TIAM2S-positive microglia enhance inflammation and neurotoxicity through soluble ICAM-1-mediated immune priming.

TIAM Rac1-associated GEF 2 short form (TIAM2S) as an oncoprotein alters the immunity of peripheral immune cells to construct an inflammatory tumor microenvironment. However, its role in the activation of microglia, the primary innate immune cells of the brain, and neuroinflammation remains unknown. This study investigated the mechanism underlying TIAM2S shapes immune properties of microglia to facilitate neuron damage. Human microglial clone 3 cell line (HMC3) and human brain samples were applied to determine the presence of TIAM2S in microglia by western blots and double immunostaining. Furthermore, TIAM2S transgenic mice combined with multiple reconstituted primary neuron-glial culture systems and a cytokine array were performed to explore how TIAM2S shaped immune priming of microglia and participated in lipopolysaccharide (LPS)-induced neuron damage. TIAM2S protein was detectable in HMC3 cells and presented in a small portion (~11.1%) of microglia in human brains referred to as TIAM2S-positive microglia. With the property of secreted soluble factor-mediated immune priming, TIAM2S-positive microglia enhanced LPS-induced neuroinflammation and neural damage in vivo and in vitro. The gain- and loss-of-function experiments showed soluble intercellular adhesion molecule-1 (sICAM-1) participated in neurotoxic immune priming of TIAM2S+ microglia. Together, this study demonstrated a novel TIAM2S-positive microglia subpopulation enhances inflammation and neurotoxicity through sICAM-1-mediated immune priming.

DNA-induced 2'3'-cGAMP enhances haplotype-specific human STING cleavage by dengue protease.

The cytosolic DNA sensor cGMP-AMP synthase (cGAS) synthesizes the noncanonical cyclic dinucleotide 2'3'-cGAMP to activate the adaptor protein stimulator of IFN genes (STING), thus awakening host immunity in response to DNA pathogen infection. However, dengue virus (DENV), an RNA virus without a DNA stage in its life cycle, also manipulates cGAS-STING-mediated innate immunity by proteolytic degradation of STING. Here, we found that the sensitivity of STING to DENV protease varied with different human STING haplotypes. Exogenous DNA further enhanced DENV protease's ability to interact and cleave protease-sensitive STING. DNA-enhanced STING cleavage was reduced in cGAS-knockdown cells and triggered by the cGAS product 2'3'-cGAMP. The source of DNA may not be endogenous mitochondrial DNA but rather exogenous reactivated viral DNA. Cells producing 2'3'-cGAMP by overexpressing cGAS or with DNA virus reactivation enhanced STING cleavage in neighboring cells harboring DENV protease. DENV infection reduced host innate immunity in cells with the protease-sensitive STING haplotype, whose homozygote genotype frequency was found significantly reduced in Taiwanese people with dengue fever. Therefore, the human STING genetic background and DNA pathogen coinfection may be the missing links contributing to DENV pathogenesis.

Behavioral and brain- transcriptomic synchronization between the two opponents of a fighting pair of the fish Betta splendens.

Conspecific male animals fight for resources such as food and mating opportunities but typically stop fighting after assessing their relative fighting abilities to avoid serious injuries. Physiologically, how the fighting behavior is controlled remains unknown. Using the fighting fish Betta splendens, we studied behavioral and brain-transcriptomic changes during the fight between the two opponents. At the behavioral level, surface-breathing, and biting/striking occurred only during intervals between mouth-locking. Eventually, the behaviors of the two opponents became synchronized, with each pair showing a unique behavioral pattern. At the physiological level, we examined the expression patterns of 23,306 brain transcripts using RNA-sequencing data from brains of fighting pairs after a 20-min (D20) and a 60-min (D60) fight. The two opponents in each D60 fighting pair showed a strong gene expression correlation, whereas those in D20 fighting pairs showed a weak correlation. Moreover, each fighting pair in the D60 group showed pair-specific gene expression patterns in a grade of membership analysis (GoM) and were grouped as a pair in the heatmap clustering. The observed pair-specific individualization in brain-transcriptomic synchronization (PIBS) suggested that this synchronization provides a physiological basis for the behavioral synchronization. An analysis using the synchronized genes in fighting pairs of the D60 group found genes enriched for ion transport, synaptic function, and learning and memory. Brain-transcriptomic synchronization could be a general phenomenon and may provide a new cornerstone with which to investigate coordinating and sustaining social interactions between two interacting partners of vertebrates.

Estradiol-mediated inhibition of Sp1 decreases miR-3194-5p expression to enhance CD44 expression during lung cancer progression.

BACKGROUND: Sp1, an important transcription factor, is involved in the progression of various cancers. Our previous studies have indicated that Sp1 levels are increased in the early stage of lung cancer progression but decrease during the late stage, leading to poor prognosis. In addition, estrogen has been shown to be involved in lung cancer progression. According to previous studies, Sp1 can interact with the estrogen receptor (ER) to coregulate gene expression. The role of interaction between Sp1 and ER in lung cancer progression is still unknown and will be clarified in this study. METHODS: The clinical relevance between Sp1 levels and survival rates in young women with lung cancer was studied by immunohistochemistry. We validated the sex dependence of lung cancer progression in EGFRL858R-induced lung cancer mice. Wound healing assays, chamber assays and sphere formation assays in A549 cells, Taxol-induced drug-resistant A549 (A549-T24) and estradiol (E2)-treated A549 (E2-A549) cells were performed to investigate the roles of Taxol and E2 in lung cancer progression. Luciferase reporter assays, immunoblot and q-PCR were performed to evaluate the interaction between Sp1, microRNAs and CD44. Tail vein-injected xenograft experiments were performed to study lung metastasis. Samples obtained from lung cancer patients were used to study the mRNA level of CD44 by q-PCR and the protein levels of Sp1 and CD44 by immunoblot and immunohistochemistry. RESULTS: In this study, we found that Sp1 expression was decreased in premenopausal women with late-stage lung cancer, resulting in a poor prognosis. Tumor formation was more substantial in female EGFRL858R mice than in male mice and ovariectomized female mice, indicating that E2 might be involved in the poor prognosis of lung cancer. We herein report that Sp1 negatively regulates metastasis and cancer stemness in E2-A549 and A549-T24 cells. Furthermore, E2 increases the mRNA and protein levels of RING finger protein 4 (RNF4), which is the E3-ligase of Sp1, and thereby decreases Sp1 levels by promoting Sp1 degradation. Sp1 can be recruited to the promoter of miR-3194-5p, and positively regulate its expression. Furthermore, there was a strong inverse correlation between Sp1 and CD44 levels in clinical lung cancer specimens. Sp1 inhibited CD44 expression by increasing the expression of miR-3194-5p, miR-218-5p, miR-193-5p, miR-182-5p and miR-135-5p, ultimately resulting in lung cancer malignancy. CONCLUSION: Premenopausal women with lung cancer and decreased Sp1 levels have a poor prognosis. E2 increases RNF4 expression to repress Sp1 levels in premenopausal women with lung cancer, thus decreasing the expression of several miRNAs that can target CD44 and ultimately leading to cancer malignancy.

TIAM2S as a novel regulator for serotonin level enhances brain plasticity and locomotion behavior.

TIAM2S, the short form of human T-cell lymphoma invasion and metastasis 2, can have oncogenic effects when aberrantly expressed in the liver or lungs. However, it is also abundant in healthy, non-neoplastic brain tissue, in which its primary function is still unknown. Here, we examined the neurobiological and behavioral significance of human TIAM2S using the human brain protein panels, a human NT2/D1-derived neuronal cell line model (NT2/N), and transgenic mice that overexpress human TIAM2S (TIAM2S-TG). Our data reveal that TIAM2S exists primarily in neurons of the restricted brain areas around the limbic system and in well-differentiated NT2/N cells. Functional studies revealed that TIAM2S has no guanine nucleotide exchange factor (GEF) activity and is mainly located in the nucleus. Furthermore, whole-transcriptome and enrichment analysis with total RNA sequencing revealed that TIAM2S-knockdown (TIAM2S-KD) was strongly associated with the cellular processes of the brain structural development and differentiation, serotonin-related signaling, and the diseases markers representing neurobehavioral developmental disorders. Moreover, TIAM2S-KD cells display decreased neurite outgrowth and reduced serotonin levels. Moreover, TIAM2S overexpressing TG mice show increased number and length of serotonergic fibers at early postnatal stage, results in higher serotonin levels at both the serum and brain regions, and higher neuroplasticity and hyperlocomotion in latter adulthood. Taken together, our results illustrate the non-oncogenic functions of human TIAM2S and demonstrate that TIAM2S is a novel regulator of serotonin level, brain neuroplasticity, and locomotion behavior.

FGF9 is a downstream target of SRY and sufficient to determine male sex fate in ex vivo XX gonad culture.

Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor that plays critical roles in embryonic and organ developments and is involved in diverse physiological events. Loss of function of FGF9 exhibits male-to-female sex reversal in the transgenic mouse model and gain of FGF9 copy number was found in human 46, XX sex reversal patient with disorders of sex development. These results suggested that FGF9 plays a vital role in male sex development. Nevertheless, how FGF9/Fgf9 expression is regulated during testis determination remains unclear. In this study, we demonstrated that human and mouse SRY bind to -833 to -821 of human FGF9 and -1010 to -998 of mouse Fgf9, respectively, and control FGF9/Fgf9 mRNA expression. Interestingly, we showed that mouse SRY cooperates with SF1 to regulate Fgf9 expression, whereas human SRY-mediated FGF9 expression is SF1 independent. Furthermore, using an ex vivo gonadal culture system, we showed that FGF9 expression is sufficient to switch cell fate from female to male sex development in 12-16 tail somite XX mouse gonads. Taken together, our findings provide evidence to support the SRY-dependent, fate-determining role of FGF9 in male sex development.

Fibroblast growth factors: Potential novel targets for regenerative therapy of osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disorder and is the leading cause of disability of people, which negatively impact people's physical and mental health. Although OA causes great socioeconomic burden and individual suffering, no effective treatment options are provided so far. This is partially resulted from poor regenerative activity of articular cartilage and our incomplete understanding of the underlying mechanism of OA. Traditional drug therapies such as acetaminophen and opioids are effective in relieving pain but do not reverse cartilage damage and are often associated with adverse events. Therefore, it is necessary to find effective OA drugs. In recent years, novel regenerative therapies have received much attention because they can effectively promote tissue repair and regeneration. The fibroblast growth factor (FGF) signaling has been suggested to involve in cartilage homeostasis for decades. The current research shows that sprifermin/recombinant FGF18 significantly reduces the loss of cartilage thickness and volume without serious side effects, thus warrants a continued research for potential new medications of OA. This review mainly highlights the current research progress on FGFs and FGF receptors as a potential therapeutic target for OA.

FGF9/FGFR2 increase cell proliferation by activating ERK1/2, Rb/E2F1, and cell cycle pathways in mouse Leydig tumor cells.

Fibroblast growth factor 9 (FGF9) promotes cancer progression; however, its role in cell proliferation related to tumorigenesis remains elusive. We investigated how FGF9 affected MA-10 mouse Leydig tumor cell proliferation and found that FGF9 significantly induced cell proliferation by activating ERK1/2 and retinoblastoma (Rb) phosphorylations within 15 minutes. Subsequently, the expressions of E2F1 and the cell cycle regulators: cyclin D1, cyclin E1 and cyclin-dependent kinase 4 (CDK4) in G1 phase and cyclin A1, CDK2 and CDK1 in S-G2 /M phases were increased at 12 hours after FGF9 treatment; and cyclin B1 in G2 /M phases were induced at 24 hours after FGF9 stimulation, whereas the phosphorylations of p53, p21 and p27 were not affected by FGF9. Moreover, FGF9-induced effects were inhibited by MEK inhibitor PD98059, indicating FGF9 activated the Rb/E2F pathway to accelerate MA-10 cell proliferation by activating ERK1/2. Immunoprecipitation assay and ChIP-quantitative PCR results showed that FGF9-induced Rb phosphorylation led to the dissociation of Rb-E2F1 complexes and thereby enhanced the transactivations of E2F1 target genes, Cyclin D1, Cyclin E1 and Cyclin A1. Silencing of FGF receptor 2 (FGFR2) using lentiviral shRNA inhibited FGF9-induced ERK1/2 phosphorylation and cell proliferation, indicating that FGFR2 is the obligate receptor for FGF9 to bind and activate the signaling pathway in MA-10 cells. Furthermore, in a severe combined immunodeficiency mouse xenograft model, FGF9 significantly promoted MA-10 tumor growth, a consequence of increased cell proliferation and decreased apoptosis. Conclusively, FGF9 interacts with FGFR2 to activate ERK1/2, Rb/E2F1 and cell cycle pathways to induce MA-10 cell proliferation in vitro and tumor growth in vivo.

Fibroblast Growth Factor 9 Suppresses Striatal Cell Death Dominantly Through ERK Signaling in Huntington's Disease.

BACKGROUND/AIMS: Huntington's disease (HD) is a heritable neurodegenerative disorder, and there is no cure for HD to date. A type of fibroblast growth factor (FGF), FGF9, has been reported to play prosurvival roles in other neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. However, the effects of FGF9 on HD is still unknown. With many similarities in the cellular and pathological mechanisms that eventually cause cell death in neurodegenerative diseases, we hypothesize that FGF9 might provide neuroprotective functions in HD. METHODS: In this study, STHdhQ7/Q7 (WT) and STHdhQ111/Q111 (HD) striatal knock-in cell lines were used to evaluate the neuroprotective effects of FGF9. Cell proliferation, cell death and neuroprotective markers were determined via the MTT assay, propidium iodide staining and Western blotting, respectively. The signaling pathways regulated by FGF9 were demonstrated using Western blotting. Additionally, HD transgenic mouse models were used to further confirm the neuroprotective effects of FGF9 via ELISA, Western blotting and immunostaining. RESULTS: Results show that FGF9 not only enhances cell proliferation, but also alleviates cell death as cells under starvation stress. In addition, FGF9 significantly upregulates glial cell line-derived neurotrophic factor (GDNF) and an anti-apoptotic marker, Bcl-xL, and decreases the expression level of an apoptotic marker, cleaved caspase 3. Furthermore, FGF9 functions through ERK, AKT and JNK pathways. Especially, ERK pathway plays a critical role to influence the effects of FGF9 toward cell survival and GDNF production. CONCLUSIONS: These results not only show the neuroprotective effects of FGF9, but also clarify the critical mechanisms in HD cells, further providing an insight for the therapeutic potential of FGF9 in HD.

Low-cell-number, single-tube amplification (STA) of total RNA revealed transcriptome changes from pluripotency to endothelium.

BACKGROUND: In addition to messenger RNA (mRNA), noncoding RNAs (ncRNAs) are essential components in cellular machineries for translation and splicing. Besides their housekeeping functions, ncRNAs are involved in cell type-specific regulation of translation, mRNA stability, genome structure, and accessibility. To have a comprehensive understanding of the identities and functions of different cell types, a method to comprehensively quantify both mRNA and ncRNA in a sensitive manner is highly desirable. METHODS: Here we tried to develop a system capable of concurrently profiling both mRNA and ncRNA by polyadenylating RNA in samples before reverse transcription. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. The single-tube amplification (STA) system was applied to single to 100 cells of 293T cells, human pluripotent stem cells (hPSCs) and their differentiated endothelial progenies to validate its quantitative power and sensitivity by qPCR and high-throughput sequencing. RESULTS: Using microRNA (miRNA) as an example, we showed that complementary DNA (cDNA) from ncRNAs could be amplified and specifically detected from a few cells within a single tube. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. With 100 human embryonic stem cells (hESCs) and their differentiated endothelial cells as input for high-throughput sequencing, the single-tube amplification (STA) system revealed both well-known and other miRNAs selectively enriched in each cell type. The selective enrichment of the miRNAs was further verified by qPCR with 293FT cells and a human induced pluripotent stem cell (hiPSC) line. In addition, the detection of other non-miRNA transcripts indicated that the STA target was not limited to miRNA, but extended to other ncRNAs and mRNAs as well. Finally, the STA system was capable of detecting miRNA and mRNA expression down to single cells, albeit with some loss of sensitivity and power. CONCLUSIONS: Overall, STA offered a simple and sensitive way to concurrently quantify both mRNA and ncRNA expression in low-cell-number samples for both qPCR and high-throughput sequencing.

Validation of simplified postoperative nausea and vomiting intensity scale in Taiwan.

This study determined whether the Simplified Postoperative Nausea and Vomiting Impact Scale (SPONVIS), could be used to predict clinically important PONV in Taiwanese. In this prospective, observational study, SPONVIS, simplified Apfel PONV Risk Scores, post-operative anti-emetic drug use, total PONV score, and 3-month recall score for PONV were recorded from Taiwanese patients who had undergone general anesthesia and surgery. With antiemetic use and 3-month recall score as validations of clinical significance, we determined whether the elements and cut-off points used in the original SPONVIS study could be used in Taiwanese patients. A total of 378 patients were included in the analysis. One hundred forty (37.1%) patients had PONV. Forty-eight patients (12.7%) had clinically important PONV (SPONVIS score ≥ 5). The odds ratios were 14.26 (CI 6.91-29.43; P < 0.001) and 4.95 (CI 2.42 to 10.11; P < 0.001), respectively, for prediction of anti-emetic drug use and 3-month recall. The SPONVIS and its construct elements were significantly related to anti-emetic drug use, 3-month recall score for PONV, total PONV score, and Apfel risk score (all P ≤ 0.005), results similar to those reported in the original Australian PONV impact score study. The SPONVIS cut-off points 3 and 5 were statistically significant predictors of anti-emetic drug use. However, a cut-off point of 3 had a higher OR (24.08) than a cut-off of 5 (14.26) for prediction of anti-emetic drug use. SPONVIS and both construct elements (the nausea and vomiting impact scores) are useful predictors of clinically important PONV in Taiwanese.

Global Oct4 target gene analysis reveals novel downstream PTEN and TNC genes required for drug-resistance and metastasis in lung cancer.

Overexpression of Oct4, a stemness gene encoding a transcription factor, has been reported in several cancers. However, the mechanism by which Oct4 directs transcriptional program that leads to somatic cancer progression remains unclear. In this study, we provide mechanistic insight into Oct4-driven transcriptional network promoting drug-resistance and metastasis in lung cancer cell, animal and clinical studies. Through an integrative approach combining our Oct4 chromatin-immunoprecipitation sequencing and ENCODE datasets, we identified the genome-wide binding regions of Oct4 in lung cancer at promoter and enhancer of numerous genes involved in critical pathways which promote tumorigenesis. Notably, PTEN and TNC were previously undefined targets of Oct4. In addition, novel Oct4-binding motifs were found to overlap with DNA elements for Sp1 transcription factor. We provided evidence that Oct4 suppressed PTEN in an Sp1-dependent manner by recruitment of HDAC1/2, leading to activation of AKT signaling and drug-resistance. In contrast, Oct4 transactivated TNC independent of Sp1 and resulted in cancer metastasis. Clinically, lung cancer patients with Oct4 high, PTEN low and TNC high expression profile significantly correlated with poor disease-free survival. Our study reveals a critical Oct4-driven transcriptional program that promotes lung cancer progression, illustrating the therapeutic potential of targeting Oc4 transcriptionally regulated genes.

MicroRNAs regulate gene plasticity during cold shock in zebrafish larvae.

BACKGROUND: MicroRNAs (miRNAs) are critical regulators responding to acute environmental stresses in both plants and animals. By modulating gene expression, miRNAs either restore or reconstitute a new expression program to enhance cell tolerance to stresses. Cold shock is one of the stresses that can induce acute physiological responses and transcriptional changes in aquatic creatures. Previous genomic studies have revealed many cold-affected genes in fish larvae and adults, however, the role of miRNAs in acute cold response is still ambiguous. To elucidate the regulatory roles of miRNAs in the cold-inducible responses, we performed small RNA-seq and RNA-seq analyses and found potential cold regulatory miRNAs and genes. We further investigated their interactions and involvements in cold tolerance. RESULTS: Small RNA-seq and RNA-seq identified 29 up-/26 down-regulated miRNAs and 908 up-/468 down-regulated genes, respectively, in responding to cold shock for 4 h at 18 °C. miRNA and transcriptomic analyses showed these miRNAs and mRNAs are involved in similar biological processes and pathways. Gene ontology enrichment analyses revealed the cold-induced genes were enriched in pathways, including melanogenesis, GnRH pathway, circadian rhythm, etc. We were particularly interested in the changes in circadian clock genes that affect daily metabolism. The enrichment of circadian clock genes was also observed in previous fish cold acclimation studies, but have not been characterized. To characterize the functional roles of circadian clock genes in cold tolerance, we individually overexpressed selected clock genes in zebrafish larvae and found one of the core clock genes per2 resulted in better recovery from cold shock. In addition, we validated the interaction of per2 with its associate miRNA, dre-mir-29b, which is also cold-inducible. It suggests the transcription of per2 can be modulated by miRNA upon cold shock. CONCLUSIONS: Collectively, our observations suggest that miRNAs are fine turners for regulating genomic plasticity against cold shock. We further showed that the fine tuning of core clock gene per2 via its associated miRNA, dre-mir-29b, can enhance the cold tolerance of zebrafish larvae.

Effectiveness and Mechanism of Preoperative Lugol Solution for Reducing Thyroid Blood Flow in Patients with Euthyroid Graves' Disease.

BACKGROUND: To reduce intraoperative and postoperative complications, using Lugol solution to preoperatively prepare patients with Graves' disease has (1) rapidly reduced the severity of thyrotoxicosis and (2) reduced the vascularity of the thyroid gland. The vascularity reduction normally accompanies reducing the severity of thyrotoxicosis. However, the effects and mechanism of Lugol solution for reducing blood flow have not been well investigated in the patients with euthyroid (normally functioning thyroid) Graves' disease. METHODS: Twenty-five patients with euthyroid Graves' disease being preoperatively treated with Lugol solution for 10 days were measured, at baseline and on the operative day, for (1) superior thyroid artery blood flow; (2) systemic angiogenic factor (VEGF); and (3) systemic inflammatory factor [interleukin (IL)-16]. RESULTS: All three parameters were significantly (p < 0.0001) lower after 10 days of Lugol solution treatment. The average reductions were blood flow: 60% (0.294 vs. 0.117 L/min), serum VEGF: 55% (169.8 vs. 76.7 pg/mL), and serum IL-16: 50% (427.2 vs. 214.2; pg/mL). CONCLUSION: Lugol solution significantly reduced thyroid arterial blood flow, VEGF, and IL-16, even in patients with euthyroid Graves' disease. We recommend routine preoperative Lugol solution treatment for all patients with Graves' disease.

Overexpression of FGF9 in colon cancer cells is mediated by hypoxia-induced translational activation.

Human fibroblast growth factor 9 (FGF9) is a potent mitogen involved in many physiological processes. Although FGF9 messenger RNA (mRNA) is ubiquitously expressed in embryos, FGF9 protein expression is generally low and restricted to a few adult organs. Aberrant expression of FGF9 usually results in human malignancies including cancers, but the mechanism remains largely unknown. Here, we report that FGF9 protein, but not mRNA, was increased in hypoxia. Two sequence elements, the upstream open reading frame (uORF) and the internal ribosome entry site (IRES), were identified in the 5' UTR of FGF9 mRNA. Functional assays indicated that FGF9 protein synthesis was normally controlled by uORF-mediated translational repression, which kept the protein at a low level, but was upregulated in response to hypoxia through a switch to IRES-dependent translational control. Our data demonstrate that FGF9 IRES functions as a cellular switch to turn FGF9 protein synthesis 'on' during hypoxia, a likely mechanism underlying FGF9 overexpression in cancer cells. Finally, we provide evidence to show that hypoxia-induced translational activation promotes FGF9 protein expression in colon cancer cells. Altogether, this dynamic working model may provide a new direction in anti-tumor therapies and cancer intervention.

Coordination of AUF1 and miR-148a destabilizes DNA methyltransferase 1 mRNA under hypoxia in endometriosis.

STUDY HYPOTHESIS: DNA methylation is regulated by hypoxia in endometriosis. STUDY FINDING: Hypoxia causes global hypomethylation through AU-rich element binding factor 1 (AUF1)/microRNA-148a (miR-148a)-mediated destabilization of DNA methyltransferase 1 (DNMT1) mRNA. WHAT IS KNOWN ALREADY: Eutopic endometrial and ectopic endometriotic stromal cells have the same genetic background, but differ in several cellular and molecular responses. Both hypoxia and DNA methylation regulate several genes involved in the development of endometriosis. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: This laboratory study included 15 patients of reproductive age with endometriosis or normal menstrual cycles. Paired endometrial and endometriotic tissues were collected for assaying the levels of DNMT1, 3a and 3b using quantitative RT-PCR, western blot and immunohistochemical (IHC) staining. Primary cultured endometrial stromal cells maintained in normoxia/hypoxia (1% O2) or treated with hypoxia-mimetic compounds were also assayed. The levels of DNA 5-methylcytosine were assayed by using IHC in clinical specimens and murine tissues, and by ELISA in cultured stromal cells. The 3'-untranslated region reporter assay was used to evaluate the effect of hypoxia, microRNAs (miRNAs) and human antigen R (HuR)/AUF1 on DNMT1 mRNA stability. RNA immunoprecipitation was used to assess the interaction of HuR/AUF1 and miR-148a/DNMT1 mRNA under hypoxia. Finally, a transplant-induced mouse model of endometriosis using 20 mice was used to elucidate the alteration of Dnmt1 levels and DNA methylation in the endometriotic tissues. MAIN RESULTS AND THE ROLE OF CHANCE: Levels of DNMT1 mRNA and protein and 5-methylcytosine were lower in the ectopic stromal cells (P < 0.05) than in the eutopic cells. Treatment with hypoxia and its mimetic compounds recapitulated the reduced levels of DNMT1 and 5-methylcytosine levels (P < 0.05 versus control). Hypoxia treatment destabilized DNMT1 mRNA through recruitment of miR-148a and AUF1. Mutations introduced to the miR-148a targeting site or AU-rich element (ARE) restored the hypoxia-suppressed DNMT1 3'-untranslated region (3'-UTR) reporter activity (P < 0.05 versus control). Levels of proteins of three hypermethylated genes in endometrial stroma cells, GATA6, HOXA3 and SLC16A5, were elevated after 72 h of hypoxia treatment (P < 0.05 versus control). Finally, a transplant-induced model of endometriosis demonstrated the down-regulation of DNMT1 and a decrease in 5-methylcytosine in the endometriotic tissues (P < 0.05, eutopic versus ectopic). LIMITATIONS, REASONS FOR CAUTION: Primary human cell cultures and a murine model were used in this study, and thus the results may not fully represent the situation in vivo. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to elucidate how microenvironmental hypoxia links to the epigenetic effects of DNA methylation in the endometriosis, and to delineate the molecular mechanism of hypoxia-coordinated AUF1/miR-148a interaction and recruitment to DNMT1 mRNA during the pathogenesis of endometriosis. The development of future therapeutics in endometriosis may aim at disrupting this specific interaction and eventually restore the epigenetic regulation. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by the National Science Council of Taiwan (NSC101-2320-B-006-030-MY3). The author declares that there are no conflicts of interest.

Rapid identification of bacteria and Candida pathogens in peritoneal dialysis effluent from patients with peritoneal dialysis-related peritonitis by use of multilocus PCR coupled with electrospray ionization mass spectrometry.

PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was compared with culture for pathogen detection in peritoneal dialysis (PD)-related peritonitis. Of 21 samples of PD effluent, PCR/ESI-MS identified microorganisms in 18 (86%) samples, including Mycobacterium tuberculosis in 1 culture-negative sample. Of 15 double-positive samples, PCR/ESI-MS and culture reached levels of agreement of 100% (15/15) and 87.5% (7/8) at the genus and species levels, respectively. PCR/ESI-MS can be used for rapid pathogen detection in PD-related peritonitis.

In silico identification of oncogenic potential of fyn-related kinase in hepatocellular carcinoma.

MOTIVATION: Cancer development is a complex and heterogeneous process. It is estimated that 5-10% of human genes probably contribute to oncogenesis, whereas current experimentally validated cancer genes only cover 1% of the human genome. Thus hundreds of cancer genes may still remain to be identified. To search for new genes that play roles in carcinogenesis and facilitate cancer research, we developed a systematic workflow to use information saved in a previously established tumor-associated gene (TAG) database. RESULTS: By exploiting the information of conserved protein domains from the TAG, we identified 183 potential new TAGs. As a proof-of-concept, one predicted oncogene, fyn-related kinase (FRK), which shows an aberrant digital expression pattern in liver cancer cells, was selected for further investigation. Using 68 paired hepatocellular carcinoma samples, we found that FRK was up-regulated in 52% of cases (P < 0.001). Tumorigenic assays performed in Hep3B and HepG2 cell lines revealed a significant correlation between the level of FRK expression and invasiveness, suggesting that FRK is a positive regulator of invasiveness in liver cancer cells. CONCLUSION: These findings implied that FRK is a multitalented signal transduction molecule that produces diverse biological responses in different cell types in various microenvironments. In addition, our data demonstrated the accuracy of computational prediction and suggested that other predicted TAGs can be potential targets for future cancer research. AVAILABILITY: The TAG database is available online at the Bioinformatics Center website: http://www.binfo.ncku.edu.tw/TAG/.

Sequence variants of the HTR3A gene contribute to the genetic prediction of postoperative nausea in Taiwan.

Postoperative nausea (PON) is a common complication, and therefore, it is important to identify the associated genetic factors and the candidate predictive markers. Current clinical and basic research suggests that the 5-hydroxytryptamine type 3A receptor (HTR3A) may be important in the occurrence of PON. The association between three single nucleotide polymorphisms (SNPs) of the HTR3A gene and PON was examined to determine whether this can be used to predict the incidence of PON in a unique Taiwanese population without any reported postoperative nausea and vomiting (PONV) risk factors associated with PON occurrence. One thousand adult surgical patients who received general anesthesia were included in this analysis. A total of 369 patients were finally selected for a two-stage association study. Significant single-locus associations for all three HTR3A SNPs and PON were identified in both stages. In addition, two of the most common haplotypes, CTT and TAG, showed both a significant risk for and a protective effect against PON, respectively. Our findings support the notion that different haplotypes of HTR3A have reciprocal effects in the etiology of PON. Therefore specific haplotypes of HTR3A may be useful as predictors of PON for 24 h immediately after surgery in our population.