Transcription factors regulating human CDT2-INTS7 intergenic region.

<p>(A) Luciferase expression of pGL3-ABCD and pGL3-ABCD Rev constructs co-transfected with indicated expression vectors in A549 cells are shown as fold induction with respect to the pGL3-Basic vector together with pcDNA3 as 1. The values reported for transfection experiments are the means±standard deviation (n = 3; asterisk, <i>P</i><0.05 for ABCD versus ABCD Rev). (B) Luciferase expression of pGL3-ABCD co-transfected with indicated E2F members in cells are shown as fold induction with respect to the pGL3-Basic vector together with pcDNA3 as 1. The values reported for transfection experiments are the means±standard deviation (n = 3; asterisk, <i>P</i><0.05 for pcDNA3 versus E2Fs). (C) Overexpression of E2F1 in A549 cells by adenovirus transfer resulted in the upregulation of CDT2 but not INTS7 mRNA. Western blot analysis of E2F1 and GAPDH in A549 cells infected with Ad-Control or Ad-E2F1. GAPDH was detected as a control. RT-PCR analysis of CDT2 and INTS7 mRNA expression levels are shown with or without E2F1 overexpression. GAPDH was detected as a control. RT-PCR products were derived from amplifications in the log range.</p

<i> In silico</i> analysis of the CDT2-INTS7 intergenic region.

<p>(A) Nucleotide sequence of the intergenic region of CDT2 and INTS7 genes. The sequences of seven mammalian genes are aligned and conserved nucleotides are marked with asterisks under the alignment. The bent arrows indicate the transcription start sites and direction of human genes (5′-CGATA— and 5′-AGCGC— for CDT2 and INTS7, respectively), canine genes, (5′-TCAGT— and 5′-AACAG— for CDT2 and INTS7, respectively), and mouse genes (5′-GGCGG— and 5′-CGCGG— for CDT2 and INTS7, respectively). The bent arrows positioned on the sequences are for CDT2. The bent arrows positioned under the sequences are for INTS7. Transfac software (threshold >80) predicts four E2F consensus sites (E2F A∼D, marked with dotted boxes), and Sp1 (5′-GAGGCGGGGA), NF-Y (5′-AAGCCAATCAG), CREB (5′-TGACGTCA), and Myb (5′-CCAAACTGAC) transcription factor-binding sites (marked by arrows with dotted lines). (B) Computer predicted threshold (Transfac software) of E2F A∼D were summarized for seven mammalian genes.</p

RT-PCR analysis of the CDT2 and INTS7 genes in various human (A), mouse (B), and rat (C) tissues and cell lines.

<p>The lower panels in each part show the G3PDH bands of the ethidium bromide-stained gels as a control. The source of the cDNA is indicated at the top. RT-PCR products were derived from amplifications in the log range.</p

Deletion analyses of the human CDT2 promoter to identify the E2F responsive site.

<p>(A) Structure of the human CDT2 gene and location of a series of deleted constructs. Translation start codons (represented by ATG) of CDT2 and INTS7 genes are marked by bold arrows in white. Transcription start sites are indicated by the bent arrows. The transcription start site of CDT2 is designated as “+1”. Positive (negative) numbers are assigned to nucleotides downstream (upstream) of nucleotide +1. Arrowheads indicate E2F consensus sites (threshold >85). Arrows with numbers represent the region and direction used for the luciferase (Luc) assay. (B) The levels of luciferase expression of human CDT2 deleted promoter constructs in A549 cells were tested with E2F1 coexpression, and are shown as fold induction with respect to the pcDNA3 vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3; asterisk, <i>P</i><0.05 for pcDNA3 versus E2F1).</p

Promoter analysis of the CDT2-INTS7 intergenic region.

<p>(A) The structure of human, mouse, and canine CDT2 and INTS7 genes in the head-to-head orientation. Translation start codons (represented by ATG) of the CDT2 and INTS7 genes are marked by bold arrows in white. Transcription start sites are indicated by the bent arrows. The transcription start site of CDT2 is designated as “+1”. Positive (negative) numbers are assigned to nucleotides downstream (upstream) of nucleotide +1. Arrowheads indicate the E2F consensus sites (threshold >85). Arrows with numbers were the region and direction used for the luciferase (Luc) assay. (B) Luciferase expression of pGL3-human −363/+1, pGL3-mouse −335/+32, and pGL3-canine −312/+54 constructs in A549 cells are shown as fold induction with respect to the pGL3-Basic vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3). (C) Luciferase expression of pGL3-human −363/+1 (hereafter denoted as ABCD), pGL3-mouse −335/+32, and pGL3-canine −312/+54 constructs in A549 cells were up-regulated by co-expressing the E2F1, and are shown as fold induction with respect to the pcDNA3 vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3).</p

The human CDT2-INTS7 intergenic region acts as a bidirectional promoter.

<p>(A) Structure of human CDT2 and INTS7 genes in the head-to-head orientation. Translation start codons (represented by ATG) of CDT2 and INTS7 genes are marked by bold arrows in white. Transcription start sites are indicated by the bent arrows. The transcription start site of CDT2 is designated as “+1”. Positive (negative) numbers are assigned to nucleotides downstream (upstream) of nucleotide +1. Arrowheads indicate E2F consensus sites (threshold >85). Arrows with numbers represent the region and direction used for the luciferase (Luc) assay. (B) Luciferase expression of pGL3 constructs are summarized in (A) in A549 cells and are shown as fold induction with respect to the pGL3-Basic vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3).</p

Additional file 1 of Accuracy evaluation of mainstream and sidestream end-tidal carbon dioxide monitoring during noninvasive ventilation: a randomized crossover trial (MASCAT-NIV trial)

Additional file 1. Appendix S1. Potential risks of post-extubation respiratory failure. Appendix S2. Weaning group according to the WIND criteria. Appendix S3. Updated Charlson comorbidity index. Appendix S4. The Smart Capnoline ® Plus. Table S1. Details of updated Charlson comorbidity index. Fig. S1. Correlations between PaCO2 and PETCO2 according to leakage. Fig. S2. Bland–Altman plot of agreements between PaCO2 and PETCO2 according to leakage. Fig. S3. Sensitivity analyses for correlations with difference (PaCO2—PETCO2)

Clinical Heterogeneity of Acquired Idiopathic Isolated Adrenocorticotropic Hormone Deficiency - Supplementary Table

Based on clinical characteristics, patients were classified using Principal Component Analysis(PCA). For this analysis, 6 clinical parameters were included. PCA could reduce these 6 clinical parameters into 3 new parameters, known as the principal components (PCs). These 3 PCs could explain approximately 70% (cumulative contribution ratio) of all data

DataSheet_1_The Effect of Aging on Quality of Life in Acromegaly Patients Under Treatment.docx

ContextWith the increasing number of older patients with acromegaly, it is important to understand the effects of aging on the quality of life (QoL) in acromegaly.ObjectiveTo investigate the factors associated with the QoL of older acromegaly patients.DesignThis was a single-center, retrospective, cross-sectional study conducted between 2014 and 2019.MethodsAmong 90 acromegaly patients at Kobe University Hospital, 74 who had completed the QoL evaluation under treatment were enrolled (age = 62.0 [50.7–70.0], female 52%). SF-36 and the AcroQoL questionnaire were used to quantify QoL. The patients were divided into two groups: the young and middle-aged group, aged ResultsThe scores for the physical component summary of SF-36 were negatively associated with age (P ConclusionWe showed that the factors associated with impaired QoL differed in the young and middle-aged, and older patients with acromegaly. In older patients, arthropathy and higher BMI were associated with poor QoL. These suggest the importance of early diagnosis and appropriate treatment in preventing arthropathy in acromegaly.</p

IGF-I enhanced p53-p21 pathway in human fibroblasts.

<p><b>A, B,</b> p53 and p21 mRNA expression in human fibroblasts treated with GH or IGF-I for 27 days (PDL of 20–21). The expressions were measured using quantitative RT-PCR and normalized to β-actin. <b>C,</b> Immunoblotting analysis of p53, serine-phosphorylated-p53 (p-p53), and p21 proteins in human fibroblasts treated with GH or IGF-I for 27 days (PDL of 20–21). Anti-p-p53 protein at serine 15 antibody was used for the detection of p-p53. <b>D, E,</b> Densitometric analysis of p53, p-p53, and p21 protein levels normalized to β-actin. All data are expressed as mean ± standard deviation. Data were compared using one-way analysis of variance followed by post hoc Fisher’s least significant difference test. *<i>P</i> < 0.05, **<i>P</i> < 0.01; PDL, population doubling levels.</p