Epigenetic regulation of REG1A and chemosensitivity of cutaneous melanoma

Regenerating gene 1A (REG1A) plays an important role in tissue regeneration and in cell proliferation in epithelium origin tumors; however, its role in melanoma has not been explored in details. The objective of this study was to identify whether REG1A is expressed in cutaneous melanoma and if REG1A expression status can predict prognosis in cutaneous melanoma patients with metastasis. We also determined whether epigenetic regulation of the promoter region regulates REG1A expression. AJCC stage III cutaneous melanoma specimens with clinically well annotated stage III lymph node melanoma metastasis tissue microarray were assessed by IHC. MALDI-TOF-mass spectrometry and HM450K array were used to identify REG1A promoter region CpG site methylation. Chemotherapeutic agent response by melanoma cells as related to REG1A protein expression was assessed. Post-surgery melanoma patients followed by adjuvant chemotherapy with high REG1A expression had a significantly better prognosis (disease-specific survival) compared with patients with low REG1A expression (log rank test; p = 0.0013). The demethylating reagent 5-Aza-2′-deoxycytidine activated REG1A promoter region resulting in enhanced REG1A mRNA and protein expression in melanoma cell lines. Promoter region CpG methylation was shown to regulate REG1A expression in melanoma cells. Moreover, melanoma lines with high REG1A mRNA expression were more susceptible to Dacarbazine and Cisplatin, as compared with those with low REG1A mRNA expression. In conclusion, REG1A expression status may be useful as a biomarker in melanoma patients for sensitivity to these chemotherapeutic agents. The epigenetic regulation of the REG1A promoter region may offer a potential therapeutic approach to improve chemotherapy for metastatic melanoma patients.     

Rilonacept in the treatment of acute gouty arthritis: a randomized,controlled clinical trial using indomethacin as the active comparator

Introduction

In phase-3 clinical trials, the interleukin (IL-1) blocker, rilonacept (IL-1 Trap), demonstrated efficacy for gout flare prevention during initiation of urate-lowering therapy. This trial evaluated rilonacept added to a standard-of-care, indomethacin, for treatment of acute gout flares.

Methods

Adults, aged 18-70 years, with gout presenting within 48 hours of flare onset and having at least moderate pain as well as swelling and tenderness in the index joint were randomized to subcutaneous (SC) rilonacept 320 mg at baseline plus oral indomethacin 50 mg TID for 3 days followed by 25 mg TID for up to 9 days (n = 74); SC placebo at baseline plus oral indomethacin as above (n = 76); or SC rilonacept 320 mg at baseline plus oral placebo (n = 75). The primary efficacy endpoint was change in pain in the index joint (patient-reported using a Likert scale (0 = none; 4 = extreme)) from baseline to the average of values at 24, 48 and 72 hours (composite time point) for rilonacept plus indomethacin versus indomethacin alone. Comparison of rilonacept monotherapy with indomethacin monotherapy was dependent on demonstration of significance for the primary endpoint. Safety evaluation included clinical laboratory and adverse event (AE) assessments.

Results

Patient characteristics were comparable among the groups; the population was predominantly male (94.1%), white (75.7%), with mean ± SD age of 50.3 ± 10.6 years. All treatment groups reported within-group pain reductions from baseline (P < 0.0001). Although primary endpoint pain reduction was greater with rilonacept plus indomethacin (-1.55 ± 0.92) relative to indomethacin alone (-1.40 ± 0.96), the difference was not statistically significant (P = 0.33), so formal comparison between monotherapy groups was not performed. Pain reduction over the 72-hour period with rilonacept alone (-0.69 ± 0.97) was less than that in the other groups, but pain reduction was similar among groups at 72 hours. Treatment with rilonacept was well-tolerated with no reported serious AEs related to rilonacept. Across all groups, the most frequent AEs were headache and dizziness.

Conclusions

Although generally well-tolerated, rilonacept in combination with indomethacin and rilonacept alone did not provide additional pain relief over 72 hours relative to indomethacin alone in patients with acute gout flare.

Trial registration

ClinicalTrials.gov registration number {"type":"clinical-trial","attrs":{"text":"NCT00855920","term_id":"NCT00855920"}}NCT00855920.     

Mitochondrial function in response to cardiac ischemia-reperfusion after oral treatment with quercetin

Polyphenolic compounds present in red wines, such as the flavonol quercetin, are thought capable of cardioprotection through mechanisms not yet clearly defined. It has been established that mitochondria play a critical role in myocardial recovery from ischemia-reperfusion (I-R) damage, and in vitro experiments indicate that quercetin can exert a variety of direct effects on mitochondrial function. The effects of quercetin at concentrations typically found in 1-2 glasses of red wine on cardiac I-R and mitochondrial function in vivo are not known. Quercetin was administered to rats (0.033 mg/kg per day by gavage for 4 d). Isolated Langendorff perfused hearts were subjected to I-R, and cardiac functional parameters determined both before and after I-R. Mitochondria were isolated from post-I-R hearts and their function assessed. Compared to an untreated control group, quercetin treatment significantly decreased the impairment of cardiac function following I-R. This protective effect was associated with improved mitochondrial function after I-R. These results indicate that oral low dose quercetin is cardioprotective, possibly via a mechanism involving protection of mitochondrial function during I-R.     

COPI–TRAPPII activates Rab18 and regulates its lipid droplet association

The transport protein particle (TRAPP) was initially identified as a vesicle tethering factor in yeast and as a guanine nucleotide exchange factor (GEF) for Ypt1/Rab1. In mammals, structures and functions of various TRAPP complexes are beginning to be understood. We found that mammalian TRAPPII was a GEF for both Rab18 and Rab1. Inactivation of TRAPPII‐specific subunits by various methods including siRNA depletion and CRISPR–Cas9‐mediated deletion reduced lipolysis and resulted in aberrantly large lipid droplets. Recruitment of Rab18 onto lipid droplet (LD) surface was defective in TRAPPII‐deleted cells, but the localization of Rab1 on Golgi was not affected. COPI regulates LD homeostasis. We found that the previously documented interaction between TRAPPII and COPI was also required for the recruitment of Rab18 to the LD. We hypothesize that the interaction between COPI and TRAPPII helps bring TRAPPII onto LD surface, and TRAPPII, in turn, activates Rab18 and recruits it on the LD surface to facilitate its functions in LD homeostasis.     

Evidence for independent recruitment of zeta-crystallin/quinone reductase (CRYZ) as a crystallin in camelids and hystricomorph rodents

Zeta-crystallin/quinone reductase (CRYZ) is an NADPH oxidoreductase expressed at very high levels in the lenses of two groups of mammals: camelids and some hystricomorph rodents. It is also expressed at very low levels in all other species tested. Comparative analysis of the mechanisms mediating the high expression of this enzyme/crystallin in the lens of the Ilama (Lama guanacoe) and the guinea pig (Cavia porcellus) provided evidence for independent recruitment of this enzyme as a lens crystallin in both species and allowed us to elucidate for the first time the mechanism of lens recruitment of an enzyme- crystallin. The data presented here show that in both species such recruitment most likely occurred through the generation of new lens promoters from nonfunctional intron sequences by the accumulation of point mutations and/or small deletions and insertions. These results further support the idea that recruitment of CRYZ resulted from an adaptive process in which the high expression of CRYZ in the lens provides some selective advantage rather than from a purely neutral evolutionary process.      

Evidence of independent gene duplications during the evolution of archaeal and eukaryotic family B DNA polymerases

Eukaryotes and archaea both possess multiple genes coding for family B DNA polymerases. In animals and fungi, three family B DNA polymerases, alpha, delta, and epsilon, are responsible for replication of nuclear DNA. We used a PCR-based approach to amplify and sequence phylogenetically conserved regions of these three DNA polymerases from Giardia intestinalis and Trichomonas vaginalis, representatives of early-diverging eukaryotic lineages. Phylogenetic analysis of eukaryotic and archaeal paralogs suggests that the gene duplications that gave rise to the three replicative paralogs occurred before the divergence of the earliest eukaryotic lineages, and that all eukaryotes are likely to possess these paralogs. One eukaryotic paralog, epsilon, consistently branches within archaeal sequences to the exclusion of other eukaryotic paralogs, suggesting that an epsilon-like family B DNA polymerase was ancestral to both archaea and eukaryotes. Because crenarchaeote and euryarchaeote paralogs do not form monophyletic groups in phylogenetic analysis, it is possible that archaeal family B paralogs themselves evolved by a series of gene duplications independent of the gene duplications that gave rise to eukaryotic paralogs.      

Evidence of cardiovascular protection by moderate alcohol: role of nitric oxide

Epidemiological evidence indicates that moderate alcohol consumption reduces the incidence of heart disease. Endothelial nitric oxide synthase (eNOS) is a key regulator of vascular homeostasis and myocardial functions through the controlled production of nitric oxide (*NO). These studies were conducted to determine if the apparent alcohol-associated cardioprotection is mediated, in part, through modulation of the eNOS protein and activity in the cardiovascular system. Rats were fed alcohol and eNOS protein and *NO production were evaluated at the end of 8 weeks. Myocardial and vascular function was assessed ex vivo in a subset of animals. Moderate alcohol improved postischemic myocardial systolic and diastolic function and attenuated the postischemic reduction in coronary vascular resistance. Moderate alcohol also enhanced maximum vascular relaxation by 26 +/- 0.2% and increased plasma *NO production concomitant with a greater than 2.5-fold increase in eNOS protein. Higher levels of alcohol impaired maximum vascular relaxation by 22 +/- 0.1%. These results suggest that moderate alcohol improves postischemic myocardial functions and increases *NO production by vascular endothelium. An increase in *NO may explain, at least in part, the cardioprotective benefits of moderate alcohol consumption.     

Variations in lymphokine generation by individual lymph nodes draining human malignant tumors

Summary Individual lymph nodes draining tumors vary in their degree of immunological activity. Cell suspensions from tumor-free nodes located relatively near to tumors are spontaneously less reactive and respond poorly to exogenous stimulation by mitogens and lymphokines. Diminished spontaneous uptake of tritiated thymidine by lymph node cells not exposed to exogenous stimulation suggests that tumor-proximate immune suppression exists in vivo and is not purely a laboratory artefact. The present study was undertaken to explore that possibility further. Fluid in which cell suspensions from tumor-free nodes were prepared, and supernatants from short-term cultures of nodes located at different distances from tumors were compared for their capacity to inhibit the in vitro migration of the human lymphoblastoid cell line QIMR-WIL. Inhibitory activity of fluids from individual nodes was related to their position relative to the tumor and their immune competence, assessed by the responses to mitogens of cell suspensions prepared from them. Cell suspension fluids from 92/111 nodes (83%) significantly inhibited the migration of QIMR-WIL, at a level similar (44±14%) to that induced by the supernatants of mixed lymphocyte cultures (43±17%). Fluids from the nodes of melanoma patients were more inhibitory than those from breast cancer patients (49±12% and 37±13%, respectively,P = 0.003). The inhibitory activity of the different nodes of individual node groups varied significantly in 25 of 33 patients (76%), the node nearest the tumor generating least inhibitory activity (indexing the greatest immune suppression) in 20 of these 25 patients (80%). The strength of migration-inhibitory activity was concordant with the responsiveness to mitogen stimulation in up to 14 of 18 patients (78%). Studies of molecular size and heat stability indicated that the inhibitory factors had characteristics consistent with common migration-inhibitory lymphokines such as leukocyte-migration-inhibitory factor, macrophage-inhibitory factor and interleukin-2. Our findings further support the hypothesis that lymph nodes nearest to tumors are relatively immune-suppressed in vivo.Supported by grants CA 29938 and CA 43658, awarded by the National Cancer Institute, DHHS and a grant from the Candle Foundation, Los Angeles     

Mitochondrial dysfunction in cardiac ischemia-reperfusion injury: ROS from complex I, without inhibition

A key pathologic event in cardiac ischemia reperfusion (I-R) injury is mitochondrial energetic dysfunction, and several studies have attributed this to complex I (CxI) inhibition. In isolated perfused rat hearts, following I-R, we found that CxI-linked respiration was inhibited, but isolated CxI enzymatic activity was not. Using the mitochondrial thiol probe iodobutyl-triphenylphosphonium in conjunction with proteomic tools, thiol modifications were identified in several subunits of the matrix-facing 1alpha sub-complex of CxI. These thiol modifications were accompanied by enhanced ROS generation from CxI, but not complex III. Implications for the pathology of cardiac I-R injury are discussed.