Novel regulation of the transcription factor ZHX2 by N-terminal methylation.

N-terminal methylation (Nα-methylation) by the methyltransferase NRMT1 is an important post-translational modification that regulates protein-DNA interactions. Accordingly, its loss impairs functions that are reliant on such interactions, including DNA repair and transcriptional regulation. The global loss of Nα-methylation results in severe developmental and premature aging phenotypes, but given over 300 predicted substrates, it is hard to discern which physiological substrates contribute to each phenotype. One of the most striking phenotypes in NRMT1 knockout (Nrmt1-/-) mice is early liver degeneration. To identify the disrupted signaling pathways leading to this phenotype and the NRMT1 substrates involved, we performed RNA-sequencing analysis of control and Nrmt1-/- adult mouse livers. We found both a significant upregulation of transcripts in the cytochrome P450 (CYP) family and downregulation of transcripts in the major urinary protein (MUP) family. Interestingly, transcription of both families is inversely regulated by the transcription factor zinc fingers and homeoboxes 2 (ZHX2). ZHX2 contains a non-canonical NRMT1 consensus sequence, indicating that its function could be directly regulated by Nα-methylation. We confirmed misregulation of CYP and MUP mRNA and protein levels in Nrmt1-/- livers and verified NRMT1 can methylate ZHX2 in vitro. In addition, we used a mutant of ZHX2 that cannot be methylated to directly demonstrate Nα-methylation promotes ZHX2 transcription factor activity and target promoter occupancy. Finally, we show Nrmt1-/- mice also exhibit early postnatal de-repression of ZHX2 targets involved in fetal liver development. Taken together, these data implicate ZHX2 misregulation as a driving force behind the liver phenotype seen in Nrmt1-/- mice.

Performance Characteristics of Photoacoustic Imaging Probes with Varying Frequencies and Light-delivery Schemes.

Photoacoustic imaging (PAI) is an emerging biomedical imaging technique that utilizes a combination of light and ultrasound to detect photoabsorbers embedded within tissues. While the clinical utility of PAI has been widely explored for several applications, limitations in light penetration and detector sensitivity have restricted these studies to mostly superficial sites. Given the importance of PA signal generation and detection on light delivery and ultrasound detector frequency, there is an ongoing effort to optimize these parameters to enhance photoabsorber detection at increased depths. With this in mind, in this study we examined performance benchmarks of a commercially available PAI/ultrasound linear array system when using different imaging frequencies and light delivery schemes. A modified light fiber jacket providing focused light delivery (FLD) at the center of the probe was compared with the built-in fiber optics lining the length of the probe. Studies were performed in vitro to compare performance characteristics such as imaging resolution, maximum imaging depth, and sensitivity to varying hematocrit concentration for each frequency and light delivery method. Monte Carlo simulations of each light delivery method revealed increased light penetration with FLD. In tissue-mimicking phantoms, vascular channels used to simulate blood vessels could be visualized at a depth of 2.4 cm when lowering imaging frequency and utilizing FLD. Imaging at lower frequencies with FLD also enabled enhanced detection of varying hematocrit concentration levels at increased depths, although lateral imaging resolution was reduced. Finally, a proof of concept in vivo probe comparison study in a mouse tumor model provided supportive evidence of our in vitro results. Collectively, our findings show that adjusting imaging frequency and applying FLD can be a straightforward approach for improving PAI performance.

Theophylline action on primary human bronchial epithelial cells under proinflammatory stimuli and steroidal drugs: a therapeutic rationale approach.

Theophylline is a natural compound present in tea. Because of its property to relax smooth muscle it is used in pharmacology for the treatment of airway diseases (ie, chronic obstructive pulmonary disease, asthma). However, this effect on smooth muscle is dose dependent and it is related to the development of side effects. Recently, an increasing body of evidence suggests that theophylline, at low concentrations, also has anti-inflammatory effects related to the activation of histone deacetylases. In this study, we evaluated the effects of theophylline alone and in combination with corticosteroids on human bronchial epithelial cells under inflammatory stimuli. Theophylline administrated alone was not able to reduce growth-stimulating signaling via extracellular signal-regulated kinases activation and matrix metalloproteases release, whereas it strongly counteracts this biochemical behavior when administered in the presence of corticosteroids. These data provide scientific evidence for supporting the rationale for the pharmacological use of theophylline and corticosteroid combined drug.

Effects of simvastatin on cell viability and proinflammatory pathways in lung adenocarcinoma cells exposed to hydrogen peroxide.

Lung cancer is characterized by a high mortality rate probably attributable to early metastasis. Oxidative stress is involved in development and progression of lung cancer, through cellular and molecular mechanisms which at least in part overlap with proinflammatory pathways. Simvastatin is a statin with pleiotropic effects that can also act as an anti-oxidant agent, and these pharmacologic properties may contribute to its potential anti-cancer activity. Therefore, the aim of this study was to evaluate, in the human lung adenocarcinoma cell line GLC-82, the effects of a 24-hour treatment with simvastatin on hydrogen peroxide (H2O2)-induced changes in cell viability, ERK phosphorylation, matrix metalloproteinase (MMP) expression, innate immunity signaling, NF-κB activation and IL-8 secretion. Cell counting was performed after trypan blue staining, cell proliferation was assessed using MTT assay, and apoptosis was evaluated through caspase-3 activation and Tunel assay. Western blotting was used to analyze protein extracts, and IL-8 release into cell culture supernatants was assessed by ELISA. Our results show that simvastatin (30 µM) significantly (P <0.01) inhibited the proliferative effect of H2O2 (0.5 mM) and its stimulatory actions on ERK1/2 phosphorylation, NF-κB activation and IL-8 production. Furthermore, simvastatin decreased H2O2-mediated induction of the cellular expression of MMP-2 and MMP-9, as well as of several components of the signaling complex activated by innate immune responses, including MyD88, TRAF2, TRAF6 and TRADD. In conclusion, these findings suggest that simvastatin could play a role in prevention and treatment of lung cancer via modulation of important proinflammatory and tumorigenic events promoted by oxidative stress.

Assessment of pre-analytical and analytical variables affecting peptidome profiling of gingival crevicular fluid by MALDI-TOF mass spectrometry.

BACKGROUND: Gingival crevicular fluid (GCF) may be a source of new biomarkers of periodontitis/gingivitis. However, the minute volumes of GCF harvested in healthy sites are a serious drawback. We evaluated how pre-analytical and analytical variables concerning GCF collection and processing, could significantly influence quality and reproducibility of MALDI-TOF profiles. METHODS: GCF was collected from healthy sites. The use of paper strips vs paper points was compared. Peptides and proteins were extracted by centrifugal elution in different solutions, at different accelerations, with and without protease inhibitor cocktail (PIC). Finally, we evaluated how matrix composition and matrix/sample volume ratio affect the reproducibility of MALDI-TOF profiles. RESULTS: Trifluoroacetic acid elution generated richer gingival fingerprints compared to acetic acid, independently of the collection device. Centrifugation speed and PIC supplementation did not change GCF profiles. A fine modulation of matrix composition and matrix/sample volume ratio resulted in a satisfactory reproducibility (CV less than 10% for peak area and signal-to-noise ratio). CONCLUSION: An optimized procedure, enabling generation of reproducible MALDI-TOF profiles from limited volume of GCF, is proposed. These fingerprints may serve as reference for future studies oriented to the maintenance and preservation of good gingival status and to discovery biomarkers of periodontitis/gingivitis.

Effects of acetaminophen and ibuprofen in children with migraine receiving preventive treatment with magnesium.

AIM: The purpose of this study was to evaluate both the effects of ibuprofen and/or acetaminophen for the acute treatment of primary migraine in children in or out prophylactic treatment with magnesium. METHODS: Children ranging from the ages of 5 to 16 years with at least 4 attack/month of primary migraine were eligible for participation the study. A visual analog scale was used to evaluate pain intensity at the moment of admission to the study (start of the study) and every month up to 18 months later (end of the study). RESULTS: One hundred sixty children of both sexes aged 5-16 years were enrolled and assigned in 4 groups to receive a treatment with acetaminophen or ibuprofen without or with magnesium. Migraine pain endurance and monthly frequency were similar in the 4 groups. Both acetaminophen and ibuprofen induced a significant decrease in pain intensity (P < .01), without a time-dependent correlation, but did not modify its frequency. Magnesium pretreatment induced a significant decrease in pain intensity (P < .01) without a time-dependent correlation in both acetaminophen- and ibuprofen-treated children and also significantly reduced (P < .01) the pain relief timing during acetaminophen but not during ibuprofen treatment (P < .01). In both acetaminophen and ibuprofen groups, magnesium pretreatment significantly reduced the pain frequency (P < .01). CONCLUSIONS: Magnesium increased the efficacy of ibuprofen and acetaminophen with not age-related effects.

Effects of ambrisentan in a patient affected by combined pulmonary fibrosis and emphysema and by severe pulmonary hypertension: clinical, functional, and biomolecular findings.

Combined pulmonary fibrosis and emphysema (CPFE) is a computed tomography (CT)-defined syndrome of combined pulmonary fibrosis and emphysema, characterized by subnormal spirometry, impairment of gas exchange, and high prevalence of pulmonary hypertension. Although endothelin-1 (ET-1) plays an important role in the development of lung fibrosis as well as in pulmonary hypertension, no ET-1-targeted therapy is currently recommended. Here we report a case of CPFE successfully treated with ambrisentan, an endothelin-A receptor antagonist, and also discuss the biologic mechanisms underlying the observed therapeutic effects. A 79-year-old man with chronic obstructive pulmonary disease (COPD) was referred to our respiratory unit as an outpatient for dyspnea. Clinical, radiologic, and laboratory findings suggested a diagnosis of chronic hypoxemic, type 1 respiratory failure, due to combined pulmonary fibrosis and emphysema, complicated by severe, precapillary pulmonary hypertension. Pharmacologic treatment with ambrisentan induced an initial improvement in clinical symptoms that proved to be very relevant 9 months later. In order to investigate the biologic mechanisms underlying the clinical effects of ambrisentan, we performed an "in vitro" study on primary cultures of fibrotic human lung fibroblasts, as well as on human umbilical vein endothelial cells, incubated for 24 and 48 h with ET-1, in the absence or presence of an overnight treatment with ambrisentan. ET-1 significantly increased cell proliferation and mitogen-activated protein kinase activation (P < 0.01). These effects were significantly (P < 0.01) inhibited by ambrisentan in both cell cultures. In conclusion, we hypothesize that the clinical benefits induced by ambrisentan in this patient with CPFE can be attributed to its vasodilator and anti-proliferative actions, exerted on pulmonary the vascular bed and lung fibroblasts.

Chronic venous leg ulcers are associated with high levels of metalloproteinases-9 and neutrophil gelatinase-associated lipocalin.

Venous ulcers are related to dysfunctions in extracellular matrix. Both matrix metalloproteinases (MMP) and neutrophil gelatinase-associated lipocalin (NGAL) could play a role in the healing process in patients with chronic venous ulcers. We evaluated the role of MMP-9 and NGAL in the healing process in venous ulceration. We performed an open-label, parallel groups, single clinical center study. Patients with chronic venous leg ulcers represented the test group (Group I), whereas patients without chronic ulcers represented the control group (Group II). In Group I plasma and wound fluid samples were collected at the time of admission, at the time of the surgery, and at the follow-up, while ulcer tissues were taken at the time of the surgery. In Group II, plasma and wound fluid were collected at admission and at the time of the surgery, whereas skin tissues were collected at the time of the surgery. Enzyme-linked immunosorbent assay test was used to evaluate the levels of MMP-9 and NGAL in plasma and wound fluid, whereas Western blot analysis was performed to estimate the expression of MMP-9 and NGAL in tissues. Enzyme-linked immunosorbent assay tests revealed significantly higher levels of MMP-9 and NGAL in both plasma and wound fluid of patients with ulcers compared to patients without ulcers (p < 0.01). Moreover, Western blot analysis documented an increased expression of MMP-9 and NGAL in biopsy tissue of patients with ulcers compared to patients without ulcers (p < 0.01). In conclusion MMP-9 and NGAL may correlate with the clinical course of venous ulcers.

Derivatized mesoporous silica beads for MALDI-TOF MS profiling of human plasma and urine.

Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a promising tool for large-scale screening of body fluids for the early detection of human diseases. Proteins, peptides, and metabolites present in cells, tissues, or in body fluids constitute the molecular signatures of individuals. The design and generation of material-based platforms for capturing molecular signatures from body fluids has gained increasing interest in recent years. Highly selective materials are attractive candidates for a wide range of applications in biofluid proteomics. We have therefore developed a procedure based on mesoporous silica particles for the selective binding and enrichment of low molecular weight plasma/serum proteins by MALDI MS analysis ( Terracciano, R., Gaspari, M., Testa, F., Pasqua, L., Cuda G., Tagliaferri, P., Cheng, M. C., Nijdam, A. J., Petricoin, E. F., Liotta, L. A., Ferrari, M., and Venuta, S. ( 2006 ) Selective binding and enrichment for low-molecular weight biomarker molecules in human plasma after exposure to nanoporous silica particles . Proteomics 6, 3243-3250 ). Mesoporous silica beads (MSB) are able to harvest peptides from plasma and serum by means of nanosized porous channels with high surface area, while excluding large size proteins. Moreover, the absorption properties can be modified since the pore walls can be functionalized with different chemical species due to the high concentration of silanol groups at the surface. In this study, we performed derivatization of MSB with different functionalities, and we evaluated the derivatized materials for plasma and urine peptidomic profiling. Aminopropyl, N-(2-aminoethyl)-3-aminopropyl, and N,N,N' tris-carboxymethyl ethylene diamine, have been introduced onto the mesoporous silica surfaces in order to modulate selective peptide enrichment. We also explored various experimental conditions in order to optimize the performance of chemically modified MSB in the peptide profiling of human plasma and urine. These new derivatized mesoporous surfaces, in addition to the previous nonderivatized MSB, constitute an extended and reliable platform of five distinct chromatographic phases with defined surface functionality and porosity. Several plasma and urine peptides were extracted from derivatized MSB and then profiled by MALDI-TOF MS. The reproducibility of sample preparation by different functionalized beads was evaluated via three replicate analyses of plasma and urine samples. Lower coefficients of variation in the mass values and peak intensities resulted for plasma in comparison to those of urine samples; nevertheless, these where satisfactory for diagnostic purposes. For human urine, a linear correlation was found between spiked peptide concentrations and their peak areas (R(2) > 0.98) with a limit of detection in the low-nanogram per milliliter range, thus confirming the high sensitivity of the methodology, previously demonstrated for human plasma. Different panels of peptide repertoires have thus been collected from highly porous substrates chemically conjugated with different functional groups, and these may be used in biomarker discovery for disease diagnosis.