Abstract The AMC-HN-8 cell line and the primary human laryngeal epithelial cell lines were utilized in this work to explore the molecular mechanism of miR-548-3p regulating the gene DAG1 to induce the occurrence and malignant transformation of laryngeal carcinoma. Non-coding RNA miR-548- 3p overexpression plasmid, interference plasmid and blank plasmid were constructed, and the plasmids were transfected into AMC-HN-8 cells, respectively. Meanwhile, a non-transfected plasmid group and a human laryngeal epithelial primary cell group were set up. Five groups of cells were named as NC (Normal control), Model, Ov-miR-548-3p, Sh-miR-548-3p and Blank-plasmid group. The luciferase reporter experiment was used to analyze the regulation characteristics of hsa-miR-548-3p on dystrophin-associated glycoprotein 1 (DAG1). Immunofluorescence was used to analyze the relative expression characteristics of the protein DAG1. The cell cloning experiment was used to analyze the proliferation characteristics of AMC-HN-8. The scratch healing test was used to analyze the migration ability of AMC-HN-8. The transwell test was used to analyze the invasion ability of AMC-HN-8. The RT-PCR was used to analyze the expression level of miR-548-3p. Western blot experiments were used to analyze the expression of protein DAG1, laminin α2 (LAMA2) and utrophin (UTRN). The luciferase report experiment and immunofluorescence test found that the expression of DAG1 and miR-548-3p are positively correlated. Cell cloning, scratching and migration experiments identified that the activity of laryngeal cancer cells was positively correlated with the expression of DAG1. The results of Western blot analysis further strengthened the above conclusions. Through carrying out research on the cellular levels, our work has demonstrated that miR-548-3p regulated the content of protein DAG1, and then further induced malignant transformation of laryngeal carcinoma.

Resumen En este trabajo se utilizaron la línea celular AMC-HN-8 y la línea celular epitelial laríngea humana primaria, para explorar el mecanismo molecular regulador del miR-548-3p sobre el gen DAG1 para inducir la aparición y la transformación maligna del carcinoma laríngeo. Se construyeron el plásmido de sobreexpresión de miR-548-3p de ARN no codificante, el plásmido de interferencia y el plásmido en blanco, y los plásmidos se transfectaron en células AMCHN-8 respectivamente. Mientras tanto, se establecieron un grupo de plásmidos no transfectados y un grupo de células primarias epiteliales laríngeas humanas. Se nombraron cinco grupos de células como NC (control normal), modelo, OvmiR-548-3p, Sh-miR-548-3p y grupo de plásmido en blanco. El experimento indicador de luciferasa se utilizó para analizar las características de regulación de hsa-miR-548-3p en la glicoproteína 1 asociada a distrofina (DAG1). Se utilizó inmunofluorescencia para analizar las características de expresión relativa de la proteína DAG1. El experimento de clonación celular se utilizó para analizar las características de proliferación de AMC-HN-8. Se utilizó la prueba de cicatrización por rascado para analizar la capacidad de migración de AMC-HN-8. La prueba de transwell se utilizó para analizar la capacidad de invasión de AMCHN-8. Se utilizó RT-PCR para analizar el nivel de expresión de miR-548-3p. Se usó un experimento de transferencia Western (Western blot) para analizar las expresiones de la proteína DAG1, laminina α2 (LAMA2) y utrofina (UTRN). El experimento de reporte de luciferasa y la prueba de inmunofluorescencia encontraron que la expresión de DAG1 y miR-548-3p están positivamente correlacionadas. Los experimentos de clonación celular, rascado y migración, identificaron que la actividad de las células cancerosas de laringe se correlacionó positivamente con la expresión de DAG1. Los resultados del análisis de transferencia Western fortalecieron aún más las conclusiones anteriores. A través de la investigación a nivel celular, nuestro proyecto ha demostrado que miR-548-3p regula el contenido de la proteína DAG1 y luego induce la transformación maligna del carcinoma de laringe.

Abstract Psoriasis is a chronic skin inflammation, characterized by impaired differentiation, hyperproliferation of keratinocytes involving pro-inflammatory factors interleukin (IL)-13/17A, tumor necrosis factor (TNF)-α, interferon (IFN)-γ. Among the integrin family, α5 is important for blood vessel formation, and β4 for proliferation, differentiation of keratinocytes. To investigate the expression and regulation of integrin α5 and β4 in psoriatic keratinocytes. Skin biopsies were obtained from 14 psoriatic patients and 12 normal volunteers. We compared the immunolocalization and regulation of α5 and β4 between the psoriatic and normal ones, before and after incubation with MEK/ERK pathway inhibitor U0126 by immunohistochemistry and western blot separately. Immunohistochemistry showed psoriatic keratinocytes had higher α5 than normal ones. According to western blot, IL-17A and IL-13 increased normal keratinocytes’ α5 and β4 respectively, but psoriatic keratinocytes were the exact opposite. Incubated with U0126, normal keratinocytes’ α5 was enhanced by the 5 cytokines ; while IL-13/17A, IFN-γ suppressed β4. Psoriatic keratinocytes’ α5 was increased by IL-13/17A, decreased by IFN-γ; but β4 increased by IL-17A, IFN-γ. IL-13/17A, TNF-α, IFN-γ regulate α5 and β4 through ERK pathway whether normal or psoriasis. The normal and psoriatic keratinocytes respond to the same cytokines differently.

Abstract Background: Unsubstantiated concerns have been raised on the potential correlation between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination and infertility, leading to vaccine hesitancy in reproductive-aged population. Herein, we aim to evaluate the impact of inactivated SARS-CoV-2 vaccination on embryo ploidy, which is a critical indicator for embryo quality and pregnancy chance. Methods: This was a retrospective cohort study of 133 patients who underwent preimplantation genetic testing for aneuploidy (PGT-A) cycles with next-generation sequencing technology from June 1st 2021 to March 17th 2022 at a tertiary-care medical center in China. Women fully vaccinated with two doses of Sinopharm or Sinovac inactivated vaccines (n = 66) were compared with unvaccinated women (n = 67). The primary outcome was the euploidy rate per cycle. Multivariate linear and logistic regression analyses were performed to adjust for potential confounders. Results: The euploidy rate was similar between vaccinated and unvaccinated groups (23.2 ± 24.6% vs. 22.6 ± 25.9%, P = 0.768), with an adjusted β of 0.01 (95% confidence interval [CI]: -0.08–0.10). After frozen-thawed single euploid blastocyst transfer, the two groups were also comparable in clinical pregnancy rate (75.0% vs. 60.0%, P = 0.289), with an adjusted odds ratio of 6.21 (95% CI: 0.76–50.88). No significant associations were observed between vaccination and cycle characteristics or other laboratory and pregnancy outcomes. Conclusions: Inactivated SARS-CoV-2 vaccination had no detrimental impact on embryo ploidy during in vitro fertilization treatment. Our finding provides further reassurance for vaccinated women who are planning to conceive. Future prospective cohort studies with larger datasets and longer follow-up are needed to confirm the conclusion.

Abstract Vascular hypo-responsiveness to vasopressors in patients with obstructive jaundice (OJ) is a common anesthetic event, which leads to perioperative complications and increased mortality. The cause of this clinical issue remains unclear. In this study, we estimated the actin cytoskeleton and arterial protein level in the artery of OJ patients by proteomic analysis. Ten patients with OJ due to bile duct diseases or pancreatic head carcinoma were enrolled, while another ten non-jaundice patients with chronic cholecystitis or liver hemangioma as the control group. Vascular reactivity to noradrenaline was measured before anesthesia on the day of surgery. Artery samples in adjacent tissues of removed tumor were collected and evaluated by 2-dimensional electrophoresis. Proteins with differential expression were detected by MALDI-TOF mass spectrometry with immunoblot confirmation. The results confirmed the phenomenon of vascular hypo-reactivity in OJ patients as suppressed aortic response to noradrenaline were existed in these patients. We also found that actin cytoskeleton and several actin-binding proteins were up- or down-regulated in the artery of OJ patients. These proteins changed in OJ patents might be the basic mechanism of vascular hypo-reactivity, further studies to uncover the role of these proteins in OJ is critical for clinical treatment of these patients.

Abstract Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1 mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18 CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10 h, which is a promising rapid method to detect Salmonella in emergency.

Resumo Introdução: Diversas mutações do DNA mitocondrial tem sido descritas, em diferentes famílias, associadas à deficiência auditiva não sindrômica. No entanto, pouco se sabe sobrea prevalência dessas mutações em pacientes esporádicos com deficiência auditiva sensorioneural não sindrômica. Objetivo: A finalidade do nosso estudo foi investigar a incidência dessas mutações no DNA mitocondrial nessa população. Método: No total, 178 pacientes esporádicos com deficiência auditiva sensorioneural não sindrômica foram recrutados para participação no estudo. O DNA genômico foi extraído de amostra, de sangue periférico. Utilizamos o método de sequenciamento SNaPshot(r) para detecção de cinco mutações do DNA mitocondrial: A1555G e A827G no gene 12S rRNA e A7445G, 7472insCe T7511C no gene tRNASerUCN. Paralelamente, utilizamos a reação de polimerase em cadeia e sequenciamos os produtos para triagem das mutações no gene GJB2 nos pacientes portadores de mutações no DNA mitocondrial. Resultados: Em nossa população, não conseguimos detectar a presença da mutação A1555G no gene 12S rRNA e nem as mutações A7445G, 7472insC e T7511C no gene tRNASerUCN. Entretanto, constatamos que seis pacientes (3,37%) eram portadores da mutação homozigota A827G; e um deles também portava a mutação homozigota GJB2 235delC. Conclusão: Nossos achados no presente estudo indicam que, mesmo em pacientes esporádicos com deficiência auditiva sensorioneural não sindrômica, as mutações do DNA mitocondrial também podem contribuir para o fenótipo clínico.

ABSTRACT INTRODUCTION: Several mitochondrial DNA mutations have been reported to be associated with nonsyndromic hearing loss in several families. However, little is known about the prevalence of these mutations in sporadic patients with nonsyndromic sensorineural hearing loss. OBJECTIVE: The purpose of our study was to investigate the incidence of these mitochondrial DNA mutations in such population. METHODS: A total of 178 sporadic patients with nonsyndromic sensorineural hearing loss were enrolled in this study. Genomic DNA was extracted from the peripheral blood sample. We employed the SNaPshot(r) sequencing method to detect five mitochondrial DNA mutations, including A1555G and A827G in 12S rRNA gene and A7445G, 7472insC, and T7511C in tRNASerUCN gene. Meanwhile, we used polymerase chain reaction and sequenced the products to screen GJB2 gene mutations in patients carrying mitochondrial DNA mutations. RESULTS: We failed to detect the presence of A1555G mutation in 12S rRNA gene, and of A7445G, 7472insC, T7511C mutations in tRNASerUCN gene in our population. However, we found that 6 patients (3.37%) were carriers of a homozygous A827G mutation and one of them also carried homozygous GJB2 235delC mutation. CONCLUSION: Our findings in the present study indicate that even in sporadic patients with nonsyndromic sensorineural hearing loss, mitochondrial DNA mutations might also contribute to the clinical phenotype.

A toxoplasmose é uma zoonose global causada pelo Toxoplasma gondii, o qual pode infectar animais de sangue quente e seres humanos. Este estudo foi realizado com o objetivo de investigar a soroprevalência em cavalos para T. gondii, na região de Xinjiang, no Noroeste da China. Em 2011, foram recolhidas 637 amostras de sangue em sete distritos da Prefeitura Autônoma de Changji Hui do Xinjiang, as quais foram testadas para a presença de anticorpos, utilizando-se o teste de aglutinação modificado (MAT). Foram estimados fatores de risco relacionados com a soroprevalência (idade, sexo e distrito), através de uma análise de regressão logística multivariada. Um total de 200 equinos (31,4%, 95% IC 27,79 – 35,00) foi positivo para T. gondii. Idade, sexo e região estudada não apresentaram associação com a soroprevalência (p>0,05) na análise de regressão logística. Os resultados revelam que a infecção por T. gondii tem uma prevalência generalizada em todo o território de Xinjiang, no Noroeste da China, constituindo uma séria ameaça à saúde de animais e de humanos. Consequentemente, propõe-se que sejam adotadas medidas reforçadas para o controle e prevenção da infecção de cavalos por T. gondii, no Xinjiang, Noroeste da China.

Toxoplasmosis is a worldwide zoonosis caused by Toxoplasma gondii, which can infect warm-blooded animals and humans. The present study was performed to investigate the seroprevalence of T. gondii in horses in Xinjiang, northwestern China. A total of 637 blood samples were collected from seven regions in Changji Hui Autonomous Prefecture, Xinjiang in 2011 and assayed for T. gondiiantibodies using the modified agglutination test (MAT). Risk factors (age, gender, and region) related to seroprevalence were determined by a multivariate logistic regression analysis. A total of 200 horses (31.4%, 95% CI 27.79–35.00) were seropositive for T. gondii. Age, gender, and region present no association with seroprevalence (p>0.05) in the logistic regression analysis. The results indicated that T. gondii is widely prevalent in horses in Xinjiang, northwestern China, representing a serious threat to animal and human health. Therefore, more careful measures should be performed to control and prevent T. gondii infection in horses from Xinjiang, northwestern China.

Polymorphisms in the nicotinic acetylcholine receptor subunit CHRNA5 gene have been associated with lung cancer positive susceptibility in European and American populations. In the present hospital-based, case-control study, we determined whether polymorphism in rs503464 of CHRNA5 is associated with lung cancer risk in Chinese individuals. A single nucleotide polymorphism in CHRNA5 rs503464, c.-166T>A (hereafter T>A), was identified using TaqMan-MGB probes with sequencing via PCR in 600 lung cancer cases and 600 healthy individuals. Genotype frequencies for rs503464 (T>A) were in Hardy-Weinberg equilibrium for the control population. However, genotype frequencies were significantly different between cases and controls (P < 0.05), while allele frequencies were not significantly different between groups. Compared to homozygous genotypes (TT or AA), the risk of lung cancer in those with the heterozygous genotype (TA) was significantly lower (OR = 0.611, 95%CI = 0.486-0.768, P = 0.001). Using genotype AA as a reference, the risk of lung cancer for those with genotype TA was increased 1.5 times (OR = 1.496, 95%CI = 1.120-1.997, P = 0.006). However, no difference in risk was observed between T allele carriers and A allele carriers (OR = 0.914, 95%CI = 0.779-1.073, P = 0.270). Stratification analysis showed that the protective effect of TA was more pronounced in those younger than 60 years, nonsmokers, or those without a family history of cancer, as well as in patients with adenocarcinoma or squamous cell carcinoma in clinical stages III or IV (P < 0.05). Therefore, the heterozygous genotype c.-166T>A at rs503464 of CHRNA5 may be associated with reduced risk of lung cancer, thus representing a susceptibility allele in Chinese individuals.

OBJECTIVE: To asses the association between intake of folate and B vitamins and the incidence of spontaneous abortion (SA) according to the maternal methylenetetrahydrofolate reductase (MTHFR) polymorphisms (677 C>T and 1298 A>C). MATERIAL AND METHODS: We conducted a nested case-control study within a perinatal cohort of women recruited in the state of Morelos, Mexico. Twenty-three women with SA were compared to 74 women whose pregnancy survived beyond week 20th. Intake of folate and B vitamins respectively, was estimated using a validated food frequency questionnaire. Maternal MTHFR polymorphisms were determined by PCR-RFLP and serum homocysteine levels by HPLC. RESULTS: Carriers of MTHFR 677TT and 1298AC genotypes respectively showed an increased risk of SA (OR 677TT vs. CC/CT=5.0; 95% CI: 1.2, 20.9 and OR 1298 AC vs. AA=5.5; 95% CI: 1.1, 26.6). CONCLUSIONS: Our results support the role of MTHFR polymorphisms as a risk factor for SA, regardless of dietary intake of B vitamins.

OBJETIVO: Evaluar la asociación entre aborto espontáneo (AE) y el consumo dietético de vitaminas B en mujeres mexicanas portadoras de los polimorfismos de la metilentetrahidrofolato reductasa (MTHFR) (677 C>T y 1298 A>C). MATERIAL Y MÉTODOS: Mediante un diseño de casos y controles anidados en una cohorte, se comparó la ingesta dietética materna de vitaminas B y folato, los polimorfismos maternos de la MTHFR y la concentración sérica de homocisteina de 23 casos de AE (< 20 semanas) y 74 controles (mujeres con embarazos > 20 semanas). RESULTADOS: Las portadoras de los genotipos MTHFR 677TT y 1298AC presentaron un incremento significativo en el riesgo de AE (RM 677TT vs. CC/CT=5.0; IC 95%: 1.2, 20.9 RM 1298 AC vs. AA=5.5; IC95%: 1.1, 26.6), respectivamente. CONCLUSIONES: Nuestros resultados apoyan el papel de la mutación de la MTHFR como posible factor de riesgo para el AE, independientemente del consumo de vitaminas B.

OBJECTIVE: To asses the association between intake of folate and B vitamins and the incidence of spontaneous abortion (SA) according to the maternal methylenetetrahydrofolate reductase (MTHFR) polymorphisms (677 C>T and 1298 A>C). MATERIAL AND METHODS: We conducted a nested case-control study within a perinatal cohort of women recruited in the state of Morelos, Mexico. Twenty-three women with SA were compared to 74 women whose pregnancy survived beyond week 20th. Intake of folate and B vitamins respectively, was estimated using a validated food frequency questionnaire. Maternal MTHFR polymorphisms were determined by PCR-RFLP and serum homocysteine levels by HPLC. RESULTS: Carriers of MTHFR 677TT and 1298AC genotypes respectively showed an increased risk of SA (OR 677TT vs. CC/CT=5.0; 95% CI: 1.2, 20.9 and OR 1298 AC vs. AA=5.5; 95% CI: 1.1, 26.6). CONCLUSIONS: Our results support the role of MTHFR polymorphisms as a risk factor for SA, regardless of dietary intake of B vitamins.

OBJETIVO: Evaluar la asociación entre aborto espontáneo (AE) y el consumo dietético de vitaminas B en mujeres mexicanas portadoras de los polimorfismos de la metilentetrahidrofolato reductasa (MTHFR) (677 C>T y 1298 A>C). MATERIAL Y MÉTODOS: Mediante un diseño de casos y controles anidados en una cohorte, se comparó la ingesta dietética materna de vitaminas B y folato, los polimorfismos maternos de la MTHFR y la concentración sérica de homocisteina de 23 casos de AE (< 20 semanas) y 74 controles (mujeres con embarazos > 20 semanas). RESULTADOS: Las portadoras de los genotipos MTHFR 677TT y 1298AC presentaron un incremento significativo en el riesgo de AE (RM 677TT vs. CC/CT=5.0; IC 95%: 1.2, 20.9 RM 1298 AC vs. AA=5.5; IC95%: 1.1, 26.6), respectivamente. CONCLUSIONES: Nuestros resultados apoyan el papel de la mutación de la MTHFR como posible factor de riesgo para el AE, independientemente del consumo de vitaminas B.