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I have been seeing some really great pizza stick recipes on my timeline on Facebook. They use string cheese in them. Since string cheese can get kinda pricey it may be a good time to pick these up at Kroger. Checkout the following deals you can grab…
At Kroger:
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use one $2/1 Turkey Hill All Natural Gelato or Gelato Swirls 32 oz RP 7/24
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use one $1/1 Tasty Bite Product printable coupon
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use one $.55/1 Sargento Natural String or Stick Cheese Snack 4.5 oz or larger printable coupon (preclipped)
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This high resolution camera allows you to capture movement and still gives you pristine images. The lens focuses and zooms automatically thereby enabling you to easily identify intruders or suspicious vechiles with their number plates. Suitable for warehouses, petrol stations or parking lots. With a range of 35 metres and a weather-proof body, this camera is the the ideal deterrent.
This is our most advanced DVR. This DVR gives you all the facilities you need when it comes to surveillance. Super imagery and real-time recording, it does not miss a shot!! Built-in DVD writer and USB port allows easy storage. If you want quality, this is the best when it comes to performance.
The design of the product shown may change without prior notice. The specification is not affected.

subset: commoncrawl


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We are always focused on the latest development of reliable testing technologies, and also value the importance of upgrading the technologies of the equipment. Requirement for "Environment Simulation" also helps the clients to realize the importance of vibration platform and test chambers provided by supplier. We can provide broad range of combined environment testing systems according to the clients' requirements. For example: 3 to 4 integrated testing setup scheme (air-cooled vibration test system complimented by low pressure test chamber), multi-axis vibration test and latest testing technologies.
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Model Mayhem Admin
HellonHighHeelsModel Female Palmdale, California, US
Mayhem # 595131
About Me
Hi my name is Kristin im 19 and im from Palmdale, CA. I just redently kind of fell into modeling, hooked up with a couple local photographers to my area who liked my look. I like to consider myself a cross between a modernized Elvira mistress of the Dark and Bettie Page. I love pin ups, 1940's and 50's glamour, and anything dark. Modeling is more of a hobby of mine right now do to my working and school schedule but I would absolutely love to turn it into more of a career. I idolize people like sabina kelley, masuimi max, and the beautiful dita von teese and one day i hope i can be on their level.
Check out my Myspace www.myspace.com/ushouldntkissmelikethis
Have you worked with HellonHighHeels? Add Credits for HellonHighHeels! >>
Credit Notes
Greaser Tom- Pinup Shots Photography, Lancaster, CA
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text:
Obama Mocks House Dem — But Ends Up Looking Like Jerk
Michigan Democratic Rep. Dan Kildee has been in Congress for a little more than two months, and today the leader of the free world gave him a gentle ribbing about just how new he is to Capitol Hill.
At a closed-door meeting between President Barack Obama and House Democrats, Kildee introduced himself as a freshman, to which Obama said, "Wow, you're really classing up the place then."
Kildee then proceeded to ask what one person described as a somewhat "long-winded" question about the importance of creating jobs for his economically distraught district.
http://www3.hoh.rollcall.com/a-kildee-comeuppance-or-more-obama-aloofness/1 views
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text:
Frequently Asked Questions
What are your store hours?
Where are you located?
Do you carry SAS?
What is your return policy?
Do you dye shoes?
Do you do outside fittings?
Do you have shoes that will accommodate my orthotics?
How do I know my size?
Frequently Asked Questions
What are your store hours?We are open Monday - Friday, 9:30 am - 8:00 pm, Saturdays 9:30 am - 5:30 pm, and Sundays 12 pm -5 pm. We are open 24/7 on the world wide web.
Where are you located?We are located at 1880 Henderson Road, in the Northwest Center, at the northwest corner of Reed and Henderson Road, 2 miles west of 315 and 1 mile east of Sawmill Road in the northwest portion of Columbus. Our store is between the Lifestyle Family Fitness and The Fresh Market.
Do you carry SAS?We handle the full line of SAS products. Visit our store or www.sasshoes.com to view all shoes and handbags in production, including size ranges and colors for each product.
What is your return policy?Unworn shoes may be returned within 14 days of purchase for like credit or exchange if in original box or package with original receipt. All sale items shall be treated as final sales. No refunds on non-shoe items. For any other inquiries please refer to our policies page.
Do you dye shoes?We dye fabric shoes made of satin, luxe or other dyeable materials in our dyeable lab for your special occassion. We can fulfill your entire dyeable needs with shoes and handbags to match.
Do you do outside fittings?Yes, within our geographical area. We will send one of our expert shoe fitting professionals to your residence. Please call for details.
Do you have shoes that will accommodate my orthotics?Yes, most of our shoes are constructed with removeable footbeds to accommodate orthotics and our staff is highly trained in fitting orthotics to our shoes. Bring your orthotics with you for your fitting.
How do I know my size?Our shoes are sized in American, European and UK sizing. Each manufacturer fits differently and on a particular size scale. Please email or call us before ordering and we will help you determine your correct size, or stop in and we will fit you the the proper size.


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Music Emotion Recognition
by Yi-Hsuan Yang, Homer H. Chen
English | 2011 | ISBN: 1439850461 | 261 pages | PDF | 3.23 MB
Providing a complete review of existing work in music emotion developed in psychology and engineering, Music Emotion Recognition explains how to account for the subjective nature of emotion perception in the development of automatic music emotion recognition (MER) systems. Among the first publications dedicated to automatic MER, it begins with a comprehensive introduction to the essential aspects of MERвЂ"including background, key techniques, and applications.
This ground-breaking reference examines emotion from a dimensional perspective. It defines emotions in music as points in a 2D plane in terms of two of the most fundamental emotion dimensions according to psychologistsвЂ"valence and arousal. The authors present a computational framework that generalizes emotion recognition from the categorical domain to real-valued 2D space. They also:
Introduce novel emotion-based music retrieval and organization methods
Describe a ranking-base emotion annotation and model training method
Present methods that integrate information extracted from lyrics, chord sequence, and genre metadata for improved accuracy
Consider an emotion-based music retrieval system that is particularly useful for mobile devices
The book details techniques for addressing the issues related to: the ambiguity and granularity of emotion description, heavy cognitive load of emotion annotation, subjectivity of emotion perception, and the semantic gap between low-level audio signal and high-level emotion perception. Complete with more than 360 useful references, 12 example MATLABВ® codes, and a listing of key abbreviations and acronyms, this cutting-edge guide supplies the technical understanding and tools needed to develop your own automatic MER system based on the automatic recognition model.
Download link:
(Buy premium account for maximum speed and resumming ability)
(Buy premium account for maximum speed and resumming ability)
Links are Interchangeable - No Password


text:
What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
Can a user view and edit form fields and view and delete uploaded files?Asked by jasondgray on January 04, 2013 at 05:01 PM
I would like to create a form whereby the user can view previous form submissions and make changes:
1) Change previous responses to questions
2) Change uploads - this would require the ability to delete the old one and upload a new file. For example, I would like the user to upload their performance review and if changes are made later that result in a revised performance review, they can delete/replace the old one through upload.
You can share submissions page in order to let your users view, print, edit or delete submissions (on the upper right corner buttons) or download the entire form submissions to Excel, CSV or even PDF.
To share your form's submissions page, please follow the guide below:1. Go to My Forms page and select the form that you'd like its Submissions to be shared.2. Click Submissions button3. While on submissions page, click the Gear Icon to open submissions page settings4. Check "Make This Page Public"5. Enter a Password for Submissions Page protection and copy the provided Submissions Page URL.6. Share! Note that you have to provide the password as well as the URL before your form users could access the submissions page.However, on regards to deleting uploads, and in order to keep a backup, you could also integrate the form with dropboxLet us know if we can be of further help
I've shared the public page with my client, but there doesn't seem to be a way for them to delete anything once they've downloaded the information. Is there a way they can do this?
heroweb, I can see that you have your own thread. Kindly stick to it so that we could assist you better and to avoid any confusion.
Thank you


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Bringing a sense of mystery to Elle Romania's September issue, photographer Sylvie Malfray captures cover girl Diana Dondoe wearing Chanel's Paris Shanghai collection. With the perfect balance of darkness and seduction, Diana dons the elegant stylings of Domnica Margescu for one tantalizing spread.
Lots of Chanel pre-fall pieces. Lovely
uninspired. But I guess everyone's gotta get a paycheck, so it could be worse.
So sexy…
-diary of a fashion stylist-
those jackets are amazing xoxox
Louise Petit
I want to like this piece, but her face looks plastic. I don't understand why people are so attracted to removing all skin detail in the human face and making models look plastic. What's the appeal? It looks obvious that the skin was removed. Do retouchers think people will better relate to plastic subjects? The removing of skin gives the appearance of sharp, unnatural jawlines, as in the first photo, the black and white one.
The thing is, sometimes the original photos, especially in fashion full body images and location, depending on lighting, etc., don't have that much texture to begin with, so it is not always that it is removed in post.
I love the Chanel bibnecklace in the first pic!!!
So Russian tsar style!!
xo from Milan, gaia
She looks lovely! Diana Dondoe is a fantastic model!
meh. it could be a lot worse.
she looks cross-eyed.
Like the clothes.
why is it uninspired?I think it's pretty good.All the clothes are from Chanel.They wanted to do it so.They could have chosen a better model but overall I think it's not bad at all.
i think i fell asleep…
Im loving the clothes and the location! but the model kills it a little for me…. the styling is great anyways


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We have used BG 3.0s for the last year to great sucess... but the last few months my son has gotten a recurring yeast infection. It clears up with lotrimin & disposables, but as soon as we put the BGs on him it is back.
We've washed the BGs every way we could imagine, over and over and over and over (vinegar, bleach, dawn stripping, just hot water, just cold water....), but to no avail. I don't want to stop cloth diapering him and we don't have the money to keep shelling out for disposables.
Anyone have this happen to you? Any advice for what we should do? I really want him back in his BGs.


text:
de Figueiredo, CMH, Klein, S and Vuskovic, K (2002) The graph sandwich problem for 1-join composition is NP-complete. Discrete Applied Mathematics, 121 (1-3). 73 - 82 . ISSN 0166-218X
A graph is a 1-join composition if its vertex set can be partitioned into four nonempty sets and SR such that: every vertex of AL is adjacent to every vertex of AR; no vertex of SL is adjacent to vertex of AR∪SR; no vertex of SR is adjacent to a vertex of AL∪SL. The graph sandwich problem for 1-join composition is defined as follows: Given a vertex set V, a forced edge set E1, and a forbidden edge set E3, is there a graph G=(V,E) such that E1⊆E and E∩E3=∅, which is a 1-join composition graph? We prove that the graph sandwich problem for 1-join composition is NP-complete. This result stands in contrast to the case where SL=∅ (SR=∅), namely, the graph sandwich problem for homogeneous set, which has a polynomial-time solution.
|The University of Leeds
|Academic Units:
|The University of Leeds > Faculty of Engineering (Leeds) > School of Computing (Leeds)
|Depositing User:
|Symplectic Publications
|Date Deposited:
|22 Jun 2012 10:17
|Last Modified:
|04 Nov 2016 00:35
|Published Version:
|Elsevier for North-Holland Publishing
|Identification Number:


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Development of a high-throughput method for the systematic identification of human proteins nuclear translocation potential
Important clues to the function of novel and uncharacterized proteins can be obtained by identifying their ability to translocate in the nucleus. In addition, a comprehensive definition of the nuclear proteome undoubtedly represents a key step toward a better understanding of the biology of this organelle. Although several high-throughput experimental methods have been developed to explore the sub-cellular localization of proteins, these methods tend to focus on the predominant localizations of gene products and may fail to provide a complete catalog of proteins that are able to transiently locate into the nucleus.
We have developed a method for examining the nuclear localization potential of human gene products at the proteome scale by adapting a mammalian two-hybrid system we have previously developed. Our system is composed of three constructs co-transfected into a mammalian cell line. First, it contains a PCR construct encoding a fusion protein composed of a tested protein, the PDZ-protein TIP-1, and the transactivation domain of TNNC2 (referred to as ACT construct). Second, our system contains a PCR construct encoding a fusion protein composed of the DNA binding domain of GAL4 and the PDZ binding domain of rhotekin (referred to as the BIND construct). Third, a GAL4-responsive luciferase reporter is used to detect the reconstitution of a transcriptionally active BIND-ACT complex through the interaction of TIP-1 and rhotekin, which indicates the ability of the tested protein to translocate into the nucleus. We validated our method in a small-scale feasibility study by comparing it to green fluorescent protein (GFP) fusion-based sub-cellular localization assays, sequence-based computational prediction of protein sub-cellular localization, and current sub-cellular localization data available from the literature for 22 gene products.
Our reporter-based system can rapidly screen gene products for their ability to be translocated to the nucleus. Large-scale applications of the system presented herein should provide invaluable information for a more complete biological atlas.
Mammalian nuclei are extremely dynamic organelles. They are structured into domains and contain numerous distinct architectural features related to their function
Nuclear protein import in mammalian cells requires soluble cytoplasmic co-factors
Important clues to the function of novel and uncharacterized proteins can be obtained by identifying the potential nuclear translocation of a protein
There are several high-throughput experimental screening methods used to examine the sub-cellular localization of proteins and their nuclear localization: gene trap screening
Here, we report the development of a reporter-based system to systematically analyze the nuclear translocation potential of proteins. Our system is based on a modification of our high-throughput mammalian two-hybrid system
Development of the nuclear translocation assay
We have developed a high-throughput assay to systematically identify a protein's potential for nuclear translocation according to the level of luciferase reporter activity (Figure
Figure 1
Schematic representation of the system
Schematic representation of the system. The pG5
Optimization of interacting partners in ACT and BIND constructs
A key feature of the system is the interaction of the ACT and BIND fusion proteins in the nucleus via domains A and B. This interacting pair, A and B, must satisfy the following criteria: 1) their interaction is well-characterized, 2) both domains are as small as possible so as not to be a limiting factor for the generation of fusion protein constructs containing large investigated CDSs, 3) the interaction is easily detected by the luciferase reporter expression, yet its affinity is weak enough that the ACT fusion proteins are seldom transported into the nucleus by associating with the BIND protein, 4) domain A does not possess any NLSs, and 5) domain B does not possess transactivation activity.
Satisfying criteria 1 and 2, we selected TIP-1 and rhotekin as the domains A and B, in which the reported interaction is mediated by the small domains, the PDZ domain of TIP-1 and the C-terminus sequence of rhotekin
Selection of the transactivation protein
We selected a transactivation domain (TA) to fuse to the TIP-1 PDZ domain that would 1) result in a small fusion protein and not interfere with the translocation potential of the added CDS, 2) possess a strong transactivation activity inducing the expression of the luciferase reporter, and 3) not induce translocation to the nucleus except when fused with a tested CDS possessing a NLS. We turned to our previous protein-protein interaction work in which we had systematically screened for protein self-activity: that is, a protein that when fused to Gal4 DNA-binding domain is able to interact with the transcriptional machinery and induce the expression of the reporter gene in the mammalian two-hybrid system
BIND construct and high-throughput ACT construct preparation
Each ACT construct bearing a CDS of interest was created by a two-step PCR reaction. The CDS of each target gene was amplified with specific forward and reverse primers (Figure
Additional file 1
Agarose gel electrophoresis of six ACT samples. (A) CDSs of six representative target genes with various ORF length were amplified by gene-specific forward and reverse primers and confirmed on 1.5% agarose gel (the first PCR). λ-
Click here for file
Figure 2
Strategy for the high-throughput
Strategy for the high-throughput
To generate BIND constructs, we employed a similar strategy; the DNA fragments for CMV-Gal4, and SV40 were amplified from the pBIND vector, purified, and used in an overlapping PCR to connect the DNA fragments of CMV-Gal4, Rhot20aa, and SV40 (Figure
Selection of cells and conditions for the assay
To test if CDSs of interest can translocate to the nucleus, we rely on the detection of the interaction between TIP-1 and Rhotekin (fused to the queried CDS), both of which can be expressed only transiently. Thus, the assay only requires the transfection of PCR products, which is a process that is easily automated and systematic. As a proof of concept, we tested the system using MT1M, a metallothionein protein annotated to predominantly localize in the nucleus, and SNX3, a member of the sorting nexin family involved in cytoplasmic trafficking of proteins. The ACT, BIND, and luciferase reporter constructs were transfected into the CHO-K1 cell line using lipofection. As we expected, we found that MT1M containing ACT constructs induced high reporter activity, while the induction of the luciferase reporter gene was marginal for the ACT construct containing SNX3 CDS (Figure
Figure 3
Selection of cells using a nuclear localized protein, MT1M, and a non-nuclear protein, SNX3
Selection of cells using a nuclear localized protein, MT1M, and a non-nuclear protein, SNX3. Reported values are luciferase activity; the error bars represent standard deviations. (A) MT1M and SNX3 were used to test the system using CHO-K1 cells. (B) Influence of cell lines was explored using hamster CHO-K1 cells and human HeLa cells.
Next we explored whether the type of cell line in which we performed our assay influenced the results. The ACT constructs for MT1M and SNX3, together with the BIND and luciferase reporter constructs, were transfected into the same number of CHO-K1 and HeLa cells. We observed that MT1M shows higher luciferase activity than SNX3 in both cell lines although CHO-K1 cells shows higher luciferase counts than HeLa cells (Figure
Large proteins generally translocate to the nucleus more slowly than smaller ones. We therefore evaluated the adequacy of incubating for 20 hours post-transfection before lysis of cells in the luciferase reporter assay (see Additional file
Additional file 2
Effect of incubation time on the readout. Three assay-positive constructs with various protein sizes (NANOG (305 aa), ARNT2 (717 aa) and CRIP1 (77 aa)) were selected from Additional File
Click here for file
Next, we investigated if the presence of a strong nuclear exclusion signal affected the assay read-out (see Additional File
Additional file 3
Effect of NES addition on nuclear localized proteins. (A) We fused two assay-positive proteins (NANOG and ELK1 in Additional File
Click here for file
Additional file 4
Sub-cellular localization of the tested proteins in the in vivo assay. Table summarizing the tub-cellular localization of the tested proteins in the in vivo assay
Click here for file
Figure 4
Small-scale validation of the assay
Small-scale validation of the assay. Luciferase-based nuclear translocation assay and GFP-fusion nuclear localization assay were compared for 22 constructs. Histogram represents the log10 of the average luciferase ratio for three independent assays. Error bars are standard deviation. The black line represents the 5-fold threshold above which a given construct is qualified as able to translocate into the nucleus; histograms in blue highlight positive luciferase results and those in orange negative results. The (#) and (x) signs, respectively, highlight the false-positive and false-negative results when compared to GFP-fusion-based nuclear localization. A representative picture of the GFP-fusion assay with blue DAPI straining and green GFP is positioned under each tested construct. The red line and error bars represent the ratio of GFP intensity in the nucleus to that of the cytoplasm computed from the GFP-fusion-based nuclear localization images. Values are also summarized in Additional File
Small-scale validation of the assay
To test the ability to detect the translocation of proteins in the nucleus, we analyzed two sets of genes with nuclear localization reported in HPRD
We considered assays reporting an average 5-fold ratio of the luciferase signal with the BIND construct co-transfected to luciferase signal without the co-transfected BIND construct to represent confident nuclear translocation potential, based on empirical results. Eight of the 22 genes were observed exclusively in the cytoplasm, 5 were exclusively in the nucleus, and 9 were diffusively localized both in the cytoplasm and the nucleus when GFP fusions were transiently expressed in CHO-K1 cells. Our mammalian two-hybrid derived assay was designed to detect the nuclear translocation potential of a CDS; therefore, we considered GFP sub-cellular localization assay reporting diffuse localization of encoded fusion protein both in the cytoplasm and the nucleus to represent true positive results. Though the luciferase ratio was 5.30 (± 1.08), LGALS4-GFP fusions localized exclusively in the cytoplasm. Therefore, compared to the GFP sub-cellular localization assay, the false-positive rate was 7% (1/13). Reciprocally, while DLX6 and TLX2 appeared to be located in the nucleus when fused to GFP, the luciferase ratios of those two genes were only 3.77 (± 0.42) and 4.4 (± 0.53), respectively. As a result, we can conclude that compared with a GFP sub-cellular localization assay, our system performed with a false-negative rate of 22% (2/9).
We also used the program PSORT II to predict the sub-cellular localization of those 22 genes and compared the most probable localization reported by the program to our assay. Again, although we predicted from the results of our assay that DLX6 and TLX2 are unable to translocate to the nucleus, PSORT II predicts them to be nuclear proteins, thus yielding a 22% (2/9) false-negative rate when compared with computational predictions. Four proteins with luciferase ratios ranging from 5.2 (± 0.79) to 7.9 (± 2.27) were predictd by PSORT II to be cytoplasmic proteins which results in a false-positive rate of 30% (4/13).
Sub-cellular localization annotations reported in HPRD agreed poorly with our assay. Under the 5-fold luciferase signal threshold that we used to define proteins as able or not to translocate to the nucleus, our comparison of the reporter-based system with the HPRD annotations showed a 46% (6/13) false-positive rate and a 55% (5/9) false-negative rate. It is important to note that this poor false-positive rate was in large part due to proteins for which our assay gave results that were very close to the 5-fold threshold we defined; 4 out of 6 false-positive results arose from luciferase assay in the 5.13 (± 1.54) to 5.33 (± 0.8) range. Thus, under a stricter definition of the cut off for which a protein is considered to be able to translocate into the nucleus, comparisons of our assay to HPRD annotations would result in a more reasonable 14% false-positive rate. Additionally, our observations of CRIP1 nuclear localization in the GFP-fusion and luciferase reporter-based assays as well as PSORT II prediction contrasts with the lack of nuclear annotation noted for CRIP1 in HPRD. Similarly, the relatively high false-negative rate can be counter-balanced by the observation that 1) both ANKRD2 and IRF3 that were also consistently predicted by our luciferase assay, our GFP fusion assays, and PSORT II as not localized in the nucleus and 2) TLX2 and DLX6 were also mistakenly characterized in our assay when compared to our own GFP-fusion assay.
Finally for each of the 22 GFP fusions, we conducted a quantitative analysis of the distribution of GFP signal located over the nuclear versus that distributed in the cytoplasm. For 5 to 7 single-cell images per construct, the DAPI and GFP signals were used to locate, respectively, the nucleus boundary and the extent of the cytoplasmic compartments. The average intensity of GFP within the nucleus boundary was then computed and compared to that of the cytoplasm. A good correlation between those GFP signal intensity ratios and luciferase activities was observed, providing yet another line of evidence that the luciferase activity measured in our assay accurately reflects the nuclear translocation potential of a particular coding sequence (Figure
Additional file 5
Correlation between the ratio of GFP intensity in the nucleus to that of the cytoplasm computed from the GFP-fusion-based nuclear localization images and the log10 of the average luciferase ratio for three independent assays for 22 tested constructs. Standard deviations are represented by the horizontal and vertical bars for the luciferase and GFP fusion quantitative analysis respectively. In blue the gene product that we detected as being able to translocate in the nucleus, in orange the gene product that did not translocate into the nucleus.
Click here for file
To test the capacity of our method to detect the translocation potential of proteins located in the cytoplasm during steady state but known to shuttle between the nucleus and the cytoplasm, we selected three known cases and assayed their nuclear translocation: GTSE-1
Additional file 6
Measure of the translocation potential of known steady state cytoplasmic proteins. Selected steady state cytoplasmic proteins that can shuttle between the nucleus and the cytoplasm: GTSE-1, DVL2 and survivin/BIRC5. Reported values are average luciferase ratios for three independent assays. Error bars are standard deviation.
Click here for file
The assay described here can be used to systematically characterize human gene products ability to translocate in the nucleus. It is easy to prepare samples by designing the gene-specific forward and reverse primers, in which no purification or cloning steps are also required The assay uses a luciferase reporter to directly and quantitatively measure if the easily engineered hybrid protein is able to undergo nuclear translocation. Since both the PCR-based sample preparation and the luciferase-based reporter assay can easily be manipulated in 96- or 384-well plate formats, we believe that our system can achieve the throughput required for a proteome-scale analysis of nuclear translocation potential. We demonstrated the validity of our approach using HeLa and CHO-K1 cells, but as long as lipofection of PCR products is tolerated, a wide range of alternative cell types can be employed.
While our luciferase reporter system gave results relatively consistent with those obtained by GFP fusion assay, our results did not correlate well with literature-derived localization reported in HPRD. Perhaps protein localizations that are too often reported in the literature are limited to the predominant localizations of a protein, with minor sub-cellular localizations often hardly accessible to search for location and therefore are poorly described. In addition, methods relying on ectopic protein over-expression to report sub-cellular localization may overlook minor localizations of proteins, or even induce unusual sub-cellular localization. On the other hand, a fusion with TIP-1 PDZ domain and TNNC2 trans-activation domain, which our system relies on, may also hamper the genuine localization of the protein. In some cases, the binding of rohtekin to the TIP1 PDZ domain could be altered by the fusion of particular CDS, thus preventing the reconstitution of a transcriptionally active BIND-ACT construct and the detection of the effective translocation of ACT in the nucleus by the luciferase reporter gene. This scenario is probably the most plausible reason for failing to detect the nuclear translocation of TLX2.
Methods used to characterize sub-cellular localizations of proteins usually focus on a description of cellular compartments where the proteins are predominantly localized. On the other hand, our system can provide information concerning the localization or function of a gene product that is not apparent from previous studies
We have described a new method of analyzing the nuclear translocation potential of a given coding sequence. Our method can easily be employed in parallelized settings to analyze nuclear translocation potentials upon different cellular conditions and treatments. The major advantages of the method are its ease of use and the scalability of both sample preparation and final read-out. Since the PCR-based sample preparation and the luciferase-based reporter assay can be used in 96- or 384-well plates, we believe that our system can achieve the throughput required for proteome-scale analysis.
PCR primers
Gene-specific forward and reverse primers for amplification of target genes were designed as described previously
Additional file 7
List of primers used. Primers name and sequence used in this study
Click here for file
Constructs and assay samples
PCR procedures were performed as described previously
Sub-cellular localization assay using luciferase reporter
Sub-cellular localization assay was carried out in 384-well assay plates and the M2H assay was assayed as described previously
Protein sub-cellular localization using GFP fusion proteins
To construct a model for sub-cellular localization experiments, we first amplify the fragment CMV-EGFP-Tag1 with CMV_GFP1 and pEGFP-C1-CMVR-Tag1 primers, and the fragment for Tag2-SV40 with Tag2-pEGFP-C1-SV40F and SV40_GFPRev1 primers from the plasmid pEGFP-C1 (CLONTECH), where Tag1 and Tag2 are the two common sequences used to connect the DNA fragments during sample preparation (see the Figure
The PCR products were transfected into HeLa and CHO-K1 cells, then grown on 24-well plates for 20 h. Twenty-four hours after transfection, cells were washed with 1× PBS and fixed in 0.5 ml of 1× PBS (Sigma) containing 4% of paraformadehyde (Wako Pure Chemical Industries, Ltd) for 10 minutes at room temperature followed by a wash with 1× PBS three times. The cells were stained with 200 μl of twice pre-diluted of VECTASHILED Mounting Medium with diamidino-2-phenyl-indole (DAPI) (Vector Laboratories). Fluorescence images were acquired using the inverted research microscope DM IRE2 (Leica, Wetzlar, Germany) equipped with N PLAN L 20× 0.40 NA CORR (Leica) or PL Fluotar L 40× 0.40 NA CORR (Leica) lens. Image acquisitions were performed with Leica's FW4000 software. For each image, a 359 nm and a 490 nm wavelength fluorescence filter were used for DAPI and EGFP imaging, respectively. For these assays, a minimum of two independent transfections were performed.
Quantitative analysis of GFP fusion protein sub-cellular localization
In order to quantify the nuclear localization of GFP fusion proteins, we wrote a custom Perl PDL script to analyze microscopic images of DAPI-stained, GFP fusion protein in transfected cells. For each fusion protein tested, 5 to 7 single cell images were delineated manually. For each image, we first applied a smoothening filter of 10 × 10 pixels on the DAPI signal, and selected the area corresponding to 1.5 deviations from the mean DAPI value as representative of the nucleus boundary. The rational behind this filtering process was confirmed by manual comparisons with direct cell imaging. A similar filter was applied to the GFP signal to delineate the extent of the area where GFP could be confidently detected. We then computed the average intensity of GFP within the nucleus boundary and compared it to that of the whole area delineated by the GFP signal. The reported values are the average ratios of the GFP intensity for each pixel of the single cell isolated images in arbitrary units.
List of abbreviations
NLSs: nuclear localizations signals; NES: nuclear exclusion sequence; CDS: coding sequence; GFP: green fluorescent protein; PCR: polymerase chain reaction; TA: trans-activation domain.
Authors' contributions
TXH, NN and SF produced the experimental data; NB and HS wrote the paper; JK, YH and HS managed the research. All the authors read and approved the final manuscript.
We especially thank members of the protein interaction assay team for their technical assistance. We would like to thank the members of the Omics Science Center, RIKEN Yokohama for their kind support and discussions. We also thank Teasdale R. and Imamoto N. for their helpful advice, as well as Rodgers T. for proof reading the manuscript. This study was supported by a grant of the Genome Network Project from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) to Yoshihide Hayashizaki (Y.H.) and a Research Grant for RIKEN Omics Science Center from MEXT to Y.H. and a Cell Innovation Project from MEXT to YH."
Structure and function in the nucleus
Cell biology of transcription and pre-mRNA splicing: nuclear architecture meets nuclear function
Functional architecture in the cell nucleus
Pictures in cell biology. Structures of nuclear-transport components
Transport between the cell nucleus and the cytoplasm
Nucleocytoplasmic transport: Ran, beta and beyond
Transport into and out of the nucleus
Nucleocytoplasmic transport: taking an inventory
Nuclear pores form de novo from both sides of the nuclear envelope
Nuclear pore complex structure and plasticity revealed by electron and atomic force microscopy
Dynamic nuclear pore complexes: life on the edge
Structural biology of nucleocytoplasmic transport
Karyopherins: from nuclear-transport mediators to nuclear-function regulators
Distinct functions for the two importin subunits in nuclear protein import
In vivo evidence for involvement of a 58 kDa component of nuclear pore-targeting complex in nuclear protein import
A short amino acid sequence able to specify nuclear location
Protein import into the nucleus: an integrated view
Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen
The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen
Targeting of E. coli beta-galactosidase to the nucleus in yeast
Large-scale identification of mammalian proteins localized to nuclear sub-compartments
The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line
Global analysis of protein localization in budding yeast
Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae
Towards defining the nuclear proteome
Identification of fission yeast nuclear markers using random polypeptide fusions with green fluorescent protein
Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency
High-throughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata
Protein-protein interaction panel using mouse full-length cDNAs
Amino terminus of the yeast GAL4 gene product is sufficient for nuclear localization
DNA binding is not sufficient for nuclear localization of regulatory proteins in Saccharomyces cerevisiae
Context affects nuclear protein localization in Saccharomyces cerevisiae
The PDZ protein TIP-1 interacts with the Rho effector rhotekin and is involved in Rho signaling to the serum response element
PDZ domains: folding and binding
The PDZ protein tax-interacting protein-1 inhibits beta-catenin transcriptional activity and growth of colorectal cancer cells
Human protein reference database--2006 update
WoLF PSORT: protein localization predictor
hGTSE-1 expression stimulates cytoplasmic localization of p53
Dishevelled nuclear shuttling
CRM1-mediated nuclear export determines the cytoplasmic localization of the antiapoptotic protein Survivin
Nuclear or cytoplasmic expression of survivin: what is the significance?
Being in the right location at the right time
Protein-protein interactions of the hyperthermophilic archaeon Pyrococcus horikoshii OT3


text:
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NB! Kirjelduses võib esineda ebatäpsusi või olla muus keeles. Palume võtta ühendust, kui soovite saada täiendavat lisainfot selle toote kohta eesti keeles.
HP H3W58AA#ABB. Recommended usage: PC, kõlar type: 1-way, kõlar placement: Portable. Frequency range: 150 - 20000. Colour of product: valge, Housing material: Plastic. Connectivity technology: Wired, kõlar interface type: USB, Headphone connectivity: 3.5 mm. Power source type: USB
Lightweight and elegant, HP Multimedia Speakers 2.0 produce crisp, clear sound – perfect for listening to all your music, playing games and watching movies. Just plug it into the USB, and play!
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text:
Meaning and Origin of: Clement
Search for Names & Meanings Top 100 Names for Boys Top 100 Names for Girls Last Name History & Meaning Name Popularity Tracker Celebrity Names Search for Names by Trait Browse First Names A-Z Browse Last Names A-Z Browse First Names by Origin Browse Last Names by Origin Baby Name Lists How to Choose a Name
Save Name
Boy name origins & meanings:
- Latin : Merciful
Boy name variations
Family name origins & meanings:
- English, French, and Dutch : from the Latin personal name Clemens meaning 'merciful' (genitive Clementis). This achieved popularity firstly through having been borne by an early saint who was a disciple of St. Paul, and later because it was selected as a symbolic name by a number of early popes. There has also been some confusion with the personal name Clemence (Latin Clementia, meaning 'mercy', an abstract noun derived from the adjective; in part a masculine name from Latin Clementius, a later derivative of Clemens). As an American family name, Clement has absorbed cognates in other continental European languages. (For forms, see Hanks and Hodges 1988.)


text:
NServiceBus takes instances of .NET objects (messages, events and commands) and then sends/receives them over a specified Transport. As part of this the object needs to be serialized and deserialized. To achieve this NServiceBus uses Serializers.
Supported Serializers
- Newtonsoft
- Xml
- ProtoBuf-Net
- ProtoBuf-Google
- Message Pack
- Wire
- Hyperion
- Jil
- Bond
- ZeroFormatter
- JSON/BSON (deprecated in Versions 7 and above)
- Binary (deprecated in Versions 6 and above)
Using an existing serializer
The default serializer used in NServiceBus projects is the custom XmlSerializer.
The pages dedicated to particular Serializers show how to configure the endpoint to use each of them. Unless explicitly configured otherwise, NServiceBus will use XmlSerializer for serializing and deserializing all messages.
In order to register community run serializer or custom serializer, refer to the Custom serializers - Register the serializer section.
Specifying additional deserializers
To support sending and receiving messages between endpoints using different serializers, additional deserialization capability may be specified. It is possible to register additional deserializers to process incoming messages. Additionally, if a deserializer requires custom settings, they can be provided during its registration.
// configures XML serialization as default
// configures additional deserialization
var mySerializer = endpointConfiguration.AddDeserializer<MyCustomSerializerDefinition>();


text:
How do I know which size is which?
The different sizers are easy to identify. Size 12 will look noticeably smaller than size 15 … and size 15 also has a black dot in the middle of the dome.
Keywords: sizes, purchase, sizer
Source: Sizing Guide
User Guide Part #: LBL0020
Revision: REV_A


text:
Project Snapshot: Godrej E-city is a low rise residential construction being developed by Godrej Developer, located at Electronic City Phase-1, Bangalore. The elegant project offers 1, 2 & 3 BHK configuration designed for a smart and comfortable living. Currently under construction, Godrej E City apartment sizes ranges from 638 sqft to 1,652 sqft and is expected to be ready next year end.
Launched in 2012, the overall project is being developed in an area of 15 acres and encompasses a total of 860 units. The project is equipped with a range of amenities such as gymnasium, swimming pool, tennis court, squash, health club, landscaped garden and party hall etc.
When launched the project was priced at INR 3,300 per sqft, however thanks to the trusted brand name of Godrej and the elegant design of the development, the project has experienced an appreciation of over 30% in the last two years and is presently priced at INR 4,300 per sqft, which puts in the higher bracket, when compared to numerous other properties in the location such as Krishna Gold Fields, Concorde Group Tech Turf, Concorde Group Epitome and Prestige Birchwood.
Location Analysis
The project is located in Electronic City Phase-1, and enjoys a close proximity to NICE road and elevated expressway on Hosur Road. Additionally major junctions such as Sarjapur / ORR and Silk boards etc. are easily accessible through the well-developed road network. Thanks to the close proximity of most of the major IT/ITes/Industrial work places like Infosys, Wipro, TCS, HCL, Bomassandra industrial area and Sipcot Industrial area, the location is touted for being situated inside a vibrant economic landscape. Its closeness to the major IT companies and other industrial areas make it a suitable accommodation option for professionals working in these areas.
Coupled with the economic significance of the location, it is also at a center stage of vibrantly up- swinging social infrastructure. The project involves a slew popular schools, restaurants and hospitals. Not to mention banks, ATM and bus stations. Some of the prominent schools in the nearby vicinity are Delhi Public School (DPS), Ebenezer International School, Treamis World School and Indus International School. Metro is also expected to enter into electronic city in phase II, which will further add value to the location.
– Distance from Major Landmark
Expected Appreciation
Large volume of IT and ITes companies are in close vicinity of the project, makes it a suitable option for individuals working in these companies. A robust IT industry integrated with a rapidly evolving residential market has also resulted in a vibrant social and retail infrastructure in the form of plethora of schools, hospitals, colleges, restaurants and retail outlets, thereby adding further value to the location.
As mentioned above the price of the project has appreciated by 30% in the last two years. In the coming time on the backdrop of the economic significance of the location integrated with robust social infrastructure the present prices are expected to be rise with rate of 13-16% annually- according to the estimates made by the Kin Housing research team.
3 Reasons to Invest in Godrej E-City
Mentioned below are 3 reasons to invest into the project
– High appreciation expected in the coming time on the backdrop of rise in IT companies, retail and residential activities.
– Trusted brand name of Godrej.
– In close proximity to the vast IT space of Bangalore and hence will be preferred by individuals working over there. It also has Powerful retail and social infrastructure along with upcoming infrastructure projects.
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My Philosophy and Approach to Wellness...The MDVIP model allows me to provide the level of individualized and personalized primary care that I think everyone deserves. This is very difficult to accomplish with the ongoing changes in the traditional medical practice in America. In this practice model, my patients and I cultivate a doctor-patient relationship that fosters trust and allows me to tailor recommendations unique to you. These include recommendations for health and prevention that fit your life and goals based on the most current screening and predictive tests available. Complementing what is available through local medical resources, I also have additional referral options, tools, and tests through MDVIP's connections with national medical centers of excellence. This allows me to manage any ongoing medical needs based on the most current medical academics.
Physician Profile
- Undergraduate
- University of Southwestern Louisiana
- Lafayette, LA
- Bachelor of Science, Biology & Chemistry 1976
- Medical School
- Tulane University School of Medicine
- New Orleans, LA
- Doctorate of Medicine 1979
- Internship
- Harvard University, New England Deaconess Hospital
- Boston, MA
- Internal Medicine 1979-1980
- Residency
- Harvard University, New England Deaconess Hospital
- Boston, MA
- Internal Medicine 1980-1982
Professional Society Memberships
- Metropolitan Medical Society of Greater Kansas City
- American College of Physicians
- American College of Preventive Medicine
Professional Affiliations and Appointments
- Medical Director, Pegasus Recovery Center, Trinity Lutheran Hospital 1990-1992
- Treasurer, Jack R. Hill Medical Education Foundation 2002-present
- Kansas City Internal Medicine
- - Executive Committee 2003-present
- - President 2012-present
- Assistant Clinical Professor of Medicine, University of Kansas Medical School 2004- present
Hospital Affiliations
- Menorah Medical Center and St. Joseph Hospital
Honors and Awards
- Tulane Hospital Affiliates Award for Excellence 1979
- Kansas City Super Doctors 2008-2015
- Ingram's Top Doctor January 2015
- Diplomate, American Board of Internal Medicine
- Diplomate, American Board of Internal Medicine, Geriatric Medicine
Patient Testimonials
Member: Cindy
Dr. Delcambre has given me my life back! I went to her after loosing my younger brother and my mother to heart disease. I did no...
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This week's episode of Fuse's new digital series The Cue starts off by discussing the pop star at the forefront of everyone's mind: Justin Bieber, who's on this week's cover of Rolling Stone next to the headline "Bad Boy." Fuse hosts Elaine Moran, Dan Brown and Zuri Hall debate whether the public is piling on the Canadian pop star and if he really is "all about the music," as he so frequently says.
Then they move on to discussing the high-profile break-ups of Katy Perry/John Mayer (allegedly) and Robin Thicke/Paula Patton, as well as Drake and Rihanna reconnecting in Paris, the budding trend of surprise album releases thanks to Beyonce and whether the title of Lily Allen's new album, Sheezus, is cool or corny.
Watch new episodes of Fuse's new digital series The Cue every Friday at 1PM ET right here on Fuse.tv.


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Question about Nintendo Wii Console
I have heard that the game cubes discs will work for Wii, but I am wondering if the games designed for Wii will work in the game cube console. The game cube we have belongs to my six year old grandson, and we are not ready to buy him an expensive console just yet, until he is more responsible.
Posted by twimikal on
No. The gamecube discs are more the size of a minidisc and the ones for wii are full size discs like a DVD. My son is also 6 (going on 7 of course!) I decided to take the plunge after a long time battling with this decision. I just put the wii up high so he gets my help to load/change discs. He LOVES it! He is so great at it too! He loves the WiiSports, which the console comes with. (He even beats me at bowling!) It's a fun set up. I love that there are many games we can play together as a family, Cranium Kadooi, WiiSports, Playground, SmartyPants, as well as Active Life is due out soon and that looks to be awesome for some indoor activity! It is hard to make the decision to spend that much. I have been hanging onto the idea the last 2 years, that the price would drop and I would buy it then. Well, the price does not go down. In fact, it's hard to find at the retail price as it is hard for the stores to keep on the shelf! Good luck with the gamecube. If you still aren't ready to switch, maybe just let him pic out a few new games. This is sure to renew his interest in Gamecube! AS it is hard to find gamecube games in stores new, definately look on ebay.com for new and slightly used (which are great buys and I've never had a bad experience) gamecube games. Barnyard is a super cute one!
Please leave me a great rating if you found my reply helpful. Thank you! :)
Posted on Sep 03, 2008
SOURCE: I have freestanding Series 8 dishwasher. Lately during the filling cycle water hammer is occurring. How can this be resolved
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Posted on Jan 02, 2017
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|There's a saying, "at some point, infrastructure does matter". You have to invest to understand deeper what the issues, build a solid foundation and then move up. To stay competitive and survive in the long haul in Retail, there are no quick routes.
Reliance Retail India: Challenges for Indian RetailersAt the BuildUp 2008 conference in Bangalore, Retailers discussed the various challenges ahead of them. The consensus was, Retail cannot develop unless infrastructure develops.


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You can find us here.
For further questions, suggestions and wishes please don't hesitate to contact us.
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We are available for an individual consultation by phone 24 hours a day.
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Car & Bus
Since parking is very difficult here, we recommend taking the U-Bahn, which has a stop very close to Schweizer Platz. From the station "Schweizer Straße", walk about 50 meters towards city centre to our IQ location in Frankfurt.


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Black Throat LoveFavorites [+]
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Running Time: 126 minutes
Released: 07/2009
Studio Name: Juicy Entertainment D.E.F.
Director: John E. Depth
Description: Soft & slow, then the fireworks blast off! J. Depth brings you the finest bitches with the best lips in the business, slurping and sucking your stiff tool like you've never had it before. Making your eyes roll and your mind explode as they go down on your long Johnson and lick it like a lollipop, wrapping their lips tight around your stick 'til you pop like a volcano!
8 Scenes from Black Throat Love
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What Happened to the People Who Died in the Flash-sideways World
(Season 6 Spoilers Below)
Keamy, Mikhail and Omar died on the Island in the following ways:
1. Keamy was killed by Ben.
2. Mikhail was killed by a grenade he himself detonated, successfully killing Charlie.
3. Omar exploded, also by a grenade.
So, since they died in real life, on The Island, how could they also die in the afterlife if they are already dead? Here's a theory...or two.
Theory One. If they were killed On-Island, and escaped becoming the Whispers, before "dying" in the Flash-sideways world; they could have simply been reborn in the afterlife again, or the bodies in the mourge presumably disappeared.
Theory Two. If they did, in fact, become the Whispers, does that mean that, when Hurley took over as the leader, he somehow gave them some closeure. They lived in the Flash-sideways but, ater dying, simply became the Whispers again, due to events that happened in the afterlife.
Anyone have any other ideas?


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This luxury resort balances traditional Lanna and hip boutique styles and is housed in a new fully furnished building where designers have paid great attention to detail. Renovated in 2009, this family-friendly city hotel is ideal for business travellers and holidaymakers alike and comprises a total of 76 rooms. Facilities on offer to guests at this air-conditioned establishment include a lobby area with a 24-hour reception, a hotel safe, lift access and a newspaper stand. There is a bar and a breakfast/dining room and guests have WLAN Internet access at their disposal. They can also take advantage of the room and laundry services. There is a car park for those arriving by car and it is possible to hire bicycles on the premises.
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This hotel serves international, Thai and speciality dishes. There is a continental breakfast buffet each morning and the midday and evening meals may be enjoyed � la carte.
MasterCard, JCB and VISA are accepted at this hotel.
The hotel located in Chiang Mai on Charoen Muang Road. The hotel is just 300 meters away form Chiangmai Railway Station.
Room Facilities:
Satellite cable TV
Internet access
Mini fridge
Tea Coffee
Double bed
King size bed
Individual airconditioned


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1 answer
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text: Foodservice operators use beverage menu to intrigue consumers Seasonal alcohol and nonalcohol drinks help innovate menu Jessica Jacobsen September 16, 2015 No Comments SKU proliferation has left its mark on the consumer packaged goods (CPG) market. But innovative new products aren't just for the retail shelves. Foodservice operations also are developing new beverages to entice consumers. Read More


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GD Awarded $40 M for Abrams Tank Production
General Dynamics Land Systems has been awarded an additional $40 million for the procurement and production of Saudi M1A2 (M1A2S) Abrams tanks for the Kingdom of Saudi Arabia. This modification is part of an existing contract to upgrade the Kingdom of Saudi Arabia's fleet of tanks.
The Foreign Military Sales contract was awarded by the U.S. Army TACOM Life Cycle Management Command on behalf of the Royal Saudi Land Forces.
Global Amphibious Combat Vehicles (ACV) Market Research Report 2017
This contract extends work started in 2008 to update M1A1 and M1A2 tanks to the M1A2S configuration for the Kingdom of Saudi Arabia. The M1A2S conversion increases the efficiency and capability of the tank.
The work will be performed by current employees at the Joint Systems Manufacturing Center in Lima, Ohio, with an estimated completion date of July 31, 2014.
Source : General Dynamics Corporation (NYSE: GD)
Future Ground Combat Vehicles Conference
Nov 13 - 15, 2017 - Detroit, United States
Interactive Export Controls Workshop
Sep 12 - 13, 2017 - Washington, United States


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Learn more about E.J.HOWELL & SON(TRANSPORT CONTRACTORS)LIMITED. Check the company's details for free and view the Companies House information, company documents and list of directors.
Know about every new company document that is added!
Take advantage of our watch this company feature free of charge and have notifications sent directly to your inbox. Whenever a new document appears on this website, or if their particulars change, you will be immediately informed about such changes. To monitor these changes, all you need to do is click on watch this company.
|Tangible Fixed Assets
|£ 9,543
|Current Assets
|£ 459,184
|Tangible Fixed Assets Depreciation
|£ 23,230
|£ 46,534
|Shareholder Funds
|£ 334,078
|Profit Loss Account Reserve
|£ 333,078
|Called Up Share Capital
|£ 1,000
|Total Assets Less Current Liabilities
|£ 334,078
|Net Current Assets Liabilities
|£ 324,535
|Creditors Due Within One Year
|£ 134,649
|Cash Bank In Hand
|£ 412,200
|Stocks Inventory
|£ 450
|Share Capital Allotted Called Up Paid
|£ 1,000
|Number Shares Allotted
|£ 1,000
|Tangible Fixed Assets Cost Or Valuation
|£ 30,402
|Tangible Fixed Assets Depreciation Charged In Period
|£ 2,371
Information about the Private Limited Company E.J.HOWELL & SON(TRANSPORT CONTRACTORS)LIMITED has been prepared for information purposes only. It is not intended to be nor does it constitute legal advice. This is public information provided by the official company register.


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Salary : $500,000
Date of Birth : May 23, 1987
Country / Birthplace : Brooksville, Florida, United States
Source of Income : WWE Wrestling
Age : 28 Year Old
Real Name : Windham Rotunda
He is the main leader of the "The Wyatt Family" . He fight from 2009-2010 in the Florida Championship Wrestling and also get trained from the franchise . Then later he is moved to the WWE NXT where start wrestling the group called "Nexus".
In 2012 he made his way to Raw and created his own wrestling group "The Wyatt Family" with Luke Harper . He shared a great rivalry with wrestlers like "Dean Ambrose and Roman Regins" .
- FCW Florida Tag Team Championship
- PWI - Most Hated Wrestler of the Year (2010) – As part of The Nexus
- Worst Worked Match of the Year (2014) vs. John Cena at Extreme Rules on May 4


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Absolutely stunning game.. I loved the visuals, the music and the atmosphere! A very big well done to the creator! :) If you would like to see what this game is about or would just like to enjoy a let's play on this game; then please feel free to check out my video here: :)
A downloadable game for Windows and Linux
Afterlight is an ethereal puzzle and it features unique gameplay with mind-blowing riddles.
The game is split up into levels, and each one is a riddle itself. You start with an empty world and an exit door, but it's not so simple. You reveal the path by shooting magic and gathering the shards to make a complete picture.
The game is suitable for all ages and has English and Russian language support. 20 levels form a gameplay with duration for an hour (or even more!). All levels can be replayed in either order if unlocked.
Linux version
After a week of fighting with engine I made it! *hooray* Test it, send to your friends and please tell me if something bad happens. If not, please tell me too :)
Known issue: the game path should contain only standard ASCII symbols (that means no diactric symbols, no non-english letters, only latin ones and digits). A desktop folder should work well.
You need keyboard and a mouse.
- Use your mouse to look around;
- WASD to move;
- Left mouse button to shoot magic ✨
- Spacebar to jump;
- Esc or P key to pause;
System requirements
- Supported platforms: Windows Vista and higher, Linux;
- Minimal required storage: ~320 Mb for Windows, 700+ Mb for Linux;
- The game requires video card that supports DirectX 10 or above (this is for Windows; Linux binaries are shipped with OpenGL support);
- Recommended video card is Nvidia GeForce GT 650m and its analogs (works smoothly with high quality on my laptop);
- The minimal requirements for video card are NVIDIA GeForce 470 GTX or AMD Radeon 6870 HD series card or higher
- Windows requires Engine Prerequisites which you will find in Game Folder\Engine\Extras\Redist\en-us\UE4PrereqSetup_x64.exe (or x32).
|1 year ago
|In development
|Windows, Linux
|Cosmo Myzrail Gorynych
|3D, Atmospheric, Colorful, Difficult, Exploration, First-Person, Minimalist, Puzzle-Platformer, Relaxing, unreal-engine
|All rights reserved
|Asset license
|All rights reserved
|Average session
|A few minutes
|English, Russian
|Keyboard, Mouse
Click download now to get access to the following files:
Log in with your itch.io account to leave a comment.
Beautiful game you have here. I loved the piano.
Afterlight looks very nice and well done but the pixel (well, polygon) hunting gets very annoying nearly immediately to me I'm afraid (which seems to be the main mechanic?).
I like to throw around the "this game is an experience/work of art" card on this channel. It's not that I'm loose in throwing it out left and right, but because I mean it when I say it.
That said, Afterlight is an experience.
It's a refreshing game. It still presents a bit of a puzzle challenge, but it doesn't challenge you the way most puzzlers do. It's like yoga compared to wind sprints: you end up mentally refreshed instead of mentally wasted.
The graphics are minimalistic to the point it's almost art. The controls are basic, nothing to write home about by any means, but it works for a game like this. What makes this game is the music though. The ethereal piano of hitting the orbs mixed with the relaxing background music perfectly sets the atmosphere.
An escape from reality. That's Afterlight in a nutshell.