text:
From design to delivery, Moneysaver Print Shop has got you covered. Our production department can create almost anything and output to almost any media. If you don't see it, just ask—we can probably do it.
Moneysaver Printshop
626 Thain Road
Lewiston, Idaho 83501
Phone: 208-298-0161
Fax: 208-746-8507
Toll-Free: 800-473-4158
Email: firstname.lastname@example.com
- Announcements – Baby, Graduation, Engagement, and Wedding
- Bookmarks
- Booklets & Magazines
- Brochures & Rack Cards
- Business Cards
- Business Forms
- Calendars
- Campaign Materials
- Carbonless/NCR Printing
- Catalogs
- Coupons
- Doorhangers
- Envelopes
- Flyers
- Labels
- Letterhead
- Logos
- Manuals/Handbooks
- Membership Booklets
- Memorials
- Newsletters
- Notepads & Memo Pads
- Personal Stationery
- Postcards
- Posters
- Programs
- Property Listing Flyers
- Rack Cards
- Raffle Tickets/Books
- Resumes
- Rubber Stamps
- Signs & Banners
- Tent Cards
- Tickets
- Vehicle Magnets
- Wide format printing
- Vinyl outdoor banners
- Black & White Copies
- Color Copies
- Comb Binding
- Design & Typesetting
- Direct Mail
- Cutting
- Digital File Transfer
- Enlargements & Reductions
- Folding
- High Quality Digital Scanning
- Lamination
- Logo Design
- Numbering
- Offset Printing
- On-Demand Printing
- Online Ordering
- Perforation
- Pickup and Delivery
- Saddle Stitching
- Scoring
- Variable Data Printing
7 Reasons Why You Shouldn't Buy Printing Online
Online shopping has opened doors for shoppers everywhere, bringing once unavailable products to their doorstep and discounted prices. While this may be a great idea for certain products, there are multiple reasons why you may get more than you bargained for when buying printed products online.
1. You Don't Know What You're Getting Until You've Got It.
Nothing is worse than waiting for a package in the mail, and then being surprised at what lies inside the box. You've already spent time, energy and money on the printing – now what do you do? Purchasing printed products online, like business cards and wedding invitations, means you're taking a risk at what your final product is going to look like. Local print shops like ours have samples ready for you to view, and paper swatches to help you determine what would work best for your project. No surprises, except for how easy it can be!
2. You Get What You Pay For.
500 business cards for $7.99 sounds like a great deal, but if it sounds too good to be true, it probably is. What they're not telling you is your cards will be printed on a thin, flexible index weight paper. Premium business cards – ones that you're proud to hand out to your customers – should be printed on quality paper, not economy paper. Our business cards are printed on bright white cardstock to make your card beautiful as well as durable. Want your card to REALLY stand out? Pick from an array of colored and textured papers to make your card even more unique – something the online guys don't offer.
3. Your Cards Look Like Everyone Else's.
Have you ever opened a Christmas card in the mail to find that someone else used the same design you did? Have you ever been handed a business card that looks just like your co-worker's? Another downside to using online printers is the chance that YOUR design might also be used by someone else in the same field of work in the very same town you live in. Templates make it simple and easy to design a card quickly, but they are generic. Local print shops like ours staff graphic designers that are ready to custom design your next project, and often times the design work is included in the price of the product.
4. Surprise! Your Order Comes With Extra Fees!
If paying $8 for a box of business cards still sounds good to you, consider this – you'll pay at least that much in the shipping and handling. Online retailers create a lot of buzz for listing products at discounted prices, banking on the idea that once you get it in your cart, you won't notice the extra fees they tack on during check out. Printed products are much the same. You're taking all the risks listed here, plus you're paying extra to have it shipped from out-of-state. Moneysaver Printshop offers free local delivery to businesses. And free is always a good thing, right?
5. Customer Service
So you got your printed product in the mail and it was wrong – now what do you do? You'll have to call their national customer service line, sit on hold for a while and then talk to a person who has never met you face-to-face and likely never will. Is that person going to work hard for you? Probably not. Spending money locally comes with the added benefit of seeing your graphic designer in person, and being able to view hard copies of proofs. Have a question about a job in progress? Pop into the shop and talk directly to the people who work for you.
6. Long Turnaround Time
Buying online means added wait time. Often, online printers have a standard 7-day long turnaround time, which is more than reasonable, but add in added time in transit to your mailbox and you're looking at 2-3 weeks total. Ordering from Moneysaver Printshop means you'll have your product ready in a fraction of that time.
7. Supporting Your Local Economy.
The Lewis-Clark Valley is a great place to live and a great place to spend your money! Stimulate the local economy by spending it here. More money spent at local businesses means better shopping choices for you.
Still not convinced? Give us call and we can talk more about how we can go the extra mile for you.


text:
(TRINIDAD EXPRESS) — A 75-year-old medical doctor was killed in a robbery near his home before dawn on Wednesday.
Dr Vin Sein Chiang of Lover's Lane, Wallerfield, was found dead next to his vehicle.
The Express was told that Chiang had left home at 5a.m., telling his wife that he was heading to Port of Spain.
Moments later, a commotion was heard outside.
Two men were seen confronting the 75-year-old as he stood outside his silver Mitsubishi Sportero pick up van.
The doctor was shot dead and the men fled.
In an unrelated incident, two men were shot along Dibe Road, Long Circular, St James.
At about 5a.m. police received a report of gunshots in an area along Dibe Road known as "The Creek".
The victims, Trevon Lewis and Mark Ellis, were taken to the St James Infirmary by PC Goddard and PC Lewis of the Emergency Response Patrol Unit.

subset: commoncrawl


text:
Results of the DACUM workshop led by Annett Hilpert , INBAS, June 2013: What is DACUM? DACUM stands for Develop A CUrriculuM. It is a method to analyze a job in order to find out what to teach. In the DACUM…
- about PIRATE Project
- PIRATE news
- Partners
- Previous projects
- Earth news
- Downloads
- Contact


text:
Featured Post
Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor - Journal of Virology
- Get link
- Other Apps
Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor - Journal of Virology
Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor - Journal of Virology
Posted: 21 Aug 2018 12:37 AM PDT
Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor.
IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection, are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor (AAVR) is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discrete interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.
Adeno-associated viruses (AAVs) are nonenveloped, single-stranded DNA viruses of the Dependoparvovirus genus of the Parvoviridae family (1). Unlike most viruses, AAVs are innately nonpathogenic, poorly immunogenic, and broadly tropic, making them attractive gene delivery candidates for virus-based gene therapies (2). Despite the extensive utility of AAV vectors in several ongoing clinical trials and preclinical studies for severe, monogenic disorders (2–5), we understand little about how AAV initiates infection and penetrates the cell barrier (6). Additionally, it is unclear how this might differ among AAV serotypes, many of which display significant differences in transduction efficiency and tissue tropism (7, 8).
Most naturally occurring AAVs utilize glycan moieties for initial attachment to the cell surface, and these interactions have been well characterized for a number of serotypes (9). The interacting glycan moieties identified to date include heparan sulfate proteoglycans for AAV serotype 2 (AAV2), AAV3, and AAV6 (10); N-terminal galactose for AAV9 (11, 12); and specific N- or O-linked sialic acid moieties for AAV1, -4, -5, and -6 (9, 13–16). Postattachment, AAV is thought to engage a proteinaceous receptor(s) to mediate cellular entry, although less is known about these interactions. For AAV2, the most well-studied AAV serotype, several studies have investigated possible cellular receptors. Mizukami et al. (17) were the first to report characteristics of a putative AAV2 receptor, describing a 150-kDa glycoprotein detected in membrane fractions of AAV-permissive cells, which bound AAV2 particles in a virus overlay assay. Following this work, several proteinaceous coreceptors, such as fibroblast growth factor receptor 1 (FGFR-1) (18), integrin αVβ5 (19), and the hepatocyte growth factor receptor (c-Met) (20), were implicated in AAV2 entry. However, clustered regularly interspaced short palindromic repeat (CRISPR)-mediated knockout of FGFR-1 and c-Met in several cell lines did not substantially affect AAV2 transduction (21), suggesting that these coreceptors have accessory rather than essential roles in AAV2 transduction.
We recently identified the AAV receptor (AAVR) (also known as KIAA0319L) as an essential receptor for AAV transduction of human cells derived from a broad range of tissues and in an in vivo mouse model (21). Multiple serotypes, including AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, and AAV9, require AAVR for transduction. AAVR is a glycosylated membrane protein that is capable of recycling from the plasma membrane to the trans-Golgi network using the cellular endosomal network. The ectodomain of AAVR comprises of a MANEC (motif at the N terminus with eight cysteines) domain as well as five Ig-like domains known as PKD (polycystic kidney disease) domains (22). Ig-like domains have been associated with cell adhesion functions and are often exploited by viruses during cell entry (23). AAV also utilizes these Ig-like structures to facilitate transduction, specifically PKD domains 1 (PKD1) to PKD3 within the ectodomain of AAVR (21). Here, we show that AAVR is identical to the receptor previously implicated in the study by Mizukami et al. and further characterize the AAV-AAVR interaction. Our findings demonstrate that there are serotype-specific interactions with AAVR, which may suggest evolutionary differences in receptor usage between serotypes.
The major AAV2 binding protein in membrane preparations is identical to AAVR.It was previously found that AAV2 binds a 150-kDa cell membrane glycoprotein (which we call AAV binding protein [AAV-BP] here) in a virus overlay assay using 35S-labeled AAV2 (17). We confirmed this virus-cellular protein interaction utilizing a modified virus overlay assay that makes use of unlabeled AAV2 and a monoclonal antibody (A20) against the intact AAV2 capsid. Cell membrane fractions taken from immortalized cell lines derived from a variety of human tissues were isolated and probed in this assay (Fig. 1A). The 150-kDa AAV-BP was detected strongly in membrane fractions of AAV2-permissive cells (HeLa S3, K562, HeLa, HEK293, KB, and Hep-2 cells) but was weakly detected or undetectable in cell lines known to be less permissive to AAV2 transduction (HL60 and UT7/Epo cells) (17, 21, 24). We went on to purify AAV-BP from the cell membrane fraction of HeLa cells, optimizing the purification process by using Pisum sativum agglutinin (PSA) lectin-coated beads and then jacalin-conjugated beads, which bound AAV-BP well. Upon mass spectrometry (MS) analysis of the protein excised from the gel in a region corresponding to where AAV binding activity was detected, we identified peptide sequences from a number of proteins, including the low-density lipoprotein receptor precursor (LDLR), apolipoprotein E receptor 2 (ApoER2), AAVR (KIAA0319L), oxygen-regulated protein 150 (ORP150), and integrin α5 (Fig. 1B). By using specific antibodies against each of the proteins identified, only those against AAVR precisely overlapped the region that displayed AAV2 binding activity in the virus overlay assay (Fig. 1C and D). These results indicate that the protein with the strongest binding activity in a virus overlay assay (AAV-BP) is identical to AAVR, the multiserotype receptor identified in an unbiased genetic screen for AAV2 transduction (21). This was further validated by an AAV2 virus overlay assay using isogenic knockout cells lines (created by using CRISPR-mediated genome editing), where the 150-kDa band of AAV-BP was detectable in wild-type (WT) but not in AAVR knockout (AAVRKO) cells (Fig. 2A and B).
AAVR glycosylation is not essential for AAV2-AAVR interactions or AAV2 transduction.AAVR has a predicted protein molecular mass of 108 kDa, but it is detectable at 150 kDa after immunoblotting (21), likely due to the effect of N- and O-linked glycosylation on protein migration during gel electrophoresis. To assess the importance of AAVR glycosylation on its interaction with AAV2, we treated cellular membrane preparations with deglycosylation enzymes (Fig. 3A and B). N-Glycosidase treatment led to a shift in migration, implying that AAVR is an N-linked glycosylated protein. Concomitant treatment with N- and O-linked glycosidases and neuraminidase (both of the latter two enzymes are required to cleave O-linked disaccharides) led to a further shift, suggesting that AAVR is also an O-linked glycosylated protein. None of the deglycosylation treatments reduced the binding of AAV2 in a virus overlay assay (Fig. 3A), demonstrating that glycosylation is not required for binding. To test the effect of N-linked glycosylation in a functional transduction assay, we used the stable expression of AAVR mutants in HeLa AAVRKO cells. We have previously shown that the expression of an AAVR minimal mutant (miniAAVR), consisting of a signal peptide, PKD domains 1 to 3 of the ectodomain, the transmembrane domain, and the C-terminal cytoplasmic region, is able to efficiently rescue AAV2 transduction in AAVRKO cells (21). This indicates that these PKD domains are sufficient for binding AAV2 and mediating entry. Within the ectodomain of miniAAVR, there are 5 putative asparagine glycosylation sites (indicated in Fig. 3C), which were mutated to alanine (individually or in combination) to prevent N-linked glycosylation. Mutation N525A and the quintuple mutant mildly affected AAV2 transduction, whereas the other mutations did not appreciably affect the transduction efficiency compared to that of the wild-type construct (Fig. 3D). Thus, by this cellular assay, N-linked glycosylation of AAVR facilitates optimal AAV2 transduction but is not strictly required.
AAVR PKD2 is critical for the interaction of AAV2 with AAVR.We previously identified AAVR PKD domains 1 to 3 to include the binding domain for AAV2 (21). To further characterize the AAVR-AAV2-interacting region, we created glutathione S-transferase (GST)-tagged AAVR ectodomain mutants consisting of individual PKD domains or combinations of sequential domains (Fig. 4A). We expressed the respective mutants in Escherichia coli and performed a virus overlay assay, where we observed binding of AAV2 to PKD domains 1 to 5, PKD domains 1 and 2, and PKD domain 2 alone (Fig. 4B and C). In addition, AAV2 transduction of AAVRKO cells stably expressing AAVR deletion mutants consisting of individual domain deletions (Fig. 4D and F) demonstrated that only the deletion of PKD2 completely abrogated AAV2 transduction (Fig. 4E). The AAVR deletion mutant missing PKD1 showed a less pronounced decrease in transduction efficiency. These data indicate that PKD2 is the critical region for AAV2 binding and transduction, with PKD1 playing a possible accessory role.
To further define the region of PKD2 necessary for AAV2 transduction, we constructed domain swap mutants in the AAVR homologue, KIAA0319, which shares approximately 60% amino acid sequence similarity with AAVR. AAVR is known to be KIAA0319-like because of its structural likeness to KIAA0319 (Fig. 5A). The two proteins both comprise a MANEC domain along with 5 PKD domains in their ectodomain and are both glycosylated, type I transmembrane proteins (25). Moreover, PKD1 and PKD2 of KIAA0319 share 72% and 80% similarities, respectively, with the corresponding PKD domains in AAVR (Fig. 5B). Importantly, despite its similarity to AAVR, KIAA0319 cannot be utilized by AAV2 to successfully transduce cells (Fig. 5C). Thus, we utilized KIAA0319 as a backbone in which regions of KIAA0319 were replaced with AAVR (Fig. 5A, D, and E). We then stably expressed these mutants in AAVRKO cells and assessed AAV2 transduction. A swap-in of AAVR PKD domains 1 to 3 into KIAA0319 (mutant a) and PKD2 alone (mutant b) fully rescued AAV2 transduction, whereas a swap-in of PKD1 alone (mutant g) did not rescue transduction (Fig. 5C). This is consistent with the binding and deletion mutant data and reinforces the major role of AAVR PKD2 for AAV2. Additionally, a swap-in of only an N-terminal region of AAVR PKD2 (∼30 amino acids [aa]) partially rescued transduction, indicating that this region is particularly important for AAV2 transduction (mutant c). Reiterating this point, swapping of the same region of KIAA0319 into AAVR (mutant f) eliminated the capacity of AAVR to rescue AAV2 transduction. Swapping of the C-terminal region of AAVR PKD2 into KIAA0319 (mutants d and e) did not rescue transduction. Using a collection of deletion constructs of PKD2, we observed that none of the mutants, even the ones containing small N- and C-terminal PKD2 deletions (Fig. 6A and B), were able to bind to AAV2 in a virus overlay assay (Fig. 6C and D). Thus, N-terminal of AAVR PKD2 is able to partially rescue AAV2 transduction in the domain swaps yet is not able to bind AAV2 particles in the in vitro binding assay. This suggests that full PKD2 of AAVR is required for optimal binding and transduction.
AAV5 requires AAVR PKD1 for binding and functional transduction.Our previous study demonstrated that AAVR is an essential receptor for multiple AAV serotypes (21), including AAV5, which is evolutionarily distant from AAV2 and shows only 57% identity in the capsid amino acid sequence (26–28). To explore the hypothesis that the interaction interfaces of different AAV serotypes with AAVR are distinct, we used the collection of domain swap mutants and probed AAV5 transduction. In stark contrast to the results obtained with AAV2, neither full-length PKD2 (mutant b) nor the N-terminal mutant (mutant c) rescued AAV5 transduction when swapped into KIAA0319 (Fig. 7A). Additionally, the AAVR mutant with a partial PKD2 swap of KIAA0319 (mutant f), which completely lost its ability to facilitate AAV2 transduction, was fully competent in mediating AAV5 transduction. Interestingly, when the most membrane-distal PKD domain, PKD1, was swapped into KIAA0319 (mutant g), we observed a rescue of transduction to the same levels as those with full-length AAVR, suggesting that AAV5 evolved to require PKD1 rather than PKD2 for functional transduction (Fig. 7A). In agreement with this, an AAV5 virus overlay assay using individual AAVR PKD ectodomain mutants demonstrated that AAV5 bound specifically to AAVR PKD1 and not PKD2 (Fig. 7B). Consistent with data from the virus overlay assay and swap experiments, AAV5 transduction of AAVRKO cells stably expressing AAVR single deletion mutants confirmed the strict requirement of AAVR PKD1 for AAV5 transduction (Fig. 7C). To test the differential requirements for PKD domain usage further, we used a virus transduction assay, where increasing amounts of soluble variants of AAVR domains were incubated with cells during AAV2 or AAV5 transduction. The transduction of both AAV2 and AAV5 was inhibited by increasing amounts of PKD1-5, while the addition of PKD3 had no effect on either virus (Fig. 7D). AAV2 transduction was potently blocked by PKD2 and, to a lesser degree, by PKD1, in concurrence with the deletion mutant data (Fig. 4E). Conversely, AAV5 transduction was potently blocked by PKD1 and unaffected by the presence of PKD2 at low concentrations, in agreement with the transduction and virus overlay data (Fig. 7D). However, at a higher concentration of soluble PKD2, we observed a significant decrease in AAV5 transduction (Fig. 7E), suggesting that PKD2 may play a minor role in the AAV5-AAVR interaction. The functional relevance of this is unclear because the deletion of PKD2 does not reduce AAV5 transduction (Fig. 7C). Together, these results imply complexity in the interaction of AAVR with different serotypes, where AAV2 utilizes PKD2 and, to a lesser degree, PKD1 to enable vector transduction, whereas AAV5 primarily requires PKD1.
Serotype-specific utilization of AAVR PKD domains 1 and 2.In view of the disparity in AAVR domain dependence between AAV2 and AAV5, we evaluated the transduction of AAV1 (Fig. 8A) and AAV8 (Fig. 8B) in AAVRKO cells stably expressing AAVR single deletion mutants. Notably, AAV1 and AAV8 were similar to AAV2 in their critical requirement for PKD2, although they appeared to depend more strongly on PKD1 than AAV2 did for transduction. Both AAV1 and AAV8 bound to only PKD2 in a virus overlay assay (Fig. 8C and D), suggesting a higher affinity for PKD2 in this assay.
A thorough understanding of how AAV interacts with its multiserotype receptor AAVR has the potential to lead to improvements in future vector design and modulations in tissue-targeting strategies. In this study, we establish that AAV-BP, the component of cell membrane extracts reported 2 decades ago to have the greatest AAV2 binding activity, is identical to the multiserotype receptor, AAVR. We further uncover that several different AAV serotypes have evolved distinct interactions for engaging the same receptor, AAVR, upon which they all have a strict dependence.
It has long been established that AAV2 attaches to cells via interactions with heparan sulfate proteoglycan (10). However, it was unclear if a proteinaceous receptor was a critical requirement for AAV2 infection. We recently identified AAVR in an unbiased genetic screening approach as being an essential receptor required for infection by AAV2 as well as by several other AAV serotypes (21). To further characterize AAVR, we revisited the first reported AAV receptor study (17) to determine if characteristics of the previously described AAV2 binding protein (AAV-BP) matched those of AAVR. AAV-BP, a highly glycosylated protein with a molecular mass of 150 kDa, was found to have dominant binding to AAV2 in cell membrane extracts from several human cell types. We show here, using mass spectrometry, modified virus overlay assays, and deglycosylation treatments, that AAVR is identical to the previously described AAV-BP. Moreover, using isogenic AAVR knockout cell lines, we unambiguously demonstrate that AAV-BP is present only in cells that express AAVR. This study thus provides an independent yet complementary approach to the identification of AAVR as an essential AAV2 receptor, demonstrating that the traditional virus overlay assay combined with a membrane protein purification procedure can still be a powerful approach to identify viral receptors. Notably, several other proteinaceous coreceptors for AAV2 have been described (18–20), but none scored in the genetic screens or are implicated in the current proteomic analysis of virus overlays.
Although the role of AAVR in cellular physiology is unclear, the structure indicates that it is a glycoprotein, containing Ig-like PKD domains in its ectodomain (21, 29). Many glycoproteins that contain repeats of Ig-like domains have been identified as viral receptors, including the human rhinovirus (HRV) major receptor, ICAM-1, and the coxsackievirus/adenovirus receptor, CAR (23). Here, we observed that the enzymatic removal of both O- and N-glycosylation decreased AAVR to a molecular mass of around 100 kDa, approximately the predicted molecular mass without modifications, which suggests that AAVR itself is not a heparan sulfate proteoglycan. We show that different AAV serotypes specifically interact with the Ig-like PKD domains of AAVR but demonstrate that glycosylation is not a prerequisite for AAV2 binding and transduction. This is similar to what has been noted for other nonenveloped viruses such as HRV and coxsackievirus, where the interactions between the virus and Ig-like domains in their receptors occur independently of N-linked glycosylation (30, 31). Interestingly, our glycosylation studies showed a slight reduction in functional transduction upon mutation of the Asp525 residue located in AAVR PKD3 as well as in the mutant carrying all 5 putative glycosylation sites. Because PKD3 is not critical for AAV2 transduction per se (Fig. 4E), we hypothesize that this observed reduction could be due to a disturbed folding or trafficking of AAVR, which potentially might require N-linked glycosylation at this site.
AAVR shares striking homology with KIAA0319, a candidate gene that may contribute to dyslexia (25, 32). Specifically, the ectodomain of KIAA0319 also contains 5 Ig-like PKD domains, which have an implied role in cell-to-cell adhesion (22, 33), and KIAA0319 is a highly glycosylated, transmembrane protein. Despite these similarities, AAV cannot utilize KIAA0319 instead of AAVR to gain entry into the cell. Thus, to determine which domain of AAVR is critical for transduction, we took advantage of the homology between AAVR and KIAA0319 and swapped regions of AAVR into KIAA0319, and vice versa, evaluating which mutants would rescue AAV transduction. Notably, we found that although AAVR is required for multiple serotypes, its interaction with AAV differs between serotypes. We show that AAV has evolved to interact specifically with the most membrane-distal domains (PKD1 and PKD2). In particular, AAV2 functionally interacts mainly with PKD2, with some contribution by PKD1, while AAV5 predominantly interacts with PKD1. It is interesting to note that, similarly to AAV, other viruses that engage receptors with Ig-like domains also utilize the most membrane-distal domain (34–39) as their contact site. For example, ICAM-1 contains 5 Ig-like domains (D1 to D5), of which the most membrane-distal domain (D1) directly interacts with HRV14, D2 contributes somewhat to binding, and D3 to D5 provide spacing to allow accessibility to the binding sites (38, 39).
The difference in AAVR interactions among the various serotypes is intriguing, as we have tested serotypes from different clades. AAV1 (clade A) and AAV2 (clade B) functionally interact mainly with PKD2, with some contribution by PKD1, while AAV5 (no designated clade) predominantly interacts with PKD1, and AAV8 (clade E) requires both PKD1 and -2 to transduce cells. These striking differences in AAV-AAVR functional interactions could be related to the origin from which the serotype was initially isolated, as AAV5 was isolated from human tissues (40, 41), while many other AAVs, including AAV1 and AAV2, were first found as contaminants in laboratory adenovirus stocks (42–46), and AAV8 was isolated from nonhuman primate samples (47). Additionally, AAV5 speciated early and is quite distinct from the evolutionary lineage to which the other AAV serotypes belong (28). As sequence diversity between AAV serotypes has allowed AAV to evolve footprints that preferentially interact with different glycan receptors, such as heparan sulfate and sialic acid (9), our results suggest that AAV serotypes also evolved distinct interactions with AAVR. This may or may not be related to their glycan dependence, something to be investigated in future studies for a more in-depth understanding of virus-receptor engagement. Moving forward, structural studies that complement the data presented here will further define the AAV-AAVR interaction domain in greater molecular detail and will be necessary to establish the exact binding sites, both in AAVR and on the viral capsids.
Cell lines and primary cells.All cell lines were grown in medium supplemented with 10% fetal calf serum (FCS) (Sigma, St. Louis, MO), 100 IU/ml penicillin-streptomycin (Sigma, St. Louis, MO), and 2 mM l-glutamine (Sigma, St. Louis, MO) and grown in a humidified incubator at 37°C with 5% CO2. KB, HeLa, HeLa S3, 293, Hep-2, K562, and HL60 cells were purchased from the American Type Culture Collection (ATCC) (Manassas, VA). HeLa S3 cells were cultured as a suspension in F-12 medium, and other cells were maintained as a monolayer in Dulbecco's modified Eagle's minimal medium (DMEM). UT7/Epo cells were provided by N. Komatsu (Jichi Medical School, Japan) and cultured in DMEM with the same supplements as those described above plus erythropoietin (EPO) at 1 U/ml. The isogenic AAVR knockout cell lines used in this study were generated by using CRISPR-Cas9 technology and were described previously (21). They were maintained in the same way as parental cell lines. The AAVRKO cells referred to in the genetic experiments shown in Fig. 3 to 8 are HeLa AAVRKO cells.
Viruses and recombinant vectors.WT AAV2 was propagated and purified by CsCl ultracentrifugation, performed as previously reported (48). The protein concentration of the virus preparation was measured by using the bicinchoninic acid (BCA) protein assay reagent (Pierce Co., Rockford, IL).
Recombinant AAV (rAAV) vectors carrying reporter genes, rAAV1(Luc/mCherry), rAAV2(Luc/mCherry), rAAV5(Luc/mCherry), or rAAV8(Luc/mCherry), were produced by cotransfecting 293 cells with pAVF5tg83luc-CMVmChery, an adenoviral helper plasmid (pHelper; Agilent, Santa Clara, CA), and pAAV2RepAV1Cap, pAAV2RepCap, pAAV2RepAV5Cap, or pAAV2RepAV8Cap. At 48 h posttransfection, rAAV vectors were purified by two consecutive rounds of CsCl ultracentrifugation as previously reported (49). The titers of rAAV vector stocks were estimated to be ∼5 × 1012 viral genomic copies (vgc)/ml by quantitative PCR. For the rAAV transduction assays with the rescued AAVRKO cell lines, recombinant self-complementary reporter vectors scAAV2-CMV-RFP and scAAV5-CMV-GFP were used, which were purchased from the University of North Carolina, Chapel Hill, Gene Therapy Center Vector Core.
Antibodies.The following antibodies were used in this study: mouse polyclonal anti-KIAA0319L (catalogue number ab105385) from Abcam (Cambridge, CA); mouse monoclonal anti-FLAG antibody (catalogue number F3165) from Sigma-Aldrich (St. Louis, MO); mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (catalogue number GT239) from Genetex (Irvine, CA); mouse monoclonal anti-GST antibody (catalogue number A00865) from GenScript (Piscataway, NJ); anti-intact AAV1 (clone ADK1a, catalogue number 03-651150), anti-intact AAV2 (clone A20, catalogue number 03-61055), anti-intact AAV5 (clone ADK5a, catalogue number 03-651148), and anti-intact AAV8/9 (clone ADK8/9, catalogue number 03-651161) antibodies from American Research Products, Inc. (Waltham, MA); anti-LDLR (catalogue number A304-417A) from Bethyl Laboratories, Inc. (Montgomery, TX); anti-ApoE2 (catalogue number NB100-2216) from Novus Biologicals (Littleton, CO); anti-ORP150 (catalogue number AF5558) from R&D Systems (Minneapolis, MN); and anti-integrin α5 antibody (catalogue number AB1921) from EMD Millipore (Billerica, MA). Antibodies were used at dilutions suggested by the manufacturers.
Enzymatic deglycosylation of cell membrane proteins.Peptide-N-glycosidase F (PNGase F) (catalogue number P0705S), O-glycosidase (catalogue number P0733S), and α2-3,6,8,9-neuraminidase A (catalogue number P0722S) were purchased from New England BioLabs (NEB) (Ipswich, MA). Eighteen micrograms of 2% n-dodecyl-β-d-maltoside (DMT)-solubilized HeLa S3 membrane proteins (1.4 mg/ml) was first incubated with 10× denaturing buffer in a volume of 20 μl at 100°C for 10 min. The reaction mixture was then incubated with GlycoBuffer 2 and N-glycosidase F (1,000 U); O-glycosidase (80,000 U); both N-glycosidase F (1,000 U) and O-glycosidase (80,000 U); or N-glycosidase F (1,000 U), O-glycosidase (80,000 U), and α2-3,6,8,9-neuraminidase A (80 U) at 37°C for 1 h in a volume of 40 μl. Mock digestion reaction mixtures were set up without the addition of any enzymes.
Purification of AAV-BP and mass spectrometry.Cell membrane proteins were prepared from 200 ml of HeLa S3 cell pellets as previously reported (17), solubilized with PB–2% DMT buffer (20 mM phosphate buffer [pH 7.2], 1 M NaCl, 1 mM CaCl2, 1 mM MgCl2, protease inhibitors cocktails, and 2% DMT), and loaded onto an XK 16/70 column (Pharmacia) containing ∼100 ml of PSA lectin-conjugated agarose (E-Y Lab, Inc., San Mateo, CA). After washing with PB–0.1% DMT, PSA binding proteins were eluted with elution buffer (100 mM methyl-α-d-glucopyranoside and 100 mM methyl-β-d-mannopyranoside in PB–0.1% DMT), followed by dialysis against TE–0.05% DMT buffer (20 mM Tris-HCl [pH 7.2], 1 mM ETDA, 0.05% DMT). NaCl was added to the dialyzed protein solution to 1 M and loaded onto a jacalin lectin-immobilized agarose column. After extensive washing with wash buffer (20 mM Tris-HCl [pH 7.2], 1 M NaCl, 1 mM ETDA, 0.05% DMT), the jacalin lectin binding protein was eluted with 0.4 M melibiose in wash buffer. The eluted protein was dialyzed against TE–0.01% DMT and concentrated by ultrafiltration with stirred cells (Amicon, Inc., Beverly, MA) to a concentration of 4 mg/ml. Approximately 10 mg of the lectin-purified protein was loaded onto a Prep Cell (model 491; Bio-Rad Laboratories, Hercules, CA) each time, followed by preparative electroelution. The fractions with AAV2 binding activity were combined and concentrated by ultrafiltration. The above-described concentrated protein preparation was further separated by isoelectric focusing (IEF) at pH 4 to 6, followed by a second sodium dodecyl sulfate (SDS)–6% polyacrylamide gel electrophoresis (PAGE) step. AAV-BP was located by performing a virus overlay assay. The protein spot with binding activity (at pI ∼4.5) was cut off and subjected to protein identification. Protein identification was performed at the Harvard Microchemistry Facility (Cambridge, MA) by microcapillary reverse high-performance liquid chromatography (HPLC)–nanoscale electrospray tandem mass spectrometry (μLC-MS/MS) on a Finnigan LCQ quadrupole ion trap mass spectrometer.
Two-dimensional gel electrophoresis of cell membrane proteins.Each aliquot of the sample (100 μl) was dialyzed overnight against 5 mM Tris-HCl (pH 6.8) using a 6,000- to 8,000-molecular-weight (MW)-cutoff (MWCO) membrane at 5°C. Each aliquot was lyophilized and redissolved in 50 μl of a 1:1 dilution of SDS boiling buffer-urea sample buffer before loading. Two-dimensional (2-D) electrophoresis was performed according to the carrier ampholyte method of isoelectric focusing (50, 51) by Kendrick Labs, Inc. (Madison, WI), as follows. Isoelectric focusing was carried out in a glass tube with an inner diameter of 2.3 mm using 2% pH 3 to 10 or pH 4 to 6 Servalytes (Serva, Heidelberg, Germany) for 9,600 V · h. One microgram of an IEF internal standard, tropomyosin, was added to the sample. This protein migrates as a doublet with a lower polypeptide spot at a MW of 33,000 and pI 5.2. The enclosed tube gel pH gradient plot for this set of Servalytes was determined with a surface pH electrode. After equilibration for 10 min in buffer "O" (10% glycerol, 50 mM dithiothreitol, 2.3% SDS, and 0.0625 M Tris [pH 6.8]), each tube gel was sealed to the top of a stacking gel, which was on top of an 8% acrylamide slab gel (0.75 mm thick) poured with lower-gel buffer at pH 8.0. SDS slab gel electrophoresis was carried out for about 10 h at 15 mA/gel. After slab gel electrophoresis, the gel was stained Coomassie brilliant blue R-250 or silver stained. The gels for blotting were placed into transfer buffer (10 mM N-cyclohexyl-3-aminopropanesulfonic acid [CAPS] [pH 11], 10% methanol [MeOH]) and transblotted onto a polyvinylidene difluoride (PVDF) membrane overnight at 200 mA and approximately 100 V per two gels.
Plasmid construction. (i) pLenti-based mammalian expression constructs.To generate all recombinant constructs for genetic experiments, a Gibson assembly reaction kit (New England BioLabs, UK) was used to insert the gene of interest into a lentivirus-based destination (DEST) vector, pLenti-CMV-Puro-DEST (w118-1) (Addgene plasmid 17452) digested with EcoRV to remove the DEST cassette (a gift from Eric Campeau) (52). AAVR and derived AAVR genes were amplified from AAVR-FLAG or miniAAVR, constructed as described previously (21). The KIAA0319 gene was amplified from a cDNA clone (GenBank accession number BC140821) (clone 9021689; GE Dharmacon, Lafayette, CO) and Gibson cloned into pLenti-CMV-Puro-DEST by using primers 5′-TGTGGTGGAATTCTGCAGATACCATGGCGCCCCCCACAG-3′ and 5′-CGGCCGCCACTGTGCTGGATTTACTTATCGTCGTCATCCTTGTAATCTCTGTCCTTTGAGCAATAACTG-3′, introducing a FLAG epitope in frame with the C-terminal end of KIA0319.
To generate the PKD deletion constructs of AAVR, two PCR products were generated for Gibson cloning. The extreme 5′-end primer (5′-GACTCTAGTCCAGTGTGGTG-3′) and 3′-end primer (5′-ATCCAGAGGTTGATTGTCGAG-3′) were similar for the constructs. Primers used to amplify the fragments for the constructs were 5′-CCATACCCAGTTATAAAGGAACTGCGGCCCCCCATTGCTATTG-3′ and 5′-CAGTTCCTTTATAACTGGGTATGG-3′ for ΔPKD1, 5′-GCCAGAGCCCCGTAAGCCCCCTGTGGCCAACG-3′ and 5′-CTTACGGGGCTCTGGC-3′ for ΔPKD2, 5′-GTGAACAAAGCTGTGGATTACCCTCCTCAGGCAGATGC-3′ and 5′-GTAATCCACAGCTTTGTTCAC-3′ for ΔPKD3, 5′-ATTGTGCAACCTGAAAACAATAAGCCACCTATAGCCAAGATAACTG-3′ and 5′-CTTATTGTTTTCAGGTTGCACAAT-3′ for ΔPKD4, and 5′-GTCATTGTCAAAGAAGAAATAAACAAAAACCTG GTGGAGATCATCTTG-3′ and 5′-TTTGTTTATTTCTTCTTTGACAATGAC-3′ for ΔPKD5.
To generate the miniAAVR glycosylation mutants, N-linked glycosylation sites in miniAAVR were identified by using UniProt (http://www.uniprot.org/). Five sites were found, namely, N395, N472, N487, N492, and N525. These asparagine residues were mutated to alanine residues in order to prevent N-linked glycosylation from taking place at these sites. Gibson cloning was used to make these mutations. The extreme 5′-end primer (5′-GACTCTAGTCCAGTGTGGTG-3′) and 3′-end primer (5′-ATCCAGAGGTTGATTGTCGAG-3′) were similar for the constructs. The following primers were used to amplify the mutated fragment in the respective constructs: 5′-CATGGGGAAGGCTATGTGGCCGTGACAGTCAAGCCAGAG-3′ and 5′-CTCTGGCTTGACTGTCACGGCCACATAGCCTTCCCCATG-3′ for the N395A mutant, 5′-GTAAACTCGTCCCTGGGGCCTACACTTTCAGCTTGACTGTAG-3′ and 5′-CTACAGTCAAGCTGAAAGTGTAGGCCCCAGGGACGAGTTTAC-3′ for the N472A mutant, 5′-GACTCTGATGGAGCTACCGCCTCTACTACTGCAAACCTGAC-3′ and 5′-GTCAGGTTTGCAGTAGTAGAGGCGGTAGCTCCATCAGAGTC-3′ for the N487A mutant, 5′-GCTACCAACTCTACTACTGCAGCCCTGACAGTGAACAAAGCTGTG-3′ and 5′-CACAGCTTTGTTCACTGTCAGGGCTGCAGTAGTAGAGTTGGTAGC-3′ for the N492A mutant, and 5′-CCATCACCCTCTTTGGGGCCCAGAGCACTGATGATCATGG-3′ and 5′-CCATGATCATCAGTGCTCTGGGCCCCAAAGAGGGTGATGG-3′ for the N525A mutant.
To generate the swap-in constructs of KIAA0319 and AAVR, the following primers were used: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-GACACTCTCTCCAGCAGATACCGTAAGTTCTTTCACTGTC-3′ for fragment 1, 5′-GTATCTGCTGGAGAGAGTGTC-3′ and 5′-CTTATTGTTTTCAGGTTGCACAAT-3′ for fragment 2, and 5′-ATTGTGCAACCTGAAAACAATAAGCCTCCAGTGGCTGTGG-3′ and 5′-ATCCAGAGGTTGATTGTCGAG-3′ for fragment 3 for PKD1-3 AAVR swap-in mutant a; 5′-GGTGGAATTCTGCAGATACCATGGCGCCCCCCACAGG-3′ and 5′-CAATAGCAATGGGGGGCCGATTGACTCTTCTGGCAGGCTTAACAGTGAC-3′ for fragment 1, 5′-ATCGGCCCCCCATTGCTATTG-3′ and 5′-CTGACAGTGAACAAAGCTGTGGA-3′ for fragment 2, and 5′-GACAGTGAACAAAGCTGTGGATTACCCACCAGTTGCTAATGC-3′ and 5′-CGGCCGCCACTGTGCTGGATTTACTTATCGTCGTCATCCTTG-3′ for fragment 3 for PKD2 AAVR swap-in mutant b; 5′-GGTGGAATTCTGCAGATACCATGGCGCCCCCCACAGG-3′ and 5′-CAATAGCAATGGGGGGCCGATTGACTCTTCTGGCAGGCTTAACAGTGAC-3′ for fragment 1, 5′-ATCGGCCCCCCATTGCTATTG-3′ and 5′-TTTATCATCATCAGTGCTTTGACTGCC-3′ for fragment 2, and 5′-GTCAAAGCACTGATGATGATAAAATAGTGAGTTATCATTGGGAAG-3′ and 5′-CGGCCGCCACTGTGCTGGATTTACTTATCGTCGTCATCCTTG-3′ for fragment 3 for PKD2 AAVR swap-in mutant c (aa −4 to 30); 5′-GGTGGAATTCTGCAGATACCATGGCGCCCCCCACAGG-3′ and 5′-CTTCCCAATGGTACTGAACGATTTCAGTATCATCTGTACTTTG-3′ for fragment 1, 5′-ATCGTTCAGTACCATGGGAAG-3′ and 5′-GAGTTTACTTAGTTTTAATATGGCTGTA-3′ for fragment 2, and 5′-CATATTAAAACTAAGTAAACTCGATCCTGGTAACTATAGTTTCAG-3′ and 5′-CGGCCGCCACTGTGCTGGATTTACTTATCGTCGTCATCCTTG-3′ for fragment 3 for PKD 2 AAVR swap-in mutant d (aa 31 to 60); 5′-GGTGGAATTCTGCAGATACCATGGCGCCCCCCACAGG-3′ and 5′-GAAAGTGTAGTTCCCAGGGACAAGGTTAGACAAGCGTAAGACGGGAGAG-3′ for fragment 1, 5′-GTCCCTGGGAACTACACTTTCAG-3′ and 5′-AGCTTTGTTCACTGTCAGGTTTG-3′ for fragment 2, and 5′-GGTGGAATTCTGCAGATACCATGGAGAAGAGGCTGGGAGT-3′ and 5′-AACTGCTACAGGTGGCAGGTTCTTACGGGGCTCTGGCTTGAC-3′ for fragment 3 for PKD2 AAVR swap-in mutant e (aa 61 to 90); and 5′-GGTGGAATTCTGCAGATACCATGGAGAAGAGGCTGGGAGT-3′ and 5′-AACTGCTACAGGTGGCAGGTTCTTACGGGGCTCTGGCTTGAC-3′ for fragment 1, 5′-AACCTGCCACCTGTAGCAGTT-3′ and 5′-TTCAGTATCATCTGTACTTTGGCTGCCA-3′ for fragment 2, and 5′-AAAGTACAGATGATACTGAAATCGTTCAGTACCATTGGGAAG-3′ and 5′-CGGCCGCCACTGTGCTGGATTTACTTATCGTCGTCATCCTTGTAATCC-3′ for PKD2 KIAA0319 swap-in mutant f (aa −4 to 30).
(ii) Bacterial expression constructs.For GST-fused PKD expression, the pGEX-4T-3 vector (GE Healthcare Bio-Sciences, Pittsburgh, PA) was used to clone various AAVR PKD-encoding regions, which are diagrammed in Fig. 3A and 6A and B, into BamHI/XhoI sites.
For the expression of polyhistidine (His)-tagged PKD1, PKD2, and PKD3, the PET30a(+) vector (Novagen/EMD Millipore, Billerica, MA) was used to clone PKD1-, PKD2-, and PKD3-encoding sequences through the NdeI/XhoI sites. The coding regions of PKD1, PKD2, and PKD3 are diagrammed in Fig. 3A.
(iii) rAAV vector constructs.pAV2RepAV5Cap was constructed by replacing the AAV2 VP-encoding sequence with the AAV5 VP-encoding sequence of pAV5RepCap (53) in pAV2RepCap (psK45) (53). pAV2RepAV1Cap and pAV2AV8Cap were described previously (54, 55). pAV2F5tg83luc-CMVmChery (4.6 kb) is an rAAV2 cis transfer plasmid containing the AAV2 inverted terminal repeats (ITR), the simian virus 40 (SV40) poly(A)-firefly luciferase gene-F5tg83 promoter (luciferase expression cassette) (49), the cytomegalovirus (CMV) immediate early (IE) promoter-mCherry gene-bGH (bovine growth hormone) poly(A) (mCherry expression cassette), and the AAV2 ITR, in order.
Protein expression and purification.PKD-cloned pGEX-4T-3 or pET30 plasmids were transformed in E. coli strains BL21 and BL21(DE3)/pLysS, respectively. GST-fused PKD and His-tagged PKD proteins were expressed by the induction of isopropyl-β-d-1-thiogalactopyranoside (IPTG) at 1 mM (at an optical density [OD] of 0.6). His-tagged PKD1, PKD2, and PKD3 proteins were purified by using Ni-nitrilotriacetic acid (NTA) agarose (Qiagen, Germantown, MD) under native conditions according to the manufacturer's instructions. Purified proteins were separated on an SDS–12% PAGE gel to ensure a purity of >90%.
Generation of stable cell lines.Lentiviral transduction was used to create stable cell lines expressing a selected gene of interest under a CMV promoter. By using the Gibson assembly reaction, the respective genes of interest (see the section on the construction of plasmids, above) were inserted into the pLenti-CMV-Puro-DEST vector and used as described previously (52). Lentivirus was produced by using HEK293 cells and was utilized to transduce the respective cell lines overnight. Cells stably expressing the gene of interest were selected by treatment with 1 to 3 μg/ml puromycin over 2 days (InvivoGen).
Virus overlay assay.Proteins were separated by SDS-PAGE or 2-D electrophoresis and electrotransferred onto a nitrocellulose or PVDF membrane. The virus overlay assay was performed as previously described (56). Briefly, the membrane was sequentially incubated with Tris-buffered saline–0.1% Tween 20 (TBS-T) buffer with 10% nonfat milk (NFT), purified WT AAV or rAAV at ∼5 × 1011 vgc/ml in TBST–2% NFT, an anti-intact AAV2 or AAV5 antibody at a 1:100 dilution in TBST–2% NFT, a horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch, Inc., West Grove, PA), and then SuperSignal West Pico chemiluminescent substrate (Thermo-Fisher) for signal development under a Fujifilm LAS 4000 imager (Fujifilm Life Sciences). Images were processed with Multi Gauge version 2.3 software (Fujifilm Life Sciences).
Immunoblot analysis.Proteins were separated by SDS-PAGE or 2-D electrophoresis and electrotransferred onto a nitrocellulose or PVDF membrane. Membranes were blocked by incubation with 1× phosphate-buffered saline (PBS) containing 5% NFT for 1 h at room temperature (RT). Membranes were subsequently incubated overnight at 4°C with primary antibodies in blocking buffer. Membranes were then washed 3 times for 5 min by using wash buffer (1× PBS with 0.1% Tween 20) and further incubated with HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit at 1:5,000 in blocking buffer) (GeneTex) for 1 h at RT. After another set of three washes, antibody-bound proteins were visualized on film by using West Pico and Extended Duration chemiluminescence peroxide solutions (Thermo-Scientific, USA).
rAAV transduction assay.Cells were seeded at 10,000 cells/well (96-well plate) overnight. They were then infected with scAAV2-CMV-RFP or scAAV5-CMV-GFP at a multiplicity of infection (MOI) of 20,000 viral genomes (vg)/cell, in complete DMEM. Virus infectivity was determined at 48 h postransduction by measuring transgene expression (red fluorescent protein [RFP] or green fluorescent protein [GFP]) by using flow cytometry. All transductions were performed in triplicate. To measure virus transgene expression (RFP or GFP) in all other experiments, cells were trypsinized 48 h after infection, and a BD LSR Fortessa flow cytometer (BD, Franklin Lakes, NJ, USA) was used to detect fluorescent cells. Data were analyzed and assembled by using FlowJo software (TreeStar, Inc., Ashland, OR, USA).
Virus neutralization assays. (i) Luciferase assay (Fig. 7D).HeLa cells were seeded into a 96-well plate at 1 × 104 cells per well 1 day prior to transduction. Purified PKD or the GST protein control was mixed with rAAV(Luc/mCherry) in a volume of 100 μl of PBS (pH 7.4) at various concentrations for 30 min at room temperature. At the time of infection, the medium was removed, and 100 μl of the rAAV(Luc/mCherry)-protein mixture was added to the wells of cells (at an MOI of 7,500 vg per cell). At 3.5 h postinfection at 37°C with 5% CO2, the inoculum was removed, the cells were washed with DMEM with 10% FCS briefly, and 100 μl of medium was added to each well. The cells were incubated for 48 h, followed by quantification of luciferase expression. Luciferase activity was quantified by using a luciferase assay system kit (catalogue number E1500; Promega, Madison, WI) according to the manufacturer's instructions, with measurements being taken on a Synergy H1 microplate reader (BioTek, Winooski, VT). The construction and purification of the His-tagged PKD1, PKD2, and PKD3 proteins used in this experiment are described above. The maltose binding protein–PKD1-5 fusion protein used as a control was described previously (21).
(ii) Fluorescence assay (Fig. 7E).HeLa cells were seeded into 96-well plates at 10,000 cells per well overnight. Purified, soluble His-tagged AAVR PKD domain constructs were introduced to the medium at the specified concentrations. Cells were then transduced with scAAV-CMV-GFP or scAAV5-CMV-GFP at an MOI of 12,000 vg per cell and incubated for 24 h at 37°C. This was followed by trypsinization and measurement of transgene expression by flow cytometry.
We thank members of the Carette, Qiu, and Chapman laboratories for discussions and support and Juliana Idoyaga (Stanford University) for flow cytometer access and discussions.
S.P., W.Z., M.S.C., J.Q., K.E.B., J.F.E., and J.E.C. were responsible for the overall design of the study. S.P. designed and constructed swap mutants and performed all genetic complementation and mutant studies. J.E.C. designed deletion and glycosylation mutants, J.E.W. constructed glycosylation mutants, A.S.P. performed mutant immunoblots, and O.D. contributed to the production of E. coli ectodomain constructs. W.Z. and J.Q. prepared membrane proteins and purified AAV-BP. W.Z. prepared purified AAV stocks, and J.Q. and F.C. constructed PKD-expressing plasmids in E. coli and performed all virus overlay assays. S.S.G. purified His-tagged PKD proteins. X.D. and Z.Y. performed virus neutralization luciferase assays, and N.L.M. performed the fluorescence neutralization assay. S.P., J.E.C., and J.Q. wrote the manuscript, with intellectual input from M.S.C., J.F.E., and Z.Y., and W.Z. revised the manuscript.
The work was funded in part by NIH grants R01 GM066875 (M.S.C.), R35 GM122564 (M.S.C.), DP2 AI104557 (J.E.C.), U19 AI109662 (J.E.C.), R01 AI070723 (J.Q.), R21 AI112803 (J.Q.), and P01 HL051670 (J.F.E.); Cystic Fibrosis Foundation grant YAN15XX0 (Z.Y.); the Weston Havens Foundation (J.E.C., S.P., and A.S.P.); and the Stanford SPARK program (S.P. and A.S.P.). J.E.C. is a David and Lucile Packard Foundation fellow. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
S.P., A.S.P., N.L.M., O.D., M.S.C., and J.E.C. are inventors on a patent describing the utilization of AAVR to modulate AAV transduction.
|You are subscribed to email updates from "parvoviridae,cmv" - Google News.
To stop receiving these emails, you may unsubscribe now.
|Email delivery powered by Google
|Google, 1600 Amphitheatre Parkway, Mountain View, CA 94043, United States
- Get link
- Other Apps


text:
Rosie Jane
Rollerball Perfume - Rosie
Regular price $39.00
A rollerball perfum with no parabens, sulfates, phthalates. totally cruelty free. vegan.
ingredients: coconut oil, essential oil, fragrance
- Leila Lou effortlessly captures the essence of Spring with notes of pear, jasmine and fresh cut grass. Clean and fresh.
- Tilly effortlessly captures the essence of summer with notes of grapefruit, pineapple and tropical gardenia. Warm, sensual and delicate.
- James has notes of fig, amber and gardenia. James is light, earthy and romantic. Our best seller!
- Rosie is inspired by the cooler months; with layers of red rose, moroccan rose, nude musk and a touch of vanilla.


text:
Here is the team I'm going to in Match Day 2 with, I called all of my guys and asked them to put on a much better performance than last week! Legend: (*) – starting XI, ⇐ – incoming (transfer), ⇒ – outgoing (transfer).
- *David de Gea – £6 – Week 2 points: 1 — As noted in our Week 2: Post-Game Thoughts (Part 2 of 2), this has got to be the surprise of the week. I had written about de Gea having a good shot at a clean sheet, but apparently Manchester United's kryptonite struck again. Despite the less than stellar performances for de Gea, I don't plan on moving him anywhere.
- Jonas Lössl – £4.5 – Week 2 points: 0 — Lössl v. Man City was an absolute non-starter, I think Lössl will have some good games this year, but this was never going to be one.
- *Schindler – £4.5 – Week 2 points: -2 — I didn't expect a huge night from Schindler, but I also didn't expect Huddersfield to ship 6 goals.
- * – £5.5 Week 2 points: 1 — Monreal made it back into the side and it was just not a great team performance, I feel pretty comfortable about holding to him for the game against West Ham and beyond — Arsenal should run into a string of easy fixtures.
- *Wan-Bissaka – £4.5 – Week 2 points: -2 — Wan-Bissaka got sent off towards the end of a game for denying Mo Salah a goal scoring opportunity. It's a shame really because the rest of his game was marvelous. He will miss the next game (vs. Watford) because of the red card, but I would be absolutely shocked if the disciplinary committee gave him anything but a one-week ban. Best case scenario is they rescind the ban, but I think this is unlikely.
- Fredericks – £5 – Week 2 points: 0 — Fredericks didn't make the team for Match Day 2 after a rough showing for West Ham in their decimation against Liverpool. West Ham has a really rough schedule going forward, I'm considering moving him out until closer to their stretch of easier fixtures (might allow me to buy him back for even less).
- Lovren – £5 – Week 2 points: 0 — Lovren expected to come back 1 Sep, and he should work his way back into the side. I don't think he'll even need to play the full 90′ to make an impact on the pitch.
- *Mo Salah – £13 – Week 2 points: 9 (18 if he was your captain) — Salah stacked the sheet yet again, he had a hand in two separate goals and was just generally dominant. No reason to come off him, probably for the whole season.
- *Pogba – £8 – Week 2 points: 8 — Manchester United's performance definitely hindered Pogba's value this week, he managed to put points on the board because of a converted penalty.
- *Fred – £6 – Week 2 points: 2– United played terribly, and with spurs coming up — I'm having some reservations about continuing with him on my lineup.
- Jahanbakhsh – £7 – Week 2 points: 0 — I'm starting to have reservations about holding on Jahanbakshsh, he made the bench, but I'm struggling with whether or not to sell keep him on my roster. He's almost the same price as Pogba who's actually been contributing to my side, Jahanbakhsh has only played 20 minutes in the league and featured yesterday in the Premier League 2 side. That's not looking promising for a start this weekend.
- *Jack Wilshere – £5 – Week 2 points: 1 — Wilshere didn't contribute much to West Ham's eventual loss to Bournemouth.
- *Harry Kane – £12 – Week 2 points: 7 — Kane broke his August drought and put a goal in support for us. He very nearly bagged a second, but I'm thrilled about his potential for the season.
- *Marko Arnautovic – £7 – Week 2 points: 8 — We got a little bit from Arnautovic this weekend, and I think he's still going to be the focal point for West Ham going forward, so he won't be leaving my team.
- *Joshua King – £6.5 – Week 2 points: 1 — Although he didn't really contribute, King had a good game and Bournemouth has an easy run, with the exception of Chelsea in week 4, until week 11 (which is against Man Utd).
Well, I guess technically I got my wish as the score was higher. This week I got hurt by the crazy Manchester United upset and the 6-goal shellacking by Man City. This season has started off rough, but we'll have a better sense of what's to come in the next little bit.
Week 1 score: 38 Points 2018-2019 Fantasy Premier Week 1: Recap
Week 2 score: 43 Points
While we desire to keep our content free for our readers and free from ads, there are costs associated with this project. If you would like to help offset these costs we have partnered with some merchants through affiliate links, you can find them here.


text:
Classic Bedroom
Our classic range of bedrooms offer guests a well equipped room with Freeview television, comfy bed and bathroom. All bedrooms have beautiful views overlooking our hotel grounds or our picturesque courtyards.
Room Amenities
- Freeview television
- En-suite bathroom with shower or bath
- Complimentary toiletries
- Free WiFi
- Coffee and tea facilities
- Telephone
- Hairdryer
Our Classic Rooms types include Twin and Interconnecting rooms


text:
Copendium: Julian Cope / Various [Import]
- (United Kingdom - Import)
- Artist: Various Artists
- Format: CD
- Release Date: 11/6/2012
|your cart is empty
2012 three CD collection, the 'soundtrack' to the recently published collection of writings from musician/author/musicologist Julian Cope. Never less than real, this compilation is as refreshing as it is challenging. It launches from the opening riff of Lord Buckley into a chronological trip through the dawn of hard rock/Psychedelia/soundscapes of the late 60s/early 70s, arriving at the outer limits of punk with the final track on Disc One.. with a great leap across the 80s, Disc Two is an intense, relentless destructo-guitar fest in the age of grunge. By Disc Three anything goes, acoustically or electrically driven, but the intensity does not abate. Not for the faint-hearted, the whole set is held together by the Cope doctrine of music. All of the music, sequencing and audio have been overseen by Julian Cope.


text:
As a business owner, you already know everything there is to know about shipping and delivery services, right? What if there was some new ways or methods of shipping products that not only shipped things to your customers faster but cheaper too? It saves you and the customers money and the products still get to them in record time. That is the kind of shipping you would want to offer, right? Well, those kinds of solutions are now available.
Shipping Companies Teaming Up with the Postal Service
Some of the most popular and widely used shipping companies in the U.S. have recently teamed up with the United States Postal Service. The benefits of these unions is two-fold. One, your customers get expedited shipping that costs less than it would if the products were sent via a single delivery service, and two, customers have the option of receiving the package via their local postal delivery by waiting one extra day or they can take the tracking number to the post office on the day that the post office receives the package from the other shipping company and pick their packages up there.
Flat Rates for Shipping
Another inexpensive and speedy shipping option is the flat rate method. Regardless of what the product is or even the weight of the products being shipped, if you can squeeze it into a specially marked shipping box or envelope, it ships at a flat rate. This flat rate also provides one- to two-day shipping anywhere in the continental United States (Hawaii and Alaska may require an extra day or two given their distance and the fact that a plane may be delivering the packages there). The only restriction that comes with this method of shipping is that you cannot send anything fragile, perishable, toxic or otherwise dangerous. Everything else, regardless of size or weight, is acceptable and accepted so long as it fits in the box or envelope.
International Expedited Air Mail
If your packages are heading out of the country and going around the world, no one wants to wait two weeks to a month to get what they ordered. You can abbreviate the wait time while still getting their packages to them for an affordable amount of shipping. This is done through international expedited air mail, a service commonly provided by certain shippers who have direct access to international airports and flights and can schedule your pick-ups from your office and drop them off directly at the airport where they will await the next outgoing international flight.
For more information about quick shipping options, contact a company like Kidd Curry Express.Share


text:
Commerce Server 220.127.116.11 released
For who keep track of CommerceServer releases on 28th October has been released the latest version of CommerceServer, you can read here more about it
testing Commerce Server Pipelines
this is a great resource to learn how to instrument your own project to test PipeLines…
if your CS implementation is correct, you will be able to test 95% of your business logic testing your pipelines, testing pipelines, it could be very useful to verify that a new discount is interacting correctly with the existing ones, and that's a way to automate your discount testing procedure.
3X2 Discounts on Commerce Server
As several you know, Commerce Server discounts are very cool, but sometime, if you don't know what you are doing, they can make you crazy…
the most frequent question that I receive from my clients is about the 3 x 2 Discounts or better called N x M… generally CS newbies can configure correctly the discount on Commerce Server, but they complain about the fact that the award item is the most expensive and not the cheapest one (as it make sense for a 3×2 discount).
well Commerce Server support it from long time, the only things that you have to do is configure the Order Discount Pipeline Component properly to satisfy your discount condition. In the specific case, to let it works 3×2 properly customers HAVE to change the option to the Order Discount so that the default award is NOT the MOST EXPENSIVE FIRST but the cheapest one first.
Unfortunately this option belong at pipeline level, it means, that all basket that will run that pipeline, will follow that business rule… if you really want to avoid it, you should implemnt several pipelines and try to understand witch pipeline to run for each specific basket.
Place Orders outside of the webContext
99% of developers still look at Commerce Server as Web Platform…
in my project experience, I understood to appreciate Commerce Server as an application platform for E-Commerce architectures….
well the difference between a Web Platform and an Application platform is quite big, and this post want to demonstrate and explain hot to run the CS MultiChannel foundation outside from a web application.
More specifically I will explain how to place an order (running pipelines and so on…) in a console application.
you can get the full source code, the exe and all the proper documentation here
this kind of scenario could be very useful to test your schema changes, Order Components, Commerce Query, Pipeline, Pipeline Components and so on much more quickly than in a classic web Context.
this project could also used to instrument your own Unit – Integration – Load test environment running test on the CS Components.
the most important configuration setting belong in the channel configuration
as you can see the code is quite simple and use a lot of the CSSamples
and the contemporary site.
//Add LineItem to the Basket Basket mybskt= BasketManager.AddLineItem( new Microsoft.Samples.Commerce.Order.CommerceEntities.LineItem("Adventure Works Catalog","AW075-03","64",1)); var myShippingAddress = new Address(); myShippingAddress.AddressName = "any"; myShippingAddress.AddressType = 2; myShippingAddress.Id = Guid.NewGuid().ToString(); var myBillingAddress = new Address(); myBillingAddress.AddressName = "any name"; myBillingAddress.AddressType = 2; myBillingAddress.Id = Guid.NewGuid().ToString(); var myCreditCard = new CreditCard(); myCreditCard.AuthorizationCode = "AuthCode"; myCreditCard.CustomerName = "custName"; myCreditCard.CreditCardNumber = "4111111111111111"; myCreditCard.DisplayName = "test displayname"; myCreditCard.ExpirationMonth = 1; myCreditCard.ExpirationYear = 2015; myCreditCard.BillingAddressId = myBillingAddress.Id; myCreditCard.PaymentMethod = new CommerceRelationship(); myCreditCard.PaymentMethod.Id = paymentMethodId; //Set the Shipping, BillingAddress, ShippingMethod,CreditCard and run basket and checkoutPipeline BasketManager.PrepareForCheckout(myShippingAddress, myBillingAddress, myCreditCard, shippingMethodId, mystr.ToArray(), paymentMethodId); //Place the Order and run the CheckoutPipeline BasketManager.Checkout();
this post was also inspired by the following post http://notepuddle.com/2009/02/10/using-commerce-server-2009-outside-of-a-web-application/
you can check also the following reosurces for CS 2007 http://msdn.microsoft.com/en-us/library/bb219215.aspx
and this application that do the same thing on CS 2007http://blogs.msdn.com/nihitk/archive/2006/08/07/RunningPipelinesFromAConsoleApp.aspx
Commerce Server Profile Adapter – Useful Information
As most of you already know, commerce server come with a set of Biztalk Adapters that rely on Commerce Server Web Services (the same ones used by the Business User Applications)
to create your own schema for the profile system you can use the ExportProfileXsd.exe tool
but before starting to work on the Profile Adapter there are few things that you should keep in mind:
- it does not support multiple profile import, it means one profile for each message….
- it support message types CommerceServerProfilesImport and CommerceServerProfilesUpdate on the port level and you HAVE to use Import to Add a profile and update to UPDATE a profile
the first point means, that you will have a bigger number of messages to handle (it could affect the monitoring of your solution – transaction handling and your orchestration complexity)
the second point means that in a typical UPSERT scenario you will have to query the profile system to decide witch message type to use (you can specify it in the context)
these 2 limitations are quite big and it could affect your implementation and the decision to use the Profile Adapter to integrate your profile system in your Architecture….
obviously you could consider to workaround it extending the profile system web service with your own logic to handle the UPSERT scenario, or write your own custom WCF or Biztalk adapter to support your specific requirements or consider using the SQL adapter or consider a different integration pattern (SSIS!?)
as usual there are several approaches when it comes to integrate systems and each one has his own pro and contra….
Commerce Server Catalog Adapter, common Issue with GlobalPropertySchemas
this is one of the most common issue that I had working with Commerce Server catalog Adapter…
after a succesfull installation and configuration you will receive the follwing mesage:
The Messaging Engine received an error from transport adapter "Commerce Server Catalog Adapter" when notifying the adapter with the BatchComplete event. Reason "Exception from HRESULT: 0xC0C01620".
The problem is quite simple and easy to solve….
and it's related to the fllowing assembly:
For some reason is not installed correctly in your biztalk application (where you are using the Commerce Server Catalog Adapter)
so what you need to do to fix it is just add it to your application using the Biztalk administrative console…
just go on the root, select Add Biztalk Assemblies and select Microsoft.CommerceServer.Adapter.GlobalPropertySchemas.dll in the Commerce Server 2007 installation directory.
Once you finish the installation everything will work as expected.


text:
Getting past the myths that persist
2019 is shaping up to be an awesome year for people who walk and bike in Traverse City. In June, MDOT will install HAWK (High-Intensity Activated CrossWalk) signals on Grandview Parkway. These will go in at Elmwood and Hall street. This fall, Traverse City will cut the ribbon for the reconstructed Eighth Street. The new Eighth Street is narrower (slower) and even includes one of Michigan's first separated protected bike lanes.
All too often, good news for the pro-walk, pro-bike team attracts naysayers. These two projects won't be any different (they already haven't). This inspired Norte board member, Chris Hinze, to examine a few of the usual suspects and offer a little Myth Busting.
"Bikes don't belong on the road…get on the sidewalk!"
People have a right to bike on both the roadway or the sidewalk. For people new to riding or those still building up confidence, riding in the roadway is intimidating. For them, riding on the sidewalk may be an attractive option. But, there are reasons that this isn't the safest decision.
First, riding a bike on the sidewalk can lead to conflicts with people on foot. Second, in certain locations riding on the sidewalk is against the law. Downtown Traverse City is an example.
Last, and most important, riding on the sidewalk is more dangerous. The risk of a crash between someone on a bike and a car is higher when the former is on the sidewalk. The person driving is often caught off guard because of the sight lines and speed of the person on a bike. Protected bike lanes and trails are designed to reduce this conflict. Sidewalks are not.
Again, the streets are multimodal. Outside of limited access highways, everyone has a right to bike in the streets. To brush up on your Michigan law, visit the Michigan Vehicle Code.
"Traverse City has a perfectly good TART trail…get on the trail!"
A close cousin to the "get on the sidewalk" denouncement is "get on the trail." What "get on the trail" fails to understand is that, by and large, trails are for recreational purposes. When people bike they tend to do so to get somewhere. And, to get somewhere with the least amount of hassle and in the least amount of time. We don't ask people to only drive on highways so why ask bicyclists to only use trails. People on bikes have places to go. When they do so on a bike, they are freeing up street space that can help relieve congestion–besides to freeing up a parking space.
"Bikes don't pay taxes!"
Well, this one is true; bikes don't pay taxes. But, neither do cars. Or any other inanimate object for that matter. People pay taxes; we all pay taxes. Those taxes pay for the expense of constructing, maintaining, and policing streets. The majority of this comes from property taxes, which we pay regardless of how we move around. For local streets, less than 10% comes from user fees like gas taxes and registration fees. The user fee dollars predominantly go towards big projects like highways.
Countless studies have shown that walking and biking break even as a cost to society. Meaning, for every $1 you spend on biking, society chips in less than a dime. For driving, every private dollar spent on motordom, society is paying around $9. That's a Hefty subsidy no matter how you slice it.
"Bikers don't obey the rules so they don't deserve nice things!"
This is the ultimate "straw man" fallacy. This argument references the time someone saw that one person on a bike run a stop sign (confirmation bias). As a result, the community doesn't deserve better bicycle infrastructure. If bicyclists don't follow the rules and thus don't deserve bike lanes, what then of drivers and streets? Local law enforcement agencies dedicate entire portions of their department to "traffic enforcement." Does that mean because some people in cars speed or run red lights that we should not allow cars to use the streets? As someone who uses cars as well as bikes and walks, that doesn't seem logical.
Here at Norte, we encourage everyone to be courteous regardless of how they move around town. When we walk, bike, drive, or otherwise roll we need to be aware of our surroundings and understanding of others who, like us, are simply trying to get through their day. Safer sidewalks and better streets will only be that much sweeter when do.
What myths and biases do you see at play in the community?


text:
Apple is hard at work trying to get Samsung's Galaxy Tab 10.1 banned in the European Union, and a lawsuit happening in The Hague might be enough to block the tablet where a German lawsuit has failed. The Galaxy Tab 10.1 case rages all over the world, with Apple claiming that Samsung copied its iPad […]
Apple is hard at work trying to get Samsung's Galaxy Tab 10.1 banned in the European Union, and a lawsuit happening in The Hague might be enough to block the tablet where a German lawsuit has failed.
The Galaxy Tab 10.1 case rages all over the world, with Apple claiming that Samsung copied its iPad 2 and it's actively suing Samsung in multiple countries to get imports of the Korean company's tablet banned. It was working in the European Union after an injunction was handed down from a Dusseldorf district court, banning the sale of the Galaxy Tab 10.1 in the European Union – until a question of jurisdiction suspended enforcement of the indictment until a hearing that takes place next week.
Another suit going forward in The Netherlands could reinstate the ban, however, according to Ars Technica. That suit could have a serious effect on Samsung's tablets and smartphone handsets because The Hague is where Samsung's devices enter Europe.
The Ars Technica story also mentions that suit in The Netherlands is actually broader than those against Samsung in other places:
Apple's case in The Netherlands not only concerns the EU Community Design, but is much broader in scope and much more demanding in terms of injunctive relief. In addition to allegations that Samsung copied the iPad's physical design, the complaint includes allegations of infringement of three European functional patents, including one for mobile photo management, one for interpreting touch events, and Apple's 'swipe to unlock' patent.
The EU Community Design registration for the iPad is the document Apple claimed Samsung violated in its German suit. Samsung could have a harder time with its devices should The Netherlands suit go against it; and that's to say nothing of the German suit, or suits that are taking place in other places. According to Ars, Apple is suing Samsung in nine countries and 11 courts: France, Italy, U.K., Japan, The Netherlands, South Korea, Germany and Australia, as well as the U.S.So far, Apple hasn't made much forward motion in the U.S., and the Dutch case's ruling is expected to come down on Sept. 15. Meanwhile, even if things don't go Samsung's way with the Galaxy Tab 10.1 as it stands, the company may have another device waiting in the wings to replace it.
Samsung has already agreed to release a different version of its Galaxy Tab in Australia in order to avoid a lawsuit with Apple. It's even allowing Apple to examine the device for a week in order to satisfy the company that the new device doesn't infringe any of Apple's patents. If it already has that different device, which it refers to as the Australian Galaxy Tab model, Samsung could realistically release another tab to dodge Apple's complaints and still maintain a foot in the market.
We'll have to wait and see how all these cases shake out, both for Samsung and Apple. If the Galaxy Tab 10.1 is allowed to be sold as-is, it may very well be able to take a bite out of the iPad's market share.


text:
I would like to live on a Desert Island in a beach hut! Where would you like to live if you could choose?
Desert IslandMarch 27th, 2015 | Posted by in Year 1 Archive - (7 Comments)
Houses and Homes Summer 1March 27th, 2015 | Posted by in Year 1 Archive - (39 Comments)
In our new project we will be exploring all learning about Houses and Homes. We will be using History, Geography, Art and DT in our project. Here are some starting points for discussion on the school website. Try to answer in full sentences with punctuation and check before you post! Remember Blogger of the …
Read more


text:
PRODUCTS > Rope & Rope Equipment > Rope Care & Maintenance > PMI Spiroll Rope Protector
Your lifespan is directly proportional to that of your rope's. Love your rope.
Price: $12.00 Qty:
Questions? | Quote | Email
Effective, economical protection for your stationary ropes, Spiroll® Rope Protectors employ a unique patent pending technology. Rugged polyurethane material wraps snugly around a single rope several times to secure itself anywhere it is placed without being tied.
PMI Spiroll Rope Protector
Sponsored Links:
See item for pricing
Price: $15.00
Price: $8.00
Home | Products | About Us | Contact Us | Search | Site Map
Privacy Policy | Promo Policy | Shipping Policy | Tax Exempt Policy & W-9 Form | Disclaimer | Feedback
© Copyright 1997-2020 Columbus Supply. All Rights Reserved.
Font Size: Switch views: What's this?


text:
OTTAWA — Canada's science minister says more than half the scientists nominated by universities for lucrative Canada 150 research jobs are female.
Kirsty Duncan is gleeful universities heard loud and clear her ultimatum earlier this year that if they didn't step up efforts to find more women to fill research chair seats they would lose federal funding for future positions.
In the opening round of Canada 150 research chair nominations, 52% of the candidates submitted by schools were women.
The latest round of Canada Research Chair nominations included 41% women which Duncan says is the highest percentage of women ever submitted for the jobs.
Duncan says the names are still going through the approval process but she still sees this as a sign of change and a positive advancement for diversity in science.
During the April round of Canada Research Chair applications, Duncan was appalled when twice as many men were nominated as women, prompting her ultimatum to the schools.


text:
Refs & annos
Baldwin's Kentucky Revised Statutes AnnotatedRules of Criminal Procedure
Kentucky Rules of Criminal Procedure (RCr) Refs & annos
Editors' Notes
A. Introduction
1. Derivation and scope
The rules of procedure in criminal cases incorporate refinements of procedural reform without significant departure from the traditional scheme of criminal procedure as outlined in Blackstone's Commentaries, limited in constitutional bills of rights, and enacted in the Criminal Code. A few of the refinements already had become part of the practice as a result of judicial application or changes in other practice, but many improvements universally desired had been unattainable because of rigid requirements of a practice designed more to implement technical concepts than to promote the interests of justice.
The rules were derived from a section by section examination of the 473 sections of the Criminal Code and a comparison of these sections with similar rules in the American Law Institute Code of Criminal Procedure, the Uniform Rules of Criminal Procedure promulgated by the National Conference of Commissioners on Uniform State Laws, recent revisions of the codes of other states, the Kentucky Rules of Civil Procedure, and the Federal Rules of Criminal Procedure. Common law principles and case law applications also were studied, as were related statutes, particularly those that overlapped the code provisions. No changes were made for the sake of change. On the other hand, there was no hesitancy to make a change where it was desirable, as little of the Criminal Code had been reevaluated since 1876.
2. Arrangement
While the arrangement of the rules is very similar to the Criminal Code, changes in scope and form offer much clarification, simplification and condensation. Without changing the practice, the four titles relating to the classification of offenses, jurisdiction and venue, impeachment, and removal of court clerks were eliminated as matters governed by the constitution, statutes or other court rules, or as not essential to general criminal practice. With few changes in the practice, the three titles relating to police courts, justice's courts, prevention of offenses, and habeas corpus were eliminated as covered by other rules or as more properly statutory. The chapters on bill of exceptions and supersedeas bonds were eliminated as a result of substantive changes, as were several series of sections. Recognized ambiguities and anomalies were eliminated, and restatement of the other provisions makes them briefer and groups them in a manner more easily understood. The practice is made more complete in several phases, particularly the production of evidence.
B. Synopsis of Changes in Practice
The form of the rules makes the changes in substance seem greater than they are. Except as to the requirements for indictments and the pleading practice, most of the changes are not major. There are many changes in detail but some are for the sole purpose of reducing unnecessary differences between civil and criminal practice, and between state and federal courts. The summary of changes here presents only those changes that are significant.
1. General provisions
All criminal proceedings will be governed by rules designed to obtain better administration of justice. Penal actions are not recognized as a separate class, but governed entirely by the criminal rules.
2. Arrest
Institution of the complaint as a charging instrument is basically a formalization of existing practice but, with the change allowing magistrates to issue summons in felony cases, and officers to issue citations in any misdemeanor, should result in more complete satisfaction of constitutional requirements and fewer physical arrests. The taking of bail by arresting officers is eliminated and an immediate hearing required in all cases.
3. Preliminary hearings
Prescribing the alternative of an inferior court trial, and incidental powers to require peace bonds, does not change the practice, but a requirement that makes a full transcript of recorded testimony available to the defendant is intended to insure fair play at this stage of the proceedings.
4. Bail
The change to allow the making of one bail bond for appearance at all stages of the proceedings should reduce administrative steps and remove what in many cases is a hardship on defendants. It will be possible for a case to originate in magistrate's court, be tried in circuit court either de novo or upon an indictment, and be retried following a reversal by the Court of Appeals while the defendant remains on bail through execution of two bonds, one at the beginning and the other following his conviction in circuit court. Provisions for broader qualifications and better justification of sureties, and more effective forfeiture procedures, should do much to reduce abuses in bond making. Standards governing the amount of bail are prescribed for the first time, and changes in provisions governing deposit of money and surrender of principal modernize the practice considerably.
5. Grand jury
The only substantial practice change in grand jury proceedings is that if the grand jury to which a defendant is held fails to indict him he cannot be retained in custody or under bond pending submission of the charge to the next grand jury. Here also, a transcript of all testimony relating to his indictment is made available to the defendant. Modern recording devices are authorized and the matter of other persons with the grand jury clarified.
Editor's Note
Amendment of section 5 by Court of Appeals, June 26, 1964.
6. Indictment and information
The provision allowing use of the information to prosecute any offense that is not required to be prosecuted by indictment is designed to encourage the use of this process. A federal court type indictment, in which is contained only the name of the offense and the essential facts alleged to constitute it, will replace the old indictment, which was hedged about with several technicalities that served only as sources of trouble. The bill of particulars is made a part of the practice.
7. Process on indictment and information
The only change in the process on indictment and information is the addition of provisions making the practice complete as to information process.
8. Production of evidence
A more complete deposition practice is provided by allowing the Commonwealth to take depositions with protection of the defendant's right of confrontation. Provision for securing the attendance of material witnesses is added. The defendant is permitted to discover and inspect documents seized from him, giving him an opportunity to prepare his explanation of the documents.
9. Arraignment and pleadings
The reduction of technical pleadings, such as demurrers and motions to set aside, to the pleas of guilty and not guilty, and the substitution of a simplified motion practice under which the defendant may move directly for the relief he seeks should result in settlement of preliminary issues without delay.
10. Trial
All jurors in circuit court will be drawn from the jury drum, eliminating the use of bystanders except by consent of the parties. To obtain full attention of all jurors who might participate in the verdict, extra jurors impaneled in felony cases will sit as regular jurors until time for retirement for deliberation, at which time a drawing determines the final makeup of the jury. Elimination of classes and specifications of bias does not change the practice.
Many of the changes in trial provisions make details of records, witnesses, and instructions like the practice in civil cases or like the practice in federal courts. One such change authorizes the court to call expert witnesses. More significant is the elimination of the need to take formal exceptions to the decisions of the court in order to preserve objections for purposes of appeal--formality already abolished in civil procedure. Consequently, the bill of exceptions also is abolished. Other important changes would put matters of joinder and severance largely in the discretion of the court. There are no changes in the practice relating to verdict.
11. New trial and arrest of judgment
The prescription of grounds for new trial is generalized. The time for making the motion is changed from during the term to within five days after the verdict. Requirement that grounds must be stated in the motion in order to preserve them for review is reduced to only those not previously considered by the court.
12. Judgment and execution
Sentence is to be imposed without delay instead of waiting two days in felony cases. The writ of coram nobis is abolished. The presumption that multiple sentences are to be served concurrently is abolished. Provisions as to disability of judge and setting aside sentences make the practice more complete.
13. Appeals
The only changes in appeals to circuit court are changes in detail making the practice more uniform. Similar changes are made in provisions relating to appeals to the Court of Appeals. The time for taking appeal is measured from the entry of judgment instead of the term of court. The certificate of appeal is replaced by a notice of appeal; the bill of exceptions is replaced by the transcript of proceedings. Bail on appeal is simplified, and other provisions make familiar provisions of civil appeals, such as agreed statement and narrative statement, a part of appellate procedure in criminal cases.
Rules Crim. Proc., Refs & annos, KY ST RCRP Refs & annos
Current with amendments received through May 1, 2020.
|End of Document
|© 2020 Thomson Reuters. No claim to original U.S. Government Works.


text:
- Nonkanyiso Conco, Jacob Zuma's 24-year-old fiancée, has apparently joined Twitter
- She immediately caused a stir with her posts, some of them declaring her love for Zuma
- She has also retweeted several of Zuma's tweets
PAY ATTENTION: Click "See first" under the "Following" tab to see Briefly News on your newsfeed!
Nonkanyiso Conco, the 24-year-old fiancée of former President Jacob Zuma, has apparently joined Twitter.
This comes almost a month after Zuma also joined the social media platform. Just like her fiancé, Nonkanyiso immediately caused a stir with her posts.
In one of them, she declared, "My hubby is fit and fresh, I love you bae @PresJGZuma."
PAY ATTENTION: Save mobile data with FreeBasics: Briefly is now available on the app
Nonkanyiso's first post was on 6 January, Briefly.co.za gathered. Apparently speaking for the Zuma family, she wrote:
Good morning South Africa. On behalf of Zuma family we wish you a prosperous 2019. This year is a game changer for us, as me and hubby we are on this platform #ngiyabonga.
READ ALSO: Woman airs her relationship drama about her bae and ex-bf on Twitter
In another post, Nonkanyiso shared a photo of Zuma superimposed on hers and declared that she's "wife number 7." She also urged people to mind their own business.
The young fiancée has retweeted several of Zuma's posts and videos. In her Twitter bio, she describes herself as:
A graduate in journalism, VumaFm employee and lastly but not least, I'm Jacob Zuma's youngest wife.
READ ALSO: Mother completes her LLB degree on domestic worker's salary
Enjoyed reading our story? Download BRIEFLY's news app on Google Play now and stay up-to-date with major South African news!


text:
Rising Aussie star "stoked" over title sponsor EFG's invite as she looks forward to pro debut
EFG Bank has confirmed one of Australia's leading amateurs, Rebecca Kay, as its sponsor invite for the 2019 EFG Hong Kong Ladies Open.
The 20-year-old from the Gold Coast will follow in the footsteps of Singapore's Koh Sock-hwee and Malaysian Winnie Ng, whose careers benefited from invitations in 2017 and 2018 respectively, gaining invaluable competitive experience ahead of joining the professional ranks later this year.
Kay, who first represented Australia as a 13-year-old, will compete against a record 123-player field headlined by LPGA Tour member and EFG Global Sport Ambassador Tiffany Chan Tsz-ching.
Chan, the pride of Hong Kong has been supported by EFG Bank for over a decade, first through the bank's sponsorship of the Hong Kong Golf Association Junior Programme, and then as one of the first beneficiaries of the EFG Young Athletes Foundation. She became an EFG Global Sport Ambassador when she turned professional in May 2017.
Kay will look to turn professional in the coming months following a decorated amateur career highlighted by a T6 finish at the World Amateur Championships last year in Ireland. Just like Tiffany two years ago, she will attempt to reach the gold standard of women's golf, the LPGA tour, at Q-School in the US in the autumn.
The rising star, who led after round one of the Women's Amateur Asia-Pacific in Japan, is relishing the opportunity to compete against seasoned professionals in Hong Kong, and looks forward to meeting Tiffany for the first time.
"I couldn't be happier when I first learned about this wonderful opportunity to play at the EFG Hong Kong Ladies Open and must thank EFG for this generous invitation.
"It feels awesome to know that the effort I have put in is being recognised internationally, and I am stoked and humbled to be given this chance by EFG when there are so many talented players they could have offered the invite to. I played the Hong Kong Golf Club in Fanling once before when I competed for Australia at the Queen Sirikit Cup in 2015, and cannot wait to return to play with Tiffany and some of the best golfers in the region," said Kay.
Mr. Albert Chiu, Executive Chairman, EFG Bank, Asia Pacific, commented:
"We are delighted to offer our 2019 EFG Hong Kong Ladies Open sponsor invite to Rebecca Kay. EFG has been a passionate supporter of golf for over a decade, especially in the development and support of young and talented local players, giving them every opportunity to reach their full potential with the hope that one day they will do their hometowns proud, just like what Tiffany has done for Hong Kong."
"Rebecca's achievements on and off the course over the past few years have caught our attention, and we see similarities between her career path and Tiffany's. We hope the experience she will gain at the EFG Hong Kong Ladies Open will put her in good stead to achieving her goals as a professional."
In addition to Tiffany Chan, the high-quality field also includes Thai stars Parinda Phokan, 2017 champion Supamas Sanchan, and Renuka Suksukont. Liu Wen-bo and Liu Yi-yi will spearhead China's challenge, while the LPGA of Taiwan will once again be fielding a strong contingent, led by Chen Yu-ju and Kuo Ai-chen. 15-year-old Chloe Chan Cheuk-yee, a scholar of the EFG Young Athletes Foundation, will be among eight home-grown talents flying the flag for Hong Kong this year.
The EFG Hong Kong Ladies Open tees off on Hong Kong Golf Club's Old Course in Fanling from 10th to 12th May. EFG Bank has been the title sponsor of this prestigious and popular event since 2017, which is sanctioned by the China LPGA Tour, Taiwan LPGA and Ladies Asian Golf Tour, and supported by the Hong Kong Professional Golfers' Association and Hong Kong Golf Association. Inaugurated in 2015, the EFG Hong Kong Ladies Open is the only golf tournament in Hong Kong in which players can earn Rolex World Ranking points.


text:
Category Archives: Food&Drink
The Food Collection #1: Christine's Classmates' Pot Luck Lunch Party
I don't usually photograph food… but the discussions were flowing around me. Rather than interrupt with my own poor Chinese conversation skills, I picked up the camera instead. I had no control of the lighting, and apparently no control of my ISO either. The only one that wasn't shot at ISO3200 was the Taro Cake (BTW, it was… Read More »
Beer Boxes with Empties: Wakayama Photo
Photos from Our Kitchen: Aunty's Quiche
I had a lot of fun making this, it's the first time I've really made Quiche. The recipe is from the Beeb, of course. Roasted Tomato, Basil & Parmesan. Thanks, Aunty! Alain Rakamr't suggested a slightly different pastry recipe, so I'm tempted to remake it this way. If you give it a go to DIY the dough: 250… Read More »


text:
Disney Winnie The Pooh Geo Eeyore Ceramic Mug£10.95
Disney Winnie The Pooh Geo Eeyore iPhone Case
Starting at: £13.99
iPhone Case - Winnie The Pooh Best Friends
Starting at: £13.99
Samsung Case - Winnie The Pooh Best Friends
Starting at: £14.99
About Eeyore:
Eeyore is a reliable friend; someone you can lean on in times of trouble. Eeyore can sometimes be quite insightful and is arguably the wisest and most down to earth resident of the Hundred Acre Woods. He is a blue and grey donkey, about 3-years in age and stuffed with sawdust. Eeyore can sometimes be a gloomy donkey that has a very bad habit of losing his tail with its small pink bow.


text:
Using ez-link card to mark school attendance
Madrasah students will need to scan their ez-link cards to mark their attendance, with a new app developed by Al-Amin Mosque and a group of volunteers called Void Deck Ventures. The pilot scheme, which will be tested at the mosque in Telok Blangah, was announced by Dr Yaacob Ibrahim, Minister-in-charge of Muslim Affairs, at the mosque's 25th anniversary dinner last night.
An SMS will be sent to parents when students are late or absent. They will also be notified when classes end.
Over 500 guests attended last night's dinner.
4 nabbed, contraband cigs found on speedboat
Four foreigners aged between 23 and 39 years old were nabbed after they tried to enter Singapore illegally on a speedboat heading towards Tanah Merah on Saturday.
A total of 532 cartons of contraband cigarettes were seized just before midnight, after their speedboat was intercepted. The coast guard detected the vessel in Singapore waters and alerted various land and sea resources, said the police.
Suspect arrested over alleged car theft
A 47-year-old man has been caught for allegedly stealing a motor vehicle in Turf Club Road on Saturday.
Police received a report that a car was stolen at 11.52 am. The suspect was arrested at East Coast Park less than 12 hours later.


text:
As a member of Global Compact and a main driver of energy transition, CNOOC actively takes low-carbon action to build a clean, safe and efficient energy system. Natural gas and power generation segment has become an important part of the mid- and downstream business of the Company, and accelerating the development of natural gas and power generation is in line with the restructuring of national energy consumption.
CNOOC Gas & Power Group concentrates on the Yangtze River Delta, the Pearl River Delta and the Bohai Bay Area, optimizes the layout of the industry, extends natural gas supply network and actively builds LNG receiving terminals, natural gas pipelines and gas stations. Through the efforts, it enhances coastal natural gas pipeline network to enhance supply capacity of clean energy and promote sustainable development in the surrounding areas.


text:
Article: Getting The ROI With AI
Looking back over the past couple of years, the one subject that seems to have captured communications professionals' imagination more than any other is Artificial intelligence (AI). A quick Google search for Contact Center AI yields over 770 million results. This demonstrates that a lot of people are talking about it but is everyone making sense? Does everyone even agree on what AI is?
In this Article, we will cover:
- The Background of AI
- What is AI?
- AI in action
- An ROI for AI