SNP analysis of the inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) gene by a fluorescence-adapted SSCP method

BACKGROUND: Single-nucleotide polymorphisms (SNPs) are considered to be useful polymorphic markers for genetic studies of polygenic traits. Single-stranded conformational polymorphism (SSCP) analysis has been widely applied to detect SNPs, including point mutations in cancer and congenital diseases. In this study, we describe an application of the fluorescent labeling of PCR fragments using a fluorescent-adapted primer for SSCP analysis as a novel method. METHODS: Single-nucleotide polymorphisms (SNPs) of the inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) gene were analyzed using a fluorescence-adapted SSCP method. The method was constructed from two procedures: 1) a fluorescent labeling reaction of PCR fragments using fluorescence-adapted primers in a single tube, and 2) electrophoresis on a non-denaturing polyacrylamide gel. RESULTS: This method was more economical and convenient than the single-stranded conformational polymorphism (SSCP) methods previously reported in the detection of the labeled fragments obtained. In this study, eight SNPs of the IHRP gene were detected by the fluorescence-adapted SSCP. One of the SNPs was a new SNP resulting in an amino acid substitution, while the other SNPs have already been reported in the public databases. Six SNPs of the IHRP were associated with two haplotypes. CONCLUSIONS: The fluorescence-adapted SSCP was useful for detecting and genotyping SNPs

Methodology for evaluating thermal track buckling in dual gauge tracks with continuous welded rail

In the National Spanish railway network, two types of track gauge with continuous welded rails are currently in use: the Iberian wide gauge (1668 mm) and the standard gauge (1435 mm). In order to improve links and freight traffic between different lines and with the rest of Europe, a dual gauge track with three rails was developed. This solution modifies the classical track configuration, so it is necessary to develop new methodologies and studies to understand its behavior. Among other loads applied on a continuous welded rail track, a considerable rise in temperature induces compressive stresses in the three rails that can lead to lateral track buckling. Moreover, on dual gauge tracks, the addition of the third rail increases the axial compression, which may lead to track instability. For this reason, a three-dimensional continuous welded rail model is developed in this study to be used for dual gauge track buckling analysis on straight tracks subjected to temperature load. The continuous welded rail dual gauge track model consists of beam, solid and spring elements, in which a non-linear behaviour of the ballast is considered. The results obtained may be used to predict the buckling capacity of the continuous welded rail on dual gauge tracks with respect to different parameters such as lateral resist-ance, lateral imperfections, sleeper spacing or torsional stiffness.Villalba Sanchis, I.; Insa Franco, R.; Salvador Zuriaga, P.; Martínez Fernández, P. (2017). Methodology for evaluating thermal track buckling in dual gauge tracks with continuous welded rail. Proceedings of the Institution of Mechanical Engineers, Part F: Journal of Rail and Rapid Transit. 231(3):269-279. doi:10.1177/0954409715626957S269279231

Fibronectin-Containing Extracellular Vesicles Protect Melanocytes against Ultraviolet Radiation-Induced Cytotoxicity.

Skin melanocytes are activated by exposure to UV radiation to secrete melanin-containing melanosomes to protect the skin from UV-induced damage. Despite the continuous renewal of the epidermis, the turnover rate of melanocytes is very slow, and they survive for long periods. However, the mechanisms underlying the survival of melanocytes exposed to UV radiation are not known. Here, we investigated the role of melanocyte-derived extracellular vesicles in melanocyte survival. Network analysis of the melanocyte extracellular vesicle proteome identified the extracellular matrix component fibronectin at a central node, and the release of fibronectin-containing extracellular vesicles was increased after exposure of melanocytes to UVB radiation. Using an anti-fibronectin neutralizing antibody and specific inhibitors of extracellular vesicle secretion, we demonstrated that extracellular vesicles enriched in fibronectin were involved in melanocyte survival after UVB radiation. Furthermore, we observed that in the hyperpigmented lesions of patients with melasma, the extracellular space around melanocytes contained more fibronectin compared with normal skin, suggesting that fibronectin is involved in maintaining melanocytes in pathological conditions. Collectively, our findings suggest that melanocytes secrete fibronectin-containing extracellular vesicles to increase their survival after UVB radiation. These data provide important insight into how constantly stimulated melanocytes can be maintained in pathological conditions such as melasma.1166Ysciescopu

Calcified multilocular thymic cyst associated with thymoma: a case report

<p>Abstract</p> <p>Introduction</p> <p>There are few case reports of thymoma with a thymic cyst. Such an association renders it difficult for any pathologist to differentiate from other neoplasms, such as a cystic thymoma.</p> <p>Case presentation</p> <p>A 50-year-old Berber woman from Morocco was admitted with a chronic cough of more than 10 years duration. Her medical history and physical examination were normal. Anterior chest radiography demonstrated a calcified opacity in her right anterior mediastinum. A chest-computed tomogram revealed a round cystic tumor, with significant calcification in her right anterior mediastinum. A surgical exploration was performed. The tumor seemed to be a well-encapsulated and totally calcified lesion, arising from the right lobe of her thymus. It was removed by partial resection of her thymus. Through histology, the calcified tumor exhibited some areas of multilocular fibrous-wall cysts. These cysts were partially lined by small cuboidal cells with severe chronic inflammation and an AB thymoma that arose from the wall of the cyst.</p> <p>Conclusion</p> <p>Greater attention should be given to multilocular thymic cysts, to exclude the possibility of neoplasm, especially when the cyst wall is thickened.</p

Spread of Mutant Middle East Respiratory Syndrome Coronavirus with Reduced Affinity to Human CD26 during the South Korean Outbreak

The newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes a severe respiratory infection with a high mortality rate (similar to 35%). MERS-CoV has been a global threat due to continuous outbreaks in the Arabian peninsula and international spread by infected travelers since 2012. From May to July 2015, a large outbreak initiated by an infected traveler from the Arabian peninsula swept South Korea and resulted in 186 confirmed cases with 38 deaths (case fatality rate, 20.4%). Here, we show the rapid emergence and spread of a mutant MERS-CoV with reduced affinity to the human CD26 receptor during the South Korean outbreak. We isolated 13 new viral genomes from 14 infected patients treated at a hospital and found that 12 of these genomes possess a point mutation in the receptor-binding domain (RBD) of viral spike (S) protein. Specifically, 11 of these genomes have an I529T mutation in RBD, and 1 has a D510G mutation. Strikingly, both mutations result in reduced affinity of RBD to human CD26 compared to wild-type RBD, as measured by surface plasmon resonance analysis and cellular binding assay. Additionally, pseudotyped virus bearing an I529T mutation in S protein showed reduced entry into host cells compared to virus with wild-type S protein. These unexpected findings suggest that MERS-CoV adaptation during human-to-human spread may be driven by host immunological pressure such as neutralizing antibodies, resulting in reduced affinity to host receptor, and thereby impairs viral fitness and virulence, rather than positive selection for a better affinity to CD26. IMPORTANCE Recently, a large outbreak initiated by an MERS-CoV-infected traveler from the Middle East swept South Korea and resulted in 186 confirmed cases with 38 deaths. This is the largest outbreak outside the Middle East, and it raised strong concerns about the possible emergence of MERS-CoV mutations. Here, we isolated 13 new viral genomes and found that 12 of them possess a point mutation in the receptor-binding domain of viral spike protein, resulting in reduced affinity to the human cognate receptor, CD26, compared to the wild-type virus. These unexpected findings suggest that MERS-CoV adaptation in humans may be driven by host immunological pressure.111819Ysciescopu

Anatomical significance of a posterior horn of medial meniscus: the relationship between its radial tear and cartilage degradation of joint surface

<p>Abstract</p> <p>Background</p> <p>Traumatic injury and surgical meniscectomy of a medial meniscus are known to cause subsequent knee osteoarthritis. However, the difference in the prevalence of osteoarthritis caused by the individual type of the medial meniscal tear has not been elucidated. The aim of this study was to investigate what type of tear is predominantly responsible for the degradation of articular cartilage in the medial compartment of knee joints.</p> <p>Methods</p> <p>Five hundred and forty eight cadaveric knees (290 male and 258 female) were registered in this study. The average age of cadavers at death was 78.8 years old (range: 52-103 years). The knees were macroscopically examined and their medial menisci were classified into four groups according to types of tears: "no tear", "radial tear of posterior horn", "other types of tear" and "worn-out meniscus" groups. The severity of cartilage degradation in their medial compartment of knee joints was evaluated using the international cartilage repair society (ICRS) grading system. We statistically compared the ICRS grades among the groups using Mann-Whitney U test.</p> <p>Results</p> <p>The knees were assigned into the four groups: 416 "no tear" knees, 51 "radial tear of posterior horn" knees, 71 "other types of tear" knees, and 10 "worn-out meniscus" knees. The knees with substantial meniscal tears showed the severer ICRS grades of cartilage degradation than those without meniscal tears. In addition, the ICRS grades were significantly severer in the "radial tear of posterior horn" group than in the "other types of tear" group, suggesting that the radial tear of posterior horn in the medial meniscus is one of the risk factors for cartilage degradation of joint surface.</p> <p>Conclusions</p> <p>We have clarified the relationship between the radial tear of posterior horn in the medial meniscus and the severer grade of cartilage degradation. This study indicates that the efforts should be made to restore the anatomical role of the posterior horn in keeping the hoop strain, when patients' physical activity levels are high and the tear pattern is simple enough to be securely sutured.</p

Tumor necrosis factor-α-mediated threonine 435 phosphorylation of p65 nuclear factor-κB subunit in endothelial cells induces vasogenic edema and neutrophil infiltration in the rat piriform cortex following status epilepticus

<p>Abstract</p> <p>Background</p> <p>Status epilepticus (SE) induces severe vasogenic edema in the piriform cortex (PC) accompanied by neuronal and astroglial damages. To elucidate the mechanism of SE-induced vasogenic edema, we investigated the roles of tumor necrosis factor (TNF)-α in blood-brain barrier (BBB) disruption during vasogenic edema and its related events in rat epilepsy models provoked by pilocarpine-induced SE.</p> <p>Methods</p> <p>SE was induced by pilocarpine in rats that were intracerebroventricularly infused with saline-, and soluble TNF p55 receptor (sTNFp55R) prior to SE induction. Thereafter, we performed Fluoro-Jade B staining and immunohistochemical studies for TNF-α and NF-κB subunits.</p> <p>Results</p> <p>Following SE, most activated microglia showed strong TNF-α immunoreactivity. In addition, TNF p75 receptor expression was detected in endothelial cells as well as astrocytes. In addition, only p65-Thr435 phosphorylation was increased in endothelial cells accompanied by SMI-71 expression (an endothelial barrier antigen). Neutralization of TNF-α by soluble TNF p55 receptor (sTNFp55R) infusion attenuated SE-induced vasogenic edema and neuronal damages via inhibition of p65-Thr435 phosphorylation in endothelial cells. Furthermore, sTNFp55R infusion reduced SE-induced neutrophil infiltration in the PC.</p> <p>Conclusion</p> <p>These findings suggest that impairments of endothelial cell functions via TNF-α-mediated p65-Thr 485 NF-κB phosphorylation may be involved in SE-induced vasogenic edema. Subsequently, vasogenic edema results in extensive neutrophil infiltration and neuronal-astroglial loss.</p

Laser microdissection and mass spectrometry–based proteomics aids the diagnosis and typing of renal amyloidosis

Accurate diagnosis and typing of renal amyloidosis is critical for prognosis, genetic counseling, and treatment. Laser microdissection and mass spectrometry are emerging techniques for the analysis and diagnosis of many renal diseases. Here we present the results of laser microdissection and mass spectrometry performed on 127 cases of renal amyloidosis during 2008–2010. We found the following proteins in the amyloid deposits: immunoglobulin light and heavy chains, secondary reactive serum amyloid A protein, leukocyte cell–derived chemotaxin-2, fibrinogen-α chain, transthyretin, apolipoprotein A-I and A-IV, gelsolin, and β-2 microglobulin. Thus, laser microdissection of affected areas within the kidney followed by mass spectrometry provides a direct test of the composition of the deposit and forms a useful ancillary technique for the accurate diagnosis and typing of renal amyloidosis in a single procedure

A serine proteinase from the sarcoplasmic fraction of red sea bream Pagrus major is possibly derived from blood

Collagen degradation is known to be involved in the post mortem tenderization of fish muscle. A serine proteinase that is assumed to be related to collagen degradation after fish death was purified from the sarcoplasmic fraction of red sea bream Pagrus major by ammonium sulfate fractionation and column chromatography on Sephacryl S-300, Q Sepharose and Phenyl Sepharose CL-4B. The enzyme hydrolyzed gelatin and was obtained as a protein band of approximately 38 kDa upon sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. The N-terminal amino acid sequence of the enzyme was determined for 32 residues. A protein that had the same N-terminal amino acid sequence as the enzyme for ten residues was purified from the serum of red sea bream and showed the same characteristics as the enzyme. Therefore, it is suggested that the serine proteinase migrates from the blood to muscle and degrades muscle proteins after the death of the fish