Decreased Extracellular Adenosine Levels Lead to Loss of Hypoxia-Induced Neuroprotection after Repeated Episodes of Exposure to Hypoxia

Achieving a prolonged neuroprotective state following transient ischemic attacks (TIAs) is likely to effectively reduce the brain damage and neurological dysfunction associated with recurrent stroke. HPC is a phenomenon in which advanced exposure to mild hypoxia reduces the stroke volume produced by a subsequent TIA. However, this neuroprotection is not long-lasting, with the effects reaching a peak after 3 days. Therefore, in this study, we investigated the use of multiple episodes of hypoxic exposure at different time intervals to induce longer-term protection in a mouse stroke model. C57BL/6 mice were subjected to different hypoxic preconditioning protocols: a single episode of HPC or five identical episodes at intervals of 3 days (E3d HPC) or 6 days (E6d HPC). Three days after the last hypoxic exposure, temporary middle cerebral artery occlusion (MCAO) was induced. The effects of these HPC protocols on hypoxia-inducible factor (HIF) regulated gene mRNA expression were measured by quantitative PCR. Changes in extracellular adenosine concentrations, known to exert neuroprotective effects, were also measured using in vivo microdialysis and high pressure liquid chromatography (HPLC). Neuroprotection was provided by E6d HPC but not E3d HPC. HIF-regulated target gene expression increased significantly following all HPC protocols. However, E3d HPC significantly decreased extracellular adenosine and reduced cerebral blood flow in the ischemic region with upregulated expression of the adenosine transporter, equilibrative nucleoside transporter 1 (ENT1). An ENT1 inhibitor, propentofylline increased the cerebral blood flow and re-established neuroprotection in E3d HPC. Adenosine receptor specific antagonists showed that adenosine mainly through A1 receptor mediates HPC induced neuroprotection. Our data indicate that cooperation of HIF-regulated genes and extracellular adenosine is necessary for HPC-induced neuroprotection.     

Up-Regulation of Hepatoma-Derived Growth Factor Facilities Tumor Progression in Malignant Melanoma

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression in developing melanoma remains unclear. In this study, human melanoma cell lines (A375, A2058, MEL-RM and MM200) showed higher levels of HDGF gene expression, whereas human epidermal melanocytes (HEMn) expressed less. Exogenous application of HDGF stimulated colony formation and invasion of human melanoma cells. Moreover, HDGF overexpression stimulated the degree of invasion and colony formation of B16–F10 melanoma cells whereas HDGF knockdown exerted opposite effects in vitro. To evaluate the effects of HDGF on tumour growth and metastasis in vivo, syngeneic mouse melanoma and metastatic melanoma models were performed by manipulating the gene expression of HDGF in melanoma cells. It was found that mice injected with HDGF-overexpressing melanoma cells had greater tumour growth and higher metastatic capability. In contrast, mice implanted with HDGF-depleted melanoma cells exhibited reduced tumor burden and lung metastasis. Histological analysis of excised tumors revealed higher degree of cell proliferation and neovascularization in HDGF-overexpressing melanoma. The present study provides evidence that HDGF promotes tumor progression of melanoma and targeting HDGF may constitute a novel strategy for the treatment of melanoma.     

The DAO Gene Is Associated with Schizophrenia and Interacts with Other Genes in the Taiwan Han Chinese Population

Background

Schizophrenia is a highly heritable disease with a polygenic mode of inheritance. Many studies have contributed to our understanding of the genetic underpinnings of schizophrenia, but little is known about how interactions among genes affect the risk of schizophrenia. This study aimed to assess the associations and interactions among genes that confer vulnerability to schizophrenia and to examine the moderating effect of neuropsychological impairment.

Methods

We analyzed 99 SNPs from 10 candidate genes in 1,512 subject samples. The permutation-based single-locus, multi-locus association tests, and a gene-based multifactorial dimension reduction procedure were used to examine genetic associations and interactions to schizophrenia.

Results

We found that no single SNP was significantly associated with schizophrenia. However, a risk haplotype, namely A-T-C of the SNP triplet rsDAO7-rsDAO8-rsDAO13 of the DAO gene, was strongly associated with schizophrenia. Interaction analyses identified multiple between-gene and within-gene interactions. Between-gene interactions including DAO*DISC1 , DAO*NRG1 and DAO*RASD2 and a within-gene interaction for CACNG2 were found among schizophrenia subjects with severe sustained attention deficits, suggesting a modifying effect of impaired neuropsychological functioning. Other interactions such as the within-gene interaction of DAO and the between-gene interaction of DAO and PTK2B were consistently identified regardless of stratification by neuropsychological dysfunction. Importantly, except for the within-gene interaction of CACNG2, all of the identified risk haplotypes and interactions involved SNPs from DAO.

Conclusions

These results suggest that DAO, which is involved in the N-methyl-d-aspartate receptor regulation, signaling and glutamate metabolism, is the master gene of the genetic associations and interactions underlying schizophrenia. Besides, the interaction between DAO and RASD2 has provided an insight in integrating the glutamate and dopamine hypotheses of schizophrenia.     

New Approaches of PARP-1 Inhibitors in Human Lung Cancer Cells and Cancer Stem-Like Cells by Some Selected Anthraquinone-Derived Small Molecules

Poly (ADP-ribose) polymerase-1 (PARP-1) and telomerase, as well as DNA damage response pathways are targets for anticancer drug development, and specific inhibitors are currently under clinical investigation. The purpose of this work is to evaluate anticancer activities of anthraquinone-derived tricyclic and tetracyclic small molecules and their structure-activity relationships with PARP-1 inhibition in non-small cell lung cancer (NSCLC) and NSCLC-overexpressing Oct4 and Nanog clone, which show high-expression of PARP-1 and more resistance to anticancer drug. We applied our library selected compounds to NCI''s 60 human cancer cell-lines (NCI-60) in order to generate systematic profiling data. Based on our analysis, it is hypothesized that these drugs might be, directly and indirectly, target components to induce mitochondrial permeability transition and the release of pro-apoptotic factors as potential anti-NSCLC or PARP inhibitor candidates. Altogether, the most active NSC747854 showed its cytotoxicity and dose-dependent PARP inhibitory manner, thus it emerges as a promising structure for anti-cancer therapy with no significant negative influence on normal cells. Our studies present evidence that telomere maintenance should be taken into consideration in efforts not only to overcome drug resistance, but also to optimize the use of telomere-based therapeutics. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and telomerase inhibitors, in particular. Together, the data presented here expand our insight into the PARP inhibitors and support the resource-demanding lead optimization of structurally related small molecules for human cancer therapy.     

Interleukin-19 Mediates Tissue Damage in Murine Ischemic Acute Kidney Injury

Inflammation and renal tubular injury are major features of acute kidney injury (AKI). Many cytokines and chemokines are released from injured tubular cells and acts as proinflammatory mediators. However, the role of IL-19 in the pathogenesis of AKI is not defined yet. In bilateral renal ischemia/reperfusion injury (IRI)-induced and HgCl₂-induced AKI animal models, real-time quantitative (RTQ)-PCR showed that the kidneys, livers, and lungs of AKI mice expressed significantly higher IL-19 and its receptors than did sham control mice. Immunohistochemical staining showed that IL-19 and its receptors were strongly stained in the kidney, liver, and lung tissue of AKI mice. In vitro, IL-19 upregulated MCP-1, TGF-β1, and IL-19, and induced mitochondria-dependent apoptosis in murine renal tubular epithelial M-1 cells. IL-19 upregulated TNF-α and IL-10 in cultured HepG2 cells, and it increased IL-1β and TNF-α expression in cultured A549 cells. In vivo, after renal IRI or a nephrotoxic dose of HgCl₂ treatment, IL-20R1-deficient mice (the deficiency blocks IL-19 signaling) showed lower levels of blood urea nitrogen (BUN) in serum and less tubular damage than did wild-type mice. Therefore, we conclude that IL-19 mediates kidney, liver, and lung tissue damage in murine AKI and that blocking IL-19 signaling may provide a potent therapeutic strategy for treating AKI.     

Imputing Amino Acid Polymorphisms in Human Leukocyte Antigens

DNA sequence variation within human leukocyte antigen (HLA) genes mediate susceptibility to a wide range of human diseases. The complex genetic structure of the major histocompatibility complex (MHC) makes it difficult, however, to collect genotyping data in large cohorts. Long-range linkage disequilibrium between HLA loci and SNP markers across the major histocompatibility complex (MHC) region offers an alternative approach through imputation to interrogate HLA variation in existing GWAS data sets. Here we describe a computational strategy, SNP2HLA, to impute classical alleles and amino acid polymorphisms at class I (HLA-A, -B, -C) and class II (-DPA1, -DPB1, -DQA1, -DQB1, and -DRB1) loci. To characterize performance of SNP2HLA, we constructed two European ancestry reference panels, one based on data collected in HapMap-CEPH pedigrees (90 individuals) and another based on data collected by the Type 1 Diabetes Genetics Consortium (T1DGC, 5,225 individuals). We imputed HLA alleles in an independent data set from the British 1958 Birth Cohort (N = 918) with gold standard four-digit HLA types and SNPs genotyped using the Affymetrix GeneChip 500 K and Illumina Immunochip microarrays. We demonstrate that the sample size of the reference panel, rather than SNP density of the genotyping platform, is critical to achieve high imputation accuracy. Using the larger T1DGC reference panel, the average accuracy at four-digit resolution is 94.7% using the low-density Affymetrix GeneChip 500 K, and 96.7% using the high-density Illumina Immunochip. For amino acid polymorphisms within HLA genes, we achieve 98.6% and 99.3% accuracy using the Affymetrix GeneChip 500 K and Illumina Immunochip, respectively. Finally, we demonstrate how imputation and association testing at amino acid resolution can facilitate fine-mapping of primary MHC association signals, giving a specific example from type 1 diabetes.     

Pyrosequencing to Identify Homogeneous Phenomenon When Using Recipients/Donors with Different CYP3A5*3 Genotypes in Living Donor Liver Transplantation

This study used pyrosequencing to determine the proportional distribution of CYP3A5*3 genotypes to further confirm the homogeneous phenomenon that is observed when recipients and donors in living donor liver transplantation (LDLT) have a different single nucleotide polymorphism (SNP) genotype. We enrolled 42 recipient/living donor pairs and the SNPs of CYP3A5*3 were identified by polymerase chain reaction-restriction fragment length polymorphism. We performed 120 liver graft biopsies as part of clinical investigations after LDLT. Pyrosequencing of the CYP3A5*3 SNPs revealed that among the 16 recipients with the G/G genotype, 94.68% had the G and 5.32% the A allele. Among the 14 recipients with the A/G genotype, 78.08% had the G and 21.92% the A allele, and among the 12 recipients with the A/A genotype, 18.45% had the G and 81.55% the A allele. Among the 12 donors with the G/G genotype, 93.85% had the G and 6.14% the A allele. Among the 26 donors with the A/G genotype, 75.73% had the G and 24.27% the A allele, and among the 4 donors with the A/A genotype, 11.09% had the G and 88.91% the A allele. There were a total of 120 liver graft biopsy samples; among the 37 recipients with the G/G genotype, 89.74% had the G and 10.26% the A allele, among the 70 recipients with the A/G genotype, 71.57% had the G and 28.43% the A allele, and among the 13 recipients with the A/A genotype, 48.25% had the G and 51.75% the A allele. The proportional distribution of G and A alleles of the CYP3A5*3 SNP between recipients/donors and liver grafts after LDLT was significantly different (p<0.001). Pyrosequencing was useful in identifying detailed proportional changes of the CYP3A5*3 SNP allele distribution, and to confirm the homogeneous phenomenon when recipients and donors in LDLT have a different genotype.     

Antidepressant-Like Activity of YL-0919: A Novel Combined Selective Serotonin Reuptake Inhibitor and 5-HT1A Receptor Agonist

It has been suggested that drugs combining activities of selective serotonin reuptake inhibitor and 5-HT_1A receptor agonist may form a novel strategy for higher therapeutic efficacy of antidepressant. The present study aimed to examine the pharmacology of YL-0919, a novel synthetic compound with combined high affinity and selectivity for serotonin transporter and 5-HT_1A receptors. We performed in vitro binding and function assays and in vivo behavioral tests to assess the pharmacological properties and antidepressant-like efficacy of YL-0919. YL-0919 displayed high affinity in vitro to both 5-HT_1A receptor and 5-HT transporter prepared from rat cortical tissue. It exerted an inhibitory effect on forskolin-stimulated cAMP formation and potently inhibited 5-HT uptake in both rat cortical synaptosomes and recombinant cells. After acute p.o. administration, very low doses of YL-0919 reduced the immobility time in tail suspension test and forced swimming test in mice and rats, with no significant effect on locomotor activity in open field test. Furthermore, WAY-100635 (a selective 5-HT_1A receptor antagonist, 0.3 mg/kg) significantly blocked the effect of YL-0919 in tail suspension test and forced swimming test. In addition, chronic YL-0919 treatment significantly reversed the depressive-like behaviors in chronically stressed rats. These findings suggest that YL-0919, a novel structure compound, exerts dual effect on the serotonergic system, as both 5-HT_1A receptor agonist and 5-HT uptake blocker, showing remarkable antidepressant effects in animal models. Therefore, YL-0919 may be used as a new option for the treatment of major depressive disorder.     

Epidemiological Characteristics of Lower Extremity Cellulitis after a Typhoon Flood

Objective

The flood after a typhoon may lead to increase in patients with cellulitis of lower limbs. However, the microbiological features of these cases are rarely reported. We conducted a study of patients with lower extremity cellulitis after a typhoon followed in southern Taiwan to study the risk factors of cellulitis and the bacteriological features of the patients.

Methods

We reviewed all the medical records of cellulitis at emergency departments of two teaching hospitals in southern Taiwan 30 days before and after the landing of Typhoon Morakot and collected data on the demographic and bacteriological characteristics. In addition, we evaluated the relationship between the daily number of patients and the rainfall in the Tainan area.

Results

The number of cellulitis patients increased from 183 to 344 during the 30-day period after the typhoon. The number peaked in the third and fourth days and lasted for 3 weeks. The proportion of patients with water immersion of the affected limb was higher after the typhoon (6% vs. 37%, odds ratio [OR]: 9.0, 95% Confidence interval [CI]: 4.7–17.2). We found cultures from the infected limbs with immersion had more polymicrobial (73% vs. 26%, OR: 7.8, 95% CI: 3.2–19.2) and Gram-negative bacilli infection (86% vs. 34%, OR: 11.8, 95% CI: 4.1–34.5).

Conclusions

Flood arose from Typhoon Morakot caused increases in cellulitis patients, which lasted for 3 weeks. Antibiotic treatment that were effective to both Gram-positive cocci and Gram-negative bacilli are recommended for patients with limbs emerged in the water.