Supplementary Material for: Meta-Analysis Shows Strong Positive Association of the TNF-α Gene with Tumor Stage in Bladder Cancer

There is no consensus on the association between the <i>tumor necrosis factor-</i>α<i> (TNF-</i>α<i>)</i> gene promoter –308 A/G single nucleotide polymorphisms and bladder cancer risk. To obtain a more precise estimation of this correlation, we conducted a meta-analysis. The PubMed, MEDLINE, Cochrane Library and China National Knowledge Infrastructure (CNKI) databases were searched for relevant published studies. Seven case-control studies with a total of 1,311 cases and 1,436 controls were identified and analyzed. A notable correlation was observed between the <i>TNF-</i>α genotype and bladder cancer grade (<i>AA+GA </i>vs. <i>GG;</i> odds ratio 1.96, 95% confidence interval 1.37–2.80, p = 0.0002). In summary, this meta-analysis demonstrates that the <i>TNF-</i>α –308 <i>AA+GA</i> genotype may be a marker to the tumor-invasive stage of bladder cancer

Supplementary Material for: Uniparental Disomy of Chromosome 15 in Two Cases by Chromosome Microarray: A Lesson Worth Thinking

<p>Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurogenetic disorders caused by loss of function of the imprinted genes at 15q11q13. A 5-7 Mb paternal/maternal deletion of chromosomal region 15q11.2q13 is the major genetic cause of PWS/AS, but in a small group of patients, the PWS/AS phenotype can result from maternal/paternal uniparental disomy (UPD) of chromosome 15. Various mechanisms leading to UPD include gametic complementation, trisomy rescue, and compensatory UPD, which can be inferred from the pattern of uniparental heterodisomy (heteroUPD) or uniparental isodisomy (isoUPD). However, heteroUPD and isoUPD, especially mixed heteroUPD and isoUPD, are very rare in patients with PWS/AS. Here, we report 2 children with PWS/AS caused by mixed segmental heteroUPD 15 and isoUPD 15 which failed to be identified by chromosome microarray (CMA) but could be detected by other molecular genetic methods. The present report unravels the mechanism of mixed iso/heteroUPD 15 in PWS/AS and phenotype-genotype correlations. Moreover, our study suggests that CMA is prone to misdiagnosis for imprinting disorders such as PWS/AS, though it is considered a highly useful tool for copy number variations. As a result, other molecular detection methods, such as methylation analysis and STR marker analysis for UPD, should be supplementary used in this situation.</p

Supplementary Material for: Perilipin1 deficiency prompts lipolysis in lipid droplets and aggravates the pathogenesis of persistent immune activation in Drosophila

Lipid droplets (LDs) are highly dynamic intracellular organelles, which are involved in lots of biological processes. However, the dynamic morphogenesis and functions of intracellular LDs during persistent innate immune responses remain obscure. In this study, we induce long-term systemic immune activation in Drosophila through genetic manipulation. Then, the dynamic pattern of LDs is traced in the Drosophila fat body. We find that deficiency of Plin1, a key regulator of LDs’ reconfiguration, blocks LDs minimization at the initial stage of immune hyperactivation but enhances LDs breakdown at the later stage of sustained immune activation via recruiting the lipase Brummer (Bmm, homologous to human ATGL). The high wasting in LDs shortens the lifespan of flies with high-energy-cost immune hyperactivation. Therefore, these results suggest a critical function of LDs during long-term immune activation and provide a potential treatment for the resolution of persistent inflammation

Supplementary Material for: Perilipin1 deficiency prompts lipolysis in lipid droplets and aggravates the pathogenesis of persistent immune activation in Drosophila

Lipid droplets (LDs) are highly dynamic intracellular organelles, which are involved in lots of biological processes. However, the dynamic morphogenesis and functions of intracellular LDs during persistent innate immune responses remain obscure. In this study, we induce long-term systemic immune activation in Drosophila through genetic manipulation. Then, the dynamic pattern of LDs is traced in the Drosophila fat body. We find that deficiency of Plin1, a key regulator of LDs’ reconfiguration, blocks LDs minimization at the initial stage of immune hyperactivation but enhances LDs breakdown at the later stage of sustained immune activation via recruiting the lipase Brummer (Bmm, homologous to human ATGL). The high wasting in LDs shortens the lifespan of flies with high-energy-cost immune hyperactivation. Therefore, these results suggest a critical function of LDs during long-term immune activation and provide a potential treatment for the resolution of persistent inflammation

Supplementary Material for: Blood eosinophil count and its determinants in a Chinese population-based cohort

Introduction: Blood eosinophil count has been shown markedly variable across different populations. However, its distribution in Chinese general population remains unclear. We aimed to investigate blood eosinophil count and its determinants in a Chinese general population. Methods: In this population-based study, general citizens of Sichuan province in China were extracted from the China Pulmonary Health study. Data on demographics, personal and family history, living condition, lifestyle, spirometry and complete blood count test were obtained and analyzed. A stepwise multivariate binary logistic regression analysis was performed to identify determinants of high blood eosinophils (>75th percentile). Results: A total of 3310 participants were included, with a mean age (SD) of 47.0 (15.6) years. In total population, the median blood eosinophil count was 110.0 (IQR 67.2-192.9) cells/μL, lower than that in smokers (133.4 cells/μL, IQR 79.3-228.4) and patients with asthma (140.7 cells/μL, IQR 79.6-218.2) or post-bronchodilator airflow limitation (141.5 cells/μL, IQR 82.6-230.1), with a right-skewed distribution. Multivariate analyses revealed that oldness (aged ≥60 years) (OR 1.66, 95% CI 1.11-2.48), smoking ≥ 20 pack-years (OR 1.90, 95% CI 1.20-3.00), raising dog/cat (OR 1.72, 95% CI 1.17-2.52) and occupational exposure to dust, allergen and harmful gas (OR 1.58, 95% CI 1.15-2.15) were significantly associated with high blood eosinophils. Conclusion: This study identifies a median blood eosinophil count of 110.0 cells/μL and determinants of high blood eosinophils in a Chinese general population, including oldness (aged ≥60 years), smoking ≥ 20 pack-years, raising dog/cat and occupational exposure to dust, allergen and harmful gas

Supplementary Material for: Relative Strengths and Regulation of Different Promoter-Associated Sequences for Various blaSHV Genes and Their Relationships to β-Lactam Resistance

<p><b><i>Aims:</i></b> This work investigated the relative strengths of different <i>bla</i>_SHV promoter-associated sequences and their regulation function in <i>bla</i>_SHV expression and β-lactam resistance. <b><i>Methods:</i></b> Recombinant plasmids with the promoter-associated sequences (P-W, P-S, P-IS, and P-WPD), <i>tac</i> promoter, and combined fragments of promoter and <i>bla</i>_SHV were separately constructed and transformed into <i>Escherichia coli</i> DH5α. The relative strengths of the promoters indicated by the intensities of green fluorescent protein and the mRNA expression levels of <i>bla</i>_SHV were compared. The minimum inhibitory concentration and extended spectrum β-lactamase phenotypes were evaluated. <b><i>Results:</i></b> The relative strengths were ranked as P-<i>tac</i> > P-WPD > P-IS > P-S > P-W. The mRNA expression and β-lactam resistance levels of the different promoter-associated sequence groups were generally consistent with the strength rank, but the extent of <i>gfp</i> and <i>bla</i>_SHV mRNA levels varied significantly in each group. The β-lactam resistance levels were inconsistent with the strength rank in certain <i>bla</i>_SHV groups. In relation to the different promoter-associated sequences,<i> bla</i>_SHV-ESBLs displayed significantly different change modes of β-lactam resistance compared with <i>bla</i>_{SHV-non-ESBLs}. <b><i>Conclusion:</i></b> The mRNA expression and β-lactam resistance of the <i>bla</i>_SHV showed consistencies and inconsistencies with the strengths of the promoter-associated sequences. The mechanisms accounting for these discrepancies need further investigation.</p

Supplementary Material for: 5’-AMP-Activated Protein Kinase Regulates Goat Sperm Functions via Energy Metabolism <b><i>In Vitro</i></b>

<b><i>Background/Aims:</i></b> ATP is essential for mammalian sperm to survive and maintain fertilizing capacity. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. The aims of the present study were to explore the localization of AMPK in goat sperm and to investigate whether and how AMPK regulates sperm functions <i>in vitro</i>. <b><i>Methods:</i></b> Sperm were treated with AMPK modulators (AICAR, metformin and Compound C) during incubation. Sperm motility was assessed with a computer-assisted spermatozoa analysis system (CASA). Membrane integrity, acrosome reaction and mitochondrial membrane potentials were detected by SYBR-14/PI, FITC-PNA and JC-1 staining, respectively. And the lactate content, ATP content, AMPK activity, activity of pyruvate kinase (PK) and lactate dehydrogenase (LDH) were also measured with the commercial assay kits. Immunofluorescence staining was used to analyze the distribution of PK, LDH, AMPK and phospho-Thr172-AMPK in sperm. The role of AMPK was further studied during induction of capacitation and acrosome reaction. <b><i>Results:</i></b> We found that AMPKα was localized in the entire acrosomal region, the midpiece and the flagellum, while the phospho-Thr172-AMPK was distributed in the head, the midpiece and flagellum. Activation of AMPK by AICAR and metformin significantly improved sperm motility, membrane integrity and acrosome reaction, largely maintained sperm mitochondrial membrane potentials, lactate content and ATP content, and enhanced the activity of AMPK, PK and LDH, whereas inhibition by Compound C triggered the converse effects. Moreover, PK was localized in the acrosomal area and the midpiece, while LDH was distributed in the tail. Induction of capacitation and acrosome reaction led to AMPK phosphorylation. AMPK phosphorylation regulated the activity of energetic enzymes. <b><i>Conclusion:</i></b> This study for the first time provides evidence that AMPK governs goat sperm functions through energy metabolism <i>in vitro</i>. This finding will help to improve assisted reproductive techniques in goats and the other species

Supplementary Material for: LncRNA HANR Promotes Tumorigenesis and Increase of Chemoresistance in Hepatocellular Carcinoma

<p><b><i>Background/Aims:</i></b> Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and the third leading cause of cancer-related death. Critical roles for long non-coding RNAs (lncRNAs) have recently been demonstrated for a variety of cancers, including hepatocellular carcinoma. However, the effect and mechanism of lncRNAs in HCC tumorigenesis and chemoresistance have not been extensively characterized. <b><i>Methods:</i></b> In the current study, we have identified a HCC-expressed lncRNA termed as <i>HANR</i> (HCC associated long non-coding RNA). We identified <i>HANR</i> by microarray analysis and validated its up-regulated expression by quantitative PCR. RNA pull-down and pathway analyses were conducted to evaluate physical and functional interactions with <i>HANR</i>. <i>In vivo</i> experiments were performed to assess tumorigenesis and increase of chemoresistance. In addition, the <i>HANR</i> expression in HCC specimens was detected by FISH. Xenograft and orthotopic mice model was constructed to observe the effect of <i>HANR</i> on tumorigenesis and chemoresistance <i>in vivo</i>. <b><i>Results:</i></b> <i>HANR</i> was demonstrated to be up-regulated in HCC patients and HCC cell lines. Increased <i>HANR</i> expression in HCC predicted short survival of patients. Knock-down of <i>HANR</i> markedly retarded cell proliferation, suppressed HCC xenograft/orthotopic tumor growth, induced apoptosis and enhanced chemosensitivity to doxorubicin, while over-expression of <i>HANR</i> showed the opposite effects. It was found that <i>HANR</i> bind to GSKIP for regulating the phosphorylation of GSK3β in HCC. <b><i>Conclusion:</i></b> Our results demonstrate that <i>HANR</i> contributes to the development of HCC and is a promising therapeutic target for chemosensitization of HCC cells to doxorubicin, which may represent a promising therapeutic target in the future.</p