BackgroundThe model eukaryote, Tetrahymena thermophila, is the first ciliated protozoan whose genome has been sequenced, enabling genome-wide analysis of gene expression.Methodology/Principal FindingsA genome-wide microarray platform containing the predicted coding sequences (putative genes) for T. thermophila is described, validated and used to study gene expression during the three major stages of the organism's life cycle: growth, starvation and conjugation.Conclusions/SignificanceOf the ∼27,000 predicted open reading frames, transcripts homologous to only ∼5900 are not detectable in any of these life cycle stages, indicating that this single-celled organism does indeed contain a large number of functional genes. Transcripts from over 5000 predicted genes are expressed at levels >5× corrected background and 95 genes are expressed at >250× corrected background in all stages. Transcripts homologous to 91 predicted genes are specifically expressed and 155 more are highly up-regulated in growing cells, while 90 are specifically expressed and 616 are up-regulated during starvation. Strikingly, transcripts homologous to 1068 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation. The patterns of gene expression during conjugation correlate well with the developmental stages of meiosis, nuclear differentiation and DNA elimination. The relationship between gene expression and chromosome fragmentation is analyzed. Genes encoding proteins known to interact or to function in complexes show similar expression patterns, indicating that co-ordinate expression with putative genes of known function can identify genes with related functions. New candidate genes associated with the RNAi-like process of DNA elimination and with meiosis are identified and the late stages of conjugation are shown to be characterized by specific expression of an unexpectedly large and diverse number of genes not involved in nuclear functions.

The regulation of sympathetic nerve activity in chronic heart failure (CHF) has been an area of renewed investigation. Understanding the central mechanisms that are responsible for sympatho-excitation in this disease state may help in reducing the deleterious effects of chronic sympatho-excitation. This review will summarize our understanding of abnormal reflex control of the circulation in CHF. The roles of the arterial baroreflex, the chemoreflex, the cardiac sympathetic afferent reflex and the cardiopulmonary reflex are discussed. New experimental techniques that allow genetic manipulation of substances such as nitric oxide synthase in discrete areas of the brain aid in clarifying the role of NO in the modulation of sympathetic tone in the CHF state. Lastly, clinical implications of this work are discussed.

Chronic kidney disease (CKD) is a prevalent life-threatening disease frequently associated with hypertension, progression to renal fibrosis and eventual renal failure. While the pathogenesis of CKD remains largely unknown, an increased inflammatory response is known to be associated with the disease and has long been speculated to contribute to disease development. However, the causative factors, the exact role of the increased inflammatory cascade in CKD and the underlying mechanisms for its progression remain unidentified. Here we report that interleukin-6 (IL-6) expression levels were significantly increased in the kidneys collected from CKD patients and further elevated in CKD patients characterized with hypertension. Functionally, we determined that angiotensin II (Ang II) is a causative factor responsible for IL-6 induction in the mouse kidney and that genetic deletion of IL-6 significantly reduced hypertension and key features of CKD including renal injury and progression to renal fibrosis in Ang II-infused mice. Mechanistically, we provide both human and mouse evidence that IL-6 is a key cytokine functioning downstream of Ang II signaling to directly induce fibrotic gene expression and preproendothelin-1 (prepro-ET-1) mRNA expression in the kidney. Overall, both the mouse and human studies reported here provide evidence that Ang II induces IL-6 production in the kidney and that, in addition to its role in hypertension, increased IL-6 may play an important pathogenic role in CKD by inducing fibrotic gene expression and ET-1 gene expression. These findings immediately suggest the IL-6 signaling is a novel therapeutic target to manage this devastating disorder affecting millions worldwide.

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Human sperm cryopreservation in assisted reproductive technology is the only proven method that enables infertile men to father their own children. However, freezing and thawing reduces spermatozoon motility, viability, and fertilizing ability. An association between dysfunctional spermatozoa due to cryoinjury and protein changes has not been established. We investigated through proteomic analysis the differential protein characteristics between freeze-thawed and fresh sperm samples obtained from nine normozoospermic donors. Twenty-seven proteins differed in abundance between the two groups, and results were verified for four proteins via Western blot and immunofluorescent staining. These proteins are putatively involved in sperm motility, viability, acrosomal integrity, ATP and isocitrate content, mitochondrial membrane potential, capacitation, acrosome reaction, and intracellular calcium concentration. These marked differences suggest that dysfunctional spermatozoon after cryopreservation may be due to protein degradation and protein phosphorylation.

5-Lipoxygenase (5-LOX) inhibitors have been shown to be protective in several neurodegenerative disease models; however, the underlying mechanisms remain unclear. We investigated whether 5-LOX inhibitor zileuton conferred direct neuroprotection against glutamate oxidative toxicity by inhibiting ferroptosis, a newly identified iron-dependent programmed cell death. Treatment of HT22 mouse neuronal cell line with glutamate resulted in significant cell death, which was inhibited by zileuton in a dose-dependent manner. Consistently, zileuton decreased glutamate-induced production of reactive oxygen species but did not restore glutamate-induced depletion of glutathione. Moreover, the pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (ZVAD-fmk) neither prevented HT22 cell death induced by glutamate nor affected zileuton protection against glutamate oxidative toxicity, suggesting that zileuton did not confer neuroprotection by inhibiting caspase-dependent apoptosis. Interestingly, glutamate-induced HT22 cell death was significantly inhibited by the ferroptosis inhibitor ferrostatin-1. Moreover, zileuton protected HT22 neuronal cells from erastin-induced ferroptosis. However, we did not observe synergic protective effects of zileuton and ferrostatin-1 on glutamate-induced cell death. These results suggested that both the 5-LOX inhibitor zileuton and the ferropotosis inhibitor ferrostatin-1 acted through the same cascade to protect against glutamate oxidative toxicity. In conclusion, our results suggested that zileuton protected neurons from glutamate-induced oxidative stress at least in part by inhibiting ferroptosis.Key words 5-lipoxygenase inhibitor; neuroprotection; glutamate oxidative toxicity; ferroptosis Glutamate, an important excitatory transmitter in the central nervous system, essentially contributed to the pathogenesis of a variety of neurological diseases. When excessively released, glutamate induces both receptor-dependent excitotoxicity and non-receptor-mediated oxidative toxicity, which are implicated in a number of neurodegeneration diseases, such as stroke.1-6) Thus, targeting glutamate-induced oxidative toxicity is a promising strategy for treating neurological diseases.

Objective. To explore the influence of M2-polarized tumor-associated macrophages (TAMs) on high-risk human papillomavirus (hr-HPV)-related cervical carcinogenesis and metastasis.Methods. CD68+ and CD163+ macrophages were examined immunohistochemically in a series of 130 samples, including 26 cases of normal cervical tissues, 59 cases of cervical intraepithelial neoplasia (CIN), and 45 cases of squamous cell carcinoma (SCC), and the results were statistically analyzed. The macrophage count was corrected for the epithelial and stromal compartments respectively. Clinical data were also obtained.Results. High counts of CD68+ and CD163+ macrophages were associated with hr-HPV infection (both p < 0.05) and positively correlated with cervical carcinogenesis (Spearman's rho = 0.478, p = 0.000; Spearman's rho = 0.676, p =0.000, respectively). The immunostaining pattern of CD163 exhibited clearer background than that of CD68. CD163+ macrophages showed a more obviously increasing migration into the epithelium along with the progression of CIN to invasive cancer. Notably, a high index of CD163+ macrophages was significantly associated with higher FIGO stages (p = 0.009) and lymph node metastasis (p = 0.012), but a similar finding was not found for CD68+ macrophages (p = 0.067, p = 0.079, respectively).Conclusions. Our study supported a critical role of TAMs as a prospective predictor for hr-HPV-related cervical carcinogenesis. CD163, as a promising TAMs marker, is superior to CD68 for predicting the malignant transformation and metastatic potential of cervical cancer.

Aim: To investigate the regulatory effect of microRNA-221 (miR-221) on CDKN1C/p57 expression in colorectal carcinoma (CRC). Methods: Thirty four CRC and adjacent non-tumorous tissue samples were collected individually. Total RNA and protein were isolatedand from these samples and four human CRC-derived cell lines (including HT-29, Lovo, SW-480 and Caco2). MiR-221 expression was examined using real-time RT-PCR. CRC cells were treated with or without anti-p57-siRNA prior to the addition of premiR-221 or anti-miR-221. The mRNA and protein levels of CDKN1C/p57 were examined using semi-quantitative RT-PCR and Western blot, respectively. CRC cell proliferation and apoptosis were assessed using MTT assay and flow cytometry, respectively. The CDKN1C/p57 3′-UTR fragment was amplified using PCR from the genomic DNA of human colon cells and inserted into a luciferase reporter construct. The reporter construct was then transfected into CRC cells together with pre-miR-221 or anti-miR-221, and the luciferase activity in the transfected cells was examined. Results: MiR-221 expression was significantly up-regulated in 90% of CRC samples compared to that in the adjacent non-tumorous tissue, and the expression level was positively correlated to an advanced TNM stage and local invasion. There was no significant difference in CDKN1C/p57 mRNA expression between CRC and corresponding non-tumorous tissues, whereas CDKN1C/p57 protein expression was markedly decreased in the CRC samples. A significant inverse correlation between miR-221 and CDKN1C/p57 expression was found in CRC cells. Moreover, a miR-221-specific inhibitor significantly increased CDKN1C/p57 protein expression in CRC cells. Anti-miR-221 markedly inhibited CRC cell proliferation and induced apoptosis. This inhibitory effect was abolished by pretreatment with anti-p57-siRNA, suggesting that the inhibition was mediated by CDKN1C/p57. A significant increase of the luciferase activity was observed in CRC cells co-transfected with the luciferase reporter construct and anti-miR-221. Conclusion: MiR-221 binds to the target site in the 3′-UTR of the CDKN1C/p57 mRNA to inhibit CDKN1C/p57 expression by posttranscriptional gene silencing to promote CRC occurrence and progress, therefore serving as a potential therapeutic target for the prevention and treatment of CRC.