Secretome analysis of atherosclerotic and non-atherosclerotic arteries reveals dynamic extracellular remodeling during pathogenesis

Aims

Early detection of cardiovascular diseases and knowledge of underlying mechanisms is essential. Tissue secretome studies resemble more closely to the in vivo situation, showing a much narrower protein concentrations dynamic range than plasma. This study was aimed to the analysis of human arterial tissue secretome and to the quantitative comparison of healthy and atherosclerotic secretome to discover proteins with key roles in atherosclerosis development.

Methods and results

Secretomes from three biological replicates of human atherosclerotic coronary arteries (APC), preatherosclerotic coronaries (PC) and mammaries (M) were analyzed by LC-MS/MS. The identified proteins were submitted to Ingenuity Pathway Analysis (IPA) tool. Label-free MS/MS based quantification was performed and validated by immunohistochemistry. 64 proteins were identified in the 3 replicates of at least one of the 3 groups and 15 secreted proteins have not been previously reported in plasma. Four proteins were significantly released in higher amounts by mammary tissue: gelsolin, vinculin, lamin A/C and phosphoglucomutase 5.

Conclusion

The study of tissue secretome reveals key proteins involved in atherosclerosis which have not been previously reported in plasma. Novel proteins are here highlighted which could be potential therapeutic targets in clinical practice. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

The effect of marriage on utilization of colorectal endoscopy exam in the United States

Purpose: This study examines the association between marriage and colorectal endoscopy exam, and whether this association varies by gender and financial benefits of marriage including improved access to health insurance and pooled family income. Methods: Representative survey data of the non-institutionalized United States population were used from the 2000, 2005, and 2008 National Health Interview Survey. Analyses targeted persons 50-85 years of age without a personal history of cancer and with complete information on all study variables (n=21,760). Multivariate logistic regression was used to model marital status differences in the probability of undergoing a colorectal endoscopy exam with interaction effects used to model variation over time by gender, health insurance, and poverty level. Results: Married persons were more likely than unmarried persons to report ever having undergone a colorectal endoscopy exam (odds ratio [OR], 1.21; 95% confidence interval [CI], 1.12-1.29), and the difference between married and unmarried persons in the probability of undergoing a colorectal endoscopy exam remained stable over time. Married persons were more likely than unmarried persons to report having undergone a colorectal endoscopy exam within the past 10 years (OR, 1.49; 95% CI, 1.15-1.95). For each survey year, married men were significantly more likely than women and unmarried men to report having undergone a colorectal endoscopy exam. For example, in 2008, 56% of married men reported having undergone a colorectal endoscopy exam, compared to 49% of unmarried men, 52% of married women, and 50% of unmarried women. Among persons with health insurance, married persons were significantly more likely than unmarried persons to have undergone a colorectal endoscopy exam. Among persons who were poor, there was no difference by marital status in the likelihood of having undergone a colorectal endoscopy exam. However, among persons who were not poor, married persons were more likely than unmarried persons to have undergone a colorectal endoscopy exam. Conclusion: Given that colorectal endoscopy exams are a potentially life-saving procedure, persistently higher uptake of colorectal endoscopy for married persons over time may be an important health promoting benefit of marriage. Therefore, clinicians and policy makers should focus on improving the use of cancer prevention services among unmarried persons.     

BF integrase genes of HIV-1 circulating in São Paulo, Brazil, with a recurrent recombination region

Although some studies have shown diversity in HIV integrase (IN) genes, none has focused particularly on the gene evolving in epidemics in the context of recombination. The IN gene in 157 HIV-1 integrase inhibitor-na?ve patients from the S?o Paulo State, Brazil, were sequenced tallying 128 of subtype B (23 of which were found in non-B genomes), 17 of subtype F (8 of which were found in recombinant genomes), 11 integrases were BF recombinants, and 1 from subtype C. Crucially, we found that 4 BF recombinant viruses shared a recurrent recombination breakpoint region between positions 4900 and 4924 (relative to the HXB2) that includes 2 gRNA loops, where the RT may stutter. Since these recombinants had independent phylogenetic origin, we argue that these results suggest a possible recombination hotspot not observed so far in BF CRF in particular, or in any other HIV-1 CRF in general. Additionally, 40% of the drug-na?ve and 45% of the drug-treated patients had at least 1 raltegravir (RAL) or elvitegravir (EVG) resistance-associated amino acid change, but no major resistance mutations were found, in line with other studies. Importantly, V151I was the most common minor resistance mutation among B, F, and BF IN genes. Most codon sites of the IN genes had higher rates of synonymous substitutions (dS) indicative of a strong negative selection. Nevertheless, several codon sites mainly in the subtype B were found under positive selection. Consequently, we observed a higher genetic diversity in the B portions of the mosaics, possibly due to the more recent introduction of subtype F on top of an ongoing subtype B epidemics and a fast spread of subtype F alleles among the B population.     

A new approach to antiglaucoma drugs: carbonic anhydrase inhibitors with or without NO donating moieties. Mechanism of action and preliminary pharmacology

The clinically used sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor dorzolamide (DRZ), a new sulfonamide CA inhibitor also incorporating NO-donating moieties, NCX250, and isosorbide mononitrate (ISMN) (an NO-donating compound with no CA inhibitory properties) were investigated for their intraocular pressure (IOP) lowering effects in rabbits with carbomer-induced glaucoma. NCX250 was more effective than DRZ or ISMN on lowering IOP, increasing ocular hemodynamics, decreasing the inflammatory processes and ocular apoptosis in this animal model of glaucoma. NO participate to the regulation of IOP in glaucoma, having also antiapoptotic and anti-inflammatory effects. The ophthalmic artery, both systolic and diastolic velocities, were significantly reduced in NCX250-treated eyes in comparison to DRZ treated ones, suggesting thus a beneficial effect of NCX250 on the blood supply to the optic nerve. Combining CA inhibition with NO-donating moieties in the same compound offers an excellent approach for the management of glaucoma.     

Centrally necrotizing breast carcinoma: a rare histological subtype, which was cause of misdiagnosis in an evident clinical local recurrence

ABSTRACT: Centrally necrotizing carcinoma is a rare subtype of breast carcinoma, which is characterized by an extensive central necrotic zone accounting for at least 70% of the cross-sectional area of the neoplasm. This central necrotic zone, in turn, is surrounded by a narrow rim of proliferative viable tumor cells. We report an unusual clinical situation in which a patient whose evident breast mass suggested an ipsilateral local recurrence and for which numerous attempts to confirm the histological diagnosis had failed. The patient was treated with a radical mastectomy based on clinical suspicion of breast cancer recurrence after an undesirable delay. In this case, the narrow rim of viable malignant tissue had a thickness of 0.5 to 8 mm, and the centrally necrotizing carcinoma had a central zone with a predominance of fibrosis. The special features of this case led to a misdiagnosis and to an evident clinical local recurrence.     

Developmental origin of a major difference in sensory patterning between zebrafish and bluefin tuna

The posterior lateral line system (PLL) of teleost fish comprises a number of mechanosensory organs arranged in defined patterns on the body surface. Embryonic patterns are largely conserved among teleosts, yet adult patterns are highly diverse. Although changes in pattern modify the perceptual abilities of the system, their developmental origin remains unknown. Here we compare the processes that underlie the formation of the juvenile PLL pattern in Thunnus thynnus, the bluefin tuna, to the processes that were elucidated in Danio rerio, the zebrafish. In both cases, the embryonic PLL comprises five neuromasts regularly spaced along the horizontal myoseptum, but the juvenile PLL comprises four roughly parallel anteroposterior lines in zebrafish, whereas it is a simple dorsally arched line in tuna fish. We examined whether this difference involves evolutionary novelties, and show that the same mechanisms mediate the transition from embryonic to juvenile patterns in both species. We conclude that the marked difference in juveniles depends on a single change (dorsal vs. ventral migration of neuromasts) in the first days of larval life.     

Polymeric Display of Proteins through High Affinity Leucine Zipper Peptide Adaptors

The polymeric display of proteins is a method that could be used to increase the immunogenicity of antigens and to enhance the interaction strength of binding domains for their target ligands through an avidity effect. However, the coupling of proteins to oligomeric scaffolds is challenging. The chemical conjugation and recombinant fusion techniques have limitations that prevent their general use. In this work we describe a simple and effective method for coupling proteins to the decameric structure of Brucella abortus Lumazine Synthase based on the use of a pair of high affinity heterodimeric coiled coil peptides complementary fused to the scaffold and the target protein. Results obtained with a series of proteins demonstrate the capability of this approach to generate polyvalent particles. Furthermore, we show that the method is able to increase the immunogenicity of antigens and produce polyfunctional particles with promising biomedical and nanotechnological applications.     

Dominant activation of the hedgehog signaling pathway alters development of the female reproductive tract

The role of hedgehog (HH) signaling in reproductive tract development was studied in mice in which a dominant active allele of the signal transducer smoothened (SmoM2) was conditionally expressed in the Müllerian duct and ovary. Mutant females are infertile, primarily because they fail to ovulate. Levels of mRNA for targets of HH signaling, Gli1, Ptch1, and Hhip, were elevated in reproductive tracts of 24-day-old mutant mice, confirming overactivation of HH signaling. The tracts of mutant mice developed abnormally. The uterine luminal epithelium had a simple columnar morphology in control mice, but in mutants contained stratified squamous cells typical of the cervix and vagina. In mutant mice, the number of uterine glands were reduced and the oviducts were not coiled. Expression of genes within the Hox and Wnt families that regulate patterning of the reproductive tract were altered. Hoxa13, which is normally expressed primarily in the vagina and cervix, was expressed at 12-fold higher levels in the uterus of mutant mice compared with controls. Wnt5a, which is required for development of the cervix and vagina and postnatal differentiation of the uterus, was expressed at higher levels in the oviduct and uterus of mutant mice compared with controls. Mating mutant females with fertile or vasectomized males induced a severe inflammatory response in the tract. In summary, overactivation of HH signaling causes aberrant development of the reproductive tract. The phenotype observed could be mediated by ectopic expression of Hoxa13 in the uterus and elevated levels of Wnt5a in the oviducts and uterus.     

Improvement of tablet coating uniformity using a quality by design approach

A combination of analytical and statistical methods is used to improve a tablet coating process guided by quality by design (QbD) principles. A solid dosage form product was found to intermittently exhibit bad taste. A suspected cause was the variability in coating thickness which could lead to the subject tasting the active ingredient in some tablets. A number of samples were analyzed using a laser-induced breakdown spectroscopy (LIBS)-based analytical method, and it was found that the main variability component was the tablet-to-tablet variability within a lot. Hence, it was inferred that the coating process (performed in a perforated rotating pan) required optimization. A set of designed experiments along with response surface modeling and kriging method were used to arrive at an optimal set of operating conditions. Effects of the amount of coating imparted, spray rate, pan rotation speed, and spray temperature were characterized. The results were quantified in terms of the relative standard deviation of tablet-averaged LIBS score and a coating variability index which was the ratio of the standard deviation of the tablet-averaged LIBS score and the weight gain of the tablets. The data-driven models developed based on the designed experiments predicted that the minimum value of this index would be obtained for a 6% weight gain for a pan operating at the highest speed at the maximum fill level while using the lowest spraying rate and temperature from the chosen parametric space. This systematic application of the QbD-based method resulted in an enhanced process understanding and reducing the coating variability by more than half.     

Can Cone Scales and Bark of Aleppo Pine Be Used in the Biomonitoring of Some Heavy Metals?

The highest levels of Pb in soils and needles were detected in the Lisbon Botanical Garden, reflecting the intense car circulation since the end of the late nineteenth century, and the presence of tetraethyl lead in petrol. Levels of Pb in Aleppo bark were at or above the threshold values for plant tissues as defined by the UN-ECE/EC (>4–30 μg/g). Nickel was easily washed by distilled water from the bark, indicating a predominant adsorption due to atmospheric deposition, while only Pb was water-extracted from cone scales, but no more than 0.88 μg/g (4.3% of the total). If we take into account the dry weight of each cone, we can assess the amount of extractable lead present in a given time (ranges between 3.0 and 15.5 μg), although its eventual transfer to the ground through rainfall will be hampered by the cone architecture. The average levels of total Pb in the cones (12.7–35.4 μg/g) fall within the range defined by the UN-ECE/EC, generally; only the reference value is below 4 μg/g. Due to the high capabilities of allocating Pb inside the structure and through adsorption, Aleppo bark and cones can be very interesting tools in the monitoring of Pb.