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Merkel cell polyomavirus (MCV), a previously unrecognized component of the human viral skin flora, was discovered as a mutated and clonally-integrated virus inserted into Merkel cell carcinoma (MCC) genomes. We reconstructed a replicating MCV clone (MCV-HF), and then mutated viral sites required for replication or interaction with cellular proteins to examine replication efficiency and viral gene expression. Three days after MCV-HF transfection into 293 cells, although replication is not robust, encapsidated viral DNA and protein can be readily isolated by density gradient centrifugation and typical ∼40 nm diameter polyomavirus virions are identified by electron microscopy. The virus has an orderly gene expression cascade during replication in which large T (LT) and 57kT proteins are first expressed by day 2, followed by expression of small T (sT) and VP1 proteins. VP1 and sT proteins are not detected, and spliced 57kT is markedly diminished, in the replication-defective virus suggesting that early gene splicing and late gene transcription may be dependent on viral DNA replication. MCV replication and encapsidation is increased by overexpression of MCV sT, consistent with sT being a limiting factor during virus replication. Mutation of the MCV LT vacuolar sorting protein hVam6p (Vps39) binding site also enhances MCV replication while exogenous hVam6p overexpression reduces MCV virion production by >90%. Although MCV-HF generates encapsidated wild-type MCV virions, we did not find conditions for persistent transmission to recipient cell lines suggesting that MCV has a highly restricted tropism. These studies identify and highlight the role of polyomavirus DNA replication in viral gene expression and show that viral sT and cellular hVam6p are important factors regulating MCV replication. MCV-HF is a molecular clone that can be readily manipulated to investigate factors affecting MCV replication.
Citation: Feng H, Kwun HJ, Liu X, Gjoerup O, Stolz DB, Chang Y, et al. (2011) Cellular and Viral Factors Regulating Merkel Cell Polyomavirus Replication. PLoS ONE 6(7): e22468. https://doi.org/10.1371/journal.pone.0022468
Editor: Jianming Qiu, University of Kansas Medical Center, United States of America
Received: April 20, 2011; Accepted: June 22, 2011; Published: July 22, 2011
Copyright: © 2011 Feng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health (NIH) CA136363, CA120726, the Al Copeland Foundation, and University of Pittsburgh EXPLORER grants to Patrick S. Moore and Yuan Chang, who are American Cancer Society Research Professors; and by NIH AI078926 to Ole Gjoerup. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have read the journal's policy and have the following conflicts: PSM and YC are inventors on products and assays developed from MCV. These rights have been assigned to the University of Pittsburgh. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Merkel cell polyomavirus (MCV) was identified by digital transcriptome subtraction from Merkel cell carcinoma (MCC), a rare but aggressive human skin cancer [1], [2]. MCV is a double-stranded DNA virus belonging to the Polyomaviridae family, members of which share conserved early, late, and regulatory regions. The polyomavirus early viral tumor (T) antigens play key roles in viral genome replication as well as tumorigenesis. Large T (LT) antigen-encoded helicase activity, for example, unwinds the viral replication origin [3], [4] and enhances the polyomavirus late promoter leading to an early-to-late switch in gene expression. For murine polyomavirus, this switch has been shown to depend on LT-initiated viral DNA replication [5]. The late region encodes viral capsid proteins (VP1 and VP2) that self-assemble into virus-like particles (VLP) when expressed in cells [6], [7], [8], [9], [10]. MCV VLP have been used to infect cells and can be used in neutralization experiments [6] but replication of full MCV genome has not been described. The concerted regulation and interaction of both early and late polyomavirus proteins are necessary to produce viral particles.
Loss of viral replication capacity, or permissivity, is a common feature of virus-initiated tumors [11], [12]. Approximately 80% of MCC are infected with MCV in which the viral genome is clonally-integrated into the host tumor cell genome, preventing viral replication [1], [13], [14]. MCV obtained from tumors also possess LT gene mutations that are a central feature of MCV-driven human tumor formation [13]. LT normally binds a specific site in the viral replication origin and initiates DNA replication through its C-terminal helicase domain. Tumor-specific mutations prevent LT-initiated DNA replication at the integrated genome thus preventing independent and unlicensed DNA replication from the viral genome that could lead to catastrophic replication fork collisions and DNA breakage when multiple virus-initiated replication forks proceed onto the cellular DNA template [13].
The minimal MCV replication origin has been mapped to a 71 bp fragment in a non-coding region that LT protein binds in order to initiate viral DNA replication. Among MCV proteins, LT protein alone is sufficient for this process but MCV small T (sT) protein acts as an accessory factor that greatly increases the efficiency of MCV origin firing [3]. We have recently described MCV sT as a transforming oncoprotein in MCC that inactivates the Akt-mTOR pathway protein 4E-BP1 and activates cap-dependent protein translation in MCV-positive tumors [15]. In one MCV tumor strain (MCV350), a point mutation in its replication origin prevents proper assembly of the LT helicase complex, also rendering the tumor-derived virus nonpermissive [3]. Additional virus mutations in capsid genes, including in the MCV350 strain, have been described that are predicted to prevent virion self-assembly and replication [16]. The sT and the N-terminal portions of LT, however, are unaffected by tumor-specific mutations, suggesting that they may play a key role in MCC tumorigenesis. The importance of viral early gene contributions to this cancer are shown by knockdown of the common T antigen exon 1 sequence, which leads to cell cycle arrest and cell death of MCV-positive MCC cells [17].
More than 50% of the healthy adult population is serologically positive for MCV antibodies [6], [7], [18], [19] and most adult MCV infections are asymptomatic [20]. In contrast to MCC tumors, only very low level MCV genomic DNA is present in healthy tissues, including skin, peripheral blood mononuclear cells, gastrointestinal tract [1], [21], [22], human respiratory tract secretions [23], and other tissues [21]. Using rolling circle amplification, Schowalter et al. have recently isolated several encapsidated strains of wild-type MCV from healthy skin [22]. Nonetheless, the limiting amounts of wild-type MCV flora present in healthy tissues have been a significant barrier to isolation of replication-competent MCV.
To search for novel cellular factors binding to MCV early proteins, we performed tandem-affinity pulldown assays with a unique region of the MCV LT [24]. An MCV LT domain that is conserved in both tumor-derived and wild-type MCV strains interacts with the cytoplasmic vacuolar sorting protein, hVam6p (also known as Vps39), a component of the HOPS (homotypic fusion and protein sorting) complex involved in late endosomal and lysosomal fusion [25]. Coexpression of MCV LT with hVam6p causes relocalization of hVam6p from the cytoplasm, where it is normally found, to the nucleus and to perinuclear bodies. This interaction can be abrogated by a single alanine substitution at tryptophan 209 (LT.W209A) in the hVam6p binding domain of LT. No differences in cell viability or cell cycling have been detected for the wild-type LT and LT.W209A expression and so the role for this LT interaction remains unknown. MCV LT antagonizes the ability of hVam6p to induce lysosomal clustering, raising the possibility that hVam6p might modulate MCV replication or egress [24].
Development of an MCV molecular clone allows engineering the viral genome, such as introducing a mutation at the hVam6p interaction site in LT, to examine effects on virus replication and assembly. To generate an MCV clone, we aligned MCV genomes from MCC tumors as well as non-tumor tissues and designed a consensus MCV molecular genome (MCV-HF). Subsequent to construction of this consensus clone, the same viral sequence was identified in several naturally occurring MCV strain obtained from healthy human skin samples [22], supporting the notion that MCV-HF is a permissive viral clone.
We show here that the MCV-HF clone is replication competent and sequentially expresses early and late viral proteins to generate packaged virions after transfection of the molecular clone DNA into 293 cells. MCV-HF has modest but reproducible replication capacity in a variety of tissue culture cell lines that is enhanced by MCV sT coexpression. In contrast, MCV engineered with the MCV350 origin point mutation is replication-deficient, only transiently expresses LT protein, has diminished or absent expression of other early spliced transcript protein forms and does not express late MCV protein. Overexpression of hVam6p is potent in inhibiting MCV replication whereas engineering MCV-HF with the LT.W209A mutation amplifies virus replication suggesting a key role for this vacuolar sorting protein in MCV virion production.
Design and construction of a consensus MCV genome
At the initiation of this study, no full-length viral genomes from nontumor sources had been isolated. We originally found 8 of 10 (80%) MCC tumors to be positive for MCV DNA [1]. We sequenced full-length MCV genomes by PCR-direct sequencing from 5 of the 8 virus-positive tumors (MCV350, MCV339, MCV344, MCV349, MCV352), as well as from 1 MCV positive cell line (MKL-1), which were compared to a single whole virus sequence (MCV85) obtained from a peripheral blood sample (Fig. 1A). All tumor-derived sequences have T antigen truncations, including mutations or short genomic deletions. For some strains, tumor-derived mutations are also observed in late gene regions, as reported by A. Zur Hausen's group [16]. For example, a 200-bp deletion is present in the VP1 locus of MCC352, generates a truncated VP1 protein that is likely to lead to incomplete viral assembly for this strain (Fig. 1A).
(A) Full-length of MCV genomes identified from 5 MCC tumors (MCV350, MCV339, MCV344, MCV349, MCV352), 1 MCC cell line (MCVMKL-1) and 1 PBMC sample (MCV85). T antigen ORFs are shown in blue arrows, VP ORFs in pink arrows. Numbers stand for positions in MCV genome. Black solid boxes indicate genomic deletions in MCV genome. (B) Phylogenetic tree of MCV genomes. The consensus genome (MCV-HF, JF813003) is located in the center of a tree including MCV350 (EU375803), MCV339 (EU375804), MCV344 (JF812999), MCV349 (JF813000), MCV352 (JF813001), MCVMKL-1 (FJ173815), MCV85 (JF813002) and other MCV sequences obtained from human skin [22] and Kaposi's sarcoma [40]. (C) The consensus MCV-HF genome can be linearized at BsrFI site (4,596 nt) and cloned for propagation in E. coli. Sites for mutations engineered into two MCV-HF genomes (MCV-Rep− (C/A) and MCV-hVam6p− (TG/GC)) are shown.
To address the issue of polymorphisms between and within individual cases, we designed a consensus genome (MCV-HF, GenBank ID: JF813003). This cloned genomic DNA (available through our website, www.tumorvirology.pitt.edu/mcvtools.html) is based on the 7 full-length MCV genomes. Compared to these genomes, the MCV-HF genome is located centrally in the phylogenetic tree (Fig. 1B), closest to wild-type R17a strain and has the same nucleotide sequence as the 17b, 18b and 20b strains identified by Schowalter et al. from human normal skin [22]. The consensus MCV-HF was synthesized and cloned into a kanamycin selectable vector (Fig. 1C). An MCV genome variant possessing the MCV350 mutation in the replication origin (MCV-Rep−) was generated by site-directed mutagenesis to serve as a negative control for viral replication [3].
MCV-HF and MCV-Rep− viral protein expression in 293 cells
MCV-HF or MCV-Rep− circular genome DNAs were transfected into 293 cells and viral protein expression was determined by immunoblotting for LT, 57kT, sT antigen and VP1 proteins (Fig. 2). The defective replication origin in MCV-Rep− genome does not affect initial LT antigen expression, indicating that this mutation does not directly alter early gene transcription. LT protein is readily detected 24 hrs after transfection for both MCV-HF and MCV-Rep− and equal amounts of LT expressed by day 2 for both clones. This reveals that the early promoter regulating LT is intact in both viruses and similarly active at time points prior to active viral replication and amplification. Surprisingly, expression of the alternatively spliced 57kT form is reduced with MCV-Rep− at day 2. This is not due to differences in detection since both LT and 57kT are determined on the same blots with the same CM2B4 antibody. LT protein expression continues over 5 days for the MCV-HF virus. LT protein levels peak at 48 hrs for MCV-Rep− and then subsequently decline. Notably, the spliced 57kT antigen protein is diminished at all time points for the MCV-Rep− clone. In contrast to LT and 57kT, no sT (an alternate spliced form from the early Tag locus) expression is detected after transfection of MCV-Rep−, but sT is readily detected by day 3 after transfection with the replication-competent genome. These findings suggest that splicing efficiency among early MCV genes may be dependent on viral genome replication.
One microgram of recircularized MCV genomes (wild-type MCV-HF or replication-defective MCV-Rep−) was transfected into 293 cells. Immunoblotting was performed to examine T antigen expression over 5 days (indicated by hollow arrows) and VP1 protein (indicated by solid arrow) using CM2B4 (LT, 57kT), CM8E6 (sT) and CM9B2 (VP1) antibodies, respectively. Alpha-tubulin detection was used as a protein loading control. LT protein is expressed equally at day 2 for both viruses but decreases for MCV-Rep− on days 3–5. VP1 increases on days 3–5 only for MCV-HF, corresponding to viral DNA replication (Fig. 3D). Other early proteins are also diminished (57kT) or absent (sT) in the replication deficient MCV-Rep−.
VP1 structural protein also increases in abundance from day 3 through 5 post MCV-HF transfection (Fig. 2), consistent with a switch from early to late gene expression to generate spontaneously assembling virus particles. VP1 expression, however, is not detected after MCV-Rep− transfection. Thus, in 293 cells, transient transfection of only the wild-type MCV-HF genome produces both early and late proteins required for virus replication and assembly. Alternatively-spliced early proteins (sT and 57kT) are diminished or absent, as is the late VP1 protein, for the MCV-Rep− virus despite similar initial levels of LT expression with both permissive and nonpermissive viruses.
Detection of MCV-HF virus in 293 cells
We next examined virion production in 293 cells transfected with MCV-HF. At day 4 post MCV-HF transfection, cells were harvested, lysed, matured overnight and treated with benzonase, RNase A and Plasmid-safe™ nuclease. Fractions were collected from an ultracentrifuged Optiprep™ (iodixanol) gradient and immunoblotted for VP1 protein (Fig. 3A). High molecular weight aggregates of the ∼45 kDa VP1 protein are present in fraction 4 having a 1.24 g/ml buoyant density. A high molecular weight VP1 form (∼90 kDa) is also present polyacrylamide gels that may represent covalently-crosslinked dimeric VP1 protein. VP1 is also present in the lowest density fractions 9 and 10, that represent unassembled, free VP1 protein. Fraction 4 contains typical 38–43 nm diameter polyomavirus particles detected by transmission electron microscopy with uranyl acetate negative staining (Fig. 3B). Quantitative real-time PCR for DNA isolated from Optiprep gradients reveals highest copy numbers of nuclease-resistant DNA in fraction 4 (Fig. 3C). In contrast, nuclease-resistant MCV genome is not detected in fractions 9 and 10, consistent with digestion of unencapsidated DNA. Encapsidated viral DNA increases up to day 4 after MCV-HF transfection and then plateaus (representative results are shown in Fig. 3D), correlating with VP1 protein expression levels. As expected for the nonreplicating virus, viral DNA is present immediately after MCV-Rep− transfection but diminishes below the level of detectability by day 5.
(A) Twelve fractions were collected from highest to lowest density, and analyzed by immunoblotting with CM9B2 antibody to detect VP1 capsid protein. Assembled 45 kDa VP1 protein is isolated in fraction 4. Unassembled, free VP1 protein is present in Fractions 9 and 10. The positive control (+) is virus-like particle (VLP) prepared from 293TT cells by MCV VP1 and VP2 transfection. (B) Typical 40 nm diameter icosahedral Merkel cell polyomavirus particles present in fraction 4 (upper panel) and VP1/VP2-containing MCV virus-like particles for comparison (bottom panel). (C) Nuclease-resistant MCV DNA in various gradient fractions quantitated by real time PCR. Highest levels of encapsidated DNA are present in fraction 4, corresponding to the fraction having MCV virions. (D) Time-course for MCV virion production after transfection of 1 µg replication competent (MCV-HF) or incompetent (MCV-Rep−) genomes into 293 cells was determined on lysed cells by quantitative PCR after nuclease treatment. Genome replication and packaging of MCV-HF is evident by day 3.
These findings are confirmed by Southern blotting for viral DNA (Fig. 4). In this experiment, DNA from 293 cells 4 days post-transfection, with either MCV-HF (lane 1) or MCV-Rep− (lane 2), were treated with DpnI to digest unreplicated DNA and with BamHI to linearize MCV genome. No bands are present for MCV-Rep−, while a weak 5.4 kb DpnI-resistant band representing full-length genome is present in extracts from MCV-HF transfected 293 cells. Significantly, a subgenomic 0.3–2 kb smear of DpnI-resistant MCV DNA is also present, consistent with a large fraction replicated MCV DNA being composed of either abortively-replicated or partially-digested genome fragments.
Southern blot (right panel) for MCV for MCV-HF (lane 1), MCV-Rep− (lane 2) and MCV-hVam6p− (lane 3) viruses four days after transfection of 1 µg circular genomic DNA into 293 cells. Panel on left shows the ethidium bromide-stained gel prior to transfer indicating equal DNA loading. Bands for the full-length 5.4 kb MCV genome are present as DpnI-resistant bands in MCV-HF and MCV-hVam6p− viruses (lanes 1 and 3) but not in the replication deficient MCV-Rep− virus (lane 2). The replication efficiency was measured by the ratio between the DpnI-resistant 5.4 kb band and the DpnI-sensitive band. The MCV-hVam6p− virus generates ∼2-fold more full length genome compared to wild-type MCV-HF virus. Replicated viral DNAs also show the presence of extensive subgenomic fragments.
Optimization of MCV clone replication
To determine whether coexpression of MCV early genes might enhance MCV replication, 293 cells were stably transduced with MCV sT, LT or empty vector expression constructs. MCV-HF and MCV-Rep− genomes were then transfected into these cells and MCV virion production was quantitated by real-time PCR. Cells coexpressing MCV sT generate approximately 5-fold increased nuclease-protected MCV DNA compared to cells without sT coexpression (Fig. 5). In contrast, only a small increase in MCV DNA is present in cells stably expressing LT protein, suggesting that sT but not LT protein levels are limiting for genome replication after transfection. Several other cell lines (UISO, 293TT, NIH3T3 and COS7) were also examined, with and without various MCV gene coexpressions, in an attempt to optimize MCV virion production (Table 1). None of these conditions led to appreciably greater MCV virion production compared to 293 cells alone.
One microgram MCV clone DNAs were transfected into 293 cells stably transduced to express MCV sT or LT proteins (not shown). DNA was extracted and treated with benzonase and RNase to discriminate packaged viral DNA. The nuclease-resistant MCV genome was precipitated and measured by quantitative PCR after proteinase K treatment. Cellular sT expression increases virion production for both MCV-HF and MCV-hVam6p− viruses. Comparison of MCV-HF and MCV-hVam6p− shows that loss of the hVam6p binding site also increases virus production. Coexpression of sT and mutation of the hVam6p binding site in the MCV genome are additive in virion production compared to MCV-HF without sT coexpression.
hVam6p interaction with MCV LT diminishes MCV replication
Tandem-affinity pulldown studies have found that MCV LT binds to hVam6p and relocalizes it from the cytoplasm to the nucleus, but the biological importance of this interaction is unknown [24]. A mutant MCV-HF, designated as MCV-hVam6p−, was engineered to encode a W209A substitution in the LT gene to prevent hVam6p interaction and to test its role in MCV replication. As seen in Fig. 5, mutation of the hVam6p-binding site leads to a 4–6 fold increase in nuclease-resistant virion production compared to the wild-type MCV-HF virus. Enhanced virion replication is additive with sT coexpression, suggesting that LT.W209A and sT coexpression are independent of each other and act at different stages of replication. Increased virion and subgenomic DNA production for MCV-hVam6p− was confirmed by Southern blotting (Fig. 4, lane 3). The comparable levels of DpnI-sensitive bands in all lanes on the Southern blot demonstrate that this effect is unlikely to be due to differences in transfection efficiency. Immunoblotting for encapsidated VP1 after gradient purification also reveals markedly increased viral particle production with MCV-hVam6p− genome (Fig. 6). In contrast, hVam6p coexpression inhibits nuclease-resistant, encapsidated DNA production from MCV-HF to levels comparable to the nonpermissive MCV-Rep− virus (Fig. 7). The MCV-hVam6p− clone replication is relatively resistant to the effects of hVam6p coexpression compared to MCV-HF but also declines in a dose-dependent fashion. hVam6p overexpression does not decrease 293 cell viability or alter cell cycling, nor does it alter transfection efficiency (data not shown).
Optiprep™ density gradient fractions from wild type (MCV-HF) and mutant viruses (MCV-Rep−, MCV-hVam6p−) generated from transfected 293 cells were used for Western blotting. Dilutions of MCV virus-like particles (VLP) provide a marker for the relative abundance of VP1 protein in each fraction. Assembled MCV-hVam6p− virus VP1 expression is ∼10 fold increased in fraction 4 compared to MCV-HF.
293 cells cotransfected with hVam6p and MCV genomes at day 4 as measured by nuclease-resistant DNA by quantitative PCR. Circularized viral plasmids (1 µg) together with varying amounts of hVam6p expression plasmid were simultaneously transfected during this experiment.
Since hVam6p is normally a cytoplasmic protein that relocalizes to nuclear and perinulclear sites when MCV LT is co-expressed, we sought to determine whether hVam6p might directly inhibit viral DNA synthesis in the presence of LT (Fig. 8). An in vitro origin replication assay was performed with either the entire T antigen gene locus, TAg (expressing LT, 57kT and sT), or the LT cDNA alone. These plasmids were transfected together with the MCV origin cloned into the pCR2.1 vector, with or without hVam6p expression. Southern blotting for the origin plasmid measures T antigen-initiated DNA replication (DpnI-resistant band) relative to unreplicated, transfected origin DNA (DpnI-sensitive band) [3].
293 cells were transfected with plasmids containing MCV origin and equal amounts of either wild type of genomic T antigen (TAg), LT cDNA, or the corresponding constructs containing the hVam6p-binding site mutations (TAg.W209A and LT.W209A). Origin replication was assessed through Southern blotting by comparing the ratio of DpnI-resistant (replicated) to DpnI-sensitive (unreplicated) DNA. For each condition, replication in the absence or presence of simultaneously cotransfected hVam6p expression plasmid was determined. Expression of the genomic TAg containing both sT and LT showed increased replication of the MCV origin compared to the LT cDNA regardless of hVam6p coexpression. Neither mutation of the hVam6p binding site on LT nor coexpression of exogenous hVam6p significantly altered MCV origin replication.
As previously reported [3], the genomic TAg locus (Fig. 8 , lane 3) expressing all early proteins is markedly more efficient at initiating origin replication than the LT cDNA alone (Fig. 8, lane 7), consistent with an accessory role for sT in LT-mediated DNA replication. Unlike virion production, however, coexpression of hVam6p does not significantly change in vitro MCV origin replication by TAg (Fig. 8, lane 5) or LT cDNA (Fig. 8, lane 9). Further, the LT.W209A substitution in either TAg or LT cDNA does not appreciably affect origin replication efficiency (Fig. 8, lanes 4, 6, 8 and 10).
Failure to achieve secondary MCV-HF transmission
We used fraction 4 (Fig. 3A), which contains encapsidated MCV virions to infect a variety of cell types including 293, 293TT, UISO, A549, BJAB, Raji, BSC40 and PBMC purified from whole blood with or without polybrene treatment. No cytopathic effect (CPE) was observed in long-term culture (4 weeks). Immunoblotting and immunofluorescence staining for T antigen and VP1 proteins were not positive for cells exposed to virus (data not shown). We also did not detect viral transcripts by RT-PCR (not shown), suggesting no secondary detectable infection occurred. Co-culture of MCV-HF transfected 293 cells with 293, A549 and UISO cells separated by 0.2 or 0.45-µm membranes on a transwell plate also failed to demonstrate secondary virus infection. These results may be due in part to the limited amount of infectious virus generated after transfection.
We generated a replicating MCV molecular clone that can be manipulated to assess effects on virus DNA replication and encapsidation. As previously seen in origin-replication studies [3], introduction of the MCV350 strain point mutation into the MCV-HF replication origin abolishes the clone's ability to replicate. We also confirm that MCV sT protein expression together with LT expression is required for optimal MCV replication, a feature of viral genome replication that MCV shares with the human JC polyomavirus [26].
Fully-encapsidated MCV virions from MCV-HF were isolated in fraction 4 at 1.24 g/ml on iodixanol (Optiprep™) gradients. Evidence that these are fully-encapsidated viruses include isolation of nuclease-protected DNA and ultra-high molecular weight aggregates of VP1 protein specific to this fraction. In the 1970s, hyperosmolar CsCl isopycnic gradients were used to isolate polyomaviruses having higher apparent buoyant densities (e.g., 1.34 g/ml [27]). It is now well-established that hyperosmolar CsCl gradients overestimate the densities of large macromolecular structures, such as viruses, since CsCl gradients dehydrate virions, replacing water with heavy salts, which reduces viability and artifactually increases the buoyant density. JC virus has a buoyant density of 1.20 g/ml on linear sucrose gradients and 1.35 g/ml in CsCl gradients [28]. Similarly, goose hemorrhagic polyomavirus virion has a 1.20 g/ml density in sucrose and an apparent 1.34–1.35 g/ml density with CsCl gradients [29], [30]. MCV DNA has been isolated from skin at 1.22 g/ml density using iodixanol gradients, which is in agreement with our findings [22]. The encapsidated MCV we identify in fraction 4 using uranyl acetate negative staining have the same size (∼40 nm) as similarly prepared MCV VLP but are smaller than MCV VLP (55–58 nm) visualized using phosphotungstic acid staining [7].
MCV, like other polyomaviruses, has a strictly regulated order of viral gene expression that leads to encapsidation of the viral genome. MCV LT and 57kT proteins are expressed early after MCV-HF transfection. Subsequently sT and VP1 (and presumably other virion protein components) are detectably expressed, leading to generation of self-assembling viral particles. Our study reveals an unexpected and interesting complexity for MCV gene expression. MCV LT, sT and 57kT are generated from alternatively-spliced, overlapping genes driven by the same early promoter. MCV-Rep− expression for LT is initially identical to MCV-HF, indicating that the early promoter is intact, but other early proteins are diminished (57kT) or absent (sT) suggesting the possibility that viral DNA replication also regulates early TAg splicing. Late VP1 protein expression also is not detected using the MCV-Rep− genome indicating that it is likely the early-to-late promoter switch also depends on viral DNA replication. This is similar to mouse polyomavirus, a close relative to MCV in the murine polyomavirus clade [5]. Well-established late lytic expression and packaging cascades for other viruses, such as herpesviruses, are also dependent on active viral DNA replication [31]. Although our results are consistent with DNA replication-dependent viral transcription, MCV-Rep− is mutated with a single pentamer sequence (PS) 7/8 substitution in the MCV origin and so we cannot exclude the possibility that this point mutation also affects late promoter activity in addition to genome replication. Additional studies are needed to determine if DNA replication also regulates MCV splicing patterns.
One key factor determining MCV-HF virion production is the abundance of sT protein, which our data suggests is in turn increased in a positive feedback loop during MCV replication. When sT is coexpressed with the transfected MCV-HF genome, there is 5–6 fold increased virus production. For mouse polyomavirus, sT can signal to AP-1 and PEA3 factors to promote viral replication and late gene expression [32]. Based on SV40 studies, the most prominent role of sT involves inhibition or retargeting of substrates for the major cellular phosphatase, PP2A [33]. We previously identified MCV sT as an important accessory factor for efficient MCV DNA replication [3], but it is unknown whether this is due to PP2A or 4E-BP1 targeting, or due to targeting of our other cellular factors [15]. The Southern blot in Figure 4 reveals that MCV DNA replication is sparse and most of the replicated DNA is fragmented. This suggests that MCV genome replication, at least under conditions of plasmid transfection, is inefficient and small increases in successful MCV genome replication may have a large impact on virion replication and encapsidation. Whether the same is true during natural infection will only be determined through development of a successful MCV transmission system.
Our findings that hVam6p inhibits MCV production are unexpected and, to a degree, paradoxical. MCV LT retains a conserved hVam6p-binding domain that represses virion production. Other polyomaviruses also encode gene sequences that repress viral replication [34], which might reflect the need for these small DNA viruses to suppress virus replication to sustain a chronic, persistent infection without virion production (e.g., latency). Although the effects of hVam6p on MCV replication are clear, the mechanism by which hVam6p suppresses replication is not. Our origin replication studies in the presence and absence of hVam6p overexpression, together with LT.W209A mutations in LT coding region, show no direct effect of hVam6p on initiation of viral DNA replication. Additional studies are also needed to investigate this effect, however, since Southern blotting reveals amplified viral DNA replication for MCV-hVam6p− virus compared to MCV-HF, suggesting hVam6p might be inhibitory to viral DNA replication in the context of the full viral genome.
It is noteworthy that overexpression of this vacuolar sorting protein has a profound antiviral effect on MCV replication. hVam6p possesses citron and clathrin homology domains, the latter being involved in MCV LT binding [24], that are important to its functions in the HOPS-CORVET complex as an accessory factor for endosomal fusion [35]. In yeast, the hVam6p homolog also has been reported to act as a guanine nucleotide exchange factor for Gtr1 that contributes to TORC1 activation [36]. An isoform of hVam6p, TRAP-1-like protein (TLP) regulates the balance between Smad2 and Smad3 in TGF-ß signaling [37]. Previous studies did not find either mTOR activation or TGF-ß signaling, however, to be appreciably altered by MCV LT expression [24]. In our current study, even low levels of hVam6p transfection reduce MCV-HF virus production to levels similar to the replication-deficient clone demonstrating that it is a potent factor restricting MCV replication. For the MCV-hVam6p− virus, hVam6p inhibits replication in a dose-dependent manner, which may be due to either the W209A substitution incompletely disabling the hVam6p-binding site or hVam6p acting in other steps of MCV replication beyond those depending on LT. If hVam6p plays a role in inhibiting egress of MCV, this might represent a novel component of innate immunity. Tetherin, for example, is a recently-discovered innate immune component that prevents enveloped viral budding from cells [38]. Detailed analysis of hVam6p's role in antiviral responses is beyond our current study but development of a replicating MCV clone that can be genetically manipulated provides a critical reagent for use in these follow-on investigations.
Secondary MCV transmission was not detected in our study suggesting that MCV may have a tissue tropism that is not easily modeled in undifferentiated tissue culture. MCV resembles some other small DNA viruses in this way, such as JCV as well as other human tumor viruses including human papillomaviruses (HPV), hepatitis B and C viruses and KSHV. Poor MCV transmissibility could be due in part to low virus yields from our molecular clone system that might be improved with other MCV strains or cell culture settings. Cloning MCV-HF, however, provides a useful tool for these testing conditions to optimize virus yield that may ultimately allow a laboratory transmission model.
Materials and Methods
Cell lines and clinical samples
293 cell line (ATCC), 293FT (Invitrogen Inc.), 293TT [7], NIH3T3 (ATCC), A549 (ATCC), COS7 (ATCC) and BSC40 (ATCC) were maintained in DMEM medium supplemented with 10% FBS, penicillin and streptomycin (pen/strep). MKL-1 [13], UISO [13], BJAB (ATCC), Raji (ATCC) and peripheral blood mononuclear cell (PBMC) were cultured in RPMI 1640 medium with 10% FBS and pen/strep. MCC clinical specimens (MCC350, MCC337, MCC339, MCC344, MCC345, MCC347, MCC349 and MCC352) and PBMC sample have been described [1]. Clinical samples were collected under University of Pittsburgh Institutional Review Board (IRB) guidelines for study of human tissues.
Characterization of MCV genomes and construct consensus MCV genomes
MCV genomes in MCC cases were directly sequenced with 13 pairs of contig primer sets as previously described [1]. Long PCR was performed to amplify the whole genome in a MCV positive PBMC sample with two primer sets (contig.1f-8r and contig.9f-1r). The consensus genome (MCV-HF) was generated from 6 tumor-type MCV genomes and 1 wild-type MCV genome using MacVector program (MacVector Inc.). The whole genome was synthesized by the DNA 2.0 Inc (Menlo Park, CA) and cloned into a Kanamycin selectable vector. The consensus MCV genome was linearized at a BsrFI restriction site (RCCGGY) in the VP region. A replication-defective MCV genome (MCV-Rep−) was mutagenesized from consensus MCV-HF with 5′-GAA AAA AAA GAG AGA GGA CTC TGA GGC TTA AGA G-3′ and 5′-CTC TTA AGC CTC AGA GTC CTC TCT CTT TTT TTT C-3′ primers, using the QuikChange Lightning Site-Directed Mutagenesis kit (Stratagene). MCV-hVam6p− was generated with the primer set (W209A.S: 5′-GAA CGG ATG GCA CCG CGG AGG ATC TCT TCT GC-3′, W209A.AS: 5′- GCA GAA GAG ATC CTC CGC GGT GCC ATC CGT TC-3′) to eliminate the hVam6p binding to T antigen [24]. Plasmids containing MCV-HF, MCV-Rep− or MCV-hVam6p− were propagated in E. coli JM109 and purified using the Qiagen maxiprep kit (Valencia, CA). Linear MCV was digested out with BsrFI and re-ligated into circular form under low concentration of T4 ligase (1 U/µl, New England Biolabs) overnight at 16°C. Circular DNA was further digested with AvaI to linearize non-MCV DNA, and treated with Plasmid-Safe™ Exonuclease (Epicentre, Madison, WI) to isolate circular MCV genomes. All three genomes were sequenced confirmed.
Nuclease-protection assay
Cells transfected with MCV-HF or MCV-Rep− or MCV-hVam6p− were collected and lysed with 3 freeze-thaw cycles in DPBS-Mg2+ buffer. After centrifugation, supernatants were treated with 250 units of benzonase (Promega) and 5 units of RNase A (Ambion) at 37°C for 4 hrs. EDTA was then added to inactivate nuclease. Proteinase K was used to digest capsid proteins at 56°C for 1 hr. The capsid-protected MCV DNA was prepared by phenol-chloroform extraction and dissolved in 50 µl TE buffer. One microliter of DNA was used for PCR quantification with VP2 primers and TaqMan probe as previously described [39].
293 cells and stably infected 293 cells lines with LT or sT lentiviral vector (293-LT or 293-sT) were seeded in 6-well plates and transfected with 1 µg circularized MCV genomes using Lipofectamine 2000 (Invitrogen). Radio immunoprecipitation assay buffer (RIPA) was used to lyse cells. Monoclonal antibody CM2B4 [39] was used to examine LT antigen and 57kT antigen expression at 1∶2000 dilution. Monoclonal antibody CM8E6 [17] was used to detect sT antigen expression at a dilution of 1∶250. Late gene expression of VP1 was examined with monoclonal antibody CM9B2 at dilution of 1∶2000. The CM9B2 antibody (IgG2b isotype) was generated by immunizing mice with KLH-derivatized (DKGKAPLKGPQKASQKES) peptide from MCV VP1 using standard methods (Epitope Recognition Immunoreagent Core Facility, University of Alabama) and tested for reactivity to the MCV VP1 protein [7]. Tubulin (Sigma) was used to quantitate sample input at dilution of 1∶2000.
Lentivirus production and infection
293FT cells (Invitrogen) were transfected with lentiviral construct containing MCV LT or sT antigen together with packaging plasmids, psPAX2 and pMD2.G (Addgene) in 100 mm dish by Lipofectamine 2000 (Invitrogen). 293 cells were infected with lentivirus in the presence of 1 µg/ml polybrene. At day 3 after infection, puromycin (3 µg/ml) was added, and infected cells were selected in bulk for 7 days for stably expression of MCV LT and sT antigen. Expression was confirmed by immunoblotting.
MCV origin replication
For MCV origin replication assay, 293 cells were transfected with a plasmid containing MCV origin (Ori339(589)) [3] together with wild type (genomic T (TAg), LT) or mutant type (TAg.W209A, LT.W209A) of T antigens as well as hVam6p gene [25]. These cells were harvested at day 2 after transfection. Low molecular weight DNA was extracted with modified Hirt-extraction and Southern blot analysis was performed by method previously described [3] with a MCV origin probe (5,074–65 nt) amplified by PCR with primers: 5′-CTC GAG AGC AAT TTC ACC AAT ATT GGC C-3′ and 5′- GAT ATC TAA GCC TCT TAA GCC TCA GAG TCC-3′. To measure MCV clone replication, 2.5 µg of extracted DNA was digested overnight with 5 units of DpnI and BamHI and subjected to Southern blotting. Same MCV origin probe was used to measure replication efficiency. The blot was analyzed by using a PhosphorImager (Typhoon 9400, GE Healthcare) and ImageJ software (National Institute of Health, USA).
Virion purification and cell infection
At day 4 after post-transfection, cells were harvested and matured overnight with benzonase, RNase A and Plasmid-safe™ nuclease as previously described [7]. Cell lysate was separated on a 27-33-39% Optiprep (Sigma, St. Louis, MO) gradient after ultracentrifugation for 3.5 hrs at 234,000 g. Fractions were collected after puncturing the bottom of ultracentrifuge tube using a 25-G needle and stored at −80°C. Viral particles were stained with 1% uranyl acetate negative staining and observed on JEOL JEM-1011 (Tokyo, Japan) electron microscope at 80 kV and compared to self-assembling VP1-VP2 MCV virus-like particles generated as previously described [7]. In MCV infection assays, various cells (293, 293TT, UISO, A549, BJAB, Raji, BSC40 and PBMC purified from whole blood) were cultured with 4 µl ultracentrifuged fractions containing MCV virions together with or without 1 µg/ml polybrene (Sigma) treatment. For transwell experiments, 6-well transwell plates were used (Corning, New York, USA) with a 0.2 or 0.45-µm pore size polycarbonate membrane. Transfected 293 cells with MCV-HF genomes were labeled with intracellular fluorescent dye 5- (and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) to monitor any cell contamination in transwell experiments. 293, A549 and UISO were seeded in the lower chamber to co-culture with transfected 293 cells for 6 days.
We thank C.B. Buck for sharing wild-type of MCV genome sequences and providing A549 and 293TT cells, E.D. Mirvish for comments, and Herman Poole Blount (Sun Ra) for help on the manuscript.
Author Contributions
Conceived and designed the experiments: HCF HJK XL YC PSM. Performed the experiments: HCF HJK XL DBS. Analyzed the data: HCF HJK XL OG YC PSM. Wrote the paper: HCF HJK XL YC PSM.
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Have you ever seen a phone the size of a person? That is basically what I had beside my table at this years Fieldays. This interactive screen that anyone could use to view our training videos was a very nice bonus. I think I even learned something from it playing over and over so many times.
The Way To A Man's Heart
Honda has allowed me to represent Broxy Rider Coaching at their stand for 15 years now. I love this opportunity to catch up with friendly faces, and everything else about it just keeps getting better. A few years ago they really ramped up their hospitality with incredible food and coffee for Honda customers, and you know how food is the best present a man can get.
This year they included the race bike of HRC Honda rider Todd Waters alongside Cody Coopers blinged up machine, with Mitchell Rees' Superbike sporting his new number 2. All of the bikes were set up for you to hop aboard, with the kids range once again the obvious favorites.
Head Swell
Lining the walls was a completely new display of images showing bikes and their riders in epic locations and various states of awesomeness. I was especially stoked with the canvas style display of me wheelying my favorite Africa Twin right next to a massive cliff. It will look great on my garage wall at home if they let me take it when they are not using it any more!
Of course being able to sign stuff is the real highlight for me. We had another very cool poster to give away, but my favorite was signing the back of someones iPhone, reminding me that this definitely is the Millennial Generation.

subset: commoncrawl


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1) What is the difference between American and National Conferences in Valley League (VL)?
There is no difference between American & National Conferences, it's for naming purposes only. Both conferences include teams playing 35 game season & teams playing 45 game season. Standings are based on winning percentages & not points. All teams make the playoffs.
2) At the end of the season will each team within a division end up having played the same number of games?
Yes, except for mites, where there are American and National teams mixed together.
3) The names of our teams on VL are different from wysa.net is there a particular logic behind the new name assignments?
The Valley League (VL) has classified all teams as Elite, AAA, AA or A within each age group. After each realignment, a team may be moved to a different division (ie AAA to AA), however their name would not change again to avoid further confusion. That is why you may see AAA and AA teams in the same division.
4) In the "full standings" there is and RPI #, what does that mean?
The RPI is a ranking system used within each division; the higher the RPI the better the team is ranked. It is used to rank teams when the VL is trying to determine if a team needs to be moved to another division (it takes into account strength of schedule, scores, etc). Once a team is moved to another division, however the RPI is no longer meaningful until you play games in your new division. It is not used at all for standings, playoff seedings, etc- it is only used internally by the VL to put teams in the correct divisions to keep play competitive.
5) When do Valley League playoffs begin?
Playoffs typically begin in late March and end before Easter Sunday.
6) How do I download my team's schedule onto my iphone?
Click on "teams" and choose your team from the pull down menu.
7) What are Playdowns?
During the month of March each year, Massachusetts Hockey conducts State Tournaments at each level (see level classifications below). Mass Hockey breaks the state into 10 districts - WYSA is considered a part of District 10 –Middlesex County. Each District Champion advances to the State Tournament. On occasion, a District Tournament Runner Up also advances to the State tournament, but typically just the District Champion. For those teams fortunate enough to advance, it can be a truly great experience for players and parents.
In order to determine the District champ, each of the ten Districts conducts what are called "Playdowns". For each district, all of the teams that enter the playdown for that particular district play hockey games in a tournament. The number of Playdown games is simply a function of how many teams from other Programs within District 10 sign up for the Playdowns. The Playdowns are a single elimination tournament. You have to win to advance to the next round.
District 10 Playdowns have traditionally been over Martin Luther King weekend. The games and venues are set by the District Registrar and games typically cannot be rescheduled. WYSA will work with existing league games to take care of any existing conflicts. All WYSA teams are entered into the Playdowns as WYSA feels all players should have the opportunity to participate. Teams are expected to show for their game. The District takes it very serious if a team is a no-show and will impose a heavy fine on the association. A no-show can also impact another WYSA team's participation in the State Tournament should they advance. WYSA typically covers the costs of the first playdown game for all teams as part of the tuition (see financial contract). Subsequent games, should a team advance, would be the responsibility of the team to pay. Generally, each player on the team pays about $10-$20 towards the game depending on the cost that year. Game fees must be paid in advance of the game.
The teams can only play the players officially listed on their rosters (no call ups from lower teams). In addition, each Coach must meet the coaching certification requirements as well. Coaches who are not "patched" at the appropriate level will not be allowed on the bench. Coaches must have their CEP levels up to date by 12/31 of the current season. No player or coach will be allowed on the bench if they are not listed on the official, stamped USA roster. Any changes that need to be made to the roster prior to playdowns must be approved by the WYSA board and require certain fees to the District.
The Mass Hockey State tournaments are for different levels broken down as follows:
• Mites. There is no State Tournament at the Mite Level.
• Squirts
• Peewees
• Bantams


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We all know that first impressions aren't always correct.
Earlier today, I wrote that there didn't seem to be as many women out on the streets in Varanasi as there were in the south. Well, it turns out that they don't come out until later in the day.
When we made our second trek down to the riverside today, this time to watch the Ganga Aarti ceremony,* there were many more women out than there had been in the morning. Presumably, in the morning they were taking care of getting-their-families-ready-for-the-day things. And, as it was slightly warmer this evening, there were fewer coats and much more color to be seen. So, I apologize for any misinformation I have spread in the guise of my hasty impressions.
*Blog post coming soon. I have to wait until Joan air drops some photos.


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Product Description
TR100 Mul-T-Lock Hockey Puck Padlock.
Solid machined hardened steel body fitted with a highest level Interactive+ pin in pin cylinder*.
Mul-T-Lock, an ASSA ABLOY company, is famous for its high security protected key systems.
This TR-100 puck lock is fitted with Mul-T-Lock's unique Interactive keyway with floating pin and 5+5 telescopic security pins.
A second level of security is in the restricted keyway.
Keys can't be duplicated without presentation of the personal key code card.
The cut-out back (stepped) allows for the lock to be safely used direct on trailer or container door closing arm brackets but it also fits in our PL770 and PL775 hasps.
Standard amount of keys per lock is 2 keys and one code card.
Keyed Alike sets are always available and ship with 2 keys and code card for the entire set.
Extra keys can be ordered.
- High Security Platform
- Multiple Locking Elements Including the Patented Interactive Disc
- Pick, Drill and Bump Resistant
- Key Order Cards for Enhanced Key Control
*Many cheaper puck locks have a die-cast metal body and are fitted with cheap China made cylinders.
Write an online review and share your thoughts with others.


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The end of I-O-Us is upon us!
The rise of social media has finally brought payment technologies to the masses, and while some of the most popular mediums have features that can be seen as intrusive, nothing is scaring new users away from the convenience of touch and pay. Here are some companies that are leading the pack when it comes to social payments:
Parent company: Braintree / eBay
Launch: Winter 2012
Perhaps THE app that brought payments to the social media generation, Venmo simplifies payments and invoicing in a way that just didn't exist before. Venmo risked being too social by implementing a public comment feature for every exchange, but despite it being a massive example of overshare in this day and age, it took off. It seemed to take off even more after a much maligned ad-campaign nearly took over NYC. It just goes to show that the best way to convert a client is having a good product.
Parent company: eBay
Est.: Summer 1998
eBay's child is still leader of the pack when it comes to e-commerce, online payments, and online exchanges. It has had years and years of lead-time on a lot of the other platforms to embed itself in online culture. With the growing popularity of Venmo, PayPal has started to put more attention into mobile payments, instant payments, and making its app into something that is as easy to use as Lucas' favorite. Whether PayPal can gain back some ground with the youth that Venmo has started filling remains to be seen, but one wonders if they really mind, being that they share a parent company with Venmo and all.


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4.3 out of 5 stars with 11 reviews
LUFFY Coco Petite Nana -Plant on coconut shell Suitable for Nano Tanks Best Placed on Rock or Wood for Beautiful Tanks
List Price
Our Price
* Pricing Information Updated Less Then A Minute Ago
How do these petite nana plant grow?
As the petite version of the anubias plant family, the Luffy Petite Nana is slow growing; however the plant will begin to spread out as new leaves sprout. If you would like to add more greenery to your tank, anubias nana petite plants are easily propagated as they grow from cuttings and rhizome division. The rhizome is the stem of this aquatic plant and can be cut up and regrown to reproduce the petite nana. When re-planting, remember to keep the roots from submerging in the substrate to prevent your new plants from rotting.
What types of conditions are ideal for this plant?
Adaptable to a range of moderate water temperatures from 70-85° Fahrenheit (20-30° Celsius) and pH levels between 6 and 8, the Luffy Petite Nana Plant on a coconut shell can be used as a mid-ground or foreground plant. Although the anubias nana petite plant can live in light levels from low to moderate to high, it is important to keep the plant out of direct lighting or else they could become covered with algae.
Can I put the anubias nana petite plant in any aquarium?
Because of their size, petite nana plants from Luffy are perfect for freshwater aquariums of all sizes. These versatile plants are resilient in different temperatures and water conditions, making them excellent for nano tanks with an array of fish from goldfish and guppies to common plecos and angelfish. The petite size of these beautiful green aquatic plants is ideal for small aquariums such as betta fish tanks.
QUICK AND SIMPLE TO DEPOSIT INTO YOUR TANK --- Also known as the Anubias Nana Petite, this beautiful petite nana plant from Luffy comes already attached to an all natural coconut shell for easy installation. As the smallest member of the Anubias family, the Luffy Petite Nana is approximately 2" tall by 2.5" wide (5.5cm x 6.4cm) with round, dark green ½" (1cm) leaves. It is ideal for nano tanks and water vessels.
NATURALLY ENHANCES WATER QUALITY AND BENEFIT YOUR PETS --- Fish and other aquatic life require oxygen in order to breathe and survive. Because the petite nana is a living aquatic plant, it naturally produces essential oxygen. Luffy Petite Nana plants absorb undesired carbon dioxide and nitrates through their leaves, therefore removing them from your aquarium water. So, the more such plants your tank has, the more benefits your carp, tetra, platies, black molly and other smaller fishes will get.
LOVED BY ALL TYPES OF MARINE LIFE --- Easy to take care of and adaptable to a wide range of water, pH and lighting conditions, the Petite Nana Plant on a Coconut Shell by Luffy is a great aquatic plant for many species. Herbivorous fish, guppies, cichlids and more love to nibble on the plants and hide in the leaves.
LIVENS UP AQUARIUMS AND WATER DECORATIONS --- A favorite of hobbyists because of its hardiness, the petite nana plant adds a natural, beautiful splash of rich green color to your aquarium, fish tank or unique water decoration. Just like marimo moss balls, this adorable aquatic plant requires little maintenance and thrives without any special care. Plants like these are perfect for making creative handmade gifts such as mini centerpieces or charming desktop adornment.
COCONUT SHELL PROTECTS ROOTS OF PLANT --- The all natural coconut shell is domed to protect the hard fibrous stem (called a rhizome) of the Anubias Nana Petite plant. This convenient shell also holds the plant securely in place at the bottom of your tank and has very little effect on the pH levels of your aquarium water. If you prefer a look other than the coconut husk, you can cover it with a substrate, but be sure not to bury the rhizome or the plant will begin to rot.
Product Information Specifications for LUFFY Coco Petite Nana -Plant on coconut shell Suitable for Nano Tanks Best Placed on Rock or Wood for Beautiful Tanks Below:
|Marimo Pet Company
|Package Weight
|0.05 pounds
|Package Size
|4.7 x 1.3 x 1.3 inches
|Units in Package
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paramyotonia congenita(redirected from paramyotonia)
Also found in: Dictionary, Medical.
Related to paramyotonia: Paralysis periodica paramyotonia
paramyotonia congenita['par·ə'mī·ə′tō·nē·ə kən′jen·əd·ə]
A heredofamilial condition characterized by recurrent muscular stiffness and weakness (myotonia) on exposure to cold, as well as on mechanical irritation; transmitted as an autosomal dominant and considered to be a variety of the hyperkalemic form of periodic paralysis. Also known as Eulenburg's disease; myotonia congenita intermittens.


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It's now time to renew your North Coast Boardriders membership! To kick off 2019 we are offering NCB t-shirts at cost to anyone signing up again. Membership fees for 2019 will be $30 for a family or $20 for an individual and $15 per tee per member.
Other perks include:
– Casual gym membership to Coastal Fitness in Ngunguru for $13 per week
– Free entry to club comps, with a BBQ put on for members
– Access to Grom Squad and Little Rippers Training at no cost, pending numbers (contact Tia or Josh for more details)
– Support to attend national competitions
– Access to training programs like judging courses and surf workshops at little to no cost
– A platform for community development, kaitiakitanga, sharing the love of surfing, youth development and heaps more.
Head over to the 'memberships' tab for all the info on how to join/renew your membership.
We have some cool stuff planned for this year so don't miss out!


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Double Dhamaka Offer:
To counter Airtel, Jio has announced Jio Double Dhamaka Offer. This offer provide 1.5GB extra data per day for select prepaid recharge packs.
This Jio Double dhamaka offfer will be effective right from today – June 12 , till June 30.
Jio is lainching a fresh Rs. 499 recharge along with the offer that offers 3.5GB data per day for 3 months.
What is the offer:
Under this Offer, subscribers will get exrra 1.5GB data per day at no additional charges on recharges purchased before June 30.
Hence, the recharge packs with 1.5GB data per day ( Rs. 149, Rs. 349, Rs. 399, and Rs. 449) will get 3GB per day now.
Similarly 2GB data packs will get 3.5GB per day for packs that offer 2GB data per day
Other recharge packs – Rs. 299 – 3GB per day, Rs. 509 -4GB per day, and Rs. 799- 5GB per day packs will also get the additional 1.5GB data daily, so their per day cap increased to 4.5GB, 5.5GB, and 6.5GB respectively.
There is no change in the validity of the packs and they will continue to come bundled with post-FUP speed of 64kbps, unlimited calls and access to Jio apps.
All new Rs.499 Recharge pack:
Jio launches all new new Rs. 499 recharge pack. This pack offers 3.5GB data per day for 91 days.
Additionally, Jio subscribers can use the Rs. 50 vouchers which they received earlier while buying packs with this offer.
Users can avail 20% discount upto Rs.100 if they recharge via MyJio app. The transaction is made using PhonePe.


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To order images and/or obtain permission to use them commercially, please contact the KSHS Reference Desk at firstname.lastname@example.com or 785-272-8681, ext. 117.
For more information see the Copyright and Permission FAQ.
Viebrock enlisted in the Army (Air Force) in 1942 and served until 1946. Interviewed by Brian Grubbs on May 30, 2007, Viebrock talked about military experiences in the Second World War. The 2005 Kansas Legislature passed a bill funding the WWII Veterans Oral History grant program. This transcript is from one of the community institutions receiving grants. The transcript of the interview is presented here; the original video copy of the interview is available through the Watkins Community Museum of History (Lawrence) and through the Kansas State Historical Society.
Creator: Viebrock, Fred
Date: May 30, 2007
Item Number: 212788
Call Number: World War II Oral Histories Project
KSHS Identifier: DaRT ID: 212788
Collections - Oral History - Kansas Veterans of World War II - Watkins Community Museum of History, Lawrence, KS
Date - 2000s - 2007
Military - Service - Air Force
Military - Wars - World War II
Objects and Artifacts - Communication Artifacts - Documentary Artifact - Film
Places - Cities and towns - Lawrence
Places - Counties - Douglas
Places - Other States - Missouri
Thematic Time Period - World War II, 1939 - 1945
Type of Material - Film and video


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The Dolphin, Newbury
Muddy says: Shoreditch House meets Miami Beach in Newbury town centre, this pub with stylish mid-century rooms is grown-up, glamorous and fun.
The Dolphin in Newbury may sound like your typical boozer but, believe us, it isn't. The show-stopping orange velvet sofa, , the walls peppered with tongue-in-cheek but cool art, kitsch knick knacks (try saying that after a few bevvies), brass palm tree floor lamps. craft beers and cocktails. Once your cross the traditional threshold, you're in a house of fun.
The five fresh out of th box B&B rooms are equally stylish – with their deliciously crispy bed linen, mid century furniture and pillow menu, they're city boutique hotel standard. What's more, the Modern British menu is uniformly excellent. Book a beach hut in the garden for a party, private dining or meetings can be booked in The Fish Tank and the restaurant is perfect for a bigger do.


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She does something new everyday.....
Today she pulled all her books off her shelf. I silently watched her do it because I think that is what you do with your first born!
Of course you let her do it! It probably won't be so much fun after she does it a dozen more times, but every "first" is fascinating time in the life of a baby. So glad you documented it!
She is such a cutie! Look at that sense of accomplishment on her face! Can you believe her 1st birthday will be here before you know it. I was thinking about Brenner turning 1 yesterday..CRAZY!Miss Lucy will be getting an invite!!


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Ex-footy star Campbell Brown has shared horrific photos of his father-in-law's feet after his dog chewed off his big toe.
Brown's father-in-law, Tom Johnson, who can't feel his feet because of diabetes, had his right toe bitten off by the feisty dog – named Savel - two years ago.
But two days ago, the same thing happened again.
Mr Johnson woke to find his left toe was nibbled half off.
He was rushed to hospital, where doctors amputated the toe.
"Quick update. Toe amputated. Chihuahua winning 2 toes to nil," Brown tweeted rather light-heartedly.
Brown then shared a photo of the innocent looking dog.
"Savel the cute little Chihuahua," he tweeted.
Doctors told Mr Johnson it will take weeks to make a full recovery, as he now has to learn to regain his balance.
"He wanted to give the dog away the first time but my mother-in-law said no," Brown told the Herald Sun.
"There's a fair chance the dog may now get the boot."
Brown, 34, played for Hawthorn until 2010 before leaving the club to join the Gold Coast Suns until 2013.


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Understanding pain and its management
Pain is perhaps the most common symptom of human ailments. Pain is an unpleasant sensation that may be due to physical injury, disease or psychological i.e. without any reason. Usually pain classified as visceral pain and somatic pain. Visceral pain arises from internal organs and is experienced as burning pain as in gastric ulcers, spasmodic pain and spasmodic pain as in intestine colic or as angina pain that arises from heart. Whereas somatic pain arises from body parts like muscles, bones and joints. It may be superficial or deep musculo-skeletal pain.
Being a leading pain management clinic Singapore, we at Specialist Pain International (SPI), strive to provide effective pain management is provided through our team of pain management specialists.


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Lee Strobel is a bestselling author and the former award-winning legal editor of The Chicago Tribune. With a journalism degree from the University of Missouri and a Master of Studies in Law degree from Yale, he has won four Gold Medallions for publishing excellence, and he co-authored the Christian Book of the Year. He serves as a professor of Christian thought at Houston Baptist University. For more information, visit leestrobel.com.
El caso de los milagros (The Case for Miracles)
By Lee Strobel
Read by Mauricio Perez
Un periodista investiga la evidencia de lo sobrenatural (A Journalist Investigates Evidence for the Supernatural)
How do we differentiate between a miracle and a coincidence?
Released April 24, 2018
Spanish, Unabridged


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A jury's knowledge of a rental payment prosecutors kept hidden wouldn't have changed the verdict in the case of a man convicted of killing a woman with a machete in 2007, a Clark County judge ruled Friday.
Search results for:
Sierra Oeste, considered for years one of the most violent neighborhoods in Las Vegas, has undergone a year-long makeover that has already shown serious benefits for residents and police alike.
In a more than two-hour discussion at Ebenezer Church of God in Christ led by outgoing Rep. Steven Horsford, D-Nev., on Friday, more than 15 black faith leaders and seven North Las Vegas police officers and the chief of police talked about what the department can do to build trust between law enforcement and communities of color.
A judge increased bail Friday for a woman accused of leaving the scene of a hit-and-run accident that killed a man while he was pushing his granddaughter in stroller in November.


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The highly-touted Department of Energy's grid study is anodyne and irrelevant but worthwhile nonetheless. Commissioned by new energy secretary Rick Perry in April, the order for the study charged the Department of Energy staff to examine whether current policy was responsible for base load coal and nuclear plant retirements that jeopardize the reliability of the nation's electric grid.
Many expected that the study would be a political screed, beginning with unwarranted assumptions and ending with foregone conclusions, aimed at candidate Donald Trump's claim that he would reverse the Obama administration's "war on coal."
That isn't what happened. Rather, the long-awaited study released this week found what virtually every analyst who has looked at the events on the ground over the last decade has concluded. Cheap natural gas, weak demand for power, and a push for renewables has doomed high-cost coal and nuclear generation.
Industry interests, particularly the National Mining Association and the Nuclear Energy Institute, have cherry-picked the report to argue that it supports major changes to the current electricity markets. The nukes said the report supports valuing atomic power for qualities that aren't recognized in competitive markets, such as low carbon dioxide emissions and 24/7 reliability. The coal industry wrapped itself in the national security flag. West Virginia Governor Jim Justice, erstwhile Democrat and newly-minted Republican, proposed a $15/ton subsidy for Eastern coal, claiming that terrorists could blow up gas pipelines and jeopardize the grid.
Independent experts have greeted the industry spin with scorn. Ari Peskoe at Harvard's Environmental Law Program wrote, "There is nothing new" in the DOE grid study. He noted that "DOE's 2016 Quadrennial Energy Review (QER) already recognized these trends, and aimed to ensure that regulators and market operators had the tools to ensure reliability in the face of dramatic changes that challenge many of the industry's long-held assumptions."
Peskoe criticizes Perry's cover memo unveiling the study, which focused on the Trump theme of changing rules and markets. That, focus, Peskoe said, attempts to "shoehorn old, inflexible generators into a rapidly evolving and more nimble grid.
Veteran energy economist and analyst Sue Tierney, who served as energy policy chief in the Clinton administration's DOE, said the high-profile DOE grid study "ends with a thud." She commented, "Now that the report is actually out, what's surprising is that there are not many surprises. In keeping with other recent actions by the DOE, it provides little technical support for bailing out financially struggling coal-fired power plants. And that, in itself, is the main eye-opener."
Tierney notes that the DOE study, which breaks no new ground, is notable for avoiding any references to global warming. She wrote, "This omission undermines the department's call to provide proper compensation for certain power plants―notably, existing nuclear plants and hydroelectric facilities which, like coal-fired plants, are currently under financial stress in various wholesale markets. Without a willingness to point out that nuclear and hydro facilities produce power with zero carbon emissions, and that few wholesale markets properly compensate generators for their zero-carbon attribute, the DOE grid study misses an opportunity to support reasonable reforms to market rules. And it underscores the impression that this report's goal is to support coal-fired generation, consistent with the Trump administration's pro-coal agenda."
The report is anodyne. It largely reports that conventional wisdom and breaks no new ground. It is irrelevant. The policy changes it suggests are out of the DOE's authority and would require major action by Congress, a non-starter. But it is worthwhile, because Perry accepted the report and does appear to have exerted little political muscle to alter it. Maybe Perry is intellectually honest (unlike his boss)?


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As a fully-functioning e-commerce marketplace, customers can purchase products and services directly on the website, all while having access to browse business information, custom services, upcoming events, and more.
Customer Benefits:
Shop & Buy Online
Save time and find great local products. No longer will you have to visit every storefront or website of businesses in your community to find what you're looking for. You'll be able to shop and buy from many local businesses right in the comfort of your own home. No store hours, we're always open!
Orders Fulfilled the Way You Like
Choose the order fulfillment the way you want it, with options from in-store pickup, to delivery, to shipping, or instant download – get the products on your terms.
Keep $ Local
When you shop local you're contributing to the economic growth of your own community.
Similar to other online marketplaces, businesses have their own shop on the website and their products/services can also be found through the general product feeds and searches.
- Businesses can choose to have a full e-commerce shop and sell their products/services directly on the website, take orders, and fulfill them as indicated by the customer (in-store pick up, delivery, shipping, or instant download).
- Businesses can also choose to make some or all of their products lead-only products, where they can highlight their product or service offerings, show examples of their past work, and get lead inquiries right to their inbox.
- Businesses have a back-end login to manage their shop, products/services, respond to inquiries, and fulfill orders.
Business Benefits:
Community of Businesses
Selling together online, in partnership with your local Chamber of Commerce and other business owners, creates even more exposure for your business vs. having to drive traffic to your individual website.
Statistic – In the last year, shoppers have spent the most money online with e-commerce marketplaces – source.
Guided Selling Support
We know there are a lot of ways to sell online and that can be overwhelming. We've simplified the process and have made it easy for you to sell online with our website/shop support, knowledge base resources, and customer service.
Low Cost Website/E-Commerce Solution
A shop on our website could serve as your business website (if you don't have one). Otherwise you can link to your shop from your website for purchasing your products/services, giving you online e-commerce functionality without paying a web developer to set up your shopping cart and related processes.
Before we add on a new community, we're looking to partner with a local Chamber of Commerce, Main Street Community, or Economic Development Group in the area. We know that having your local expertise, business support, and community trust will help to ensure the site is successful in your community. We want your help to attract local businesses and the community to utilize the website.
Site partners have a small annual investment to bring the Shop Where I Live platform to their community. This investment is based on the number of businesses supported by your chamber/main street. This investment may be 50% – 100% discounted in subsequent years if target benchmarks for the number of businesses that are selling on the site are met.
Chamber/Main Street Benefits:
Managed Website
We do it all! We know you are busy and are often limited on time and resources. We handle everything related to the website from design and maintenance, to business support, and even customer service to your community.
Member Discounts
As a benefit for your support of the website, your member businesses receive a hefty discount to sell on the site, creating another great benefit your members receive for belonging to the Chamber/Main Street.
Attract New Members and Create Non-Dues Revenue Through Sponsorships
Give non-members a reason to join the chamber when they are able to learn about and see the support you give to your members. Your logo, messaging, and information is infused into the website.
Chambers/Main Streets can also sell sponsorship opportunities/advertisements for the website. A Shop Where I Live community website is a great way for businesses to gain exposure to a local audience. We'll help to add your sponsor logos or advertisements to the site.
We identified e-commerce as a priority initiative, as e-commerce is one of bricks and mortar's greatest competitors. We are excited to announce a partnership with Shop Where I Live.com that will help Marion's small businesses to maintain an e-commerce platform together and take advantage of each other's customer bases – there is power in numbers" – Jill Ackerman, President, Marion Chamber of Commerce


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Monday, August 8, 2011
Tralee had to give the scripture in Primary yesterday. We worked on it all week, and by Sunday she had it memorized perfectly. When her time came to give the scripture, she got up to the podium and. . . nothing. She froze. Jim got up to help her (I had a child from my class on my lap), but he later told me Tralee had it down so well prior to this, that he didn't bother to memorize it himself and he left his own scriptures on his seat. So, then there were two frozen people looking out into the sea of tiny Primary faces. Jim looked at me and asked, "how does it go?" I mouthed to him the beginning, and then pointed out on the wall where the scripture was posted. Eventually Jim and Tralee got through, but it sure didn't go smoothly. Right after church we asked Tralee to say the scripture again, and she nailed it. I guess the pressure got to her this time, I mean, she's only four! Plus, it was really noisy in the Primary room; too many distractions don't lend well to recitation. The good news is, Tralee looked perfectly darling up there, and we're proud she powered through, even though she looked like she wanted to be anywhere but up on the stand. Sweet little girl.
2 comments:
- Boom said...
Awww, I'm sorry she froze. Maybe she will get another chance soon. And hopefully it won't be a whole long missive like it was this time.
Love Grandma Charlene
- August 8, 2011 at 5:58 PM
- Necha said...
So cute. The first time Tessa gave a talk she got up there and hid behind the podium for half the talk while I gave it, and then she poked her head up and said the second half. Then when she was done she SCREAMED...."I DID IT!" It was pretty funny. Kids are the best! :)
- August 9, 2011 at 7:04 AM
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See series description for full review.
A Beka does not sell their materials to retail bookstores, preferring instead to sell their products through representatives and through their website. As a result, we can offer only used A Beka, and can't guarantee we'll get it. We offer here the current retail price of the book for your information, but if our page does not say "Add Used to Cart," we do not have a copy.
World History Maps are designed for teaching and reviewing the information presented in the 7th and 10th grade texts History of the World and World History and Cultures. They are correlated with World Atlas and Geography Studies: Eastern Hemisphere and with World History and Cultures Tests/Map Projects. The daily plans for 7th and 10th grade history indicate which maps to use and when to use them. The Scope and Sequence for each grade gives the teacher an overview of student map work.
The physical, political, and historical maps may be used to point out important locations during the history lesson, to teach and review the Geography Studies, and to go over the Geography Projects. They may also be used for quizzes and review games and contests. The physical and political maps are labeled on one side for teacher reference and unlabeled on the other for student review.
Learn more about our store location and hours
19146 Molalla Ave,
Oregon City, OR 97045


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Groupe PublicActif il y a 1 an et 9 mois
You'll most likely get interested in investing in a pc. That enthusiasm can rapidly use dread as you are searching for your computer you wish to purchase. How could you get the best computer feasible? The following advice will help you find out what you need to know about investing in a computer.If you are going to get doing a little updates in your computer be sure you terrain yourself before you begin caring for your computer. A simple jolt of static electric power could be more than sufficient to fry your motherboard. Unless you ground on your own, you will be getting your whole system at an increased risk.Creating your own personal laptop or computer can save a ton of money, when as well making a program that is certainly doubly powerful as you would get if you purchased it coming from a producer. Creating your own pc saves time, dollars, and reduces the center man. 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Anyone Can Use A Computer With The Proper Guidance
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Symptoms of a traumatic brain injury (TBI) do not always show up immediately after the accident. The physical and psychological damage can take days or even weeks to appear. Symptoms may include:
The above symptoms are just a few of the many signs and symptoms of a traumatic brain injury. A detailed list of symptoms can be found on the Mayo Clinic website.
One of the leading causes of traumatic brain injuries are motor vehicle accidents. TBI's are caused by a sudden blow or jolt to the head, which often happens when you're in a car accident. If you've been injured in an accident and believe you may have suffered a traumatic brain injury, you should seek medical attention. A traumatic brain injury is not an injury to be taken lightly and should be treated as soon as possible by a medical professional.
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We decided to do a hike in Lake Mead Recreation Area, a place we haven't really explored near las Vegas.
This hike has a little bit of everything - rock hounding, mining ruins, canyon narrows, red rocks, a peak, and really fun route finding.
The hike was a loop so it didn't really matter where we left the truck. Could have driven this first mile and a half but would have had to walk it at the end. Decided to do the road in the first part of the hike.
About a mile from where we parked we came across this sign.
minesignore car mine
Some tailing piles and the wash at the exit of Anniversary Narrows. We'll drop down into there and walk a couple miles up the canyon.
Muddy wash
anniversary narrowscanyonwash
Starting up the canyon.
Right from the start the patterns in the rock were really striking.
anniversary narrowscanyon
A short ways up the canyon we came across these ruins and some tunnels.
anniversary narrowscanyonminetunnel
Finally we get to the narrows section. Really nice rock through here, but bad lighting. Oh well.
Towards the end of the narrows there is a short section of this angled and wavy really pink rock. Haven't seen anything like this before.
Emerging from the narrows.
After a long walk through the wide upper canyon, we turn out of it by scrambling up this dry fall. Finding the proper exit is tricky - there are lots of hills and walls an canyons running through here and it's easy to get walled out. I was very thankful for a detailed GPS track, but even then it was a bit complicated.
After finding our way across the more complicated terrain, we gain this ridge and follow it to the low point on the range.
The ridge to the saddle
anniversary peaksaddle
David following the ridge. You can see the maze-like terrain behind us. The canyon we came from is to the left.
View from the saddle into the Bowl of Fire
anniversary peakbowl of fire
Bowl of Fire
To complete the loop we head along the range ridge to the high point "Anniversary Peak". We can descend into the bowl of fire from the less insanely steep slope on the other side of the peak.
View into the Muddy Mountains from the climb to Anniversary
muddy mountainsanniversary peak
David camo'd against the rock on the final piece of the scramble to the summit. This was a fun one!
anniversary peakscramble
On the summit of Anniversary Peak - I don't think it's an official name (nothing labeled on any maps I have found) but hey, there was a register.
Looking back along the ridge. Canyon approach to the left of this image, Bowl of Fire on the right.
Lake mead in the distance. The terrain was much gentler on this side of the peak, nice for the descent.
anniversary peaklake mead