotroph adenoma cells. METHODS: Reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were conducted with AtT20 cells to confirm the effects of OXT on hormonal activity; flow cytometry was used to assess changes in the cell cycle after OXT treatment. Moreover, the impact of OXT on proliferating cell nuclear antigen (PCNA), nuclear factor κB, and mitogen-activated protein kinase signaling pathway was analyzed by Western blot. RESULTS: OXT treatment of 50 nM changed the gene expression of OXT receptor and pro-opiomelanocortin within a short time. In addition, OXT significantly reduced adrenocorticotropic hormone secretion within 1 hour. S and G2/M populations of AtT20 cells treated with OXT for 24 hours were significantly decreased compared to the control. Furthermore, OXT treatment decreased the protein levels of PCNA and phosphorylated extracellular-signal-regulated kinase (P-ERK) in AtT20 cells. CONCLUSION: Although the cytotoxic effect of OXT in AtT20 cells was not definite, OXT may blunt cell proliferation of corticotroph adenomas by altering the cell cycle or reducing PCNA and P-ERK levels. Further research is required to investigate the role of OXT as a potential therapeutic target in corticotroph adenomas.

Platelet-Rich Plasma Pretreatment on Grit-Blasted Titanium Alloy for Enhanced Osteogenic Differentiation of Human Adipose-Derived Stem Cells

BACKGROUND: Adequate bone formation around titanium alloy implants is integral to successful implantation surgery. Stem cell-coated implants may accelerate peri-implant bone formation. This study investigates the effect of platelet-rich plasma (PRP) pretreatment on a titanium-alloy surface in terms of proliferation and osteogenic differentiation of human adipose-derived stem cells (hADSCs). METHODS: Allogenic leukocyte-depleted PRP was obtained from blood supernatants. The hADSCs were isolated from thigh subcutaneous fat tissue. Grit-blasted titanium plugs were used in two different groups. In one group, 200 µL of PRP was added to the grit-blasted titanium plugs. The hADSCs were seeded in two groups: grit-blasted titanium plugs with or without PRP. The number of hADSCs was measured after 4 hours, 3 days, and 7 days of culture using Cell Counting Kit-8. Osteogenesis of hADSCs was measured by using an alkaline phosphatase activity assay on days 7 and 14, and a calcium assay on days 14 and 21. Osteogenic gene expression was measured by using reverse transcription polymerase chain reaction analysis of alkaline phosphatase, osteocalcin, and type I collagen mRNA. The microscopic morphology of grit-blasted titanium plugs with or without PRP was examined with a field-emission scanning electron microscope using a JSM-7401F apparatus on days 1 and 7. RESULTS: Proliferation and osteogenic differentiation of hADSCs were found to be significantly higher on the grit-blasted titanium alloy preprocessed with PRP than the same alloy without pretreatment. Furthermore, a structural fibrillar mesh developed compactly on the grit-blasted titanium alloy with the PRP pretreatment. CONCLUSIONS: Our results demonstrate that a hADSC-based approach can be used for tissue-engineered peri-implant bone formation and that PRP pretreatment on the grit-blasted titanium alloy can improve proliferation and osteogenic differentiation of hADSCs.

Loss of LKB1 Protein Expression Correlates with Increased Risk of Recurrence and Death in Patients with Resected, Stage II or III Colon Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The purpose of this study was to investigate the prognostic significance of liver kinase b1 (LKB1) loss in patients with operable colon cancer (CC). MATERIALS AND METHODS: Two hundred sixty-two specimens from consecutive patients with stage III or high-risk stage II CC, who underwent surgical resection with curative intent and received adjuvant chemotherapy with fluoropyrimidine and oxaliplatin, were analyzed for LKB1 protein expression loss, by immunohistochemistry as well as for KRAS exon 2 and BRAF(V600E) mutations by Sanger sequencing and TS, ERCC1, MYC, and NEDD9 mRNA expression by real-time quantitative reverse transcription polymerase chain reaction. RESULTS: LKB1 expression loss was observed in 117 patients (44.7%) patients and correlated with right-sided located primaries (p=0.032), and pericolic lymph nodes involvement (p=0.003), BRAF(V600E) mutations (p=0.024), and TS mRNA expression (p=0.041). Patients with LKB1 expression loss experienced significantly lower disease-free survival (DFS) (hazard ratio [HR], 1.287; 95% confidence interval [CI], 1.093 to 1.654; p=0.021) and overall survival (OS) (HR, 1.541; 95% CI, 1.197 to 1.932; p=0.002), compared to patients with LKB1 expressing expressing tumors. Multivariate analysis revealed LKB1 expression loss as independent prognostic factor for both decreased DFS (HR, 1.217; 95% CI, 1.074 to 1.812; p=0.034) and decreased OS (HR, 1.467; 95% CI, 1.226 to 2.122; p=0.019). CONCLUSION: Loss of tumoral LKB1 protein expression, constitutes an adverse prognostic factor in patients with operable CC.

Upregulation of PITX2 Promotes Letrozole Resistance Via Transcriptional Activation of IFITM1 Signaling in Breast Cancer Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Although the interferon α (IFNα) signaling and the paired-like homeodomain transcription factor 2 (PITX2) have both been implicated in the progression of breast cancer (BCa), it remains obscure whether these two pathways act in a coordinated manner. We therefore aimed to elucidate the expression and function of PITX2 during the pathogenesis of endocrine resistance in BCa. MATERIALS AND METHODS: PITX2 expression was assessed in BCa tissues using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunohistochemistry and in experimentally induced letrozole-resistant BCa cells using RT-qPCR and immunoblotting. Effects of PITX2 deregulation on BCa progression was determined by assessing MTT, apoptosis and xenograft model. Finally, using multiple assays, the transcriptional regulation of interferon-inducible transmembrane protein 1 (IFITM1) by PITX2 was studied at both molecular and functional levels. RESULTS: PITX2 expression was induced in letrozole-resistant BCa tissues and cells, and PITX2 induction by IFNα signaling powerfully protected BCa cells against letrozole insult and potentiated letrozole-resistance. Mechanistically, PITX2 enhanced IFNα-induced AKT activation by transactivating the transcription of IFITM1, thus rendering BCa cells unresponsive to letrozoleelicited cell death. Additionally, ablation of IFITM1 expression using siRNA substantially abolished IFNα-elicited AKT phosphorylation, even in the presence of PITX2 overexpression, thus sensitizing BCa cells to letrozole treatment. CONCLUSION: These results demonstrate that constitutive upregulation of PITX2/IFITM1 cascade is an intrinsic adaptive mechanism during the pathogenesis of letrozole-resistance, and modulation of PITX2/IFITM1 level using different genetic and pharmacological means would thus have a novel therapeutic potential against letrozole resistance in BCa.

Changes of Bax, Bcl-2, CCR-2, MCP-1, and TGF-β1 genes in the left ventricle of spontaneously hypertensive rat after losartan treatment / 소아과

PURPOSE: Increased apoptosis was recently found in the hypertrophied left ventricle of spontaneously hypertensive rats (SHRs). Although the available evidence suggests that apoptosis can be induced in cardiac cells by various insults including pressure overload, cardiac apoptosis appears to result from an exaggerated local production of angiotensin in adult SHRs. Altered expressions of Bcl associated X (Bax), Bcl-2, chemokine receptor (CCR)-2, monocyte chemoattractant protein (MCP)-1, transforming growth factor (TGF)-β1, phosphorylated extracellular signal-regulated kinases (PERK), and connexin 43 proteins, and kallikrein mRNA were investigated to explore the effects of losartan on the SHR model. METHODS: Twelve-week-old male rats were grouped as follows: control (C), SHR (hypertension: H), and losartan (L; SHRs were treated with losartan [10 mg/kg/day] for 5 weeks). Western blot and reverse transcription polymerase chain reaction assays were performed. RESULTS: Expression of Bax, CCR-2, MCP-1, TGF-β1, PERK, and connexin 43 proteins, and kallikrein mRNA was significantly increased in the H group compared to that in the C group at weeks 3 and 5. Expression of Bax, CCR-2, MCP-1, TGF-β1, and connexin 43 proteins and kallikrein mRNA was significantly decreased after losartan treatment at week 5. PERK protein expression was significantly decreased after losartan treatment at weeks 3 and 5. Bcl-2 protein expression was significantly decreased in the H group compared to that in the C group at weeks 3 and 5. CONCLUSION: Losartan treatment reduced expression of Bax, CCR-2, MCP-1, TGF-β1, PERK, and connexin 43 proteins, and kallikrein mRNA in SHRs, along with decreased inflammation and apoptosis.

C-Type Natriuretic Peptide/Natriuretic Peptide Receptor 2 Is Involved in Cell Proliferation and Testosterone Production in Mouse Leydig Cells

PURPOSE: This study investigated the role of natriuretic peptide receptor 2 (NPR2) on cell proliferation and testosterone secretion in mouse Leydig cells. MATERIALS AND METHODS: Mouse testis of different postnatal stages was isolated to detect the expression C-type natriuretic peptide (CNP) and its receptor NPR2 by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Leydig cells isolated from mouse testis were cultured and treated with shNPR2 lentiviruses or CNP. And then the cyclic guanosine monophosphate production, testosterone secretion, cell proliferation, cell cycle and cell apoptosis in mouse Leydig cells were analyzed by ELISA, RT-qPCR, Cell Counting Kit-8, and flow cytometry. Moreover, the expression of NPR2, cell cycle, apoptosis proliferation and cell cycle related gene were detected by RT-qPCR and Western blot. RESULTS: Knockdown of NPR2 by RNAi resulted in S phase cell cycle arrest, cell apoptosis, and decreased testosterone secretion in mouse Leydig cells. CONCLUSIONS: Our study provides more evidences to better understand the function of CNP/NPR2 pathway in male reproduction, which may help us to treat male infertility.