LncRNA-599554 sponges miR-15a-5p to contribute inductive ability of dermal papilla cells through positive regulation of the expression of Wnt3a in cashmere goat

BACKGROUND: Long non-coding RNAs (lncRNAs), as post-transcriptional regulators, were thought to function in the inductive property of dermal papilla cells (DPCs) in cashmere goat. Previously, lncRNA-599554 was identified in secondary hair follicle (SHF) of cashmere goat, but its functional significance is unknown. RESULTS: In the present investigation, we verified that lncRNA-599554 had significantly higher expression at the anagen dermal papilla of cashmere goat SHF than that at telogen. Based on overexpression and knockdown techniques, we found that lncRNA-599554 contributes the inductive property of DPCs of cashmere goat, which was assessed by detecting the changes in the expression of several typical indictor genes in DPCs including ET-1, SCF, Versican, ALP, Lef1 and Ptc-1. Based on RNA pull-down assay, we verified that lncRNA-599554 directly interacted with chi-miR-15a-5p. Also, we showed that lncRNA-599554 positively regulated the Wnt3a expression in DPCs but which did not appear to involve its modulating of promoter methylation. Based on the use of Dual-luciferase reporter assays, our data indicated that lncRNA-599554 regulated the Wnt3a expression through chi-miR-15a-5p-mediated post-transcriptional level. CONCLUSIONS: We showed that lncRNA-599554 contributes the inductive property of DPCs in cashmere goat which might be achieved through sponging chi-miR-15b-5p to promote the Wnt3a expression. The results from the present investigation provided a novel insight into the functional mechanism of lncRNA-599554 in the SHF regeneration of cashmere goat along with the formation and growth of cashmere fiber.

The luciferase reporter system of the MMP12 endogenous promoter for investigating transcriptional regulation of the human MMP12 gene

Background: Matrix metalloproteinase 12 (MMP12), a member of MMPs, can take lots of roles including extracellular matrix component degradation, viral infection, inflammation, tissue remodeling and tumorigenesis. To explore the transcriptional regulation of MMP12 gene, a sensitive luciferase reporter HEK293 cell line for endogenous MMP12 promoter was generated by CRISPR/Cas9 technology. Results: The HEK293-MMP12-T2A-luciferase-KI cell line was successfully established by CRISPR/Cas9 technology. The sequencing results indicated that one allele of the genome was proven to have a site-directed insertion of luciferase gene and another allele of the genome was confirmed to have additional 48 bp insertion in this cell line. The cell line was further demonstrated to be a sensitive reporter of the endogenous MMP12 promoter by applying transcription factors STAT3, AP-1 and SP-1 to the cell line. The reporter cell line was then screened with bioactive small molecule library, and a small molecule Tanshinone I was found to significantly inhibit the transcriptional activity of MMP12 gene in HEK293-MMP12-T2A-luciferase-KI cell line by luciferase activity assay, which was further confirmed to inhibit the expression of MMP12 mRNA in wild-type HEK293 cells. Conclusions: This novel luciferase knock-in reporter system will be helpful for investigating the transcriptional regulation of MMP12 gene and screening the drugs targeting MMP12 gene.

LncRNA LINC00483 promotes gastric cancer development through regulating MAPK1 expression by sponging miR-490-3p

BACKGROUND: Previous studies have shown that long noncoding RNA (IncRNA) LINC00483 was aberrantly expressed in human cancers, including gastric cancer. However, the regulatory mechanism of this IncRNA in gastric cancer remains largely unknown. The present study aimed to investigate the effect of LINC00483 on gastric cancer development and explore the potential regulatory network of LINC00483/microRNA (miR)-490-3p/mitogen-activated protein kinase 1 (MAPK1). METHODS: Thirty patients with gastric cancer were recruited for tissues collection. The expression levels of LINC00483, miR-490-3p and MAPK1 were detected by quantitative real-time polymerase chain reaction or western blot. Cell viability, apoptosis, migration and invasion were determined by MTT, flow cytometry, transwell assays and western blot, respectively. The target association between miR-490-3p and LINC00483 or MAPK1 was confirmed by luciferase reporter assay. Xenograft model was established to assess the function of LINC00483 in vivo. RESULTS: LINC00483 and MAPK1 levels were increased in gastric cancer tissues and cells. Knockdown of LINC00483 or MAPK1 inhibited cells viability, migration and invasion but promoted apoptosis in gastric cancer cells. Moreover, MAPK1 overexpression attenuated the effect of LINC00483 knockdown on gastric cancer development. LINC00483 could increase MAPK1 expression by competitively sponging miR-490-3p. miR-490-3p overexpression suppressed gastric cancer development, which was abated by introduction of LINC00483. Besides, inhibition of LINC00483 decreased xenograft tumor growth by regulating miR-490-3p/MAPK1 axis. CONCLUSION: Knockdown of LINC00483 inhibited gastric cancer development in vitro and in vivo by increasing miR- 490-3p and decreasing MAPK1, elucidating a novel mechanism for understanding the development of gastric cancer.

Effect of lncRNA HULC knockdown on rat secreting pituitary adenoma GH3 cells

Pituitary adenoma is one of the most common tumors in the neuroendocrine system. This study investigated the effects of long non-coding RNAs (lncRNAs) highly up-regulated in liver cancer (HULC) on rat secreting pituitary adenoma GH3 cell viability, migration, invasion, apoptosis, and hormone secretion, as well as the underlying potential mechanisms. Cell transfection and qRT-PCR were used to change and measure the expression levels of HULC, miR-130b, and FOXM1. Cell viability, migration, invasion, and apoptosis were assessed using trypan blue staining assay, MTT assay, two-chamber transwell assay, Guava Nexin assay, and western blotting. The concentrations of prolactin (PRL) and growth hormone (GH) in culture supernatant of GH3 cells were assessed using ELISA. The targeting relationship between miR-130b and FOXM1 was verified using dual luciferase activity. Finally, the expression levels of key factors involved in PI3K/AKT/mTOR and JAK1/STAT3 pathways were evaluated using western blotting. We found that HULC was highly expressed in GH3 cells. Overexpression of HULC promoted GH3 cell viability, migration, invasion, PRL and GH secretion, as well as activated PI3K/AKT/mTOR and JAK1/STAT3 pathways. Knockdown of HULC had opposite effects and induced cell apoptosis. HULC negatively regulated the expression of miR-130b, and miR-130b participated in the effects of HULC on GH3 cells. FOXM1 was a target gene of miR-130b, which was involved in the regulation of GH3 cell viability, migration, invasion, and apoptosis, as well as PI3K/AKT/mTOR and JAK1/STAT3 pathways. In conclusion, HULC tumor-promoting roles in secreting pituitary adenoma might be via down-regulating miR-130b, up-regulating FOXM1, and activating PI3K/AKT/mTOR and JAK1/STAT3 pathways.

miR-29b-3p promotes progression of MDA-mB-231 triple-negative breast cancer cells through downregulating TRAF3

BACKGROUND: Breast cancer is the second common malignant cancer among females worldwide. Accumulating studies have indicated that deregulation of miRNA expression in breast cancer will contribute to tumorigenesis and form different cancer subtypes. However, the reported studies on miR-29b-3p-regulated breast cancer are limited so far. Herein, we investigated the role and mechanism of miR-29b-3p in the triple negative breast cancer cell line MDA-MB-231. METHODS: The relative miR-29b-3p expression in different breast cancer cell lines were determined by qRT-PCR. CCK8 and colony formation assay were used to determine the influence of miR-29b-3p on cell proliferation. Migration assay and invasion assay were performed for cell migration and invasion respectively. To study the cell integrity immunofluorescence was performed. TUNEL assay, flow cytometry assay, hoechst staining and western blot were conducted to determine the influence of miR-29b-3p inhibitor on cell apoptosis. TRAF3 was found to be the target gene of miR-29b-3p using bioinformatics predictions. Dual-luciferase assay was performed to determine the relative luciferase activity in NC, miR-29b-3p mimic, miR-29b-3p inhibitor with TRAF3 3'-UTR wt or TRAF3 3'-UTR mt reporter plasmids. The proteins expression of NF-κB signaling pathway in MDA-MB-231 after transfection with NC, miR-29b-3p mimic, miR-29b-3p inhibitor were determined by western blot. RESULTS: The miR-29b-3p expression was significantly increased in MDA-MB-231 compare with MCF-10A. miR-29b-3p inhibitor reduced the cell viability of MDA-MB-231 and inhibited cell migration and invasion. Cell cytoskeleton integrity destroyed after miR-29b-3p inhibitor treatment. Furthermore, we identified the mechanism and found miR-29b-3p targets the TRAF3 and activates NF-κB signaling pathway. CONCLUSIONS: From the above studies, our results indicated that miR-29b-3p acts as a promoter for the development of MDA-MB-231.

Brazilian Bioluminescent Beetles: Reflections on Catching Glimpses of Light in the Atlantic Forest and Cerrado

ABSTRACT Bioluminescence - visible and cold light emission by living organisms - is a worldwide phenomenon, reported in terrestrial and marine environments since ancient times. Light emission from microorganisms, fungi, plants and animals may have arisen as an evolutionary response against oxygen toxicity and was appropriated for sexual attraction, predation, aposematism, and camouflage. Light emission results from the oxidation of a substrate, luciferin, by molecular oxygen, catalyzed by a luciferase, producing oxyluciferin in the excited singlet state, which decays to the ground state by fluorescence emission. Brazilian Atlantic forests and Cerrados are rich in luminescent beetles, which produce the same luciferin but slightly mutated luciferases, which result in distinct color emissions from green to red depending on the species. This review focuses on chemical and biological aspects of Brazilian luminescent beetles (Coleoptera) belonging to the Lampyridae (fireflies), Elateridae (click-beetles), and Phengodidae (railroad-worms) families. The ATP-dependent mechanism of bioluminescence, the role of luciferase tuning the color of light emission, the "luminous termite mounds" in Central Brazil, the cooperative roles of luciferase and superoxide dismutase against oxygen toxicity, and the hypothesis on the evolutionary origin of luciferases are highlighted. Finally, we point out analytical uses of beetle bioluminescence for biological, clinical, environmental, and industrial samples.

NLRP3 inflammasome signaling as an early molecular response is negatively controlled by miR-186 in CFA-induced prosopalgia mice

The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is the most frequently studied in the central nervous system and has been linked to neuropathic pain. In this study, a post-translational mechanism of microRNA (miR)-186 via regulating the expression of NLRP3 in the complete Freund's adjuvant (CFA)-treated mice was investigated. The injection of CFA was used to induce trigeminal neuropathic pain in mice. miRs microarray chip assay was performed in trigeminal ganglions (TGs). CFA treatment significantly increased the mRNA expression of NLRP3, interleukin (IL)-1β, and IL-18 in TGs compared to the control group. Moreover, 26 miRs were differentially expressed in TGs from trigeminal neuropathic pain mice, and the expression of miR-186 showed the lowest level of all the miRs. Further examination revealed that NLRP3 was a candidate target gene of miR-186. We delivered miR-186 mimics to CFA-treated mice. The head withdrawal thresholds of the CFA-treated mice were significantly increased by miR-186 mimics injection compared with CFA single treatment. The mRNA and protein expression of NLRP3, IL-1β, and IL-18 in TGs from trigeminal neuropathic pain mice were significantly inhibited by miR-186 mimics treatment compared to the CFA group. miR-186 was able to suppress the neuropathic pain via regulating the NLRP3 inflammasome signaling.

Modulation of the prostaglandin-endoperoxide synthase 2 gene expression by variant haplotypes: influence of the 3'-untranslated region

The inducible inflammatory enzyme cycloxigenase-2 is up-regulated in cancer, and favors tumor progression. Cycloxigenase-2 is encoded by the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which presents sequence variations in the promoter region (PR) and in the 3′-untranslated region (3′-UTR). Different PR (rs689465, rs689466, rs20417) and 3′-UTR (rs5275) variants were generated by site-directed mutagenesis, and combined in haplotypes to access expression levels using a reporter system (luciferase) in human cells (MCF-7 and HEK293FT). Luciferase activity did not differ significantly among PTGS2 PR constructs, except for pAAC (containing variant allele rs20417 C), with 40% less activity than pAAG (wild-type sequence) in MCF-7 cells (P<0.01). Despite the lack of individual significant differences, PTGS2 PR constructs enclosing rs689466 G (pAGG and pAGC) showed an approximate two-fold increase in luciferase activity when compared to those containing rs689466 A (pAAG, pGAC, pAAC and pGAG) in both cell lines (P<0.001 for MCF-7 and P=0.03 for HEK293FT). The effect of PTGS2 3′-UTR sequences varied between MCF-7 and HEK293FT: MCF-7 cells showed significant reduction (40-60%) in luciferase activity (at least P<0.01), whereas HEK293FT cells showed more diverse results, with an average 2-fold increase when combined constructs (PR and 3′-UTR) were compared to respective parental PR sequences. The contribution of 3′-UTR variant (rs5275) was not consistent in either cell line. Despite the modulation of the 3′-UTR, with variable effects of rs5275, the enhancing transcriptional effect of rs689466 G was still detectable (P<0.0001 in MCF-7 or P=0.03 in HEK293FT cells).

Effect of miR-125b on dermal papilla cells of goat secondary hair follicle

Background: MicroRNAs (miRNAs) are endogenous noncoding RNAs that regulate various biological processes. miR-125b is a miRNA that has been reported to be critical for hair follicle (HF) morphogenesis and development. We identified that the expression of miR-125b varies during an individual hair cycle (anagen, catagen, and telogen) in the skin of cashmere goats. We constructed a gain model (by overexpressing miR-125b) and a loss model (by inhibiting endogenous miR-125b) based on dermal papilla cells (DPCs) to further investigate the role of miR-125b in HF cycle. In addition, we used a dual-luciferase system to highlight the predicated target genes of miR-125b. Results: We found that miR-125b affects the expression of FGF5, IGF-1, SHH, TNF-α, MSX2, LEF-1, FGF7, NOGGIN, BMP2, BMP4, TGF-ß1, and ß-catenin. The dual-luciferase assay further validated a direct interaction between miR-125b and FGF5 and TNF-α. Conclusion: miR-125b affects the expression levels of genes related to hair cycle and may also play a critical role in regulating the periodic development of HF.

MicroRNA-340-5p modulates cisplatin resistance by targeting LPAATβ in osteosarcoma

MicroRNAs (miRNAs) play an important role in drug resistance and modulate the efficiency of chemotherapy. A recent study indicated that miR-340 functions as a tumor suppressor in various types of cancer. However, the role of miR-340 in chemotherapy has not been reported yet. In this study, we found that miR-340 enhanced cisplatin (CDDP)-induced cell death. Induction of miR-340-5p expression decreased the IC50 of CDDP and increased the apoptosis of CDDP-resistant MG-63 and Saos-2 cells. Moreover, miR-340-5p decreased the accumulation of MRP1 and MDR1. We further explored the mechanism underlying the promoting effects of miR-340-5p on CDDP-induced cell death. We identified a potential target of miR-340 in the 3′ untranslated region of lysophosphatidic acid acyltransferase (LPAATβ) using the online program Targetscan (http://www.microrna.org). Luciferase reporter assays showed that miR-340 binds to the 3′UTR of LPAATβ. Enforced expression of miR-340-5p decreased the accumulation of LPAATβ in both MG-63 and Saos-2 cells. Silencing LPAATβ decreased the IC50 of CDDP and increased the apoptosis of CDDP-resistant MG-63 and Saos-2 cells, which is consistent with the effect of miR-340-5p on CDDP-induced cell death. Moreover, induced expression of LPAATβ compromised the effects of miR-340-5p on CDDP-induced cell death and accumulation of MRP1 and MDR1. Taken together, our data indicated that miR-340-5p enhanced the sensitivity to CDDP by targeting LPAATβ.

Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase

ABSTRACT Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV) expressing Gaussia luciferase (GLuc) (YFV-GLuc). We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r2=0.9967), indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct.

Ectasia ductal mamaria en niños, a propósito de un caso clínico: A case report / Mammary duct ectasia in children

Introducción: La telorragia es un síntoma poco frecuente en pacientes pediátricos, la causa más frecuente en esta población es la ectasia ductal mamaria (EDM), que es una afección benigna y autolimitada, caracterizada por la dilatación del conducto mamario, fibrosis e inflamación periductal. Objetivo: Presentar un caso de EDM, para facilitar el rápido reconocimiento por parte de los médicos, y evitar estudios y tratamientos agresivos. Caso clínico: Lactante de sexo masculino de 6 meses de edad, sano, alimentado por lactancia materna exclusiva; consultó por un nódulo retroareolar derecho y telorragia unilateral. Se realizó una ecografía Doppler que mostró una lesión multiquística, sugerente de una EDM. Se planteó tratamiento expectante y acudió a control a los 6 meses con excelente evolución. Conclusiones: La EDM es la principal causa de telorragia en niños, corresponde a una afección benigna, y la resolución generalmente es espontánea, antes de los 9 meses. Por lo que su conocimiento es de gran relevancia para el adecuado diagnóstico y manejo de estos pacientes.

Introduction: Bloody nipple discharge is an infrequent symptom during childhood. The most common cause in this population is mammary duct ectasia (MDE), which is a benign and self-limiting condition, that is characterized by dilatation of the mammary ducts, fibrosis and periductal inflammation. Objective: Report of a case of MDE in order to improve physicians’ diagnosis accuracy and avoid aggressive studies and treatments. Case report: Six-months old male healthy infant, exclusively breastfeeded, that visited our clinic with a lump beneath his right nipple and bloody discharge from the same nipple. An ultrasound was performed which showed a multicystic lesion suggestive of MDE. Watchful waiting was decided as treatment, with good evolution after six months of follow up. Conclusions: The MDE is the leading cause of bloody discharge in pediatric population, being a benign condition that resolves spontaneously before nine months. The knowledge of this condition is essential so as to accurately diagnose and treat it.

Prevalência de dor crônica e sua associação com a situação sociodemográfica e atividade física no lazer em idosos de Florianópolis, Santa Catarina: estudo de base populacional / Prevalence of chronic pain and its association with the sociodemographic situation and physical activity in leisure of elderly in Florianópolis, Santa Catarina: population-based study

OBJETIVO: Estimar a prevalência de dor crônica e sua associação com a situação socioeconômica, demográfica e atividade física no lazer em idosos. MÉTODOS: Este estudo é parte do inquérito epidemiológico e transversal de base populacional e domiciliar EpiFloripa Idoso 2009-2010 realizado com 1.705 idosos (≥ 60 anos), residentes em Florianópolis, Santa Catarina. A partir da resposta afirmativa de dor crônica, foram investigadas as associações com as variáveis obtidas por meio de entrevista estruturada. Realizou-se a estatística descritiva, incluindo cálculos de proporções e intervalos de confiança 95% (IC95%). Na análise bruta e ajustada, empregou-se regressão de Poisson, estimando-se as razões de prevalência, com intervalos de confiança de 95% e valores p ≤ 0,05. RESULTADOS: Dentre os idosos investigados, 29,3% (IC95% 26,5 - 32,2) relataram dor crônica. Na análise ajustada, observou-se que as variáveis sexo feminino, menor escolaridade e pior situação econômica ficaram associadas significativamente com maior prevalência de dor crônica; ser fisicamente ativo no lazer ficou associado significativamente com menor prevalência do desfecho. CONCLUSÕES: Percebe-se que a dor crônica é um agravo que acomete considerável parcela de idosos, havendo desigualdades sociais na sua frequência e sendo beneficamente afetada pela atividade física no lazer. É necessário que políticas públicas de saúde subsidiem programas multidisciplinares de controle da dor incluindo a prática regular de atividade física, voltada especificamente à promoção da saúde do idoso, evitando assim que a dor crônica comprometa a qualidade de vida desta população. .

OBJECTIVE: To estimate the prevalence of chronic pain and its association with socioeconomic and demographic status, and leisure physical activity in the elderly population. METHODS: This study is part of an epidemiological cross-sectional population-based household survey called EpiFloripa Elderly 2009-2010, which was conducted with 1,705 elderly individuals (≥ 60 years) residents of Florianópolis, Santa Catarina. From the positive response to chronic pain, the associations with the variables were investigated through a structured interview. Descriptive statistics were conducted, including ratio calculation and 95% confidence intervals. In crude and adjusted analysis, Poisson regression was utilized, estimating prevalence ratios, with 95% confidence intervals and ≤ 0.05 p-values. RESULTS: Among the subjects, 29.3% (IC95% 26.5 - 32.2) reported chronic pain. Adjusted analysis showed that being female, having less years of schooling, and being in worse economic situation were significantly associated with a higher prevalence of chronic pain. Being physically active during leisure time was significantly associated with lower prevalence of the outcome. CONCLUSIONS: Therefore, it is clear that chronic pain affects a considerable amount of elderly individuals. Social inequalities are a harmful influence in these individuals' quality of life, inasmuch as those inequalities increase the frequency with which chronic pain afflicts them. At the same time, physical activity during leisure time decreases chronic pain frequency. It is fundamental that public health policies subsidize multidisciplinary pain management programs, which should include health targeted physical activity for the elderly, thus preventing the decrease in quality of life that chronic pain brings to this population. .

Validating of the pre-clinical mouse model for metastatic breast cancer to the mandible

Metastatic breast carcinoma has a great tendency to spread to the mandible. It is concomitantly associated with bone destruction, food intake disorder, and a poorer prognosis. Appropriate animal models need to be developed for a better understanding of the mechanisms underlying the metastatic process of breast cancer cells to mandible and to test the effects of potential lead compounds. Here, we assessed the metastasis model of intracardiac injection using luciferase-transfected metastatic breast cancer cells (MDA-MB-231Luc+) by determining the incidences of metastasis, mCT images, and histopathological results. A high bioluminescence signal mainly detected mandibular lesions with less frequent distal femora and proximal tibiae lesions. Extensive mandibular bone destruction occurred in nude mice grafted with metastatic breast cancer cells. This type of animal model might be a useful tool in assessing therapeutic implications and the efficacy of anti-cancer drugs for osteolytic cancers.

Correlação e associação de renda e escolaridade com condições de saúde e nutrição em obesos graves / Correlation and association of income and educational level with health and nutritional conditions among the morbidly obese

O objetivo deste artigo é investigar relações entre renda e escolaridade com condições de saúde e nutrição em obesos graves. Estudo transversal ambulatorial com 79 pacientes de primeira consulta, com Índice de Massa Corporal (IMC) ≥ 35 kg/m2 e idade ≥ 20 anos. Coletaram-se dados: sociodemográficos, antropométricos, estilo de vida, exames bioquímicos e consumo alimentar. O IMC médio foi 48,3 ± 6,9 kg/m2. Observou-se correlação negativa significante de escolaridade com variáveis peso (r = -0,234) e IMC (r = -0,364) e de renda familiar per capita com consumo diário de vegetal A (r = -0,263). Após análise multivariada maior renda familiar per capita se associou à ausência de cardiopatia (RP: 0,51, IC95%: 0,32-0,81), maior consumo diário de vegetal A (RP: 1,79, IC95%: 1,16-2,75) e doces (RP: 3,12, IC95%: 1,21-8,04). Em obesos graves a maior renda familiar per capita se associou à ausência de cardiopatia e maior consumo de vegetais folhosos e doces. Já a escolaridade não se manteve associada às condições de saúde e nutrição.

This article seeks to investigate the relationship between income and educational level and health and nutritional conditions among the morbidly obese. A cross-sectional study was conducted with 79 patients at first appointment, with Body Mass Index (BMI) ≥ 35 kg/m2 and age ≥ 20 years. The following data was collected: demographic, socioeconomic, anthropometric, lifestyle, biochemical and food intake data. Average BMI was 48.3 ± 6.9 kg/m2. There was a significant negative correlation between education level and the variables of weight (r = -0.234) and BMI (r = -0.364) and per capita family income with daily consumption of leafy vegetables (r = -0.263). After multivariate analysis, higher per capita family income was associated with the absence of heart disease (PR: 0.51, CI95%: 0.32-0.81), higher daily consumption of leafy vegetables (PR: 1.79, CI95%: 1.16-2.75) and candy (PR: 3.12, CI95%: 1.21-8.04). In the morbidly obese, per capita household income was associated with absence of heart disease and higher consumption of leafy vegetables and candy. On the other hand, education level was not associated with health and nutrition conditions.

Suppression of miR-221 inhibits glioma cells proliferation and invasion via targeting SEMA3B

BACKGROUND: Gliomas are the most common primary tumors in the central nervous system. Due to complicated signaling pathways involved in glioma progression, effective targets for treatment and biomarkers for prognosis prediction are still scant. RESULTS: In this study we revealed that a new microRNA (miR), the miR-221, was highly expressed in the glioma cells, and suppression of miR-221 resulted in decreased cellular proliferation, migration, and invasion in glioma cells. Mechanistic experiments validated that miR-221 participates in regulating glioma cells proliferation and invasion via suppression of a direct target gene, the Semaphorin 3B (SEMA3B). The rescue experiment with miR-221 and SEMA3B both knockdown results in significant reversion of miR-221 induced phenotypes. CONCLUSION: Taken together, our findings highlight an unappreciated role for miR-221 and SEMA3B in glioma.

Sox2 function as a negative regulator to control HAMP expression

BACKGROUND: Hepcidin, encoding by HAMP gene, is the pivotal regulator of iron metabolism, controlling the systemic absorption and transportation of irons from intracellular stores. Abnormal levels of HAMP expression alter plasma iron parameters and lead to iron metabolism disorders. Therefore,itis animportant goal to understand the mechanisms controlling HAMP gene expression. RESULTS: Overexpression of Sox2 decrease basal expression of HAMP or induced by IL-6 or BMP-2, whereas, knockdown of Sox2 can increase HAMP expression, furthermore, two potential Sox2-binding sites were identified within the human HAMP promoter. Indeed, luciferase experiments demonstrated that deletion of any Sox2-binding site impaired the negative regulation of Sox2 on HAMP promoter transcriptional activity in basal conditions. ChIP experiments showed that Sox2 could directly bind to these sites. Finally, we verified the role of Sox2 to negatively regulate HAMP expression in human primary hepatocytes. CONCLUSION: We found that Sox2 as a novel factor to bind with HAMP promoter to negatively regulate HAMP expression, which may be further implicated as a therapeutic option for the amelioration of HAMP-overexpression-related diseases, including iron deficiency anemia.

Effects of eugenol on resting tension of rat atria

In cardiac and skeletal muscle, eugenol (μM range) blocks excitation-contraction coupling. In skeletal muscle, however, larger doses of eugenol (mM range) induce calcium release from the sarcoplasmic reticulum. The effects of eugenol are therefore dependent on its concentration. In this study, we evaluated the effects of eugenol on the contractility of isolated, quiescent atrial trabeculae from male Wistar rats (250-300 g; n=131) and measured atrial ATP content. Eugenol (1, 3, 5, 7, and 10 mM) increased resting tension in a dose-dependent manner. Ryanodine [100 µM; a specific ryanodine receptor (RyR) blocker] and procaine (30 mM; a nonspecific RyR blocker) did not block the increased resting tension induced by eugenol regardless of whether extracellular calcium was present. The myosin-specific inhibitor 2,3-butanedione monoxime (BDM), however, reversed the increase in resting tension induced by eugenol. In Triton-skinned atrial trabeculae, in which all membranes were solubilized, eugenol did not change resting tension, maximum force produced, or the force vs pCa relationship (pCa=-log [Ca2+]). Given that eugenol reduced ATP concentration, the increase in resting tension observed in this study may have resulted from cooperative activation of cardiac thin filaments by strongly attached cross-bridges (rigor state).

Optimization of transgenesis conditions for the generation of CXCL2-luciferase reporter mice line

Background: Transgenesis by microinjection has been widely used for the generation of different mouse models. Different variables of the procedure may critically affect the efficiency of the process. A DNA construction that carries the CXCL2 promoter gene and firefly luciferase has been used to optimize aspects of the procedure. Three different concentrations (0.5, 1.0 and 4.0 ng/µl) of the DNA construction to microinject a total of 1981 zygotes has been tested. Intact/injected embryos, pregnancy and birth rate, survival of pups 7 days after birth, number of transgenic pups and overall transgenic efficiency was registered and analyzed by Z test of proportions for each group. Results: A total of seven transgenic founders were detected for the three DNA concentrations used, 1 in 46 alive pups in the 0.5 ng/µl group, 5 in 38 alive pups in the 1 ng/µl group and 1 in 21 alive pups in the 4 ng/µl group ( p < 0.1). The overall transgenic efficiency was higher for the 1 ng/µl concentration, with a transgenic rate of 13.2%. Conclusions: In conclusion, we have selected the best operative conditions to maximize the transgenesis efficiency. Furthermore, the transgenic lines developed could be used as a reporter model of innate immunity activation with many different applications in the fields of immunology, cancer and neurodegenerative diseases.

Prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation

OBJECTIVE: The aim of this study was to evaluate the prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation (AF). METHODS: A cohort of 136 unrelated patients with idiopathic atrial fibrillation and 200 unrelated, ethnically matched healthy controls were enrolled. The coding exons and splice junctions of the Nkx2.5 gene were sequenced in 136 atrial fibrillation patients, and the available relatives of mutation carriers and 200 controls were subsequently genotyped for the identified mutations. The functional characteristics of the mutated Nkx2.5 gene were analyzed using a dual-luciferase reporter assay system. RESULTS: Two novel heterozygous Nkx2.5 mutations (p.N19D and p.F186S) were identified in 2 of the 136 unrelated atrial fibrillation cases, with a mutational prevalence of approximately 1.47%. These missense mutations co-segregated with atrial fibrillation in the families and were absent in the 400 control chromosomes. Notably, 2 mutation carriers also had congenital atrial septal defects and atrioventricular block. Multiple alignments of the Nkx2.5 protein sequences across various species revealed that the altered amino acids were completely conserved evolutionarily. Functional analysis demonstrated that the mutant Nkx2.5 proteins were associated with significantly reduced transcriptional activity compared to their wild-type counterpart. CONCLUSION: These findings associate the Nkx2.5 loss-of-function mutation with atrial fibrillation and atrioventricular block and provide novel insights into the molecular mechanism involved in the pathogenesis of atrial fibrillation. These results also have potential implications for early prophylaxis and allele-specific therapy of this common arrhythmia. .