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RT @BLMDetroit: #BLMDetroit Are you coming to" #Detroit to #Ferguson" 630PM Tonight @MGCollege ? Live tweet using #D2Ferguson http://t.co/J…
Welcome to the 2015 Commencement Information Website. On behalf of Marygrove College, we would like to extend our warmest congratulations on your upcoming graduation! The completion of your degree requirements and your participation in commencement are important events. We are very proud of you, and look forward to celebrating with you at the Commencement Ceremony on Saturday, May 9, 2015.
Please take some time to review the 2015 Marygrove Commencement Information Booklets and other important information as soon as possible, since they contain important, time-sensitive information.
Commencement Ceremony
Saturday, May 9 • 11:00 a.m.
Marygrove College • Outside of Liberal Arts Building
Associate, Bachelor's, and Master's degree candidates who have completed degree requirements for Spring/Summer 2014, Fall 2014, or Winter 2015 semesters are eligible to participate.
Tickets are not required. Please plan to attend with your guests. You must arrive at Marygrove College, Madame Cadillac Building no later than 9:30 a.m. (Continental breakfast 8:00-9:30 a.m. in Madame Cadillac Building). Marshals, identified by green robes, will direct you to the area where you will robe and line-up.
When you assemble for the procession, you will receive a card with your name and degree. During the ceremony, designated readers will be at two podiums on stage. You will follow the Marshals to the podium, hand your name card to the reader, and then proceed to the middle of the stage. If you have an unusual pronunciation to your name, you may write it phonetically on your card and make sure you let them know so your name can be pronounced correctly. You will receive your diploma case and congratulations from the Chairperson of the Board or the Marygrove College President.
Any candidate with an outstanding balance will be able to participate (walk) in the Commencement Ceremony. All outstanding balances must be paid by Tuesday, April 28, 2015 or your diploma and official transcripts will be withheld until all outstanding balances are paid. If you have any questions regarding your account, contact the Cashier in the Business Office or call (313) 927-1227.
Please call the Commencement Hotline at (313) 927-1471.
(Associate and Bachelor's Degree Candidates Only) Thursday, May 7 • 7:00 p.m.
Sacred Heart Chapel, Liberal Arts Building
The Baccalaureate Ceremony consists of the procession, Mass, hooding and the sending off of students. To ensure candidates celebrate and enjoy this ceremony, please follow the guidelines below.
Read more: Undergraduate Degree Candidate Baccalaureate Ceremony
RT @BLMDetroit: #BLMDetroit Are you coming to" #Detroit to #Ferguson" 630PM Tonight @MGCollege ? Live tweet using #D2Ferguson http://t.co/J…
RT @IndependentsMCA: Out: Joe Buck. In: Jeremy Otto. Meet the voice of the @MGroveAthletics Mustangs, @MGCollege sophomore @JeremyOtto17: h…
Happy #MartinLutherKing Day from Marygrove College. http://t.co/AUm50LySKV
RT @jbrownroosevelt: Congrats Dallas Chicone of PHN signed with Marygrove to play ball! Proud @PHSchools @PHNAthletics @MGroveAthletics htt…
Marygrove College will be closed Monday, January 19th in observance of #MartinLutherKing Day. http://t.co/1lOSJoTZTx


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subset: commoncrawl


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Arizona Diamondbacks
D-backs targeting INF Aubrey Huff
While a lot can change next week at the MLB Winter Meetings in Nashville, as it stands now the Arizona Diamondbacks are probably slated to start two right-handed bats at first (Paul Goldschmidt) and third base (Chris Johnson) going into Spring Training.
Seeking a left-handed bat to platoon with either Goldschmidt or Johnson, it apparently hasn't taken long for general manager Kevin Towers to reach out to an available veteran.
In need of a backup corner IF, #Dbacks have made contact with Aubrey Huff, source said.— Jack Magruder (@JackMagruder) December 1, 2012
Huff, a 12-year veteran, is very familiar with the NL West, having played the past three-plus seasons with the San Francisco Giants. Although the former fifth-round selection has won two World Series rings with the Giants (2010 and 2012), Huff's numbers have dramatically decreased over the past few seasons.
In 2011, he finished last among NL first baseman in OPS (.676), and saw his batting average (.246), home runs (12), RBI (57) and walks (47) all take significant dives from the 2010 season.
Last year in San Francisco, Huff began the season as Bruce Bochy's opening day left fielder, but injuries and a lack of production (.192 batting average, one home run and seven RBI), reduced him to a minor role during the Giants' championship run.
As a Giant, Huff has made a living against the Arizona pitching, hitting .321 with seven home runs and 22 RBI.
Most Popular
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- HardwareThe Justin Bieber award, the You're Just Mean award, and more of the best/worst of the weekend
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- 6-26-15 The Sports Kabob with Jarrett CarlenTopics include the NBA Draft and Pete Rose. It's sports news skewered.
- Derrick Hall, Diamondbacks President & CEODerrick Hall says the media is driving the story of reaching .500.


text: Pop Culture New Museum Charts the History of the Cocktail February 26, 2005 A group of passionate bartenders from all over America are in the process of establishing the world's first museum devoted exclusively to high balls, low balls, fizzes and other mixed drinks. Co-founder Dale DeGroff describes the Museum of the American Cocktail. Listen Loading… Playlist Download Embed Embed Close embed modal <iframe src="http://www.npr.org/player/embed/4513707/4513714" width="100%" height="290" frameborder="0" scrolling="no">


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The Natural Resource Reserve is managed as a sustainable wood lot over the long term. A selective timber harvest took place in 2007, and will continue at, planned long intervals to promote forest health.
Meanwhile, the primary thrust of economic production on ICNL is the management and sale of non-timber forest products. South Hill Forest Products, a student-run business based in South Hill Natural Area East, produces maple syrup, edible mushrooms, honey, and other hive products. One of the primary aims of these projects is to demonstrate that intact forest can be used as a working landscape, delivering revenue for landowners who do not wish to harvest their trees for timber.


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WARPED X - What Do We Want To Do?
View Single Post
single track rider
Joined: Oct 2009
Location: lost in space
Oddometer: 1,021
Originally Posted by
there's never been a problem before with our trucks/vans. we tend to share between 2-4 trucks.
if you have a problem with 8AM riders (I'm definitely not one of then since I'm still in my tent hung over) go up to the rider and tell him in person. it's not a small bike vs big bike issue.
keep in mind... people on street bikes.... I've been joking throughout this thread about our small bikes but Kernville has some of the best Singletrack trails in SoCal. we've stayed at MRA a few times in the summer and fall throughout the years and they're always very nice to us.
there is so much riding in this area, none of you have ridden all the trails. no one. Sawmill Road and Jawbone Road and Rancheria Road are not trails.
there is NO adventure in riding a street bike hundreds of miles on pavement to a rally then sit around and drink for days.
we (small bikes) go to WARPED to ride AND drink. and drink.
so lets bury this issue.
well said charles, just cause we ride little pussy bikes does not make us any less irresponsible than the sleds that others ride!
this is a riding and drinking event!
best of both worlds for both kinds of riding!
so bring AND ride what ud like!
I am in not associated with happy-trails.com
I am the original happytrails
View Public Profile
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Audio Samples
Album Summary
- Vebjorn Anvik (Piano)
- Lars Tomter (Viola)
- Iona Brown (Violin)
- H�vard Gimse (Piano)
Notes & Reviews:
This recording by the Norwegian Chamber Orchestra was directed by Iona Brown and was a Gramophone "Critic's Choice" on first release. The reviewer commented, "performances at once scintillating and polished". Classic FM wrote of the performances, "Iona Brown has the full measure of these works individually and her direction, always alert to the implications of modulations, tempo and dynamic markings, is outstanding".
ReviewsThere are currently no reviews, be the first one!
Also Purchased
Works DetailsMozart, Wolfgang Amadeus : Concerto for 2 Pianos in E flat major, K 365 (316a)
- Performers: Håvard Gimse (Piano); Vebjorn Anvik (Piano)
- Notes: Lommedalen Church, Norway (10/06/1996-10/07/1996)
- Running Time: 23 min. 38 sec.
- Period Time: Classical
- Written: 1779
Mozart, Wolfgang Amadeus : Adagio for Piano in B minor, K 540
- Performers: Lars Tomter (Viola); Iona Brown (Violin)
- Notes: Eidsvoll Church, Eidsvoll, Norway (11/13/1995-11/14/1995)
- Running Time: 30 min. 14 sec.
- Period Time: Classical
- Written: 1779


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Creative Inspire T6100 Speaker System - 51MF4086AA000 - Surround Yourself with Sound
Learn more about Creative Inspire T6100 Speaker System:
|Duck and cover as explosions rock your room. Hear the crack of gunshots in the distance. Hear every word of dialog. Connect the Inspire T6100 speaker system to your PC or notebook and breing your games and movies to life.
Place satellite speakers all around your room -- two in front, two behind you, a center channel for powerfully clear and clean dialog, and a subwoofer for booming lows. Fine tune your sound with the built-in controls.
Sounds good, looks good. You'll love the sleek, flat design, whether the speakers are sitting on the included stands or mounted on your walls. For a dramatic experience when gaming or watching movies, grab your Inspire T6100 speaker system today.
Customer Reviews of Creative Inspire T6100 Speaker System
Worth Spending9/26/2008
This is a good speaker system... i bought this to connect it to my HP laptop... however the downside of this is that is has three input plugs... so i have to buy an external sound card... however, for the time being i m connecting it to my laptop to the regular sound output n one to the SPDIF output... i know i m not getting the sound that i sud be getting but it works for me... i ordered a x-fi external sound card n cant wait to connect the speaker to that.... but i really like it...
Great Speakers1/3/2008
For the price, you cant go wrong with these speakers. Great for games, music, and movies. These sound better than my 5.1 surround sound speakers Home Theater System I bought from circuit City
great for those who don't care too much about qual6/16/2007
The speakers are great, especially for the price, except for a couple of things. The sub does not reach as low as almost any other subwoofer which is very annoying for me because it is recognizable even for the average user. Also volume control is on the right speaker and bass control is seperated onto the back (hard to reach) part of the sub. In other words, it lacks a remote which most speakers come with now. But everything else works great, including quality at high volumes, without distortion
Cheap for 5.1 but far from the best5/15/2007
Pros: Cheap, speakers look simple and stylish, compact sub puts out plenty of bass, wall mountable, decent sound quality and volume. Cons: Speaker construction on the cheap side, wires very thin and short, too short to hide in most computer setups. Sub is overpowered even on minimum setting. No external base and volume control, only volume on front right speaker and bass knob on sub. P.S. for those needing longer wires, simply cut the wire and splice in whatever length of regular speaker you need. Just be careful to mark the two wires and connect the same sides w/ the same wire. Mind sound great like this.


text:
Apostles Instruct Peruvian Saints
- Date Featured:20 August 2012
Understand why President Uchtdorf teaches that making the scriptures a part of our lives is so important.
Welcome Home…Changing lives
- Date Featured:20 August 2012
Bringing about a mighty change of heart.
Fulfilling Your Duty to God
- Date Featured:15 August 2012
In this youth video, learn how the goals in Duty to God reach far beyond the young men who fulfill them.
A FamilySearch Indexing Challenge
- Date Featured:5 August 2012
Calgary Saints take on a FamilySearch Indexing challenge to prepare for a temple in their midst.


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Patagonia Adze Jacket
Where to Buy
Retailers' Descriptions
Here's what other sites are saying:
With looks that lets you wear it on the street and protection that lets you sport it during outdoor activity, the new Patagonia Women's Adze Softshell Jacket is sure to be your most versatile outer piece. A polyurethane membrane between the two layers of stretchy polyester completely blocks wind and provides a substantial water-resistant barrier that, you'll quickly realize, make the Adze the ideal piece for just about all of your year-round activities.
- Backcountry.com
In anything from a breeze to a tempest, stay agile and protected whether climbing desert spires or scrambling alpine ridges. The Adze Jacket uses a breathable, windproof Polartec(R) Windbloc(R) stretch-woven polyester shell that has a soft, supple hand and a moisture-shedding DWR (durable water repellent) finish to fend-off wet weather. The microgrid fleece interior traps heat, moves moisture and feels great next to your skin, allowing total versatility with short- or long-sleeved layers. Features include two reverse-coil, zippered handwarmer pockets and one internal zippered chest pocket, hook-and-loop cuff closures and a drawcord hem to seal out the elements and lock in heat.
- Patagonia.com
Patagonia's Adze Jacket features Polartec Windbloc stretch-woven fabric for mobility and durability. This Jacket is a windproof performer that offers protection from even the worst wind.
- CampSaver.com
On Sale. Free Shipping. Patagonia Women's Adze Jacket FEATURES of the Patagonia Women's Adze Jacket Polartec Wind bloc stretch-woven polyester soft-shell fabric, with a membrane construction and DWR (durable water repellent) finish, stops wind and resists precip A warm fleece grid backer wicks moisture Microfleece-lined neck and wind flap for next-to-skin comfort Two harness- and pack-compatible handwarmer pockets and one internal chest pocket, all with reverse-coil zippers Low-profile, hook-and-loop self-tab cuffs create a tight wrist seal Drawcord hem SPECIFICATIONS of the Patagonia Women's Adze Jacket Weight: 21.1 oz / 598 g Regular fit 9-oz Polartec Wind bloc 93% polyester / 7% spandex, with a DWR (durable water repellent) finish This product can only be shipped within the United States. Please don't hate us.
- Moosejaw
Hit the trails for your daily run and cut through the strongest wind with the Adze Jacket from Patagonia. Constructed with Polartec Windbloc stretch-woven polyester, not only are you completely shielded from the wind but you have ultimate mobility and durable while you move. Stay comfortable while you move with microfleece-lined neck and wind flap. Low-profile gusseted cuffs and drawcord hem creates a tight seal to lock heat in and wind out.
- Mountain Gear
Patagonia Women's Adze Jacket
currently retails for:
$55.60 - $139.00
$55.60 - $139.00


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Leukocyte infiltration plays an important role in the pathogenesis and progression of myositis, and is highly associated with disease severity. Currently, there is a lack of: efficacious therapies for myositis; understanding of the molecular features important for disease pathogenesis; and potential molecular biomarkers for characterizing inflammatory myopathies to aid in clinical development.
In this study, we developed a simple model and predicted that 1) leukocyte-specific transcripts (including both protein-coding transcripts and microRNAs) should be coherently overexpressed in myositis muscle and 2) the level of over-expression of these transcripts should be correlated with leukocyte infiltration. We applied this model to assess immune cell infiltration in myositis by examining mRNA and microRNA (miRNA) expression profiles in muscle biopsies from 31 myositis patients and 5 normal controls.
Several gene signatures, including a leukocyte index, type 1 interferon (IFN), MHC class I, and immunoglobulin signature, were developed to characterize myositis patients at the molecular level. The leukocyte index, consisting of genes predominantly associated with immune function, displayed strong concordance with pathological assessment of immune cell infiltration. This leukocyte index was subsequently utilized to differentiate transcriptional changes due to leukocyte infiltration from other alterations in myositis muscle. Results from this differentiation revealed biologically relevant differences in the relationship between the type 1 IFN pathway, miR-146a, and leukocyte infiltration within various myositis subtypes.
Results indicate that a likely interaction between miR-146a expression and the type 1 IFN pathway is confounded by the level of leukocyte infiltration into muscle tissue. Although the role of miR-146a in myositis remains uncertain, our results highlight the potential benefit of deconvoluting the source of transcriptional changes in myositis muscle or other heterogeneous tissue samples. Taken together, the leukocyte index and other gene signatures developed in this study may be potential molecular biomarkers to help to further characterize inflammatory myopathies and aid in clinical development. These hypotheses need to be confirmed in separate and sufficiently powered clinical trials.
Keywords:Myositis; Genomics; Leukocyte infiltration; Type 1 interferon; miR-146a
Myositis is characterized clinically by skeletal muscle weakness and histopathologically by the presence of inflammatory cells in muscle tissue. There are several major subclasses of myositis, including dermatomyositis (DM), polymyositis (PM), inclusion body myositis (IBM), and immune mediated necrotizing myopathy (NM). The leukocyte infiltration present in myositis muscle is believed to contribute to disease pathogenesis [1-5]. The types of immune cells present in myositis muscle were originally identified in the 1980s as predominantly CD4+ T cells and B cells in DM, and CD4+ and CD8+ T cells in IBM [1,6-8], with more recent identification of plasmacytoid dendritic cells in DM [9], myeloid dendritic cells in PM and IBM [3], and plasma cells in all three disorders [2]. Unlike DM, PM or IBM, NM is characterized by myofiber necrosis associated with macrophages and minimal T cell infiltration or MHC Class I expression [10]. Given the differences in clinical manifestations between these subtypes of myositis and the lack of optimal efficacious therapies for these diseases, understanding the molecular characteristics underlying their subtypes may facilitate the development of novel therapeutics that could benefit patients with myositis.
Technologies such as whole genome microarray have advanced our understanding of the disease pathogenesis of myositis [11-13]. A large number of type 1 interferon-stimulated genes (ISGs) were identified to be strongly overexpressed in DM muscle and this molecular signature has been further confirmed by recent studies [4,14]. The activation of type 1 interferon (IFN) signaling has been observed in many autoimmune diseases [15,16], such as systemic lupus erythematosus (SLE) [17-19], systemic sclerosis [20], rheumatoid arthritis [21], and psoriasis [22]. A type 1 IFN signature is not only present in DM muscle but also expressed in DM skin [23], as well as peripheral blood of DM and PM, reflecting disease activity [24-26]. Based on the accumulating evidence from recent microarray studies and other complementary experiments, disease models have been proposed to emphasize the central role of type 1 IFN pathway activation in the pathogenesis of DM, suggesting that blockade of type 1 IFN might provide clinical benefit to DM patients [27].
In addition to previous studies focused on altered mRNA expression in myositis, the roles of microRNAs (miRNAs) in regulating immune responses, muscle development, and regeneration are also emerging [28-31]. miRNAs including miR-146a, miR-155, and miR-101 have been shown to be aberrantly expressed in rheumatic diseases [32-35] and miR-1, miR-133a/b, and miR-206 have been identified as muscle-specific miRNAs critical for muscle development and function [28-31]. Though the role of miRNAs in the pathogenesis of myositis has yet to be evaluated extensively, it is worth noting that interactions between miRNAs and type 1 IFN have been identified [36]. Specifically, miR-146a suppresses the innate immune response not only via the TLR-mediated NFκB pathway [37], but also negatively regulates the type 1 IFN pathway in SLE by targeting STAT1 and IFN regulatory factor 5 (IRF5) [38].
Despite the aforementioned studies, there still lacks a clear understanding of the disease pathogenesis that underlies myositis. Meanwhile, few molecular biomarkers have been identified to aid in stratifying myositis patients or objectively quantifying the leukocyte abundance in inflammatory muscle and the corresponding muscle fiber damage. To address the unmet needs in these areas, we performed both genome-wide mRNA and miRNA expression profiling in muscle biopsies from myositis patients and normal controls. Our studies revealed gene expression signatures specific for myositis and distinct for each subclass of myositis, as well as multiple pairs of mRNA:miRNA displaying anti-correlation expression patterns in line with predicted relationships. Additionally, expression data from this study indicated that miR-146a displayed a positive correlation with the type 1 IFN signature rather than the expected negative correlation in myositis muscle. We postulated that such positive correlation could be driven by infiltrated leukocytes; therefore, we developed an invasion model to account for transcriptional changes due to leukocyte infiltration. Further analyses with this invasion model indicated that the source of ISG expression may differ between subtypes of myositis, such that in PM and IBM, ISG expression is associated with infiltrated leukocytes, whereas in DM, non-leukocyte cells (e.g., muscle cells) might contribute significantly to ISG expression. Collectively, our results revealed multiple gene expression signatures that can potentially advance our understanding of the pathologic characteristics of myositis and provide utility as molecular biomarkers for identifying the right therapeutics for myositis patients.
Altered mRNA and miRNA expression reveals upregulation of immune pathways and downregulation of muscle contraction pathways in myositis
A total of 837 probesets that represent 606 unique genes were differentially expressed by two-group t-tests in muscle specimens of myositis patients compared with non-neuromuscular disease patients (Additional file 1: Table S1). Most differentially expressed transcripts (88%, 740 of 837) were over-expressed, with only 97 (12%) down-regulated. Functional analysis using IPA (Ingenuity Pathway Analysis, http://www.ingenuity.com/ webcite) and gene set enrichment showed that the most upregulated genes are involved in the immune and inflammatory responses, including a large number of ISGs, MHC class I/II proteins, and genes associated with natural killer cell mediated cytotoxicity. In contrast, muscle contraction and calcium signaling pathway-related genes are enriched in the down-regulated transcripts (p-value < 0.001 and p-value = 0.001, respectively). In addition to the identification of differentially expressed transcripts in myositis compared to normal controls, we also identified subclass-specific differentially expressed mRNAs by comparing each myositis subtype to the normal controls (Additional file 2: Supplementary Text; Additional file 2: Figure S2; Additional file 3: Table S2).
Additional file 1. Table S1. 837 differentially expressed transcripts in myositis compared to normal controls.
Format: XLS Size: 214KB Download file
This file can be viewed with: Microsoft Excel Viewer
Additional file 2. Supplementary material. Supplementary information and figures for the main manuscript.
Format: DOC Size: 582KB Download file
This file can be viewed with: Microsoft Word Viewer
Additional file 3. Table S2. Numbers of the differentially expressed transcripts in the subclasses of myositis compared to normal controls.
Format: XLS Size: 27KB Download file
This file can be viewed with: Microsoft Excel Viewer
A similar statistical analysis to determine differentially expressed mRNAs was applied to miRNA expression data to identify differentially expressed miRNAs. Of 69 differentially expressed miRNAs identified when comparing myositis muscle with normal controls, only five were over-expressed (miR-146a/b, miR-155, miR-21 and miR-432). The remaining 64 miRNAs were down-regulated, including miR-133a/b, miR-1, and miR-29c (Additional file 4: Table S3). Notably, both miR-146a and miR-155 are predominantly expressed in leukocytes and also play important roles in regulating immune responses [32-35].
Additional file 4. Table S3. 69 differentially expressed miRNAs in myositis compared to normal controls.
Format: XLS Size: 32KB Download file
This file can be viewed with: Microsoft Excel Viewer
Unsupervised hierarchical clustering of the 837 differentially regulated mRNA transcripts revealed two main transcriptionally-defined subcategories, with all normal and NM samples clustered within one branch (Figure 1, branch 1), and all IBM samples clustered within the other branch (Figure 1, branch 2). DM and PM samples are divided into both branches. Although unavailable for the patients within this study, additional information such as myositis-specific antibodies and other serology data could have contributed to a more optimal phenotyping of the myositis specimens. The lack of these data may explain some of the heterogeneity observed within the groups defined strictly by the diagnostic criteria NM, DM, PM, and IBM. Nevertheless, our results suggest that specific molecular expression patterns, in addition to others previously defined for myositis, might provide insight into classifying and understanding the pathophysiology of these different subtypes of myositis.
Figure 1. Heatmap of 837 differentially expressed transcripts. Transcript expression fold change is represented by the color scale increasing from blue to white to red; the color key is shown in the top left corner. The 36 subjects are shown horizontally and represented by the color bar on the top of the heatmap. The color that corresponds to each disease type is displayed in the upper right corner. At the first bifurcation of the horizontal dendrogram, the subjects are classified into 2 groups from left to right: group 1 and group 2.
Consensus clustering reveals five gene signatures that characterize myositis
In addition to identifying molecular expression patterns specific for myositis as a whole and distinct for its individual subtypes, we also applied and merged multiple clustering algorithms to identify robust clusters of genes that may provide unique insight into the molecular components of myositis (see Methods for the details). To rule out uninformative genes, we selected the top 200 genes with the most varied expression from the 606 differentially expressed genes, among which, only three were down-regulated. Therefore, we focused on the remaining 197 up-regulated genes and clustering produced five clusters with robust gene members (i.e., cluster A-E; Additional file 2: Figure S2C and S3; also see Methods).
Subsequent functional analyses of the five clusters revealed that clusters A and B were enriched with type 1 ISGs and immunoglobulins, respectively; clusters C and D had very similar profiles (Figure 2A) and were enriched with immune response-related genes (Additional file 5: Table S4); and cluster E was dominated by MHC class I genes. These observations led us to define five gene signatures enriched in myositis muscle tissue: type 1 IFN signature (from cluster A), immunoglobulin signature (from cluster B), leukocyte I and II indices (derived from clusters C and D, respectively) and MHC class I signature (from cluster E). The similarity in cluster results shared by the different clustering methods confirmed the scientific rigor of this analysis (Additional file 5: Table S4). We then proceeded to analyze disease heterogeneity within each myositis subclass by utilizing these five gene signatures (Figure 2B-C). In strong agreement with results shown in Figure 1, NM displayed homogeneous gene signature profiles similar to normal muscle, with a slightly elevated leukocyte II signature observed (see Figure 2B-C). IBM exhibited clear over-expression of all five gene signatures. Gene signatures in PM and DM varied from normal-like signatures to patterns similar to IBM. Most myositis patients showed coordinate overexpression across all modules (shown as an equilateral right pentagon in the radial plot; Figure 2B), but DM showed some heterogeneity of relative overexpression of the type 1 IFN module (i.e., cluster A).
Additional file 5. Table S4. List of the gene members of the clusters A-E.
Format: XLS Size: 35KB Download file
This file can be viewed with: Microsoft Excel Viewer
Figure 2. Gene signatures derived from clusters A-E from the consensus cluster. (A) Pairwise scatter plots of the five gene signatures. The scatter plots of the signatures of the clusters A-E are displayed in the lower panels, where data points are colored by the sample type (same as the Figure 1) and red lines represent the lowess (Locally Weighted Scatterplot Smoothing) lines. The corresponding spearman correlation coefficients are displayed in the upper panels, with significance levels indicated by the stars (*** p < 0.001, ** p < 0.01, * p < 0.05). (B) Subclasses of myositis characterized by the five gene signatures are shown in the star plots. Each subject is represented with a specific pentagon in the star plot and the vertices of one pentagon along the spokes (A-E) represent enrichment of the signature scores from clusters A-E. Colors of pentagons are randomly assigned to distinguish subjects and star plots are grouped by the myositis subclasses. (C) Subjects can be further characterized by the five gene signatures using a heatmap. The signature scores are represented by the rainbow color in the heatmap, ranging between 0 (colored in blue) and 4 (colored in red); the color key for each sample's disease type is displayed horizontally between the sample identifiers and the heatmap, where, (from left to right), green denotes normal, gray for NM, blue for DM, magenta for PM and red for IBM. The four DM patients with perifascicular atrophy (PFA) are marked by the star symbol.
Both leukocyte index I and II are comprised of over-expressed genes related to immune function (Additional file 5: Table S4); displayed differences in magnitude between myositis patients; and are highly correlated with each other (Spearman rank test; r = 0.97, p < 0.001; see Figure 2A). Therefore, we selected leukocyte index I (generated from cluster C) as the leukocyte infiltration index for subsequent analyses examining altered gene expression due to increased immune cell infiltration into muscle.
A leukocyte index distinguishes altered expression of transcripts due to immune cell infiltration and correlates with histopathology results
In myositis, leukocyte infiltration and/or clonal expansion results in a marked increase in the abundance of leukocytes in muscle. Consequently, the interpretation of gene expression studies in myositis tissue biopsies can be greatly influenced by immune cell infiltration. In a principal components analysis of the 837 differentially expressed transcripts in this study, we found that the first principal component (PC1) accounted for 75.6% of the variance in the data. PC1 had a significant linear association with our proposed leukocyte index (R2=0.92, p<0.001), suggesting that the majority of the overall expression variation was due to the change of leukocyte cell numbers and could be accounted for by this index. This computational representation of leukocyte infiltration could eliminate the transcriptional variation generated solely from the increased immune cell proportion in muscle samples and allow gene expression changes to be attributed to the appropriate source.
The ability of the leukocyte index to quantify the extent of leukocyte infiltration is indirectly supported by the significant enrichment of immune response-related transcripts among the differentially expressed transcripts (hypergeometric test, p< 0.001), as well as the robust clustering of these genes. Additionally, there is a significant linear correlation between the leukocyte index and immune cell infiltration identified and scored by hematoxylin and eosin (H&E) staining (R2=0.79, p<0.001; Figure 3). This provides direct evidence to support the leukocyte infiltration index described here by confirming that the index accurately reflects leukocyte abundance in muscle.
Figure 3. Correlation between leukocyte infiltration index and H&E staining. A significant linear correlation is displayed in the scatter plot between the leukocyte infiltration index and the degree of inflammation assessed by H&E staining (0=no inflammation, 1=sparse inflammation, 2=mild inflammation, 3=moderate inflammation, and 4=severe inflammation). Data points are colored by the sample type; color key is displayed in the upper right corner of the plot. The regression line is denoted by the blue dotted line (intercept=0.07, slope=1.00; R2=0.79, p<0.001).
In addition to this ability of the leukocyte index to correctly reflect the abundance of immune cells in muscle tissue, it may also inversely correlate with the expression of muscle-specific mRNA or miRNA due to either a decreased proportion of muscle cells in the sample or muscle damage. For instance, TTN is a structural protein abundant in myofibers whose deficiency was found to be associated with DM perifascicular atrophy (DM-PFA) [14]; miR-1 is a muscle specific miRNA [39] that has an important role in muscle development [28-31]. Both TTN and miR-1 exhibited a marked anti-correlation with the leukocyte index (Spearman rank test; r = −0.70, p<0.001 and r = −0.64, p<0.001, respectively), providing additional evidence to support the validity of our invasion model and the derived leukocyte index.
Multiple mRNAs dysregulated in myositis muscle are not due to leukocyte infiltration
The development of the leukocyte infiltration index revealed that most of the over-expressed transcripts in myositis muscle compared to normal muscle specimens were not truly up-regulated due to transcript over-expression, but rather these expression changes were actually due to leukocyte cells infiltrating the muscle tissue. Accordingly, we adjusted the expression data matrix using the infiltration index to identify over-expressed transcripts unaccounted for by leukocyte infiltration. Consequently, only 41 (5.5%) out of 740 over-expressed mRNAs remained over-expressed after adjustment with the infiltration index (Additional file 6: Table S5). These miRNAs included many immunoglobulins (IGK, IGH, IGL), chemokines (CCL8, CCL11, CCL18), myosin complex genes, and cytoskeletal genes (POSTN, TNNT2 and MYH8). The dramatic reduction of over-expressed transcripts after adjusting for leukocyte infiltration confirmed that most of the over-expression is driven by increased leukocyte abundance in myositis muscle.
Additional file 6. Table S5. Overexpressed transcripts after adjustment with the leukocyte index.
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Subclass-specific transcript analysis following adjustment for leukocyte infiltration (Additional file 7: Table S6) revealed that immunoglobulin proteins and some chemokines were over-expressed in both IBM and PM, whereas ISGs were heavily over-expressed in DM muscle. Overall, our results suggested that myofibril and chemokine signatures exist in DM, PM and IBM, and likely resulted from changes in gene expression in the muscle cells themselves rather than alterations due to leukocyte infiltration. Understanding the contribution of these pathways altered in muscle tissue may provide keen insight into the pathogenesis of specific subclasses of myositis.
Additional file 7. Table S6. Subclass-specific overexpressed transcripts after adjustment with the leukocyte index.
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The leukocyte index and the type 1 IFN signature in myositis muscle
After re-assessing gene expression changes to account for leukocyte infiltration, we observed that ISGs remained highly over-expressed in muscle biopsies particularly from DM patients. Accordingly, we investigated the relationship between over-expression of ISGs and the leukocyte index in more depth. Strikingly, the type 1 IFN signature showed a significant linear correlation with the leukocyte index with exception of four outliers (i.e., the DM subjects GEIM 12, 272, 382 and 383; see Figure 4). The linear correlation suggests that leukocyte infiltration might have a more pronounced effect on ISG over-expression in IBM, PM, and DM without perifascicular atrophy (PFA; see Table 1); whereas in a subset of DM patients with marked ISG over-expression, especially those with PFA, other cell types (e.g., muscle fiber cells) might contribute to type 1 IFN pathway activation.
Figure 4. Correlation between type 1 IFN signature score and leukocyte infiltration index in myositis. A significant linear correlation is displayed in the scatter plot between type 1 IFN index and leukocyte infiltration index. Data points are colored by the sample type; color key is displayed in the upper right corner of the plot. The regression line (R2=0.41, p<0.001) is denoted by the black dotted line and 95% confidence interval is outlined by the red dotted line and gray shaded background. The subject identifiers of the four outliers are labeled. When excluding the four outliers, the R-squared value in the regression analysis escalates from 0.41 to 0.91.
Table 1. Patient information
Observations that a correlation between ISG over-expression and leukocyte infiltration varied between myositis subtypes prompted us to also investigate the correlation of miR-146a with the leukocyte index as this miRNA has been previously reported to negatively regulate the type 1 IFN pathway in SLE [38]. Our results indicated that both miR-146a and the type 1 IFN signature display an overall positive correlation with the leukocyte index (Spearman rank test; r = 0.64, p < 0.001; r = 0.72, p < 0.001, respectively; also see Figure 4), and therefore they are likely to be positively correlated with each other as well. Upon further evaluation, this positive pattern of association was evident primarily in IBM and PM (Figure 5), where leukocyte infiltration may be responsible for the observed alterations in miR-146a expression. In contrast, 6 out of 8 DM samples displayed a negative pattern of association between the expression level of miR-146a and the type 1 IFN signature, similar to what has been shown previously in SLE [38].
Figure 5. Intricate association between the expression of miR-146a and type 1 IFN signature score in a subclass of myositis. Data points are colored by the sample type in this scatter plot; color key is displayed in the upper right corner of the plot. The identity reference line y=x is denoted by the blue dotted line. Type 1 IFN signature displays a positive pattern of association in most subjects including the normal control, NM, PM, IBM and some DM, as highlighted in the light blue shaded oval. On the other hand, the type 1 IFN signature displays a negative pattern of association with the expression level of miR-146a in most DM (highlighted in the pink shaded oval). The subject identifiers of the eight DM subjects are all labeled in this plot.
To examine the possibility that miR-146a could negatively regulate the type 1 IFN signature in muscle tissue, we utilized an in vitro muscle cell model. The level of miR-146a expression was altered in C2C12 muscle cells by transfecting either miR-146a mimics or miR-146a inhibitors into this cell line prior to stimulation with type 1 IFN. Measuring the resulting expression levels of ISGs illustrated that miR-146a is able to negatively regulate the type 1 IFN pathway in muscle cells (Figure 6), as demonstrated by the increased ISG expression following inhibition of miR-146a and the decreased ISG expression following increased miR-146a levels. These trends and degree of effect on ISGs by miR-146a are in agreement with what has been reported previously in SLE [38], and suggest a possible role for miR-146a in regulating the type 1 IFN pathway in muscle, although this remains to be verified by in vivo studies.
Figure 6. miR-146a alters the expression of type 1 IFN inducible genes in C2C12 muscle cells. C2C12 cells were transfected with miR-146a mimics and miR-146a inhibitors, as well as the appropriate scrambled controls. 24 hours after transfection, cells were stimulated with mouse IFN-alpha for 4 hours before RNA was isolated and profiled for alterations in ISGs. Regulation of the IFN pathway was demonstrated by increased ISG transcript levels following inhibition of miR-146a, as well as decreased ISG transcript levels following addition of miR-146a. Values are shown as mean ± SEM and represent results from 3 independent experiments.
Most biological tissue specimens are heterogeneous, often consisting of multiple cell types. This heterogeneity is evident in myositis muscle biopsies and confounds transcriptional analyses, as the source of an observed expression change is unclear. We developed a leukocyte index to differentiate the transcriptional alterations due to an increased proportion of inflammatory cells from other changes at the expression level. The positive correlation of this computational leukocyte index with immune cell infiltration scored from H&E staining and the expression of lymphocyte-specific mRNAs/miRNAs validated our invasion model and further confirmed the use of this index as a surrogate for leukocyte abundance in muscle. The ability to distinguish between muscle-derived transcripts and genes over-expressed due to leukocyte infiltration may improve the interpretation of gene expression results and increase existing knowledge of molecular pathways critical for myositis.
In addition to the leukocyte index, we have also identified several other gene signatures (each from a consensus cluster with coherently expressed gene members): MHC class I, type 1 IFN, and immunoglobulin signatures. The type 1 IFN, MHC class I, and immunoglobulin signatures have been reported previously in various subclasses of myositis, in good agreement with the results in this study. Also, the current study is the first to systematically evaluate these genomic signatures together, which could provide important insight into the pathogenesis and potential therapeutic targets for myositis. Several transcripts displaying a good correlation with the leukocyte index and/or the type 1 IFN signature in muscle were also identified in this study, such as TTN, miR-1, miR-155 and amyloid beta (A4) precursor protein-binding, member B and member 1 interacting protein (APBB1IP). However, the small sample size and insufficient clinical information preclude us from evaluating the association of these molecular characteristics with the disease activity/severity within this study. Additionally, there was not adequate material to conduct extensive immunohistochemistry studies on patients in this study to correlate the presence of immunoglobulin and MHC class I protein levels with the corresponding gene signature scores. Continued effort to evaluate these molecular features in clinical studies where detailed clinical/demographic information is available will allow for further characterization of the utility of these potential biomarkers to aid in the patient stratification or to predict therapeutic efficacy.
In contrast to the positive correlation identified between the type 1 IFN signature and leukocyte infiltration index in PM and IBM, a positive association was not observed in most DM samples. One possible explanation is that the type 1 IFN signature in PM and IBM is mainly driven by leukocyte infiltration into muscle, while distinct drivers might be present in DM. Our results support the previous finding [14] that the type 1 IFN signature in DM might be connected with PFA (Figure 4), although a larger sample size is desired. Notably, this association between PFA and the type 1 IFN signature was also accompanied by the presence of the MHC class I signature (see Figure 2B, C). Additional studies need to be carried out in the future to confirm these observations.
The ability to distinguish the role of different cell types at disease sites is important in identifying anti-correlations between miRNA and mRNA expression, such as the expression of miR-29c and collagen genes (Additional file 2: Supplementary Text and Supplementary Methods) and other anti-correlations that may be important to the pathogenesis of myositis. Anti-correlations comparing mRNA and miRNA expression levels could also be complicated by the effect of increased leukocyte cell abundance, potentially masking true correlations between mRNAs/miRNAs in various disease settings. The positive correlation identified between miR-146a and the type 1 IFN signature in PM and IBM is one example where the increased number of leukocytes in muscle influenced the resulting gene expression comparison between myositis patients and normal subjects. Taken together, these results strongly support the application of the leukocyte infiltration index to transcriptomic studies to appropriately characterize altered mRNA and/or miRNA expression from heterogeneous biological samples, thus allowing for more meaningful interpretation of microarray data with the aim of increasing our understanding of the fundamental mechanisms of disease pathogenesis.
Evaluation of the effect of miR-146a in the suppression of the type 1 IFN pathway in myositis muscle revealed an interaction that was confounded by leukocyte infiltration. Nevertheless, our results indicated that a subset of DM patients had high type 1 IFN signatures and low miR-146a expression in muscle. Additional in vitro results showed that altered miR-146a levels in muscle cells could regulate the activation of the type 1 IFN pathway. Due to the small number of myositis samples displaying an anti-correlation between miR-146a and the type 1 IFN pathway and the lack of in vivo studies confirming this relationship, the exact functional role of miR-146a in myositis remains uncertain. However, continued work on characterizing this relationship in myositis is warranted.
In this study comparing transcriptional changes between myositis muscle biopsies and normal controls, we identified several gene signatures, including a leukocyte index and a type 1 IFN signature, as genomic biomarkers to characterize myositis subjects at the molecular level. Additionally, use of the leukocyte index to account for transcriptional changes due to increased lymphocyte infiltration into muscle revealed that the majority of these changes are driven by increased leukocyte abundance. Investigation into the relationship between leukocyte infiltration and a type 1 IFN signature suggested that increased ISGs might have two distinct sources at the myositis disease site: one is closely associated with leukocyte infiltration in IBM, PM and some DM; and the other source is not certain, but may be due to MHC class I-expressing myofibers, especially in some DM patients. Further understanding of the relationship between these signatures in vivo, as well as any association with myositis disease severity or activity will be explored in future clinical studies to evaluate their utilities as biomarkers. The ability to differentiate the transcriptional alterations due to an increased proportion of inflammatory cells from other changes at the expression level increases our ability to connect changes in specific cellular subsets with important biological phenomena critical for disease development or progression.
Patients and tissue samples
Muscle samples from 36 patients (5 normal controls, 5 NM, 8 DM, 8 PM and 10 IBM) were studied (Table 1). Normal controls are those subjects who were not suspected clinically to have neuromuscular disease; had normal muscle strength by examination; and showed normal serum CK levels. Diagnostic criteria for IBM, DM, and PM were as previously described [11]. Patients with NM had an acute or subacute myopathy responsive to immunotherapy, with pathology showing necrotic muscle fibers without inflammatory cells other than macrophages. Myositis-specific antibodies may also be highly predictive of the clinical pheno-subtype of myositis; however, these data were not available for the patients utilized in this study and could not be incorporated into the overall phenotyping of the samples.
Open muscle biopsies were performed at the time of a diagnostic biopsy (the biopsy site is listed in Table 1) and immediately frozen in liquid nitrogen for RNA and miRNA. A separate piece of muscle was used for hematoxylin and eosin (H&E) staining. Patients provided informed consent for research and institutional review boards approved all studies.
Hematoxylin and eosin (H&E) staining and assessment
H&E stained frozen sections were examined microscopically by a single examiner blinded to diagnosis. The degree of inflammation graded between 0–4 (0=no inflammation, 1=sparse inflammation, 2=mild inflammation, 3=moderate inflammation, and 4=severe inflammation).
Total RNA extraction
miRNA-RNA was extracted from muscle biopsies using the mirVana miRNA Isolation kit (Applied Biosystems/Ambion, Austin, TX), according to manufacturer's instructions.
mRNA profiling by Affymetrix microarray
Small species RNAs (e.g. miRNA, snRNA, tRNA) were removed from a volume of each miRNA-RNA sample using Agencourt RNAclean magnetic beads (Beckman-Coulter, Brea, CA). The resulting total RNA was profiled using Affymetrix Human Genome U133 plus 2.0 GeneChips® (Affymetrix, Santa Clara, CA). A selection of genes with high expression intensities identified by microarray were validated by quantitative Real-Time PCR (Additional file 2: Supplementary Methods; Additional file 2: Figure S4).
miRNA profiling by ABI TaqMan Low-Density Array (TLDA)
miRNAs were profiled using TLDA microRNA Cards v1.0 (Applied Biosystems, Foster City, CA). Single-stranded cDNA synthesis from 100 ng of total miRNA-RNA and pre-amplification of specific miRNA targets was performed according to the manufacturer's protocol. The microRNA cards were loaded and run on an Applied Biosystems 7900HT Real-Time PCR system.
Normalization of microarray and TLDA expression data
Probe-level summaries of microarray expression data were calculated using fRMA, which was implemented in R Bioconductor (http://bioconductor.org webcite). The microarray expression data and the normalized expression data matrix were deposited at the NCBI Gene Expression Omnibus (accession number GSE39454). For quantitative RT-PCR, data analysis of Ct values was conducted with SDS v2.2.2 software (Applied Biosystems). All samples were normalized to the mean Ct value of the endogenous control transcript RNU48.
Statistical analysis
All statistical analyses were performed using R (http://www.r-project.org webcite). Two group t-test was applied to identify differentially expressed mRNA/miRNAs in myositis muscle biopsies compared to the normal controls. The empirical Bayes method (from Bioconductor limma package) was also employed to identify myositis subclass-specific expression patterns [37]. In both of the overall and subclass-specific comparisons, the transcripts with average fold change > 4 and adjusted p-value for Benjamini-Hochberg < 0.05 are considered significant.
Invasion model
Measurements of gene expression from heterogeneous samples are typically confounded by the abundance of the constituent cell types, which generally can be formulated as a linear model. In this study, we considered a special case of the linear model, where the proportion of a certain subset t of cell types increases significantly in a certain condition (e.g., the disease state) compared to a normal condition. We named this model the invasion model. On the basis of some simple interpolation (see Additional file 2: Supplementary Methods for details), the invasion model characterizes several valuable outcomes when the special case holds: 1) variations of the gene expression will result predominantly from the alterations in proportions of the cell type t; 2) the t cell type-specific gene will be co-overexpressed even if there is either no or negative correlation between gene expression; 3) the average of the fold changes of the t cell type-specific gene expression is a good indicator of the increase in the fraction of the cell type t in a state change, for example, from the normal state to a disease state. The portions of invading cell types (or subsets) may change in distinct patterns and thus multiple cell type-specific gene signatures will be identified in that case.
The invasion model can be readily applied to myositis to assess the leukocyte infiltration into the disease site. Abundance of leukocytes is low in normal skeletal muscles and may increase dramatically in inflammatory myopathies. Given the existence of some mRNAs and/or miRNAs predominantly expressed in leukocytes, we should observe strong concordance among the overexpression of those particular protein-coding and/or non-protein coding transcripts. In practice, constant expression of those transcripts is not a requisite in the invasion model as long as the variation of the expression is minor relative to the variation of the lymphocyte cell abundance due to the infiltration, according to the equation (5) in Additional file 2: Supplementary Methods. It is anticipated that the leukocyte-specific transcripts should display "coherent" overexpression compared to the normal muscle biopsies and form one or more large clusters. As a result, the overexpression of those mRNA/miRNAs could serve as a gene signature to quantify the level of the infiltration of one or multiple subtypes of dominant leukocytes in myositis muscle.
Consensus clustering and gene signatures
A consensus clustering approach was employed to determine the proper cluster numbers and the robust members within each cluster by using R clusterCons package [40]. Multiple clustering algorithms (including agglomerative nesting clustering (agnes), divisive analysis clustering (diana), k-means, partitioning around medoids (pam), and hierarchical clustering (hclust)) were applied with a bootstrapping approach. The clustering results were further merged to identify the consensus clusters and their robust members. The membership robustness of each gene ranged from 0 to 1, defined as the average connectivity between a gene and all other members of the cluster [40]. Thus, a robust member, by definition, should have a high score (i.e., close to 1). We empirically used 0.6 as the cutoff value of membership robustness and took any cluster with at least five robust members to define the gene signature. For each cluster, a gene signature was defined as the median of the fold changes of the robust members relative to the normal controls.
To reduce the scale of the clustering problem and exclude uninformative genes, we applied the clustering only on a subset (i.e., ~200) of the differentially expressed genes that had the highest expression variance across subjects as recommended by the clusterCons developers [40].
Functional analysis of differentially expressed mRNAs
The differentially expressed transcripts are annotated in multiple ways: Ingenuity Pathway Analysis Tool (IPA; Redwood City, CA), hypergeometric enrichment of gene ontology (GO) terms, KEGG pathways (using Bioconductor GOstats package) and the gene sets from MSigDB (Molecular Signatures Database, http://www.broadinstitute.org/gsea/msigdb/index.jsp webcite). IPA microRNA Target Filter is also utilized to explore the likely interaction between differentially expressed mRNAs and miRNAs (see Additional file 2: Supplementary Methods; Additional file 8: Table S7).
Additional file 8. Table S7. miRNA-target mRNA pair candidates.
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C2C12 cell maintenance and transfection
C2C12 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultivated in recommended media at 37°C in a humidified atmosphere with 5% CO2. Artificial miR-146a mimics (Dharmacon) and miR-146a inhibitors (Dharmacon), as well as the appropriate scrambled controls, were transfected into C2C12 cells with PrimeFect siRNA (Lonza) according to manufacturer's protocol. 24 hours after transfection, cells were stimulated with mouse IFN-alpha (PBL interferon source) for 4 hours before RNA was isolated and profiled for alterations in IFN-inducible genes.
Competing interests
The work described in this article was supported by MedImmune, LLC. WZ, KS, BWH, CM, LG, KR, LR, BJ and YY are full-time employees of AstraZeneca/MedImmune. NS and SAG have served as consultants for AstraZeneca/MedImmune. There is no significant financial or other competing interest in the work.
Authors' contributions
YY, NS, and SAG designed research; AAA consented subjects and performed the biopsies; KS, CM, LG, SAG performed research; WZ, BWH analyzed data; and WZ, KS, BWH, KR, LR, DF, BJ, SAG, YY wrote the paper. All authors' read and approved the final manuscript.
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All tax information is summarized by KPMG Alpen-Treuhand AG Wirtschaftsprüfungs- und Steuerberatungsgesellschaft (Wien), the Austrian member firm of KPMG International, based on the Austrian Tax Book 2014 presented by the Austrian Federal Ministry of Finance.
When are tax returns due? That is, what is the tax return due date?
In general, income tax is assessed for the calendar year on the basis of an individual's tax return, which should be filed by 30 April (if a paper version is filed) or 30 June (if filed electronically), respectively 30 September, in case salary is taxed via payroll, of the following year. An automatic extension up to 31 March of the next following year is granted if the individual is represented by a tax adviser. Further extensions are available on request in special circumstances.
What is the tax year-end?
31 December.
What are the compliance requirements for tax returns in Austria?
Generally, a tax return is required if the individual's taxable income exceeds EUR11,000. Income tax on employment income is generally withheld at the source. Still, a tax return is required in case the individual has additional income, not previously subject to employer withholdings, in excess of EUR730. Please note that for foreign income from investment a threshold of only EUR22 applies.
Non-resident individuals must file an income tax return whenever they have taxable income in excess of EUR2,000 from a taxable source, unless the withholding tax applied represents the final settlement of the tax liability. For calculation of the tax rate the income will be increased by a hypothetical figure of EUR9,000.
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What are the current income tax rates for residents and non-residents in Austria?
In the case of unlimited tax liability, the tax brackets with progressive tax rates for 2014 are as follows:
Income tax table for 2012
Taxable income bracket
The Austrian tax law foresees a beneficiary tax rate (flat tax rate of 6 percent) for separate/irregular payments (such as bonus payments). The maximum amount of the tax-favorable payment is one-sixth of the total annual regular payments. Therefore, and also due to collective agreements, it is very common to pay the yearly salary in 14 installments, 12 current payments (one each month) and a holiday and a Christmas payment as bonus.
Otherwise to calculate the bonus, it would be most tax-effective, to pay one-seventh of the total yearly gross compensation as bonus. Please be aware, that there are several prerequisites to apply the one-seventh rule. For the years 2013 to 2016 a so called solidarity surcharge was implemented with tax rates for special payments from 0% - 50% depending on the amount of special payments and the total employment income.
Please note that it is very important that the special payments are actually paid separately. It is not possible to calculate them only hypothetically.
The income tax is reduced by the following deductions:
* There is a phase out for this deduction from EUR17,000 – EUR25,000. For pensioners with regular pension income of below EUR19,930 and who are sole-earners without a child, an increased deduction of EUR764 could be claimed. The increased deduction is phased out between EUR19,930 – EUR25,000.
** Increased to EUR669 with two children and additionally EUR220 for every additional child.
Furthermore, transfer payments per month are granted for each child entitled to a child allowance (EUR58.40 for each child). The child allowance is paid together with the following family allowance and is tax-free:
The total amount of family allowance received is augmented by EUR50 for every further child.
In July 2014 the family allowance will be increased by 4%.
Starting in 2011, an addition in the amount of EUR100 to the family allowance is paid in September for children between 6 and 15.
Income tax rates for non-residents are the same as for residents, but the hypothetical amount of EUR9,000 has to be added to the actual income. However, withholding tax rates may differ. Furthermore, various tax deductions are not available.
For the purposes of taxation, how is an individual defined as a resident of Austria?
An individual is deemed to be resident in case he/she either maintains any kind of accommodation, or is physically present, under circumstances indicating that his/her abode will not be temporary.
As a general rule, tax residence is deemed to exist if the individual's stay in Austria exceeds six months (183 days). Once these six months have expired, tax residence is deemed to have commenced at the beginning of the stay in Austria. Citizenship is not relevant in determining residence.
Is there a de minimus number of days rule when it comes to residency start and end date? For example, a taxpayer cannot come back to the host country for more than 10 days after their assignment is over and they repatriate.
What if the assignee enters the country before the assignment begins?
It is possible to have a period of limited and a period of unlimited tax liability. If business trips occur before Austrian residence is taken, these days are not allocated to the assignment.
Furthermore in terms of labor law, necessary preparations for the assignment are permitted such as apartment-hunting.
Are there any tax compliance requirements when leaving Austria?
The taxpayer's tax office should be notified of a termination of residence.
What if the assignee comes back for a trip after residency has terminated?
Depending on the purpose of the trip (business or vacation) and on the employer (foreign or Austrian), the income will be prorated and allocated to Austria. Furthermore, it is possible to have limited tax liability during this period.
Communication between immigration and taxation authorities
Do the immigration authorities in Austria provide information to the local taxation authorities regarding when a person enters or leaves Austria?
Usually not.
Filing requirements
Will an assignee have a filing requirement in the host country after they leave the country and repatriate?
The assignee may have a filing requirement after repatriation if he/she still receives any compensation in connection to his/her ended assignment (such as Austrian tax payments paid by employer).
Do the taxation authorities in Austria adopt the economic employer approach to interpreting Article 15 of the OECD treaty? If no, are the taxation authorities in Austria considering the adoption of this interpretation of economic employer in the future?
Up to now, the Austrian Tax Authorities recognize the legal employer approach, which means that the employer must fulfill typical employer functions which can be checked by an edict of the Austrian Ministry of Finance. If the receiving host company fulfills these requirements, it may be considered as the economic employer.
Nevertheless, there has been a switch to the economic employer approach in 2013 due to decisions of the Higher Administrative Court. In the first quarter of 2014, we are expecting changes in the guidelines and practice of the Austrian Tax Authorities.
De minimus number of days2
Are there a de minimus number of days before the local taxation authorities will apply the economic employer approach? If yes, what is the de minimus number of days?
What categories are subject to income tax in general situations?
All types of remuneration and benefits received by an employee for services rendered under an employment agreement are deemed to be taxable income, regardless of where they are paid and by whom. The following typical items of an expatriate compensation package are fully taxable unless otherwise indicated.
An employer contribution to rent is taxable as income. Free or below-market-value use of accommodation provided by the employer is taxable as income. The benefit is initially valued at fixed monthly rates per square meter, determined according to category of accommodation and year of construction.
This notional rent is compared with the actual rent paid reduced by 25 percent. The taxable value is the larger of the two figures.
Are there any areas of income that are exempt from taxation in Austria? If so, please provide a general definition of these areas.
Moving expenses
Moving expense reimbursements are generally not taxable if the actual expenses were reimbursed and the employee was transferred within the same enterprise or between group-related companies (if relocation is solely in the interest of the entrepreneur).
Moving bonus
If certain requirements are met, a moving bonus of up to one-fifteenth of the total annual salary may be granted tax-free.
Per diems
Employer may pay tax-free per diems up to certain limits.
Are there any concessions made for expatriates in Austria?
There is only one expatriate concession, which allows the employer to exempt specified expenses (double housing expenses, lump sum school tuition fee, and moving expenses) up front from payroll withholding tax. Several requirements have to be met.
Is salary earned from working abroad taxed in Austria? If so, how?
An Austrian resident is taxed on his/her worldwide income in Austria, however there may be an exemption according to double tax treaties.
Are investment income and capital gains taxed in Austria? If so, how?
Austrian residents are subject to income tax on their worldwide income derived from capital and investment, including dividends, interest, profit shares of dormant partners, rents and royalties, capital gains.
Non-residents are subject to tax on income derived from Austrian sources, such as dividends from Austrian companies, interest on loans secured by mortgages on real estate located in Austria, of foreign dormant partners in Austria, rents and royalties from real estate located or patents utilized in Austria, transactions involving sales of Austrian real estate.
Dividends, interest, and rental income
Dividends and interest received by an Austrian resident recipient are subject to a withholding tax of 25 percent.
Interest income as well as dividend income derived from bank deposits and fixed interest-bearing securities subject to the 25 percent withholding tax are not subject to income tax at the recipient (final taxation). Furthermore, any obligation to pay any inheritance tax that might accrue on this income (also gift tax from 1 June 2001 until 31 December 2003), is deemed to be discharged. This final taxation only applies if the recipients are individuals (partners of partnerships included).
If the dividend or interest is paid by an agency abroad, no Austrian withholding tax can be withheld. In this case the income has to be reported in Austrian income tax return and a flat tax rate of 25 percent can be applied.
Income from investment funds is treated differently depending on whether the fund is an Austrian or a foreign fund and whether it is considered to be white (registered and represented) or black (neither registered, nor represented).
Basically the taxation of foreign funds is the same as of Austrian investment funds.
A special taxation regime exists for black funds, which are taxed on a lump-sum basis. Besides the actual payments, 90 percent of the difference between the redemption price at the beginning and the end of the year (at least a minimum of 10 percent of the last redemption price) are taxable.
For all types of foreign investment funds deposited at an Austrian bank, a special withholding tax (Sicherungssteuer) is applicable. Fifteen percent of the last redemption price has to be withheld in case the taxpayer does not confirm tax registration to the bank.
Rental income from properties located in Austria will be taxed at normal rates. Rental income from foreign countries may be considered as (progression) income depending on the respective double tax treaty.
The sale of private assets is taxed as follows:
The sales gain (difference between the sales proceeds and the purchase price) should be subject to capital gain tax under the following circumstances:
A loss on the sale of shares does not trigger capital gain tax, but may be available to offset against other capital gains made during the tax year.
Rental income from properties located in Austria will be taxed at normal rates. Rental income from foreign countries may be considered as (progression) income depending on the respective double tax treaty.
Gains from stock option exercises
The value of a stock option, granted in respect of services rendered in Austria, is included in the taxable income, regardless of when the option is exercised. The taxable value of an option is the difference between the fair market value of the shares at the date of exercise and the acquisition costs paid by the employee.
However, the Austrian tax law foresees some tax benefits which are granted if certain restrictions are met.
The following dates of taxation apply to restricted stock options:
Taxable at:
Foreign exchange gains and losses
The Austrian income tax return has to be filed in Euros. So gain or loss in a foreign currency resulting from a source of income is included in the tax return and taxed at the normal tax rates.
Principal residence gains and losses
Since April 1, 2012, Austria has implemented a taxation regime on the sale of non-business property with grandfathering-rules.
Taxation of "new property":
In case the real estate would still have been qualified as "speculative" on March 31, 2012 (eg the ten years holding period was not over), or the real estate is bought after that date, the sale of this real estate is always considered as taxable event on which a special tax rate of 25% can be applied.
Taxation of "old property":
The sale of real estate that did not fulfill the requisitions of speculative transaction on March 31, 2012 (eg as it was held for more than 10 years) is now also considered as a taxable income on which a special tax rate of 25% can be applied. There is the possibility to apply a lump-sum taxation on property which has not been rededicated: 14% of the sales price can be taxed at 25% tax rate (effective tax burden of 3,5%).
Since April 1, 2012 two tax exemptions for the sale of a real estate which was used as primary residence are applicable:
Capital losses
Capital losses may only be set off against capital gains within the same year and cannot be carried forward.
Personal use items
The sale of personal use items is taxable if sold within one year after acquisition and certain tax free amounts are exceeded.
Following a decision of the Austrian constitutional court, inheritance and gift tax are abolished from 1 August 2008 onwards.
However, in order to prevent avoidance of income tax it is required to report gifts over EUR15,000 within five years (EUR50,000 within one year for relatives) to the tax authorities. There is a penalty of up to 10 percent of the value if the report was not done deliberately.
Are there additional capital gains tax (CGT) issues in Austria? If so, please discuss?
Currently, the Austrian Ministry of Finance is discussing a concept of wealth tax. However, it is uncertain if and when this concept will apply.
Are there capital gains tax exceptions in Austria? If so, please discuss?
Pre-CGT assets
Not applicable
Deemed disposal and acquisition
What are the general deductions from income allowed in Austria?
There are only minor general standard deductions for income-related expenses, such as the general standard deduction from taxable employment income of EUR132. Non-recurring payments of salaries and wages, such as 13th and 14th month salary payments and bonus payments, enjoy a tax-free allowance of EUR620 per year in aggregate; a tax deduction of EUR54 from employees and EUR291 (note that this is a reduction of the tax due) are granted as a flat refund of expenses for clothing, and so on and for travel between the residence and the place of employment. If the distance between the private residence and the place of employment exceeds certain limits, additional standard amounts can be deducted from taxable income and from the computed income tax (different commuter deductions).
The following items can also reduce taxable income.
What are the tax reimbursement methods generally used by employers in Austria?
From KPMG Austria's experience, net agreements are usually concluded. In case payroll withholding is obligatory, the net salary is grossed up. Otherwise, the net income is declared in the income tax return and the assessed tax is paid by the employer in the following year. Since the tax payment is qualified as benefit, it has to be added to the income of the respective year.
How are estimates/prepayments/withholding of tax handled in Austria? For example, Pay-As-You-Earn (PAYE), Pay-As-You-Go (PAYG), and so on.
Basically, there is the obligation for employers to withhold payroll tax from their employees and to forward it to the responsible tax office. If there is no permanent establishment for the payroll administration, which could withhold the wage tax or there is no withholding obligation for other reasons, the tax is paid to the tax office by the individual himself/herself.
If tax return filing is applicable, quarterly prepayments of the expected tax have to be made.
When are estimates/prepayments/withholding of tax due in Austria? For example, monthly, annually, both, and so on.
Taxes are collected during the tax year either by the tax office or through withholding at the source. Prepayments must be effected by 15 February, 15 May, 15 August, and 15 November each year for income tax purposes. These payments are generally based on the prior year's tax. Payroll tax will be withheld by the employer and paid monthly (until the 15th of the following month) to the financial authorities.
Withheld or prepaid taxes are credited against the final tax liability. If the taxpayer's final tax liability exceeds the aggregate of the amounts already withheld or paid, tax is refunded. The final tax is payable within one month after the tax assessment.
Is there any Relief for Foreign Taxes in Austria? For example, a foreign tax credit (FTC) system, double taxation treaties, and so on?
Austria has concluded about 90 double taxation treaties based on the OECD model tax convention, thus providing relief against double taxation to taxpayers resident in the contracting states.
In the absence of a tax treaty, the Austrian Income Tax Act grants credit or exemption under certain circumstances in relation to actual double taxation.
What are the general tax credits that may be claimed in Austria? Please list below.
There are only some tax credits, which may be claimed against the taxpayer's tax liability. Several requirements have to be met:
This calculation assumes a married taxpayer resident in Austria with two children whose three-year assignment begins 1 January 2014 and ends 31 December 2016. The taxpayer's base salary is USD100,000 and the calculation covers three years.
Exchange rate used for calculation: USD1.00 = EUR0.75.
Other assumptions
Calculation of taxable income (EUR)
Calculation of tax liability (EUR)
1Certain tax authorities adopt an 'economic employer' approach to interpreting Article 15 of the OECD model treaty which deals with the Dependent Services Article. In summary, this means that if an employee is assigned to work for an entity in the host country for a period of less than 183 days in the fiscal year (or, a calendar year of a 12-month period), the employee remains employed by the home country employer but the employee's salary and costs are recharged to the host entity, then the host country tax authority will treat the host entity as being the 'economic employer' and therefore the employer for the purposes of interpreting Article 15. In this case, Article 15 relief would be denied and the employee would be subject to tax in the host country.
2For example, an employee can be physically present in the country for up to 60 days before the tax authorities will apply the 'economic employer' approach.
3Sample calculation is generated by KPMG Alpen-Treuhand AG Wirtschaftsprüfungs- und Steuerberatungsgesellschaft (Wien), the Austrian member firm of KPMG International, based on the Austrian Tax Book 2014 presented by the Austrian Federal Ministry of Finance.
© 2014 KPMG Alpen-Treuhand AG Wirtschaftsprfungs - und Steuerberatungsgesellschaft (Wien), an Austrian limited liability company and a member firm of the KPMG network of independent member firms affiliated with KPMG International Cooperative (KPMG International), a Swiss entity. All rights reserved.
© 2015
KPMG International Cooperative ("KPMG International"), a Swiss entity. Member firms of the KPMG network of independent firms are affiliated with KPMG International. KPMG International provides no client services. No member firm has any authority to obligate or bind KPMG International or any other member firm vis-à-vis third parties, nor does KPMG International have any such authority to obligate or bind any member firm. All rights reserved.