Pixathlon/REX/Shutterstock, APCaleb Stanko of Holly has joined Major League Soccer's FC Cincinnati from Bundesliga side side SC Freiburg on a transfer.The MLS club, which starts play this spring, announced the move Wednesday.' We are happy to add Caleb to our roster,' FC Cincinnati coach Alan Koch said in a written statement. 'We look for versatility in our central midfielders and we feel he can fill a number of roles depending on the personnel we have on the pitch. We look forward to Caleb competing immediately with our group.' More: Holly's Caleb Stanko forges path in German gameStanko, 25, who starred at Oakland Christian High for four years, bypassed a chance to play at the University of Michigan to pursue a pro career in Europe.The midfielder played eight seasons with SC Freiburg and one season on loan with Swiss League side FC Vaduz (2016-17).The Vardar SC product made his first team debut in a German Cup match against HSV Barmbek-Uhlenhorst in 2015.
1970 STANKO 6P 8 in. Location: Brno, Czech Republic. Price: Contact Seller for Price. Manufacturer: Stanko machining centers. This seller has. A new way of investigating rape could transform attitudes to sex crime. Stanko's research, which will be presented in a lecture at the London School of Economics on Tuesday, is very troubling.
Last season, he appeared in his first Bundesliga contest, playing 90 minutes against Schalke on Nov. 4, 2017.Stanko made six Bundesliga appearances with SC Freiburg. He has three goals and 10 assists in 141 appearances in 141 matches, most of those with the German club's reserve and U19 sides.This season, he made one appearance with SC Freiburg II in a Regionalliga Sudwest match.Stanko has one cap with the U.S. National Team, appearing in a World Cup qualifying match against Trinidad and Tobago Sept. He's also captained the U.S. U-20 team in Concacaf and World Cup competitions.
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The MEP1A gene, located on human chromosome 6p (mouse chromosome 17) in a susceptibility region for inflammatory bowel disease (IBD), encodes the α-subunit of metalloproteinase meprin A, which is expressed in the intestinal epithelium. This study shows a genetic association of MEP1A with IBD in a cohort of ulcerative colitis (UC) patients. There were four single-nucleotide polymorphisms in the coding region ( P=0.0012–0.04), and one in the 3′-untranslated region ( P=2 × 10 −7) that displayed associations with UC.
Moreover, meprin-α mRNA was decreased in inflamed mucosa of IBD patients. Meprin-α knockout mice exhibited a more severe intestinal injury and inflammation than their wild-type counterparts following oral administration of dextran sulfate sodium. Collectively, the data implicate MEP1A as a UC susceptibility gene and indicate that decreased meprin-α expression is associated with intestinal inflammation in IBD patients and in a mouse experimental model of IBD.
The two major forms of inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohn's disease (CD), are caused by genetic and environmental factors that affect the interaction between host and microflora. Genetic association of MEP1A with IBDTo identify polymorphisms in the MEP1A gene, all 14 exons were sequenced in 24 patients: 6 patients with UC, 6 with CD (none of whom carried any of the three common CD-associated NOD2/CARD15 variants), and 12 celiac disease patients. Eight single-nucleotide polymorphisms (SNPs) and one polymorphism consisting of a 12-nucleotide (12 nt) insert were found (, ). All nine polymorphisms were genotyped in 379 UC and 380 CD patients as well as in 372 healthy controls. The human genome contains multiple copies of MEP1A-like sequences on chromosome 9. Owing to the presence of these pseudogenes that share high sequence similarities with the meprin-α gene on chromosome 6, a MEP1A-specific sequencing strategy was developed.
The genotype data and detailed information are summarized in online. A pairwise linkage disequilibrium analysis indicated strong linkage disequilibrium across the MEP1A gene ( online). Analysis of allele frequencies showed moderate, but statistically significant, associations with UC for four synonymous SNPs in the coding regions of the gene ( P=0.0012, 0.03, 0.04, and 0.002, in exons 8, 11, 12, and 12, respectively). One SNP in the 3′-untranslated region (UTR) (rs1059276 in exon 14) was most significantly associated with UC ( P=2 × 10 −7). The evidence for an association with CD was weaker, supported by the 3′-UTR only with a P-value of 0.003. An overview of MEP1A polymorphisms.
The polymorphic sites in the human MEP1A gene are shown in the context of the multidomain structure of the meprin-α subunit. Meprin protein domains are S, signal peptide; Pro, propeptide; Astacin, catalytic protease domain; MAM, meprin/ A5-protein/protein-tyrosine phosphate μ; TRAF, TNF-α receptor-associated factor; I, inserted; EGF, epidermal growth factor; TM, transmembrane; and C, cytosolic tail. The AUG start and UGA stop codons and Poly A are italicized.
The SNPs and the 12-bp insert are indicated by their nucleotide positions. Affected amino-acid residues and their position in the protein sequence are shown below the subunit structure. UC-associated SNPs are highlighted in bold. Reference mRNA sequence, GenBank, which lacks the 12-bp insert. SNP, single-nucleotide polymorphism; TNF, tumor necrosis factor; UC, ulcerative colitis.Allele frequencies for rs1059276 in our control group (risk allele frequency, 0.61) differed from those for Caucasians (the CEU population) in Hapmap (risk allele frequency, 0.64) and average frequency in dbSNP (risk allele frequency, 0.47). However, the association with UC was also evident considering control allele frequencies given in these databases ( P=5 × 10 −5.Expression of meprin-α mRNA in the intestine of IBD patientsThe mRNA levels for both meprin-α and villin-1 (a marker of differentiated epithelial cells) were measured using quantitative reverse transcription PCR (RT-PCR) in endoscopic biopsies from IBD patients and healthy controls.
Meprin-α and meprin-β are expressed abundantly in the terminal ileum in healthy patients (, left panel). Meprin-α is also expressed in the entire large intestine (, right panel), albeit at two- to sixfold lower levels than in the ileum. Villin-1 displays a similar expression pattern that probably reflects a decrease in the relative number of differentiated epithelial cells in the colon.
In contrast to meprin-α, meprin-β mRNA expression is markedly decreased in proximal parts of the colon and is undetectable in distal parts. Expression of meprin-α, meprin-β, and villin-1 in the intestinal mucosa of IBD patients and healthy controls. MRNA levels, as determined by quantitative RT-PCR, are shown relative to the average colonic expression levels of meprin-α in healthy controls. ( a) Endoscopic biopsies of three control individuals collected from the terminal ileum, cecum, proximal and distal transverse colon, sigmoid colon, and rectum. ( b) Endoscopic biopsies from the sigmoid colon from not affected and inflamed mucosal areas of CD patients, UC patients, and healthy controls ( n=14 in each group). Shown are individual values and mean.To determine whether meprin subunit expression correlates with inflammation, meprin mRNA levels were compared in affected and unaffected regions of the sigmoid colon of IBD patients and healthy controls.
Meprin-α expression was significantly lower in inflamed mucosa of IBD patients compared with the other groups (, top panel). Little-to-no meprin-β mRNA was detectable in either unaffected or inflamed mucosa (data not shown). Expression levels of meprin-α in CD and UC relative to average values for healthy colon were between 0.03 and 0.7 (average, 0.34) and 0.04 and 0.5 (average, 0.24), respectively. A concomitant decrease of villin-1 mRNA indicated a decreased number of differentiated epithelial cells in affected mucosal areas, which could account for some of the decreased meprin-α expression (, bottom panel). If the loss of differentiated epithelial cells was the only cause for the decreased expression of meprin-α mRNA, the meprin-α/villin-1 ratio would be expected to remain unchanged in the inflamed samples. However, a significantly decreased ratio of meprin-α to villin-1 mRNA was found in affected mucosa.
Although the meprin-α to villin-1 mean ratio was 0.79 (range, 0.51–1.04) in healthy controls, it was lower in the affected regions to 0.48 (range, 0.11–0.73) in CD and 0.43 (range, 0.13–0.76) in UC groups, respectively, indicating that the expression of meprin-α was affected to a greater extent than of villin-1. Interestingly, meprin-α mRNA levels in unaffected mucosa of CD patients were similar to those of healthy controls (, top panel).
However, in UC patients, we observed a trend of lower meprin-α mRNA levels in unaffected mucosa (mean, 0.64; range, 0.1–1.2). In summary, mucosal inflammation in IBD correlates strongly with low meprin-α mRNA levels in CD and UC.
Generation and characterization of meprin αKO miceTo determine whether meprin-α affects the course of IBD in mice, meprin-α knockout mice were generated and an experimental model of IBD was used. Targeted disruption of the Mep1a gene was achieved by inserting a neomycin cassette in exon 7, the segment that codes for the zinc-catalytic center of meprin-α. F 1 heterozygote matings gave the expected Mendelian genotype distribution ( online). The homozygous meprin αKO mice did not show any overt physical, anatomical, or histological abnormalities (data not shown). Conventional serum chemistries were determined and all the parameters tested for meprin αKO mice lay within the normal ranges and were similar to those of the WT counterparts ( online).
The αKO and WT mice also showed no difference in growth rates, as determined by body weights during different the phases of growth. Strategy for Mep1a gene disruption on mouse chromosome 17. ( a) Schematic diagram of a portion of the exon–intron structure of the WT and meprin αKO alleles.
Exons (6–9) are represented as black boxes. The neomycin cassette derived from the targeting vector (Osdupdel: gift of O.
Smithies) is depicted as a gray box in exon 7 of the KO allele. The 19-amino-acid consensus sequence for the catalytic center of astacin family metalloproteases is also shown. ( b) Southern blot analysis of tail-derived genomic DNA. The probe used for Southern blotting detects two HindIII-generated fragments: 3.5 kb corresponding to the WT allele and 4.7 kb from the αKO allele.
Lanes: −/−, meprin αKO DNA; +/+, WT DNA; +/−, meprin-α heterozygous DNA. WT, wild type.Matings of meprin αKO mice produced significantly smaller litters than their WT counterparts ( P. Meprin αKO mice are more severely affected than WT mice by DSS treatment. ( a) Body weight loss in WT and meprin αKO mice was monitored over a 7-day period ( n=7 mice per group). The meprin αKO DSS group lost a greater percent of body weight than the WT DSS group over days 4–7 (. P.When fecal matter of DSS-treated and control mice was monitored for occult blood to assess rectal bleeding, the control groups had an occult score less than 1 (WT values, 0.86±0.14; meprin αKO values, 0.71±0.29).
There was an increase in fecal occult blood at 7 days in mice treated with DSS, with meprin αKO mice (mean values, 2.8±0.37) bleeding more than the WT mice (mean values, 2.0±0.22).Disease activity index (DAI) values were calculated as described in the Methods section considering weight loss, stool formation, and rectal bleeding. These scores reflect the degree of inflammation and injury in mice.
The DAI values were greater for the meprin αKO mice than for WT mice from days 4 to 7. Although the DAI score for the DSS-treated meprin αKO mice (1.86±0.3) was significantly higher than that for controls (0.1±0.1) at day 4 ( P. Greater colon injury and inflammation in meprin αKO compared with WT mice. ( a) Colon shortening at day 7 for the mice treated with DSS. The average colon length for both WT and meprin αKO mice administered 3.5% DSS was shorter than that for their respective controls at day 7 (.
P.Sections of proximal colon of all the groups were examined histologically after hematoxylin–eosin staining (, panels i–iv). Both the WT (i) and meprin αKO (ii) controls showed normal colon morphology. DSS treatment caused marked changes in colon structure in both the genotypes with respect to their control groups. In addition, obvious differences between the two genotypes were observed. The DSS-treated WT mice showed some crypt destruction and infiltrating leukocytes (iii). Greater damage in the DSS-treated meprin αKO mice was observed, as evidenced by massive crypt destruction and ulceration.
Heavy leukocytic infiltration was seen both in the lamina propria and submucosal regions (iv).The colon sections were scored for injury using a multifactorial scoring system. The DSS-treated mice had a greater injury score than the controls of both the genotypes, with αKO having a higher score (WT, 14.5±2.3; αKO, 21.1±1.1) ( P. Colon cytokine and chemokine levels of WT and αKO mice treated with or without DSS were measured on day 5 ( n=5 per group). Both the DSS-treated groups had significant elevations in their cytokine levels compared with their corresponding controls (WT DSS treated vs. Control,. P.Meprin αKO mice show higher systemic inflammation in response to DSS treatmentAs colitis induction elicited a greater inflammatory response in the αKO mice, the degree of systemic inflammation was investigated by measuring the serum nitric oxide (NO 2/NO 3) levels.
The total nitric oxide levels in control mice of both the genotypes were low (5–7 μ M). The levels in WT DSS-treated mice did not show any significant elevation until day 5, and the increase was modest (10–15 μ M). By contrast, the nitric oxide levels in the DSS-treated meprin αKO group was increased as early as day 1 ( P. Meprin αKO mice have greater systemic inflammation than WT mice.
( a) Serum nitric oxide levels of all the four groups were measured throughout the course of the study from days 1 to 5. Compared with its control group, DSS-treated meprin αKO mice showed significantly higher levels as early as day 1( # P.The same panel of 16 cytokines and chemokines, measured in the colon, was measured in the sera of these mice to define the inflammatory environment further. In contrast to the colon cytokines, significant differences between the two genotypes were limited to four cytokines: IL-1β, IL-6, MCP-1, and RANTES. IL-1β levels in the DSS-treated WT mice were ninefold higher than in controls, whereas in the αKO mice, the elevation was over 200-fold.
Serum IL-6 levels in the WT mice were approximately fourfold increased after DSS treatment compared with controls, whereas in the αKO mice, levels increased over 16-fold. Modest increases in MCP-1 and RANTES levels were also observed in the αKO DSS-treated group. The positive association of the five exonic SNPs at the MEP1A locus with UC reported in this study implicates this metalloprotease gene as a genetic determinant in this disease. Of these five SNPs, only rs1059276 was statistically significant in CD in our study, and the P-value for the SNP was much lower in CD (3 × 10 −3) than in UC (2 × 10 −7).
An “ in silico” replication was performed with the publicly available data from the recent CD meta-analysis, incorporating the data of three previous genome-wide studies. Although the SNPs presented here were not specifically analyzed in these studies, “imputed” P-values for the SNPs were not significant. Therefore, we conclude that although there is a strong association of meprin-α SNPs with UC, evidence for such associations with CD remains unclear.The two earlier genome-wide studies for UC by Fisher et al.
And Franke et al. Did not report SNPs at or nearby the MEP1A locus. Notably, Fisher et al. Analyzed only a subset of non-synonymous SNPs. The UC-associated SNPs are all synonymous, which points to expression levels being affected rather than enzyme function. We cannot exclude the possibility that the MEP1A locus contains causal intronic SNPs linked to the significantly associated exonic SNPs identified in this study. The most significant genetic association between MEP1A SNPs and UC was the 3′-UTR C2417A SNP (dbSNP ID rs1059276).
Important functions mediated by the 3′-UTR are mRNA stability and translation regulation. It is, therefore, possible that the 3′-UTR polymorphism affects constitutive meprin-α expression levels and/or the regulation of meprin-α expression in inflamed mucosa. Consistent with this idea is the evidence provided by the expression studies in human IBD patients in this study as well as by a microarray study reported earlier in UC patients showing that there are decreased levels of meprin-α mRNA associated with mucosal inflammation in the intestine.
Our studies show that mice lacking meprin-α protein develop a heightened inflammatory response and greater intestinal injury than WT mice when challenged with DSS.
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